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Detection of KMT2A-PTDs in healthy donor and myeloid malignant samples using next generation sequencing
Jiajie Huanga, Haigang Gu b, Janet Orton b, Marina Sedovaa, Amir Marcovitza, Jennifer Burkea, Sarah Brozio a, Paul Williamsc, Scott Myrand c, Nate Olowo a, Adam Broomerd, Brendan Deala, Collyn Seegere, Seth Sadisc, Sophie Rozenzhakd, Fiona Hyland a, Guang Liu b
a: Thermo Fisher ScientificTM , South San Francisco, CA; b: Sonora Quest Laboratories, Phoenix, AZ; c: Thermo Fisher ScientificTM , Ann Arbor, MI; d: Thermo Fisher ScientificTM , Carlsbad, CA; e: Thermo Fisher ScientificTM , Guilford, CT

INTRODUCTION MATERIALS AND METHODS RESULTS Fig 5. IGV view of KMT2A-KMT2A.K9K2 in healthy donor samples vs myeloid samples
KMT2A (MLL) fusions and KMT2A-PTD (partial tandem duplication) are vital biomarkers in We sequenced 8483 research samples with known myeloid malignancies in both Sonora The mean read length of this data set is 90 – 120 bp and the mapped fusion reads is 20,000 –
myeloid malignancies traditionally detected by RT-qPCR (quantitative real-time PCR). This Quest Laboratories TM and at Thermo Fisher Scientific TM South San Francisco site. We 30,000. KMT2A-PTDs were detected in both healthy donors and myeloid samples. Healthy (a)
study utilizes next generation sequencing (NGS) with Oncomine TM Myeloid Assay GX v2 to also acquired 20 healthy donor whole blood samples (total 127 replicates) from Stanford TM donor PTD read counts were consistently <2000 and averaged 1/3 of myeloid samples. About
report the detection of KMT2A-PTDs in both healthy donors and myeloid malignancy Blood Center and Discovery Life Sciences TM and sequenced them at 3 different sites of 33% of myeloid samples had higher PTD read counts than any healthy donor sample. BLAT
Healthy donor sample #H1
samples. KMT2A fusions in myeloid malignant samples are also reported. Thermo Fisher Scientific TM (South San Francisco, CA; Guilford, CT; Carlsbad, CA). (BLAST TM–like Alignment Tool) analysis confirmed specific exon matching on the KMT2A gene KMT2A-KMT2A.K9K2 = 514 reads
Samples were processed on the Ion TorrentTM GenexusTM Software 6.6 and analyzed using in both cohorts. Among the 8503 myeloid samples, 162 contained a total of 5 unique KMT2A
Fig 1. Estimated new cases (%) of Leukemia, Lymphoma and Myeloma in USA, 2021 the Oncomine TM Myeloid Assay GX v2 for fusion profiling targeting 6 different KMT2A-PTD PTDs, and 105 contained a total of 30 unique KMT2A fusion isoforms with KMT2A-MLLT1 and
Healthy donor sample #H2
variants and 199 KMT2A fusion isoforms. KMT2A-MLLT3 being the most prevalent KMT2A fusion gene pairs. KMT2A-KMT2A.K9K2 = 796 reads

