Doua Bakry

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

DNA replication−associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10−13). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

BRAF Mutation and CDKN2A Deletion Define a Clinically Distinct Subgroup of Childhood Secondary High-Grade Glioma

PURPOSE To uncover the genetic events leading to transformation of pediatric low-grade glioma (PLGG) to secondary high-grade glioma (sHGG). PATIENTS AND METHODS We retrospectively identified patients with sHGG from a population-based cohort of 886 patients with PLGG with long clinical follow-up. Exome sequencing and array CGH were performed on available samples followed by detailed genetic analysis of the entire sHGG cohort. Clinical and outcome data of genetically distinct subgroups were obtained. RESULTS sHGG was observed in 2.9% of PLGGs (26 of 886 patients). Patients with sHGG had a high frequency of nonsilent somatic mutations compared with patients with primary pediatric high-grade glioma (HGG; median, 25 mutations per exome; P = .0042). Alterations in chromatin-modifying genes and telomere-maintenance pathways were commonly observed, whereas no sHGG harbored the BRAF-KIAA1549 fusion. The most recurrent alterations were BRAF V600E and CDKN2A deletion in 39% and 57% of sHGGs, respectively. Importantly, all BRAF V600E and 80% of CDKN2A alterations could be traced back to their PLGG counterparts. BRAF V600E distinguished sHGG from primary HGG (P = .0023), whereas BRAF and CDKN2A alterations were less commonly observed in PLGG that did not transform (P < .001 and P < .001 respectively). PLGGs with BRAF mutations had longer latency to transformation than wild-type PLGG (median, 6.65 years [range, 3.5 to 20.3 years] v 1.59 years [range, 0.32 to 15.9 years], respectively; P = .0389). Furthermore, 5-year overall survival was 75% ± 15% and 29% ± 12% for children with BRAF mutant and wild-type tumors, respectively (P = .024). CONCLUSION BRAF V600E mutations and CDKN2A deletions constitute a clinically distinct subtype of sHGG. The prolonged course to transformation for BRAF V600E PLGGs provides an opportunity for surgical interventions, surveillance, and targeted therapies to mitigate the outcome of sHGG.

Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: Report from the constitutional mismatch repair deficiency consortium

BACKGROUND Constitutional mismatch repair deficiency (CMMRD) is a devastating cancer predisposition syndrome for which data regarding clinical manifestations, molecular screening tools and management are limited. METHODS We established an international CMMRD consortium and collected comprehensive clinical and genetic data. Molecular diagnosis of tumour and germline biospecimens was performed. A surveillance protocol was developed and implemented. RESULTS Overall, 22/23 (96%) of children with CMMRD developed 40 different tumours. While childhood CMMRD related tumours were observed in all families, Lynch related tumours in adults were observed in only 2/14 families (p=0.0007). All children with CMMRD had café-au-lait spots and 11/14 came from consanguineous families. Brain tumours were the most common cancers reported (48%) followed by gastrointestinal (32%) and haematological malignancies (15%). Importantly, 12 (30%) of these were low grade and resectable cancers. Tumour immunohistochemistry was 100% sensitive and specific in diagnosing mismatch repair (MMR) deficiency of the corresponding gene while microsatellite instability was neither sensitive nor specific as a diagnostic tool (p<0.0001). Furthermore, screening of normal tissue by immunohistochemistry correlated with genetic confirmation of CMMRD. The surveillance protocol detected 39 lesions which included asymptomatic malignant gliomas and gastrointestinal carcinomas. All tumours were amenable to complete resection and all patients undergoing surveillance are alive. DISCUSSION CMMRD is a highly penetrant syndrome where family history of cancer may not be contributory. Screening tumours and normal tissues using immunohistochemistry for abnormal expression of MMR gene products may help in diagnosis and early implementation of surveillance for these children.

