Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients.

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett's esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

Detection of Loss of Heterozygosity in cfDNA of Advanced EGFR- or KRAS-Mutated Non-Small-Cell Lung Cancer Patients.

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.

ERCC1 C8092A (rs3212986) polymorphism as a predictive marker in esophageal cancer patients treated with cisplatin/5-FU-based neoadjuvant therapy.

OBJECTIVE: At present, no consensus exists on the beneficial effect of preoperative cisplatin/5-fluorouracil (5-FU)-based chemotherapy versus primary surgery in the management of patients with esophageal cancer. The aim of this study was to evaluate the impact of some relevant genetic polymorphisms, within drug-related and DNA repair genes, on the clinical outcome of esophageal cancer patients subjected to cisplatin/5-FU-based neoadjuvant treatment. METHODS: DNA from 143 esophageal cancer patients, 63 receiving neoadjuvant therapy and 80 receiving primary surgery, was analyzed for the following polymorphisms: the GSTM1 null, GSTT1 null, and GSTP1 Ile105Val (rs16953) in glutathione S-transferase (GST) family, 2 in thymidylate synthase (TS) gene, and the ERCC1 Asn118Asn (rs11615), ERCC1 C8092A (rs3212986), XPD/ERCC2 Asp312Asn (rs1799793), and XPD/ERCC2 Lys751Gln (rs13181) of the nucleotide excision repair pathway. RESULTS: We found that the ERCC1 rs3212986, although not associated with therapeutic response, is an independent predictive marker of better outcome in a cisplatin/5-FU-based neoadjuvant setting (hazard ratio: 0.38, 95% confidence interval: 0.2-0.73, P=0.008). In contrast, no association with clinical outcome was observed for this polymorphism in the primary surgery group. CONCLUSION: Our study indicates the ERCC1 rs3212986 as a predictive marker in the cisplatin/5-FU-based neoadjuvant setting, and also suggests its use as a marker to select the appropriate therapeutic approach in esophageal cancer patients.

Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells.

Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.

Redox regulation of T-cell turnover by the p13 protein of human T-cell leukemia virus type 1: distinct effects in primary versus transformed cells.

The present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1-infected cells. ROS measurements showed an increasing gradient from resting to mitogen-activated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of "normal" infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host.

Allelic Imbalance Analysis in Liquid Biopsy to Monitor Locally Advanced Esophageal Cancer Patients During Treatment.

Esophageal cancer (EC) is a highly aggressive tumor, and the current monitoring procedures are partially inadequate to evaluate treatment efficacy. The aim of this study was to investigate whether allelic imbalance analysis in liquid biopsy could be used as an additional tool to monitor tumor burden in EC patients. For this purpose, circulating cell-free DNA (cfDNA) from 52 patients with a locally advanced EC, which underwent neoadjuvant treatment and resection, was analyzed. Data from four representative longitudinally followed patients are also reported. Furthermore, 17 DNAs from formalin-fixed paraffin-embedded (FFPE) tumor samples were analyzed and compared to time-matched cfDNAs. To look for allelic imbalance, which is the main genetic alteration in both EC histotypes, we used a panel of five microsatellites (MSs) and three single-nucleotide polymorphisms (SNPs) near genes described as frequently altered. The Fisher exact and Mann-Whitney U tests were used to analyze categorical and continuous data, respectively. The correlation coefficient between cfDNA and FFPE-DNA was calculated with the Pearson's correlation test. We found that the selected tumor-related alterations are present in cfDNA of both adenocarcinoma (EADC) and squamous cell carcinoma (ESCC) with similar frequencies. The only exception were the MSs, one downstream and one upstream, of SMAD4 of which the loss was only observed in EADC (26 vs. 0%, P = 0.018). More interestingly, longitudinal studies disclosed that in patients with disease progression, tumor-related alterations were present in cfDNA before overt clinical or instrumental signs of relapse. In conclusion, our data indicate that the evaluation of tumor-related gene allelic imbalance in cfDNA might be a useful tool to complement the current monitoring procedures for EC patients and to guide their management.

Identification of host variants associated with overall survival of esophageal cancer patients receiving platinum-based therapy.

Aim: Clinical features of esophageal cancer (EC) patients have poor prognostic power. Thus, it is paramount to discover biomarkers that can allow a more accurate survival prediction. Methods: To detect genetic variants associated with survival, DNA from 120 patients treated with cisplatin-based neoadjuvant therapy were genotyped using drug metabolism enzymes and transporters array. Results: We identified two variants: the rs2038067 in PPARD (p = 0.0004) and the rs683369 (F160L) in SLC22A1 (p = 0.001). Their prognostic power was greater than that of clinical stage alone (p = 0.017) and comparable to that of response to neoadjuvant therapy (p = 0.71). Interestingly, the prognostic accuracy of response models increased significantly when genetic variables were included (p = 0.003). Conclusion: Our data, though preliminary, strengthen the potential utility of germline variants for a better-tailored management of EC patients.

HTLV-1 Tax protein cooperates with Ras in protecting cells from apoptosis.

Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) plays a critical role in HTLV-I-correlated diseases through its ability to deregulate the expression of a vast array of cellular genes. We have previously shown that Tax counteracts apoptosis induced by stimuli triggering mitochondria apoptotic pathway, most likely by activating CREB-mediated transcription and affecting the phosphorylation levels of CREB at Ser-133. Here, we report data that indicate the oncoprotein Ras as a possible mediator of Tax-induced apoptosis protection and suggest a possible role of Tax in Ras activation. In addition, using inhibitors of down stream effectors of Ras, we found that ERK signaling is the most relevant for Tax-mediated apoptosis protection. As a whole, our findings provide intriguing evidence of a possible link between Ras signaling and Tax capability to counteract apoptosis and to enhance P-CREB levels, and implicates a potential role for Ras in HTLV-1-induced diseases.

DNA copy number alterations correlate with survival of esophageal adenocarcinoma patients.

Despite recent advances in surgical and multidisciplinary treatment, prognosis for patients with esophageal adenocarcinoma remains poor, and the low prognostic significance of pTNM staging suggests that additional parameters are needed. To identify genomic abnormalities characteristic of esophageal adenocarcinoma, a panel of 33 samples obtained at surgery from previously untreated patients were analyzed by muliplex ligation-dependent probe amplification technique. We detected frequent gains of 6p, 8q, 13q, 17q, 20q, and losses of 4q, 5q, 15q, and 18q. When DNA copy number changes were correlated to clinicopathological features of patients no association was found between the number of chromosomal aberrations and gender, age, tumor grade or pTNM staging. However, interestingly, a significant correlation between patient survival and total number of chromosomal aberrations was found when esophageal adenocarcinoma cases were stratified according to the median of survival (20 months) (P=0.002) or the median of aberrations (12 aberrations) (P=0.014). Evaluation of the distribution of gains and losses at the level of single chromosomes indicated that gains on chromosomes 5, 6, 8, 11, 20 and losses on chromosomes 1, 3, 5, 11, and 18 were significantly different in the two survival groups. Furthermore, when single gene imbalances were analyzed in further details, we found that besides alterations that involve genes shared by both survival groups, a few genes (KIAA0170, EMS1, ABCC4, F3, and MIF) were altered only in samples from patients with poor survival. Thus, we established a good correlation between the total number of chromosomal alterations and survival, suggesting that the estimation of total imbalances might represent an additional indicator of disease outcome. In addition, the finding of alterations specific for the more aggressive esophageal adenocarcinoma subset might represent promising biomarkers to increase the accuracy of clinical outcome prediction.

Association Between ERCC1 rs3212986 and ERCC2/XPD rs1799793 and OS in Patients With Advanced Esophageal Cancer.

