Supplemental Table 1. Biomarker Associations with Outcome

Legend for Supplementary Figures 1-2

Supplemental Figure 1. Number of cycles received

Supplemental Figure 2. Representative biomarker staining

Background Palbociclib is highly active in oestrogen-receptor positive (ER+) metastatic breast cancer, but neutropenia is dose limiting. The goal of this study was to determine whether early neutropenia is associated with disease response to single-agent palbociclib. Methods Blood count and disease-response data were analysed from two Phase 2 clinical trials at different institutions using single-agent palbociclib: advanced solid tumours positive for retinoblastoma protein and advanced liposarcoma. The primary endpoint was PFS. The primary exposure variable was the nadir absolute neutrophil count (ANC) during the first two cycles of treatment. Results One hundred and ninety-six patients (61 breast, 135 non-breast) were evaluated between the two trials. Development of any grade neutropenia was significantly associated with longer median PFS in both the breast cancer (HR 0.29, 95% CI 0.11–0.74, p = 0.010) and non-breast cancer (HR 0.57, 95% CI 0.38–0.85, p = 0.006) cohorts. Grade 3–4 neutropenia was significantly associated with prolonged PFS in the non-breast cohort (HR 0.57, 95% CI 0.38–0.85, p = 0.006) but not in the breast cohort (HR 0.87, 95% CI 0.51–1.47, p = 0.596). Multivariate analysis yielded similar results. Conclusions Treatment-related neutropenia in the first two cycles was significantly and independently associated with prolonged PFS, suggesting that neutropenia may be a useful pharmacodynamic marker to guide individualised palbociclib dosing. Clinical trials registration information Basket Trial: NCT01037790; Sarcoma Trial: NCT01209598.

1043Background: CDK 4/6 inhibitors have been practice-changing in the treatment of metastatic breast cancer. Their activity against retinoblastoma protein positive (Rb+) breast cancer cells is mediated through cell-cycle arrest at the G1/S checkpoint. The most frequent dose-limiting toxicity is neutropenia (NTP), which, in preclinical studies, also occurs via reversible cell-cycle inhibition. The goal of this study is to determine whether NTP is associated with disease response to single agent palbociclib (PAL) as a predictive biomarker of efficacy. Methods: Blood count and response data were analyzed from two phase II clinical trials at different institutions using single agent PAL in 1) advanced Rb+ solid tumors (the “Penn trial”, n = 137) and 2) advanced liposarcoma (the “MSK trial”, n = 59). The majority of subjects in the Penn trial had breast cancer (45%). The primary endpoint was progression free survival (PFS). For each patient, NTP was examined as cycle 1 nadir absolute neutrophil count (C1ANC) a...

Breast cancer metastases differ biologically from primary disease; therefore, metastatic biopsies may assist in treatment decision making. Commercial genomic testing of both tumor and circulating tumor DNA have become available clinically, but utility of these tests in breast cancer management remains unclear.Patients undergoing a clinically indicated metastatic tumor biopsy were consented to the ongoing METAMORPH registry. Tumor and blood were collected at the time of disease progression before subsequent therapy, and patients were followed for response on subsequent treatment. Tumor testing (n = 53) and concurrent cell-free DNA (n = 32) in a subset of patients was performed using CLIA-approved assays.The proportion of patients with a genomic alteration was lower in tumor than in blood (69 vs. 91%; p = 0.06). After restricting analysis to alterations covered on both platforms, 83% of tumor alterations were detected in blood, while 90% of blood alterations were detected in tumor. Mutational load specific for the panel genes was calculated for both tumor and blood. Time to progression on subsequent treatment was significantly shorter for patients whose tumors had high panel-specific mutational load (HR 0.31, 95% CI 0.12-0.78) or a TP53 mutation (HR 0.35, 95% CI 0.20-0.79), after adjusting for stage at presentation, hormone receptor status, prior treatment type, and number of lines of metastatic treatment.Treating oncologists must distinguish platform differences from true biological heterogeneity when comparing tumor and cfDNA genomic testing results. Tumor and concurrent cfDNA contribute unique genomic information in metastatic breast cancer patients, providing potentially useful biomarkers for aggressive metastatic disease.

