Afatinib versus placebo as adjuvant therapy after chemoradiation in a double-blind, phase III study (LUX-Head & Neck 2) in patients with primary unresected, clinically intermediate-to-high-risk head and neck cancer: study protocol for a randomized controlled trial.

BACKGROUND: Over 50% of patients with head and neck squamous cell carcinoma (HNSCC) present with locoregionally advanced disease. Those at intermediate-to-high risk of recurrence after definitive therapy exhibit advanced disease based on tumour size or lymph node involvement, non-oropharynx primary sites, human papillomavirus (HPV)-negative oropharyngeal cancer, or HPV-positive oropharynx cancer with smoking history (>10-pack-years). Non-surgical approaches include concurrent chemoradiotherapy, induction chemotherapy followed by definitive radiotherapy or chemoradiotherapy, or radiotherapy alone. Following locoregional therapies (including surgical salvage of residual cervical nodes), no standard intervention exists. Overexpression of epidermal growth factor receptor (EGFR), an ErbB family member, is associated with poor prognosis in HNSCC. EGFR-targeted cetuximab is the only targeted therapy that impacts overall survival and is approved for HNSCC in the USA or Europe. However, resistance often occurs, and new approaches, such as targeting multiple ErbB family members, may be required. Afatinib, an irreversible ErbB family blocker, demonstrated antiproliferative activity in preclinical models and comparable clinical efficacy with cetuximab in a randomized phase II trial in recurrent or metastatic HNSCC. LUX-Head & Neck 2, a phase III study, will assess adjuvant afatinib versus placebo following chemoradiotherapy in primary unresected locoregionally advanced intermediate-to-high-risk HNSCC. METHODS/DESIGN: Patients with primary unresected locoregionally advanced HNSCC, in good clinical condition with unfavourable risk of recurrence, and no evidence of disease after chemoradiotherapy will be randomized 2:1 to oral once-daily afatinib (40 mg starting dose) or placebo. As HPV status will not be determined for eligibility, unfavourable risk is defined as non-oropharynx primary site or oropharynx cancer in patients with a smoking history (>10 pack-years). Treatment will continue for 18 months or until recurrence or unacceptable adverse events occur. The primary endpoint measure is duration of disease-free survival; secondary endpoint measures are disease-free survival rate at 2 years, overall survival, health-related quality of life and safety. DISCUSSION: Given the unmet need in the adjuvant treatment of intermediate-to-high-risk HNSCC patients, it is expected that LUX-Head & Neck 2 will provide new insights into treatment in this setting and might demonstrate the ability of afatinib to significantly improve disease-free survival, compared with placebo. TRIAL REGISTRATION: ClinicalTrials.gov NCT01345669.

Risk factors for young-onset colorectal cancer.

PURPOSE: We investigated risk factors for colorectal cancer in early-onset cancers, to provide quantitative estimates for major selected risk factors. METHODS: We analyzed data from three Italian and Swiss case-control studies conducted between 1985 and 2009, including 329 colorectal cancer cases and 1,361 controls aged ≤45 years. We computed odds ratios (ORs) from unconditional logistic regression models, adjusted for major confounding factors. RESULTS: The OR of young-onset colorectal cancer was 4.50 for family history of colorectal cancer in first-degree relatives, the association being higher in subjects with affected siblings (OR 11.68) than parents (OR 3.75). The ORs of young-onset colorectal cancer were 1.56 for ≥14 drinks/week of alcohol, 1.56 for the highest tertile of processed meat, 0.40 for vegetables, 0.75 for fruit, and 0.78 for fish intake. Among micronutrients, the ORs were 0.52 for β-carotene, 0.68 for vitamin C, 0.38 for vitamin E, and 0.59 for folate. No significant associations emerged for physical activity, overweight, and diabetes. CONCLUSIONS: This study-the largest on young-onset colorectal cancer-confirms that several recognized risk factors for colorectal cancer are also relevant determinants of young-onset colorectal cancer. Family history of colorectal cancer in particular is a stronger risk factor in young subjects, as compared to middle age and elderly ones.

Lack of EGFR-activating mutations in European patients with triple-negative breast cancer could emphasise geographic and ethnic variations in breast cancer mutation profiles.

