Search

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

Seventy percent of breast cancers express the estrogen receptor (ER), and agents that target the ER are the mainstay of treatment. However, virtually all people with ER

Inhibition of proliferation in estrogen receptor-positive (ER+) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER+/human epidermal growth factor receptor 2-negative (HER2-) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including FGFR1 and CCND1, respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER+ tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER+FGFR1/CCND1 coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal ESR1 fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation.

The use of targeted therapeutics directed against BRAFV600-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAFV600-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor–resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents. Cancer Res; 74(23); 7079–89. ©2014 AACR.

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

In estrogen-receptor positive (ER+) breast cancer, therapies that target the ER have led to reductions in recurrence and mortality. However, in the metastatic setting, resistance to ER-targeted therapies is near universal. Various resistance mechanisms have been proposed, including ESR1 activating mutations and upregulation of alternative signal transduction pathways, yet clinically relevant resistance mechanisms have not been clearly defined. We conducted a genome-scale gain-of-function screen in ER+ breast cancer cell lines, spanning 17,255 overexpressed open reading frames (ORFs), to investigate genes whose overexpression is sufficient to confer resistance to selective estrogen receptor degraders (SERDs). Among several validated resistance genes, the fibroblast growth factor receptor (FGF/FGFR) pathway was among the top pathways, with several FGF ligands scoring as top resistance genes in this screen. In parallel, we performed whole exome sequencing (WES) in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-targeted therapy. The FGFR pathway was altered in 40% (24 of 60) of the post-resistance biopsies; alterations included FGFR1 amplifications (9 patients), FGFR2 amplifications and activating mutations (4 patients), and FGF3 amplifications (17 patients). In 12 out of the 24 cases (5 FGFR1, 4 FGFR2, 3 FGF3), the FGF/FGFR pathway alterations were present in the resistant tumors and not detected in the pre-treatment tumors, suggesting that these alterations were acquired under the selective pressure of ER-directed therapy. Overexpression of wildtype FGFR1, FGFR2, FGF3 in ER+ breast cancer cells (T47D and MCF7) led to resistance to fulvestrant as well as the combination of fulvestrant and the CDK4/6 inhibitor palbociclib. This resistance phenotype was reversed by the FGFR inhibitor PD173074, and, to a lesser extent, the MEK inhibitor trametinib and mTOR inhibitor everolimus, suggesting several potential treatment strategies. The three FGFR2 mutations (M538I, N550K and K660N) identified in these patients induced hyperactive FGFR signaling and conferred resistance to fulvestrant, which was abrogated by the irreversible FGFR inhibitors FIIN-2 and FIIN-3. Together, these results offer new insights into endocrine resistance and suggest new clinical approaches to overcome or preempt therapeutic resistance. Citation Format: Pingping Mao, Ofir Cohen, Kailey Kowalski, Justin Kusiel, Jorge Buendia, Pedro Exman, Seth A. Wander, Adrienne G. Waks, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired FGF and FGFR alterations confer resistance to estrogen receptor (ER) targeted therapy in metastatic ER+ breast cancer [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 314.

Durable control of invasive solid tumors is thwarted by the lack of knowledge of effective drug combinations and of the acquired and intrinsic resistance mechanisms of drugs. In an effort to tackle this problem, the SU2C-NSF-TVF Drug Combination Convergence Team is using mechanistic models of cancer cell signaling based on therapeutic and cell line data in order to identify elements within cancer cells that might eventually be exploited through therapeutic combinations. Here we present a comprehensive mechanistic network model of signal transduction in ER+ PIK3CA-mutant breast cancer. Focusing on PI3K inhibitors, the model recapitulates known resistance mechanisms and predicts other possibilities for resistance: loss of RB1, FOXO3, P27, or PRAS40. To test these predictions, we analyzed genome-wide CRISPR screens of two breast cell lines in the presence of PI3K inhibitors (BYL719 and GDC0032) and found that the predicted genes (RB1, FOXO3, P27, and PRAS40) were significantly enriched in the screens. Some of these resistance genes (e.g. loss of RB1) were found to be cell-line specific and follow-up experiments in RB1-KO cells confirmed the cell-line-specific nature of PI3K-inhibitor resistance. The model also reveals known and novel combinatorial interventions that are more effective than PI3K inhibition alone. For example, the model predicts that the combination of PI3K inhibitors with inhibitors of anti-apoptotic protein MCL1 would be effective. Follow up experiments in cell lines using cell viability assays, cell death analyses, and dynamic BH3 profiling experiments to determine increases in apoptotic priming upon treatment confirmed that MCL1 inhibitors (S63845) enhance the effect of PI3K inhibitors (BYL719) and that this combinatorial effect is cell-line-specific, similarly to what was found in the resistance genes case. In conclusion, the model predicted drug resistance mechanisms and effective drug combinations, some of which were verified experimentally and found to be cell-line-specific. Next iterations of the model will incorporate the identified discrepancies and newly identified resistance mechanisms to drugs of clinical interest. Citation Format: Jorge Gómez Tejeda Zañudo, Pingping Mao, Joan Montero, Guotai Xu, Kailey J. Kowalski, Gabriela N. Johnson, José Baselga, Maurizio Scaltriti, Anthony G. Letai, Nikhil Wagle, Reka Albert. Network modeling of drug resistance mechanisms and drug combinations in breast cancer [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 675.

554 Background: HER2+ breast cancer (BC) is particularly common in young women. Genomic features of HER2+ tumors before and after H-based therapy have not been described in a population of young women and may point to clinically targetable mechanisms of resistance. Methods: From a large prospective cohort of women diagnosed with BC age ≤40 years, we identified those with HER2+ BC and tumor tissue available for sequencing before and after chemo+H. Whole exome sequencing (WES) was performed on each tumor and on germline DNA from blood. Tumor-normal pairs were analyzed for mutations and copy number (CN) changes. Evolutionary analysis was performed for patients with both pre- and post-treatment (tx) samples. Results: 22 women had successful WES samples from at least one timepoint; 13 of these had paired sequencing results both before and after chemo+H. For the majority of women, post-tx sample was following neoadjuvant chemo + H, though post-tx timepoint for other women represented locoregional or distant metastasis (Table). TP53 was the only gene that was significantly recurrently mutated in both pre- and post-tx samples. Comparison of matched pre-tx and post-tx samples demonstrated that large changes in HER2 CN over the course of tx were uncommon, only 2/13 pts had > 2-fold change in HER2 CN. Other clonal and subclonal genomic alterations were found to be acquired in the post-tx sample compared to the pre-tx sample. One patient acquired a putative activating mutation in ERBB2. Another patient acquired a clonal hotpsot mutation in TP53. MYC gene amplification was observed in 4 post-tx tumors. NOTCH2 alterations were found in post-tx biopsies from 2 different patients, and mutations in STIL were also found in post-tx biopsies from 2 patients, though the function of these mutations is not known. Conclusions: HER2+ breast tumors in young women display genomic evolution following tx with chemo+H. HER2 CN changes are uncommon, but we identified several genes that warrant exploration as potential mechanisms of resistance to therapy in this population.[Table: see text]

1085 Background: Little is known about MoR to PARPi and platinum chemotx in MBC pts with BRCA1/2 mutations. Biomarkers predictive of response/resistance have not been identified, but could have clinical utility. Methods: We obtained 8 BRCA-mutant metastatic tumor biopsies from MBC pts with acquired resistance to DNA-damaging tx (PARPi/platinum) on a prospective tissue collection protocol. In 7/8 patients, we also obtained pre-tx biopsies. Whole exome sequencing (WES) was performed on each tumor and on germline DNA from blood. We performed immunohistochemical (IHC) staining for RAD51 foci for functional assessment of intact homologous recombination (HR). Results: 4/7 pts with complete WES analysis acquired a somatic reversion mutation likely to result in functional BRCA1/2 protein in the post-tx tumor specimen after platinum (2 pts) or PARPi (2 pts; Table). 4/7 pts had plausible non-reversion MoR identified by WES, including alterations in genes involved in replication fork protection and DNA end resection. As expected, in all pts with genomic reversion, RAD51 foci were acquired in the post-resistance tumor, consistent with reconstitution of HR. In 2 pts without reversion, presence of RAD51 foci post-resistance was mixed. Reversion mutations occurred both with and without other alterations that could possibly lead to fork protection, suggesting > 1 MoR could occur in the same tumor. 3 pts whose tumors demonstrated RAD51 foci post-resistance were later re-exposed to DNA-damaging tx, to which all had intrinsic resistance. Conclusions: BRCA1/2 reversion was identified as a MoR in the majority of pts. WES identified potential novel MoR in fork protection and end resection genes. The presence of RAD51 foci by IHC was consistent with BRCA protein functional status from genomic data and predicted response to later DNA-damaging tx, suggesting RAD51 IHC may be a clinically useful biomarker. [Table: see text]