Background : While several comprehensive genomic sequencing tests are clinically available for breast cancer(BC), little is known about the spectrum of findings reported in the general population and clinical utility of findings for patients(pts). Here we report tumor sequencing from the METAMORPH study, a comprehensive genomic testing approach in pts with metastatic(met) BC. Methods : Pts with either known or suspected BC mets consented to and clinically underwent concurrent diagnostic and research tumor biopsies(bx). FFPE specimens were profiled via Illumina TruSeq Cancer Panel next generation sequencing platform covering 212 amplicons in 47 cancer genes. Pathology, treatment and outcome data were prospectively collected and tracked. Aside from Her2-directed treatment, therapy was not mutation (mut)-matched. Results : 64 pts enrolled between 11/2013 – 05/2015. Of these, 48 had bx successfully sequenced (75%). Of those without sequencing, 5 had negative/insufficient tissue, 2 had insufficient DNA, remainder no bx/pending. Median age of those sequenced was 56 (range 31-78); 81% Caucasian, 17% African American. 25% (12 pts) presented with de novo stage IV disease. Of those with recurrence (n=36), 83% had prior adjuvant chemotherapy; 81% hormone receptor positive(HR+) had prior endocrine therapy. Median # prior lines of therapy for met disease was 2 (IQR 0 – 8). Tumor characteristics, including mut analyses, are shown in Table 1. # muts did not differ significantly by subtype(p=0.22). Frequency of TP53 and PIK3CA hotspot muts was nearly identical to TCGA. Median # muts was 1 for pts with both de novo mets and recurrence(p=0.79). # of muts was not associated with time to recurrence(p=0.80). Excluding pts found to have TP53 mut only or ERBB2 alterations in known Her2+ disease, 42% of pts were identified as having at least one potentially actionable alteration ( PIK3CA mut, AKT1 mut or EGFR amplification). Median time to treatment failure(TTF) on subsequent therapy was 4.1 months for overall group, and 4.1, 6.2, and 1.6 months for HR+/Her2-, any Her2+ and TN, respectively, adjusted for line of therapy(p=0.03). After adjustment for # lines of prior met therapy, TTF was 4.7 vs. 4.1 months for pts with any mut vs. none(p=0.89); 5.7 vs 4.1 months for PIK3CA + vs. not (p=0.94); 3.3 vs. 6.5 months for TP53 + vs. not (p=0.03). Conclusion : Pts with met BC have frequent and potentially actionable muts.While overall # of muts did not affect response, tumors with TP53 muts had shorter response to subsequent therapy in this cohort. Additional data are needed to determine the clinical utility of mut testing in met BC, for both standard and mut-matched therapy. Citation Format: Soucier-Ernst D, Colameco C, Troxel AB, Clark C, Shih N, Maxwell KN, Morrissette J, Lieberman D, Feldman M, Goodman N, Bradbury A, Clark A, Domchek S, Fox K, Glick J, Matro J, Nathanson K, Chodosh L, DeMichele A. Mutational spectrum and tumor response in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-05.

The mechanisms of metastatic progression from hormonal therapy (HT) are largely unknown in luminal breast cancer. Here we demonstrate the enrichment of CD133 hi /ER lo cancer cells in clinical specimens following neoadjuvant endocrine therapy and in HT refractory metastatic disease. We develop experimental models of metastatic luminal breast cancer and demonstrate that HT can promote the generation of HT-resistant, self-renewing CD133 hi /ER lo /IL6 hi cancer stem cells (CSCs). HT initially abrogates oxidative phosphorylation (OXPHOS) generating self-renewal-deficient cancer cells, CD133 hi /ER lo /OXPHOS lo . These cells exit metabolic dormancy via an IL6-driven feed-forward ER lo -IL6 hi -Notch hi loop, activating OXPHOS, in the absence of ER activity. The inhibition of IL6R/IL6-Notch pathways switches the self-renewal of CD133 hi CSCs, from an IL6/Notch-dependent one to an ER-dependent one, through the re-expression of ER. Thus, HT induces an OXPHOS metabolic editing of luminal breast cancers, paradoxically establishing HT-driven self-renewal of dormant CD133 hi /ER lo cells mediating metastatic progression, which is sensitive to dual targeted therapy.

