TYPE Original Research

PUBLISHED 14 October 2022

DOI 10.3389/fonc.2022.941676

Prognostic role of HPV

OPEN ACCESS integration status and molecular
EDITED BY
Sridhar Muthusami,
Karpagam Academy of Higher
profile in advanced anal
Education, India

REVIEWED BY
carcinoma: An ancillary study to
Ulrike Wieland,
University Hospital of Cologne,
Germany
the epitopes-HPV02 trial
Cathy Eng,
Vanderbilt University, United States
Alice Debernardi 1, Aurélia Meurisse 2,3, Jean-Luc Prétet 1,4,
*CORRESPONDENCE
Christophe Borg David Guenat 1,5, Franck Monnien 6, Laurie Spehner 3,7,
[email protected] Angélique Vienot 3,7, Patrick Roncarati 8, Thierry André 9,
SPECIALTY SECTION
Laurent Abramowitz 10,11, Chloé Molimard 12,
This article was submitted to
Molecular and Cellular Oncology, Christiane Mougin 3,4, Michael Herfs 8, Stefano Kim 3,13,14
a section of the journal
Frontiers in Oncology and Christophe Borg 3,5,13*
RECEIVED 11 May 2022 1
EA3181, University of Bourgogne Franche-Comté, LabEx LipSTIC ANR-11-LABX-0021, Besançon,
ACCEPTED 20 September 2022 France, 2 Methodology and Quality of Life in Oncology Unit, University Hospital of Besançon,
PUBLISHED 14 October 2022 Besançon, France, 3 INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté,
Besançon, France, 4 Papillomavirus National Reference Center, University Hospital, Besançon,
CITATION France, 5 'Molecular Biology and Microbiology Department, Anamed SA Laboratory, Lausanne,
Debernardi A, Meurisse A, Prétet J, Switzerland, 6 Department of Pathology, University Hospital of Besançon, Besançon, France,
Guenat D, Monnien F, Spehner L, 7
Department of Medical Oncology, University Hospital of Besançon, Besançon, France, 8 Laboratory
Vienot A, Roncarati P, André T, of Experimental Pathology, GIGA-Cancer, University of Liege, Liege, Belgium, 9 Department of
Abramowitz L, Molimard C, Mougin C, Medical Oncology, University Hospital Saint Antoine, Paris, France, 10Division of Gastroenterology
Herfs M, Kim S and Borg C (2022) and Hepatology and Proctology, University Hospital Bichat, Paris, France, 11 Ramsay GDS, Blomet
Prognostic role of HPV integration Clinic, Paris, France, 12 Department of Anatomopathology, University Hospital of Besançon,
status and molecular profile in Besançon, France, 13 Clinical investigation center, CIC-1403 University Hospital of Besançon,
advanced anal carcinoma: Besançon, France, 14 Department of Medical Oncology, Sanatorio Allende, Cordoba, Argentina
An ancillary study to the
epitopes-HPV02 trial.
Front. Oncol. 12:941676.
doi: 10.3389/fonc.2022.941676

COPYRIGHT Squamous Cell Carcinoma of the Anal canal (SCCA) is a rare disease associated
© 2022 Debernardi, Meurisse, Prétet, with a Human Papillomavirus (HPV) infection in most cases, predominantly the
Guenat, Monnien, Spehner, Vienot,
Roncarati, André, Abramowitz,
HPV16 genotype. About 15% of SCCA are diagnosed in metastatic stage and
Molimard, Mougin, Herfs, Kim and Borg. some will relapse after initial chemoradiotherapy (CRT). Treatment of patients
This is an open-access article by Docetaxel, Cisplatin and 5-fluorouracil (DCF) has been recently shown to
distributed under the terms of the
Creative Commons Attribution License improve their complete remission and progression-free survival. The aim of this
(CC BY). The use, distribution or retrospective study was to explore the impact of HPV infection, HPV DNA
reproduction in other forums is
integration, TERT promoter mutational status and somatic mutations of
permitted, provided the original author
(s) and the copyright owner(s) are oncogenes on both progression-free (PFS) and overall survivals (OS) of
credited and that the original patients treated by DCF. Samples obtained from 49 patients included in the
publication in this journal is cited, in
accordance with accepted academic Epitopes-HPV02 clinical trial, diagnosed with metastatic or non-resectable
practice. No use, distribution or local recurrent SCCA treated by DCF, were used for analyses. Median PFS and
reproduction is permitted which does
OS were not associated with HPV status. Patients with episomal HPV had an
not comply with these terms.
improved PFS compared with SCCA patients with integrated HPV genome
(p=0.07). TERT promoter mutations were rarely observed and did not
specifically distribute in a subset of SCCA and did not impact DCF efficacy.
Among the 42 genes investigated, few gene alterations were observed, and
were in majority amplifications (68.4%), but none were significantly correlated

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Debernardi et al. 10.3389/fonc.2022.941676

to PFS. As no biomarker is significantly associated with patients’ survival, it

prompts us to include every patient failing CRT or with metastatic disease in
DCF strategy.