1022 Background: Genomic profiling of primary and recurrent metastatic breast cancer (rMBC) has revealed potential resistance mechanisms to therapy. In contrast, de novo stage IV breast cancer (DNIV) represents an opportunity to elucidate metastatic drivers in the absence of treatment selection. Methods: Targeted NGS (Oncopanel, OP) using multiplexed copy number variation (CNV) and mutation (mut) detection across the full coding regions of 300 genes and selected intronic regions of 35 genes was performed on either primary or metastatic samples collected in patients (pts) with DNIV or rMBC. Mut/CNV in primary and metastatic tumors were compared per subtype between DNIV and rMBC using Fisher´s exact test (significant p

1019 Background: Diverse mutational processes are active at different stages of cancer development and cause the mutational burden in cancer cells. These processes leave distinctive signatures, characterized by varying frequencies of nucleotide substitutions and their flanking base-pairs. Mutational signatures found in early stage breast cancer have been described, but less is known about the mutational processes in metastatic breast cancer (MBC). Methods: We performed whole exome sequencing (WES) on 209 metastatic tumor biopsies from patients with ER+ MBC. In a subset of patients, we obtained and performed WES on the matched primary biopsy (n = 62). Decomposition of mutational signatures was done by determining the linear combination of 30 previously reported mutational signatures (Alexandrov et al., 2013) that most accurately reconstructs the mutational profile of each tumor sample. For comparison, signature decomposition was also performed on 695 treatment-naïve primary ER+ breast cancers form The Cancer Genome Atlas (TCGA). Results: Cohort-level comparison of signature activity between our cohort of MBC biopsies versus the primary ER+ breast cancers from TCGA demonstrated enrichment of Signature 13 in MBC samples (q = 0.002), attributed to activity of the APOBEC family of cytidine deaminases and implicated in resistance to endocrine therapies. Activity of Signature 22, associated to activity of the nucleotide-excision repair mechanism, was depleted in MBC tumors relative to TCGA ER+ breast tumors (q = 0.02). Decomposition of mutational signatures at the clone level in the subset of patients with both metastatic and matched primary samples allowed us to infer signatures acquired in the metastatic setting. Among mutations in acquired clones, we found enrichment of APOBEC signatures (Sig. 2, q = 0.018; Sig. 13, q = 0.003). Conversely, activity of Signature 1, attributed to the normal aging process, was enriched in mutations shared between primary and MBC samples (q = 0.004). This suggests that mutations developed early in tumor evolution may be driven primarily by natural processes, while later mutations may reflect selective pressures imposed by therapies and cellular defects acquired during disease progression. Conclusions: The profile of somatic mutations in MBC differs from early stage breast cancer. APOBEC-related mutations arising late during tumor evolution are one possible mechanism for this difference. Further studying the associations between clinical features and therapies to mutational processes could inform design of clinical trials and management of MBC.

Background: Approximately 5-10% of newly diagnosed breast cancers (BC) are de novo MBC, which means that metastatic disease was identified at the time of initial diagnosis. Patients with de novo MBC are underrepresented in currently available genomic studies. In The Cancer Genome Atlas (TCGA) dataset, only 15 out of ˜980 BC patients can be classified as having de novo MBC. The objective of this study is to analyze the genomic landscape of de novo MBC and to study the genomic differences of this cohort with early stage BC. To enhance our ability to study de novo MBC, we utilized data from the Metastatic Breast Cancer Project (MBCproject), a patient-partnered research project that aims to generate a large public database of clinical, genomic, and patient reported data (PRD) from patients with MBC. Methods: We defined de novo MBC as patients diagnosed with metastatic disease less than 4 months after their initial diagnosis with BC.Out of 127 patients in the MBCproject with publicly released whole exome sequencing (WES) data, we identified 33 patients with de novo MBC. We combined this data with 15 de novo MBC patients in TCGA. For patients with de novo MBC with multiple tumor biopsies available, we used WES from breast biopsies to enable appropriate comparison to the early stage biopsies. Somatic mutations were evaluated and significantly recurring genes were identified using MutSig2CV. We compared the mutations found in the de novo cohort with early stage tumors. 10 patients in the de novo MBC cohort had paired simultaneous primary and metastatic biopsies; genomic alterations in these samples were compared. Finally, we used RNA sequencing data to compare cytolytic signatures among the de novo and early stage biopsies. Results: Among the 48 patients analyzed the receptor subtype distribution was: HR+/HER2-(23), HR+/HER2+(13), HR-/HER2+(2), HR-/HER2-(3), HR+/HER2 unknown(5), and HR-/HER2 unknown(2). Histology subtype distribution was as follows: IDC(39), MDLC(6), ILC(2) and Other (1). Significantly recurrent genes in the de novo MBC cohort (q Gene% Mutation Rate in De Novo MBC (N=48)% Mutation rate in Early Stage BC (N= 997)p-valuePTEN10.403.510.0324EGFR6.250.500.00435MDM44.170.300.0189 Conclusions: Our initial results highlight genomic differences between de novo MBC and early stage BC, including increased frequency of PTEN, EGFR, and MDM4 mutations. Enrichment of PTEN mutations (implicated in tumor immune surveillance), and downregulation of cytolytic signature potentially suggests that de novo MBC may have immunosuppressive tumor microenvironment. To date, ˜1200 patients with self-reported de novo MBC have registered for the MBCproject. We anticipate that additional study of genomic and clinical data from these patients will greatly improve our understanding of de novo MBC. Citation Format: Jain E, Kim D, Buendia JB, Cohen O, Sousa RB, Anastasio E, Dunphy M, McGillicuddy M, Stoddard R, Balch S, Thomas B, Di Lascio S, Tomson BN, Nguyen C, Painter C, Wagle N. The genomic landscape of de novo metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-03.

Comprehensive molecular characterization of cancer that has metastasized to bone has proved challenging, which may limit the diagnostic and potential therapeutic opportunities for patients with bone-only metastatic disease. We describe successful tissue acquisition, DNA extraction, and whole-exome sequencing from a bone metastasis of a patient with metastatic, castration-resistant prostate cancer (PCa). The resulting high-quality tumor sequencing identified plausibly actionable somatic genomic alterations that dysregulate the phosphoinostide 3-kinase pathway, as well as a theoretically actionable germline variant in the BRCA2 gene. We demonstrate the feasibility of diagnostic bone metastases profiling and analysis that will be required for the widespread application of prospective ‘precision medicine’ to men with advanced PCa.

Background: Although the spectrum of genomic alterations in primary, treatment-naïve breast tumors has been described, the genomic landscape of HER2+ MBC remains underexplored. Furthermore, tumor genomic alterations that arise after progression on anti-HER2 therapy are largely unknown. Methods: We prospectively collected metastatic tumor biopsies from patients (pts) enrolled on TBCRC003 (NCT00470704), a phase II study evaluating the combination of lapatinib (L) and T in pts with HER2+ MBC who had varying degrees of prior T exposure. We performed WES on baseline metastatic biopsies and normal DNA from 57 pts. In 36 pts, we also performed WES on pre-treatment primary tumors. Tumors were analyzed for point mutations, insertions/deletions, and copy number alterations. Results: Total accrual was 116 pts. 87 pts were registered in one of two efficacy cohorts: Cohort 1 included pts w no prior T for MBC. Pts with prior adjuvant T were included if the interval from last T to 1st recurrence > 12 months. Cohort 2 included pts with 1-2 prior lines of T for MBC or recurrence within 12 months of adjuvant T. An additional 29 pts were enrolled in a biomarker cohort (Cohort 3). Per-protocol efficacy analyses for 85 pts deemed evaluable are shown below: Objective Response RateClinical Benefit RateMedian Time to ProgressionCohort 150% (90% CI 33.8-66.2%)57.5% (95% CI 40.9-73.0)7.4 monthsCohort 222.2% (90% CI 11.2-37.1%)42.2% (95% CI 27.7-57.8)5.3 months As we previously reported (Wagle et al, ASCO 2014), across 57 metastatic tumors, significant recurrently mutated genes were TP53 (n=30; 53%) and PIK3CA (n=19; 33%). The frequency of mutant TP53 and PIK3CA was not significantly different from 119 primary, treatment-naïve HER2+ tumors sequenced in the TCGA study (50%, p=0.8 and 27%, p=0.5, respectively). Recurrent copy number alterations were also similar to TCGA data. Comparing the 38 pts who received any prior T with the 19 pts who did not, there was no significant difference in the incidence of mutant TP53 (53% vs 53%, p=1.0) and PIK3CA (37% vs 26%, p=0.6). We identified mutations in the HER2 kinase domain in 4/38 pts who received prior T (11%), as compared to 0/19 T-naïve pts. HER2 kinase domain mutations have been identified in ∼2% of HER2-negative cancers but An analysis comparing paired archival primary tumors and baseline metastatic biopsies from 36 pts to identify genomic alterations acquired or enriched in the metastatic tumors will be presented. Conclusions: We present an analysis of the genomic landscape of HER2+ MBC, including comparisons between matched primary tumors and metastatic biopsies. Somatic HER2 kinase mutations in pts with HER2+ MBC treated with prior T suggests that these mutations may be involved in resistance to T, and may predict poor response to additional anti-HER2 therapy with combined L and T. Novel therapeutic approaches may be required for these pts. Citation Format: Nikhil Wagle, Nancy U Lin, Andrea L Richardson, Ignaty Leshchiner, Ingrid A Mayer, Andres Forero-Torres, Timothy J Hobday, Elizabeth C Dees, Rita Nanda, Mothaffar F Rimawi, Hao Guo, William T Barry, Ron Bose, Wei Shen, Antonio C Wolff, Stacey B Gabriel, Levi A Garraway, Eric P Winer, Ian E Krop. Whole exome sequencing (WES) of HER2+ metastatic breast cancer (MBC) from patients with or without prior trastuzumab (T): A correlative analysis of TBCRC003 [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 PD3-5.