Total cases: 186,400 Table 1. Oncomine TM Myeloid Assay GX v2 Panel Table 4. KMT2A fusions & PTDs existed in ~3% samples
Found in # of unique Found in % of all samples
DNA Panel RNA Panel Gene Pair Myeloid malignant sample #M1
samples (N=8503) KMT2A-KMT2A.K9K2 = 2659 reads
KMT2A-AFF4 2 0.02%
M yelom a , 19% Expression
Hotspot genes Full genes Fusion Driver Expression Myeloid malignant sample #M2
34,920 control KMT2A-CASC5 2 0.02% KMT2A-KMT2A.K9K2 = 6394 reads
(28) (17) Genes (30) genes (5)
Lym phom a , 48% genes (5)
cases KMT2A-CBL 4 0.05%
90,390 A N K R D 26 KRAS A S X L1 P R P F8 A B L1 MECOM BAALC EIF2B1 KMT2A-ELL 11 0.13%
cases A B L1 MPL BCOR RB1 A B L2 MET
MECOM FBXW2 KMT2A-EPS15 4 0.05% (b)
BRAF M Y D 88 C A LR RUNX1 B C L2 M LLT10 KMT2A fusions
CBL NPM1 C E B PA S H 2B 3 BRAF M RTFA
MYC PSMB2 KMT2A-MLLT1 30 0.35%
C S F3R NRAS E TV 6 S TA G 2 CCND1 (M K L1) SMC1A PUM1
WT1 TRIM27 KMT2A-MLLT10 9 0.11% On IGV view (Fig 5(a)), the
61,090 D D X 41 P P M 1D E ZH 2 TE T2 CREBBP M Y B L1
Leukem ia, 33% D N M T3A P TP N 11 IK ZF1 TP 53 E G FR E TV 6 M Y H 11 alignment of KMT2A-
cases FLT3 (ITD S M C 1A N F1 ZR S R 2 FG FR 1 N TR K 2
KMT2A-MLLT3 29 0.34%
KMT2A.K9K2 looks clean with
+ TK D ) SMC3 P H F6 FG FR 2 FU S N TR K 3 KMT2A-MLLT4 14 0.16%
some insertions and
G ATA 2 S E TB P 1 HMGA2 N U P 214 Total 105 1.23%
HRAS S F3B 1 JA K 2 N U P 98 mismatches in both healthy
ID H 1 S R S F2 K AT6A PA X 5 KMT2A-KMT2A 162 1.91% donor and myeloid malignant
KMT2A-PTDs
Source: Cancer Facts & Figures 2021. American Cancer Society, 2021. ID H 2 U 2A F1 (M O Z) P D G FR A Total 162 1.91% samples. This is confirmed by
JA K 2 W T1 K AT6B P D G FR B BLAT (Fig 5(b)). One insertion
K IT K M T2A (M L RARA
Samples w/ ≥ 1 (c)
L) RUNX1 KMT2A fusion or Total 265 3.12% of 18 bps is observed at the
Fig 2. Oncomine TM Myeloid assay enables rapid and efficient multi-biomarker testing K M T2A - TC F3 PTD break point of KMT2A-
P TD s (M LL- TFE 3 ~2% samples had at least one KMT2A-PTD, ~1% samples had at least one KMT2A fusion. KMT2A.K9K2. BLAT shows
One test for All key biomarkers across Multiple samples P TD s) ZN F384 that it is from intron 1 (Fig 5(c)).
PML-RARA RUNX1
Fig 5. Read count differentiates myeloid cancer PTDs
FLT3-ITD BCR-ABL
CALR JAK2
Fig 4. KMT2A-PTDs: a relevant biomarker in myeloid malignancies
KIT IDH1
TP53 IDH2 1 2 3 4 5 6 7 8 9 10 11 KMT2A WT
NPM1 MPL
ASXL1 KMT2A 1 2 3 4 5 6 7 8 2 3 4 5 6 7 8 9 10 11

KMT2A-PTD CONCLUSIONS
Translocations
Mutations (DNA)
(RNA)
In this study, we describe the detection of KMT2A fusions in myeloid malignant samples. Our
study also describes the detection of KMT2A-PTDs in both healthy donor and myeloid
Table 2. Oncomine TM Myeloid assay is designed to detect six KMT2A-PTD variants samples, with myeloid cases showing significantly higher PTD read counts. additional studies
Full mutational profile in 2 days Free up time for lab techs and bioinformaticians to understand the relevant expression level of PTD are in progress. This intriguing finding
Variant Length (bp) opens opportunities for prospective studies to monitor individuals with elevated PTD levels for
Consolidate testing of individual biomarkers Reduce the need for NGS expertise n = 162
KMT2A-KMT2A.nt51.K11K2 178 myeloid malignancy development and retrospective studies to explore whether healthy donors
KMT2A-KMT2A.K9K2 172 identified with this alteration years ago after blood donation were subsequently recorded in the
KMT2A-KMT2A.K7K2 169 national health system with myeloid malignancies.
KMT2A-KMT2A.K11K2 116
KMT2A-KMT2A.K8K2 105 n = 127 ACKNOWLEDGEMENTS
KMT2A-KMT2A.K10K2 105
Fig 3. Genexus Instruments Thank you to all the individuals involved in the development of the Oncomine TM Myeloid
Assays.
Table 3. Report only the PTDs associated with myeloid malignancy with high confidence
Example: KMT2A-PTDs @ Genexus software 6.6 OMAv2 w4.3.2 (a past release)
1 2 Sample name Isoforms detected Read count DISCLAIMER
Healthy donor sample #A KMT2A-KMT2A.K8K2 9 For Research Use Only. Not for use in diagnostic procedures. © 2024 Thermo Fisher Scientific
KMT2A-KMT2A.K10K2 147 Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its
KMT2A-KMT2A.K7K2 201 REFERENCES subsidiaries unless otherwise specified. Sonora Quest Laboratories is a trademark of Sonora
KMT2A-KMT2A.K9K2 343 Schnittger S, Wörmann B, Hiddemann W, Griesinger F. Partial tandem duplications of the MLL Quest Laboratories, LLC. Discovery Life Sciences is a trademark of Discovery Life Sciences,
gene are detectable in peripheral blood and bone marrow of nearly all healthy donors. Blood. LLC. BLAST is a trademark of The National Library of Medicine.
Myeloid malignant sample #B KMT2A-KMT2A.K10K2 551 1998 Sep 1;92(5):1728-34. PMID: 9716602.
KMT2A-KMT2A.K7K2 722 Dai B, Yu H, Ma T, Lei Y, Wang J, Zhang Y, Lu J, Yan H, Jiang L, Chen B. The Application of
KMT2A-KMT2A.K8K2 311 Targeted RNA Sequencing for KMT2A-Partial Tandem Duplication Identification and Integrated
KMT2A-KMT2A.K9K2 2466
Is there a differentiation in PTD read count in myeloid malignant vs healthy donor samples? Analysis of Molecular Characterization in Acute Myeloid Leukemia. J Mol Diagn. 2021
Nov;23(11):1478-1490. doi: 10.1016/j.jmoldx.2021.07.019. Epub 2021 Aug 10. PMID: 34384895.