KEYWORDS

NGS - next generation sequencing, SCCA, Somatic mutation analysis, TERT promoter
mutation, HPV integration

Introduction progression free survival rate was 47%. Therefore, DCF

became one of the standard regimen at first-line in advanced
Squamous cell carcinoma of the anal canal is a rare disease, SCCA (13, 15–17).
representing less than 3% of all gastrointestinal malignancies in Here, we describe the molecular characterization of anal
the world (1). Its incidence has been steadily increasing in recent cancer biopsies with advanced SCCA, included in the Epitopes-
decades in men as well as in women with 50 000 new cases HPV02 trial.
diagnosed each year worldwide and it is estimated that it will
continue to increase in the next future (2). This increase is likely
due to its association with human papillomavirus infection (3),
Material and methods
predominantly genotype HPV16, since HPV-related
oncoproteins (E6 and E7) are expressed in more than 90% of
Patients and cell lines
patients with SCCA (4).
The cohort is constituted of patients included in the
The great majority of SCCA patients are diagnosed at a
prospective multicenter phase II Epitopes-HPV02 study
localized stage. However, about 15% of patients are diagnosed at
(NCT02402842). Patients with diagnosis of metastatic or non-
metastatic stage in US (https://seer.cancer.gov/statfacts/html/
resectable local recurrent SCCA were included, and were treated
anus.html, accessed July 25th 2022), and between 25% to 40%
by DCF. The study was described in detail elsewhere (15–17).
of patients treated initially by chemoradiotherapy will develop
Among 66 patients included in the trial, 49 patients had available
locally advanced recurrences or metastases in Western countries
material for molecular analysis. The EDITH V cohort
(5–7). Treatment of patients with nonresectable local
constituted of patients with diagnosis of SCCA at early stage
recurrences or with distant metastases relies on systemic
was used as a validation cohort. The study was described
chemotherapy. The combination of cisplatin (CDDP) and 5-
elsewhere (18). Overall, 381 patients had available material for
Fluorouracil (5FU) was historically considered as the
molecular analysis.
recommended treatment for advanced SCCA based in
retrospective analysis (8, 9). However, complete remission was
a rare event, and only about 15% of patients were progression-
free at 1 year (10, 11).
DNA extraction
Docetaxel is an anticancer agent which exerts cytotoxic
Prior to DNA extraction, separate hematoxylin-eosin-stained
functions by stabilizing tubulin polymerization leading to
slides were reviewed by an experienced histopathologist and
mitosis arrest and cell death. It has been previously proposed
manually macro-dissected when appropriate to ensure tumor
that a loss of normal p53 function confers sensitization to taxane
content greater than 20%. Depending on the size of the fixed
chemotherapy by increasing G2/M arrest and apoptosis (12).
tissue, between 3 and 8 formalin-fixed paraffin-embedded (FFPE)
Because the association between SCCA and HPV infection is
tissue sections of 10 µm thickness were processed for DNA
strong and E6 oncoprotein encoded by High Risk (HR) HPV,
extraction with the QIAamp DNA mini kit (QIAGEN)
such as HPV16 and 18, induces the degradation of p53, we
according to manufacturer’s instructions.
previously hypothesized that SCCA might be sensitive to taxane-
containing chemotherapies such as docetaxel (13). In addition,
docetaxel has been shown to increase endoplasmic reticulum
(ER) stress and to induce immunogenic cell death of cancer cells HPV genotyping
(14). In 2018, Epitopes-HPV02 trial confirmed the benefit of the
addition of docetaxel to CDDP and 5FU (DCF) (15). A complete HPV genotyping was performed locally [Department of
response was observed in 45% of patients, and the 1-year Cellular and Molecular Biology, University Hospital of