INTRODUCTION: Triple-negative breast cancers (TNBCs) are characterised by lack of expression of hormone receptors and epidermal growth factor receptor 2 (HER-2). As they frequently express epidermal growth factor receptors (EGFRs), anti-EGFR therapies are currently assessed for this breast cancer subtype as an alternative to treatments that target HER-2 or hormone receptors. Recently, EGFR-activating mutations have been reported in TNBC specimens in an East Asian population. Because variations in the frequency of EGFR-activating mutations in East Asians and other patients with lung cancer have been described, we evaluated the EGFR mutational profile in tumour samples from European patients with TNBC. METHODS: We selected from a DNA tumour bank 229 DNA samples isolated from frozen, histologically proven and macrodissected invasive TNBC specimens from European patients. PCR and high-resolution melting (HRM) analyses were used to detect mutations in exons 19 and 21 of EGFR. The results were then confirmed by bidirectional sequencing of all samples. RESULTS: HRM analysis allowed the detection of three EGFR exon 21 mutations, but no exon 19 mutations. There was 100% concordance between the HRM and sequencing results. The three patients with EGFR exon 21 abnormal HRM profiles harboured the rare R836R SNP, but no EGFR-activating mutation was identified. CONCLUSIONS: This study highlights variations in the prevalence of EGFR mutations in TNBC. These variations have crucial implications for the design of clinical trials involving anti-EGFR treatments in TNBC and for identifying the potential target population.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

The HER2 amplicon in breast cancer: Topoisomerase IIA and beyond

HER2 gene amplification is observed in about 15% of breast cancers. The subgroup of HER2-positive breast cancers appears to be heterogeneous and presents complex patterns of gene amplification at the locus on chromosome 17q12-21. The molecular variations within the chromosome 17q amplicon and their clinical implications remain largely unknown. Besides the well-known TOP2A gene encoding Topoisomerase IIA, other genes might also be amplified and could play functional roles in breast cancer development and progression. This review will focus on the current knowledge concerning the HER2 amplicon heterogeneity, its clinical and biological impact and the pitfalls associated with the evaluation of gene amplifications at this locus, with particular attention to TOP2A and the link between TOP2A and anthracycline benefit. In addition it will discuss the clinical and biological implications of the amplification of ten other genes at this locus (MED1, STARD3, GRB7, THRA, RARA, IGFPB4, CCR7, KRT20, KRT19 and GAST) in breast cancer.

Characterization of the effect of mammary gland irridiation on experimental brest cancer progression and analysis of the implicated mechanisms

