Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies

Abstract

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.

JAK2 , CALR , MPL and ASXL1 mutational status correlates with distinct histological features in Philadelphia chromosome-negative myeloproliferative neoplasms

“…Next-generation sequencing 6 was performed on all biopsies and showed that 72 (62.6%) patients carried a JAK2 V617F mutation, 21 (18.3%) a CALR mutation, and 6 (5.2%) an MPL W515 mutation, while 16 (13.9%) had nonmutated JAK2, CALR, and MPL ('triple-negative', TN) ( Table 1). CALR mutations were more common in ET patients (P=0.004) and were associated with a higher platelet count in all MPN patients (P=0.001) (Online Supplementary Table S2), consistent with previous studies.…”

Maternal iAMP21 acute lymphoblastic leukemia detected on prenatal cell-free DNA genetic screening

“…Our institution's 95-gene hematologic malignancy-focused targeted DNA sequencing panel 1 revealed an insertion in IL7R (p.L242_L243delinsFGLRGCP) as well as mutations in CSF3R (p.809fs*) and LUC7L2 (p.R205*). Read count analysis ( Figure 1A) was significant for loss of IKZF1 (on 7p), loss of CTCF (16q), multiple copy gain of RUNX1 (21q), deletion of U2AF1 (21q), as well as gain of CRLF2, PIGA, ZRSR2, and BCOR (all on Xp).…”

“…Genetic characterization of the leukemia was notable for a complex karyotype 46,X,add(X)(p22.1),del(9) (p21),add(10)(q25),-21,1mar[cp19]/46,XX [1]. Our institution's 95-gene hematologic malignancy-focused targeted DNA sequencing panel 1 revealed an insertion in IL7R (p.L242_L243delinsFGLRGCP) as well as mutations in CSF3R (p.809fs*) and LUC7L2 (p.R205*).…”

Many faces of the same myeloid neoplasm: a case of leukaemia cutis with mixed histiocytic and Langerhans cell differentiation

“…Molecular analysis2 at multiple time points following initial AML diagnosis revealed variability in somatic variants detected in the neoplastic cells infiltrating skin and/or bone marrow. Using allele frequencies, we constructed a possible hierarchical model of clonal evolution (table 1; figure 3).…”