Citations

Abstract Estrogen receptor positive (ER+) breast cancer accounts for the majority of breast cancers diagnosed worldwide but fortunately, outcomes are markedly improved by pharmacological interventions that interrupt ER function. Unfortunately, suppression of relapse risk from early stage disease with endocrine therapy (anti-estrogens or aromatase inhibitors) is only ∼50%, and for advanced disease, pan endocrine therapy resistance is almost inevitable. While many mechanisms for intrinsic and acquired endocrine resistance have been explored, links between defects in DNA repair, the fundamental drivers of cancer pathogenesis, and endocrine therapy resistance have been understudied. Here we link mismatch repair (MMR) deficiency to poor clinical outcomes in ER+ breast cancer using whole exome DNA sequencing data and mRNA expression analysis. We subsequently demonstrate that MMR deficiency bypasses ER dependent cell cycle regulation through disruption of Chk2/p21-mediated feedback inhibition of CDK4 in breast cancer cell lines and tumor samples, as well as through correlations with human clinical data. We also show that pharmacological targeting of CDK4 significantly inhibits growth of MMR-deficient ER+ breast cancer cells in vitro and in vivo. This mechanism provides a new explanation for why endocrine therapy resistant ER+ breast cancers can respond to CDK inhibition and suggests that primary tumors exhibiting MMR deficiency are good candidates for adjuvant CDK4 inhibitor treatment. Citation Format: Haricharan S, Schmelz J, Schmidt C, Singh P, Holloway K, Anurag M, Suman V, Olson JA, Hunt K, Bainbridge MN, Ellis MJ. Mismatch repair deficiency induces endocrine therapy resistance in 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 PD2-06.

Abstract Background. We previously reported an alternative ESR1 somatic gain-of-function chromosomal translocation event in a patient presenting with aggressive, endocrine therapy resistant estrogen receptor (ER) positive disease, producing an in-frame fusion gene consisting of N-terminal ESR1 and the C-terminus of the Hippo pathway coactivator YAP1 (ESR1-YAP1). We recently identified another ESR1 fusion through RNA sequencing (RNA-seq) in advanced stage ER+ disease from a chest wall recurrence in a male patient that was refractory to multiple lines of treatment. Two examples of fusions discovered in primary breast cancer samples include ESR1 fused in-frame to C-terminal sequences from NOP2 (ESR1-NOP2), identified in a resistant cohort from a RNA-seq screen focused on 81 primary breast cancers from aromatase inhibitor clinical trials, and a second ESR1 fusion, fused in-frame to the entire coding sequence of POLH (ESR1-POLH), that was identified from RNA-seq analysis of 728 Cancer Genome Atlas breast samples. This current study extends our previous characterization of ESR1-YAP1 by comparing functional and pharmacological properties of these three additional ESR1 gene fusion events of both early stage and advanced breast cancers. Methods. In vitro and in vivo experiments were conducted to test ESR1 fusions to induce therapeutic resistance, and metastasis. The transcriptional and binding properties of each fusion was also examined. Pharmacological inhibition with Palbociclib, a cyclin-dependent kinase 4/6 inhibitor, was utilized to assess drug sensitivity in ESR1 fusion containing breast cancer cells and in a patient derived xenograft (PDX) model expressing ESR1-YAP1 (WHIM18). Results. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth. Immunohistochemistry revealed significantly higher numbers of ER+ cells in lungs of mice xenografted with T47D cells expressing the YAP1 and PCDH11x fusions compared to YFP control, NOP2 and POLH fusions. Results from ChIP-seq and microarray studies suggest that these two fusions promote proliferation and metastasis through genomic action by binding estrogen response elements (ERE) and subsequent gene activation. We thereby define these fusions as “canonical” fusions compared to “non-canonical” NOP2 and POLH fusions, which demonstrated dramatically decreased genomic binding ability. The non-canonical fusions induced genes associated with basal-like breast cancer and promoted HER2, EGFR, and MAPK gene expression signatures in contrast to genes associated with cell cycle/proliferation induced by canonical fusions. The proliferative ability of canonical fusion-containing ER+ cells was inhibited by Palbociclib in a dose-dependent manner. In vivo WHIM18 tumors in mice fed with Palbociclib-containing chow demonstrated significantly reduced tumor volume, growth rate, and weight compared to tumors in mice on control chow. Conclusions. In-frame ERE activating canonical fusions occur in end-stage drug resistant advanced breast cancer and can be added to ESR1 point mutations as a class of recurrent somatic mutation that may cause acquired resistance. Growth induced by these fusions can be antagonized by Palbociclib and is potentially clinically helpful. Citation Format: Lei JT, Shao J, Zhang J, Iglesia M, Cao J, Chan DW, He X, Kosaka Y, Schmidt C, Matsunuma R, Haricharan S, Crowder R, Hoog J, Phommaly C, Goncalves R, Ramalho S, Lai W-C, Hampton O, Rogers A, Tobias E, Parikh P, Davies S, Ma C, Suman V, Hunt K, Watson M, Hoadley KA, Thompson A, Chen X, Perou CM, Creighton CJ, Maher C, Ellis MJ. Recurrent functionally diverse in-frame ESR1 gene fusions drive endocrine resistance in 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 PD2-03.

Abstract BACKGROUND: Based on prospective clinical trials, there is no evidence for differences in efficacy between the 3 aromatase inhibitors (AIs) anastrozole, exemestane, and letrozole. The purpose of this study was to identify germline genetic variants associated with response to AIs and to help identify novel mechanisms associated with drug disease efficacy. METHODS: A genome-wide association study (GWAS) was performed for 624 patients (Steroids 2015;99:32-38) to identify SNPs associated with estrogen level change in women with estrogen receptor (ER) positive breast cancer treated with anastrozole. Replication of associated SNPs was performed in a GWAS from the MA.27 trial that compared adjuvant anastrozole and exemestane treatment of post-menopausal women with ER+ breast cancer. Functional studies were subsequently performed to determine SNP effects and underlying mechanisms. RESULTS: Our initial GWAS identified SNPs within CSMD1 that were associated with changes in estrogen levels during anastrozole therapy. An additional SNP in CSMD1 was also associated with breast cancer events in CCTG MA.27. Functionally, we showed that CSMD1 regulates CYP19 expression in a SNP-, and in an anastrozole- dependent fashion. These phenomena were not observed for either letrozole or exemestane. In MA.27, an anastrozole- specific effect was also seen with the minor allele having a protective effect on time to distant metastasis (HR=0.49, p=0.00259), but this was not the case for exemestane (HR=0.71, p=0.111). Our in vitro functional studies indicated that overexpression of CSMD1 sensitized anastrozole or letrozole resistant cells to anastrozole but not to the other two AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane in lymphoblastoid cell lines (LCLs) homozygous for either WT or variant CSMD1 SNP genotypes. Based on these observations, we explored whether anastrozole has additional mechanisms beyond its function as a CYP19 inhibitor. Utilizing an estrogen response element (ERE) luciferase reporter assay in a CYP19 CRISPR knockout breast cancer T47D cell line and a surface plasmon resonance (SPR) assay, we found that anastrozole can also function as an ERα agonist, and can bind to, and result in, proteasome dependent ERα degradation, especially in the presence of E2. Treatment of these CYP19 CRISPR knockout cells with anastrozole in the presence of increasing concentrations of E2 results in greater sensitivity compared with anastrozole alone, while the addition of E2, as expected, does not improve letrozole or exemestane sensitivity. These same observations were also seen in letrozole and anastrazole resistant cells. CONCLUSIONS: Our findings suggest that anastrozole might be more effective than letrozole or exemestane in patients with the CSMD1 SNP. Furthermore, anastrozole can function as an ERα agonist, binding to ERα and resulting in its degradation, especially in the presence of E2. These findings should help to make it possible to develop precision endocrine therapies for women who are candidates for AIs. Citation Format: Cairns J, Ingle J, Dudenkov T, Kalari K, Buzdar A, Kubo M, Robson M, Ellis M, Goss P, Shepherd L, Goetz M, Weinshilboum R, Wang L. CSMD1 SNPs selectively affect anastrozole response in postmenopausal breast cancer patients [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 PD1-04.

94 Background: Technetium (Tc99m) trofolastat (USANC) is a novel urea-based small molecule SPECT radiotracer with utility in imaging overexpression of PSMA in prostate cancer (PCa). Unlike earlier agents targeting PSMA, this ligand is rapidly internalized and retained in tumors. We conducted an open-label, multicenter phase 2 study in the US and Europe (NCT01667536). An interim analysis was performed at approximately 50% accrual. Methods: Patients (pts) with confirmed adenocarcinoma of the prostate scheduled for RP with EPLND at high risk for disease outside of the prostate gland were eligible. High risk pts were ≥cT3 or Kattan nomogram score ≥130. Within 30 days of screening, pts required a bone scan and pelvic MRI. After enrollment, pts received trofolastat followed by whole-body planar and SPECT/CT imaging 3 to 6 hrs later. Pts then underwent RP with EPLND within 21 days. SPECT/CT images were evaluated centrally by 3 readers blinded to clinical information and compared to on-site pathology assessments using a common scoring template. The primary endpoint was the ability of trofolastat to detect PCa within the gland. Secondary endpoints included detection of extent and location within the gland, pelvic lymph nodes and comparative performance against MRI. Results: 84 pts were enrolled to date from 16 centers. Interim data is available for 54 pts. A majority (≥2/3) of SPECT/CT readers correctly identified the presence or absence of primary PCa in 51/54 (94%, 85-98 95% CI) patients including 2 true-negative cases treated with neoadjuvant enzalutamide. Sensitivity and specificity were 94% (84-98 95% CI) and 100% (34-100 95% CI) respectively. Conclusions: Based on the interim data available, trofolastat has accurately detected primary prostate carcinoma within the gland with high sensitivity and specificity in high-risk pts prior to surgery. Updated results, analyses of secondary endpoints, pelvic lymph nodes, and comparative performance vs. MRI from this ongoing study will be presented. Clinical trial information: NCT01667536. [Table: see text]

442 Background: For patients with advanced or locally recurrent (LR) renal cell carcinoma (RCC), the role of intra-operative radiation therapy (IORT) remains controversial. We analyzed outcomes in a multi-institutional cohort of patients with RCC who received IORT. Methods: Between 1985 and 2010, 98 patients were treated with IORT for advanced or LR RCC at 9 institutions. We collected demographic, clinical, treatment, and outcomes data for all patients. Overall (OS), disease specific (DSS), and disease-free survival (DFS) was estimated using the Kaplan-Meier method. A multivariate Cox hazards regression was used to test significance. Results: IORT was delivered at nephrectomy for advanced disease (28%) or during resection of LR RCC in the renal fossa (72%). 69% of patients were male and mean age at RCC diagnosis was 57. At initial nephrectomy, primary T-stage was 17% T1, 12% T2, 55% T3, and 16% T4. Prior to IORT, 87% had visibly complete surgical resection of tumor. Mean IORT dose delivered was 14.7Gy (range 9-20Gy). Average post-op hospitalization was 10.7 days. Median follow-up after IORT was 1.6 years. OS at 1 and 5 years after IORT was 69% and 37% for advanced patients and 94% and 55% for LR patients, respectively. DFS at 1 and 5 years was 72% and 39% for advanced patients and 96% and 52% for LR patients, respectively. DSS at 1 and 5 years was 72% and 41% for advanced patients and 96% and 60% for LR patients, respectively. For the entire cohort, higher IORT dose (HR 1.3, p < 0.001), positive initial node status (HR 2.9-3.6, p < 0.01), and presence of sarcomatoid features (HR 3.7-6.9, p < 0.05) had a significant association with decreased OS, DFS and DSS. Patients who received adjuvant systemic therapy after IORT showed decreased DSS (HR 2.4, p = 0.03). When analyzing LR tumors alone, positive margin (HR 2.6, p = 0.01) was associated with decreased OS. Conclusions: We report on the largest known cohort of patients with RCC managed with IORT and we have identified factors associated with survival. Outcomes in our cohort with LR RCC treated with local resection and IORT compare favorably to similar groups treated with resection alone suggesting improved DSS may be possible with IORT.

PURPOSE: The current American Joint Committee on Cancer (AJCC) staging system for hepatocellular carcinoma (HCC) fails to stratify patients adequately with respect to prognosis. PATIENTS AND METHODS: The ability of the currently proposed tumor (T) categories to effectively stratify the survival of 557 patients who underwent complete resection for HCC at four centers was examined. Independent predictors of survival were combined into a new staging system. RESULTS: Using the current AJCC T classification, patients with T1 and T2 tumors had similar 5-year survivals (P = .6). In addition, the survival of patients with multiple bilobar tumors (T4) matched that of T3 patients (P = .5). Independent predictors of death were major vascular invasion (P < .001), microvascular invasion (P = .001), severe fibrosis/cirrhosis of the host liver (P = .001), multiple tumors (P = .007), and tumor size greater than 5 cm (P = .01). Based on our results, a simplified stratification is proposed: (a) patients with a single tumor and no microvascular invasion, (b) patients with a single tumor and microvascular invasion or multiple tumors, none more than 5 cm, and (c) patients with either multiple tumors, any more than 5 cm, or tumor with major vascular invasion (P < .001). Severe fibrosis/cirrhosis had a negative impact on survival within all categories. The survival of patients with lymph node involvement matched that of patients with major vascular invasion (P = .3). CONCLUSION: The current AJCC staging system for HCC is unnecessarily complex. We propose a simplified model of stratification that is based on vascular invasion, tumor number, and tumor size and incorporates the effect of fibrosis on survival.

205 Background: Central review is an important trial management and validation tool. Multisite trials require data systems to accommodate diverse image acquisition and review. Breast cancer is challenging as its imaging objects are not always easily shared across departments. Quality Assurance Review Center (QARC) has a diverse portfolio of facile data acquisition and powerful informatics support that meets these needs. We report on the role of QARC in three of the current breast cancer trials from the American College of Surgeons Oncology Group. Methods: QARC data management system includes secure network infrastructure and a validated relational operating database (MAX). QARC has data acquisition/imaging re-distribution expertise for real time response reviews, response measuring tools and corresponding data archive for secondary reviews. MAX includes query ability; records link to digital information. QARC underwent systematic information systems validation process for 21 CFR Part 11. DICOM/non-DICOM files are stored in the QARC PACS. On-site/remote reviewers use MAX to retrieve, view, annotate and save images. Data extracted is securely sent to partner statistical centers. Results: In Z1031, a neoadjuvant endocrine therapy trial, all of the mammographic exams are archived at QARC. For Z1071, a sentinel lymph node trial, pre- and post-treatment ultrasound (US) images of 321 patients were remotely reviewed. For Z1072, the cryoablation US, pre and post-cryoablation MRI for 40 of 47 cases were remotely archived, retrieved and transferred to investigator workstation for post-processing and review. The other 7 patients did not have evaluable imaging studies. In all three trials, the costs of shipping hardcopies of exams and travel to QARC for central review functions were eliminated. Conclusions: QARC data management systems provide diverse informatics supports for multi-institutional trials, ranging from archives of images, provide remote access and download of data, and central review. The vibrant informatics supports meet the growing needs of clinical trials. Future directions include radiation field review for breast cancer trials and the incorporation of pathology microarray analysis as DICOM objects.