Frontiers in Oncology 02 frontiersin.org

Debernardi et al. 10.3389/fonc.2022.941676

Besanç on, France as described previously (19)]. Briefly, TABLE 2 Primer sequence used for TERT sequencing.
genotyping was performed with the INNO-LiPA HPV
Primer Sequence
Genotyping Extra® test (Fujirebio) allowing the identification
of 28 different HPV genotypes as well as the HLA-DPB1 gene as TERT Fw 5’-CGTCCTGCCCCTTCACCT-3’
internal control for sample quality for DNA detection. As TERT Rev 5’-AGCGCTGCCTGAAACTCG-3’
recommended by the manufacturer, samples negative for the
HLA-DPB1 gene and negative for HPV were excluded from
the analysis. using the online Primer-BLAST software (www.ncbi.nlm.nih.
gov/tools/primer-blast/) (22). Targeted sequences were
amplified by PCR using the Qiagen Multiplex PCR kit
In situ hybridization (QIAGEN) and 5% DMSO. PCR conditions were as follows:
94°C for 15 min, 40 cycles at 94°C for 1 min, then 64°C for 30
To confirm the HPV infection, in situ hybridization sec, 72°C for 45 sec and finally 7 min at 72°C. PCR products
experiments were performed according to the manufacturer’s were purified using the gel extraction kit NucleoSpin Gel and
instructions. The INFORM HPV III Family 16 Probe (Ventana PCR Clean-up (Macherey-Nagel). Bidirectional sequencing
Medical Systems) allowed the detection of 12 high-risk reaction was performed using the BigDye Terminator v3.1
HPV genotypes. Cycle Sequencing Kit (Life technologies-Thermofisher). The
reactions were run according to the following protocol: one
cycle at 96°C for 1 min; 15 cycles at 96°C for 10 s, 50°C for 5 s,
HPV physical status 60°C for 1 min 15 s; 5 cycles at 96°C for 10 s, 50°C for 5 s, 60°C
for 1 min 30 s; 5 cycles of 96°C for 10 s, 50°C for 5 s and 60°C
As routinely performed, the physical status of HPV for 2 min. After purification with a NucleoSEQ kit
(episomal or mixed/integrated) was determined by assessing (Macherey-Nagel), samples were run and analyzed on an
the disruption of the viral E2 gene (Table 1). Briefly, after DNA ABI 3130 sequencer (Life technologies-Thermofisher).
extraction and concentration measurement with NanoDrop Finally, the sequences obtained were compared with the
1000 spectrophotometer (Thermo Fisher Scientific), reference sequence of TERT promoter using GeneScan
quantitative real-time PCR experiments were performed as analysis software.
follows: 95°C for 15 min, 40 cycles at 95°C for 30 sec, then 50°
C for 1 min and 72°C for 1 min. Each experiment was performed
in triplicate and the E6/E2 ratio cut-off value was determined, as TERT mutations analysis by SNaPshot
previously described (20, 21). Importantly, to be able to compare
the collected results, the amplification efficiency of each PCR SNaPshot analysis was performed from the purified
reaction was determined (qPCR efficiency calculator, Thermo amplicons used for Sanger sequencing with the ABI Prism
Fisher Scientific). SNaPshot Multiplex kit (AB Life Technologies). Amplified
TERT promoter was analyzed for the presence of mutations at
position C228 and C250 using two primers that contained an
TERT mutations analysis by Sanger additional poly(dC) tail at their 5’ end, allowing for their
sequencing simultaneous detection (Table 3). Reactions were performed in
a final volume of 5 µL, containing 1.5 µL of purified multiplex
Genomic sequence of promoter flanking region of TERT PCR product (2 to 10 ng/µL), 2.5 µL of SNaPshot Ready
(ENSR00001274355) was obtained from Ensembl database Multiplex Ready Reaction Mix, 0.5 µL of probe equimolar mix
(www.ensembl.org). Specific primers were designed (Table 2) (each probe at 0.2 pmol/L final), and 0.5 µL of double-distilled
water. Multiplex single base extensions were carried out for 25
TABLE 1 Primer sequence used for E2, E6 and GAPDH qPCR. cycles according to the following program: 10 seconds at 96°C, 5
seconds at 52°C, and 30 seconds at 60°C. SNaPshot products
Primer Sequence
were then treated at 37°C for 15 min with 0.5µL of shrimp
HPV16 E2 Fw 5’- TTTAGCAGCAACGAAGTATCC-3’
alkaline phosphatase at 1 U/µL diluted in 2.5 µL of shrimp
HPV16 E2 Rev 5’- AGTCTCTGTGCAACAACTTAG-3’
alkaline phosphatase buffer 10X and 11.5 µL of double-distilled
HPV16 E6 Fw 5’- AAAGCCACTGTGTCCTGAAGA-3’
water. After heat inactivation of shrimp alkaline phosphatase for
HPV16 E6 Rev 5’-CTGGGTTTCTCTACGTGTTCT -3’
10 minutes at 75°C, 2 µL of the labelled products were mixed
GAPDH Fw 5’-ACCAGGTGGTCTCCTCTGAC-3’
with 9.5 µL of HiDi formamide and 0.5 µL of Genescan-120LIZ
GAPDH Rev 5’-TGCTGTAGCCAAATTGGTTG-3’ size standard. They were then separated using a 25 min run on
an ABI Prism 3130 DNA sequencer with POP-7 matrix and 14

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Debernardi et al. 10.3389/fonc.2022.941676

TABLE 3 SNaPshot primers used for detection of TERT promoter

mutation.
Statistical analysis

Primer Sequence Size (bp) Mutation Log-rank (Mantel-Cox) test was used for the analysis of PFS
and OS according to HPV integration status and PIK3CA
C228 5’-T23GGCTGGGAGGGCCCGGA-3’ 40 C228T
mutational status. PFS was determined at 12 months from the
C228A
first DCF cycle. Fisher’s exact test was used for all other analysis.
C250 5’-T39CTGGGCCGGGGACCCGG-3’ 56 C250T

seconds for injection. The analysis was performed using Results

GeneMapper ID software version 3.2.1 (Applied Biosystems).
Influence of HPV status and integration
in SCCA patients included in Epitopes
Libraries preparation and NGS HPV02 study

