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RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections.We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC).The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods.Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER+) breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation, respectively. Although ER+ breast cancers typically express a high ratio of ERα to ERβ, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers, ERβ has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERβ agonists, WAY-20070 and a novel “A-CD” estrogen called L17, produce a marked reduction in G2–M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERβ agonists recruited both the ERα and ERβ to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERβ-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERβ. Exposure to the ERβ ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERβ agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERβ agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers. Mol Cancer Ther; 13(7); 1882–93. ©2014 AACR.

PDF - 130K, Effects of tamoxifen and fulvestrant on LCC9 and MCF-7 cell proliferation.

PDF - 154K, Effects of ER ligands on cell cycle in MCF-7 and LCC9 cells.

PDF - 84K, ERalpha knockdown in ERbeta overexpressing cells.

PDF - 174K, Examples of DNA histograms from LCC9 and MCF-7 cells treated with combinations of ERbeta-selective ligands and chloroquine.

PDF - 441K, LCC9 and MCF-7 ChIP assays.

PDF - 80K, Supplementary Materials and Methods

Background The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. Methods We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. Results Intralaboratory (CCC 0.96–0.99) and interlaboratory (CCC 0.98–0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83–0.93) and between laboratories (Kappa 0.87–0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). Conclusion Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Background Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SETER/PR and PI3Kges), and measures expression of selected activating point mutations and key breast cancer genes. Methods Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin’s concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene’s) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers. Results The modified (NEW) SET4 assay measured single transcripts (pER/PR (p=0.002) more reproducibly in technical replicates from FFPE samples. The modified SET4 assay was more precise for measuring single transcripts (Rho 0.966 vs 0.888, pER/PR (Rho 0.985 vs 0.968) or PI3Kges (Rho 0.985 vs 0.946) in FFPE, compared to FF samples. It was also more precise than wtRNAseq of FFPE for measuring transcripts (Rho 0.986 vs 0.934, pER/PR (Rho 0.993 vs 0.915, p=0.004), but not PI3Kges (Rho 0.988 vs 0.945, p=0.051). Accuracy (Bias) was comparable between protocols. Two samples carried a PIK3CA mutation, and measurements of transcribed mutant allele fraction was similar in FF and FFPE samples and appeared more precise with the modified SET4 assay. Amplification efficiency (reads per UMI) was consistent in FF and FFPE samples, and close to the theoretically expected value, when the library size exceeded 400,000 aligned reads. Conclusions Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.

Background We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II–III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. Methods We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin’s concordance correlation coefficient (CCC) was used to measure concordance between measurements. Results SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1–9% coefficient of variation over 20 weekly tests, linear range 3.0–11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). Conclusions Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript., SCOPUS: ar.j, info:eu-repo/semantics/published

Triple-negative breast cancer is a highly diverse group of cancers, with poor prognosis, and currently, there are no targeted drugs available in the clinic. In TNBC around 50% percent of the patients respond to chemotherapy, while, the other 50% percent relapse with poor prognosis. There is a need to understand better the targetable mechanisms driving TNBC via integrative analysis of gene-expression, copy-number, and mutational data. Samples from 220 triple-negative breast cancer (TNBC) pts treated with NACT were prioritized for transcriptomic and genomic profiling. Non-negative matrix factorization was used on array-based profiling to identify six robust (ARTEMIS) subtypes. Comparing ARTEMIS subtypes with Vanderbilt subtypes, revealed significant overlap with 4/6 clusters while identifying two new clusters. Logistic regression on ssGSEA scores vs. subtypes revealed several pathways, selectively enriched specific subtypes. CL1/IM (Immune subtype), was enriched in INFg and INFa, while CL2 (MYC/mTOR), showed enrichment of several proliferation-related pathways. In addition, LAR and M (Mesenchymal) pts formed overlapping clusters, using either method. Two new subtypes did not associate significantly with any of the previous subtypes. The majority of the tumors from the Vanderbilt BL2 and MSL were reclassified into a CL5 (ANGIO) cluster, which was enriched in angiogenesis geneset, including targetable genes like VEGF and FGFR. Also, an MYO (CL3) subtype was identified, with myogenesis-related genes. Of note, TIL (tumor infiltrating lymphocytes) and LAR quantification using IHC were associated with respective ARTEMIS subtypes. Finally, the IM subtype was significantly associated with higher rates of RCB 0-I and the M (CL4) subtype was associated with higher rates of RCB II-III, irrespective of the neoadjuvant treatment regimen. ARTEMIS subtypes are a novel classification system for TNBC that is focused on therapeutic translation. Further, we show a possibility to classify previously un-classified (UNS) tumors, which will be validated using additional cohorts (TCGA/METABRIC). Citation Format: Seth S, Huo L, Rauch G, Lau R, Gilcrease M, Adrada B, Piwnica-Worms H, Symmans WF, Draetta G, Futreal AP, Moulder S, Chang JT. Towards a therapeutically relevant subtyping scheme for triple-negative breast cancer (TNBC), profiling results from A Randomized, TNBC Enrolling trial to confirm Molecular profiling Improves Survival (ARTEMIS) [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 P3-07-01.

