Tiffany Avery

Population and target considerations for triple-negative breast cancer clinical trials

Triple-negative breast cancer (TNBC) is an aggressive disease subtype that has a poor prognosis. Extensive epidemiological evidence demonstrates clear socioeconomic and demographic associations with increased likelihood of TNBC in both poorer and minority populations. Thus, biological aggressiveness with few known therapeutic directions generates disparities in breast cancer outcomes for vulnerable populations. Emerging molecular evidence of potential targets in triple-negative subpopulations offers great potential for future clinical trial directions. However, trials must appropriately consider populations at risk for aggressive subtypes of disease in order to address this disparity most completely. New US FDA draft guidance documents provide both flexible outcomes for accelerated approvals as well as flexibility in design with adaptive trials. Careful planning with design, potential patient population and choices of molecular targets informed by biomarkers will be critical to address TNBC clinical care.

Clinical-pathological features and treatment modalities associated with recurrence in DCIS and micro-invasive carcinoma: Who to treat more and who to treat less

The primary aim in the management of DCIS is the prevention of recurrence and contralateral tumor. Risk factors for DCIS recurrence and appropriate treatments are still widely debated. Adjuvant therapies after surgical resection reduce recurrences and contralateral disease, but these treatments have significant financial costs, side effects and there is a group of low-risk patients who would not gain additional benefit. The aim of our analysis was to identify clinical-pathological features and treatment modalities associated with recurrence in DCIS and microinvasive carcinoma. In the Thomas Jefferson University Cancer Registry of Philadelphia, we identified 865 patients with DCIS or micro-invasive carcinoma treated between 2003 and 2013. Associations between recurrence and demographic factors (age at diagnosis, ethnicity), biological features (ER, PR and HER2) and treatment modalities (surgery, radiotherapy and endocrine treatment) were assessed. Our single institution register-based study showed that distribution of age at diagnosis and biological features did not significantly differ among ethnic groups. Younger women and micro-invasive carcinoma patients were more likely to undergo mastectomy, while African Americans were more likely to take endocrine therapy and undergo radiotherapy. In our sample only ER/PR negative DCIS were associated with significantly higher recurrence rate. Moreover, we reported a high rate of HER2 positive recurrences, suggesting that expression of this oncogene may represent a potential biomarker for DCIS at high risk of recurrence. To better define the molecular profile of the subgroup at worse prognosis might help to identify biomarkers predictive of recurrence or second tumors, identifying patients candidates for more appropriate treatments.

Abstract P6-03-01: A phase Ib study of the CXCR1/2 inhibitor reparixin in combination with weekly paclitaxel in metastatic HER2 negative breast cancer – First analysis

