Marcia Eisenberg

Comparison of KRAS Mutation Assessment in Tumor DNA and Circulating Free DNA in Plasma and Serum Samples

Testing for mutations in the KRAS oncogene for patients with metastatic colorectal cancer (mCRC) is generally performed using DNA from formalin-fixed paraffin-embedded tumor tissue; however, access to specimens can be limited and analysis challenging. This study assessed the identification of KRAS mutations in circulating free DNA (cfDNA) using a commercially available KRAS polymerase chain reaction (PCR) kit. Matched plasma, serum and tumor samples were available from 71 patients with mCRC who had received prior therapy but whose disease progressed following therapy. Yields of cfDNA from plasma and serum samples were comparable. Analyses were successful in 70/71 plasma-extracted samples (specificity: 97%, sensitivity: 31%) and 67/71 serum-extracted samples (specificity: 100%, sensitivity: 25%). This study demonstrates that KRAS mutations can be detected in cfDNA using a commercially available KRAS PCR kit, confirming cfDNA as a potential alternative source of tumor DNA in a diagnostic setting if access to archival tumor specimens is limited.

A Single-Tube Quantitative Assay for mRNA Levels of Hormonal and Growth Factor Receptors in Breast Cancer Specimens

Knowledge of estrogen receptor (ER) and progesterone receptor (PR) status has been critical in the evolution of modern targeted therapy of breast cancer and remains essential for making informed therapeutic decisions. Recently, growth factor receptor HER2/neu (ERBB2) status has made it possible to provide another form of targeted therapy linked to the overexpression of this protein. Presently, pathologists determine the receptor status in formalin-fixed, paraffin-embedded sections using subjective, semiquantitative immunohistochemistry (IHC) assays and quantitative fluorescence in situ hybridization for HER2. We developed a single-tube multiplex TaqMan (mERPR+HER2) assay to quantitate mRNA levels of ER, PR, HER2, and two housekeeping genes for breast cancer formalin-fixed, paraffin-embedded sections. Using data from the discovery sample sets, we evaluated IHC-status-dependent cutoff-point and IHC-status-independent clustering methods for the classification of receptor status and then validated these results with independent sample sets. Compared with IHC-status, the accuracies of the mERPR+HER2 assay with the cutoff-point classification method were 0.98 (95% CI: 0.97-1.00), 0.92 (95% CI: 0.88-0.95), and 0.97 (95% CI: 0.95-0.99) for ER, PR, and HER2, respectively, for the validation sets. Furthermore, the areas under the receiver operating-characteristic curves were 0.997 (95% CI: 0.994-1.000), 0.967 (95% CI: 0.949-0.985), and 0.968 (95% CI: 0.915-1.000) for ER, PR, and HER2, respectively. This multiplex assay provides a sensitive and reliable method to quantitate hormonal and growth factor receptors.

Global transcriptome dysregulation in second trimester fetuses with FMR1 expansions

We tested the hypothesis that FMR1 expansions would result in global gene dysregulation as early as the second trimester of human fetal development.

Validation of the Lung Subtyping Panel in Multiple Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Lung Tumor Gene Expression Data Sets.

