High-Throughput Characterization of Cytogenomic Heterogeneity of MDS Using High-Resolution Optical Genome Mapping

Introduction Introduction of next-generation sequencing has defined the somatic mutational landscape in MDS. Comprehensive high-throughput structural variant profiling (SVP) is as important as mutation profiling in characterizing MDS clonal architecture since these large genomic aberrations have already shown to be critical for diagnosis and risk-stratification of MDS. A subset (MECOM, KMT2A rearrangements) are therapeutic targets in clinical trials. At this time, technical advances in SVP for copy number alterations (CNAs) and fusions have not been congruent with mutation profiling due to the inability of short-read (150bp) NGS to detect SVs. Currently available long-read (10-20Kbp) and whole genome sequencing cannot detect all SVs due to the presence of repeat sequences. Hence, conventional karyotyping (CK) remains the gold standard. Optical genome mapping (OGM) is a novel single-platform technique that measures ultra-long-range sequence patterns (>300Kbp), thereby unaffected by repeat sequences, enabling unbiased evaluation of all types of SVs at a high resolution. Here, we performed comprehensive SVP and mutation profiling in a large well-characterized cohort of MDS. Methods We selected samples with available fresh/frozen BM cells from consecutive treatment-naïve MDS pts who also underwent standard-of-care tests (CK, FISH, targeted 81-gene NGS for mutations). For OGM, ultra-high-molecular-weight-DNA was extracted, followed by labeling, linearization and imaging of DNA (Saphyr, Bionano) [median coverage:>300X]. The results were analyzed using de novo (>500 bp), rare variant (>5000 bp) and copy number (>500,000 bp) pipelines. The data was compared against 200 healthy controls to exclude common germline SVs. Clinical significance of the SVs was determined based on the location/overlap with the coding region of myeloid malignancy associated genes. The detection sensitivity was 10%. Results There were 76 treatment naïve MDS patients. Baseline characteristics, comprehensive cytogenetic scoring system (CCSS) and R-IPPS risk categories and somatic mutations are in Fig 1. OGM identified all clonal abnormalities detected by CK [CNAs, inversions, inter/intra-chromosomal translocations, dicentric, complex derivative chromosomes]. Precise mapping of SVs by OGM at gene-level allowed determining the status of clinically informative biomarkers such as TET2, MECOM, TP53 and KMT2A, without the need for confirmatory assays. Detailed gene-level characterization of different SVs included KMT2A-ELL [t(11;19)] in MDS with WT1 mut, t(9;11) with SYTL2 fusion (and not KMT2A), der(1;7) leading to del(7q) in MDS with GATA2 mut/IDH2 mutand t(1;3)(p36;q21) rearrangements with potential PRDM16 disruption in SF3B1 mut/RUNX1 mutMDS, among others. Using OGM, we mapped the sequence patterns in both samples with IM with high level of confidence. Additionally, OGM identified 23 cryptic, clinically significant SVs in 14 (18%) of 76 pts. These included deletions of TET2, KMT2A, and del(5q), KMT2A amplification in MDS with FLT3-ITD/DNMT3A mut/RAS mut, NUP98-PRRX2, MECOM rearrangement in TET mut mutated NK-MDS. In addition, there were SVs of uncertain significance: duplications of chr1 (PDE41P), deletions of chr21 (involving RUNX1), chr2 (DNMT3A, ASXL2), chr12 (ETV6) and chr22 (EP300) and der(16)t(12;16)(q21.1;q12.1). These cryptic SVs were noted across all R-IPSS risk categories (highest yield in very-low and low R-IPSS) and across all cytogenetic risk-groups (very-good to very-poor). In complex karyotype setting, OGM could resolve the markers and additional genetic material, and in most cases, showed a much higher the degree of complexity within the genome than was apparent by CK. Four pts showed SV patterns typical of chromothripsis/chromoplexy. The median number of mutations per pt was 1 (0-6). When compared to mutation subsets, cryptic SVs were only identified in pts with ≤3 mutations. Majority represented either MDS with TP53 mut (6, 29%) or SF3B1 mut/TET mut (deletions of TET2, KMT2A, NOTCH1 and EP300 genes). Conclusions Unbiased, high-throughput whole genome SVP revealed cryptic, clinically significant SVs in ~18% of MDS pts. OGM is a single-platform cytogenomic tool that can facilitate SVP at a gene-level resolution. This study provides strong support for further validation in expanded cohorts to guide clinical implementation and integration of SVP for routine work-up. Figure 1 Figure 1. Disclosures Wei: Daiichi Sanko: Research Funding. Kantarjian: Ipsen Pharmaceuticals: Honoraria; Amgen: Honoraria, Research Funding; Astellas Health: Honoraria; Astra Zeneca: Honoraria; AbbVie: Honoraria, Research Funding; KAHR Medical Ltd: Honoraria; NOVA Research: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Diverse ‘just-right’ levels of chromosomal instability and their clinical implications in neoadjuvant treated gastric cancer

