Erica Schleifman

Next generation MUT-MAP, a high-sensitivity high-throughput microfluidics chip-based mutation analysis panel.

Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development.

The molecular landscape of high-risk early breast cancer: comprehensive biomarker analysis of a phase III adjuvant population

Breast cancer is a heterogeneous disease and patients are managed clinically based on ER, PR, HER2 expression, and key risk factors. We sought to characterize the molecular landscape of high-risk breast cancer patients enrolled onto an adjuvant chemotherapy study to understand how disease subsets and tumor immune status impact survival. DNA and RNA were extracted from 861 breast cancer samples from patients enrolled onto the United States Oncology trial 01062. Samples were characterized using multiplex gene expression, copy number, and qPCR mutation assays. HR+ patients with a PIK3CA mutant tumor had a favorable disease-free survival (DFS; HR 0.66, P=0.05), however, the prognostic effect was specific to luminal A patients (Luminal A: HR 0.67, P=0.1; Luminal B: HR 1.01, P=0.98). Molecular subtyping of triple-negative breast cancers (TNBCs) suggested that the mesenchymal subtype had the worst DFS, whereas the immunomodulatory subtype had the best DFS. Profiling of immunologic genes revealed that TNBC tumors (n=280) displaying an activated T-cell signature had a longer DFS following adjuvant chemotherapy (HR 0.59, P=0.04), while a distinct set of immune genes was associated with DFS in HR+ cancers. Utilizing a discovery approach, we identified genes associated with a high risk of recurrence in HR+ patients, which were validated in an independent data set. Molecular classification based on PAM50 and TNBC subtyping stratified clinical high-risk patients into distinct prognostic subsets. Patients with high expression of immune-related genes showed superior DFS in both HR+ and TNBC. These results may inform patient management and drug development in early breast cancer.

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.

Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab

Although programmed death-ligand 1–programmed death 1 (PD-L1–PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.A blood-based DNA sequencing assay to infer tumor mutational burden in the absence of tumor biopsy predicts response to PD-L1 blockade in patients with non-small-cell lung cancer.

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.

Development and Application of a Microfluidics-Based Panel in the Basal/Luminal Transcriptional Characterization of Archival Bladder Cancers.

In the age of personalized medicine stratifying tumors into molecularly defined subtypes associated with distinctive clinical behaviors and predictable responses to therapies holds tremendous value. Towards this end, we developed a custom microfluidics-based bladder cancer gene expression panel for characterization of archival clinical samples. In silico analysis indicated that the content of our panel was capable of accurately segregating bladder cancers from several public datasets into the clinically relevant basal and luminal subtypes. On a technical level, our bladder cancer panel yielded robust and reproducible results when analyzing formalin-fixed, paraffin-embedded (FFPE) tissues. We applied our panel in the analysis of a novel set of 204 FFPE samples that included non-muscle invasive bladder cancers (NMIBCs), muscle invasive disease (MIBCs), and bladder cancer metastases (METs). We found NMIBCs to be mostly luminal-like, MIBCs to include both luminal- and basal-like types, and METs to be predominantly of a basal-like transcriptional profile. Mutational analysis confirmed the expected enrichment of FGFR3 mutations in luminal samples, and, consistently, FGFR3 IHC showed high protein expression levels of the receptor in these tumors. Our bladder cancer panel enables basal/luminal characterization of FFPE tissues and with further development could be used for stratification of bladder cancer samples in the clinic.

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 4901: Comparison of gene expression platforms: RNA-Seq, Fluidigm, and Nanostring

Accurately measuring the expression of genes in formalin-fixed paraffin embedded (FFPE) tumor tissues has long been a struggle due to the inherent degradation of RNA isolated from these materials. Accurate quantification of gene expression levels in FFPE samples can enable the testing of biomarker hypotheses in the clinic and can potentially be used for patient stratification or selection in clinical trials. Platforms such as Fluidigm, Nanostring and microarrays are currently the high-throughput technologies utilized. Each of these platforms has different drawbacks and challenges such as lack of sensitivity, reproducibility or dynamic range when working with degraded FFPE samples. Currently, whole transcriptome RNA sequencing (RNA-Seq) is the only platform that offers a truly high-throughput, sensitive, and reproducible method to accurately quantify mRNA levels in RNA derived from FFPE samples. RNA-Seq, however has not yet been widely evaluated and adapted for use with degraded FFPE samples. Here we report the direct comparison of whole transcriptome RNA-Seq with two platforms that are currently compatible with FFPE derived RNA, Fluidigm and Nanostring, to determine the accuracy and feasibility of using this technology on degraded RNA. Utilizing a collection of matched fresh frozen (FF) and FFPE samples we analyzed gene expression using all three platforms to allow for a direct evaluation of each technology. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE derived RNA. By titrating down the input of FFPE RNA into the RNA-Seq library prep we were also able to define the optimal input of FFPE RNA needed to accurately and reproducibly quantify gene expression. This work allows for a systematic comparison of different gene expression platforms for their use with degraded FFPE RNA. Citation Format: Erica B. Schleifman, Maipelo Motlhabi, Craig Cummings, Rin Nakamura, Linda Bosch, Rajesh Patel, An Do, Andrew Watson, Thomas Sandmann, Walter Darbonne, Ian McCaffery, Eric Peters, Rajiv Raja. Comparison of gene expression platforms: RNA-Seq, Fluidigm, and Nanostring. [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 4901. doi:10.1158/1538-7445.AM2015-4901

Abstract B024: Comprehensive biomarker profiling of matched primary and metastatic estrogen receptor positive breast cancers

Patients with newly diagnosed, early stage estrogen receptor positive (ER+) breast cancer often show disease free survival in excess of five years following surgery and systemic adjuvant therapy. As such, an important question is whether diagnostic tumor tissue from the primary lesion offers an accurate molecular portrait of the cancer post recurrence and thus may be used for predictive diagnostic purposes for patients with relapsed, metastatic disease. To address this question, we performed detailed biomarker analyses on matched, asynchronous primary and metastatic tumors from 77 patients with ER+ breast cancer. The class I phosphatidylinositol 39 kinase (PI3K) is thought to be an important driver in ER+ breast cancer and has been linked to acquired resistance to hormonal therapy. We thus examined whether mutations in PIK3CA and loss of PTEN showed differences in primary and metastatic samples. We also sought to look more broadly at markers reflective of proliferation, molecular subtype, and key receptors and signaling pathways. To accomplish this, we developed an analysis platform using the Fluidigm BioMark™ microfluidics system to measure the relative expression of 90 breast cancer related genes in formalin-fixed paraffin-embedded (FFPE) tissue. Application of this panel of assays to matched tumor pairs showed a very high concordance between primary and metastatic tissue, with generally few changes in mutation status, proliferative markers, or gene expression between matched samples. Thus, archival primary tumor tissue may still provide an accurate portrait of biomarker status in patients with disease recurrence. Citation Format: Jill M. Spoerke, Erica B. Schleifman, Rupal Desai, Yuanyuan Xiao, Cheryl Wong, Ilma Abbas, Carol O9Brien, Rajesh Patel, Teiko Sumiyoshi, Ling Fu, Rachel Tam, Hartmut Koeppen, Timothy Wilson, Rajiv Raja, Garret M. Hampton, Mark R. Lackner. Comprehensive biomarker profiling of matched primary and metastatic estrogen receptor positive breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B024.

Abstract 3462: High-throughput qRT-PCR expression profiling of estrogen receptor positive breast tumors.

The class I phosphatidylinositol 3’ kinases (PI3K) play a major role in proliferation and survival in a wide variety of human cancers, and activation of the PI3K pathway is thought to be an important driver in estrogen receptor positive (ER+) breast cancer. A key factor in successful development of drugs targeting this pathway will be development in appropriate molecular subsets. Important questions relevant to PI3K inhibitor development in ER+ breast cancers are whether these inhibitors will work equally well in luminal A compared to luminal B tumors, and whether gene expression signatures of pathway activation may have additional utility in patient stratification beyond PIK3CA mutation status alone. The goal of this study was to develop a methodology for high throughput profiling of ER+ breast cancers, in order to enable molecular subtyping of patients enrolled in clinical studies. To accomplish this, we developed an analysis platform to measure the relative expression of 90 breast cancer and PI3K pathway specific genes in formalin-fixed paraffin-embedded (FFPE) tissue. The content for this panel consists of genes known to be important for epithelial-mesenchymal biology, proliferation rate, and transcriptional output of the PI3K pathway. The 96 assay panel (including 6 housekeeping genes) was developed on the Fluidigm Biomark microfluidics platform and was extensively validated using well-characterized breast cancer cell lines and FFPE breast cancer samples of known subtypes based on immunohistochemistry for HER2, ER, and PR. All assays showed high levels of inter-and intra-chip reproducibility and were sensitive on standard curves down to 3ng RNA input. Using this method we were able to separate breast cancers into distinct molecular subtypes, as well as identify more proliferative luminal B type tumors. In addition, PIK3CA mutation status, a potential biomarker, was determined using a highly specific and sensitive qRT-PCR mutation assay, in order to allow comparison with the PI3K pathway activation signature. We extended these analyses to a small cohort of patient samples consisting of matched primary and metastatic tumor tissues, and report here the correlation of primary and matched metastatic ER+ breast cancer FFPE tumor samples at both the gene expression and mutational levels. We found that the majority of matched pairs were concordant for both mutation status and gene expression, though a subset did show differences. Future studies will examine the prognostic significance and clinical relevance of this gene signature. Citation Format: Erica B. Schleifman, Rupal M. Desai, Jill Spoerke, Cheryl Victoria Wong, Ilma Abbas, Carol O9Brien, Garret Hampton, Timothy Wilson, Hartmut Koeppen, Rajesh Patel, Teiko Sumiyoshi, Ling Fu, Rachel Tam, Rajiv Raja, Mark Lackner. High-throughput qRT-PCR expression profiling of estrogen receptor positive breast tumors. [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 3462. doi:10.1158/1538-7445.AM2013-3462