Annals of Medicine & Surgery Publish Ahead of Print

DOI: 10.1097/MS9.0000000000003015

The Neurobiological Impact of Oxytocin in Mental

Health Disorders: A Comprehensive Review

*1 1 2 3 4
Ram Prasad Chaulagain , Yelona Shrestha , Harisharan Shrestha , Rameshor Bhandari , Praful Gurung

*1. National Academy of Medical Sciences, Bir Hospital, Mahaboudha, Kathmandu, Nepal. [Link]@[Link]

1. National Academy of Medical Sciences, Bir Hospital, Mahaboudha, Kathmandu, Nepal. yelnastha@[Link]

2. Civil Service Hospital of Nepal, Minbhawan Marg, Kathmandu, Nepal. sthaharisharan@[Link]

3. Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. drrameshorbhandari@[Link]

4 National Academy of Medical Sciences, Bir Hospital, Mahaboudha, Kathmandu, Nepal. prafulgrg53@[Link]

The Neurobiological Impact of Oxytocin in Mental

Health Disorders: A Comprehensive Review

Abstract
Oxytocin, a neuropeptide, plays a significant role in modulating social behavior and has been
widely studied for its potential impact on mental health disorders. This review examines the
neurobiological mechanisms through which oxytocin influences mental health and its therapeutic
potential in conditions such as autism spectrum disorder, schizophrenia, PTSD, anxiety, and

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depression. Oxytocin enhances social bonding, trust, and empathy by modulating neural circuits
linked to social interactions. Studies indicate that oxytocin's ability to regulate the hypothalamic-
pituitary-adrenal (HPA) axis plays a vital role in stress response and emotional regulation.
Therapeutic applications, particularly intranasal administration of oxytocin, have shown promise
in alleviating symptoms and improving patient outcomes. However, personalized approaches are
essential to optimize treatment effectiveness. Despite its potential, challenges remain in
understanding the mechanisms underlying its effects and identifying the patient populations that
would benefit most from such therapies. Future research should focus on elucidating these
mechanisms, exploring the long-term efficacy of oxytocin-based interventions, and advancing
personalized medicine to maximize its clinical utility.

Keywords: Oxytocin, Mental Disorders, Neurobiology, Social Behavior, Therapeutic Potential,

HPA Axis

Short Title: Oxytocin in Mental Health.

Introduction:
The magnocellular nuclei of the hypothalamus synthesize the hormone oxytocin and
subsequently store and release it by the posterior pituitary gland. Oxytocin, a nine-amino acid
polypeptide, is primarily a neuromodulator in the brain and peripheral tissues. It is synthesized
from prooxyfizine, an inactive precursor. The precursor is subjected to enzymatic degradation,
resulting in the formation of oxytocin and neurophysin. Neurophysin plays a role in facilitating
the transportation and storage of oxytocin 1. Oxytocin is activated through further enzymatic
hydrolysis of ascorbic acid and distributed for physiological functions 2.
The degradation of oxytocin primarily occurs in the liver and plasma via oxytocinases.
The phenomenon substantially impacts a range of physiological systems, including but not
limited to the cardiovascular, renal, muscular, and reproductive organs 3–5. The objective of this
study is to consolidate existing research on the involvement of oxytocin in mental diseases,
examine its influence on social and emotional mechanisms, and evaluate its potential therapeutic
uses in the context of mental health. To support our objective, we have included Table 1, which
summarizes key findings regarding oxytocin's role in various neurological and psychological
disorders.

Localization and synthesis:

 Neurohypophysial hormones are classified into the AVP and OT families based on the
amino acid at position 8, which determines receptor binding efficiency. OT, a nonapeptide with a
disulfide bridge between Cys1 and Cys6, includes a COOH-terminal alpha-amidated tail.
Vertebrates universally possess OT-like and AVP-like peptides, reflecting their evolutionary roles
in reproduction (OT) and water homeostasis (AVP) 18,19.
OT and AVP are synthesized in the paraventricular (PVN) and supraoptic (SON) nuclei of
the hypothalamus. Precursors, such as preprooxytocin, are processed in neurosecretory vesicles
through glycosylation, amidation, and proteolytic cleavage, yielding OT, neurophysin, and a
glycoprotein. These products are transported to the posterior pituitary for release 20,21. OT, when
secreted, acts on distant organs like the kidney and mammary gland, mediating uterine
contraction, lactation, and water regulation 22,23.
Triggers such as nursing, labor, dehydration, and stress release OT locally in the PVN and
SON, where it acts as a self-neuromodulator. OT influences anterior pituitary hormones,
including prolactin, ACTH, and luteinizing hormone (LH). For instance, OT enhances LH
secretion and may accelerate ovulation, while it suppresses ACTH under stress, potentially
modulating the hypothalamic-pituitary-adrenal axis 24–29.
Peripheral OT is also produced in tissues like the uterus, testis, and heart, with its exact
roles and interactions, particularly regarding other hormones, still under investigation 30,31.

THE OXYTOCIN-GENERATING SYSTEM

Hypothalamus-Hypophysis:
The hypothalamic-neurohypophysial system, composed of the paraventricular (PVN) and
supraoptic (SON) nuclei [Fig.1], is the primary oxy-neurosecretory system 32,33. Oxytocin is
mainly synthesized in the magnocellular regions of the PVN and SON, with their neurons
projecting to areas like the medial amygdala and the lateral septum 24. Magnocellular
oxytocinergic neurons release oxytocin into the posterior pituitary, which then enters circulation
to act on peripheral organs 34. Oxytocin does not easily cross the blood-brain barrier, and
peripheral stimulation (e.g., milk sucking) may not affect central oxytocin levels 34. Electrical
stimulation of the neurohypophysis releases oxytocin peripherally, while stimulation of the PVN
releases it centrally and peripherally 35. Following hypophysectomy, oxytocin concentration
decreases in the blood but increases in the cerebrospinal fluid (CSF), where its half-life is longer
than in plasma 36–38. Local dendritic release of oxytocin also occurs in the PVN and SON, crucial
for breastfeeding and post-partum feedback loops 25. Parvocellular neurons, smaller than
magnocellular neurons, project to areas like the vagus motor nucleus and sympathetic spinal
centers 30,31,39. Oxytocin may also play a role in regulating adenohypophysial hormones such as
gonadotropins, prolactin, and ACTH, supported by its fibers passing through the hypophyseal
portal system to the anterior pituitary40,41 42
Receptor for Oxytocin:
The oxytocin receptor is a G protein-coupled receptor with seven transmembrane
domains. Its activation triggers phospholipase C-β, leading to the production of inositol
trisphosphate (IP3) and diacylglycerol (DAG) 42. IP3 facilitates calcium release, while DAG
activates protein kinase C (PKC), phosphorylating downstream proteins. Elevated calcium
activates calmodulin, stimulating nitric oxide synthase and guanylate cyclase, which enhance
smooth muscle contraction in the myometrium and mammary gland to support parturition and
milk ejection 42

Oxytocin and Social Behavior:

Oxytocin regulates several neuroendocrine reflexes and is essential in developing
complicated social behaviors or coordinating behaviors linked to reproduction, partner selection,
childcare, or community life. For instance, male and female rats and mice's sexual behaviors are
controlled by oxytocin receptors in the paraventricular nucleus 43. Additionally, oxytocin has
potent anti-stress properties and modulates the hypothalamic-pituitary-adrenal axis 44,45. By
stimulating affiliative behavior, the oxytocin system contributes to social behaviors. Oxytocin
has a role in group-related behaviors, such as deciding how to react to a stranger or another
group member [Fig. 2].

Oxytocin's primary effects include regulating neuroendocrine responses and generating

intricate social and bonding behaviors associated with reproduction and childrearing 46.
Additionally, oxytocin has potent anti-stress properties that may promote pair bonding. One of
the most crucial aspects of the oxytocin system is its ability to modulate sex hormones.

Autism and oxytocin:

The relationship between oxytocin and autism has been the subject of much investigation
over the past 20 years. When Modahl examined the amount of oxytocin in autistic children's
blood in 1998, she found that it was much lower than it was in the control group 47. After
oxytocin delivery, autistic repetitive behaviors subsided in 2003 in a different experimental
environment. Adults with autism also benefited from this, although these benefits were
particularly noticeable in their capacity to comprehend and interpret speech tone 48 accurately.
 Functional neuroimaging supports the role of oxytocin in autism, particularly in the
mesocorticolimbic system. Studies show that dopamine and oxytocin are linked in these brain
areas, crucial for oxytocin's effects in autism. Intranasal oxytocin treatment increased activity in
regions like the left premotor cortex and ventral striatum during socio-emotional recognition
tasks in autistic individuals, but reduced activity in non-social tasks involving only objects 7.
Additionally, oxytocin enhanced functional connectivity between the ventral striatum and the
ventromedial prefrontal cortex, highlighting the importance of mesocorticolimbic areas in its
mechanism [Link] autism, where social connection is crucial, the social aspect of oxytocin's
activity may have a significant effect. Numerous investigations have been unsuccessful in
determining the therapeutic advantages of oxytocin for individuals with autism in terms of
general social behavior 50. Nevertheless, a recent study demonstrated that oxytocin has positive
benefits on social functioning in autistic people and that these effects are also impacted by
endogenous vasopressin levels 51. How oxytocin affects social behavior still needs to be fully
understood. The subject of specific theories is how oxytocin affects the dynamics of the social
response. The capacity of oxytocin to affect sensitivity to external incentives and, hence, directly
promote reward-based learning behavior may be a mechanism of action. Preclinical research
suggests that oxytocin exerts prosocial effects via the mesocorticolimbic dopamine pathway 52.
The interaction between oxytocin and dopamine in the mesocorticolimbic system causes
dopaminergic activity to rise when oxytocin neurons in the ventral tegmental region are
activated.
Additionally, after receiving an oxytocin receptor agonist, mice exhibit a reduction in
dopaminergic release in the nucleus accumbens, indicating the importance of oxytocin in the
mesocorticolimbic transmission of dopamine 53. Studies have shown that oxytocin does not
instantly play a prosocial function, but it does modify how people and animal models respond to
certain social cues connected to the conservation instinct. Growing interest in oxytocin's ability
to improve social communication deficits has arisen from the need for effective therapies for the
symptoms of autism spectrum diseases.
Research indicates that oxytocin administration positively impacts various aspects of
social functioning in autism, such as enhanced emotional receptivity and self-confidence 54.
However, core symptoms like restrictive and repetitive behaviors (e.g., hand flapping, object
lining, or echolalia) that disrupt social interactions show no significant improvement with
oxytocin. Most studies favor antipsychotics for managing these behaviors 55. A recent meta-
analysis also strongly associates low oxytocin levels with autism in children.
Additionally, oxytocin levels in people with autism were identical to those in neurotypical adults,
indicating that there may be a higher amount of oxytocin in neurotypical children that may
support social interaction and growth, with a reduction in age. In future research, oxytocin
therapy in childhood may be the top contender for addressing significant social deficiencies and
enhancing cerebral growth in autistic individuals [Link] investigations, including those
carried out by various experiments, showed that intranasal oxytocin enhanced social cognition
and decreased repetitive behaviors in people with autism, in addition to reducing repetitive
behaviors in mice. These results highlight the possibility of using oxytocin as a treatment for
ASD 57.

Schizophrenia and oxytocin:

Other forms of mental disease, including schizophrenia, may include oxytocin.
Considering the social deficiencies connected to severe mental disease, this is not altogether
unexpected. Significant evidence suggests that oxytocin may be a critical factor in the etiology of
schizophrenia because of the inverse relationship between plasma oxytocin levels and the
severity of schizophrenia symptoms. More severe symptoms have been linked to much lower
oxytocin levels.
Schizophrenia symptoms are linked to lower plasma oxytocin levels, particularly
negative and cognitive symptoms, though the exact relevance remains unclear. Low oxytocin
levels may be causative, a result of the disorder, or a response to antipsychotic medication 58.
Studies also found no consistent correlation between plasma oxytocin levels and the severity of
positive symptoms. Interestingly, female patients with positive symptoms had higher plasma
oxytocin levels than male participants 59.
Positive symptoms correlate constantly with oxytocin levels in the cerebrospinal fluid
and plasma, whereas negative symptoms have a consistent negative correlation with both 60,61.
This is in contrast to positive symptoms. Furthermore, several genetic studies have linked low
oxytocin levels to unpleasant sensations. For instance, a variation of the oxytocin gene
(rs2740204) has been linked to negative symptoms in people with schizophrenia. The oxytocin
receptor mutations rs53576 and rs237885 have been shown to significantly correlate with
negative symptom scores as determined by the PANSS scale (positive and negative
schizophrenia symptoms scale) 62. The rs53576 oxytocin receptor gene variation has been linked
to the emotional disengagement characteristic of schizophrenia in several investigations 63.
Higher peripheral oxytocin levels are frequently linked to enhanced social cognition,
prosocial behavior, and more excellent emotion perception, which is relevant to the cognitive
aspect of schizophrenia. Higher plasma oxytocin levels have also been linked to more excellent
emotion perception, avoidant behavior towards angry faces, and more accurate socially relevant
information coding in schizophrenia64,65 . Additionally, a greater oxytocin level boosts working
memory and the efficiency with which information is processed 66. Intranasal oxytocin may thus
be a contender as an addition to antipsychotic therapy for improving negative and positive
feelings as well as potentially reversing social cognitive deficiencies 67. One cannot yet draw a
broad conclusion because not all research has found these links. As a result, the importance of
oxytocin in schizophrenia is still not entirely understood.
Several studies have shown that oxytocin treatment results in symptom improvement in a
range of mental health conditions. For instance, individuals with schizophrenia demonstrated
enhanced emotion recognition and social cognition after oxytocin administration 68. Similarly, in
patients with PTSD, oxytocin was effective in reducing conditioned fear responses and
improving emotional regulation 69. These findings suggest that oxytocin may play a significant
role in modulating mental health symptoms beyond its well-documented effects on social
behavior.

Post-traumatic stress disorder and oxytocin:

OT promotes the extinction of an activated avoidance reflex and reduces passive
avoidance behavior, as well as memory consolidation and retrieval 70. Patients with post-
traumatic stress disorder (PTSD) were shown to have reduced memory recall and conditioned
response after receiving intranasal OT 71. It appears that alterations to the OT system following
early traumatic stress and abuse may block brain development and raise the chance of PTSD and,
more generally, mental illnesses later in life 72. It was also hypothesized that increased P.E.P.
activity might contribute to the pathophysiology of the behavioral and affective symptoms of
PTSD through an increased degradation of different neuropeptides because it was discovered that
P.E.P. activity was increased in PTSD patients, especially in those with a concurrent major
depression 73.

Disorders of anxiety and oxytocin:

Because OT has been shown to impact affiliative behavior in non-human animals, several
ideas have been proposed regarding its potential involvement in human emotions and
relationships 74. Additionally, it is thought that pregnancy, a time when OT levels are elevated,
might help avoid several anxiety disorders, including panic disorder. It has been shown that
mothers' OT levels are positively correlated with social skills, calmness, and tolerance 75, as well
as a decrease in the prevalence of stress and anxiety disorders7. Stress-related OT release, which
mostly has anxiolytic effects, appears to be an essential regulator of the fear and anxiety response
76–78
. It is interesting to note that OT significantly impacts how the amygdala functions, as it can
reduce amygdala activity and its linkage to brain areas responsible for the autonomic and
behavioral response to fear 79 when administered intravenously. This is due to the amygdala's
role in the biological reaction to threat cues in social interaction. Recent research has connected
the pathophysiology of social anxiety disorder to the downregulation of OT receptors, which
may assist in explaining the cognitive distortions seen often in this illness's victims 7.

Depression and oxytocin:

Studies suggest that neuropeptides regulate affect and are crucial for mood stability and
anxiety prevention. Research into the relationship between neuropeptides and monoamines
highlights oxytocin's potential role in the pathophysiology of affective disorders. Clinical and
animal studies link oxytocin imbalances to depression, demonstrating its positive role in
depression management and its frequent dysregulation in depressive states80 . Oxytocin levels are
often lower in postpartum depression compared to controls, and low oxytocin during pregnancy
may predict postpartum depression81,82 . However, the impact of oxytocin administration on the
emotional state of pregnant women is inconsistent, with postpartum depression being more
commonly associated with oxytocin delivery.
When examining the effects of oxytocin, it's important to consider the multifactorial
influences, including hormonal, genetic, and social factors. Some pregnant women may require
perinatal oxytocin treatment, which could indicate existing hormonal or neuropeptide
deficiencies that make them more prone to depression 83. Insufficient oxytocin release has been
linked to early breastfeeding cessation and postpartum depression 84. A recent study showed that
in women with depressive symptoms, oxytocin released during breastfeeding helps moderate
cortisol levels in response to stress 85. Research suggests that oxytocin may play a role in
depressive disorders beyond the postpartum period by promoting social behavior and reducing
stress responses. Clinical evidence indicates that severe depression may lead to an imbalanced
oxytocin system.
The outcomes of clinical research comparing peripheral cortisol levels with serum
oxytocin levels in depressed individuals were dissimilar. While some studies found no
appreciable changes between these parameters in depressive patients and control participants 86–
88
, others found a drop or even an increase in oxytocin levels in depressed patients 89–91. The
decline in oxytocin was significantly more dramatic when fibromyalgia and depression were
present. Due to these variations in clinical expression, certain forms of depression may be
connected to various amounts of oxytocin. Additionally, even when depressed, somatoform
manifestations are associated with a greater oxytocin level. In addition, individuals with social
withdrawal as the primary symptom of their depression may have lower levels of oxytocin than
other patients, particularly those with impulsivity as their primary symptom. Another crucial
factor in understanding variations in the peripheral level of oxytocin is sexuality.
Both in the context of unipolar depression and the presence of bipolar depression, female
patients' oxytocin levels were shown to be lower than those of male subjects 92. Addiction,
motivation, survival, nutrition, and sexual activity are all behaviors connected with rewards, and
the mesocorticolimbic pathway—where dopamine and oxytocin interact with one another as a
primary function—is crucial in accomplishing these behaviors 93. Oxytocin may be responsible
for altering how the depressed person perceives reality, making social interactions appear hostile
or unpleasant. As a result, oxytocin system failure may be a risk factor for asocial behavior 94.
According to observations in the literature, oxytocin levels in depressive patients alter both
before and after citalopram therapy. After the onset of the therapeutic response, the authors saw
considerable alterations. According to the evidence, oxytocin could be a vital mediator of the
therapeutic impact of SSRIs 95.
Evidence suggests that sildenafil, commonly prescribed for erectile dysfunction, can also
stimulate oxytocin release, producing an antidepressant effect 16. Similarly, the oxytocin agonist
carbetocin has demonstrated antidepressant effects in animal studies, with results comparable to
 the tricyclic antidepressant imipramine, as indicated by behavioral changes (e.g., swimming and
immobility time) 96. Additionally, oxytocin treatment may have beneficial effects in treating
various mental disorders, including anxiety, schizophrenia, autism, drug addiction, and anorexia.

Despite promising outcomes, several limitations must be considered when evaluating

oxytocin as a therapeutic agent. Individual response variability, influenced by genetic factors and
receptor sensitivity, makes it difficult to predict who will benefit most from treatment.
Additionally, measuring central oxytocin levels complicates understanding its precise
mechanisms of action. These challenges highlight the need for more accurate tools and methods
in future research.

Conclusion:
This review highlights novel insights into the oxytocinergic system's role in mental
disorders, emphasizing its multifaceted impact on conditions like autism, schizophrenia, PTSD,
anxiety disorders, and depression. We propose innovative perspectives on oxytocin's functions
across these disorders, suggesting that its modulation can uniquely influence social behavior,
emotional responses, and cognitive processes in each condition. Importantly, our analysis
underscores the potential of personalized oxytocin-based therapies tailored to specific symptom
profiles in mental disorders. We advocate for further research on elucidating oxytocin's
mechanisms in mental health and identifying patient subgroups most likely to benefit from such
interventions. This approach could revolutionize treatment strategies in mental health care,
moving towards more targeted and effective therapies.

Conflict of Interest:
There is no conflict of interest.

Acknowledgement:
I really appreciate the Journal Editor’s valuable feedback.

References

1. Brownstein MJ, Russell JT, Gainer H. Synthesis, transport, and release of posterior pituitary hormones.
Science. 1980;207(4429):373-378. doi:10.1126/science.6153132
2. Florea T, Palimariciuc M, Cristofor AC, et al. Oxytocin: Narrative Expert Review of Current Perspectives on
the Relationship with Other Neurotransmitters and the Impact on the Main Psychiatric Disorders.
Medicina (Kaunas). 2022;58(7). doi:10.3390/medicina58070923

3. Sheldrick EL, Flint AP. Post-translational processing of oxytocin-neurophysin prohormone in the ovine
corpus luteum: activity of peptidyl glycine alpha-amidating mono-oxygenase and concentrations of its
cofactor, ascorbic acid. J Endocrinol. 1989;122(1):313-322. doi:10.1677/joe.0.1220313

4. Liu N, Yang H, Han L, Ma M. Oxytocin in Women’s Health and Disease. Front Endocrinol (Lausanne).
2022;13:786271. doi:10.3389/fendo.2022.786271

5. Kerem L, Lawson EA. The Effects of Oxytocin on Appetite Regulation, Food Intake and Metabolism in
Humans. Int J Mol Sci. 2021;22(14). doi:10.3390/ijms22147737

6. Husarova VM, Lakatosova S, Pivovarciova A, et al. Plasma Oxytocin in Children with Autism and Its
Correlations with Behavioral Parameters in Children and Parents. Psychiatry Investig. 2016;13(2):174-
183. doi:10.4306/pi.2016.13.2.174

7. Gordon I, Vander Wyk BC, Bennett RH, et al. Oxytocin enhances brain function in children with autism.
Proc Natl Acad Sci U S A. 2013;110(52):20953-20958. doi:10.1073/pnas.1312857110

8. László K, Vörös D, Correia P, et al. Vasopressin as Possible Treatment Option in Autism Spectrum Disorder.
Biomedicines. 2023;11(10). doi:10.3390/biomedicines11102603

9. Strauss GP, Keller WR, Koenig JI, Sullivan SK, Gold JM, Buchanan RW. Endogenous oxytocin levels are
associated with the perception of emotion in dynamic body expressions in schizophrenia. Schizophr Res.
2015;162(1-3):52-56. doi:10.1016/[Link].2015.01.022

10. Papadea D, Dalla C, Tata DA. Exploring a Possible Interplay between Schizophrenia, Oxytocin, and
Estrogens: A Narrative Review. Brain Sci. 2023;13(3). doi:10.3390/brainsci13030461

11. Maslahati T, Wingenfeld K, Hellmann-Regen J, et al. Oxytocin vs. placebo effects on intrusive memory
consolidation using a trauma film paradigm: a randomized, controlled experimental study in healthy
women. Transl Psychiatry. 2023;13(1):42. doi:10.1038/s41398-023-02339-z

12. Sack M, Spieler D, Wizelman L, et al. Intranasal oxytocin reduces provoked symptoms in female patients
with posttraumatic stress disorder despite exerting sympathomimetic and positive chronotropic effects
in a randomized controlled trial. BMC Med. 2017;15(1):40. doi:10.1186/s12916-017-0801-0

13. Neumann ID, Slattery DA. Oxytocin in General Anxiety and Social Fear: A Translational Approach. Biol
Psychiatry. 2016;79(3):213-221. doi:10.1016/[Link].2015.06.004

14. Zelkowitz P, Gold I, Feeley N, et al. Psychosocial stress moderates the relationships between oxytocin,
perinatal depression, and maternal behavior. Horm Behav. 2014;66(2):351-360.
doi:10.1016/[Link].2014.06.014

15. Heinrichs M, Domes G. Neuropeptides and social behaviour: effects of oxytocin and vasopressin in
humans. Prog Brain Res. 2008;170:337-350. doi:10.1016/S0079-6123(08)00428-7

16. Matsuzaki M, Matsushita H, Tomizawa K, Matsui H. Oxytocin: a therapeutic target for mental disorders. J
Physiol Sci. 2012;62(6):441-444. doi:10.1007/s12576-012-0232-9
17. Nurnberg HG, Hensley PL, Gelenberg AJ, Fava M, Lauriello J, Paine S. Treatment of antidepressant-
associated sexual dysfunction with sildenafil: a randomized controlled trial. JAMA. 2003;289(1):56-64.
doi:10.1001/jama.289.1.56

18. Barberis C, Mouillac B, Durroux T. Structural bases of vasopressin/oxytocin receptor function. J

Endocrinol. 1998;156(2):223-229. doi:10.1677/joe.0.1560223

19. Swaab DF, Pool CW, Nijveldt F. Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-
neurohypophypopseal system. J Neural Transm. 1975;36(3-4):195-215. doi:10.1007/BF01253126

20. Holmgren S, Jensen J. Evolution of vertebrate neuropeptides. Brain Res Bull. 2001;55(6):723-735.
doi:10.1016/s0361-9230(01)00556-1

21. Arvan P, Castle D. Sorting and storage during secretory granule biogenesis: looking backward and looking
forward. Biochem J. 1998;332 ( Pt 3)(Pt 3):593-610. doi:10.1042/bj3320593

22. Pretel S, Piekut D. Coexistence of corticotropin-releasing factor and enkephalin in the paraventricular
nucleus of the rat. J Comp Neurol. 1990;294(2):192-201. doi:10.1002/cne.902940204

23. Xiao M, Ding J, Wu L, et al. The distribution of neural nitric oxide synthase-positive cerebrospinal fluid-
contacting neurons in the third ventricular wall of male rats and coexistence with vasopressin or
oxytocin. Brain Res. 2005;1038(2):150-162. doi:10.1016/[Link].2005.01.032

24. Pittman QJ, Blume HW, Renaud LP. Connections of the hypothalamic paraventricular nucleus with the
neurohypophysis, median eminence, amygdala, lateral septum and midbrain periaqueductal gray: an
electrophysiological study in the rat. Brain Res. 1981;215(1-2):15-28. doi:10.1016/0006-8993(81)90488-1

25. Neumann I, Douglas AJ, Pittman QJ, Russell JA, Landgraf R. Oxytocin released within the supraoptic
nucleus of the rat brain by positive feedback action is involved in parturition-related events. J
Neuroendocrinol. 1996;8(3):227-233. doi:10.1046/j.1365-2826.1996.04557.x

26. Breton C, Pechoux C, Morel G, Zingg HH. Oxytocin receptor messenger ribonucleic acid: characterization,
regulation, and cellular localization in the rat pituitary gland. Endocrinology. 1995;136(7):2928-2936.
doi:10.1210/endo.136.7.7540544

27. Boyle LL, Brownfield MS, Lent SJ, et al. Intensive venous sampling of adrenocorticotropic hormone in rats
with sham or paraventricular nucleus lesions. J Endocrinol. 1997;153(1):159-167.
doi:10.1677/joe.0.1530159

28. Hull ML, Reid RA, Evans JJ, Benny PS, Aickin DR. Pre-ovulatory oxytocin administration promotes the
onset of the luteinizing hormone surge in human females. Hum Reprod. 1995;10(9):2266-2269.
doi:10.1093/[Link].a136282

29. Evans JJ. Oxytocin and the control of LH. J Endocrinol. 1996;151(2):169-174. doi:10.1677/joe.0.1510169

30. Amico JA, Challinor SM, Cameron JL. Pattern of oxytocin concentrations in the plasma and cerebrospinal
fluid of lactating rhesus monkeys (Macaca mulatta): evidence for functionally independent oxytocinergic
pathways in primates. J Clin Endocrinol Metab. 1990;71(6):1531-1535. doi:10.1210/jcem-71-6-1531

31. Tóth ZE, Gallatz K, Fodor M, Palkovits M. Decussations of the descending paraventricular pathways to the
brainstem and spinal cord autonomic centers. J Comp Neurol. 1999;414(2):255-266.
32. Rhodes CH, Morrell JI, Pfaff DW. Immunohistochemical analysis of magnocellular elements in rat
hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin. J
Comp Neurol. 1981;198(1):45-64. doi:10.1002/cne.901980106

33. Swanson LW, Kuypers HG. The paraventricular nucleus of the hypothalamus: cytoarchitectonic
subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as
demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol. 1980;194(3):555-
570. doi:10.1002/cne.901940306

34. Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev.
2001;81(2):629-683. doi:10.1152/physrev.2001.81.2.629

35. Jones PM, Robinson IC, Harris MC. Release of oxytocin into blood and cerebrospinal fluid by electrical
stimulation of the hypothalamus or neural lobe in the rat. Neuroendocrinology. 1983;37(6):454-458.
doi:10.1159/000123592

36. Dogterom J, Van Wimersma Greidanus TB, Swabb DF. Evidence for the release of vasopressin and
oxytocin into cerebrospinal fluid: measurements in plasma and CSF of intact and hypophysectomized
rats. Neuroendocrinology. 1977;24(2):108-118. doi:10.1159/000122702

37. Jones PM, Robinson IC. Differential clearance of neurophysin and neurohypophysial peptides from the
cerebrospinal fluid in conscious guinea pigs. Neuroendocrinology. 1982;34(4):297-302.
doi:10.1159/000123316

38. Meyer C, Freund-Mercier MJ, Guerné Y, Richard P. Relationship between oxytocin release and amplitude
of oxytocin cell neurosecretory bursts during suckling in the rat. J Endocrinol. 1987;114(2):263-270.
doi:10.1677/joe.0.1140263

39. Rinaman L. Oxytocinergic inputs to the nucleus of the solitary tract and dorsal motor nucleus of the
vagus in neonatal rats. J Comp Neurol. 1998;399(1):101-109. doi:10.1002/(sici)1096-
9861(19980914)399:1<101::aid-cne8>[Link];2-5

40. Amar AP, Weiss MH. Pituitary anatomy and physiology. Neurosurg Clin N Am. 2003;14(1):11-23, v.
doi:10.1016/s1042-3680(02)00017-7

41. Robinson G, Evans JJ. Oxytocin has a role in gonadotrophin regulation in rats. J Endocrinol.
1990;125(3):425-432. doi:10.1677/joe.0.1250425

42. Bakos J, Srancikova A, Havranek T, Bacova Z. Molecular Mechanisms of Oxytocin Signaling at the Synaptic
Connection. Neural Plast. 2018;2018:4864107. doi:10.1155/2018/4864107

43. Melis MR, Argiolas A. Oxytocin, Erectile Function and Sexual Behavior: Last Discoveries and Possible
Advances. Int J Mol Sci. 2021;22(19). doi:10.3390/ijms221910376

44. Churchland PS, Winkielman P. Modulating social behavior with oxytocin: how does it work? What does it
mean? Horm Behav. 2012;61(3):392-399. doi:10.1016/[Link].2011.12.003

45. Ochedalski T, Subburaju S, Wynn PC, Aguilera G. Interaction between oestrogen and oxytocin on
hypothalamic-pituitary-adrenal axis activity. J Neuroendocrinol. 2007;19(3):189-197. doi:10.1111/j.1365-
2826.2006.01525.x
46. Takayanagi Y, Onaka T. Roles of Oxytocin in Stress Responses, Allostasis and Resilience. Int J Mol Sci.
2021;23(1). doi:10.3390/ijms23010150

47. Opar A. Search for potential autism treatments turns to “trust hormone”. Nat Med. 2008;14(4):353.
doi:10.1038/nm0408-353

48. Young LJ. Being human: love: neuroscience reveals all. Nature. 2009;457(7226):148.
doi:10.1038/457148a

49. Gordon I, Jack A, Pretzsch CM, et al. Intranasal Oxytocin Enhances Connectivity in the Neural Circuitry
Supporting Social Motivation and Social Perception in Children with Autism. Sci Rep. 2016;6:35054.
doi:10.1038/srep35054

50. Kovács GL, Sarnyai Z, Szabó G. Oxytocin and addiction: a review. Psychoneuroendocrinology.
1998;23(8):945-962. doi:10.1016/s0306-4530(98)00064-x

51. Anagnostou E, Soorya L, Chaplin W, et al. Intranasal oxytocin versus placebo in the treatment of adults
with autism spectrum disorders: a randomized controlled trial. Mol Autism. 2012;3(1):16.
doi:10.1186/2040-2392-3-16

52. Hung LW, Neuner S, Polepalli JS, et al. Gating of social reward by oxytocin in the ventral tegmental area.
Science. 2017;357(6358):1406-1411. doi:10.1126/science.aan4994

53. Melis MR, Melis T, Cocco C, et al. Oxytocin injected into the ventral tegmental area induces penile
erection and increases extracellular dopamine in the nucleus accumbens and paraventricular nucleus of
the hypothalamus of male rats. Eur J Neurosci. 2007;26(4):1026-1035. doi:10.1111/j.1460-
9568.2007.05721.x

54. Preti A, Melis M, Siddi S, Vellante M, Doneddu G, Fadda R. Oxytocin and autism: a systematic review of
randomized controlled trials. J Child Adolesc Psychopharmacol. 2014;24(2):54-68.
doi:10.1089/cap.2013.0040

55. Zhou MS, Nasir M, Farhat LC, Kook M, Artukoglu BB, Bloch MH. Meta-analysis: Pharmacologic Treatment
of Restricted and Repetitive Behaviors in Autism Spectrum Disorders. J Am Acad Child Adolesc Psychiatry.
2021;60(1):35-45. doi:10.1016/[Link].2020.03.007

56. John S, Jaeggi A V. Oxytocin levels tend to be lower in autistic children: A meta-analysis of 31 studies.
Autism. 2021;25(8):2152-2161. doi:10.1177/13623613211034375

57. Anagnostou E, Soorya L, Brian J, et al. Intranasal oxytocin in the treatment of autism spectrum disorders:
a review of literature and early safety and efficacy data in youth. Brain Res. 2014;1580:188-198.
doi:10.1016/[Link].2014.01.049

58. Rubin LH, Carter CS, Drogos L, Pournajafi-Nazarloo H, Sweeney JA, Maki PM. Peripheral oxytocin is
associated with reduced symptom severity in schizophrenia. Schizophr Res. 2010;124(1-3):13-21.
doi:10.1016/[Link].2010.09.014

59. Rubin LH, Carter CS, Bishop JR, et al. Reduced levels of vasopressin and reduced behavioral modulation
of oxytocin in psychotic disorders. Schizophr Bull. 2014;40(6):1374-1384. doi:10.1093/schbul/sbu027
60. Sasayama D, Hattori K, Teraishi T, et al. Negative correlation between cerebrospinal fluid oxytocin levels
and negative symptoms of male patients with schizophrenia. Schizophr Res. 2012;139(1-3):201-206.
doi:10.1016/[Link].2012.06.016

61. Strauss GP, Keller WR, Koenig JI, Gold JM, Ossenfort KL, Buchanan RW. Plasma oxytocin levels predict
olfactory identification and negative symptoms in individuals with schizophrenia. Schizophr Res.
2015;162(1-3):57-61. doi:10.1016/[Link].2014.12.023

62. Kéri S, Kiss I, Kelemen O. Sharing secrets: oxytocin and trust in schizophrenia. Soc Neurosci.
2009;4(4):287-293. doi:10.1080/17470910802319710

63. Brown EC, Tas C, Kuzu D, Esen-Danaci A, Roelofs K, Brüne M. Social approach and avoidance behaviour
for negative emotions is modulated by endogenous oxytocin and paranoia in schizophrenia. Psychiatry
Res. 2014;219(3):436-442. doi:10.1016/[Link].2014.06.038

64. Strauss GP, Keller WR, Koenig JI, Sullivan SK, Gold JM, Buchanan RW. Endogenous oxytocin levels are
associated with the perception of emotion in dynamic body expressions in schizophrenia. Schizophr Res.
2015;162(1-3):52-56. doi:10.1016/[Link].2015.01.022

65. Strauss GP, Keller WR, Koenig JI, Gold JM, Frost KH, Buchanan RW. Plasma oxytocin levels predict social
cue recognition in individuals with schizophrenia. Schizophr Res. 2015;162(1-3):47-51.
doi:10.1016/[Link].2015.01.034

66. Strauss GP, Chapman HC, Keller WR, et al. Endogenous oxytocin levels are associated with impaired
social cognition and neurocognition in schizophrenia. J Psychiatr Res. 2019;112:38-43.
doi:10.1016/[Link].2019.02.017

67. Goh KK, Chen CH, Lane HY. Oxytocin in Schizophrenia: Pathophysiology and Implications for Future
Treatment. Int J Mol Sci. 2021;22(4):2146. doi:10.3390/ijms22042146

68. Rubin LH, Carter CS, Drogos LL, Pournajafi-Nazarloo H, Sweeney JA, Maki PM. Effects of sex, menstrual
cycle phase, and endogenous hormones on cognition in schizophrenia. Schizophr Res. 2015;166(1-
3):269-275. doi:10.1016/[Link].2015.04.039

69. Neumann ID, Slattery DA. Oxytocin in General Anxiety and Social Fear: A Translational Approach. Biol
Psychiatry. 2016;79(3):213-221. doi:10.1016/[Link].2015.06.004

70. Bohus B, Kovács GL, de Wied D. Oxytocin, vasopressin and memory: opposite effects on consolidation
and retrieval processes. Brain Res. 1978;157(2):414-417. doi:10.1016/0006-8993(78)90052-5

71. Pitman RK, Orr SP, Lasko NB. Effects of intranasal vasopressin and oxytocin on physiologic responding
during personal combat imagery in Vietnam veterans with posttraumatic stress disorder. Psychiatry Res.
1993;48(2):107-117. doi:10.1016/0165-1781(93)90035-f

72. Teicher MH, Andersen SL, Polcari A, Anderson CM, Navalta CP. Developmental neurobiology of childhood
stress and trauma. Psychiatr Clin North Am. 2002;25(2):397-426, vii-viii. doi:10.1016/s0193-
953x(01)00003-x

73. Maes M, Lin AH, Bonaccorso S, et al. Higher serum prolyl endopeptidase activity in patients with post-
traumatic stress disorder. J Affect Disord. 1999;53(1):27-34. doi:10.1016/s0165-0327(98)00086-x
74. Insel TR, Fernald RD. How the brain processes social information: searching for the social brain. Annu Rev
Neurosci. 2004;27:697-722. doi:10.1146/[Link].27.070203.144148

75. Nissen E, Gustavsson P, Widström AM, Uvnäs-Moberg K. Oxytocin, prolactin, milk production and their
relationship with personality traits in women after vaginal delivery or Cesarean section. J Psychosom
Obstet Gynaecol. 1998;19(1):49-58. doi:10.3109/01674829809044221

76. Jezova D, Skultetyova I, Tokarev DI, Bakos P, Vigas M. Vasopressin and oxytocin in stress. Ann N Y Acad
Sci. 1995;771:192-203. doi:10.1111/j.1749-6632.1995.tb44681.x

77. McCarthy MM, McDonald CH, Brooks PJ, Goldman D. An anxiolytic action of oxytocin is enhanced by
estrogen in the mouse. Physiol Behav. 1996;60(5):1209-1215. doi:10.1016/s0031-9384(96)00212-0

78. Marazziti D, Dell’Osso B, Baroni S, et al. A relationship between oxytocin and anxiety of romantic
attachment. Clin Pract Epidemiol Ment Health. 2006;2:28. doi:10.1186/1745-0179-2-28

79. Kirsch P, Esslinger C, Chen Q, et al. Oxytocin modulates neural circuitry for social cognition and fear in
humans. J Neurosci. 2005;25(49):11489-11493. doi:10.1523/JNEUROSCI.3984-05.2005

80. Cochran DM, Fallon D, Hill M, Frazier JA. The role of oxytocin in psychiatric disorders: a review of
biological and therapeutic research findings. Harv Rev Psychiatry. 2013;21(5):219-247.
doi:10.1097/HRP.0b013e3182a75b7d

81. Jobst A, Krause D, Maiwald C, et al. Oxytocin course over pregnancy and postpartum period and the
association with postpartum depressive symptoms. Arch Womens Ment Health. 2016;19(4):571-579.
doi:10.1007/s00737-016-0644-2

82. Skrundz M, Bolten M, Nast I, Hellhammer DH, Meinlschmidt G. Plasma oxytocin concentration during
pregnancy is associated with development of postpartum depression. Neuropsychopharmacology.
2011;36(9):1886-1893. doi:10.1038/npp.2011.74

83. Cardaillac C, Rua C, Simon EG, El-Hage W. [Oxytocin and postpartum depression]. J Gynecol Obstet Biol
Reprod (Paris). 2016;45(8):786-795. doi:10.1016/[Link].2016.05.002

84. Hamdan A, Tamim H. The relationship between postpartum depression and breastfeeding. Int J
Psychiatry Med. 2012;43(3):243-259. doi:10.2190/PM.43.3.d

85. Cox EQ, Stuebe A, Pearson B, Grewen K, Rubinow D, Meltzer-Brody S. Oxytocin and HPA stress axis
reactivity in postpartum women. Psychoneuroendocrinology. 2015;55:164-172.
doi:10.1016/[Link].2015.02.009

86. Linkowski P, Geenen V, Kerkhofs M, Mendlewicz J, Legros JJ. Cerebrospinal fluid neurophysins in affective
illness and in schizophrenia. Eur Arch Psychiatry Neurol Sci. 1984;234(3):162-165.
doi:10.1007/BF00461555

87. Pitts AF, Samuelson SD, Meller WH, Bissette G, Nemeroff CB, Kathol RG. Cerebrospinal fluid corticotropin-
releasing hormone, vasopressin, and oxytocin concentrations in treated patients with major depression
and controls. Biol Psychiatry. 1995;38(5):330-335. doi:10.1016/0006-3223(95)00229-A
88. van Londen L, Goekoop JG, van Kempen GM, et al. Plasma levels of arginine vasopressin elevated in
patients with major depression. Neuropsychopharmacology. 1997;17(4):284-292. doi:10.1016/S0893-
133X(97)00054-7

89. Frasch A, Zetzsche T, Steiger A, Jirikowski GF. Reduction of plasma oxytocin levels in patients suffering
from major depression. Adv Exp Med Biol. 1995;395:257-258.

90. Bell CJ, Nicholson H, Mulder RT, Luty SE, Joyce PR. Plasma oxytocin levels in depression and their
correlation with the temperament dimension of reward dependence. J Psychopharmacol.
2006;20(5):656-660. doi:10.1177/0269881106060512

91. Parker KJ, Kenna HA, Zeitzer JM, et al. Preliminary evidence that plasma oxytocin levels are elevated in
major depression. Psychiatry Res. 2010;178(2):359-362. doi:10.1016/[Link].2009.09.017

92. Ozsoy S, Esel E, Kula M. Serum oxytocin levels in patients with depression and the effects of gender and
antidepressant treatment. Psychiatry Res. 2009;169(3):249-252. doi:10.1016/[Link].2008.06.034

93. Kelley AE, Berridge KC. The neuroscience of natural rewards: relevance to addictive drugs. J Neurosci.
2002;22(9):3306-3311. doi:10.1523/JNEUROSCI.22-09-03306.2002

94. Ciobica A, Balmus IM, Padurariu M. IS OXYTOCIN RELEVANT FOR THE AFFECTIVE DISORDERS? Acta
Endocrinol (Buchar). 2016;12(1):65-71. doi:10.4183/aeb.2016.65

95. Uvnäs-Moberg K, Hillegaart V, Alster P, Ahlenius S. Effects of 5-HT agonists, selective for different
receptor subtypes, on oxytocin, CCK, gastrin and somatostatin plasma levels in the rat.
Neuropharmacology. 1996;35(11):1635-1640. doi:10.1016/s0028-3908(96)00078-0

96. Chaviaras S, Mak P, Ralph D, Krishnan L, Broadbear JH. Assessing the antidepressant-like effects of
carbetocin, an oxytocin agonist, using a modification of the forced swimming test. Psychopharmacology
(Berl). 2010;210(1):35-43. doi:10.1007/s00213-010-1815-x
Figure 1:

Figure 2:
Highlights

 Oxytocin enhances social bonding, trust, and empathy through its influence on neural
circuits.

 Oxytocin plays a role in autism, schizophrenia, PTSD, anxiety, and depression,

modulating symptoms and improving outcomes.

 Intranasal oxytocin shows promise as a treatment for various mental health conditions,
with personalized approaches being crucial.

 Oxytocin affects the hypothalamic-pituitary-adrenal (HPA) axis, playing a key role in

stress response and emotional regulation.

Table 1. Summary of Key Findings on Oxytocin's Role in Neurological and Psychological

Disorders.
Topic Key Findings
Autism Oxytocin levels are lower in autistic individuals, and its administration
reduces repetitive behaviors and improves speech tone comprehension 6.
Functional neuroimaging supports oxytocin's impact on brain areas
relevant to social behaviors in autism 7.
Endogenous vasopressin levels influence the positive effects of oxytocin
on social functioning in autism, although therapeutic benefits for general
social behavior are uncertain 8.
Schizophrenia Lower oxytocin levels are associated with more severe symptoms of
schizophrenia, particularly negative and cognitive symptoms9.
Genetic studies link variations in oxytocin-related genes to negative
symptoms of schizophrenia.
Higher oxytocin levels are linked to improved social cognition, emotion
perception, and working memory, suggesting potential as a supplement to
antipsychotic therapy 10.
Post-traumatic Oxytocin promotes the extinction of avoidance reflexes, reduces passive
stress disorder avoidance behavior, and affects memory consolidation and retrieval 11.
Reduced memory recall and conditioned response in PTSD patients after
oxytocin administration suggest alterations in the oxytocin system
following trauma may contribute to PTSD development 12.
Disorders of Oxytocin's impact on affiliative behavior in animals suggests potential
Anxiety involvement in human emotions and relationships, with implications for
anxiety disorders 13.
Elevated oxytocin levels during pregnancy are associated with reduced
 prevalence of stress and anxiety disorders, and stress-related oxytocin
release may regulate fear and anxiety responses.
Depression Oxytocin imbalance is prevalent in depression, with lower levels in
postpartum depression and potential therapeutic benefits in depressive
disorders 14.
Clinical evidence suggests oxytocin's role in modulating stress response
and social behavior, with implications for depressive symptoms and
therapeutic response to antidepressant drugs 15.
Oxytocin's impact on social withdrawal, perception of reality, and
therapeutic response to SSRIs indicates its potential as a mediator of
depressive symptoms and treatment outcomes.
Other treatments, such as sildenafil and oxytocin agonists, show promise
in alleviating depressive symptoms, possibly by affecting oxytocin release
and function 16,17
 Gene 933 (2025) 148937

Contents lists available at ScienceDirect

Gene
journal homepage: [Link]/locate/gene

Exploring extrahepatic metastasis of hepatocellular carcinoma based on

methylation driver genes and establishing a prognostic model for
hepatocellular carcinoma
ShiLing Huang a , Yang Yang b, BoShu Ji c , Ubaid Ullah a , Ram Prasad Chaulagain a,
YingYing Tian a , JiaWei Qiu a , FeiYang Gao a , PengChao Deng a , HongLiang Chen a , JiHan Qi a ,
XueYu Cang a , LiNa Liu a , ShiZhu Jin a,*
a
Department of Gastroenterology and Hepatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150086, China
b
Department of Graduation, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150086, China
c
Department of Pathology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150086, China

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Hepatocellular carcinoma (HCC), the seventh most common cancer worldwide, is characterized by a
Hepatocellular carcinoma high mortality rate, advanced diagnosis, and susceptibility to extrahepatic metastasis. Numerous studies have
Extrahepatic metastasis shown that DNA methylation is a crucial factor in epigenetic modifications and regulation of carcinogenesis.
Methylation driver genes
Methods: HCC patient data were sourced from the TCGA dataset as a training set, while GSE116174 was used as
Biomarkers
Prognosis model
an external validation set for verification. Differential methylation and expression analyses were performed on
HCC samples with and without extrahepatic metastasis. In the intersecting genes, the relationship between
methylation and expression levels of the intersecting genes was analyzed. Genes with a correlation coefficient≥|
0.30| and P<0.05 were identified as methylation driver genes. Cox regression analysis was conducted to identify
genes associated with HCC prognosis and establish a risk score. Subsequently, a prognostic model was established
and validated using Cox regression analysis incorporating the risk score and other clinical factors. Using
immunohistochemistry to evaluate the expression of DHX58 and EIF5A2 in HCC tissues with and without
extrahepatic metastasis. Immunoinfiltration analysis was performed on the HCC samples using CIBERSORT.
Results: Our research identified eight methylation driver genes for HCC extrahepatic metastasis, of which two
genes (DHX58 and EIF5A2) were associated with HCC patient prognosis. And the study further constructed and
validated the risk score and prognostic model. Immunoinfiltration analysis showed that M0 macrophage abun­
dance was correlated with the prognosis of HCC patients. Immunohistochemistry revealed differences in DHX58
and EIF5A2 expression between HCC tissues with and without extrahepatic metastasis, consistent with our
bioinformatics findings.

1. Introduction hepatocytes, which are the main parenchymal cells of the liver, are
HCCs. More than 75 % of primary liver cancer cases worldwide are
Primary liver cancer consists of two main categories: hepatocellular categorized as HCC (Petrick et al., 2020). As the seventh most common
carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Malig­ tumor, HCC is also the second main reason of cancer-related deaths
nancies that originate in the bile duct are ICCs and those that originate in (Mcglynn et al., 2021). Studies have shown that mortality and morbidity

Abbreviations: HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; MRI, magnetic resonance imaging; CT, computed tomography; US, ultra­
sound; DSA, digital subtraction angiography; BP, biological process; CC, cellular component; MF, molecular function; GO, gene ontology; KEGG, kyoto encyclopedia
of genes and genomes; FC, fold change; OS, overall survival; ROC, operating characteristic; AUC, area under the curve; GSEA, gene set enrichment analysis; PBS,
phosphate buffer saline; DAB, diaminobenzidine; AOD, average optical density; EMT, epithelial–mesenchymal transition; IHC, immunohistochemical; RLRs, RIG-I
receptors; ECM, extracellular matrix; ROS, reactive oxygen species; DMGs, differentially methylated genes; DEGs, differentially expressed genes.
* Corresponding author: Department of Gastroenterology and Hepatology, The Second Affiliated Hospital of Harbin Medical University, No. 246 XueFu Road,
Nangang District, Harbin, Heilongjiang Province 150086, China.
E-mail address: drshizhujin@[Link] (S. Jin).

[Link]
Received 4 May 2024; Received in revised form 7 September 2024; Accepted 9 September 2024
Available online 11 September 2024
0378-1119/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ([Link]
S. Huang et al. Gene 933 (2025) 148937

rates are nearly equal in HCC patients (Mcglynn et al., 2021). HCC is 2. Material and methods
often diagnosed at a later stage and therefore has a higher mortality rate.
Studies have shown that 13.5 % to 42 % of HCC patients typically 2.1. Data preprocessing
develop extrahepatic metastases (Uka et al., 2007; Katyal et al., 2000; Si
et al., 2003; Shuto et al., 2001). HCC can metastasize outside the liver The paired DNA methylation profile and RNA expression data for
via direct spread, hematogenous dissemination, or lymphatic invasion. 347 HCC patients were gathered from the TCGA dataset, along with
Katyal et al. reported that HCC patients mostly had intrahepatic stage III relevant clinical information, such as age, sex, pathological stage, and
or IVA tumors when extrahepatic lesions metastasized, and the common overall survival (OS). The methylation data of the TCGA dataset were
metastasis sites of HCC included the lungs, lymph nodes, bone, adrenal obtained from the Illumina Infinium Human Methylation 450 platform
glands, peritoneum, and omentum (Katyal et al., 2000). With the in the “TCGAbiolinks” R package, and the gene expression data were
continuous development of imaging techniques, an increasing number acquired in the “TCGAbiolinks” R package. The mRNA expression and
of methods can be used to detect asymptomatic extrahepatic metastases, clinical data of 64 HCC samples were gathered from the GSE116174
including magnetic resonance imaging, computed tomography, ultra­ dataset as the validation set. HCC with and without extrahepatic
sound, and digital subtraction angiography (Becker et al., 2014). metastasis was classified according to the 7th edition of the ACJJ. In
Although the detection and treatment methods for HCC have improved, version 7 of the ACJJ, the development of tumors in stages I, II, IIIA, and
the diagnosis of HCC patients typically occurs at the terminal stage. The IIIB is confined to the inside of the liver, while cancer tissues in stages
survival time of patients has not significantly increased, and the 5-year IIIC and IV gradually and distantly invade the liver.
survival rate remains < 12 % (El-Serag, 2011). By studying HCC patients
with extrahepatic metastasis, Lee et al. reported that 48.8 % of patients 2.2. Enrichment analysis
who responded to treatment were alive after 1 year, 12.1 % were alive
after 2 years, and the survival rate of 1-year was 11.4 % for patients with “DAVID” is an integrated system of enrichment analysis and anno­
stable or progressive disease (Lee et al., 2014). Surgical treatment can tation tools that can reveal gene enrichment data. Biological process
improve the survival rate; however, only about 20 % of patients are (BP), cellular component (CC), and molecular function (MF) together
suitable for this option at the initial diagnosis (Clark et al., 2005). constitute GO analysis. GO analysis and KEGG pathway enrichment
Spreading to the regional lymph nodes and distant metastases are clear analysis were performed for the aberrantly expressed genes and differ­
contraindications for resection (Bruix and Sherman, 2011). Patients entially methylated genes, respectively.
with limited hepatocyte reserves or advanced tumors are not candidates
for curative surgical treatment and can only receive palliative care.
2.3. Methylation driver genes
These patients exhibit a survival rate that is lower in comparison to
patients who have been diagnosed with HCC at an early stage. There­
Aberrantly expressed mRNAs between HCC extrahepatic metastasis
fore, to select appropriate treatment and avoid unnecessary surgical
and HCC non-extrahepatic metastasis were calculated using the “limma”
intervention, it is important to detect extrahepatic metastatic disease in
package. Genes with |logFC|≥0.6 and P<0.05 were considered to be
HCC patients.
aberrantly expressed. Differentially methylated genes were identified
Methylation is a reversible process catalyzed by certain enzymes,
employing “ChAMP” package. Genes with |logFC|≥0.1 and P<0.05
such as methyltransferases (writers) and demethylases (erasers), and is
were regarded as aberrantly methylated. To determine the correlation
required for the recognition of methylation-dependent binding proteins
between gene methylation and expression levels, the “Pearson” method
(readeryyygx) (Dai et al., 2021). DNA methylation is a considerable
was employed to calculate the correlation coefficient. Genes with
component of epigenetic modification, which either expresses or si­
correlation≥|0.30| and P<0.05 were considered as methylation driver
lences the function of the genome by altering chromatin structure, thus
genes.
playing a crucial role in cell physiological function, metabolism, and the
regulation of carcinogenesis. Recently, researchers have focused on the
methylation of pathogenic factors during cancer progression and inva­ 2.4. Prognostic model
sion, and the correlation between DNA methylation and mRNA
expression has also been explored in detail. In HCC samples, low Samples with survival time more than 30 days in the TCGA dataset
expression of TMEM106A is significantly associated with hyper­ were selected as the training set and those in the GSE116174 dataset
methylation of TMEM106A. In highly aggressive and metastatic HCC cell were selected as the validation set. The training and validation sets
lines, the TMEM106A promoter is hypermethylated; however, the contained 312 and 64 samples, respectively. The clinicopathological
mRNA and protein levels of TMEM106A are reduced (Shi et al., 2022). features obtained from the TCGA and GSE116174 datasets are shown in
Hamada et al. collected normal and tumor tissues from patients with Table 1. Univariate Cox regression analysis was employed to identify
primary lung adenocarcinoma, who underwent complete pneumonec­ methylation driver genes associated with extrahepatic metastasis that
tomy, and reported that ZNF132 is silenced in lung adenocarcinoma due could affect the prognosis of HCC patients. Subsequently, multivariate
to DNA hypermethylation and that DNA hypermethylation of this gene Cox regression analysis was conducted to analyze the genes identified by
during normal-to-tumor tissue transition is significantly associated with univariate Cox regression (P<0.05), and a risk score model was built.
vascular infiltration (Hamada et al., 2021). Sun et al. sequenced and
analyzed paired DNA methylation and expression in lung cancer sam­ Table 1
ples, calculated the correlation coefficient between the methylation Clinicopathological features of patients in GSE116174 and TCGA sets.
changes of intersecting gene pairs and gene expression after screening, Overall GSE116174 TCGA
and identified 20 high-methylation and 11 low-methylation driver genes N 376 64 312
(Sun et al., 2021). Gender (%)
Therefore, it can be concluded that methylation of driver genes is Male 269 (71.5) 58 (90.6) 211 (67.6)
highly important for cancer metastasis. This study aimed to identify the Female 107 (28.5) 6 (9.4) 101 (32.4)
Age (median 59.00 [50.75, 53.50 [49.00, 61.00 [51.00,
mechanistic markers of extrahepatic metastasis of HCC and establish a [IQR]) 68.00] 62.00] 68.00]
prognostic model for HCC based on methylation driver genes and clin­ Stage (%)
icopathological factors. Our study also attempted to explore the mo­ I 164 (43.6) 8 (12.5) 156 (50.0)
lecular mechanism by which abnormal gene methylation affects the II 120 (31.9) 45 (70.3) 75 (24.0)
III-IV 92 (24.5) 11 (17.2) 81 (26.0)
progression of extrahepatic HCC metastasis.