Background DNA aptamers represent a novel strategy in anti-cancer medicine. AS1411, a DNA aptamer targeting nucleolin (a protein which is overexpressed in many tumor types), was evaluated in patients with metastatic, clear-cell, renal cell carcinoma (RCC) who had failed treatment with ≥1 prior tyrosine kinase inhibitor. Methods In this phase II, single-arm study, AS1411 was administered at 40 mg/kg/day by continuous intravenous infusion on days 1–4 of a 28-day cycle, for two cycles. Primary endpoint was overall response rate; progression-free survival (PFS) and safety were secondary endpoints. Results 35 patients were enrolled and treated. One patient (2.9 %) had a response to treatment. The response was dramatic (84 % reduction in tumor burden by RECIST 1.0 criteria) and durable (patient remains free of progression 2 years after completing therapy). Whole exome sequencing of this patient’s tumor revealed missense mutations in the mTOR and FGFR2 genes which is of interest because nucleolin is known to upregulate mTOR pathway activity by enhancing AKT1 mRNA translation. No other responses were seen. Thirty-four percent of patients had an AS1411-related adverse event, all of which were mild or moderate. Conclusions AS1411 appears to have minimal activity in unselected patients with metastatic RCC. However, rare, dramatic and durable responses can be observed and toxicity is low. One patient in this study had an excellent response and was found to have FGFR2 and mTOR mutations which will be of interest in future efforts to discover and validate predictive biomarkers of response to nucleolin targeted compounds. DNA aptamers represent a novel way to target cancer cells at a molecular level and continue to be developed with a view to improving treatment and imaging in cancer medicine.

Cervical cancer is the second leading cause of cancer deaths among women worldwide. The objective of this study was to describe the most common oncogenic mutations in cervical cancers and to explore genomic differences between the 2 most common histologic subtypes: adenocarcinoma and squamous cell carcinoma.A high-throughput genotyping platform, termed Oncomap, was used to interrogate 80 cervical tumors for 1250 known mutations in 139 cancer genes. Samples were analyzed using a mass spectrometry-based genotyping platform and were validated using orthogonal chemistry. Epidermal growth factor receptor (EGFR) mutations were further validated by massive parallel sequencing. Human papilloma virus (HPV) genotyping also was performed.Validated mutations were detected in 48 of 80 tumors (60%) examined. The highest mutation rates were in the genes phosphatidylinositol 3-kinase, catalytic subunit α (PIK3CA) (31.3%); Kirsten rat sarcoma viral oncogene homolog (KRAS) (8.8%); and EGFR (3.8%). PIK3CA mutation rates did not differ significantly between adenocarcinomas and squamous cell carcinomas (25% vs 37.5%, respectively; P = .33). In contrast, KRAS mutations were identified only in adenocarcinomas (17.5% vs 0%; P = .01), and a novel EGFR mutation was detected only in squamous cell carcinomas (0% vs 7.5%; P = .24). There were no associations between HPV-16 or HPV-18 and somatic mutations or overall survival. In adjusted analyses, PIK3CA mutations were associated with shorter survival (67.1 months vs 90.3 months; hazard ratio, 9.1; 95% confidence interval, 2.8-29.5 months; P.001).Cervical cancers harbor high rates of potentially targetable oncogenic mutations. In addition, cervical squamous cell carcinoma and adenocarcinoma have distinct molecular profiles, suggesting that clinical outcomes may be improved with the use of more tailored treatment strategies, including PI3K and MEK inhibitors.

Low frequency clonal mutations recoverable by deep sequencing in patients with aplastic anemia

Background: HER2-positivity is a predictor of benefit from trastuzumab (TRZ), but fails to depict the observed interpatient variability in terms of treatment (tx) duration. In this study we described the relationship between clinicopathological features and TRZ tx duration. Methods: A retrospective consecutive series of 343 HER2+ breast cancer (BC) patients (pts) treated with TRZ at the Dana-Farber Cancer Institute from 1999–2008 was identified. 139 pts treated with 1st line TRZ-based tx were selected for analysis. Pts who received any non-TRZ prior tx for metastatic disease were excluded. TRZ tx duration was defined as time from start of 1st line therapy to the 1st day of 2nd line therapy or death. Central nervous system (CNS) progression with TRZ maintenance was not considered change of tx. Pts were divided equally into 3 groups based on the duration of 1st line tx distribution. Short-term responders (STR) were on the 1st line tx for 15m. An additional group of extremely LTR (ELTR) was defined as being in the 90th percentile of tx duration (>37m). Descriptive analysis was performed; fisher exact test, Kruskal-Wallis and logistic regression methods were used to compare groups. Results: Median follow-up time since metastatic diagnosis was 4 years (y) (range 0–11). Median age at diagnosis was 47y (22–83), 25% of stage I-III pts at diagnosis received adjuvant/neoadjuvant TRZ. The median disease free interval (DFI) was 20m (0–172), median number of metastatic sites was 2(1–5), 68% of pts had visceral disease. Median duration of 1st line tx was 10m (2–105). TRZ was given with CT in 86%, hormone tx in 6% and as monotherapy in 9%. 25% of pts developed CNS progression and continued tx. There were only small absolute differences for clinicopathological characteristics among STR, IR and LTR. ELTR had a median 1st line TRZ tx duration of 49m (37–105) and similar clinicopathological features to LTR. A higher proportion of LTR had hormone receptor (HR)-positive disease compared with STR, however no significant association between LTR and STR was found for HR status, DFI and visceral involvement. Conclusions: TRZ tx duration varies widely in the 1st-line advanced setting. No clinicopathological features were associated with TRZ tx duration. Our results suggest that despite CNS progression some pts continue to have long term benefit to TRZ tx. A major research priority is to identify molecular predictors of benefit and resistance to anti-HER2-based therapies. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-18-03.

Aim: To understand how a cancer precision medicine tumor board (CPM-TB) made choices about return of results. Materials & methods: Observed CPM-TB deliberations and completed in-depth interviews with committee members. Results: Responding to complex evidence of ambiguous significance, deliberations of the CPM-TB were predicated on analytic validity and clinical utility. Members had concerns both about potential harms due to returning results based on weak evidence and about withholding potentially meaningful results. Group dynamics and the clinical experiences of individual committee members shaped their work. Conclusion: Both scientific evidence and the social context surrounding deliberations of a CPM-TB influenced decisions about return of results. Subjective elements, while present in any scientific endeavor, may carry more weight in the face of ambiguous findings.

Inhibition of proliferation in estrogen receptor-positive (ER

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine.

Background The diversity of clinical tumor profiling approaches (small panels to whole exomes with matched or unmatched germline analysis) may engender uncertainty about their benefits and liabilities, particularly in light of reported germline false positives in tumor-only profiling and use of global mutational and/or neoantigen data. The goal of this study was to determine the impact of genomic analysis strategies on error rates and data interpretation across contexts and ancestries. Methods We modeled common tumor profiling modalities—large (n = 300 genes), medium (n = 48 genes), and small (n = 15 genes) panels—using clinical whole exomes (WES) from 157 patients with lung or colon adenocarcinoma. We created a tumor-only analysis algorithm to assess germline false positive rates, the impact of patient ancestry on tumor-only results, and neoantigen detection. Results After optimizing a germline filtering strategy, the germline false positive rate with tumor-only large panel sequencing was 14 % (144/1012 variants). For patients whose tumor-only results underwent molecular pathologist review (n = 91), 50/54 (93 %) false positives were correctly interpreted as uncertain variants. Increased germline false positives were observed in tumor-only sequencing of non-European compared with European ancestry patients (p

Summary Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes ( PPP6C , RAC1 , SNX31 , TACC1 , STK19 , and ARID2 ), three of which— RAC1 , PPP6C , and STK19 —harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS / BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.

Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. Through whole-exome sequencing of metastatic tumor biopsies from patients with endocrine resistant ER+ metastatic breast cancer, we identified 13 different HER2 mutations, including five in the kinase domain, four in the signaling domain, three in the extracellular domain, and one in the transmembrane region of the protein. Two of the kinase domain mutations (L755S and V777L) have been previously described and shown to be activating and resistant to reversible anti-HER2 targeted therapies; the remaining mutations have not been reported. In several of these patients, whole exome sequencing of a pre-treatment primary tumor did not identify the HER2 mutations seen in the corresponding metastatic tumor, suggesting that they were acquired during therapy. To examine the role of HER2 mutations in endocrine resistance, we generated ER+ breast cancer cell lines (MCF7 and T47D) stably expressing the HER2 mutants observed in our clinical data. Several mutants promoted enhanced growth in charcoal dextran-stripped media, which lacks estradiol and mimics treatment with aromatase inhibitor. In addition, several mutants conferred varying degrees of resistance to fulvestrant and tamoxifen. Taken together, these results suggest that HER2 mutations are associated with acquired resistance to endocrine therapies in patients with ER+ breast cancer. The ability of irreversible anti-HER2 agents as well as other agents that target the HER2 pathway to overcome this resistance is being tested for individual HER2 mutations in vitro. The results from these studies may provide a clinical rationale for therapeutic combination strategies in patients with refractory tumors that have acquired endocrine resistance through HER2 mutations. Citation Format: Nayar U, Cohen O, Oh C, Wagle N. The role of HER2 mutations in resistance to endocrine therapy in ER+ breast cancer [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 P3-04-08.

A detailed understanding of the mechanisms by which tumors acquire resistance to targeted anticancer agents should speed the development of treatment strategies with lasting clinical efficacy. RAF inhibition in BRAF-mutant melanoma exemplifies the promise and challenge of many targeted drugs; although response rates are high, resistance invariably develops. Here, we articulate overarching principles of resistance to kinase inhibitors, as well as a translational approach to characterize resistance in the clinical setting through tumor mutation profiling. As a proof of principle, we performed targeted, massively parallel sequencing of 138 cancer genes in a tumor obtained from a patient with melanoma who developed resistance to PLX4032 after an initial dramatic response. The resulting profile identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the corresponding pretreatment tumor. The MEK1 C121S mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Thus, MEK1 C121S or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. These results provide an instructive framework for assessing mechanisms of acquired resistance to kinase inhibition and illustrate the use of emerging technologies in a manner that may accelerate personalized cancer medicine. J Clin Oncol 29:3085-3096. © 2011 by American Society of Clinical Oncology

The diagnosis and management of colorectal cancer (CRC) has been impacted by the discovery and validation of a wide variety of biomarkers designed to facilitate a personalized approach for the treatment of the disease. Recently, CRC has been reclassified based on molecular analyses of various genes and proteins capable of separating morphologic types of tumors into molecular categories. At the same time, a number of new prognostic and predictive single genes and proteins have been discovered that are designed to reflect sensitivity and/or resistance to existing therapies. Multigene predictors have also been developed to predict the risk of relapse for intermediate-stage CRC after completion of surgical extirpation. More recently, a number of biomarkers tested by a variety of methods have been proposed as specific predictors of chemotherapy and radiotherapy response. Other markers have been successfully used to predict toxic effects of standard therapies. In this review, a series of novel biomarkers are considered and compared with standard-of-care markers for their potential use as pharmacogenomic and pharmacogenetic predictors of disease outcome.

Despite the reduction in cancer recurrence and mortality provided by therapies that target the estrogen receptor (ER), resistant ER+ breast cancer remains the most common cause of breast cancer death. Beyond mutations in ER in 25-30% of patients treated with aromatase inhibitors, which deplete circulating estrogen, our understanding of clinical mechanisms of resistance to agents that target the ER remains incomplete. We used whole exome sequencing (WES) of metastatic biopsies to identify mechanisms of resistance in patients with ER+ metastatic breast cancer (MBC) who had developed resistance to ER-directed agents, including aromatase inhibitors, tamoxifen, and fulvestrant. We noted mutations in human epidermal growth factor receptor 2 (HER2) in metastatic biopsies from 12 patients out of 168. Of these, eight mutations had previously been described as activating. Examination of the treatment-naïve primary tumors in the five patients with activating mutants where WES from the matched treatment-naïve primary tumor was available revealed no evidence of pre-existing mutations in four of the five patients, suggesting that these four mutations were acquired under the selective pressure of ER-directed therapy. These acquired HER2 mutations were mutually exclusive with ER mutations, suggesting a distinct mechanism of resistance to ER-directed therapies. In vitro analysis through expression in ER+/HER2- cell lines confirmed that these mutations conferred estrogen independence. In addition, and in contrast to ER mutations, these HER2 mutations resulted in resistance to tamoxifen, fulvestrant, and the CDK4/6 inhibitor palbociclib. The mutants result in increased AKT and ERK phosphorylation in ER+ cell lines, while simultaneously repressing ER levels and ER-dependent transcriptional targets. One mutation observed in two patients, S653C, occurring in the transmembrane domain and not previously observed in breast cancer, was shown to likely function through constitutive dimerization, a mechanism of activation not previously described for this mutation. Resistance caused by all four mutations was overcome by combining ER-directed therapy with the irreversible HER2 kinase inhibitor neratinib, suggesting a novel effective treatment strategy in these patients. Thus, we have shown that acquired activating HER2 mutations can confer endocrine resistance in ER+/HER2- breast cancer, and that such resistance can be effectively reversed by combination therapies that include an anti-HER2 inhibitor. Citation Format: Utthara Nayar, Ofir Cohen, Christian Kapstad, Adrienne Waks, Seth A. Wander, Corrie Painter, Samuel Freeman, Priyanka Ram, Nicole Persky, Lori Marini, Karla Helvie, Nelly Oliver, Cynthia X. Ma, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to ER-directed therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4952.

Angiosarcoma (AS) is an exceedingly rare soft tissue sarcoma, with an incidence of 300 cases/yr and a 5-year disease-specific survival of 30%. The low incidence has impeded large-scale research efforts that may lead to improved clinical outcomes. To address this, we launched a nationwide clinical-genomics study in order to empower patients to accelerate research by sharing their normal and tumor samples and clinical information remotely. Patients can access the study through an online portal (ASCproject.org). Enrolled patients are mailed saliva and blood draw kits. The study team obtains medical records and stored FFPE tumor samples. All received FFPE samples are examined by an expert pathologist to confirm a diagnosis of angiosarcoma. In order to validate that our processes would enable the generation of a robust dataset from tissues acquired from multiple institutions, we sought to characterize previously described genes known to be altered in angiosarcoma (e.g., TP53, NF1, KDR, BRCA2, MET, ARID1A, POT1, BRCA1, ASXL1, KDM6A, BRAF, SETD2, PTPRB, NRAS). A total of 251 patients have enrolled since the project launched in March of 2017. Primary locations of AS are primary breast 59 (25%), breast with prior radiation 45 (19%), head/face/neck/scalp 52 (22%), bone/limb 26 (11%), abdomen 5 (2%), heart 5 (2%), lung 2 (1%), liver 1 (1%), lymph 1 (0.4%), multiple locations 25 (11%), and other locations 12 (5%); 107 (52%) reported being disease free at the time of enrollment. To date, we have received 129 saliva kits, 106 medical records, 19 blood samples, and 36 tissue samples. Whole-exome sequencing (WES) was performed on 21 FFPE/saliva matched pairs with a goal mean target coverage of 150x for tumors. Ultra-low pass whole-genome sequencing (0.1x) was performed on cell free DNA (cfDNA) from plasma in order to determine tumor fraction. Of 10 cfDNA samples sequenced, 4 samples met criteria to perform WES. Additionally, transcriptome sequencing was performed on 9 FFPE samples. Sequence data processing and analysis has been completed on the first 10 samples and is in progress for the subsequent samples. Alterations were detected in genes previously described to be affected in angiosarcoma. Recurrent mutations in TP53 were detected in 50% (5/10) of analyzed samples, comprising 3 missense mutations, 1 frameshift deletion, and 1 frameshift insertion. Alterations were seen in at least one sample in all other genes selected for this initial analysis. This initiative demonstrates the feasibility of studying tissues from geographically dispersed patients and serves as proof of concept that patient-driven genomics efforts can democratize research for exceedingly rare cancers. Enrollment is still in progress, and additional samples will be sequenced and analyzed at scale. The data generated from these studies will be deposited into the public domain in six-month intervals. Citation Format: Michael Dunphy, Esha Jain, 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 Hendrey Lapan, Jason Hornick, Chandrajit Raut, George Demetri, Eric Lander, Todd Golub, Nikhil Wagle, Corrie Painter. The Angiosarcoma Project: Generating the genomic landscape of an exceedingly rare cancer through a nationwide patient-driven initiative [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5384.