Background: The clinical management of patients with metastatic HR-positive breast cancer is often uncertain due to decreased sensitivity to anti-estrogen therapy over time. Recently, we developed a targeted RNAseq based 18-transcript SET ER/PR assay of endocrine sensitivity from biopsies of metastatic cancer. In this work we assess the effect of pre-analytical factors, specifically RNA extraction methods for FFPE tissue samples, on the reliability of the targeted RNAseq assay. Methods: FFPE blocks and matched fresh frozen (FF) sections from 12 tumors were collected at MD Anderson Cancer Center. RNA from FFPE slides was extracted in duplicate using three kits (Norgen, Qiagen, Roche), and RNAseq libraries from all samples were prepared using Kapa Total RNAseq kit. Targeted RNA libraries were prepared using droplet-based PCR (RainDance), and also by transcriptome-wide RNAseq for comparison. Reads were mapped to genomic sequence using STAR and expression was quantified using RSEM. Expression data were normalized based on expression of 10 reference genes. The effect of FFPE RNA extraction kit on the reliability of the SET index was assessed using linear mixed effects model (LME) analysis, and agreement with FF was assessed using the concordance correlation coefficient (CCC). Results: Analysis of the whole transcriptome RNAseq data confirmed minimal 3'-end transcript bias from FFPE samples, irrespective of transcript size or FFPE kit. All 18 genes included in the SET index had high overall concordance between FFPE and FF (median CCC percentile=98.8, range 57.2-99.9 for Norgen; similar for the other two kits) and relatively consistent bias across genes, as estimated by the random effects of the LME model. Furthermore, compared to random 18-gene indices, concordance in the SET index values between FF and FFPE was higher than 99.8% of the random samples, verifying the analytical reliability of the selected genes. For the targeted RNAseq assay, RNA from FFPE extracted with the Norgen kit showed the highest concordance compared to FF (CCC=0.956, 95%CI 0.871-0.985). In general, the analytical variation of SET from FFPE samples was greater than that from FF (1.71-2.71 fold greater), with the lowest variation associated with the Norgen kit. The SET index values from targeted RNAseq for both FF and FFPE samples were consistently lower compared to transcriptome-wide RNAseq but were highly correlated, with the Norgen kit having the highest correlation between targeted and transcriptome-wide RNAseq (rho=0.915). Conclusions: All three FFPE RNA extraction kits have excellent analytical performance compared to FF samples. The Norgen kit may be marginally better yielding higher concordance with FF and lower analytical variation between replicates. All genes in the SET ER/PR showed very good analytical performance in comparison to random indices and individual genes. Targeted gene RNA sequencing appears very promising as a platform for clinical deployment of quantitative assays, showing only a small (fixable) bias compared to RNAseq. Citation Format: Marczyk M, Fu C, Lau R, Du L, Trevarton AJ, Sinn BV, Gould RE, Symmans WF, Hatzis C. Pre-analytical effects of FFPE extraction methods on targeted and whole transcriptome sequencing assays for endocrine sensitivity in metastatic breast cancer [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 P4-08-20.

Cellular inhibitor of apoptosis proteins-1 and -2 (cIAP1/2) are integral to regulation of apoptosis and signaling by the tumor necrosis factor (TNF) and related family of receptors. The expression of cIAP2 in tissues is typically low and considered functionally redundant with cIAP1, however cIAP2 can be activated by a variety of cellular stresses. Members of the TNFR family and their ligands have essential roles in mammary gland biology. We have found that cIAP2−/− virgin mammary glands have reduced ductal branching and delayed lobuloalveogenesis in early pregnancy. Post-lactational involution involves two phases where the first phase is reversible and is mediated, in part, by TNFR family ligands. In cIAP2−/− mice mammary glands appeared engorged at mid-lactation accompanied by enhanced autophagic flux and decreased cIAP1 protein expression. Severely stretched myoepithelium was associated with BIM-EL expression and other indicators of anoikis. Within 24 h after forced or natural weaning, cIAP2−/− glands had nearly completed involution. The TNF-related weak inducer of apoptosis (Tweak) which results in degradation of cIAP1 through its receptor, Fn14, began to increase in late lactation and was significantly increased in cIAP2−/− relative to WT mice by 12 h post weaning accompanied by decreased cIAP1 protein expression. Carcinogen/progesterone-induced mammary tumorigenesis was significantly delayed in cIAP2−/− mice and tumors contained high numbers of apoptotic cells. We conclude that cIAP2 has a critical role in the mammary gland wherein it prevents rapid involution induced by milk stasis-induced stress associated with Tweak activation and contributes to the survival of mammary tumor cells.

SRC-3/AIB1 (Amplified in Breast Cancer-1) is a nuclear receptor coactivator for the estrogen receptor in breast cancer cells. It is also an intrinsically disordered protein when not engaged with transcriptional binding partners and degraded upon transcriptional coactivation. Given the amplified expression of SRC-3 in breast cancers, the objective of this study was to determine how increasing SRC-3 protein levels are regulated in MCF-7 breast cancer cells. We found that endogenous SRC-3 was expelled from the nucleus in vesicle-like spheres under normal growth conditions suggesting that this form of nuclear exclusion of SRC-3 is a homeostatic mechanism for regulating nuclear SRC-3 protein. Only SRC-3 not associated with CREB-binding protein (CBP) was extruded from the nucleus. We found that overexpression in MCF-7 cells results in aneuploid senescence and cell death with frequent formation of nuclear aggregates which were consistently juxtaposed to perinuclear microtubules. Transfected SRC-3 was SUMOylated and caused redistribution of nuclear promyelocytic leukemia (PML) bodies and perturbation of the nuclear membrane lamin B1, hallmarks of nucleophagy. Increased SRC-3 protein-induced autophagy and resulted in SUMO-1 localization to the nuclear membrane and formation of protrusions variously containing SRC-3 and chromatin. Aspects of SRC-3 overexpression and toxicity were recapitulated following treatment with clinically relevant agents that stabilize SRC-3 in breast cancer cells. We conclude that amplified SRC-3 levels have major impacts on nuclear protein quality control pathways and may mark cancer cells for sensitivity to protein stabilizing therapeutics.

Eradicating triple negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard front-line chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Taken together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes as well as maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Finally, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase I clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by non-selective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.

Rationale: A robust index for gene expression related to activity of estrogen (ESR1) and progesterone (PGR) receptors could predict sensitivity to endocrine therapy in metastatic breast cancer. Methods: Transcripts correlated with ESR1 and PGR expression in 389 hormone receptor-positive breast cancer samples (Affymetrix U133A microarrays) were ranked for reliability according to their pre-analytical (intratumoral heterogeneity, biopsy type) and analytical reproducibility. Eighteen target and ten reference genes were selected and summarized as the SETER/PR index. The SETER/PR index was evaluated in a different set of 140 biopsies from distant metastases of hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer, and in additional pre-analytical and analytical sample cohorts. Thereafter, SETER/PR was translated to a customized format for application to formalin-fixed and paraffin-embedded (FFPE) sections. Results: Higher SETER/PR in a metastasis was associated with longer progression-free survival (PFS, 9 vs. 2 months) and overall survival (OS, 50 vs. 19 months) following endocrine therapy in the cohort with metastatic breast cancer (MBC) and relapsed disease (n=79), so a cut point was defined in that cohort. SETER/PR was also significantly associated with PFS after adjusting for PR status of the metastasis, presence of visceral metastases, number of previous relapse events, and clinical history of previous sensitivity to endocrine therapy (HR 0.485, 95%CI 0.265 – 0.889, p = 0.019). Technically, SETER/PR was highly reproducible under different pre-analytical and analytical conditions, including host organ contamination. The translated SETER/PR assay used a single 10 µm FFPE tissue section, did not require RNA purification, and represented the microarray results from matched fresh samples with excellent agreement (correlation = 0.980, n = 31). Conclusion: The SETER/PR index is a new biomarker to predict PFS and OS for patients with HR+/HER2- MBC who receive endocrine therapy. The assay is applicable to FFPE tissue sections from small biopsies of metastases. Additional independent (blinded) validation studies will be necessary to confirm these results.Rationale: A robust index for gene expression related to activity of estrogen (ESR1) and progesterone (PGR) receptors could predict sensitivity to endocrine therapy in metastatic breast cancer. Methods: Transcripts correlated with ESR1 and PGR expression in 389 hormone receptor-positive breast cancer samples (Affymetrix U133A microarrays) were ranked for reliability according to their pre-analytical (intratumoral heterogeneity, biopsy type) and analytical reproducibility. Eighteen target and ten reference genes were selected and summarized as the SETER/PR index. The SETER/PR index was evaluated in a different set of 140 biopsies from distant metastases of hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer, and in additional pre-analytical and analytical sample cohorts. Thereafter, SETER/PR was translated to a customized format for application to formalin-fixed and paraffin-embedded (FFPE) sections. Results: Higher SETER/PR in a metastasis was associated with longer progression-free survival (PFS, 9 vs. 2 months) and overall survival (OS, 50 vs. 19 months) following endocrine therapy in the cohort with metastatic breast cancer (MBC) and relapsed disease (n=79), so a cut point was defined in that cohort. SETER/PR was also significantly associated with PFS after adjusting for PR status of the metastasis, presence of visceral metastases, number of previous relapse events, and clinical history of previous sensitivity to endocrine therapy (HR 0.485, 95%CI 0.265 – 0.889, p = 0.019). Technically, SETER/PR was highly reproducible under different pre-analytical and analytical conditions, including host organ contamination. The translated SETER/PR assay used a single 10 µm FFPE tissue section, did not require RNA purification, and represented the microarray results from matched fresh samples with excellent agreement (correlation = 0.980, n = 31). Conclusion: The SETER/PR index is a new biomarker to predict PFS and OS for patients with HR+/HER2- MBC who receive endocrine therapy. The assay is applicable to FFPE tissue sections from small biopsies of metastases. Additional independent (blinded) validation studies will be necessary to confirm these results. Citation Format: Sinn BV, Tsai T-H, Lau R, Fu C, Gould R, Murthy R, King TA, Hatzis C, Kwiatkowski DN, Valero V, Symmans WF. SETER/PR - A robust 18-gene predictor of sensitivity to endocrine therapy in metastatic 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 P6-09-23.

