Profiling Genomic Alterations Of Diffuse Large B-Cell Lymphoma (DLBCL) At Diagnosis, Relapse, and Transformation, Using a Novel Clinical Diagnostic Targeted Sequencing Platform

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1761-1761
Author(s):  
Andrew M. Intlekofer ◽  
Venkatraman E. Sheshan ◽  
Ross L. Levine ◽  
Andrew D. Zelenetz ◽  
Julie Teruya-Feldstein ◽  
...  
Keyword(s):  
Tumor Suppressors ◽  
Copy Number ◽  
De Novo ◽  
Ffpe Tissue ◽  
Copy Number Alterations ◽  
Loss Of Function ◽  
Genomic Alterations ◽  
Sequencing Platform ◽  
Next Generation Sequencing Platform ◽  
Generation Sequencing

Abstract Whole genome and exome sequencing studies have identified numerous genomic alterations in DLBCL, but these methods have limited applicability for the clinical care of lymphoma patients due to cost, specific tissue requirements, and laborious bioinformatic analysis. FoundationOne-Heme (FOH) is a novel next-generation sequencing platform designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of mutations within specific genes, copy number changes, and translocations. FOH can be performed on small quantities of formalin-fixed paraffin-embedded (FFPE) tissue, detect rare variants due to extensive depth of sequencing coverage, and rapidly provide results via streamlined bioinformatic interpretation. Here we report the first experience using this novel platform to evaluate the genetic landscape of DLBCL. Genomic DNA and total RNA were isolated from FFPE tissue on a cohort of 53 cases of DLBCL, including de novo (n=30), relapsed/refractory (n=12), and large cell transformation from low-grade lymphoma (n=11). The cohort included 25 cases with combined MYC and BCL2 overexpression by IHC (criteria for positivity: >40% MYC, >70% BCL2), of which only one had a known translocation involving MYC. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 frequently-rearranged genes for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >658x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. The DNA sequencing component of FOH detected translocations in BCL2, BCL6, and MYC, while the RNA sequencing component detected fusion transcripts involving BLC6 and MYC, in agreement with independent cytogenetic analysis via karyotype and FISH where available. The assay detected copy number alterations of 44 different genes, most commonly amplification of REL (15%) or loss of CDKN2A/CDKN2B (17%). The most frequent alterations of known significance are detailed in Figure 1. The most commonly altered gene was CDKN2A, exhibiting either homozygous deletion or loss of function mutation in 28% of cases. Chromatin modifying factors (e.g. MLL2, CREBBP, EZH2) represented the most frequently altered biologic category with alterations occurring in >50% of cases. Recurrent alterations in components of the Notch pathway (NOTCH1/2/4, FBXW7, SPEN), each predicted to activate the pathway, were identified in 23% of cases. Cell-of-origin was determined as per the Hans model using IHC for CD10, BCL6, and IRF4/MUM1; CD79B mutations were detected exclusively in non-GCB and EZH2 mutations were found exclusively in GCB-phenotype cases. Furthermore, IHC MYC+/BCL2+ de novo DLBCL cases (n=11) exhibited more frequent hypermutation of PIM1 (46%) compared with the 19 cases of IHC MYC-/BCL2- de novo DLBCL (11%). When comparing the various clinical categories, we found that mutations in tumor suppressors were significantly more common in relapsed/refractory than de novo DLBCL (47% vs 75%, p=0.02). Alterations in TP53 were most frequently observed in transformed lymphoma (55%). Our results demonstrate the feasibility of using a targeted next-generation sequencing platform on FFPE clinical specimens from patients with DLBCL as a means of providing an integrated analysis of gene mutations, copy number alterations, and translocations. This streamlined approach combines multiple molecular and cytogenetic tests into a single platform and uses a small amount of tissue to perform a multifaceted assessment of genomic alterations with potential diagnostic, prognostic, and therapeutic implications. Future efforts will be directed at analyzing additional cases of DLBCL to better establish the biologic and clinical significance of the observed genetic alterations, and to prospectively incorporate this novel platform to select patients for mechanism-based targeted therapy. Disclosures: Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Genomic Profiling Combining DNA and RNA Analysis of 112 Formalin-Fixed Paraffin-Embedded Diffuse Large B Cell Lymphoma Specimens Identifies a High Frequency of Clinically Relevant Genomic Alterations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 704-704
Author(s):  
Andrew M. Intlekofer ◽  
Patrick Hilden ◽  
Venkatraman E. Seshan ◽  
Amanda R. Copeland ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
B Cell ◽  
Copy Number ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Copy Number Alterations ◽  
Genomic Alterations ◽  
Large B Cell Lymphoma ◽  
Chi Squared ◽  
Generation Sequencing