Libraries were prepared from 50 ng of DNA or by using Among patients included in the Epitopes-HPV02 study,
KAPA Hyperplus Library Preparation (KAPA Biosystem) and there was a majority of female (83.7%) compared to male who
Solid Tumor Solution capture kits and protocol by SOPHIA represented only 16.3% (Table 5). Most of the patients (91.8%,
GENETICS. They were sequenced on MiSeq sequencer 45 out of the 49 patients with available tumor material)
(Illumina). Criteria used to select mutations were depth (≥100) presented an HPV16 infection. Two patients were infected
and allele frequency (≥10%). Allele frequency variants ≈100% or with other HPV genotypes (HPV33 and HPV33-45) and two
described as benign in ClinVar database, with intronic, patients had SCCA without detectable HPV genome. Median
frameshift, splicing or synonymous mutations were excluded. PFS was 10.7 months (95% CI: 9.9-16.0) for patients displaying
A list of the targeted 42 genes is shown in Table 4. HPV16+ SCCA and 12.6 months (95% CI: 6.2-18.9) when HPV
was not detected. Median OS was 36.3 months (95% CI: 24.2-
NE) for HPV16+ SCCA and 26 months for HPV negative SCCA.
We next explored if the presence of HPV genome in an
TABLE 4 List of the genes and their exons targeted by NGS.
episomal or integrated form could influence clinical outcomes of
SCCA patients treated with DCF (Table 6). There was a majority
Gene Exon Gene Exon of integrated/mixed forms (51%, n=25) in all SCCA compared to
episomal forms (28.6%, n=14). In about 20% (n=10/49, 20.4%)
AKT1 3 HIST1H3B 1
of patients, the HPV integration status was not determined (2 of
ALK 21-25 HRAS 2-4
them were actually HPV negative, 2 were infected with other
BRAF 11,15 IDH1 4
HR-HPV infection than HPV16 and the others had no
CDK4 2 IDH2 4
remaining material to perform analysis). Median PFS was 19.5
CDKN2A 1*,2,3 KIT 8-11,13,17,18
months (95% CI: 8.3-NE) for patients with HPV DNA under
CTNNB1 3 KRAS 2-4
episomal form and 10.6 months (95% CI: 9.9-12.9) for patients
DDR2 18 MAP2K1 2,3
with integrated/mixed forms (p=0.0734). Median OS was 32.3
DICER1 24,25 MET 2,14-20
months for patients with integrated/mixed forms and was not
EGFR 18-21 MYOD1 1
reached for SCCA with episomal HPV DNA (Figure 1). The
ERBB2 8,17,20 NRAS 2-4
possible correlation between episomal HPV DNA in DCF
ERBB4 10,12 PDGFRA 12,14,18
efficacy is outlined by the 57% complete response rate
FBXW7 8-12 PIK3CA 2*,3,6*,8,10,21
observed in this population vs 44% for SCCA wherein HPV
FGFR1 13,15 PTPN11 3
DNA was integrated or in a mixed form.
FGFR2 7,12,14 RAC1 3
FGFR3 7,9,14,16 RAF1 7,10,12,13*,14*,15*
FOXL2 1* RET 11,13,15,16
GNA11 4,5 ROS1 38*,41* Distribution of TERT promoter mutations
GNAQ 4,5 SF3B1 15-17 in SCCA patients
GNAS 8 SMAD4 8-12
H3F3A 2* TERT promoter*,1*,8*,9*,13* Transactivation of TERT is a critical signaling for HPV-
H3F3B 2* TP53 full coding region mediated oncogenesis. E6-E6AP ubiquitin ligase complex are
A dash (-) means “from exon X to exon X”.
known to bind to TERT promoter, activating TERT gene
A star (*) means hotspots only. transcription (23). Mutations of the TERT promoter are the

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Debernardi et al. 10.3389/fonc.2022.941676

TABLE 5 Characteristics of the HPV02 cohort. to HPV negative SCCA and are not specifically correlated to a
specific HPV type infection.
HPV02 cohort (n=49)

