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Background: Recurrent gene fusions in breast cancer have been rarely reported suggesting that they either are not present or are not easily detected by standard sequencing methods. Comprehensive genomic profiling (CGP) by hybrid capture-based, high depth next-generation sequencing approaches, can be used to detect recurrent rearrangements and other genomic alterations involving target genes. We found that CGP can identify recurrent alterations involving RET, a known oncogenic tyrosine receptor kinase, in a subset of breast cancer. Methods: CGP using FoundationOne platform was performed interrogating the entire coding region for up to 315 cancer-related genes and introns of up to 28 genes involved in rearrangements at a depth of 500-1000X in formalin-fixed, paraffin embedded tumor tissue (Foundation Medicine, MA). Engineered representative RET fusion vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts), and cells were evaluated for RET kinase signaling, drug response, and tumorigenicity. Patient-derived xenografts (PDX) generated from two triple negative breast cancers (TNBCs) were used in an ex vivo assay (Response3DXTM, Molecular Response LLC, San Diego, CA). Results: Twenty-two RET rearrangements were identified in 8119 (0.27%) breast cancer cases. Of these, 5 rearrangements were activating RET fusions including CCDC6-RET (n=4) and NCOA4-RET (n=1), that have been described in other cancer types. Five other cases had clear evidence of genomic rearrangement involving RET, but the 5' partners could not be definitively identified. The remaining twelve cases had complex rearrangements of RET including internal duplications. RET amplification was also observed, both in TNBC and in a HER2+ breast cancer at onset of resistance to HER2-targeted therapy. Both NCOA4-RET and a novel RASGEF1A-RET fusion were characterized in vitro. Non-tumorigenic cells engineered to stably overexpress either RET fusions demonstrated transformed phenotypes. The fusions were constitutively active, as shown by endogenous phosphorylation of the kinase domain, and drove activation of downstream signaling as shown by increased phosphorylation of ERK and AKT. Cells transformed by RET-fusions were exquisitely sensitive to treatment with RET inhibitors. Interestingly, a PDX model of RET-amplified TNBC was sensitive to treatment with a PIK3CA inhibitor. An index case of ER+/PR-/HER2+, metastatic breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole was found to have a NCOA4-RET fusion by CGP. Subsequent treatment with with cabozantinib plus anastrazole led a rapid clinical and radiographic response. Conclusions: CGP can identify recurrent RET rearrangements in breast cancer that act as primary oncogenic drivers and can be therapeutically targeted. RET alterations may also play a role in acquired resistance to HER2-targeted therapies, suggesting a role for combined RET and HER2-targeted therapy in this setting. Our data demonstrate that RET alterations can be identified by clinical-grade CGP and are promising candidates as therapeutic targets in selected breast cancer patients. Citation Format: Hirshfield KM, Paratala BS, Hindoyan A, Dolfi SC, Yilmazel B, Schrock A, Gay L, Ali SM, Ross JS, Williams CB, Nair P, Ganesan S, Leyland-Jones B. Are we missing actionable targets in breast cancer? Novel insights into recurrent Ret alterations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-02.

Introduction: PARP inhibition has emerged as one of the most exciting and promising ‘targeted’ therapeutic strategies in the treatment of advanced triple negative breast cancer (TNBC) – the intended ‘target,’ being DNA repair (Anders CK, 2010). Although olaparib is known to have antitumor activity in BRCA-related TNBC cells, a limited number of preclinical and clinical studies have shown antitumor efficacy of olaparib in non-BRCA-related BC (Shimo T, 2012). Understanding the biology of TNBC cells has identified molecular targets including RTK(s), such as EGFR. Purpose: Here we tested the combination of a PARP inhibitor, olaparib (O) (AstraZeneca) plus carboplatin (C) with the EGFR/VEGFR inhibitor, vandetanib (V) (AstraZeneca) in a BRCA-wt TNBC model. Methods: Athymic mice bearing TNBC (BRCA-wt VEGFR expressing MDA-MB231& MDA-MB468 [EGFR amplified/overexpressed]) xenograft tumors (200 mm3) were treated with O plus C in combination with V (Arms: vehicle control 1, vehicle control 2, C+O, V, C+O+V). In vitro effects of V in combination with O plus C (or temozolomide) on clonogenic growth (3D on-TOP assay), proliferation (MTT and CelltiterGLO), apoptosis (Apoptosis Array), cell signaling marker(s) (Western Blot), and tumor cell phenotypes (fibronectin-directed migration, matrigel-invasion, and vascular mimicry) were investigated in a panel of five BRCA-wt and BRCA-mutated TNBC cell lines. The effects of V were tested on (a) cell signaling marker(s), (b) angiogenesis marker (HIF-1alpha), and (c) angiogenesis related phenotypes (vitronectin-directed migration, and cord formation) in HUVEC. Results: (1) O plus C in combination with V caused a regression of the in vivo growth of established tumors by 50% which was evident in both the BRCA-wt TNBC models tested. Interestingly, a marked suppression of the progression of tumor-growth was observed in the O plus C arm. (2) In vitro, V alone (10 µM) inhibited baseline as well as EGF-induced phosphorylation of AKT (S473/T308), S6RP, 4EBP1 and ERK. (3) TNBC cells exhibited higher sensitivity to V in clonogenic assays when combined with a 10 µM fixed dose of O and C/temozolomide. (4) A combination of V with O plus C increased cleaved caspase-3, PARP cleavage, and pro-apoptotic signals, while inhibiting vascular mimicry, migration, and invasion in MDA-MB231, MDA-MB468, and SUM149 cells. (5) Treatment with V blocked cord formation, migration, EGF-induced HIF-1α accumulation, and phosphorylation of AKT, 4EBP1, and ERK in HUVEC. Conclusion: A profound anti-tumor efficacy of O plus C in combination with V in BRCA-wt TNBC model can be explained by a significant anti-proliferative/pro-apoptotic and anti-migratory/anti-invasive actions of the drugs (alone or in combination), as observed both in tumor cells as well as in endothelial compartments. The combination of O plus C plus V merits further investigation in TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-19-03.

While monthly infusions of intravenous (i.v.) bisphosphonates effectively reduce skeletal complications of metastatic bone disease, regular hospital visits are inconvenient for patients and may reduce their quality of life. Daily oral bisphosphonate therapy would allow long-term patient management in the community setting. However, the use of oral bisphosphonate therapy (e.g. oral clodronate) is limited in clinical practice, due to its relatively low efficacy in comparison with i.v. agents, poor gastrointestinal tolerability, and a large tablet size that is difficult for patients to administer. Ibandronate is a highly potent, third-generation aminobisphosphonate that has been developed in an oral formulation with a small, easy-to-swallow tablet and convenient once-daily dosing. Pharmacokinetic evaluations of oral ibandronate have shown a linear dose-dependent increase in plasma concentrations that is non-saturable, and is proportional to its effect on bone-resorption markers. This ensures predictability of response to a given oral dose of ibandronate, reducing safety concerns. Bioavailability analysis suggests that oral ibandronate (50 mg) taken once daily, 30 min before food, provides comparable bone-surface exposure to i.v. ibandronate (6 mg) infused every 3–4 weeks i.e. the two formulations are dose-equivalent. The clinical equivalence of oral ibandronate (50 mg) and i.v. ibandronate (6 mg) is indicated by comparable reductions in the relative risk of skeletal events in phase III trials of patients with bone metastases from breast cancer.

A competitive antigen ELISA was previously developed for NAT2 phenotyping, using caffeine as the probe drug. The ELISA phenotypes by measuring the ratio of 5-acetamido-6-amino-3-methyluracil (AAMU) and 1-methylxanthine (1X) after transformation of 5-acetamido-6-formylamino-3-methyluracil (AFMU) to AAMU, in contrast to capillary electrophoresis high-pressure liquid chromatography (HPLC) which phenotype by measuring the AFMU/1X ratio. The ELISA phenotyping was previously determined in 30 samples and correlated well with phenotypes determined by capillary electrophoresis (29/30). The correlation was extended with the standard HPLC methodology by expanding the data set by 146 in order to test the validity of the ELISA methodology. The correlation with HPLC in this larger sample size was 96%; whereas the correlation between the two methods for determination of 1X was high ( r 2 =0.90), that for determination of AAMU by ELISA and AFMU by HPLC was low ( r 2 =0.53). The poor correlation between the two methodologies could not be attributed to the age of urine samples, nor to a significant decomposition of AFMU in the body prior to collection of the urine sample. The addition of a simple caffeine metabolite extraction method, originally developed for HPLC analysis of metabolites, to the ELISA phenotyping protocol produced a methodology with absolute correlation to the standard HPLC method.

Growth factors are a diverse group of polypeptides that stimulate cell proliferation through binding to specific high-affinity cell membrane-bound receptors. Growth factors typically act as signaling molecules between the cells. Growth factors and growth factor receptors play a major role in growth and development, wound healing, angiogenesis, and have many physiological functions. Derangement in the functions of these molecules plays a critical role in cancer progression. Both tumor and stromal/endothelial cells utilize important, in some cases redundant, groups of growth factor receptors to encode signals required for (1) proliferation/survival, (2) migration, (3) invasion and matrix degradation, (4) actin cytoskeletal rearrangement, (5) angiogenesis, and (6) chemoradiosensitivity in vivo . This article reviews the role of growth factors and their receptors in cancer, particularly the member of the receptor tyrosine kinase.

Fludarabine phosphate is the 2-fluoro, 5'-monophosphate derivative of vidarabine (ara-A) with the advantages of resistance to deamination by adenosine deaminase (ADA) and improved solubility. The mechanism of cytotoxic action of the compound appears to involve metabolic conversion to the active triphosphate. Fludarabine phosphate has substantial activity against lymphoid malignancies, particularly chronic lymphocytic leukemia (CLL) and low-grade non-Hodgkin's lymphoma (NHL). Its single-agent activity in CLL appears at least comparable to those of other conventional combination regimens. Its activity in Hodgkin's disease, mycosis fungoides, and macroglobulinemia, although suggestive, needs to be further defined and clinical trials are warranted in hairy cell leukemia, prolymphocytic leukemia, and previously untreated myeloma. The compound does not appear active against most common solid tumors. Early clinical trials indicated significant myelosuppression and the potential for severe neurotoxicity. Toxicity on the currently used low-dose schedules includes transient and reversible myelosuppression, nausea and vomiting, diarrhea, somnolence/fatigue, and elevations of liver enzymes and/or serum creatinine. Possible pulmonary toxicity has been suggested in several patients. The currently used low-doses of fludarabine phosphate, even with repeated administration, are well tolerated and appear safe with a negligible risk for severe neurotoxicity. Based on its single-agent activity and tolerability, the Food and Drug Administration recently granted group C designation of the drug for the treatment of patients with refractory CLL outside the clinical trials setting. The use of fludarabine phosphate in combination regimens and its impact on the natural history of the lymphoid malignancies is yet to be determined. Fludarabine phosphate may well occupy a pivotal role in the management of CLL and low-grade NHL.

Background: Personalized healthcare tailors treatments to patients and their disease characteristics through the use of genetics and other biomarkers. Genetic differences among individuals may explain variations in drug treatment response, including side effects. With such information physicians could make more informed decisions about drugs and dosing for a given individual, thereby improving patient care. Although there has been some success, to a large extent genetic variation related to drug response remains unexplained. Bevacizumab, a humanized monoclonal antibody against the angiogenic factor VEGF, has demonstrated activity in patients with metastatic breast cancer (MBC). The InVite study will evaluate the feasibility of performing genomewide association studies using self-reported information collected via an online platform from patients with MBC who have been treated with bevacizumab. Using novel methodology in a convenient, user-friendly, and scientifically rigorous format, InVite ultimately aims to identify potential pharmacogenetic associations in this patient population. Trial design: InVite is a pilot, non-interventional, observational, web-based, prospective cohort study designed to collect patient-reported safety, efficacy, and genetic data from patients with MBC treated with bevacizumab. Data on demographics, breast cancer disease status, cancer treatment history, bevacizumab-related outcomes, and certain safety events will be collected directly from patients entirely via online surveys. Patients will be asked to complete surveys at the time of enrollment and then every 3 months for 1 year after enrollment. A saliva sample for DNA collection will be gathered using an at-home kit. Evaluations of data quality and collection feasibility will be conducted intermittently. There will be an optional substudy to allow for blood sample collection for DNA analysis. Eligibility criteria: ≥18 years of age, residing in the US, locally recurrent breast cancer or MBC, currently being or having been treated with bevacizumab starting on or before Dec 31, 2011, fluent in English, and access to a computer with an internet connection. Specific aims: The primary objective is to assess the feasibility of recruiting subjects and collecting biospecimens and self-reported data online. The secondary objective is to characterize the patient population. Exploratory objectives include analyzing potential associations between genetic polymorphisms and 1) bevacizumab-induced hypertension, the most common bevacizumab-related adverse event, and 2) patient-reported time-to-progression. Statistical methods: Baseline demographics, clinical and treatment characteristics of enrolled patients will be summarized. Each polymorphism genotyped will be tested for association with the defined endpoint using appropriate statistical modeling. Present and target accrual: Accrual as of May 23, 2012 is 82 patients. Target accrual is 1000 patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-4-04.

Background: Triple-negative breast cancer (TNBC) is an aggressive cancer with inferior survival outcomes. Although weekly paclitaxel (WP) is effective in the treatment (tx) of metastatic breast cancer (MBC), optimization of therapies for patients (pts) with TNBC is essential. Angiogenesis is a hallmark of advanced cancer, with subset analyses suggesting activity of angiogenesis inhibitors in TNBC. Tivozanib (TIVO) is a potent and selective inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3 with a promising role in metastatic renal cell carcinoma, and established safety in Phase I combination with WP in MBC. Purpose: This Phase II trial will assess the efficacy and safety of TIVO + WP in the first-line setting for pts with advanced or metastatic TNBC and evaluate the performance of candidate angiogenesis biomarkers. Objectives: The primary objective of this study is to compare progression-free survival (PFS) of pts treated with TIVO + WP vs pts treated with placebo (PB) + WP. Secondary objectives include objective response rate (ORR), overall survival (OS), safety and tolerability, quality of life, and correlative candidate biomarker endpoints. The pharmacokinetics of TIVO + WP also will be characterized. Study Design and Methods: This multicenter, randomized, PB-controlled, two-arm study will enroll pts with metastatic or unresectable TNBC (evaluable per RECIST) and no prior systemic therapy. Pts must have confirmed available archival tumor tissue. Pts will be stratified by ECOG performance score and number of metastatic sites, then randomized to receive either oral TIVO 1.5 mg once daily for 3 weeks (wks) on/1 wk off and intravenous WP 90 mg/m2 for 3 wks on/1 wk off, or PB + WP. One cycle will be 4 wks; tx will continue until disease progression or unacceptable toxicity. Archival tumor tissue and blood samples will be evaluated for response biomarkers, including a hypoxia sensitivity gene signature, a myeloid resistance gene signature, and angiogenic ligands. All pts will be followed for survival until death. Adverse events will be monitored throughout the study. Pharmacokinetic samples will be collected during cycles 1 and 2. PAM-50–defined intrinsic molecular subtype populations also will be evaluated retrospectively. Recruitment of 130 patients is planned, with an interim analysis after 80 pts to measure ORR (130 pts with a total of 82 investigator-assessed PFS events provides 80% power to detect statistically significant PFS differences between tx arms). Endpoint analyses will use the intent-to-treat population. The primary efficacy analysis will use investigator assessments of response and a two-sided 95% confidence interval for the hazard ratio produced using Cox proportional hazards regression models. OS will be compared using the log-rank test. Analyses of candidate biomarkers and determination of an optimal predictive cutoff for response also are planned. Trial enrollment will commence in fall 2012. Conclusion: This study will determine whether TIVO, a selective and potent VEGFR inhibitor, combined with WP improves clinical outcomes in pts with TNBC, and whether clinical activity is associated with candidate angiogenesis biomarkers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT2-3-11.

The aggressiveness of human epidermal growth factor receptor-2 (HER2)-positive breast cancer and the poor prognosis of women with this disease demand the availability of accurate and reliable tests for HER2 status and the optimization of HER2-targeted therapy. The distinctive clinical pattern of HER2-positive breast cancer underlines the importance of testing for HER2 status and efforts are ongoing to validate the two major methods in use-immunohistochemistry (IHC), which measures cell membrane HER2 expression, and fluorescence in situ hybridization (FISH), which measures gene copy number. Clinical trial results demonstrate that there is an association between strong HER2 overexpression (IHC 3+) and optimal response to therapy with the novel recombinant HER2 antibody Herceptin. High levels of concordance between IHC 3+ and FISH-positive status have been observed, and response to treatment with Herceptin is similar for patients whose breast cancers are IHC 3+ and those who are FISH-positive. Observations to date have led to the formulation of an algorithm for HER2 status determination and Herceptin use which recommends that: (i) the HER2 status of all women with breast cancer be determined at presentation, (ii) all IHC 3+ and FISH-positive patients with metastatic disease should receive Herceptin, (iii) Herceptin should be used early in the course of metastatic breast cancer and preferably first line, and (iv) Herceptin therapy should be continued until disease progression.

Material and Methods: For in vitro experiments, cells were exposed to paclitaxel and/or TAS-119. Cell viability was determined by measuring cellular ATP concentration. For in vivo experiments, antitumor efficacy was assessed in subcutaneous HCC-1806-luc (triple negative breast cancer) and NCI-H460-luc (non-small cell lung cancer) xenograft models. The potential of taxane therapy with TAS-119 was evaluated in non-small cell lung cancer orthotopic models of H460-luc tumor in nude mice. To monitor tumor burden, in vivo bioluminescence imaging in individual mice was monitored weekly. Results: TAS-119 selectively inhibited phosphorylation of Aurora A without affecting the activity of Aurora B and Aurora C in NCI-H460 cells. TAS119 showed synergistic cell growth inhibition in combination with paclitaxel in NCI-H460 and HCC1806 cells. TAS-119 clearly enhanced antitumor efficacy of docetaxel in NCI-H460-luc and paclitaxel in HCC-1806-luc nude mice with sc tumors. In the lung orthotopic models, TAS-119 with docetaxel improved median survival, with significant benefit of the combination (155% ILS) over docetaxel alone (67% ILS). Conclusions: TAS-119 has a potential as an enhancer of taxane therapy in NSCLC and TNBC cells both in vitro and in vivo.

Current therapeutic strategies for primary breast cancer aim to provide improvements in outcome with minimal toxicity to the patient. However, annual relapse rates of up to 12 to 13% during the first 10 years after treatment are seen, and although toxicity has been reduced, it remains a problem in a patient population that is largely asymptomatic. Thus, there is a clear need for more effective therapies. Amplification/overexpression of the human epidermal growth factor receptor-2 (HER2) is an early event in the development of a significant proportion of breast tumors. This abnormality has been shown to have a detrimental effect on prognosis, may predict the outcome of therapies such as tamoxifen and anthracyclines, and provides a target for the novel therapy, Herceptin. Herceptin is effective and well tolerated in the metastatic setting, making it an ideal candidate for use in adjuvant breast cancer therapy. This has led to the design of a number of trials that aim to provide conclusive evidence as rapidly as possible that Herceptin is well tolerated and effective in the adjuvant setting while also addressing the question of which regimen provides greatest benefit. This review describes these trials and explains how differences in practice between North America and Europe have influenced trial design.

Preclinical and phase I/II studies of Herceptin demonstrated a dose-related, non-linear pharmacokinetic profile. The results of a dose-finding study supported a regimen comprising an initial intravenous (i.v.) dose of 4 mg/kg with subsequent weekly doses of 2 mg/kg. However, pharmacokinetic and safety data suggested that increased dose and reduced frequency of Herceptin administration are feasible. In addition, evidence shows that Herceptin plus paclitaxel has additive antitumor efficacy in vitro, and this regimen produces significant clinical benefits. These observations form the rationale for conducting a phase I/II trial of Herceptin (8 mg/kg initial dose, 6 mg/kg maintenance dose) plus paclitaxel, both given 3-weekly. Preliminary results are promising, with serum trough Herceptin concentrations being similar and AUC being greater than those observed with weekly Herceptin plus 3-weekly paclitaxel. Two further trials are proposed: 3-weekly Herceptin as first-line monotherapy of metastatic breast cancer; and 3-weekly Herceptin with the oral 5-fluorouracil derivative, Xeloda (capecitabine). Subcutaneous (s.c.) administration of Herceptin would further simplify administration and studies are also underway to clinically evaluate s.c. administration of Herceptin in combination with paclitaxel. With the recent development of oral taxanes, it is predicted that combinations including an oral taxane, Xeloda and either 3-weekly or possibly s.c. Herceptin may become future therapies for breast cancer patients.

The purpose of this study was to evaluate the efficacy and the toxicity of mitomycin, ifosfamide, and cisplatin in patients with recurrent carcinoma of the cervix. Between July 1992 and March 1995, 20 patients with recurrent cervical cancer were enrolled in this study. No patients had received prior chemotherapy for metastatic disease, except some were exposed to cisplatin as a radiosensitizer at the time of their diagnosis. Mitomycin-C 6 mg/m2, ifosfamide 3 g/m2, and cisplatin 50 mg/m2 were given intravenously every 3 weeks. All patients were assessible for response and toxicity, and none was lost to follow-up. All patients except one had squamous cell carcinoma. The overall response rate was 45% (2 complete remissions and 7 partial remissions). The mean response duration was 35 months, and the median survival from treatment for the whole group was 14 months. Fifteen percent of all cycles produced grade 3 or 4 myelosuppression, and the main nonhematologic toxicity was nausea and vomiting, which was reported in 11.5% of all cycles. One death occurred secondary to chemotherapy-induced septicemia. Three patients are still alive, two with a complete response and one with a partial response. In conclusion, mitomycin, ifosfamide, and cisplatin have a good activity in recurrent carcinoma of the cervix and are comparable to other combination chemotherapy.

Human brain and colon tumor cell lines SF-188 (Mer+) and WiDR (Mer+), which express the DNA repair protein O6-methylguanine-DNA methyl transferase (MGMT), were 3- to 30-fold less sensitive to temozolomide, mitozolomide, and N, N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) than the MGMT-deficient tumor cells SF-126 (Mer-) and BE (Mer-). This differential sensitivity was not observed when these cells were exposed to the novel tetrazepinones PYRZ, NIME, QUINCL, and PYRCL, which contain, like temozolomide and mitozolomide, a ureido-triazene moiety. Flow cytometric studies revealed that temozolomide induced G2-M arrest in the Mer- cells, but exerted a minor effect on the cycle of the Mer+ cells. Similarly, mitozolomide (25-100 microM) induced a stronger S-phase arrest in the SF-126 cells than in the SF-188 cells. In the same dose range (25-100) BCNU induced a significant cell cycle accumulation in G22-M in the SF-126 cells but little in the SF-188 cell line. In contrast, the cell cycle effects of the tetrazepinones were independent of the cell phenotypes. When O6-benzylguanine (O6-BG) was used to deplete MGMT activity in the SF brain tumor cell lines, significant potentiation of temozolomide (67-fold), mitozolomide (7-fold), and BCNU (3-fold) was observed in the SF-188 cell line. By contrast, O6-BG did not potentiate PYRZ, PYRCL, QUINCL, and NIME. Moreover, an MGMT inhibitory assay showed that all the tetrazepinones were capable of inactivating MGMT in the SF-188 cell line, the strongest inhibitor being PYRCL. The results suggest that, unlike temozolomide, mitozolomide, and BCNU, the cytotoxicity of the tetrazepinones does not correlate with the alkylation of the O6 position of guanine and that the mechanism of MGMT inactivation by tetrazepinones may differ from that of hitherto known inhibitors.

Background Tumor EGFR and KRas mutations are both predictive and prognostic biomarkers in patients with advanced NSCLC. We analyzed the correlation between these biomarkers and treatment outcomes in a phase III trial of 3rd/4th line sorafenib in patients with NSCLC. Methods The global, randomized, placebo-controlled MISSION trial enrolled 703 patients with advanced relapsed/refractory NSCLC of predominantly non-squamous histology. The primary study endpoint was overall survival (OS). EGFR and KRas mutations were analyzed in archival tumor samples and in circulating tumor DNA isolated from plasma. Results Tumor and/or plasma mutation data were available from 347 patients (49%). EGFR and KRas mutations were detected in 89 (26%) and 68 (20%) patients, respectively, and were well balanced between treatment arms. Analysis of the interaction between EGFR mutation status and treatment effect on survival suggested that patients with EGFR mutations (mEGFR) benefitted from sorafenib, while those with wild-type EGFR (wtEGFR) did not (p = 0.023). Median OS was two-fold longer in mEGFR patients receiving sorafenib versus placebo (423 vs 197 days, HR 0.48, p = 0.002). There was no significant difference in OS between patients with wtEGFR receiving sorafenib or placebo (253 vs 256 days, HR 0.92, p = 0.559). An interaction was also seen between EGFR mutation status and the sorafenib effect on PFS (p = 0.015). Patients with mEGFR treated with sorafenib had better outcomes compared to placebo based on Cox regression analysis (HR 0.27, p Conclusion Post-hoc analyses of efficacy outcomes in MISSION suggest that advanced NSCLC patients with EGFR mutations may derive a survival benefit from receiving 3rd/4th line sorafenib. These results must be interpreted with caution due to the small, non-representative nature of the genetic biomarker subpopulation analyzed in this trial. Further prospective investigation may be warranted. Disclosure T.S.K. Mok: Honoraria: AstraZeneca, Roche, Eli Lilly, Merck Serono, Eisai, BMS, BeiGene, AVEO, Pfizer, Taiho, Boehringer Ingelheim, and GSK Biologicals Speaker: Astrazeneca, Roche, Eli Lilly, Boehringer Ingelheim, and Merck Serono Research funding: Astrazeneca. L. Paz-Ares: Dr. Paz-Ares has received honoraria from Bayer HealthCare Pharmaceuticals, Lilly, Roche and Pfizer. Y. Wu: Dr. Wu has received lecture fees from Roche, AstraZeneca, Eli Lilly, and Pfizer. V. Hirsh: Member of the steering committee for the MISSION trial. C. Lathia: Dr. Lathia is an employee of Bayer HealthCare. T.J. Ong: Dr. Ong is an employee of, and owns shares in, Bayer HealthCare. C. Pena: Dr. Pena is an employee of Bayer HealthCare. All other authors have declared no conflicts of interest.

e12019 Background: SU, an oral multitargeted tyrosine kinase inhibitor of VEGFRs, PDGFRs, KIT, FLT3, CSF-1R and RET has activity in heavily pretreated pts with MBC. The aromatase inhibitor (AI), E, has proven 1st-line activity that compares favorably with tamoxifen in pts with HR+ BC in the adjuvant setting (Jones et al. 2008). Combining agents that target different signaling pathways may have additive/synergistic activity; combining the AI letrozole with the anti-VEGF agent bevacizumab prolonged progression-free survival to >14 mos as 1st-line therapy for HR+ MBC (Dickler et al. 2008). An open-label, phase I, dose-finding study of first-line SU + E was conducted in HR+ MBC pts. Methods: Eligible pts (postmenopausal; female; ≥18 yrs) had an ECOG PS ≤1, LVEF ≥50% and locally recurrent (unresectable) or MBC. Exclusion criteria included HER2+ BC (unless pt had progressed after trastuzumab) and prior treatment in the metastatic setting. Pts received SU 37.5 mg/d + E 25 mg/d on a continuous daily dosing regimen; if dose-limiting toxicities (DLTs) were experienced by >1/6 pts in the first 8 wks then further pts would be enrolled at SU 25 mg/d + E 25 mg/d. Pharmacokinetic (PK) analyses were performed for each drug and the active SU metabolite SU12662. Results: As of December 2008, enrollment was completed (N=6; mean age 59 ± 11 yrs; 50% of pts had ≥3 metastatic sites). No DLTs were observed and no dose reductions were required throughout the treatment period. An overview of key data is shown below. One death occurred on study (non treatment-related Enterobacter sepsis). No treatment-related G4/5 AEs occurred. PK parameter values determined for SU and E suggested increases in the systemic exposure of both drugs when administered concurrently. Conclusions: These data indicate that SU + E was tolerable with manageable toxicities, with increases in PK parameters and a similar AE profile to that of either single agent alone. This combination should be considered in future clinical trials. [Table: see text] [Table: see text]

Background: Trastuzumab, a recombinant monoclonal antibody against the HER-2 receptor, has been shown to significantly improve disease-free and overall survival in women with HER-2 positive early breast cancer when given concurrently with or sequentially after chemotherapy. Materials and Methods: HERA is an ongoing international, multicenter, randomised phase III trial comparing one or two years of adjuvant trastuzumab given every three weeks with observation in 5,102 patients with HER-2 positive either node-negative (tumour size 1.0 cm) or nodepositive early breast cancer who had completed locoregional therapy and had received at least four cycles of standard neoadjuvant or adjuvant chemotherapy. Central confirmation of HER-2 positivity and LVEF 55% after completing chemotherapy and radiotherapy were required prior to randomisation. The second planned interim analysis of the 1 yr vs 2 yr trastuzumab comparison in the HERA trial was conducted on October 20, 2008. The Independent Data Monitoring Committee (IDMC) recommended that the study continue as planned without disclosing data on the 2 yr trastuzumab group. In the observation and the 1 yr trastuzumab groups, 827 disease-free survival events (DFS) have been observed at a median follow up of 48.4 months after randomization, and are the subject to the present update. Results: After release of the HERA and the combined B31 and N9831 results in 2005, a protocol amendment allowed for eligible observation patients to receive trastuzumab and data were continuously collected. Out of the 1698 observation patients, there were 885 observation patients who crossed over to trastuzumab. These late starter patients started trastuzumab at a median of 22.8 months (

Abstract #4058 Background: The HER2 receptor is overexpressed in 20-25% of breast cancer patients and is associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody directed against the HER2 receptor, used alone or in combination with chemotherapy, has shown significant clinical benefit in improving survival in metastatic patients, as well as improving survival in early breast cancer. However, resistance to trastuzumab often develops within 1 year of treatment initiation. Recent studies have shown that trastuzumab in combination with pertuzumab or bevacizumab may have clinical benefit in selected HER2 overexpressing breast cancer patients. In the present study, we investigated whether blockade of HER homo- and heterodimer pairs in combination with an anti-VEGF, could more effectively inhibit tumor growth in a HER2 overexpressing breast cancer model. Material and Methods: Nude mice bearing subcutaneous BT474 HER2-R (a resistant derivative cell line) xenograft tumors were treated with trastuzumab (T) (10 or 20 mg/kg), pertuzumab (P) (15 mg/kg once weekly after an initial 30 mg/kg loading dose), bevacizumab (B) (5mg/kg), as single agents or in doublets and triplets combination therapies with T at 10 mg/kg. Endpoints include tumor volume, HER signaling, angiogenic biomarkers and occurrence of metastasis. Results: T (at its highest dose) and B, as single courses, showed significant anti-tumor activity as compared with the non-treated group. P or T (at its lowest dose) were not effective at inhibiting tumor growth. Combined treatment with P and B significantly inhibited tumor growth as compared with either agent alone or T alone. Time to tumor progression (tumors reaching 2.5 times baseline size) was 26 days for T + B & T + P, and 30 days for P+B as compared with 15 for P, 20 for H (20mg/kg) and 25 days for B, as single agents. In the mice treated with the triple combination, tumor volumes decreased after treatment initiation achieving a complete tumor regression on day 47 with no tumor regrowth up to day 80. Preliminary mechanistic studies showed that P and T as single agents or in combination caused a decrease of HER2 phosphorylation and downstream AKT activation. No further decrease of HER2 signaling was observed with the triple combination. Discussion: Our results confirm that inhibiting tumour angiogenesis by targeting VEGF has anti-tumor effects. Time to progression was delayed in mice treated with T+P compared with mice treated with a double dose of T, confirming that increasing the dosage of a single anti-HER agent is not as effective as using a combination regimen of inhibitors with different mechanisms of action. Complete tumor regression was achieved when B was combined with P and T at its lower dose, suggesting crosstalk between the VEGF pathway with both the epidermal growth factor receptor (EGFR) and HER2 pathways. Ongoing experiments, IHC and angiogenic biomarker analysis, will provide additional insight into the mechanism of action of T+P+B in this tumor model. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4058.

A Phase I clinical trial and pharmacological study of nasogastrically administered hexamethylene bisacetamide, a polar-planar compound with in vitro differentiating activity, was conducted in 14 adult patients with refractory cancer. Hexamethylene bisacetamide was administered as a 5% (w/v) solution via a nasogastric or gastrostomy tube every 4 h for 5 days, followed in 21 days by a 5-day continuous i.v. infusion at the same daily dose. Parenteral drug administration was then continued at the same interval in the absence of disease progression or unacceptable toxicity. Three patients each were treated at doses of 12 and 24 g/m2/day, while eight patients received a dose of 30 g/m2/day. Toxicity was comparable for both routes of drug administration at the above doses. Nasogastrically administered hexamethylene bisacetamide was well tolerated at the lower doses, whereas neurotoxicity and nausea and vomiting were the major, but manageable, toxicities at 30 g/m2/day. Metabolic acidosis, renal dysfunction, mucositis, and thrombocytopenia were the other commonly observed drug toxicities at this dose. No objective tumor responses were observed. Hexamethylene bisacetamide was rapidly absorbed from the gastrointestinal tract with a mean measured bioavailability of 99 +/- 15%. Pharmacokinetic parameters for hexamethylene bisacetamide and plasma concentrations of the two major metabolites, N-acetyl-1,6-diaminohexane and 6-acetamidohexanoic acid, were similar for either route of administration in individual patients. Hexamethylene bisacetamide exhibited apparent monoexponential plasma elimination after either nasogastric or parenteral administration with 27 to 60% of the administered dose being excreted in the urine as parent compound. Based on its demonstrated complete bioavailability and tolerability, nasogastric administration of hexamethylene bisacetamide can be directly and safely substituted for the comparable i.v. dose.

743 Background: Prior to trastuzumab (Herceptin; H) as specific therapy for HER2-positive MBC, anthracyclines (A) had established a major role in this aggressive cancer. Slamon et al. demonstrated that adding H to A+Cyclophosphamide (AC) led to a statistically significant survival benefit, despite increased cardiac toxicity, and the combination was not filed for clinical use. This analysis addresses the question: is there greater benefit for pts eligible for AC who cannot be treated with AC+H in offering AC followed by H + taxane upon progression, compared with H + taxane first-line? Preliminary data from M77001 suggested that H should be used upfront. Methods: Results from the 2 randomised pivotal trials were compared: Slamon et al. [H0648g; AC±H and paclitaxel (P)±H] and Marty et al. [M77001; docetaxel (D)±H]. Results: AC+H and AC alone and P+H results refer to pts with IHC 3+ Results are presented in the table. Slamon et al. found a similar outcome in pts treated with AC alone and in pts receiving P+H....

Goal To improve local control by accelerating the delivery of TRT. Design Inoperable and measurable stages II, Ill-A and B NSCLC, KS ≥ 70. Prior weight loss allowed. Treatment cisplatin 100 mg/m2 week 1 and 5, vinorelbine 30 mg/m2 weekly × 5 (15 mg/m2 on week 2 followed by TRT, 30 fractions of 2 Gy in four weeks, once daily weeks 1 and 2, BID weeks 3 and 4 (same biological dose as 30 daily fractions of 2 Gy). Results From 11–92 to 11–94, 42 eligible patients entered and 39 are evaluable. Response rate 46.2% (18/39) after CT and 71.8% (28/39) after TRT. 24 patients have progressed (1st relapse: 6 in RT field, 16 outside RT field [7 in brain], 2 unknown), and 20 have died. Actuarial median survival 12.0 months. ≥ grade III toxicities post TRT in 23.1% (9/39:7 oesophagus [1 grade 5], 1 lung, 1 skin). Median weight loss during treatment was 2.5 kg. Conclusions Cisplatin and vinorelbine is an active induction CT regimen. Accelerated TRT to 60 Gy is well tolerated and may yield better local control than standard TRT.

5-methyltetrahydrohomofolate was developed in the 1970's as an antifolate with the potential to overcome methotrexate resistance. This review summarizes the preclinical and clinical data which have accumulated to date. It is concluded that more recent, better characterized antifolates offer greater potential in achieving the goals for which this drug was introduced.

Enzymuria and beta 2-microglobulinuria are sensitive markers of injury to renal tubular cells. We evaluated these markers in 41 patients with advanced malignant lymphoma who received gallium nitrate administered as a continuous infusion during a recent phase I-II study. In contrast to our findings with cisplatin, we observed no significant increase in enzyme excretion or beta 2-microglobulinuria in these patients even after long-term treatment. Our results indicate that gallium nitrate administered by infusion does not produce cumulative renal tubular toxicity.

4'-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) is a new, second generation platinum analog which had demonstrated in vitro activity in L1210 cell lines resistant to cisplatin and had less nephrotoxicity than did cisplatin in preclinical animal testing. A Phase I trial with this agent has been performed in 45 patients with advanced refractory cancers. Nine dosage levels, ranging from 40 to 800 mg/sq m, were studied. Major toxicities seen were myelosuppression, nephrotoxicity (which was generally mild), nausea and vomiting (which was quantitatively less than that seen with cis-platin), allergic reactions, and a peripheral neuropathy. The dose-limiting toxicity was thrombocytopenia. Pharmacokinetics performed at three dosage levels indicates that 4'-carboxyphthalato-(1,2-diaminocyclohexane)platinum(II) has a long t1/2 of 20 to 30 hr (total platinum) and is only partially excreted in the urine and that a high proportion of the drug is nonfilterable within 30 to 60 min of administration. Therapeutic responses were seen in nasopharyngeal carcinoma, adenocarcinoma of the cervix, and lung and gastric cancer. As a starting dose for Phase II studies, which are planned for patients with ovarian, testicular, lung, gastric, and esophageal cancers, 640 mg/sq m given every 3 to 4 weeks is recommended.

We analyzed the therapeutic effect reported in 187 phase I trials of 54 anticancer drugs introduced into National Cancer Institute-sponsored clinical trial from 1974 to 1982. An additional 12 drugs entering clinical trial prior to 1974 were also examined. Objective responses (partial and complete) were reported in 271 of the 6447 patients (4.2%). Thirty-nine percent of the patients were treated at an initial dose level greater than or equal to the dose ultimately recommended for phase II trials. Patients in phase I trials generally had adequate performance status (Karnofsky scale greater than 80% or Eastern Cooperative Oncology Group 0, 1, or 2). More than 90% of the patients had received prior chemotherapy. We next examined the therapeutic effect observed in phase II trials for each of the drugs. Thirty-eight of the 54 drugs had completed at least one phase II trial. Twenty-one drugs were rated active, and nine were rated inactive. For the drugs entering clinical trial since 1974, the median phase I response rate for those active in phase II was 4.3%, compared to 2.7% for the drugs not found active in phase II. This difference was not statistically significant. The tabulated response rate in phase I trials reported here should be interpreted cautiously. There are many factors which may contribute to significant variability in the reported response rates, including the heterogeneity of patient population with respect to prior therapy and tumor type, lack of requirement for measurable/evaluable disease on entry into many studies, and variations in the rigor with which tumor sites are followed for response. These factors may lead to either an overestimate or underestimate of the true response rate in phase I. Although little therapeutic effect is produced in phase I, we discuss several measures which might increase patient benefit during these trials.

Promising results in the treatment of indolent lymphomas have been reported with the use of 2'deoxycoformycin. This antimetabolite, an adenosine deaminase inhibitor, also shows particular efficacy in hairy cell leukemia. Of 65 patients treated to date, 44 have achieved complete and lasting remission after a limited course of deoxycoformycin. While results are not as striking as those in hairy cell leukemia, deoxycoformycin may also be a valuable adjunct to alkylating agents in the treatment of CLL. The drug also is being studied in the treatment of mycosis fungoides and other lymphoid neoplasms. Side effects in all neoplasms are dose- and schedule-dependent.