Abstract Background: Long non-coding RNAs (lncRNAs) are pervasively transcribed in the genome yet their role in human disease is not well understood. LncRNAs can have regulatory effects on coding mRNAs through a number of mechanisms, including repressing their sense-strand protein-coding partners. There is also emerging evidence that estrogen signaling affects the expression of a wide variety of non-coding functional RNA transcripts in addition to protein-coding transcripts. We performed an RNA-seq study to characterize changes in lncRNA expression and evaluate their association to estrogen receptor (ER) status and estrogen signaling. Methods: We sequenced transcriptomes of core biopsy RNA from 45 breast tumors obtained from neoadjuvant clinical trials BrUOG 211A/211B. RNA was derived from biopsy samples obtained before exposure to run-in monotherapy with either nab-paclitaxel, bevacizumab or trastuzumab. Paired-end sequencing was performed using amplified total RNA with 74bp read length, yielding genome-wide transcriptomic data. Transcriptomic abundance and differential expression were estimated assuming Poisson-distributed read-counts. Paired-end sequence data was aligned to a lncRNA database containing 14,572 unique lncRNAs. Changes in relative abundance of lncRNA transcripts were tested for association with estrogen receptor status using the Wilcoxon rank-sum test. Expression levels of the ER-associated lncRNAs were investigated in RNA-seq data from ER-positive MCF7 cells in response to treatment with E2 and tamoxifen (3hr, 12 hr and a non-treated control). ER-responsive binding sites on or near the ER-associated lncRNAs were investigated in a ChIP-seq study in MCF7 cells following estrogen treatment. MCF7 cell-line RNA-seq and ChIP-seq data were obtained from publicly available Short Read Archive (ERX030990 and SRX113365) at NCBI. Results: On average, in each patient 5000 lncRNAs were detectable. Expression of 22 lncRNAs were associated ER status (p<0.05) in breast tumors. 17 of the 22 ER-associated lncRNAs were detectable in ER-positive MCF7 breast cancer cells and were all responsive to E2 (estradiol) treatment. Interestingly, all 17 lncRNAs showed significant downregulation (> = 2 fold) upon tamoxifen treatment. To further evaluate the regulatory relationship between ER and the 22 lncRNAs, we identified the binding sites of ER in estrogen-treated MCF7 cells using ChIP-seq. We found that >50% of the ER-associated lncRNAs had ER binding site either overlapping or neighboring the lncRNA. Discussion: We have shown that lncRNA expression levels are associated with ER status in breast tumors. We have further established that they are estrogen-responsive with a majority being direct targets of ER using cell-line data. Further functional validation studies are ongoing. We are also exploring lncRNA-mRNA expression for coding partners of lncRNAs to identify coding/noncoding gene regulatory networks important in estrogen response. Understanding the regulatory effects of lncRNA expression opens up new opportunities for stratification and management of breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-02.
Abstract Background: HER2+ breast cancers are heterogeneous at both clinical and molecular levels. We and others have determined that the HER2-Enriched subtype exhibits the highest rate of pathologic complete response (pCR) to neoadjuvant chemotherapy and trastuzumab (T), while the HER2-Basal subtype is resistant to anti-HER2 therapy (Carey et al, JCO 2015;Varadan et al, CCR 2016). Additionally, we reported that signatures of immune cell infiltration and immune cell subsets evaluated after one dose of T can predict pCR to preoperative T and chemotherapy (Varadan et al, CCR 2016). Given recent evidence for improved immune response with increasing mutational load, we chose to characterize the association of somatic mutations and copy-number alterations with subtypes of HER2+ breast cancer and immune modulation after one dose of T. Methods: Fresh tumor core biopsies were taken at baseline and 2 weeks after one dose of either T or nab-paclitaxel (N) from 60 patients with stage II-III HER2+ cancers enrolled on a multicenter trial (BrUOG 211B). All patients then received 18 weeks of T+N+carboplatin. PAM50 subtyping was performed using gene expression data from patient tumor biopsies and tumors were classified into HER2-Enriched, HER2-Luminal and HER2-Basal subtypes. Whole-exome sequencing (WES) was performed on a total of 86 samples (49 