Sexe
Female 83.7% (n=41) Distribution of other gene mutations in
Male 16.3% (n=8) SCCA patients
HPV status
HPV16 91.8% (n=45) Since HPV genotype, integration status or TERT promoter
Other HR-HPV 4.1% (n=2) mutations did not account for DCF efficacy and SCCA patients’
HPV negative 4.1% (n=2) prognosis in the Epitopes-HPV02 study, we next assessed the
Integration status distribution of the main oncogenic alterations in SCCA metastatic
Episomal 28.6% (n=14) or relapsing patients treated by DCF. NGS analysis targeting 42
Integrated/mixed forms 51% (n=25) oncogenic alterations was realized among patients of the HPV02
NR or HPV negative 20.4% (n=10) cohort. No alterations were found in AKT1, ALK, BRAF, DDR2,
PFS (months) DICER1, ERBB4, FOXL2, GNAQ, GNAS, H3F3A, H3F3B,
Median 10.7 HIST1H3B, IDH1, IDH2, KIT, KRAS, MAP2K1, MET, PDGFRA,
Min 1.8 PTPN11, RAC1, RET, ROS1, SF3B1, SMAD4 and TP53. PIK3CA
Max 42.6 was the most altered gene with 10 amplifications and 6 mutations,
OS (months) followed by ERBB2 (3 amplifications and 3 mutations) and CDK4
Median 33 (5 amplifications) (Table 7).
Min 3.8 Nineteen out of the 49 (38.8%) patients with SCCA tested
Max 46.9 presented no alterations among the 42 screened genes. Most of
them were HPV positive, with 89% (n=17/19) of HPV16 and 11%
(n=2/19) of other HR-HPV. Forty-seven percent (n=9/19) of SCCA
major genomic alterations leading to TERT overactivation in most harbored integrated or mixed forms of HPV DNA, whereas 32%
cancer types. However, the occurrence of TERT promoter (n=6/19) harbored only episomal forms of oncogenic HPV DNA.
mutations was never investigated in SCCA. We hypothesized that On the contrary, 61.2% (n=30/49) patients with SCCA presented
TERT promoter mutations might sustain resistance to DCF one alteration or more. A majority of them had an HPV16 infection
therapy. Therefore, these analyses were performed in 63 patients (90%, n=27/30), one had an HPV16+33 infection and 2 were HPV
with available tumor materials. TERT promoter mutations occurred negative. Almost 53% (n=16/30) had integrated or mixed forms,
rarely and were observed in 5 patients (7.9%). Three types of TERT 26.6% (n=8/30) had an episomal HPV and 20% (n=6/30) were not
promoter mutations investigated have been observed. One HPV16+ identified. No difference was observed between the 2 groups
SCCA patient had a C250T mutation, and another HPV16+ had a concerning HPV status (p-values: 1 and 0.1581 between HPV
C228A mutation. Three other patients (2 HPV16+ and one HPV- positive/negative and HPV16/other HR-HPV respectively) nor
SCCA) had C228T mutations. Three of the SCCA patients integration status (p-value: 0.7397).
displaying TERT promoter mutations showed partial responses A heatmap was realized to cluster genomic alteration
after exposition to DCF. These results addressed the question of occurrence to PFS (more or less than 12 months) (Figure 3;
the overall distribution of TERT mutations in SCCA population. To Table 8). There was clearly no aggregation of a genomic
validate the prevalence of TERT mutations in SCCA, sequencing alteration subset with the probability to be progression free
analysis of the telomerase catalytic subunit TERT was performed in after 12 months of follow up. Since PI3KCA amplification and
the EDITH V cohort. Among the 381 patients with available DNA, mutations were the most frequent, we assessed the influence of
TERT promoter mutations were identified in 30 patients (7.8%). these genomic alterations on SCCA patient overall survival. No
The C228T mutation was predominant (73.3%, n=22), while the difference of OS was observed between patients harboring
mutations C228A and C250T were observed in 1 and 7 patients PIK3CA alterations or WT PIK3CA (Figure 4, p-value: 0.1828).
respectively. Of note, one homozygote mutation of C228T was
detected. Among the EDITH V cohort, there was a majority of
patients with HPV positive SCCA (68%, n=259) including 210 Discussion
(81%) patients with HPV16 or 18 infections and 49 (19%) patients
with other HPV genotypes; 10 (2.6%) patients represented HPV Epitopes-HPV02 study was the first prospective clinical trial
negative SCCA. HPV status was not available for 112 (29.4%) that included SCCA patients with advanced diseases and
patients. TERT promoter mutations were not specifically correlated demonstrated the ability of Docetaxel-based polychemotherapy
to HPV status in SCCA (Figure 2). These results showed that the to induce long term remissions (15). An ancillary study was
rare TERT promoter mutations observed in SCCA are not restricted performed to analyze the distribution of HPV genotypes,

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Debernardi et al. 10.3389/fonc.2022.941676

TABLE 6 Clinical characteristics of SCCA patients according to HPV genome status.

Overall population n=39 Episomal n=14 Integrated/mixed n=25 p-value

Gender
Male 7 (17.9%) 2 (14.3%) 5 (20%) 1
Female 32 (82.1%) 12 (85.7%) 20 (80%)
Age
Mean (std) 57.5 (8.4) 58.6 (6.6) 56.8 (9.3)
Median (min-max) 58.2 (38.6-74.9) 59.8 (46.1-71.7) 56.1 (38.6-74.9) 0.6318
Q1-Q3 51.4-63.8 52.1-63.8 49.4-63.7
Age ≥ 65
No 32 (82.1%) 13 (92.9%) 19 (76%) 0.3863
Yes 7 (17.9%) 1 (7.1%) 6 (24%)
ECOG
0 27 (69.2%) 9 (64.3%) 18 (72%) 0.7232
1 12 (30.8%) 5 (35.7%) 7 (28%)
HIV positivity
No 38 (97.4%) 14 (100%) 24 (96%) 1
Yes 1 (2.6%) 0 (0%) 1 (4%)
T
Missing (or x) 8 3 5
0 1 (3.2%) 1 (9.1%) 0 (0%) 0.0590
1 1 (3.2%) 0 (0%) 1 (5%)
2 13 (41.9%) 3 (27.3%) 10 (50%)
3 7 (22.6%) 1 (9.1%) 6 (30%)
4 9 (29%) 6 (54.5%) 3 (15%)
N
Missing (or x) 11 5 6
0 8 (28.6%) 3 (33.3%) 5 (26.3%) 0.4424
1 5 (17.9%) 0 (0%) 5 (26.3%)
2 5 (17.9%) 2 (22.2%) 3 (15.8%)
3 10 (35.7%) 4 (44.4%) 6 (31.6%)
M
Missing 8 3 5
0 21 (67.7%) 6 (54.5%) 15 (75%) 0.4232
1 10 (32.3%) 5 (45.5%) 5 (25%)
RTCT
No 15 (38.5%) 8 (57.1%) 7 (28%) 0.0727
Yes 24 (61.5%) 6 (42.9%) 18 (72%)
Surgery
No 35 (89.7%) 13 (92.9%) 22 (88%) 1
Yes 4 (10.3%) 1 (7.1%) 3 (12%)
Stage
Locally advanced 4 (10.3%) 1 (7.1%) 3 (12%) 0.0351
Synchronous metastasis 12 (30.8%) 8 (57.1%) 4 (16%)
Metachronous metastasis 23 (59%) 5 (35.7%) 18 (72%)
Invaded sites
Mean (std) 2.4 (1.3) 2.1 (0.9) 2.6 (1.4)
Median (min-max) 2 (1-5) 2 (1-4) 2 (1-5) 0.3314
Q1-Q3 01-mars 01-mars 01-mars
Invaded sites

(Continued)

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Debernardi et al. 10.3389/fonc.2022.941676

TABLE 6 Continued

Overall population n=39 Episomal n=14 Integrated/mixed n=25 p-value

1 11 (28.2%) 4 (28.6%) 7 (28%) 0.5195

2 12 (30.8%) 6 (42.9%) 6 (24%)
3 9 (23.1%) 3 (21.4%) 6 (24%)
≥4 7 (17.9%) 1 (7.1%) 6 (24%)
Best L1 response
Complete response 19 (48.7%) 8 (57.1%) 11 (44%) 0.4310
Partial response 18 (46.2%) 5 (35.7%) 13 (52%)
Stability 1 (2.6%) 0 (0%) 1 (4%)
Progression 1 (2.6%) 1 (7.1%) 0 (0%)

A B

FIGURE 1
Kaplan-Meier diagrams representing (A) PFS and (B) OS according to the integration of HPV genome. Blue line symbolizes integrated/mixed
forms, black line episomal forms.

A B

FIGURE 2
TERT promoter mutation distribution in human SCCA from the HPV02 and EDITH V cohorts (444 samples). (A) Wild-type (WT) TERT promoter
prevalence versus C228T, C228A and C250T mutations. (B) Distribution of HPV status according to TERT promoter mutations.

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Debernardi et al. 10.3389/fonc.2022.941676

TABLE 7 Summary of somatic mutations retrieved among SCCA from the HPV02 cohort.

Gene Exon Alteration Protein Occurrence

PIK3CA Amplification n=10

10 c.1633G>A p.Glu545Lys n=6
ERBB2 Amplification n=3
17 c.1960-1963delATCAinsGTCG p.Ile654_Ile655delinsValVal n=3
CDK4 Amplification n=5
HRAS Amplification n=3
3 c.121C>T p.Arg41Trp n=1
3 c.194G>T p.Ser65Ile n=1
MYOD1 Amplification n=4
1 c.122G>T p.Arg41Leu n=1
CDKN2A Amplification n=4
TERT 0 c.-143C>T (C247T) n=1
0 c.-124G>A (C228T) n=1
EGFR Amplification n=2
FBXW7 10 c.1513C>G p.Arg505Gly n=1
11 c.1805C>T p.Thr602Ile n=1
FGFR1 Amplification n=2
FGFR2 Amplification n=2
FGFR3 Amplification n=2
CTNNB1 3 c.110C>T p.Ser37Phe n=1
GNA11 4 c.600C>G P.Ile200Met n=1
NRAS Amplification n=1
RAF1 Amplification n=1