Background: Despite the advent of new targeted- and immuno-therapies, chemotherapy continues to be a mainstay in most tumor types. In lung and colorectal cancer, platinum-based regimens are the standard of care as part of front-line systemic, neoadjuvant, or adjuvant chemotherapy regimens. Response to these agents varies widely among patients, and currently there are no mature or reliable biomarkers of clinical response to platinum therapies for these cancer types. We hypothesized that a systematic integrated genomic analysis of pre-treatment tumors from a large cohort of platinum-treated cancer patients would reveal genomic determinants of resistance and response, which may guide a precision medicine-based approach to choosing effective treatments. Methods: 238 metastatic lung and colorectal adenocarcinoma patients were enrolled in the CanSeq study from February 2013 to July 2015. We performed whole-exome sequencing (WES) on pre-treatment tumors, and called variants including single nucleotide variants (SNVs), small insertions and deletions, copy-number alterations, purity and ploidy of tumors, and inferred clonality and subclonality of individual mutations. Clinical records were reviewed to determine response to therapy, progression-free (PFS), and overall survival. Patients were characterized as having clinical benefit (CB), intermediate benefit (IB), and no clinical benefit (NCB) based on the best radiologic response to therapy and PFS. CB included all patients with complete response or partial response + a long PFS in the top quartile of responders, while NCB experienced progressive disease. IB included all other patients. We evaluated associations between genomic features and response using standard statistical methods. Results: 119 patients received platinum-based systemic chemotherapy and had pre-treatment tumors. Our initial analysis comparing CB vs. NCB suggests that mutations in PTPRD, a receptor protein tyrosine phosphatase previously associated with platinum resistance in cancer cell lines, may be significantly associated with chemoresistance (3/9 NCB, 0/23 CB, p < 0.05, Fisher's Exact) and shorter progression-free survival (p < 0.001, Log-Rank). Further, we found that lower mutational burden is associated with response to platinum chemotherapy (p < 0.05, Wilcoxon Rank Sum). Conclusion: Our approach demonstrates the power and potential of harmonizing genomic and clinical data in an unbiased way to identify biomarkers of platinum chemosensitivity. Our findings suggest that lower mutational burden correlates with CB from platinum therapy, while specific gene alterations potentially offer additional predictive value. Citation Format: Shirley S. Mo, David Liu, Stacy W. Gray, Steven Joffe, Nikhil Wagle, Judy Garber, Levi A. Garraway, Lynette Sholl, Pasi A. Janne, Eliezer M. Van Allen. Dissecting genomic determinants of response to platinum-based chemotherapy in advanced NSCLC and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3254.

The Metastatic Breast Cancer Project (MBCproject) is a research study that directly engages patients (pts) through social media and advocacy groups, and empowers them to share samples, clinical data, and experiences. The goal is to create a publicly available database of genomic, molecular, clinical, and patient-reported data to enable research. Working with pts and advocates, a website (MBCproject.org) was developed that allows pts with metastatic breast cancer (MBC) to register. Registered pts are sent an online consent form that asks for permission to obtain and analyze their medical records and samples. Once enrolled, pts 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 a portion of their stored tumor biopsies. Pts may be asked to mail in a blood sample, which is used to extract cell free DNA (cfDNA). Whole-exome sequencing (WES) is performed on tumor DNA, germline DNA, and cfNDA; transcriptome sequencing is performed on tumor RNA. Clinically annotated genomic data are used to study specific pt cohorts (including outliers) and to identify mechanisms of response and resistance to therapies. All de-identified data are shared via public databases. Study updates are shared with participants regularly. From 10/2015-11/2017, 4237 MBC pts registered, representing over 1,000 institutions. 95% answered the 16-question survey about their cancer, treatments, and demographic information. 2471 (58%) completed the consent form. 2,136 saliva kits were mailed to pts and 1,523 saliva samples were sent in (71%). 408 blood kits were mailed to pts and 175 blood samples have been received for cfDNA analysis. To date, we have obtained medical records from 311 pts and 190 tumors from 127 pts. In 10/2017, all data generated so far were publicly released on cbioportal.org, including WES for 103 tumors from 78 pts linked to clinical data including pathology (22 elements), medical record abstraction including all treatments and timelines/durations (67 elements), and patient-reported data (11 elements). 81% of biopsies included in this release were from the breast and 19% from metastatic sites. 75% were obtained prior to any therapy, 24% following therapy. New data will be released 4/2018 and every six months thereafter, as they are generated. Additional patient-reported data, including treatments, side effects, quality of life, family history, pregnancies, and sites of metastasis, will also be collected and shared. In summary, a patient-driven approach enabled rapid identification of thousands of MBC pts willing to share samples and clinical data. Remote acquisition of medical records, saliva, blood, and tumor tissue for pts across the U.S. is feasible. This shared clinico-genomic database should enable research in MBC and may serve as a model for patient-driven research in other cancers. Citation Format: Nikhil Wagle, Corrie Painter, Elana Anastasio, Michael Dunphy, Mary McGillicuddy, Rachel Stoddard, Esha Jain, Dewey Kim, Simona Di Lascio, Brett N. Tompson, Sara Balch, Beena Thomas, Priti Kumari, Shawn Johnson, Jamie Holloway, Ofir Cohen, Erik H. Knelson, Katie Larkin, Sam Pollock, Alicia Wong, Samira Bahl, Simone Maiwald, Andrew Zimmer, Esme O. Baker, Jen Hendry Lapan, Scott Sutherland, Scott Sassone, Viktor Adalsteinsson, Eric S. Lander, Todd R. Golub. The Metastatic Breast Cancer Project: Partnering with patients to accelerate progress in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5371.

1068Background: Although adding a CDK4/6i to endocrine therapy (ET) has proved to prolong progression-free survival in patients (pts) with HR+/HER2- MBC, it is unclear if continuing a CDK4/6i, toge...

1054Background: HR+/HER2- primary breast tumors demonstrate less anti-tumor immune activity than HER2+ or triple-negative BC. However, minimal data exist about the TME of HR+/HER2- met tumors, part...

e13501Background: A challenge in cancer research is the availability of tumor samples linked to clinical information. To address this, we worked with patients (pts) to develop patient-driven studie...

1010Background: High TMB correlates with high neoantigen burden, and this is thought to be predictive of response to immunotherapy. The aim of this work is to evaluate frequency, mutational pattern...

12016Background: Combining CDK4/6i with endocrine therapy results in prolongation of disease control in HR+ MBC, though resistance invariably occurs. There is a critical need to understand mechanis...

Lessons Learned A phase I study of the pan-class I phosphoinositide 3-kinase inhibitor pilaralisib (in capsule formulation) in advanced solid tumors established the maximum tolerated dose as 600 mg once daily. The current study investigated pilaralisib in tablet formulation. Pilaralisib tablets were associated with a favorable safety profile and preliminary antitumor activity. Based on pharmacokinetic data, the recommended phase II dose of pilaralisib tablets was established as 400 mg once daily. Background A phase I trial of pilaralisib, an oral pan-class I phosphoinositide 3-kinase (PI3K) inhibitor, established the maximum tolerated dose (MTD) of the capsule formulation in patients with advanced solid tumors as 600 mg once daily. This phase I study investigated pilaralisib in tablet formulation. Materials and Methods Patients with advanced solid tumors received pilaralisib tablets (100–600 mg once daily). Primary endpoints were MTD and safety; secondary and exploratory endpoints included pharmacokinetics (PK), pharmacodynamics, and efficacy. Results Twenty-two patients were enrolled. No dose-limiting toxicities (DLTs) were reported. The most common treatment-related adverse events were diarrhea (40.9%), fatigue (40.9%), decreased appetite (22.7%), and hyperglycemia (22.7%). Pilaralisib plasma exposure did not appear to increase dose-proportionally. Steady-state exposure was higher with pilaralisib tablet formulation at 400 mg than with pilaralisib capsule formulation at 400 or 600 mg (mean area under the curve [AUC0–24] 2,820,000 ng × h/mL vs. 2,653,000 and 1,930,000 ng × h/mL, respectively). Of 18 evaluable patients, 2 (11.1%) had a partial response (PR). Conclusion Pilaralisib tablets were associated with a favorable safety profile and preliminary antitumor activity. MTD was not determined. The recommended phase II dose for pilaralisib tablets, based on PK data, was 400 mg once daily.