Abstract Resistance to aromatase inhibitors in ER+ breast cancer leads to recurrence and progression in the metastatic setting. JAK/STAT pathway activation is a resistance mechanism that could potentially be overcome with the use of JAK inhibitor therapy. Methods: We performed a phase II trial of exemestane, 25 mg daily, and ruxolitinib, 25 mg BID, in postmenopausal women with advanced, ER+ breast cancer who had progressed on a non-steroidal aromatase inhibitor and had either measureable or bone-only disease. A Simon 2-stage design was employed. A “go” decision to second stage would occur if fewer than 5/15 patients experienced any grade 3/4 toxicity requiring discontinuation from the study within the first treatment cycle. Results: Fifteen patients were enrolled; during cycle 1, no patient discontinued for toxicity and 1 patient went off study for progression of bone disease. 36 grade 3 events occurred; anemia was most common (n = 5), requiring transfusion in all patients. 47% required dose reduction. No partial or complete responses occurred; 3/15 (20%) had stable disease ≥6 months (clinical benefit, CB). Baseline CRP ≥8 was significantly associated with CB (3/3 CB vs. 1/11 non-CB; p = 0.011); other markers, including baseline ESR, IL-6 genotype status and primary tumor phosphoSTAT3 expression were not associated with CB in this small sample, though high tumoral pSTAT3 was seen in 66% of CB and 33% of non-CB. A novel pharmacodynamic (PD) assay to assess STAT3 phosphorylation in peripheral blood mononuclear cells after ruxolitinib exposure demonstrated differential effects in patients with CB vs. those without CB. Conclusions: The combination of exemestane and the JAK2 inhibitor ruxolitinib met safety criteria for continued enrollment. Anemia, an expected toxicity of R, was common and the high rate of severe anemia and need for dose reductions has led to a decision to reduce the starting dose of ruxolitinib to 15 mg BID moving forward. Promising predictive markers, including CRP, tumor pSTAT3 and a novel PD assay for pSTAT3 will be further evaluated. Citation Format: Angela M. DeMichele, Christopher B. Colameco, Anna Kalota, Andrea B. Troxel, Robin Holmes, Rebecca Cimildoro, Kelly Zafman, Kevin R. Fox, Susan M. Domchek, Keerthi Gogineni, Angela R. Bradbury, Jennifer M. Matro, Natalie Shih, Michael D. Feldman, Amy S. Clark, Elizabeth O. Hexner, Jacqueline F. Bromberg. A phase II trial of exemestane and ruxolitinib for aI-resistant ER+ breast cancer: Interim safety, efficacy, and biomarker analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT114. doi:10.1158/1538-7445.AM2015-CT114

9569 Background: Over 35% of breast cancer survivors face upper extremity disability due to breast cancer surgery, radiation, or chemotherapy to the upper body region. Race may be a key patient characteristic in disability, given that race is associated with factors related to poor functioning, including weight gain after breast cancer, disease stage, and treatment type. Studies of breast cancer survivors have not explicitly evaluated the associations between race and upper extremity disability using validated tools. Methods: The Quick-DASH (Disabilities of the Arm, Shoulder and Hand) is an 11-item self-administered questionnaire that has been validated for breast cancer survivors to assess global arm function over the past 7 days. The cross-sectional analysis assessed whether or not Black or White women had greater upper extremity disability, evidenced by higher Quick-DASH scores, in a population of 697 breast cancer survivors in the Wellness After Breast Cancer-II study. Linear regression estimated the relationship between race and Quick-DASH score, adjusting for demographics, body mass index (BMI), treatment type (lumpectomy, mastectomy, radiation, chemotherapy, and reconstruction), and other physical factors. Results: Black women had significantly higher BMI and age, were less likely to have had mastectomy, and had 7.7 points higher average Quick-DASH score than White (p < 0.001) women. After adjustment for demographics, BMI, treatment type, and other physical factors, Black women had an average 4.1-point (95% CI: 0.96-7.92) higher Quick-DASH score (p = 0.01) compared with White women. Mediation analysis suggested that BMI reduced the association between race and disability by 29.7%. Conclusions: Black women’s Quick-DASH scores indicated clinically significant worse functioning, which were partially mediated by higher BMIs. Black patients may present in clinic with both worse functioning and higher BMI. Findings suggest that more research is needed to elucidate the relationship between race, BMI, and upper extremity disability, including determining additional factors beyond BMI that account for the relationship between race and disability.

Abstract While massively parallel sequencing technology has greatly expanded the number of molecular genetic tests available in oncology, little is known about the spectrum and clinical utility of findings obtained from testing tumors and circulating tumor material in specific patient populations. Here we report findings from the METAMORPH study, in which stage IV breast cancer patients had metastatic tumor biopsies (metDNA) and concurrently collected cell-free circulating tumor DNA (cfDNA). Illumina TruSeq Cancer Panel (for metDNA) and Guardant360 (for cfDNA) were performed. 28 patients had both tests; results are shown in the Table. 68% of patients had at least one alteration in metDNA and 86% in cfDNA. PIK3CA mutations were most common, occurring in 43% and 36% of patients’ metDNA and cfDNA, respectively. Overall, 16 of 28 (57%) of patients had the same alterations identified in both metDNA and cfDNA. Excluding ERBB2 amplifications in HER2+ patients, 43% of patients’ metDNA and 57% of patients’ cfDNA contained pathogenic mutations or variants of uncertain significance (VUS) for which there are approved targeted therapies or clinical trials. Overall, 80 alterations were identified, 23 of which were detected by both assays. Multiple reasons for discordance in calls between metDNA and cfDNA assays were identified. While biological phenomena (e.g. tumor heterogeneity) may contribute to discordance, technical issues played an important role. Additional studies using whole exome sequencing and other platforms to further assess biological evolution of metastatic disease and clinical utility of molecular profiling of metastatic tumors and cell-free DNA are needed. Table 1 Tumor DNA (metDNA)Cell-free DNA (cfDNA)# pts with alteration (%)19/28 (68%)24/28 (86%)ER+/Her2- (n = 17)10/1715/17Her2+ (n = 4)4/42/4TNBC (n = 7)5/77/7Total # alterations in # genes31 in 7 genes72 in 19 genesGenes w/alterations (total); Bold: genes for which exists a possible targeted therapeuticPIK3CA (13), TP53 (10), ERBB2 (4), EGFR, RB1, SMAD4, STK11PIK3CA (14), TP53 (14), EGFR (9), ERBB2 (6), BRAF (6), MET (6), JAK2 (3), NOTCH1 (2), FBXW7 (2), ARAD, FGFR2, JAK3, KRAS, MYC, NPM1, PROC, RET, SMAD4, SMARCB1Variants only covered by one assay033Variants detected in both but only reported by one assay3 (2 indels, 1 VUS)1 (1 synonymous)Variants detected by only one assay1 amplification at 7-fold; 4 SNVs (AF range 19-75%)2 amplifications at <3-fold; 13 SNVs (AF range 0.1-0.8%) Citation Format: Kara N. Maxwell, Danielle J. Soucier-Ernst, Erica L. Carpenter, Andrea B. Troxel, Christopher Colameco, Candace Clark, Michael D. Feldman, Bijal Kakrecha, Melissa Langer, Joy Lee, David A. Lewis, David Lieberman, Jennifer Morrissette, Tien-chi Pan, Stephanie S. Yee, Natalie Shih, Lewis A. Chodosh, Angela M. DeMichele. Comparison of mutational spectra in metastatic tumors and cell-free DNA in breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2015-618

Abstract Purpose: The G1–S checkpoint of the cell cycle is frequently dysregulated in breast cancer. Palbociclib (PD0332991) is an oral inhibitor of CDK4/6. Based upon preclinical/phase I activity, we performed a phase II, single-arm trial of palbociclib in advanced breast cancer. Experimental Design: Eligible patients had histologically confirmed, metastatic breast cancer positive for retinoblastoma (Rb) protein and measureable disease. Palbociclib was given at 125 mg orally on days 1 to 21 of a 28-day cycle. Primary objectives were tumor response and tolerability. Secondary objectives included progression-free survival (PFS) and assessment of Rb expression/localization, KI-67, p16 loss, and CCND1 amplification. Results: Thirty-seven patients were enrolled; 84% hormone-receptor (HR)+/Her2−, 5% HR+/Her2+, and 11% HR−/Her2−, with a median of 2 prior cytotoxic regimens. Two patients had partial response (PR) and 5 had stable disease ≥ 6 months for a clinical benefit rate (CBR = PR + 6moSD) of 19% overall, 21% in HR+, and 29% in HR+/Her2− who had progressed through ≥2 prior lines of hormonal therapy. Median PFS overall was 3.7 months [95% confidence interval (CI), 1.9–5.1], but significantly longer for those with HR+ versus HR− disease (P = 0.03) and those who had previously progressed through endocrine therapy for advanced disease (P = 0.02). Grade 3/4 toxicities included neutropenia (51%), anemia (5%), and thrombocytopenia (22%). Twenty-four percent had treatment interruption and 51% had dose reduction, all for cytopenias. No biomarker identified a sensitive tumor population. Conclusions: Single-agent palbociclib is well tolerated and active in patients with endocrine-resistant, HR+, Rb-positive breast cancer. Cytopenias were uncomplicated and easily managed with dose reduction. Clin Cancer Res; 21(5); 995–1001. ©2014 AACR.

