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PDF file - 3 MB, Relative expression of DNA repair genes in cell lines

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PDF file - 2 MB, CUL4A mRNA and protein expression in cell lines

PDF file - 117 KB, Supplementary Table 1: Immunohistopathological characteristics of primary breast tumours; Supplementary Table 2: Characteristics of sarcoma patients; Supplementary Table 3: Sequence of qRT-PCR primers and probes and shRNAs used for CUL4A silencing

PDF file - 6 MB, Trabectedin cell cycle effects

Supplementary Fig. S3 from 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°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. [Mol Cancer Ther 2008;7(5):1309–18]

Supplementary Fig. S1 from Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin

Supplementary Fig. S2 from Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin

Supplementary Figure Legends 1-6 from Antitumor and Anti-inflammatory Effects of Trabectedin on Human Myxoid Liposarcoma Cells

Supplementary Figures 1-6 from Antitumor and Anti-inflammatory Effects of Trabectedin on Human Myxoid Liposarcoma Cells

Background The Adjuvant Zoledronic Acid (ZA) study in early breast cancer (AZURE) showed correlation between a non-amplified MAF gene in the primary tumor and benefit from adjuvant zoledronic acid (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 (1600mg/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 non-amplified patients. All statistical tests were 2-sided. Results . MAF status was assessed in 2,533 available primary tumor samples from 3,311 patients. Of these, 37 withdrew consent; in 77 samples no tumor was found; 536 assays did not meet quality standards, leaving 1,883 (77.8%) evaluable for MAF assay by fluorescence in situ hybridization (947 from placebo, and 936 from clodronate arms). At 5 years, in MAF non-amplified patients receiving clodronate, DFS improved by 30% (hazard ratio =0.70, 95% confidence interval = 0.51-0.94, P=0.02). OS improved at 5 years (hazard ratio =0.59, 95% confidence interval = 0.37–0.93, P=0.02) remaining statistically significant for clodronate throughout study follow-up. Conversely, adjuvant clodronate in women with MAF-amplified tumors was not associated with benefit, but possible harm in some subgroups. Association between MAF status and menopausal status was not seen. Conclusions Non-amplified MAF showed statistically significant benefits (DFS and OS) with oral clodronate, supporting validation of the AZURE study.

Background: Meta-analysis of clinical trials has shown that adjuvant bisphosphonates reduce bone metastases and improve survival in postmenopausal (PM) breast cancer and are now recommended for routine clinical use by international guidelines1. However, evaluation of menopause is imprecise and the biological rationale for lack of benefit in premenopausal women unclear. To address this, the biomarker, transcription factor MAF on 16q23 was tested retrospectively in the prospective randomized AZURE trial of standard adjuvant therapy +/- zoledronic acid (ZOL). Initial evaluation indicated that women with MAF negative tumors treated with ZOL had a lower rate of disease relapse irrespective of menopausal status.2 Here we present the long-term findings of this predictive biomarker on 10 year overall survival. Materials and methods: The biomarker analysis was completed on TMAs from primary tumors. Quadruplicate cores of breast tumor tissue were arrayed across replicate TMAs. MAF+ was detected using a validated (MAF/D16Z3) FISH test (Inbiomotion SL, Spain). A central laboratory (Targos, Germany) validated the assay for analytic and diagnostic performance, established acceptance criteria, included appropriate quality controls for each assay, and performed the analyses in a blinded fashion. A copy number cut-off ≥2.5 was preset for MAF+ for both prognostic and predictive testing. Interactions between MAF+ and effects of ZOL on Invasive disease free (IDFS), overall (OS) survival and time to bone metastases by menopausal status were evaluated using a Cox proportional hazards model. Results: 1769 of the 3360 AZURE pts donated primary tumor samples. Median follow-up was 117 (interquartile range 70.4-120) months. 865 pts (49%) had 2 FISH evaluable cores and were included in the analysis. These pts had similar disease and treatment characteristics to the overall study population as well as similar IDFS and OS at 10 years. 184 (21%) had MAF+ tumors and these tumors were more likely to be of higher grade, ER-ve and HER2+. In 680 pts with MAF- tumors, ZOL was associated with improved IDFS (HR=0.75; 95%CI:0.58-0.97, [P=0.02]), reduced relapse in bone (HR=0.65; 95%CI:0.45-0.94, [P=0.022] and, most importantly, better OS (HR=0.69; 95%CI:0.50-0.94, [P=0.019]). In the 185 patients with MAF+ tumors, there was a suggestion of worse outcome (IDFS HR=1.54; 95%CI:0.96-2.47 and OS HR 1.40; 95%CI:0.83-2.33), with a strong interaction between treatment effects and menopausal status. Outcomes in ZOL treated MAF+ pts who were non-PM appeared to be much worse (IDFS HR=2.31; 95%CI:1.18-4.42] and OS HR=2.28; 95%CI:1.07-4.82) due predominantly to an excess of extra-skeletal metastases in ZOL treated patients (HR=4.47; 95%CI:1.66-12.57). Conclusions: Adjuvant ZOL significantly improved disease outcomes in 79% of patients with MAF negative tumors, irrespective of menopausal status and other clinico-pathologic features. Conversely, more extra-skeletal metastases and breast cancer deaths were seen in women with MAF+ tumors who were not PM at the start of treatment. If validated in ongoing studies, the MAF FISH test could provide a clinically useful biomarker for selection of patients for adjuvant bisphosphonate treatment. 1EBCTCG, Lancet 2015; 2Coleman RE et al, Lancet Oncol 2017 Citation Format: Coleman R, Gregory W, Jean-Mairet J, Tercero JC, Torres-Martin J, Gomis R. Long term survival benefits of adjuvant zoledronic acid associated with maf status of primary tumor [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-17-01.