Phenotypic and genotypic characterisation of biallelic mismatch repair deficiency (BMMR-D) syndrome

Lynch syndrome, the most common inherited colorectal cancer syndrome in adults, is an autosomal dominant condition caused by heterozygous germ-line mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Inheriting biallelic (homozygous) mutations in any of the MMR genes results in a different clinical syndrome termed biallelic mismatch repair deficiency (BMMR-D) that is characterised by gastrointestinal tumours, skin lesions, brain tumours and haematologic malignancies. This recently described and under-recognised syndrome can present with adenomatous polyps leading to early-onset small bowel and colorectal adenocarcinoma. An important clue in the family history that suggests underling BMMR-D is consanguinity. Interestingly, pedigrees of BMMR-D patients typically show a paucity of Lynch syndrome cancers and most parents are unaffected. Therefore, a family history of cancers is often non-contributory. Detection of BMMR-D can lead to more appropriate genetic counselling and the implementation of targeted surveillance protocols to achieve earlier tumour detection that will allow surgical resection. This review describes an approach for diagnosis and management of these patients and their families.

Gastrointestinal Findings in the Largest Series of Patients With Hereditary Biallelic Mismatch Repair Deficiency Syndrome: Report from the International Consortium

Objectives:Hereditary biallelic mismatch repair deficiency (BMMRD) is caused by biallelic mutations in the mismatch repair (MMR) genes and manifests features of neurofibromatosis type 1, gastrointestinal (GI) polyposis, and GI, brain, and hematological cancers. This is the first study to characterize the GI phenotype in BMMRD using both retrospective and prospective surveillance data.Methods:The International BMMRD Consortium was created to collect information on BMMRD families referred from around the world. All patients had germline biallelic MMR mutations or lack of MMR protein staining in normal and tumor tissue. GI screening data were obtained through medical records with annual updates.Results:Thirty-five individuals from seven countries were identified with BMMRD. GI data were available on 24 of 33 individuals (73%) of screening age, totaling 53 person-years. The youngest age of colonic adenomas was 7, and small bowel adenoma was 11. Eight patients had 19 colorectal adenocarcinomas (CRC; median age 16.7 years, range 8–25), and 11 of 18 (61%) CRC were distal to the splenic flexure. Eleven patients had 15 colorectal surgeries (median 14 years, range 9–25). Four patients had five small bowel adenocarcinomas (SBC; median 18 years, range 11–33). Two CRC and two SBC were detected during surveillance within 6–11 months and 9–16 months, respectively, of last consecutive endoscopy. No patient undergoing surveillance died of a GI malignancy. Familial clustering of GI cancer was observed.Conclusions:The prevalence and penetrance of GI neoplasia in children with BMMRD is high, with rapid development of carcinoma. Colorectal and small bowel surveillance should commence at ages 3–5 and 8 years, respectively.

TP53 Germline Mutations: Genetics of Li–Fraumeni Syndrome

Li–Fraumeni syndrome (LFS) is a rare autosomal dominantly inherited cancer predisposition disorder commonly associated with germline TP53 mutations. The syndrome classically presents with a wide spectrum of childhood and adult-onset cancers. Penetrance of the cancer phenotype is high with a lifetime risk of >75 % to develop cancer in TP53 mutation carriers. The spectrum of germline TP53 mutations is similar to that observed for somatic TP53 mutations in sporadic cancers. Studies to explore the genetic and epigenetic events that modify the cancer phenotype in LFS families have identified several polymorphisms in TP53, as well as in other genes that regulate TP53 function. In addition, other global genetic/genomic features including excessive DNA copy number variation and accelerated telomere attrition have been implicated in modifying phenotype conferred by the primary TP53 defect. Genetic testing for TP53 mutations in LFS family members is now commonly used to identify at-risk individuals. Based on this information, and in the context of multidisciplinary genetic counseling teams, novel approaches to clinical surveillance for early cancer detection are being implemented. In the 20 years from discovery of the link between germline TP53 mutations and etiology of LFS, translation of basic discoveries in TP53 biology to clinical application in terms of tumor prognosis, therapy, and early detection has made significant impact on the lives of patients in these families.

Abstract 35: Novel genetic and clinical determinants of Constitutional Mismatch Repair Deficiency syndrome: Report from the CMMRD consortium

Purpose: Constitutional mismatch repair deficiency (CMMRD) is a devastating cancer predisposition syndrome affecting children born with two mutated alleles in one of four mismatch repair genes. Data regarding clinical manifestations, molecular screening tools and management are limited. Patients and methods: We established an international CMMRD consortium and collected comprehensive clinical and genetic data. Molecular diagnosis of tumor and germline biospecimens were performed. A surveillance protocol was developed and implemented. Results: Overall, 27/30 (90%) of children with CMMR-D developed 48 different tumors. While childhood CMMR-D related tumors were observed in all families, Lynch related tumors in adults were observed in only 2/17 families (p We detected 17 different germline MMR mutations. These included mutations in PMS2(8), MSH6(7) and MLH1(2). Importantly 7/17 mutations were previously unreported. Brain tumors were the most common cancers reported (44%) followed by gastrointestinal (33%) and hematological malignancies (17%). Importantly, 14 (29%) of these were low grade and respectable cancers. Tumor immunohistochemistry was 100% sensitive and specific in diagnosing MMR deficiency of the corresponding gene while microsatellite instability was neither sensitive nor specific as a diagnostic tool (p Conclusion: CMMR-D is a highly penetrant syndrome where family history of cancer may not be contributory. Screening tumors and normal tissues using immunohistochemistry for abnormal expression of MMR gene products helps in diagnosis and early implementation of surveillance for these children. Citation Format: Doua Bakry, Brittany Campbell, Carol Durno, Melyssa Aronson, Qasim Alharbi, Musa Alharbi, Shlomi Constantini, Aaron Pollett, Shay Ben-Shachar, Jordan Lerner-Ellis, Steven Gallinger, Ronit Elhasid, Roula Farah, Ibrahim Qaddoumi, Matthew Mistry, Ramyar Lily, Steve Keiles, Rina Dvir, Derek Stephens, David Malkin, Eric Bouffet, Cynthia Hawkins, Uri Tabori. Novel genetic and clinical determinants of Constitutional Mismatch Repair Deficiency syndrome: Report from the CMMRD consortium. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 35. doi:10.1158/1538-7445.CANSUSC14-35

Abstract 1435: Phenotype:Genotype correlations of p53 mutation carriers: The 20-year Toronto experience

Introduction: Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disorder associated with germline mutations of the p53 gene. LFS is associated with early age of onset of breast cancer in women, childhood sarcomas and other malignancies. Previous studies have suggested potential correlations between p53 mutation type and clinical cancer phenotypes. For example, missense mutations within the DNA binding loop were found to be associated with higher prevalence of brain tumors and breast cancer whereas missense mutations outside the DNA binding loops were associated with higher prevalence of adrenocortical carcinoma. Through our Cancer Genetics Program (CGP), we have ascertained and conducted p53 mutation analysis on LFS patients/families from 1992 to the present. This represents one of the largest collections in the world. Here we set out to collate and characterize potentially novel genotype:phenotype associations from this database. Methods: TissueMetrix (Artificial Intelligence in Medicine (AIM), Toronto) was used to develop the CGP database. Clincal-pathologic data including family pedigrees, age of tumor onset, tumor type (confirmed with pathology reports where available were collected. From 1992-1998, Sanger sequencing of p53 encompassing the DNA binding domain (exons 5-8) was performed. Since 1998, the entire coding region and additional 50-100 bases spanning intron/exon splice sites have been sequenced and since 2007, MLPA testing has been performed to look for allelic deletion/duplication. Results: p53 mutations have been identified in 110 individuals, of whom 61 (55%) are less than 18years of age. Missense mutations the most common sequence alteration (83/110= 75%). The most commonly observed mutation was the dominant-negative Arg248Gln substitution (n=12). Monoallelic complete or partial gene deletion was observed in 10 cases, several of whom exhibited a non-cancer, developmental delay phenotype. The most common cancer were brain tumors (n=21), of which 10 were choroid plexus carcinomas. ADCC, breast cancer and sarcomas were commonly observed; non-classical LFS tumors, including neuroblastoma, papillary carcinoma of the thyroid and Wilms tumor are also seen. Conclusion: Our program, being situated in a pediatric tertiary centre has an inherent ascertainment bias which likely explains the high frequency of young p53 mutation carriers, as well as a disproportionate prevalence of childhood cancers. Nevertheless, unique correlations emerge from this rich resource including that of p53 deletion and hypotonia/developmental delay, as well as de novo and novel non-coding mutations. These correlations provide the foundation on which to develop practical genetic screening and clinical surveillance approaches to at-risk children and young adults. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1435. doi:1538-7445.AM2012-1435