Lipidomics by Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-High-Resolution Mass Spectrometry in Osteosarcoma: A Pilot Study.

Cancer is a complex disease that can also affect the younger population; however, it is responsible for a relatively high mortality rate of children and youth, especially in low- and middle-income countries (LMICs). Besides that, lipidomic studies in this age range are scarce. Therefore, we analyzed blood serum samples from young patients (12 to 35 years) with bone sarcoma (osteosarcoma) and compared their lipidomics to the ones from the control group of samples, named healthy control (HC group), using NMR and LC-MS techniques. Furthermore, differences in the lipidomic profiles between OS patients with and without metastasis indicate higher glycerophosphocholine (GPC) and glycerophospholipid (GPL) levels in osteosarcoma and increased cholesterol, choline, polyunsaturated fatty acids (PUFAs), and glycerols during the metastasis. These differences, detected in the peripheral blood, could be used as biomarkers for liquid biopsy.

Analysis of the Mutational Landscape of Osteosarcomas Identifies Genes Related to Metastasis and Prognosis and Disrupted Biological Pathways of Immune Response and Bone Development.

Osteosarcoma (OS) is the most prevalent type of bone tumor, but slow progress has been achieved in disentangling the full set of genomic events involved in its initiation and progression. We assessed by NGS the mutational spectrum of 28 primary OSs from Brazilian patients, and identified 445 potentially deleterious SNVs/indels and 1176 copy number alterations (CNAs). TP53 was the most recurrently mutated gene, with an overall rate of ~60%, considering SNVs/indels and CNAs. The most frequent CNAs (~60%) were gains at 1q21.2q21.3, 6p21.1, and 8q13.3q24.22, and losses at 10q26 and 13q14.3q21.1. Seven cases presented CNA patterns reminiscent of complex events (chromothripsis and chromoanasynthesis). Putative RB1 and TP53 germline variants were found in five samples associated with metastasis at diagnosis along with complex genomic patterns of CNAs. PTPRQ, KNL1, ZFHX4, and DMD alterations were prevalent in metastatic or deceased patients, being potentially indicative of poor prognosis. TNFRSF11B, involved in skeletal system development and maintenance, emerged as a candidate for osteosarcomagenesis due to its biological function and a high frequency of copy number gains. A protein-protein network enrichment highlighted biological pathways involved in immunity and bone development. Our findings reinforced the high genomic OS instability and heterogeneity, and led to the identification of novel disrupted genes deserving further evaluation as biomarkers due to their association with poor outcomes.

DNA methylation patterns suggest the involvement of DNMT3B and TET1 in osteosarcoma development.

DNA methylation may be involved in the development of osteosarcomas. Osteosarcomas commonly arise during the bone growth and remodeling in puberty, making it plausible to infer the involvement of epigenetic alterations in their development. As a highly studied epigenetic mechanism, we investigated DNA methylation and related genetic variants in 28 primary osteosarcomas aiming to identify deregulated driver alterations. Methylation and genomic data were obtained using the Illumina HM450K beadchips and the TruSight One sequencing panel, respectively. Aberrant DNA methylation was spread throughout the osteosarcomas genomes. We identified 3146 differentially methylated CpGs comparing osteosarcomas and bone tissue samples, with high methylation heterogeneity, global hypomethylation and focal hypermethylation at CpG islands. Differentially methylated regions (DMR) were detected in 585 loci (319 hypomethylated and 266 hypermethylated), mapped to the promoter regions of 350 genes. These DMR genes were enriched for biological processes related to skeletal system morphogenesis, proliferation, inflammatory response, and signal transduction. Both methylation and expression data were validated in independent groups of cases. Six tumor suppressor genes harbored deletions or promoter hypermethylation (DLEC1, GJB2, HIC1, MIR149, PAX6, and WNT5A), and four oncogenes presented gains or hypomethylation (ASPSCR1, NOTCH4, PRDM16, and RUNX3). Our analysis also revealed hypomethylation at 6p22, a region that contains several histone genes. Copy-number changes in DNMT3B (gain) and TET1 (loss), as well as overexpression of DNMT3B in osteosarcomas provide a possible explanation for the observed phenotype of CpG island hypermethylation. While the detected open-sea hypomethylation likely contributes to the well-known osteosarcoma genomic instability, enriched CpG island hypermethylation suggests an underlying mechanism possibly driven by overexpression of DNMT3B likely resulting in silencing of tumor suppressors and DNA repair genes.

High-dose chemotherapy with autologous stem cell transplantation for patients with extracranial germ cell tumors - experience of two Brazilian pediatric centers.

Malignant extracranial germ cell tumors (GCTs) are rare in pediatric patients and are usually extremely sensitive to chemotherapy. Relapsed or refractory tumors, although rare, established the need for second-line therapies, including high-dose chemotherapy with autologous stem cell transplantation (HDCT/ASCT). However, there are few data on its use in children with GCTs. We present a retrospective analysis of all patients diagnosed with extracranial GCTs who received HDCT/ASCT at two Brazilian pediatric cancer centers from May 1999 to December 2019. We identified a total of 34 patients with a median age at diagnosis of 2.8 years (range, 0 to 18.8), who received HDCT/ASCT. Most patients (73%) received carboplatin, etoposide and melphalan (CEM) as a HDCT regimen. Fourteen patients received a second-line conventional dose chemotherapy (CDCT), 14 received a third-line CDCT and five received even a fourth-line CDCT prior to HDCT/ASCT. After a median follow-up of 22.7 months (range, 0.3 to 198.1), 16 patients had died after tumor relapse/progression and 2 patients died from HDCT/ASCT toxicity. We observed a 5-year OS of 47.1% and 5-year EFS of 44.1%. The 5-year OS for patients referred for HDCT/ASCT with progressive disease was 10% compared to 62.5% for those who achieved disease control before HDCT/ASCT (p = 0.001). In our experience, heavily pretreated children and adolescents with extracranial GCTs achieved considerable survival rates with HDCT/ASCT since, at least, partial control of their disease was possible before starting HDCT/ASCT. The role of HDCT/ASCT in pediatric patients with GCTs should be investigated in prospective trials.

Potential New Therapeutic Approaches for Cisplatin-Resistant Testicular Germ Cell Tumors.

BACKGROUND: Testicular germ cell tumors (TGCTs), a group of heterogeneous neoplasms, are the most frequent tumors of teenagers and young men, with the incidence rising worldwide. High cure rates can be achieved through cisplatin (CDDP)-based treatment, but approximately 10% of patients present refractory disease and virtually no treatment alternatives. Here, we explored new strategies to treat CDDP-resistant. METHODS: In vitro TGCT CDDP-resistance model was established and differential mRNA expression profiles were evaluated using NanoString technology. Then, TGCT cell lines were treated with four potential drugs (PCNA-I1, ML323, T2AA, and MG-132) to overcome CDDP-resistance. RESULTS: We found several differentially expressed genes related to DNA repair and cell cycle regulation on CDDP-resistant cell line (NTERA-2R) compared to parental cell line (NTERA-2P), and the proteasome inhibitor MG-132 demonstrated cytotoxic activity in all cell lines evaluated, even at a nanomolar range. MG-132 also enhanced cell lines' sensitivity to CDDP, increasing apoptosis in both NTERA-2P and NTERA-2R. CONCLUSIONS: MG-132 emerges as a potential new drug to treat CDDP-resistant TGCT. Targeted therapy based on molecular mechanism insights may contribute to overcome acquired chemotherapy CDDP-resistance.

ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.

Insights in Osteosarcoma by Proton Nuclear Magnetic Resonance Serum Metabonomics.

Pediatric osteosarcoma outcomes have improved over the last decades; however, patients who do not achieve a full resection of the tumor, even after aggressive chemotherapy, have the worst prognosis. At a genetic level, osteosarcoma presents many alterations, but there is scarce information on alterations at metabolomic levels. Therefore, an untargeted nuclear magnetic resonance metabonomic approach was used to reveal blood serum alterations, when samples were taken from 21 patients with osteosarcoma aged from 12-20 (18, 86%) to 43 (3, 14%) years before any anticancer therapy were collected. The results showed that metabolites differed greatly between osteosarcoma and healthy control serum samples, especially in lipids, aromatic amino acids (phenylalanine and tyrosine), and histidine concentrations. Besides, most of the loading plots point to protons of the fatty acyls (-CH3 and -CH2-) from very-low- and low-density lipoproteins and cholesterol, as crucial metabolites for discrimination of the patients with osteosarcoma from the healthy samples. The relevance of blood lipids in osteosarcoma was highlighted when analyzed together with the somatic mutations disclosed in tumor samples from the same cohort of patients, where six genes linked to the cholesterol metabolism were found being altered too. The high consistency of the discrimination between osteosarcoma and healthy control blood serum suggests that nuclear magnetic resonance could be successfully applied for osteosarcoma diagnostic and prognostic purposes, which could ameliorate the clinical efficacy of therapy.

The Brazilian TP53 mutation (R337H) and sarcomas.

Sarcomas represent less than 1% of all solid neoplasms in adults and over 20% in children. Their etiology is unclear, but genetic susceptibility plays an important role in this scenario. Sarcoma is central in Li-Fraumeni Syndrome (LFS), a familial predisposition cancer syndrome. In Brazil, the high prevalence of p.Arg337His mutations in the TP53 gene brings about a unique condition: a cluster of LFS. In the present work, we studied 502 sarcoma patients not selected by age or family history in an attempt to assess the impact of the so-called "Brazilian germline TP53 mutation" (p.Arg337His) on this tumor type. We found that 8% of patients are carriers, with leiomyosarcoma being the main histologic type of sarcoma, corresponding to 52.5% of the patients with the mutated TP53 gene. These findings emphasize the importance of genetic counseling and can better guide the management of sarcoma patients.

miR-450a Acts as a Tumor Suppressor in Ovarian Cancer by Regulating Energy Metabolism.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. SIGNIFICANCE: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3294/F1.large.jpg.

Highly expressed placental miRNAs control key biological processes in human cancer cell lines.

Despite being a healthy tissue, the constituent cells of the placenta, share similar characteristics with tumor cells, such as increased cell growth, migration, and invasion. However, while these processes are stochastic and uncontrolled in cancer cells, in placenta they are precisely controlled. Since miRNAs have been reported to regulate genes that control the molecular mechanisms necessary for the development of both human placenta and cancer, we addressed for miRNAs highly expressed in the placenta that could be involved in tumorigenesis. Here, we assessed the miRNA profile in placenta samples using microarray analysis. The results showed that miR-451 and miR-720, highly expressed placental miRNAs, presented very low or undetectable expression in cancer cell lines compared to the normal placenta and healthy tissues. Additionally, transfection of miR-451 or miR-720 mimics in choriocarcinoma cell line (JEG3) and colorectal adenocarcinoma cell line (HT-29) resulted in impaired cell proliferation, decreased cell migration and invasion and reduced ability of colony formation. These findings provide evidence that placenta may work as an alternative model to identify novel miRNAs involved in pathways controlling tumorigenesis.

MGMT and CALCA promoter methylation are associated with poor prognosis in testicular germ cell tumor patients.

Testicular germ cell tumors (TGCT) represent the second main cause of cancer-related death in young men. Despite high cure rates, refractory disease results in poor prognosis. Epigenetic reprogramming occurs during the development of seminomas and non-seminomas. Understanding the molecular and genetic basis of these tumors would represent an important advance in the search for new TGCT molecular markers. Hence the frequency of methylation of a gene panel (VGF, MGMT, ADAMTS1, CALCA, HOXA9, CDKN2B, CDO1 and NANOG) was evaluated in 72 primary TGCT by quantitative methylation specific PCR. A high frequency of MGMT (90.9%, 20/22; p=0.019) and CALCA (90.5%, 19/21; p<0.026) methylation was associated with non-seminomatous tumors while CALCA methylation was also associated with refractory disease (47.4%, 09/19; p=0.005). Moreover, promoter methylation of both genes predicts poor clinical outcome for TGCT patients (5-year EFS: 50.5% vs 77.1%; p=0.032 for MGMT and 51.3% vs 77.0%; p=0.029 for CALCA). The findings of this study indicate that methylation of MGMT and CALCA are frequent and could be used as new molecular markers of prognosis in TGCT.

High Expression of HULC Is Associated with Poor Prognosis in Osteosarcoma Patients.

Osteosarcoma (OS) is the most common primary bone cancer in childhood. OS is an aggressive disease, and metastatic patients evolve with very poor clinical outcomes. Genetically, OSs are extremely complex tumors, and the related metastatic process is not well understood in terms of the biology of the disease. In this context, long non-coding RNAs (lncRNAs) have emerged as an important class of gene expression regulators that play key roles in the invasion and metastasis of several human tumors. Here, we evaluated the expression of HULC, which is an lncRNA that is associated with the tumor metastatic process, and assessed its potential role as a prognostic marker in OS. HULC expression was evaluated in primary OS samples using real-time RT-PCR. HULC expression status was determined by receiver operating characteristic (ROC) analysis, and its association with survival was assessed using the Kaplan-Meier method. The HULC expression level was not significantly associated with the clinicopathological characteristics of the OS patients. However, our data demonstrated that higher levels of expression of HULC were associated with lower survival rates in OS patients, both in terms of overall and event-free survival. Elevated HULC expression was associated with poor clinical outcomes among the OS patients, which suggests that HULC could be a potential prognostic biomarker in OS.