baseline, 37 brief-exposure), sequenced at an average depth of 90X. Somatic mutations were detected by applying multiple mutation-detection algorithms on the WES data, followed by stringent quality control using public and in-house variant databases, and mutation data curated from 11,000 tumors sequenced by the TCGA. Somatic copy-number alterations were estimated using a published algorithm, ENVE (Varadan et al, Genome Med 2015) that robustly detects somatic copy-number alterations in WES tumor profiles. We employed previously defined gene-expression signatures (Varadan et al, CCR 2016) of total immune infiltration and immune cell subsets, to assess for association with genomic aberrations. Results: HER2-Basal tumors exhibited lower average copy number for HER2 and were less likely to have high-level amplifications of co-amplicons (e.g. 11q13, 20q13) with the exception of the MYC amplicon (8q24). They also exhibited a non-significant (P=0.33) trend towards higher mutational burden (Avg=85) compared to HER2-Luminals (Avg=79). A majority of somatic mutations (62%, 2282/3666) persisted after a single-dose of either T or N, while 17% (624/3666) were not detectable after brief-exposure. There was no association between immune infiltration and mutational burden in any HER2 subtype. Tumors harboring FGFR1 (8p11) amplifications exhibited higher gene-signature levels for macrophages (P=0.0073) and T-cells (P=0.0493) but not B-cells (P=0.213). Conclusions: The HER2-Basal subtype is less likely to respond to trastuzumab-based neoadjuvant therapy and exhibits lower numbers of common amplicons. The disappearance of mutations after brief-exposure to therapy may be due to either tumor heterogeneity/sampling or clonal selection. The association of 8p11 amplifications with increased T-cell infiltration suggests that this amplicon may play an immunogenic role in HER2+ breast cancer. These results warrant further investigation in larger cohorts. Citation Format: Singh S, Gilmore H, Somlo G, Abu-Khalaf M, Sikov W, Harris L, Varadan V. Association of co-amplicons with immune infiltration in subtypes of HER2-Positive 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 P1-05-09.
785 Background: Gemcitabine (G) and capecitabine (C) have antitumor activity as single agents in MBC. Each inhibits DNA synthesis, though by different mechanisms. By increasing the activity of pyrimidine nucleoside phosphorylase and depleting intracellular pools of deoxyuridine monophosphate, G promotes conversion of C to its active metabolite in tumor cells. Dose-limiting toxicities do not overlap. Based on a phase I study of the combination (Schilsky et al, JCO 2002), we studied the G + C combination as 1st- or 2nd- line chemotherapy in MBC. Methods: Eligible patients (pts) were age ≥18, ECOG PS 0–1, with measurable or evaluable disease, ≤1 prior chemo regimens for MBC, no prior G or C, ANC≥1200, plts≥100,000, bili≤2.0 mg/dl, CrCl≥30 ml/min. Pts received G 1000 mg/m2 d1&8 with C 830 mg/m2 PO BID (625 mg/m2 PO BID if CrCl 30–50 ml/min) d1–14 q21d. Treatment was held for ANC<1000, plts<75,000, grade(gr) ≥2 diarrhea (D), mucositis (M) or hand-foot syndrome (HFS). Pts were restaged every 3 cycles; those with complete response (CR), partial response (PR) or stable disease (SD) continued on treatment. Results: 25 pts entered the study. Median age 58 (39–78), 18/25 had visceral disease, ER+/ER-/ERunknown = 10/11/4, 9 had prior chemo for MBC. Pts received median 6 cycles of treatment (range 2–19). Day 8 G was held for neutropenia in 17.7% (21/113) of cycles 1–6. C dose was reduced for HFS (3 pts) and gr 3 M (1 pt). Gr 3/4 toxicities included ANC 12/2, plts 0, LFTs 3, M 1, HFS 1, and rash 1. No febrile neutropenia, gr 3 thrombocytopenia, nausea or D were seen. Of 20 evaluable pts (5 pts either didn’t receive the prescribed treatment (3) or were taken off study without progressive disease (PD) before their first re-staging (2)), 10 achieved a PR (50%, 95% CI 27–73%), including 6/8 2nd-line pts, 7 SD (minimum 6 cycles), and 3 PD. Median TTP was 5 months, median DOR 5.5 months, and median survival has not been reached at 12+ months. Conclusions: The G + C regimen is well tolerated in MBC with an encouraging response rate in this phase II study. It may warrant additional study, especially as more pts are exposed to both anthracyclines and taxanes in the adjuvant setting. Supported by grants from Roche Laboratories, Inc. and Eli Lilly & Co. No significant financial relationships to disclose.