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript.

Understanding the biological mechanisms underlying the initiation and progression of breast cancer is an important step for prevention and treatment. Individuals with mutations in the breast cancer-associated gene 1 (BRCA1) have a lifetime increased risk of up to 85% of developing breast cancer. BRCA1 participates in DNA damage repair and cell cycle checkpoint control, serving as a tumor suppressor gene to maintain global genomic stability. However, BRCA1 has also been shown to play a key role in the expansion of mammary stem/progenitor cells, which are the targets for carcinogenesis in individuals who have undergone loss of heterozygosity (LOH) for BRCA1. We have used in vitro and in vivo models to demonstrate that BRCA1 loss or mutation induces alternative NF-κB pathway activation and leads to the expansion of a genetically unstable progenitor cell population in the mammary gland. Our data showed that BRCA1 loss or mutation is responsible for activation of the alternative NF-κB pathway evidenced by IκB kinase-α (IKKα) phosphorylation, processing of p100 to p52 and p52/RelB nuclear localization. Remarkably, we found that RelB and p100/p52 were highly expressed in 20-50% of the lobular structures in histologically normal breast tissue obtained from human BRCA1 mutation carriers. After DNA damage, ATM directs the nuclear export of NEMO (NF-κB essential modifier) that in turn activates the alternative NF-κB pathway. We found high levels of phospho-ATM in breast tissue from human BRCA1-mutation carriers and in progenitor cells from BRCA1-knockout mouse mammary glands. Moreover, co-immunoprecipitation studies showed increased ATM/NEMO-containing complexes in BRCA1-deficient cells. Progesterone injections into MMTV-cre;BRCA1f/f mice induced an increase in Ki-67 and phospho-H2AX levels, demonstrating a role for progesterone in DNA damage amplification in mouse mammary glands. Mammary epithelial progenitor cells obtained from BRCA1-mutated carriers as well as BRCA1-/- mouse glands can form acini in Matrigel in a progesterone-independent manner. We found that in vivo inhibition of IKKα/β using the IKK inhibitor dimethylaminoparthenolide (DMAPT) completely blocked mammary acini formation in Matrigel, requiring several estrus cycles post-injection to recover. Importantly, knockdown studies showed that p100/p52 was necessary for progenitor cell proliferation in Matrigel. Consistent with the continued dependence on alternative NF-κB activity in tumors derived from BRCA1-deficient progenitor cells, human breast cancer xenografts derived from BRCA1-mutation carriers infected with lenti-shp100/p52 showed a significant growth inhibition. Overall, these results suggest an exciting new approach for the prevention of breast cancer in patients wherein intermittent or cyclic therapy using DMAPT (generic name LC-1) has the potential to mitigate breast cancer risk in BRCA1 mutation carriers through acute reduction of aberrant progenitor activity. As such, NF-κB-directed chemopreventive therapies may provide promising alternatives to prophylactic mastectomy in this high risk patient population. Citation Format: Andrea Sau, Rosanna Lau, Emma Nolan, Peter A Crooks, Jane E Visvader, Christine MA Pratt. Inhibition of alternative NF-κB activity prevents the expansion of genetically unstable progenitor cells in BRCA1-deficient mammary glands [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 P5-11-04.

