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Background: The Metastatic Breast Cancer Project is a nationwide research initiative that directly engages patients through social media and advocacy groups and seeks to empower them to share their samples and clinical information to accelerate research. Because the vast majority of patients are treated in the community setting, we sought to determine the feasibility of remotely obtaining tumor and saliva samples as well as medical records from a large cohort of metastatic breast cancer (MBC) patients who receive their care in diverse settings around the country. Methods: In collaboration with patients and advocacy groups, we developed a website to allow MBC patients across the U.S. to participate. Enrolled patients are sent a saliva kit and asked to mail back a saliva sample, which is used to extract germline DNA. We contact participants' medical providers and obtain medical records and part of their tumor biopsy. Whole exome and transcriptome sequencing is performed on tumor and germline samples. Clinically annotated genomic data are used to identify mechanisms of response and resistance to therapies. The database will be shared widely with researchers. Study updates and discoveries are shared with participants regularly. Results: In the first 8 months, 2285 MBC patients from all 50 states enrolled. 2163 (95%) completed the 16-question survey about their cancer, treatments, and demographic information. 1232 completed the online consent form permitting acquisition and analysis of medical records, tumor tissue, and saliva samples. 556 saliva samples have been received. Initial medical record and tumor sample requests have been made for patients who have provided saliva samples. To date, we have obtained medical records from 102 patients (93% success rate) and tumor samples from 32 patients (77% success rate). Whole exome and transcriptome sequencing has been successfully completed on initial samples received and is ongoing for additional samples. Conclusions: Partnering directly with patients through social media and advocacy groups enables rapid identification of thousands of patients willing to share tumors, saliva, and medical records to accelerate research. This approach allows for rapid identification of patients with rare phenotypes such as extraordinary responders, who have been challenging to identify with traditional approaches. Remote acquisition of medical records, saliva samples, and tumor tissue for patients located throughout the U.S. is feasible. Genomic analysis and medical record abstraction for these patients is underway. As data is generated, a clinically annotated database will be shared widely with the research community. Citation Format: Painter CA, Anastasio E, Krevalin M, Kim D, Larken K, Lennon N, Frank E, Winer EP, Lander ES, Golub T. The metastatic breast cancer project: Translational genomics through direct patient engagement [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 P1-05-13.

Introduction: Circulating tumor DNA (ctDNA) offers the ability to repeatedly interrogate tumor genomic information, providing an opportunity for real-time monitoring of tumor genomic dynamics. In this study, we deeply analyzed multiple ctDNA samples collected over narrow time frames (days-to-weeks) from seven patients with metastatic triple-negative breast cancer (mTNBC), a cancer type known to have high ctDNA content. Methods: Patients with mTNBC were enrolled in a clinical trial of multi-kinase inhibitor cabozantinib, providing uniform and targeted treatment, and samples were collected day 1, day 8, then every 21 to 42 days on study. ctDNA was extracted from each plasma sample and underwent ultra-low pass whole genome sequencing (ULP-WGS; average depth 0.1x; n=42 samples), deep targeted panel sequencing (TPS) of 402 cancer-related genes with unique molecular identifier indexing (depth 10,000x; n=42 samples), and samples with tumor fraction (TFx) >10% underwent whole exome sequencing (WES; depth 200x; n=31 samples), with whole blood germline sequencing of both TPS and WES for subsequent analyses. Somatic copy number alterations (SCNAs) were identified from ULP-WGS and WES. PyClone with TPS was employed for clonal dynamic analyses. Predicted neoantigens were determined from WES using HLAthena. Results: A total of 42 total plasma samples from 7 patients (range 4-8 samples per patient) were collected at narrow time intervals, median 21 days (range 6 to 42 days) between samples. The median TFx across all samples was 18.1% (range 2.5% to 44.3%). TFx estimates were concordant when comparing orthogonal sequencing approaches (ULP-WGS, WES) and tumor fraction estimation algorithms (ichorCNA, FACETS). Despite all seven patients having ‘stable disease’ as best objective response, TFx dynamics were widely variable with TFx declining to lower limit of detection in three of seven patients. Of all samples, 31/42 (73.8%) had tumor fraction >10% and underwent WES; each patient had at least 3 samples that underwent both WES and TPS. There was strong agreement between TPS and WES: across all 31 shared samples, mutation recall in TPS versus WES (gold standard) was 95.5%. Variant allele frequency across all mutations detected in both TPS and WES was highly concordant (Pearson’s r=0.949). Clonal mutations were consistently detected across multiple samples within patients. When comparing genome-wide copy number from last to first available sample within each patient, copy number log ratios were largely stable within patients (union Pearson’s r=0.924) and there were not recurrent shifts in SCNAs across patients. Through statistical modeling of TPS data, we tracked distinct clonal populations for each patient over their sampling windows. Modeled clonal architecture in most patients revealed stable, polyclonal profiles, with important breast cancer driver alterations (e.g. TP53 and PIK3CA) recurrently presenting at high prevalence. Infrequently, we also detected emergence and expansion of clones over narrow time frames (weeks) containing acquired alterations poorly annotated in the breast cancer literature. We successfully predicted neoantigens from ctDNA WES at multiple time points in each patient, with evidence that patients acquired new mutations predicted to be ‘strong binder’ neoantigens over time on therapy. Conclusions: Analysis of serial ctDNA samples collected at narrow time intervals (days-to-weeks) provides unique insight into the dynamics of ctDNA. We demonstrate strong concordance across ctDNA sequencing appraoches. Evolving genomic features of tumor populations can be identified via ctDNA while on treatment, potentially providing real time insight for clinical decision-making. Citation Format: Daniel G Stover, Katharine A Collier, David Tallman, Juliet Forman, Sachet Shukla, Sarah Asad, Justin Rhoades, Samuel Freeman, Mathew Cherian, Sagar Sardesai, Romualdo Barroso-Sousa, Carrie Cibulskis, Niall Lennon, Gavin Ha, Sara M Tolaney, Viktor A Adalsteinsson, Zachary Weber. Modeling clonal structure over narrow time frames via circulating tumor DNA in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-08.

BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance. Significance: RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance. Cancer Discov; 5(4); 358–67. ©2015 AACR. See related commentary by Meador and Pao, p. 348 This article is highlighted in the In This Issue feature, p. 333

Broad Genomics offers a comprehensive liquid biopsy sequencing platform designed to provide the optimal flexibility for conducting research studies in a broad range of applications including: biomarker discovery, treatment resistance monitoring, and clinical grade ctDNA profiling. By utilizing low cost, low coverage whole genome sequencing in conjunction with dual unique molecular indexed (UMI) libraries we can offer a range of analysis that allow researchers to select the most appropriate samples for whole exome profiling or for deeper coverage, higher sensitivity targeted gene panels. To date we have generated over 3000 liquid biopsy whole genome copy number profiles and purity estimates and are supporting driver projects including the Broad/IBM Cancer Resistance Project and Count Me In. The study design for the Broad/IBM effort takes advantage of the discovery potential of tissue-based sequencing combined with serial liquid biopsy analysis to elucidate resistance events by tracking clonal and subclonal populations in patient samples over time. Sourcing samples for this and other similar efforts is a major undertaking and a combination of methods for maximally broad and deep genomic profiling are required to assay patients throughout the course of care, as tumor fraction in blood fluctuates. Responding to this need, and other applications requiring increased sensitivity we have developed a high throughput, automated workflow to efficiently assay cfDNA samples with lower tumor content. Benchmarking data using healthy donor pooled cfDNA samples indicates our assay is capable of detecting > 90% of variants present at ~1% minor allele fraction with less than 1 false positive variant called per megabase. This established laboratory and analytic process forms the basis of our 2Mb, 400 gene CLIA targeted assay currently undergoing validation. Through this suite of products we hope to enable an expansion of cfDNA sequencing efforts in support of clinical and research applications. Early results from emerging studies utilizing this platform to be presented. Citation Format: Carrie Cibulskis, Brendan Blumenstiel, Matthew DeFelice, Mark Fleharty, Justin Abreu, Viktor Adalsteinsson, Laxmi Parida, Susanna Hamilton, Gad Getz, Niall Lennon. Innovations in large scale liquid biopsy analysis and the Broad/IBM Cancer Resistance Project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 455.

Whole genome sequencing (WGS) offers the greatest potential to comprehensively and accurately identify all forms of human genetic variation. The accessibility of WGS data for diagnostic use has increased due to cost reductions in recent years, driving the need for a high quality, clinically validated offering. Our platform has completed an extensive benchmarking study of the Illumina PCR-free whole genome pipeline to establish clinical validity for the end-to-end laboratory, analytical, and computational processes. Our benchmarking study is comprehensive in nature, including measuring the precision, robustness, limits of detection for variant classes, and contamination estimates. A cohort of well-characterized reference controls and clinical samples with previously identified pathogenic variants were used to establish the performance characteristics of our clinical WGS test, which at 30X coverage has >99% analytical sensitivity for SNVs and >98% analytical sensitivity for small insertions and deletions. Our platform is capable of operating at a scale that supports applications from individual clinics (cancer and medical genetics related applications) as well as large scale ambitious collaborative projects such as the All Of Us program which aims to generate clinical grade whole genome variant calls for 1 million healthy research participants. Citation Format: Alyssa MacBeth, Tera Bowers, Betty Woolf, Maegan Harden, Niall Lennon, Stacey Gabriel. Clinical whole genome sequencing at scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3543.