Background: Meta-analysis of clinical trials has shown that adjuvant bisphosphonates reduce bone metastases and improve survival in postmenopausal (PM) pts1. However, we are unable to select pts most likely to benefit. To address this, the recently identified early breast cancer bone relapse biomarker, 16q23(MAF) gain (MAF+)2, was tested retrospectively in the large prospectively randomized AZURE trial3 of standard adjuvant therapy +/- zoledronic acid (ZOL) to determine the prognostic value of MAF and its potential to predict the effects of ZOL on disease outcomes. Materials and methods: All analyses were performed with ethics approval and consent. The biomarker analysis was completed on TMAs from primary tumors. Quadruplicate cores of breast tumor tissue were arrayed across replicate TMAs. MAF+ was detected using a validated (MAF/D16Z3) FISH test (Inbiomotion SL, Barcelona, Spain). A central laboratory (Targos, Kassel, Germany) validated the assay for analytic and diagnostic performance, established acceptance criteria, included appropriate quality controls for each assay, and performed the analyses in a blinded fashion. A copy number cut-off ≥2.5 was preset for MAF+. Invasive disease free (IDFS), overall (OS) survival and time to bone metastases multivariate analyses were performed in control and ZOL pts separately. Subsequently, interactions between MAF+ and effects of ZOL on disease outcomes by menopausal status were evaluated. Results: 1769 of the 3360 AZURE pts donated primary tumor samples. Median follow-up was 84 months. 865 pts (49%) had 2 FISH evaluable cores and were included in the analysis of which 184 (21%) had MAF+ tumors. Tumors that were MAF+ were more likely to be of higher grade, ER-ve and Her2+. In control pts, MAF was not prognostic for IDFS or OS although there were differences in IDFS by menopause (HR for MAF-/MAF+ in PM=0.47 [95%CI 0.25-0.88]; HR in non-PM=1.58 [0.82-3.03], test for interaction (TFI) by menopause P=0.007). In ZOL pts, MAF was prognostic for IDFS (HR=0.52 [0.36-0.75] and OS (HR=0.48 [0.31-0.75]). There were insufficient bone events (19 MAF+, 73 MAF-) in this sample set to reliably assess the impact of MAF+ on relapse in bone. In pts with MAF- tumors, ZOL was associated with improved IDFS (HR=0.74 [0.56-0.98]) and OS (HR=0.78 [0.55-1.10]). However, the effects of ZOL in MAF+ were profoundly influenced by menopausal status with possibly better outcomes in PM women (HR for IDFS=0.74 [0.35-1.58]) but clearly worse IDFS and OS outcomes in ZOL treated MAF+ pts who were non-PM (HR for IDFS 2.46 [1.23-4.92], TFI by treatment P=0.002 and HR for OS=2.27 [1.04-4.93], TFI by treatment P=0.032). The interactions between disease outcomes, ZOL use and menopause were driven largely by an association between MAF+ and an increased risk of extra-skeletal recurrence with the use of ZOL in women who were not PM. Conclusions: Absence of MAF amplification is associated with improved disease outcomes with adjuvant ZOL. However, the use of adjuvant ZOL in women with MAF+ tumors who are not PM at the start of treatment is associated with extraskeletal spread and worse DFS and OS. 1EBCTCG Lancet 2015;386:1353–1361; 2Pavlovic M et al JNCI 2015;107(12):djv256; 3Coleman RE et al Lancet Oncol 2014;15:997-1006. Citation Format: Coleman R, Hall A, Bell R, Cameron D, Marshall H, Jean-Mairet J, Tercero J, Rojo F, Albanell J, Gomis R. Impact of MAF gene amplification on disease recurrence and effects of adjuvant zoledronic acid in early breast cancer [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 P1-09-01.

Adjuvant bisphosphonates improve disease outcomes in postmenopausal early breast cancer (EBC) but the long-term effects are poorly described. The AZURE trial (ISRCTN79831382) was designed to determine whether adjuvant zoledronic acid (ZOL) improves disease outcomes in EBC. Previous analyses showed no effect on overall outcomes but identified benefits in postmenopausal women. Here we present the long-term risks and benefits of adjuvant ZOL with 10-years follow-up. Patients and methods: 3360 patients with stage II/III breast cancer were included in an academic, international, phase III, randomized, open label trial. Patients were followed up on a regular schedule until 10 years. Patients were randomized on a 1:1 basis to standard adjuvant systemic therapy +/− intravenous ZOL 4 mg every 3–4 weeks x6, and then at reduced frequency to complete 5 years treatment. The primary outcome was disease free survival (DFS). Secondary outcomes included invasive DFS (IDFS), overall survival (OS), sites of recurrence, skeletal morbidity and treatment outcomes according to primary tumor amplification of the transcription factor, MAF. Pre-planned subgroup analyses focused on interactions between menopausal status and treatment effects. Results: With a median follow up of 117 months [IQR 70.4–120.4), DFS and IDFS were similar in both arms (HRDFS = 0.94, 95%CI = 0.84–1.06, p = 0.340; HRIDFS = 0.91, 95%CI = 0.82–1.02, p = 0.116). However, outcomes remain improved with ZOL in postmenopausal women (HRDFS = 0.82, 95%CI = 0.67–1.00; HRIDFS = 0.78, 95%CI = 0.64–0.94). In the 79% of tested women with a MAF FISH negative tumor, ZOL improved IDFS (HRIDFS = 0.75, 95%CI = 0.58–0.97) and OS HROS = 0.69, 95%CI = 0.50–0.94), irrespective of menopause. ZOL did not improve disease outcomes in MAF FISH + tumors. Bone metastases as a first DFS recurrence (BDFS) were reduced with ZOL (HRB-DFS = 0.76, 95%CI = 0.63–0.92, p = 0.005). ZOL reduced skeletal morbidity with fewer fractures and skeletal events after disease recurrence. 30 cases of osteonecrosis of the jaw in the ZOL arm (1.8%) have occurred. Conclusions: Disease benefits with adjuvant ZOL in postmenopausal early breast cancer persist at 10 years of follow-up. The biomarker MAF identified a patient subgroup that derived benefit from ZOL irrespective of menopausal status.

Summary 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.

Background and Purpose We have previously shown that cells with a defective Fanconi anaemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anti-cancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand cross link (ICL). Here we expand these observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells. Experimental Approach Initially, the sensitivity of transformed and untransformed cells, deficient or not in one component of the FA pathway, to mitomycin C (MMC) and three DBATs, trabectedin, Zalypsis and PM01183, was assessed. Then, the functional interaction of these drugs with the FA pathway was comparatively investigated. Key Results While untransformed FA-deficient haematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells were hypersensitive to MMC. Furthermore, while MMC always activated the FA pathway, the DBATs inhibited the FA pathway in the cancer cell lines tested and this enhanced their response to MMC. Conclusions and Implications Our data show that although DBATs functionally interact with DNA as do agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors rather than activators. Moreover, this effect was most significant in a variety of cancer cells. These inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of ‘fanconizing’ cancer cells in order to make them more sensitive to other anti-tumour drugs.

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. Mol Cancer Ther; 12(4); 530–41. ©2013 AACR.

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. Clin Cancer Res; 16(20); 4958–67. ©2010 AACR.

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. Cancer Res; 70(6); 2235–44

Summary 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.

Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.

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. Cancer 2011;117:3445-56. (C) 2011 American Cancer Society.

Studies of the kinetics of peptide synthesis catalysed by trypsin derivatives have been carried out. We have studied equilibrium (ECS) and kinetically (KCS) controlled synthesis using benzoyl arginine or benzoyl arginine ethyl ester as acyl donors and various amino acid derivatives as nucleophiles. In both cases, kinetics studies were carried out under conditions of industrial interest: (a) by using very active and very stabilized enzyme derivatives as catalysts, (b) under the extreme experimental conditions necessary for improving the performance of these synthetic processes (high concentrations of apolar organic co-solvents in ECS or high concentrations of ammonium sulfate in KCS), and (c) by using high concentrations of substrates. The hydrophobic adsorption of nucleophile substrates on the active center of the enzyme seems to be the key kinetic step in both of these synthetic processes, as synthetic activity in both ECS and KCS greatly increases with pH and with nucleophile concentration. In ECS, the presence of high concentrations of apolar organic solvents (necessary for achieving important synthetic yields) greatly inhibits the adsorption of nucleophiles, and hence the best performance of these processes would be obtained using a moderate excess of the nucleophile substrate in order to quantitatively convert the acyl donor substrate into synthetic product at the highest reaction rates. In KCS, we found that the use of a ‘salting out’ agent ( e.g. , ammonium sulfate) greatly improves the hydrophobic adsorption of nucleophiles. In addition, the presence of ammonium sulfate, perhaps as a ‘water-ordering agent’, promotes a dramatic reduction in the rate of hydrolysis of the acyl-enzyme complex when the active center of each trypsin molecule is partially blocked by the adsorbed nucleophile at saturating concentrations. Thus synthetic/hydrolytic ratios are dramatically improved by the presence of this salt, and consequently synthetic yields approach 100%. By using these two different synthetic strategies we have been able to quantitatively convert arginyl derivatives into dipeptides under extreme conditions in which our stabilized trypsin derivatives are very active and very stable. We have calculated catalyst productivities of 2 and 300 tons of dipeptide per year per liter of our trypsin catalysts by using ECS or KCS synthetic strategies, respectively.

The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.80, 0.3 ionic strength, 25 degrees C, and 19.5 mg/mL final fibrinogen concentration. At pH 6.80 and 7.80, the reaction was first order, with rate constant k1. At pH 8.80, a first-order reaction of the release of H+ (k1) was followed by a partial rebinding of these in a reaction consecutive to the first one (k2). At each of the above pH values, k1 was proportional to thrombin concentration in the 0.05-3.0 min-1 range investigated. The k1 constants were 0.111 +/- 0.001, 0.250 +/- 0.005, and 0.190 +/- 0.002 min-1 (NIH thrombin units)-1 mL-1 at pH 6.80, 7.80, and 8.80, respectively. Plots of log rate vs log thrombin concentration of these data were linear with slopes close to 1 at all three pH values. The rate of the second reaction (k2) was independent of both the thrombin and the initial fibrinogen concentration. The pH dependence of k1 exhibited a bell-shaped curve that could be resolved into the effect of one group with a pK of 7.27 that increased the rate and another with a pK of 9.22 that decreased the rate. With constant thrombin concentration but varying fibrinogen concentration, plots of 1/k1 vs [fibrinogen] were linear, but the lines did not pass through the origin. From the slope and intercept, kcat and KM of the Michaelis-Menten equation could be calculated. The same parameters were obtained also from initial velocity vs [fibrinogen] plots. Values of kcat were consistent and accurate; those of KM were more scattered. KM was (22.4-34.2) X 10(-6) M at pH 6.80 and approximately 7 X 10(-6) M in the pH 7.26-8.80 range. The latter value, pertaining to the release of H+ ions, is in agreement with values in the literature for KM of the release of fibrinopeptide A by thrombin in the 7.4-8.0 pH range. The value of kcat s-1 (unit of thrombin)-1 mL-1 increases from 1.2 X 10(-10) s-1 unit of thrombin-1 mL-1 at pH 6.80 to 2.46 X 10(-10) at pH 7.80 and then decreases to 2.01 X 10(-10) 10(-1) (units of thrombin)-1 mL-1 at pH 8.80. The kcat values are significantly lower than those in the literature for the release of fibrinopeptide A.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Aplidin (plitidepsin) is a novel anticancer drug isolated from the marine tunicate Aplidium albicans. Aplidin shows potent antitumor activity in preclinical models against a wide variety of human tumors. Aplidin is currently in phase II clinical trials in a variety of solid tumors and hematologic malignancies. Moreover, clinical studies of Aplidin in combination with other agents are ongoing because it generally lacks cross-resistance with other known cytotoxic drugs. The mode of action of Aplidin in tumor cells is only partially understood. Aplidin induces an early oxidative stress response, which results in a rapid and sustained activation of the epidermal growth factor receptor, the nonreceptor protein tyrosine kinase Src, and the serine threonine kinases c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase. Here, we show that sensitivity to Aplidin correlates inversely with the levels of expression of the cyclin-dependent kinase inhibitor p27kip1 (p27) in a panel of low passaged human sarcoma cell lines. Aplidin induces p27 through an oxidation-dependent mechanism and the reduction of p27 levels by specific short hairpin RNA increases Aplidin sensitivity. We confirmed these results in p27 null mouse embryonic fibroblasts corroborating the specificity of the p27 role in Aplidin response because p21waf1 null mouse embryonic fibroblasts do not show this increased sensitivity. We propose a mechanism of action of Aplidin involving p27 and support the analysis of p27 in the response to Aplidin in currently ongoing clinical trials to establish the levels of this protein as response predictor. [Mol Cancer Ther 2007;6(4):1310–6]

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.

Ecteinascidin 743 (ET-743; Yondelis, Trabectedin) is a marine anticancer agent that induces long-lasting objective remissions and tumor control in a subset of patients with pretreated/resistant soft-tissue sarcoma. Drug-induced tumor control is achievable in 22% of such patients, but there is no clear indication of the molecular features correlated with clinical sensitivity/resistance to ET-743. Nine low-passage, soft-tissue sarcoma cell lines, explanted from chemo-naïve patients with different patterns of sensitivity, have been profiled with a cDNA microarray containing 6,700 cancer-related genes. The molecular signature of these cell lines was analyzed at baseline and at four different times after ET-743 exposure. The association of levels of TP53 mutation and TP73 expression with ET-743 sensitivity and cell cycle kinetics after treatment was also analyzed. Gene expression profile analysis revealed up-regulation of 86 genes and down-regulation of 244 genes in response to ET-743. The ET-743 gene expression signature identified a group of genes related with cell cycle control, stress, and DNA-damage response (JUNB, ATF3, CS-1, SAT, GADD45B, and ID2) that were up-regulated in all the cell lines studied. The transcriptional signature 72 hours after ET-743 administration, associated with ET-743 sensitivity, showed a more efficient induction of genes involved in DNA-damage response and apoptosis, such as RAD17, BRCA1, PAR4, CDKN1A, and P53DINP1, in the sensitive cell line group. The transcriptional signature described here may lead to the identification of ET-743 downstream mediators and transcription regulators and the proposal of strategies by which ET-743–sensitive tumors may be identified.

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.

A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).

[Background] Zalypsis® is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing’s sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy., [Methods] Using in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies., [Results] Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/β, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug., [Conclusions] RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials., The AC lab was supported by grants to from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028), Consejeria de Ciencia e Innovacion (CTS-6844), and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rif r ) clinical isolates of Mycobacterium tuberculosis complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoB mutations was evaluated with isolates of the M. tuberculosis complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different rpoB mutations among the Rif r isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rif r allele within each strain, 10 of the 11 Rif r genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosis complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rif r organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods.

The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis ( P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens. © Munksgaard, 1998.

Breast cancer is a complex and heterogeneous disease and one of the most frequent causes of cancer deaths in developed countries. DNA copy number alterations occur frequently in breast tumours. Regions of DNA amplification are of major interest since often contain oncogenes whose increased expression confers tumour cells a selective advantage and contributes to the carcinogenesis process. In our laboratory prior analysis of familiar and sporadic breast tumours by using array-CGH identified an amplification event at the 13q34 region with an overall frequency of 4.5% that increased to 8.1% in BRCA1-associated tumours. Comprehensive characterization of the amplicon allowed us to define CUL4A as one of the most likely putative oncogenes. CUL4A is an E3 Ubiquitin Ligase which is amplified and/or overexpressed in primary breast tumours and involved in functions such as chromatin regulation, cell cycle regulation or DNA repair through ubiquitylation of NER pathway members. Its deregulation could have implications for oncogenic transformation and treatment response to DNA-damaging agents. One of such compounds is Trabectedin a marine-derived drug that presents antitumor activity in sarcomas and ovarian cancer. To elucidate the possible implication of CUL4A in the initial steps of the tumorigenic process we conducted the stable up-regulation of CUL4A in human mammary epithelial cells (184B5), and in NIH3T3 mouse-derived cell line by lentiviral infection. We also stably silenced CUL4A in the human breast cancer cell lines MDA-MB-157 and HCC1937 that present amplification and/or overexpression of CUL4A. With these modified cell lines we performed functional assays in different in vitro systems such as viability experiments, colony formation both in plastic and in soft agar and 3-D cultures in Matrigel. In order to measure sensitivity to Trabectedin we used silenced CUL4A breast cancer cell lines to perform clonogenic assays. Our preliminary results in CUL4A overexpression in 184B5 model system showed increase in proliferation and colony formation ability. On the other hand, CUL4A silencing in breast cancer cell lines resulted in diminished proliferation and decreased colony-forming abilities in both anchorage dependent and independent conditions. Concerning DNA-repair implication, it seems that high levels of CUL4A could be a good predictor of Trabectedin response. Our results indicate that CUL4A would play a role in promoting oncogenesis and/or in modulating breast cancer progression. Further evaluation of CUL4A amplification/overexpression impact on the maintenance of genome integrity will help elucidate its full potential for therapeutic intervention in breast and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 91. doi:1538-7445.AM2012-91