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A comprehensive genomic profiling of myeloid malignancies demonstrates mutational spectrum of DNA
variants, FLT3-ITDs, and gene fusions
Jiajie Huanga, Haigang Gub, Janet Ortonb, Marina Sedovaa, Scott Myrandc, Sophie Rozenzhakd, Fiona Hylanda, Guang Liub
a: Thermo Fisher ScientificTM, South San Francisco, CA; b: Sonora Quest LaboratoriesTM, Phoenix, AZ; c: Thermo Fisher ScientificTM, Ann Arbor, MI; d: Thermo Fisher ScientificTM, Carlsbad, CA

INTRODUCTION MATERIALS AND METHODS RESULTS Fig 4. DNA variant allele frequency (VAF) by gene Fig 7. % samples with different fusion drivers, gene pairs, and isoforms
Myeloid malignancies encompass diverse hematopoietic disorders, including acute myeloid Here we describe the genomic profiles of 8503 research samples (including AML, MPN, MDS,
leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), CML, CMML, or JMML). A total of 4723 samples were run on the Ion GeneStudioTM S5 System
chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile and analyzed using the OncomineTM Myeloid Research workflow on Ion ReporterTM 5.18. A total
myelomonocytic leukemia (JMML). We developed two research assays to address the of 3780 samples were run on the Ion TorrentTM Genexus Software 6.6 and analyzed using the
genetic complexity of myeloid malignancies, OncomineTM Myeloid Assay and OncomineTM OncomineTM Myeloid Assay GX v2 (Table 3).
Myeloid Assay GX v2 (Table 1), detecting mutations in 45 DNA genes and >30 fusion driver
genes, including >700 fusion isoforms. Our panel includes genetic alterations such as Table 3. Data set sequenced on Genexus and GeneStudio platforms
FLT3-Internal Tandem Duplications (ITDs), IDH1/2, CEBPA, CALR, and TP53. Sonora Quest # samples w/ # samples w/ # samples w/
# samples total
Laboratories DNA + Fusions DNA only Fusions only
Table 1. Oncomine Myeloid Assay GX v2 Panel Targets
GeneStudio +
Expression 4695 23 5 4723
Hotspot genes Full genes Fusion Driver Expression Ion Reporter
control
(28) (17) Genes (30) genes (5) Genexus 3780 0 0 3780
genes (5)

ANKRD26​ KRAS ASXL1 PRPF8 ABL1 MECOM BAALC EIF2B1 Both systems 8475 23 5 8503
ABL1 MPL BCOR RB1 ABL2 MET MECOM FBXW2 Fig 5. Fusion read count by driver gene
BRAF MYD88 CALR RUNX1 BCL2 MLLT10 MYC PSMB2
Fig 2. OncoPrint: visualize SNVs, InDels, & Fusions by heatmap
CBL NPM1 CEBP SH2B3 BRAF MRTFA SMC1A PUM1
mutations by
CSF3R NRAS A STAG2 CCND1 (MKL1) WT1 TRIM27
Fraction of