integration status and oncogenic-related alterations in mechanisms of viral integration into host genome have been
this population. described in the literature (29) but, most often, HPV integration
In our study, a majority of SCCA was HPV positive, mostly involves a break in the E2 gene, leading to the overexpression of
HPV16 which is the most frequent high risk type in this disease E6 et E7. Integrated/mixed forms were approximately twice
(24–26). Although HPV-negative SCCA are well-known for more frequent (51% vs 28.6%) in our study compared to
being associated with a poor outcome compared to their HPV- episomal forms. Of note, the percentage of “pure” episomal
positive counterparts (24–26), no significant difference of PFS HPV DNA is likely to be slightly overestimated in this study
and OS between HPV-positive and -negative SCCA was given that, in a minority of cases, HPV could be integrated in
observed in the present study, very likely due to the weak host genome without E2 disruption (these latter are actually only
number (n=2) of HPV-unrelated samples contained in the detectable by sequencing) (30). Despite this bias, the percentage
present cohort. of integrated/mixed forms in our study composed of relapsing
Integration of HPV genome into the host genome has been and metastatic SCCA was similar to what can be observed in
shown to be correlated with disease progression in the context of localized HPV-associated cancers (27). The better PFS observed
both anal and cervical (pre-)cancers (21). However, it is in the case of patients harboring episomal HPV DNA raises the
important to notice that all HPV-mediated (pre-)cancers do hypothesis of a predictive impact of HPV integration status on
not contain integrated forms of HPV. Indeed, while episomal DCF efficacy in advanced SCCA. Further investigations should,
HPV DNA is observed in the large majority (>90%) of low-grade however, be undertaken to confirm this observation.
squamous intraepithelial lesion, it can also be detected in up to Telomeres are nucleoprotein complexes playing a critical role
70% of high-grade intraepithelial lesions and invasive carcinoma in chromosome stability. Loss of telomere functions results in
(depending on both tumor stage and anatomical site) (27). E6 genetic instability and impairs cell viability. Telomeric complexes
and E7 can be expressed in this case via the inhibition of the also participate to chromosome repair. Then, telomere
binding between E2 and its binding sites (E2BS) at the viral nucleoprotein dysfunctions impaired DNA break repair
promoter due to the methylation of said E2BS (28). Several capacities conferring to primary or cancer cells an enhanced

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Debernardi et al. 10.3389/fonc.2022.941676

FIGURE 3
Heatmap of gene alteration frequency clustered by PFS (whether it reaches 12 months or not). The type of alterations is described in the
heatmap legend, with the list of genes on the right, and PFS of patients is found at the bottom of the heatmap.

sensitivity to ionizing radiation (31). The telomerase is the enzyme oligodendroglioma (70.0%), medulloblastoma (33.3%) and
reconstituting telomeres and is constituted of several subunits in hepatocellular carcinoma (31.4%) (33). Of note, these mutations
which the catalytic reverse transcriptase TERT is essential to were not detected in most gastrointestinal cancers including
telomerase activity, conferring in fine cellular immortalization gastric, cholangiocarcinoma and pancreatic cancers. The C228T
by preventing replicative senescence. TERT promoter mutations and C250T mutations were the most frequent in TERT promoter
are correlated to increased TERT expression and a worse [77.5% and 20.8% in glioma respectively (34)], inducing a
prognosis as confirmed by a recent meta-analysis in glioma consensus sequence bound by E-Twenty-Six (ETS) transcription
patients (32). TERT promoter mutations have been previously factors (35). A similar distribution was observed in our study in
identified in glioblastoma (84%), urothelial carcinoma (64.5%), TERT mutated SCCA wherein C228T and C250T were detected in

TABLE 8 Patient characteristics according to time of PFS.

PFS<12 months PFS>12 months p-value

n=29 n=20 0.1055

Sexe
Female n=23 n=18 0.4446
Male n=6 n=2
HPV status
HPV16 n=27 n=18 1
Other HPV and HPV negative n=2 n=2
Integration status
Episomal n=6 n=8 0.0952
Integrated/mixed n=18 n=7
NR or HPV negative n=5 n=5
TERT status
WT n=28 n=19 1
Mutated n=1 n=1
PIK3CA status
WT n=19 n=16 0.3444
Altered n=10 n=4

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Debernardi et al. 10.3389/fonc.2022.941676

FIGURE 4
OS in presence of PIK3CA alteration. Blue line symbolizes no alteration, black line symbolizes the presence of a mutation and/or amplification.

