Cell-free DNA analysis in current cancer clinical trials: a review.

Cell-free DNA (cfDNA) analysis represents a promising method for the diagnosis, treatment selection and clinical follow-up of cancer patients. Although its general methodological feasibility and usefulness has been demonstrated, several issues related to standardisation and technical validation must be addressed for its routine clinical application in cancer. In this regard, most cfDNA clinical applications are still limited to clinical trials, proving its value in several settings. In this paper, we review the current clinical trials involving cfDNA/ctDNA analysis and highlight those where it has been useful for patient stratification, treatment follow-up or development of novel approaches for early diagnosis. Our query included clinical trials, including the terms 'cfDNA', 'ctDNA', 'liquid biopsy' AND 'cancer OR neoplasm' in the FDA and EMA public databases. We identified 1370 clinical trials (FDA = 1129, EMA = 241) involving liquid-biopsy analysis in cancer. These clinical trials show promising results for the early detection of cancer and confirm cfDNA as a tool for real-time monitoring of acquired therapy resistance, accurate disease-progression surveillance and improvement of treatment, situations that result in a better quality of life and extended overall survival for cancer patients.

Population and breast cancer patients' analysis reveals the diversity of genomic variation of the BRCA genes in the Mexican population.

Interpretation of variants of unknown significance (VUS) in genetic tests is complicated in ethnically diverse populations, given the lack of information regarding the common spectrum of genetic variation in clinically relevant genes. Public availability of data obtained from high-throughput genotyping and/or exome massive parallel sequencing (MPS)-based projects from several thousands of outbred samples might become useful tools to evaluate the pathogenicity of a VUS, based on its frequency in different populations. In the case of the Mexican and other Latino populations, several thousands of samples have been genotyped or sequenced during the last few years as part of different efforts to identify common variants associated to common diseases. In this report, we analyzed Mexican population data from a sample of 3985 outbred individuals, and additional 66 hereditary breast cancer patients were analyzed in order to better define the spectrum of common genomic variation of the BRCA1 and BRCA2 genes. Our analyses identified the most common genetic variants in these clinically relevant genes as well as the presence and frequency of specific pathogenic mutations present in the Mexican population. Analysis of the 3985 population samples by MPS identified three pathogenic mutations in BRCA1, only one population sample showed a BRCA1 exon 16-17 deletion by MLPA. This resulted in a basal prevalence of deleterious mutations of 0.10% (1:996) for BRCA1 and 11 pathogenic mutations in BRCA2, resulting in a basal prevalence of deleterious mutations of 0.276% (1:362) for BRCA2, combined of 0.376% (1:265). Separate analysis of the breast cancer patients identified the presence of pathogenic mutations in 18% (12 pathogenic mutations in 66 patients) of the samples by MPS and 13 additional alterations by MLPA. These results will support a better interpretation of clinical studies focused on the detection of BRCA mutations in Mexican and Latino populations and will help to define the general prevalence of deleterious mutations within these populations.

MHC2TA and FCRL3 genes are not associated with rheumatoid arthritis in Mexican patients.

Rheumatoid arthritis (RA) is a multifactorial disease. A combination of genetic and environmental risk factors contributes to its etiology. Several genes have been reported to be associated with susceptibility to the development of RA. The MHC2TA and FCRL3 genes have been associated previously with RA in Swedish and Japanese populations, respectively. In two recent reports, we show an association between FCRL3 and juvenile rheumatoid arthritis (JRA), and MHC2TA and acute coronary syndrome (ACS) in Mexican population. We assessed the association between three single nucleotide polymorphisms (SNPs) of the MHC2TA (-168G/A; rs3087456, and +16G/C; rs4774) and FCRL3 (-169T/C; rs7528684) genes and rheumatoid arthritis in Mexican population through a genotyping method using allelic discrimination assays with TaqMan probes. Our case-control study included 249 patients with RA and 314 controls. We found no evidence of an association between the MHC2TA -168G/A and +1614G/C or FCRL3 -169T/C polymorphisms and RA in this Mexican population. In this cohort of Mexican patients with RA, we observed no association between the MHC2TA or FCRL3 genes and this autoimmune disease.

SPINK5 and ADRB2 haplotypes are risk factors for asthma in Mexican pediatric patients.

BACKGROUND: Asthma is one of the most common respiratory diseases worldwide, and the complexity of its etiology has been widely documented. Chromosome 5q31-33 is one of the main loci implicated in asthma and asthma-related traits. IL13, CD14 and ADRB2, which are located in this risk locus, are among the genes most strongly associated with asthma susceptibility. OBJECTIVES: This study evaluated whether single-nucleotide polymorphisms or haplotypes at 5q31-33 conferred risk for asthma in Mexican-Mestizo pediatric patients. METHODS: We performed a case-controlled study including 851 individuals, 421 of them affected with childhood-onset asthma and 430 ethnically matched unaffected subjects. We used the TaqMan Allelic Discrimination Assay to genotype 20 single-nucleotide polymorphisms within IL5, RAD50, IL13, IL4, CD14, SPINK5, HTR4, ADRB2 and IL12B. RESULTS: Although no association was detected for any risk allele, three SPINK5 haplotypes (GGCT: p = 6 × 10(-6); AATC: p = 0.0001; AGTT: p = 0.0001) and five ADRB2 haplotypes (AGGACC: p = 0.0014; AGGAAG: p = 0.0002; TGAGAG: p = 0.0001; AGGAAC: p = 0.0002; AAGGAG: p = 0.003) were associated with asthma. Notably, the AGTT SPINK5 haplotype exhibited a male gender-dependent association (p = 7.6 × 10(-5)). CONCLUSION: Our results suggest that SPINK5 and ADRB2 haplotypes might play a role in the susceptibility to childhood-onset asthma.

Juvenile rheumatoid arthritis and asthma, but not childhood-onset systemic lupus erythematosus are associated with FCRL3 polymorphisms in Mexicans.

A regulatory single nucleotide polymorphism located in the 5' region (-169T/C) of the Fc receptor-like 3 (FCRL3_3) gene has been associated with both susceptibility and protection in immune diseases. This case-control study aimed to evaluate the association between FCRL3 polymorphisms and juvenile rheumatoid arthritis (JRA), asthma, and childhood-onset systemic lupus erythematosus (SLE) in a Mexican population. We performed PCR-based genotyping to identify four FCRL3 single nucleotide polymorphisms (FCRL3_3 to FCRL3_6) in patients with JRA (n=202), asthma (n=239), or childhood-onset SLE (n=377), and healthy controls (n=400). The case-control analysis showed a male-gender dependent association between the FCRL3_3C, FCRL3_5C, and FCRL3_6A alleles and either JRA (OR=0.57, p=0.003; OR=0.55, p=0.002; OR=0.53, p=0.0007, respectively) or asthma (OR=0.72, p=0.04; OR=0.74, p=0.05; OR=0.70, p=0.02, respectively). As expected, minor alleles of these SNPs with the CGCA haplotype were also significantly associated with JRA (OR=0.35, p=0.00005) and asthma (OR=0.61, p=0.007). We found no association between FCRL3 SNPs or haplotypes and childhood-onset SLE. These results supported the notion that FCRL3 is involved in the etiology of several immune diseases. Our results also suggested that SNPs located in the FCRL3 gene were protective against JRA and asthma in male Mexican patients.

MiR-146a polymorphism is associated with asthma but not with systemic lupus erythematosus and juvenile rheumatoid arthritis in Mexican patients.

Extensive research has shown that aberrant expression of microRNAs (miRNAs) plays an important role in innate and adaptive immune responses. The rs2910164 polymorphism has been identified as a functional variant, which affects the transcription and expression level of miR-146a and, thereby, contributes to the pathogenesis of several inflammatory and autoimmune diseases. To investigate whether the rs2910164 G/C polymorphism was associated with asthma, systemic lupus erythematosus (SLE) or juvenile rheumatoid arthritis (JRA), we performed an association study in a pediatric Mexican cohort. We included 979 pediatric patients (asthma: 402, SLE: 367 and JRA: 210) and 531 control subjects without inflammatory or immune diseases. Genotyping was performed using the 5' exonuclease technique. The genotype distribution of the rs2910164 polymorphism was in Hardy-Weinberg equilibrium in each group. No significant differences were detected in the distribution of this polymorphism between cases and controls (P = 0.108, 0.609 and 0.553 for subjects with asthma, JRA and SLE, respectively). However, stratification by gender showed a statistically significant difference between asthmatic and control females, where the C allele was significantly associated with protection to asthma (odds ratio = 0.694, 95% confidence interval 0.519-0.929, P = 0.0138). Our results provide evidence that rs2910164 may play a role in the susceptibility to childhood-onset asthma, but not SLE or JRA in Mexicans. Further association studies may contribute to determining the role of miR-146a single-nucleotide polymorphisms in immune-mediated diseases.

Spectrum of RB1 gene mutations and loss of heterozygosity in Mexican patients with retinoblastoma: identification of six novel mutations.

RB1 mutation detection has greatly improved the clinical management of retinoblastoma and provides critical information to predict the risk of inheriting the disease. We screened for RB1 gene sequence alterations in both peripheral blood and tumor specimens from a total of 48 Mexican retinoblastoma patients using an SSCP-based screening approach followed by sequencing. Overall, 21 (43.8%) cases were bilateral and 27 (56.2%) were unilateral. Interestingly, 51.8% of unilateral patients developed the tumor before age 1 year and 10 of which (71.4%) were diagnosed before the age of 6 months. Thirteen different oncogenic mutations were detected in 14/48 (29.2%) patients, 9 of which were germline (64.3%). Six of these mutations are novel (IVS3-1G>T, 125X, 389X, 610X, 750X and -149G>T). The most frequent types of mutation were frameshift and nonsense (30.8% each). Moreover, 5 intronic variants were identified, two of which are novel (g.41908 C/A and g.161976del6T). Loss of heterozygosity of the RB1 gene as assessed by intron1/BamHI and intron17/XbaI intragenic markers was 50.0% (18 of 36 informative cases), being higher in tumors with known mutations (76.9% vs 34.8%). This low mutation detection rate and the earlier age at diagnosis in unilateral retinoblastoma cases suggest that other RB1 inactivating mechanisms could be present in the retinoblastoma development. In this study, mutation analysis was not helpful to distinguish sporadic and hereditary retinoblastoma, so, other approaches are needed to improve the molecular diagnosis of retinoblastoma and supports further investigations of Mexican retinoblastoma patients.

BCR-ABL, ETV6-RUNX1 and E2A-PBX1: prevalence of the most common acute lymphoblastic leukemia fusion genes in Mexican patients.

This study was conducted to determine the frequency of the most common fusion genes in Mexican pediatric patients with acute lymphoblastic leukemia (ALL). Molecular analysis using RT-PCR was carried out in 53-blood samples: 52 patients with de novo ALL and one with relapsed ALL. The ETV6-RUNX1 fusion was found in 7 cases (13.5%), BCR-ABL fusion was detected in 2 cases (3.8%), and 6 patients (11.5%) expressed the chimeric gene E2A-PBX1. The prevalence of E2A-PBX1 is one of the highest that has been described thus far in childhood ALL. Furthermore, we detected both the BCR-ABL, and E2A-PBX1 fusion in the relapsed patient. With regards to the immunophenotype, ETV6-RUNX1 was expressed in both pre-B and T-cell cases, while the presence of E2A-PBX1 and BCR-ABL was associated with the pre-B ALL phenotype. The prevalence of E2A-PBX1 in Mexican pediatric cases supports the existence of ethnic differences in the frequency of molecular markers of ALL.