Universal germline genetic testing in patients with hematologic malignancies using DNA isolated from nail clippings.

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Cell-free DNA from nail clippings as source of normal control for genomic studies in hematologic malignancies.

Comprehensive genomic sequencing is becoming a critical component in the assessment of hematologic malignancies, with broad implications for patient management. In this context, unequivocally discriminating somatic from germline events is challenging but greatly facilitated by matched analysis of tumor:normal pairs. In contrast to solid tumors, conventional sources of normal control (peripheral blood, buccal swabs, saliva) could be highly involved by the neoplastic process, rendering them unsuitable. In this work we describe our real-world experience using cell free DNA (cfDNA) isolated from nail clippings as an alternate source of normal control, through the dedicated review of 2,610 tumor:nail pairs comprehensively sequenced by MSK-IMPACT-heme. Overall, we find nail cfDNA is a robust source of germline control for paired genomic studies. In a subset of patients, nail DNA may have tumor DNA contamination, reflecting unique attributes of the hematologic disease and transplant history. Contamination is generally low level, but significantly more common among patients with myeloid neoplasms (20.5%; 304/1482) compared to lymphoid diseases (5.4%; 61/1128) and particularly enriched in myeloproliferative neoplasms with marked myelofibrosis. When identified in patients with lymphoid and plasma-cell neoplasms, mutations commonly reflected a myeloid profile and correlated with a concurrent/evolving clonal myeloid neoplasm. For nails collected after allogeneic stem-cell transplantation, donor DNA was identified in 22% (11/50). In this cohort, an association with recent history of graft-vs-host disease was identified. These findings should be considered as a potential limitation for the use of nail as normal control but could also provide important diagnostic information regarding the disease process.

Enhanced clinical assessment of hematologic malignancies through routine paired tumor and normal sequencing.

Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.

Validation of a High-Sensitivity Assay for Detection of Chimeric Antigen Receptor T-Cell Vectors Using Low-Partition Digital PCR Technology.

Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.

Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison.

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

Distinct IDH1/2-associated Methylation Profile and Enrichment of TP53 and TERT Mutations Distinguish Dedifferentiated Chondrosarcoma from Conventional Chondrosarcoma.

Dedifferentiated chondrosarcoma (DDCS) is a rare high-grade chondrosarcoma characterized by a well-differentiated chondrosarcoma (WDCS) component that abruptly transitions to a high-grade, noncartilaginous sarcomatous component. To date, the molecular pathogenesis of DDCS and its distinction from conventional chondrosarcoma remain poorly understood. By targeted sequencing, we examined the mutational and copy-number profiles of 18 DDCS, including macrodissected WDCS components, compared with 55 clinically sequenced conventional chondrosarcomas. In conjunction with publicly available external data, we analyzed the methylation and expression profiles of 34 DDCS and 94 conventional chondrosarcomas. Isocitrate dehydrogenase 1/isocitrate dehydrogenase 2 (IDH1/IDH2) mutations were present in 36% conventional chondrosarcomas and 71% DDCS. Compared with conventional chondrosarcomas, DDCS had higher frequencies of TP53 and TERT promoter mutations and CDKN2A/B copy-number losses. Paired analysis of macrodissected WDCS and the high-grade components revealed TERT promoter mutations as early events. Despite phenotypic similarities, the percentage of genome with copy-number alterations in DDCS was significantly lower than that in other high-grade sarcomas. Differential methylation analysis revealed reduction of IDH1/IDH2-associated global hypermethylation characteristically seen in conventional chondrosarcoma and a distinct methylation profile in DDCS. The WDCS and high-grade components in DDCS showed similar methylation profiles. These CpG sites were associated with upregulated expression of genes involved in G2-M checkpoints and E2F targets. Genomic profiling revealed enrichment of TP53, TERT promoter, and CDKN2A/B alterations in DDCS. Integrated methylation and gene expression analysis revealed distinct IDH1/IDH2-associated methylation and transcriptional profiles as early events in DDCS, which may underlie the pathogenesis of dedifferentiation in chondrosarcomas. Significance: DDCS is a rare, high-grade chondrosarcoma with a dismal prognosis. About 50%-80% of DDCS harbor IDH1/IDH2 mutations. We uncover a significant alteration of IDH-associated methylation profile in DDCS, which we propose is key to the progression to dedifferentiation. In this context, the potential effect of the use of IDH inhibitors is unclear but important to address, as clinical trials of selective IDH1 inhibitors showed worse outcome in DDCS.

Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS.

Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

Rapid EGFR Mutation Detection Using the Idylla Platform: Single-Institution Experience of 1200 Cases Analyzed by an In-House Developed Pipeline and Comparison with Concurrent Next-Generation Sequencing Results.

Mutations in the epidermal growth factor receptor (EGFR) are the most common targetable alterations in lung adenocarcinoma. To facilitate rapid testing, the Idylla EGFR assay was incorporated as a screening method before next-generation sequencing (NGS). Validation and experience using an in-house developed analysis pipeline, enhanced with a manual review algorithm is described. Results are compared with corresponding NGS results. In all, 1249 samples were studied. Validation demonstrated 98.57% (69/70) concordance with the reference methods. The limit of detection varied from 2% to 5% variant allele frequency for total EGFR quantitation cycle between 20 and 23. Of the 1179 clinical cases, 23.41% were EGFR-positive by Idylla. Concurrent NGS was successfully performed on 94.9% (799/842) requests. Concordance of Idylla with NGS was 98.62% (788/799) and 98.50% (787/799) using our in-house and Idylla analysis pipelines, respectively. Discordances involved missed mutations by both assays associated with low tumor/low input. Incorporating a manual review algorithm to supplement automated calls improved accuracy from 98.62% to 99.37% and sensitivity from 94.68% to 97.58%. Overall reporting time, from receipt of material to official clinical report, ranged from 1 to 3 days. Therefore, Idylla EGFR testing enables rapid and sensitive screening without compromising subsequent comprehensive NGS, when required. Automated calling, enhanced with a manual review algorithm, reduces false-negative calls associated with low tumor/low input samples.

Phase 2 Study of Iodine-131 Tositumomab Plus Chemotherapy in Patients With Previously Untreated Mantle-Cell Lymphoma.

BACKGROUND: Mantle-cell lymphoma (MCL) is sensitive to radiotherapy, and the CD20 antigen is relatively highly expressed in MCL. Therefore, radioimmunotherapy using radiolabeled anti-CD20 monoclonal antibodies has the potential to treat MCL. The objective of this study was to investigate the efficacy, pharmacokinetics, and safety of tositumomab (TST) and iodine-131 tositumomab (I-131 TST) followed by 6 cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in patients with previously untreated MCL (ClinicalTrials.govNCT00022945). PATIENTS AND METHODS: In this phase 2 open-label study, patients received dosimetric (day 0: 450 mg TST, then 35 mg I-131 TST [5 mCi]) and therapeutic (between days 7 and 14: 450 mg TST, then an individualized dose of I-131 TST [65-75 cGy]) TST/I-131 TST, with CHOP treatment commencing approximately 13 weeks after the therapeutic dose. The primary end point was the MCL response rate to treatment; secondary end points included confirmed complete response rate and total body residence time. RESULTS: Twenty-six patients were enrolled, and 25 were included in the intent-to-treat population. The overall unconfirmed response rate was 84%, and the confirmed complete response rate was 44%. The median progression free-survival was 27.6 months. The median total body residence time was 94.5 hours. No new or unexpected safety signals were identified. CONCLUSION: Patients with previously untreated MCL who received radioimmunotherapy with TST/I-131 TST followed by CHOP had a high response rate and a long duration of response, indicating that radioimmunotherapy is a therapeutic option in this patient population.

Establishment of Immunoglobulin Heavy (IGH) Chain Clonality Testing by Next-Generation Sequencing for Routine Characterization of B-Cell and Plasma Cell Neoplasms.

Immunoglobulin heavy chain (IGH) clonality testing by next-generation sequencing (NGS) offers unique advantages over current low-throughput methods in the assessment of B-cell lineage neoplasms. Clinical use remains limited because assays are not standardized and validation/implementation guidelines are not yet developed. Herein, we describe our clinical validation and implementation of NGS IGH clonality testing and summarize our experience based on extensive routine use. NGS-based clonality testing targeting IGH FR1, FR2, FR3, and the conserved leader sequence upstream of FR1 was validated using commercially available kits. Data were analyzed by commercial and in-house-developed bioinformatics pipelines. Performance characteristics were evaluated directly comparing with capillary electrophoresis (CE) assays (BIOMED-2 primers). Assays were monitored after implementation (>1.5 years), concurrently testing by CE methods. A total of 1189 clinical samples were studied (94 validation, 1095 postimplementation). NGS showed superior performance compared with CE assays. For initial assessment, clonality detection rate was >97% for all malignancy types. Concordance with CE was 96%; discordances were related to higher sensitivity/resolution of NGS and improved detection in cases with high somatic hypermutation. Routine NGS clonality assessment is feasible and superior to existing assays, enabling accurate and specific index clone assessment and future tracking of all rearrangements in a patient sample. Successful implementation requires new standardization, validation, and implementation processes, which should be performed as a multicenter and multidisciplinary collaboration.

Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of chronic lymphocytic leukemia B cells.

PURPOSE: Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation. EXPERIMENTAL DESIGN: We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients. RESULTS: We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. CONCLUSION: Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets.

CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.

Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD(+) disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD(-) complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell-mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.