Background . Experimental models and retrospective clinical observations point to cancer stem cells (CSCs) as the culprits for tumor recurrence and metastasis. CSCs account for a small proportion of tumor cells, suggesting that their elimination through pharmacological targeting would not necessarily translate in any sizeable tumor regression. Thus, an ideal CSCs targeting agent should be a nontoxic molecule that can be safely administered in combination with chemotherapy to reduce tumor burden and improve disease control. CXCR1, one of the receptors for CXCL8, has been identified on breast cancer (BC) CSCs ( Ginestier C et al., JCI 2010 ). Reparixin, an allosteric inhibitor of CXCR1 with a large safety database in non-cancer patients, effectively targeted CSC in BC xenografts ( Ginestier C et al., JCI 2010 ). Methods . Patients were female aged > 18 years with HER-2 neg metastatic breast cancer (MBC), eligible for treatment with paclitaxel (not taxane-refractory), had received up to 3 prior CT lines for advanced BC (not including neo/adjuvant chemotherapy), had measurable disease according to RECIST 1.1, had ECOG PS of 0-1, had adequate organ function, and had no brain metastases. Patients received a 3-day run-in with reparixin oral tablets 3 times daily (tid) followed by paclitaxel 80 mg/m2/week (Days 1, 8, and 15 for 28-day cycle) + reparixin oral tablets tid for 21 days. Three dose levels of 3-6 subjects were explored: 400 mg, 800 mg and 1200 mg oral reparixin tid. A further 17 subjects were enrolled at the highest tolerated dose (total 20 patients). Safety was assessed following one cycle. Treatment continued until disease progression, unacceptable toxicity or withdrawal of consent. Primary endpoints were safety and tolerability, and pharmacokinetic (PK) profile of the combination treatment. Among secondary endpoints, assessment of disease response every 2 cycles for indication of efficacy and correlative evaluations on peripheral blood samples were conducted. Results . From 02/12 to 04/14, 33 patients entered the study. PK of reparixin at 400 mg tid, t max = 0.5-1.5 hr, t 1/2 = 1.7 hr; at 800 mg tid, t max = 0.5-3 hr, t 1/2 = 4.6 hr; at 1200 mg tid, t max = 0.5-2 hr, t 1/2 = 1.6 h. Co-administration of reparixin on days 1 and 8 had no effect on paclitaxel kinetics. Fifteen SAEs were recorded, none of which was related to Reparixin. Grade 3-4 adverse reactions were recorded in 30% (10/33) patients including haematological toxicity (5/10). Only one patient discontinued treatment for a reversible GI adverse reaction due to reparixin at the 1200 mg dose level. To date, 5 confirmed responses (2 CR, 3 PR) were recorded among 18 patients who underwent at least 1 tumor assessment (at 8 weeks). Response duration was 20m+ and 3m+(for CR) and 9m+, 6m+, 2m+ (for PR). Final data will be presented at the meeting. Conclusions . Combination treatment was safe and well tolerated at all dose levels without evidence of pharmacologic interactions and the recommended dose for subsequent studies is 1200 mg tid. Efficacy was demonstrated both in hormone receptor positive and triple receptor negative disease. A randomized phase II study of the combination versus single agent weekly paclitaxel in patients with MBC is warranted. Citation Format: Anne F Schott, Max S Wicha, Raymond P Perez, Giraldo Kato, Tiffany Avery, Massimo Cristofanilli, James M Reuben, R Katherine Alpaugh, Susan McCanna, Pier Adelchi Ruffini, Lori J Goldstein. A phase Ib study of the CXCR1/2 inhibitor reparixin in combination with weekly paclitaxel in metastatic HER2 negative breast cancer – First analysis [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 P6-03-01.

Abstract C22: A phase Ib study of the CXCR1/2 inhibitor Reparixin in combination with weekly paclitaxel in metastatic HER2 negative breast cancer - final analysis

Background. Cancer Stem Cells (CSC) have the ability to self-renew and generate the full range of cells that make up a bulk tumor. Experimental models and retrospective clinical observations point to CSC as responsible for tumor recurrence and metastasis. An ideal CSC targeting agent should be a non toxic molecule that can be safely administered also in combination with chemotherapy to improve disease control. CXCR1, one of the receptors for CXCL8, has been identified on breast cancer CSC. Reparixin, an allosteric inhibitor of CXCR1, reduced CSC in breast cancer (BC) xenografts (Ginestier C et al., JCI 2010) both as single agent and in combination with taxane chemotherapy. Methods. Patients were female aged ≥ 18 years with HER-2 negative metastatic BC, non taxane-refractory, who had received up to 3 prior chemotherapy (CT) lines for advanced BC (not including neo/adjuvant CT), had measurable disease according to RECIST 1.1, ECOG PS of 0-1, adequate organ function, and no brain metastases. Patients received a 3-day run-in with reparixin oral tablets 3 times daily (tid) followed by paclitaxel 80 mg/m2 (days 1, 8, and 15 for 28-day cycle) + reparixin oral tablets tid for 21 days. Three dose levels of 3-6 subjects were explored: 400 mg, 800 mg and 1200 mg oral reparixin tid. The highest safe dose level was expanded twice to gain additional safety and activity data. Treatment continued until disease progression, unacceptable toxicity or withdrawal of consent. Primary endpoints were safety and tolerability, and pharmacokinetic (PK) profile of the combination treatment. Among secondary endpoints, assessment of disease response every 2 cycles for indication of efficacy and correlative evaluations on peripheral blood samples were conducted. First analysis (i.e., 60 days post last patient in, LPI) of the results from this trial was reported earlier (Schott AF et al., SABC 2014). Results. Herein we report data at 6 months post LPI. From 02/2012 to 04/2014, 33 patients entered the study (4 in cohort 1, 3 in cohort 2 and 26 in cohort 3). 30 patients were evaluable for safety. Neither grade 4 adverse events (AE) nor Serious AE related to reparixin were reported. 9/23 patients at the highest dose level reported Grade 3 AE among which granulocytopenia (3 patients) and peripheral neuropathy (2 patients) that are commonly seen with paclitaxel alone. Overall, 8 confirmed responses (2 CR, 6 PR) were observed among 26 patients who underwent at least 1 tumor assessment (every 8 weeks). Response duration (days) was 645+, 466+ (for CR) and 280+, 169, 141, 113, 113+, 47 (for PR). Two additional patients experienced SD > 6 months (318 and 288 days, respectively). Of responding patients, all but one was from cohort 3. Median TTP (days) in the safety population was 58, 67 and 170 in cohorts 1, 2 and 3, respectively. Conclusions. Combination treatment demonstrated good tolerability with low incidence and severity of adverse reactions. The recommended dose of reparixin for the combination was established at 1200 mg tid. A sizeable response rate and mTTP was recorded, with some interesting long term responders. A randomized phase II study of the combination versus single agent weekly paclitaxel in frontline treatment of patients with metastatic triple-negative BC is ongoing (NCT02370238). Citation Format: Anne F. Schott, Max S. Wicha, Raymond P. Perez, Giraldo Kato, Tiffany Avery, Massimo Cristofanilli, James M. Reuben, R. Katherine Alpaugh, Pier Adelchi Ruffini, Susan Mccanna, Lori J. Goldstein. A phase Ib study of the CXCR1/2 inhibitor Reparixin in combination with weekly paclitaxel in metastatic HER2 negative breast cancer - final analysis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C22.

Abstract P4-13-18: A phase I study of romidepsin in combination with nab-paclitaxel in patients with metastatic HER-2 negative inflammatory breast cancer (IBC)

Inflammatory breast carcinoma (IBC) is the most aggressive form of breast cancer. The hallmark of IBC is regional extension into dermal lymphatics as tumor emboli causing breast edema and erythema. Pathologic characteristics of IBC include high grade, negative hormone receptor status and overexpression of HER2 and E-cadherin. The latter is the most attractive therapeutic target in IBC. In preclinical studies the histone deacetylase inhibitor, romidepsin targeted E-cadherin, affecting tumor emboli and increasing taxane sensitivity. Rationale: In vitro studies show that histone deacetylase inhibitors (HDACi) with taxanes provide synergy to enhance cell death. HDACi alter expression of AIRH1, a regulator of autophagy, typically silenced in breast cancer. In vitro treatment with HDACi induces expression of AIRH1, resulting in enhanced cell death with taxanes. In vitro studies of IBC have demonstrated the utility of HDACi and romidepsin in IBC cell lines. SAHA and romidespin, HDACis, inhibited self-renewal of IBC tumor spheroids from IBC cell lines. This trial combines romidepsin with a taxane proven in metastatic breast cancer to explore whether the combination will be effective in IBC. Design: This is a phase I trial to assess the safety of romidepsin plus nab-paclitaxel in patients with recurrent or metastatic IBC. The maximum tolerated dose (MTD) of romidepsin + weekly nab-paclitaxel was determined to define the dose for the phase II trial. Secondary objectives included describing the adverse event profile and assessing the overall response rate (ORR) and Clinical Benefit Rate (CBR). This study employed a 3+3 design. DLTs included febrile neutropenia or non-hematologic grade 3 or 4 toxicities. Patients were treated with nab-paclitaxel 100 mg/m2 iv with romidepsin, 7 mg/m2 iv (1st cohort) and 10 mg/m2 iv (2nd cohort), on days 1, 8, 15 of a 28 day cycle. Results: Nine patients were treated. The median age was 52. Three patients were treated in the first cohort. Two patients showed progressive disease (PD). One patient has had stable disease (SD) over 10 cycles and continues treatment. DLT was not reached at 7 mg. Toxicities related to romidepsin included neutropenia, anemia and fatigue. Six patients were treated in the 2nd cohort. Grade 3 hypophosphatemia, a DLT, was reached. One patient had complete response (CR). One patient had SD; four patients had PD. Toxicities related to romidepsin were anemia, neutropenia, GI upset, edema, hyperglycemia, fatigue, hypophosphatemia, pruritis, dry mouth, and increased lab values. The overall response rate (ORR) was 33% (3/9). The table below shows results. Conclusions: This phase I trial shows that romidepsin and nab-paclitaxel are well-tolerated in patients with advanced IBC. The MTD and recommended dose of romidepsin is 10 mg/m2 with nab-paclitaxel 100 mg/ m2 days 1, 8, 15 of a 28 day cycle. A phase II trial is planned in recurrent HER negative IBC patients. Citation Format: Avery TP, Jaslow R, Basu-Mallick A, Zibelli A, Fellin F, Cristofanilli M. A phase I study of romidepsin in combination with nab-paclitaxel in patients with metastatic HER-2 negative inflammatory breast cancer (IBC). [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 P4-13-18.

Abstract P6-05-07: Improving personalized management of primary breast cancer: Mammaprint® risk stratification and blueprint® molecular subtyping

Background: Historically, breast cancer (BC) patients were offered cytotoxic or endocrine therapy based on factors such as tumor size, stage, and immunohistochemistry (IHC) markers for estrogen receptor (ER) and HER2-positivity. In 2010 the College of American Pathologists revised the breast cancer guidelines on endocrine therapy (ET) to include a lower threshold of ER positivity by immunohistochemistry, changing the definition from 10% to 1% [Hammond et al]. As a result, although a larger number of patients are offered ET, not all may benefit from this expanded definition of ER positivity if their disease is not truly estrogen driven. More recently, sensitive gene profiling assays, such as Blueprint®, can determine intrinsic molecular subtype which may be more sensitive in predicting which patients will benefit from ET. Additionally, Mammaprint® provides risk stratification which can aid in determining which patients could benefit from neoadjuvant therapy. Methods This is an observational analysis of 60 patients with stage I-IV BC. Tissue analysis for ER, PR and HER2 status were determined by IHC/FISH. mRNA expression profiles of 80 genes for Blueprint® (Agendia) analysis provided molecular subtyping: luminal, basal or her2. Moreover, Mammaprint® (Agendia) analysis of 70 genes subdivided patients into low risk or high risk providing further stratification for Luminal-type. Results By IHC staining, 48% of patients were ER+/HER2-, 10% were ER+/HER2+, 8.3% were ER-/HER2+, and the remaining patients (20%) were triple negative (TN) BC. By comparison, molecular profiling classified 21% as luminal A, 18% luminal B, 11.6% Her2 and 35% basal subtype. The 35 ER+ patients were heterogeneous by subtype: 13 were classified as molecular luminal A, 16 were luminal B, 4 were reclassified as HER2 and 2 were basal-like (one of whom had 40% ER positivity). Of the ER+ patients whose IHC quantitative staining was known, 29% with low positivity (less than 10%) were reclassified as basal subtype. Of the 5 patients who are ER+/HER2+, 2 were luminal B and 3 were of the HER2-subtype. Two patients who were TN were reclassified as luminal B, and an ER-/HER2+ was classified as a basal subtype. One patient with ER+/HER2- disease had evidence of both HER2 and luminal B subtype. Of the patients who received neo-adjuvant therapy, pCR was obtained in 33% of luminal, 60% of HER2 and 50% of basal-type patients. Conclusions BluePrint® and Mammaprint® Molecular profiling are useful diagnostic tools which further characterize tumors to predict risk of recurrence and response to treatment. About one third of ER+ patients with low positivity (less than 10%) were reclassified as basal subtype, suggesting that there is a proportion of patients who are exposed to the morbidity of hormonal therapy with little therapeutic benefit. Additionally, the test is predictive of pCR, with the highest rates in the basal and Her2 subtypes, thus enabling clinicians to predict and improve clinical outcomes through more personalized treatment decisions. Citation Format: Mikkilineni L, Austin LK, Limentani K, Jaslow RJ, Avery TP, Palazzo J, Cristofanilli M. Improving personalized management of primary breast cancer: Mammaprint® risk stratification and blueprint® molecular subtyping. [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 P6-05-07.