Abstract Background: A variety of next-generation sequencing methods have been used to investigate the genomic landscape of primary lymphoma patient samples, including whole-genome, whole-exome, and RNA sequencing. In diffuse large B cell lymphoma (DLBCL), sequencing studies identified numerous genomic alterations (GAs) of potential clinical relevance, but the distribution and frequency of GAs have not been precisely determined. Discrepancies in existing data likely arise from variability in types of specimens examined, sequencing technologies employed, and depth of coverage utilized to identify GAs. In this study, we performed comprehensive DNA/RNA sequencing of genes known to be important across the spectrum of hematologic malignancies in order to determine the nature and prevalence of GAs with potential diagnostic, prognostic, or therapeutic implications in a cohort of 112 well-annotated clinical DLBCL cases. Methods: We performed hybridization capture of 405 cancer-related genes and 31 genes commonly rearranged in cancer (FoundationOne Heme) and 265 frequently rearranged genes for RNA-seq applied to ³50ng of DNA and sequenced to high, uniform coverage. Genomic alterations, including short variants (small indels and base substitutions), rearrangements, and copy number alterations, were determined. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >500x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of GAs. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. Results: GAs with diagnostic, prognostic, or therapeutic relevance were identified in 96% of cases, with a median of 5 (range 0 to 12) alterations per sample. Figure 1 shows the frequencies of different GAs. De novo DLBCL tended to exhibit fewer GAs (median 4) than relapsed/refractory (median 6) or transformed disease (median 6) (p=0.179; Wilcoxon rank sum). Compared with reported frequencies ranging from 5 to 44%, we detected alterations in CREBBP/EP300 in 21% of cases, with CREBBP mutations preferentially found in germinal-center B cell-like (GCB) compared to non-GCB DLBCL (26% vs. 8%; p=0.02; Pearson's chi-squared). Although previously described only in Burkitt lymphoma, we identified mutations in ID3 (L70P, P98R, Q100P) and TCF3 (N551K) in GCB-phenotype DLBCL cases with aggressive pathologic features and no MYC expression by IHC. Additional novel findings included alterations of genes involved in cellular metabolism in 18% of cases, including one IDH2 R172M mutation and two cases with SDHA L649fs* truncating mutations. We also identified several mutations more commonly found in solid tumors and leukemias, including BRAF V600E and KRAS G13D. With respect to genomic rearrangements, combined DNA/RNA capture and sequencing detected translocations/fusions in MYC, BCL2, and BCL6, which were concordant with cytogenetics/FISH analysis. We also identified rearrangements involving TBXL1XR1-P63, NOTCH2, SOCS1, and ETV6. Copy number alterations were detected in 26 different genes, including amplifications of CD274/PDCD1LG2 (PD-L1/PD-L2) (n=4) that were restricted to non-GCB cases. Alterations in TP53 were more frequently observed in transformed (42%) and relapsed/refractory (26%) compared to de novo DLBCL (12%; p=0.007; Pearson's chi-squared). TP53 mutations predicted for lack of response to chemotherapy, with chemo-refractory disease occurring in 48% of TP53-mutant patients compared to 10% of patients with intact TP53 (p=0.0004; Fisher's exact). Truncating mutations or deletions of RB1, albeit rare (5% of cases), were the most significant negative prognostic factor and were associated with therapeutic resistance and poor overall survival. Conclusions: Our results demonstrate the utility of comprehensive combined DNA/RNA next-generation sequencing as a promising method to identify clinically relevant GAs in clinical lymphoma specimens. This streamlined approach has the potential to combine multiple molecular and cytogenetic tests into a single platform. Future efforts will be directed at incorporating this approach both retrospectively and prospectively into clinical trials to identify predictive biomarkers to guide therapeutic decisions. Figure 1 Figure 1. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Targeted next-generation sequencing (NGS) of advanced prostate cancer (PCA) using formalin-fixed tissue.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 4649-4649
Author(s):  
Himisha Beltran ◽  
Roman Yelensky ◽  
Garrett Frampton ◽  
Kyung Park ◽  
Sean Downing ◽  
...  
Keyword(s):  
Copy Number ◽  
Dna Analysis ◽  
Ffpe Tissue ◽  
Copy Number Alterations ◽  
Genomic Alterations ◽  
Biopsy Material ◽  
Fixed Tissue ◽  
Similar Frequency ◽  
Recurrent Mutations ◽  
Formalin Fixed