279 Background: While there has been substantial advancement in the genomic understanding of metastatic prostate cancer (MPC), there is still much to be discovered. Additional progress is dependent upon obtaining a large amount of clinically-annotated genomic data. Therefore, we piloted a direct-to-patient nationwide research initiative where patients can contribute their medical records and biospecimens to accelerate research ( mpcproject.org ). Methods: In collaboration with patients and advocacy groups, we have developed a website ( mpcproject.org ). Participants are asked to complete a 17-question survey about their experiences with prostate cancer and an electronic informed consent. All participants receive a saliva kit for germline DNA and blood kit for circulating tumor DNA (ctDNA). Additionally, medical records are collected and archived tissue samples are requested if available. Ultra low pass whole genome sequencing (ULP-WGS) and whole exome sequencing (WES) are performed on the whole blood samples. WES is performed on saliva samples. Genomic, clinical, and patient-reported data will be shared widely with the research community. Aggregate study results will be reported to patients. Results: As of October 2017, 12 pilot patients aged 47-74 from 7 states, provided informed consent. 7 saliva kits, 4 blood kits, and 2 medical records were received. 4 patients were diagnosed with de novo metastatic disease, 8 reported a family history of breast and/or prostate cancer, 6 reported a secondary malignancy. All blood kits were submitted for ULP-WGS and WES. Updated genomic, clinical, and patient-reported data will be presented. Conclusions: We have provided preliminary evidence that partnering directly with MPC patients enabled the remote collection of saliva and blood samples, medical records, and patient-reported data. At the conclusion of the pilot phase, the MPC Project will open enrollment for all men with metastatic and advanced prostate cancer in the US and Canada.

Approximately 70% of breast cancers express the estrogen receptor (ER), and estrogen signaling drives breast cancer cell growth and progression. ER-directed therapies are commonly used to treat ER+ breast cancer and have improved survival for patients, yet resistance to those therapies inevitably occurs. Mutations in the estrogen receptor itself occur in ∼25-30% of patients with ER+ metastatic breast cancer that has developed resistance to aromatase inhibitors. Beyond these ER mutations, other resistance mechanisms are not well described. Moreover, clinical mechanisms of resistance to another class of ER-targeted agents, selective estrogen receptor degraders (SERDs), such as fulvestrant have not been clearly identified. Here we report two FGFR2 mutations identified in patients with resistant ER+ metastatic breast cancer, N550K and M538I. N550K is a well-known activating FGFR2 mutation; M538I stabilizes the active kinase conformation and it has not previously been described in breast cancer. When expressed in ER+ T47D cells, FGFR2 M538I and N550K led to resistance to fulvestrant, and the CDK4/6 inhibitor palbociclib and the combination of the two agents. FGFR2 M538I induced hyperactivity of p-FRS2, p-ERK and p-AKT, which is higher than wildtype FGFR2 and comparable to other known activating mutations N550K and K660N. In addition, overexpression of M538I mutant reduced sensitivity to FGFR inhibitors PD173074 and dovitinib in T47D cells, suggesting M538I is also functionally activating. Due to the hyperactive downstream signaling elicited by the mutation, cells overexpressing FGFR2 M538I achieved optimal growth in the presence of low dose of FGFR inhibitor. Under such conditions, FGFR2 M538I conferred more potent resistance to fulvestrant as compared to wildtype FGFR2. However, drug resistance resulting from M538I mutant can be fully resensitized to fulvestrant and/or palbociclib with high dose of FGFR inhibitors. In summary, we have identified activating FGFR2 mutations (M538I and N550K) in ER+ breast cancer patients, which may contribute to the development of resistance to SERDs and CDK4/6 inhibitors. Additional FGFR2 mutations have been recently identified in other cohorts of patients with resistant ER+ metastatic breast cancer, suggesting that this may be a clinical mechanism of resistance in some patients. Patients with activating FGFR2 mutations may benefit from the treatment with an FGFR inhibitor in combination with SERDs and CDK4/6 inhibitors. Citation Format: Mao P, Kusiel J, Cohen O, Wagle N. The role of FGF/FGFR axis in resistance to SERDs and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-01.

Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER+) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets.

Importance Randomized clinical trials demonstrate no benefit for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in unselected patients with head and neck squamous cell carcinoma (HNSCC). However, a patient with stage IVA HNSCC received 13 days of neoadjuvant erlotinib and experienced a near-complete histologic response. Objective To determine a mechanism of exceptional response to erlotinib therapy in HNSCC. Design, Setting, and Participants Single patient with locally advanced HNSCC who received erlotinib monotherapy in a window-of-opportunity clinical trial (patients scheduled to undergo primary cancer surgery are treated briefly with an investigational agent). Whole-exome sequencing of pretreatment tumor and germline patient samples was performed at a quaternary care academic medical center, and a candidate somatic variant was experimentally investigated for mediating erlotinib response. Intervention A brief course of erlotinib monotherapy followed by surgical resection. Main Outcomes and Measures Identification of pretreatment tumor somatic alterations that may contribute to the exceptional response to erlotinib. Hypotheses were formulated regarding enhanced erlotinib response in preclinical models harboring the patient tumor somatic variant MAPK1 E322K following the identification of tumor somatic variants. Results No EGFR alterations were observed in the pretreatment tumor DNA. Paradoxically, the tumor harbored an activating MAPK1 E322K mutation (allelic fraction 0.13), which predicts ERK activation and erlotinib resistance in EGFR -mutant lung cancer. The HNSCC cells with MAPK1 E322K exhibited enhanced EGFR phosphorylation and erlotinib sensitivity compared with wild-type MAPK1 cells. Conclusions and Relevance Selective erlotinib use in HNSCC may be informed by precision oncology approaches.

Remarkable progress in sequencing technology over the past twenty years has made it possible to comprehensively profile tumors and identify clinically relevant genomic alterations. In breast cancer, the most common malignancy affecting women, we are now increasingly able to use this technology to help specify the use of therapies that target key molecular and genetic dependencies. Large sequencing studies have confirmed the role of well-known cancer-related genes but have also revealed numerous other genes that are recurrently mutated in breast cancer. This growing understanding of patient-to-patient variability at the genomic level in breast cancer is advancing our ability to direct the appropriate treatment to the appropriate patient at the appropriate time – a hallmark of ‘precision cancer medicine.’ This review focuses on the technological advances that have catalyzed these developments, the landscape of mutations in breast cancer, the clinical impact of genomic profiling, and the incorporation of genomic information into clinical care and clinical trials.

The development of new cancer therapeutics requires sufficient genetic and phenotypic diversity of cancer models. Current collections of human cancer cell lines are limited and for many rare cancer types, zero models exist that are broadly available. Here, we report results from the pilot phase of the Cancer Cell Line Factory (CCLF) project that aims to overcome this obstacle by systematically creating next-generation in vitro cancer models from adult and pediatric cancer patients' specimens and making these models broadly available. We first developed a workflow of laboratory, genomics and informatics tools that make it possible to systematically compare published ex vivo culture conditions for each individual tumor to enable the scientific community to iterate towards disease-specific culture recipes. Based on sample volume and rarity, 4-100 conditions were applied to each sample and all data was captured in a custom Laboratory Information Management System to enhance subsequent predictions. We developed a $150, 5-day turnaround genomics panel to validate cultures based on genomics. Importantly, we show that tumor genomics can be retained in such patient-derived models and tumor genomics are generally stable across 20 passages. Since the inception of this project, we have processed over 600 patient cancer specimens from 450 patients across 16 tumor types and report the successful generation of over 100 genomically characterized adult and pediatric cancer and normal models. We next hypothesized that novel patient-derived cell models could be used to enhance dependency predictions. To do so, we tested 72 cell lines against the informer set of 440 compounds developed by the Broad Cancer Target Discovery and Development (CTD2) Center. We show that generating cell lines and testing their sensitivities within 3 months is feasible and the high-throughput drug responses are reproducible. Moreover, to strengthen relationships between drug sensitivities and cellular features, we compared results with recently published data on the identical compounds tested against 860 existing cell lines. With this approach, we show that many chemical-genetic interaction vulnerabilities can be rapidly assessed. Importantly, adding more cancer models with the dimensions of quantity and diversity increases the predictive power of chemical-genetic interaction map. We are currently evaluating these drug sensitivity predictors for novel co-dependencies. Overall, our proof-of-concept framework demonstrates initial feasibility of rapidly generating cancer models at scale and expanding the chemical-genetic interaction map to identify new cancer vulnerability. Citation Format: Yuen-Yi (Moony) Tseng, Andrew Hong, Shubhroz Gill, Paula Keskula, Srivatsan Raghavan, Jaime Cheah, Aviad Tsherniak, Francisca Vazquez, Sahar Alkhairy, Anson Peng, Abeer Sayeed, Rebecca Deasy, Peter Ronning, Philip Kantoff, Levi Garraway, Mark Rubin, Calvin Kuo, Sidharth Puram, Adi Gazdar, Nikhil Wagle, Adam Bass, Keith Ligon, Katherine Janeway, David Root, Stuart Schreiber, Paul Clemons, Todd Golub, William Hahn, Jesse Boehm. Expanding tumor chemical-genetic interaction map using next-generation cancer models [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A02.

