Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Front Mol Biosci ; 7: 169, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33195396

RESUMO

In recent years it has become clear that pathogenic variants in PALB2 are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of uncertain significance (VUS) in PALB2, which are increasingly identified through clinical genetic testing, is unclear. Here we review recent advances in the functional characterization of VUS in PALB2. A combination of assays has been used to assess the impact of PALB2 VUS on its function in DNA repair by homologous recombination, cell cycle regulation and the control of cellular levels of reactive oxygen species (ROS). We discuss the outcome of this comprehensive analysis of PALB2 VUS, which showed that VUS in PALB2's Coiled-Coil (CC) domain can impair the interaction with BRCA1, whereas VUS in its WD40 domain affect PALB2 protein stability. Accordingly, the CC and WD40 domains of PALB2 represent hotspots for variants that impair PALB2 protein function. We also provide a future perspective on the high-throughput analysis of VUS in PALB2, as well as the functional characterization of variants that affect PALB2 RNA splicing. Finally, we discuss how results from these functional assays can be valuable for predicting cancer risk and responsiveness to cancer therapy, such as treatment with PARP inhibitor- or platinum-based chemotherapy.

2.
Cancers (Basel) ; 12(10)2020 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003546

RESUMO

Recent studies have shown that the efficacy of PARP inhibitors in epithelial ovarian carcinoma (EOC) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2 deficient EOC. A robust method with which to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we investigated the proficiency of a functional HR assay based on the detection of RAD51 foci, the REcombination CAPacity (RECAP) test, in identifying HRD tumors in a cohort of prospectively collected epithelial ovarian carcinomas (EOCs). Of the 39 high-grade serous ovarian carcinomas (HGSOC), the RECAP test detected 26% (10/39) to be HRD, whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Of the HRD tumors that could be sequenced, 8/9 showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation, indicating that the RECAP test reliably identifies HRD, including but not limited to tumors related to BRCA1/2 deficiency. Furthermore, we found a trend towards better overall survival (OS) of HGSOC patients with RECAP-identified HRD tumors compared to patients with HRP tumors. This study shows that the RECAP test is an attractive alternative to DNA-based HRD tests, and further development of a clinical grade RECAP test is clearly warranted.

3.
Clin Cancer Res ; 26(17): 4559-4568, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32546644

RESUMO

PURPOSE: Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway. EXPERIMENTAL DESIGN: Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. RESULTS: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. CONCLUSIONS: We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.

4.
Nat Commun ; 11(1): 3083, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576828

RESUMO

Short-term fasting protects tumor-bearing mice against the toxic effects of chemotherapy while enhancing therapeutic efficacy. We randomized 131 patients with HER2-negative stage II/III breast cancer, without diabetes and a BMI over 18 kg m-2, to receive either a fasting mimicking diet (FMD) or their regular diet for 3 days prior to and during neoadjuvant chemotherapy. Here we show that there was no difference in toxicity between both groups, despite the fact that dexamethasone was omitted in the FMD group. A radiologically complete or partial response occurs more often in patients using the FMD (OR 3.168, P = 0.039). Moreover, per-protocol analysis reveals that the Miller&Payne 4/5 pathological response, indicating 90-100% tumor-cell loss, is more likely to occur in patients using the FMD (OR 4.109, P = 0.016). Also, the FMD significantly curtails chemotherapy-induced DNA damage in T-lymphocytes. These positive findings encourage further exploration of the benefits of fasting/FMD in cancer therapy. Trial number: NCT02126449.


Assuntos
Neoplasias da Mama/dietoterapia , Neoplasias da Mama/tratamento farmacológico , Dieta , Jejum , Adulto , Idoso , Animais , Índice de Massa Corporal , Quimioterapia Adjuvante , Dano ao DNA , Dexametasona/uso terapêutico , Feminino , Glucose/química , Humanos , Análise de Intenção de Tratamento , Menopausa , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Países Baixos , Qualidade de Vida , Receptor ErbB-2/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia
5.
Genet Med ; 22(8): 1427-1428, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32576986

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

6.
Genet Med ; 22(8): 1355-1365, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32398771

RESUMO

PURPOSE: Current interpretation guidelines for germline variants in high-risk cancer susceptibility genes consider predicted loss-of-function (LoF) variants, such as nonsense variants and variants in the canonical splice site sequences ofBRCA2, to be associated with high cancer risk. However, some variant alleles produce alternative transcripts that encode (partially) functional protein isoforms leading to possible incorrect risk estimations. For accurate classification of variants it is therefore essential that alternative transcripts are identified and functionally characterized. METHODS: We systematically evaluated a large panel of human BRCA2 variants for the production of alternative transcripts and assessed their capacity to exert BRCA2 protein functionality. Evaluated variants included all single-exon deletions, various multiple-exon deletions, intronic variants at the canonical splice donor and acceptor sequences, and variants that previously have been shown to affect messenger RNA (mRNA) splicing in carriers. RESULTS: Multiple alternative transcripts encoding (partially) functional protein isoforms were identified (e.g., ∆[E4-E7], ∆[E6-E7], ∆E[6q39_E8], ∆[E10], ∆[E12], ∆E[12-14]). Expression of these transcripts did attenuate the impact of predicted LoF variants such as the canonical splice site variants c.631+2T>G, c.517-2A>G, c.6842-2A>G, c.6937+1G>A, and nonsense variants c.491T>A, c.581G>A, and c.6901G>T. CONCLUSION: These results allow refinement of variant interpretation guidelines for BRCA2 by providing insight into the functional consequences of naturally occurring and variant-related alternative splicing events.

8.
Cancer Res ; 80(7): 1374-1386, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046981

RESUMO

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.


Assuntos
Processamento Alternativo , Proteína BRCA2/genética , Predisposição Genética para Doença , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Éxons/genética , Feminino , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/genética
9.
Nat Commun ; 10(1): 5296, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757951

RESUMO

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.


Assuntos
Neoplasias da Mama/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Técnicas Genéticas , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Animais , DNA Complementar , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Citometria de Fluxo , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , Camundongos , Camundongos Knockout
10.
Clin Cancer Res ; 25(24): 7517-7526, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31492746

RESUMO

PURPOSE: Whether endometrial carcinoma (EC) should be considered part of the gBRCA1/2-associated hereditary breast and ovarian cancer (HBOC) syndrome is topic of debate. We sought to assess whether ECs occurring in gBRCA carriers are enriched for clinicopathologic and molecular characteristics, thereby supporting a causal relationship. EXPERIMENTAL DESIGN: Thirty-eight gBRCA carriers that developed EC were selected from the nationwide cohort study on hereditary breast and ovarian cancer in the Netherlands (HEBON), and these were supplemented with four institutional cases. Tumor tissue was retrieved via PALGA (Dutch Pathology Registry). Nineteen morphologic features were scored and histotype was determined by three expert gynecologic pathologists, blinded for molecular analyses (UCM-OncoPlus Assay including 1213 genes). ECs with LOH of the gBRCA-wild-type allele (gBRCA/LOHpos) were defined "gBRCA-associated," those without LOH (gBRCA/LOHneg) were defined "sporadic." RESULTS: LOH could be assessed for 40 ECs (30 gBRCA1, 10 gBRCA2), of which 60% were gBRCA/LOHpos. gBRCA/LOHpos ECs were more frequently of nonendometrioid (58%, P = 0.001) and grade 3 histology (79%, P < 0.001). All but two were in the TP53-mutated TCGA-subgroup (91.7%, P < 0.001). In contrast, gBRCA/LOHneg ECs were mainly grade 1 endometrioid EC (94%) and showed a more heterogeneous distribution of TCGA-molecular subgroups: POLE-mutated (6.3%), MSI-high (25%), NSMP (62.5%), and TP53-mutated (6.3%). CONCLUSIONS: We provide novel evidence in favor of EC being part of the gBRCA-associated HBOC-syndrome. gBRCA-associated ECs are enriched for EC subtypes associated with unfavorable clinical outcome. These findings have profound therapeutic consequences as these patients may benefit from treatment strategies such as PARP inhibitors. In addition, it should influence counseling and surveillance of gBRCA carriers.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Mutação em Linhagem Germinativa , Perda de Heterozigosidade , Adulto , Idoso , Estudos de Coortes , Neoplasias do Endométrio/classificação , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores
11.
J Med Genet ; 56(9): 581-589, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31186341

RESUMO

BACKGROUND: The currently known breast cancer-associated single nucleotide polymorphisms (SNPs) are presently not used to guide clinical management. We explored whether a genetic test that incorporates a SNP-based polygenic risk score (PRS) is clinically meaningful in non-BRCA1/2 high-risk breast cancer families. METHODS: 101 non-BRCA1/2 high-risk breast cancer families were included; 323 cases and 262 unaffected female relatives were genotyped. The 161-SNP PRS was calculated and standardised to 327 population controls (sPRS). Association analysis was performed using a Cox-type random effect regression model adjusted by family history. Updated individualised breast cancer lifetime risk scores were derived by combining the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm breast cancer lifetime risk with the effect of the sPRS. RESULTS: The mean sPRS for cases and their unaffected relatives was 0.70 (SD=0.9) and 0.53 (SD=0.9), respectively. A significant association was found between sPRS and breast cancer, HR=1.16, 95% CI 1.03 to 1.28, p=0.026. Addition of the sPRS to risk prediction based on family history alone changed screening recommendations in 11.5%, 14.7% and 19.8 % of the women according to breast screening guidelines from the USA (National Comprehensive Cancer Network), UK (National Institute for Health and Care Excellence and the Netherlands (Netherlands Comprehensive Cancer Organisation), respectively. CONCLUSION: Our results support the application of the PRS in risk prediction and clinical management of women from genetically unexplained breast cancer families.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Estudos de Casos e Controles , Tomada de Decisão Clínica , Gerenciamento Clínico , Genótipo , Humanos , Pessoa de Meia-Idade , Linhagem , Prognóstico , Modelos de Riscos Proporcionais , Medição de Risco , Adulto Jovem
12.
Nat Med ; 25(5): 838-849, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011202

RESUMO

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.


Assuntos
Organoides/patologia , Neoplasias Ovarianas/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Genômica , Xenoenxertos , Humanos , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Medicina de Precisão
13.
J Med Genet ; 56(7): 453-460, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30890586

RESUMO

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.


Assuntos
Processamento Alternativo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Alelos , Perfilação da Expressão Gênica , Estudos de Associação Genética/métodos , Mutação em Linhagem Germinativa , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido , Sítios de Splice de RNA
14.
Clin Cancer Res ; 25(3): 1087-1097, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30413523

RESUMO

PURPOSE: The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics. EXPERIMENTAL DESIGN: Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort. RESULTS: Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001). CONCLUSIONS: HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.


Assuntos
Neoplasias do Endométrio/genética , Endométrio/metabolismo , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Idoso , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Hibridização Genômica Comparativa , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Endométrio/efeitos dos fármacos , Endométrio/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Estudos Prospectivos , Rad51 Recombinase/metabolismo
15.
Genet Med ; 21(2): 293-302, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29988080

RESUMO

PURPOSE: Genetic testing has uncovered large numbers of variants in the BRCA2 gene for which the clinical significance is unclear. Cancer risk prediction of these variants of uncertain significance (VUS) can be improved by reliable assessment of the extent of impairment of the tumor suppressor function(s) of BRCA2. METHODS: Here, we evaluated the performance of the mouse embryonic stem cell (mESC)-based functional assay on an extensive set of BRCA2 missense variants. RESULTS: Whereas all 20 nonpathogenic (class 1/2) variants were able to complement the cell lethal phenotype induced by loss of endogenous mouse Brca2, only 1 out of 15 pathogenic (class 4/5) variants (p.Gly2609Asp) was able to do so. However, in this variant the major tumor suppressive activity of BRCA2, i.e., homology directed repair (HDR), was severely abrogated. Among 43 evaluated VUS (class 3), 7 were unable to complement the lethal phenotype of mouse Brca2 loss while 7 other variants displayed a more severe reduction of HDR activity than observed for class 1/ 2 variants. CONCLUSION: The mESC-based BRCA2 functional assay can reliably determine the functional impact of VUS, distinguish between pathogenic and nonpathogenic variants, and may contribute to improved cancer risk estimation for BRCA2 VUS carriers.


Assuntos
Neoplasias da Mama/genética , Genes BRCA2 , Variação Genética , Células-Tronco Embrionárias Murinas , Animais , Antineoplásicos/farmacologia , Western Blotting , Ciclo Celular , Células Cultivadas , Cisplatino/farmacologia , Fluorbenzenos/farmacologia , Teste de Complementação Genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Mutação de Sentido Incorreto , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
16.
J Mol Diagn ; 20(5): 600-611, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29936257

RESUMO

BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1 promoter hypermethylation and pedigree analysis were also performed. In the training cohort, 45 of 46 germline BRCA1/2 variants were detected (sensitivity, 98%). In the prospective cohort (n = 62), all six germline variants were identified (sensitivity, 100%), together with five somatic BRCA1/2 variants and eight cases with BRCA1 promoter hypermethylation. In four BRCA1/2 variant-negative patients, surveillance or prophylactic management options were offered on the basis of positive family histories. We conclude that BRCA1/2 formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2 variants. When taking family history of BRCA1/2 variant-negative patients into account, tumor BRCA1/2 variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counseling and simultaneously selects patients who benefit most from targeted treatment.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Testes Genéticos , Variação Genética , Neoplasias Ovarianas/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Metilação de DNA/genética , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética
17.
BMC Cancer ; 16: 78, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26860465

RESUMO

BACKGROUND: The high incidence of breast cancer has sparked the development of novel targeted and personalized therapies. Personalization of cancer treatment requires reliable prediction of chemotherapy responses in individual patients. Effective selection can prevent unnecessary treatment that would mainly result in the unwanted side effects of the therapy. This selection can be facilitated by characterization of individual tumors using robust and specific functional assays, which requires development of powerful ex vivo culture systems and procedures to analyze the response to treatment. METHODS: We optimized culture methods for primary breast tumor samples that allowed propagation of tissue ex vivo. We combined several tissue culture strategies, including defined tissue slicing technology, growth medium optimization and use of a rotating platform to increase nutrient exchange. RESULTS: We could maintain tissue cultures for at least 7 days without losing tissue morphology, viability or cell proliferation. We also developed methods to determine the cytotoxic response of individual tumors to the chemotherapeutic treatment FAC (5-FU, Adriamycin [Doxorubicin] and Cyclophosphamide). Using this tool we designated tumors as sensitive or resistant and distinguished a clinically proven resistant tumor from other tumors. CONCLUSION: This method defines conditions that allow ex vivo testing of individual tumor responses to anti-cancer drugs and therefore might improve personalization of breast cancer treatment.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Cultura de Tecidos/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/patologia , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fluoruracila/administração & dosagem , Humanos , Medicina de Precisão , Células Tumorais Cultivadas/efeitos dos fármacos
18.
Hum Mutat ; 37(4): 331-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26777316

RESUMO

In 2008, the International Agency for Research on Cancer (IARC) proposed a system for classifying sequence variants in highly penetrant breast and colon cancer susceptibility genes, linked to clinical actions. This system uses a multifactorial likelihood model to calculate the posterior probability that an altered DNA sequence is pathogenic. Variants between 5%-94.9% (class 3) are categorized as variants of uncertain significance (VUS). This interval is wide and might include variants with a substantial difference in pathogenicity at either end of the spectrum. We think that carriers of class 3 variants would benefit from a fine-tuning of this classification. Classification of VUS to a category with a defined clinical significance is very important because for carriers of a pathogenic mutation full surveillance and risk-reducing surgery can reduce cancer incidence. Counselees who are not carriers of a pathogenic mutation can be discharged from intensive follow-up and avoid unnecessary risk-reducing surgery. By means of examples, we show how, in selected cases, additional data can lead to reclassification of some variants to a different class with different recommendations for surveillance and therapy. To improve the clinical utility of this classification system, we suggest a pragmatic adaptation to clinical practice.


Assuntos
Predisposição Genética para Doença , Variação Genética , Neoplasias/diagnóstico , Neoplasias/genética , Penetrância , Incerteza , Gerenciamento Clínico , Aconselhamento Genético , Testes Genéticos , Humanos , Modelos Estatísticos , Mutação , Guias de Prática Clínica como Assunto
19.
BMC Cancer ; 15: 652, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26438237

RESUMO

BACKGROUND: Preclinical evidence shows that short-term fasting (STF) protects healthy cells against side effects of chemotherapy and makes cancer cells more vulnerable to it. This pilot study examines the feasibility of STF and its effects on tolerance of chemotherapy in a homogeneous patient group with early breast cancer (BC). METHODS: Eligible patients had HER2-negative, stage II/III BC. Women receiving (neo)-adjuvant TAC (docetaxel/doxorubicin/cyclophosphamide) were randomized to fast 24 h before and after commencing chemotherapy, or to eat according to the guidelines for healthy nutrition. Toxicity in the two groups was compared. Chemotherapy-induced DNA damage in peripheral blood mononuclear cells (PBMCs) was quantified by the level of γ-H2AX analyzed by flow cytometry. RESULTS: Thirteen patients were included of whom seven were randomized to the STF arm. STF was well tolerated. Mean erythrocyte- and thrombocyte counts 7 days post-chemotherapy were significantly higher (P = 0.007, 95 % CI 0.106-0.638 and P = 0.00007, 95 % CI 38.7-104, respectively) in the STF group compared to the non-STF group. Non-hematological toxicity did not differ between the groups. Levels of γ-H2AX were significantly increased 30 min post-chemotherapy in CD45 + CD3- cells in non-STF, but not in STF patients. CONCLUSIONS: STF during chemotherapy was well tolerated and reduced hematological toxicity of TAC in HER2-negative BC patients. Moreover, STF may reduce a transient increase in, and/or induce a faster recovery of DNA damage in PBMCs after chemotherapy. Larger studies, investigating a longer fasting period, are required to generate more insight into the possible benefits of STF during chemotherapy. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01304251 , March 2011.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Jejum , Receptor ErbB-2/deficiência , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Dano ao DNA , Feminino , Histonas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Terapia Neoadjuvante , Gradação de Tumores , Estadiamento de Neoplasias , Projetos Piloto , Fatores de Tempo , Resultado do Tratamento
20.
J Med Genet ; 52(4): 224-30, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25643705

RESUMO

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.


Assuntos
Proteína BRCA1/genética , Biologia Computacional/métodos , Gráficos por Computador , Internet , Mutação de Sentido Incorreto , Software , Neoplasias da Mama/genética , Análise Mutacional de DNA , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Conjuntos de Dados como Assunto , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Neoplasias Ovarianas/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA