Citations

Abstract Purpose/Objectives: The optimal clinical target volume (CTV) for internal mammary node irradiation (IMNI) is uncertain in an era of increasingly conformal volume-based treatment planning for breast cancer. We mapped the location of gross internal mammary lymph node (IMN) metastases in order to identify areas at highest risk for harboring occult disease. Methods and Materials: Patients with axial imaging of IMN disease, including fluorodeoxyglucose F-18 (FDG18) positron emission tomography (PET-CT) and magnetic resonance imaging (MRI), were identified from a breast cancer registry. The IMN location was transferred by a radiation oncologist and breast radiologist onto the corresponding anatomic position on representative axial CT images of a patient in the treatment position. Distribution of lymph nodes, and their location was compared with consensus group guidelines of IMN target delineation. Results: Sixty-seven patients with 130 IMN metastases were mapped. The location was in the first three intercostal spaces in 102 of 130 (78%) nodal metastases. Eighteen of 130 (14%) IMN were located caudal to the third intercostal space, while 10 of 130 (8%) IMN were located cranial to the first intercostal space, including 3 patients with isolated IMN metastases at that location in the absence of distant disease. Of the 102 nodal metastases within the first three intercostal spaces, 54 (53%) were located within the RTOG consensus volume. Relative to the internal mammary (IM) vessels, 19 (19%) nodal metastases were located medially with a mean distance of 2.2 mm (SD 2.9 mm), while 29 (28%) were located laterally with a mean distance of 3.6 mm (SD 2.5 mm). Ninety and ninety-five percent of lymph nodes within the first three intercostal spaces would have been encompassed within a 4 mm and 6 mm medial and lateral expansion on the IM vessels, respectively. Conclusions: For women with indications for elective IMNI, a 4 mm medial and lateral expansion on the IM vessels within the first 3 intercostal spaces may be appropriate. In women with known IMN involvement, cranial extension to the confluence of the IM vein with the brachiocephalic vein +/- caudal extension to the 4th or 5th interspace with a 6 mm medial and lateral expansion may be considered, provided that normal tissue constraints are met. Citation Format: Jethwa KR, Kahila MM, Hunt KN, Brown LC, Corbin KS, Park SS, Yan ES, Boughey JC, Mutter RW. Delineation of internal mammary nodal target volumes in breast cancer radiotherapy [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 P1-10-09.

Abstract Background: DNA methylation is a promising prognostic and predictive biomarker of response to treatment in advanced breast cancer (Fackler MJ. Can Res 2014). We evaluated whether baseline and change in tissue and serum methylation would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer treated with PST. Methods: TBCRC008 was a multicenter placebo-controlled trial that investigated pCR (no invasive cancer in breast/axilla) following 12 weeks of preoperative carboplatin and albumin-bound paclitaxel with or without the histone deacetylase (HDAC) inhibitor vorinostat in 62 patients with HER2-negative breast cancer. We performed an exploratory planned biomarker study correlating baseline and change (D15) in tumor tissue and serum methylation with pCR. Methylation was measured using cumulative methylation index (CMI) in quantitative multiplexed methylation-specific polymerase chain reaction (QM-MSP) assays; cMethDNA assay for serum (Fackler MJ. Can Res 2014) and QM-MSP for tissue (Fackler MJ. Can Res 2004). The analysis population included all patients with available methylation and pCR data. Association between CMI level (log transformed baseline, D15, change) and pCR was evaluated using univariate as well as multivariable logistic regression models controlling for treatment arm and estrogen receptor (ER) status. Additional subgroup analyses assessed association of CMI level with pCR stratified by ER status and treatment arms, respectively. Spearman's correlation coefficient was performed to evaluate the correlation between tissue and serum CMI. Results: pCR data were available for 61 of 62 patients. One patient with unknown response was treated as a non-responder. The pCR rate was similar in both arms (vorinostat 25.8%, placebo 29%). Both tissue and serum CMI data were available in 55 (baseline), 50 (D15) and 46 (change) patients. Elevated serum methylation based on a predefined threshold (6.9 units) was identified in 23/59 (39%) patients. In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with a 40% lower chance of obtaining a pCR (odds ratio, OR=0.60, 95% CI 0.37-0.97; p=0.037). The multivariable analysis did not show a statistically significant association of tissue or serum