Objective: Angiosarcoma (AS) is a rare soft tissue sarcoma, with an incidence of 300 cases/yr and a 5-year DSS of 30%. The low incidence has impeded large-scale research efforts. To address this, we launched a patient-partnered genomics study which seeks to empower patients to accelerate research by remotely sharing their samples and clinical information. Methods: We developed a website (ASCproject.org) to allow remote acquisition of medical records (MR), saliva, blood, and archival tissue from patients in the US and Canada. Whole-exome sequencing (WES) of ~20,000 genes is performed on tumor and matched germline DNA. Transcriptome analysis is performed on tumor RNA. Ultra-low pass whole-genome sequencing (ULP-WGS) and in some cases WES is performed on cell free DNA (cfDNA) obtained from blood samples. Clinical data including information about demographics, diagnosis, treatments, and responses are obtained via patient-reported data (PRD) and through MR abstraction. The resulting clinically annotated genomic database is shared widely to identify genomic drivers and mechanisms of response and resistance to therapies. Results: Since launch on March 13 2017, 321 patients with AS have registered. The average age of patients is 56 yrs (range 22-89). Primary locations of AS were primary breast (24%), breast with prior radiation (20%), head/face/neck/scalp (HFNS) (21%), bone/limb (9%), abdominal (3%), heart (3%), lung (1%), liver (1%), lymph (0.5%), multiple locations (11%), and other locations (5%). 142 (48%) reported being disease free at the time of enrollment. To date, 153 saliva kits, 167 MRs, 43 blood samples, and 97 tissue samples have been obtained. WES analysis is complete for 14 samples.ULP-WGS is complete for 10 cfDNA samples, and WES on 4 cfDNA samples. Transcriptome sequencing is complete for 9 tumor samples. We identified several previously described genes known to be altered in AS, including recurrent alterations in KDR and TP53. Tumor mutational burden (TMB) and mutational signature activities were quantified for each tumor sample. All three of the AS from the HFNS in the initial cohort exhibited a high TMB (>150 mutations) and dominant UV light signature (COSMIC Signature 7). Based on this, we hypothesized that HFNS AS might respond well to immune checkpoint inhibitors. We identified through PRD 56 patients with HFNS AS who reported what medications they received. Of these, 2 reported receiving immune checkpoint inhibitors for the treatment of metastatic disease. Both patients had refractory metastatic HFNS AS and reported receiving off-label anti-PD1 therapy. Both had complete or near-complete responses following immunotherapy, and currently report having no evidence of disease. Clinical responses were confirmed through review of MRs. Sequencing is currently being performed on tumor samples from both patients. Conclusion: A patient-partnered approach enabled rapid identification and enrollment of over 300 patients with AS, an exceedingly rare cancer, in 15 months. We were able to obtain tumor, blood, saliva samples to perform genomic analyses, which were then merged with detailed clinical information. PRD, clinical, and genomic data generated from the first 12 patients and 14 samples have been released on cbioportal.org. Additional data will be released in six-month intervals. Initial results show high TMB and a UV signature in 3 out of 3 patients with HFNS AS. In addition, we identified 2 patients with HFNS AS who had extraordinary responses to immunotherapy. These findings suggest a common genomic basis for HFNS AS and could provide rationale for clinical interventions using checkpoint inhibitors for these AS. Analyses of additional samples are under way to further characterize mutational signatures in HFNS AS and implications for patient care. This study serves as proof of principle that patient-partnered genomics efforts can democratize cancer research for exceedingly rare cancers. Citation Format: Corrie Painter, Esha Jain, Michael Dunphy, Elana Anastasio, Mary McGillicuddy, Rachel Stoddard, Beena Thomas, Sara Balch, Kristin Anderka, Katie Larkin, Niall Lennon, Yen-Lin Chen, Andrew Zimmer, Esme O. Baker, Simone Maiwald, Jen Lapan, Jason L. Hornick, Chandrajit Raut, George Demetri, Eric S. Lander, Todd Golub. High mutation burden and response to immune checkpoint inhibitors in angiosarcomas of the scalp and face [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B085.