Context .- A histologic classification of lung cancer subtypes is essential in guiding therapeutic management. Objective .- To complement morphology-based classification of lung tumors, a previously developed lung subtyping panel (LSP) of 57 genes was tested using multiple public fresh-frozen gene-expression data sets and a prospectively collected set of formalin-fixed, paraffin-embedded lung tumor samples. Design .- The LSP gene-expression signature was evaluated in multiple lung cancer gene-expression data sets totaling 2177 patients collected from 4 platforms: Illumina RNAseq (San Diego, California), Agilent (Santa Clara, California) and Affymetrix (Santa Clara) microarrays, and quantitative reverse transcription-polymerase chain reaction. Gene centroids were calculated for each of 3 genomic-defined subtypes: adenocarcinoma, squamous cell carcinoma, and neuroendocrine, the latter of which encompassed both small cell carcinoma and carcinoid. Classification by LSP into 3 subtypes was evaluated in both fresh-frozen and formalin-fixed, paraffin-embedded tumor samples, and agreement with the original morphology-based diagnosis was determined. Results .- The LSP-based classifications demonstrated overall agreement with the original clinical diagnosis ranging from 78% (251 of 322) to 91% (492 of 538 and 869 of 951) in the fresh-frozen public data sets and 84% (65 of 77) in the formalin-fixed, paraffin-embedded data set. The LSP performance was independent of tissue-preservation method and gene-expression platform. Secondary, blinded pathology review of formalin-fixed, paraffin-embedded samples demonstrated concordance of 82% (63 of 77) with the original morphology diagnosis. Conclusions .- The LSP gene-expression signature is a reproducible and objective method for classifying lung tumors and demonstrates good concordance with morphology-based classification across multiple data sets. The LSP panel can supplement morphologic assessment of lung cancers, particularly when classification by standard methods is challenging.

Epi proColon andreg; : Use of a Non-Invasive SEPT 9 Gene Methylation Blood Test for Colorectal Cancer Screening: A National Laboratory Experience

The Epi proColon® test is the first and only FDA-approved molecular blood test for colorectal cancer (CRC) screening. The high-sensitivity Real-Time PCR method detects a hypermethylated promoter region of the Septin 9 gene (SEPT9) shown to be associated with colorectal cancer when present in cell-free plasma. Following established protocols for new test assessments, the Epi proColon® test validation results were found to be comparable to the manufacturer’s stated performance characteristics. With familiar PCR methodology and minimal operator to operator variability, the test has been easily integrated into clinical molecular laboratory workflows. To accommodate our standardized process logistics, we validated the substitution of three 4 mL EDTA Vacutainer® collection tubes (Becton Dickinson) for the manufacturer-recommended single 10 mL tube for use in all patient specimen collection centers. By mid-year 2017, 2,238 Epi proColon® tests were ordered by our clinical provider-base for individuals of recommended screening ages 50-74. Positivity rates in this cohort were comparable to published clinical trial rates for the test. For those people who remain resistant to other methods of CRC screening, the availability of a blood test that may be conveniently drawn at a local community collection center may help surmount barriers of nonparticipation. This report summarizes LabCorp’s validation, implementation and clinical experience for on-boarding and offering the Epi-proColon test to healthcare providers and patients.

Predicting response to checkpoint inhibitors in melanoma beyond PD-L1 and mutational burden

BackgroundImmune checkpoint inhibitors (ICIs) have changed the clinical management of melanoma. However, not all patients respond, and current biomarkers including PD-L1 and mutational burden show incomplete predictive performance. The clinical validity and utility of complex biomarkers have not been studied in melanoma.MethodsCutaneous metastatic melanoma patients at eight institutions were evaluated for PD-L1 expression, CD8+ T-cell infiltration pattern, mutational burden, and 394 immune transcript expression. PD-L1 IHC and mutational burden were assessed for association with overall survival (OS) in 94 patients treated prior to ICI approval by the FDA (historical-controls), and in 137 patients treated with ICIs. Unsupervised analysis revealed distinct immune-clusters with separate response rates. This comprehensive immune profiling data were then integrated to generate a continuous Response Score (RS) based upon response criteria (RECIST v.1.1). RS was developed using a single institution training cohort (n = 48) and subsequently tested in a separate eight institution validation cohort (n = 29) to mimic a real-world clinical scenario.ResultsPD-L1 positivity ≥1% correlated with response and OS in ICI-treated patients, but demonstrated limited predictive performance. High mutational burden was associated with response in ICI-treated patients, but not with OS. Comprehensive immune profiling using RS demonstrated higher sensitivity (72.2%) compared to PD-L1 IHC (34.25%) and tumor mutational burden (32.5%), but with similar specificity.ConclusionsIn this study, the response score derived from comprehensive immune profiling in a limited melanoma cohort showed improved predictive performance as compared to PD-L1 IHC and tumor mutational burden.

Abstract 2757: Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets

The ability to characterize molecular features of cancer from liquid biopsies is resulting in the development of innovative health care for patients. Longitudinal changes in the mutational profiles of DNA isolated from liquid biopsies are being used to better understand and monitor the development, progression, and evolution of therapy resistance in cancer patients. To define changes in the mutational landscape and predict drug susceptibilities in Triple Negative Breast Cancer (TNBC) patients, we used whole exome analysis to profile circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from eight selected time points of a patient enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001). The patient initially received weekly cisplatin infusions followed by additional targeted therapy. Peripheral blood samples were collected at specific time points over a period of 272 days following enrollment in the clinical trial. Our data indicates that the identified mutations in genomic DNA isolated from CTCs and ctDNA can be used to understand and mitigate the impact of tumor heterogeneity in addition to identifying clinically relevant mutations at these selected time points. To further increase the resolution of our analysis, we profiled ctDNA from these samples to a higher depth targeting only clinically relevant genes. These analyses increased the sensitivity of detection and identified additional targets that could have been used for therapeutic intervention. In addition to sequence variants, copy number variations (CNVs) have also been significantly associated with the development of metastasis and changes in CNVs have been used to monitor disease progression. We performed a bioinformatics analysis of genomic instability and CNVs across 32 different time points from ctDNA from the same patient throughout the treatment period. The genomic instability number (GIN) calculated for each of the 32 time points seems to mirror the overall CTC burden in the patient at each time point tested. CNV analysis is ongoing and these data sets are being further analyzed in combination with TCGA data to define possible cancer driver genes for the functional prediction of significant TNBC candidate alterations and the results of these analyses will be presented. Citation Format: Kellie Howard, Kimberly Kruse, Brianna Greenwood, Elliott Swanson, Mathias Ehrich, Christopher K. Ellison, Taylor Jensen, Sharon Austin, Arturo Ramirez, Debbie Boles, John Pruitt, Elisabeth Mahen, Jackie L. Stilwell, Eric P. Kaldjian, Michael Dorschner, Sibel Blau, Marcia Eisenberg, Steve Anderson, Anup Madan. Using liquid biopsies and NGS as tools to analyze mutation burden and copy number variation in the blood of a patient with triple negative breast cancer to better inform therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2757. doi:10.1158/1538-7445.AM2017-2757

Abstract 498: Meta-analysis of genomic aberrations identified in CTCs andctDNA in triple negative breast cancer

Technological innovation and scientific advances in understanding cancer at the molecular level have accelerated the discovery and development of both diagnostics and therapeutics. Circulating tumor cells (CTCs) and plasma circulating tumor DNA (ctDNA) are non-invasive prognostic markers that have been associated with metastatic and aggressive disease. Both CTCs and ctDNA allow molecular characterization of a tumor that is inaccessible or too risky to biopsy. The analysis of genomic aberrations in both sample types provides insights into drug resistance and can help determine appropriate, targeted cancer treatments. Mutations found in the primary or metastatic tumor can be identified in both CTCs and ctDNA as well as novel mutations that may reflect intratumoral and intermetastatic heterogeneity. When collected and evaluated over an extended period of time, changes in the CTC and/or ctDNA mutational profile can offer guidance into the effectiveness of a treatment, indicate the progression of disease, and detect recurrence of disease earlier. We have performed whole exome sequencing of CTCs and ctDNA from a metastatic triple negative breast cancer (TNBC) patient to better understand the evolution of tumor heterogeneity during therapy. The patient was enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001) and initially received weekly cisplatin infusions followed by additional targeted therapy. Longitudinal peripheral blood samples were collected over a period of 272 days following enrollment in the clinical trial. CTCs were identified using the AccuCyte-CyteFinder system (RareCyte, Seattle WA). We used next generation sequencing, and computational biology tools to analyze genomic DNA from multiple CTCs, white blood cells (WBCs) and ctDNA from various time points. We observed similar genomic aberrations in both CTCs and ctDNA that could be classified into three groups: a) a static group that remains unchanged during the course of therapy, b) a sample-specific group that is unique to each time point and c) an intermediate group that has variants that are short-lived but are present across multiple time points. Variants identified in the liquid biopsy samples were compared with variants observed in primary breast tumor, metastatic bone marrow tumor and publically available pan-cancer datasets. We then performed meta-analysis on somatic variants to identify changes in affected networks in response to therapy over time. Several key nodes were identified that could rationally have been targeted for therapy using compounds currently in clinical trials. We then compared and combined the perturbed networks obtained from the CTCs and ctDNA to better understand the etiology of TNBC. These studies represent the first step of a synergistic partnership between the genetic information obtained from the analysis of CTCs and ctDNA with innovative health care for patients with metastatic breast cancer. Citation Format: Kellie Howard, Sharon Austin, Fang Yin Lo, Arturo Ramirez, Debbie Boles, John Pruitt, Elisabeth Mahen, Heather Collins, Amanda Leonti, Lindsey Maassel, Christopher Subia, Tuuli Saloranta, Nicole Christopherson, Kerry Deutsch, Jackie Stilwell, Eric Kaldjian, Michael Dorschner, Sibel Blau, Anthony Blau, Marcia Eisenberg, Steven Anderson, Anup Madan. Meta-analysis of genomic aberrations identified in CTCs andctDNA in triple negative breast cancer. [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 498.

Abstract P2-02-21: Longitudinal analysis of circulating tumor cells and cell free tumor DNA by next generation sequencing in triple negative breast cancer

As the practice of genetically profiling patient tumors is considered for making clinical treatment decisions, recent methodologies for screening of genomic aberrations in circulating tumor cells (CTCs) and cell-free plasma DNA (cfDNA) may provide non-invasive tools for such applications. Genomic analysis of DNA from CTCs and plasma can also provide useful insight into tumor heterogeneity and thus disease progression by revealing sub-populations of tumor cells that evolve during treatment, have novel drug-resistant genotypes, or carry alternate cancer driver mutations not identified by the sequencing of primary tumors. Comprehensive evaluation of DNA isolated from CTCs and cfDNA from a breast cancer patient by whole exome sequencing was performed to better understand the role of liquid biopsies in investigating the etiology of tumor progression. The patient was diagnosed with metastatic triple negative breast cancer (TNBC) six years after remission from estrogen receptor (ER-3+), progesterone receptor (PR-1+), human epidermal receptor growth factor 2 negative (Her2-), grade 3 intra-ductal carcinoma of the right breast. Metastatic lesions were found in the spine, pelvis and sacrum and bone-marrow. The patient was enrolled in the Intensive Trial of OMics in Cancer clinical Trial (ITOMIC-001; ClinicalTrials.gov ID NCT01957514) and initially received weekly cisplatin infusions followed by additional targeted therapy. Peripheral blood was obtained during regular clinic visits over the 272 days the patient was enrolled in the clinical trial. CTCs were identified and enumerated from each blood draw using the AccuCyte® -CyteFinder® (AC/CF) system (RareCyte, Seattle WA). Multiple CTCs along with white blood cells (WBCs) were picked from various time points throughout the treatment regimen. The selected CTCs and WBCs were whole genome amplified and whole exome sequencing was performed to identify tumor specific variants. A comparative analysis with variants present in genomic DNA isolated from the bone-marrow metastasis tissue biopsy samples and cfDNA revealed the evolution of tumor-specific variants during therapy. Each CTC had somatic alterations in genes associated with therapies in current use or those in the clinical trials setting. Sequencing analysis of cfDNA provided similar information on potential therapeutic approaches. The monitoring of disease over time through genomic analysis of CTCs and cfDNA can identify novel sub-populations related to disease progression for the tailoring of cancer treatment regimens. Further analysis is being performed to better understand the evolution of the genomic heterogeneity among CTCs at the same time point and across different time points and therefore better understand the etiology of progression of metastatic breast cancer in this patient. Citation Format: Howard K, Austin S, Ramirez AB, Ritter L, Boles D, Pruitt J, Collins H, Mahen E, Leonti A, Maassel L, Subia C, Tuuli S, Heying N, Deutsch K, Cox J, Lo FY, Stilwell JL, Kaldjian EP, Dorschner M, Blau S, Blau A, Eisenberg M, Anderson S, Madan A. Longitudinal analysis of circulating tumor cells and cell free tumor DNA by next generation sequencing in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-21.

Abstract 1609: Comprehensive multi-omic analysis of circulating tumor cells isolated from a metastatic triple-negative breast cancer patient to identify pathogenic genomic aberrations

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Increasing evidence confirms the prognostic relevance of Circulating Tumor Cells (CTCs) in a variety of cancers including advanced breast cancer. Recent data also suggests that CTCs are a useful tool for monitoring treatment and identifying potential targets for therapeutic intervention. The objective of this study was to investigate technologies that can be used for defining the genomic landscape of CTCs in order to compare the derived genomic information (1) among CTCs to assess genetic heterogeneity and (2) with that derived from bone-marrow metastasis tissue biopsy (BMM) samples to assess how reflective the molecular profile from CTCs is to the metastasis. To evaluate these potential applications, positive CTCs were identified using the AccuCyte -CyteFinder (AC/CF) system (RareCyte, Seattle WA) from the blood of a patient with triple negative breast cancer (TNBC). Twenty CTCs and twenty white blood cells (WBCs) were picked from slides using the AC/CF system. To determine whether tumor specific genomic characteristics are reflected in the isolated cells, we used whole genome amplification (WGA) followed by next-generation sequencing to perform a comprehensive analysis using WBCs as genome controls. Whole genome, exome and targeted sequencing of known cancer-associated genes using Illumina and Life Tech panels identified mutations in TP53, PTEN, ERBB2, STK11, ABL1, HRAS, MLL2 and INPPL1 which were present in the CTCs alone and not in the WBCs. Further analysis comparing the results between CTCs revealed that the majority of the identified mutations were specific to individual CTCs revealing a high degree of genetic heterogeneity. Only 5% of mutations were shared between at least 40% of the CTCs examined and included mutations in ATM, ALK, BRAF, NOTCH1, ATR, JAK3, COL1A1 and XPC. Additionally, all of the profiled CTCs contained two novel mutations in LPP and HLA-A which were not present in the WBCs. Mutations in these genes have recently been associated with aggressive solid tumors. Genetic heterogeneity was also observed in the WBC population enabling the calculation and subsequent subtraction of background noise associated with WGA of single cells. Molecular information derived from the CTCs is being compared to multiple BMM samples from the same patient. Additional analyses of copy number and structural variations and transcriptomic analysis are being performed in order to gain further insights into the genetic heterogeneity of CTCs and identify genomic markers to establish the utility of CTCs as a non-invasive real-time liquid biopsy for breast cancer. Citation Format: Kellie Howard, Sharon Austin, Arturo Ramirez, Leila Ritter, Debbie Boles, James Cox, Fang Yin Lo, Kerry Deutsch, Christopher Subia, Tuuli Saloranta, Nicole Heying, Heather Collins, Amanda Leonti, Lindsey Maassel, Jackie Stilwell, Eric Kaldjian, Michael Dorschner, Anthony Blau, Marcia Eisenberg, Steven Anderson, Anup Madan. Comprehensive multi-omic analysis of circulating tumor cells isolated from a metastatic triple-negative breast cancer patient to identify pathogenic genomic aberrations. [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 1609. doi:10.1158/1538-7445.AM2015-1609