Application of next-generation sequencing in detection of BRCA1/2 and homologous recombination repair pathway multi-genes germline mutation and correlation analysis / 中华预防医学杂志

Objective: To investigate the germline mutation status of related genes in breast cancer patients and high-risk individuals by next-generation sequencing. To analyze the correlations between homologous recombination repair (HR) pathway gene mutation status and clinicopathological characteristics of breast cancer patients. To supplement the database of breast cancer related gene mutations in Chinese population. Methods: This study is a cross-sectional study. From October 2020 to September 2021, whole blood samples were collected from 350 breast cancer patients and 49 high-risk individuals, admitted to Peking University People's Hospital and accepted genetic testing voluntarily. Germline mutations in 32 breast cancer related genes were detected by NGS. The clinicopathological characteristics, including age at the onset, family history, unilateral/bilateral tumor, Luminal typing (Luminal A subtype, Luminal B subtype, HER2-enriched subtype and triple negative breast cancer), tumor size and metastasis, were analyzed, and the correlations between HR pathway gene mutation status and clinicopathological characteristics were analyzed by Chi-squared test and Fisher's exact probability test. Results: Among 350 breast cancer patients, 64 (18.3%) cases carried gene pathogenic mutations (including pathogenic and likely pathogenic mutations), including 47 (13.4%) in BRCA1/2, 16 (4.6%) in non-BRCA1/2 genes, 1 (0.3%) in BRCA2 and FANCL. Among 49 high-risk individuals, 7 (14.3%) cases carried gene pathogenic mutations, including 6 (12.3%) in BRCA1/2 and 1 (2%) in ATM genes. BRCA1/2 pathogenic mutations were associated with age at the onset (18%, 8.7%, χ²=6.346, P=0.012), and the BRCA1/2 pathogenic mutation frequency was higher in patients diagnosed at age ≤45 years. HR pathway gene mutations (including pathogenic, likely pathogenic and uncertain significance mutations) were correlated with unilateral/bilateral tumor (49.5%, 68.4%, χ²=4.841, P=0.028) and Luminal typing (45.7%, 62.2%, 32%, 60%, χ²=12.004, P=0.007), and the HR mutation frequencies were higher in patients with bilateral tumor, Luminal B breast cancer and triple negative breast cancer (TNBC). Conclusion: The BRCA1/2 pathogenic mutation frequency in high-risk individuals is similar to that in breast cancer patients, and BRCA1/2 testing is helpful to guide breast cancer screening and prevention in high-risk individuals. Patients with early onset breast cancer, bilateral breast cancer, Luminal B breast cancer and TNBC have higher mutation frequencies of HR pathway genes, and HR pathway genes testing should be conducted as soon as possible to provide laboratory evidence for diagnosis, treatment, prognosis and risk evaluation of breast cancer.

A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.

Alteração da via da recombinação homóloga de reparo de DNA no câncer epitelial de ovário / Homologous recombination deficiency in ovarian carcinoma

Justificativa:Os carcinomas de ovário apresentam deficiência da via da recombinação homóloga de reparo de DNA em aproximadamente 50% dos casos, em 15 a 20% dos casos atribuída a mutações germinativas em BRCA1 ou BRCA2. Tumores com mutações germinativas em BRCA1 ou BRCA2 são mais sensíveis aos tratamentos com inibidores de PARP e com esquemas quimioterápicos baseados em platina. O papel de outras alterações da via da recombinação homóloga na sensibilidade a essas modalidades de tratamento bem como a forma de identificar os tumores com deficiência dessa via ainda não estão definidos. Escores avaliando o perfil de alterações estruturais genômicas causadas especificamente por deficiência da via da recombinação homóloga têm sido estudados como potenciais marcadores da deficiência desta via. Nosso objetivo foi caracterizar uma coorte de pacientes com carcinoma de ovário quanto à deficiência da via da recombinação homóloga utilizando escores calculados a partir das alterações do número de cópias genômicas e de perdas de heterozigose e avaliar o papel destes escores na identificação de pacientes com sensibilidade prolongada à platina. Métodos: Selecionamos uma coorte de 31 pacientes com carcinoma de ovário com recidiva classificada clinicamente com platina resistente e que foram reexpostas ao tratamento com quimioterapia baseada em platina. Amostras de tumores armazenados em parafina de 14 pacientes foram submetidas ao ensaio ONCOSCAN para avaliação de alterações de números de cópias, perda de heterozigose e identificação de mutações específicas em 9 genes relacionados a neoplasias. A partir destes dados caracterizamos os escores de Telomeric Allelic Imbalance (TAI), escore de perda de heterozigose (cnLOH+L) escore de Large Scale Transition (LST) e escore de deficiência da recombinação homóloga (HRD) em cada tumor e avaliamos a sua associação com a taxa de resposta à quimioterapia, sobrevida global e fatores clínico patológicos. Resultados: Na coorte de 31 pacientes, DNA foi extraído de 28 amostras e 15 amostras de 14 pacientes foram analisadas de forma efetiva. Na maioria dos casos encontramos o padrão esperado de grande instabilidade genômica com diversas regiões de ganhos e perdas do genoma e regiões de perda de heterozigose. Todos os casos analisados mostraram cnLOH ou delLOH no cromossomo 17, 12 de 14 pacientes apresentaram cnLOH ou delLOH no braço longo do cromossomo 13 e o cromossomo 22 apresentou perdas ou ganhos na maioria dos casos. Mutação em TP53 foi encontrada em 2 casos, mutação em NRAS em 2 casos, e um caso com mutação em TP53apresentou mutação em PTEN associada a deleção da região do PTEN. A mediana dos escores foi de 19,5 para TAI, 12,5 para cnLOH+L, 26,0 para LST e 6,3 para HRD. Levando-se em conta os escores com pontos de corte previamente definidos na literatura encontramos 10 de 14 (escore cnLOH+L) e 9 de 14 (escore LST) pacientes com tumores com escores altos sugerindo deficiência da via da recombinação homóloga. Tanto a coorte como um todo quanto as 14 pacientes submetidas à análise molecular apresentaram taxa de resposta acima do esperado sendo 16 de 31 pacientes (51,6%) e 7 de 14 pacientes (50,0) respectivamente. Não houve diferença estatisticamente significativa entre as taxas de resposta das pacientes de com escores altos versus baixos, embora 6 de 10 pacientes com escore cnLOH+L elevado tenham apresentado resposta e 1 de 4 pacientes com escore cnLOH+L baixo tenham apresentado resposta e 6 de 9 pacientes com escore LST elevado tenham apresentado resposta e 1 de 5 pacientes com escore LST baixo tenham apresentado resposta. Numericamente os escores cnLOH+L, LST e HDR foram mais altos nas pacientes que responderam quando comparados com os escores das pacientes que não responderam, com medianas deTAI = 19,0 vs 24,0, TAIm = 16,0 vs 19,0, cnLOH+L 13 vs 12, LST = 28,0 vs 18,0, LSTm = -4,0 vs -20,0, HRD 8,7 vs 0,3 para respondedoras versus não respondedoras. Essa diferença não foi estatisticamente significativa. A sobrevida global mediana foi 13,4 meses desde o inicio do tratamento de reexposição à platina e não houve diferença de acordo com os valores dos escores. Dentre os fatores clinicopatológicos a presença de história familiar de câncer de mama ou ovário ou a história pessoal de câncer de mama foi o único fator associado a uma maior taxa de resposta. Os tumores primários apresentaram escore TAI mais elevado que os tumores coletados no momento da recidiva e comparando duas amostras de uma mesma paciente houve diferença na classificação da deficiência da via da recombinação homóloga de acordo com os escores cnLOH+L e LST. Conclusões: Os escores de deficiência da via da recombinação homóloga se mostraram potenciais marcadores de resposta à reexposição à platina no cenário de doença platina resistente, necessitando melhor definição dos pontos de corte e do impacto da heterogeneidade tumoral e da necessidade de avaliação do material coletado no momento da recidiva. História familiar positiva é um fator clínico capaz de identificar pacientes com mais chance de resposta á reexposição á platina. Nossos dados fortalecem a hipótese da contribuição de mutações em PTEN para o desenvolvimento de deficiência da via da recombinação homóloga e o papel das mutações de NRAS nos carcinomas serosos de ovário.

Background: Homologous recombination deficiency s presente in up to 50% of ovarian carcinomas, in 15 to 20% due to germline BRCA1 or BRCA2 mutations. BRCA mutated tumors are more sensitive to PARP inhibitors and platinum based chemotherapy. The role of other homologous recombination mechanisms on PARP inhibitors and platinum sensitivity as well as the methods to identify homologous recombination deficiency is not clear yet. Scores evaluating structural genomic abnormalities caused specifically by homologous recombination deficiency have been studied as markers of deficiency of this DNA repair pathway. The main objective of the present study was to characterize a cohort of ovarian cancer patients regarding homologous recombination deficiency using scores based on copy number alterations and loss of heterozygosity and to evaluate the impact of these scores in prolonged platinum sensitivity. Methods: We selected a cohort of 31 ovarian cancer patients with platinum resistant recurrence reexposed to platinum based chemotherapy. Paraffin embedded tumor samples from 14 patients were analyzed through ONCOSCAN assay for copy number alterations, loss of heterozygosity and specific point mutations in 9 cancer related genes. Based on these alterations we calculated the scores Telomeric Allelic Imbalance (TAI), loss of heterozygosity score (cnLOH+L) Large Scale Transition score (LST) and homologous recombination deficiency score (HRD) for each tumor sample and tested their association to response rate to platinum rechallenge, overall survival and to the clinical pathologic factors. Results: From the cohort of 31 patients, DNA was extracted from 28 samples and 15 samples from 14 patients were effectively analyzed. Most cases presented the expected high genomic instability pattern with lots of genomic losses, genomic gains and loss of heterozygosity segments. All cases showed cnLOH or delLOH in chromosome 17, 12 of 14 patients showed cnLOH or delLOH in the long arm of chromosome 13, and chromosome 22 presented losses or gains in most of cases.TP53 mutations were present in 2 cases, NRAS mutations in 2 cases, and one patient with TP53 mutation showed also a PTEN mutation together with a genomic loss in PTEN region. Median scores were 19,5 for TAI, 12,5 for cnLOH+L, 26,0 for LST and 6,3 for HRD. Considering previously literature defined cutoffs we found 10 out of 14 (for cnLOH+L score) and 9 out of 14 (for LST score) patients with tumors with high scores suggesting homologous recombination deficiency. In the cohort of 31 patients 16 had response to platinum reexposition and in the cohort of 14 patients analyzed by ONCOSCAN assay 7 patients had response. There was no statistically significant difference between response rates for high versus low scores, even though 6 out of 10 patient with high cnLOH+L score while 1 out of 4 patients with low cnLOH+L score had response and 6 out of 9 patient with high cnLOH+L score while 1 out of 5 patients with low cnLOH+L score had response. Numerically, the scores cnLOH+L, LST and HDR were higher in patients with response to treatment compared to those without a response, with medians of TAI = 19,0 vs 24,0, TAIm = 16,0 vs 19,0, cnLOH+L 13 vs 12, LST = 28,0 vs 18,0, LSTm = -4,0 vs -20,0, HRD 8,7 vs 0,3 for responders versus non responders respectively. These differences were not statistically significant. Median overall survival was 13,4 months from the beginning of platinum rechallenge and there was no difference in survival according to scores. Among clinical pathologic factors, family history of breast or ovarian cancer or personal history of breast cancer was associated to a higher response rate to platinum rechallenge. Primary tumors had a higher TAI score compared to recurrent tumors and comparing two different samples from the same patient there was divergence on the homologous recombination deficiency classification according to cnLOH+L and LST scores. Conclusions: Homologous recombination deficiency scores showed to be potential markers of response to platinum rechallenge in the platinum resistant setting. It is still necessary to clarify the best cutoffs for each score, the impact of tumor heterogeneity and the need of tumor samples to be collected in the moment of treatment. Positive family history is a clinical factor predictvie of platinum rechallenge response. Our data support the hypothesis of a role for PTEN in homologous recombination deficiency as well as a role of NRAS mutations in ovarian serous carcinomas.

Assessment of adherence to the guidelines for the management of nausea and vomiting induced by chemotherapy / Avaliação da aderência à diretriz de cuidados para náuseas e vômitos induzidos por quimioterapia

ABSTRACT Objective: To assess adherence of the prescribing physicians in a private cancer care center to the American Society of Clinical Oncology guideline for antiemetic prophylaxis, in the first cycle of antineoplastic chemotherapy. Methods: A total of 139 chemotherapy regimens, of 105 patients, were evaluated retrospectively from 2011 to 2013. Results: We observed 78% of non-adherence to the guideline rate. The main disagreements with the directive were the prescription of higher doses of dexamethasone and excessive use of 5-HT3 antagonist for low risk emetogenic chemotherapy regimens. On univariate analysis, hematological malignancies (p=0.005), the use of two or more chemotherapy (p=0.05) and high emetogenic risk regimes (p=0.012) were factors statistically associated with greater adherence to guidelines. Treatment based on paclitaxel was the only significant risk factor for non-adherence (p=0.02). By multivariate analysis, the chemotherapy of high emetogenic risk most correlated with adherence to guideline (p=0.05). Conclusion: We concluded that the adherence to guidelines is greater if the chemotherapy regime has high emetogenic risk. Educational efforts should focus more intensely on the management of chemotherapy regimens with low and moderate emetogenic potential. Perhaps the development of a computer generated reminder may improve the adherence to guidelines. .

RESUMO Objetivo: Avaliar a adesão dos médicos prescritores, de um centro privado especializado em oncologia, à diretriz de antiêmese profilática da American Society of Clinical Oncology, no primeiro ciclo de quimioterapia antineoplásica. Métodos: Foram avaliados retrospectivamente 139 esquemas de quimioterapia, de 105 pacientes, tratados no período de 2011 a 2013. Resultados: Foram observados 78% de taxa de não adesão à diretriz. As principais discordâncias com a diretriz foram prescrição de doses mais elevadas de dexametasona e uso excessivo de antagonista 5-HT3 para regimes de quimioterapia de risco emetogênico baixo. Pela análise univariada, malignidades hematológicas (p=0,005), uso de dois ou mais quimioterápicos (p=0,05) e regimes de alto risco emetogênico (p=0,012) foram fatores estatisticamente associados a maior adesão à diretriz. O tratamento baseado em paclitaxel foi o único fator estatisticamente significativo para a não adesão (p=0,02). Pela análise multivariada, a quimioterapia de alto risco emetogênico apresentou maior correlação com a adesão à diretriz (p=0,05). Conclusão: Houve maior aderência para a quimioterapia de alto risco emetogênico. Esforços educacionais devem se concentrar mais intensamente na gestão de regimes de quimioterapia com potencial emetogênico baixo e moderado. Talvez o desenvolvimento de lembretes gerados por sistemas informatizados possa melhorar a aderência à diretriz. .

Single nucleotide polymorphisms of nucleotide excision repair and homologous recombination repair pathways and their role in the risk of osteosarcoma

To evaluate the influence of polymorphisms in nucleotide excision repair [NER] and homologous recombination repair [HRR] pathways on the development of osteosarcoma patients. Genotypes of ERCC1 rs11615 and rs3212986, ERCC2 rs1799793 and rs13181, NBN rs709816 and rs1805794, RAD51 rs1801320, rs1801321 and rs12593359, and XRCC3 rs861539 were conducted by Polymerase Chain Reaction Restriction Fragment Length Polymorphism [PCR-RFLP] assay. Total 148 osteosarcoma patients and 296 control subjects were collected from Taizhou First People's Hospital. Conditional logistic regression analyses found that individuals carrying with GA+AA genotype of ERCC2 rs1799793 and GC+CC genotype of NBN rs1805794 were significantly associated with increased risk of osteosarcoma, and the ORs [95%CI] were 1.58 [1.03-2.41] and 2.66 [1.73-4.08], respectively. We found that GA+AA genotype of ERCC2 rs1799793 or GC+CC genotype of NBN rs1805794 were associated with an increased risk of osteosarcoma in females, with ORs [95%CI] of 2.42 [1.20-4.87] and 2.01 [1.07-4.23], respectively. Our results suggest that ERCC2 rs1799793 and NBN rs1805794 polymorphisms were associated with an increased risk for osteosarcoma, which suggests that NER and HRR pathways modulate the risk of developing osteosarcoma

Current insights into inherited bone marrow failure syndromes / 소아과

Inherited bone marrow failure syndrome (IBMFS) encompasses a heterogeneous and complex group of genetic disorders characterized by physical malformations, insufficient blood cell production, and increased risk of malignancies. They often have substantial phenotype overlap, and therefore, genotyping is often a critical means of establishing a diagnosis. Current advances in the field of IBMFSs have identified multiple genes associated with IBMFSs and their pathways: genes involved in ribosome biogenesis, such as those associated with Diamond-Blackfan anemia and Shwachman-Diamond syndrome; genes involved in telomere maintenance, such as dyskeratosis congenita genes; genes encoding neutrophil elastase or neutrophil adhesion and mobility associated with severe congenital neutropenia; and genes involved in DNA recombination repair, such as those associated with Fanconi anemia. Early and adequate genetic diagnosis is required for proper management and follow-up in clinical practice. Recent advances using new molecular technologies, including next generation sequencing (NGS), have helped identify new candidate genes associated with the development of bone marrow failure. Targeted NGS using panels of large numbers of genes is rapidly gaining potential for use as a cost-effective diagnostic tool for the identification of mutations in newly diagnosed patients. In this review, we have described recent insights into IBMFS and how they are advancing our understanding of the disease's pathophysiology; we have also discussed the possible implications they will have in clinical practice for Korean patients.

Type III familial hemophagocytic lymphohistiocytosis susceptibility gene UNC13D involves in homologous recombination repair / 中国实验血液学杂志

This study was aimed to explore the pathogenesis of type III familial hemophagocytic lymphohistiocytosis (FHL3) via susceptibility gene UNC13D involving in homologous recombination repair (HRR) of DNA double-strand break (DSB). By means of DNA homologous recombination repair, the change of homologous recombination repair rate of normal control cells and DR-U2OS cells after down-regulation of UNC13D was detected; the UNC13D gene related function was explored. The results showed that DR-U2OS cells displayed a significant reduction in homologous recombination repair of DNA DSB after siRNA knockdown of UNC13D, compared to its normal control cell counterparts (P < 0.05), suggesting that UNC13D was involved in DNA double-stranded breakage repair. It is concluded that UNC13D gene mutation may be involved in the pathogenesis of FHL3 via its dual effects of both the cytotoxic granule exocytosis and decrease of homologous recombination repair rate after the DNA double-strand break, therefore, providing a new theoretical basis to reveal the pathogenesis of FHL3.

Expression of Several Cell Cycle Regulatory Genes in Cultured AT-1 Cardiomyocytes after Formation of Synchronously Beating Sheets

BACKGROUND AND OBJECTIVE: AT-1 cells have been derived from the left atrial tissue in which the ANF promoter targeted SV40 large T antigen expression. When cultured, clusters of spontaneously contracting cells were observed after 4-5 days and contiguous sheets of synchronously beating cardiomyocytes were formed after 10 days. In this study, expression of several cell cycle regulatory genes were monitored through Northern blot analyses in AT-1 cells during beating and after formation of beating sheets (BS). MATERIALS AND METHOD: AT-1 RNAs were obtained in 3 days after plating, during beating and after formation of BS, and used for Northern blot analyses. RESULTS: alpha-Cardiac myosin heavy chain expression was prominent in beating cells, as would be expected for this contractile protein isoform but ANF was decreased after beating. Gax was not expressed in cultured AT-1 cells but in AT-1 tumor and murine heart. p53 and p21 were decreased after beating which indicate transcription level of p53 and p21 correlated well in AT-1 cells. In contrast, pRB and p107 were increased after beating but p68 (2.4 kb) which arose by alternative splicing of p107 and lacks the pocket domain B was decreased in beating cells. pTCS2, murine tuberous sclerosis gene, represented similar levels during beating but a little was decreased after formation of BS. mRAD50, the murine homologue of yeast DNA recombinational repair gene RAD50, was increased in beating cells, a similar pattern to p107 and pRB. But the p50 arose by alternative splicing of mRAD50 and has 3' half of mRAD50 had unexpectedly appeared and maintained after beating. CONCLUSION: The expression of cell cycle regulatory genes after beating and formation of BS in AT-1 cells showed gene-specific pattern and the p50 which has homology to the mRAD50 may participate in differentiation of cardiomyocytes.