2
S. Huang et al. Gene 933 (2025) 148937

The risk score was determined by the sum of the expression levels of 2.7. Immune infiltration analyses
methylation driver genes multiplied by the corresponding multiple Cox
regression coefficients: risk score = expressionmRNA1 × coef­ CIBERSORT is a classical deconvolution method rooted in linear
ficientmRNA1 + expressionmRNA2 × coefficientmRNA2 + …expres­ support vector regression, in which mRNAs expression levels are used to
sionmRNAn × coefficientmRNAn. Using X-Tile software, a two-thirds quantitatively analyze the population of different immune cell subsets in
truncation of the risk score was obtained to divide the samples into a tumor tissue (Chen et al., 1711). We used “CIBERSORT ([Link]
different risk groups. [Link])”, “parallel,” ”e1071,” and “preprocessCore” R
Employing the “survminer” and “survival” R packages, the packages to perform immune infiltration analysis. The relative per­
“Kaplan–Meier” approach was seleted to assess OS. The “timeROC” centages of the immune cell subpopulations in a single cancer sample are
package was used to analyze the subjects’ operating characteristic shown as stacked histograms. The bar charts were constructed to reflect
(ROC) curves and the area under the ROC curves (AUCs) to estimate the the difference in immune cell infiltration in HCC extrahepatic metas­
accuracy of the risk score model prediction. To investigate independent tasis, HCC non-extrahepatic metastasis, HCC, and GSE116174 cohorts
prognostic factors, cox regression analyses were subsequently conduct­ between different groups.
ed, taking into account the risk model as well as other clinicopatho­
logical features. A prognostic model was established and a nomogram
was drawn. Using concordance index (C-index), calibration charts, and 2.8. Statistical analysis
ROC curves to evaluate the model.
All statistical analyses were performed with GraphPad Prism 9.5.0
and R. 4.3.0.
2.5. Gene set enrichment analysis (GSEA)
3. Results
GSEA is a genetic approach for interpreting gene expression data,
further distinguishing epigenetics and studying epigenetic mechanisms 3.1. Identification of differentially cpgs and mRNAs
by focusing on a set of genes with large differences (Subramanian et al.,
2005). GSEA was conducted to identify the potential molecular mech­ All 251 abnormally methylated cpgs, including 171 hypermethylated
anisms or functional pathways involved in the characterization of HCC and 80 hypomethylated cpgs, were detected in the difference analysis
extrahepatic metastasis-associated methylation driver genes. To identify between HCC samples from patients with and without extrahepatic
the different enriched pathways in the different risk groups, symbols metastasis. The cpgs were annotated as genes and 198 aberrantly
from the KEGG dataset “[Link]” were analyzed in R Studio. methylated genes were identified, including 65 hypomethylated and
133 hypermethylated genes. Compared with non-HCC metastatic tis­
sues, 369 genes were overexpressed and 354 genes were downregulated
2.6. Immunohistochemistry (IHC) in HCC metastatic tissues. Volcano plots were used to show differentially
cpgs and mRNAs (Fig. 1A, B).
Paraffin-embedded HCC samples were obtained from the Second
Affiliated Hospital of Harbin Medical University. With the consent and
3.2. Functional and pathway enrichment analyses
approval of the subjects and the Second Affiliated Hospital of Harbin
Medical University Ethical Committee (ID: KY2023-049), we collected
Using “DAVID” to conduct GO and KEGG enrichment analyses of
samples from 3 HCC patients with extrahepatic metastasis and 3 HCC
aberrantly methylated and aberrantly expressed genes obtained from
patients without extrahepatic metastasis for immunohistochemical
HCC patients with and without extrahepatic metastasis. P<0.05 was
analysis. Detailed samples information can be found in Table 2. Paraffin-
considered critical values, and p-values were selected to select the top 8.
embedded sections from HCC patients were dewaxed using xylene and
For BP, differentially methylated genes were mainly enriched in the
dehydrated in ethanol. The slices were then cleaned with distilled water
regulation of transcription from the RNA polymerase II promoter and
and pre-treated with EDTA. The sections were soaked in 3.0 % H2O2-
cell–cell signaling. Differentially expressed genes were involved in cell
inhibited endogenous peroxidases and washed with phosphate buffer
adhesion, chemical synaptic transmission, and response to xenobiotic
saline (PBS). The slices were incubated overnight after adding primary
stimuli. For CC, differentially methylated genes were significantly
antibodies and on the second day after adding asecondary antibody.
enriched in extracellular exosomes, chromatin, and intracellular
Rabbit anti-EIF5A2 (Proteintech, 17069–1-AP) and anti-DHX58 anti­
membrane-bound organelles. Differentially expressed genes in the cell
bodies (Proteintech, 11355–1-AP) were used as the primary antibodies,
surface and apical plasma membrane. For MF, differentially methylated
and a 2-step plus poly-HRP anti-mouse/rabbit IgG detection system
genes were enriched in metal ion binding and tau protein kinase activity.
(MAXIN EliVisionTM SuperHRP [Mouse/Rabbit] IHC Kit, KIT-9921)
Differentially expressed genes were related to serine-type endopepti­
was used as the secondary antibody. After the secondary antibody was
dase, metalloendopeptidase, and sulfotransferase activities. In KEGG
added and incubated, the slices were washed with PBS and dia­
analysis, differentially methylated genes were significantly enriched in
minobenzidine staining was performed for color reactions. After the
vascular smooth muscle contraction, the relaxin signaling pathway, and
sections were counterstained with hematoxylin, they were stained and
the PI3K-Akt signaling pathway. Aberrantly expressed genes were
visualized. Finally, the sections were observed under a microscope, and
involved in the PPAR signaling pathway, glycine and serine metabolism,
the average optical density (AOD) was measured using ImageJ software.
and other pathways. (Fig. 1C-F).

Table 2
Clinical information and pathology ID of HCC patients. 3.3. Selection of candidate methylation driver genes
Sample Gender Age (year) Extrahepatic metastasis Pathology ID
Fifteen overlapping aberrantly expressed and aberrantly methylated
#1 Male 66 Yes 202,218,248
#2 Female 57 Yes 202,308,206 genes were observed (Fig. 1G). The correlation between the methylation
#3 Female 35 Yes 202,220,743 and expression levels of the genes was further calculated, and cor≥|
#4 Male 62 No 202,230,858 0.30| and P<0.05 were identified as the cut-off levels. Eight methylation
#5 Female 57 No 202,245,151 driver genes were identified: ANXA6, CRIP3, DHX58, DPP4, EIF5A2,
#6 Male 76 No 202,138,765
ETNK2, FOXL1, and PIPOX (Fig. 2A-H).

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S. Huang et al. Gene 933 (2025) 148937

Fig. 1. DMGs and DEGs in HCC extrahepatic metastases and HCC non-extrahepatic metastases. (A, B) Volcano plots of the DMGs and DEGs. (C, D) The top 8 terms in
GO enrichment analysis of DEGs and DEGs. (E, F) The top 8 KEGG pathways enriched by DMGs and DEGs. (G) The Venn diagram of differentially methylated genes
and differentially expressed genes.

3.4. Formulation and validation of the prognostic risk score 0.63 (Fig. 4B, D, F).

Univariate Cox analysis was conducted to identify for genes highly

associated with extrahepatic metastasis of HCC (P<0.05, Fig. 3A). 3.5. Construction and verification of the prognostic model
Multivariate Cox regression analysis was performed to further refine the
HCC risk score model, with the risk score being calculated using the The clinicopathological features obtained from the TCGA and
formula: risk score = DHX58 × -0.308119276952731 + EIF5A2 × GSE116174 datasets are presented in Table 1. Univariate and multi­
0.276246679542202. Using a cutoff level of two-thirds of the risk score, variate Cox regression analyses were conducted to assess whether the
patients in the TCGA and GSE116174 sets were grouped into different risk score and clinicopathological factors could served as HCC inde­
risk groups, with the high-risk group showing shorter OS (Fig. 3B, C). pendent prognostic factors. Cox analysis showed that the prognostic risk
Fig. 3D-F and Fig. 3G-I show the gene expression and risk score distri­ score and pathological stage exhibited strong associations with OS
butions in the TCGA and GEO datasets. The AUCs of the risk score model (P<0.005; Fig. 4G, H). A Cox regression model, including risk score and
for 1-, 3-, and 5-year OS in the training set were 0.62, 0.67, and 0.65 pathological stage, was built to predict HCC prognosis, and a nomogram
(Fig. 4A, C, E). Moreover, in the validation set, HCC samples were was drawn (Fig. 5A). Using the training and validation sets, C-index
further evaluated, and the AUCs at 1-, 3- and 5-year were 0.62, 0.59 and (Fig. 5B, C), calibration (Fig. 5D, E), and ROC curves (Fig. 5F, G) were
plotted to evaluate the prognostic model. Fig. 5B and C show that the C-

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S. Huang et al. Gene 933 (2025) 148937

Fig. 2. Correlation analysis between gene methylation level and gene expression level. (A–H) 8 methylation driver genes: ANXA6, CRIP3, DHX58, DPP4, EIF5A2,
ETNK2, FOXL1, PIPOX.

index in the TCGA set was 0.668 and that in the GEO set was 0.608. The analysis indicated that signaling pathways such as cell cycle and extra­
calibration curves of the training and verification sets were close to the cellular matrix (ECM) receptor interactions were activated in the high-
ideal curves (45◦ ), and the measured values agreed reasonably well with risk group, whereas cytochrome P450 and fatty acid metabolism were
the predicted values (Fig. 5D, E). The 1-, 3-, and 5-year AUCs were 0.72, activated in the low-risk group (Fig. 5H, I). In general, these enriched
0.74, and 0.69 in the TCGA set and 0.66, 0.62, and 0.68 in the GEO set. signaling pathways were mainly related to cytokines, receptors, and
These outcomes shown that the prognostic model possessed good pre­ substance metabolism and played a crucial role in HCC progression.
dictive power.

3.7. Immune microenvironment

3.6. GSEA
To identify the expression of the key hub genes, DHX58 and EIF5A2,
To determine the mechanism of HCC invasion and metastasis, GSEA pathological specimens from HCC patients were subjected to IHC
was used to analyze the enrichment of genes related to signaling path­ staining. Pathology sections were taken and processed using Motic
ways in samples from different TCGA risk groups. KEGG gene set-based DSAssistant; images of three representative regions in each section were

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S. Huang et al. Gene 933 (2025) 148937

Fig. 3. Establishment and validation of HCC prognostic risk score constructed by 2 methylation driver genes. (A) Univariate Cox proportional hazards regression
model screened prognostic related methylation driver genes. (B, C) Comparison of OS between the highrisk group and lowrisk group in TCGA set and GEO set. (D, G)
Risk heatmaps of the 2 methylation driver genes expression profiles in TCGA set and GEO set. (E, H) Risk score distributions of 2 methylation driver genes expression
profiles in TCGA set and GEO set. (F, I) The survival time and survival status between the high-risk group and low-risk group in TCGA set and GEO set.

taken at 20 × magnification, and uniform settings were applied to all the prognosis. To further evaluate this observation, the “Kendall”
sections for each antibody-stained readout. The AOD of each image was method was applied to assess the correlation between the risk score and
then measured using the ImageJ software. The cumulative optical den­ M0 macrophage abundance in HCC extrahepatic metastasis samples,
sity was divided by the total area to obtain the AOD. Immunohisto­ yielding a result of cor = 0.48 and P=0.031 (Fig. 6D).
chemical and TCGA data analyses demonstrated that the expression Therefore, it can be inferred that a higher proportion of M0 macro­
level of EIF5A2 in extrahepatic metastatic specimens was found to be phages in HCC extrahepatic metastasis samples is associated with an
higher compared to in HCC non-liver metastatic specimens, and the unfavorable prognosis. A previous study has confirmed this finding,
expression of DHX58 in extrahepatic metastatic specimens was lower demonstrating that in stages III and IV of HCC, a higher fraction of M0
than that in HCC non-hepatic metastatic specimens (Fig. 5J, L,K, M). In macrophages is associated with a poorer prognosis (P=0.001) (Zhang
the TCGA cohort, the expression levels of DHX58 and EIF5A2 were et al., 2021).
notably different among the different risk groups, with low expression Further consideration was given to whether the conclusion regarding
levels of DHX58 and high expression levels of EIF5A2 in the high-risk the prognostic value of M0 macrophage abundance applies to HCC
group (Fig. 5N, O). Low DHX58 and high EIF5A2 expression have samples without extrahepatic metastasis. HCC non-extrahepatic metas­
been shown to be risk factors for poor prognosis (Fig. 5P, Q). tasis samples from the TCGA cohort were analyzed for validation. Fig. 6F
shows that the abundance of M0 macrophages in the high-risk group was
3.8. Immune infiltration analysis higher than that in the low-risk group (P<0.05). Moreover, a positive
correlation was observed between M0 macrophage abundance and the
The immune microenvironment is important in the progression and expression of EIF5A2 (Fig. 6G). In HCC non-extrahepatic metastasis
prognosis of HCC, and Zhang et al. pointed out that the fraction of im­ samples, the correlation between the proportion of M0 macrophages and
mune cells is different at different clinical stages of HCC (P<0.05) the risk score was analyzed, yielding a result of cor = 0.2, P<0.05
(Zhang et al., 2021). To explain the immune microenvironment of HCC (Fig. 6H). This suggests that the higher the abundance of M0 macro­
extrahepatic metastasis, samples from the TCGA set were selected for phages in HCC non-extrahepatic metastasis samples, the worse is the
immunoinfiltration analysis using the “CIBERSORT” package. The prognosis.
findings revealed that the abundance of M0 macrophages with HCC Therefore, it can be inferred that a greater proportion of M0 mac­
extrahepatic metastasis was higher in the high-risk group than in the rophages is indicative of poor prognosis in HCC patients. To further
low-risk group (P<0.05; Fig. 6B). M0 macrophage abundance was evaluate this, HCC samples from the TCGA cohort were analyzed,
positively correlated with the expression of the HCC prognostic gene, revealing that the abundance of M0 macrophages was high in the high-
EIF5A2 (Fig. 6C). In HCC extrahepatic metastasis samples, it was risk group (P<0.05, Fig. 6J) and was positively related to EIF5A2
concluded that the higher the proportion of M0 macrophages, the poorer expression (P<0.05, Fig. 6K). Additionally, Fig. 6L demonstrates that

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S. Huang et al. Gene 933 (2025) 148937

Fig. 4. The survival assessment of risk score and screening independent prognostic features to build prognostic model. (A, C, E) The 1, 3, and 5-year time-dependent
ROC curves for OS in the TCGA training set. (B, D, F) The 1, 3, and 5-year time-dependent ROC curves for OS in the GEO validation set. (G, H) Screening the in­
dependent prognostors for OS including risk score and clinicopathological features by univariate and multivariate Cox proportional hazards regression model.

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S. Huang et al. Gene 933 (2025) 148937

Fig. 5. Construction and validation of prognostic model. (A) A nomogram including risk score and pathological stage was constructed to predict 1, 3, and 5-year OS
for HCC patients. (B, C) The prognostic model was evaluated by drawing C-inex of TCGA training set and GEO validation set. (D, E) The prognostic model was
evaluated by drawing Calibration curves of TCGA training set and GEO validation set. (F, G) The prognostic model was evaluated by drawing ROC curves of TCGA
training set and GEO validation set. (H, I) Top 5 pathways of KEGG enrichment analysis between the high-risk group and low-risk group in TCGA set. (J,K) Protein
levels of DHX58 and EIF5A2 in HCC metastasis tissues and HCC non-metastasis tissues were analyzed by IHC assay(20×, n = 3). (L, M) AOD(IOD/Area) analysis of
DHX58 and EIF5A2 immunohistochemical staining in HCC extrahepatic and non-extrahepatic metastases was performed using ImageJ. (N, O) The expressed level of
DHX58 and EIF5A2 shows significant differentially between high-risk and lowrisk groups. In highrisk group, DHX58 expression was low while EIF5A2 expression was
high. (P, Q) Low DHX58 expression and high EIF5A2 expression were risk factors for poor prognosis.

M0 macrophage abundance and risk score were positively correlated in and influence the prognosis of HCC patients. However, verifying this
HCC samples (cor = 0.21, P<0.05). HCC samples from the GSE116174 inference and hypothesis requires more samples and in vitro experi­
dataset were validated. As shown in Fig. 6N, although the difference was ments, which are the limitations of our study.
not significant, the abundance of M0 macrophages in the GSE116174
dataset was higher in the high-risk group compared to the low-risk 4. Discussion
group. The remaining results were in agreement with these conclu­
sions (Fig. 6O, P). As one of the most prevalent malignant tumors worldwide, HCC is
It can be inferred that a higher abundance of M0 macrophages is usually diagnosed at an advanced stage and has high mortality (Toh
related to a poor prognosis in HCC samples, which is supported by et al., 2023). Therefore, identifying reliable and effective prognostic
previous studies (Zhou et al., 2021; Zhang et al., 2022; Cao et al., 2024). biomarkers for HCC extrahepatic metastasis and constructing prognostic
Additionally, we hypothesized that EIF5A2 expression works synergis­ models for HCC are vital.
tically with M0 macrophage abundance to promote HCC progression Many studies have shown that DNA methylation is vital for the

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S. Huang et al. Gene 933 (2025) 148937

Fig. 6. The role of immune cell infiltration in HCC were analyzed using CIBERSORT. (A, E, I, M) Bar chart displaying the proportion of immune cell subsets in HCC
extrahepatic metastasis cohort, HCC non-extrahepatic metastasis cohort, HCC cohort, GEO cohort. (B, F, J, N) Proportion of immune cell subsets between high-risk
group and low-risk group in HCC extrahepatic metastasis cohort, HCC non-extrahepatic metastasis cohort, HCC cohort, GEO cohort. *P<0.05, **P<0.01,
***P<0.001. (C, G, K, O) Heatmaps of correlation between 8 HCC extrahepatic metastasis methylation driver genes and immune cells in HCC extrahepatic metastasis
cohort, HCC non-extrahepatic metastasis cohort, HCC cohort, GEO cohort. *P<0.05, **P<0.01, ***P<0.001. (D, H, L, P) Correlation between M0 macrophage
abundance and risk score with HCC extrahepatic metastasis cohort, HCC non-extrahepatic metastasis cohort, HCC cohort, GEO cohort.

epigenetic regulation of gene expression. Hypermethylation can lead to in HCC patients at different stages. In the tissues of HCC patients, DPP4
changes in chromatin activity, directly leading to the inhibition of expression was reported to be lower in TNM stage II and III-V patients
transcription levels and further leading to the downregulation or than that in TNM stage I patients. Although DPP4 was not an indepen­
silencing of tumor suppressor genes, thus promoting the occurrence and dent prognostic factor, patients with lower DPP4 expression in HCC
development of cancer (Razin and Kantor, 2005). Studies have demon­ tissues had a much shorter mean survival time (Yu et al., 2020). Zheng
strated that abnormal methylation is significant in the diagnosis, prog­ et al. indicated that EIF5A2 expression could lead to the migration and
nosis, and treatment of various cancers (Bhootra et al., 2023). Moreover, invasion of PCa cells by promoting epithelial–mesenchymal transition
DNA methylation is important for cancer metastasis as it is implicated in (EMT) (Zheng et al., 2021). Therefore, these methylation driver genes
the transcriptional inactivation of key genes. Research has shown that are involved in extrahepatic metastasis of HCC, and further studies are
abnormal methylation of EZH2 leads to decreased expression of EZH2 necessary to determine the relevance of methylation driver genes in HCC
target genes and increased invasion and metastasis of breast cancer (Li prognosis.
et al., 2020). Methylated Tiam1 has also been shown to promote Rac1 The obtained DNA methylation driver genes were associated with
signaling activation and malignant colon tumor metastasis (Bhootra HCC prognosis. After single-factor Cox screening and multifactor Cox
et al., 2023). modelling, a risk score constructed with two genes (EIF5A2 and DHX58)
In this study, differential methylation and differential expression was obtained. Cox regression analysis was performed to establish a
analyses were performed on pairs of samples obtained from the TCGA prognostic model that incorporated the risk score and clinicopatholog­
cohort. The methylation level and methylation level were used to study ical factors. Our study also attempted to explain the correlation between
the correlation between the two crossover genes, and eight DNA the different risk groups and immune cell infiltration and showed the
methylation driver genes were screened (ANXA6, CRIP3, DHX58, DPP4, correlation between the differentially methylated driver genes and im­
EIF5A2, ETNK2, FOXL1, and PIPOX). For eight genes, the methylation mune cell types using heatmaps. To verify that the two DNA methylation
levels of FOXL1 were positively correlated with gene expression levels, driver genes were associated with prognosis, IHC analysis was per­
whereas the methylation levels of the remaining seven genes were formed on liver cancer samples from patients with and without extra­
negatively correlated with gene expression levels. In addition to CRIP3, hepatic metastasis; semi-quantitative IHC and statistical triple-repeat
DHX58, and PIPOX, other genes have been shown to play significant analyses were conducted using ImageJ and GraphPad Prism, respec­
roles in tumor development (Zheng et al., 2021; Cao et al., 2023; Chu tively. Compared with liver cancer without extrahepatic metastasis,
et al., 2023; Ertao et al., 2016; Yu et al., 2020; Hong et al., 2023). Several EIF5A2 was highly expressed in extrahepatic metastasis samples
of these genes play key roles in cancer metastasis and prognosis. For (P<0.05), whereas DHX58 was expressed at low levels in extrahepatic
example, studies have explored the differences in the expression of DPP4 metastasis samples (P<0.05).

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S. Huang et al. Gene 933 (2025) 148937

RIG-I receptors (RLRs) are a family of DExD/H-box RNA helicases cancer progression, invasion, and metastasis (Babaei et al., 2021; Huang
that can recognize exogenous cytoplasmic RNA. The RLR family com­ et al., 2022). Reactive oxygen species (ROS), byproducts of aerobic
prises of three members: RIG-I, LGP2 (DHX58), and MDA5 (Loo and metabolism from multiple cell sources, are recognized as markers of
Gale, 2011). Satoh et al. reported that LGP2 positively regulates RIG-1- various cancers owing to their increased production. Increasing evi­
mediated signaling (Satoh et al., 2010). Liu et al. reported that lncRNA dence indicates that ROS promotes cell invasion, metastasis, and pro­
Ftx regulates the proliferation, tumorigenicity, and cell cycle of HCC liferation (Moloney and Cotter, 2018). Many stimuli that cause REDOX-
tissues through miR-545, and that RIG-I serves as a crucial functional dependent invasion/movement are actually related to EMT, and many
downstream mediator of the lncRNA Ftx/miR-545 axis. Moreover, this studies have further revealed that ROS are key mediators of EMT pro­
axis negatively regulates RIG-I expression in HCC cells (Liu et al., 2016). gression and have proposed a large number of mechanisms (Giannoni
RIG-I expression activates PI3K/Akt signal transduction (Yeon et al., et al., 2012). Liu et al. reported that EIF5A2 further induces EMT pro­
2015), and activation of the PI3K/Akt pathway is closely related to the gression by activating the ROS pathway, thereby promoting HCC inva­
proliferation, invasion, and metastasis of HCC cancer tissues (Courtney sion and metastasis (Liu et al., 2016). Therefore, our study proposed a
et al., 2010). Liu et al. suggested that the lncRNA Ftx/miR-545 axis hypothesized mechanism by which EIF5A2 promotes HCC extrahepatic
activates PI3K/Akt by targeting RIGi, which is crucial for the malignant metastasis: EIF5A2 activates the activity of Rho GTPases (RhoA and
progression and metastasis of HCC (Liu et al., 2016). Therefore, in Rac1) to stimulate actin cytoskeletal rearrangement and further induces
combination with our own studies, it can be speculated that LGP2, as a the progression of EMT through activation of the ROS pathway, thereby
positive regulator of RIG-1, participates in PI3K/Akt activation via the promoting HCC extrahepatic metastasis (Fig. 7).
Ftx/miR-545 axis of ncRNA by targeting RIG-1, leading to HCC extra­ In summary, our study identified DNA methylation driver genes for
hepatic metastasis (Fig. 7). the extrahepatic metastasis of liver cancer using a network database,
Previous studies have shown that Rho GTPases regulate actin cyto­ screened prognostic genes, and constructed a risk score. Cox regression
skeleton remodeling and cell movement and are important for the analysis was conducted on the risk score and clinicopathological factors
migration, invasion, and metastasis of tumor cells (Van Golen, 2003; to develop and validate a HCC prognostic model. By studying the role of
Wang et al., 2022). Rho GTPases can be divided into eight subfamilies the immune microenvironment in HCC samples, we concluded that the
according to their sequence homology, and the two major subclasses, higher the abundance of M0 macrophages, the worse is the prognosis.
Rho and Rac, play important roles in regulating actin polymerization Finally, combined with previous studies, the hypothesis that DHX58 and
required by biological processes (Hanna and El-Sibai, 2013). Many EIF5A2 lead to extrahepatic metastasis of HCC was proposed. However,
studies have shown that the upregulation of RHOA and Rac1 promotes there are some limitations to the research. This research was based on
the migration and invasion of HCC cells (Lee et al., 2005; Wang et al., the integration of bioinformatics and IHC and was limited by the number
2007; Wang et al., 2015; Zhan et al., 2017; Li et al., 2019). Tang et al. of samples and insufficient experimental validation. Therefore, the ac­
revealed that EIF5A2 activates Rho GTPases (RhoA and Rac1), stimu­ curacy of the constructed prognostic model of HCC patients remains an
lates actin cytoskeletal rearrangement (formation of stress fibers and important clinical issue. In particular, the mechanism underlying the
lamellar feet), and promotes the invasion and metastasis of HCC (Tang influence of M0 macrophages on prognosis and the hypothetical
et al., 2010). EMT is a biological process in which cells with an inter­ mechanism of extrahepatic metastasis of HCC caused by DHX58 and
stitial phenotype are transformed through specific processes, such as EIF5A2 require further experimental verification.
increased ECM protein levels, increased resistance to stress, and induced
apoptotic death. In addition, it enhances tissue migration and invasion
(Giannoni et al., 2012). Studies have indicated that EMT is important for

Fig. 7. Mechanism diagram of DHX58 and EIF5A2 leading to extrahepatic metastasis.

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S. Huang et al. Gene 933 (2025) 148937

