Amy Donahue

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non–small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8–3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.

Clinical next‐generation sequencing successfully applied to fine‐needle aspirations of pulmonary and pancreatic neoplasms

Next‐generation sequencing was performed on pulmonary and pancreatic fine‐needle aspirations (FNAs) and on paired FNAs and resected primary tumors from the same patient.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

A High Frequency of Activating Extracellular Domain ERBB2 (HER2) mutation in Micropapillary Urothelial Carcinoma

Purpose: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. Experimental Design: DNA was extracted from 40 μm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation–based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. Results: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). Conclusions: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients. Clin Cancer Res; 20(1); 68–75. ©2013 AACR.

Patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias.

Genomic studies have identified recurrent somatic mutations in acute leukemias. However, current murine models do not sufficiently encompass the genomic complexity of human leukemias. To develop preclinical models, we transplanted 160 samples from patients with acute leukemia (acute myeloid leukemia, mixed lineage leukemia, B-cell acute lymphoblastic leukemia, T-cell ALL) into immunodeficient mice. Of these, 119 engrafted with expected immunophenotype. Targeted sequencing of 374 genes and 265 frequently rearranged RNAs detected recurrent and novel genetic lesions in 48 paired primary tumor (PT) and patient-derived xenotransplant (PDX) samples. Overall, the frequencies of 274 somatic variant alleles correlated between PT and PDX samples, although the data were highly variable for variant alleles present at 0–10%. Seventeen percent of variant alleles were detected in either PT or PDX samples only. Based on variant allele frequency changes, 24 PT-PDX pairs were classified as concordant while the other 24 pairs showed various degree of clonal discordance. There was no correlation of clonal concordance with clinical parameters of diseases. Significantly more bone marrow samples than peripheral blood samples engrafted discordantly. These data demonstrate the utility of developing PDX banks for modeling human leukemia, and emphasize the importance of genomic profiling of PDX and patient samples to ensure concordance before performing mechanistic or therapeutic studies.

Analytical Validation of a Hybrid Capture-Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA.

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture–based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode–based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%–99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%–95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%–71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments–certified/College of American Pathologists–accredited/New York State–approved laboratory.

Abstract 4737: Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies

Background: Sequencing the genes encoding immunoglobulins is critical in detecting clonal cell populations as well as determining prognosis and therapeutic decisions in patients with lymphoid malignancies including B-cell leukemias, lymphomas and multiple myeloma. Current assays for identifying the rearranged immunoglobulin sequence in B-cell malignancies rely on sequence specific PCR based amplification of conserved immunoglobulin (IG) regions. We have developed a novel, hybrid capture-based approach to sequencing the immunoglobulin chains that enables identification of the heavy and light chain variable domains, complementarity-determining region (CDR) sequences and somatic hypermutation (SHM) status. Method: RNA and DNA were successfully extracted from 60 specimens, including 7 mantle cell lymphoma cell (MCL) lines and 53 clinical chronic lymphocytic leukemia (CLL) bone marrow aspirates. Adaptor-ligated DNA and cDNA sequencing libraries were captured by solution hybridization using custom bait-sets targeting the immunoglobulin variable, joining and class segments. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine). Results: The capture-based approach was validated using 7 MCL cell lines and 53 CLL samples profiled using a CLIA-certified commercial PCR-based assay (Invivoscribe). The immunoglobulin sequences derived from the 7 MCL cell lines were 100% concordant with identifying the published heavy and light chain variable domain and percent SHM. Comparison to 53 previously clinically tested samples showed 98% (52/53) concordance for identifying the presence of a clonal population, and 100% (39/39) concordance for identifying the IGHV domain. Comparison to 50 CLL samples previously tested for SHM resulted in 96% (48/50) overall concordance with 94% (31/33) concordant for no SHM and 100% (17/17) concordant for the presence of SHM. Additionally, secondary clones were identified in 11 samples. Conclusions: We have demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to comprehensively characterize the immunoglobulin sequence of clonal tumor B cell populations. This capability enables quantification of SHM and identification of the variable domain, CDR3 sequence and class restriction. Integration of this methodology with comprehensive genomic profiling approaches will expand the clinical utility of such assays in patients with hematological malignancies and may provide important insights in immune oncology and response of patients to immunotherapies, including patients with solid tumors. Citation Format: Michelle K. Nahas, Lauren E. Young, Jeff Gardner, Omar Abdel-Wahab, Jie He, Amy L. Donahue, Kristina M. Knapp, Geoff A. Otto, Doron Lipson, Vincent A. Miller, Ross L. Levine, Philip J. Stephens. Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2015-4737

Abstract 3570: Development and validation of a clinical next generation sequencing-based assay for hematologic malignancies

Background: Next-generation sequencing (NGS) is rapidly becoming an indispensable cancer diagnostic, as it can detect most genomic alterations in a single assay from limited tissue. We developed a novel, NGS-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events which are common in these tumors. Methods: The high accuracy of the assay for detection of substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across all alteration types with high specificity (PPV>99%) [Frampton et al, Nat Biotech, 2013]. To validate assay performance in detecting gene fusions we created reference samples by mixing 21 cell-lines with previously characterized fusions in 39 combinations representing 167 fusion events in 10-50% tumor cell fractions. In addition, we confirmed accuracy in 76 clinical hematologic FFPE and bone-marrow samples profiled for 212 substitutions, indels and fusions in 11 genes by Sanger sequencing, PCR, fragment sizing and FISH. DNA and RNA were extracted from all samples; adaptor ligated sequencing libraries were captured by solution hybridization using custom baits targeting 405 cancer related genes by DNA-seq, and 265 frequently rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 467x for DNA and 6M unique pairs for RNA. Results: On reference samples, sensitivity for detection of fusions events reached >99% for tumor cell fractions of 20-50%, and 97% for tumor fraction of 10%, all with high specificity (PPV>95%). Robust performance translated to the clinical samples: we observed a concordance rate of 98.6% relative to prior calls, with only 3/212 differing calls (2+, 1-) by NGS. 129 additional known oncogenic alterations in 57 different genes were detected in these samples, for a total of 3.1 alterations per sample. Analysis of 290 additional leukemia, lymphoma and myeloma patient samples revealed known and novel gene fusions in 15% of cases, more than half of which were identifiable only by RNA-seq. Conclusions: We describe the analytic validation of a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used to identify a spectrum of somatic alterations from blood, bone marrow and paraffin embedded patient samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations, including gene fusions, with high accuracy. This approach offers the opportunity to streamline the characterization of genomic alterations in hematologic malignancies and to expand targeted treatment options for patients with liquid tumors. Citation Format: Doron Lipson, Michelle K. Nahas, Geoff A. Otto, Jie He, Kai Wang, Kristina M. Knapp, Kristina W. Brennan, Amy L. Donahue, Lauren E. Young, Geneva Young, Alex Fichtenholtz, Jeffrey S. Ross, Roman Yelensky, Philip J. Stephens, Vincent A. Miller, Ross Levine. Development and validation of a clinical next generation sequencing-based assay for hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2014-3570

Abstract 4699: Analytical validation of solid tumor fusion gene detection in a comprehensive NGS-based clinical cancer genomic test

Background: As the number of clinically actionable cancer genes grows and the size of most diagnostic biopsies decreases, next-generation sequencing (NGS) becomes increasingly attractive as a diagnostic tool, as it can detect all classes of genomic alterations in all cancer genes in a single test. However, for NGS to achieve its full utility in the clinic, robust analytical validation and performance comparisons against established detection methodologies are required for each class of targetable genomic alteration. Methods: Previously, we reported on the development and validation of an NGS-based diagnostic test for accurate detection of clinically-relevant genomic alterations across all exons of 287 cancer genes in routine FFPE specimens (Frampton, et. al, Nat. Biotech. Oct 2013). Here, we present systematic validation of fusion gene detection in the test, enabled by hybrid-selection and deep sequencing of commonly rearranged introns in 19 (or, in an alternative version, 31) genes. We created reference samples reflecting key determinants of detection accuracy for gene fusions, including extent of stromal admixture and variety of partner genes: We obtained 5 solid tumor fusion-bearing cell-lines (2 ALK, 1 RET, 1 ROS1, 1 TMPRSS2) and mixed these into 23 variably sized pools, such that each fusion was represented at 100%, 50%, 33%, 25%, and 20% simulated cellular fraction at least once. Gene fusions were called if a well-mapped cluster of 5 chimeric reads or greater was observed in a targeted intron. We then verified that the observed performance translated to FFPE clinical samples by examining test concordance in 43 tumors (20+/23-) characterized for ALK rearrangement by FISH. Finally, we evaluated the test broadly by assessing detection prevalence of the three most common gene fusions (ALK, ROS1, RET) in 724 lung adenocarcinoma FFPE patient samples, including needle biopsies. Results: Of 32 tested gene fusion instances in the 28 cell-lines samples, all were successfully detected (sensitivity 100%, 95% CI 89%-100%), with no false positive calls. Robust performance translated to FFPE: of the 20 ALK FISH+ specimens, 18 were unequivocally (+) by NGS, with remaining 2 specimens showing sub-threshold evidence of the events. 22/23 FISH- specimens were NGS-, with the remaining specimen an apparent false (-) by FISH. Clinical lung adenocarcinoma samples showed 5% ALK, 3% RET, and 2% ROS1 rearrangement rate respectively, in line with published data. Conclusions: We present rigorous validation and performance benchmarks for efficient targeted fusion gene detection for solid tumors in an NGS-based test for use in clinical oncology. Given the ability of NGS to detect a much broader range of genomic alterations than currently available technologies, particularly on limited tissue, this type of testing can be a direct component of patient care and potentially expand targeted treatment options. Citation Format: Roman Yelensky, Amy Donahue, Geoff Otto, Michelle Nahas, Jie He, Frank Juhn, Sean Downing, Garrett M. Frampton, Juliann Chmielecki, Jeffrey S. Ross, Maureen Zakowski, Marc Ladanyi, Vincent A. Miller, Philip J. Stephens, Doron Lipson. Analytical validation of solid tumor fusion gene detection in a comprehensive NGS-based clinical cancer genomic test. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2014-4699