4649 Background: The genomic landscape of advanced PCA is not well characterized, partly due to limited availability of frozen metastatic tissue. This has created a gap in our knowledge of treatment related and potentially targetable genomic alterations. The purpose of this study was to demonstrate feasibility of performing NGS to molecularly characterize advanced PCA using formalin-fixed paraffin embedded (FFPE) tissue. Methods: 25 metastatic CRPC, 4 metastatic hormone naïve PCA, 3 primary localized PCA, and 18 benign matched prostates were evaluated (40mm FFPE tissue per case). High-density foci were captured and sequenced for 3230 exons of 182 cancer-related genes and 37 introns of 14 genes often rearranged in cancer to an average depth of >800x in a CLIA lab (Foundation Medicine). Recurrent mutations, copy number alterations, and fusions were validated using PCR and FISH. Results: In >90% of samples, there was sufficient DNA (≥50 ng) for analysis. Recurrent high confidence cancer alterations in CRPC included: TMPRSS2-ERG fusion (44%), PTEN loss (44%), TP53 mutation (40%), AR mutation (24%), AR gain (24%), RB mutation (28%), MYC gain (12%), and BRCA2 loss (12%). Overall 48% of CRPC harbored AR gene alterations. Additionally, there were mutations in CTNNB1 (12%), ATM (8%) and PIK3CA (4%). Copy number alterations not previously described in PCA included CCND1, CDK4/6 gains and CDKN2A/CDKN2B deletions. Hormone naïve metastatic and high risk localized PCA demonstrated similar frequency of TMPRSS2-ERG gene fusion, BRCA2 deletion, and TP53 mutations as CRPC, but AR alterations and MYC gain were not seen. Conclusions: This study demonstrates feasibility of in-depth, NGS based DNA analysis using FFPE tissue, even biopsy material. Frequent AR alterations in CRPC, mutations associated with disease progression, and potential drug targets were identified. Focused NGS has clinical potential to identify actionable genomic alterations in advanced PCA that can impact patient participation in trials as well as treatment and outcome. Treatment options include PARP inhibitors for patients with BRCA2 and ATM alterations (20% of cases) and PI3K/AKT inhibitors for PIK3CA mutations.

scholarly journals Copy number alterations in cancer: detection and precision medicine

2021 ◽  
Vol 41 ◽  
pp. 02005
Author(s):  
Arief Gusnanto
Keyword(s):  
Next Generation Sequencing ◽  
Precision Medicine ◽  
Cancer Patients ◽  
Cancer Detection ◽  
Copy Number ◽  
Copy Number Alterations ◽  
Genomic Alterations ◽  
Genomic Markers ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Copy number alterations (CNAs) are genomic alterations where some regions exhibit more or less copy number than the normal two copies. In this talk, I will describe two ideas: (1) how CNAs are estimated from data generated by next generation sequencing (NGS) and what steps are required to make the data interpretable, (2) how the CNA can be utilised for precision medicine in terms of prediction of tumour subtypes and prediction of cancer patients’ survival. If time permits, I will also discuss how to estimate genomic markers from CNA profile across cancer patients.

scholarly journals Performance Characteristics of a Targeted Sequencing Platform for Simultaneous Detection of Single Nucleotide Variants, Insertions/Deletions, Copy Number Alterations, and Gene Fusions in Cancer Genome

2020 ◽  
Vol 144 (12) ◽  
pp. 1535-1546
Author(s):  
Kyung Park ◽  
Hung Tran ◽  
Kenneth W. Eng ◽  
Sinan Ramazanoglu ◽  
Rebecca M. Marrero Rolon ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Precision Oncology ◽  
Gene Fusions ◽  
Copy Number Alterations ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Sequencing Platform ◽  
Generation Sequencing

Context.— An increasing number of molecular laboratories are implementing next-generation sequencing platforms to identify clinically actionable and relevant genomic alterations for precision oncology. Objective.— To describe the validation studies as per New York State–Department of Health (NYS-DOH) guidelines for the Oncomine Comprehensive Panel v2, which was originally tailored to the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial. Design.— Accuracy, precision, and reproducibility were investigated by using 130 DNA and 18 RNA samples from cytology cell blocks; formalin-fixed, paraffin-embedded tissues; and frozen samples. Analytic sensitivity and specificity were tested by using ATCC and HapMap cell lines. Results.— High accuracy and precision/reproducibility were observed for single nucleotide variants and insertion/deletions. We also share our experience in the detection of gene fusions and copy number alterations from an amplicon-based sequencing platform. After sequencing analysis, variant annotation and report generation were performed by using the institutional knowledgebase. Conclusions.— This study serves as an example for validating a comprehensive targeted next-generation sequencing assay with both DNASeq and RNASeq components for NYS-DOH.

scholarly journals Next-Generation Sequencing–Based Assessment of JAK2, PD-L1, and PD-L2 Copy Number Alterations at 9p24.1 in Breast Cancer

2019 ◽  
Vol 21 (2) ◽  
pp. 307-317 ◽  
Author(s):  
Sounak Gupta ◽  
Chad M. Vanderbilt ◽  
Paolo Cotzia ◽  
Javier A. Arias-Stella ◽  
Jason C. Chang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Copy Number ◽  
Copy Number Alterations ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Old gene, new phenotype: splice-altering variants in CEACAM16 cause recessive non-syndromic hearing impairment

2018 ◽  
Vol 55 (8) ◽  
pp. 555-560 ◽  
Author(s):  
Kevin T Booth ◽  
Kimia Kahrizi ◽  
Hossein Najmabadi ◽  
Hela Azaiez ◽  
Richard JH Smith
Keyword(s):  
Hearing Loss ◽  
Hearing Impairment ◽  
Loss Of Function ◽  
Bioinformatics Pipeline ◽  
Exon 2 ◽  
Reading Frame ◽  
Sequencing Platform ◽  
Syndromic Hearing Impairment ◽  
Generation Sequencing

BackgroundHearing loss is a genetically and phenotypically heterogeneous disorder.ObjectivesThe purpose of this study was to determine the genetic cause underlying the postlingual progressive hearing loss in two Iranian families.MethodsWe used OtoSCOPE, a next-generation sequencing platform targeting >150 genes causally linked to deafness, to screen two deaf probands. Data analysis was completed using a custom bioinformatics pipeline, and variants were functionally assessed using minigene splicing assays.ResultsWe identified two homozygous splice-altering variants (c.37G>T and c.662–1G>C) in the CEACAM16 gene, segregating with the deafness in each family. The minigene splicing results revealed the c.37G>T results in complete skipping of exon 2 and loss of the AUG start site. The c.662–1G>C activates a cryptic splice site inside exon 5 resulting in a shift in the mRNA reading frame.ConclusionsThese results suggest that loss-of-function mutations in CEACAM16 result in postlingual progressive hearing impairment and further support the role of CEACAM16 in auditory function.

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