BACKGROUND: Patient-derived xenograft (PDX) models of breast cancer replicate the diverse histologic and molecular features of patient tumors and provide a renewable source of human tumor tissue; however collection of tissue by core needle biopsy is problematic due to patient discomfort, bleeding risk and the limited number of passes a patient can tolerate. In addition, FDA guidelines caution that multiple core needle biopsies could lead to an overestimation of the true pCR rate in neoadjuvant trials. METHODS: To support the neoadjuvant molecular diagnostic and drug development program in TNBC, a pilot study was conducted to determine if fine needle aspiration (FNA) could be used for building PDX models. Prior to engraftment, FNA samples were analysed for cell number and viability. RESULTS: Six PDX models were successfully generated from eight individual tumor samples. These models retain histologic and molecular features of the original patient tumors as determined by immunohistochemistry, RNA expression profiling, and deep whole-exome and targeted gene sequencing. In addition, the tested PDX models recapitulate the responses to therapies across multiple chemotherapeutic agents. Based on this success, we have standardized the use of FNAs to generate PDX models both pre- and post-therapy in two ongoing neoadjuvant clinical trials: 1. MDACC 2014-0185 (PI Stacy Moulder, 360 patients), 'Improving outcomes in TNBC using molecular triaging and diagnostic imaging to guide neoadjuvant therapy' 2. MDACC 2014-0045 (PI Jennifer Litton, 20+ patients), 'A pilot study of BMN673 as a neoadjuvant study in patients with a diagnosis of invasive breast cancer and a deleterious BRCA mutation' FNA cells (x10^4)Cell viability (%)Total viable cells (x10^4)Study entry biopsy (n=67)144.5050.6544.14Post treatment biopsy (n=16)47.0732.5428.38 To date, treatment-naïve primary tumor samples from 67 patients enrolled onto these neoadjuvant trials, and 16 matched non-responsive post treatment tumor samples have been analysed for cell count and viability (table below) prior to being engrafted into the humanized mammary fat pads of NOD/SCID mice. CONCLUSION: We have demonstrated success in using FNAs to build PDX models that recapitulate the biology and clinical course of the original tumor. In our pilot study, we successfully generated six PDX models using FNA for TNBC, including some harboring deleterious BRCA1/2 mutations. Because of the high concordance in histologic, genomic, and clinical attributes, we are now using this approach to develop a rich resource of pre- and post-treatment PDX models for the investigation of therapeutic resistance. Citation Format: Echeverria GV, Chang JT, Cai S, Tu Y, McCoy A, Lau R, Redwood A, Kaffiabasabadi S, Rauch GM, Adrada BE, Jennifer L, Moulder SL, Symmans WF, Piwnica-Worms H. An annotated collection of pre- and post-therapy breast cancer patient-derived xenograft models built from fine needle aspiration samples aligned with ongoing clinical trials documenting response to treatment [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 P4-06-03.

Cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2) function to prevent apoptosis and are often overexpressed in various cancers. However, mutations in cIAP1/2 can activate the alternative NFκB pathway through IκBα-kinase-α (IKKα) and are associated with hematopoetic malignancies. In the current study, we found that knockdown of cIAP2 in human mammary epithelial cells resulted in activation of MDM2 through increased SUMOylation and profound reduction of the pool of MDM2 not phosphorylated at Ser166. cIAP2 siRNA markedly decreased p53 levels, which were rescued by addition of the MDM2 inhibitor, Nutlin3a. An IAP antagonist, which induces cIAP degradation, transiently increased MDM2 mRNA. Simultaneous transfection of siRNA for cIAP2 and IKKα reduced MDM2 protein, while expression of a kinase-dead IKKβ strongly increased non-Ser166 P-MDM2. Inhibition of either IKKα or -β partially rescued p53 levels, while concomitant IKKα/β inhibition fully rescued p53 after cIAP2 knockdown. Surprisingly, IKKα knockdown alone increased SUMO-MDM2, suggesting that in the absence of activation, IKKα can prevent MDM2 SUMOylation. cIAP2 knockdown disrupted the interaction between the MDM2 SUMO ligase, PIAS1 and IKKα. Partial knockdown of cIAP2 cooperated with (V12) H-ras-transfected mammary epithelial cells to enhance colony formation. In summary, our data identify a novel role for cIAP2 in maintaining wild-type p53 levels by preventing both an NFκB-mediated increase and IKKα/-β-dependent transcriptional and post-translational modifications of MDM2. Thus, mutations or reductions in cIAP2 could contribute to cancer promotion, in part, through downregulation of p53.

588Background: TNBC cell lines characterized as mesenchymal (M) and luminal androgen receptor (LAR) commonly have aberrations in the PI3K pathway. However, the incidence in human tumors and impact on response to therapy is less clear. Methods: Pre-treatment biopsies were collected from TNBC patients (pts) prior to NACT. Tumors were categorized into 5 groups using the Pietenpol criteria (Lehmann JCI 2011): basal-like (BL) comprised of BL-1 and BL-2, M comprised of M and mesenchymal stem-like, immunomodulatory (IM), LAR, or unspecified (UNS). Using RNAseq data, variants were identified in 16 PI3K pathway genes: AKT1, AKT2, AKT3, CRKL, IRS2, MTOR, PIK3CA, PIK3CG, PIK3R1, PTEN, RICTOR, RPTOR, RNF43, TSC1, TSC2. Results: Data was available in 63 pts (N = 4 stage I; N = 41 stage II; N = 18 stage III). There was no significant association between stage and subtype. 67 PI3K pathway variants were identified in 39 (62%) tumors. The incidence of mutated tumors and their differential response to NACT defined by resid...

Human BRCA1 mutation carriers and BRCA1-deficient mouse mammary glands contain an abnormal population of mammary luminal progenitors that can form 3D colonies in a hormone-independent manner. The intrinsic cellular regulatory defect in these presumptive breast cancer precursors is not known. We have discovered that nuclear factor kappaB (NF-κB) (p52/RelB) is persistently activated in a subset of BRCA1-deficient mammary luminal progenitors. Hormone-independent luminal progenitor colony formation required NF-κB, ataxia telangiectasia-mutated (ATM), and the inhibitor of kappaB kinase, IKKα. Progesterone (P4)-stimulated proliferation resulted in a marked enhancement of DNA damage foci in Brca1(-/-) mouse mammary. In vivo, NF-κB inhibition prevented recovery of Brca1(-/-) hormone-independent colony-forming cells. The majority of human BRCA1(mut/+) mammary glands showed marked lobular expression of nuclear NF-κB. We conclude that the aberrant proliferative capacity of Brca1(-/-) luminal progenitor cells is linked to the replication-associated DNA damage response, where proliferation of mammary progenitors is perpetuated by damage-induced, autologous NF-κB signaling.

Fifty percent of all triple negative breast cancer (TNBC) patients harbor significant residual tumor burden following treatment with standard neoadjuvant chemotherapy (NACT), resulting in poor prognosis. Recent studies in TNBC have revealed extensive intra-tumoral heterogeneity at the time of diagnosis and throughout disease progression, but the relative contributions of these heterogeneous populations of tumor cells to chemoresistance are not well understood. The primary tumor, dermal metastasis, and germline reference were obtained from a patient with untreated metastatic TNBC. Tumor cells were engrafted into the humanized mammary fat pads of NOD/SCID mice to establish PDX models of the primary (PIM001-P) and metastatic (PIM001-M) tumors. RNA sequencing and whole-exome sequencing (WES), performed on the patient's primary and metastatic tumors and the first- and third- passage PDX models revealed transcriptomic profiles and subclonal heterogeneity of the patient's tumors were recapitulated in the PDX models. Treatment of mice engrafted with PIM001-P tumors with NACT (Adriamycin plus cyclophosphamide, AC) resulted in partial response, the magnitude of which was diminished in mice bearing PIM001-M tumors. Tumor subclones were tracked during chemotherapy treatment in mice engrafted with PIM001-P tumors using lentiviral non-targeting DNA barcodes. Residual tumors maintained the clonal architecture of untreated tumors, and deep WES revealed stable maintenance of somatic mutant allele frequencies throughout treatment. Therefore, selection of pre-existing resistant clones did not lead to AC resistance in this model. Interestingly, only 25% of residual tumor clones contributed to primary relapse once treatment was halted, suggesting only a subpopulation of tumor cells was able to reconstitute the tumor. RNA sequencing and reverse phase protein array revealed that while vehicle-treated and regrown tumors were highly similar, residual tumors harbored a unique profile characterized by numerous significant alterations in RNA and protein levels. Together, these results suggest that residual tumors enter into a transient drug-resistant state that is reversible. Residual tumors were enriched for alterations in pathways such as metabolism, extracellular matrix remodeling, and cell-cell communication. Pharmacologic targeting of the residual tumor state with an inhibitor of mitochondrial oxidative phosphorylation led to significant inhibition of tumor regrowth following AC treatment. Additional vulnerabilities identified in residual tumors are being targeted therapeutically with the goal of eradicating residual tumor cells. Citation Format: Echeverria GV, Seth S, Ge Z, Sun Y, DiFrancesco E, Lau R, Marszalek J, Moulder S, Symmans F, Heffernan TP, Chang JT, Piwnica-Worms H. Characterizing and targeting chemoresistant subclones in patient-derived xenograft models of 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 P4-03-02.

BACKGROUND: Patient-derived xenograft (PDX) models of breast cancer replicate the diverse histologic and molecular features of patient tumors and provide a renewable source of human tumor tissue. However, collection of tissue by core needle biopsy is problematic due to patient discomfort, bleeding risk and the limited number of passes a patient can tolerate. Several studies have catalogued the maintenance of molecular features of patient tumors in PDX models of breast cancer. METHODS: To support the neoadjuvant molecular diagnostic and drug development program in triple negative breast cancer (TNBC), a pilot study was conducted to determine if fine needle aspiration (FNA) could be used for building PDX models. Subsequently, PDX models are being established in alignment with ongoing clinical trials at MDACC. The molecular evolution of patient's tumors, matched with PDXs engrafted from their tumors, is under study throughout the neoadjuvant treatment of TNBC using RNA sequencing, whole-exome sequencing, deep sequencing of cancer genes, and histologic analyses. RESULTS: To date, 20 established PDX models have been developed and stable PDX models continue to be generated at a rate of 2-3 per month. Several of these models are derived from serial FNAs derived from patients throughout neoadjuvant treatment. These models retain histologic and molecular features of the original patient tumors. Serial patient biopsies, matched with PDX models, have enabled measurement of the mutational and transcriptomic evolution in vivo of TNBC undergoing neoadjuvant treatment. We have standardized the use of FNAs to generate PDX models both pre- and post-neoadjuvant therapy in the following ongoing neoadjuvant clinical trials: 1. MDACC 2014-0185 (PI Stacy Moulder, 360 patients), 'ARTEMIS: A Randomized TNBC-Enrolling trial to confirm Molecular profiling Improves Survival' 2. MDACC 2014-0045 (PI Jennifer Litton, 20+ patients), 'A pilot study of BMN673 as a neoadjuvant study in patients with a diagnosis of invasive breast cancer and a deleterious BRCA mutation' CONCLUSION: We demonstrated that PDX models from tissue collected by FNA recapitulate the biology and clinical course of the patient's tumor. Sequencing analyses revealed that neoadjuvant chemotherapy and PDX engraftment enrich for cancer gene mutations. We observe association of the rate of successful PDX engraftment with clinical parameters such as the patient's residual cancer burden (RCB) status at the time of surgery (upon completion of neoadjuvant treatment). In addition, we observe that PDX models derived from serial patient biopsies throughout treatment are more resistant to chemotherapy treatment. These models recapitulate the variety of chemotherapy responses observed in patients with TNBC and serve as powerful tools for preclinical biomarker and discovery studies. Citation Format: Echeverria GV, Cai S, Tu Y, McCoy A, Lau R, Redwood A, Rauch G, Adrada B, Candelaria R, Santiago L, Thompson A, Litton J, Moulder S, Symmans F, Chang JT, Piwnica-Worms H. A molecularly annotated collection of breast cancer patient-derived xenograft models aligned with ongoing clinical trials built from fine needle aspiration samples throughout neoadjuvant treatment [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 P5-05-01.

