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1.
Cancers (Basel) ; 11(12)2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31795313

RESUMO

Early-onset colorectal cancer (EOCRC) is an increasing and worrisome entity. The aim of this study was to analyze its association with polyps concerning prognosis and surveillance. EOCRC cases were compared regarding the presence or absence of associated polyps (clinical and molecular features), during a minimum of 7 years of follow-up. Of 119 cases, 56 (47%) did not develop polyps (NP group), while 63 (53%) did (P group). The NP group showed a predominant location of the CRC in the rectum (50%), of sporadic cases (54%), and diagnosis at advanced stages: Only P53 and SMARCB1 mutations were statistically linked to this group. The P group, including mainly early-diagnosed tumors, was linked with the most frequent and differential altered chromosomal regions in the array comparative genomic hybridization. The two most frequent groups according to the follow-up were the NP group (40%), and patients developing polyps in the first 5 years of follow-up (P < 5FU) (34%) (these last groups predominantly diagnosed at the earliest stage and with adenomatous polyps (45%)). EOCRC with polyps that developed during the entire follow-up (PDFU group) were mainly located in the right colon (53%), diagnosed in earlier stages, and 75% had a familial history of CRC. Patients developing polyps after the first 5 years (P > 5FU) showed a mucinous component (50%). Our results show that the absence or presence of polyps in EOCRC is an important prognostic factor with differential phenotypes. The development of polyps during surveillance shows that it is necessary to extend the follow-up time, also in those cases with microsatellite-stable EOCRC.

2.
J Med Genet ; 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31494577

RESUMO

INTRODUCTION: Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are hereditary cancer syndromes associated with mismatch repair (MMR) deficiency. Tumours show microsatellite instability (MSI), also reported at low levels in non-neoplastic tissues. Our aim was to evaluate the performance of high-sensitivity MSI (hs-MSI) assessment for the identification of LS and CMMRD in non-neoplastic tissues. MATERIALS AND METHODS: Blood DNA samples from 131 individuals were grouped into three cohorts: baseline (22 controls), training (11 CMMRD, 48 LS and 15 controls) and validation (18 CMMRD and 18 controls). Custom next generation sequencing panel and bioinformatics pipeline were used to detect insertions and deletions in microsatellite markers. An hs-MSI score was calculated representing the percentage of unstable markers. RESULTS: The hs-MSI score was significantly higher in CMMRD blood samples when compared with controls in the training cohort (p<0.001). This finding was confirmed in the validation set, reaching 100% specificity and sensitivity. Higher hs-MSI scores were detected in biallelic MSH2 carriers (n=5) compared with MSH6 carriers (n=15). The hs-MSI analysis did not detect a difference between LS and control blood samples (p=0.564). CONCLUSIONS: The hs-MSI approach is a valuable tool for CMMRD diagnosis, especially in suspected patients harbouring MMR variants of unknown significance or non-detected biallelic germline mutations.

3.
Sci Rep ; 9(1): 9814, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285513

RESUMO

Attenuated adenomatous polyposis (AAP) is a heterogeneous syndrome in terms of clinical manifestations, heritability and etiology of the disease. Genetic heterogeneity and low penetrance alleles are probably the best explanation for this variability. Certainly, it is known that APC and MUTYH are high penetrance predisposition genes for adenomatous polyposis, but they only account for 5-10% of AAP. Other new predisposition genes, such as POLE, POLD1, NTHL1, AXIN2 or MSH3, have been recently described and have been associated with AAP, but their relative contribution is still not well defined. In order to evaluate the genetic predisposition to AAP in a hospital based population, germline DNAs from 158 AAP subjects were screened for genetic variants in the coding regions and intron-exon boundaries of seven associated genes through a next-generation sequencing (NGS) custom gene panel. Splicing, segregation studies, somatic mutational screening and RNA quantitative expression assays were conducted for selected variants. In four of the probands the adenoma susceptibility could be explained by actionable mutations in APC or MUTYH, and one other patient was a double carrier of two truncating variants in both POLE and NTHL1. Furthermore, 16 additional patients harbored uncertain significance variants in the remaining tested genes. This report gives information about the contribution of the newly described adenomatous polyposis predisposition genes in a Spanish attenuated polyposis cohort. Our results highly support the convenience of NGS multigene panels for attenuated polyposis genetic screening and reveals POLE frameshift variants as a plausible susceptibility mechanism for AAP.

4.
Sci Rep ; 9(1): 10516, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324877

RESUMO

Colorectal cancer (CRC) with CpG island methylator phenotype (CIMP) is recognized as a subgroup of CRC that shows association with particular genetic defects and patient outcomes. We analyzed CIMP status of 229 individuals with CRC using an eight-marker panel (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); CIMP-(+) tumors were defined as having ≥ 5 methylated markers. Patients were divided into individuals who developed a "unique" CRC, which were subclassified into early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and patients with multiple primary CRCs subclassified into synchronous CRC (SCRC) and metachronous CRC (MCRC). We found 9 (15.2%) CIMP-(+) EOCRC patients related with the proximal colon (p = 0.008), and 19 (26.8%) CIMP-(+) LOCRC patients associated with tumor differentiation (p = 0.045), MSI status (p = 0.021) and BRAF mutation (p = 0.001). Thirty-five (64.8%) SCRC patients had at least one CIMP-(+) tumor and 20 (44.4%) MCRC patients presented their first tumor as CIMP-(+). Thirty-nine (72.2%) SCRC patients showed concordant CIMP status in their simultaneous tumors. The differences in CIMP-(+) frequency between groups may reflect the importance of taking into account several criteria for the development of multiple primary neoplasms. Additionally, the concordance between synchronous tumors suggests CIMP status is generally maintained in SCRC patients.

5.
Genet Med ; 21(12): 2706-2712, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31204389

RESUMO

PURPOSE: Biallelic pathogenic variants in the mismatch repair (MMR) genes cause a recessive childhood cancer predisposition syndrome known as constitutional mismatch repair deficiency (CMMRD). Family members with a heterozygous MMR variant have Lynch syndrome. We aimed at estimating cancer risk in these heterozygous carriers as a novel approach to avoid complicated statistical methods to correct for ascertainment bias. METHODS: Cumulative colorectal cancer incidence was estimated in a cohort of PMS2- and MSH6-associated families, ascertained by the CMMRD phenotype of the index, by using mutation probabilities based on kinship coefficients as analytical weights in a proportional hazard regression on the cause-specific hazards. Confidence intervals (CIs) were obtained by bootstrapping at the family level. RESULTS: The estimated cumulative colorectal cancer risk at age 70 years for heterozygous PMS2 variant carriers was 8.7% (95% CI 4.3-12.7%) for both sexes combined, and 9.9% (95% CI 4.9-15.3%) for men and 5.9% (95% CI 1.6-11.1%) for women separately. For heterozygous MSH6 variant carriers these estimates are 11.8% (95% CI 4.5-22.7%) for both sexes combined, 10.0% (95% CI 1.83-24.5%) for men and 11.7% (95% CI 2.10-26.5%) for women. CONCLUSION: Our findings are consistent with previous reports that used more complex statistical methods to correct for ascertainment bias. These results underline the need for MMR gene-specific surveillance protocols for Lynch syndrome.

6.
PLoS One ; 14(5): e0216472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095598

RESUMO

Comparative studies of colorectal cancer (CRC) according to the age of onset have found differences between early-onset CRC (EOCRC) and late-onset CRC (LOCRC). Using this as a starting point, we wished to determine whether intermediate-onset CRC (IOCRC) might also be considered as an independent group within CRC. We performed a retrospective comparative study of the clinicopathological and familial features, as well as of the symptoms and their duration, of a total of 272 subjects diagnosed with CRC classified into three groups according to the age-of-onset (98 EOCRC, 83 IOCRC and 91 LOCRC). The results show that from a clinicopathological point of view, IOCRC shared certain features with EOCRC (gender, prognosis), and with LOCRC (multiple primary CRCs), whereas it also had characteristics that were specific for IOCRC (mean number of associated polyps). A gradual progression was observed from EOCRC to LOCRC from a greater family aggregation to sporadic cases, in parallel with a change of Lynch Syndrome cases to the sporadic microsatellite instability pathway, with the IOCRC being a boundary group that is more related to EOCRC. With respect to symptoms, duration and correlation with stages, IOCRC appeared more similar to EOCRC. Clinically, IOCRC behaves as a transitional group between EOCRC and LOCRC, with features in common with both groups, but also with IOCRC-specific features. Excluding cases with familial cancer history, the awareness for EOCRC diagnosis should be extended to IOCRC.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais Hereditárias sem Polipose , Instabilidade de Microssatélites , Adulto , Idade de Início , Idoso , Neoplasias do Colo/genética , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/mortalidade , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Sobrevida
7.
Int J Mol Sci ; 20(4)2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30813366

RESUMO

Our aim was to characterize and validate that the location and age of onset of the tumor are both important criteria to classify colorectal cancer (CRC). We analyzed clinical and molecular characteristics of early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and we compared each tumor location between both ages-of-onset. In right-sided colon tumors, early-onset cases showed extensive Lynch syndrome (LS) features, with a relatively low frequency of chromosomal instability (CIN), but a high CpG island methylation phenotype. Nevertheless, late-onset cases showed predominantly sporadic features and microsatellite instability cases due to BRAF mutations. In left colon cancers, the most reliable clinical features were the tendency to develop polyps as well as multiple primary CRC associated with the late-onset subset. Apart from the higher degree of CIN in left-sided early-onset cancers, differential copy number alterations were also observed. Differences among rectal cancers showed that early-onset rectal cancers were diagnosed at later stages, had less association with polyps, and more than half of them were associated with a familial LS component. Stratifying CRC according to both location and age-of-onset criteria is meaningful, not only because it correlates the resulting categories with certain molecular bases, but with the confirmation across larger studies, new therapeutical algorithms could be defined according to this subclassification.


Assuntos
Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , Idade de Início , Neoplasias Colorretais/genética , Dosagem de Genes , Humanos
8.
Hum Mutat ; 40(5): 649-655, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30740824

RESUMO

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.

9.
Int J Cancer ; 144(7): 1596-1608, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30151896

RESUMO

To analyze the possible clonal origin of a part of Synchronous colorectal cancer (SCRC), we studied 104 paired-SCRCs from 52 consecutive patients without hereditary forms of CRC. We used a Single-Nucleotide Polymorphism array to characterize the genomic profiles, and subsequently used a statistical application to define them according to clonality within the same individual. We categorized the ensuing groups according to colonic location to identify differential phenotypes. The SCRC Monoclonal group (M) (19 cases) was divided into Monosegmental (MM) and Pancolonic (MP) groups. The SCRC Polyclonal group (P) (33 cases) was also divided into Monosegmental (PM) and Pancolonic (PP), the first exhibiting preference for left colon. The MM group showed a high rate of mucinous tumors, the lowest mean-number of tumors and associated-polyps, and the worst prognosis. The MP group included the largest mean-number of associated-polyps, best prognosis and familial cancer component. The PM group seemed to be a "frontier" group. Finally, the PP group also exhibited a mucin component, the highest mean-number of tumors (4.6) compared with the mean-number of polyps (7.7), poor prognosis and sporadic cases. Most relevant differential genomic regions within M groups were gains on 1q24 and 8q24, and deletions on 1p21 and 1p23 for MM, while within P were the gains on 7q36 and deletions on 1p36 for PM. The statistical application employed seems to define clonality more accurately in SCRC -more likely to be polyclonal in origin-, and together with the tumor locations, helped us to configure a classification with prognostic and clinical value.


Assuntos
Neoplasias Colorretais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Primárias Múltiplas/genética , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodos , Idoso , Idoso de 80 Anos ou mais , Evolução Clonal , Neoplasias Colorretais/classificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/classificação , Prognóstico
10.
Br J Cancer ; 119(8): 978-987, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30283143

RESUMO

BACKGROUND: Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. METHODS: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. RESULTS: No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter-generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. CONCLUSION: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Metilação de DNA/genética , Epigênese Genética/genética , Predisposição Genética para Doença/genética , Proteína 1 Homóloga a MutL/genética , Sequência de Bases , Neoplasias Colorretais/epidemiologia , Ilhas de CpG/genética , Feminino , Haplótipos/genética , Humanos , Masculino , Mutação/genética , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA
11.
Int J Mycobacteriol ; 7(3): 275-281, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30198510

RESUMO

Introduction: Tuberculosis (TB) is a significant cause of morbidity and mortality worldwide. The patient compliance with the long treatment regimens is essential for successful eradication. Pyrazinamide (PZA) shortens these regimens from 9 to 6 months, and therefore, improves treatment completion rates. Although PZA is a first-line medication for the treatment of TB, no simple or reliable assay to determine PZA resistance is yet available. In the presence of PZA, only susceptible Mycobacterium tuberculosis strains release pyrazinoic acid (POA). Therefore, the measurement and quantification of released POA is an indicator of PZA resistance. Methods: Two electrochemical sensors were constructed and tested with alternative working electrodes in conjunction with a portable potentiostat to measure the current produced when a potential difference of 2 V is applied to varying concentrations of POA in controlled solutions. Results: The large (13.2 mm) electrochemical sensor was able to detect POA at a minimum concentration of 40 µM to a statistically significant level (P = 0.0190). Similar graphical trends were obtained when testing the electrochemical sensor in the supernatant of a negative microscopic observation drug susceptibility assay culture, irrespective of the presence of PZA. Conclusion: Inexpensive and reusable electrochemical sensors with a portable potentiostat are a promising tool for the detection of POA, a biomarker of PZA susceptible M. Tuberculosis.


Assuntos
Farmacorresistência Bacteriana , Técnicas Eletroquímicas , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Antituberculosos/farmacologia , Meios de Cultura , Eletrodos , Humanos , Testes de Sensibilidade Microbiana , Potenciometria , Pirazinamida/análogos & derivados , Pirazinamida/isolamento & purificação , Tuberculose/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
12.
Front Immunol ; 9: 1506, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30013564

RESUMO

Immunoglobulin class-switch recombination (CSR) and somatic hypermutations (SHMs) are prerequisites for antibody and immunoglobulin receptor maturation and adaptive immune diversity. The mismatch repair (MMR) machinery, consisting of homologs of MutSα, MutLα, and MutSß (MSH2/MSH6, MLH1/PMS2, and MSH2/MSH3, respectively) and other proteins, is involved in CSR, primarily acting as a backup for nonhomologous end-joining repair of activation-induced cytidine deaminase-induced DNA mismatches and, furthermore, in addition to error-prone polymerases, in the repair of SHM-induced DNA breaks. A varying degree of antibody formation defect, from IgA or selective IgG subclass deficiency to common variable immunodeficiency and hyper-IgM syndrome, has been detected in a small number of patients with constitutional mismatch repair deficiency (CMMRD) due to biallelic loss-of-function mutations in one of the MMR genes (PMS2, MSH6, MLH1, or MSH2). To elucidate the clinical relevance of a presumed primary immunodeficiency (PID) in CMMRD, we systematically collected clinical history and laboratory data of a cohort of 15 consecutive, unrelated patients (10 not previously reported) with homozygous/compound heterozygous mutations in PMS2 (n = 8), MSH6 (n = 5), and MLH1 (n = 2), most of whom manifested with typical malignancies during childhood. Detailed descriptions of their genotypes, phenotypes, and family histories are provided. Importantly, none of the patients showed any clinical warning signs of PID (infections, immune dysregulation, inflammation, failure to thrive, etc.). Furthermore, we could not detect uniform or specific patterns of laboratory abnormalities. The concentration of IgM was increased in 3 out of 12, reduced in 3 out of 12, and normal in 6 out of 12 patients, while concentrations of IgG and IgG subclasses, except IgG4, and of IgA, and specific antibody formation were normal in most. Class-switched B memory cells were reduced in 5 out of 12 patients, and in 9 out of 12 also the CD38hiIgM- plasmablasts were reduced. Furthermore, results of next generation sequencing-based analyses of antigen-selected B-cell receptor rearrangements showed a significantly reduced frequency of SHM and an increased number of rearranged immunoglobulin heavy chain (IGH) transcripts that use IGHG3, IGHG1, and IGHA1 subclasses. T cell subsets and receptor repertoires were unaffected. Together, neither clinical nor routine immunological laboratory parameters were consistently suggestive of PID in these CMMRD patients, but previously shown abnormalities in SHM and rearranged heavy chain transcripts were confirmed.

13.
Oncotarget ; 9(20): 15302-15311, 2018 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-29632645

RESUMO

Background: Since there is a predilection of some clinical and molecular features for a given tumor location, we assessed whether this can be confirmed in late-onset colorectal cancer (LOCRC). Results: Right colon cancers showed features associated with sporadic Microsatellite Instability: predominance of female cases and BRAF mutations, and an important mucinous component. Left colon cancers developed a higher number of polyps and multiple primary CRCs, showed the strongest familial component, and had better prognosis. Rectal cancers showed a predominantly sporadic phenotype, with worse prognosis and a CpG Island Methylator Phenotype (CIMP)-High. No copy number alterations (CNAs) greater than or equal to 50% were observed in this LOCRC group, and the most recurrent alterations were losses at 5q13 and 14q11, and gains at 7q11, 7q21-q22, 19p13-p12, 19q13 and 20p11-q11. KRAS and PIK3CA were the only mutated genes showing differences according to the tumor location, mainly for right colon cancers. Materials and Methods: We analyzed clinical and molecular characteristics of LOCRC at different tumor locations in order to determine if there are differential phenotypes related with the location in the colon. Conclusions: Categorizing LOCRC according to tumor location appears to be an adequate first step to resolving the heterogeneity of this subset of CRC.

14.
Int J Mycobacteriol ; 7(1): 16-25, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29516881

RESUMO

Background: The main mechanism of resistance to PZA in Mycobacterium tuberculosis relies on mutations on its pyrazinamidase/nicotinamidase. Recently, a rapid colorimetric test relying on the PCR-based in vitro-synthesized-PZase assay has been reported for PZase activity determination from clinical M. tuberculosis isolates but the assay has not been compared with other tests to evaluate PZA susceptibility in M. tuberculosis isolates. Methods: In this study, we have used the PCR-based in vitro-synthesized-PZase assay to analyze the specific pyrazinamidase (PZase) activity of PncA mutants and have correlated the results to the PZA susceptibility phenotype determined by culture in acidic agar medium at pH 6.0. A set of 23 clinical isolates displaying mutated pncA genes (11 PZA-resistant and 12 PZA-susceptible) and 55 PZA-susceptible clinical strains displaying a wild-type pncA gene were tested. Results: Among the 23 mutants tested, 4 corresponded to mutations not reported before (I5T, Y99S, T142R and P77L+V131G). Of the 11 PncA mutants expressed from PZA-resistant clinical isolates, 9 were expressed in vitro at yields > 50% relative to the wild type enzyme. Among them, 6 enzymes (T47P, H51P, H51R, H57D, L85R and T142R) showed no detectable activity, while the relative activities for the 3 others, V9A (27%), G97D (10%) and A146V (28%) were low compared to the wild-type PZase. The remaining two mutants, I5T and V9G, presented very low relative expression (5%) and relative activities values of 12 and 1%, respectively. Twelve mutants were expressed from PZA-susceptible isolates. Their expression was similar to the wild type enzyme and behaved as active pyrazinamidase with specific relative activities ranging from 34 to 314%. Finally, discrepant results were observed for two mutants, V7A and P62T. Conclusion: Thus, this study provides the proof of concept that the PCR-based in vitro-synthesized-PZase assay represents a promising rapid approach for the evaluation of PZA susceptibility based on the estimation of the relative PZase activity from clinical isolates.


Assuntos
Amidoidrolases/metabolismo , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Amidoidrolases/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mutação
16.
Clin Lung Cancer ; 19(1): 65-73.e7, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28780976

RESUMO

INTRODUCTION: A substantial fraction of non-small-cell lung cancers (NSCLCs) harbor targetable genetic alterations. In this study, we analyzed the feasibility and clinical utility of integrating a next-generation sequencing (NGS) panel into our routine lung cancer molecular subtyping algorithm. PATIENTS AND METHODS: After routine pathologic and molecular subtyping, we implemented an amplicon-based gene panel for DNA analysis covering mutational hot spots in 22 cancer genes in consecutive advanced-stage NSCLCs. RESULTS: We analyzed 109 tumors using NGS between December 2014 and January 2016. Fifty-six patients (51%) were treatment-naive and 82 (75%) had lung adenocarcinomas. In 89 cases (82%), we used samples derived from lung cancer diagnostic procedures. We obtained successful sequencing results in 95 cases (87%). As part of our routine lung cancer molecular subtyping protocol, single-gene testing for EGFR, ALK, and ROS1 was attempted in nonsquamous and 3 squamous-cell cancers (n = 92). Sixty-nine of 92 samples (75%) had sufficient tissue to complete ALK and ROS1 immunohistochemistry (IHC) and NGS. With the integration of the gene panel, 40 NSCLCs (37%) in the entire cohort and 30 NSCLCs (40%) fully tested for ALK and ROS1 IHC and NGS had actionable mutations. KRAS (24%) and EGFR (10%) were the most frequently mutated actionable genes. Ten patients (9%) received matched targeted therapies, 6 (5%) in clinical trials. CONCLUSION: The combination of IHC tests for ALK and ROS1 and amplicon-based NGS is applicable in routine clinical practice, enabling patient selection for genotype-tailored treatments.


Assuntos
Quinase do Linfoma Anaplásico/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/diagnóstico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Idoso , Estudos de Coortes , Testes Diagnósticos de Rotina , Amplificação de Genes , Humanos , Neoplasias Pulmonares/genética , Estadiamento de Neoplasias , Patologia Molecular , Seleção de Pacientes , Prognóstico , Carcinoma de Pequenas Células do Pulmão/genética
17.
Gastroenterology ; 154(1): 181-194.e20, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28912018

RESUMO

BACKGROUND & AIMS: Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC. METHODS: We collected blood samples from 3 relatives with CRC in Spain (65, 62, and 40 years old at diagnosis) and performed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases (502 additional families). We carried out protein-dependent yeast growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants. RESULTS: A total of 42 unique or rare (population minor allele frequency below 1%) nonsynonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11 of 503 CRC families, a significantly greater proportion than in the control population (34 of 4300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability, or expression and/or function. CONCLUSIONS: In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC.


Assuntos
Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Adulto , Idoso , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Espanha
18.
PLoS One ; 12(11): e0187312, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29095867

RESUMO

The involvement of GALNT12 in colorectal carcinogenesis has been demonstrated but it is not clear to what extent it is implicated in familial CRC susceptibility. Partially inactivating variant, NM_024642.4:c.907G>A, p.(D303N), has been previously detected in familial CRC and proposed as the causative risk allele. Since phenotypes of the described carrier families showed not only CRC but also a polyp history, we hypothesized that GALNT12 could be involved in adenoma predisposition and consequently, in hereditary polyposis CRC syndromes. For that purpose, we have screened the GALNT12 gene in germline DNA from 183 unrelated attenuated polyposis patients. c.907G>A, p.(D303N) was detected in 4 cases (MAF = 1.1%) and no other candidate variants were found. After segregation studies, LOH analyses, glycosylation pattern tests and case-control studies, our results did not support the role of c.907G>A, p.(D303N) as a high-penetrance risk allele for polyposis CRC.


Assuntos
Síndrome de Gardner/genética , Predisposição Genética para Doença , N-Acetilgalactosaminiltransferases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Linhagem
19.
Pharmacogenomics ; 18(13): 1215-1223, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28745575

RESUMO

AIM: To identify genetic variants associated with capecitabine toxicity in fluoropyrimidine pathway genes using exome sequencing. PATIENTS & METHODS: Exomes from eight capecitabine-treated patients with severe adverse reactions (grade >2), among a population of 319, were sequenced (Ion Proton). SNPs in genes classified as potentially damaging (Sorting Intolerant from Tolerant and Polymorphism Phenotyping v2) were tested for association with toxicity in a validation cohort of 319 capecitabine-treated patients. RESULTS: A total of 17 nonsynonymous genetic variants were identified. Of these, five putative damaging SNPs in DPYD, ABCC4 and MTHFR were genotyped in the validation cohort. DPYD rs1801160 was associated with the risk of toxicity (p = 0.029) and MTHFR rs1801133 with delayed administration of chemotherapy due to toxicity (p = 0.047). CONCLUSION: Exome sequencing revealed two specific biomarkers of the risk of toxicity to capecitabine.


Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Capecitabina/efeitos adversos , Neoplasias Colorretais/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Exoma/genética , Polimorfismo de Nucleotídeo Único/genética , Pirimidinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Biomarcadores/metabolismo , Capecitabina/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade
20.
Oncotarget ; 8(15): 24429-24436, 2017 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-28416736

RESUMO

To characterize clinical features of a recurrent alteration in 16p13.12-p13.11 in Colorectal Cancer (CRC), mainly in Early-onset subgroup (EOCRC), and to assess the status of NOMO1, a gene located in that region, we analyzed differential clinicopathological, familial and molecular features of CRC subsets with and without alterations in the 16p13.12-p13.11, in global and EOCRC groups. We confirmed the region by fluorescence in-situ hybridization, and Quantitative Real-Time PCR analyzed the status of NOMO1 in different age-of-onset and Microsatellite Instability (MSI)-status CRC subsets. Both age-of-onset subsets were subsequently extended to further confirm NOMO1 gene changes. 16p13.12-p13.11 alterations were observed in 23.3% of CRCs, and was detected more frequently in EOCRC (33.3%) than in late-onset CRC (16.3%). The group with deletion in 16p showed a higher frequency of females and left-colon locations; a better prognosis; and higher Chromosomal Instability. Within the primary EOCRC population, 34 out of 34 of tumours showed a homozygous deletion in NOMO1, while in the late-onset population only 2 of the 17 tumours (11.7%) showed it. In the extended group, we found 61 out of 75 EOCRC patients (81.3%) with homozygous deletion and 7 patients (9.3%) with heterozygous deletion of NOMO1; moreover, in the new 50 late-onset patients, the proportions of deletions decreased. Microsatellite-Stable (MSS) EOCRC showed a very high proportion of homozygous loss of NOMO1 (54 of 59 cases, 91.5%), while the deletion was observed in only 7 out of 16 MSI cases. Deletion of NOMO1 is a molecular marker predominantly associated with EOCRC, particularly MSS subtypes.


Assuntos
Neoplasias Colorretais/genética , Proteína Nodal/genética , Adulto , Neoplasias Colorretais/patologia , Feminino , Deleção de Genes , Humanos , Masculino , Prognóstico
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