5. Conclusion Bhootra, S., Jill, N., Shanmugam, G., Rakshit, S., Sarkar, K., 2023. DNA methylation and
cancer: transcriptional regulation, prognostic, and therapeutic perspective. Med.
Oncol. 40, 71.
This study identified biomarkers of HCC extrahepatic metastasis Bruix, J., Sherman, M., 2011. D American Association for the Study of Liver,
based on methylation driver genes. A prognostic model of HCC patients Management of hepatocellular carcinoma: an update. Hepatology 53, 1020–1022.
was constructed through network database validation and immunohis­ Cao, H., Huang, P., Qiu, J., Gong, X., Cao, H., 2024. Immune landscape of hepatocellular
carcinoma tumor microenvironment identifies a prognostic relevant model. Heliyon
tochemical analysis, which revealed the validity of the model and po­ 10, e24861.
tential of EIF5A2 and DHX58 as prognostic biomarkers. By studying the Cao, J., Wan, S., Chen, S., Yang, L., 2023. ANXA6: a key molecular player in cancer
immunoinfiltration of the samples, we concluded that M0 macrophages progression and drug resistance. Discov Oncol 14, 53.
B. Chen, M. S. Khodadoust, C. L. Liu, A. M. Newman, A. A. Alizadeh, Profiling Tumor
were related to the prognosis of HCC patients. Drawing from previous Infiltrating Immune Cells with CIBERSORT, Methods in molecular biology (Clifton,
studies, it is hypothesized that markers promote HCC extrahepatic N.J.) 1711 (2018) 243-59.
metastasis. DHX58, as a positive regulator of RIG-1, participates in the Chu, J., Qian, X.X., Zhang, X.M., Jiang, T., Li, X.J., Sun, W., 2023. ETNK2 Low-
Expression Predicts Poor Prognosis in Renal Cell Carcinoma with
PI3K/Akt activation process of the Ftx/miR-545 axis of ncRNA by tar­ Immunosuppressive Tumor Microenvironment. J. Oncol. 2023, 1743357.
geting RIG-1, leading to HCC extrahepatic metastasis. EIF5A2 activates H. P. Clark, W. F. Carson, P. V. Kavanagh, C. P. Ho, P. Shen, R. J. Zagoria, Staging and
Rho GTPases (RhoA and Rac1), stimulates actin cytoskeletal rear­ current treatment of hepatocellular carcinoma, Radiographics : a review publication
of the Radiological Society of North America, Inc 25 Suppl 1 (2005) S3-23.
rangement, and induces EMT progression by activating the ROS Courtney, K.D., Corcoran, R.B., Engelman, J.A., 2010. The PI3K pathway as drug target
pathway, thereby promoting HCC extrahepatic metastasis. However, the in human cancer. J. Clin. Oncol. 28, 1075–1083.
role of M0 macrophages in HCC prognosis and the hypothesized Dai, X., Ren, T., Zhang, Y., Nan, N., 2021. Methylation multiplicity and its clinical values
in cancer. Expert Rev. Mol. Med. 23.
mechanism by which DHX58 and EIF5A2 promote HCC extrahepatic
El-Serag, H.B., 2011. Hepatocellular carcinoma. N. Engl. J. Med. 365, 1118–1127.
metastasis need further verification. Ertao, Z., Jianhui, C., Chuangqi, C., et al., 2016. Low level of FOXL1 indicates a worse
prognosis for gastric cancer patients. Tumour Biol. 37, 11331–11337.
Giannoni, E., Parri, M., Chiarugi, P., 2012. EMT and oxidative stress: a bidirectional
Ethics approval and consent to participate interplay affecting tumor malignancy. Antioxid. Redox Signal. 16, 1248–1263.
Hamada, K., Tian, Y., Fujimoto, M., et al., 2021. DNA hypermethylation of the ZNF132
The study was authorized by the Second Affiliated Hospital of Harbin gene participates in the clinicopathological aggressiveness of ’pan-negative’-type
lung adenocarcinomas. Carcinogenesis 42, 169–179.
Medical University Ethical Committee (ID: KY2023-049) and was car­ Hanna, S., El-Sibai, M., 2013. Signaling networks of Rho GTPases in cell motility. Cell.
ried out in line with the Declaration of Helsinki. Consent was given Signal. 25, 1955–1961.
voluntarily by all participants and volunteered to allow researchers to Hong, K., Yang, Q., Yin, H., Zhang, J., Yu, B., 2023. SDR16C5 promotes proliferation and
migration and inhibits apoptosis in pancreatic cancer. Open Life Sci. 18, 20220630.
use their pathological wax mass of liver cancer tissue for study.
Huang, Y., Hong, W., Wei, X., 2022. The molecular mechanisms and therapeutic
strategies of EMT in tumor progression and metastasis. J. Hematol. Oncol. 15, 129.
CRediT authorship contribution statement Katyal, S., Oliver 3rd, J.H., Peterson, M.S., Ferris, J.V., Carr, B.S., Baron, R.L., 2000.
Extrahepatic metastases of hepatocellular carcinoma. Radiology 216, 698–703.
Lee, J.I., Kim, J.K., Kim, D.Y., et al., 2014. Prognosis of hepatocellular carcinoma patients
ShiLing Huang: Writing – original draft, Methodology, Conceptu­ with extrahepatic metastasis and the controllability of intrahepatic lesions. Clin. Exp.
alization. Yang Yang: Software, Resources. BoShu Ji: Software, Re­ Metastasis 31, 475–482.
Lee, T.K., Man, K., Ho, J.W., et al., 2005. Significance of the Rac signaling pathway in
sources. Ubaid Ullah: Writing – review & editing. Ram Prasad HCC cell motility: implications for a new therapeutic target. Carcinogenesis 26,
Chaulagain: Writing – review & editing. YingYing Tian: Writing – 681–687.
original draft, Data curation. JiaWei Qiu: Writing – original draft, Data Li, C.F., Chen, J.Y., Ho, Y.H., et al., 2019. Snail-induced claudin-11 prompts collective
migration for tumour progression. Nat. Cell Biol. 21, 251–262.
curation. FeiYang Gao: Visualization, Formal analysis. PengChao Li, Z., Wang, D., Lu, J., et al., 2020. Methylation of EZH2 by PRMT1 regulates its stability
Deng: Visualization, Formal analysis. HongLiang Chen: Validation. and promotes breast cancer metastasis. Cell Death Differ. 27, 3226–3242.
JiHan Qi: Validation. XueYu Cang: Validation. LiNa Liu: Writing – Liu, Z., Dou, C., Yao, B., et al., 2016. Ftx non coding RNA-derived miR-545 promotes cell
proliferation by targeting RIG-I in hepatocellular carcinoma. Oncotarget 7,
review & editing, Investigation. ShiZhu Jin: Writing – review & editing,
25350–25365.
Supervision, Project administration. Liu, R.R., Lv, Y.S., Tang, Y.X., et al., 2016. Eukaryotic translation initiation factor 5A2
regulates the migration and invasion of hepatocellular carcinoma cells via pathways
involving reactive oxygen species. Oncotarget 7, 24348–24360.
Loo, Y.M., Gale Jr., M., 2011. Immune signaling by RIG-I-like receptors. Immunity 34,
Declaration of competing interest
680–692.
Mcglynn, K.A., Petrick, J.L., El-Serag, H.B., 2021. Epidemiology of Hepatocellular
The authors declare that they have no known competing financial Carcinoma. Hepatology 73 (Suppl 1), 4–13.
interests or personal relationships that could have appeared to influence Moloney, J.N., Cotter, T.G., 2018. ROS signalling in the biology of cancer. Semin. Cell
Dev. Biol. 80, 50–64.
the work reported in this paper. Petrick, J.L., Florio, A.A., Znaor, A., et al., 2020. International trends in hepatocellular
carcinoma incidence, 1978–2012. Int. J. Cancer 147, 317–330.
Data availability Razin, A., Kantor, B., 2005. DNA methylation in epigenetic control of gene expression.
Prog. Mol. Subcell. Biol. 38, 151–167.
Satoh, T., Kato, H., Kumagai, Y., et al., 2010. LGP2 is a positive regulator of RIG-I- and
Data will be made available on request. MDA5-mediated antiviral responses. PNAS 107, 1512–1517.
Shi, S., Wang, B., Wan, J., et al., 2022. TMEM106A transcriptionally regulated by
promoter methylation is involved in invasion and metastasis of hepatocellular
Acknowledgments carcinoma. Acta Biochim. Biophys. Sin. (Shanghai) 54, 1008–1020.
Shuto, T., Hirohashi, K., Kubo, S., et al., 2001. Treatment of adrenal metastases after
hepatic resection of a hepatocellular carcinoma. Dig. Surg. 18, 294–297.
First, we would like to thank all the authors for their dedication to
Si, M.S., Amersi, F., Golish, S.R., et al., 2003. Prevalence of metastases in hepatocellular
this review. Second, we would also like to thank the permission of carcinoma: risk factors and impact on survival. Am. Surg. 69, 879–885.
medpeer and Fig. 7 should be accompanied by the following citation: A. Subramanian, P. Tamayo, V. K. Mootha, et al., Gene set enrichment analysis: a
“Created With MedPeer ([Link])”. knowledge-based approach for interpreting genome-wide expression profiles,
Proceedings of the National Academy of Sciences of the United States of America 102
(2005) 15545-50.
References Sun, X., Yi, J., Yang, J., et al., 2021. An integrated epigenomic-transcriptomic landscape
of lung cancer reveals novel methylation driver genes of diagnostic and therapeutic
relevance. Theranostics 11, 5346–5364.
G. Babaei, S. G. Aziz, N. Z. Z. Jaghi, EMT, cancer stem cells and autophagy; The three
Tang, D.J., Dong, S.S., Ma, N.F., et al., 2010. Overexpression of eukaryotic initiation
main axes of metastasis, Biomedicine & pharmacotherapy = Biomedecine &
factor 5A2 enhances cell motility and promotes tumor metastasis in hepatocellular
pharmacotherapie 133 (2021) 110909.
carcinoma. Hepatology 51, 1255–1263.
Becker, A.K., Tso, D.K., Harris, A.C., Malfair, D., Chang, S.D., 2014. Extrahepatic
Toh, M.R., Wong, E.Y.T., Wong, S.H., et al., 2023. Global Epidemiology and Genetics of
metastases of hepatocellular carcinoma: A spectrum of imaging findings. Can. Assoc.
Hepatocellular Carcinoma. Gastroenterology 164, 766–782.
Radiol. J. 65, 60–66.

11
S. Huang et al. Gene 933 (2025) 148937

Uka, K., Aikata, H., Takaki, S., et al., 2007. Clinical features and prognosis of patients Yu, H., Mei, X.P., Su, P.F., Jin, G.Z., Zhou, H.K., 2020. A poor prognosis in human
with extrahepatic metastases from hepatocellular carcinoma. World J. Gastroenterol. hepatocellular carcinoma is associated with low expression of DPP4. Braz. J. Med.
13, 414–420. Biol. Res. 53, e9114.
Van Golen, K.L., 2003. Inflammatory breast cancer: relationship between growth factor Zhan, Y., Zheng, N., Teng, F., et al., 2017. MiR-199a/b-5p inhibits hepatocellular
signaling and motility in aggressive cancers. Breast Cancer Research : BCR 5, carcinoma progression by post-transcriptionally suppressing ROCK1. Oncotarget 8,
174–179. 67169–67180.
Wang, S.J., Cui, H.Y., Liu, Y.M., et al., 2015. CD147 promotes Src-dependent activation Zhang, Z., Wang, Z., Huang, Y., 2021. Comprehensive analyses of the infiltrating immune
of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular cell landscape and its clinical significance in hepatocellular carcinoma. Int. J. Gen.
carcinoma cells. Oncotarget 6, 243–257. Med. 14, 4695–4704.
Wang, D., Dou, K., Xiang, H., et al., 2007. Involvement of RhoA in progression of human Zhang, Y., Zou, J., Chen, R., 2022. An M0 macrophage-related prognostic model for
hepatocellular carcinoma. J. Gastroenterol. Hepatol. 22, 1916–1920. hepatocellular carcinoma. BMC Cancer 22, 791.
Wang, T., Rao, D., Yu, C., et al., 2022. RHO GTPase family in hepatocellular carcinoma. Zheng, Y., Li, P., Huang, H., et al., 2021. Androgen receptor regulates eIF5A2 expression
Exp. Hematol. Oncol. 11, 91. and promotes prostate cancer metastasis via EMT. Cell Death Discov 7, 373.
Yeon, S.H., Song, M.J., Kang, H.R., Lee, J.Y., 2015. Phosphatidylinositol-3-kinase and Zhou, W., Fang, D., He, Y., Wei, J., 2021. Correlation analysis of tumor mutation burden
Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS- of hepatocellular carcinoma based on data mining. J. Gastrointest. Oncol. 12,
1. Immunology 144, 312–320. 1117–1131.

12
’ Case Report
Bilateral vestibular schwannoma with a cooccurring
meningioma in a child: a case report and review of
literature
Ram P. Chaulagain, MBBSa, Yelona Shrestha, MBBSb, Kusha K.C., MBBSc, Abal Baral, MBBSd,*

Introduction: Meningioma and vestibular schwannoma (VS) are the first and second most common benign central nervous system
tumors. The coexistence of VS and meningioma presents a rare clinical scenario, particularly in pediatric patients. This report
presents a case of bilateral VS with a cooccurring meningioma in a Nepali child and provides an overview of the literature on this
condition.
Case report: A 15-year-old male presented with bilateral sensorineural hearing loss, seizures, and neurological deficits and was
ultimately diagnosed with concomitant bilateral acoustic neuroma and meningioma. The patient underwent radiosurgery for bilateral
VS and nonoperative management of the meningioma. Long-term follow-up revealed symptomatic improvement, emphasizing the
importance of a multidisciplinary approach in managing such complex cases. The management of these tumors requires tailored
treatment strategies guided by tumor characteristics and associated risks.
Discussion: Meningioma and VS are common tumors of the central nervous system. Their coexistence is possible in
neurofibromatosis type 2 but is exceedingly rare in pediatric age group. The tumors, often coexisting, pose diagnostic challenges.
Diagnosis relies on clinical and genetic features, with multidisciplinary management involving various specialists. Treatment aims to
preserve function and quality of life, utilizing approaches such as bevacizumab and surgical intervention. The role of radiation therapy
remains uncertain. Genetic testing and regular monitoring are vital for early detection and intervention.
Conclusion: The cooccurrence of acoustic neuromas and meningiomas is poorly understood, with limited reported cases and
unclear pathophysiological mechanisms. Further research into the genetic and molecular mechanisms underlying the coexistence of
these tumors is needed to optimize patient outcomes in this rare clinical entity.
Keywords: case report, meningioma, Nepal, neurofibromatois type 2, vestibular schwannoma

Introduction HIGHLIGHTS
VS is a benign intracranial tumor that originates from schwann • Meningioma and vestibular schwannoma (VS) are the first
cells that reside along the vestibular portion of the eighth cranial and second most common benign central nervous system
nerve[1]. VS accounts for 10% of all CNS tumors and 80% of the (CNS) tumors.
tumors in the cerebellopontine angle[2,3]. Meningioma derives • Their coexistence is possible in neurofibromatosis type 2
from the meningothelial cells of the arachnoid layer and is the (NF2) but is exceedingly rare in the pediatric age group.
most common benign CNS tumor, accounting for 13–26% of all • Diagnosis relies on clinical and genetic features, with
multidisciplinary management involving various
specialists.
a
Department: Department of Internal Medicine The Second Affiliated Hospital of • Further research into the genetic and molecular mechan-
Harbin Medical University, Harbin City, China, bDepartment: Department of Internal isms underlying the coexistence of these tumors is needed
Medicine Bir Hospital, cDepartment of Child HealthTribhuvan University Teaching
Hospital and dNo department Ministry of Health and Population, Kathmandu, Nepal to optimize patient outcomes in this rare clinical entity.
Sponsorships or competing interests that may be relevant to content are disclosed at
the end of this article.
brain tumors overall. More than 90% of all meningiomas are
*Corresponding author. Address: Ministry of Health and Population, Ramshah Path,
Kathmandu 44600, Nepal. Tel.: + 977 984 322 9124. E-mail: abalbaral@[Link] solitary[1]. However, in some extremely rare situations, VS can
(A. Baral). coexist with a meningioma in an adjacent anatomical position.
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. This is an This situation is most often reported in patients with NF2 or those
open access article distributed under the terms of the Creative Commons who have a history of previous irradiation[4–7]. NF2 is an auto-
Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is
permissible to download and share the work provided it is properly cited. The work
somal dominantly inherited syndrome that predisposes indivi-
cannot be changed in any way or used commercially without permission from the duals to bilateral VSs as well as multiple other tumors of the
journal. nervous system[8–10]. Except for those circumstances, the coex-
Annals of Medicine & Surgery (2024) 86:4247–4254 istence of these tumors is exceedingly rare. Children diagnosed
Received 24 April 2024; Accepted 15 May 2024 with NF2 often have an atypical but more severe presentation.
Published online 22 May 2024 Such a combination is exceptionally rare in this age group and
[Link] emphasizes some unique aspects of diagnosis and management.

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Herein, we present a rare case of a 15-year-old male with con- or calcification. These features were suggestive of a menin-
comitant bilateral VS and meningioma, which is also the first such gioma (differential diagnosis: choroid plexus papilloma).
reported case from Nepal, and a review of literature of similar Pure-tone audiometry demonstrated bilateral severe sensor-
other cases. This is reported in line with the Surgical CAse REport ineural hearing loss. MRI of the spine was normal. Other
(SCARE) 2023 criteria[11]. laboratory investigations, including complete blood count, renal
and liver function tests, and serology, were normal.

Case report Treatment and follow-up

History The patient’s parents were counseled regarding the possible
A 15-year-old male presented to the outpatient department with diagnosis of NF2. Subsequently, they expressed interest in seek-
the complaint of bilateral hearing loss, occasional imbalance, and ing CyberKnife Radiosurgery in India. It was chosen due to the
risk of neurological deficits after surgical resection and other
several episodes of generalized tonic–clonic seizures. The hearing
surgical risks to the patient. An informal follow-up via phone call
loss initially started almost 2 years back from the left ear and
revealed that the patient underwent radiosurgery for bilateral
progressed to involve both ears. There were multiple episodes of
VSs. Following a comprehensive assessment of the meningioma’s
tinnitus during the progression of hearing loss. He also com-
characteristics and location, a nonsurgical approach was selected
plained of blurred and double vision and early morning head-
due to the potential risks and absence of acute symptoms. This
aches. He had several episodes of generalized tonic–clonic
decision was reached through collaborative efforts involving
seizures in the last 2 months, which led his parents to seek medical
neurosurgeons, neurologists, and neuroradiologists. Following
care. He was not treated in any other center for such complaints
surgical intervention for the VS and conservative management of
in the past. There was no known family history of NF2. There
the concurrent meningioma, the patient reported improvement in
was no history of skull irradiation in the past.
symptoms, particularly in hearing and balance. Any kind of
genetic testing was not done due to limited access and financial
Examination reasons. A follow-up MRI of the brain conducted 32 months later
On examination, he was well oriented to time, place, and person, revealed the following findings (Fig. 2):
had a Glasgow Coma Scale of 15 (E4M5V6), had normal vital • Well-defined altered signal intensity lesion (T1 hypointense
signs, and there was no pallor, icterus, clubbing, cyanosis, or and T2/FLAIR heterogeneously hyperintense) of size
edema. Neurological examination revealed bilateral sensor- 22 × 20 mm in the atrium of right lateral ventricle with
ineural hearing loss (positive Rinne test and no lateralization of homogenous enhancement on postcontrast study and areas
Weber test), papilledema, nystagmus, diplopia on lateral gaze, of diffusion restriction on DWI within the lesion likely an
decreased corneal reflexes, facial twitching, and hyperesthesia. intraventricular meningioma (white arrowhead).
Other cranial nerves, motor and sensory systems, and cerebellar • Well-defined homogenously enhancing multilobulated T1
examination were normal. Physical examination did not reveal hypointense and T2/FLAIR isointense lesions of size
any findings suggestive of NF2. 24 × 23 mm and 25 × 12 mm were present in the right and
left cerebellopontine angles, respectively, with evidence of
extension into the internal auditory angle and prepontine
Investigations
cistern and homogenous enhancement postcontrast likely
Preoperative MRI of the brain revealed the following findings bilateral VSs (broad and thin white arrows respectively).
(Fig. 1): • Two well-defined homogenously lobulated altered signal
• Well-defined multilobulated extra-axial lesions of altered intensity lesions measuring 18 × 16 mm and 19 × 10 mm in
signal intensity (hypointense in T1, heterogeneously isointense the right and left prepontine cisterns, extending into adjacent
to gray matter) in bilateral cerebellopontine angle cisterns Meckel’s cave and heterogenous enhancement postcontrast
extending into the internal acoustic meatus measuring likely bilateral trigeminal schwannomas.
30 × 25 mm and 28 × 17 mm in left (thin white arrow) and • T2 hypointense lesion of size 26 × 8.3 mm arising from the
right side (broad white arrow), respectively, were present. The posterior duramater at D6–D7 level likely a spinal menin-
lesions were abutting both sides of the pons and anteromedial gioma (differential diagnosis: spinal schwannoma) with com-
aspect of bilateral cerebellar hemispheres. Similar lesions pression of the adjacent spinal cord with T2 hyperintensity.
measuring 17 × 14 mm and 13 × 11 mm were extending into • Multiple T2 hypointense nodular lesions arising from the
bilateral Meckel’s cave. The lesions showed mild homogenous cauda equina and filum terminale are likely to be
enhancement after contrast. There was no evidence of restric- neurofibromas
tion of diffusion. These lesions were suggestive of bilateral
vestibular and trigeminal schwannomas.
Discussion
• A well-defined intraventricular lesion of altered signal inten-
sity (isointense to gray matter in both T1 and T2) measuring Meningioma and VS are the first and second most common
22 × 21 mm is noted in the posterior horn of the right lateral benign CNS tumors. The coexistence of these tumors is seen
ventricle, and expanding it with restriction of diffusion was relatively often in patients with NF2, which is an autosomal
present (white arrowhead). Mild edema in the adjacent brain dominantly inherited syndrome that predisposes individuals to
parenchyma was also noted. In the postcontrast study, the bilateral VSs as well as multiple other tumors of the nervous
lesion showed near-homogenous and moderate enhancement. system[8–10]. A previous history of irradiation is another known
There was no evidence of intralesional necrosis, hemorrhage, cause of the occurrence of multiple primary brain tumors[12].

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Figure 1. Preoperative MRI of the brain.

Generally, acoustic neuromas are diagnosed between the fourth commonly present with a constellation of symptoms, including
and sixth decades of life. However, individuals with NF2 tend to eye problems, hearing loss, weakness, pain, mononeuropathy,
present earlier, with the peak incidence occurring in the third cutaneous tumors, and seizures[15–17]. In contrast, adults with
decade of life[13]. Children diagnosed with NF2 often have an NF2 commonly present with hearing loss and tinnitus as the
atypical but more severe presentation[14]. In childhood, patients predominant symptoms. The hallmark features of NF2

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Figure 2. Postoperative MRI of the brain.

encompass a spectrum of neurologic and ocular lesions, including common presenting symptom in patients with VSs[26–34]. The
bilateral VSs, schwannomas of other cranial nerves, intracranial onset of hearing loss is typically gradual and progressive, leading
meningiomas, intramedullary and extramedullary spinal tumors, to deafness over time, although sudden hearing loss can also
and peripheral neuropathy[18–20]. Additionally, ocular manifes- occur. NF2-related VSs are usually multifocal, affecting both the
tations such as cortical and posterior subcapsular cataracts, superior and inferior vestibular nerves[35]. Despite their pre-
epiretinal membranes, and retinal hamartomas are frequently valence, the mechanism underlying hearing loss in NF2-related
observed[21–23]. Cutaneous manifestations, including cutaneous VSs remains poorly understood, with no clear correlation
tumors, skin plaques, and subcutaneous tumors, further con- between tumor size, growth rate, and the degree of hearing loss
tribute to the clinical complexity of NF2[18,20,24,25]. observed[36–39]. If left untreated, VSs can extend medially and
VSs, the primary hallmark lesion of NF2, are typically bilateral cause brainstem compression and hydrocephalus.
and cause symptoms such as tinnitus, hearing loss, and balance Individuals with NF2 commonly develop meningiomas, with
dysfunction[10]. Hearing loss is well-documented as the most about half of NF2 patients affected[40]. The incidence of

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meningiomas increases with age, potentially reaching a lifetime risk improving hearing in NF2-associated VSs. Some centers use
of up to 75%[20]. While most meningiomas are found within the bevacizumab as a first-line therapy for rapidly growing schwan-
cranium, spinal meningiomas are also observed. NF2 patients nomas threatening function before considering surgery[66]. The
typically develop meningiomas at a younger age compared to efficacy of bevacizumab is supported by retrospective case series
sporadic cases, with approximately 20% of childhood meningioma and limited prospective studies[66–76].
diagnoses being associated with NF2[14,41]. Histopathologically, Surgical management is often necessary for VSs, although
NF2-associated meningiomas are more frequently atypical or bevacizumab may be used initially in some cases. Surgical pro-
anaplastic compared to sporadic cases[42,43]. cedures in NF2 patients can be more complex due to multifocal
When meningiomas and VSs occur simultaneously, they are tumors[35,77]. VSs may involve facial nerve fibers, increasing the
termed concomitant, collision[44], concurrent[45,46], coexisting[45–47], risk of facial nerve damage during surgery[8].
or coincidental tumors[2]. Several hypotheses have been proposed to The role of radiation therapy in NF2-related VSs is
explain the formation of these collision tumors: uncertain[78–81], with variable outcomes reported in studies.
1. Schwannomas may influence the local microenvironment, Long-term follow-up data are lacking, and concerns exist
promoting the occurrence of meningiomas[5,48–50]. regarding the potential for increased risk of second malignancies
2. Mutations in the Merlin gene, located on chromosome 22q, after radiation therapy[82–84]. Additionally, surgical resection
are associated with meningioma development[51]. The may be more challenging following stereotactic radiosurgery.
absence or inactivation of merlin is also implicated in sporadic Hearing impairment in NF2 patients significantly impacts their
VSs[52]. quality of life and social well-being[85]. Strategies such as cochlear
3. Both neoplasms may originate from the same mesenchymal or brainstem implants may offer benefits for those with severe
progenitor cells, differentiating into distinct tumor cell hearing impairment, helping to mitigate social avoidance,
types[48,53–57]. unemployment, and decreased social support associated with
4. Exposure of two different tumor cell types to the same hearing loss in NF2[86].
oncogenic stimulus at the same site may lead to the develop- Meningiomas can develop intracranially or within the spinal
ment of a collision tumor[2,55,56]. cord’s intradural, extramedullary sites. Many NF2-associated
5. The independent development of both neoplasms, with their meningiomas reach a stable size without needing active treat-
concurrent appearance, is considered a coincidence[2,55,58]. ment. Factors like tumor size, peritumoral edema, absence of
The diagnosis of NF2 relies on characteristic clinical and calcifications, and isointense or hyperintense MRI signals are risk
molecular genetic features. The diagnostic criteria are recom-
factors for rapid growth, similar to sporadic meningiomas[87].
mended by an international consensus group[59], a refinement of
Rapidly growing or functionally threatening meningiomas are
the Manchester criteria. Diagnosis involves a comprehensive
typically managed surgically, with radiation therapy considered
evaluation, including clinical and family history, neurological
for inoperable cases[88]. Lapatinib has shown some efficacy in
examination, and imaging studies such as contrast-enhanced
progressive NF2-associated meningiomas[89], but evidence sup-
MRI of the brain and spine[8]. MRI with gadolinium contrast is
porting bevacizumab’s effectiveness is limited[90].
crucial for assessing meningiomas and VSs[45], although it may
Intramedullary spinal tumors, primarily ependymomas, are
not always detect coexisting tumor types[45–47,60,61]. VSs often
often asymptomatic and grow slowly. Surgical resection is pre-
invade the internal auditory canal, exhibit cystic changes, and
ferred over radiation therapy if intervention is necessary[91],
show reduced internal homogeneity on imaging[2]. In contrast,
although cystic components may respond to bevacizumab[92].
meningiomas typically present with a dural tail sign and
Genetic testing is vital for suspected schwannomatosis and NF2
calcifications[53,62], lacking in VSs. Imaging characteristics
diagnosis, especially in younger patients and first-degree relatives.
include intense to slight hypointensity on T1-weighted images
and higher intensity on T2-weighted images for VSs compared to Regular monitoring, including audiology, ophthalmologic eva-
meningiomas[2,53,62,63]. Schwannomas are heterogeneously luation, cutaneous examination, and MRI scans, is recommended
enhanced on contrast enhancement and frequently extend into for individuals with NF2 pathogenic variants[8,10,65,93]. The lim-
the internal auditory canal, while meningiomas show homo- itations of our study include lack of long-term follow-up data,
genous enhancement[2,58]. Pathological examination reveals that lack of genetic testing of the patient, and for familial cases.
schwannomas express S-100 protein and anti-Leu 7 but are
negative for EMA, with possible focal EMA reactivity[5,46,64]. In
contrast, meningiomas are positive for EMA and negative for Conclusion
S-100 and anti-Leu 7[2,5,64]. The cooccurrence of VSs and meningiomas is poorly understood,
The management of NF2 is complex and requires a multi- with limited reported cases and unclear pathophysiological
disciplinary approach involving various specialists to prevent or mechanisms. While our study contributes to this knowledge gap,
address potential complications. A multidisciplinary team typi- long-term follow-up data are needed, and genetic testing for
cally includes neurosurgeons, neuro-otologists, neuroradiolo- familial causes remains unavailable. Further research into the
gists, neurologists or neuro-oncologists, audiologists, specialist genetic and molecular mechanisms underlying the coexistence of
nurses, and geneticists with expertise in NF2 care[65]. these tumors is needed to optimize patient outcomes in this rare
Treatment of VSs in NF2 patients aims at preserving function
clinical entity.
and maintaining quality of life[8]. Treatment is typically initiated
when there is a risk of brainstem compression, hearing dete-
rioration, or facial nerve dysfunction[10]. Bevacizumab, a Ethical approval
monoclonal antibody targeting vascular endothelial growth fac-
tor, has shown promising results in inducing tumor shrinkage and Not applicable.

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Chaulagain et al. Annals of Medicine & Surgery (2024) Annals of Medicine & Surgery

Consent [8] Evans DGR. Neurofibromatosis type 2 (NF2): a clinical and molecular
review. Orphanet J Rare Dis 2009;4:16.
Written informed consent was obtained from the patient’s par- [9] Asthagiri AR, Parry DM, Butman JA, et al. Neurofibromatosis type 2.
ents for the publication of this case report and accompanying Lancet Lond Engl 2009;373:1974–86.
[10] Evans DGR. Neurofibromatosis 2 [Bilateral acoustic neurofibromatosis,
images. A copy of written consent is available for review by the
central neurofibromatosis, NF2, neurofibromatosis type II. Genet Med
editor-in-chief of this journal on request. Off J Am Coll Med Genet 2009;11:599–610.
[11] Sohrabi C, Mathew G, Maria N, et al. The SCARE 2023 guideline:
updating consensus Surgical CAse REport (SCARE) guidelines. Int J Surg
Source of funding Lond Engl 2023;109:1136–40.
[12] Chen JC, Tseng SH, Chen Y, et al. Cervical dumbbell meningioma and
Not applicable. thoracic dumbbell schwannoma in a patient with neurofibromatosis. Clin
Neurol Neurosurg 2005;107:253–7.
[13] Greene J, Al-Dhahir MA. Acoustic neuroma In: StatPearls. StatPearls
Author contribution Publishing; 2024.
[14] Evans DG, Birch JM, Ramsden RT. Paediatric presentation of type 2
R.P.C.: wrote the original manuscript, reviewed and edited the neurofibromatosis. Arch Dis Child 1999;81:496–9.
[15] Forde C, King AT, Rutherford SA, et al. Disease course of neurofi-
original manuscript; K.K.C.: wrote the original manuscript,
bromatosis type 2: a 30-year follow-up study of 353 patients seen at a
reviewed and edited the original manuscript; A.B.: wrote the single institution. Neuro-Oncology 2021;23:1113–24.
original manuscript, reviewed and edited the original manuscript; [16] Gaudioso C, Listernick R, Fisher MJ, et al. Neurofibromatosis 2 in
Y.S.: edited the original manuscript. children presenting during the first decade of life. Neurology 2019;93:
e964–7.
[17] Anand G, Vasallo G, Spanou M, et al. Diagnosis of sporadic neurofi-
Conflict of interest disclosure bromatosis type 2 in the paediatric population. Arch Dis Child 2018;103:
463–9.
There are no conflicts of interest. [18] Parry DM, Eldridge R, Kaiser-Kupfer MI, et al. Neurofibromatosis 2
(NF2): clinical characteristics of 63 affected individuals and clinical evi-
dence for heterogeneity. Am J Med Genet 1994;52:450–61.
[19] Mautner VF, Lindenau M, Baser ME, et al. The neuroimaging and clin-
Research registration unique identifying number ical spectrum of neurofibromatosis 2. Neurosurgery 1996;38:
(UIN) 880–5; discussion 885–886.
[20] Evans DG, Huson SM, Donnai D, et al. A clinical study of type 2 neu-
Not applicable. rofibromatosis. Q J Med 1992;84:603–18.
[21] Bosch MM, Boltshauser E, Harpes P, et al. Ophthalmologic findings and
long-term course in patients with neurofibromatosis type 2. Am J
Guarantor Ophthalmol 2006;141:1068–77.
[22] Ragge NK, Baser ME, Riccardi VM, et al. The ocular presentation of
Abal Baral. neurofibromatosis 2. Eye Lond Engl 1997;11(Pt 1):12–8.
[23] Ragge NK, Baser ME, Klein J, et al. Ocular abnormalities in neurofi-
bromatosis 2. Am J Ophthalmol 1995;120:634–41.
Data availability statement [24] MacCollin M, Mautner VF. The diagnosis and management of neurofi-
bromatosis 2 in childhood. Semin Pediatr Neurol 1998;5:243–52.
Not applicable. [25] Mautner VF, Lindenau M, Baser ME, et al. Skin abnormalities in neu-
rofibromatosis 2. Arch Dermatol 1997;133:1539–43.
[26] Strasnick B, Glasscock ME, Haynes D, et al. The natural history of
untreated acoustic neuromas. Laryngoscope 1994;104:1115–9.
Provenance and peer review [27] Fucci MJ, Buchman CA, Brackmann DE, et al. Acoustic tumor
growth: implications for treatment choices. Am J Otol 1999;20:
Non-commissioned, externally peer-reviewed. 495–9.
[28] Brackmann DE, Owens RM, Friedman RA, et al. Prognostic factors for
hearing preservation in vestibular schwannoma surgery. Am J Otol 2000;
References 21:417–24.
[1] Louis DN, Ohgaki H, Wiestler OD, et al. The 2007 WHO classification of [29] Briggs RJ, Brackmann DE, Baser ME, et al. Comprehensive management
tumours of the central nervous system. Acta Neuropathol (Berl) 2007; of bilateral acoustic neuromas. Current perspectives. Arch Otolaryngol
114:97–109. Head Neck Surg 1994;120:1307–14.
[2] Izci Y, Secer HI, Gönül E, et al. Simultaneously occurring vestibular [30] Evans DG, Huson SM, Donnai D, et al. A genetic study of type 2 neu-
schwannoma and meningioma in the cerebellopontine angle: case report rofibromatosis in the United Kingdom. I. Prevalence, mutation rate, fit-
and literature review. Clin Neuropathol 2007;26:219–23. ness, and confirmation of maternal transmission effect on severity. J Med
[3] Chen AF, Samy RN, Gantz BJ. Cerebellopontine angle tumor composed Genet 1992;29:841–6.
of Schwann and meningeal proliferations. Arch Otolaryngol Head Neck [31] Gadre AK, Kwartler JA, Brackmann DE, et al. Middle fossa decom-
Surg 2001;127:1385–9. pression of the internal auditory canal in acoustic neuroma surgery: a
[4] Verma SK, Kumar S, Deb P, et al. Rare case of radiologically distinct but therapeutic alternative. Laryngoscope 1990;100:948–52.
pathologically admixed vestibular schwannoma and meningioma in the [32] Kesterson L, Shelton C, Dressler L, et al. Clinical behavior of acoustic
cerebellopontine angle: a case report. J Cancer Res Ther 2015;11:1029. tumors. A flow cytometric analysis. Arch Otolaryngol Head Neck Surg
[5] Matyja E, Kunert P, Grajkowska W, et al. Coexistence of meningioma 1993;119:269–71.
and schwannoma in the same cerebellopontine angle in a patients with [33] Saunders JE, Luxford WM, Devgan KK, et al. Sudden hearing loss in
NF2. Folia Neuropathol 2012;50:166–72. acoustic neuroma patients. Otolaryngol Head Neck Surg Off J Am Acad
[6] Elizabeth J, Menon G, Nair S, et al. Mixed tumour of schwannoma and Otolaryngol-Head Neck Surg 1995;113:23–31.
meningioma in a patient with neurofibromatosis-2 : a case report. Neurol [34] Slattery WH, Brackmann DE, Hitselberger W. Middle fossa approach for
India 2001;49:398–400. hearing preservation with acoustic neuromas. Am J Otol 1997;18:
[7] Kim DG, Paek SH, Chi JG, et al. Mixed tumour of schwannoma and 596–601.
meningioma components in a patient with NF-2. Acta Neurochir (Wien) [35] Evans DGR, Stivaros SM. Multifocality in neurofibromatosis type 2.
1997;139:1061–4; discussion 1064–1065. Neuro-Oncology 2015;17:481–2.

4252
Chaulagain et al. Annals of Medicine & Surgery (2024)

[36] Abaza MM, Makariou E, Armstrong M, et al. Growth rate character- [59] Plotkin SR, Messiaen L, Legius E, et al. Updated diagnostic criteria and
istics of acoustic neuromas associated with neurofibromatosis type 2. nomenclature for neurofibromatosis type 2 and schwannomatosis: an
Laryngoscope 1996;106:694–9. international consensus recommendation. Genet Med Off J Am Coll Med
[37] Fisher LM, Doherty JK, Lev MH, et al. Concordance of bilateral ves- Genet 2022;24:1967–77.
tibular schwannoma growth and hearing changes in neurofibromatosis 2: [60] Jain A, Suri V, Sharma BS, et al. Mixed schwannoma with meningioma of
neurofibromatosis 2 natural history consortium. Otol Neurotol Off Publ the trigeminal nerve. Indian J Pathol Microbiol 2010;53:769–71.
Am Otol Soc Am Neurotol Soc Eur Acad Otol Neurotol 2009;30: [61] Lüdemann W, Stan AC, Tatagiba M, et al. Sporadic unilateral vestibular
835–41. schwannoma with islets of meningioma: case report. Neurosurgery 2000;
[38] Masuda A, Fisher LM, Oppenheimer ML, et al. Natural History 47:451–2; discussion 452–454.
Consortium. Hearing changes after diagnosis in neurofibromatosis type [62] Lalwani AK, Jackler RK. Preoperative differentiation between menin-
2. Otol Neurotol Off Publ Am Otol Soc Am Neurotol Soc Eur Acad Otol gioma of the cerebellopontine angle and acoustic neuroma using MRI.
Neurotol 2004;25:150–4. Otolaryngol Head Neck Surg Off J Am Acad Otolaryngol-Head Neck
[39] Graamans K, Van Dijk JE, Janssen LW. Hearing deterioration in patients Surg 1993;109:88–95.
with a non-growing vestibular schwannoma. Acta Otolaryngol (Stockh) [63] Wilms G, Plets C, Goossens L, et al. The radiological differentiation of
2003;123:51–4. acoustic neurinoma and meningioma occurring together in the cere-
[40] Goutagny S, Kalamarides M. Meningiomas and neurofibromatosis. J bellopontine angle. Neurosurgery 1992;30:443–5; discussion 445–446.
Neurooncol 2010;99:341–7. [64] Buerki RA, Horbinski CM, Kruser T, et al. An overview of meningiomas.
[41] Evans DGR, Watson C, King A, et al. Multiple meningiomas: differential Future Oncol Lond Engl 2018;14:2161–77.
involvement of the NF2 gene in children and adults. J Med Genet 2005; [65] Evans DGR, Salvador H, Chang VY, et al. Cancer and Central Nervous
42:45–8. System Tumor Surveillance in Pediatric Neurofibromatosis 2 and Related
[42] Perry A, Giannini C, Raghavan R, et al. Aggressive phenotypic and Disorders. Clin Cancer Res Off J Am Assoc Cancer Res 2017;23:e54–61.
genotypic features in pediatric and NF2-associated meningiomas: a [66] Morris KA, Golding JF, Axon PR, et al. Bevacizumab in neurofi-
clinicopathologic study of 53 cases. J Neuropathol Exp Neurol 2001;60: bromatosis type 2 (NF2) related vestibular schwannomas: a nationally
994–1003. coordinated approach to delivery and prospective evaluation.
[43] Larson JJ, van Loveren HR, Balko MG, et al. Evidence of meningioma Neuro-Oncol Pract 2016;3:281–9.
infiltration into cranial nerves: clinical implications for cavernous sinus [67] Mautner VF, Nguyen R, Kutta H, et al. Bevacizumab induces regression
meningiomas. J Neurosurg 1995;83:596–9. of vestibular schwannomas in patients with neurofibromatosis type 2.
[44] Muzumdar DP, Goel A. Acoustic schwannoma and petroclival menin- Neuro-Oncol 2010;12:14–8.
gioma occurring as collision tumours: a case report. J Clin Neurosci Off J [68] Plotkin SR, Stemmer-Rachamimov AO, Barker FG, et al. Hearing
Neurosurg Soc Australas 2004;11:207–10. improvement after bevacizumab in patients with neurofibromatosis type
[45] Kutz JW, Barnett SL, Hatanpaa KJ, et al. Concurrent vestibular 2. N Engl J Med 2009;361:358–67.
schwannoma and meningioma mimicking a single cerebellopontine angle [69] Plotkin SR, Merker VL, Halpin C, et al. Bevacizumab for progressive
tumor. Skull Base Off J North Am Skull Base Soc Al 2009;19:443–6. vestibular schwannoma in neurofibromatosis type 2: a retrospective
[46] Grauvogel J, Grauvogel TD, Taschner C, et al. A rare case of radi- review of 31 patients. Otol Neurotol Off Publ Am Otol Soc Am Neurotol
ologically not distinguishable coexistent meningioma and vestibular Soc Eur Acad Otol Neurotol 2012;33:1046–52.
schwannoma in the cerebellopontine angle – case report and literature [70] Hochart A, Gaillard V, Baroncini M, et al. Bevacizumab decreases ves-
review. Case Rep Neurol 2010;2:111–7. tibular schwannomas growth rate in children and teenagers with neuro-
[47] Chandra PS, Hegde T. A case of coexisting cerebellopontine angle fibromatosis type 2. J Neurooncol 2015;124:229–36.
meningioma and schwannoma. Neurol India 2000;48:198. [71] Blakeley JO, Ye X, Duda DG, et al. Efficacy and biomarker study of
[48] Nakamizo A, Suzuki SO, Shimogawa T, et al. Concurrent spinal nerve bevacizumab for hearing loss resulting from neurofibromatosis type 2-
root schwannoma and meningioma mimicking single-component associated vestibular schwannomas. J Clin Oncol Off J Am Soc Clin
schwannoma. Neuropathol Off J Jpn Soc Neuropathol 2012;32:190–5. Oncol 2016;34:1669–75.
[49] Geddes JF, Sutcliffe JC, King TT. Mixed cranial nerve tumors in neuro- [72] Sverak P, Adams ME, Haines SJ, et al. Bevacizumab for hearing pre-
fibromatosis type 2. Clin Neuropathol 1995;14:310–3. servation in neurofibromatosis type 2: emphasis on patient-reported
[50] Sobel RA. Vestibular (acoustic) schwannomas: histologic features in outcomes and toxicities. Otolaryngol Head Neck Surg Off J Am Acad
neurofibromatosis 2 and in unilateral cases. J Neuropathol Exp Neurol Otolaryngol-Head Neck Surg 2019;160:526–32.
1993;52:106–13. [73] Li KL, Djoukhadar I, Zhu X, et al. Vascular biomarkers derived from
[51] Lee S, Karas PJ, Hadley CC, et al. The role of merlin/NF2 loss in dynamic contrast-enhanced MRI predict response of vestibular schwan-
meningioma biology. Cancers 2019;11:1633. noma to antiangiogenic therapy in type 2 neurofibromatosis.
[52] Sass H, Cayé-Thomasen P. Contemporary molecular biology of sporadic Neuro-Oncology 2016;18:275–82.
vestibular schwannomas: a systematic review and clinical implications. J [74] Lu VM, Ravindran K, Graffeo CS, et al. Efficacy and safety of bev-
Int Adv Otol 2018;14:322–9. acizumab for vestibular schwannoma in neurofibromatosis type 2: a
[53] Porčnik A, Žele T, Prestor B. Concurrent intradural meningioma and systematic review and meta-analysis of treatment outcomes. J
schwannoma at the same lumbar level in a patient without neurofi- Neurooncol 2019;144:239–48.
bromatosis: a case report. Br J Neurosurg 2020;34:104–6. [75] Plotkin SR, Duda DG, Muzikansky A, et al. Multicenter, prospective,
[54] Oichi T, Chikuda H, Morikawa T, et al. Concurrent spinal schwannoma phase II and biomarker study of high-dose bevacizumab as induction
and meningioma mimicking a single cervical dumbbell-shaped tumor: therapy in patients with neurofibromatosis type 2 and progressive ves-
case report. J Neurosurg Spine 2015;23:784–7. tibular schwannoma. J Clin Oncol Off J Am Soc Clin Oncol 2019;37:
[55] Nehete L, Nandeesh BN, Bharath RD, et al. Cerebellopontine angle 3446–54.
schwannoma and meningioma in contiguity: surgical implications in [76] Plotkin SR, Allen J, Dhall G, et al. Multicenter, prospective, phase II study of
neurofibromatosis. J Neurol Surg Part Cent Eur Neurosurg 2018;79: maintenance bevacizumab for children and adults with NF2-related schwan-
177–80. nomatosis and progressive vestibular schwannoma. Neuro-Oncology 2023;
[56] Liebelt BD, Haider AS, Steele WJ, et al. Spinal schwannoma and 25:1498–506.
meningioma mimicking a single mass at the craniocervical junction [77] Dewan R, Pemov A, Kim HJ, et al. Evidence of polyclonality in neuro-
subsequent to remote radiation therapy for acne vulgaris. World fibromatosis type 2-associated multilobulated vestibular schwannomas.
Neurosurg 2016;93:484.e13–16. Neuro-Oncology 2015;17:566–73.
[57] Matsuda S, Kajihara Y, Abiko M, et al. Concurrent schwannoma and [78] Kida Y, Kobayashi T, Tanaka T, et al. Radiosurgery for bilateral neur-
meningioma arising in the same spinal level: a report of two cases. NMC inomas associated with neurofibromatosis type 2. Surg Neurol 2000;53:
Case Rep J 2018;5:105–9. 383–9; discussion 389–390.
[58] Zhan Z, Yan X, Nie W, et al. Neurofibroma and meningioma within a [79] Mathieu D, Kondziolka D, Flickinger JC, et al. Stereotactic radiosurgery
single dumbbell-shaped tumor at the same cervical level without neuro- for vestibular schwannomas in patients with neurofibromatosis type 2: an
fibromatosis: a case report and literature review. World Neurosurg 2019; analysis of tumor control, complications, and hearing preservation rates.
130:1–6. Neurosurgery 2007;60:460–8; discussion 468–470.

4253
Chaulagain et al. Annals of Medicine & Surgery (2024) Annals of Medicine & Surgery

[80] Roche PH, Régis J, Pellet W, et al. Neurofibromatosis type 2. Preliminary [86] Neff BA, Wiet RM, Lasak JM, et al. Cochlear implantation in the neu-
results of gamma knife radiosurgery of vestibular schwannomas. rofibromatosis type 2 patient: long-term follow-up. Laryngoscope 2007;
Neurochirurgie 2000;46:339–53; discussion 354. 117:1069–72.
[81] Rowe JG, Radatz MWR, Walton L, et al. Clinical experience with gamma [87] Abi Jaoude S, Peyre M, Degos V, et al. Validation of a scoring system to
knife stereotactic radiosurgery in the management of vestibular evaluate the risk of rapid growth of intracranial meningiomas in neuro-
schwannomas secondary to type 2 neurofibromatosis. J Neurol fibromatosis type 2 patients. J Neurosurg 2020;134:1377–85.
Neurosurg Psychiatry 2003;74:1288–93. [88] Wentworth S, Pinn M, Bourland JD, et al. Clinical experience with radiation
[82] Baser ME, Evans DG, Jackler RK, et al. Neurofibromatosis 2, therapy in the management of neurofibromatosis-associated central nervous
radiosurgery and malignant nervous system tumours. Br J Cancer system tumors. Int J Radiat Oncol Biol Phys 2009;73:208–13.
2000;82:998. [89] Osorio DS, Hu J, Mitchell C, et al. Effect of lapatinib on meningioma growth
[83] Evans DGR, Birch JM, Ramsden RT, et al. Malignant transformation in adults with neurofibromatosis type 2. J Neurooncol 2018;139:749–55.
and new primary tumours after therapeutic radiation for benign disease: [90] Nunes FP, Merker VL, Jennings D, et al. Bevacizumab treatment for
substantial risks in certain tumour prone syndromes. J Med Genet 2006; meningiomas in NF2: a retrospective analysis of 15 patients. PloS One
43:289–94. 2013;8:e59941.
[84] Balasubramaniam A, Shannon P, Hodaie M, et al. Glioblastoma multi- [91] Kalamarides M, Essayed W, Lejeune JP, et al. Spinal ependymomas in
forme after stereotactic radiotherapy for acoustic neuroma: case report NF2: a surgical disease? J Neurooncol 2018;136:605–11.
and review of the literature. Neuro-Oncol 2007;9:447–53. [92] Farschtschi S, Merker VL, Wolf D, et al. Bevacizumab treatment for
[85] Merker VL, Bergner AL, Vranceanu AM, et al. Health-related quality of symptomatic spinal ependymomas in neurofibromatosis type 2. Acta
life of individuals with neurofibromatosis type 2: results from the NF2 Neurol Scand 2016;133:475–80.
natural history study. Otol Neurotol Off Publ Am Otol Soc Am Neurotol [93] Evans DG, Raymond FL, Barwell JG, et al. Genetic testing and screening
Soc Eur Acad Otol Neurotol 2016;37:574–9. of individuals at risk of NF2. Clin Genet 2012;82:416–24.

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 TYPE Original Research
PUBLISHED 07 August 2024
DOI 10.3389/fcimb.2024.1434677

Antifungal susceptibility pattern

OPEN ACCESS of Candida species isolated from
EDITED BY
Kunlong Yang,
Jiangsu Normal University, China
pregnant women
REVIEWED BY
Su Qu,
Maqsood Ali 1*, Wadhah Hassan Edrees 2,3,
Sun Yat-Sen University, China Wadee Abdullah Al-Shehari 4, Gao Xue 1, Safa Al-Hammadi 5,
Guang Yang,
Xinyang Normal University, China Eglal Ahmed Qasem 4, Ram Prasad Chaulagain 6 and Nand Lal 7
*CORRESPONDENCE 1
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Harbin Medical
Maqsood Ali University, Harbin, Heilongjiang, China, 2 Medical Microbiology Department, Faculty of Applied
[Link]@[Link] Science, Hajjah University, Hajjah, Yemen, 3 Medical Laboratory Department, Faculty of Medical
Sciences, Al-Razi University, Sana’a, Yemen, 4 Medical Microbiology Department, Faculty of Medical
RECEIVED 18 May 2024 Sciences, Ibb University, Ibb, Yemen, 5 Department Oral Medicine, Harbin Medical University, Harbin,
ACCEPTED 10 July 2024 Heilongjiang, China, 6 Department of Gastroenterology and Hepatology, Second Affiliated Hospital of
PUBLISHED 07 August 2024 Harbin Medical University, Harbin, Heilongjiang, China, 7 Department of Physiology, School of
Biomedical Sciences, Harbin Medical University, Harbin, Heilongjiang, China
CITATION
Ali M, Edrees WH, Al-Shehari WA, Xue G,
Al-Hammadi S, Qasem EA, Chaulagain RP
and Lal N (2024) Antifungal susceptibility
pattern of Candida species isolated from
Introduction: Candida species, opportunistic yeast, are the second most
pregnant women. common cause of female vulvovaginal candidiasis. This study aimed to
Front. Cell. Infect. Microbiol. 14:1434677. evaluate the antifungal susceptibility profile of the isolated Candida species in
doi: 10.3389/fcimb.2024.1434677
pregnant women in Hajjah governorate, Yemen.
COPYRIGHT
© 2024 Ali, Edrees, Al-Shehari, Xue,
Al-Hammadi, Qasem, Chaulagain and Lal. This Methods: A hospital-based cross-sectional study was conducted among 396
is an open-access article distributed under the pregnant women attending Authority AL-Gumhorri Hospital Hajjah between
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
February and July 2023. Vaginal swabs were collected, and Candida species
reproduction in other forums is permitted, were isolated and identified based on the standard laboratory method.
provided the original author(s) and the Furthermore, the antifungal drug susceptibility of Candida species was
copyright owner(s) are credited and that the
original publication in this journal is cited, in determined by the Kirby-Bauer technique.
accordance with accepted academic
practice. No use, distribution or reproduction Results and discussion: The prevalence of vaginal Candida infection among
is permitted which does not comply with
these terms. pregnant women was 61.4%. Candida albicans was the most predominant
species (59.26%), followed by Candida krusei(13.58%), Candida Tropicalis
(11.12%), Candida Grabata (9.87%), and Candida dubliniensis (6.17%). The
highest rate of Candida infections was among women aged 24–30 years
(71.9%) who finished primary school (77.8%), with the third trimester (80%),
multigravida (66.1%), and recurrent infection (67.7%) showing significant
differences (P < 0.05). The Candida albicans isolates were resistant to
clotrimazole and itraconazole at 34.7% and 23.6%, [Link] addition, the
resistance of Candida krusei, Candida tropicalis, Candida glabrata, and Candida
dublinensis isolates to fluconazole, voriconazole, voriconazole, and nystatin was
57.6%, 63%, 43.8%, and 60%, respectively. Additionally, approximately 46.2% of
isolated Candida albicans exhibited one kind of antifungal drug resistance,
whereas 38.7% of isolated non-albicans exhibited resistance to three different

Abbreviations: CDC, Centre for Disease Control and Prevention; CI, Confidence Interval; CLSI, Clinical and
Laboratory Standard Institute; MHA, Mueller Hinton Agar; OR, Odds Ratio; SD, Standard Division; SPSS,
Statistical Package for the Social Sciences; VVC, Vulvovaginal Candidiasis; c2, Chi-square.

Frontiers in Cellular and Infection Microbiology 01 [Link]

Ali et al. 10.3389/fcimb.2024.1434677

antifungal agents. According to the above findings, Candida infection is highly

prevalent in Yemen and quite widespread. Interventions in health education are
advised to increase women’s knowledge of vaginitis and its prevention. The
antifungal susceptibility test may also be helpful in determining the best
medication for each patient.

KEYWORDS

antifungal, Candida albicans, pregnant women, vulvo vaginal candidiasis, Yemen

Introduction The issue is particularly significant during pregnancy since

Candida colonization is linked to infant mortality and premature
Vulvovaginal candidiasis (VVC) commonly infects the mucous delivery and because pregnant women can contaminate their
membrane of the lower genital tract in females. It is usually caused unborn children to a degree of up to 65%, leading to invasive
by Candida albicans but may occasionally be caused by other neonatal candidiasis. Due to the emergence of Candida species that
Candida species or yeasts. It is also known to have severe or are resistant to routinely used antifungal medications and repeated
persistent daily symptoms and is linked to an increased rate of infections, the prevalence of vaginal candidiasis has recently
antifungal resistance (Central for Diseases Control and Prevention substantially increased (Roberts et al., 2011; Masri et al., 2015).
(CDC), 2021). Increased Candida species resistance to various antifungal
Furthermore, it is common for Candida species, which are a treatments is a result of improper use of antifungal medications
part of the vulvovagina’s natural flora, to produce opportunistic and a lack of adequate strategies to govern their usage, particularly
infections when the host’s immune system is impaired. Because in the treatment of vulvovaginal candidiasis. Azoles are the first-line
Candida sp. coexist in harmony with the vaginal microbiota, medications for treating VVC initially, but fluconazole’s prolonged
asymptomatic colonization is frequent and can last for years. usage has led to the emergence of multidrug-resistant infections and
Young women without symptoms have a genital Candida recurring infections, which is a serious healthcare issue. To combat
colonization rate of 20%, whereas pregnant women have a rate of the medication resistance issue, it is urgently necessary to find novel
up to 30% (Ghaddar et al., 2020; Sule-Odu et al., 2020). compounds with antifungal capabilities due to various restrictions
Pruritus, vaginal discomfort, dyspareunia, external dysuria, and on the accessibility of current antifungal agents, ineffective therapy,
atypical vaginal discharge are typical symptoms of VVC. According high toxicity, low tolerability, and drug interaction (Lı́rio et al.,
to estimates, 75% of women will experience at least one episode of 2019; Rosati et al., 2020; Nikoomanesh et al., 2023; Zaman
VVC, and 40%–45% of them may experience two or more episodes. et al., 2024).
VVC is characterized as both complicated and uncomplicated. A One of the Yemeni governorates, Hajjah, lacks information on
complicated VVC will affect 10%–20% of women, necessitating the antifungal susceptibility pattern of Candida species that cause
particular diagnostic and treatment considerations (Sule-Odu et al., vulvovaginal candidiasis in pregnant women. Therefore, the
2020; Central for Diseases Control and Prevention (CDC), 2021). objective of this study was to evaluate the antifungal susceptibility
The predominant species historically has been Candida profile of the isolated Candida species in pregnant women in Hajjah
albicans, which accounts for 85–95% of Candida vaginal governorate, Yemen.
infections. However, non-albicans Candida (NAC) species have
been reported more frequently globally, with Candida glabrata,
Candida tropicalis, Candida parapsilosis, Candida krusei, and Materials and methods
Candida dupliniesis being the most common (Makanjuola et al.,
2018; Sule-Odu et al., 2020). Study design and period
A key risk factor is the age at which sexual engagement begins.
Host-related factors include immunologic alterations, increased A hospital-based cross-sectional study was conducted in
estrogen levels, diabetes mellitus, immunodeficiency states, obstetrics and gynecology clinics at Authority Al-Gumhorri
antibiotic use, treatment with glucocorticoids, genetic Hospital-Hajjah, which is situated in the Hajjah City. Hajjah is
predispositions, and behavioral factors such as birth control pills, located in the northwest of Sana’a capital with a distance of 123
intrauterine devices, spermicides, and condoms. Hygiene practices, kilometers and an elevation of almost 1800 meters. Additionally, it
tight-fitting clothing, and sexual behavior are thought to be risk is divided into 31 administrative districts on its border and
factors linked to an increased rate of VVC (Disha and Haque, 2022). consists of 2,630,678 inhabitants (Figure 1). The period of study

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Ali et al. 10.3389/fcimb.2024.1434677

FIGURE 1
Map of Yemen showing study area in Hajjah governorate.

was 6 months, from February to July 2023, and the laboratory those who were currently receiving antimicrobial medicine
analysis was performed at the hospital laboratory. treatment or refused to provide vaginal samples were not
included in the study.

Population size
Sample collection and examination
The population size consisted of 396 pregnant women attending
obstetrics and gynecology clinics with symptoms suggestive of A well-qualified obstetrician collected vaginal swabs from each
vaginitis. The age of the participants ranged between 17 and 44 pregnant woman by using a sterile cotton swab. The swab was
years, with an average age of 28.6 years. inserted approximately 5 cm into the vaginal opening, gently turned
for approximately 30 seconds, and rubbed against the wall of the
vagina. The collected specimens were labelled with patient
Data collection information and sent directly to the medical laboratory for
further examination (Mahon et al., 2015; Tille, 2017).
A pretested questionnaire was developed based on multiple past
studies (Khan et al., 2018; Abdul-Aziz et al., 2019), and some
adjustments were made to gather the necessary data separately Culture and identification of
from each participant. Age, residence area, educational level, Candida species
trimester, gravidity, frequency of infection, and medical signs and
symptoms of vaginal abnormalities (burning, itching, and vaginal The vaginal swabs were streaked individually under aseptic
discharge) were all information that was included in the conditions onto Sabouraud Dextrose Agar (SDA) plate agar
questionnaire. Research teams completed the questionnaire. The (HiMedia Lab., India) supplemented with chloramphenicol
age categories were divided into four groups: 18–23 years, 24–30 (250 mg/L). The inoculated plates were incubated aerobically at
years, 31–37 years, and 38–44 years. 37°C for 48 h. The growth colonies were inoculated onto
HiCrome agar (HiMedia Lab., India) and incubated at 37°C for
48 h. In addition, the identification of Candida species was
Inclusion and exclusion criteria performed based on the Candida colony’s characterization of
the medium. In addition, all isolates were further confirmed using
Women who gave vaginal specimens and had vaginal the API 20C AUX strip, as recommended by the manufacturer
inflammation with discharge, burning, itching, and other (BioMereaux, Marcy-l`É toile, France). The green colonies were
symptoms during pregnancy were included. On the other hand, identified as C. albicans, the purple colonies as C. glabrata, the

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Ali et al. 10.3389/fcimb.2024.1434677

pink colonies as C. krusei, the blue colonies as C. tropicalis, and addition, the validity of the results was guaranteed through the
the darker green colonies as C. dublinensis. Additionally, the implementation of standards of quality control during the pre-
germ tube test was used to distinguish between C. albicans and analytical, analytical, and post-analytical stages. All prepared
non-albicans (Mahon et al., 2015; Tille, 2017). culture media (plates and tubes) were checked for sterility and
performance before being used for the isolation and identification of
the studied Candida species. Positive controls included standard
Antifungal susceptibility testing fungi and patient specimens. while negative control plates were un-
inoculated. The positive and negative controls were employed to
The disk diffusion test of the Kirby–Bauer technique was used validate the quality of the isolation and identification results and to
to determine the antifungal drug susceptibility profiles of Candida avoid the possibility of cross-contamination.
species on Mueller Hinton agar (MHA) medium (HiMedia Lab.,
India) containing glucose (2%) and methylene blue dye (0.5 µg/
mL) according to the guidelines of the Clinical and Laboratory Ethics approval
Standards Institute (CLSI) (2021). A suspension of overnight
cultures of C. albicans, C. krusei, C. tropicalis, C. glabrata, and The Ethical Review Board of the Faculty of Applied Sciences
C. dublinensis were prepared separately in 5 mL of sterile 0.85% at Hajjah University granted ethical clearance on January 24,
saline. The turbidity containing 1-5×106 CFU/mL of isolated 2023. Additionally, the Faculty of Applied Sciences presented a
Candida species was adjusted to McFarland 0.5 standard by written document to the Authority Al-Gumhorri Hospital’s
using a DensiChek (bioMerieux) turbidity meter. A sterile administration to approve the collection and analysis of
cotton swab was moistened in the prepared suspension, and specimens. Before collecting data and specimens, study
excess fluid was removed. Under aseptic conditions, the surface volunteers were fully informed of the objectives and
of Müller-Hinton agar (MHA) plate was streaked by the swab for justification for their participation in the study. The informed
three times and kept at room temperature for 15 minutes to dry. consent form was obtained from educated individuals by taking
The antifungal discs, namely, amphotericin-B (50 µg), their signatures, while the illiterate participants took a fingerprint
itraconazole (30 µg), fluconazole (25 µg), ketoconazole (10 µg), instead of a signature. Additionally, the study’s confidentiality of
miconazole (30 µg), voriconazole (l0 µg), clotrimazole (10 µg), and data was maintained.
nystatin (100 units) (HiMedia Lab., India), were placed over the
surface of the MHA plate and incubated at 37°C for 48 h.
According to CLSI (2021) recommendations, the zone of Statistical analysis
inhibition was measured in millimeters, and the results were
interpreted as sensitive (S) and resistant (S) (Table 1) (Clinical The statistical analysis was performed using SPSS version 20
and Laboratory Standards Institute (CLSI), 2021). (Statistical Package for Social Science). Descriptive statistics,
numbers, frequencies, percentages, and tables were used to
describe the findings. Moreover, chi-square (c2), confidence
Quality control interval (95% CI), and odds ratio (OR) were used to assess the
association between dependent and independent variables. Finally,
The reference strains of Candida albicans ATCC 10231 and a p value less than 0.05 was considered statistically significant.
Candida krusei ATCC 6258 were used for quality control. In

TABLE 1 Interpretative breakpoints of antifungal agents.