CMI with pCR, although a similar trend in magnitude towards association with pCR was observed for D15 and change in tumor tissue CMI (OR 0.67 and 0.57, p=0.129 and 0.147 respectively). Subgroup analyses suggested a significant association between tissue CMI levels at D15 and pCR in the group treated with vorinostat [tissue OR: 0.44 (0.20, 0.93), p=0.03; serum OR 0.37 (0.13, 1.07), p=0.07] but not in the placebo group. No significant correlation was observed between tissue and serum CMI. Conclusion: This is the first study to evaluate the predictive role of tissue and serum methylation (CMI) in patients with early breast cancer treated with PST ± an HDAC inhibitor. Methylation was detected more frequently in tissue than serum. Tissue CMI levels at D15 may predict poor response to this regimen. Further evaluation of the predictive role of methylation in early breast cancer is warranted. Citation Format: Connolly RM, Fackler MJ, Zhang Z, Zhou XC, Goetz MP, Boughey JC, Walsh B, Carpenter J, Storniolo AM, Watkins S, Barielson E, Sukumar S, Stearns V. Tumor and serum DNA methylation in women receiving preoperative chemotherapy (PST) with or without vorinostat in primary operable HER2-negative breast cancer in TBCRC008 [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 P1-09-15.

Abstract Background: The primary mechanism by which tamoxifen (Tam) and its metabolites exert their biologic effects is through estrogen receptor (ER) binding and inhibition of ER signaling. We and others demonstrated that endoxifen (Endx) has greater antitumor activity in vitro and in vivo compared to Tam and the first-in-human Endx phase I study demonstrated its antitumor activity in patients with prior progression on Tam (Goetz SABC 2015). PKCs are a family of serine/threonine-specific protein kinases that regulate signaling pathways involved in cell proliferation and tumorigenic transformation. Our prior protein docking studies suggested endoxifen may be a substrate for PKCs. Here we report the effects of Tam and Endx on PKCβ1 binding, kinase activity, as well as interactions between PKCβ1 and ERα. Methods: Surface Plasmon Resonance (SPR, Biacore T200, GE Healthcare) was used to evaluate binding of Tam, N-desmethyl Tam (NDMT), 4-HT, and Endx to PKCβ1 and PKCβ2. The effects of Tam and Endx on PKCβ1 kinase activity were determined. Proliferation and colony formation in MCF7 parental and PKCβ1 overexpressing cells were evaluated. siRNA silencing was used to knockdown PKCβ1 expression in the following cells: MCF7 aromatase expressing cells that were either sensitive (MCF7/AC1) or resistant to letrozole (MCF7/AC1 L-resistant); T47D; and MDA-MB-361. Coimmunoprecipitation assay and DUOlink in situ proximity ligation were used to investigate the interaction between PKCβ1 and ERα. Results: Endx more potently inhibited PKCβ1 kinase activity compared to Tam ( IC50 350 nM vs 47.8 μM ) with KDs for PKCβ1 binding as follows: Endx (100 nM ), Tam ( 2 μM ), 4-HT ( 2 μM ) and NDMT (> 7 μM ). None of the SERMs exhibited PKCβ2 binding. In the MCF7/AC1 and MCF7/AC1 L-resistant cells, PKCβ1 knockdown resulted in ERα degradation and potently inhibited cell proliferation. These results were confirmed in T47D and MDA-MB-361 cells. Notably, PKCβ1 knockdown in MCF7/AC1 cells resulted in significantly greater E2 induced proliferation comparing siRNA knockdown vs. control. To further explore these effects, we evaluated the effects of PKCβ1 overexpression in MCF7 cells and demonstrated that PKCβ1 overexpression reduced cell proliferation and colony formation compared to parental MCF-7 cells without affecting ERα protein stability. Coimmunoprecipitation assays in transient transfected MCF-7 cells with exogenous PKCβ1 as well as PKCβ1 expressing MDA-MB-231 cells transiently or stably transfected with ERα demonstrated PKCβ1 and ERα interaction, with confirmation by Duolink assay that this interaction occurs in the cytoplasm. Conclusions: Our findings demonstrated that endoxifen binds and inhibits PKCβ1 at relevant concentrations achieved in the endoxifen clinical trial studies. PKCβ1 interacts with cytoplasmic ERα and PKCβ1 knockdown inhibits cell proliferation and enhances ERα turnover. However, in PKCβ1 overexpressing cells, PKCβ1 may exhibit tumor suppressive effects. These data suggest a complex interaction between PKCβ1 and ERα and that endoxifen's effects on PKCβ1 may alter drug response of endocrine therapy. Further studies are ongoing to characterize the role of PKCβ1 and its role in ER biology and response to endoxifen. Citation Format: Guo C, Kuffel MJ, Kudgus RA, Huang Z, Bode AM, Cheng J, Suman VJ, Reid JM, Bruinsma ES, Subramaniam M, Ames MM, Hawse JR, Goetz MP. Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor [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 P1-08-03.