SUMMARY Radiotherapy is commonly and efficiently used to treat solid cancer in the clinic. Experimental evidence however suggests that radiation can promote tumor progression by inducing chronic modifications of the tumor microenvironment. Clinically, these observations are highly relevant to aggressive tumoral lesions relapsing after radiation therapy, a leading cause of patients' death. The investigation and understanding of the biological mechanisms implicated in the malignant progression of post-radiation relapses are therefore of major importance. Here we used a syngeneic (immunocompetent) breast cancer orthotopic xenograft model, to show that local irradiation of the mammary gland promotes the appearance of an invasive and metastatic tumor phenotype. Previous studies in our laboratory revealed that inhibition of tumor-induced angiogenesis and consequent increase in tumor hypoxia promotes metastasis formation through the activation of pro-invasive programs in the tumor cells. Our results extend these observations suggesting that mammary gland irradiation induces the recruitment of CD11b+ cells to both the primary tumor and the lungs at pre-metastatic stages through the hypoxia-dependent induction of Kit-ligand (KITL) expression in primary tumors. Abrogation of KITL expression in tumor cells prevented CD11 b+ cells accumulation in both the primary tumor and lungs and significantly reduced metastases of tumors growing in irradiated mammary gland. Importantly, irradiated mammary gland enhanced tumor-induced mobilization of circulating CD11b+cKit+ myelomonocytic cells through a HIF1- and KITL-dependent process. By cell transfer experiments, mobilized circulating CD11b+cKit+ cells were shown to supply both tumor- and lungs infiltrating CD11b+ cells. Using a blocking antibody against cKit (the KITL receptor), the mobilization of CD11b+cKit+ ceils was prevented as well as lung metastases derived from tumors growing in irradiated mammary gland. Taken together, these results indicate that tumors growing in a pre-irradiated mammary gland partially promote their malignant progression through the distant mobilization of circulating myelomonocytic precursor cells. They identify KITL inhibition and/or cKit receptor neutralization as potentially promising therapeutic approaches for post-radiation relapses. RESUME La radiothérapie est largement utilisée comme traitement de choix de nombreux types de cancers. L'agressivité des récidives tumorales observée en clinique après radiothérapie suggère cependant que le recours à l'irradiation pourrait dans certains cas accélérer la progression tumorale. De récents travaux expérimentaux ont en effet permis d'appuyer cette hypothèse, en montrant notamment l'effet néfaste des modifications chroniques de l'environnement induites par l'irradiation sur la progression tumorale. A l'aide d'un modèle murin syngénique orthotopique de cancer de sein, nous avons pu montrer que l'irradiation locale de la glande mammaire facilite l'invasion et la dissémination métastatique des cellules tumorales en favorisant le recrutement de cellules myéloïdes CD11 b+ vers la tumeur primaire et les poumons à un stade pré-métastatique. Comme mécanisme impliqué dans le recrutement des cellules CD11b+, nous avons pu observer après irradiation locale de la glande mammaire une expression augmentée de Kit-ligand (KITL) dans la tumeur (induite par l'hypoxie) ainsi que la mobilisation de cellules myéloïdes circulantes exprimant le récepteur cKit et précurseurs des cellules CD11b+ infiltrant la tumeur et les poumons. En empêchant la mobilisation par la tumeur de cellules circulantes cKit+ par des approches à la fois génétique et pharmacologique nous avons pu prévenir l'accumulation de cellules myéloïdes CD11 b+ dans la tumeur primaire et les poumons ainsi que la dissémination métastatique induites par' l'irradiation de la glande mammaire. De façon générale, ces résultats montrent que la progression agressive des tumeurs qui se développent dans un environnement irradié repose à la fois sur l'expression tumorale de KITL et la mobilisation de cellules myéloïdes précurseurs cKit*. Ils auront permis d'identifier KITL et/ou cKit comme des cibles thérapeutiques potentielles intéressantes pour le traitement des récidives tumorales après radiothérapie.

Fatigue and weight loss predict survival on circadian chemotherapy for metastatic colorectal cancer.

BACKGROUND: Chemotherapy-induced neutropenia has been associated with prolonged survival selectively in patients on a conventional schedule (combined 5-fluorouracil, leucovorin, and oxaliplatin [FOLFOX2]) but not on a chronomodulated schedule of the same drugs administered at specific circadian times (chronoFLO4). The authors hypothesized that the early occurrence of chemotherapy-induced symptoms correlated with circadian disruption would selectively hinder the efficacy of chronotherapy. METHODS: Fatigue and weight loss (FWL) were considered to be associated with circadian disruption based on previous data. Patients with metastatic colorectal cancer (nâeuro0/00=âeuro0/00543) from an international phase 3 trial comparing FOLFOX2 with chronoFLO4 were categorized into 4 subgroups according to the occurrence of FWL or other clinically relevant toxicities during the initial 2 courses of chemotherapy. Multivariate Cox models were used to assess the role of toxicity on the time to progression (TTP) and overall survival (OS). RESULTS: The proportions of patients in the 4 subgroups were comparable in both treatment arms (Pâeuro0/00=âeuro0/00.77). No toxicity was associated with TTP or OS on FOLFOX2. The median OS on FOLFOX2 ranged from 16.4 (95% confidence limits [CL], 7.2-25.6 months) to 19.8 months (95% CL, 17.7-22.0 months) according to toxicity subgroup (Pâeuro0/00=âeuro0/00.45). Conversely, FWL, but no other toxicity, independently predicted for significantly shorter TTP (Pâeuro0/00<âeuro0/00.0001) and OS (Pâeuro0/00=âeuro0/00.001) on chronoFLO4. The median OS on chronoFLO4 was 13.8 months (95% CL, 10.4-17.2 months) or 21.1 months (95% CL, 19.0-23.1 months) according to presence or absence of chemotherapy-induced FWL, respectively. CONCLUSIONS: Early onset chemotherapy-induced FWL was an independent predictor of poor TTP and OS only on chronotherapy. Dynamic monitoring to detect early chemotherapy-induced circadian disruption could allow the optimization of rapid chronotherapy and concomitant improvements in safety and efficacy.

