Multiple endocrine neoplasia type 1 (MEN1) is an

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1 JCEM ONLINE Advances in Genetics Endocrine Care Higher Risk of Aggressive Pancreatic Neuroendocrine Tumors in MEN1 Patients With MEN1 Mutations Affecting the CHES1 Interacting MENIN Domain Detlef K. Bartsch, Emily P. Slater, Max Albers, Richard Knoop, Brunhilde Chaloupka, Caroline L. Lopez, Volker Fendrich, Peter H. Kann, and Jens Waldmann Departments of Visceral, Thoracic, and Vascular Surgery (D.K.B., E.P.S., M.A., R.K., B.C., C.L.L., V.F., J.W.) and Gastroenterology (P.H.K.), Division of Endocrinology and Diabetology, University Hospital Giessen and Marburg, Campus Marburg, Marburg, Germany Context: Sixty to 80% of multiple endocrine neoplasia type 1 (MEN1) patients develop pancreatic neuroendocrine neoplasias (s), which reveal an aggressive behavior in 10%-20% of patients. Causative MEN1 mutations in the interacting domains of the encoded Menin protein directly alter its regulation abilities and may influence the phenotype. Objective: The objective of the study was the evaluation of an association between MEN1 mutations in different interacting domains of Menin and the phenotype of s. Design: This was a retrospective analysis of a prospectively collected cohort of 71 genetically confirmed MEN1 patients at a tertiary referral center. Main Outcome Measures: Analysis of patients characteristics and clinical phenotype of s regarding the mutation type and its location in Menin interacting domains was measured. Results: Sixty-seven patients (93%) developed s after a median follow-up of 134 months. Patients with mutations leading to loss of interaction (LOI) with the checkpoint kinase 1 (CHES1) interacting domain codons ( ) compared with patients with mutations resulting in LOI with other domains (eg, JunD, Smad3) had significantly higher rates of functioning s (70% vs 34%), malignant s (59% vs 16%), and aggressive s (37% vs 9%), respectively. Patients with CHES1-LOI also had an increased -related mortality (20% vs 4.5%). Neither gender, age, nor the ABO blood types were associated with the phenotype of s. Conclusions: MEN1 patients with MEN1 mutations leading to CHES1-LOI have a higher risk of malignant s with an aggressive course of disease and disease-related death. (J Clin Endocrinol Metab 99: E2387 E2391, 2014) Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited disorder classically characterized by the development of neuroendocrine neoplasms in the endocrine pancreas (s), the pituitary, and parathyroid glands (1, 2). The syndrome is caused by germline mutations in the MEN1 gene, mapped to the 11q13 (3). More than 1300 different mutations without a ISSN Print X ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received December 16, Accepted July 15, First Published Online September 11, 2014 mutation hotspot have been reported (4). The MEN1 gene encodes for the Menin protein, which has been demonstrated to interact with a number of proteins that are involved in transcriptional regulation [JunD, phosphorylated mothers against decapentaplegic 3 (Smad3), nuclear factor- B (NF- B), checkpoint kinase 1 (CHES1)], genome stability (RPA2, FANCD2), cell division (NMHCII-A), Abbreviations: CHES1, checkpoint suppressor 1; CT, computed tomography; EUS, endoscopic ultrasonography; HDAC1, histone deacetylase 1; LOI, loss of interaction; MEN1, multiple endocrine neoplasia type 1; NF- B, nuclear factor- B;, neuroendocrine neoplasms in the endocrine pancreas; Smad3, phosphorylated mothers against decapentaplegic 3. doi: /jc J Clin Endocrinol Metab, November 2014, 99(11):E2387 E2391 jcem.endojournals.org E2387