Results
Disk concentration Diameter of zone of This research included approximately 396 pregnant women.
Antifungals inhibition (mm) Table 2 lists the participants’ characteristics, including the fact that
128 (32.3%) of them were between the ages of 24 and 30 years, 279
Sensitive Resistant
(70.5%) were from urban areas, 206 (52%) had completed their
Amphotericin-B 50 µg ≥15 ≤12
secondary education, 180 (45.5%) were in the third trimester, 207
Itraconazole 30 µg ≥22 ≤18 (52.3%) were primigravida, and 279 (70.5%) were in the first time
Fluconazole 25 µg ≥39 ≤29
of infection.
Figure 2A shows that out of 396 vaginal swabs, 243 (61.4%)
Ketoconazole 10 µg ≥32 ≤20
were positive for growth in culture media, while 153 (38.6%) were
Miconazole 30 µg ≥26 ≤22 negative. Additionally, the frequency rates of isolates of C. albicans
Voriconazole l0 µg ≥19 ≤14
were 144 (59.26%) followed by C. krusei 33 (13.58%), C. tropicalis
27 (11.12%), C. glabrata 24 (9.87%), and C. dublinensis 15 (6.17%)
Clotrimazole 10 µg ≥20 ≤11
as figured in Figure 2B.
Nystatin 100 units ≥ 15 ≤10 According to this finding, the highest rate of Candida infections
was recorded among pregnant women aged between 24 and 30

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TABLE 2 Sociodemographic characteristics of the pregnant women infected (66.1%) than primigravida participants (63.8%), with a
participating in the study.
statistically significant difference (P = 0.000). Moreover, recurrent
infections showed a high rate among participants (67.7%; P = 0.117)
Variables Frequency Rate (%)
compared to the first time of infection (46.2%), as listed in Table 3.
17-23 99 25.0
The current research found that participants’ clinical signs and
24-30 128 32.3 symptoms and Candida sp. prevalence were statistically significant
Age (in years)
(P < 0.05). Additionally, as shown in Table 4, pregnant women who
31-37 106 26.8
experienced discharge (72.7%; OR= 2.667; 95% CI=1.21–1.29) and
38-44 63 15.9
itching (64.5%; OR= 0.335; 95% CI=1.03–1.07) had the greatest
Urban 279 70.5 proportion of Candida sp. positivity.
Resident area
Rural 117 29.5
According to the antifungal susceptibility data, C. albicans
isolates were highly susceptible to amphotericin B, which had a
Illiterate 45 11.4
92.4% sensitivity rate, followed by ketoconazole (91.7%),
Primary 81 20.5 miconazole (85.4%), voriconazole (81.9%), and fluconazole
Educational status
Secondary 206 52.0
(80.6%). In contrast, clotrimazole and itraconazole were both
ineffective against 34.7% and 23.6% of C. albicans isolates,
Graduate 64 16.2
respectively. In addition, the C. krusei, C. tropicalis, C. glabrata,
1st trimester 90 22.7 and C. dublinensis isolates were resistant to fluconazole (57.6%),
Gestational
2nd trimester 126 31.8
voriconazole (63%), voriconazole (43.8%), and nystatin (60%),
trimester
respectively, (Table 5, Figure 3).
3rd trimester 180 45.5
Of the nine tested antifungal drugs, the high-sensitivity
Primigravida 207 52.3 proportion of isolated Candida sp. was to ketoconazole at 218
Gravidity
Multigravida 189 47.7
(89.7%), while the highest rate of Candida species resistance was for
clotrimazoleat 74 (30.5%), followed by Voriconazole 65 (26.7%),
First time 117 29.5
Frequency Nystatin 62 (25.5%), and lowest resistant was for Ketoconazole 25
of infection
Recurrent 279 70.5 (10.3%) (Table 6).
According to Table 7, the majority of isolated Candida albicans
(46.2%) exhibited only one kind of antifungal drug resistance,
years (71.9%), and the lowest rate was among individuals aged 38–
compared to 38.7% of isolated non-albicans Candida, which
44 years (36.5%), with a statistically significant difference (95%
exhibited resistance to three different antifungal drug types (38.7%).
CI =2.23–2.44; P = 0.000). Similarly, the participants who lived in
urban areas had the highest rate (64.5%) compared with those who
lived in rural areas (53.8%). Furthermore, the study subjects who
completed primary school had a higher rate of Candida infections Discussion
(77.8%), whereas the uneducated participants had a lower rate
(40.0%), with a statistically significant difference (P = 0.000). The present finding revealed that the prevalence proportion of
Additionally, a high rate of infection was significantly observed Candida sp. was 61.4% among pregnant women, which is close to
among pregnant women in the third trimester (80.0%) compared to the finding reported among pregnant women in Ibb City (Edrees
others, with a statistically significant difference (P = 0.004). et al., 2020). This outcome is less than a report conducted in Kenya,
Furthermore, the multigravida mothers were found to be more which found that 90.38% of pregnant women were infected by

A B

FIGURE 2
Growth in culture media and isolated Candida species. (A) Positive and negative growth of vaginal swabs in culture media. (B) Frequency of Candida
species isolated from pregnant women.

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TABLE 3 Association between positive Candida infection and socio-demographic and risk factors.

Mean Positive Negative OR

Variables No. (%) X P value
± S.D No. (%) No. (%) 95%CI
Age (in years)

17-23 99 (25) 63 (63.6) 36 (36.4)

24-30 128 (32.3) 92 (71.9) 36 (28.1)

NA
2.34 ± 1.022 39.914 0.000
(2.23–2.44)
31-37 106 (26.8) 65 (61.3) 41 (38.7)

38-44 63 (15.9) 23 (36.5) 40 (63.5)

Resident area

Urban 279 (70.5) 180 (64.5) 99 (35.5)

0.835
1.70 ± 0.457 56.333 0.693
(1.66–1.75)
Rural 117 (29.5) 63 (53.8) 54 (46.2)

Educational status

Illiterate 45 (11.4) 18 (40) 27 (60)

Primary 81 (20.5) 63 (77.8) 18 (22.2)

NA
2.73 ± 0.866 125.971 0.004
(2.64–2.82)
Secondary 206 (52) 131 (63.6) 75 (36.4)

Graduate 64 (16.2) 31 (48.4) 33 (51.6)

Gestational trimester

1st trimester 90 (22.7) 36 (40) 54 (60)

NA
2nd trimester 126 (31.8) 2.23 ± 0.795 63 (50) 63 (50) 78.000 0.000
(2.15–2.31)
3rd trimester 180 (45.5) 144 (80) 36 (20)

Gravidity

Primigravida 207 (52.3) 132 (63.8) 75 (36.2)

1.086
1.48 ± 0.500 1.815 0.000
(1.43–1.53)
Multigravida 189 (47.7) 125 (66.1) 63 (33.9)

Frequency of infection

First time 117 (29.5) 54 (46.2) 63 (53.8)

1.7045 0.681
75.000 0.117
± 0.45682 (1.66–1.75)
Recurrent 279 (70.5) 189 (67.7) 90 (32.3)

SD, Standard Division; c2, Chi-square; CI, Confidence interval; OR, Odds ratio, P value (p< 0.05: significant); NA, Not Applicable.

TABLE 4 Clinical signs and symptoms associated with positive Candida sp.

Mean OR
Variables No. Positive Negative X P value
± S.D 95%CI
Itching

Yes 377 (95.2) 243 (64.5) 134 (34.5)

0.355
1.05 ± 0.214 NA 0.000
(1.03–1.07)
No 19 (4.8) 0 (0) 19 (100)

Burning

Yes 342 (86.4) 198 (57.9) 144 (42.1)

0.695
1.14 ± 0.344 96.333 0.003
(1.10–1.17)
No 54 (13.6) 45 (83.3) 9 (16.7)

Discharge

Yes 297 (75) 216 (72.7) 81 (27.3)

2.667
1.25 ± 0.434 147.00 0.000
(1.21–1.29)
No 99 (25.0) 27 (27.3) 72 (72.7)

SD, Standard Division; c2, Chi-square; CI, Confidence interval; OR, Odds ratio, P value (p< 0.05: significant); NA, Not Applicable.

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TABLE 5 Antifungal susceptibility pattern of isolated Candida species.

C. albicans C. dublinensis
C. krusei (n=33) C. tropicalis (n=27) C. glabrata (n=24)
(n=144) (n=15)
Antifungal drug
S R S R S R S R S R
No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%)
Amphotericin B 133 (92.4) 11 (7.6) 17 (51.5) 15 (49.5) 25 (92.6) 2 (7.4) 15 (62.5) 9 (37.5) 15(100) 0 (0)

Fluconazole 116 (80.6) 28 (19.4) 14 (42.4) 19 (57.6) 25 (92.6) 2 (7.4) 23 (95.8) 1 (4.2) 14 (93.3) 1 (6.7)

Nystatin 111 (77.1) 33 (22.9) 22 (66.7) 11 (23.3) 21 (77.8) 6 (22.2) 21 (87.5) 3 (12.5) 6 (40.0) 9 (60.0)

Voriconazole 118 (81.9) 26 (18.1) 25 (75.7) 8 (24.3) 10 (37.0) 17 (63.0) 13 (54.2) 11 (43.8) 12 (80.0) 3 (20.0)

Clotrimazole 94 (65.3) 50 (34.7) 23 (79.7) 10 (20.3) 22 (81.5) 5 (18.5) 19 (79.2) 5 (20.8) 11 (73.3) 4 (16.7)

Ketoconazole 132 (91.7) 12 (8.3) 31 (93.9) 2 (6.1) 21 (77.8) 6 (22.2) 22 (91.7) 2 (8.3) 12 (80.0) 3 (20.0)

Itraconazole 110 (76.4) 34 (23.6) 32 (96.9) 1 (3.1) 27 (100) 0 (0) 24 (100) 0 (0) 9 (60.0) 5 (40.0)

Miconazole 123 (85.4) 21 (14.6) 28 (84.8) 5 (15.2) 19 (70.4) 8 (19.6) 22 (91.7) 2 (8.3) 14 (93.3) 1 (6.7)

S, sensitive; R, Resistant.

FIGURE 3
Antifungal resistant pattern of isolated Candida species.

TABLE 6 Total susceptibility pattern of isolated Candida species.

Sensitive Resistant
Antifungal drug
No. % No. %
Amphotericin_B 206 84.8 37 15.2

Fluconazole 192 79.0 51 21.0

Nystatin 181 74.5 62 25.5

Voriconazole 178 73.3 65 26.7

Clotrimazole 169 69.5 74 30.5

Ketoconazole 218 89.7 25 10.3

Itraconazole 203 83.5 40 16.5

Miconazole 206 84.8 37 15.2

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TABLE 7 Multiple-antifungal resistance profiles of Candida isolates.

Antibiogram patterns
Species No. (%)
R1 R2 R3 R4 R5
[Link] 144 (59.3) 54 (46.2%) 37 (31.6%) 20 (17.1%) 4 (3.4%) 2 (1.7%)

Non-albicans Candida 99 (40.7) 23 (30.7%) 14 (18.7%) 29 (38.7%) 8 (10.7%) 1 (1.3%)

Total 243 (100) 77 (31.68%) 51 (20.98%) 49 (20.16%) 12 (4.94%) 3 (1.23%)

R1/R2/R3/R4/R5, Resistant to one/two/three/four/five antifungal agents.

Candida sp (Nelson et al., 2013). In contrast, the current finding is pregnant women in the urban area, at 88.44%. The prevalence of the
lower than several reports that showed that the prevalence rate of infection was higher in uneducated women than in patients with
Candida species among pregnant women was 55.4% in Cameroon basic school education and above, and there was a statistically
(Toua et al., 2013), 60.8% in Egypt (Abbas et al., 2016), 51.6% in significant correlation between vulvovaginal candidiasis and
Sana’a, Yemen (Al-Rukeimi et al., 2020), 34% in Saudi Arabia educational level (P = 0.004). This finding was compatible with
(Venugopal et al., 2021), and 25% in north-western Ethiopia (Tsega earlier reports (Konadu et al., 2019; Waikhom et al., 2020) that
and Mekonnen, 2019). The great variation in frequency rate might revealed a high frequency of Candida sp. among those with basic
be due to the differences in geographical locations, study education. The difference in infection rates between illiterate
population, sample size, hygienic conditions, socioeconomic individuals and those with more education could be explained by
status, and diagnostic methods employed by the participants. improvements in personal cleanliness and/or economic position
The present study revealed that the frequency rates of brought on by education (Bitew and Abebaw, 2018).
C. albicans, C. krusei, C. tropicalis, C. glabrata, and C. dublinensis Compared to primigravidae (63.8%), multigravidae (66.1%)
were reported to be 59.26%, 13.58%, 11.12%, 9.87%, and 6.17%, women exhibited a higher rate of Candida colonization, with
respectively. This result is similar to some reports performed in significant differences (P = 0.000) in the present study. Similarly,
different countries (Tsega and Mekonnen, 2019; Ghaddar et al., research conducted in Pakistan found that multigravidae women
2020; Venugopal et al., 2021). The widespread use of antifungal experienced the condition more frequently than primigravidae
drugs over-the-counter, inappropriate use, incomplete description women, with results of 60% and 40%, respectively (Aslam et al.,
of treatment, longer treatment for recurrent candidiasis, and use of 2008). Further research from Nepal (Shrestha et al., 2011) supports
effective agents to eliminate C. albicans have all been proposed as our findings. The rationale is that the rate of infection rises with the
potential explanations for the increased isolation of non-albicans frequency of pregnancies (3rd> 2nd> 1st), which lowers immunity
Candida species from vulvovaginitis patients. Given the high and may lead to extensive Candida colonization. Additionally, a
frequency of VVC among women in the study area, it is study by Tsega and Mekonnen (2019) showed that Candida
important to screen or treat pregnant women for VVC to prevent infection was more common among women with multigravidae
its detrimental health effects (Sule-Odu et al., 2020; Zaman (61.5%) than among those with primigravidae (38.5%).
et al., 2024). According to these data, females with recurrent infections had a
Remarkably, the highest rate of Candida infections in this study greater prevalence rate of vulvovaginal candidiasis (67.7%) than
was noticed in pregnant women aged between 24 and 30 years those who had it for the first time (29.5%). This result conflicts with
(71.9%). Similar reports documented that a high frequency of research performed in Nigeria by Sobel (2007). The significantly
Candida species was found at 60% among the ages 26–35 (Nelson rising rate of recurrent vaginal candidiasis infections in this research
et al., 2013), 38.5% in the ages 34–40 (Tsega and Mekonnen, 2019), may be due to an increase in Candida species that are resistant to
37.4% in the ages 20–34 (Konadu et al., 2019), and 44.4% in the ages widely used antifungal medications.
26–35 (Waikhom et al., 2020). The current results revealed that there were significant
The cause of this might be explained by the fact that women in differences between the gestational period and Candida
this age range release many reproductive hormones, which can colonization (80%; P = 0.000). The highest rate of vaginal
inhibit the immune system and foster Candida infection. The use of Candida species was detected in the third-trimester participants
antibiotics, which kill bacteria, including natural flora, is another at 80%, while the lowest was in the first trimester (40%). A similar
factor that might be involved. This will give Candida a chance to finding was further reported: the highest rate of Candida species
attack the vaginal wall (Tsega and Mekonnen, 2019). Additionally, was in the third trimester, at 68.09% in Kenya (Nelson et al., 2013)
this high proportion is caused by the vagina’s higher glycogen and 57.4% in Ghana (Waikhom et al., 2020). According to research,
content and high estrogen hormone levels. It offers a reliable source third-trimester pregnant women were most likely to develop an
of carbon, which helps with Candida proliferation. illness. Pregnancy raises the risk of VVC, and the risk might reach
The present outcome showed that the highest rate of Candida 50% during the final trimester (Konadu et al., 2019; Tsega and
infections was recorded among those living in urban areas (64.5%). Mekonnen, 2019; Waikhom et al., 2020).
The outcome, consistent with Abdul-Aziz et al. (Abdul-Aziz et al., This is because during pregnancy, particularly in the third
2019), revealed that the highest distribution of VVC was among trimester, elevated estrogen levels generate larger glycogen stores

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Ali et al. 10.3389/fcimb.2024.1434677

in the vagina, which serve as an excellent source of carbon and 9.6%, respectively. In addition, itraconazole (24.1%) and
encourage the growth of Candida sp. Additionally, estrogen makes posaconazole (14.5%) were the only two drugs for which
vaginal epithelial cells’ yeast cytosol receptors more attractive to resistance rates were noted to C. glabrata. Whereas C. krusei
Candida species (Babić and Hukić , 2010). So, to prevent vaginal exhibited the highest resistance rates to itraconazole at 81.5%
microorganisms’ infections, particularly in the last trimester, it is (Peman et al., 2012). Variations in the research populations and
imperative that health education interventions be enhanced and geographic settings, as well as variations in the usage of antifungals,
women’s awareness raised about the disease (Abruquah, 2012; could be an explanation for the varying resistance rates observed in
Holzer et al., 2017). this investigation.
The current findings showed that 92.4% of C. albicans were According to the multidrug resistance results, a high proportion
sensitive to amphotericin B. This finding was similar to reports that of Candida albicans (46.2%) were resistant to one type of antifungal
recorded 93.8% in Ethiopia (Tsega and Mekonnen, 2019) and 87.2% drug, while 38.7% of isolated non-albicans Candida were resistant
in Ghana (Kan et al., 2023). Amphotericin-B is more sensitive than to three types of antifungal drugs. A study by Tsega and Mekonnen
other antibiotics because it is not frequently given and used (2019) revealed that 46.29% of isolated C. albicans were resistant to
extensively due to its high cost, difficulties in administration, and three types of antifungal agents, and non-albicans Candida,
severe renal toxicity. Therefore, the less a drug is used, the less likely including C. glabrata (35.29%) and C. krusei (57.17%), were
it is to develop resistance to it (Tsega and Mekonnen, 2019). resistant to one type of antifungal. Therefore, healthcare settings
Consequently, to avoid the emergence and spread of drug- should strongly consider implementing an antibiogram as part of
resistant bacterial strains, it is critical to conduct periodic their infection control programs to guide their decision-making on
surveillance of antimicrobial susceptibility testing and proper appropriate empirical treatment and infection control programs
management of pregnant women (Abruquah, 2012). (Brandão et al., 2018).
The overall rate of Candida species in the present investigation
was highly resistant to clotrimazole (30.5%), voriconazole (26.7%),
nystatin (25.5%), and fluconazole (21.0%). These results are lower Strength and limitation of the study
than the results of Khan et al (Khan et al., 2018), who detected a
high resistance rate of Candida sp. against fluconazole (62%), This study is the first to try to shed some light on the prevalence
clotrimazole (59.3%), itraconazole (40.7%), and voriconazole of Candida species and their antifungal susceptibility profiles
(10.2%). Another study by Tsega and Mekonnen (2019) revealed among pregnant women in Hajjah governorate, Yemen, which is
that a high resistance rate of Candida sp. (57.3%) was found for an important public health issue that has not been previously
ketoconazole and itraconazole. In addition, approximately 17.2% studied in this region. Additionally, the results of this
and 5.7% of Candida species were resistant to fluconazole and investigation are expected to yield valuable insights that will
flucytosine, respectively (Bitew and Abebaw, 2018). Hence, more provide decision makers with antifungal susceptibility profiles in
research on family health strategies is required for developing a the study area and encourage other researchers to conduct further
better understanding of how drug-resistant microbes spread and studies in this field. However, the limitations of this work include
their role in health-related infections. the fact that modern and advanced techniques such as polymerase
The current study showed that ketoconazole was the most chain reaction (PCR) for genotypes identifying the isolated Candida
effective antifungal drug when compared to tested antifungal species are lacking due to limited resources. In addition, this study is
agents that had 89.7% effectiveness. In different reports, Bitew and unable to cover the behavioral risk factors that may be associated
Abebaw (2018) reported that fluconazole was the most effective with the prevalence of Candida species among study participants.
antifungal drug, while Tsega and Mekonnen (2019) documented
clotrimazole as an effective antifungal agent. Earlier studies
demonstrated that the resistance rate in Candida sp. to Conclusion
voriconazole and fluconazole has remained constant over a decade
(Pfaller et al., 2007; Lyon et al., 2010; Bitew and Abebaw, 2018). The greater frequency of Candida species isolates in this study is
According to Gualco et al., (2007), C. albicans exhibited resistance to considered a serious health problem for pregnant women. The high
fluconazole and itraconazole at rates of 0.7% and 2.7%, respectively. humidity in Hajjah may have contributed to the spread of Candida
To determine the most effective antibiotic, establishing a national species among women. Therefore, to increase women’s knowledge
antibiotic policy is crucial for regulating the administration of of vaginal candidiasis and its prevention, health education
antibiotics to patients prior to conducting an antibiotic sensitivity interventions are highly recommended. Furthermore, screening
test, which aims to identify the most optimal antibiotic. women for vaginal infections prior to treatment is recommended,
Furthermore, the isolated C. krusei, C. tropicalis, C. glabrata, as well as educating spouses and partners on the transmission and
and C. dublinensis in this finding were resistant to fluconazole prevention of sexually transmitted illnesses. The consequences of
(57.6%), voriconazole (63%), voriconazole (43.8%), and nystatin candidiasis can be avoided with proper and immediate treatment
(60%), respectively. A report by Luo et al. (Luo et al., 2016) found and diagnosis. Moreover, the antifungal susceptibility test may help
that the resistance levels of C. albicans, C. glabrata, and C. tropicalis determine the best drug therapy for each patient. Accordingly,
to amphotericin B, voriconazole, fluconazole, 5-fluorocytosine, and future investigations should focus on the occurrence of drug-
itraconazole were found to be 0.5% to 6.4%, 0% to 7.7%, and 0% to resistant Candida strains and their emergence.

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Data availability statement Funding

The raw data supporting the conclusions of this article will be The author(s) declare that no financial support was received for
made available by the authors, without undue reservation. the research, authorship, and/or publication of this article.

Ethics statement Acknowledgments

The studies involving humans were approved by Authority AL- We are appreciative of the females who agreed to voluntarily
Gumhorri Hospital Hajjah. The studies were conducted in enroll in this study. We want to express our thanks to the laboratory
accordance with the local legislation and institutional requirements. staff of the Authority Al-Gumhorri Hospital-Hajjah for their
Written informed consent for participation in this study was technical assistance.
provided by the participants’ legal guardians/next of kin.

Conflict of interest
Author contributions
The authors declare that the research was conducted in the
MA: Writing – original draft, Writing – review & editing, absence of any commercial or financial relationships that could be
Conceptualization, Data curation, Formal analysis, Investigation, construed as a potential conflict of interest.
Methodology, Funding acquisition, Project administration,
Resources, Software, Supervision, Validation, Visualization. WE:
Conceptualization, Data curation, Writing – review & editing, Publisher’s note
Methodology, Formal analysis, Resources, Validation. WA-S:
Investigation, Resources, Software, Writing – review & editing. All claims expressed in this article are solely those of the
GX: Writing – review & editing, Supervision. SH: Writing – authors and do not necessarily represent those of their affiliated
review & editing, Formal analysis, Software. EQ: Writing – review organizations, or those of the publisher, the editors and the
& editing, Resources. RC: Investigation, Visualization, Writing – reviewers. Any product that may be evaluated in this article, or
review & editing, Software. NL: Writing – review & editing, Data claim that may be made by its manufacturer, is not guaranteed or
curation, Investigation, Visualization. endorsed by the publisher.

References
Abbas, A. M., Shaaban, O. M., Badran, S. M., Shaltout, A. S., Nasr, A., and Abdullah, Clinical and Laboratory Standards Institute (CLSI) (2021). Performance standards for
S. A. (2016). Risk factors and health hazards of vaginal infections in upper Egypt: A antimicrobial susceptibility testing. 31st ed (Wayne, PA: Clinical and Laboratory
cross sectional study. Thai J. Obstet Gynecol. 30, 50–56. doi: 10.14456/tjog.2016.14 Standards Institute).
Abdul-Aziz, M., Mahdy, M. A. K., Abdul-Ghani, R., Alhilali, N. A., Al-Mujahed, L. Disha, T., and Haque, F. (2022). Prevalence and risk factors of vulvovaginal
K.A., Alabsi, S. A., et al. (2019). Bacterial vaginosis, vulvovaginal candidiasis and candidosis during pregnancy: A review. Infect. Dis. Obstet Gynecol. 20, 6195712.
Trichomonal vaginitis among reproductive-aged women seeking primary healthcare in doi: 10.1155/2022/6195712
Sana’a city, Yemen. BMC Infect. Dis. 19, 1–10. doi: 10.1186/s12879-019-4549-3 Edrees, W. H., Al-Asbahi, A. A., Al-Shehari, W. A., and Qasem, E. A. (2020).
Abruquah, H. H. (2012). Prevalence and antifungal susceptibility of Candida species Vulvovaginal candidiasis prevalence among pregnant women in different hospitals in
isolated from women attending a gynecological clinic in Kumasi, Ghana. J. Sci. Techno. Ibb, Yemen. Universal J. Pharm. Res. 5, 1–5. doi: 10.22270/ujpr.v5i4.431
32, 39–45. doi: 10.4314/just.v32i2.6 Ghaddar, N., Anastasiadis, E., Halimeh, R., Ghaddar, A., Dhar, R., AlFouzan, W.,
Al-Rukeimi, A. D. A., Al-Hatami, S. M. M., AL-Danany, D. A., Al-Shamahy, H. A., et al. (2020). Prevalence and antifungal susceptibility of Candida albicans causing
and Al Rukeimi, R. A. A. (2020). Prevalence and risk factors associated with vaginal discharge among pregnant women in Lebanon. BMC Infect. Dis. 20, 32.
vulvovaginal candidiasis during pregnancy in Sana'a, Yemen. Universal J. Pharm. doi: 10.1186/s12879-019-4736-2
Res. 5, 1–5. doi: 10.22270/ujpr.v5i3.407 Gualco, L., Debbia, E. A., Bandettini, R., Pescetto, L., Cavallero, A., Ossi, M. C., et al.
Aslam, M., Hafeez, R., Ijaz, S., and Tahir, M. (2008). Vulvovaginal candidiasis in (2007). Antifungal resistance in Candida spp. isolated in Italy between 2002 and 2005
pregnancy. Biomedica 24, 54–56. Available at: [Link] from children and adults. Int. J. Antimicrob. Agents 29, 179–184. doi: 10.1093/jac/
repid=rep1&type=pdf&doi=357bef2e32f8ac66f7290b06df4e dbe4db4385a6. dks019
Babić , M., and Hukić , M. (2010). Candida albicans and non-albicans species as Holzer, I., Farr, A., Kiss, H., Hagmann, M., and Petricevic, L. (2017). The
etiological agent of vaginitis in pregnant and nonpregnant women. Bosnian J. Basic colonization with Candida species is more harmful in the second trimester of
Med. Sci. 10, 89–97. doi: 10.17305/bjbms.2010.2744 pregnancy. Arch. Gynecol. Obstet 295, 891–895. doi: 10.1007/s00404-017-4331-y
Bitew, A., and Abebaw, Y. (2018). Vulvovaginal candidiasis: Species distribution of Kan, S., Song, N., Pang, Q., Mei, H., Zheng, H., Li, D., et al. (2023). In vitro antifungal
Candida and their antifungal susceptibility pattern. BMC Women's Health 18, 94. activity of azoles and other antifungal agents against pathogenic yeasts from
doi: 10.1186/s12905-018-0607-z vulvovaginal candidiasis in China. Mycopathologia 188, 99–109. doi: 10.1007/s11046-
Brandão, L. S., Boniek, D., Stoianoff, M. A.R., Da Mata, F. M.R., De Azevedo, P. R.M., 022-00687-w
Fernandes, J. V., et al. (2018). Prevalence and antifungal susceptibility of Candida Khan, M., Ahmed, J., Gul, A., Ikram, A., and Lalani, F. K. (2018). Antifungal
species among pregnant women attending a school maternity at Natal, Brazil. Lett. susceptibility testing of vulvovaginal Candida species among women attending
Appl. Microbiol. 67, 285–291. doi: 10.1111/[Link]-3 antenatal clinic in tertiary care hospitals of Peshawar. Infection Drug Resistance 11,
Central for Diseases Control and Prevention (CDC) (2021). Sexually transmitted 447–456. doi: 10.2147/IDR.S153116
infections (STI) treatment guidelines, 2021 (Atlanta, US: Vulvovaginal Candidiasis Konadu, D. G., Owusu-Ofori, A., Yidana, Z., Boadu, F., Iddrisu, L. F., Adu-Gyasi, D.,
(VVC). Available at: [Link] et al. (2019). Prevalence of vulvovaginal candidiasis, bacterial vaginosis and

Frontiers in Cellular and Infection Microbiology 10 [Link]

Ali et al. 10.3389/fcimb.2024.1434677

Trichomoniasis in pregnant women attending antenatal clinic in the middle belt of Roberts, C. L., Rickard, K., Kotsiou, G., et al. (2011). Treatment of asymptomatic
Ghana. BMC Pregnancy Childbirth 19, 1–10. doi: 10.1186/s12884-019-2488-z vaginal candidiasis in pregnancy to prevent preterm birth: an open-label pilot
Lı́rio, J., Giraldo, P. C., Amaral, R. L., Sarmento, A. C. A., Costa, A. P. F., Gonçalves, randomized controlled trial. BMC Pregnancy Childbirth 11, 1–6. doi: 10.1186/1471-
A. K., et al. (2019). Antifungal (oral and vaginal) therapy for recurrent vulvovaginal 2393-11-18
candidiasis: A systematic review protocol. BMJ Open 9, e027489. doi: 10.1136/ Rosati, D., Bruno, M., Jaeger, M., ten Oever, J., and Netea, M. G. (2020). Recurrent
bmjopen-2018-027489 vulvovaginal candidiasis: An Immunological Perspective. Microorganisms 8, 2, 144.
Luo, X., Dong, X., and Pen, Z. (2016). Distribution and drug susceptibility of doi: 10.3390/microorganisms8020144
Candida spp. associated with female genital tract infection, Chongqing, China. Shrestha, S., Tuladhar, N. R., Basnyat, S., Acharya, G. P., Shrestha, P., and Kumar, P.
Jundishapur. J. Microbiol. 9, e19386. doi: 10.5812/jjm.19386 (2011). Prevalence of vaginitis among pregnant women attending Paropakar maternity
Lyon, G. M., Karatela, S., Sunay, S., and Adiri, Y. (2010). Antifungal susceptibility and Women's hospital, Thapathali, Kathmandu, Nepal. Nepal. Med. Coll. J. 13, 293–
testing of Candida isolates from the Candida surveillance study. J. Clin. Microbiol. 48, 296.
1270–1275. doi: 10.1128/JCM.02363-09 Sobel, J. D. (2007). Vulvovaginal candidosis. Lancet 369, 1961–1971. doi: 10.1016/
Mahon, R. C., Lehman, C. D., and Manuselis, G. (2015). Textbook of diagnostic S0140-6736(07)60917-9
microbiology. 5th Edition (USA: Saunders, An Imprint of Elsevier, Inc), 37–115. Sule-Odu, A., Akadri, A. A., Oluwole, A. A., Osinupebi, O. A., Andu, B. A., Akiseku,
Makanjuola, O., Bongomin, F., and Fayemiwo, S. A. (2018). An update on the roles of A. K., et al. (2020). Vaginal Candida infection in pregnancy and its implications for
non-albicans Candida species in Vulvovaginitis. J. Fungi (Basel) 4, 121. doi: 10.3390/ fetal well-being. Afr. J. Reprod. Health 24, 33–40. Available at Vaginal Candida
jof4040121 infection in pregnancy and its implications for fetal well-being on JSTOR.

Masri, S. N., Noor, S. M., Nor, L. A., Osman, M., and Rahman, M. M. (2015). Tille, P. M. (2017). Bailey and Scott’s diagnostic microbiology. 14th edition (St. Louis,
Candida isolates from pregnant women and their antifungal susceptibility in a Missouri: Elsevier).
Malaysian tertiary-care hospital. Pak J. Med. Sci. 31, 658–661. doi: 10.12669/ Toua, V., Djaouda, M., Gaké , B., Menye, D. E., Christie, E. A., Tambe, E., et al. (2013).
pjms.313.7072 Prevalence of vulvovaginal candidiasis amongst pregnant women in Maroua
Nelson, M., Wanjiru, W., and Margaret, M. W. (2013). Prevalence of vaginal (Cameroon) and the sensitivity of Candida albicans to extracts of six locally used
candidiasis and determination of the occurrence of Candida species in pregnant antifungal plants. Int. Res. J. Microbiol. 4, 89–97.
women attending the antenatal Clinic of Thika District Hospital, Kenya. OJMM 3, Tsega, A., and Mekonnen, F. (2019). Prevalence, risk factors and antifungal
264–272. doi: 10.4236/ojmm.2013.34040 susceptibility pattern of Candida species among pregnant women at Debre Markos
Nikoomanesh, F., Falahatinejad, M., Č erná ková , L., dos Santos, A. L., Mohammadi, Referral Hospital, Northwest Ethiopia. BMC Pregnancy Childbirth 19, 1–8.
S. R., Rafiee, M., et al. (2023). Combination of Farnesol with common antifungal drugs: doi: 10.1186/s12884-019-2494-1
Inhibitory effect against Candida species isolated from women with RVVC. Medicina Venugopal, D., Husain, K., Mustafa, S. A., and Sabeen, S. (2021). Epidemiology, risk
59, 743. doi: 10.3390/medicina59040743 factors and antimicrobial profile of vulvovaginal candidiasis (VVC): A study among
Peman, J., Canton, E., Quindos, G., Eraso, E., Alcoba, J., Guinea, J., et al. (2012). women in the central region of Saudi Arabia. J. Mycol. Med. 31, 101049. doi: 10.17305/
Epidemiology, species distribution and in vitro antifungal susceptibility of fungaemia in bjbms.2010.2744
a Spanish multicentre prospective survey. J. Antimicrobial. Chemother. 67, 1181–1187. Waikhom, S. D., Afeke, I., Kwawu, G. S., Mbroh, H. K., Osei, G. Y., Louis, B., et al.
doi: 10.1093/jac/dks019 (2020). Prevalence of vulvovaginal candidiasis among pregnant women in the Ho
Pfaller, M. D., Diekema, D., Gibbs, V., Newell, J., Meis, I., Gould, W., et al. (2007). municipality, Ghana: species identification and antifungal susceptibility of Candida
Results from the ARTEMIS DISK global antifungal surveillance study, 1997 to 2005: An isolates. BMC Pregnancy Childbirth 20, 266. doi: 10.1186/s12884-020-02963-3
8.5-year analysis of susceptibilities of Candida species and other yeast species to Zaman, R., Ullah, I., and Arif, A. (2024). Antifungal resistance in Candida species isolated
fluconazole and voriconazole determined by CLSI 30 standardized disk diffusion from patients presenting with vulvovaginal candidiasis during different trimesters of
testing. J. Clin. Microbiol. 45, 1735–1745. doi: 10.1128/JCM.00409-07 pregnancy. J. Population Ther. Clin. Pharmacol. 31, 57–65. doi: 10.53555/jptcp.v31i1.3796

Frontiers in Cellular and Infection Microbiology 11 [Link]

 TYPE Original Research
PUBLISHED 08 November 2024
DOI 10.3389/fonc.2024.1475189

Identification of immune-
OPEN ACCESS associated genes for the
EDITED BY
Zhen Dong,
Southwest University, China
diagnosis of ulcerative
REVIEWED BY
Vaibhavi V. Gujar,
colitis-associated carcinogenesis
National Institutes of Health (NIH),
United States
Yingcheng Charles Wu,
via integrated
Fudan University, China
Yuanming Yang, bioinformatics analysis
Guangzhou University of Chinese Medicine,
China
Jiaming Su, Xueyu Cang 1†, Ning Li 1†, Jihan Qi 1, Hongliang Chen 1, Hui Xing 1,
Beijing University of Chinese Medicine, China
Jiawei Qiu 1, Yingying Tian 1, Shiling Huang 1, Pengchao Deng 1,
*CORRESPONDENCE
Shizhu Jin
Feiyang Gao 1, Ram Prasad Chaulagain 1, Ubaid Ullah 1,
drshizhujin@[Link] Chunjing Wang 2, Lina Liu 3 and Shizhu Jin 1*
†
These authors have contributed 1
Department of Gastroenterology and Hepatology, The Second Affiliated Hospital of Harbin Medical
equally to this work and share University, Harbin, China, 2 Department of General Surgery, The Second Affiliated Hospital of Harbin
first authorship Medical University, Harbin, China, 3 Department of Endoscopic Center, The Second Affiliated Hospital
RECEIVED 03 August 2024 of Harbin Medical University, Harbin, China
ACCEPTED 23 October 2024
PUBLISHED 08 November 2024

CITATION Background: UC patients suffer more from colorectal cancer (CRC) than the
Cang X, Li N, Qi J, Chen H, Xing H, Qiu J, general population, which increases with disease duration. Early colonoscopy is
Tian Y, Huang S, Deng P, Gao F,
Chaulagain RP, Ullah U, Wang C, Liu L and difficult because ulcerative colitis-associated colorectal cancer (UCAC) lesions
Jin S (2024) Identification of immune- are flat and multifocal. Our study aimed to identify promising UCAC biomarkers
associated genes for the diagnosis of
ulcerative colitis-associated carcinogenesis
that are complementary endoscopy strategies in the early stages.
via integrated bioinformatics analysis.
Front. Oncol. 14:1475189. Methods: The datasets may be accessed from the Gene Expression Omnibus and
doi: 10.3389/fonc.2024.1475189
The Cancer Genome Atlas databases. The co-expressed modules of UC and CRC
COPYRIGHT
were determined via weighted co-expression network analysis (WGCNA). The
© 2024 Cang, Li, Qi, Chen, Xing, Qiu, Tian,
Huang, Deng, Gao, Chaulagain, Ullah, Wang, biological mechanisms of the shared genes were exported for analysis using the
Liu and Jin. This is an open-access article Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To identify
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
protein interactions and hub genes, a protein-protein interaction network and
use, distribution or reproduction in other CytoHubba analysis were conducted. To evaluate gene expression, external
forums is permitted, provided the original datasets and experimental validation of human colon tissues were utilized. The
author(s) and the copyright owner(s) are
credited and that the original publication in diagnostic value of core genes was examined through receiver operating
this journal is cited, in accordance with characteristic (ROC) curves. Immune infiltration analysis was employed to
accepted academic practice. No use,
investigate the associations between immune cell populations and hub genes.
distribution or reproduction is permitted
which does not comply with these terms.
Results: Three crucial modules were identified from the WGCNA of UC and CRC
tissues, and 33 coexpressed genes that were predominantly enriched in the NF-
kB pathway were identified. Two biomarkers (CXCL1 and BCL6) were identified
via Cytoscape and validated in external datasets and human colon tissues. CRC
patients expressed CXCL1 at the highest level, whereas UC and CRC patients
showed higher levels than the controls. The UC cohort expressed BCL6 at the
highest level, whereas the UC and CRC cohorts expressed it more highly than the
controls. The hub genes exhibited significant diagnostic potential (ROC curve >
0.7). The immune infiltration results revealed a correlation among the hub genes
and macrophages, neutrophils and B cells.

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Cang et al. 10.3389/fonc.2024.1475189

Conclusions: The findings of our research suggest that BCL6 and CXCL1 could
serve as effective biomarkers for UCAC surveillance. Additionally, they
demonstrated a robust correlation with immune cell populations within the
CRC tumour microenvironment (TME). Our findings provide a valuable insight
about diagnosis and therapy of UCAC.

KEYWORDS

ulcerative colitis, ulcerative colitis-associated carcinogenesis, bioinformatics analysis,

immune infiltration, diagnosis biomarker

Introduction the disease, its stages, the extent of the lesion, the age at onset,
genders, and whether or not primary sclerosing cholangitis (PSC) is
Ulcerative colitis (UC), known as inflammatory bowel disorder, present (12, 13). However, a variety of factors affected
is characterized by mucopurulent and bloody stools and relapsing- tumorigenesis; these studies only focused on the disease itself or
remitting diarrhea (1–3). Studies have consistently shown a the comparison between the tumor cohort and the controls. The
correlation between UC and the risk of colorectal cancer (CRC) application of bioinformatics methods has provided unparalleled
(4). Chronic inflammation in the intestinal tract generates insight into disease mechanisms and biomarker identification in
proinflammatory mediators and causes cell proliferation, which recent years (14). A variety of studies have been carried out in the
influences the immune system and increases cancer risk in UC detection of UC-CRC fields, such as biomarkers from blood, fecal,
patients, according to growing evidence (5). In the case of intestinal and miRNA, as well as the alteration of intestinal flora et al. (15–19).
barrier impairment, exposure to luminal antigens can lead to However, there are also some deficiencies, including poor
excessive infiltration of immune-related cells and heightened specificity, detection complexity, lower acceptance of patients, and
production of chemokines in the intestinal lamina propria (5–7). high cost.
The infiltration cell population comprises dendritic cells, In our research, using database mining, we analyzed multiple
neutrophils, and macrophages. Activated immune cells interact microarray datasets correlated with UC and CRC. We identified
with each other through direct or indirect contact by releasing gene modules related to the UC-CRC via weighted gene
various cytokines (6). The complex intestinal microenvironment coexpression network analysis (WGCNA) and Venn diagram
complicates the exploration of pathogenesis behind UCAC (8, 9). analysis. Next, we applied enrichment analyses to the module
Meanwhile, the specific genes responsible for regulating UC genes, revealing important associated biological processes.
progression and the extent of their expression alterations during Cytoscape identified two key genes, CXCL1 and BCL6. The
colitis-associated malignant transformation remain unclear. immune correlation analysis of key genes reveals a strong
Although colonoscopic detection has served as the standard relationship between these genes and immune cells (macrophages,
strategy for screening for malignancies in UC patients for a long neutrophils, and B cells) involved in colitis-associated cancer.
time, early diagnosis remains tricky due to the flat appearance and Furthermore, we verified the presence of CXCL1 and BCL6 in
presence of multifocal lesions (10, 11). It is therefore necessary to external datasets and human colon tissues. Instead of concentrating
ascertain the potential mechanisms that may led to the development solely on exploring the relationship between healthy and diseased
of UC-CRC and to investigate promising biomarkers as a means of groups or a single immune cell, our research utilized bioinformatics
facilitating early diagnosis. techniques to extract and analyze transcriptome data. This
Studies on the progression from UC to CRC over the past few approach yielded disease-related biomarkers that exhibit a strong
decades have examined a variety of factors, including the length of relation with immune cell populations in the TME.

Abbreviations: CRC, colorectal cancer; TNF, tumour necrosis factor; IL,

interleukin; UC, ulcerative colitis. UC-CRC, Ulcerative colitis-associated Materials and methods
carcinogenesis; GEO DataSets, Gene Expression Omnibus database; TCGA,
The Cancer Genome Atlas; RNA, Ribonucleic Acid; CRC, Colorectal Cancer; Data sources
WGCNA, weighted coexpression network analysis; GO enrichment analysis, gene
ontology enrichment analysis; KEGG enrichment analysis, Kyoto Encyclopedia Figure 1 exhibitied the workflow of our study. Microarray datasets
of Genes and Genomes enrichment analysis; PPI network, protein–protein of UC came from the GEO database ([Link]
interaction network; ROC curve, receiver operating characteristic curve; IBD, geo), and GEO include numerous high-throughput datasets
inflammatory bowel disease. and expression microarray datasets. We searched related gene

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Cang et al. 10.3389/fonc.2024.1475189

FIGURE 1
Research design flowchart. The RNA sequences of colon tissues from UC, CRC cohorts and the controls were obtained from the GEO and TCGA
databases. The datasets were pretreated via R software. WGCNA was used to identify the significant coexpression modules between UC and CRC.
Then, the coexpressed genes were visualized with a Venn diagram. GO and KEGG analyses were applied to coexpressed genes. The top 5 hub genes
related to UC-CRC were screened via cytoHubba. The expression of the five hub genes was verified via validation cohorts, and the hub genes was
estimated via ROC curve and immune infiltration analyses. Finally, experimental verification (HE, WB, IHC and IF analysis) of human colon tissues was
applied to affirm the differential expression of hub genes among UC patients, CRC patients and controls.

expression profiles and UC patients information with the key words omit unqualified genes and samples, Hclust (hierarchical clustering)
“colon mucosal” and “ulcerative colitis” in the GEO database. We analysis was performed in R prior to analysis. First, based on the
got the transcriptome data, patients information and healthy criterion of R2 > 0.85, a candidate soft-thresholding power b
control data from the TCGA database. First, gene expression (ranging from 1-20) was computed for the scale-free topology
profiling should include case and control groups. Second, to using the pickSoftThreshold function. The subsequent step
maintain the accuracy of WGCNA, more than 10 samples were involved the construction of an adjacency matrix utilizing the soft
included in each dataset. Third, all the samples were colon tissues. power value and gene correlation matrix derived from Pearson
Fourth, these gene matrices must provide raw data for further analysis. This adjacency matrix was then transformed into a
analysis. Finally, TCGA-COAD in the TCGA dataset and topological overlap and corresponding dissimilarity matrix.
GSE87473, GSE87466, GSE39582, GSE92415 and GSE20916 in Subsequently, coexpressed gene modules were determined via
the GEO dataset were chosen for subsequent research. The probe hierarchical clustering, and subsequently, we acquired a dynamic
expression matrix was converted into a gene expression matrix tree. The minimum number of modules was 30. Finally, to
based on platform annotation files. determine each module’s expression profile, we calculated the
module eigengene and its association with clinical features. Other
parameters were as follows: mergeCutHeight = 0.2, deepSplit = 2
Co-expression module identification and networkType = “unsigned”.

WGCNA explored gene module associations with disease traits

by analyzing coexpressed gene modules with high biological Functional enrichment analysis
significance (20). Hence, modules associated with UC and CRC
were detected in the GSE87473 and TCGA-COAD datasets by using For acquiring the potential inner mechanisms and functions of
“WGCNA package”. In our study, we selected the first 10,000 genes coexpressed genes, Gene Ontology (GO) annotation and Kyoto
with large variation based on their variance for further analysis. To Encyclopedia of Genes and Genomes (KEGG) analysis were

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Cang et al. 10.3389/fonc.2024.1475189

performed with the “ClusterProfiler package”. GO included the UC and healthy cohorts. Finally, Spearman’s correlation was
biological process (BP), cellular component (CC) and molecular performed to reveal the relationships between hub biomarker
function (MF) (21). KEGG is a comprehensive database containing expression and infiltrating immune cells.
valuable information on gene pathways (22). Furthermore, barplots
and circos plots were employed to display the outcome of
enrichment analysis. Verification of the human colonic mucosa

Collection of clinical samples

Construction of the PPI network In this study, clinical partial intestinal biopsy tissue samples,
including adjacent cancerous tissue from colorectal cancer patients,
The online search tool STRING ([Link] (version colon tissue from UC patients, and cancer tissue from colorectal
12.0) was applied to search for interrelationship among protein- cancer patients, were available from The Second Affiliated Hospital
coding genes of interest (minimum required interaction score: of Harbin Medical University. All patients suffered from UC and
0.15). PPI network could be visualized via the utilization of CRC after endoscopic examination and pathological diagnosis. The
Cytoscape (version 3.9.1) (23). As a plugin, MCODE was applied remaining colon tissue from UC and CRC patients was collected as
to filter critical functional interaction genes, with K-core=2, degree our validation cohort. The protocols were approved via the Ethics
cut-off=2, max depth=100 and node score cut-off=0.2 (24). Committee of the Second Affiliated Hospital, Harbin Medical
CytoHubba was utilized to figure out the hub genes. For the University (KY2023-051).
purpose of evaluating and selecting hub genes, five common
algorithms (MNC, DMNC, degree, MCC and EPC) were applied.
The subsequent step involved the utilization of GeneMANIA Hematoxylin-eosin staining analysis
([Link] which is used for internal
association detection within gene sets and for establishing The fresh colon tissues were fixed in 4% paraformaldehyde
coexpression networks for central genes (25). solution overnight, dehydrated and embedded in paraffin.
Subsequently, paraffin section were cut into 5-mm sections and
immersed in a hematoxylin-eosin blend for uniform staining. Using
Expression verification of hub genes an microscope (Olympus, Japan), we observed and photographed
colon tissue slices.
Expression validation of these genes in GSE87466 and
GSE39582 datasets, respectively. The GSE87466 dataset consisted
of 21 normal people and 87 ulcerative colitis patients. GSE39582 is Immunohistochemical analysis
composed of 19 normal people and 566 CRC patients. The
GSE87466 and GSE39582 datasets were obtained from human After fixation in 4% paraformaldehyde for 12 hours, Paraffin
intestinal tract tissue. We used the t test to compare the sections of the colon tissues were prepared. A thermostat set at 80°C
two datasets. was applied to heat the sections overnight. Afterwards, the sections
were dewaxed in xylene and dehydrated in graded ethanol, followed
by subsequent inactivation of endogenous peroxidase activity
Receiver operating characteristic curves using a 3% H2O2 solution and then antigen retrieval with
ethylenediaminetetraacetic acid. Subsequently, they were blocked with
With the “pROC” package, Receiver Operating Characteristic 5% goat serum (ABS933, Absin, Shanghai, China) for 30 minutes and
(ROC) curves were established in order to estimate the diagnosis incubated overnight at 4°C with Primary antibodies (anti-CXCL1[1:100,
capacity of key genes (26). The AUC values and corresponding 95% 12335-1-AP, Proteintech] and anti-BCL6 [1:1000, 66340-1-Ig,
CIs were calculated, with an AUC > 0.7 indicating high Proteintech]). The following day, the sections were incubated at 37°C
diagnostic value. without light for 1 hour with secondary antibodies (goat anti-rabbit
[RGAR011, Proteintech] and anti-mouse [RGAM011, Proteintech]).
After washing again with PBS, the chromogenic substrate
Analysis of immune infiltration diaminobenzidine (ZLI-9017, ZSGB-BIO, China) was applied to the
sections. Staining was finished when positive cells exhibited deep
To evaluate disease immune microenvironment and immune staining while maintaining an unstained or lightly stained background
cell infiltration, the “cibersort” package was utilized (27). The under a microscope (Olympus, Japan). The paraffin sections were
distribution of immune cells across different samples was visually subsequently counterstained with hematoxylin followed by thorough
represented using a bar plot. Additionally, a correlation heatmap rinsing in water. Dehydration was achieved using gradient alcohol and
depicting the interrelationships among 22 immune cell types was xylene before the samples were sealed appropriately in resin. Finally, the
generated utilizing the “corrplot” package. Vioplotting was dried sections were photographed for analysis by ImageJ software and
conducted to compare the proportions of diverse immune cells in GraphPad Prism 8 software.

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Cang et al. 10.3389/fonc.2024.1475189

Western blot analysis microscope (Zeiss, Germany). The results were semiquantitatively
analyzed with GraphPad Prism 8.2.1 and Image J software.
In liquid nitrogen, samples of colon tissues were thoroughly
ground and in lysis buffer, contain protease inhibitors, samples of
colon tissues were homogenized. The tissues were placed in a Statistical analysis
centrifuge tube and centrifuged at 12,000 rpm for 20 min at 4°C.
The BCA approach was applied to measure the total protein The statistical analysis and image preparation were completed
concentration in the supernatant. We heated and denatured the via R studio (version 4.1.3) and GraphPad Prism 8.2.1, respectively.
protein solution at 100°C for 5 minutes. After removing solution, Continuous variables with a normal distribution are described as
placed them on ice and stored at a temperature of 20°C. We further the mean ± standard deviation (SD). One-way analysis of variance
transferred the protein samples to a polyvinylidene fluoride (PVDF) was employed for data analysis involving more than two groups.
membranes. The PVDF membrane was blocked with 5% skim milk. Figures were edited using Adobe Illustrator (AI), PhotoShop (PS),
The membrane was washed twice with TBST for 5 minutes each and and Image J. Statistical significance was defined when P < 0.05.
subsequently incubated overnight at 4°C with primary antibodies
against CXCL1 (1:500, 12335-1-AP, Proteintech), BCL6 (1:1000,
66340-1-Ig, Proteintech), and b-actin (1:1000, 7A-09, ab8226, Results
ZSGB-BIO). On the following day, the PVDF membrane was
exposed to the secondary antibodies (goat anti-rabbit [RGAR001, Dataset information
Proteintech] and anti-mouse [RGAM001, Proteintech]) for 1 hour
at 37°C. Subsequently, the protein bands were immersed in Five GEO datasets and one TCGA dataset, namely, GSE87473,
enhanced chemiluminescence solution, and images were captured TCGA-COAD, GSE87466, GSE92415, GSE39582 and GSE20916,
using a gel imaging system. were selected. Information on each dataset is shown in Table 1. We
further coupled the GSE87473 and TCGA-COAD cohorts as
training sets for WGCNA and the other cohorts as verification
Immunofluorescence staining sets to validate the expression of hub genes.

After the colon tissues were rinsed with saline solution, they
were gently dried using filter paper to eliminate any residual WGCNA and key module
moisture. The tissue blocks were then carefully sectioned into 1 gene identification
cm3 pieces and embedded in optimal cutting temperature
compound (OCT). Frozen colon tissues were sliced into 5-mm We identified 11 functional modules in GSE87473 by WGCNA
sections, followed by treatment with 0.1% Triton X-100 to between the UC and control groups. We chose an optimal b value =
enhance cell permeability. After blocking with 5% goat serum 20 (scale-free R2 = 0.85) as the soft threshold according to the scale-
(ABS933, Absin, China) for 30 minutes at a temperature of 37°C, free topology criterion and average connectivity (Figure 2A). The
primary antibodies, including anti-CXCL1 (1:100, 12335-1-AP, Spearman correlation coefficient was used to create a heatmap that
Proteintech) and anti-BCL6 (1:1000, 66340-1-Ig, Proteintech), illustrates the relationships between modules and traits. This
were added and incubated overnight at 4°C. On the following allowed for an evaluation of the associations between the modules
day, goat anti-rabbit secondary antibody (RGAR002; Proteintech, and the disease (Figures 2C, E). Three modules, namely, ‘skyblue’,
China) and anti-mouse secondary antibody (RGAM002; ‘plum1’, and ‘gray60’, showed significantly positive correlations
Proteintech, China) were applied to the colon sections, which with UC and were considered UC-associated modules. (sky blue
were then incubated for one hour at a temperature of 37°C in the module: r= 0.52, p = 3e−10, genes= 116; plum1: r=0.52, p= 5e−10,
dark. Nuclei staining was performed using DAPI (Beyotime, China) genes= 90; grey60: r= 0.59, p = 5e−13, genes=169). By setting the
for 5 min before images were captured under a laser confocal soft threshold to 7 (Figure 2B), 32 modules were identified

TABLE 1 Dataset information used in this study.

ID Dataset number Platform Sample Diseases Group

1 GSE87473 GPL13158 106 patients and 21 controls UC Discovery cohort

2 TCGA-COAD TCGA 456 patients and 41 controls CRC Discovery cohort

3 GSE87466 GPL13158 87 patients and 21 controls UC Validation cohort

4 GSE39582 GPL570 566 patients and 19 controls CRC Validation cohort

5 GSE92415 GPL13158 87 patients and 21 controls UC Validation cohort

6 GSE20916 GPL570 66 patients and 34 controls CRC Validation cohort

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in TCGA-COAD, with the modules “blue” (r = 0.81, p = 2e−119, Functional analysis of co-expressed genes
genes = 480), “pink” (r = 0.45, p = 1e−26, genes = 834) and
“darkturquoise” (r=0.45, p = 2e−26, genes = 206) being significantly GO and KEGG analysis were applied to explore the underlying
correlated with CRC (Figures 2D, F). A Venn diagram was information and pathways associated with coexpressed genes.
constructed, and 33 intersecting genes were obtained (Figure 3A). The genes were predominantly enriched in humoral immune

FIGURE 2
WGCNA of the UC group (GSE87473) and CRC group (TCGA-COAD). (A) b= 20 is the soft threshold in UC patients according to the combined
analysis of scale independence and average connectivity. (B) b = 7 was selected as the soft threshold in CRC. (C, D) Gene dendrograms were
obtained by hierarchical clustering. The different colored rows under the dendrogram show the gene coexpression module assignments determined
by the dynamic tree cut method. (E, F) Relationships between modules and traits visualized as a heatmap. Correlations and p values are displayed in
each cell. Each row corresponds to the gene module, and each column is related to a clinical trait.

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FIGURE 3
PPI network and enrichment analysis results. (A) A total of 33 overlapping genes were identified from the gene intersections in UC and CRC patients
via WGCNA. (B) GO circle representing the GO enrichment analysis of the overlapping genes. (C) GO functional enrichment analysis of the
overlapping genes, comprising BP, CC, and MF. The different GO terms are displayed on the y-axis. Gene ratios enriched in terms are shown on the
x-axis. (D) The 15 most significantly enriched KEGG pathways. (E, F) Sixteen interacting genes and important modules visualized via MCODE. (G)
Identification of the top five key genes by multiple MCC, DMNC, MNC, Degree and EPC methods. (H) GeneMANIA was applied to explore internal
association of overlapping genes and their coexpressed genes.

response, molecular mediators of immune response production Molecular function analysis demonstrated significant enrichment in
and biological processes related to immunoglobulin production. antigen binding and immunoglobulin receptor binding (Figures 3B,
In CC, the genes were predominantly enriched in immunoglobulin C). Furthermore, the coexpressed genes were mainly involved in the
complexes, immunoglobulin complexes and blood microparticles. NF−kappa B pathway, chemokine signaling pathway, TNF

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signaling pathway, and cytokine–cytokine receptor interaction 0.809−0.947) (Figures 4M–P). The AUC values of four of the five
(Figure 3D). Supplementary Tables S1, S2 show the GO and hub genes exceeded 0.85, which indicates that these genes have high
KEGG analyses. These findings strongly suggest the relationship diagnostic value.
of proinflammatory factors and disease development.

Correlations of hub genes and immune

PPI network construction cell infiltration

We constructed a network of interactions among 33 In UC samples of GSE87473, macrophage infiltration was

overlapping gene proteins, with combined scores exceeding 0.15 strongly correlated with CXCL1, BCL6 and CXCL2. In addition,
(Supplementary Figure S1). Eighteen nodes and twenty-six edges in CXCL1, BCL6 and CXCL2 were associated with the expression of
the interaction network were visualized with Cytoscape (Figure 3E). activated dendritic cells and T regulatory cells (Figure 5A).
The MCODE was applied to find subnetworks and gene cluster Specifically, these genes were strongly related to M0 macrophages
modules (scores: 4.0, 5 nodes and 10 edges) (Figure 3F). (BCL6, R=0.598450467, p=1.36E-13; CXCL1, R=0.487848406,
p=6.87E-09; and CXCL2, R=0.402050537, p=3.06E-06)
(Figure 5B). Moreover, all genes were positively linked to activated
Selection and analysis of hub genes dendritic cells (BCL6, R=0.365708474, p=2.54E-05; CXCL1,
R=0.426961846, p=6.17E-07; and CXCL2, R=0.349592704,
We identified the top five crucial genes (CXCL1, BCL6, CXCL2, p=6.01E-05) (Figure 5B). As shown in Figure 5D, the bar plot
LCN2, and IRF4) from the PPI network (Figure 3G). Topological shows 22 different immune cell type proportions of each sample
analysis algorithms (MCC, DMNC, EPC, MNC and Degree) were from the UC and control groups (Figure 5D). Violin plots revealed
applied to comprehensively estimate and identify crucial genes in PPI that the UC group had greater numbers of neutrophils, M0
networks. Detailed information on these genes is provided in macrophages, M1 macrophages, activated memory CD4+ T cells
Supplementary Table S3 (Supplementary Table S3). Using the and activated mast cells and lower numbers of M2 macrophages,
GeneMANIA database, we analyzed the coexpression networks and resting mast cells, CD8+ T cells and resting dendritic cells (Figure 5C).
functions of these genes. Our findings revealed a complex protein- In addition, resting mast cells were positively related to M2
protein interaction (PPI) network comprising physical interactions macrophages (r=0.54), and Monocytes were positively related
(77.64%), coexpression patterns (8.01%), predicted associations to resting dendritic cells (r=0.53) and Regulatory T Cells
(5.37%), colocalization relationships (3.63%), genetic interactions (r=0.62) (Figure 5E). Moreover, M0 macrophages were negatively
(2.87%), and pathway connections (1.88%), as depicted in correlated with M2 macrophages (=-0.58), and activated mast cells
Supplementary Figure S2. The predicted genes could be found in the were negatively linked to resting mast cells (r=-0.55) (Figure 5E).
outer ring, whereas core genes were located in the inner ring. In
Figure 3H, these genes were enriched in neutrophil migration, humoral
immune response, cellular response to chemokines, regulation of T-cell Verification of the expression of hub genes
differentiation, response to chemokines and cytokine activity. in human colonic mucosa

Endoscopic diagnosis and histological evaluation

Expression verification of hub genes Intestinal manifestation of healthy person, ulcerative colitis
patients and colon cancer patients under endoscopy (Figures 6A–
Differential expression verification was performed in the D). Histologic evaluation revealed crypt abscess, mucosal damage
validation cohort GSE87466 for UC and GSE39582 for CRC. We and neutrophil infiltration in the intestinal tract of UC patients and
found that all hub genes (CXCL1, BCL6, CXCL2, and LCN2) mucosal structure disorder and nuclear swelling in CRC patients
showed predominant up-regulation in the UC and CAC patient (Figures 6E–J).
cohorts in contrast with the controls (Figures 4A–H).

Western blot analysis

Diagnostic value evaluation of hub genes
Compared to the controls, the UC group expressed more
ROC curve analysis of the GSE92415 dataset revealed BCL6 CXCL1 (P < 0.001), while the CRC group expressed more CXCL1
(AUC: 0.883, CI: 0.817−0.939), CXCL1 (AUC: 0.955, CI: 0.918 (P<0.0001), and the CRC group expressed more CXCL1 than UC
−0.984), CXCL2 (AUC: 0.935, CI: 0.878−0.977) and LCN2 (AUC: cohort did (P<0.01) (Figure 6Q). Moreover, the level of BCL6 in
0.965, CI: 0.925−0.995) (Figures 4I–L). Furthermore, GSE20916 was colon tissues demonstrated that the BCL6 in UC patients was
used to confirm the diagnostic ability of the hub genes BCL6 (AUC: markedly greater than CRC patients (P <0.001), CRC patients
0.772, CI: 0.661−0.872), CXCL1 (AUC: 0.948, CI: 0.905−0.981), was greater than the control group (P <0.001) and in UC patients
CXCL2 (AUC: 0.947, CI: 0.902−0.983) and LCN2 (AUC: 0.884, CI: was greater than the controls (P<0.0001) (Figure 6R).

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FIGURE 4
Verification and ROC curves of the hub genes. (A–D) The expression verification in the GSE87466 dataset. (E–H) Hub gene expression in the
GSE39582 dataset. (I–L) ROC curves of BCL6, CXCL1, LCN2 and CXCL2 in the GSE92415 dataset. (M–P) ROC curves of BCL6, CXCL1, LCN2 and
CXCL2 in the GSE20916 dataset. *p<0.05, ***p<0.001.

IHC analysis markedly greater than controls (P<0.001), and in UC was

greater than the controls (P<0.05) (Figures 6K–M, S). The levels
We applied immunohistochemical staining to determine the of BCL6 in UC patients were markedly greater than the CRC
mRNA expressions of CXCL1 and BCL6 in nontumor adjacent, patients (P < 0.05) and the controls (P < 0.001). Moreover, the
UC and cancer tissues (Figures 6K–P). CXCL1 in CRC group level of BCL6 in CRC patients was higher than controls (P < 0.01)
expressed greater than the UC group (P<0.05), CRC group was (Figures 6N–P, T).

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FIGURE 5
Immune cell infiltration analysis. (A) According to the Spearman correlation analysis, the hub genes were strongly correlated with immune cells. (B)
Correlation results of hub genes and immune cells. (C) Violin plot of the proportion of immune cells infiltrating the UC cohort compared to that in
the control cohort. (D) Proportions of 22 immune cells visualized from the bar plot. (E) Heatmap of correlations of different immune cells in UC
samples. Red: positive correlation; blue: negative correlation.

Protein expression verification of core upregulated. The level of CXCL1 in the UC patients was markedly
genes in the intestinal mucosa greater than the controls (P < 0.0001) (Figures 7A–J). The level of
BCL6 in the UC patients was greater than the cancer patients (P <
Immunofluorescence assays demonstrated that, compared with 0.0001). In contrast with the controls, the level of BCL6 in the UC
that in the UC cohort (P<0.001) and control group (P<0.0001), and cancer patients dramatically increased (P < 0.0001)
CXCL1 expression in cancer patients was greater and markedly (Figures 7K–T).

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FIGURE 6
Verification of gene expression in colon tissues (A) Images of a normal intestinal tract obtained via white light endoscopy. (B) Intestinal tract images
of UC patients via white light endoscopy. (C) Intestinal tract images of CRC patients via white light endoscopy. (D) NBI images of the intestinal tract
of CRC patients. (E, H) H&E staining of human normal colon sections. (F, I) H&E staining of intestinal tissues from UC patients. (G, J) H&E staining of
intestinal sections from CRC patients. (K–P) Expression of CXCL1 (K–M) and BCL6 (N–P) in the control cohort, UC patient cohort and cancer
patient cohort determined by IHC analysis. (S, T) Statistical comparison of the IHC analysis of the mean densities of CXCL1 (S) and BCL6 (T) in the
control cohort, UC patient cohort and cancer patient cohort. CXCL1 was expressed at the highest level in the cancer patient cohort. BCL6 was
expressed at the highest level in the UC patient cohort. (Q, R) Western blot analysis of CXCL1 and BCL6 expression in human intestinal tissues. In the
cancer patients group, the CXCL1 expressed the highest than others. In the UC patient group, the BCL6 expression was greater than that in the
other groups. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Discussion leads to increased mortality in UCAC patients (28–30). Thus, we

identified two biomarkers (CXCL1 and BCL6) for the diagnosis of
UCAC often occurs at a more advanced stage in clinical case colitis-related cancer. The chemokine CXCL1, known as growth-
reports than in other patients with CRC (10). Endoscopies are regulated oncogene a, belongs to the CXC chemokine family (31).
difficult in the early stages due to the flat UCAC lesion, which Chemokines play a vital part in each process of immune system, for

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FIGURE 7
Immunofluorescence images of CXCL1 and BCL6. (A–I) Immunofluorescence images of CXCL1 in colon tissues from the control, UC and cancer
cohorts. (J) Quantitative analysis of CXCL1 immunofluorescence. (K–S) Immunofluorescence images of BCL6 in colon tissues from the control, UC
and cancer groups. (T) Quantitative analysis of BCL6 immunofluorescence. ***p<0.001 and ****p<0.0001.

instance immune system development and homeostasis (32, 33). gastrointestinal cancers (31, 34). Moreover, in many cancers, the
Numerous studies have found that tumor cells and tumor expression of CXCL1 may correlate with tumor dimensions, tumor
microenvironment cells produce chemokines (32). Chemokines can grade, and tumor-node-metastasis (TNM) stage (34). L. Han et al.
influence the growth of tumor cells, cell survival, angiogenesis, and found a link between CXCL1 and TNM stage in laryngeal squamous
metastasis (34). Recent extensive research has showed the cell carcinoma (35). Shi Liu et al. also found a relation between
participation of CXCL1 in various tumorigenesis processes, CXCL1 expression and lymph node metastasis in patients with
including lung cancer, melanoma, reproductive cancers, and laryngeal squamous cell carcinoma (36). Comprehensive studies on

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FIGURE 8
Effects of hub genes on the transformation from ulcerative colitis to colorectal cancer.

CXCL1’s involvement in colitis-related cancer are relatively rare. This between mice and humans. Furthermore, because UC and UCAC
research attempted to address the aforementioned gap by have low incidence rates, we included a small sample size.
investigating the role of CXCL1 in colitis-related cancer. Studies The NF-kB family is made up of five transcription factors that
have demonstrated a connection between the pathogenesis of UC and play a part in immunity, inflammation, cell growth, and the
follicular helper T (Tfh) cells (37). Bcl-6 plays a crucial role in Tfh cell development of cancer (40, 41). NF-kB activation is pivotal in
development, as it is essential for its generation (37). According to UCAC pathogenesis because it orchestrates the transcriptional
research by Youguang Yang et al., Bcl-6 changes Tfh/Tfr, promoting upregulation of proinflammatory cytokines, facilitates tumor
the occurrence of IBD (38). However, studies comparing BCL6 and growth by promoting angiogenesis-related genes, and enhances
colitis-related cancer are rare. Youguang Yang et al. collected blood cell survival via the induction of antiapoptotic genes (41). In
samples of IBD patients and detected the level of BCL6 in the normal, mouse models of UCAC, tumor incidence was reduced by
UC, and CD groups via PCR. They observed significant increases in ablation of IkB kinase that resulted in NF-kB pathway
BCL6 expression in the ulcerative colitis group (38). In their study, inactivation (42). In a different study, a UCAC mouse model that
Jiwei Wang et al. found that decreasing BCL6 makes TNF-a-induced had NLRP12 cut out was more likely to get severe colitis and even
apoptosis worse in colonic epithelial cells, which makes UC tumors linked to colitis than wild-type mice. NLRP12 belongs to the
symptoms worse. Additionally, Wang Team demonstrated that Nod-like receptor family and negatively regulates NF-kB signaling
IRF4 exerts a negative regulatory effect on BCL6, facilitating Treg (43, 44). These studies further demonstrated that NF-kB plays a
cell differentiation into macrophage-like cells and consequently significant role in promoting inflammation-driven colon
impeding colon cancer cell proliferation (39). The Jiwei Wang tumorigenesis. In our research, we conducted an enrichment
team’s results partially mirrored our own. Our research indicated analysis of core genes and found that they were enriched in the
that BCL6 acts as a protective factor against the progression of CRC, NF-kB pathways, which indicates that our core genes likely led to
which is consistent with Jiwei Wang’s results. However, our study the tumorigenesis of UCAC via the NF-kB pathway.
found that BCL6 expression in the UC cohort was higher than in the The core genes (CXCL1 and BCL6) showed a strong link with
controls, which differs from the findings of Jiwei Wang’s team. On the macrophages, neutrophils, and B cells in our research. These
one hand, Jiwei Wang’s team used an acute ulcerative colitis UC immune cells were a significant component of the UCAC’s TME
mouse model, which contrasts with our long-term tissues from UC (Figure 8) (45). The Gao team discovered that TME’s CXC
patients. On the other hand, there are differences in gene expression chemokines drive neutrophil production, recruitment, and

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presence, while also promoting tumor cell survival and [Link] and TCGA database https://
extravasation (46). They were consistent with our finding that [Link]/.
CXCL1 is positively associated with CRC risk and strongly
correlated with neutrophils. Inducing neutrophil polarization
toward anti-tumor is then an effective means of improving the Ethics statement
CRC prognosis. The Gao team’s results provided direction and a
solid foundation for our immunotherapy in the future. In the pan- The protocols used for human sample collection were reviewed
cancer B-cell Atlas, the Gao team described and investigated the and approved by the Ethics Committee of the Second Affiliated
role of BCL6 in the TME of CRC (47). They demonstrated that Tfh Hospital of Harbin Medical University (ethics review batch
played an auxiliary role in B cell proliferation and differentiation number: KY2023-051). The studies were conducted in accordance
and that Bcl-6 was essential for Tfh cell development (47). Given B with the local legislation and institutional requirements. The human
cells’ role in cancer immunity, we believe that BCL6 could be used samples used in this study were acquired from the Second Affiliated
for cancer surveillance and treatment in next time. Hospital of Harbin Medical University. Written informed consent
Using scMetabolism (45), the Gao team demonstrated that for participation was not required from the participants or the
macrophages were heavily enriched in CRC and had extremely participants’ legal guardians/next of kin in accordance with the
high metabolic activity. In our research, there was a strong relation national legislation and institutional requirements.
between our markers and macrophages in TME. It seems that the
core genes suppress the immune system by activating macrophages,
which can facilitate CRC development, progression, and even Author contributions
metastasis. Next, we will explore the metabolic landscape of the
CRC immune microenvironment using scMetabolism. Meanwhile, XC: Conceptualization, Data curation, Methodology, Supervision,
the Gao team studied the systemic metabolism and local TME (48). Validation, Visualization, Writing – original draft, Writing – review &
A metabolic competition existed between immune cells and tumors editing. NL: Methodology, Supervision, Validation, Writing – original
in the TME (48). Metabolism plays an instrumental part in cancer draft, Writing – review & editing. JHQ: Data curation, Supervision,
development, and local metabolism intervention represents a Validation, Visualization, Writing – review & editing. HC: Data
promising treatment for CRC. curation, Supervision, Validation, Visualization, Writing – review &
Although bioinformatic methods predicted the core genes editing. HX: Supervision, Validation, Writing – review & editing.
involved in the conversion from UC to CRC, the morbidity of JWQ: Supervision, Validation, Writing – review & editing. YT:
UC and UCAC patients was low, and the sample sizes were small. Supervision, Validation, Writing – review & editing. SH: Data
Therefore, more detailed studies and larger sample sizes are curation, Supervision, Validation, Writing – review & editing. PD:
necessary. Multiple factors affected the disease. Thus, advanced Data curation, Supervision, Validation, Writing – review & editing.
comprehensive multiomics studies, such as single-cell sequencing, FG: Data curation, Supervision, Validation, Writing – review & editing.
spatial genomics, and scMetabolism et al., should be performed in RC: Data curation, Supervision, Validation, Writing – original draft,
our next studies. Writing – review & editing. UU: Data curation, Supervision,
Validation, Writing – review & editing. CW: Supervision, Validation,
Writing – review & editing. LL: Supervision, Validation, Writing –
Conclusion review & editing. SJ: Conceptualization, Data curation, Investigation,
Methodology, Supervision, Validation, Visualization, Writing –
In conclusion, core genes (CXCL1 and BCL6) are effectiveness original draft, Writing – review & editing.
biomarkers for evaluating the progression from ulcerative colitis to
cancer. The level of CXCL1 exhibited a positive relation with UCAC,
whereas the level of BCL6 showed a negative relation with UCAC.
BCL6 and CXCL1 showed a strong link with macrophages, Funding
neutrophils and B cells, which is an important component in TME
of UCAC. Our findings provide basic research that targets immune The author(s) declare that no financial support was received for
the research, authorship, and/or publication of this article.
checkpoints to block tumorigenesis and intervene in the
heterogeneity of immune cells to play a role in anti-tumor.

Acknowledgments
Data availability statement
We thank all of authors contributing to our article. We sincerely
Publicly available datasets were analyzed in this study. The appreciate the GEO and TCGA databases for providing meaningful
data supporting our study are available in the GEO database, datasets and clinical information.

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Conflict of interest organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
The authors declare that the research was conducted in the claim that may be made by its manufacturer, is not guaranteed or
absence of any commercial or financial relationships that could be endorsed by the publisher.
construed as a potential conflict of interest.

Supplementary material
Publisher’s note
The Supplementary Material for this article can be found online
All claims expressed in this article are solely those of the authors at: [Link]
and do not necessarily represent those of their affiliated full#supplementary-material

References
1. Kobayashi T, Siegmund B, Le Berre C, Wei SC, Ferrante M, Shen B, et al. 19. Takahashi K, Shigeyasu K, Kondo Y, Gotoh K, Yano S, Umeda Y, et al. RNA
Ulcerative colitis. Nat Rev Dis Primers. (2020) 6:74. doi: 10.1038/s41572-020-0205-x editing is a valuable biomarker for predicting carcinogenesis in ulcerative colitis.
2. Chen H, Lin X, Pan X, Xu H, Zhang X, Liang G, et al. Development and validation J Crohns Colitis. (2023) 17:754–66. doi: 10.1093/ecco-jcc/jjac186
of a blood routine-based extent and severity clinical decision support tool for ulcerative 20. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation
colitis. Sci Rep. (2023) 13:21368. doi: 10.1038/s41598-023-48569-5 network analysis. BMC Bioinf. (2008) 9:559. doi: 10.1186/1471-2105-9-559
3. Le Berre C, Honap S, Peyrin-Biroulet L. Ulcerative colitis. Lancet. (2023) 402:571– 21. Carbon S, Thomas PD, Albou LP, Hill DP, Gaudet P, Van Auken, et al. The
84. doi: 10.1016/S0140-6736(23)00966-2 Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. (2019) 47:
4. Jess T, Rungoe C, Peyrin-Biroulet L. Risk of colorectal cancer in patients with D330–330D338. doi: 10.1093/nar/gky1055
ulcerative colitis: a meta-analysis of population-based cohort studies. Clin Gastroenterol 22. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic
Hepatol. (2012) 10:639–45. doi: 10.1016/[Link].2012.01.010 Acids Res. (2000) 28:27–30. doi: 10.1093/nar/28.1.27
5. Amoroso C, Perillo F, Strati F, Fantini MC, Caprioli F, Facciotti F. The role of gut 23. Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, et al. The
microbiota biomodulators on mucosal immunity and intestinal inflammation. Cells. STRING database in 2021: customizable protein-protein networks, and functional
(2020) 9:1234. doi: 10.3390/cells9051234 characterization of user-uploaded gene/measurement sets. Nucleic Acids Res. (2021) 49:
6. Frigerio S, Lartey DA, D'Haens GR, Grootjans J. The role of the immune system in D605–605D612. doi: 10.1093/nar/gkaa1074
IBD-associated colorectal cancer: from pro to anti-tumorigenic mechanisms. Int J Mol 24. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape:
Sci. (2021) 22:12739. doi: 10.3390/ijms222312739 a software environment for integrated models of biomolecular interaction networks.
7. Li N, Zhao L, Geng X, Liu J, Zhang X, Hu Y, et al. Stimulation by exosomes from Genome Res. (2003) 13:2498–504. doi: 10.1101/gr.1239303
hypoxia-preconditioned hair follicle mesenchymal stem cells facilitates mitophagy by 25. Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P, et al.
inhibiting the PI3K/AKT/mTOR signaling pathway to alleviate ulcerative colitis. The GeneMANIA prediction server: biological network integration for gene
Theranostics. (2024) 14:4278–96. doi: 10.7150/thno.96038 prioritization and predicting gene function. Nucleic Acids Res. (2010) 38:W214–20.
8. Luo C, Zhang H. The role of proinflammatory pathways in the pathogenesis of doi: 10.1093/nar/gkq537
colitis-associated colorectal cancer. Mediators Inflamm. (2017) 2017:5126048. 26. Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez JC, et al. pROC: an
doi: 10.1155/2017/5126048 open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinf.
9. Hirano T, Hirayama D, Wagatsuma K, Yamakawa T, Yokoyama Y, Nakase H. (2011) 12:77. doi: 10.1186/1471-2105-12-77
Immunological mechanisms in inflammation-associated colon carcinogenesis. Int J 27. Newman AM, Liu CL, Green MR, Gentles AJ, Feng W, Xu Y, et al. Robust
Mol Sci. (2020) 21:3062. doi: 10.3390/ijms21093062 enumeration of cell subsets from tissue expression profiles. Nat Methods. (2015)
10. Laine L, Kaltenbach T, Barkun A, McQuaid KR, Subramanian V, Soetikno R. 12:453–7. doi: 10.1038/nmeth.3337
SCENIC international consensus statement on surveillance and management of 28. Matkowskyj KA, Chen ZE, Rao MS, Yang GY. Dysplastic lesions in
dysplasia in inflammatory bowel disease. Gastroenterology. (2015) 148:639–51.e28. inflammatory bowel disease: molecular pathogenesis to morphology. Arch Pathol
doi: 10.1053/[Link].2015.01.031 Lab Med. (2013) 137:338–50. doi: 10.5858/arpa.2012-0086-RA
11. Rubin DT, Ananthakrishnan AN, Siegel CA, Sauer BG, Long MD. ACG clinical 29. Romano M, Francesco F DE, Zarantonello L, Ruffolo C, Ferraro GA, Zanus G,
guideline: ulcerative colitis in adults. Am J Gastroenterol. (2019) 114:384–413. et al. From inflammation to cancer in inflammatory bowel disease: molecular
doi: 10.14309/ajg.0000000000000152 perspectives. Anticancer Res. (2016) 36:1447–60.
12. Lutgens MW, van Oijen MG, van der Heijden GJ, Vleggaar FP, Siersema PD, 30. Faye AS, Holmer AK, Axelrad JE. Cancer in inflammatory bowel disease.
Oldenburg B. Declining risk of colorectal cancer in inflammatory bowel disease: an Gastroenterol Clin North Am. (2022) 51:649–66. doi: 10.1016/[Link].2022.05.003
updated meta-analysis of population-based cohort studies. Inflammation Bowel Dis. 31. Korbecki J, Bosiacki M, Barczak K, Łagocka R, Brodowska A, Chlubek D, et al.
(2013) 19:789–99. doi: 10.1097/MIB.0b013e31828029c0 Involvement in tumorigenesis and clinical significance of CXCL1 in reproductive
13. Roda G, Narula N, Pinotti R, Skamnelos A, Katsanos KH, Ungaro R, et al. cancers: breast cancer, cervical cancer, endometrial cancer, ovarian cancer and prostate
Systematic review with meta-analysis: proximal disease extension in limited ulcerative cancer. Int J Mol Sci. (2023) 24:7262. doi: 10.3390/ijms24087262
colitis. Aliment Pharmacol Ther. (2017) 45:1481–92. doi: 10.1111/apt.14063 32. Lazennec G, Richmond A. Chemokines and chemokine receptors: new insights
14. Sudhakar P, Alsoud D, Wellens J, Verstockt S, Arnauts K, Verstockt B, et al. into cancer-related inflammation. Trends Mol Med. (2010) 16:133–44. doi: 10.1016/
Tailoring multi-omics to inflammatory bowel diseases: all for one and one for all. J [Link].2010.01.003
Crohns Colitis. (2022) 16:1306–20. doi: 10.1093/ecco-jcc/jjac027
33. Hughes CE, Nibbs R. A guide to chemokines and their receptors. FEBS J. (2018)
15. Arthur JC, Perez-Chanona E, Mühlbauer M, Tomkovich S, Uronis JM, Fan TJ, 285:2944–71. doi: 10.1111/febs.14466
et al. Intestinal inflammation targets cancer-inducing activity of the microbiota.
Science. (2012) 338:120–3. doi: 10.1126/science.1224820 34. Korbecki J, Bosiacki M, Barczak K, Łagocka R, Chlubek D, Baranowska-Bosiacka
I. The clinical significance and role of CXCL1 chemokine in gastrointestinal cancers.
16. Ananthakrishnan AN, Cheng SC, Cai T, Cagan A, Gainer VS, Szolovits P, et al. Cells. (2023) 12:1406. doi: 10.3390/cells12101406
Serum inflammatory markers and risk of colorectal cancer in patients with
inflammatory bowel diseases. Clin Gastroenterol Hepatol. (2014) 12:1342–8.e1. 35. Gros B, Kaplan GG. Ulcerative colitis in adults: A review. JAMA. (2023)
doi: 10.1016/[Link].2013.12.030 330:951–65. doi: 10.1001/jama.2023.15389
17. Benderska N, Dittrich AL, Knaup S, Rau TT, Neufert C, Wach S, et al. miRNA- 36. Liu S, Wang X, Xu X. Expression of GRO-1 and its relationship with VEGF in
26b overexpression in ulcerative colitis-associated carcinogenesis. Inflammation Bowel squamous cell carcinoma of larynx. Lin Chuang Er Bi Yan Hou Ke Za Zhi. (2006) 20:541–4.
Dis. (2015) 21:2039–51. doi: 10.1097/MIB.0000000000000453 37. Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, et al.
18. Ricciuto A, Griffiths AM. Clinical value of fecal calprotectin. Crit Rev Clin Lab Bcl6 mediates the development of T follicular helper cells. Science. (2009) 325:1001–5.
Sci. (2019) 56:307–20. doi: 10.1080/10408363.2019.1619159 doi: 10.1126/science.1176676

Frontiers in Oncology 15 [Link]

Cang et al. 10.3389/fonc.2024.1475189

38. Yang Y, Lv X, Zhan L, Chen L, Jin H, Tang X, et al. Case report: IL-21 and bcl-6 during colitis-associated cancer. J Exp Med. (2010) 207:1045–56. doi: 10.1084/
regulate the proliferation and secretion of tfh and tfr cells in the intestinal germinal jem.20100050
center of patients with inflammatory bowel disease. Front Pharmacol. (2020) 44. Allen IC, Wilson JE, Schneider M, Lich JD, Roberts RA, Arthur JC, et al. NLRP12
11:587445. doi: 10.3389/fphar.2020.587445 suppresses colon inflammation and tumorigenesis through the negative regulation of
39. Wang J, Wu Z, Huang Y, Jin L, Xu J, Yao Z, et al. IRF4 induces M1 macrophage noncanonical NF-kB signaling. Immunity. (2012) 36:742–54. doi: 10.1016/
polarization and aggravates ulcerative colitis progression by the Bcl6-dependent STAT3 [Link].2012.03.012
pathway. Environ Toxicol. (2024) 39:2390–404. doi: 10.1002/tox.24106 45. Wu Y, Yang S, Ma J, Chen Z, Song G, Rao D, et al. Spatiotemporal immune
40. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK landscape of colorectal cancer liver metastasis at single-cell level. Cancer Discovery.
function. Nat Rev Mol Cell Biol. (2007) 8:49–62. doi: 10.1038/nrm2083 (2022) 12:134–53. doi: 10.1158/[Link]-21-0316
41. Viennois E, Chen F, Merlin D. NF-kB pathway in colitis-associated 46. Wu Y, Ma J, Yang X, Nan F, Zhang T, Ji S, et al. Neutrophil profiling illuminates
cancers. Transl Gastrointest Cancer. (2013) 2:21–9. doi: 10.3978/[Link].2224- anti-tumor antigen-presenting potency. Cell. (2024) 187:1422–39.e24. doi: 10.1016/
4778.2012.11.01 [Link].2024.02.005
42. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, et al. IKKbeta links 47. Ma J, Wu Y, Ma L, Yang X, Zhang T, Song G, et al. A blueprint for tumor-infiltrating
inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell. B cells across human cancers. Science. (2024) 384:eadj4857. doi: 10.1126/science.adj4857
(2004) 118:285–96. doi: 10.1016/[Link].2004.07.013 48. Wu Y, Zou Q, Jiang P, Gao Q. Tumor-host cometabolism collaborates to
43. Allen IC, TeKippe EM, Woodford RM, Uronis JM, Holl EK, Rogers AB, shape cancer immunity. Cancer Discovery. (2024) 14:653–7. doi: 10.1158/2159-
et al. The NLRP3 inflammasome functions as a negative regulator of tumorigenesis [Link]-23-1509

Frontiers in Oncology 16 [Link]