Changing patterns of cancer incidence in the early- and late-HAART periods: the Swiss HIV Cohort Study.

BACKGROUND: The advent of highly active antiretroviral therapy (HAART) in 1996 led to a decrease in the incidence of Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma (NHL), but not of other cancers, among people with HIV or AIDS (PWHA). It also led to marked increases in their life expectancy. METHODS: We conducted a record-linkage study between the Swiss HIV Cohort Study and nine Swiss cantonal cancer registries. In total, 9429 PWHA provided 20,615, 17,690, and 15,410 person-years in the pre-, early-, and late-HAART periods, respectively. Standardised incidence ratios in PWHA vs the general population, as well as age-standardised, and age-specific incidence rates were computed for different periods. RESULTS: Incidence of KS and NHL decreased by several fold between the pre- and early-HAART periods, and additionally declined from the early- to the late-HAART period. Incidence of cancers of the anus, liver, non-melanomatous skin, and Hodgkin's lymphoma increased in the early- compared with the pre-HAART period, but not during the late-HAART period. The incidence of all non-AIDS-defining cancers (NADCs) combined was similar in all periods, and approximately double that in the general population. CONCLUSIONS: Increases in the incidence of selected NADCs after the introduction of HAART were largely accounted for by the ageing of PWHA.

Survival after lung metastasectomy in colorectal cancer patients with previously resected liver metastases

Objective: Resection of hepatic metastases is indicated in selected Stage IV colorectal cancer (CRC) patients. A minority will eventually develop pulmonary metastases and may be candidates for lung surgery. The aim of this study was to assess clinical outcome, and identify parameters predicting survival after pulmonary metastasectomy, in patients who underwent prior resection of hepatic CRC metastases.Methods: We performed a retrospective analysis of 27 consecutive patients (median age 62 [range 33-75] years) who underwent resection of pulmonary metastases from CRC in two institutions from 1996 to 2009. All patients considered in the analysis had previously undergone colorectal and hepatic surgery with curative intent, and were considered cured in both locations (R0).Results: Median follow-up was 32 (range 3-69) months after resection of lung metastases and 65 (range 19-146) months after resection of primary CRC. Eleven (40?7%) patients had their primary tumors located in the rectum, and 12 (44%) patients presented initially with synchronous liver metastases (stage IV). Median disease-free interval between primary colorectal tumor and development of first metastasis was 6 (range 0-50) months. At the time of last follow-up, seven patients only (26%) were alive without evidence of recurrence. Three- and 5-year overall survival rates after lung surgery were 56% and 39%, respectively. Median survival after pulmonary metastasectomy was 46 months. In multivariate analysis, the number of pulmonary lesions was significantly correlated with survival (log-rank test, p = 0?035).Conclusion: Resection of lung metastases from CRC patients is compatible with prolonged (median = 4 years) survival, even when those patients had undergone prior resection of liver metastases. While prolonged, disease-free survival remains the exception, patients presentin

Exposure to radio-frequency electromagnetic fields from broadcast transmitters and risk of childhood cancer: a census-based cohort study.

We investigated the association between exposure to radio-frequency electromagnetic fields (RF-EMFs) from broadcast transmitters and childhood cancer. First, we conducted a time-to-event analysis including children under age 16 years living in Switzerland on December 5, 2000. Follow-up lasted until December 31, 2008. Second, all children living in Switzerland for some time between 1985 and 2008 were included in an incidence density cohort. RF-EMF exposure from broadcast transmitters was modeled. Based on 997 cancer cases, adjusted hazard ratios in the time-to-event analysis for the highest exposure category (>0.2 V/m) as compared with the reference category (<0.05 V/m) were 1.03 (95% confidence interval (CI): 0.74, 1.43) for all cancers, 0.55 (95% CI: 0.26, 1.19) for childhood leukemia, and 1.68 (95% CI: 0.98, 2.91) for childhood central nervous system (CNS) tumors. Results of the incidence density analysis, based on 4,246 cancer cases, were similar for all types of cancer and leukemia but did not indicate a CNS tumor risk (incidence rate ratio = 1.03, 95% CI: 0.73, 1.46). This large census-based cohort study did not suggest an association between predicted RF-EMF exposure from broadcasting and childhood leukemia. Results for CNS tumors were less consistent, but the most comprehensive analysis did not suggest an association.

Gene expression profiling of rat spermatogonia and Sertoli cells reveals signaling pathways from stem cells to niche and testicular cancer cells to surrounding stroma.

Background: Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells. Results: The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance. Conclusions: Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-enviroment in testicular cancer.

Early stage primary bone lymphoma : a retrospective, multicenter rare cancer network (RCN) study

IntroductionLe lymphome primaire de l'os (LPO) représente moins de 1% de tous les lymphomes malins. Dans cette étude, nous avons évalué le profil de la maladie, les résultats thérapeutiques, et les facteurs pronostiques d'une série consécutive de patients atteints de LPO de stade I et II. Matériel et méthodeDans treize institutions du Réseau des Cancers Rares (Rare Cancer Network), 116 patients ont été traités pour un LPO entre 1987 et 2008, et sont l'objet de cette étude rétrospective. Quatre-vingt-sept patients ont subi une chimioradiothérapie (CXRT) sans (78), ou avec (9) une chirurgie, 15 ont bénéficié de radiothérapie (RT) sans (13), ou avec (2) chirurgie, 14 d'une chimiothérapie (CXT) sans (9), ou avec (5) chirurgie. La dose médiane de RT était de 40 Gy (4-60). Le nombre médian de cycles de CXT était de 6 (2-8). Le suivi médian était de 41 mois (6-242). RésultatsLe taux de réponse global à la fin du traitement était de 91% (74% de réponses complètes et 17% de réponses partielles). Une récidive locale ou une progression ont été observées chez 12 (10%) patients et une récidive systémique chez 17 (15%) patients. La survie globale, la survie spécifique, et le contrôle local à 5 ans ont été de 76%, 78% et 92%, respectivement. En analyse univariée (log-rank test), les facteurs pronostiques favorables pour la survie globale et la survie spécifique étaient: un indice pronostique international (IPI) inférieur ou égale à 1 (P = 0.009), un grade histologique élevé (P = 0.04), une CXRT (P = 0.05), une CXT (P = 0.0004), une réponse complète (P <0.0001), et une dose de supérieure à 40 Gy (p = 0.005). Concernant le contrôle local, seules la rémission complète et stade I ont été des facteurs favorables. En analyse multivariée, le score IPI, la dose de RT, la rémission complète, et la CXT ont influencé le résultat de façon indépendante en ce qui concerne la survie globale et la survie spécifique. La rémission complète a été le seul facteur prédictif pour le contrôle local. ConclusionCette étude multicentrique rétrospective confirme le bon pronostic du LPO de stade précoce traité par une combinaison de chimio-radiothérapie. Une dose de suffisant de radiothérapie et un nombre adéquat de cycles de chimiothérapie ont été suivis des résultats les plus favorables.

Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype.

The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.

European cancer mortality predictions for the year 2012.

BACKGROUND: Estimating current cancer mortality figures is important for defining priorities for prevention and treatment.Materials and methods:Using logarithmic Poisson count data joinpoint models on mortality and population data from the World Health Organization database, we estimated numbers of deaths and age-standardized rates in 2012 from all cancers and selected cancer sites for the whole European Union (EU) and its six more populated countries. RESULTS: Cancer deaths in the EU in 2012 are estimated to be 1 283 101 (717 398 men and 565 703 women) corresponding to standardized overall cancer death rates of 139/100 000 men and 85/100 000 women. The fall from 2007 was 10% in men and 7% in women. In men, declines are predicted for stomach (-20%), leukemias (-11%), lung and prostate (-10%) and colorectal (-7%) cancers, and for stomach (-23%), leukemias (-12%), uterus and colorectum (-11%) and breast (-9%) in women. Almost stable rates are expected for pancreatic cancer (+2-3%) and increases for female lung cancer (+7%). Younger women show the greatest falls in breast cancer mortality rates in the EU (-17%), and declines are expected in all individual countries, except Poland. CONCLUSION: Apart for lung cancer in women and pancreatic cancer, continuing falls are expected in mortality from major cancers in the EU.

The redox state of cytochrome C modulates resistance to methotrexate in human MCF7 breast cancer cells

Background: Methotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells. Results: Cell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexateinduced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells. Conclusions: We conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.