Esophageal cancer (EC) is a very aggressive tumor, and no reliable prognostic markers exist especially for resectable advanced neoplasia. The principal aim of this study was to investigate the association of germline polymorphisms in nucleotide excision repair (NER) pathway genes with the overall survival (OS) of patients with advanced EC. As a second aim, we also studied the association of NER gene variants with response to cisplatin-based chemotherapy. Among the EC patients referred to our Institution between 2004 and 2012, we selected a cohort of 180 patients diagnosed with a clinical tumor stage ranging from IIB and IVA. Patients were genotyped for four NER variants, two in the ERCC1 (rs11615 and rs3212986) and two in the ERCC2/XPD (rs1799793 and rs13181) genes. Kaplan-Meier analyses and Cox proportional hazards model were used to evaluate the associations of the selected variants with OS; association with response to neoadjuvant therapy was investigated using logistic regression. Results showed that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 were significantly associated with shorter OS. On the contrary, response association analysis displayed that, while rs11615 and rs3212986 in ERCC1 were associated with response, both ERCC2/XPD variants were not. By creating survival prediction models, we showed that the rs3212986 and the rs1799793 have a better predictability of the tumor stage alone. Furthermore, they were able to improve the power of the clinical model (AUC = 0.660 vs. AUC = 0.548, p = 0.004). In conclusion, our results indicate that the ERCC1 rs3212986 and the ERCC2/XPD rs1799793 could be used as surrogate markers for a better stratification of EC patients with advanced resectable tumor.

Gold(III)-dithiocarbamato complexes induce cancer cell death triggered by thioredoxin redox system inhibition and activation of ERK pathway.

Although gold compounds are now recognized as promising anticancer agents, so far only gold(I) derivatives have been investigated for this purpose, whereas the use of gold(III) complexes has been hampered by their poor stability under physiological conditions. We have therefore carried out studies on selected gold(III) anticancer agents, showing enhanced stability due to the presence of chelating dithiocarbamato ligands. We found that they induce cancer cell death through both apoptotic and nonapoptotic mechanisms. They also inhibit thioredoxin reductase activity, generate free radicals, modify some mitochondrial functions, and increase ERK1/2 phosphorylation. Based on our results, we propose and discuss a working model suggesting that deregulation of the thioredoxin reductase/thioredoxin redox system is a major mechanism involved in the anticancer activity of the investigated gold(III)-dithiocarbamato complexes.

Detection of genetic alterations in cfDNA as a possible strategy to monitor the neoplastic progression of Barrett's esophagus.

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. Despite the low absolute risk of neoplastic progression of BE, probability increases with the diagnosis of dysplasia. For this reason, BE patients undergo an endoscopy-based surveillance that is, however, burdensome for patients, subject to inter-observer subjectivity, and expensive for national health systems. Thus, less invasive and low-cost diagnostic tools are needed. This study is aimed at finding a simple and reliable method to detect in the circulating cell-free DNA (cfDNA) of BE patients evidence of the molecular instability that accompanies BE carcinogenesis. We chose the loss of heterozygosity analysis because chromosomal region gains or losses have been described in BE and esophageal adenocarcinoma. Furthermore, this analysis does not require an a priori knowledge of tumor specific mutations and/or rearrangements. Previous data showed a good consistency between tissue and cfDNA alterations. Here, we report that, in the cfDNA of dysplastic BE patients, the frequency of genetic alterations is statistically higher than that of metaplastic BE patients (P = 0.005). Interestingly, after endoscopic treatment, the alteration frequency dropped, suggesting that cfDNA can also be used to monitor curative effects. Among the used markers, those that map nearby TP53 gene were the most discriminant between metaplastic and dysplastic BE. Furthermore, longitudinal follow-up cases showed that genetic alterations can be found in cfDNA before the appearance of a detectable lesion. Altogether, our data suggest that the use of liquid biopsy could become a minimally invasive diagnostic tool to implement BE patient monitoring.

Predictive role of host constitutive variants in neoadjuvant therapy of esophageal cancer.

Chemoradiotherapy followed by surgery is at present the standard therapeutic approach for esophageal cancer (EC) in patients with resectable tumor. However, response to neoadjuvant therapy is characterized by a strong interpatient variability, and the identification of markers predictive of outcome is mandatory. In this review, taking into account the currently available literature, we report the impact that host genetic variables can have on EC neoadjuvant therapy. We mainly focused on the gene variants involved in the pharmacokinetics and pharmacodynamics of the common chemotherapeutic drugs used to treat EC patients, commented on the weakness of the present knowledge, and discussed the future strategies to achieve a more personalized and effective EC treatment.

Liquid biopsy as a novel tool to monitor the carcinogenesis of Barrett's esophagus.

Barrett's esophagus (BE) is associated with an increased risk of developing esophageal adenocarcinoma. For this reason, endoscopic-based surveillance protocols have been developed. This prevention program is, however, burdensome for the patients and expensive for the national health systems. Thus, diagnostic strategies with a low invasiveness and a reduced economic impact are required. This study investigated the power of plasma circulating free DNA (cfDNA) in predicting neoplastic transformation in the natural history of two BE patients who progressed to esophageal adenocarcinoma. Longitudinally collected DNAs from plasma and paired formalin fixed paraffin embedded samples were examined for both loss of heterozygosity (LOH) in areas proximal to TP53, FHIT and BRCA2 genes, and mutations in TP53 gene. Results showed that: (i) early BE molecular alterations are mainly localized proximal to, or within, TP53 gene; (ii) LOH events present in cfDNA not only retrace the time-matched biopsy profile but better represent the total alterations of the BE epithelium. In conclusion, our findings suggested that LOH analysis in plasma cfDNA could represent an additional, less invasive, diagnostic tool to monitor neoplastic progression of BE epithelium.

A germline predictive signature of response to platinum chemotherapy in esophageal cancer.

Platinum-based neoadjuvant therapy is the standard treatment for esophageal cancer (EC). At present, no reliable response markers exist, and patient therapeutic outcome is variable and very often unpredictable. The aim of this study was to understand the contribution of host constitutive DNA polymorphisms in discriminating between responder and nonresponder patients. DNA collected from 120 EC patients treated with platinum-based neoadjuvant chemotherapy was analyzed using drug metabolism enzymes and transporters (DMET) array platform that interrogates polymorphisms in 225 genes of drug metabolism and disposition. Four gene variants of DNA repair machinery, 2 in ERCC1 (rs11615; rs3212986), and 2 in XPD (rs1799793; rs13181) were also studied. Association analysis was performed with pTest software and corrected by permutation test. Predictive models of response were created using the receiver-operating characteristics curve approach and adjusted by the bootstrap procedure. Sixteen single nucleotide polymorphisms (SNPs) of the DMET array resulted significantly associated with either good or poor response; no association was found for the 4 variants mapping in DNA repair genes. The predictive power of 5 DMET SNPs mapping in ABCC2, ABCC3, CYP2A6, PPARG, and SLC7A8 genes was greater than that of clinical factors alone (area under the curve [AUC] = 0.74 vs 0.62). Interestingly, their combination with the clinical variables significantly increased the predictivity of the model (AUC = 0.78 vs 0.62, P = 0.0016). In conclusion, we identified a genetic signature of response to platinum-based neoadjuvant chemotherapy in EC patients. Our results also disclose the potential benefit of combining genetic and clinical variables for personalized EC management.

Genetic features of metachronous esophageal cancer developed in Hodgkin's lymphoma or breast cancer long-term survivors: an exploratory study.

BACKGROUND: Development of novel therapeutic drugs and regimens for cancer treatment has led to improvements in patient long-term survival. This success has, however, been accompanied by the increased occurrence of second primary cancers. Indeed, patients who received regional radiotherapy for Hodgkin's Lymphoma (HL) or breast cancer may develop, many years later, a solid metachronous tumor in the irradiated field. Despite extensive epidemiological studies, little information is available on the genetic changes involved in the pathogenesis of these solid therapy-related neoplasms. METHODS: Using microsatellite markers located in 7 chromosomal regions frequently deleted in sporadic esophageal cancer, we investigated loss of heterozygosity (LOH) and microsatellite instability (MSI) in 46 paired (normal and tumor) samples. Twenty samples were of esophageal carcinoma developed in HL or breast cancer long-term survivors: 14 squamous cell carcinomas (ESCC) and 6 adenocarcinomas (EADC), while 26 samples, used as control, were of sporadic esophageal cancer (15 ESCC and 11 EADC). RESULTS: We found that, though the overall LOH frequency at the studied chromosomal regions was similar among metachronous and sporadic tumors, the latter exhibited a statistically different higher LOH frequency at 17q21.31 (p = 0.018). By stratifying for tumor histotype we observed that LOH at 3p24.1, 5q11.2 and 9p21.3 were more frequent in ESCC than in EADC suggesting a different role of the genetic determinants located nearby these regions in the development of the two esophageal cancer histotypes. CONCLUSIONS: Altogether, our results strengthen the genetic diversity among ESCC and EADC whether they occurred spontaneously or after therapeutic treatments. The presence of histotype-specific alterations in esophageal carcinoma arisen in HL or breast cancer long-term survivors suggests that their transformation process, though the putative different etiological origin, may retrace sporadic ESCC and EADC carcinogenesis.

Clinical and Genetic Factors Associated With Severe Hematological Toxicity in Glioblastoma Patients During Radiation Plus Temozolomide Treatment: A Prospective Study.

BACKGROUND: Temozolomide (TMZ) administered daily with radiation therapy (RT) for 6 weeks, followed by adjuvant TMZ for 6 cycles, is the standard therapy for newly diagnosed glioblastoma (GBM) patients. Although TMZ is considered to be a safe drug, it has been demonstrated to cause severe myelotoxicity; in particular, some case reports and small series studies have reported severe myelotoxicity developing during TMZ and concomitant RT. We performed a prospective study to analyze the incidence of early severe myelotoxicity and its possible clinical and genetic factors. PATIENTS AND METHODS: From November 2010 to July 2012, newly diagnosed GBM patients were enrolled. They were eligible for the study if they met the following criteria: pathologically proven GBM, age 18 years and older, an Eastern Cooperative Oncology Group performance status of 0 to 2, adequate renal and hepatic function, and adequate blood cell counts before starting TMZ plus RT. Grading of hematologic toxicity developing during radiation and TMZ was based on the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. Clinical factors from all patients were recorded. The methylation status and polymorphic variants of O-methylguanine-DNAmethyl-transferase gene in peripheral blood mononuclear cells, and polymorphic genetic variants of genes involved in the pharmacokinetics and pharmacodynamics of TMZ, were analyzed. For genetic analyses, patients with toxicity were matched (1:2) for age, performance status, anticonvulsants, and proton pump inhibitors with patients without myelotoxicity. RESULTS: We enrolled 87 consecutive GBM patients: 32 women and 55 men; the average age was 60 years. During TMZ and RT, 4 patients (5%) showed grade 3-4 myelotoxicity, and its median duration was 255 days. Predictor factors of severe myelotoxicity were female sex, pretreatment platelet count of ≤3,00,000/mm, methylated O-methylguanine-DNA methyltransferase promoter in the hematopoietic cell system, and specific polymorphic variants of the cytochrome P450 oxidoreductase and methionine adenosyltransferase 1A genes. CONCLUSIONS: Although we studied a small population, we suggest that both clinical and genetic factors might simultaneously be associated with severe myelosuppression developed during TMZ plus RT. However, our results deserve validation in larger prospective studies and, if the factors associated with severe myelotoxicity are validated, dose adjustments of TMZ for those patients may reduce the risk of severe myelotoxicity during the concomitant treatment.

Inhibition of apoptosis by human T-lymphotropic virus type-1 tax protein.

Deregulation of the apoptotic process can lead to pathophysiological changes that result in either degenerative diseases or cancer. Although the transactivator Tax has been established as an essential effector of human T-lymphotropic virus type-1 (HTLV-1)-mediated diseases, which include both a neurodegenerative pathology and leukemia/lymphoma, its exact role(s) in the pathogenesis remains to be clarified. Because the apoptotic potential of Tax is still being debated, we addressed this question by testing the susceptibility of Tax(-) and Tax(+) cells to apoptosis under conditions of growth factor withdrawal and Bax overexpression. Results showed that Tax(+) cells are protected from apoptosis triggered by depletion of growth factors. This protective effect seems to be the result of a block in the apoptotic program regulated by mitochondria. Furthermore, in an attempt to elucidate which transcriptional pathway activated by Tax is important in the observed Tax-induced resistance, we found that CREB/ATF activity plays a relevant role in protecting cells from apoptosis induced by Bax overexpression. All together, these data might suggest that the ability of Tax to inhibit certain apoptotic stimuli could be important in its role as a viral transforming protein.

DMET™ (Drug-Metabolizing Enzymes and Transporters) microarray analysis of colorectal cancer patients with severe 5-fluorouracil-induced toxicity.

PURPOSE: 5-fluorouracil (5-FU) has been widely used since the 1980s, and it remains the backbone of many chemotherapeutic combination regimens. However, its use is often limited by the occurrence of severe toxicity. Although several reports have shown the detrimental effect of some dihydropyrimidine dehydrogenase (DPYD) and thymidylate synthase (TYMS) gene polymorphisms in patients undergoing 5-FU-based treatment, they account for only a minority of toxicities. METHODS: Looking for new candidate genetic variants associated with 5-FU-induced toxicity, we used the innovative genotyping microarray Affymetrix Drug-Metabolizing Enzymes and Transporters (DMET)™ Plus GeneChip that interrogates 1,936 genetic variants distributed in 231 genes involved in drug metabolism, excretion, and transport. To reduce variability, we analyzed samples from colorectal cancer patients who underwent fairly homogenous treatments (i.e., Machover or Folfox) and experienced G3 or G4 toxicity; control patients were matched for therapy and selected from those who did not disclose toxicity (G0-G1). RESULTS: Pharmacogenetic genotyping showed no significant difference in DPYD and TYMS genetic variants distribution between cases and controls. However, other polymorphisms could account for 5-FU-induced toxicity, with the CHST1 rs9787901 and GSTM3 rs1799735 having the strongest association. CONCLUSIONS: Although exploratory, this study suggests that genetic polymorphisms not directly related to 5-FU pharmacokinetics and pharmacodynamics are involved in 5-FU-induced toxicity. Our data also indicates DMET™ microarray as a valid approach to discover new genetic determinants influencing chemotherapy-induced toxicity.

Anti-apoptotic effect of Tax: an NF-κB path or a CREB way?

The NF-κB pathway is intimately linked to the survival of mammalian cells, and its activation by Tax has consequently been considered important for human T-cell leukemia/lymphoma virus type 1 (HTLV-1)-infected cell resistance to death. Very little emphasis has been given to other mechanisms, although Tax regulates the expression and activity of several cellular genes. The finding that CREB protein is activated in HTLV-1 infected cells underlines the possibility that other mechanisms of survival may be implicated in HTLV-1 infection. Indeed, CREB activation or overexpression plays a role in normal hematopoiesis, as well as in leukemia development, and CREB is considered as a survival factor in various cell systems. A better understanding of the different molecular mechanisms used by Tax to counteract cell death will also help in the development of new therapeutic strategies for HTLV-1 associated diseases.