sample Fraction of
DDX41​ PPM1D ETV6 TET2 CREBBP MYBL1 mutations by
gene
DNMT3A PTPN11 EZH2 TP53 EGFR MYH11
FLT3 (ITD SMC1A​ IKZF1 ZRSR2 ETV6 NTRK2 Fig 3. Mutation spectrum of FLT3-ITDs
+ TKD) SMC3 NF1 FGFR1 NTRK3
GATA2 SETBP1 PHF6 FGFR2 NUP214
HRAS SF3B1 FUS NUP98
IDH1 SRSF2 HMGA2 PAX5
IDH2 U2AF1 JAK2 PDGFRA
JAK2 WT1 KAT6A PDGFRB
KIT (MOZ) RARA
KAT6B RUNX1 Fig 8. Interaction of
KMT2A ( TCF3 Fig 6. Co-occurring gene pairs DNA small variants and Table 4. frequency of fusion drivers
MLL) TFE3 fusions – not seen
KMT2A- ZNF384 Gene 1 Gene 2 Co-occurrence % of all co-occurences Driver # samples % samples
PTDs ASXL1 TET2 259 1.06% ABL1 432 60.85%
(MLL- SRSF2 TET2 248 1.01% KMT2A 171 24.08%
PTDs) BCR ABL1 238 0.97% MYH11 126 17.75%
RARA 117 16.48%
RESULTS RUNX1 95 13.38%
% samples On the Genexus system, the turnaround time (the time between starting the run and NGS data NUP98 32 4.51%
Table 2. Frequency of relevant mutations
Gene # samples (N=4797) report) was 23-25 hours and the hands-on time was around 1 hour. The success rate of the MLLT10 30 4.23%
TET2 1068 22.26% samples was 100%. ETV6 24 3.38%
ASXL1 787 16.41% Fig 1. Abundance of mutations in samples NUP214 12 1.69%
DNMT3A 665 13.86% Frequency of relevant mutations and variant allele frequency by gene JAK2 11 1.55%
TP53 640 13.34% Overall, 4797 of 8503 (56%) samples had a mutation in at least one gene in the panel (Table 2 MKL1 8 1.13%
SRSF2 587 12.24% and Fig 1). Mutation refers to Oncomine variants (somatic SNVs, InDels, and Fusions). Within MECOM 6 0.85%
JAK2 562 11.72% the samples that had any mutation, the average number of mutations per samples was 2.3, and FGFR1 3 0.42%
RUNX1 446 9.30% the highest percentage of detection was TET2 (22%) (Fig 2). Common co-occurring mutations HMGA2 2 0.28%
SF3B1 423 8.82% included ASXL1-TET2 and SRSF2-TET2 (Fig 6). Variant allele frequency varied greatly, e.g.,
TCF3 2 0.28%
IDH2 337 7.03% ANKRD26 had a median VAF of 66%, while MYD88 had only 7% (Fig 4).
KAT6A 1 0.14%
NRAS 318 6.63%
FLT3 292 6.09% Mutation spectrum of FLT3ITD variants
STAG2 278 5.80% FLT3-ITD was observed in 2% of samples, with an average VAF of 32%, featuring a multi-modal CONCLUSIONS
ABL1 273 5.69% length with the highest peak at around 34 bp and maximum length at 151 bp (Fig 3). The Oncomine Myeloid Assay is a fast, robust, and reproducible solution for comprehensive
KMT2A 265 5.52% genomic profiling of myeloid malignancies. We describe the mutational spectrum of DNA
BCR 238 4.96% Mutation spectrums of fusions variants and RNA fusions in a range of research samples.
BCOR 229 4.77% 710 (8.4%) samples were fusion-positive with read counts ranging 21-113,000. ABL1 was the
U2AF1 224 4.67% most common driver and BCR-ABL1 was the most common gene pair. Other common drivers DISCLAIMER
NPM1 201 4.19% included KMT2A, MYH11, RARA, and RUNX1 (Table 4 and Fig 5). 17 distinct driver genes, 49 For Research Use Only. Not for use in
PPM1D 186 3.88% gene pairs, and 110 gene isoforms were observed, including rare fusions like ZMYM2-FGFR1 diagnostic procedures. © 2024 Thermo
NF1 and KAT6A-CREBBP that are not detectable by traditional methods like qPCR (quantitative Fisher Scientific Inc. All rights reserved. All
183 3.81%
polymerase chain reaction) or FISH (fluorescence in-situ hybridization) (Fig 7). No significant trademarks are the property of Thermo
IDH1 170 3.54%
difference in # DNA small variants was detected in fusion group vs no-fusion group. Fisher Scientific and its subsidiaries unless
KRAS 154 3.21%
otherwise specified. Sonora Quest
SETBP1 151 3.15%
Laboratories is a trademark of Sonora Quest
ETV6 107 2.23%
Laboratories LLC.

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