Jay L. Patel

Clinical utility of next‐generation sequencing in the diagnosis of hereditary haemolytic anaemias

Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life‐threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next‐generation sequencing to provide a high‐throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion‐dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next‐generation sequencing panel could be a cost‐effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.

Performance characteristics of an automated assay for the quantitation of CYFRA 21-1 in human serum☆

OBJECTIVES A fragment of cytokeratin 19, CYFRA 21-1, has been reported to be a sensitive tumor marker for non-small cell lung cancer (NSCLC). We describe analytical performance characteristics of a novel CYFRA 21-1 assay and hypothesize that CYFRA 21-1 complements the clinical sensitivity of carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCCa). DESIGN AND METHODS Performance characteristics of a CYFRA 21-1 immunochemiluminescent assay included analytical sensitivity, imprecision, linearity, analyte stability, and reference interval determination. Ninety-two pretreatment NSCLC serum samples were tested for CYFRA 21-1, CEA, and SCCa. Sensitivity was determined for each marker individually and in combination, with regard to tumor stage and histology. RESULTS The analytical sensitivity was 0.01 ng/mL. Total imprecision ranged from 4.0 to 6.3% at 4.9 to 28.4 ng/mL, respectively. The assay was linear from 0.9 to 71.4 ng/mL (slope=0.995, intercept=-0.60, r(2)=0.999). CYFRA 21-1 was stable for 48 h at ambient temperature and 14 days at 4°C. The 97.5th percentile of a reference population was 1.9 ng/mL. Across disease stage, the sensitivities of CYFRA 21-1, CEA, and SCCa were 17-81%, 30-52%, and 24-39%, respectively. CYFRA 21-1 combined with CEA or SCCa increased sensitivity above that of any single marker. CONCLUSIONS An immunochemiluminescent assay for CYFRA 21-1 had favorable performance characteristics. CYFRA 21-1 was complementary to CEA and SCCa and increased clinical sensitivity in patients with NSCLC.

Coexisting and cooperating mutations in NPM1-mutated acute myeloid leukemia

NPM1 insertion mutations represent a common recurrent genetic abnormality in acute myeloid leukemia (AML) patients. The frequency of these mutations varies from approximately 30% overall up to 50% in patients with a normal karyotype. Several recent studies have exploited advances in massively parallel sequencing technology to shed light on the complex genomic landscape of AML. We hypothesize that variant allele fraction (VAF) data derived from massively parallel sequencing studies may provide further insights into the clonal architecture and pathogenesis of NPM1-driven leukemogenesis. Diagnostic peripheral blood or bone marrow samples from NPM1-mutated AML patients (n=120) were subjected to targeted sequencing using a panel of fifty-seven genes known to be commonly mutated in myeloid malignancies. NPM1 mutations were always accompanied by additional mutations and NPM1 had the highest VAF in only one case. Nearly all NPM1-mutated AML patients showed concurrent mutations in genes involved in regulation of DNA methylation (DNMT3A, TET2, IDH1, IDH2), RNA splicing (SRSF2, SF3B1), or in the cohesin complex (RAD21, SMC1A, SMC3, STAG2). Mutations in these genes had higher median VAFs that were higher (40% or greater) than the co-existing NPM1 mutations (median VAF 16.8%). Mutations associated with cell signaling pathways (FLT3, NRAS, and PTPN11) are also frequently encountered in NPM1-mutated AML cases, but had relatively low VAFs (7.0-11.9%). No cases of NPM1-mutated AML with a concurrent IDH2R172 mutation were observed, suggesting that these variants are mutually exclusive. Overall, these data suggest that NPM1 mutations are a secondary or late event in the pathogenesis of AML and are preceded by founder mutations in genes that may be associated with recently described preclinical states such as clonal hematopoiesis of indeterminate potential or clonal cytopenias of undetermined significance.

Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon‐level duplications, including so‐called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof‐of‐concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.

An unusual case of cutaneous blastic plasmacytoid dendritic cell neoplasm with concomitant B-cell lymphoproliferative disorder.

Blastic plasmacytoid dendritic cell neoplasm is a rare hematologic malignancy characterized by aggressive clinical behavior and frequent cutaneous involvement. We describe a case of a 64-year-old man with a rapidly enlarging subcutaneous forearm mass. Histologic examination of the excisional biopsy specimen revealed a diffuse proliferation of atypical hematolymphoid cells in the dermis extending to the deep subcutaneous soft tissues. Occasional aggregates of small lymphocytes were noted to be distributed within the mass. The tumor cells expressed CD4, CD45, CD56, CD123, and terminal deoxynucleotidyl transferase (TdT) but not CD3, CD20, or CD34. A diagnosis of blastic plasmacytoid dendritic cell neoplasm was rendered. Chromosome analysis revealed a 45 X, -Y karyotype. In addition, flow cytometry identified a small population of monoclonal B cells. A staging bone marrow aspirate and biopsy was performed, which showed normal cytogenetics and no evidence of involvement by blastic plasmacytoid dendritic cell neoplasm. Flow cytometric evaluation of the bone marrow revealed a CD5-negative, CD10-negative monoclonal B-cell population consistent with a B-cell lymphoproliferative disorder. This is a very unusual example of cutaneous blastic plasmacytoid dendritic cell neoplasm with a novel cytogenetic finding and concomitant B-cell lymphoproliferative disorder. Although previously not reported, our case shows that blastic plasmacytoid dendritic cell neoplasm may be associated with lymphoid malignancy. The relationship between the 2 neoplasms, however, is unclear. A high degree of suspicion and bone marrow examination in patients with a new diagnosis of blastic plasmacytoid dendritic cell neoplasm is required to avoid this potential diagnostic pitfall.

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing

Aims Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). Methods For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. Results We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. Conclusions We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay.