Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1

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1 Clin Genet 2017: 91: Printed in Singapore. All rights reserved Review 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1 Wimmer K., Rosenbaum T., Messiaen L. Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1. Clin Genet 2017: 91: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2016 Constitutional mismatch repair (MMR) deficiency (CMMRD) is a rare childhood cancer susceptibility syndrome resulting from biallelic germline loss-of-function mutations in one of the MMR genes. Individuals with CMMRD have high risk to develop a broad spectrum of malignancies and frequently display features reminiscent of neurofibromatosis type 1 (NF1). Evaluation of the clinical findings of genetically proven CMMRD patients shows that not only multiple café-au-lait macules but also any of the diagnostic features of NF1 may be present in a CMMRD patient. This phenotypic overlap may lead to misdiagnosis of CMMRD patients as having NF1, which impedes adequate management of the patients and their families. The spectrum of CMMRD-associated childhood malignancies includes high-grade glioma, acute myeloid leukaemia or rhabdomyosarcoma, also reported as associated with NF1. Reported associations between NF1 and these malignancies are to a large extent based on studies that neither proved the presence of an NF1 germline mutation nor ruled-out CMMRD in the affected. Hence, these associations are challenged by our current knowledge of the phenotypic overlap between NF1 and CMMRD and should be re-evaluated in future studies. Recent advances in the diagnostics of CMMRD should render it possible to definitely state or refute this diagnosis in these individuals. Conflictofinterest The authors have no conflicts of interest to declare. K. Wimmer a, T. Rosenbaum b and L. Messiaen c a Division of Human Genetics, Medical University Innsbruck, Innsbruck, Austria, b Department of Pediatrics, Sana Kliniken Duisburg, Wedau Kliniken, Duisburg, Germany, and c Medical Genomics Laboratory, Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, USA Key words: acute myeloid leukaemia café-au-lait spot childhood cancer constitutional mismatch repair deficiency germline mutation high-grade glioma mismatch repair gene neurofibromatosis type 1 rhabdomyosarcoma Corresponding author: Katharina Wimmer, PhD, Division of Human Genetics, Medical University Innsbruck, Peter-Mayr-Straße 1, 6020 Innsbruck, Austria. Tel.: ; fax: ; katharina.wimmer@i-med.ac.at Received 31 August 2016, revised and accepted for publication 20 October 2016 Neurofibromatosis type 1 With a birth frequency of 1 in , neurofibromatosis type 1 (NF1; #MIM ) is a frequent neurocutaneous disorder that predisposes to various benign and malignant tumours. About 50% of NF1 patients are sporadic cases without family history of NF1 (1). The hallmark features of NF1 are hyperpigmented skin spots, i.e. café-au-lait macules (CALM) and skinfold freckling, and peripheral nerve sheath tumours termed neurofibromas. Several additional characteristic features, which are present with varying frequency in NF1 patients, are included in the criteria for the clinical diagnosis of NF1 that were agreed at the NIH Consensus Conference in 1987 (Table 1). NF1 is a progressive disorder with most of the cardinal clinical features developing during childhood and adolescence. CALM are usually the first presenting sign of NF1, followed by skinfold freckling (2). About half of sporadic NF1 cases do not yet fulfil the NIH diagnostic criteria by the age of 1 year (2). Nevertheless, NF1 is the most likely diagnosis in a child with multiple CALM and/or freckling, and a disease causing NF1 mutation is found by sensitive comprehensive mutation analysis in 43% of all sporadic patients presenting with only multiple (>5) CALM with or without freckling (3). 507

2 Wimmer et al. Table 1. Adapted NIH NF1 diagnostic criteria a The NIH Consensus Development Conference Statement diagnostic criteria for NF1 are met in an individual who has two or more of the following: (1) Six or more café-au-lait macules of over 5 mm in greatest diameter in pre-pubertal individuals and over 15 mm in greatest diameter in post-pubertal individuals. (2) Two or more neurofibromas of any type or one plexiform neurofibroma. (3) Freckling in the axillary or inguinal regions. (4) Optic pathway glioma. (5) Two or more Lisch nodules (iris hamartomas). (6) A distinctive osseous lesion such as sphenoid dysplasia or thinning of the long bone cortex with or without pseudarthrosis. (7) A parent or offspring with NF1 by above criteria. a NF1, neurofibromatosis type 1. a Changed according to the suggestions of Huson (57) from A first-degree relative (parent, sibling, or offspring) with NF1 by above criteria. NF1 is caused by heterozygous mutations in the tumour suppressor gene NF1 which encodes neurofibromin, a Ras-specific GTPase activating protein (Ras-GAP) that functions as a key negative regulator of the cellular rat sarcoma (RAS) signalling pathway (4, 5). Hence, inactivation of neurofibromin increases cellular levels of active Ras-guanosine-5 -triphosphate (GTP) which is in turn responsible for deregulation of cell growth and potentially tumourigenesis (6). Consistent with Knudson s two-hit hypothesis for tumour suppressor gene inactivation, somatic second-hit NF1 mutations are found in neoplastic cells of NF1-associated tumours, e.g. in Schwann cells from neurofibromas (7), but also in non-neoplastic cells of other features, e.g. in the melanocytes cultivated from CALM (8). The tumour spectrum associated with NF1 not only contains the usually benign cutaneous and plexiform neurofibromas, but also other benign and malignant neoplasias. Malignant peripheral nerve sheath tumours (MPNSTs) occur at any age in 8 13% of NF1 patients, primarily arise from pre-existing plexiform neurofibromas and have a poor prognosis (9). A total of 15% of NF1 patients develop optic pathway gliomas (OPGs) in childhood (10). OPG are histologically WHO grade I pilocytic astrocytomas located anywhere along the optic nerves, chiasm