1511 Background: Multiplex panel testing studies evaluating cancer susceptibility in breast cancer (BC) patients suggest that between 4-11% of BRCA1/2 negative individuals have a deleterious mutation in a high or moderate penetrance gene. The range in mutation positivity is likely due to heterogeneity of the studied patient populations. Methods: We performed targeted, massively parallel sequencing of 795 BRCA1/2 negative BC patients in a research laboratory and analyzed 18 cancer susceptibility genes.Three high risk groups were studied: multiple primary cancers (MP, n = 315), early onset breast cancer (EOBC, n = 323), and familial breast cancer (FBC, n = 415). MP was defined as at least one BC and at least one other primary malignancy excluding non-melanoma skin cancer, EOBC was defined as BC under age 40, and FBC was defined as at least three first to third degree relatives with BC under age 75. Results: Overall, 78 patients (10%) were found to have a deleterious mutation. Mutations were found most commonly in CHEK2 (n = 32, 4.0%), ATM (n = 14, 1.8%), TP53 (n = 10, 1.3%), and MSH6 (n = 4, 0.5%). One to two patients each had deleterious mutations in BARD1, BRIP1, CDKN2A, MRE11A, MSH2, NBN, PALB2, PTEN, PMS2, and RAD50; no mutations were identified in CDH1, MLH1, or STK11. Deleterious mutations were more common in MP patients versus FBC patients without MP (12% versus 7%, p = 0.04) but not EOBC patients without MP (10%, p = NS). CHEK2 and MSH6 mutations were more common in MP versus non-MP patients (6% vs 3% and 1.3% vs none, respectively, p = 0.05) whereas ATM mutations were more common in non-MP patients (0.6% vs 2.5%, p = 0.05). The most common cancers in mutation carriers were a second primary breast cancer, sarcoma, melanoma, hematological malignancies, ovarian, thyroid and uterine cancer. There was a similar distribution of malignancies in MP mutation carriers versus non-mutation carriers. Sequencing is ongoing for 276 additional MP patients to validate these findings. Conclusions: Our results indicate that multiplex panel testing may find a higher rate of mutations in patients with MP malignancies as compared to patients with FBC alone; however, the type of second primary malignancy may not be predictive of mutation status.

Resistance to aromatase inhibitors in ER+ breast cancer leads to recurrence and progression in the metastatic setting. JAK/STAT pathway activation is a resistance mechanism that could potentially be overcome with the use of JAK inhibitor therapy. Methods: We performed a phase II trial of exemestane, 25 mg daily, and ruxolitinib, 25 mg BID, in postmenopausal women with advanced, ER+ breast cancer who had progressed on a non-steroidal aromatase inhibitor and had either measureable or bone-only disease. A Simon 2-stage design was employed. A “go” decision to second stage would occur if fewer than 5/15 patients experienced any grade 3/4 toxicity requiring discontinuation from the study within the first treatment cycle. Results: Fifteen patients were enrolled; during cycle 1, no patient discontinued for toxicity and 1 patient went off study for progression of bone disease. 36 grade 3 events occurred; anemia was most common (n = 5), requiring transfusion in all patients. 47% required dose reduction. No partial or complete responses occurred; 3/15 (20%) had stable disease ≥6 months (clinical benefit, CB). Baseline CRP ≥8 was significantly associated with CB (3/3 CB vs. 1/11 non-CB; p = 0.011); other markers, including baseline ESR, IL-6 genotype status and primary tumor phosphoSTAT3 expression were not associated with CB in this small sample, though high tumoral pSTAT3 was seen in 66% of CB and 33% of non-CB. A novel pharmacodynamic (PD) assay to assess STAT3 phosphorylation in peripheral blood mononuclear cells after ruxolitinib exposure demonstrated differential effects in patients with CB vs. those without CB. Conclusions: The combination of exemestane and the JAK2 inhibitor ruxolitinib met safety criteria for continued enrollment. Anemia, an expected toxicity of R, was common and the high rate of severe anemia and need for dose reductions has led to a decision to reduce the starting dose of ruxolitinib to 15 mg BID moving forward. Promising predictive markers, including CRP, tumor pSTAT3 and a novel PD assay for pSTAT3 will be further evaluated. Citation Format: Angela M. DeMichele, Christopher B. Colameco, Anna Kalota, Andrea B. Troxel, Robin Holmes, Rebecca Cimildoro, Kelly Zafman, Kevin R. Fox, Susan M. Domchek, Keerthi Gogineni, Angela R. Bradbury, Jennifer M. Matro, Natalie Shih, Michael D. Feldman, Amy S. Clark, Elizabeth O. Hexner, Jacqueline F. Bromberg. A phase II trial of exemestane and ruxolitinib for aI-resistant ER+ breast cancer: Interim safety, efficacy, and biomarker analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT114. doi:10.1158/1538-7445.AM2015-CT114