73% and 23.3% of the cases. However, TERT promoter mutation were not associated with PFS and OS in our study, we observed a
distribution remains scarce in SCCA, even in HPV negative cases high number of gene amplifications (39 amplifications on tumors
where the absence of immortalization by E6 viral oncoprotein of 23 patients), mostly in PIK3CA. Gene amplification could be a
could have had an impact on mutational frequency of TERT consequence of HPV integration into the host genome, as shown
promoter. Therefore, TERT promoter mutations are not elsewhere (45, 46), which could explain the high proportion of this
correlated to HPV status, type, nor disease progression. As the alteration in the present study. Indeed, as HPV genome is
EDITH V cohort, composed of localized SCCA, presented the integrated into host genome, regions flanking the viral genome
same profile of TERT promoter mutation than the HPV02 cohort are amplified at the same time as HPV genes in a rolling circle
composed of metastatic SCCA, TERT promoter mutations also do manner (47, 48). Six out of ten patients with a PIK3CA
not appear to be associated to SCCA status. amplification presented integrated HPV forms, which was not
Furthermore, when analyzing somatic mutations in other statistically different, whereas patients with CDK4 and CDKN2A
genes, it appeared that SCCA were not highly mutated, the amplifications presented integrated HPV forms in 5/5 and 3/4
most frequently mutated being PIK3CA. This result was similar cases respectively (p-values: 0.0079 and 0.485 respectively). The
to others (36–41). In general, most common PIK3CA mutations fact that HPV integration is sometimes located in or near
are E542K and E545K, with 75% for the last one (37, 39). In our amplification regions has been shown for other genes, like MYC
study we found 6 mutations in PIK3CA, all being E545K (45), which is in favor of the impact of viral integration, in late
(1633G>A), which is mostly linked to an APOBEC stages of SCCA, on oncogenesis by deregulating oncogenes and
(Apolipoprotein B mRNA editing enzyme catalytic polypeptide- mostly amplifying pro-oncogenes.
like) alteration (42). As most substitutions (60%, n=6/10) The main limitation of our study is its small sample size, and the
retrieved in our study for all genes were C>T/G>A, it is possible HPV integration status analysis is based in 39 patients. However, to
that some of them may in part be due to the activity of APOBEC, date, this clinical situation is in the scope of active clinical and
creating “passenger mutations” opposed to “driver” to the translational research, and several prospective trials are ongoing (49).
oncogenesis. Indeed, it has been shown in Head and Neck More robust confirmatory data are awaited in the near future.
Squamous Cell Carcinoma (HNSCC) that APOBEC activity and Targeted therapy for patients with HPV-associated cancers
mutations are concordant between viral genome and host cell resisting to standard treatments are ongoing. Bevacizumab (anti-
genome (43). We also found no TP53 mutations in HPV-positive VEGF antibody) and pembrolizumab (anti-PD1 antibody) were
SCCA, as opposed to HPV-negative SCCA (44). Even if mutations approved in progressive and metastatic cervical cancers (50),

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Debernardi et al. 10.3389/fonc.2022.941676

and were also evaluated in SCCA, as well as other anti-PD1 Funding

antibodies (nivolumab, retifanlimab) (51), and showed a
promising result in a subgroup of patients (52–54). Part of this work was funded by the Ré gion Franche-Comté
In conclusion, as HPV status, integration status, TERT and the Belgian Fund for Scientific Research (FNRS;
promoter mutations, and mutational profiling are not MIS F.4520.20).
significantly correlated to PFS and/or OS in this study, DCF
chemotherapy should be proposed to all SCCA patients failing
radio-chemotherapy (CRT) or with metastatic disease. Further Acknowledgments
investigations are required to identify SCCA-related
predictive biomarkers. The authors would like to thank Guadalupe Iné s Tizó n for
English writing assistance. We also thank the biobank
‘Tumorothèque Ré gionale de Franche-Comté ’ (registration
Data availability statement number BB-0033-00024) for biobanking.

The raw data supporting the conclusions of this article will

be made available by the authors, without undue reservation.
Conflict of interest
CB has received a research grant from companies Bayer and
Ethics statement
Roche, and was an advisory board member of Bayer, MSD and
Pierre Fabre companies. None of them had a role in the study
The studies involving human participants were reviewed and
design, analysis, or interpretation of the results.
approved by The French ethic committee (NCT02402842). The
The remaining authors declare that the research was
patients/participants provided their written informed consent to
conducted in the absence of any commercial or financial
participate in this study.
relationships that could be construed as a potential conflict
of interest.
Author contributions
CB, SK, J-LP and DG contributed to conception of the whole Publisher’s note
study or a part of it, and CB coordinated the clinical trial. AD,
DG and PR performed the experiments and CB, AD, MH and All claims expressed in this article are solely those of the
AM performed data analysis and/or statistical analysis. AD authors and do not necessarily represent those of their affiliated
wrote the first draft of the manuscript and CB, SK, AM and organizations, or those of the publisher, the editors and the
MH wrote sections of the manuscript. FM, LS, AV, AT, AL, reviewers. Any product that may be evaluated in this article, or
ChlM and ChrM allowed data collection. All authors claim that may be made by its manufacturer, is not guaranteed
contributed to the article and approved the submitted version. or endorsed by the publisher.

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