Background: We developed SET4 as a targeted, droplet-based, next-generation RNA sequencing assay to measure both the SETER/PR index of gene expression and the percent of estrogen receptor (ER) transcripts with point mutation in the ligand-binding domain (LBD) in metastatic biopsies of Stage IV breast cancer. The SETER/PR index (31 genes) is a cumulative measure of gene expression for transcripts associated with ER and progesterone receptor (PR), excluding those with a direct role in proliferation. High SETER/PR could indicate increased sensitivity to endocrine therapy, whereas LBD mutations indicate resistance but might also induce high SETER/PR. Methods: Targeted needle biopsies from a metastatic site were prospectively obtained from 82 patients with HR+/HER2- breast cancer at time of any progression event, and preserved in RNAlater. Samples were prepared for targeted sequencing on a MiSeq by combining purified total RNA with SET4 primers and RT-PCR master mix into single molecule-formatted picodroplets using a RainDrop Source instrument, followed by thermal cycling and sample indexing. Calculated SETER/PR index and percent ER transcripts with LBD mutations were evaluated as continuous variables and compared to progression-free and overall survival using Cox regression analysis with log-rank test, if the next treatment after biopsy included endocrine therapy. Results: The average read depth for the LBD of the ER transcript was 33,475X (range: 1230-180,889X), confidently detecting mutation at 1% frequency. LBD mutations were identified in 17% (14/82) of metastases (range of mutated transcripts 1%-98%). LBD mutations (>10% of transcripts) were only observed in metastases with higher SETER/PR (above the median). In patients who next received endocrine therapy (n=58), higher SETER/PR predicted longer progression-free (PFS) (HR=0.37, p=0.0004, Δ median PFS 9 months) and overall survival (OS) (HR=0.49, p=0.03). The predictions were more pronounced in patients without LBD mutation (PFS HR=0.32, p=0.0001, Δ median PFS 13 months; OS HR=0.42, p=0.01). Currently, there are insufficient cases with LBD mutation for reliable survival analysis. Conclusion: The SET4 assay measured the percent of ER transcripts with activating LBD mutation (≥1% prevalence) and also downstream ER/PR-related transcription. High SETER/PR predicted longer PFS and OS with endocrine therapy. While activating LBD mutations may be associated with endocrine resistance of Stage IV cancer, they were associated with high SETER/PR index. Consequently, metastatic cancers with high SETER/PR index and no LBD mutation in ER transcripts were particularly sensitive to endocrine therapy. This single assay unraveled a possible interaction between genotype, phenotype, and treatment outcome; and is currently being evaluated in a larger cohort of patients. Citation Format: Rosanna Lau, Lily Fu, Michael Samuels, Rashmi K. Murthy, Bruno Sinn, Jane Yu, Rebekah Gould, Jennifer Litton, Alda Tam, Stacy Moulder, Daniel Booser, Debu Tripathy, Vicente Valero, Fraser Symmans. A targeted RNA-seq assay to measure activating ER mutations and ER/PR-associated gene expression predicts sensitivity to endocrine therapy for metastatic breast cancer [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 1796. doi:10.1158/1538-7445.AM2017-1796

Acquired resistance to selective estrogen receptor (ER) modulators (SERM) and downregulators (SERD) is a significant clinical problem in the treatment of estrogen (E2) receptor-positive (ER+) breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation, respectively. Although ER+ breast cancers typically express a high ratio of ERα to ERβ, the acquisition of SERM resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers, ERβ has been shown to function in opposition to the ERα in the presence of E2. Here, we demonstrate that two different ERβ agonists, WAY-20070 and a novel “A-CD” estrogen called L17, produce a marked reduction in G2–M phase correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line. ERβ agonists recruited both the ERα and ERβ to the Bcl-2 E2-response element strongly reducing Bcl-2 mRNA and protein in an ERβ-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing ERβ. Exposure to the ERβ ligands also resulted in increased processing of LC3-I to LC3-II, indicative of enhanced autophagic flux. The coaddition of ERβ agonist and the autophagy inhibitor chloroquine resulted in a significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase inhibitor Z-VAD-FMK. We propose that combined therapies with an ERβ agonist and an inhibitor of autophagy may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers. Mol Cancer Ther; 13(7); 1882–93. ©2014 AACR.

The cold inducible RNA binding protein (CIRBP) responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC), at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6) encoded proteins important in inflammation with IL-1β apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS) was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1β expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.

The role of the canonical NF-kappaB pathway in mammary tumorigenesis was investigated using a transgenic (TG) mouse expressing a dominant-negative inhibitor of kappaB (IkappaBalpha(SR (S32A/S36A))) in the mammary gland under the control of the mouse mammary tumor virus promoter (MMTV). TG and control mice were subjected to a chemical carcinogenesis protocol. Hyperkeratinized squamous metaplasias (cytokeratin-6+/p63+) sometimes with a basaloid island component, were found in both TG and control mice whereas luminal (cytokeratin-19+/MUC1+) ErbB2+ papillary and adenomatous lesions developed almost exclusively in control mice. p65/RelA- and NF-kappaB DNA-binding activity were detected in mammary luminal lesions, but rarely in squamous metaplasias. Analysis of NF-kappaB family proteins and target genes using microarray data from a cohort of human mammary tumors revealed the expression of a canonical NF-kappaB pathway, but not non-canonical pathway proteins in HER2+ luminal cancers. HER2+ tumors also showed differential regulation of specific NF-kappaB target genes relative to basal and ER+ luminal cancers. Isolation of mammary cell populations enriched for stem and progenitor cell characteristics from an NF-kappaB-EGFP reporter mouse by fluorescence-activated cell sorting demonstrated that luminal progenitors contain activated NF-kappaB whereas the mammary stem cell-enriched population, does not. Together these data suggest that the canonical NF-kappaB pathway is active in normal luminal progenitor cells before transformation and is required for the formation of mammary luminal-type epithelial neoplasias.

Purpose: Previous efforts to develop transcriptional markers of chemotherapy sensitivity in TNBC had limited success due to the heterogeneity of this disease. The purpose of this study was to identify genomic differences between extremely chemotherapy sensitive and highly chemotherapy resistant TNBC through whole exome sequencing and to assess measures of genomic heterogeneity as predictive markers. Methods: Twenty nine cases were selected from a prospectively collected cohort of fine needle aspiration specimens obtained before preoperative chemotherapy (MDACC) to represent two extreme response cohorts including pathologic complete response (pCR, N=17) or extensive residual cancer (eRD, N=12). DNA was extracted from specimens stored in RNAlater, exomes were captured using NimbleGen SeqCap EZ Exome Library preparation and paired-end sequencing of 75 base pair fragments was performed on Illumina HiSeq 2000. Alignment and variant calling were performed with BWA and GATK haplotype caller. Variants were filtered against the 1000 Genomes and TCGA normal breast samples to identify candidate somatic variants. Fisher-exact test was used to identify variant genes associated with sensitivity to chemotherapy. We calculated overall mutational load and normal-adjusted clonal entropy of driver mutations as measures of genome heterogeneity. The chi-squared test was used to compare differences in mutational spectra and genome heterogeneity between the two response groups. Results: The mean coverage was over 150X and 93% of target regions had > 20X coverage. The number of non-silent COSMIC mutations was similar in tumors from the two response groups (pCR: 63, range 49-82; eRD: 59, range 43-78) as well as the number of novel non-silent mutations (eRD: 223, range 113-388; pCR: 192, range 125-293). Gene level aggregation of variants identified 4 genes (MUC21, SLCO5A1, LRBA, STNE1) with response-associated mutational patterns. However, mutations were non recurrent and p-values were modest, ranging from 0.04 to 0.005. We observed greater overall mutational load and subclonal heterogeneity (clonal entropy of cancer related mutations) associated with eRD compared to pCR. Both measures suggest that higher genomic DNA diversity is associated with chemotherapy resistance. However, some genes (BRCA1 and MKI67) had higher mutational load (sum of minor allele frequencies per gene) associated with pCR compared to eRD. In general, a higher proportion of C>T transition (P=0.011) and lower A>G transition (P=0.028) was associated with eRD. The same mutational spectrum shift was previously described in the comparison of trunk and branch driver events suggesting that eRD tumors may undergo heterogeneous branched evolution. Conclusion: We observed greater genomic diversity and distinctive mutational spectra in original pre-treatment samples of TNBC that were associated with extensive residual disease compared to pCR. Our analysis suggests that broad measures of genomic diversity may serve as markers of resistance to chemotherapy. Citation Format: Tingting Jiang, Weiwei Shi, William F Symmans, Charles Li, James Platt, Rosanna Lau, Vikram B Wali, Richard Lifton, Lajos Pusztai, Christos Hatzis. Broad exonic DNA diversity is associated with resistance to taxane-FAC chemotherapy in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-2.