and optic tracts (10). NF1 children also carry a 350-fold increased risk to develop juvenile myelomonocytic leukaemia (JMML) (11). However, the individual risk of this very rare myelodysplastic/myeloproliferative disorder in NF1 is still small [given as perhaps 1:2500 by Chang and Shannon (12)]. Within the paediatric age group, NF1-associated tumours also include rhabdomyosarcomas (RMSs) (13, 14) and possibly neuroblastomas (15), while a definite association between Wilms tumours and NF1 could not be established (16). Constitutional mismatch repair deficiency The highly conserved mismatch repair (MMR) system plays a key role in safeguarding genomic integrity during DNA replication by correcting replication errors, which escape the proofreading activity of the DNA polymerases (17). In the absence of a functional MMR system, replication errors, such as base base mismatches and small insertion deletion loops, remain uncorrected resulting in a mutator phenotype that is well known to contribute to the development of sporadic and familial human cancer. Five human MMR genes, MSH2 (MIM *609309), MSH6 (MIM *600678), MSH3 (MIM *600887), MLH1 (MIM *120436), and PMS2 (MIM *600259), code for proteins which play a crucial role in the MMR process. In sporadic colorectal cancers, somatic MMR deficiency primarily results from silencing of MLH1 by promoter hypermethylation. Whereas germline mutations in the MLH3 gene were only very recently shown to be associated with a recessively inherited polyposis and cancer predisposition syndrome (18), Lynch syndrome (LS; formerly named hereditary non-polyposis colorectal cancer) is a well-known autosomal dominant familial cancer syndrome resulting from heterozygous (monoallelic) loss-of-function germline mutations in one of the MMR genes MLH1, MSH2, MSH6 or PMS2. In LS-associated tumours, the wild type MMR allele is lost through somatic mutations or loss of heterozygosity (LOH), resulting in MMR deficiency. LS primarily predisposes to colorectal and endometrial cancers, but also to urinary tract, stomach, small bowel, ovarian cancer and brain tumours (19, 20). The age of cancer onset in LS patients is much younger than the age of onset of the respective cancer in the general population. In general, however, LS patients do not develop cancer before the age of 25 years. In 1999, two reports described the phenotype of five children who were born to consanguineous parents from two LS families and who carry homozygous MLH1 germline mutations (21, 22). These five individuals developed haematological malignancies (age range 14 months to 6 years) and one individual additionally developed a medulloblastoma at the age of 7 years. Importantly, these children also displayed clinical features reminiscent of NF1, i.e. >5 CALM (>15 mm) in all who were alive when the clinical phenotype was recorded (4/5); dermal neurofibromas in two individuals and a tibial pseudarthrosis in one individual. Since 508

3 Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1 these initial reports, nearly 200 individuals carrying inactivating biallelic (homozygous or compound heterozygous) germline mutations in one of the four MMR genes involved in LS have been reported and the condition is now recognized as a distinct childhood cancer susceptibility syndrome with a very broad tumour spectrum including primarily haematological malignancies, malignant brain tumours and LS-associated malignancies (OMIM #276300). The condition is known under the name constitutional MMR deficiency (CMMRD) syndrome, a designation recapitulating the underlying pathogenic mechanism of the condition (23). There is a remarkable difference in the distribution of MMR gene mutations in the two cancer syndromes, LS and CMMRD. While MLH1 and MSH2 mutations are found in the vast majority ( 75%) of LS patients, they are responsible for less than 20% of the reported CMMRD syndrome cases. Conversely, biallelic PMS2 mutations account for roughly 60% of the reported CMMRD patients, whereas heterozygous PMS2 mutations are found in only 5 15% of LS patients (24). Equally, MSH6 mutations are more frequently found in CMMRD patients ( 20%) than in LS patients ( 10%). This difference is partially explained by the lower penetrance of PMS2 and MSH6 mutations (25 27). This leads to underrepresentation of particularly monoallelic PMS2 mutation carriers among LS patients and also explains why LS-associated cancers frequently are absent in the family history of biallelic PMS2 mutation carriers. Furthermore, review of all known CMMRD cases shows that, compared with patients with biallelic germline mutation in MLH1/MSH2, more patients with PMS2-associated CMMRD syndrome develop more than one metachronous malignancy [30% (10/33) vs 48% (56/117)], suggesting that they are more likely to survive their first tumour and develop later on a second unrelated malignancy. This genotype phenotype correlation may facilitate the clinical diagnosis of PMS2-associated CMMRD and, hence, possibly lead to ascertainment bias in the reported CMMRD patients. Moreover, biallelic PMS2 mutation carriers more frequently have brain and LS-associated tumours compared with biallelic MLH1/MSH2 mutation carriers, who in turn have a higher prevalence of haematological malignancies compared with individuals with biallelic PMS2 mutations (28). Thus, biallelic PMS2 mutation carriers are more likely to resemble the Turcot-syndrome (TS) phenotype, which is clinically characterized by the association of a primary brain tumour and colorectal carcinoma and/or adenomas. It is known for long time that particularly those TS patients who have small numbers of colorectal neoplasms, colorectal carcinoma in childhood or adolescence, glioblastoma, or CALM show microsatellite instability (MSI) in tumour tissue, which is a hallmark of a MMR defect (29). Therefore, due to the more frequent phenotypic overlap between TS and PMS2-associated CMMRD, the latter diagnosis may be more readily considered and established genetically. Regardless of the reasons of a potential ascertainment bias in favour of PMS2-associated CMMRD, it may have influenced our current knowledge on CMMRD. In addition, it may be speculated that certain MLH1 and MSH2 mutations are not viable in a homozygous state, whereas this may be less likely the case for PMS2 mutations. The clinical presentation and diagnostic criteria of CMMRD The spectrum of CMMRD-associated malignancies can be separated in four main groups: (i) haematological malignancies with a predominance of T-cell non-hodgkin lymphoma [for review see (30)]; (ii) high-grade gliomas and other brain/central nervous system (CNS) tumours [for review see (31)]; (iii) colorectal and other carcinomas associated with Lynch syndrome [for review see (32 34)] and (iv) a miscellaneous group of malignancies seen less frequently in CMMRD including, amongst others, sarcomas and embryonic tumours [for review see (28, 35)]. Most malignancies diagnosed in CMMRD patients fall in one of the first three groups, however, any malignancy in the paediatric age group may be a CMMRD associated one. Table 2 lists and summarizes all malignant tumours (321) that have been observed in a cohort of 197 genetically proven CMMRD patients from 128 families collated from the medical literature or recently diagnosed in one of our laboratories. Figure 1 shows the distribution of the age at diagnosis of malignancy for the three main malignant tumour groups and subgroups in CMMRD patients. This figure illustrates that haematological tumours overall, and T-non-Hodgkin s lymphoma (NHL) in particular, tend to develop at an earlier age than brain/cns tumours and LS-associated tumours. The spectrum of tumours found in CMMRD patients includes also pre-malignancies and non-malignant tumours which are not listed in Table 2. Among these, colorectal polyps, mainly adenomatous but occasionally also juvenile, are the most prevalent (28). There is evidence that gastrointestinal adenomas in CMMRD patients rapidly transform into carcinomas (32, 34). In many patients, multiple synchronous adenomas ranging from a few up to 100 polyps are present mimicking (attenuated) familial adenomatous polyposis (32 34, 36). Hepatic adenomas have been found so far in three genetically confirmed CMMRD patients and in one of the patients originally described by Turcot (37). Holter et al. (37) provide evidence that CMMRD patients are prone to hepatic adenomas because of the presence of a hypermutable C8-monomer in exon 4 of the HNF1 gene (NM_ ) in the MMR deficient cells. Pilomatricomas (pilomatrixomas, calcifying epitheliomas of Malherbe) are slow growing benign skin tumours that generally appear within the first decades of life as a solitary lesion on the face and upper extremities (38). Multiple pilomatricomas in the same individual are very rare, but have so far been reported in four genetically confirmed CMMRD patients [(18, 39), Ilencikova personal communication]. It was shown in one of these CMMRD patients that each distinct pilomatricoma resulted from different activating mutations in CTNNB1 (ß-Catenin gene), indicating that the increased frequency 509

4 Wimmer et al. Table 2. Distribution of 321 malignancies in 197 CMMRD patients Percent of Percent of patients with all tumours malignancy (n = 321) (n = 197) 1. Haematological malignancies T-cell non-hodgkin s lymphoma (NHL) 1.2. B-cell non-hodgkin s lymphoma (NHL) 1.3. Burkitt lymphoma Other lymphoma (including NHL unspecified) 1.5. Lymphoid leukaemia Acute myeloid leukaemia Atypical chronic myeloid leukaemia 1.8. Not specified acute leukaemia Brain and central nervous system tumours High-grade gliomas Medulloblastoma (Supratentorial) primitive neuroectodermal tumour 2.4. Infiltrating cerebral angiosarcoma Cerbral anaplastic ganglioma Papillary ganglioneural tumour Pleomorphic xanthoastrocytoma Not specified brain tumour Lynch syndrome-associated carcinomas of Colon/rectum a Duodenum/jejunum/ileum a Endometrium Bladder/ureter (papillary transitional cell ca.) 3.5. Ureter/renal pelvis (unspecified) Ovarian cancer Gastric cancer Others Neuroblastoma Nephroblastoma (Wilms tumour) Ovarian neuroectodermal tumour 4.4. Borderline philloides tumour of breast 4.5. Infantile myofibromatosis Rhabdomyosarcoma Osteosarcoma Mediastinal granulocytic sarcoma 4.9. Dermatofibrosarcoma protuberans Basal cell carcinoma Muco-epidermoid ca. of parotis CMMRD, constitutional mismatch repair deficiency. a Includes adenomas with high-grade dysplasia. Fig. 1. Age range of constitutional mismatch repair deficiency (CMMRD) patients at malignancy diagnosis. Box plots show the age range (in years) at diagnosis of the three main malignancy types (1, haematological malignancies; 2, brain and central nervous system tumours; and 3, Lynch syndrome-associated carcinomas) and their main subtypes (1.1, T-cell non-hodgkin s lymphomas; 2.1, high-grade gliomas; and 3.1, colorectal carcinomas). In this graphical representation, the bottom and top of the box indicate the first and the third quartiles, and the band inside the box indicates the second quartile, i.e. the median age at diagnosis which is noted also to the left of the box plot. The ends of the whiskers indicate the youngest and oldest age at diagnosis of each malignancy (sub-)type. 510

5 Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1 of multiple pilomatricomas in CMMRD patients results from a hypermutability of CTNNB1 (ß-Catenin gene) in individuals with impaired MMR capacity (39). Multiple pilomatricomas, when present in a paediatric cancer patient, should raise a high level of suspicion for this tumour susceptibility syndrome. The most frequently described non-neoplastic features indicative of CMMRD when present in a young cancer patient are multiple ( 2) CALM or other hyperpigmented skin lesions. More details of CALM and other NF1 signs in CMMRD patients will be discussed later (Overlap of CMMRD and NF1). Besides hyperpigmented macules, hypopigmented skin areas also are frequently reported in CMMRD patients. Systematic evaluation for this feature shows that hypopigmented macules are present in roughly 20% of CMMRD patients [in 6 of 31 patients from the French cohort (35)]. Other non-neoplastic findings in CMMRD patients that are seen more frequently in CMMRD patients than in the general population and therefore considered diagnostic signposts of this childhood cancer susceptibility syndrome include agenesis of the corpus callosum, sometimes associated with grey matter heterotopia (40). Furthermore, non-therapy induced brain cavernomas have been reported in at least three CMMRD patients (35, 41) and are, therefore, also considered a diagnostic feature of CMMRD. In addition to DNA repair activity, the MMR system is also involved in other cellular processes, including immunoglobulin (Ig) class switch recombination and somatic hypermutation of variable regions of Ig genes (17). Decreased IgA and/or IgG2/4 levels, which are associated particularly in young children with increased IgM levels (hyper-igm syndrome), were reported so far in 12 CMMRD patients carrying biallelic PMS2, MSH6 or MSH2 mutations (42 44). In most of these cases, this shift in Ig class distribution, indicative of an impairment of Ig class switch recombination, was not associated with severe and persistent infections. Hence, the lack of a clinically noticeable immune defect may have precluded testing of Ig levels in other CMMRD patients, and systematic testing may reveal that this diagnostic feature may be present in many more of the CMMRD patients. Since presence of one or more of the above described non-neoplastic features may indicate CMMRD as the underlying syndrome in a paediatric/young adult cancer patient, they were included in a scoring system developed by the European consortium care for CMMRD (C4CMMRD). In this scoring system, malignancies and non-malignant features are assigned scoring points (Table S1, Supporting information) and any cancer patient reaching 3 points should be suspected of having CMMRD (28). Overlap of CMMRD and NF1 The connection between NF1 and CMMRD was established in 1999 by two seminal papers reporting CMMRD patients who clearly fulfilled the NIH criteria of NF1 with >5 CALM (>15 mm), and a first-degree relative (sibling) with signs of NF1 in all individuals, and additionally dermal neurofibromas in two and a tibial pseudarthrosis in one individual (21, 22). Of note, for one of these patients a strictly hemicorporal distribution of CALM was reported (22). A segmental distribution of CALM and freckling was also observed in several subsequently reported CMMRD patients (35, 45) and in the patient shown in Fig. 2b who is compound heterozygous for two MSH6 mutations. Presence of >5 CALM is a diagnostic criterion for NF1 (Table 1) and 99% of NF1 patients fulfil this criterion by the age of 1 year (2). Multiple CALM, usually the first presenting sign of NF1 in children, were reported in at least 62% (91/146) of the CMMRD patients evaluated as of June 2013 (28), in 71% (22/31) of patients from a French cohort (35) and in all patients with available clinical data (18/23) collected by an international consortium headed by a Canadian team (46). Only a few CMMRD patients are explicitly reported to lack CALM (35, 40, 47 50). However, the number of CALM did not reach the critical number of >5 in all CMMRD patients which is needed to fulfil one NF1 diagnostic criterion. Furthermore, several reports stress that the CALM in CMMRD patients vary in their degree of pigmentation and have jagged coast of Maine borders and, hence, differ from the typical uniformly pigmented and smooth-bordered CALM associated with NF1 (51 54). However, these subtle differences may be recognized only by clinicians with specific experience related to NF1, and, moreover, several CMMRD patients also show CALM typically found in NF1 (compare CMMRD- and NF1-associated CALM in Fig. 2c f). Roughly a fifth of the CMMRD patients [27/146 (28)] were reported to show more than one NIH NF1 feature. Most frequently, i.e. in 10% of all CMMRD patients [15/146 (28); 3/23 (46); 2/31 (35)], CALM with axillary and/or inguinal freckling were present. Other NIH diagnostic NF1 features, such as cutaneous or plexiform neurofibromas, Lisch nodules, tibial or sphenoid wing dysplasia or OPG, have been reported in one or more CMMRD patients (Table 3). In some recently diagnosed CMMRD patients focal areas of signal intensity (FASI) in brain Magnetic resonance imaging (MRI) were noticed (Fig. 2h). Brain hamartomas reported in one CMMRD patient (35) or areas of increased signal intensity (47) may represent the same lesions. FASI, also termed unidentified bright objects, although not considered a diagnostic criterion of NF1, are a frequent finding in pre-pubertal NF1 children (55, 56) and, therefore, when present in a CMMRD patient, may be interpreted as an indication for NF1 (compare the NF1- and CMMRD-associated FASI in Fig. 2g,h, respectively). Parents of CMMRD patients typically do not show signs of NF1, but siblings may also present with NF1-associated features, if they also inherited both mutant parental MMR alleles. In such cases, the NIH criterion for NF1 diagnosis first-degree affected relative would be fulfilled. In view of this potential pitfall, Huson (57) suggested to change the wording of the NIH diagnostic criterion from first-degree relative (which includes siblings) to parent or offspring (Table 1). 511

6 Wimmer et al. (a) (b) (c) (d) (e) (f) (g) (h) Fig. 2. Typical neurofibromatosis type 1 (NF1)-associated features as observed in patients with constitutional mismatch repair deficiency (CMMRD) syndrome in comparison to NF1 patients. Segmental distribution of café-au-lait macules (CALM) and freckling in an adult NF1 patient (a; photo courtesy of Prof S. Tinschert) and a 10-year-old CMMRD patient (b). Different CALM and other hyperpigmented skin pigmentation alterations in a 14-year-old NF1 patient (c, e; photos courtesy of P.D. Dr R. Gruber) and in an 8-year-old CMMRD patient (d, f; photo courtesy of Prof D. Januszkiewicz-Lewandowska). T2-weighted axial Magnetic resonance imaging (MRI) scans of the brain showing focal areas of high signal intensity (FASI) in an NF1 and CMMRD patient, respectively, (g, h). The MRI of an 8-year-old NF1 patient (g) shows a bright FASI in the left globus pallidus as well as a smaller one in the right capsula interna and the MRI of an 8-year-old CMMRD patient (h) shows a bright FASI in the right capsula interna and a subtle spot in the left thalamus. 512

7 Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1 Table 3. NIH NF1 features seen in CMMRD patients Feature Percent of CMMRD patients References CALM (28, 35, 46) CALM and freckling 6 13 (28, 35, 46) Lisch nodules 3.6 (7/197) (45, 84, ), unpublished patient Cutaneous 1.5 (3/197) (21, 22, 110) neurofibroma Plexiform neurofibroma 1 (2/197) (84, 107) Tibia pseudarthrosis 0.5 (1/197) (22) Sphenoid wing 0.5 (1/197) Unpublished dysplasia Optic pathway glioma 0.5 (1/197) (76) CALM, café-au-lait macules; CMMRD, constitutional mismatch repair deficiency; NF1, neurofibromatosis type 1. It is speculated that signs reminiscent of NF1 in CMMRD patients result from post-zygotic NF1 mutations which may occur more frequently than in the normal population due to an accelerated NF1 mutation rate in cells lacking a functional MMR system. The notion that the NF1 gene is a target of MMR deficiency is supported by identification of somatic NF1 mutations in 4 of 10 tumour cell lines and 2 of 5 primary tumours exhibiting MSI, the hallmark of a MMR defect, but in none of the five MMR-proficient tumour cell lines (58). The identified alterations reflected the typical mutational footprint of a MMR defect, as half of the alterations were frameshift mutations and four of them at monomer repeat sequences within the NF1 coding sequence. Nevertheless, a deleterious NF1 mutation in blood was found so far only in one CMMRD patient (59), who was originally described by Ricciardone et al. (21). This mutation was a recurrent C to T transition at a CpG dinucleotide (c.3721c>t, p.arg1241ter) and found in a heterozygous status in blood cells without evidence of mosaicism. Therefore, the authors speculated that it is a somatic mutation that occurred very early in embryonic development. No underlying NF1 mutation was uncovered in any of the other CMMRD patients for whom NF1 mutation analysis was performed [even after applying comprehensive and highly sensitive mutation analysis (60) in several cases] (35, 45, 61 65). In part, this may be explained by the difficulties to detect low level mosaicism for NF1 mutations in blood lymphocytes or by the absence of an NF1 mutation in blood cells in patients with segmental skin findings. However, it is also possible that the CALM and other NF1 features in these patients each represent isolated skin manifestations with each carrying two different NF1 mutations rather than a common first hit with different second hits in every lesion, as commonly seen in mosaic or segmental NF1 (66). Furthermore, CALM are also found in other hereditary syndromes like Legius syndrome, Noonan syndrome with multiple lentigines (formerly termed also LEOPARD syndrome), McCune-Albright syndrome, Nijmegen breakage syndrome, Fanconi anaemia and others (67). Hence, it is conceivable that even in one given CMMRD patient the CALM are the result of (somatic) genetic alterations in NF1 as well as possibly in other genes. This could also explain why some CALM in CMMRD patients differ in colour and shape from typical NF1-associated CALM. Regardless of the genetic basis underlying the pigment alterations found in CMMRD patients, the vast majority of patients so far reported showed two or more CALM and/or other signs indicative of NF1, although only a minority fulfilled the NIH criteria of NF1. This explains why in the past and, occasionally, still today the phenotypic overlap leads to misdiagnosis of CMMRD patients as having NF1 impeding adequate management and counselling of these patients and their families. Potential impact of CMMRD on our current knowledge of NF1-associated malignancies Misdiagnosing CMMRD patients as having NF1 also may have influenced our current knowledge of NF1 and its association with rare childhood malignancies. In NF1 children, the vast majority of brain tumours are WHO grade I pilocytic astrocytomas, primarily OPGs. NF1-associated pilocytic astrocytomas and OPG have a particularly benign behaviour and hardly ever develop into life-threatening disease (10, 68, 69). There are, however, also a few case reports (70 72) and retrospective clinical-pathological studies (73 75) that report a possible association of childhood high-grade (WHO III or IV) CNS tumours and NF1. Huttner et al. (73) searched for patients who had a childhood glioblastoma and fulfilled NIH clinical criteria of NF1 using a computerized search engine of the medical records of patients from a single institution and found five patients fulfilling these criteria. Two patients had a concurrent pilocytic astrocytoma and two other patients had a concurrent asymptomatic OPG. As an OPG is a rare tumour in CMMRD, so far reported in only one genetically proven CMMRD patient (76), finding concurrent OPGs in two of five patients in this cohort suggests that these glioblastomas indeed developed in children with NF1. However, it is noteworthy that all tumours of this study showed increased TP53 expression, a finding reported to be highly associated with loss of MMR protein expression in high-grade gliomas (77). Rosenfeld et al. (74) reviewed charts of 740 NF1 children (based on NIH clinical criteria) from a single NF1 clinic and identified 5 of 740 having a high-grade brain tumour (3 with anaplastic astrocytoma, 1 with glioblastoma and 1 with anaplastic medulloblastoma). Two of three patients with an anaplastic astrocytoma developed the high-grade glioma after treatment of an OPG with radio and/or chemotherapy. It cannot be excluded that in one patient the secondary malignancy was induced by radiotherapy. In the third patient the anaplastic astrocytoma was diagnosed after an untreated OPG. Finally, Varan et al. (75) reported that 11 of 473 (2.3%) Turkish NF1 patients followed in a single paediatric oncology department, developed childhood brain tumours other than OPG, including three high-grade gliomas and two medulloblastomas. The remaining brain tumours were reported to be low-grade gliomas, however, at least one 513

8 Wimmer et al. of the patients with low-grade glioma died from disease despite tumour treatment. In none of the patients from these three studies an NF1 diagnosis was genetically confirmed through identification of a germline NF1 mutation. As high-grade CNS tumours, primarily high-grade gliomas but also medulloblastomas, are amongst the most prevalent malignancies associated with CMMRD (Table 2), the diagnosis NF1 probably is questionable at least in some of these patients. Therefore, a possible association of NF1 and high-grade CNS tumours remains to be re-evaluated and prospective studies should exclude CMMRD and confirm a germline NF1 mutation in patients with a brain tumour other than an OPG or other pilocytic astrocytoma. NF1 children have a 350-fold increased risk to develop JMML compared with the general population (11). So far, no CMMRD patients with this rare mixed myelodysplastic/myeloproliferative disorder have been reported. The association of other hematopoietic malignancies with NF1 is less clear. Clinical observations suggested that children and adults with NF1 are at increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) after genotoxic treatment for primary malignancies. Maris et al. (78) reported five NF1 children with a secondary therapy-related MDS with monosomy 7, which is a common finding in the bone marrow of children who develop therapy-related MDS and AML (79, 80). Four of these children were sporadic NF1 cases. Two of them had a high-grade brain tumour as a primary malignancy, i.e. an anaplastic astrocytoma and a glioblastoma, and two had a Wilms tumour, a tumour which has been reported so far in one CMMRD patient (81, 82). The fifth patient presented with acute lymphoblastic leukaemia as primary malignancy. This patient had a sibling who had previously been treated for RMS and was also diagnosed with NF1 while both parents of these siblings had no signs of NF1. In none of the five patients the NF1 diagnosis was genetically confirmed and none of the tumours tested (n = 5) showed LOH at the NF1 locus, although this is the most frequent second-hit at least in NF1-related JMML (83). Conversely, AML was reported in eight genetically confirmed CMMRD patients (30, 84). In seven of these cases AML was a secondary malignancy following medulloblastoma (n = 4), astrocytoma (n = 1), anaplastic ganglioglioma and osteosarcoma (n = 1) and osteosarcoma (n = 1). In one of the patients the AML was MDS-related and showed partial loss of the long arm of chromosome 7 due to an unbalanced translocation der(7)t(2;7)(p13;q21) (61). These findings challenge the notion of an increased risk of therapy-related MDS/AML in NF1 patients. Therefore, prospectively, it is recommended to test for CMMRD in all patients with a haematological malignancy other than a JMML and signs reminiscent of NF1. The overlap in the clinical presentation of CMMRD and NF1 should also be considered when evaluating patients with sarcomas and embryonic tumours. In particular, RMS, the most common soft tissue sarcoma in children, has been reported to be associated with NF1 (13). Based on the clinical assessment of NF1 in a large cohort of 1025 patients with RMS, the prevalence of NF1 among children with RMS was reported to be 0.5%, which is approximately 20-times higher than in the general population (14). RMS was also reported in approximately 1% (5/473) of Turkish presumed NF1 patients (i.e. without molecular confirmation) followed at a paediatric oncology unit (75). RMS in patients with NF1 invariably is of embryonal histology. Although RMS is not a frequent tumour in CMMRD, the association of CMMRD and RMS is supported by one genetically confirmed and two inferred CMMRD patients with RMS (22, 85). Both CMMRD-associated RMS for whom this information is available were of embryonal histology. Therefore, it is possible that some of the reported NF1 patients with RMS actually had CMMRD syndrome. The association of other embryonic tumours and NF1 is also under debate. Varan et al. (75) report two neuroblastoma cases in their cohort of NF1 patients. It is important to keep in mind that also neuroblastoma (86) and Wilms tumour (81, 82) have been diagnosed in CMMRD patients. Diagnostic strategies to confirm or exclude CMMRD in paediatric cancer patients with NF1 signs and a malignancy that is not typically NF1 associated CMMRD should be confirmed or refuted in all paediatric patients suspected of having sporadic NF1 and develop a malignancy other than an MPNST, JMML or an OPG (or other pilocytic astrocytoma). The identification of a mosaic NF1 mutation in a sporadic patient may in itself not be sufficient to exclude CMMRD, as it is possible that a CMMRD patient carries a post-zygotic NF1 mutation which may or may not present as a mosaic mutation (59). Different strategies including assays that were recently developed specifically for the diagnosis of CMMRD may be pursued to definitely confirm or refute CMMRD in a suspected patient. Which diagnostic strategy will be pursued will to a large extent depend on the methodological set-up that is available to the medical team managing the patient. The classical approach follows the protocols developed for Lynch syndrome and includes immunohistochemistry (IHC) and/or the analysis of MSI in tumour tissue as pre-screen, followed by mutation analysis. Given the biallelic germline mutations, CMMRD leads to MMR protein expression loss in both neoplastic and non-neoplastic tissues. Bakry et al. (46) previously reported 100% sensitivity for IHC staining in their cohort of CMMRD patients. They suggest using IHC on skin biopsies as a fast, sensitive and specific pre-screening tool that may have a role if treatment decisions must be made quickly and before conclusive genetic testing is available (87). However, IHC may not detect loss of protein expression of the affected MMR gene if the mutation is non-truncating, e.g. missense or in-frame (50, 88, 89). MSI is a hallmark of replication infidelity due to defective MMR. Standard MSI analysis, which tests for shortening or lengthening of the repeated-sequence motifs of microsatellites, assesses alleles with altered size in a defined panel of 5 6 dinucleotide (90) and/or 514

9 Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1 mononucleotide (91) repeat markers. It reliably detects MSI in gastrointestinal and other LS-associated tumours of CMMRD patients, however, frequently shows false negative results in CMMRD-associated brain tumours and other malignancies, for reasons that are currently not fully understood (40, 46, 81, 92, 93). The unique mechanism underlying the development of brain and potentially other tumours in CMMRD might, however, provide more insights. Shlien et al. (94) showed that CMMRD-associated brain tumours have an extraordinarily high load of specific substitution mutations (250 mutations/mb), which results from a combination of the constitutional MMR defect and somatic loss of proofreading capacity of the replicative polymerases, resulting in complete ablation of replication error repair in the tumour. Shlien et al. (94) further provided evidence that this leads to a rapid accumulation of mutations until a threshold of mutational burden is reached that cannot be surpassed. This potentially generates a brain tumour with multiple independent sub-clones, which may prevent the detection of altered microsatellite alleles by standard techniques developed to detect MSI in clonally more uniform cancers. As altered microsatellite alleles are also present in non-neoplastic cells of an individual with CMMRD, Ingham et al. (95) developed a sensitive method to detect this germline MSI (gmsi) in constitutional DNA (e.g. extracted from blood lymphocytes). The assay relies on the analysis of stutter peaks typically associated with microsatellite polymerase chain reaction products. When quantified by their publicly available software application, the relative peak height of the n + 1-stutter peak of the larger allele of selected dinucleotide microsatellites significantly increases in DNA of CMMRD patients compared to normal controls (95). The main limitation of this assay is that the relative peak height is not altered in patients with CMMRD due to biallelic MSH6 mutations. Nevertheless, gmsi testing has been proven to be a simple and fast method to detect CMMRD in a second unrelated cohort of patients with PMS2, MLH1 or MSH2 mutations (88). Therefore, it is a useful screening test complementary or alternative to IHC, which may be used to assess CMMRD in individual cases but also in large (retrospective) cohorts of patients. Bodo et al. (88) proposed to combine two newly developed tests as diagnostic pre-screen for CMMRD. The ex vivo MSI (evmsi) test assesses classical MSI in immortalized lymphoblastoid cell lines (LCLs) from patients. The N-methyl-N -nitro-n-nitrosoguanindine (MNNG) tolerance assay assesses the resistance of MMR deficient cells to methylating genotoxic agents also using patients LCLs. As the method relies on LCLs and assesses cell growth under MNNG treatment, it is time consuming and needs specialized laboratory experience. Nevertheless, due to its high negative predictive value [97.2% (CI: %)] and high sensitivity [95% (CI: )], the method can confirm or reject CMMRD in patients where mutation analysis yields inconclusive results, e.g. in cases where an unclassified variant, none or only one clearly pathogenic mutation is identified and where gmsi and IHC are unable to provide a definite answer (88). Identification of both disease causing germline MMR gene mutations is the most reliable method to confirm the diagnosis of CMMRD, and allows offering reliable predictive testing for CMMRD and Lynch syndrome in the wider family as well as prenatal or pre-implantation genetic diagnosis after informed decision making. With the increasing availability of massive parallel sequencing (MPSeq), analysis of MPSeq data for point mutations and copy number alterations in all four MMR genes may be the most appropriate primary assay to confirm the diagnosis of CMMRD in settings where pre-screening assays are not readily available. If mutation analysis of all four MMR genes cannot identify two clearly disease causing mutations, it will depend on the mutation analysis results (none or only one clearly disease causing mutation identified together with or without unclassified variants in one of the four MMR genes) which of the described assays, IHC, gmsi, evmsi and MNNG toxicity assay, will be necessary to come to a final conclusion. When performing mutation analysis as a primary assay it is important to keep in mind that approximately 60% of CMMRD patients carry biallelic PMS2 mutations. Due to the presence of multiple PMS2 pseudogenes, in particular pseudogene PMS2CL, which has been shown to frequently exchange sequences with the functional gene leading to the formation of functional and non-functional gene-pseudogene hybrids (96 98), mutation analysis of this gene is notoriously difficult. Currently, there are no MPSeq protocols that reliably detect mutations in the 3 region of the gene. Therefore, PMS2 cdna sequencing (61, 98) in combination with refined MLPA assays (99, 100) is currently still the most reliable and robust method. Taking this and the high frequency of biallelic PMS2 mutation carriers among CMMRD patients into account, it might be the most cost-effective mutation analysis approach to start with effective and reliable targeted mutation analysis of the PMS2 gene in situations where no directive IHC results are available and the family history of the patient lacks LS-associated cancers in adults. Use of the appropriate combination of diagnostic tools should allow making a definite diagnosis in nearly all suspected CMMRD patients (88). Only patients in whom CMMRD was excluded and in whom the causative NF1 mutation was identified should be considered true NF1 patients. Making a diagnosis of CMMRD in a patient has implications not only for the management of the patient but also for the entire family. Parents of newly diagnosed CMMRD patients need to be informed, preferentially by a team of medical geneticists and (paediatric) oncologists, of their own risk for LS-associated cancer and the recurrence risk in siblings, which may lead to predictive testing and influence family planning. Considering the severe impact of this syndrome on a family, psychological support should be offered to the family during the entire process of diagnostic evaluation. The management of CMMRD patients is, however, not the topic of this review. Therefore, we here refer only to the most recent 515

10 Wimmer et al. literature. Given the high risk for a second malignancy in the patients and other affected siblings, the European consortium C4CMMRD proposed possible surveillance protocols (101). Similar suggestions came from an international consortium headed by a Canadian team (102). Treatment modalities of CMMRD-associated malignancies should be adjusted to the underlying defect. This may include avoidance of certain genotoxic agents, such as thiopurines and methylating agents which rely on a functional MMR system to be effective (35, 53, 103, 104). Due to their extraordinarily high mutation load, CMMRD-associated high-grade gliomas are predicted to produce a high number of T-cell activating neoantigens and, hence, are probably to respond to immunotherapy (105). This notion is supported by a favourable response to immune checkpoint inhibition of recurrent multifocal glioblastomas multiforme in two siblings with CMMRD syndrome (105). Concluding remarks A clinical phenotype reminiscent of NF1 in addition to any malignancy other than an MPNST, JMML or pilocytic astrocytoma, well known to be associated with NF1, in a child or young adult should raise a high level of suspicion of CMMRD syndrome. As this diagnosis has important implications for management of the patient and his/her entire family, every effort should be undertaken to confirm or refute this diagnosis. The current knowledge on the association of NF1 with certain rare childhood malignancies (e.g. high-grade brain/cns tumours, haematological malignancies other than JMML, embryonic tumours and RMSs) may be inaccurate due to inadvertent inclusion of (some) CMMRD patients in presumed NF1 cohorts. Therefore, further prospective and retrospective studies excluding CMMRD and genetically confirming NF1 in these patients are needed to re-evaluate the possible association of NF1 and these tumour entities. Clearly, a proportion of the biallelic MMR gene mutation carriers show a phenotype indistinguishable from generalized or segmental NF1 before they develop a malignancy. Based on the so far reported CMMRD cases, most of these will not carry a detectable NF1 mutation in blood lymphocytes. Conversely, the prevalence of CMMRD among sporadic children suspected of having (segmental) NF1 but no NF1 mutation is detectable in blood, is unknown. As the awareness of this childhood cancer susceptibility syndrome increases, testing for CMMRD probably needs to be considered in these children so that surveillance can be offered to biallelic MMR gene mutation carriers before developing a first malignancy and family counselling can be initiated early on. Currently, there are no recommendations as to when to consider testing for CMMRD in a child without malignancy. Given the severe impact of this rare childhood cancer syndrome on the management of the patient and its entire family, such recommendations should be developed in an interdisciplinary discussion on the advantages, limitations and ethical issues implicated with predictive CMMRD testing in individuals without malignancy. Finally, further studies are needed to establish whether somatic NF1 mutations are the underlying cause for NF1 features in CMMRD patients. Although there is support that at least in some CMMRD patients a post-zygotic NF1 mutation may be responsible for the NF1 features, it is conceivable that genetic alterations also in other genes may account for the frequent occurrence of atypical and typical CALM and freckling in CMMRD patients. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. Acknowledgements The authors wish to thank Prof Sigrid Tinschert for helpful comments on the manuscript and providing the figure of a segmental NF1 patient with a proven somatic NF1 mutation. The authors are also grateful to Prof Danuta Januszkiewicz-Lewandowska, the father of a patient and P.D. Dr Robert Gruber for providing photographs of pigmentation alterations and brain MRI scans of NF1 and CMMRD patients. Furthermore, we thank all physicians and parents who shared so far unpublished data of CMMRD patients. References 1. Friedman JM. Epidemiology of neurofibromatosis type 1. Am J Med Genet 1999: 89: DeBella K, Szudek J, Friedman JM. Use of the national institutes of health criteria for diagnosis of neurofibromatosis 1 in children. Pediatrics 2000: 105: Messiaen L, Yao S, Brems H et al. Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome. JAMA 2009: 302: Ballester R, Marchuk D, Boguski M et al. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell 1990: 63: Scheffzek K, Ahmadian MR, Wiesmuller L et al. Structural analysis of the GAP-related domain from neurofibromin and its implications. EMBO J 1998: 17: DeClue JE, Papageorge AG, Fletcher JA et al. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell 1992: 69: Serra E, Rosenbaum T, Winner U et al. Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations. Hum Mol Genet 2000: 9: De Schepper S, Maertens O, Callens T, Naeyaert JM, Lambert J, Messiaen L. Somatic mutation analysis in NF1 cafe au lait spots reveals two NF1 hits in the melanocytes. J Invest Dermatol 2008: 128: Evans DG, Baser ME, McGaughran J, Sharif S, Howard E, Moran A. Malignant peripheral nerve sheath tumours in neurofibromatosis 1. J Med Genet 2002: 39: Helfferich J, Nijmeijer R, Brouwer OF et al. Neurofibromatosis type 1 associated low grade gliomas: a comparison with sporadic low grade gliomas. Crit Rev Oncol Hematol 2016: 104: Niemeyer CM, Arico M, Basso G et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood 1997: 89: Chang T, Shannon K. NF1 mutations in hematologic cancers. In: Upadhyaya M, Cooper DN, eds. Neurofibromatosis type 1. Heidelberg; New York, NY; Dordrecht; London: Springer, 2012: Crucis A, Richer W, Brugieres L et al. Rhabdomyosarcomas in children with neurofibromatosis type I: a national historical cohort. Pediatr Blood Cancer 2015: 62: