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1.
Nature ; 630(8017): 752-761, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38867045

RESUMO

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.


Assuntos
Pareamento Incorreto de Bases , Dano ao DNA , DNA de Cadeia Simples , Análise de Sequência de DNA , Imagem Individual de Molécula , Humanos , Envelhecimento/genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Pareamento Incorreto de Bases/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Citosina/metabolismo , Desaminação , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Genoma Mitocondrial/genética , Mutação , Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Imagem Individual de Molécula/métodos , Masculino , Feminino
2.
Lancet Oncol ; 25(5): 668-682, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38552658

RESUMO

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD. METHODS: In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions. FINDINGS: We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions. INTERPRETATION: The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD. FUNDING: The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center.


Assuntos
Proteínas de Ligação a DNA , Síndromes Neoplásicas Hereditárias , Humanos , Masculino , Feminino , Criança , Pré-Escolar , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/terapia , Estudos Transversais , Adolescente , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/epidemiologia , Reparo de Erro de Pareamento de DNA , Estudos Longitudinais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/mortalidade , Incidência , Proteína 2 Homóloga a MutS/genética , Proteína 1 Homóloga a MutL/genética , Adulto , Adulto Jovem , Mutação
3.
Clin Chem ; 70(5): 737-746, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38531023

RESUMO

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. METHODS: The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). RESULTS: Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). CONCLUSIONS: Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.


Assuntos
Mutação em Linhagem Germinativa , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/diagnóstico , Criança , Neoplasias Colorretais/genética , Neoplasias Colorretais/diagnóstico , Feminino , Masculino , Reparo de Erro de Pareamento de DNA/genética , Pré-Escolar , Adolescente , Alelos
4.
NPJ Precis Oncol ; 8(1): 110, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773265

RESUMO

Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.

5.
Nat Commun ; 15(1): 5790, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987542

RESUMO

With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly characterized. Accordingly, we developed a clinical immune-oncology gene expression assay and used it to profile a diverse range of 1382 samples with detailed clinical and molecular annotation. In low-grade gliomas we identify distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors. In high-grade gliomas, we observe immune activation and T-cell infiltrates in tumors that have historically been considered immune cold, as well as genomic correlates of inflammation levels. In mismatch repair deficient high-grade gliomas, we find that high tumor inflammation signature is a significant predictor of response to immune checkpoint inhibition, and demonstrate the potential for multimodal biomarkers to improve treatment stratification. Importantly, while overall patterns of immune activation are observed for histologically and genetically defined tumor types, there is significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine.


Assuntos
Neoplasias Encefálicas , Glioma , Microambiente Tumoral , Humanos , Microambiente Tumoral/imunologia , Microambiente Tumoral/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Criança , Glioma/imunologia , Glioma/genética , Glioma/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Feminino , Masculino , Adolescente , Regulação Neoplásica da Expressão Gênica , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Pré-Escolar , Perfilação da Expressão Gênica , Imunoterapia/métodos , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Mutação , Linfócitos T/imunologia , Medicina de Precisão/métodos , Linfócitos do Interstício Tumoral/imunologia , Relevância Clínica
6.
Cancer Discov ; 14(2): 258-273, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-37823831

RESUMO

Immune checkpoint inhibition (ICI) is effective for replication-repair-deficient, high-grade gliomas (RRD-HGG). The clinical/biological impact of immune-directed approaches after failing ICI monotherapy is unknown. We performed an international study on 75 patients treated with anti-PD-1; 20 are progression free (median follow-up, 3.7 years). After second progression/recurrence (n = 55), continuing ICI-based salvage prolonged survival to 11.6 months (n = 38; P < 0.001), particularly for those with extreme mutation burden (P = 0.03). Delayed, sustained responses were observed, associated with changes in mutational spectra and the immune microenvironment. Response to reirradiation was explained by an absence of deleterious postradiation indel signatures (ID8). CTLA4 expression increased over time, and subsequent CTLA4 inhibition resulted in response/stable disease in 75%. RAS-MAPK-pathway inhibition led to the reinvigoration of peripheral immune and radiologic responses. Local (flare) and systemic immune adverse events were frequent (biallelic mismatch-repair deficiency > Lynch syndrome). We provide a mechanistic rationale for the sustained benefit in RRD-HGG from immune-directed/synergistic salvage therapies. Future approaches need to be tailored to patient and tumor biology. SIGNIFICANCE: Hypermutant RRD-HGG are susceptible to checkpoint inhibitors beyond initial progression, leading to improved survival when reirradiation and synergistic immune/targeted agents are added. This is driven by their unique biological and immune properties, which evolve over time. Future research should focus on combinatorial regimens that increase patient survival while limiting immune toxicity. This article is featured in Selected Articles from This Issue, p. 201.


Assuntos
Antineoplásicos , Neoplasias Encefálicas , Glioma , Humanos , Antígeno CTLA-4 , Glioma/tratamento farmacológico , Glioma/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Antineoplásicos/uso terapêutico , Imunoterapia , Microambiente Tumoral
7.
bioRxiv ; 2023 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-36824744

RESUMO

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases1-4. Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired5. However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging.

8.
Clin Cancer Res ; 29(23): 4770-4783, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37126021

RESUMO

PURPOSE: Checkpoint inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory nonhematologic cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch repair deficiency (MMRD). PATIENTS AND METHODS: Twenty patients were screened, and 10 were ultimately included in the response cohort of whom nine had TMB >10 mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5 and 10 mut/Mb. RESULTS: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% [95% confidence interval (CI), 27-93]. Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37 months (range, 32.4-60), culminating in 2-year OS of 43% (95% CI, 18.2-100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with an MMR-proficient tumor and TMB 7.4 mut/Mb. CONCLUSIONS: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint inhibitors, including upfront management of cancer. See related commentary by Mardis, p. 4701.


Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Criança , Nivolumabe/uso terapêutico , Estudos Prospectivos , Mutação , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Biomarcadores Tumorais/genética , Reparo de Erro de Pareamento de DNA/genética , Microambiente Tumoral
9.
J Clin Oncol ; 41(4): 766-777, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36240479

RESUMO

PURPOSE: Diagnosis of Mismatch Repair Deficiency (MMRD) is crucial for tumor management and early detection in patients with the cancer predisposition syndrome constitutional mismatch repair deficiency (CMMRD). Current diagnostic tools are cumbersome and inconsistent both in childhood cancers and in determining germline MMRD. PATIENTS AND METHODS: We developed and analyzed a functional Low-pass Genomic Instability Characterization (LOGIC) assay to detect MMRD. The diagnostic performance of LOGIC was compared with that of current established assays including tumor mutational burden, immunohistochemistry, and the microsatellite instability panel. LOGIC was then applied to various normal tissues of patients with CMMRD with comprehensive clinical data including age of cancer presentation. RESULTS: Overall, LOGIC was 100% sensitive and specific in detecting MMRD in childhood cancers (N = 376). It was more sensitive than the microsatellite instability panel (14%, P = 4.3 × 10-12), immunohistochemistry (86%, P = 4.6 × 10-3), or tumor mutational burden (80%, P = 9.1 × 10-4). LOGIC was able to distinguish CMMRD from other cancer predisposition syndromes using blood and saliva DNA (P < .0001, n = 277). In normal cells, MMRDness scores differed between tissues (GI > blood > brain), increased over time in the same individual, and revealed genotype-phenotype associations within the mismatch repair genes. Importantly, increased MMRDness score was associated with younger age of first cancer presentation in individuals with CMMRD (P = 2.2 × 10-5). CONCLUSION: LOGIC was a robust tool for the diagnosis of MMRD in multiple cancer types and in normal tissues. LOGIC may inform therapeutic cancer decisions, provide rapid diagnosis of germline MMRD, and support tailored surveillance for individuals with CMMRD.


Assuntos
Neoplasias Encefálicas , Neoplasias Colorretais , Síndromes Neoplásicas Hereditárias , Humanos , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA/genética , Genômica , Células Germinativas/patologia , Instabilidade de Microssatélites , Repetições de Microssatélites , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética
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