IJC International Journal of Cancer

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1 IJC International Journal of Cancer High frequency of mismatch repair deficiency among pediatric high grade gliomas in Jordan Nisreen Amayiri 1 *, Uri Tabori 2,3,4 *, Brittany Campbell 2,3, Doua Bakry 2,3, Melyssa Aronson 5, Carol Durno 5,6, Patricia Rakopoulos 7, David Malkin 2,3, Ibrahim Qaddoumi 8, Awni Musharbash 9, Maisa Swaidan 10, Eric Bouffet 2,3, Cynthia Hawkins 7 *, and Maysa Al-Hussaini 11 * On behalf of BMMRD consortium 1 Department of Pediatrics Hematology/Oncology, King Hussein Cancer Center, Amman, Jordan 2 Division of Hematology/Oncology, The Hospital for Sick Children, Institute of Medical Sciences, the University of Toronto, Toronto, Canada 3 Department of Pediatrics, The Hospital for Sick Children, Institute of Medical Sciences, the University of Toronto, Toronto, Canada 4 Arthur and Sonia Labbatt Brain Tumor Research Center, Toronto, Canada 5 The Familial Gastrointestinal Cancer Registry at the Zane Cohen Centre for Digestive Disease and Department of Surgery, Mount Sinai Hospital, Toronto, Canada 6 Division of Gastroenterology, Nutrition and Hepatology, The Hospital for Sick Children, Toronto, Canada 7 Division of Pathology, Arthur and Sonia Labatt Brain Tumour Research Center, The Hospital for Sick Children, Toronto, Canada 8 St. Jude Children s Research Hospital, Memphis, TN 9 Department of Surgery, King Hussein Cancer Center, Amman, Jordan 10 Department of Radiology, King Hussein Cancer Center, Amman, Jordan 11 Department of Pathology, King Hussein Cancer Center, Amman, Jordan Biallelic mismatch repair deficiency (bmmrd) is a cancer predisposition syndrome affecting primarily individuals from consanguinous families resulting in multiple childhood cancers including high grade gliomas (HGG). This is the first study to assess the prevalence of bmmrd among patients with HGG in countries where consanguinity is high. We collected molecular and clinical information on all children diagnosed with HGG and supratentorial primitive neuroectodermal tumors (spnet) between 2003 and 2013 at King Hussein Cancer Center, Jordan. Comparison was made to a similar cohort from Toronto. Clinical data regarding presence of cafe au lait macules(cal), family history of cancer, consanguinity, pathology and treatment were collected. Tumors were centrally reviewed and tested for MMRD by immunohistochemistry of the corresponding proteins. Fortytwo patients fulfilled the inclusion criteria, including 36 with HGG. MMRD was observed in 39% of HGG of whom79% also lost MMR staining in the corresponding normal cells suggestive of bmmrd. P53 dysfunction was highly enriched in MMR deficient tumors (p ).The frequency of MMRD was significantly lower in Toronto cohort (23%, p ). Both evidence of CAL and consanguinity correlated with bmmrd (p and 0.05,respectively) but family history of cancer didn t. HGG with all three bmmrd risk factors had evidence of MMRD and all children affected by multiple bmmrd related cancers had identical gene loss by immunohistochemical staining. In Jordan, the frequency of clinical and immunohistochemical alterations suggestive of bmmrd in pediatric HGG is high. Genetic testing will enable appropriate counseling and cancer screening to improve survival of these patients. Key words: biallelic mismatch repair deficiency, high grade glioma, consanguinity, cafe au lait macules, low income countries Additional Supporting Information may be found in the online version of this article. Conflicts of interest: None *N.A., U.T., C.H. and M.A.-H contributed equally to this work. Grant sponsor: King Hussein Cancer Center (Intramural grant) and B.R.A.I.N child, Canada (bmmrd consortium) DOI: /ijc History: Received 11 June 2015; Accepted 9 July 2015; Online 7 Aug 2015 Correspondence to: Nisreen Amayiri, department of pediatrics hematology oncology, King Hussein Cancer Center, 202 Al-Jubeiha, Queen Rania Al-Abdullah Street, P.O.Box 1269 Al-Jubeiha,Amman 11941, Jordan, Tel.: (962) , Fax: (962-6) , namayiri@khcc.jo Biallelic mismatch repair deficiency (bmmrd) is an autosomal recessive cancer predisposition syndrome caused by a biallelic germline mutation in the DNA mismatch repair (MMR) genes. This is one of the most devastating cancer syndromes associated with high penetrance and mortality during childhood. 1 3 The MMR genes which are known to cause this syndrome in humans are MLH1, MSH2, MSH6 and PMS2. In contrast to heterozygous carriers of mutations in these genes who are at risk to develop gastrointestinal and urogenital tumors as adults, 4,5 bmmrd is characterized by a broad spectrum of early onset tumors during childhood. The most common cancers affecting children are brain tumors followed by hematological malignancies and a variety of premalignant and malignant lesions of the gastrointestinal tract. 3 High grade gliomas (HGG) are the most common CNS tumors associated with bmmrd and are the major cause of Int. J. Cancer: 138, (2016) VC 2015 UICC

2 Amayiri et al. 381 What s new? Homozygous germline mutations in mismatch repair (MMR) genes can result in biallelic MMR deficiency (bmmrd), a devastating childhood cancer syndrome frequently associated with consanguinity. This study suggests that in Jordan, where consanguinity is common, the prevalence of MMR dysfunction among pediatric patients with high grade glioma (HGG) may be alarmingly high. In the Jordan cohort, bmmrd occurrence was correlated with consanguinity and presence of cafe au lait macules. Because bmmrd-related HGGs do not respond to conventional treatment regimens and additional tumor development is common, accurate diagnosis is essential. The findings presented here emphasize the importance of genetic testing. death in these children. Our group and others have demonstrated that immunohistochemistry for expression of the four MMR proteins is a robust screening assay in the diagnostic work-up of patients with suspected bmmrd. 6 Loss of expression of one or more MMR proteins is highly suggestive of underlying biallelic mutations in the corresponding gene and can guide genetic diagnosis. Such genetic diagnosis is especially important for children with glioma since bmmrd children present with multiple cafe au lait macules(cal) mimicking neurofibromatosis type 1 (NF1). Patients with NF1 have high rate of low grade gliomas, most of which do not progress. Therefore, misdiagnosis of individuals with bmmrd as NF1 results in late diagnosis of malignant lesions and can potentially influence survival. Other clues which distinguish NF1 from bmmrd include lack of other features of NF1 in these children, family history of Lynch syndrome associated malignancies and consanguinity. Although a number of observations suggest that bmmrd is related to the development of malignant brain tumors in children, 3,7 little is known about the prevalence of bmmrd in children presenting with malignant gliomas or spnet especially in countries where consanguinity is common. To gain insight into the burden of bmmrd in children with HGG/sPNET in a region of high consanguinity, we analyzed the frequency of MMR deficiency by immunohistochemistry in childhood HGG/sPNET-in Jordan and compared it to a North American cohort. Additionally we linked the MMR staining data with patient and family history suggestive of bmmrd. Patients and Methods We retrospectively reviewed the pediatric neurooncology database at King Hussein Cancer Center (KHCC) in Jordan between May 2003 and May 2013.We included patients younger than 18 years at referral with biopsy proven intracranial HGG, spnet or pleomorphic xanthoastrocytoma (PXA). PXA tumors were included in this study as these were recently described to be associated with bmmrd and their tendency to transform to higher grade tumors. 8,9 KHCC is the largest cancer center in Jordan treating most children with brain tumors in the country. Medical charts were reviewed for clinical presentation, presence or absence of CAL (or any other neurofibromatosis features), family history of cancer, consanguineous marriage, and the treatment received. Patients were excluded if they had different pathology than HGG, spnet or PXA on review or if their tumor paraffin blocks were not available or contained inadequate samples for further staining. In order to compare frequency of MMR deficiency in Jordan to a North American center, a cohort of pediatric patients with intracranial HGG from the Hospital for Sick Children in Toronto (SickKids) who were previously tested for MMR mutations was used as control. This study was approved by the Research Ethics Board in both institutions. Tumor analysis All tumors were reviewed by a single neuro-pathologist (M.A.H) from KHCC; all cases with discrepant diagnoses were excluded. All tumors were additionally stained for p53 and the four MMR proteins (PMS2, MSH6, MLH1, and MSH2). The stains were run according to manufacturer instructions using the Ventana Benchmark automated immunostainer (Supporting Information Table). All stains were performed with positive and negative controls placed on the same slide. The case was considered positive for p53 stain if >50% of tumor cell nuclei were strongly positive; otherwise it was considered negative. 10,11 As for the MMR stains, any nuclear staining in the tumor cells was considered to be positive regardless of the percentage of positive cell nuclei or the intensity of staining. Additional slides were then prepared and sent to the laboratory of SickKids for pathology review and re-staining by single neuropathologist (C.H). Additionally; five tumor samples from bmmrd confirmed patients (by mutation analysis) were sent from SickKids to be stained at KHCC for validation of the local stains. Statistical analysis Comparison between tumors characteristics and patient demographics was carried out using v 2 and Fisher exact tests. Analysis of age of onset was done using t test. Kaplan Meier curves were plotted for overall survival and statistical analysis was done using log rank test. All analyses were performed using SPSSversion2.0. Results Tumor characteristics In order to confirm the feasibility of MMRD detection by immunohistochemistry on brain tumors, we first analyzed five gliomas samples from patients with proven bmmrd (by mutation analysis). 1 All tumors were stained and reviewed independently by two neuropathologists (M.A.H and C.H) from the two centers. All tumors revealed negative staining

3 382 High frequency of bmmrd in pediatric HGG Table 1. Characteristics of patients with mismatch repair deficient tumors of the corresponding mutant gene in both centers. We then proceeded to analyze the sporadic brain tumors from Jordan (n 5 44) by the two centers independently. MMR staining was concordant between the two institutions in 42/44 (96%) tumors (Table 1). The final cohort for central pathology review consisted of 36 HGG including five anaplastic PXA. An additional two grade II PXA were included. Five PNET were included and a medulloblastoma from a patient with synchronous HGG (Table 2).Strikingly, seventeen tumors (39%) exhibited absence of at least one MMR stain. Of these, 82% also exhibited nommr staining in the corresponding normal endothelium suggesting a germline mutation in MMR. MLH1/PMS2 stains were absent in 8/17 of tumors followed by MSH6 loss in 6/17(Table 1). Metachronous and synchronous primary brain tumors from two patients exhibited identical MMR gene loss in both tumors and normal samples (Table 2). Interestingly, 51% of multiforme (GBM) had evidence of MMR dysfunction while only 14% of PXA tumors exhibited loss of MMR stain in this cohort. Eighteen tumors (41%) had positive P53 staining. P53 immunopositivity was highly associated with MMR protein loss (p ). Comparison of MMR deficiency between cohorts In order to test whether the high prevalence of MMR dysfunction among KHCC children with HGG is different from the expected prevalence in North America, we compared the KHCC tumor stains Patient number Consanguinity Family history of cancer CAL Pathology P53 MMR stains Stains of normal tissue Sickkids tumor stains 1 A Absent Present Present GBM P PMS2/MLH1 lost PMS2/MLH1 lost Same 1 B Absent Present Present MB P PMS2/MLH1 lost PMS2/MLH1 lost Same 2 Absent GBM P PMS2/MLH1 lost PMS2/MLH1 lost Same 3 GBM P PMS2/MLH1 lost PMS2/MLH1 lost All stains retained 4 Absent GBM P PMS2/MLH1 lost PMS2/MLH1 lost Same 