Placenta-Enriched LincRNAs MIR503HG and LINC00629 Decrease Migration and Invasion Potential of JEG-3 Cell Line.

LINC00629 and MIR503HG are long intergenic non-coding RNAs (lincRNAs) mapped on chromosome X (Xq26), a region enriched for genes associated with human reproduction. Genes highly expressed in normal reproductive tissues and cancers (CT genes) are well known as potential tumor biomarkers. This study aimed to characterize the structure, expression, function and regulation mechanism of MIR503HG and LINC00629 lincRNAs. According to our data, MIR503HG expression was almost exclusive to placenta and LINC00629 was highly expressed in placenta and other reproductive tissues. Further analysis, using a cancer cell lines panel, showed that MIR503HG and LINC00629 were expressed in 50% and 100% of the cancer cell lines, respectively. MIR503HG was expressed predominantly in the nucleus of JEG-3 choriocarcinoma cells. We observed a positively correlated expression between MIR503HG and LINC00629, and between the lincRNAs and neighboring miRNAs. Also, both LINC00629 and MIR503GH could be negatively regulated by DNA methylation in an indirect way. Additionally, we identified new transcripts for MIR503HG and LINC00629 that are relatively conserved when compared to other primates. Furthermore, we found that overexpression of MIR503HG2 and the three-exon LINC00629 new isoforms decreased invasion and migration potential of JEG-3 tumor cell line. In conclusion, our results suggest that lincRNAs MIR503HG and LINC00629 impaired migration and invasion capacities in a choriocarcinoma in vitro model, indicating a potential role in human reproduction and tumorigenesis. Moreover, the MIR503HG expression pattern found here could indicate a putative new tumor biomarker.

Hotspot TERT promoter mutations are rare events in testicular germ cell tumors.

The abnormal activation of telomerase, codified by the telomerase reverse transcriptase (TERT) gene, is related to one of cancer hallmarks. Hotspot somatic mutations in the promoter region of TERT, specifically the c.-124:C>T and c.-146:C>T, were recently identified in a range of human cancers and have been associated with a more aggressive behavior. Testicular germ cell tumors frequently exhibit a good prognosis; however, the development of refractory disease is still a clinical challenge. In this study, we aim to evaluate for the first time the presence of the hotspot telomerase reverse transcriptase gene promoter mutations in testicular germ cell tumors. A series of 150 testicular germ cell tumor cases and four germ cell tumor cell lines were evaluated by PCR followed by direct Sanger sequencing and correlated with patient's clinical pathological features. Additionally, we genotyped the telomerase reverse transcriptase gene promoter single nucleotide polymorphism rs2853669 (T>C) located at -245 position. We observed the presence of the TERT promoter mutation in four patients, one exhibited the c.-124:C>T and three the c.-146:C>T. No association between TERT mutation status and clinicopathological features could be identified. The analysis of the rs2853669 showed that variant C was present in 22.8 % of the cases. In conclusion, we showed for the first time that TERT promoter mutations occur in a small subset (~3 %) of testicular germ cell tumors.

Array-CGH as an adjuvant tool in cytogenetic diagnosis of pediatric MDS and JMML.

Myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) are rare clonal hematopoietic diseases presented in the childhood. Both diseases exhibit abnormal karyotype and/or monosomy of chromosome 7 in a subgroup of patients. We screened for copy number variations (CNVs) by array-comparative genomic hybridization (aCHG) the DNA from bone marrow of six MDS and four JMML pediatric patients. Array-CGH analysis identified five cases (50%) with monosomy 7, disclosing the chromosome 7 monosomy in two patients whose samples could not be evaluated by other methods. We identified CNVs in six patients, one of which displayed loss of LMO2, an oncogene that plays a central role in hematopoietic development. Our results suggest that array-CGH is a reliable and accurate technique to identify genomic alterations in MDS and JMML.

The role of microRNAs in medulloblastoma.

Medulloblastomas (MBs) are the most frequent brain tumors in children and remained a major therapeutic challenge. Clinical and histopathological features are used for disease classification and patient prognostication. Currently, several molecular studies using transcriptomic and genomic approaches suggested the existence of four molecular subtypes, increasing the complexity, and knowledge of MB biology. Despite these significant advances, the molecular basis of MBs is not fully understood. MicroRNAs (miRNAs) are a group of small nonprotein coding RNA molecules that target genes by inducing mRNA degradation or translational repression. They represent an evolutionary conserved mechanism that controls fundamental cellular processes, such as development, differentiation, metabolism, proliferation, and apoptosis. Aberrant expression of miRNAs correlates with various cancers. This altered expression can arise from mutation, methylation, deletion, and gain of miRNA-encoding regions. We here review the knowledge of miRNAs in MBs. The expression patterns of miRNAs in MBs were comprehensively evaluated and their diagnostic, prognostic, and therapeutic biomarker role assessed. miRNAs are important players in MB tumorigenesis and their therapeutic exploitation can constitute an alternative approach to this devastating disease.

1031-1034delTAAC (Leu125Stop): a novel familial UBE3A mutation causing Angelman syndrome in two siblings showing distinct phenotypes.

BACKGROUND: More than 50 mutations in the UBE3A gene (E6-AP ubiquitin protein ligase gene) have been found in Angelman syndrome patients with no deletion, no uniparental disomy, and no imprinting defect. CASE PRESENTATION: We here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows a milder phenotype presenting the typical AS features. CONCLUSIONS: We hypothesized that the mutation Leu125Stop causes both severe and milder phenotypes. Potential mechanisms include: i) maybe the proband has an additional problem (genetic or environmental) besides the UBE3A mutation; ii) since the two siblings have different fathers, the UBE3A mutation is interacting with a different genetic variant in the proband that, by itself, does not cause problems but in combination with the UBE3A mutation causes the severe phenotype; iii) this UBE3A mutation alone can cause either typical AS or the severe clinical picture seen in the proband.

Analysis of allelic differential expression in the human genome using allele-specific serial analysis of gene expression tags.

Recent reports have demonstrated that a significant proportion of human genes display allelic differential expression (ADE). ADE is associated with phenotypic variability and may contribute to complex genetic diseases. Here, we present a computational analysis of ADE using allele-specific serial analysis of gene expression (SAGE) tags representing 1295 human genes. We identified 472 genes for which unequal representation (>3-fold) of allele-specific SAGE tags was observed in at least one SAGE library, suggesting the occurrence of ADE. For 235 out of these 472 genes, the difference in the expression level between both allele-specific SAGE tags was statistically significant (p < 0.05). Eleven candidate genes were then subjected to experimental validation and ADE was confirmed for 8 out of these 11 genes. Our results suggest that at least 25% of the human genes display ADE and that allele-specific SAGE tags can be efficiently used for the identification of such genes.

CYP1A2*1C, CYP2E1*5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) is associated with environmental factors, especially tobacco and alcohol consumption. Most of the carcinogens present in tobacco smoke are converted into DNA-reactive metabolites by cytochrome P450 (CYPs) enzymes and detoxification of these substances is performed by glutathione S-transferases (GSTs). It has been suggested that genetic alterations, such as polymorphisms, play an important role in tumorigenesis and HNSCC progression. The aim of this study was to investigate CYP1A1, CYP1A2, CYP2E1, GSTM1, and GSTT1 polymorphisms as risk factors in HNSCC and their association with clinicopathologic data. The patients comprised 153 individuals with HNSCC (cases) and 145 with no current or previous diagnosis of cancer (controls). Genotyping of the single nucleotide polymorphisms (SNPs) of the CYP1A1, CYP1A2, and CYP2E1 genes was performed by PCR-RFLP and the GSTM1 and GSTT1 copy number polymorphisms (CNPs) were analyzed by PCR-multiplex. As expected, a significant difference was detected for tobacco and alcohol consumption between cases and controls (P<0.001). It was observed that the CYP1A2*1D (OR=16.24) variant and GSTM1 null alleles (OR=0.02) confer increased risk of HNSCC development (P<0.001). In addition, head and neck cancer alcohol consumers were more frequently associated with the CYP2E1*5B variant allele than control alcohol users (P<0.0001, OR=190.6). The CYP1A2*1C polymorphism was associated with tumor recurrence (log-rank test, P=0.0161). The CYP2E1*5B and GSTM1 null alleles were significantly associated with advanced clinical stages (T3+T4; P=0.022 and P=0.028, respectively). Overall, the findings suggested that the genetic polymorphisms studied are predictors of risk and are also associated with tumor recurrence, since they are important for determining the parameters associated with tumor progression and poor outcomes in HNSCC.