Elisabeth Mahen

Comprehensive statistical inference of the clonal structure of cancer from multiple biopsies

A comprehensive characterization of tumor genetic heterogeneity is critical for understanding how cancers evolve and escape treatment. Although many algorithms have been developed for capturing tumor heterogeneity, they are designed for analyzing either a single type of genomic aberration or individual biopsies. Here we present THEMIS (Tumor Heterogeneity Extensible Modeling via an Integrative System), which allows for the joint analysis of different types of genomic aberrations from multiple biopsies taken from the same patient, using a dynamic graphical model. Simulation experiments demonstrate higher accuracy of THEMIS over its ancestor, TITAN. The heterogeneity analysis results from THEMIS are validated with single cell DNA sequencing from a clinical tumor biopsy. When THEMIS is used to analyze tumor heterogeneity among multiple biopsies from the same patient, it helps to reveal the mutation accumulation history, track cancer progression, and identify the mutations related to treatment resistance. We implement our model via an extensible modeling platform, which makes our approach open, reproducible, and easy for others to extend.

Participant Attitudes Toward an Intensive Trial of Multiple Biopsies, Multidimensional Molecular Analysis, and Reporting of Results in Metastatic Triple-Negative Breast Cancer

Purpose Multidimensional molecular analysis of tumor tissue intensively over space and time can provide insight into how cancers evolve and escape treatment. Attitudes of participants in such trials have not been assessed. We explored patient views regarding an intensive study incorporating multiple biopsies, multidimensional molecular testing, and drug response predictions that are reported to the oncologist and patient. Patients and Methods A structured, self-administered survey was conducted among the first 15 patients enrolled in ITOMIC-001 (Intensive Trial of Omics in Cancer). Patients with metastatic triple-negative breast cancer were accrued at two sites in Washington state. Surveys containing 17 items were administered at enrollment and after the return of results. Surveys explored perceptions regarding risks, personal benefits, benefits to others, uncertainties associated with interpreting complex molecular results, concerns regarding multiple biopsies, and potential loss of confidentiality. At follow-up, three additional unique items explored patient coping. Results All participants expressed a strong desire for their experiences to benefit others, and all perceived a higher likelihood of deriving benefit than described during detailed consent discussions. Loss of confidentiality ranked lowest among patient concerns. Despite acknowledging uncertainties and risks inherent in complex molecular testing for clinical reporting, participants wanted access to findings in evaluating treatment choices, even if the best available evidence was weak. Follow-up surveys demonstrated relatively little change in attitudes, although concern about study biopsies generally declined. Study participation helped several patients cope better with their disease. Conclusion In advanced breast cancer, these findings demonstrate the feasibility of engaging motivated patients in trials that navigate the uncertainties associated with intensive spatial and longitudinal multidimensional molecular testing for the purpose of advancing precision medicine.

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

Cis-Compound Mutations are Prevalent in Triple Negative Breast Cancer and Can Drive Tumor Progression

About 16% of breast cancers fall into a clinically aggressive category designated triple negative (TNBC) due to a lack of ERBB2, estrogen receptor and progesterone receptor expression1-3. The mutational spectrum of TNBC has been characterized as part of The Cancer Genome Atlas (TCGA)4; however, snapshots of primary tumors cannot reveal the mechanisms by which TNBCs progress and spread. To address this limitation we initiated the Intensive Trial of OMics in Cancer (ITOMIC)-001, in which patients with metastatic TNBC undergo multiple biopsies over space and time5. Whole exome sequencing (WES) of 67 samples from 11 patients identified 426 genes containing multiple distinct single nucleotide variants (SNVs) within the same sample, instances we term Multiple SNVs affecting the Same Gene and Sample (MSSGS). We find that >90% of MSSGS result from cis-compound mutations (in which both SNVs affect the same allele), that MSSGS comprised of SNVs affecting adjacent nucleotides arise from single mutational events, and that most other MSSGS result from the sequential acquisition of SNVs. Some MSSGS drive cancer progression, as exemplified by a TNBC driven by FGFR2(S252W;Y375C). MSSGS are more prevalent in TNBC than other breast cancer subtypes and occur at higher-than-expected frequencies across TNBC samples within TCGA. MSSGS may denote genes that play as yet unrecognized roles in cancer progression.

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 P4-01-21: Analysis and single-cell retrieval of circulating tumor cells to monitor treatment response and assess genotype in triple-negative breast cancer

Introduction: We used a high-recovery rare cell analysis and single-cell picking system to enrich, visualize, and isolate circulating tumor cells (CTCs) for genomic analysis from the blood of patients with advanced triple-negative breast cancer (TNBC) undergoing treatment with cisplatin as part of a study to intensively characterize TNBC. CTCs were evaluated regularly during treatment to monitor CTC burden and characteristics that could be associated with treatment response or disease progression, and perform single-cell mutational analysis to inform clinical decision making. Methods: Patients were enrolled in the study at the University of Washington Center for Cancer Innovation after informed consent for participation in investigation of their disease, including molecular analysis of multiple biopsies of accessible tumor. CTCs were evaluated prior to treatment and tracked longitudinally. Density-based enrichment of blood cells was performed using the AccuCyte tube, float and collector system. Collected cells were processed and applied to microscopic slides. Fluorescently labeled antibodies to cytokeratin, CD45 and EpCAM, and a nuclear dye were applied to samples using an automated slide stainer. Slides were scanned on a digital microscope and candidate CTCs identified using image analysis software. CTCs were verified by appropriate morphology and expression of epithelial and nuclear stains without CD45 expression. Other antibodies used to characterize cells included Her2, EGFR, and Ki-67. A mutation hypothesized to lead to the activation of ROS1 was identified in the cancer cells isolated from the bone marrow of one patient. CTCs were picked using our integrated semi-automated system and evaluated for the ROS1 variant using whole genome amplification followed by nested PCR and Sanger sequencing. Results: Seven patients have been enrolled to date. At least 1 CTC/mL has been found in all patients. Pre-treatment CTC levels in the patient with the ROS1 mutation were extremely high (1500/mL). One week after treatment, CTC levels spiked to more than 5000/mL. CTC counts then dropped exponentially to 9/mL after 4 months. CTC clusters and Ki-67 positive cells also decreased during therapy. Treatment with cisplatin was discontinued in this patient due to toxicity and progression, and CTC levels increased to nearly 9000/mL over 4 months. The ROS1 mutation was found in approximately 50% of individually picked CTCs before treatment with crizotinib, a ROS1 inhibitor. A second patient was found to have somatic loss of BRCA1, and was therefore treated with the PARP inhibitor, veliparib. CTC levels increased during veliparib treatment up to 13/mL. The same patient was subsequently treated with ponatinib, an FGFR inhibitor, based on the identification of two linked somatic missense mutations involving FGFR2 (S252W and Y375C). After beginning ponatinib, CTCs fell to undetectable levels. Conclusions: Analysis of CTCs may provide a non-invasive measure of cancer progression/response and the molecular evolution of tumor cells in patients with TNBC. Single-cell CTC retrieval after slide-based immunofluorescent visualization is compatible with whole genome amplification and sequencing methods. Citation Format: Arturo Ramirez, Daniel Campton, Elisabeth Mahen, Sibel Blau, Anthony Blau, Eric Kaldjian, Jackie Stilwell. Analysis and single-cell retrieval of circulating tumor cells to monitor treatment response and assess genotype in triple-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-21.