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.