Neelima Reddy

Clinical validation of a next-generation sequencing screen for mutational hotspots in 46 cancer-related genes

Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.

Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics

Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.

Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring

Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.

Predictors of Primary Imatinib Resistance in Chronic Myelogenous Leukemia Are Distinct From Those in Secondary Imatinib Resistance

PURPOSE A subset of patients with chronic myelogenous leukemia (CML) do not respond to the tyrosine kinase inhibitor (TKI) imatinib mesylate. Such primary imatinib resistance is distinguished from secondary resistance which reemerges after attainment of cytogenetic remission. PATIENTS AND METHODS We studied gene expression patterns in total WBCs using a panel of 21 genes previously implicated in TKI handling, resistance, or progression comparing patients who had newly diagnosed TKI-naive CML that had optimal (n = 41), or suboptimal (n = 7) responses to imatinib, or primary resistance (n = 20). Expression patterns were compared to those in secondary TKI-resistant chronic phase CML without ABL1 kinase domain mutations (n = 29), and to lymphoid (n = 15) or myeloid blast phase disease (n = 12). RESULTS Fifteen genes in the panel distinguished blast phase from chronic phase disease, and 12 genes distinguished newly diagnosed CML from TKI-resistant CML without ABL1 kinase domain mutations, but only a single gene, prostaglandin-endoperoxide synthase 1/cyclooxgenase 1 (PTGS1/COX1; P = .005), differentiated imatinib-responsive from primary imatinib-resistant CML. The association of primary imatinib resistance with higher transcript levels of the drug metabolism gene PTGS1 was confirmed in a separate data set of 68 newly diagnosed, imatinib-treated CML (P = .008). In contrast, up to 11 different genes were identified in a multivariate model that optimally discriminated secondary imatinib resistance lacking ABL1 kinase domain mutation from imatinib-responsive cases, likely related to the more complex pathogenesis of secondary resistance. CONCLUSION Gene expression profiling of CML at diagnosis for PTGS1 may be useful in predicting imatinib response and in selecting alternate therapy.

TET2 mutations, myelodysplastic features, and a distinct immunoprofile characterize blastic plasmacytoid dendritic cell neoplasm in the bone marrow.

Distinguishing blastic plasmacytoid dendritic cell neoplasm (BPDCN) from acute myeloid leukemia (AML) is gaining increased importance because of emerging differences in therapeutic approaches, and this distinction can be problematic in bone marrow specimens. We identified retrospectively 16 patients with bone marrow involvement by BPDCN: 11 men and 5 women with a median age of 62.5 years (range, 19–86 years). Myelodysplastic changes were observed in five patients. Immunophenotypic analysis showed that the neoplastic cells were positive for CD4, CD123, TCL‐1, and HLA‐DR and were negative for CD3, CD8, CD13, CD19, CD34, and myeloperoxidase. Other antigens expressed by subsets of BPDCN cases included the following: CD56 (13/15; 81%), CD33 (7/10; 70%), CD7 (11/14; 69%), TdT (5/15; 33%), CD2 (5/11; 31%), CD117 (2/9; 22%), and CD5 (2/13; 15%). Conventional cytogenetic analysis showed chromosomal abnormalities in 6 of 13 (46%) cases analyzed, of which 3 cases had −13/13q−. Targeted next‐generation sequencing performed on five BPDCN cases identified TET2 (ten eleven translocation 2) mutations and no other AML‐associated mutations. In conclusion, BPDCN in the bone marrow has a characteristic immunoprofile (CD4+, CD56+, CD123+, and TCL‐1+) and appears to be commonly associated with myelodysplastic features and a high frequency of TET2 mutations in the absence of other mutations commonly observed in AML. Am. J. Hematol. 88:1055–1061, 2013.

Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours

Background:In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours.Methods:DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array.Results:The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton.Conclusions:The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

Diagnostic Testing for IDH1 and IDH2 Variants in Acute Myeloid Leukemia An Algorithmic Approach Using High-Resolution Melting Curve Analysis

Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations and polymorphism are reported in 5% to 15% of acute myeloid leukemia (AML) cases, with G105 and R132 of IDH1 and R140 and R172 of IDH2 known to be clinically significant. Current Sanger sequencing assays to detect IDH mutations are labor intensive and not cost effective for clinical testing of low-frequency mutations. Therefore, we developed clinical assays using high-resolution melting (HRM) analysis to screen for all four variants listed above, followed by Sanger sequencing confirmation. The sensitivities of the assays were 7.3% and 7.9% for the detection of IDH2 and IDH1 variants, respectively, against the background of wild-type transcripts. Comparison of HRM to Sanger sequencing on 146 AML bone marrow samples for validation showed near-perfect concordance for all positive and negative results for IDH1 (98%) and IDH2 (94%). Postvalidation clinical implementation of upfront HRM screening (N = 106), using a more conservative algorithm to avoid false-negative results, reduced the number of Sanger sequencing tests by 73% (IDH1) and 78% (IDH2). Of the variant calls made by HRM in postvalidation clinical samples, Sanger confirmed the presence of a variant in 62% (IDH1) and 44% (IDH2) of the samples. In conclusion, our HRM assays are rapid, convenient, and versatile assays for screening and confirmation of alterations in IDH1 and IDH2.

Rapid Detection and Quantitation of BRAF Mutations in Hairy Cell Leukemia Using a Sensitive Pyrosequencing Assay

BRAF protooncogene is an important mediator of cell proliferation and survival signals. BRAF p.V600E mutation was recently described as a molecular marker of hairy cell leukemia (HCL). We developed and validated a pyrosequencing-based approach that covers BRAF mutational hotspots in exons 11 (codon 468) and 15 (codons 595 to 600). The assay detects BRAF mutations at an analytical sensitivity of 5%. We screened 16 unenriched archived bone marrow aspirate samples from patients with a diagnosis of HCL (n = 12) and hairy cell leukemia-variant (HCL-v) (n = 4) using pyrosequencing. BRAF p.V600E mutation was present in all HCL cases and absent in all HCL-v. Our data support the recent finding that BRAF p.V600E mutation is universally present in HCL. Moreover, our pyrosequencing-based assay provides a convenient, rapid, sensitive, and quantitative tool for the detection of BRAF p.V600E mutations in HCL for clinical diagnostic testing.

Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

Rapid clonal shifts in response to kinase inhibitor therapy in chronic myelogenous leukemia are identified by quantitation mutation assays

Treatment of CML with the tyrosine kinase inhibitor (TKI) imatinib mesylate results in the emergence of point mutations within the kinase domain (KD) of the BCR‐ABL1 fusion transcript. The introduction of next‐generation TKIs that can overcome the effects of some BCR‐ABL1 KD mutations requires quantitative mutation profiling methods to assess responses. We report the design and validation of such quantitative assays, using pyrosequencing and mutation‐specific RT‐PCR techniques, to allow sequential monitoring and illustrate their use in tracking specific KD mutations (e.g. G250E, T315I, and M351T) following changes in therapy. Pyrosequencing and mutation‐specific RT‐PCR allows sequential monitoring of specific mutations and identification of rapid clonal shifts in response to kinase inhibitor therapy in CML. Rapid reselection of TKI‐resistant clones occurs following therapy switch in CML. (Cancer Sci 2010)