Trabectedin is approved in Europe for the treatment of advanced soft tissue sarcoma (STS) and provides objective response and disease stabilisation rates ranging from 5%-10% and 30-40%, respectively. Although the precise mechanism of action of trabectedin is not fully elucidated, various in vitro studies have shown that its activity depends, at least in part, on the nucleotide excision repair (NER) status of the cells. Indeed, NER-deficient cell lines are less sensitive to trabectedin than their wild-type counterparts. Our aim was to determine whether the status of ERCC5 (XPG), a 3′-endonuclease which plays a crucial role in the excision of the damaged DNA, was associated with the clinical activity of trabectedin in advanced STS patients. We analyzed both, the single nucleotide polymorphism (Asp1104His, G>C, exon 15) and the expression status (real-time quantitative RT-PCR) of ERCC5 in tumors from a cohort of 119 patients with advanced STS (median age at the start of treatment: 49 years old). All patients included in this retrospective analysis were treated between 1999 and 2007 in phase I-II clinical trials or in the context of a compassionate-use program. Trabectedin was given at different doses (0.5-3 mg/m2) with either a 3-h infusion or a 24-h continuous infusion schedule. The two most frequent histological subtypes were leiomyosarcoma (non-uterine: 21, uterine: 11) and liposarcoma (myxoid round cell: 24, other: 15). Overall, tumour control (CR, PR, and SD ≥ 6 months) was achieved in 42 patients (35%, 95% CI 26-44). The median progression-free survival (PFS) and overall survival (OS) of the entire patient group were 3.3 months (95% CI 2.5-4.1) and 12 months (95% CI 7.8-16.1), respectively. Variant allele carriers for ERCC5 were found in 58 cases: G/C (40; 33%), C/C (17; 15%). ERCC5 mRNA was found to be overexpressed in 48% of analyzed cases. In patients with tumors homozygous for the wild-type allele (Asp), ERRC5 mRNA overexpression was associated with significantly higher median PFS: 17.1 months (95% CI 4.7-29.4) versus 1.8 months (95% CI 1.4-2.2), p=0.002. In the group of patients with tumors heterozygous or homozygous for the variant allele (His), median PFS was poor regardless to the expression status of ERRC5 mRNA: 2.8 months (95% CI 1.3-4.2) versus 3.4 months (95% CI 1.4-5.5), p=0.50. We also compared the effect of wild-type and variant ERCC5 expression in human 94RD27 XPG-deficient fibroblasts (deletion of the last 261 aa) on the sensitivity to trabectedin. Preliminary results show that cells stably expressing wild-type ERCC5 are more sensitive to trabectedin than cells expressing the variant allele of ERCC5. Altogether, our data suggest that overexpression of wild-type ERCC5 might be predictive of clinical benefit from trabectedin in advanced STS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2699.

This work was focused on the identification of predictive biomarkers for response to Zalypsis®, a bis-tetrahydroisoquinoline derivative structurally related to the marine-derived compounds jorumycin and renieramycins. Zalypsis® binds to the minor groove of DNA through its reactive carbinolamine group, establishing covalent bonds with the amino group of selected guanines in the DNA, with preference for the triplets AGG, GGC, AGC, CGG and TGG. This particular molecular interaction of Zalypsis® with the DNA creates the equivalent to a functional interstrand crosslink that lead to a strong inhibition of the early phases of transcription. Zalypsis® showed potent antitumor activity both in vitro and in vivo, in preclinical models, against a wide variety of human tumors and is currently in phase II clinical trials for solid tumors, showing a tolerable toxicologic profile. In order to gain knowledge on the molecular basis of sensitivity/resistance to Zalypsis®, we used a panel of cell lines from untreated sarcoma tumor samples, keeping the cell lines low passaged to avoid extensive genetic alterations as a consequence of genetic instability of the tumor. These cell lines were extensively characterized for molecular markers and for response to chemotherapeutic drugs, including Zalypsis®. When analyzing the correlation of the sensitivity of these sarcoma cell lines to Zalypsis® and the expression of different molecular markers, it was found that, in general, high basal protein expression levels of PDGFR-a were associated with increased resistance to the drug in vitro. However, among the 20 cell lines studied, two of them expressed low levels of PDGFR-a and were quite resistant to the drug. After analysing other markers, it was found that these cell lines expressed constitutively phosphorylated c-kit receptor, which could have accounted for the observed resistance. To validate the predictive value of PDGFR-a in vivo, and due to the fact that the established sarcoma cells were not tumorigenic in mice, we selected a panel of 9 human epithelial tumor cell lines and tested their sensitivity to Zalypsis® in xenograft models, and, in parallel, the expression of PDGFR-a. HGC27, SW1990, Calu-6 and A2780 showed relative high levels of expression of PDGFR-a in vitro, while UM-UC-3, MX1, MDA-MB-231 and HepG2 showed relative low levels. The subset of cells expressing high levels of PDGFR-a was consistently resistant to Zalypsis®. SKOV-3 cells, which expressed low levels of the marker, were also resistant to the drug. However, after analyzing the expression of other molecular markers, we found that these cells expressed high levels of constitutively phosphorylated EGF receptor that, again, could have accounted for the relative resistance to the drug. Altogether, these results suggest that PDGFR-a could be considered as a useful predictive biomarker of response to Zalypsis®. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1685.

Trabectedin is a marine derived antitumoral agent, originally isolated from Ecteinascidia turbinata. It acts by binding to the minor groove of DNA interfering with cell division, transcription, and the DNA repair machinery. Trabectedin was approved by the EMEA as second line therapy for the treatment of advanced soft tissue sarcoma and for ovarian cancer in combination with Doxil. Further Phase II trials with Trabectedin in breast and prostate cancer are underway. We characterized Trabectedin for antitumor efficacy and selectivity in patient-derived tumor xenografts to identify target tumor types for further clinical studies. The compound was tested in 67 tumor xenografts of 15 histo types using an ex vivo clonogenic assay. Pronounced concentration dependent antitumor activity (mean IC70 = 1.3 nM) and selectivity was observed, with sensitive tumor models being on average about 7-fold more sensitive than the average of all tumors tested. Trabectedin was also given to tumor-bearing nude mice at 0.2 and 0.15 mg/kg/d iv once weekly for 3 weeks and showed substantial inhibition of tumor growth at a dose level of 0.2 mg/kg/d in tumors of lung, colon, and breast. The activity data of Trabectedin against 67 tumors in the clonogenic assay were used for further bioinformatic analysis. Subsets of tumors and their corresponding data were randomly split into a training set (n=44) and an independent validation set (n=23). By matching in vitro antitumor efficacy data (IC70) of the tumors with the corresponding gene expression profiles (determined by Affymetrix HG-U133 Plus 2.0 gene chip array), a signature of 19 gene transcripts being specific for the responsiveness towards Trabectedin was determined. The classification border for activity of Trabectedin was IC70 = 0.5 nM. The signature was validated by leave-one-out-cross-validation (LOOCV) on the training set, and prediction of tumors of the independent validation set. In the LOOCV sensitivity or resistance of tumors was predicted correctly in 13/18 (72%) and 26/26 tumors (100%), in the validation set in 4/7 (57%) and 13/16 tumors (81%), respectively. In the validation set, the predicted responders showed a 4.5-fold lower median IC70 compared to the predicted non-responders (p= 0.02). Moreover, the signature was used to predict responsiveness of 173 tumors of the Oncotest xenograft collection with unknown sensitivity to Trabectedin. In this set of tumors, the signature identified sarcoma, leukemia/lymphoma, as well as ovarian, head and neck, small cell lung, mammary and bladder cancer as Trabectedin sensitive tumor types. Experimental testing of the predicted tumors so far confirmed the predictions in 11/13 cases (85%). This study shows the feasibility of combining experimental testing and virtual prediction to identify additional tumor types as candidates for further preclinical and clinical investigations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2598.

Introduction: Trabectedin is a marine alkaloid originally isolated from Ecteinascidia turbinata approved in Europe for the second line of therapy in soft tissue sarcomas (STS). Here we characterize and compare the gene expression profile of 402-91/ET Res liposarcoma cells obtained after continuous exposure to trabectedin in vitro with that of their parental cell line 402-91. Methods: Drug sensitivity was measured by colony assay and Flow Cytometric analysis was used to analyze cell cycle perturbations. Microarray analysis between sensitive and 402-91/ET Res cells was performed using commercially available arrays onto which 44K oligos were pre-spotted. Gene expression analysis was performed with “R” package software. Pathway analysis was performed using Metacore software. qRT-PCR was used for data validation. Statistical analysis was performed using Graphpad software. Results: 402-91/ET Res cells were selected in vitro for their stable resistance to trabectedin by step-wise increase in the drug concentration (25 nM) lasting 14 months. Resistance to Trabectedin was stable over 22 months without compound exposure. Cells were cross resistant to melphalan, but more sensitive to taxanes, vinblastine, temozolomide and UV rays. Resistance was not associated to the expression of MDR proteins as well as different uptake/efflux. Cell cycle analysis revealed that trabectedin is able to induce a G2-M block in the sensitive cell line but not in the resistant one. The expression levels of the FUS-CHOP gene were comparable between the 402-91 and 402-91/ET Res cells but with a different trans-activation ability. Microarray analysis identified 511 probes set able to discriminate between sensitive and resistant cell lines. Pathway analysis revealed that most of the genes altered in the 402-91/ET Res cells were associated with liposarcoma phenotype and functionally involved in the CREB pathway as well as control of apoptosis. Conclusion: Data suggests that in the 402-91/ET Res cells FUS-CHOP trans-activation activity has been impaired. Studies are in progress to address whether these mechanisms are implicated in sensitivity of liposarcoma cell lines to trabectedin. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B64.

10575 Background: To determine the efficacy of rechallenge with trabectedin (T) in patients with myxoid liposarcoma who were responding to T at the time of discontinuation but subsequently developed progressive disease. Methods: Since September 2002, 32 patients with recurrent or advanced myxoid liposarcoma received T at our institution within an expanded access program. We report herein 8 patients who received T for a median of 10 cycles (range 5–15) prior to discontinuation. We used RECIST criteria to classify patient response to therapy. During the initial administration of T, a CR had been observed in 2 patients, a PR in 3 patients, SD in 2 patients, and one had had tumor shrinkage but did not meet RECIST definition of PR. The median PFS was 24 months from treatment start, and 14 months (range 9–27) from treatment-end. In 6 of the 8 patients, the treatment had been discontinued in the absence of any evidence of disease: 4 patients had undergone complete surgical resection (R0) of residual disease and 2 patients had achieved a radiological complete response (CR). The other two patients stopped treatment voluntarily, one with a partial response (PR) and one with stable disease (SD). All these patients resumed treatment at the time of progression. Results: Following rechallenge with T, no PD was seen at first assessment, and no patients had to discontinue or reduce the dose due to toxicity. Response rate according to RECIST was 50%. At a median follow up of 7 months (range 2.7–12), the median TTP has not yet been reached. Treatment is still ongoing in 5 patients. Conclusions: Rechallenge with T can be beneficial in some patients with myxoid liposarcoma. If further data confirm this, treatment of progressing patients previously responding to T may include this option. Furthermore, studies on optimization of treatment duration upfront may be worthwhile. [Table: see text]

10530 Background: To explore the clinical impact of T in U-LM. T has been approved in Europe for second line treatment of advanced soft tissue sarcomas (STS). Efficacy is well established in liposarcoma and leiomyosarcoma. U-LMs display peculiar clinical and genetic features compared to other STS. These differences may be responsible for the sensitivity of this subtype to therapy, thus justifying an evaluation of the activity of T in a relatively homogeneous series of U-LM patients. Methods: From April 2000, 56 patients (pts) with advanced disease, previously exposed to a median of 3 chemotherapy lines (range 1–5), received T within an expanded access programme at two European referral institutions for sarcoma. The clinical records were reviewed focusing on response and treatment outcome. Two pts were excluded from the analysis having received only 1 course of T. Median age was 56 yrs (range 29–73), median number of metastatic sites was 2 (range 1–4), the most frequent metastatic site was lung (88%), 24 patients had a local relapse. Results: A total of 252 courses were delivered (median 3, IQR2–6) and 36% of patients received more than 5 courses of T. Fifty-two patients were evaluable for response. A partial response was observed in 11 patients and stable disease in 15, for a PR rate of 21% and a tumor control rate of 50%. The median progression-free survival was 3.6 months (CI95% 2.6–6.7), with 41% of patients free from progression at 6 months. Conclusions: These results compare favourably with other systemic treatments in advanced U-LMS and support their sensitivity to T. This should prompt further studies to prospectively evaluate the efficacy of T in U-LMS and elucidate possible biological predictive factors (e.g. DNA repair protein expression). [Table: see text]

9522 Background and Methods: The sensitivity to trabectedin (Yondelis, ET-743) in experimental cancer models correlates with functional transcription-coupled nucleotide excision repair (TC-NER) and with deficient homologous recombination repair (HRR) activity. In order to assess the clinical relevance of this observation we have characterized the mRNA expression levels of ERCC1, XPD, BRCA1 and BRCA2 by RT-PCR in historical tumor samples from 92 sarcoma patients (pts) treated with the agent. Results: The overall objective response (CR+PR) rate to Yondelis in this group was 9%, which confirms the previous clinical experience with the compound in unselected, pre-treated patients with sarcoma. The Kaplan-Meier estimates for progression-free survival at 6 months (PFS6) was 23% and for median survival 8 months (mo). 26% of pts were alive at 24 mo. Correlative Study: Pts with high expression levels (> median) of ERCC1 had better PFS6 (32% vs 15%, p = 0.07) and better median survival (12 vs 7 mo) as compared to pts with low expression ( ≤ to median). Pts with low expression levels of BRCA1 had better PFS6 (33 vs 11%, p = 0.02) and superior median survival (15 vs 5 mo, p = 0.0003) than pts with high expression. Expression levels of XPD and BRCA2 (58 pts) had no significant impact on PFS and survival. The analysis of co-expression of ERCC1-BRCA1 identified a highly sensitive group of pts, characterized by high ERCC1 and low BRCA1 expression levels, with a PFS6 of 50% (p=0.0003) and median survival of 20.4 mo (p = 0.0005). A Cox regression analysis demonstrated that ERCC1 (HR=0.52, p = 0.02) and BRCA1 (HR=2.73, p = 0.0006) are independent variables for PFS, while BRCA1 (HR= 2.57, p = 0.0005) is also an independent variable for survival. Conclusion: This exploratory retrospective study supports the hypothesis that the sensitivity to Yondelis in pts with sarcoma correlates with a functional TC-NER and with deficient HRR. Prospective studies in sarcoma and other potentially sensitive tumor types are required to confirm and validate these findings. [Table: see text]

A method for the fractionation of plasmic digests of both fibrinogen and fibrin was developed by taking advantage of the different chromatographic behaviour of fibrinogen and its fragments on immobilized concanavalin A and Lens culinaris agglutinin. Columns with different lectin concentration but with the same total lectin content were tested. Fragment E was retained on all the concanavalin A-Sepharose preparations while fragment D was mostly eluted in the unbound fraction. However, the binding of fragment DD depended on the lectin concentration of the gel. Thus, the percentage of fragment DD specifically bound to concanavalin A-Sepharose increased from 5–10% to 67% as the lectin density of the gel increased from 0.9 to 8.7 mg/ml gel. On the other hand, Lens culinaris agglutinin-Sepharose retained fibrinogen and high molecular weight fragments depending on the lectin concentration of the gel while neither fragment E nor fragment D were bound to any of the columns.