Matthew Mistry

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.

Phase II Weekly Vinblastine for Chemotherapy-Naïve Children With Progressive Low-Grade Glioma: A Canadian Pediatric Brain Tumor Consortium Study

Purpose Vinblastine monotherapy has shown promising activity and a low-toxicity profile in patients with pediatric low-grade glioma (PLGG) who experienced treatment failure after initial treatment with chemotherapy and/or radiation. The aim of this study was to assess the activity of vinblastine in therapy-naïve children. Patients and Methods Patients < 18 years old with unresectable and/or progressive therapy-naïve PLGG were eligible. Vinblastine was administered once per week at a dose of 6 mg/m2 intravenously over a period of 70 weeks. Vision, quality of life, neurofibromatosis type 1 (NF1) status, and BRAF mutation/fusion status were also determined and correlated with outcome. Results Fifty-four patients were enrolled onto the study, with a median age of 8 years (range, 0.7 to 17.2 years). Most patients had chiasmatic/hypothalamic tumors (55.5%), and 13 patients (24.1%) had NF1. The most common histology was pilocytic astrocytoma (46.3%). Seventeen patients were diagnosed using radiologic criteria alone. Best response to chemotherapy was centrally reviewed with a response rate (complete, partial, or minor response) of 25.9%. Disease stabilization (complete, partial, or minor response or stable disease) was achieved in 47 patients (87.0%). Visual improvement was observed in 20% of patients with optic pathway glioma. Five-year overall survival and progression-free survival (PFS) rates were 94.4% (95% CI, 88.5% to 100%) and 53.2% (95% CI, 41.3% to 68.5%), respectively, for the entire cohort. Patients with NF1 had a significantly better PFS (85.1%; 95% CI, 68.0% to 100%) when compared with patients without NF1 (42.0%; 95% CI, 29.1% to 60.7%; P = .012). Age< 3 years or > 10 years was not associated with poor outcome. Treatment was well tolerated, and quality of life was not affected during treatment. In this trial, there was no correlation between BRAF alterations and outcome. Conclusion Vinblastine administered once per week is well tolerated in children with treatment naïve PLGG. Overall survival and PFS are comparable to current therapies, with a favorable toxicity profile and a maintained quality of life.

Clinical and treatment factors determining long-term outcomes for adult survivors of childhood low-grade glioma: A population-based study.

The determinants of outcomes for adult survivors of pediatric low‐grade glioma (PLGG) are largely unknown.

Therapeutic and prognostic implications of BRAF V600E in pediatric low-grade gliomas

Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.

Multiplex Detection of Pediatric Low-Grade Glioma Signature Fusion Transcripts and Duplications Using the NanoString nCounter System

Previous studies identified recurrent fusion and duplication events in pediatric low-grade glioma (pLGG). In addition to their role in diagnosis, the presence of these events aid in dictating therapy and predicting patient survival. Clinically, BRAF alterations are most commonly identified using fluorescent in situ hybridization (FISH). However, this method is costly, labor-intensive and does not identify nonBRAF events. Here, we evaluated the NanoString nCounter gene expression system for detecting 32 of the most commonly reported fusion/duplication events in pLGG. The assay was validated on 90 pLGG samples using FISH as the gold standard and showed sensitivity and specificity of 97% and 98%, respectively. We next profiled formalin-fixed paraffin-embedded preserved biopsy specimens from 429 pLGG cases. 171 (40%) of the cases within our cohort tested positive for a fusion or duplication event contained within our panel. These events, in order of prevalence, were KIAA1549-BRAF 16;9 (89/171, 52.0%), KIAA1549-BRAF 15;9 (42/171, 24.6%), KIAA1549-BRAF 16;11 (14/171, 8.2%), FGFR1-TACC1 17;7 (13/171, 7.6%), MYBL1 duplication (5/171, 2.9%), KIAA1549-BRAF 18;10 (4/171, 2.3%), KIAA1549-BRAF 15;11 (2/171, 1.2%), FAM131B-BRAF 2;9 (1/171, 0.6%), and RNF130-BRAF 3;9 (1/171, 0.6%). This work introduces NanoString as a viable clinical replacement for the detection of fusion and duplication events in pLGG.

Abstract B12: H3-K27M is a negative prognostic marker in high- and low-grade pediatric thalamic glioma

Pediatric gliomas are the most commonly diagnosed brain cancer in children, accounting for approximately 50% of all cases. Work by our group and others has revealed recurrent histone mutations in diffuse intrinsic pontine glioma (DIPG), astrocytomas that arise in the brainstem of children and boast a dismal median survival of less than one year. In ~70% of DIPG, a mutation in histone variant H3.3 or H3.1 at position 27 leads to substitution of a lysine by a methionine (K27M). When correlated to clinical data, patients harbouring H3-K27M mutations showed a worse prognosis in comparison to patients wild type for H3 (H3-WT), regardless of their histologic grade. Investigation into H3-K27M prevalence in pediatric gliomas revealed that they were not exclusively observed in the brainstem, but rather, in additional midline structures such as the thalamus. However, the clinical relevance of H3-K27M in other midline tumours has not yet been investigated. We hypothesized that H3-K27M would represent a negative prognostic marker in pediatric thalamic gliomas. We searched the Hospital for Sick Children9s tumour archives and obtained 86 radiologically confirmed cases of pre-treatment thalamic glioma. The cohort contained 46 male and 40 female patients with a combined median age of diagnosis and survival of 9.04 and 3.88 years, respectively. Of the 86 samples analyzed, 53 were histologically reviewed as low grade malignancies while 33 appeared high grade. Median survival for patients with high grade thalamic gliomas was 1.02 years vs 8.71 years for low grade thalamic glioma patients. Six (11.3%) low grade and 17 (51.5%) high grade gliomas tested positive for the H3-K27M mutation. Patients were clinically annotated for treatment, extent of resection, age of diagnosis and outcome (median follow-up of 10.3 years) and were used to determine the prognostic implications of H3-K27M. Kaplan-Meier survival analysis revealed significantly worse overall survival of thalamic glioma patients harbouring the H3-K27M mutation versus wild type samples (log rank p These findings provide the first evidence that H3-K27M is a negative prognostic indicator for pediatric thalamic glioma. Further, the survival difference between H3-K27M mutant low grade and high grade astrocytoma suggests differing biology and a role for “second hits” in dictating the aggressiveness of these tumours. Importantly, it supports prognostic subgrouping based on mutation status and histologic grade. Citation Format: Scott Ryall, Pawel Buczkowicz, Anthony Arnoldo, Rahul Krishnatry, Matthew Mistry, Joshua Rubin, Paul Steinbok, Uri Tabori, Cynthia Hawkins. H3-K27M is a negative prognostic marker in high- and low-grade pediatric thalamic glioma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B12.

Abstract B09: DNA polymerase mutations trigger rapid onset of ultra-hypermutant malignant brain tumors in children with biallelic mismatch repair deficiency

Background: Biallelic Mismatch Repair Deficiency (bMMRD) is a childhood cancer predisposition syndrome caused by germline mutations in MSH2, MSH6, MLH1, and PMS2. The leading cause of death is malignant brain tumors. The genomic landscape and secondary somatic mutations of bMMRD brain tumors are unknown. Methods: We analyzed 27 cancers and corresponding normal tissues from bMMRD patients using genome, exome sequencing and SNP-arrays. Additionally, we performed sequential sequencing from five primary and recurrent tumor pairs. Results: BMMRD malignant brain tumors harbored massive numbers of substitution mutations (>250/Mb), greater than all childhood and most adult cancers (>7,000 analyzed). These cancers lacked copy number alterations (p Conclusions/Significance: Early-onset brain tumors from bMMRD patients have a unique mechanism of malignant progression through secondary mutations in DNA polymerases. During transformation, brain tumors quickly reach a threshold of mutations developed in a rapid burst once a mutation in a DNA polymerase is acquired. The high mutation load and threshold of bMMRD cancers may be its Achilles9 heel, exploitable for diagnosis and therapeutic intervention. Note: This abstract was not presented at the conference. Citation Format: Adam Shlien, Brittany B. Campbell, Richard de Borja, Ludmil B. Alexandrov, Daniele Merico, David Wedge, Peter Van Loo, Patrick S. Tarpey, Paul Coupland, Aaron Pollett, Tatiana Lipman, Abolfazl Heidari, Shriya Deshmukh, Moritz Gerstung, Diana Merino, Manasa Ramakrishna, Marc Remke, Roland Arnold, Gagan B. Panigrahi, Samina Afzal, Valerie Larouche, Harriet Druker, Jordan Lerner-Ellis, Matthew Mistry, Rina Dvir, Ronald Grant, Ronit Elhasid, Roula Farah, Glenn P. Taylor, Paul C. Nathan, Sarah Alexander, Shay Ben-Shachar, Nada Jabado, Steven Gallinger, Shlohmi Constantini, Peter Dirks, Annie Huang, Steven W. Scherer, Richard G. Grundy, Carol Durno, Melyssa Aronson, M Stephen Meyn, Michael D. Taylor, Zachary F. Pursell, Christopher E. Pearson, David Malkin, P Andrew Futreal, Cynthia Hawkins, Eric Bouffet, Michael D. Taylor, Peter J. Campbell, Uri Tabori. DNA polymerase mutations trigger rapid onset of ultra-hypermutant malignant brain tumors in children with biallelic mismatch repair deficiency. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B09.

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