527 Background: Palbociclib (Palb) is an oral CDK 4/6 inhibitor that is under development in breast cancer as a single agent and in combination with endocrine therapy. Preclinical studies suggest that Palb synergizes with paclitaxel (Pac) when given on an alternating schedule. We conducted a Phase I trial investigating the combination of weekly Pac and alternating Palb to synchronize the cell cycle in the tumors of patients (pts) with metastatic breast cancer. Methods: Pts with tumors expressing Rb protein, adequate organ function, and ≤3 prior cytotoxic metastatic regimens were eligible. Prior taxane was allowed. Palb was dose-escalated in a standard 3+3 design and taken on days 2-6, 9-14, 16-20 of each 28 day cycle. Pts received Pac 80mg/m2 weekly for 3 cycles; thereafter, Pac was administered on days 1, 8 and 15. After 6 cycles of therapy, pts had the option to drop the Pac and continue on Palb alone. Toxicity was assessed weekly and response was assessed every 2 cycles using RECIST 1.0. Results: The table below shows Palb dose level, enrollment, dose limiting toxicities (DLT), number of patients with Grade 3/4 neutropenia (NTP) and response (partial response (PR), stable disease (SD) or progressive disease (PD)). 8 patients had previously received a taxane. The only DLT was grade 3 AST and ALT (LFT). Among 11 pts with PR or SD, 8 pts continued on therapy >6 months and 4 >12 months. 11 pts are off study; 10 for PD, and one for toxicity (NTP in cycle 17) and 3 remain on study. Median time on treatment is 8 cycles. Conclusions: Combination Pac and Palb is safe and well tolerated. Prolonged tumor responses were seen. However, because uncomplicated grade 3/4 NTP was common and frequently led to dose reduction or dose interruption with 5-day Palb dosing, an additional Phase 1 expansion to examine Palb 100mg on a 3-day schedule (days 2-4, 9-11 and 16-18) is underway. Clinical trial information: NCT01320592. Starting dose level Palb Number (total 15) DLT Grade 3/4 NTP (n) Final dose Palb mg (n) Dose interruption (n) Best response (n) 50 mg 3 0 0 50 (1) 50 (1) 50 (1) No (2) Yes (1) PR (1) SD (1) PD (1) 75 mg 3 0 2 75 (1) 50 (1) 25 (1) No (1) Yes (2) PR (2) SD (1) 100 mg 6 0 5 100 (2) 75 (3) 25 (1) No (1) Yes (5) PR (2) SD (1) PD (3) 125 mg 3 1-LFT 3 75 (1) 50 (2) No (0) Yes (3) PR (1) SD (2)

Abstract Background: Despite overall favorable prognosis, a subset of patients with ER+ breast cancer will subsequently relapse, and there is a need to identify mechanisms associated with recurrence. Interleukin-6 (IL-6) is a cytokine that has been implicated in progression and metastatic behavior in breast cancer. We previously demonstrated that genotypic variants in the IL-6 promotor associated with high IL-6 production are associated with poor prognosis in a subset of non-metastatic, ER+ breast cancers (Cancer Res, 2009). In the current study, we sought to determine whether patients with these “high-producer” (HP) genotypes (GT) had tumors that were enriched for IL-6 related alterations in membrane receptors, signaling pathways and cell cycle control. Methods: We identified patients with tumor blocks available from the ECOG 2190/INT0121 trial subjects in which we previously had obtained germline DNA and performed GT/haplotype (HT) analysis, and tumor microarrays (TMAs) were prepared. Expression of IL-6/gp130 membrane receptors, tumoral IL-6 content, cytoplasmic signaling through expression of phosphorylated signaling proteins (p-STAT3, p-AKT and p-ERK) and alterations in nuclear cell cycle marker expression (cyclin D, cyclin E and p27) were assessed by immunohistochemistry. Patients had previously been GT for IL-6-174, -572, -597 and the -373 variable repeat as well as assigned HT combining the HP alleles. Chi2 and Fishers exact tests were used to compare proportions. All tests were 2-sided. Results: From the previously GT ER+ patients in E2190 (n=205), we recovered assessable tumor in 84 (41%) patients. Those with tumor did not differ from the overall or ER+ GT cohorts by any tumor/patient characteristics, genotype frequencies or recurrence rate at 10 years (63% with tumor vs. 60% overall). Gp130 membrane receptor positivity was significantly associated with all IL-6 HP GT (-174GG p=0.012; -572GG p=0.01; -597GG p=0.007 and -373non8A12T p=0.008), though IL-6 receptor or IL-6 tumor content were not, and none were associated with HT. Tumor expression of p-STAT3 was seen exclusively in tumors of patients with any of the four IL-6 HP genotypes (rate 10 - 14% for HP GT vs. 0% for all other GT) and within those with the intermediate and HP HT (p=0.03), and p-AKT expression was significantly higher among those with IL6-174GG (p=0.06) and -373non8A12T (p=0.04) GT. Tumor cyclin D levels were significantly higher in those with IL6-174GG (p=0.02), -597GG (p=0.02) and HT (p=0.04). No associations were seen with p-ERK, cyclin E or p27 expression. Conclusions: These data suggest that patients with ER+ breast cancer who have “high-producer” IL-6 genotypes and poor prognosis have tumors enriched for the IL-6 gp130 receptor, JAK/STAT signaling and cyclin D overexpression, suggesting targets for intervention in these patients. Citation Format: Angela M. Demichele, Michelle Donelson, Sara Komrokian, Christopher Colameco, Jinbo Chen, Lu Chen, Robert Gray, Jennifer Nnoli, William Vaughan, Karen Anderson, Jacqueline Bromberg. Associations between IL-6 genotype and IL-6-related tumor alterations in ER+ breast cancer: results from ECOG2190/Int0121. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4719. doi:10.1158/1538-7445.AM2014-4719

2547 Background: Palbociclib (P), an oral, selective, reversible CDK4/6 inhibitor induces transient G1 arrest in hematopoietic stem/progenitor cells (HSPC), without DNA damage or apoptosis in preclinical studies. In patients (pts) with advanced malignancy, P had activity in several tumor types, but neutropenia was dose-limiting. We sought to characterize the incidence and patterns of myelosuppression to determine if the mechanisms observed preclinically were reflected in the clinical experience. Methods: Data from 2 phase 2 studies of single agent P in pts with advanced RB+ tumors (UPenn, NCT01037790) and liposarcoma (MSKCC, NCT01209598) were analyzed. Dose (125 mg P), schedule (3 wks on/1 wk off) and safety assessments (baseline, weekly cycle 1 and d 1 of subsequent cycle) were identical. Both trials required P be held for > grade (Gr) 3 myelosuppression until resolved to < Gr 2. Results: 140 patients with breast (36%), sarcoma (21%), germ cell (21%), colon (13%), gastric (7%) tumors were enrolled. Neutropenia, anemia and thrombocytopenia rates in cycle 1 by Gr are shown. Mean absolute neutrophil count was 4.98, 4.24, 2.15, 1.62 and 1.86 at weeks 1, 2, 3, 4 and 5, respectively, reflecting time to nadir. Time to Gr3 neutropenia was 3 wks in 11%, 4 wks in 30%, 5 wks in 14%. Recovery occurred after brief dose delay. Neutropenia led to dose interruption in 19% of pts in cycle 1 and 8% had Gr3 neutropenia on day 1/cycle 2. Despite the high rate of Gr 3/4 neutropenia (39%), only 1 episode of neutropenic fever occurred (in setting of disease progression), with no infections or use of G-CSF. Mucositis was rare and