2 E2388 Bartsch et al MEN1 Mutations in CHES1 Domain and Aggressive sj Clin Endocrinol Metab, November 2014, 99(11):E2387 E2391 and proliferation (NM23H1, apoptosis signal-regulating kinase) (4 7), respectively. PNENs represent the second most common manifestation of MEN1 with a prevalence up to 85% by the age of 50 years (1, 2, 8, 9). These s are nonfunctional or functional and carry low or high malignant potential. It has been shown that s together with thymic tumors represent the major cause of death in MEN1(8) and that 23% of patients with MEN1-s have an aggressive tumor growth with hepatic metastases leading to early death (10). At present there are neither clinical nor genetic parameters that reliably predict an aggressive or less aggressive phenotype of MEN1-s (11, 12). Therefore, no general consensus has been reached regarding the optimal timing and extent of surgery in MEN1-s (13, 14). However, a recent large scale analysis of the French Groupe d Etude des Tumeurs Endocrines group postulated that MEN1 patients with mutations affecting the JunD interacting domain had a higher risk of death secondary to a MEN1-associated neoplasia (15). The present study aimed to evaluate associations between the phenotype of s and the mutation type and its location in interacting domains to identify factors that predict an aggressive course of MEN1-s. Materials and Methods All MEN1 patients treated at the Department of Surgery, University Hospital Marburg, were documented in a prospective database since Patient demographics, clinical phenotypes, pathological findings, and follow-up were analyzed with special regard to s and other malignant NETs. In addition, laboratory data were reviewed to document the blood type. The study included only patients older than 18 years with a confirmed MEN1 gene germline mutation. The presence of MEN1 associated tumors was assessed by an annual routine screening program, which included the measurement of plasma hormone levels (insulin, proinsulin, gastrin, pancreatic polypeptide, vasoactive intestinal peptide, glucagon, chromogranin A) and imaging with endoscopic ultrasonography (EUS), abdominal magnetic resonance imaging, computed tomography (CT) of the thorax, somatostatin receptor scintigraphy, or Ga 68 -DOTATOC-positron emission tomography-ct, respectively (9, 16). All MEN1 patients with either biochemical evidence of functioning s or nonfunctioning s larger than 1 cm in size on imaging were recommended to undergo duodenopancreatic surgery. The diagnosis of s was established by plasma hormone levels (see above), imaging, especially EUS, and/or conventional histological and immunohistochemical examinations (chromogranin A, synaptophysin, insulin, gastrin) of resection specimens. All resected specimens were classified according to the World Health Organization criteria (17). Malignancy was determined on the strict criteria of lymph node or distant metastases. Aggressive disease was defined as the presence or development of distant metastases. The follow-up was based on the most recent screening examination until July All patients with s had annual follow-up screening examinations of the pancreas, including EUS or magnetic resonance imaging and somatostatin receptor scintigraphy or Ga 68 -DOTATOC-positron emission tomography-ct in case of Zollinger-Ellison syndrome as described previously (9, 16). The type of mutation was analyzed by comparing truncating mutations (nonsense, frame shift) vs nontruncating mutations (missense, in-frame deletions, or insertions) with special regard to the phenotype of s and also other malignant NENs. In addition, mutations affecting an interacting domain causing loss of interaction (LOI) with functional partners of Menin JunD (codons 1 40, , and ), NF- B (codons ), Smad3 (codons and ), Pem (codons ), NM23H1 (codons 1 486), RPA2 (codons 1 40 and ), FANCD2 (codons ), msin3a (codons ), histone deacetylase 1 (HDAC1; ), and CHES1 (codons ) according to the published mapping (4) were evaluated. The analysis of the locations of mutations included all types of mutations. For further analyses, the causes of death were categorized as secondary to a, secondary to a cancer of the MEN1 spectrum (eg, pancreas, thymus, bronchi, adrenal glands, parathyroid glands and pituitary gland), and death of a MEN1- unrelated cause. Statistics Time-to-event techniques were used to identify genotypephenotype correlations. Events of interest included occurrence of s, prevalence of functioning s, prevalence of malignant s, prevalence of aggressive s with distant metastases, prevalence of other malignant NETs, and cause of death of, respectively. The prognostic role of the phenotypic profiles for each phenotypic criterion was assessed using a Fisher s exact test. Odds ratios and contingency intervals were calculated for significant variables to quantify the relative risk. A value of P.05 was considered statistically significant. The intrafamilial heterogeneity could not be assessed because of the small sample size. Based on their blood types, patients were stratified into 2 groups: individual blood types (A, AB, B, or O), and 0 blood type vs non-0 blood type. To test for a general association between blood type and disease state, a Fisher s exact contingency table analysis was performed using 4 2 tables. Results Clinical characteristics and pathology Sixty-seven of 71 (93%) MEN1 patients (38 male, 33 female) of 56 families had s. The median age of diagnosis was 37 years (range 9 69 y). Fifty-eight of these patients (87%) underwent duodenopancreatic surgery, and the diagnosis of was confirmed by histopathology. The s were nonfunctional in 24 patients (41%) and functional in 34 patients (59%), including Zollinger-Ellison syndrome (n 20), insulinoma (n

3 doi: /jc jcem.endojournals.org E ), and vipoma (n 1). Of the 58 patients who underwent duodenopancreatic surgery, 44 patients (76%) had G1 tumors, 10 patients (17%) had G2 (Ki67 2% 10%) tumors, and for four patients grading was not available. Twenty-three patients (34%) had histologically verified malignant s (18 functioning, five nonfunctionings), which showed an aggressive course of disease with the development of distant metastases in 10 patients (15%). In addition, 8 of 71 patients (11%) had other malignant NETs, including thymic carcinoid (n 4) and bronchial NET (n 2) as well as gastric NET and adrenocortical cancer in one patient each, respectively (Supplemental Table 1). The patient with the gastric NET was very uncommon because it was a neuroendocrine carcinoma G3 (Ki67, 60%) with liver metastases, which developed during 2 years based on multiple small G1 gastric neuroendocrine neoplasias. After a median follow-up of 134 months (6 to 468 mo), 62 patients were alive, seven patients (9%) died due to MEN1-related cancers, and two patients died of MEN1- unrelated causes (one car accident, one pneumonia). The seven cancer deaths were caused by metastatic s in four patients, by metastatic thymic carcinoids in two patients, and by adrenocortical cancer in one patient, respectively. Gender and phenotype Male patients had a higher prevalence of s (100%) than females patients (88%, P.042). However, no significant gender difference was observed regarding the other phenotypic parameters (Supplemental Table 1). Blood type and phenotype The individual blood type was neither associated with the phenotype of s nor the risk of MEN1-related death. The same held true when comparing blood 0 vs non-blood 0 types (Supplemental Table 1). Genotype-phenotype comparisons A MEN1 gene mutation analysis identified the 45 different causative MEN1 gene mutations in the 71 patients of 56 families distributed throughout the gene. Identified mutations encompassed 36 truncating (10 nonsense, 26 frame shift mutations) and nine nontruncating mutations (eight missense, one in frame deletion). There was no statistically significant association between the genotype criteria truncating or nontruncating mutation and the phenotypic criteria of s or other malignant NETs (data not shown). Most mutations affected the interacting domains of JunD, Smad3, CHES1, and HDAC1, leading to LOI with these functional domains (Figure 1). Patients with CHES1-LOI had significantly higher rates of func- Figure 1. Location of MEN1 mutations and domains that interact with other proteins.

4 E2390 Bartsch et al MEN1 Mutations in CHES1 Domain and Aggressive sj Clin Endocrinol Metab, November 2014, 99(11):E2387 E2391 Table 1. Genotype/Phenotype Analysis of s in Genetically Confirmed MEN1 Patients LOI n Age Dx Functioning <30 y at Age >50 y Aggressive a Disease Overall Disease >50 y Aggressive Disease a DOD All (9 69) 34/71 (47%) 22/71 (31%) 23/71 (32%) 23/47 (49%) 10/71 (14.%) 27/71 (38%) 27/49 (55%) 12/71 (16.9%) 7/71 (9.9%) JunD (15 68) 12/31 (39%) 9/31 (29%) 7/31 (22.5%) 7/18 (39%) 1/31 (3.%) 8/31 (26%) 8/18 (44%) 2/31 (6.4%) 2/31 (6.4%) None JunD (9 69) 22/40 (55%) 13/40 (32.5%) 16/40 (40%) 16/32 (47%) 9/40 (22.5%) 19/40 (47.5%) 19/30 (63%) 10/40 (25%) 5/ %) Smad (9 69) 26/49 (52%) 15/49 (31%) 15/49 (31%) 15/31 (40.5%) 8/49 (16%) 19/49 (39%) 19/36 (53%) 10/49 (20%) 6/49 (12.5%) None Smad (12 50) 8/21 (38%) 7/22 (32%) 8/22 (36%) 8/14 (57.5%) 2/22 (9%) 8/22 (36%) 8/13 (61.5%) 2/22 (9%) 1/22 (4.5%) CHES (9 58) 19/27 (70%) 9/27 (33%) 16/27 (59%) 16/20 (80%) 8/27 (30%) 17/27 (63%) 17/21 (81%) 10/27 (37%) 5/25 (20%) None CHES (12 69) 15/44 (34%) 13/44 (29%) 7/44 (16%) 7/27 (26%) 2/44 (4.5%) 10/44 (23%) 10/28 (36%) 4/44 (9%) 2/44 (4.5%) HDAC (15 68) 15/29 (52%) 8/29 (27.5%) 9/29 (31%) 9/20 (45%) 3/29 (10%) 11/29 (38%) 9/19 (47%) 5/29 (17%) 2/29 (6.8%) None HDAC (9 69) 19/42 (45%) 14/42 (33%) 15/42 (36%) 15/28 (53%) 7/42 (16.5%) 1/42 (40%) 17/30 (56%) 9/42 (215%) 5/42 (12%) Abbreviations: DOD, dead of disease; Dx, diagnosis. a As determined by the development of distant metastases. tioning s (70% vs 34%), malignant s overall (59% vs 16%), and, by the age older than 50 years (80% vs 24%), aggressive s (30% vs 4.5%) and tended to a higher rate of -related death (15% vs 2.2%) than patients with LOI with other domains (Table 1). However, there was no difference regarding the grading of s between groups. Patients with CHES1-LOI also had higher rates of malignant disease overall (63% vs 23%) and aggressive course of malignant disease (37% vs 9%), leading to a tendency of a higher rate of disease-related death (20% vs 4.5%, P.089) and decreased survival (287 vs 422 months, P.01, Supplemental Figure 1). The presence of CHES1-LOI predicted a malignant, aggressive with a sensitivity and specificity of 76% and 71% (odds ratio and confidence intervals; also see Supplemental Table 2). In contrast, none of the eight other tested interacting domains, especially JunD, showed significant associations regarding the phenotype of s and other NETs (Table 1). Discussion It has been shown that truncating as well as nontruncating mutations can affect the interaction with known functional partners of Menin such as JunD, Smad3, and CHES1 (4 7). A recent French large-scale study of 806 patients reported a significantly higher risk of MEN1-related tumor death in patients with mutations affecting the JunD interacting domain (15). Using the same approach, the present study focused on a potential genotype-phenotype correlation with regard to the detailed clinicopathological characteristics of s in a well-defined, prospectively collected cohort of genetically confirmed MEN1 patients who were all treated under the same surgical philosophy. This analysis reveals for the first time that patients with mutations affecting the CHES1-interacting domain as compared with patients with mutations affecting the other domains (eg, JunD, Smad3) had significantly higher rates of functioning s, malignant s, aggressive s with the development of distant metastasis, and -related deaths, respectively. This is somehow in line with the French study (15) that also revealed a significant correlation (P.04) between CHES1-LOI and the phenotype when using the frailty model of Cox-hazard ratio. However, this correlation lost significance after the false discovery rate correction. In contrast to this study (15), a higher rate of MEN1- related tumor death in patients with JunD-LOI could not be demonstrated. The reasons for this controversial result remain speculative. It might be influenced by the heterogeneity across families, which could not be statistically determined in the present study and differing qualities of analyzed clinical data, respectively. In addition, the aggressive surgical attitude in our cohort might have changed the natural course of some aggressive s resulting in some bias. In accordance with previous studies, no mutational hot spot was evident, and there was no statistically significant association between the genotypic criteria truncating or nontruncating mutation (5, 14, 15). The presented cohort confirms the results of other studies (2, 8, 15) with regard to gender distribution; age of disease onset; rate of MEN1-related deaths; and the prevalence of primary hyperparathyroidism, pituitary tumors, adrenal neoplasms, thymic and bronchial NETs, respectively. This also holds true for the observation that thymic tumors occurred exclusively in male patients and the risk of MEN1-related death tended to be higher in male than in female patients (8, 15, 18, 19). In contrast to a previous study (20) of 105 MEN1 subjects, the presented study cannot confirm that MEN1 patients with the 0 blood type have a higher rate of GEP-NETs and NETs of the lung nor a significantly higher rate of metastatic tumors (Table 1). This controversial result might be partially caused by the high rate of s ( 90%) in both blood 0 type and nonblood 0 type in our cohort.

5 doi: /jc jcem.endojournals.org E2391 On the molecular mechanisms behind our observation can only be speculated. Menin is also required for the expression of cyclin dependent kinases such as p18 and p27 that maintain cells in a quiescent state. Menin can bind specific DNA directly and is involved in DNA repair through the CHES1 and other pathways (ATR-checkpoint kinase 1, FANCD2). At the molecular level, loss of function of CHES1 may lead to additional mutations by impairing DNA-repair mechanism that potentially accelerate cell division. Through these mechanisms, LOI with CHES1 may promote a more aggressive phenotype. Although the underlying mechanisms were not analyzed and remain unclear, identification of at-risk MEN1 mutations, such as those with CHES1-LOI, might be relevant for the indication for surgery in MEN1-s because this is a highly controversial issue. If CHES1-LOI would reliably indicate a high risk of a malignant and aggressive course of MEN1-s, it might be an important additional parameter for the decision for surgery. This hypothesis warrants confirmation in larger studies with the functional measurement of CHES1 activity in patients with mutations in codon positions The present study has some limitations as well as some strengths. It is limited by its relatively small sample size, the inability to calculate the heterogeneity across families, and the lack of molecular data on expression and function. In addition, the aggressive surgical attitude might have changed the natural course of some aggressive s, resulting in some bias. The strength of the study is the well-defined, prospectively collected patient cohort with detailed pathological and clinical follow-up data. Acknowledgments We are grateful to all the patients who participated in our screening program. Address all correspondence and requests for reprints to: Jens Waldmann, MD, Department of Visceral, Thoracic and Vascular Surgery, Philipps University Marburg, Baldingerstrasse, Marburg, Germany. jwaldmann@med.uni-marburg.de. Disclosure Summary: The authors have nothing to declare. References 1. Wermer P. Genetic aspects of adenomatosis of endocrine glands. Am J Med. 1954;16: Trump D, Farren B, Wooding C, et al. Clinical studies of multiple endocrine neoplasia type 1 (MEN1). Q J Med. 1996;89: Chandrasekharappa SC, Guru SC, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia type 1. Science. 1997;276: Lemos MC, Thakker RV. Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Hum Mutat. 2008;29: Wautot V, Vercherat C, Lespinasse J, et al. 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