Abstract 4918: Concordance of circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) as molecular monitoring tools in metastatic breast cancer (MBC)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background MBC is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tissue genomic evaluation using next-genomic sequencing (NGS). Furthermore, circulating DNA fragments with tumor-specific sequence alterations (ctDNA) found in the blood of patients with advanced disease offer the possibility of non-invasive molecular monitoring. ctDNA detects actionable mutations with the advantage of serial evaluation and allowing capture of inter- and intra-tumor heterogeneity. Methods This is a retrospective evaluation of 28 patients with MBC who failed standard therapies and had baseline plasma analyzed for ctDNA and tissue analysis (NGS) before starting new therapy. We were interested in the performance of the ctDNA test and the concordance rate of genomic alterations detected in the two tests. Selection criteria: progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of persistence of genomic abnormalities already demonstrated in tissue or blood analysis. Guardant360™(Guardant Health) involves comprehensive sequencing of a panel of 54 gene associated with solid tumors using single-molecule digital sequencing technology. FoundationOne®(Foundation Medicine) performed the NGS on tissue evaluates the entire coding sequence of 315 cancer-related genes. Results All patients had biopsy-proved metastatic disease, and had ctDNA and NGS performed. 93% of patients had ctDNA alterations detected (with 0.1%-27.8% circulating tumor fraction), and 9 patients had serial ctDNA. Overall, for the patients we detected mutations in ctDNA and tissue, 89% of patients had a specific alteration on ctDNA that matched the NGS analysis. Among all mutations detected in tumors which are in overlapped genes, 71% of alterations were common (83% excluding gene amplifications). Interestingly, for patients who had both tests done within 8 weeks, 70% of had additional alterations in the ctDNA that were not found on NGS, such as ERBB2 mutations. Conclusions Genomic analysis using ctDNA and NGS detects genomic abnormalities in all patients with MBC with high concordance. However, each method was able to detect alterations that the other did not, suggesting the two methods can be complementary in detecting actionable mutations and expanding therapeutic options. Intriguingly, this occurred more frequently with the ctDNA, demonstrating its utility as an adjunct to tissue sampling which permits capture of the inter- and intra-tumor heterogeneity of the disease, and warrants further investigation prospectively. Citation Format: Laura K. Austin, Tiffany Avery, Rebecca Jaslow, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, AmirAli Talasaz, Massimo Cristofanilli. Concordance of circulating tumor DNA (ctDNA) and next-generation sequencing (NGS) as molecular monitoring tools in metastatic breast cancer (MBC). [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 4918. doi:10.1158/1538-7445.AM2015-4918

Abstract 2788: Comprehensive high-depth target sequencing in circulating tumor DNAs of patients with inflammatory and non-inflammation breast cancers

Inflammatory breast cancer (IBC) is an extremely aggressive form of locally advanced breast cancer that affects about 5% of breast cancer patients. The prognosis of IBC patients is remarkably poor, with a three-year survival rate of approximately 30% compared to 60% for patients with non-IBC breast cancers. These facts highlight the importance of accurate characterization, early detection, and timely treatment of IBC patients. Thus, it is important to develop novel and clinically applicable non-invasive biomarkers to characterize the unique presentation of IBC. In this study, we searched for somatic mutations in the circulating tumor DNAs (ctDNAs) that could be used to non-invasively characterize IBC patients and inform their clinical management. Using ctDNAs extracted from plasma of 10 pairs of IBC and non-IBC patients that were matched on major demographic and clinical variables, we conducted a high-depth target next-generation sequencing study that interrogated a comprehensive panel of 127 TCGA (The Cancer Genome Atlas)-reported cancer-related genes with >7000 uniquely designed and validated probes. Overall, we obtained >500x coverage in >80% of the interrogated regions, and >100x coverage in >97% of the regions. We found that C>T mutations predominated in well-reported mutated genes such as TP53, PIK3CA, EGFR, and CDH1. Compared to non-IBC patients, IBC patients appeared to have a higher percentage of mutations in PIK3CA but a lower percentage in TP53. Interestingly, about 78% of mutated genes that were only detected in IBC patients encode zinc finger-related proteins, a family of transcriptional factors that have been implicated in IBC development. In comparison, about 43% of genes that were detected only in non-IBC patients encode proteins important to cell division regulation. Furthermore, network-based stratification (NBS) analysis of the mutation profile revealed clusters of IBC relative to non-IBC samples, indicating the potential of mutation profiling in identifying molecularly distinct subtypes of IBC patients. Preliminary longitudinal analysis of ctDNAs from three patients with multiple plasma samples indicated that de novo mutations in important genes including PIK3CA, RB1, and KRAS appeared in patient blood after chemotherapy and/or targeted therapy treatments. Moreover, the emergence of some of these mutations was temporally correlated with the responses of patients to the treatments they received. Overall, this study provides novel evidence that ctDNA mutation status may help to non-invasively characterize IBC tumors, and might also serve as a novel non-invasive marker to monitor treatment efficacy and prognosis of breast cancer patients. Future studies with larger sample sizes are warranted to confirm our findings and identify additional clinically useful markers for the characterization and management of IBC and non-IBC patients. Citation Format: Hushan Yang, Xue Zhong, Qiang Wei, Zhaomei Mu, Zhong Ye, Yinzhi Lai, Huei-Wen Lin, Rebecca Jaslow, Tiffany Avery, Laura Austin, Zhaohui Sun, Shengrong Lin, Grace Zhao, Ling Fang Tang, Ronald E. Myers, Juan P. Palazzo, Laura Biederman, Bingshan Li, Massimo Cristofanilli. Comprehensive high-depth target sequencing in circulating tumor DNAs of patients with inflammatory and non-inflammation breast cancers. [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 2788. doi:10.1158/1538-7445.AM2015-2788

Abstract 4928: Clinical utility of circulating tumor DNA (ctDNA) in advanced and metastatic breast cancer

Background MBC is an incurable disease characterized by genomic abnormalities including somatic mutations, amplifications and gene rearrangements. It is believed that treatment resistance and disease progression is related to genomic instability making “molecular monitoring” and consequent therapeutic management a dynamic and potentially more effective approach. The detection of circulating DNA fragments with tumor-specific sequence alterations (ctDNA) found in the blood of patients is the ideal diagnostic tool for non-invasive molecular monitoring. Methods This is a retrospective evaluation of 75 patients with advanced or MBC who both completed primary therapy and were NED (stage 1-3), or failed standard therapies and had baseline plasma analyzed for ctDN before starting new therapy (stage 4). For patients with progression, selection criteria included: progression of disease after standard therapies, need to detect novel molecular abnormalities for possible therapeutic targeting, or confirmation of persistence of genomic abnormalities already demonstrated in tissue or blood analysis. Guardant360™(Guardant Health) involves ctDNA isolation from plasma using a Qiagen circulating nucleic acid kit, then a panel of 54 gene mutations associated with solid tumors as reported in the COSMIC database sequenced using single-molecule digital sequencing technology. Results Most of the patients have metastatic disease (95%), but four patients had stage III disease and are now NED after neo-adjuvant therapy and surgery. According to subtype, 41% ER+/HER2-, 17% ER+/HER2+, 11% ER-/HER2+, and 31% TNBC. ctDNA was detected in 84% (63) of all patients; however, only 20% of patients who are now NED had ctDNA detected. Of patients with detected mutations, 52% had an actionable mutation that was incorporated into treatment planning. The most common mutations were TP53 (65%), PIK3CA (38%), ALK (21%) and NOTCH1 (17%). One patient with ER+/HER2- disease had ctDNA drawn at progression demonstrating a PIK3CA mutation, started on clinical trial with Letrozole/Palbociclib with clinical response and no ctDNA detected on two serial draws. Another patient with ER+/HER2- disease with 11 alterations noted in her blood, including PIK3CA and ATM, was started on targeted therapy with Trastuzumab based on HER2+ CTCs with clinical response but upon progression, ctDNA showed a dramatic increase in the resistant ATM clone, increasing from 1.1% to 27.8%. Conclusions ctDNA can be detected in the majority of patients with MBC irrespective of disease subtype. Actionable mutations can be identified and used for selection of targeted therapies. Longitudinal monitoring can accurately predict treatment response but also detect the onset of new mutations likely related to resistant clones. The real-time molecular monitoring and therapeutic implications hold the promise to significantly change our approach to the management of MBC with survival benefit. Citation Format: Laura K. Austin, Rebecca Jaslow, Tiffany Avery, Paolo Fortina, Dragan Sebisanovic, LaiMun Siew, Aubrey Zapanta, AmirAli Talasaz, Massimo Cristofanilli. Clinical utility of circulating tumor DNA (ctDNA) in advanced and metastatic breast cancer. [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 4928. doi:10.1158/1538-7445.AM2015-4928

Abstract 4847: Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using next-generation sequencing (NGS) and reverse-phase protein microarrays (RPMA)

Background Inflammatory Breast Cancer (IBC) is a clinicopathologic entity characterized by rapid progression and poor prognosis, even with the advent of targeted therapies and a multimodal approach. Gene expression profiles of IBC tumors vs. non-IBC demonstrated that HER2+ IBC have increased mTOR signaling compared to non-IBC (Iwamoto et al). mTOR activation is a mechanism for both HER-2 targeted and endocrine resistance. Combining the molecular diagnostics of next-generation sequencing (NGS) and reverse-phase protein microarray (RPMA) may identify additional therapeutic targets as well as mechanisms of resistance to targeted therapy. Methods This is an observational analysis of 12 IBC patients who had tissue biopsy after progression on standard therapies. All patients had tissue analysis by NGS and RPMA. NGS was performed by either FoundationOne™ or our in-house hot spot panel of 45 genes. RPMA was performed using TheraLink™ to identify expression of cancer-related phosphoproteins; it quantifies HER1, HER2, and HER3 receptor overexpression, and evaluates for phosphorylation of the receptor which indicates activation. Phosphorylation of HER downstream signaling pathways such as JAK2, AKT/mTOR and MEK1/2 are also detected. Subset of 7 patients had AR, MET, ALK and PDL1 expression evaluation. Results All patients had IBC and 75% had metastatic disease. 17% of patients were ER+/HER2-, 42% ER+/HER2+, 25% ER-/HER2+, and 17% TNBC. All HER2+ patients by IHC had activation of HER2 on RPMA, and 75% of those patients had confirmed ERBB2 amplification on NGS. 75% of patients had mTOR activation on RPMA, 78% of whom had a concomitant alteration in the PIK3CA pathway on NGS. 71% of the patients evaluated had ALK expression and 80% of those patients had concomitant mTOR activation. Also, both TNBCs had HER2 activation by RPMA. HER2 targeted therapy was continued on 2 patients after RPMA confirmed activation of HER2. 4 patients were initiated on combinations with an mTOR inhibitor, Everolimus (Afinitor®), and one of whom initially obtained a clinical response, upon progression had repeat RPMA demonstrating only persistent mild activation of mTOR with a decrease in downstream proteins. Another patient with TNBC IBC who underwent 3 lines of neoadjuvant therapy prior to surgery was found to have HER1, HER2, HER3 and mTOR activation; she was started on adjuvant lapatinib and capecitabine and remains with no recurrent disease. Conclusions Patients with IBC often have activation of members of the HER family and mTOR pathway indicating molecular targets and potential mechanisms of resistance in IBC. The concomitant use of NGS and RPMA is an intriguing approach to molecular diagnostics as they provide complimentary data on molecular pathways activation which expands therapeutic options, predicts treatment-sensitivity and selects more effective combinations. Citation Format: Laura K. Austin, Keithe Shensky, Juan Palazzo, Tiffany Avery, Rebecca Jaslow, Corrine Ramos, Nicholas Hoke, Emanuel Petricoin, Massimo Cristofanilli. Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using next-generation sequencing (NGS) and reverse-phase protein microarrays (RPMA). [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 4847. doi:10.1158/1538-7445.AM2015-4847