Effect of MAF amplification on treatment outcomes with adjuvant zoledronic acid in early breast cancer: a secondary analysis of the international, open-label, randomised, controlled, phase 3 AZURE (BIG 01/04) trial.

BACKGROUND: Adjuvant use of bisphosphonates can reduce the incidence of bone metastases in early breast cancer. Recurrence and survival seem to be improved only in postmenopausal patients, but the underlying mechanisms remain unclear. We investigated whether MAF amplification (a biomarker for bone metastasis) in primary tumours could predict the treatment outcomes of adjuvant zoledronic acid. METHODS: The study population included patients enrolled in the international, open-label, randomised, controlled, phase 3 AZURE trial at eligible UK sites who had stage II or III breast cancer and who gave consent for use of their primary tumour samples. Patients were randomly assigned (1:1) to receive standard adjuvant systemic therapy alone (control group) or with zoledronic acid every 3-4 weeks for six doses, then every 3-6 months until the end of 5 years. Minimisation took into account the number of involved axillary lymph nodes, clinical tumour stage, oestrogen-receptor status, type and timing of systemic therapy, menopausal status, statin use, and treating centre. The primary endpoint was disease-free survival; the secondary endpoint, invasive-disease-free survival, was the primary disease endpoint for the analysis in this report. MAF amplification was assessed by fluorescence in-situ hybridisation of two cores of breast tumour tissue in a microarray, done in a central laboratory by technicians unaware of treatment assignment. We used multivariate analyses to assess disease outcomes by intention to treat. We also assessed interactions between MAF-positive status and menopausal status on efficacy of zoledronic acid. The AZURE trial is registered with the International Standard Randomised Controlled Trial Registry, number ISRCTN79831382. FINDINGS: 1739 AZURE patients contributed primary tumour samples, of whom 865 (50%) had two assessable cores (445 in the control groups and 420 in the zoledronic acid group). 184 (21%) tumours were MAF positive (85 in the control groups and 99 in the zoledronic acid group) and the remaining tumours were MAF negative. At a median follow-up of 84·6 months (IQR 72·0-95·8), MAF status was not prognostic for invasive-disease-free survival in the control group (MAF-positive vs MAF-negative: hazard ratio [HR] 0·92, 95% CI 0·59-1·41), but was in the zoledronic acid group (0·52, 0·36-0·75). In patients with MAF-negative tumours, zoledronic acid was associated with higher invasive-disease-free survival than was control treatment (HR 0·74, 95% CI 0·56-0·98), but not in patients who had MAF-positive tumours. Additionally, among 121 patients not postmenopausal at randomisation with MAF-positive tumours, zoledronic acid was associated with lower invasive-disease-free survival (HR 2·47, 95% CI 1·23-4·97) and overall survival (2·27, 95% CI 1·04-4·93) than control treatment. INTERPRETATION: MAF status can predict likelihood of benefit from adjuvant zoledronic acid and merits further investigation as a potential companion diagnostic. FUNDING: Novartis Global and Inbiomotion.

ERCC5/XPG, ERCC1, and BRCA1 gene status and clinical benefit of trabectedin in patients with soft tissue sarcoma.

BACKGROUND: The objective of this study was to determine whether specific single nucleotide polymorphisms (SNPs) from nucleotide excision repair (NER) and homologous recombination (HR) DNA repair pathways are associated with sensitivity to trabectedin in patients with soft tissue sarcoma (STS). METHODS: The authors analyzed excision repair cross-complementation group 5/xeroderma pigmentosum group G (ERCC5/XPG) (NER), excision repair cross-complementation group 1 (ERCC1) (NER), and breast cancer 1 (BRCA1) (HR) SNPs and messenger RNA expression levels in tumor specimens from 113 patients with advanced STS who were enrolled in previously published phase 2 trials or in a compassionate-use program. The 6-month progression-free rate (PFR), progression-free survival (PFS), and overall survival (OS) were analyzed according to ERCC5, ERCC1, and BRCA1 status using log-rank tests. RESULTS: High expression of the common allele (aspartic acid at codon 1104) of ERCC5, high expression of ERCC1, and BRCA1 haplotype were associated significantly with improved PFR, PFS, and OS. The ERCC1 thymine-to-cytosine (T→C) SNP at codon 19007 and BRCA1 expression were not associated with outcome. On univariate analysis, tumor histology, favorable NER status (high expression of common ERCC5 and/or high ERCC1 expression status), and favorable BRCA1 haplotype (at least 1 triple-adenine plus guanine [AAAG] allele) were the sole variables associated significantly with PFS and OS. CONCLUSIONS: In the current study, ERCC5, ERCC1, and BRCA1 status represented a potential DNA repair signature that could be used for the prediction of clinical response to trabectedin in patients with STS.

Circulating tumor cells predict progression free survival and overall survival in patients with relapsed/recurrent advanced ovarian cancer.

OBJECTIVE: Serial circulating tumor cell (CTC) counts have demonstrated predictive and prognostic value in patients with metastatic breast, colorectal, and prostate cancer. In a phase III study of pegylated liposomal doxorubicin (PLD) with trabectedin vs. PLD for relapsed ovarian cancer, we evaluated the correlation, if any, between numbers of CTCs and progression free survival, (PFS) and overall survival (OS). METHODS: CTCs were isolated from peripheral blood (10 mL) using the CellSearch system and reagents (Veridex). A CTC is defined as EpCAM+, cytokeratin+, CD45-, and is positive for the nuclear stain DAPI. The normal reference range for CellSearch is <2 CTC/7.5 mL of blood. Hazard ratios adjusted for known prognostic factors were estimated by Cox regression. RESULTS: Two-hundred sixteen patients had baseline CTC measurements of which 111 (51.4%) were randomized to the trabectedin+PLD arm; 143/216 patients (66.2%) were platinum-sensitive. Thirty-one of 216 patients (14.4%) had 2 or more CTCs detected prior to the start of therapy (range 2-566). Univariate Cox regression analyses indicated that patients with ≥2 CTCs prior to therapy had 1.89- (p=0.003) and 2.06-fold (p=0.003) higher risk for progression and death respectively. Multivariate analyses that include baseline CA-125, platinum sensitivity status, largest diameter lesion, number of tumor lesions, ECOG PS, and tumor grade show that patients with elevated baseline CTC had 1.58- (p=0.058) and 1.54-fold (p=0.096) higher risk for progression and death respectively. CONCLUSIONS: Results from this study indicate that elevated numbers of CTCs impart an unfavorable prognosis for ovarian cancer patients.

MAF Amplification and Adjuvant Clodronate Outcomes in Early-Stage Breast Cancer in NSABP B-34 and Potential Impact on Clinical Practice.

Background: The Adjuvant Zoledronic Acid (ZA) study in early breast cancer (AZURE) showed correlation between a nonamplified MAF gene in the primary tumor and benefit from adjuvant ZA. Adverse ZA outcomes occurred in MAF-amplified patients. NSABP B-34 is a validation study. Methods: A retrospective analysis of MAF gene status in NSABP B-34 was performed. Eligible patients were randomly assigned to standard adjuvant systemic treatment plus 3 years oral clodronate (1600 mg/daily) or placebo. Tumors were tested for MAF gene amplification and analyzed for their relationship to clodronate for disease-free survival (DFS) and overall survival (OS) in MAF nonamplified patients. All statistical tests were 2-sided . Results: MAF status was assessed in 2533 available primary tumor samples from 3311 patients. Of these, 37 withdrew consent; in 77 samples, no tumor was found; 536 assays did not meet quality standards, leaving 1883 (77.8%) evaluable for MAF assay by fluorescence in situ hybridization (947 from placebo and 936 from clodronate arms). At 5 years, in MAF nonamplified patients receiving clodronate, DFS improved by 30% (hazard ratio = 0.70, 95% confidence interval = 0.51 to 0.94; P = .02). OS improved at 5 years (hazard ratio = 0.59, 95% confidence interval = 0.37 to 0.93; P = .02) remaining statistically significant for clodronate throughout study follow-up. Conversely, adjuvant clodronate in women with MAF-amplified tumors was not associated with benefit but rather possible harm in some subgroups. Association between MAF status and menopausal status was not seen. Conclusions: Nonamplified MAF showed statistically significant benefits (DFS and OS) with oral clodronate, supporting validation of the AZURE study.

Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin.

Trabectedin (Yondelis; ET-743) is a potent anticancer drug that binds to DNA by forming a covalent bond with a guanine in one strand and one or more hydrogen bonds with the opposite strand. Using a fluorescence-based melting assay, we show that one single trabectedin-DNA adduct increases the thermal stability of the double helix by >20 degrees C. As deduced from the analysis of phosphorylated H2AX and Rad51 foci, we observed that clinically relevant doses of trabectedin induce the formation of DNA double-strand breaks in human cells and activate homologous recombination repair in a manner similar to that evoked by the DNA interstrand cross-linking agent mitomycin C (MMC). Because one important characteristic of this drug is its marked cytotoxicity on cells lacking a functional Fanconi anemia (FA) pathway, we compared the response of different subtypes of FA cells to MMC and trabectedin. Our data clearly show that human cells with mutations in FANCA, FANCC, FANCF, FANCG, or FANCD1 genes are highly sensitive to both MMC and trabectedin. However, in marked contrast to MMC, trabectedin does not induce any significant accumulation of FA cells in G2-M. The critical relevance of FA proteins in the response of human cells to trabectedin reported herein, together with observations showing the role of the FA pathway in cancer suppression, strongly suggest that screening for mutations in FA genes may facilitate the identification of tumors displaying enhanced sensitivity to this novel anticancer drug.

Adding pharmacogenomics to the development of new marine-derived anticancer agents.

Nature has always been a highly productive tool in the development of anticancer therapies. Renewed interest in the potential of this tool has recently been sparked by the realization that the marine ecosystem can be used for the discovery and development of new compounds with clinical potential in advanced resistant tumors. These compounds can be incorporated into combination approaches in a chronic therapy scenario. Our marine anticancer program is using the sea to develop new agents with activity in resistant solid tumors and to identify new cellular targets for therapeutic intervention. This review describes the integration of different pharmacogenomic tools in the development of Yondelis, Aplidin and Kahalalide F, three marine-derived compounds currently in Phase II or III development. Our results are reinforcing the targeted selectivity of these agents and opening the gates for customized therapies in cancer patients in the near future.

Analysis of DNA repair-related genes in breast cancer reveals CUL4A ubiquitin ligase as a novel biomarker of trabectedin response.

Trabectedin is more active in nucleotide excision repair (NER)-efficient and homologous recombination repair (HRR)-deficient cells. As up to 25% of sporadic breast tumors present somatic inactivation of the HRR pathway (BRCAness phenotype), we sought to characterize trabectedin effect in BRCA1-proficient and BRCA1-null breast cancer cell lines. We evaluated whether HRR and NER gene expression correlates with trabectedin sensitivity and explored the response predictive value of the CUL4A ubiquitin ligase, which ubiquitinates NER pathway members. We characterized trabectedin cytotoxicity, cell-cycle effects, and BRCA1, BRCA2, XRCC3, XPG, ERCC1, and CUL4A expression in 10 breast cancer cell lines. Gene expression and trabectedin sensitivity association were determined in cell lines. Survival assays after trabectedin treatment were conducted in CUL4A-silenced BRCA1-proficient and -deficient cells. Because of limited phase II clinical trials evaluating trabectedin efficacy in patients with breast cancer, we assessed CUL4A immunohistochemical staining in a retrospective series of 118 sarcomas from trabectedin-treated patients to validate in vivo our in vitro observations. In cell lines, greater trabectedin sensitivity was associated with higher CUL4A expression and lower BRCA1/ERCC5, BRCA1/CUL4A, and XRCC3/CUL4A expression ratios. In agreement, BRCA1-deficient CUL4A-knockdown cells presented higher cell survival after trabectedin exposure than did scramble control cells. Lack of effect in BRCA1-proficient cells suggests that HRR impairment is key in CUL4A-mediated trabectedin sensitivity. High CUL4A expression in nontranslocation-related patients with sarcoma predicted improved progression-free survival [PFS; HR, 0.37; 95% confidence interval (CI), 0.20-0.68, P = 0.001] and overall survival (OS; HR, 0.44; 95% CI, 0.21-0.93, P = 0.026). Our observations support the notion of greater trabectedin activity in tumors exhibiting BRCAness and reveal CUL4A as a potential biomarker for definition of trabectedin target patients.

Novel models of myxoid liposarcoma xenografts mimicking the biological and pharmacologic features of human tumors.

PURPOSE: Myxoid liposarcoma is a common subtype of liposarcoma. It is associated in more than 90% of cases with the chromosomal translocation t(12;16)(q13;p11) leading to the fusion FUS-CHOP gene that is responsible for the oncogenic transformation of preadipocytes. Recently the marine natural product trabectedin has shown highly selective activity for myxoid liposarcoma, even in the most aggressive round-cell subtype. EXPERIMENTAL DESIGN: Fragments of 17 sarcomas were transplanted s.c. in female athymic NCr-nu/nu mice. Xenografts were established and characterized by morphology, fluorescence in situ hybridization analysis for the translocation and reverse transcriptase-PCR analysis for fusion transcripts. Trabectedin was injected i.v. RESULTS: Seven of 17 tumors grew as continuous xenografts, five of them being myxoid liposarcoma of the round-cell subtype. The chromosomal rearrangement and fusion transcripts in different passages were the same as in the human tumors from which they were derived. The responsiveness to trabectedin in type II myxoid liposarcoma xenografts was as high as in patients. The pathologic response was associated with the presence of the FUS-CHOP fusion gene, indicating that the drug does not totally eradicate the disease. Type III myxoid liposarcoma xenografts seemed much less sensitive to trabectedin, confirming previous clinical observations. CONCLUSIONS: This study reports for the first time the characterization of human myxoid liposarcoma xenografts that adequately mimic the biological and pharmacologic features of the human tumor. These models offer a useful tool for investigating the mechanism of selectivity of trabectedin, testing new combinations with this drug and evaluating novel therapies for myxoid liposarcoma.

Antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators.

In vitro radiosensitisation by trabectedin in human cancer cell lines.

PURPOSE: To examine the potential radiosensitising properties of trabectedin (ET-743, Yondelis). METHODS AND MATERIALS: In vitro chemosensitivity was assessed in four tumour cell lines (DU145, HeLa, HT29, HOP62) by the crystal violet method. IC10s and IC50s were established for 1-h, 24-h and 7-day (continuous) exposure times. Radiosensitisation was evaluated by conventional colony assay. BrdUrd DNA-labelling and flow cytometry were used to analyse cell cycle kinetics. The rate of apoptotic induction was assed by annexyn-V labelling. RESULTS: Mean IC50s were 18.8 nM (10.5 - 30), 2.5 nM (1.5 - 5) and 0.25 nM (0.2-0.8) for 1 h, 24 h and continuous exposure times, respectively. HT29 and HOP62 were the most sensitive cells lines to trabectedin. Radiosensitisation was observed in DU145 and HeLa cells with a dose enhancement factor (DEF) of 1.92 and 1.77 at IC50 dose level, respectively. Trabectedin induced early S phase arrest in all cell lines studied. CONCLUSIONS: Trabectedin, at pharmacologically appropriated concentrations, harbours a significant in vitro radiosensitising effect and induces cell cycle changes and apoptosis in several human cancer cell lines. Further studies to define the clinical potential of the combination of trabectedin and radiotherapy are needed.

Efficacy of trabectedin (ecteinascidin-743) in advanced pretreated myxoid liposarcomas: a retrospective study.

BACKGROUND: Previous studies have suggested that trabectedin (ecteinascidin-743) could have antitumour activity in soft-tissue sarcoma. We aimed to study the usefulness of trabectedin in the treatment of patients with myxoid liposarcomas, a subtype of liposarcoma that is associated with specific chromosomal translocations t(12;16)(q13;p11) or t(12;22)(q13;q12) that result in the formation of DDIT3-FUS or DDIT3-EWSR1 fusion proteins. METHODS: 51 patients with advanced pretreated myxoid liposarcoma who started treatment with trabectedin between April 4, 2001, and Sept 18, 2006 at five institutions in a compassionate-use programme were analysed retrospectively. Centralised radiological and pathological reviews were done for most patients. Trabectedin was given either as a 24-h continuous infusion or as a 3-h infusion, every 21 days, at 1.1-1.5 mg(2). 558 courses of trabectedin were given in total, with a median of ten courses for each patient (range 1-23). The primary endpoints were response rate and progression-free survival, and the secondary endpoint was overall survival. FINDINGS: According to Response Evaluation Criteria in Solid Tumors (RECIST), after a median follow-up of 14.0 months (IQR 8.7-20.0), two patients had complete responses (CR) and 24 patients had partial responses (PR); the overall response was 51% (95% CI 36-65). Five patients had early progressive disease. In 17 of the 23 patients who achieved PR or CR as defined by RECIST and who had centralised radiological review, tissue-density changes, consisting of a decrease in tumour density on CT scan or a decrease in contrast enhancement on MRI (or both), preceded tumour shrinkage. Median progression-free survival was 14.0 months (13.1-21.0), and progression-free survival at 6 months was 88% (79-95). INTERPRETATION: Trabectedin was associated with antitumour activity in this series of patients with myxoid liposarcoma. The noted patterns of tumour response were such that tissue density changes occurred before tumour shrinkage in several patients. In some patients, tissue-density changes only were seen. Long-lasting tumour control was noted in responsive patients. The compassionate-use programme is still ongoing. This analysis has resulted in the initiation of two prospective studies to assess the role of trabectedin in the treatment of patients with myxoid liposarcoma in preoperative and metastatic settings. Furthermore, the selective mechanism of action for trabectedin in this translocation-related sarcoma is being studied.