Background The Cancer Genome Atlas (TCGA) consortium described EBV positivity(+), high microsatellite instability (MSI-H), genomic stability (GS) and chromosomal instability (CIN) as molecular subtypes in gastric carcinomas (GC). We investigated the predictive and prognostic value of these subtypes with emphasis on CIN in the context of neoadjuvant chemotherapy (CTx) in GC. Methods TCGA subgroups were determined for 612 resected adenocarcinomas of the stomach and gastro-oesophageal junction (291 without, 321 with CTx) and 143 biopsies before CTx. EBV and MSI-H were analysed by standard assays. CIN was detected by multiplex PCRs analysing 22 microsatellite markers. Besides the TCGA classification, CIN was divided into four CIN-subgroups: low, moderate, substantial, high. Mutation profiling was performed for 52 tumours by next-generation sequencing. Results EBV(+) (HR, 0.48; 95% CI, 0.23–1.02), MSI-H (HR, 0.56; 95% CI, 0.35–0.89) and GS (HR, 0.72; 95% CI, 0.45–1.13) were associated with increased survival compared to CIN in the resected tumours. Considering the extended CIN-classification, CIN-substantial was a negative prognostic factor in uni- and multivariable analysis in resected tumours with CTx (each p < 0.05). In biopsies before CTx, CIN-high predicted tumour regression (p = 0.026), but was not prognostically relevant. Conclusion A refined CIN classification reveals tumours with different biological characteristics and potential clinical implications.

Biomarkers in Acute Myeloid Leukemia: Leveraging Next Generation Sequencing Data for Optimal Therapeutic Strategies

Next generation sequencing (NGS) is routinely used for mutation profiling of acute myeloid leukemia. The extensive application of NGS in hematologic malignancies, and its significant association with the outcomes in multiple large cohorts constituted a proof of concept that AML phenotype is driven by underlying mutational signature and is amenable for targeted therapies. These findings urged incorporation of molecular results into the latest World Health Organization (WHO) sub-classification and integration into risk-stratification and treatment guidelines by the European Leukemia Net. NGS mutation profiling provides a large amount of information that guides diagnosis and management, dependent on the type and number of gene mutations, variant allele frequency and amenability to targeted therapeutics. Hence, molecular mutational profiling is an integral component for work-up of AML and multiple leukemic entities. In addition, there is a vast amount of informative data that can be obtained from routine clinical NGS sequencing beyond diagnosis, prognostication and therapeutic targeting. These include identification of evidence regarding the ontogeny of the disease, underlying germline predisposition and clonal hematopoiesis, serial monitoring to assess the effectiveness of therapy and resistance mutations, which have broader implications for management. In this review, using a few prototypic genes in AML, we will summarize the clinical applications of NGS generated data for optimal AML management, with emphasis on the recently described entities and Food and Drug Administration approved target therapies.

Stage 4 pancreatic adenocarcinoma harbouring an FGFR2-TACC2 fusion mutation with complete response to erdafitinib a pan-fibroblastic growth factor receptor inhibitor

We report a case of a frail 68-year-old woman with stage 4 pancreatic carcinoma harbouring a fibroblastic growth factor receptor 2 (FGFR2) fusion who achieved a durable complete response after treatment with erdafitinib a pan-FGFR inhibitor. The FGFR2-TACC2 fusion was detected on comprehensive tumour somatic mutation profiling. There is ongoing complete response at 10 months after initiation of erdafitinib. Transient central serous retinopathy, grade 2 hyperphosphataemia and diarrhoea were the adverse events encountered.