Abstract Background: While genomic alterations are central players in tumor progression, proteins are the targets for precision therapy. The degree by which “actionable” genomic alterations translate into activated/altered proteins and pathway is still under investigation. Using a multi-OMIC approach from the SideOut 2 metastatic breast cancer (MBC) trial, this study explored the concordance between selected “actionable” genomic alterations and protein expression/activation. Methods: Snap frozen biopsies from 29 MBC patients enrolled in a prospective phase II trial were used for this analysis. Exome WES and RNASeq data was processed using an in-house developed pipeline and identified amplification of CCND1 (6/29), FGFR1 (4/29), and FGF 3, 4, 5, and 19 (4/29) as some of most frequent “actionable” genomic alterations in our MBC cohort. Signaling analysis of the 29 cases was performed using Reverse Phase Protein Microarray coupled with Laser Capture Microdissection. Protein expression/phosphorylation was measured in a continuous scale and classified based on quartile distribution. Concordance between CCND1 amplification and Cyclin D1 expression, along with the activation of FOXM1 T600 and Rb S780, was explored. Amplification of the FGFR1 locus or its ligands was correlated with the level of activation/phosphorylation of FGFR1 Y653/654. Results: While Cyclin D1 protein expression was greater than the population mean for 4/6 (67%) patients with CCND1 amplification, only 2/6 (33%) patients with CCND1 amplification had Cyclin D1 level within the top quartile of the population (n=29). FOXM1 T600 activation was independent from CCND1 amplification, with high levels of FOXM1 T600 predominantly in the CCND1 wild-type population. Only 1/6 (17%) patients with CCND1 amplification had FOXM1 T600 level similar to the top quartile of the population while a second patient was above the population median. Activation of Rb S780 was above the population median, but below the top quartile, in 2/6 (33%) CCND1 amplified patients. Similarly, none of the patients with activation of FGFR Y653/654 equal to the top quartile harbored an FGFR1 amplification. Only 1/4 (25%) patients carrying an FGFR1 amplification had an activation of FGFR Y653/654 above the population median. Similarly, 1/4 (25%) patients with FGF ligand amplification showed FGFR Y653/654 level within the top quartile while three patients had FGFR Y653/654 activation below the population median. No significant results were found between proteomic (below/above the median) and genomic characteristics by Fisher test (p>0.05). Conclusion: Molecular genotyping of “actionable” cancer targets alone may be insufficient in predicting whether the actual drug target protein is expressed and/or activated in any given patient's tumor. Although these results need further validation, the combination of genomic and proteomic data may represent a more informative approach for identifying real molecular drivers of individual lesions as well as “actionable” protein/phosphoprotein targets in the absence of genomic events. Multi-OMIC approaches may lead to more effective stratification in precision medicine trials. Citation Format: Pierobon M, Wong S, Reeded A, Anthony S, Robert N, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Carpten J, Petricoin EF. A multi-OMIC analysis to explore the impact of “actionable” genomic alterations on protein pathway activation: Clinical implication for precision medicine 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 P1-07-09.

