Correlation between CTMP expression levels and resistance to trastuzumab in HER2 + metastatic breast cancer.

e13008 Background: Approximately 15-20% of women diagnosed with breast cancer (BC) have HER2+ disease. Trastuzumab, the first Food and Drug Administration- approved targeted therapy for BC, serves as a turning point in the personalized therapy of HER2+ metastatic breast cancer (MBC). However, not all (HER2+MBC) patients respond to trastuzumab treatment. The attendant mechanism of trastuzumab resistance remains unclear and there are currently no conclusive biomarkers for patient response to trastuzumab. Recent studies have submitted assorted likely mechanisms leading to the resistance, including Carboxyl-terminal modulator protein (CTMP). CTMP was shown to bind to the carboxy terminus of Akt and regulate its activity. The goal of this study is to identify the role of CTMP in trastuzumab resistance in Syrian patients with (HER2+MBC). Methods: Patients received trastuzumab plus chemotherapy (either docetaxel or vinorelbine) as an adjuvant treatment until the time of disease progression. Ninety-six patients were assessable for efficacy, progressive disease (PD) and clinical benefit rate (CBR) were achieved in (32.3%) and (67.7%) respectively of the study population. Immunohistochemistry (IHC) staining was performed to examine CTMP expression in formalin-fixed paraffin-embedded (FFPE) archival tissue blocks. Results: Based on IHC scoring, 38 Cases (39.6%) had low CTMP staining, and 58 cases (60.4%) had high CTMP staining. Notably, CTMP overexpression was significantly correlated with histological grade and tumor recurrence. Moreover, the data showed that CTMP staining index is significantly higher in (PD group) than in (CBR group) (P=0.039). Intriguingly, survival analysis indicated that MBC patients with high CTMP expression had lower recurrence-free survival than those with low CTMP expression as determined by Kaplan-Meier method (p=0.048). Conclusions: Our findings confirm that (HER2+MBC) patients with high CTMP expression level tended to be refractory to trastuzumab treatment.

HER2-positive breast cancers represent approximately 20-25% of all breast cancers and are characterized by an overexpression of the growth factor receptor HER2. Trastuzumab, a monoclonal antibody, is a molecularly targeted therapeutic used in the treatment of this subtype of breast cancer. However, 30% of eligible patients have intrinsic resistance to trastuzumab and approximately 60% of patients who initially responded to this therapeutic, develop resistance within one year. Calcium transporters and modulators are known to be involved in breast cancer and in chemoresistance. However, their role has not been evaluated in HER2-positive trastuzumab resistant breast cancer cells. The aim of this project was to identify possible calcium related proteins associated with trastuzumab resistance. In the first part of this thesis, the expression of Ca2+ transporters and modulators and their role in trastuzumab activity was assessed in the HER2-positive breast cancer cell line SKBR3. Ca2+ signaling profiling was also assessed using fluorescence imaging plate reader (FLIPR) assays. Inhibition of the expression of the Ca2+ channels TPC2, TRPV1 and the Ca2+ channel modulator STIM1 using siRNA decreased SKBR3 cellular proliferation. Silencing of STIM1, the Ca2+ pump SPCA1 and the Ca2+ permeable ion channel TRPM7 increased the anti-proliferative effects of trastuzumab in SKBR3 cells. In the second part of this thesis, trastuzumab resistant and age-matched control cell lines were established from parental SKBR3 cells through seven months of continuous culturing in the presence of trastuzumab. Two trastuzumab treated colonies were selected for their resistance to trastuzumab (RT1 and RT2). Two other colonies were selected from age-matched controls because of their development of de novo resistance to trastuzumab (RV1 and RV2). Two age-matched cell lines that retained their sensitivity to trastuzumab were selected as controls (SV1 and SV2). Levels of mRNA expression of 45 Ca2+ channels, pumps and channel modulators were evaluated using quantitative RT-PCR. An siRNA screen of selected targets to identify targets that when silenced could restore trastuzumab sensitivity was also performed. Additionally Ca2+ signaling profiling and the quantitation of HER2, EGFR and IGF1R protein expression were conducted. All trastuzumab resistant cell lines maintained their overexpression of the HER2 receptor. Significantly increased mRNA levels of the voltage-gated calcium Ca2+ channel CaV3.2 was observed in both de novo resistant cell lines RV1 and RV2 compared to control cell lines SV1 and SV2. Acquired resistant cell lines RT1 and RT2 showed altered sensitivity to the purinergic receptor activator ATP, indicating a possible remodeling of Ca2+ signaling in these trastuzumab resistant cell lines. In the third part of this thesis, specific experiments were conducted to further evaluate two selected targets, the Ca2+ permeable ion channels CaV3.2 and TRPM7 channel. Pharmacological inhibition and silencing of CaV3.2 channel did not reverse trastuzumab resistance. However, CaV3.2 mRNA levels were higher in the basal HER2-positive trastuzumab resistant HCC1569 breast cancer cell line compared to the luminal HER2-positive trastuzumab sensitive SKBR3 cell line. Partial siRNA-mediated silencing of TRPM7 or pharmacological inhibition of TRPM7 channel activity did not reverse trastuzumab resistance in the trastuzumab resistant cell line RV1. However, the TRPM7 kinase inhibitor NH125 was able to promote trastuzumab activity in the trastuzumab resistant cell line RV1. Further studies are required to definitively associate TRPM7 kinase with trastuzumab resistance, given the reported sensitivity of other atypical α-kinases to NH125. In the last part of this thesis publically available data was mined to identify other potential calcium related proteins associated with trastuzumab resistance. These data sets included cDNA microarray analysis of trastuzumab resistant and sensitive SKBR3 cell lines and trastuzumab resistant breast cancer clinical samples and proteomic analysis of trastuzumab resistant and sensitive SKBR3 cell lines. These analyses indicated that the Ca2+ ATPase pump SERCA3 and galectin-3 may be associated with trastuzumab resistance. Results presented in this thesis suggest that the acquisition of trastuzumab resistance may be associated with the expression and/or activity of specific Ca2+ channels and pumps, including SERCA3, CaV3.2 channel and TRPM7 and the Ca2+-related protein galectin-3. Further studies of these proteins may help identify new approaches to reverse trastuzumab resistance and/or identify new biomarkers for predicting trastuzumab sensitivity in HER2-positive breast cancers.

Background: Preclinical and clinical studies suggest that trastuzumab resistance in HER2+ BC is mediated by cross-activation of alternative signaling pathways. Computational analysis and pooled whole-genome RNAi screens in HER2 transformed BC cell lines identified the IL6/JAK2/STAT3 axis as a master regulator pathway. The combination of trastuzumab plus ruxolitinib, a JAK1/JAK2 inhibitor, demonstrated synergistic tumor growth inhibition in mouse xenografts of HER2 transformed BC cell lines. These data provide the rationale for studying the efficacy of ruxolitinib and trastuzumab in a phase I/II trial. Design: This is an investigator-initiated, multi-center, open-label, phase I/II trial of ruxolitinib plus trastuzumab in patients (pts) with HER2+ MBC who have progressed on >2 HER2-directed therapies in the metastatic setting (including trastuzumab, pertuzumab and T-DM1, unless pt refusal). The phase I is an adaptive design with 10 pts to determine the recommended phase II dose (RP2D: 25 mg po BID, SABCS 2017). Phase II is a non-randomized open-label trial of 30 pts with trastuzumab plus ruxolitinib to compare progression free survival (PFS) against historical data with single-agent HER2-targeted therapy in HER+ MBC (Blackwell JCO 2010). The study was powered to detect a difference in median PFS of 8 weeks versus 13 weeks. Response was assessed by imaging every 9 weeks. Results: We accrued 28 pts from 10/14-8/18 and stopped early due to slow accrual. Of the 27 evaluable pts, 11 were in the phase I, of which 10 pts received 25 mg po BID (RP2D). Thus, 26 pts are included in the phase 2 study (pre-specified). Of the evaluable pts, the median age was 56 (range 32 - 77). Of these, 24 pts were postmenopausal (92%), 15 (58%) were estrogen receptor positive, and 24 (92%) had measurable disease, including 8 (33%) in lung and 4 (15%) in liver. The median number of prior lines of therapy in the metastatic setting was 4 (range: 2-9). Twenty-three pts (88.5%) received prior pertuzumab and 25 (96.1%) received prior T-DM1. As of 6/24/19, 2 pts remain on therapy at 46 and 72 weeks of follow-up. Twenty evaluable pts came off study due to progression, 3 due to death from disease, and 1 for adverse events. The median PFS was 10.7 weeks (95% CI: 7.1-15.1). The 6-month PFS is 21% (7%, 40%). The most common treatment-related adverse events (AEs) were hematologic [anemia 13 (50%), neutropenia 10 (38%), thrombocytopenia 8 (31%)], fatigue 9 (35%), aspartate aminotransferase increase 4 (15%) and diarrhea 4 (15%). The most common grade 3 AEs were anemia 7 (27%), and neutropenia 6 (23%). Grade 4 or higher events were not observed. Response data in pts with measurable disease will be presented at SABCS. Conclusion: Ruxolitinib plus trastuzumab was a well-tolerated regimen that does not include cytotoxic chemotherapy. While the combination did not achieve the primary endpoint (median PFS), there was 6-month PFS of 21% in this heavily pre-treated HER2+ MBC population. Ongoing analyses will be done to determine factors that predict clinical benefit. Citation Format: Kevin Kalinsky, Shing Lee, Amy Tiersten, Della F Makower, Tessa Cigler, Lauren Franks, Prabhjot Mundi, Dow-Chung Chi, Lauren Blumberg, Naomi Sender, Magdalena Galazyn, Kristina Hosi, Anupama Goel, Paula Klein, Eleni Andreopoulou, Joseph Sparano, Katherine D Crew, Andrea Califano, Jose Silva, Dawn L Hershman. Phase I/II trial of ruxolitinib in combination with trastuzumab in HER2+ metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-32.

Currently, the survival rate for breast cancer is more than 90%, but once the cancer cells metastasize to distal organs, the survival rate is dramatically reduced, to less than 30%. Triple-negative breast cancer accounts for 15-20% of all breast cancers. Triple-negative breast cancer (TNBC) is associated with poor prognostic and diagnostic outcomes due to the limiting therapeutic strategies, relative to non-TNBC breast cancers. Therefore, the development of targeted therapy for TNBC metastasis remains an urgent issue. In this study, high Carboxyl-terminal modulator protein (CTMP) is significantly associated with recurrence and disease-free survival rate in TNBC patients. Overexpression of CTMP promotes migration and invasion abilities in BT549 cells. Down-regulating of CTMP expression inhibits migration and invasion abilities in MDA-MB-231 cells. In vivo inoculation of high-CTMP cells enhances distant metastasis in mice. The metastasis incidence rate is decreased in mice injected with CTMP-downregulating MDA-MB-231 cells. Gene expression microarray analysis indicates the Akt-dependent pathway is significantly enhanced in CTMP overexpressing cells compared to the parental cells. Blocking Akt activation via Akt inhibitor treatment or co-expression of the dominant-negative form of Akt proteins successfully abolishes the CTMP mediating invasion in TNBC cells. Our findings suggest that CTMP is a potential diagnostic marker for recurrence and poor disease-free survival in TNBC patients. CTMP promotes TNBC metastasis via the Akt-activation-dependent pathway.

618 Background: Clinical activity of the combination of chemotherapy plus trastuzumab in HER2+ ABC has been well documented. We report the first results in terms of activity and safety of the combination of trastuzumab plus metronomic capecitabine and cyclophosphamide as first line therapy in HER-2 positive ABC. Methods: Patients (pts) at first relapse or with synchronous metastasis, were treated with trastuzumab (4 mg/kg, loading dose 6 mg/kg) plus oral capecitabine (1500mg/daily) and cyclophosphamide (50 mg/daily). Primary end-point was overall response rate (ORR), secondary end-points time to progression (TTP), clinical benefit rate (CBR; PR+ CR + prolonged SD for ≥ 24 weeks) and tolerability. The optimal two-stage design was applied. Results: A total of 31 pts with measurable ABC, tumors scored as +3 positive for HER-2 or FISH +, no pretreated with chemotherapy or trastuzumab for advanced disease have been enrolled, 28 actually valuable for response and toxicity. Median age was 59 years (range 42-87), visceral metastases were present in most patients (61%). Median number of cycles was 12 (range 1-37+). The ORR was 61 % (95% CI, 41-78%), with 1 CR (3.6 %) and 16 PR (57.1%). 9 patients had prolonged SD (32%). The CBR was 82.1% (95% CI , 63%-94%). Five progressions were observed (18%). Median TTP was 7 months (range 2- 19 + months). Ten pts received more than 20 courses. Worst toxicities were grade 2 hand-foot syndrome in 4 pts, grade 2 anemia in 4 pts, grade 2 nausea in 2 pts and diarrhea grade 3 in 1 pt. Cardiac toxicity grade 2 in 1 pts. Alopecia was not reported. Conclusions: Combination of trastuzumab and low dose metronomic oral chemotherapy in HER-2 + breast cancer has shown clinical activity. The tolerability was excellent and allowed the prolonged delivery of the combination. Thus, the patients accrual is ongoing to the pre-set target of 66 patients. Clinical trial information: 2009-017083-16.

Acquired resistance to trastuzumab is a clinical problem in the treatment of HER2-over-expressing metastatic breast cancer. Importantly, an earlier report suggested that high body mass index was associated with reduced overall survival and reduced time to progression in patients with early stage or metastatic HER2-positive breast cancer treated with trastuzumab. Adipocyte-secreted factors may stimulate growth of HER2-positive cancers, blocking the growth inhibitory action of trastuzumab. Leptin and growth differentiation factor 15 (GDF15) are two adipocytokines that have been reported to stimulate HER2-PI3K signaling. We previously showed that cells with acquired trastuzumab resistance express increased levels of GDF15, and that GDF15 knockdown restores sensitivity to trastuzumab. The objective of the current study was to identify potential molecular mechanisms by which adipocytes stimulate resistance to trastuzumab in HER2-over-expressing breast cancer cell lines. Cells were cultured in complete media or conditioned media from differentiated adipocytes (CM). Cell viability of trastuzumab-treated cells was examined under anchorage-dependent and -independent conditions. Phosphorylation of Akt was assessed by Western blotting, and response to trastuzumab was reassessed upon treatment with the PI3K inhibitor LY294002 or after transfection with kinase-dead Akt. We report that CM significantly reduced trastuzumab-mediated growth inhibition of HER2-positive cells, and stimulated rapid phosphorylation of Akt. Pharmacologic or genetic inhibition of PI3K overcame CM-mediated trastuzumab resistance. Leptin and GDF15 were both measured in CM, but only GDF15 conferred resistance to trastuzumab. Leptin, on the other hand, abrogated sensitivity to lapatinib but not trastuzumab. Our observations suggest that adipocyte-secreted factors such as GDF15 stimulate PI3K signaling, resulting in reduced response to trastuzumab. The utility of adipocytokines as predictors of drug resistance and approaches to mitigate the cancer-promoting effects of adipocyte-secreted factors should be further examined. Our work supports additional investigation into GDF15 as a potential biomarker of trastuzumab resistance, and development of approaches to therapeutically target GDF15 in HER2-positive breast cancers that have progressed on trastuzumab.

HER2 overexpression occurs in 20-30% of breast cancers and is associated with poor prognosis. Trastuzumab is a standard treatment for HER2-positive breast cancer; however, resistance develops in approximately 50% of patients within a year. The Hedgehog (Hh) signalling pathway, known for its role in maintaining stemness in various cancers, may contribute to trastuzumab resistance in HER2-positive breast cancer. This study aimed to investigate the role of Hedgehog signalling in maintaining stemness and contributing to trastuzumab resistance in HER2-positive breast cancer cell lines. Trastuzumab-resistant HER2-positive breast cancer cell lines, SKBR3 and HCC1954, were developed through continuous trastuzumab exposure. Cells were treated with GANT61 (Hh inhibitor, IC50:10 µM) or SAG21K (Hh activator, IC50:100 nM) for 24 h to evaluate the Hedgehog signalling response. Stemness marker expression (Nanog, Sox2, Bmi1, Oct4) was measured using qRT-PCR. The combination index (CI) of GANT61 with trastuzumab was calculated using CompuSyn software (version 1.0) to identify synergistic doses (CI < 1). The synergistic concentrations' impact on stemness markers was assessed. Data were analysed using two-way ANOVA and Tukey's post hoc test (p < 0.05). Trastuzumab-resistant cells exhibited increased Hedgehog signalling activity. Treatment with GANT61 significantly downregulated stemness marker expression, while SAG21K treatment led to their upregulation in both SKBR3-R and HCC1954-R cells. The combination of GANT61 and trastuzumab demonstrated a synergistic effect, markedly reducing the expression of stemness markers. These findings indicate that Hedgehog signalling plays a pivotal role in maintaining stemness in trastuzumab-resistant cells, and that the inhibition of this pathway may prevent tumour progression. Hedgehog signalling is crucial in regulating stemness in trastuzumab-resistant HER2-positive breast cancer. Targeting this pathway could overcome resistance and enhance trastuzumab efficacy. Further studies should explore the clinical potential of Hedgehog inhibitors in combination therapies.

HER2-positive breast cancer has an aggressive tumour progression among breast cancers characterized by the overexpression of HER2. Trastuzumab is an FDA-approved drug and has significantly improved outcomes for patients; however, drug resistance remains a major challenge. Tumour heterogeneity, describing genetic, epigenetic, and phenotypic differences within and between tumours, complicates tumour treatment and contributes to drug resistance. Understanding the mechanisms underlying Trastuzumab resistance, such as tumour heterogeneity, is crucial for developing new and effective therapeutic strategies. This study investigates the role of ITGB3 heterogeneity in Trastuzumab resistance, focusing on its impact on TGF-β signalling and migration marker response. It also evaluates the potential of combining Trastuzumab with the integrin β3 inhibitor cilengitide to overcome resistance associated with ITGB3 levels. Trastuzumab-resistant HER2-positive HCC1954 and SKBR3 breast cancer cell lines were generated and analysed for ITGB3 expression heterogeneity. The impact of ITGB3 on TGF-β-responsive genes (WWP1, CARM1, RASGRP1, THBS1, KCTD5, SGCA, EIF3S6, MCAM, FXR2, MTMR3, SOCS3, SLC2A4RG, MMP2, MMP9, and HSP47) and cell migration (Col4a1, fibronectin, ICAM1, Timp2, and vimentin) was analysed using luciferase reporter assays and real-time PCR. The effects of combined treatment with Trastuzumab and cilengitide were also evaluated via wound closure assay. ITGB3 expression varied significantly among resistant clones, correlating with increased expression of TGF-β-responsive genes and enhanced migration markers. Combined treatment with Trastuzumab and cilengitide significantly reduced TGF-β signalling and migration-related gene expression, particularly in high ITGB3-expressing cells. ITGB3 plays a critical role in Trastuzumab resistance through the modulation of TGF-β signalling, migration, and contributing to tumour heterogeneity. Targeting ITGβ3, alone or in combination with cilengitide, offers a promising strategy to resensitize resistant HER2-positive breast cancer cells to Trastuzumab. These findings provide valuable insights into the mechanisms of Trastuzumab resistance and suggest potential therapeutic avenues for improving patient outcomes.

Trastuzumab beyond progression: a challenge to translational oncology?

2582 Trastuzumab with or without taxanes are the cytotoxic therapies of choice for advanced Her-2 positive breast cancer. Ixabepilone and lapatinib have demonstrated clinical efficacy in advanced breast cancer that is resistant to taxanes and trastuzumab. We have compared the therapeutic potential of ixabepilone, lapatinib, paclitaxel and trastuzumab, in vitro, prior to commencing a phase I clinical trial. Three different breast cancer cell lines SK-BR3, BT-474 and MCF-7 (control; non-Her-2 amplified), were seeded 96-well plates, cultured for 24 hours and different concentrations of ixabepilone or paclitaxel and trastuzumab or lapatinib were added. Experiments were performed in triplicate. A MTT viability assay was used to measure the activity of live cells after 0, 3, 24, 48 and 120 hours. The cells and vehicle control wells were averaged and normalized to 100% for comparison to the average value of the 6 replicate wells graphed over time for each cell line, drug concentration and cell density. Student-t test was used to determine the level of significance comparing the 0 hour time point in a pair-wise, pooled variance manner to each other time point. Paclitaxel + trastuzumab significantly reduced proliferation p &lt; 0.001 at 120 hrs; Paclitaxel + lapatinib significantly reduced proliferation p &lt; 0.001 at 120 hrs; Ixabepilone + trastuzumab significantly reduced proliferation p &lt; 0.001 at 120 hrs; Ixabepilone + lapatinib significantly reduced proliferation p &lt; 0.001 at 120 hrs; Dose response curves were clearly evident for all combinations. Of note proliferation was reduced earlier and at lower drug concentrations with lapatinib combinations than with trastuzumab combinations. The drugs whose efficacy was proven in MCF-7 cells were studied in detail on the xCELLigence cell analysis system. These data recapitulated the MTT data and provided in depth detail of the rate of drug action. An international multicentre phase I trial of Ixabepilone with lapatinib ± capecitabine has commenced in patients with Her-2 positive taxane and trastuzumab resistant advanced breast cancer. Toxicity has been as previously described with fatigue, arthralgias, onycholysis and rash occurring. No DLTs have occurred. RECIST responses have been confirmed in 2 of the first 3 patients enrolled. [Table: see text]

Introduction: Targeted therapies in HER2+ MBC significantly improve outcomes but efficacy is limited by therapeutic resistance. HSP90 is a molecular chaperone involved in the stability and function of multiple signaling onco-proteins. HER2 is an acutely sensitive HSP90 client and HSP90 inhibition can overcome trastuzumab resistance. Our group reported objective responses with 17-AAG plus trastuzumab in HER2+ MBC. Ganetespib, a synthetic, second generation HSP90 inhibitor has increased potency and tolerability compared with earlier agents. We reported anti-tumor activity in metastatic HER2+ and triple negative breast cancer with single agent ganetespib. Preclinically, HSP90 inhibition has synergistic anti-tumor activity with taxanes and trastuzumab. This study will define the MTD and RP2D of ganetespib plus paclitaxel and trastuzumab in HER2+ MBC. Methods: In this 3+3 phase I dose escalation study, patients with trastuzumab-resistant HER2+ MBC receive weekly trastuzumab and paclitaxel (80mg/m2) with ganetespib on day 1, 8, 15 of a 28 day cycle. HR+ positive patients are required to have at least one prior line of endocrine therapy. DLT of ganetespib monotherapy is diarrhea and therefore patients receive prophylactic anti-motility agents. Based on prior experience with ganetespib plus docetaxel in NSCLC, only 3 dose levels of ganetespib were explored: 100mg/m2, 150mg/m2 and a 3rd cohort of 125mg/m2, if needed. Secondary endpoints include evaluation of effects of ganetespib on the pharmacokinetics (PK) of paclitaxel and preliminary efficacy assessment. Results: The dosing cohorts (100 mg/m2 (n=3) and 150 mg/m2 (n=6)) have been completed without any DLTs. Median age was 46 years (range 29-65), median prior lines of chemotherapy and anti-HER2 therapy were 3 (range 2-6) and 3 (range 2-4) respectively, including prior pertuzumab in 9/9 and T-DM1 in 8/9 patients. There were no grade 3/4 adverse events (AEs) related to ganetespib. Most common AEs related to ganetespib were diarrhea, fatigue, anemia and rash. Paclitaxel PK data available from 6/9 patients are not appreciably different from those reported in literature. Overall response rate was 25% (2/8 had PR in 150 mg/m2 cohort; 1 patient was not evaluable), SD in 63% (5/8), and clinical benefit rate (CR+PR+SD&amp;gt;24 weeks) was 50% (4/8). 3 patients remain on study. Conclusion: The RP2D of ganetespib is 150mg/m2 in combination with paclitaxel and trastuzumab. The combination was safe and well tolerated. Updated PFS and PK data will be presented. Despite prior taxanes, pertuzumab and T-DM1, clinical activity of this triplet regimen in this heavily pre-treated cohort is very promising and together with our prior experience with 17-AAG plus trastuzumab and single agent ganetespib warrants further study in HER2+ MBC. A phase 2 trial is being planned in trastuzumab-refractory HER2+ MBC who have progressed on prior pertuzumab and T-DM1. Additionally, the protocol is amended to assess the safety of ganetespib in combination with paclitaxel, trastuzumab and pertuzumab in the first-line setting. Citation Format: Jhaveri K, Teplinsky E, Chandarlapaty S, Solit D, Cadoo K, Speyer J, D'Andrea G, Adams S, Patil S, Haque S, Friedman K, Neville D, Esteva F, Hudis C, Modi S. A phase I trial of ganetespib (heat shock protein 90 inhibitor) in combination with paclitaxel and trastuzumab in patients with human epidermal growth factor receptor-2 positive (HER2+) metastatic breast cancer (MBC). [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 P4-14-21.

TPS648 Background: Up to 25% of BCs overexpress human epidermal growth factor receptor 2 (HER2). Patients with HER2+ disease have a higher rate of relapse and shorter overall survival (OS). Trastuzumab, a monoclonal antibody targeting HER2, is the standard of care and improves OS for HER2+ BC, but acquired resistance is common. The combination of trastuzumab and paclitaxel has shown good tolerability and excellent objective response rates (ORR) in up to 84% of patients with HER2+ metastatic BC (MBC) (Gasparini G, et al. BCRT. 2007;101:355-65). Everolimus is an orally bioavailable inhibitor of mammalian target of rapamycin (mTOR), a protein kinase central to multiple protein synthesis pathways and implicated in trastuzumab resistance. Everolimus-containing regimens have shown promising results in patients with ER+, HER2– advanced BC in phase II/III trials; everolimus plus trastuzumab/paclitaxel or vinorelbine has shown encouraging ORR with an acceptable safety profile in phase I/II trials in patients with HER2+ MBC. The present phase III study was undertaken to assess the effectiveness of adding everolimus to first-line standard therapy in HER2+ advanced BC. Methods: Women with HER2+, locally advanced or metastatic BC who have received no prior systemic therapy (except endocrine) are eligible. Local disease must not be amenable to resection with curative intent. Women with a history of central nervous system metastasis are excluded. Patients are randomized 2:1 (everolimus vs control) to receive standard therapy (paclitaxel and trastuzumab) plus everolimus (10 mg daily) or placebo. The primary endpoint is progression-free survival. Secondary endpoints include OS, ORR, and clinical benefit rate. Additional endpoints are safety, performance status, and biomarkers. This trial is sponsored by Novartis Pharmaceuticals and is registered (ClinicalTrials.gov: NCT00876395). Enrollment began September 2009, with a planned accrual of 717. The current accrual is 719, and the estimated primary completion date is October 2012.

Suberoylanilide hydroxamic acid (SAHA; vorinostat), a small molecule inhibitor of histone deacetylase, attenuates signaling pathways known to confer trastuzumab resistance. A combination of SAHA and trastuzumab may be a promising strategy to improve the efficacy of trastuzumab against breast cancer. In this Phase I/II study, we evaluated the toxicity and response rate after treatment with SAHA and trastuzumab in patients with HER2-overexpressing metastatic breast cancer with trastuzumab-resistant progressive disease. In Phase I, the SAHA dose was modified in cohorts of 3-6 patients to find the dose level at which 0 or 1 patients experienced a dose-limiting toxicity (DLT) during the first cycle of therapy. In the Phase II study, response to the recommended dose identified in Phase I was based on the response evaluation criteria in solid tumors. Overall survival and time to progression were also evaluated. The recommended dose was determined to be 200mg twice a day on days 1-14 and IV trastuzumab 6mg/kg on day 1 of a 21-day cycle (n=6). The Phase II study (n=10) was terminated when the pre-planned efficacy evaluation found that none of the patients in the primary analysis set responded to combination SAHA and trastuzumab treatment. In patients with HER2-positive metastatic breast cancer who had relapsed or progressed during trastuzumab therapy, we observed no DLTs with SAHA 200mg twice daily combined with trastuzumab; however, there was insufficient statistical evidence that adding SAHA reversed trastuzumab resistance in these patients.

Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues

Abstract #3148 Background: Vinflunine (VFL) is a novel microtubule inhibitor agent of the vinca alkaloid class that inhibits tubulin polymerization without stabilization, resulting in cell cycle arrest in mitosis and apoptosis. Weak tubulin binding at the vinca-binding site accounts for its reduced neurotoxicity. VFL has demonstrated activity in anthracycline and taxane pretreated patients (pts) and in combination with capecitabine. This trial evaluates the activity and safety of VFL monotherapy and in combination with trastuzumab (T) in HER2+ pts as 1st line therapy metastatic breast cancer (MBC).&amp;#x2028; Methods: Eligibility: 0 prior regimens for MBC, &amp;gt; 6 mo from adjuvant therapy, RECIST criteria measurable disease, ECOG PS 0-2, adequate organ function, peripheral neuropathy &amp;lt; G2. Treatment: HER2 unspecified: VFL 320 mg/m2 IV q3 wks; FISH HER2+ pts: VFL 280 mg/m2 plus T 6 mg/kg q3 wks. Response evaluations q9 wks; treatment continued until disease progression or toxicity.&amp;#x2028; Results: Due to termination of VFL licensing between BMS and Pierre Fabre Medicament, the study closed prematurely with only 31 evaluable pts of a planned 48 pts in each treatment arm of VFL monotherapy or VFL in combination with T. 10 pts received VFL and 21 pts were treated with VFL + T. Median age: 59 yrs (35-78). ECOG PS 0-18 pts, 1-11 pts, 2-2 pts. 48% were ER+. Prior adjuvant anthracyclines and taxanes noted in 17 and 19 pts respectively. 4 pts presented with de novo stage IV disease, all HER2 positive. 45% had 3 or more metastatic disease sites with bone (17 pts), liver (16 pts) and lung (15 pts) predominating. Median of # cycles: 4 (range 1-19). There were 10 PRs (32%), all in VFL + T, and 9 pts (29%) with PD (VFL-4 pts, VFL + T-5pts). SD was reported in 10 pts (32%). 2 pts (7%) were unevaluable, divided equally between the two arms. G3/4 neutropenia occurred in 11 pts (35%); none with fever. G3 nonhematologic toxicity consisted of pain, attributed to treatment in 5 pts (16%) (sites: abdomen-2, chest, back, and infusion site each in 1 pt), and GI toxicity characterized by N/V 3 pts (10%) as well as abdominal pain, diarrhea, constipation, occurring each in 2 pts (6%). There were no G4 events. 10 pts were hospitalized (GI -4 pts, pain 2 pts, pulmonary 2 pts, and other 2 pts). Median PFS was 3.5 months for VFL and 6.6 months for VFL + T. Median overall survival was 9 months for VFL and has not been reached for VFL + T.&amp;#x2028; Conclusions: The combination of vinflunine and trastuzumab is active in the first line treatment of MBC, producing a 48% response rate. Adverse events were as expected, manageable and consisted primarily of neutropenia, pain and GI toxicity. This encouraging activity compares favorably with other trastuzumab combination regimens and merits further evaluation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3148.