Introduction: Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNA) and mutations that are central to disease pathogenesis, prognosis, risk-stratification, and identification of response or resistance to therapies. Fluorescence in situ hybridization (FISH) is gold standard in the clinical laboratory for detecting prognostic CNAs in CLL (e.g. deletion 17p13 (del(17p), deletion 11q23 (del(11q), deletion 13q14 (del(13q), and trisomy 12). Most clinical FISH assays have high specificity and sensitivity, but the technique can detect a limited number of alterations per assay. Importantly, next-generation sequencing (NGS) techniques have become more readily available for clinical applications and are increasingly being used for screening not only mutations, but also copy number abnormalities in multiple genes and chromosomal regions of interest in hematologic malignancies. Here, we evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using a custom targeted NGS assay for detecting common prognostic chromosomal alterations in CLL and high-count monoclonal B-cell lymphocytosis (MBL), the precursor to CLL.

Methods: We designed a SureSelect DNA targeted sequencing panel, covering all exons of 59 recurrently CLL mutated genes and additional amplicons across regions affected by clinically relevant CNAs. All CLL (N=534) and MBL (N=162) patients had pre-treatment peripheral blood mononuclear cells (PBMC) collected within two years of diagnosis. DNA was extracted in cases with purity >80% of CD5+/CD19+ cells. Clinical FISH data was available within 100 days of NGS in all untreated CLL and MBL cases. PatternCNV was used to detect clinically relevant CNAs in chromosomes 11, 12, 13 and 17. We performed a principal component analysis on the CNA calls, excluding chromosomes 11, 12, 13, and 17 to identify clusters of samples. Each cluster was then independently rerun with PatternCNV and the results from chromosomes 11, 12, 13, and 17 were extracted and further analyzed. We excluded samples with low tumor metrics identified by FISH (less than 20% of cells with del(17p), del(11q), trisomy 12 and del(13q)).

Results: We sequenced a total of 696 patients of whom 162 were MBL and 534 were untreated CLL. The most commonly mutated genes were NOTCH1 (11.0%), TP53 (8.7%), SF3B1 (7.7%), ATM (4.1%), and CHD2 (3.8%). Based on CNA analyses from the NGS data, we identified 59 (9.1%) individuals with del(17p), 88 (13.4%) individuals with del(11q), 128 (20.0%) individuals with trisomy 12, and 329 (53.0%) individuals with del(13q). All 696 individuals had FISH panels conducted, with 39 (5.6%) individuals with del(17p), 68 (9.8%) individuals with (11q), 119 (17.1%) with trisomy 12, and 295 (42.4%) with del(13q). When we compared our CNA analyses with the FISH data, we found high concordance 95.0% for del(17p), 92.7% del(11p), 94.3% for trisomy 12, and 88.2% for del(13q). For del(17p) we found a sensitivity of 93.9%, specificity of 95.4%, PPV of 52.5%, and NPV of 99.7%. Del(11q) revealed a sensitivity of 88.1%, specificity of 94.0%, PPV of 59.1%, and NPV 98.8%. We found a sensitivity of 93.8%, specificity of 95.6%, PPV 82.0%, and NPV of 98.6% for trisomy 12 and for del(13q) we found a sensitivity of 92.6%, specificity of 90.9%, PPV of 91.7%, and NPV of 93.8%. We found lower PPVs in del(17p) and del(11q) likely due to lower prevalence of these chromosomal abnormalities.

Conclusion: Here we show a high sensitivity, specificity, and NPV when comparing targeted sequencing with FISH. FISH panel testing is widely used in clinical practice to characterize highly prognostic chromosomal abnormalities in CLL. Comprehensive genetic profiling with NGS has become increasingly important in the work up of hematologic malignancies and provides additional prognostic and predictive information, including clinically relevant mutations such as TP53, SF3B1, and NOTCH1, tumor mutation load and mutations associated with resistance to chemo-immunotherapy and targeted therapies, such as BTK or BCL2 inhibitors, that FISH cannot offer. We show that NGS can infer clinically relevant CNA in cases without FISH testing while also providing additional clinically relevant information.

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Disclosures

Cerhan: Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding; Genentech: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Genentech: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Acerta Pharma: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees.

© 2021 by The American Society of Hematology
2021
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