Complementing Tissue Testing with Plasma mutation Profiling Improves Therapeutic Decision Making for Lung Cancer Patients

Background: Tissue biopsy is an integral part of the diagnostic approach to lung cancer. It is however invasive and associated with limitations of tissue heterogeneity. Liquid biopsies may complement tissue testing by providing additional molecular information and may be particularly helpful in patients from whom obtaining sufficient tissue for genomic profiling is challenging. Methods: Patients with suspected lung cancer (n=71) were prospectively recruited. Blood and diagnostic tissue samples were collected within 48 hrs of each other. Plasma cell-free DNA (cfDNA) testing was done using an ultrasensitive amplicon-based next-generation sequencing (NGS) panel (plasma NGS testing). For cases diagnosed as non-small cell lung carcinoma (NSCLC) via histology or cytology, targeted testing for epidermal growth factor receptor (EGFR) mutations was performed using tissue biopsy samples, where available (tissue EGFR testing). Concordance of clinically actionable mutations between methods and sample types were assessed. Results: For confirmed NSCLC cases (n = 54), tissue EGFR test results were available only for 70.3% (38/54) due to sample inadequacies, compared to blood samples for 98.1% (53/54) cases. Tissue EGFR testing identified sensitizing EGFR (L858R or exon 19 deletion) mutation in 31.6% (12/38) of cases. Plasma NGS identified clinically actionable mutations in 37.7% (20/53) of cases, including EGFR mutations in two cases with no tissue EGFR results, and mutations in KRAS, BRAF and MET. Overall sensitivity of EGFR sensitizing mutation detection by plasma NGS was 75% (9/12), and specificity was 100% (25/25) in patients tested in both tissue EGFR and plasma NGS (n=37). In this cohort of patients, tissue EGFR testing alone informed clinical decisions in 22.2% (12/54) of cases. Adding plasma NGS to tissue EGFR testing increased the detection rate of actionable mutations to 42.6% (23/54), representing a near doubling (1.9-fold increase) of clinically relevant findings. The average turnaround time (TAT) of plasma NGS was shorter than standard tissue testing (10 days vs. 29.9 days, p-value <0.05). Conclusions: In the first-line setting, plasma NGS was highly concordant with tissue EGFR testing. Plasma NGS increases the detection of actionable findings with shorter time to results. This study outlines the clinical utility of a complementary plasma mutation profiling in the routine management of lung cancer patients.

Genomic alterations and possible druggable mutations in carcinoma of unknown primary (CUP)

Carcinoma of Unknown Primary (CUP) is a heterogeneous and metastatic disease where the primary site of origin is undetectable. Currently, chemotherapy is the only state-of-art treatment option for CUP patients. The molecular profiling of the tumour, particularly mutation detection, offers a new treatment approach for CUP in a personalized fashion using targeted agents. We analyzed the mutation and copy number alterations profile of 1709 CUP samples deposited in the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) cohort and explored potentially druggable mutations. We identified 52 significant mutated genes (SMGs) among CUP samples, in which 13 (25%) of SMGs were potentially targetable with either drugs are approved for the know primary tumour or undergoing clinical trials. The most variants detected were TP53 (43%), KRAS (19.90%), KMT2D (12.60%), and CDKN2A (10.30%). Additionally, using pan-cancer analysis, we found similar variants of TERT promoter in CUP and NSCLC samples, suggesting that these mutations may serve as a diagnostic marker for identifying the primary tumour in CUP. Taken together, the mutation profiling analysis of the CUP tumours may open a new way of identifying druggable targets and consequently administrating appropriate treatment in a personalized manner.

Correction: Earl et al. Somatic Mutation Profiling in the Liquid Biopsy and Clinical Analysis of Hereditary and Familial Pancreatic Cancer Cases Reveals KRAS Negativity and a Longer Overall Survival. Cancers 2021, 13, 1612

The authors wish to make the following corrections to this paper [...]