Abstract Introduction: Homologous recombination (HR) is essential for repairing double-stranded DNA breaks. Tumors deficient in DNA HR display high levels of copy number aberrations including amplifications, deletions, and breakpoints. Studies in BRCAmut cancers have shown that HR deficiency (HRD) renders triple negative breast carcinoma (TNBC) sensitive to carboplatin or Poly(ADP-ribose) polymerase (PARP) inhibitors via a synthetic lethal interaction, as previously described. Clinical observations in breast and other solid tumors suggest that the HRD phenotype may arise independently of BRCA1/2 mutation. The implications of HRD enrichment in PD-L1, PD-L2, Jak2 (PDJ) 9p24.1 amplified TNBC are not known. Our goal was to determine the HRD status in PDJ enriched tumors, and the potential association of the PDJ amplicon with a BRCA like HRD phenotype. Methods: Archival fresh frozen and formalin-fixed paraffin embedded (FFPE) tumor samples from 41 patients with TNBC were obtained from 1998-2015. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted using DNA content flow cytometry and interrogated with oligonucleotide array comparative genomic hybridization (aCGH). Tumor homologous recombination deficiency (HRD) scores were calculated by number of interstitial aberrations (IA) in their genomes (HRD-IA). A cut-off of < 40 (low), > 40 (high) or > 50 (very high) was assigned based on our studies of known BRCAmut tumors. Data were compared between HRD groups using chi-square, ANOVA F-test or log-rank test. Results: 14/41 (34%) TNBC patients had high HRD-IA score and 6/41 (14.6%) had very high HRD-IA score. The median age was 61.8 years (SD = 12.8), the average tumor size was 3.1cm (SD = 2.6), 42% were node positive, 24% were pre-menopausal, 19% were Black, Hispanic, or Asian/Pacific Islander. Genetic testing was negative for BRCA1/2 mutations in 9/41 patients, including 3 high and very high HRD-IA score patients. There was no difference in the OS at 5-years [74% versus 59% (p = 0.907)] in the high HRD-AI group. 12/41 (29%) patients were PDJ+. In the PDJ+ patients, 4/12 (33.3%) had HRD > 40 and 1/12 (8.3%) had HRD > 50 compared to the PDJ- patients where 10/29 (34%) had high HRD-IA score and 5/29 (17%) had a very high HRD-IA score, p=0.463. Patients with inflammatory TNBC were significantly more likely to have high HRD-IA score 4/6 (66%) compared to non-inflammatory TNBC 8/31 (26%), p=0.05. Conclusions: We describe a unique group of TNBC patients with similar patient characteristics, tumor size, nodal status, and stage when compared by low, high and very high HRD-IA score. For inflammatory TNBC, there was statistically significant percentage of patients with a high HRD-IA score. We did not see a statistically significant correlation with 9p24.1 amplification based on HRD-IA score which has been suggested in other data sets. The HRD-IA score did not statistically impact OS but there was a suggestion that those with a higher score may have an improved OS potentially due to chemotherapy sensitivity. Further study with a greater number of patients to adequately power the study is needed to determine whether our initial findings can be confirmed and whether the HRD-IA score can be used to define a specific subset of TNBC patients. Citation Format: Gawryletz CD, Anderson KS, Northfelt DW, Kosiorek HE, Linnaus ME, Ocal IT, McCullough AE, Pockaj BA, Barrett MT. Homologous recombination deficiency in PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma [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 P1-05-19.

Abstract Introduction: Clonal heterogeneity in cancer is associated with resistance to therapies and evolution of metastatic disease. Clinical management of breast cancer relies on the status of estrogen (ER), progesterone (PR), and Her2 receptors in diagnostic biopsies. Results for these biomarkers rely on IHC and FISH assays that incorporate staining intensity and percentage of positive cell numbers for each sample of interest. Heterogeneous results for one or more of these biomarkers suggest the presence of multiple tumor populations within a biopsy. However the impact of clonal heterogeneity on clinical biomarkers has not been rigorously evaluated. Methods: We interrogated the clonal composition of 3 treatment-naïve surgically excised invasive ductal carcinomas. Patient #1 cancer, (pT2, pN0) was grade 3, ER+, PR+, and Her2-- with a germ line BRCA2Q49X mutation. Patient #2 cancer (pT2, pN0) was grade 3, ER+ PR- Her2+. Patient #3 was a locally advanced cancer with both axillary and supraclavicular lymph node (LN) involvement (pT2, pN3c, and was grade 3, ER+PR-Her2+ by core but found to be ER+PR-Her2- on the resection specimen. We applied DNA content flow cytometry to multiple (8-18) mapped biopsies in each case. Each sorted tumor population was interrogated with whole exome sequencing, and whole genome array comparative genomic hybridization (aCGH). Single cell analysis of aneuploid populations present in the primary tumor and lymph nodes in patient 3 was done to further assess clonal heterogeneity. Results: Tumor from patient #1 had a single ploidy with stable copy number profiles including an interstitial 13q deletion that converted a germ line BRCA2Q49X mutation to homozygosity, and a clonal homozygous deletion in Numb in each of 4 tumor biopsies. Tumor from patient #2 had a dominant ploidy (3.2N) in each of 10 (A1-A10) primary tumor biopsies but with a second co-existing ploidy (3.6N) in one (A2) biopsy. A homozygous somatic BRCA2r3129x mutation was identified in all 10 biopsies and in both ploidies; however the genomes had heterogeneous aCGH profiles. In contrast, 12 primary biopsies from Patient #3 contained 6 distinct ploidies with highly aberrant but homogenous genomes, characterized by SARC amplification, homozygous deletion of ROBO1 and ROBO2, and clonal mutations in TP53, NF1, and PIK3CA. One of the ploidies, (5.8N) was present in an adjacent node and another (5.0N) in both the adjacent and distant nodes. Single cell analyses of the 5.0N and 5.8N populations revealed that allele frequencies of driver mutations were stable in the tumor and become homozygous in the LNs. There were no Her2 amplicons or mutations in biopsies analyzed in Patients #2 and #3 further contradicting the heterogeneous staining for ER, PR and Her2. Conclusion: Tumor heterogeneity includes variations in ploidies, copy number aberrations, and allele frequencies. However even highly aberrant aneuploid genomes can be stable within multiple primary and lymph node sites. Rigorous interrogation of flow sorted tumor populations, in contrast to inferring heterogeneity in bulk samples, identifies driver aberrations in single biopsies, challenges heterogeneous results with clinical biomarkers, and distinguishes allelic heterozygosity from tumor heterogeneity in highly aberrant aneuploid tumor genomes. Citation Format: Barrett MT, Lenkiewicz E, Malasi S, Webster T, Wison Sayres MA, McCullough AE, Anderson KS, Pockaj BA. Clonal heterogeneity in breast cancer and its impact on clinical biomarkers [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 P1-05-12.

Abstract Introduction: We have previously identified a 9p24.1 amplicon targeting PD-L1/JAK2 (PDJ) in subset (26.7%) of triple negative breast cancer (TNBC) patients. The PDJ amplicon is common in newly diagnosed and chemotherapy treated TNBCs and is associated with a worse prognosis. Our goal in this study was to determine whether immunohistochemistry (IHC) evaluation could identify those patients who harbor the PDJ amplicon. Methods: TNBC patients from 1999 to 2015 whose tumors were flow-sorted and evaluated by array-based comparative genomic hybridization (CGH) were identified; paraffin slides were obtained for IHC staining evaluation of JAK-2, phosphorylated STAT3 (pSTAT3), and PD-L1. Pathologic analysis consisted of scoring the stains for intensity (0-3+) and the relative percent of tumor cells with positive staining; positive score was defined as 3+ for any percent staining. Statistical analysis of IHC staining was performed to determine association with the PDJ amplicon defined by focal 9p24.1 copy number gain with CGH log2ratios of >1.0 for each sorted TNBC sample. Results: Eleven of 43 TNBC patients evaluable by IHC had the PDJ amplicon. There was no association between PDJ amplification and IHC staining for JAK-2, pSTAT3, or PD-L1 regardless of staining intensity or percentage tumor cells positive. Table 1 describes PDJ amplicon status and positivity for JAK2, pSTAT3 and PD-L1. Of PDJ-positive samples, 64%, stained positive for JAK2, 27% positive for pSTAT3, but 0% for PD-L1 in tumor cells. However, in the PDJ-negative group, 69% still stained positive for JAK2, 19% positive for pSTAT3, and 9% for PD-L1. Table 1: PDJ amplicon status and IHC staining JAK2 (3+) (n;%)pSTAT3 (3+) (n;%)PD-L1 (3+) (n;%)PDJ - (n=32)22;69%6;19%3;9%PDJ + (n=11)7;64%3;27%0;0% JAK2: Most PDJ-positive samples (10/11, 91%) showed some JAK-2 staining. Although one had only 1+ staining at 5% of the cells, the majority (7/11, 64%) had staining of 3+ and >10% of the cells with 2 patients demonstrating 3+ staining of 75% and 90% of the cells. However, those patients without the PDJ amplicon also exhibited JAK2 staining, with 66% (n= 22) of PDJ-negative cases staining strongly for JAK-2 (3+) and > 10% of cells. pSTAT3: All but one PDJ-positive case demonstrated some staining (1+ or greater) for pSTAT3; the staining intensity and percent positivity were much less than the JAK-2 staining with only 2 cases (18%) having 3+ staining of > 10%. Similar staining was seen in the PDJ-negative cohort; 75% of PDJ-negative patients had some pSTAT3 staining (1+ or greater) and 1 patient (5%) exhibited 3+ staining > 10%. PD-L1: Finally, the PD-L1 staining was low overall with only 18% of PDJ-positive cases demonstrating some staining for PD-L1. Notably, only one case stained 3+ with 20% positivity while the majority of the samples had a 2+ intensity encompassing 5-50% of the cells. Conclusions: IHC staining for JAK2, pSTAT3, or PD-L1 was not associated with the presence of the PDJ amplicon. Notably positive IHC staining for JAK2 was observed for both PDJ-positive and PDJ-negative tumor cells, thereby nullifying its application as a screening tool for PDJ amplification. Alternative methods, such as fluorescence in situ hybridization, are needed to identify PDJ-positive patients for further study. Citation Format: Linnaus ME, Kosiorek H, Ocal IT, Dixon L, Barrett MT, Gawryletz CD, Anderson KS, McCullough AE, McEachron TA, Pockaj BA. Immunohistochemistry cannot be used to detect PD-L1/JAK-2 amplification [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 P1-03-13.

Abstract This abstract was not presented at the symposium.

Abstract Introduction: Only half of hormone receptor positive (HR+) metastatic breast cancer (MBC) patients (pts) benefit from endocrine therapy (ET). Circulating tumor cells (CTC) are prognostic in pts with MBC using CellSearch® technology. The CTC-endocrine therapy index (CTC-ETI) provides semi-quantitative analyses of CTC-ER (estrogen receptor), BCL2, HER2, and Ki67 expression. We hypothesized that CTC-ETI high (elevated CTC number and/or low expression of ER and BCL2, and high expression of HER2 and Ki-67) might predict resistance to ET in a prospective, multi-institutional clinical trial: COMETI-P2-2012.0 (NCT01701050). Methods: 121 pts with ER+, HER2 negative (-), and progressive MBC after one or more lines of ET or within 12 months (mos) of completing adjuvant ET, who were initiating a new ET, were enrolled after informed consent. CTC and CTC-ETI were determined as previously reported (Paoletti C et al, CCR 2015) at baseline (BL), 1, 2, 3, and 12 mos, and/or at the time of progression. Imaging was performed every 3 mos. Association of CTC levels and CTC-ETI with patient outcomes (progression free survival (PFS); rapid progression (RP) defined as progression within 3 mos) was assessed using logrank and Fisher's exact tests. Trial design estimated 85 PFS and 51 RP events, providing >90% power (2-sided a=0.05); pts with unsuccessful BL CTC-ETI or ineligible were unevaluable. Only baseline (BL) data are reported in this abstract. Results: 32% of enrolled pts had progression within 12 mos of completing adjuvant ET, whereas 40%, 20%, and 8% had 1, 2, ≥3 lines of ET for MBC. CTC-ETI was successfully determined in 93% of pts (90% CI, 88% to 97%). CTC were ≥5 CTC/7.5 ml whole blood in 37/108 (34%) pts evaluable for clinical validity. Elevated CTC was associated with worse PFS (median (m) PFS: 3.3 vs. 5.9 mos; P<0.01). Low, intermediate, and high CTC-ETI were observed in 75 (69%), 6 (6%), and 27 (25%) pts, respectively. CTC-ETI was associated with PFS (logrank P<0.01): pts with low, intermediate, and high CTC-ETI had mPFS of 5.7, 8.5, and 2.8 mos, respectively. In the 96 pts eligible for determination, elevated CTC was associated with RP, (65.6% vs. 42.2%; P=0.05) as was CTC-ETI (P=0.003): 79.2% (95% CI, 57.8% to 92.9%) of pts with high CTC-ETI had RP versus 41.2% (95% CI, 29.4% to 53.8%) with low CTC-ETI; in the small group with intermediate CTC-ETI 1 of 4 pts (25%) had RP. Conclusions: In this multi-institutional, prospective study, CTC-ETI was accurately determined, confirming the previously established analytical validity of the assay, meeting the primary objective of the trial. Elevated CTC and CTC-ETI high compared to low were associated with poor outcomes to ET. CTC-ETI distribution resulted in a small number of patients assigned to the intermediate group, restricting our ability to associate this group with outcomes. These results suggest that CTC-biomarker phenotype and enumeration have clinical validity. CTC-ETI may identify ER+ HER2– MBC pts who are unlikely to benefit from ET and might be better treated with ET in combination with other therapies or proceed to chemotherapy. Further analyses including CTC-ETI at serial time points during ET are planned. Citation Format: Paoletti C, Regan MM, Liu MC, Marcom PK, Hart LL, Smith II JW, Tedesco KL, Amir E, Krop IE, DeMichele AM, Goodwin PJ, Block M, Aung K, Cannell EM, Darga EP, Baratta PJ, Brown ME, McCormack RT, Hayes DF. Circulating tumor cell number and CTC-endocrine therapy index predict clinical outcomes in ER positive metastatic breast cancer patients: Results of the COMETI Phase 2 trial [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 P1-01-01.

Abstract Background: Post-mastectomy radiotherapy improves survival in women with node-positive breast cancer. Pencil beam scanning proton therapy is attractive due to potential to reduce the dose to the heart and lungs compared with traditional photon techniques while improving conformality and limiting skin dose compared with passively scanned proton therapy. The optimal dose and fractionation for pencil-beam scanning proton therapy remains unknown. Trial Design: This is a multi-center open label phase II randomized controlled trial to determine the safety of 15 fraction vs 25 fraction pencil beam scanning proton radiotherapy after mastectomy in patients requiring regional nodal irradiation. Eligibility Criteria: Patients ≥ 18 years with primary, non-inflammatory invasive breast cancer who have undergone mastectomy with or without immediate reconstruction and chest wall and regional nodal irradiation planned. Aims: To determine whether the 24 month complication rate (defined as grade 3 or greater late adverse events, and unplanned surgical intervention in patients who undergo mastectomy with reconstruction) of 15 fraction chest wall and regional node pencil beam scanning proton radiotherapy is acceptable relative to 25 fraction chest wall and regional nodal pencil beam scanning proton radiotherapy and worthy of further investigation. Statistical methods: The study is designed as a non-inferiority/superiority “hybrid” design using the approach of Freidlin et al. It is Using a one-sided type I error rate of 0.05 (corresponding equivalently to constructing a 1-sided 95% confidence limit), 72 evaluable patients will have 80% power to reject the null hypothesis that the 24-month complication rate in the experimental arm is higher than that of the control arm by more than 10% (i.e. rule out inferiority) under the alternative hypothesis that the complication rate in the experimental arm is 5% less than that of the control arm (i.e. superiority). However, the design will have only 41% power when the two treatment arms are equivalent (i.e. the complication rate is 10% for both arms). Accrual: The study opened in June 2016. Five of a planned eighty-two patients have been accrued to date. Citation Format: Mutter RW, Park SS, Hieken TJ, Vargas CE, Mei-Yin PC, Kathryn RJ, Hector VR, Kimberly CS, Elizabeth YS, Daniel VW. A randomized trial of 15 fraction vs 25 fraction pencil beam scanning proton radiotherapy after mastectomy in patients requiring regional nodal irradiation [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 OT2-03-03.