Kalari, Krishna R.

Abstract Triple-negative breast cancer (TNBC) is characterized by elevated locoregional recurrence risk despite aggressive local therapies. New tumor-specific radiosensitizers are needed. We hypothesized that the ATR inhibitor, VX-970 (now known as M6620), would preferentially radiosensitize TNBC. Noncancerous breast epithelial and TNBC cell lines were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, patient-derived xenograft (PDX) models generated prospectively as part of a neoadjuvant chemotherapy study from either baseline tumor biopsies or surgical specimens with chemoresistant residual disease were assessed for sensitivity to fractionated radiotherapy, VX-970, or the combination. To explore potential response biomarkers, exome sequencing was assessed for germline and/or somatic alterations in homologous recombination (HR) genes and other alterations associated with ATR inhibitor sensitivity. VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the radiotherapy-induced G2–M checkpoint, delayed resolution of DNA double-strand breaks, and reduced colony formation after radiotherapy in TNBC cells relative to normal-like breast epithelial cells. In vivo, VX-970 did not exhibit significant single-agent activity at the dose administered even in the context of genomic alterations predictive of ATR inhibitor responsiveness, but significantly sensitized TNBC PDXs to radiotherapy. Exome sequencing and functional testing demonstrated that combination therapy was effective in both HR-proficient and -deficient models. PDXs established from patients with chemoresistant TNBC were also highly radiosensitized. In conclusion, VX-970 is a tumor-specific radiosensitizer for TNBC. Patients with residual TNBC after neoadjuvant chemotherapy, a subset at particularly high risk of relapse, may be ideally suited for this treatment intensification strategy. Mol Cancer Ther; 17(11); 2462–72. ©2018 AACR.

Abstract Purpose: TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. Patients with residual TNBC after neoadjuvant chemotherapy (NAC) have a high risk of locoregional recurrence despite aggressive subsequent local therapies, including RT. Thus, new radiosensitizers that address chemoresistant TNBC (i.e residual disease after NAC) are needed. ATR plays a key role in the cellular response to replication stress and DNA damage. TNBC is characterized by G1 checkpoint loss, homologous recombination deficiency (HRD), and other features which increase dependence on ATR pathway signaling. We hypothesized that VX-970, a selective inhibitor of ATR, would effectively radiosensitize TNBC. Methods: Cell lines representing varying subtypes of TNBC (MDA-MB-231, HCC1806, and BT-549) and the normal breast epithelial cell line MCF10a were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, Mayo Clinic (MC)TNBC1 and MCTNBC2, patient derived xenograft models (PDXs) generated prospectively from baseline and chemoresistant residual disease surgical specimens after NAC, respectively, of unique patients in the Breast Cancer Genome Guided Therapy Study were analyzed. Tumors were injected into hind-legs of athymic nude mice and animals were randomized to: 1) Vehicle 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX-970 was administered daily 1 hour prior to RT at a dose of 60 mg/kg. Exome sequencing was assessed for germ-line and/or somatic alterations in HR genes and an ex-vivo RAD51 foci formation assay was applied to confirm the functional status of HR in each PDX model. Results: In vitro, VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the RT-induced G2/M checkpoint, delayed resolution of γH2AX and 53BP1 foci and reduced colony formation after RT in MDA-MB-231, HCC1806, and BT-549 relative to MCF10a. In vivo, VX-970 did not exhibit single agent activity at the dose administered but markedly sensitized both MCTNBC1 and MCTNBC2 to RT, suggesting that VX-970 could overcome chemoresistant TNBC biology. For MCTNBC1 and MCTNBC2, the median time to tumor tripling was 26 vs 49 days (p=0.002) and 25 vs 43 days (p=0.006) for the RT and RT + VX-970 groups, respectively. Ex-vivo RAD51 foci formation was robust in MCTNBC1, a PDX without HR gene alterations. In contrast, there was no RT-induced RAD51 foci formation in MCTNBC2, suggesting a BRCA1 mutation in the corresponding human tumor resulted in loss of HR function in that model. Conclusion: VX-970 + RT effectively radiosensitized TNBC PDXs established from both pre-treatment biopsy specimens and chemoresistant residual tumor samples and in both HR deficient and HR proficient settings. ATR inhibition should be investigated in the clinic in combination with RT for patients with residual TNBC after NAC. Citation Format: Robert W. Mutter, Mohamed M. Kahila, Xinyi Tu, Qin Zhou, Jia Yu, Krishna R. Kalari, Liewei Wang, Judy C. Boughey, Matthew P. Goetz, Jann N. Sarkaria, Zhenkun Lou. ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC) [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 840.

Abstract Purpose: Although high glucocorticoid receptor (GR) expression in early-stage estrogen receptor (ER)-negative breast cancer is associated with shortened relapse-free survival (RFS), how associated GR transcriptional activity contributes to aggressive breast cancer behavior is not well understood. Using potent GR antagonists and primary tumor gene expression data, we sought to identify a tumor-relevant gene signature based on GR activity that would be more predictive than GR expression alone. Experimental Design: Global gene expression and GR ChIP-sequencing were performed to identify GR-regulated genes inhibited by two chemically distinct GR antagonists, mifepristone and CORT108297. Differentially expressed genes from MDA-MB-231 cells were cross-evaluated with significantly expressed genes in GR-high versus GR-low ER-negative primary breast cancers. The resulting subset of GR-targeted genes was analyzed in two independent ER-negative breast cancer cohorts to derive and then validate the GR activity signature (GRsig). Results: Gene expression pathway analysis of glucocorticoid-regulated genes (inhibited by GR antagonism) revealed cell survival and invasion functions. GR ChIP-seq analysis demonstrated that GR antagonists decreased GR chromatin association for a subset of genes. A GRsig that comprised n = 74 GR activation-associated genes (also reversed by GR antagonists) was derived from an adjuvant chemotherapy-treated Discovery cohort and found to predict probability of relapse in a separate Validation cohort (HR = 1.9; P = 0.012). Conclusions: The GRsig discovered herein identifies high-risk ER-negative/GR-positive breast cancers most likely to relapse despite administration of adjuvant chemotherapy. Because GR antagonism can reverse expression of these genes, we propose that addition of a GR antagonist to chemotherapy may improve outcome for these high-risk patients. Clin Cancer Res; 24(14); 3433–46. ©2018 AACR.

5 Background: Triple-negative breast cancer (TNBC) is an immunogenic breast cancer subtype, with a greater percentage of tumors containing tumor-infiltrating lymphocytes (TILs) compared to non-TNBC. TILs are prognostic for recurrence and predictive for chemotherapy benefit in TNBC, but not in estrogen receptor (ER)-positive tumors. A subset of TNBC expresses the androgen receptor (AR), and resembles ER-positive breast cancer in terms of demographics (older age) and clinical course (later recurrences that tend to involve bone). However, little is known regarding the association between TILs and AR expression in TNBC. Methods: From a cohort of 9982 women with surgically-treated non-metastatic breast cancer, patients who met criteria for TNBC (ER/PR < 1% and HER2-negative) by centralized-pathology review were included as previously published [Leon-Ferre et al, Breast Cancer Res Treat 2017]. Stromal TILs in these tumors were quantified on full-face hematoxylin and eosin sections from the surgical specimen, following the 2014 TIL Working Group recommendations [Salgado et al, Ann Oncol 2014]. The expression of AR [EPR1535(2), Abcam] was scored as a continuous variable (1% increments), and categorized as absent (0%), low (1-25% ), low-moderate (26-50%) moderate (51-75%) and high (> 75%) and correlated with TIL density. Results: 605 patients met criteria for TNBC. The median age was 56 years. Most tumors were T1-2 (95%), N0-1 (86%), and high grade (88%). The median stromal TIL content was 20% (0-90%). Tumor tissue was available for AR staining in 509/605 patients. 165 (32%) tumors expressed AR as follows: low: 12%, low-moderate: 7%, moderate: 5%, and high: 8%. Apocrine tumors (n = 30) expressed AR most frequently (86%) and at higher levels (AR high: 50%). For all 509 tumors, the median stromal TIL content according to AR expression was AR absent: 20%, AR low: 20%, AR low-moderate: 15%, AR high: 10%. AR and stromal TILs (both as continuous variables) demonstrated a weak inverse correlation (correlation coefficient -0.1064, p = 0.017). Conclusions: AR expression in TNBC is associated with lower levels of stromal TILs. Additional data correlating the AR, stromal TILs and outcomes in this cohort will be presented at the meeting.

Abstract Motivation RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study, we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. Results We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16 562 isoform-pairs from 4929 genes. Among those, 26% of the discovered patterns were significant (P&lt;0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. Finally, the effects of drop-out events and expression levels of isoforms on ISOP's performances were investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoform-level preference, commitment and heterogeneity in single-cell RNA-sequencing data. Availability and implementation The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. Supplementary information Supplementary data are available at Bioinformatics online.

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with substantial risks of disease recurrence. While cytotoxic chemotherapy is commonly administered and reduces recurrence, disease outcomes vary considerably and few prognostic tools are available for risk stratification for TNBC patients. We constructed and validated clinical calculators for invasive-disease free survival (IDFS) and overall survival (OS) for TNBC and compared their performance against AJCC-based models which include only tumor size and nodal status. Methods: From a surgical cohort of 9,982 patients who underwent breast cancer surgery at Mayo Clinic between January 1985 and December 2012, 605 centrally reviewed TNBC patients were identified and used to construct Cox models for IDFS and OS. Patients treated with neoadjuvant chemotherapy were excluded. Variables considered included age, menopausal status, tumor size, nodal status, Nottingham grade, type of breast surgery (mastectomy vs. lumpectomy), adjuvant radiation therapy, adjuvant chemotherapy, Ki67, stromal tumor infiltrating lymphocytes (sTILs), and neutrophil-to-lymphocyte ratio (NLR). Missing values were imputed using single imputation with all variables (including outcomes) included in the imputation model. Backward step-down procedure was used for model selections. The final models were internally validated for calibration and discrimination using bootstrapping methods and compared with AJCC-based models. Results: For both IDFS and OS, higher sTIL's, less extensive nodal involvement, use of adjuvant chemotherapy, and lower NLR were significant predictors of improved clinical outcomes. Premenopausal status and younger age were additionally predictive of improved IDFS and OS, respectively. Models for IDFS and OS have good calibration and are associated with bias-corrected C-indices of 0.68 and 0.71, respectively, as compared with C-indices of 0.59 and 0.62 for AJCC-based models. Conclusions: Our data indicate that a clinical calculator that includes sTIL's, NLR, menopausal status, age, nodal involvement as well as chemotherapy use can provide significantly greater prediction of clinical risk than tumor size and nodal status alone. These tools may be used to identify TNBC patients at elevated risk of disease relapse and to aid physician's communication with patients regarding their long-term disease outlook and planning treatment strategies. External validation is required to further evaluate broader applicability of this tool, which was developed utilizing a single-institutional experience. Citation Format: Polley M-YC, Leon-Ferre RA, Liu H, Gilbert J, Cafourek V, Hillman DW, Negron V, Boughey JC, Liu MC, Ingle JN, Kalari K, Couch F, Visscher DW, Goetz MP. Mayo clinic TNBC outcome calculator: A clinical calculator to predict disease relapse and survival in women with triple-negative 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 P1-06-07.