e11573 Background: To evaluate CbwAbAv NAC in HER2- BrCA. Methods: 60 patients (pts) with HER2- (IHC 0-1+ or FISH <2.0) invasive BrCA, clinical stage IIA-IIIC, will undergo research biopsies/MRI pre and post “priming” doses of Av or wAb, then receive q3wk Cb AUC 6, wAb 100 mg/m2 + Av 5 mg/kg/week x 12 weeks + (cohort 1)/- (cohort 2) dose-dense doxorubicin and cyclophosphamide (ddAC)Av x 4. Post-op, pts receive Av x 34 weeks; radiation, endocrine Rx, and ddACAv x 4 (cohort 2) at MD discretion. Endpoints include pathologic complete response (pCR - absence of invasive disease in breast + axillary nodes), residual cancer burden (RCB) in non-pCRs, dose delivery and toxicities. Circulating tumor/endothelial cells (CTC/CEC) at baseline and after priming are analyzed using the Veridex platform. Results: 39 pts (median age 47; range 25-69), 21 hormone receptor positive (HR+), 18 triple-negative (TN) have received a median of 11 wAb and 4 Cb doses, with dose reductions in 25% (wAb - neutropenia [ANC]) and 14% (Cb - thrombocytopenia (tcp)). Grade 3/4 toxicities include ANC (43%/39%), tcp (18%/7%), transaminitis, nausea and hypokalemia, with no grade >2 neuropathy. Serious adverse events (SAEs): febrile neutropenia (2), altered mental status, anemia, post-mastectomy tissue necrosis, GI bleed and grade 2 proteinuria. Pathologic responses are tabulated below (Table). At baseline, few pts (19%) had identifiable CTC, which did not correlate with response. Pts with higher CEC levels at baseline (median, 22 vs. 11, p=.087) or after priming (median, 31 vs. 14.5, p=.035) were more likely to achieve RCB 0-1. Conclusions: Neoadjuvant CbwAbAv is generally well tolerated and yields encouraging pathologic responses, especially in TN pts. Differences between cohorts suggest that longer duration NAC or the use of non-cross resistant regimens may improve pCR rate. Higher CEC levels at baseline or after initial therapy may be associated with improved pathologic response. Updated results will be presented. n pCR IA/IB II-III RCB 0-1 RCB 2-3 Total 39 13 (33%) 9 17 21 (54%) 18 Cohort 1 21 (12 TN) 12 (57%) 3 6 15 (71%) 6 Cohort 2 18 (6 TN) 1 (6%) 6 11 6 (33%) 12 HR+ 21 3 (14%) 2 16 6 (29%) 15 TN 18 10 (56%) 7 1 15 (83%) 3
512 Background: OF causes accelerated bone loss in premenopausal women receiving AdC of greater magnitude than natural menopause or aromatase inhibitor therapy in postmenopausal women. CALGB 79809 compared the effect of early ZA (with AdC) or late (one year after AdC) on change in BMD in the lumbar spine (LS). We report the effect of early ZA versus no ZA (Control) at 12 months after randomization. Methods: Eligible women (≥ 40 years; stages I-III breast cancer; and last menstrual period ≤ 6 months prior to entry) were randomized to either ZA 4 mg IV every 3 months beginning 1–3 months (Arm A) after the start of AdC or no ZA. BMD of LS, serum FSH, estradiol, and β-HCG were performed at baseline (≤ 28 days prior to randomization) and repeated at 12 months. OF was prospectively defined at the 12 month study visit as ≥ 3 months of amenorrhea with an FSH ≥ 30. All women were told to take 1,000 mg of calcium and 400 IU of vitamin D and compliance (by self-report) and toxicity were assessed every 3 months. Assuming an attrition rate of 20% and that only 50% of women would develop OF, a sample size of 200 per arm was required to have 80% power to detect a mean difference of 0.09 g/cm2 in LS BMD at 12 months with at a two-sided significance level of 0.05. A prespecified boundary for early stopping for superiority was crossed at the first interim analysis. Results: From 12/01 to 12/06, 439 women enrolled and 166 (38%) met the criteria for OF at 12 months. Median age was 47 years (range 40–58); white-92%; performance status 0–91%; stage I-25%; and stage II- 56%. Compliance with calcium and vitamin D was nearly 100%. The majority of ZA-related toxicities were grades 1 or 2; grade 3 included fever- 3%, fatigue-2%, and pain-3%. There was 1(0.5%) woman with possible jaw osteonecrosis after trauma to the jaw and 2 doses of ZA. The results are described in the Table. Conclusions: ZA adds minimal toxicity and prevents the accelerated bone loss that occurs in women who develop OF receiving AdC. Arm A (n=81) Control (n=85) p Mean Percent Change in LS BMD (SD) +2.6 (6.4) −6.4 (6.7) <0.0001 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis
