Geneva Young

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.

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 P6-07-08: The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples

Background: Approximately 70% of newly diagnosed breast cancers express estrogen receptor alpha (ERα), and are treated with agents that block ER signaling. Acquired mutations in ESR1, the gene that encodes ERα, have been associated with resistance to aromatase inhibitor therapy in patients with ER positive metastatic breast cancer (ER+ mBC). The most frequently occurring ESR1 mutations are clustered between amino acids 536 to 538 within the ligand binding domain (LBD), although limited data exists characterizing the full mutation profile in a large number of breast cancer samples. Methods: We surveyed the Foundation Medicine dataset of 7590 primary and metastatic breast cancer tumor samples for ESR1 short variants and copy number alterations. Hormone receptor status was unavailable, therefore two assumptions were made to provide an estimate of prevalence in the ER+ HER2- population: 70% of the tumor samples are from ER+ HER2- patients, and all ESR1 mutations from non-HER2 amplified metastatic sites are from ER+ HER2- patients. In a separate cohort of 48 ER+ mBC patients, circulating tumor DNA (ctDNA) was analyzed for ESR1 mutations using the BEAMing method by Sysmex and with Foundation Medicine9s sequencing assay, FoundationACT (Assay for Circulating Tumor DNA). Results: The prevalence of mutations in ER+ HER2- breast cancer was estimated to be 22% in samples from metastatic sites but less than 3% in samples from primary sites. ESR1 amplification was rare in samples from both primary and metastatic disease sites at 1.3% and 2.0% respectively. A total of 153 unique short variants of known and unknown status were identified. In addition to hotspot mutations at 537 and 538, previously undescribed rare mutations were identified throughout the entire length of the LBD, although 10 alterations at amino acids 380, 463, 536, 537, and 538 account for 86% of all ESR1 mutations in the ER+ HER2- metastatic sites. We also characterized the overlap of ESR1 alterations with commonly altered and clinically relevant genes in breast cancer, including PIK3CA mutations and HER2 amplification, and we report here a landscape of co-occurring alterations. In the cohort of patient samples where ctDNA was analyzed, BEAMing and FoundationAct assays both detected ESR1 mutations in 19 out of 48 samples, and overall concordance of mutation status (wild-type vs mutant) was 100%. A total of 51 individual mutations were detected with the BEAMing assay, 42 of which were detected with the FoundationACT assay. Seven mutations that were undetected by FoundationACT had mutant allele frequencies less than 0.1%. Ten ESR1 mutations were detected only by FoundationACT, 9 of which are not covered with the BEAMing assay. Alterations in PIK3CA, CDH1, TP53, ERBB2, and other breast cancer relevant genes were also detected with FoundationACT. Conclusions: Understanding the mutational landscape of ESR1 and co-occurring alterations is important for diagnostic development in conjunction with the clinical development of novel anti-endocrine therapies. Our data demonstrate a large spectrum of mutations in the LBD in addition to known hotspot mutations. In addition, the FoundationACT assay offers a robust NGS-based method to screen for mutations in ctDNA that is highly concordant with digital PCR methods. Citation Format: Spoerke JM, Schleifman E, Clark TA, Young G, Nahas M, Kennedy M, Young L, Chmielecki J, Otto GA, Lipson D, Wilson TR, Gendreau S, Lackner MR. The complete spectrum of ESR1 mutations from 7590 breast cancer tumor samples [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-08.

Abstract 3965: Rigorous validation of a clinical circulating tumor DNA assay for cancer molecular profiling

Background: Profiling cell-free circulating tumor DNA (ctDNA) for genomic alterations which drive oncogenesis in patients with cancer promises to provide information important for understanding cancer biology, informing therapy selection when conventional FFPE biopsies are unobtainable and potentially monitoring response to therapy. To allow routine use of blood-based ctDNA molecular profiling with clinical samples we developed and performed analytic validation of an accurate, targeted NGS-based assay. The analytic validation included over 400 samples demonstrating ≥99% sensitivity and ≥99% positive predictive value for base substitutions, indels and rearrangements with limit-of-detection below 1%. Methods: To ensure robust performance, the ctDNA assay was developed as part of an integrated workflow including sample collection, storage and transport, and ctDNA purification, followed by optimized construction of adaptor-ligated sequencing libraries and enrichment by solution hybridization and then sequencing to high depth (Illumina HiSeq). Computational methodologies were developed to enable sensitive and specific detection of base substitutions, indels, genomic rearrangements and high-level amplifications from ctDNA. Accuracy and reproducibility were analytically validated in a CLIA-certified laboratory using reference samples with known alterations (117 cell-line mixtures and synthetic constructs) and 268 clinical ctDNA samples. Many alterations found in clinical ctDNA samples were validated with orthogonal reference methods including a CLIA-validated NGS assay, droplet digital PCR and break-point PCR. Results: The ctDNA assay validation demonstrated ≥99% sensitivity and ≥99% positive predictive value for base substitutions, indels and rearrangements with a limit-of-detection below 1% and robust detection of high-level, focal amplifications when present at adequate tumor fraction. In addition, the assay accurately reports the allele frequency of alterations in the sample. In 48 clinical ctDNA samples, 95 alterations of all classes were 100% confirmed by orthogonal testing. As part of our extensive clinical utility study, we report results comparing alterations from patient-matched ctDNA and FFPE biopsies across more than 200 lung, breast and other cancer samples. Conclusions: Accurate clinical profiling of ctDNA enables detection of genomic alterations in patient plasma samples to provide rationale targeted therapeutic options. Our rigorous analytic validation study demonstrates high-sensitivity detection of alterations present in blood at low frequency with a very low rate of false positives, realizing the potential of ctDNA-based molecular profiling for the management of patients with cancer. This validated assay allows us to embark upon a rigorous investigation of clinical best-practices based on tumor-type specific assessment of matched ctDNA and solid biopsy specimens. Citation Format: Travis A. Clark, Mark Kennedy, Jie He, Geneva Young, Mandy Zhao, Mike Coyne, Virginia Breese, Lauren Young, Shan Zhong, Mark Bailey, Bernard Fendler, Erica Schleifman, Eric Peters, Phil J. Stephens, Geoff A. Otto, Doron Lipson. Rigorous validation of a clinical circulating tumor DNA assay for cancer molecular profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3965.

Abstract 2415: Development of a clinical cell-free DNA assay for cancer molecular profiling

Background: Profiling cell-free circulating tumor DNA (ctDNA) for the genomic alterations which drive oncogenesis in patients with cancer promises to provide information important for understanding cancer biology, informing therapy selection when conventional biopsies are unobtainable and monitoring response to therapy. Facile collection of ctDNA samples could enable recurrent molecular profiling of an individual patient9s disease with lower cost, discomfort and risk as compared to conventional biopsies. To assess the clinical validity of profiling cancer genomics in ctDNA, a highly accurate targeted NGS-based assay was developed and ctDNA results were compared to patient-matched FFPE tumor biopsies characterized by comprehensive genomic profiling using the FoundationOne assay from over 150 patients with lung, breast and other cancer types. Methods: To ensure robust performance, the ctDNA assay was developed as part of an integrated workflow including primary sample collection, storage and transport, and ctDNA purification, followed by optimized construction of adaptor-ligated sequencing libraries and enrichment by solution hybridization and then sequencing to high depth (Illumina HiSeq). Computational methodologies were developed to enable sensitive and specific detection of base substitutions, indels, genomic rearrangements and high-level amplificagtions from ctDNA. Accuracy and reproducibility was assessed using cell-line mixtures designed to simulate the limited DNA inputs and low tumor purity expected in routine clinical samples. Results: The ctDNA assay enabled accurate detection of most genomic alterations in cell-line mixtures. Short variants (base substitutions and indels) and genomic rearrangements in ALK and RET were detected with high sensitivity and specificity at Conclusions: Accurate profiling of ctDNA can enable detection of biologically and clinically relevant genomic alterations in clinical plasma samples. These results demonstrate the potential utility of ctDNA molecular profiling for the management of patients with cancer, but prior to integration into routine practice, extensive rigorous tumor-type specific studies of patient-matched ctDNA and solid biopsy specimens are required. Citation Format: Travis Clark, Mark Kennedy, Geneva Young, Lauren Young, Jie He, Roman Yelensky, Siraj Ali, Geoff Otto, Doron Lipson, Vince Miller, Phil Stephens. Development of a clinical cell-free DNA assay for cancer molecular profiling. [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 2415. doi:10.1158/1538-7445.AM2015-2415

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 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.