Sean Downing

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.

Targeted Next-generation Sequencing of Advanced Prostate Cancer Identifies Potential Therapeutic Targets and Disease Heterogeneity

BACKGROUND Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue. OBJECTIVE To demonstrate the feasibility of a novel next-generation sequencing (NGS)-based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa. DESIGN, SETTING, AND PARTICIPANTS FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression. INTERVENTION At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations. RESULTS AND LIMITATIONS We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected. CONCLUSIONS This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.

Advanced urothelial carcinoma: next-generation sequencing reveals diverse genomic alterations and targets of therapy

Although urothelial carcinoma (UC) of the urinary bladder generally portends a favorable prognosis, metastatic tumors often follow an aggressive clinical course. DNA was extracted from 40 μm of formalin-fixed, paraffin-embedded (FFPE) sections from 35 stage IV UCs that had relapsed and progressed after primary surgery and conventional chemotherapy. Next-generation sequencing (NGS) was performed on hybridization-captured, adaptor ligation-based libraries for 3320 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer to at an average sequencing depth of 1164 × and evaluated for all classes of genomic alterations (GAs). Actionable GAs were defined as those impacting the selection of targeted anticancer therapies on the market or in registered clinical trials. A total of 139 GAs were identified, with an average of 4.0 GAs per tumor (range 0–10), of which 78 (56%) were considered actionable, with an average of 2.2 per tumor (range 0–7). Twenty-nine (83%) cases harbored at least one actionable GA including: PIK3CA (9 cases; 26%); CDKN2A/B (8 cases; 23%); CCND1 (5 cases; 14%); FGFR1 (5 cases; 14%); CCND3 (4 cases; 11%); FGFR3 (4 cases; 11%); MCL1 (4 cases; 11%); MDM2 (4 cases; 11%); EGFR (2 cases, 6%); ERBB2 (HER2/neu) (2 cases, 6%); NF1 (2 cases, 6%) and TSC1 (2 cases, 6%). Notable additional alterations included TP53 (19 cases, 54%) and RB1 (6 cases; 17%). Genes involved in chromatin modification were altered by nonsense mutation, splice site mutation or frameshift indel in a mutually exclusive manner in nearly half of all cases including KDM6A (10 cases; 29%) and ARID1A (7 cases; 20%). Comprehensive NGS of 35 UCs of the bladder revealed a diverse spectrum of actionable GAs in 83% of cases, which has the potential to inform treatment decisions for patients with relapsed and metastatic disease.

Relapsed Classic E-Cadherin (CDH1)–Mutated Invasive Lobular Breast Cancer Shows a High Frequency of HER2 (ERBB2) Gene Mutations

Purpose: We queried whether comprehensive genomic profiling using a next-generation sequencing–based assay could identify novel and unanticipated targets of therapy for patients with relapsed invasive lobular carcinoma (ILC). Experimental Design: DNA sequencing (Illumina HiSeq 2000) was conducted for 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on indexed, adaptor-ligated, hybridization-captured libraries using DNA isolated from formalin-fixed paraffin-embedded sections from 22 histologically verified ILC. Results: A total of 75 genomic alterations were identified with an average of 3.4 alterations per tumor (range, 1–6), of which 35 were actionable for an average of 1.59 actionable alterations per patient (range, 0–3). Nineteen of 22 (86%) of the ILC samples harbored at least one actionable alteration. Six (27%) cases featured alterations in ERRB2 including 4 (18%) with ERBB2 mutation, 1 (5%) with an ERBB2 gene fusion, and 1 (5%) with an ERBB2 copy number gain (amplification). The enrichment of ERBB2 mutations/fusion in CDH1-mutated ILC (5 of 22, 23%) compared with the 5 ERBB2 mutations in a series of 286 non-CDH1-mutated breast cancers from which the ILC cases were obtained (5 of 286, 2%) was significant (P = 0.0006). Conclusions: Comprehensive genomic profiling of relapsed CDH1-mutated ILC revealed actionable genomic alterations in 86% of cases, featured a high incidence of ERBB2 alterations, and can reveal actionable alterations that can inform treatment decisions for patients with ILC. Clin Cancer Res; 19(10); 2668–76. ©2013 AACR.

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.

Isolation and proteomic analysis of platelets by SELDI-TOF MS.

Many growth factors, leukotrines, and biological ligands are not circulating free in plasma or serum, except in the case of late or disseminated disease. During early tumor growth and angiogenesis, platelets actively and selectively sequester regulators of angiogenesis and, as such, the platelet protein content can be used as a marker of early tumor growth or angiogenesis. With the recent increase in the clinical use of biologic modifiers in cancer and chronic disease therapy, the search for markers of early disease, therapeutic response, and/or recurrence has suggested that analysis of platelet proteins may be more relevant and accurate. We provide a guideline for the proteomic analysis of platelet proteome, placing specific emphasis on angiogenesis regulators, even though other platelet proteins may serve as markers of disease in the future. The analysis of serum/plasma has been fraught with difficulties because of the extraordinarily large number of proteins and because some of the proteins are contained in extraordinarily large amounts, masking the less abundant proteins. Thus, platelets may provide a much more biologically relevant analyte for biomarker discovery.

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

Abstract B122: A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma.

Background: Micropapillary urothelial carcinoma of the urinary bladder (MPUC) encompasses approximately 5% of all bladder cancers and comprises approximately 3,000 to 4,000 new cases diagnosed each year in the US. MPUUPC is a highly aggressive form of bladder cancer associated with distant metastases and shortened patient survival. Once MPUC recurs progresses from loco-regional and progresses to metastatic disease, there is no currently no recognized effective treatment. We conducted a genomic analysis of a series of patients with MPUC to characterize the genomic landscape of MUPUC and identify targeted treatment options for patients with this lethal form of urologic malignancy. Methods: DNA was extracted from 40 microns of formalin-fixed paraffin embedded (FFPE) sections from 15 MPUC and 64 non-MPUC. Sequencing to high, uniform coverage was performed on hybridization-captured, adaptor ligated, hybridization capturedion based libraries for for 3,230 exons of 182 cancer-related genes plus 37 introns offrom 14 genes frequently rearranged in cancer to high, uniform coverage and evaluated for all classes of genomic alteration. Results: Extracellular domain mutations of ERBB2 were identified in 6/15 (40%) MPUC including: S310F (4 cases), S310Y (1 case) and R157W (1 case). All 6 cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification which was confirmed by immunohistochemistry (IHC) in the 3 cases where additional tissue was available. In contrast, 6/64 (9.4%) of non-MPUC harbored an ERBB2 alteration: base substitutions (3 cases), amplifications (2 cases) and gene fusion (1 case), which is higher than the 2/159 (1.3%) of protein changing ERBB2 mutations reported in urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared to non-MPUC is significant inbetween this series (p < 0.0084) and for all types of urinary tract cancer in COSMIC (p < 0.001). All 9 ERBB2 WT MPUC cases harbored at least 1 actionable alteration, including alterations in AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 and RAF1. Conclusions: Comprehensive genomic profiling of MPUC revealed actionable genomic alterations in all 15 specimens including a high incidence of ERBB2 extracellular domain mutation. We conclude that genomic profiling of MUPUC samples can reveal actionable alterations that can inform potential targeted treatment decisions for the majority of patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B122. Citation Format: Kai Wang, Jeff S. Ross, Laurie M. Gay, Rami N. Al-Rohil, Tipu Nazeer, Christine E. Sheehan, Timothy A. Jennings, Geoff A. Otto, Amy Donahue, Jie He, Gary Palmer, Siraj Ali, Michelle Nahas, Geneva Young, Elaine LaBrecque, Garrett Frampton, Rachel Erlich, John A. Curran, Tina Brennan, Sean R. Downing, Roman Yelensky, Doron Lipson, Matthew J. Hawryluk, Vincent A. Miller, Philip J. Stephens. A high frequency of activating extracellular domain ERBB2 (HER2) mutation in micropapillary urothelial carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B122.

Abstract 2279: Bringing next generation sequencing (NGS) to the clinic: Analytical validation of a comprehensive NGS-based cancer gene test.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: As the number of clinically relevant cancer genes grows and the amount of tissue available for analysis 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 demonstrate its full utility in the clinic, robust analytical validation approaches and performance benchmarks against gold standard methodologies are required. Methods: We have developed a comprehensive NGS-based diagnostic test to enable accurate detection of clinically-relevant genomic alterations across 4604 exons of 289 cancer genes (∼1.5 Mb) in routine FFPE specimens, including needle biopsies. To validate the test, we created synthetic reference standards spanning key determinants of detection accuracy for somatic alterations, such as a range of stromal admixture, indel length, and amplitude of copy change: For base substitutions, we mixed 2 pools of 10 normal cell-lines, thereby testing 1035 different variants in a broad 10%-100% allele frequency range. For indels, 28 tumor cell lines with 44 alterations (1-40bp) were mixed into 41 variably sized pools, creating a validation set of 163 events. For copy alterations, 7 matched tumor/normal cell-lines bearing a total of 12 amplifications (copy number 8-15) and 7 homozygous deletions were mixed in 5 tumor content ratios 20%-75%. The test was applied blinded to all pools and alteration calls compared to expectation based on constituents in each mix. We then verified that observed performance translated to FFPE by examining test concordance on 185 tumors characterized clinically for mutations in 8 genes including KRAS, BRAF, and EGFR (Sanger, Sequenom, and PCR) and 131 specimens characterized for CNAs in ERBB2, PTEN, AR and CCND1 (IHC and FISH). Results: On synthetic standards, performance reached >99% sensitivity for substitutions (1,035/1,035) and >98% for indels (161/163) at ≥10% allele-frequency with PPV>99%. Sensitivity for CNAs was >95% for focal amplifications (46/48) and >99% for homozygous deletions (28/28) at ≥30% tumor fraction, with PPV>99%. Robust performance translated to FFPE: concordance was 98% for both substitutions and indels (164/168) and CNAs (41/42) relative to prior calls. Conclusions: We present a rigorous validation approach and performance benchmarks for a comprehensive 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, we suggest that this type of testing can be a direct component of patient care and potentially expand targeted treatment options. Citation Format: Roman Yelensky, Garrett M. Frampton, Alex Fichtenholtz, Sean Downing, Jie He, Frank Juhn, Kristina Brennan, Kai Wang, Geoff Otto, Mirna Jarosz, Alex Parker, Jeffrey S. Ross, John Curran, Maureen T. Cronin, Philip J. Stephens, Doron Lipson. Bringing next generation sequencing (NGS) to the clinic: Analytical validation of a comprehensive NGS-based cancer gene test. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2279. doi:10.1158/1538-7445.AM2013-2279