Introduction: In recent studies, constitutively active recurrent ESR1 ligand binding mutations (LBD) were found in about 20% of metastatic (met) HR+ breast cancers ( BRCAs) and rarely in primary HR+ cancers. In our previous work, we analyzed clinical tissue samples and detected an association between the number of prior endocrine treatments and the prevalence of these mutations, suggesting the emergence of the ESR1 mutations under the selective pressure of endocrine treatment. More recently, the LBD ESR1 mutations were successfully detected in plasma cell free (cf)DNA in patients with met HR+ disease. The presence of mutant ESR1 cfDNA was found to be prognostic. Here we sought to study the association between endocrine treatments in the adjuvant (adj) and met settings and the prevalence of cfDNA ESR1 and PIK3CA mutations in patients with met HR+ BRCA. Methods: Plasma samples and detailed clinical data were collected from patients with met BRCA through the Collection of Specimens and Clinical Data program of the Breast Oncology Center at the Dana Farber Cancer Institute. Droplet Digital PCR was used for the detection of the most common ESR1 LBD mutations (E380Q, Y537C, Y537N, Y537S and D538G) and the 3 most common PIK3CA mutations (E542K, E545K and H1047R) in cfDNA. Fisher’s Exact Test was used for statistical analysis. Results: Plasma samples were collected from 155 patients with met BRCA. ESR1 mutations were found in 30.1% of the patients with HR+/HER2- negative disease (34/113). PIK3CA mutations were detected in 31.8% of patients with HR+/HER2- disease. The majority of the patients had either newly diagnosed met disease or progressive met disease at the time of the blood draw. 14 patients had stable met disease and among these patients, only 1 of these patients was found to have an ESR1 mutation and no PIK3CA mutations were detected. The majority of patients with ESR1 mutations (88%) and PIK3CA mutations (75%) had progressive disease. Patients that received an aromatase inhibitor (AI) either in the adj or met setting had a higher prevalence of ESR1 mutations compared to patients that had no AI treatment, regardless of whether or not they received tamoxifen (TAM) (prevalence was 32% for adj AI only, 40.4% AI in met only, No AI and no TAM 7.1% and TAM but no AI 6.7%). In addition fulvestrant treatment in the met setting was significantly associated with ESR1 mutations (odds ratio 3.38, p-value Conclusions: Analysis of cfDNA can successfully detect ESR1 and PIK3CA mutations in newly diagnosed or progressive met BRCA patients and the emergence of the ESR1 mutations is associated with AI and fulvestrant treatment. These results support the serial monitoring of ESR1 mutations in cfDNA in met disease and highlight the need to study new agents to target these mutations. Citation Format: Yanan Kuang, Bilal Siddiqui, Jiani Hu, William T. Barry, Nancy U. Lin, Nikhil Wagle, Paul Kirschmeier, Pasi A. Jänne, Cloud Paweletz, Ian Krop, Eric P. Winer, Myles Brown, Rinath Jeselsohn. The emergence of ESR1 mutations is associated with aromatase inhibitor and fulvestrant therapy [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 4950. doi:10.1158/1538-7445.AM2017-4950

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth-leading cause of cancer-related death in the United States and is projected to become the second leading cause by 2030. Most patients present with advanced disease and die within 12 months of diagnosis. Recent genomic studies of primary pancreatic cancer resection specimens have identified several molecular alterations and genomic subtypes of the disease that may guide precision medicine approaches to clinical management. However, the molecular landscape of metastatic PDAC has been less well characterized. Moreover, biopsy-driven studies in metastatic PDAC have been historically very challenging due to the aggressive course of this disease as well as the low-volume and heterogeneous nature of biopsies that makes deep molecular characterization difficult. Insufficient genomic analysis of a patient’s tumor early in their disease course is a major barrier to enrollment on clinical trials of targeted therapies. To address these limitations, we have implemented a multi-disciplinary clinical and research biopsy protocol to enable real time comprehensive molecular characterization of metastatic PDAC biopsy specimens. We have performed core needle biopsies of metastatic lesions in the liver or peritoneal cavity in 42 patients at the time of initial presentation. A low rate of complications was observed, with only a single patient having a self-limited hemorrhagic complication after liver biopsy. On average, 4-6 separate biopsy specimens were collected from each patient for histopathology and genomic analysis. Whole exome sequencing (WES) was performed in a CLIA-certified laboratory and a comprehensive molecular report of somatic alterations and selected pathogenic germline variants was returned to the referring clinician with a typical turn-around time of 3-5 weeks. We observed a striking incidence of recurrent germline and somatic alterations in DNA-damage repair genes, such as BRCA2, ATM and CHEK2. We also observed alterations in genes with known therapeutic implications, such as BRAF, RNF43, STK11 and ROS, and in select cases, these results guided choice of second or third line therapy. In parallel to WES, we performed RNA sequencing on bulk tumor tissue and readily identified expression signatures defining multiple subtypes of tumor and stroma that may have prognostic or therapeutic implications for tumor- or stroma-directed therapies. Collectively, these results demonstrate the feasibility and value of real-time genomic characterization of metastatic PDAC and provide a path forward for improved stratification and enrollment of PDAC patients on molecularly defined clinical trials. Citation Format: Andrew J. Aguirre, Scott Carter, Nicholas Camarda, Arezou Ghazani, Jonathan Nowak, Annacarolina Da Silva, Lauren Brais, Dorisanne Ragon, Devin McCabe, Lori Marini, Kristin Anderka, Karla Helvie, Nelly Oliver, Ana Babic, Paul Shyn, Douglas Rubinson, Anuj Patel, James Cleary, Nadine McCleary, Matthew Kulke, Thomas Clancy, Leona Doyle, Jason Hornick, Christine Ardito-Abraham, Ruth Yu, Michael Downes, Ronald Evans, Richard A. Moffitt, Jen Jen Yeh, William C. Hahn, Charles Fuchs, Robert Mayer, Nikhil Wagle, David Tuveson, Levi A. Garraway, Brian M. Wolpin. Real-time genomic characterization of metastatic pancreatic cancer to enable precision medicine [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 3036. doi:10.1158/1538-7445.AM2017-3036

Ongoing pre-clinical efforts aim to deploy genome-scale CRISPR/Cas9 technology and large collections of small molecules to catalog maps of cancer vulnerabilities at scale. However, such efforts in pediatric and rare cancers have lagged behind comparable efforts in more common cancer types due to the dearth of cell models. Here, we present an update from our “Cancer Cell Line Factory” project on efforts to overcome key laboratory and biologistics challenges precluding progress in pediatric and rare cancers. This effort, now in it’s 3rd year, represents an industry scale pipeline aiming to generate, characterize and share novel cancer models of many tumor types with the scientific community. Overall, we have processed 1153 samples from 818 patients across over 16 cancer types through this pipeline with a 28% success rate overall, including over 350 patient samples from rare and pediatric cancers. To optimize conditions for each tumor type, we have systematically compared published methods including (1) next-generation 2-dimension, (2) organoid and (3) standard approaches and have captured all information with a data management system that should enhance the ability to predict optimal ex vivo propagation conditions for future samples. Among the successful cell models verified already as part of this effort, we have generated a series of over 30 unique pediatric and rare cancer models, many of which represent the first of their kind. We screened these and other models against a library of highly annotated 440 small molecules that were previously tested against 860 existing cancer cell lines. Our results suggest that dependency data generated with novel next-generation cell cultures is potentially backwards-compatible with existing small molecule dependency datasets. Furthermore, we tested the novel Broad Institute Drug Repurposing library consisting of 4100 approved therapeutics, or those under investigation for any disease, against the first cell line models of several of these rare next generation models including angioimmunoblastic T-cell lymphoma and renal medullary carcinoma, leading to several novel drug repurposing hypotheses for rare cancers. Given these proof-of-concept studies, in partnership with the Rare Cancer Research Foundation, we launched an online matchmaking platform to connect patients with rare cancers to available research studies, facilitate online consent and provide biologistics support to enable fresh tissue donation to support cancer model generation from any clinical site in the United States. We will present results from this novel direct-to-patient approach to facilitate the generation of even larger numbers of next generation models from rare and pediatric cancers, propelling the generation of pre-clinical dependency maps of these tumors for the scientific community. Citation Format: Yuen-Yi Tseng, Andrew Hong, Paula Keskula, Shubhroz Gill, Jaime Cheah, Grigoriy Kryukov, Aviad Tsherniak, Francisca Vazquez, Glenn Cowley, Sahar Alkhairy, Coyin Oh, Anson Peng, Rebecca Deasy, Abeer Sayeed, Peter Ronning, Samuel Ng, Steven Corsello, Corrie Painter, David Sandak, Levi Garraway, Mark Rubin, Calvin Kuo, Sidharth Puram, David Weinstock, Adam Bass, Nikhil Wagle, Keith Ligon, Katherine Janeway, David Root, Stuart Schreiber, Paul Clemons, Aly Shamji, Aly Shamji, William Hahn, Todd Golub, Jesse Boehm. Accelerating prediction of pediatric and rare cancer vulnerabilities using next-generation cancer models [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 1953. doi:10.1158/1538-7445.AM2017-1953

1034 Background: As a single agent, eribulin is an active agent among MBC pts. In the first-line setting, pertuzumab improves progression free and overall survival when added to trastuzumab and chemotherapy (CT); however there are limited data in the second line or beyond. The primary aim of this study was to evaluate the activity of eribulin in combination with trastuzumab and pertuzumab in pts with HER2+ MBC whose cancers are refractory to trastuzumab containing regimens. Safety and toxicity were examined and correlative studies are also planned. Methods: Single center, single-arm, phase II, 2 cohorts study planned to enroll up to 81 pts. The studied combination would be of clinical interest if the true overall response rate (ORR) was 40% among those without prior pertuzumab exposure (Cohort A) and 30% among those with prior pertuzumab exposure (Cohort B). The trial was stopped prematurely due to low accrual. Prior to the start of the phase II study, a run-in established dose of eribulin with standard dose of trastuzumab and pertuzumab (1.4mg/m2 days 1, 8 /21 days). Results: Of 32 pts enrolled, 6 were in the run-in, 19 in Cohort A and 7 in Cohort B. Most pts were heavily pre-treated (median [range] lines of CT for metastatic disease, run-in: 4[2-11], cohort A: 2.5 [0-7], cohort B: 3[1-8]). In cohort A, 5 pts had partial responses (ORR = 26% [95% CI 9-51%]), 1 (5%) experienced stable disease (SD) lasting ≥ 6 months and 11 (58%) had a SD lasting < 6 months. In cohort B, the ORR was 0% [95% CI 0-41%]), 1(14%) experienced SD lasting ≥ 6 months and 5 (75%) had a SD lasting < 6 months. The combination was well tolerated, most adverse events (AE) were mild to moderate. Hematologic toxicity was the most frequent grade 3-4 AE. Analyses looking at tissue genomic markers and cell free DNA will be presented. Conclusions: We observed modest activity of the treatment combination in those without prior pertuzumab exposure but limited activity in unselected pts with HER2+ MBC. The study was limited by small sample size. Correlative studies may help us to understand resistance and response to eribulin with anti-HER2 therapy and the interplay between CT and targeted therapy. Clinical trial information: NCT01912963.

1092 Background: Triple-negative breast cancer (TNBC) is a poor prognosis breast cancer subset characterized by relatively few mutations but extensive copy number alterations (CNAs). Cell-free DNA (cfDNA) offers the potential to overcome infrequent tumor biopsies in metastatic TNBC (mTNBC) and interrogate the genomics of chemotherapy resistance. Methods: 506 archival or fresh plasma samples were identified from 164 patients with mTNBC who had previously received chemotherapy. We performed low coverage sequencing to determine genome-wide copy number and estimate ‘tumor fraction’ of cfDNA (TFx). In patient samples with TFx >10%, we identified regions that were significantly gained or lost using GISTIC2.0. We compared CNAs of mTNBCs with primary TNBCs from a publicly-available dataset, METABRIC (TNBC n=277). Results: We successfully obtained high quality, low coverage whole genome sequencing data for 478 (94.5%) plasma samples from 158 patients, with 1 to 14 samples per patient. Archival samples had significantly higher average cfDNA per mL plasma and TFx than fresh samples, potentially due to later average line of therapy. Average TFx of first blood draw was significantly higher in patients with liver metastases (TFx 28.3% vs. 14.4%, p=1.1e-7). 101/158 patients (63.9%) had at least one sample with TFx >10%, our threshold for high confidence CNA calls. Most alterations significantly enriched in chemotherapy-resistant mTNBCs were chromosomal gains, including NOTCH2 and ERCC1. Median overall survival from time of first blood draw was 9 months, and TFx was highly correlated independent of metastatic line of therapy, age at metastatic diagnosis, BRCA status, and primary stage: adjusted hazard ratio between 4th and 1stquartiles = 4.29 (95% CI 1.66-11.1; p=0.0008). Conclusions: It is feasible to perform genome-level copy number analysis from cfDNA in both archival and fresh samples from patients with mTNBC. Copy number alterations enriched in mTNBC may have implications in the understanding of metastasis, therapeutic resistance, and novel therapeutic targets. ‘Tumor fraction’ of cfDNA is correlated with overall survival and may be an independent prognostic marker in mTNBC.

1519 Background: Angiosarcoma (AS) is a rare soft tissue sarcoma, with an incidence of 300/yr and a 5-year DSS of 30%. The low incidence has impeded large-scale research efforts that may lead to improved clinical outcomes. To address this, we launched a nationwide study, which seeks to empower patients (pts) to accelerate research by sharing their samples and clinical information remotely. Methods: With pts and advocacy groups we developed a website to allow AS pts to participate across the US. Pts are mailed a saliva and blood draw kit for germline and cell free (cf) DNA analysis. We then obtain medical records and stored tumor samples. Whole exome sequencing will be performed on tumor, cfDNA and saliva samples. Transcriptome analysis will be performed on tumor samples. A clinically annotated genomic database will be generated and shared widely to identify genomic drivers and mechanisms of response and resistance to therapies. Study updates will be shared with pts regularly. Results: We conducted a 3-week pilot study to test the feasibility of enrolling geographically dispersed AS pts through a direct-to-patient (DTP) approach. Through social media, we identified 100+ pts willing to participate, 90 within the first day of outreach. We enrolled 15 pts from 10 states to test our ability to remotely obtain pt reported data, online consent, and samples. The average age of pts is 48, ranging 23-71 yrs. Primary locations of AS are breast 6 pts (40%), cardiac 4 pts (27%), scalp 2 pts (13%), liver 1 pt (6%), bladder 1 pt (6%), forehead 1 pt (6%). 9 pts (60%) reported being disease free, 4 pts (27%) reported having AS spread to lung, lymph, bone, and hip. Requests for medical records and tissue samples are underway, and initial saliva samples have been received. We are now opening this study to all AS pts in the USA. Conclusions: A DTP approach enabled rapid identification of an initial cohort of AS pts willing to share tumors, saliva, blood and medical records. We were able to obtain detailed clinical experiences and samples to perform genomic analysis. This study serves as proof of principle that DTP genomics efforts can democratize cancer research for exceedingly rare cancers, which to date have been disproportionately understudied.

1076 Background: The Metastatic Breast Cancer Project is a nationwide research study, launched in Oct 2015 in collaboration with patients (pts) and advocacy groups, that directly engages pts through social media and seeks to empower them to share their experiences, clinical information, and samples to accelerate research. Methods: MBC pts enroll by providing their information at mbcproject.org. Pts are sent a saliva kit and asked to mail back a sample which is used to extract germline DNA. We contact pts medical providers and obtain medical records (MRs) and stored tumor samples. Pts may also submit a blood sample, used to extract cell free DNA (cfDNA). Whole exome sequencing (WES) is performed on tumor, germline, and cfDNA; transcriptome sequencing is performed on tumor. Clinical and genomic data are used to generate genomic landscapes in pt subgroups and to identify mechanisms of response and resistance to therapies. Data are shared widely through public databases. Pts receive regular study updates. Results: In 12 months, 2908 MBC pts from 50 states enrolled. 95% completed the 16-question survey about their cancer, treatments, and demographics. 1730 (60%) completed the online consent form. 100-200 pts continue to enroll monthly. To date, 1539 saliva kits were mailed and 1120 samples were received (73%). 992 unique treating institutions were reported by pts, including 733 institutions reported by only 1 pt each and 5 institutions reported by more than 40 pts each. We have obtained MRs from 253 patients (67% yield) and tumor samples from 85 pts (67% yield). WES was successfully completed for 79 tumors of 88 attempted (90%). WES has been performed on initial cfDNA samples. Conclusions: A direct-to-patient approach enabled rapid identification of thousands of MBC pts willing to share MRs, saliva, and tumor samples, including many with rare phenotypes. Remote acquisition of MRs, saliva, tumor, and blood for pts located throughout the US is feasible. We estimate that for ~33% of consenting patients, we can obtain medical records, saliva, and tumor tissue. Genomic analysis of tumor and cfDNA from subgroups including young pts, pts with extraordinary responses, and pts with de novo MBC will be presented.