5 Present PXA N PMS2/MLH1 lost PMS2/MLH1 lost Same 6 A Absent Absent Present GBM P PMS2/MLH1 lost PMS2/MLH1 lost Same 6 B Absent Absent Present GBM P PMS2/MLH1 lost PMS2 lost Same 7 Absent Present Present PNET N PMS2 lost PMS2 lost Same 8 Present Absent Present GBM with PNET P PMS2 lost PMS2 lost Same 9 GBM N PMS2 lost PMS2 lost Same 10 Present Present Present GBM P MSH6/MSH2 lost All stains retained Same 11 Absent Absent GBM N MSH6/MSH2 lost All stains retained Same 12 Absent Present Absent AA P MSH6/MSH2 lost All stains retained Same 13 Present Present Present GBM P MSH6 lost MSH6 lost Same 14 Absent Present Present GBM P MSH6 lost MSH6 lost All stains retained 15 Present Present Present GBM P MSH6 lost MSH6 lost Same AA: anaplastic astrocytoma; CAL: cafe au lait macules; GBM: multiforme; KHCC: King Hussein Cancer Center; MB: medulloblastoma; MMR: mismatch repair; N: negative; P: positive; PNET: primitive neuroectodermaltumor; PXA: pleomorphic xanthoastrocytoma KHCC cohort to a cohort of 34 HGG from SickKids institutional database. Eight (23%) of these tumors stained negative for one of the MMR proteins. Four (50%) of the negative staining tumors were from patients with bmmrd mutations in the corresponding genes. For sporadic tumors, the frequency of MMR deficiency was significantly higher in KHCC than SickKids (p ). Clinical and molecular analysis of bmmrd In order to examine the clinical association of the above molecular findings we compared these to the available demographic and outcome data (Fig. 1). 44% of children with MMR deficient HGG tumors were the offspring of firstdegree consanguineous parents compared with only 15%of children with MMR intact cancers(p ). Furthermore, CAL was observed in 91% of children with MMR deficient tumors and in 33%of patients with MMR intact tumors (p ). In contrast, family history of cancer was observed in55%of children with MMR deficient tumors and in 37% of patients with MMR proficient tumors (p 5 1). Five patients developed two tumors each (Table 2); all had evidence of CAL and negative stain in one of the MMR genes in their tumors. All patients with clinical data suggestive of bmmrd (defined as presence of CAL, consanguinity and family history of cancer) exhibited negative staining of an MMR protein in their tumor. Interestingly, median age at diagnosis was 12.2 years (range, years) and did not differ between children with MMR proficient and deficient tumors.

4 Amayiri et al. 383 Table 2. Characteristics of patients with two bmmrd related tumors Patient status*/ follow up since first intracranial tumor Family history of cancer Consanguinity Stains Interval between tumors CAL Patient number First tumor Second tumor Alive (5.3 years) 4 years Present Absent Absent PMS2/MLH1 lost Posterior fossa 1 Supratentorial Alive (2.5 years) synchronous Present Present Absent PMS2/MLH1 lost Posterior fossa medulloblastoma 2 Supratentorial Colon polyp 15 months Present Absent Present PMS2 lost Alive (1.25 years) 3 Supratentorial 1.5 years Present Present Present MSH6 lost Dead (3.5 years) Non Hodgkin lymphoma 4 Supratentorial Dead (3 months) NA NA NA NA PMS2/MLH1 lost 5 Colon polyps Supratentorial *Patient status as of June 2014, later during writing of this manuscript two patients (1 and 2) died. bmmrd: biallelic mismatch repair deficiency; CAL: cafe au lait macules; NA: no clinical data available When examining the clinical associations of all MMR deficient tumors, all but one of corresponding children had evidence of CAL. That patient came from a nonconsanguineous family with a strong history of Lynch-related cancers. MMR deficiency impact on outcome Survival data was available on all patients with high grade glioma.at last follow-up, 20 patients were dead at a median time of 1.98 years. Of the13 patients with MMR deficient tumors, six were alive. However, no significant difference in overall survival was observed between children with bmmrd intact and deficient high grade gliomas (Fig. 2). Discussion This study reveals a high prevalence of MMRD among high grade glioma in a country where consanguinity is high and bmmrd may be relatively common and under recognized. Combining the clinical findings and molecular data, up to 51% of patients with childhood in Jordan may harbor germline mutations in one of the four MMR genes. If these mutations are confirmed by genetic testing, these observations have critical implications on the management, choice of chemotherapeutic agents and screening of these and other bmmrd related cancers among patients and their extended families. The gold standard for bmmrd diagnosis is evidence of germline biallelic mutations in one of the MMR genes. However, recently developed tools 12 may allow for a functional analysis of tumors which will enable early diagnosis in challenging cases and implementation of surveillance even when genetic testing is not available or not desired by the family for cultural, or logistic reasons. Since bmmrdmay be common in countries with high rates of consanguinity, an initial screening tool may include routine immunostaining of all high grade gliomas and malignant brain tumors from children with CAL and consanguinity for the four MMR proteins. As demonstrated by our group and others 2,13 this approach is highly reproducible with 96% agreement between different pathologists and feasible in most clinical laboratories worldwide (Supporting Information Table). In contrast to Lynch related tumors where infiltrating normal cells such as lymphocytes and blood vessels stain positive, negative staining of normal cells further reinforces the suspicion of biallelic germline mutations causing complete loss of protein expression in all tissues. This initial screening is not diagnostic but can facilitate proper genetic testing and counseling. Since some of the clinical features associated with bmmrd are not specific to the syndrome, care must be given to combining careful clinical history and physical examination with the tumor and molecular findings. Indeed, in our study, both consanguinity and CAL were highly correlated with evidence of MMRD in the tumors while family history was not specific (Fig. 1). These observations are consistent with previous publications 3 and are probably due to lack of Lynch related tumors

5 384 High frequency of bmmrd in pediatric HGG Figure 1. Association of clinical markers with tumor mismatch repair status. Figure 2. Kaplan Meyer estimates of overall survival in patients with high grade glioma. Hatched line - MMR proficient tumors. Solid line - MMR deficient tumors. in many families with bmmrd on one hand and other cancer syndromes resulting in nonspecific cancers in family members in our highly consanguineous cohort. However, evidence of CAL and consanguinity in a patient with high grade glioma should raise the question of bmmrd and should not be confused with neurofibromatosis type 1 where most brain tumors are low grade gliomas and high grade tumors are rare. Finally, evidence of other bmmrd related cancers such as gastrointestinal and hematological malignancies in the index case or family members together with consanguinity and CAL are almost pathognomonic for bmmrd in a child with a diagnosis of HGG (Table 2). There is paucity of information regarding survival of children with bmmrd cancers. Some case series suggested improved survival for bmmrd patients with high grade gliomas. 14,15 We could not find any difference between suspected bmmrd and sporadic high grade glioma in our center. However, these data require longer follow up and genetic confirmation of our observations. Importantly, of the four patients diagnosed with in the SickKids cohort, two are alive with a follow-up of five and eight years, respectively. These two patients had complete resection of their tumors as a part of the surveillance protocol developed by the consortium. 16 These latter findings highlight the need for early detection, correct diagnosis and genetic counseling for children and families with bmmrd. The reasons for implementations of such guidelines are threefold. First, bmmrd related gliomas

6 Amayiri et al. 385 seem to behave differently than sporadic tumors. Recent work suggest that they do not respond to chemotherapies currently used for HGG such as temozolomide and these agents may, much to the contrary, lead to further mutation accumulation and resistance. 17 Second, recognition of bmmrd can allow for early detection of other cancers in these patients resulting in improved survival with use of less toxic therapies. Indeed, the two survivors of developed an additional four tumors in the combined 13 years of follow up. Finally, correct diagnosis and counseling can offer similar benefits for other family members of these children and hopefully allow for targeted therapies for prevention and treatment of bmmrd related cancers. In summary, this is the first study which attempts to estimate the burden of bmmrd cancers in countries where consanguinity is high. Although our cohort is small and restricted to a single national center, our results suggest that References 1. Bakry D, Aronson M, Durno C, et al. Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium. Eur J Cancer 2014;50: Wimmer K, Kratz CP. Constitutional mismatch repair-deficiency syndrome. Haematologica 2010; 95: Wimmer K, Kratz CP, Vasen HF, et al. Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium care for CMMRD (C4CMMRD). J Med Genetics 2014;51: Grover S, Syngal S. Risk assessment, genetic testing, and management of Lynch syndrome. J Natl Comprehensive Cancer Network 2010;8: Sehgal R, Sheahan K, O Connell PR, et al. Lynch syndrome: an updated review. Genes 2014;5: O Regan T, Chau K, Tatton M, et al. Immunochemistry screening for Lynch syndrome in colorectal adenocarcinoma using an initial two antibody panel can replace a four antibody panel. N Z Med J 2013;126: Bougeard G, Olivier-Faivre L, Baert-Desurmont S, et al. Diversity of the clinical presentation of the MMR gene biallelic mutations. Familial Cancer 2014;13: Marton E, Feletti A, Orvieto E, et al. Malignant progression in pleomorphic xanthoastrocytoma: personal experience and review of the literature. JNeurologSci2007;252: Vu TM, Liubinas SV, Gonzales M, et al. Malignant potential of pleomorphic xanthoastrocytoma. J Clin Neurosci 2012;19: Tabori U, Shlien A, Baskin B, et al. TP53 alterations determine clinical subgroups and survival of patients with choroid plexus tumors. J Clin Oncol 2010;28: Tabori U, Baskin B, Shago M, et al. Universal poor survival in children with medulloblastoma harboring somatic TP53 mutations. J Clin Oncol 2010;28: Shlien A, Campbell BB, de Borja R, et al. Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of among children with high grade gliomas the frequency of bmmrd may be alarmingly high. Since these patients and cancers require different treatment and approach than children with sporadic, implementation of comprehensive genetic testing and counseling may improve survival for individuals with this orphan syndrome. Molecular and functional tests may allow for early detection and interventions in patients with bmmrd. Finally, there is a need to develop new treatment protocols utilizing alternative medications to alkylating agents (in particular Temozolomide) for these patients. Acknowledgements Special thanks to Ms Reham Abu Rumman and Ms Manal Al Masri from KHCC for their laboratory technical help. Parts of this work were presented in a poster form at the International Society of Pediatric Neuro-oncology (ISPNO) meeting in 2014 in Singapore. ultra-hypermutated cancers. Nat Genetics 2015;47: Stoffel EM, Mangu PB, Gruber SB, et al. Hereditary colorectal cancer syndromes: American Society of Clinical Oncology Clinical Practice Guideline endorsement of the familial riskcolorectal cancer: European Society for Medical Oncology Clinical Practice Guidelines. J Clin Oncol 2015;33: Rutz HP, de Tribolet N, Calmes JM, et al. Longtime survival of a patient with and Turcot s syndrome. Case report. J Neurosurg 1991;74: Hamilton SR, Liu B, Parsons RE, et al. The molecular basis of Turcot s syndrome. N Engl J Med 1995;332: Durno CA, Aronson M, Tabori U, et al. Oncologic surveillance for subjects with biallelic mismatch repair gene mutations: 10 year follow-up of a kindred. Pediatr Blood Cancer 2012;59: Wick W, Platten M. Understanding and targeting alkylator resistance in. Cancer Discov 2014;4: