Melyssa Aronson

Chrystelle Colas

Andrew Shuen

Heather Hampel

Hagit William D Foulkes

Baris Feldman

Yael Goldberg

Martine Muleris

Wolfe Kami

Rose B Schneider

Mcgee

Hagit Baris Feldman

Kami Wolfe Schneider

Rose Mcgee

Kory Jasperson

Arun Rangaswami

Laurence Brugieres

Uri Tabori

Diagnostic criteria for constitutional mismatch repair deficiency (CMMRD): recommendations from the international consensus working group

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Constitutional Mismatch Repair Deficiency Syndrome (CMMRD), caused by biallelic pathogenic variants (PV) in mismatch repair (MMR) genes, is one of the most aggressive and complex hereditary cancer syndrome with a very high prevalence of synchronous and metachronous cancers resulting in patient death in early life. CMMRD can also present with benign manifestations resembling neurofibromatosis type I (NF1). [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF] While most malignancies present in childhood, there is variability in the age and presentation of cancers and benign manifestations.

Of the 4 MMR genes, PMS2 gene is responsible for over 60% of patients with CMMRD, followed by MSH6 (20-30%), MLH1 and MSH2 (10-20%). [START_REF] Vasen | Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D)[END_REF][START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF] Consanguinity has been reported in 39-45% of CMMRD families, varying slightly depending on country of origin. [START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Amayiri | High frequency of mismatch repair deficiency among pediatric high grade gliomas in Jordan[END_REF][START_REF] Durno | Phenotypic and genotypic characterisation of biallelic mismatch repair deficiency (BMMR-D) syndrome[END_REF] Homozygous variants have been reported in 10-28.5% of families that deny consanguinity suggesting common founder mutations, with higher frequency in isolated populations or those that may share a common ancestor. [START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Durno | Phenotypic and genotypic characterisation of biallelic mismatch repair deficiency (BMMR-D) syndrome[END_REF][START_REF] Li | A homozygous PMS2 founder mutation with an attenuated constitutional mismatch repair deficiency phenotype[END_REF] Genetic testing for CMMRD may not be definitive as the most commonly impacted gene, PMS2, can pose testing challenges due to pseudogenes and frequent gene conversion events that can be circumvented only by specialized assays. [START_REF] Etzler | RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference[END_REF][START_REF] Van Der Klift | Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome[END_REF] Unclassified variants, or variants of uncertain significance (VUS), pose challenges for interpretation, and PMS2 has the highest incidence of VUS among the MMR genes, comprising 49% of reported variants in this gene. [START_REF] Drost | Inactivation of DNA mismatch repair by variants of uncertain significance in the PMS2 gene[END_REF] Lack of definitive genetic confirmation in some individuals and a complex presentation of the disorder necessitates the need for diagnostic criteria. Together, the growing number of patients with clinical or tumor characteristics suggestive of CMMRD requires a robust pathway for genetic diagnosis and testing which allows for counselling, surveillance, and better tumor management.

An understanding of CMMRD requires background information on Lynch syndrome (LS), caused by monoallelic PV in the MMR genes. LS is an autosomal dominant condition predisposing primarily to adult-onset colorectal and endometrial cancer. Compared to CMMRD, there is an inverse distribution of gene mutations, with MSH2 and MLH1 accounting for 80% of families with LS, MSH6 in 13% and PMS2 in 6% of families. [START_REF] Dominguez-Valentin | Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database[END_REF] In addition, deletions of the terminal exons of the EPCAM gene that include 3'UTR sequences upstream from MSH2 cause methylation of MSH2 leading to a LS phenotype. [START_REF] Grandval | Colon-specific phenotype in Lynch syndrome associated with EPCAM deletion[END_REF][START_REF] Tutlewska | Germline deletions in the EPCAM gene as a cause of Lynch syndrome -literature review. Hereditary cancer in clinical practice[END_REF][START_REF] Ligtenberg | Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1[END_REF][START_REF] Kempers | Risk of colorectal and endometrial cancers in EPCAM deletion-positive Lynch syndrome: a cohort study[END_REF] LS is associated with a high cancer penetrance leading to a strong family history of colorectal and endometrial cancer. PMS2 and MSH6 have a lower penetrance of CRC and endometrial cancer (10% and 12% in PMS2 and 20% and 40% in MSH6, respectively), [START_REF] Dominguez-Valentin | Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database[END_REF] which is supported by lack of family history for individuals with PMS2 or MSH6 related LS. [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Durno | Phenotypic and genotypic characterisation of biallelic mismatch repair deficiency (BMMR-D) syndrome[END_REF] On the other hand, patients with CMMRD typically present in childhood with brain, lymphoma or GI cancers, although there is a broad spectrum of malignancies, including individuals presenting without cancer and/or NF1 manifestations. Given the range in phenotypical presentation, criteria for risk assessment and determining the need for genetic testing for CMMRD in pediatric or young adult patients were outlined by the European consortium CARE for CMMRD (C4CMMRD) (2014). This scoring system is based on a calculated clinical score of malignancy and benign features (Figure 1). [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF] The C4CMMRD consortium also defined CMMRD counseling and testing criteria for children suspected to have sporadic NF1 but without a detectable mutation in NF1 or SPRED1 and without a malignancy. [START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF] These scoring systems are helpful in identifying patients who should undergo genetic testing for CMMRD and are hence an entry point for evaluation in an individual. However, no diagnostic criteria currently encompass molecular results, ancillary testing and clinical presentation. Given the lack of one definitive test for CMMRD in most cases and the variable presentation, an international committee was convened to establish diagnostic criteria for CMMRD which are summarized in this paper. An accurate diagnosis has important implications for genetic counselling, tumor surveillance, [START_REF] Vasen | Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D)[END_REF][START_REF] Tabori | Clinical Management and Tumor Surveillance Recommendations of Inherited Mismatch Repair Deficiency in Childhood. Clinical cancer research : an official journal of the American Association for[END_REF][START_REF] Durno | Recommendations on Surveillance and Management of Biallelic Mismatch Repair Deficiency (BMMRD) Syndrome: A Consensus Statement by the US Multi-Society Task Force on Colorectal Cancer[END_REF] (Table 1) and access to immunotherapy cancer treatment. [START_REF] Bouffet | Immune Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline Biallelic Mismatch Repair Deficiency[END_REF][START_REF] Westdorp | Immunotherapy holds the key to cancer treatment and prevention in constitutional mismatch repair deficiency (CMMRD) syndrome[END_REF]

Methods

In order to establish diagnostic criteria, an international working group was formed, including 13 experts, with representatives from the International Replication Repair Deficiency Consortium workshop attendees, made up of 60 experts from 18 countries for input and consensus vote. The agreed upon criteria were further evaluated through an in-depth survey sent to the 13 members of the working group and completed by 8 members of the group by April 23, 2018. If consensus of 80% was reached, the criterion was adopted. A follow-up conference call with all members was arranged to discuss criteria that did not reach 80% agreement for review to eliminate or re-vote.

Hypothetical cases as well as families with typical and atypical presentations of CMMRD from the IRRD consortium were reviewed to test the draft criteria. A second survey to finalize data was completed by February 28, 2019 by all members of the working group. The focus of this working group was development of criteria to determine who has a CMMRD diagnosis, thereby necessitating surveillance. We did not determine how to definitively rule out a diagnosis of CMMRD, which may be explored in a future document.

Results

:

CMMRD diagnostic criteria

The expert panel established 6 diagnostic criteria outlined in Table 2 It is the recommendation of this working group that the diagnostic criteria established be reassessed as data emerges and as the criteria are applied to newly identified families suspected of CMMRD. The committee did not establish criteria to eliminate the suspicion of CMMRD and recognizes there may be families that meet a level of suspicion for this condition, but do not meet the strict evidence to confirm diagnosis. Clinical judgment should be used in all cases.

The internationally agreed upon diagnostic criteria, using a combination of clinical manifestations, genetic testing and ancillary molecular assays, are outlined in Table 2.

Rationale for Criteria

A. Mismatch repair genes germline testing

The American College of Medical Genetics (ACMG) outlined 5 variant classifications for germline analysis of Mendelian disorders; pathogenic (P), likely pathogenic (LP), variants of unknown significance (VUS), likely benign (LB) and benign (B) variants. [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for[END_REF] Actionable variants include P/LP, while a VUS should not be used for clinical decision-making, and LB/B variants can be assumed not to cause phenotype.

Non definitive mismatch repair (MMR) germline results have been described in multiple CMMRD patients, including cases with biallelic VUS or combinations of VUS and P/LP variants. [START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Mork | Identification of a novel PMS2 alteration c.505C>G (R169G) in trans with a PMS2 pathogenic mutation in a patient with constitutional mismatch repair deficiency[END_REF][START_REF] Poley | Biallelic germline mutations of mismatch-repair genes: a possible cause for multiple pediatric malignancies[END_REF] This suggests the VUS was misclassified and is pathogenic or that the VUS may not be clinically actionable as a monoallelic variant, it may sufficiently impact protein function when in combination with a second VUS or P/LP variant. One example of the complexity of molecular VUS results were reported by Taeubner et al (2018) who found both homozygous MSH2 and MSH6 variants in a 13 month old with desmoplastic medulloblastoma and striking skin pigmentation using ancillary assays. [START_REF] Ingham | Simple detection of germline microsatellite instability for diagnosis of constitutional mismatch repair cancer syndrome[END_REF][START_REF] Bodo | Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents[END_REF] It was concluded that CMMRD was caused by the MSH6 homozygous VUS. [START_REF] Taeubner | Diagnostic challenges in a child with early onset desmoplastic medulloblastoma and homozygous variants in MSH2 and[END_REF] A review of the literature outlined suspected CMMRD families in the absence of definitive biallelic pathogenic MMR variants, or based solely on clinical phenotype and ancillary testing. [START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Poley | Biallelic germline mutations of mismatch-repair genes: a possible cause for multiple pediatric malignancies[END_REF][START_REF] Bakry | Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium[END_REF] A study by Bakry et al (2014) reported MMR germline results for 12 CMMRD families; 67% (n=8) had P/LP biallelic MMR variants, 17% (n=2) had biallelic VUS, 8% (n=1) had monoallelic PV, and 8% (n=1) had no mutation identified. [START_REF] Bakry | Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium[END_REF] Similar distribution was seen in 38 families from the C4CMMRD consortium with 74% (n=28) P/LP biallelic MMR variants, 18% (n=7) biallelic VUS, and 8% (n=13) monoallelic PV and no monoallelic VUS. [START_REF] Guerrini-Rousseau | Constitutional mismatch repair deficiencyassociated brain tumors: report from the European C4CMMRD consortium[END_REF] This highlights the need to define criteria for ancillary assays that will diagnose CMMRD in the absence of confirmatory MMR germline testing mutation analysis.

B. Ancillary Testing

Ancillary testing, including assays showing microsatellite instability (MSI) in constitutional tissue, functional assays showing loss of MMR activity and MMR immunohistochemistry (IHC)

showing loss of MMR protein expression may be used to support a diagnosis of CMMRD.

IHC analysis in non-neoplastic tissue (i.e. skin biopsy or adjacent normal tissue to a cancer) is the most widely available and least expensive test to assess for absence of MMR protein, however, interpretation is subjective and obtaining normal tissue can be invasive. [START_REF] Bakry | Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium[END_REF] Pathologists must be aware of the suspected diagnosis as the positive stain which serves as an internal control in LS patients is often absent in all tissues of individuals with CMMRD. In addition, pathogenic missense variants, frequent in CMMRD, can lead to false normal results, where the protein is present and staining, but is non-functional, thereby leading to a false negative result. [START_REF] Gallinger | Gastrointestinal cancers and neurofibromatosis type 1 features in children with a germline homozygous MLH1 mutation[END_REF] Despite limitations, IHC of non-neoplastic tissue has higher than 90% sensitivity and specificity in experienced pathology laboratories and can be a useful tool in the diagnosis of CMMRD.

Functional assays have been developed, although widespread access at commercial laboratories may be limited. While conventional MSI testing using an NCI panel of mono-and dinucleotide repeat markers were validated for adult-onset Lynch syndrome cancers, [START_REF] Umar | Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability[END_REF] it is not sensitive at detecting MSI in CMMRD. A method to detect MSI in non-neoplastic tissue termed germline MSI (gMSI) has been developed, and this assay relies on the analysis of 'stutter' peaks typically associated with microsatellite PCR products. [START_REF] Ingham | Simple detection of germline microsatellite instability for diagnosis of constitutional mismatch repair cancer syndrome[END_REF] Its main limitation is that it uses dinucleotid microsatellites and, therefore, is insensitive to an MSH6 deficiency. It is routinely used for diagnosis purposes in some European countries and allows for rapid results.

Another functional assays includes a combination of tolerance to methylating agents (a characteristic of MMR deficient cells) and MSI from lymphoblastoid cell lines called ex-vivoMSI (evMSI). [START_REF] Bodo | Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents[END_REF] A presumptive diagnosis of CMMRD using this assay requires both tolerance to methylating agents and MSI to be concordant. A diagnosis of CMMRD can be ruled out if both tests show normal activity. The method is 100% sensitive and 100% specific. In cases of discrepancy, no conclusion can be made and another test should be considered. A limitation of this test is the timing necessary for immortalization and culture cells for evMSI (at least 120 days). This assay is available in routine diagnosis in France in an accredited laboratory and since January 2015, 77 patients have been tested prospectively with the two functional assays and 15 had both abnormal results consistent with a diagnosis of CMMRD (personal communication of M Muleris and C Colas). These assays are not easily accessible clinically in North America.

An in vitro repair assay was developed to quantify MMR activity from patient-derived lymphoblastoid cell lines. [START_REF] Shuen | Functional Repair Assay for the Diagnosis of Constitutional Mismatch Repair Deficiency From Non-Neoplastic Tissue[END_REF] Testing a series of patients with CMMRD, Lynch syndrome, NF1, Li-Fraumeni, and polymerase proofreading-associated polyposis syndrome, the assay demonstrated high specificity and sensitivity. The assay requires live cell cultures as mismatch repair proteins are maximally produced during cell division, which limits its scalability. In addition, the assay is a complementation assay which allows for the determination of the defective protein complex (MSH2-MSH6 or MLH1-PMS2). This assay is not yet established as a clinically approved test, but is available through the IRRD consortium.

More recently next-generation sequencing (NGS)-based methods have proven to be very sensitive and specific at diagnosing CMMRD as they can detect low level of MSI from constitutional tissue. [START_REF] González-Acosta | Highsensitivity microsatellite instability assessment for the detection of mismatch repair defects in normal tissue of biallelic germline mismatch repair mutation carriers[END_REF][START_REF] Gallon | A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes[END_REF] NGS-based methods for MSI detection have the advantage of being easily scalable, cost effective.

Pediatric tumors showing hypermutation (>10 mutations/Mb) and MMR deficient signatures (single base substitution signatures 6, 15, 20, 26 and 44, doublet base substitution 7 and 10, small insertion and deletion signatures 1 and 2), may raise the suspicion of CMMRD. [START_REF] Alexandrov | Signatures of mutational processes in human cancer[END_REF] Cerebral tumors related to CMMRD are also associated with concurrent somatic mutations in the proofreading domain of the polymerases POLE/POLD1, which leads to ultrahypermutation phenotypes with mutation loads in excess of 100 mutations/Mb and specific POLE/POLD1 signatures (single base substitution signature 14 and 20. [START_REF] Shlien | Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers[END_REF] It is also noted dMMR in a malignancy is not sufficient to confirm a diagnosis of CMMRD as these features can also be present in tumors from patients with Lynch syndrome and even sporadic cancers that have acquired biallelic hits in a MMR gene. [START_REF] Shlien | Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers[END_REF][START_REF] Campbell | Comprehensive Analysis of Hypermutation in Human Cancer[END_REF] As IHC deficiency in tumours, TMB and signature cannot clearly differentiate between LS, somatic biallelic MMR and CMMRD, the working group does not support using these an ancillary test to confirm CMMRD at this time. It can be used to support a diagnosis once outlined diagnostic criteria are met.

The working group recommends using ancillary testing in challenging cases, and should be used to interpret atypical presentation of CMMRD or inconclusive germline molecular results within the clinical context. If a discrepancy occurs among tests, additional ancillary tests, preferably by orthogonal methods, should be performed to reach a more conclusive decision. Consideration may be given to implement tests that are already published with high sensitivity and specificity in accredited (e.g. CAP inspected) laboratories authorised to give a clinically usable report. The working group gives a framework of current ancillary tests, however a definitive list was not incorporated as new functional assays may be developed over time. A summary of the current tests outlining pros and cons is provided in Table 3.

C. Clinical Presentation

Since genetic and molecular criteria may be insufficient in the diagnosis of CMMRD, the working group defined specific clinical parameters to add in the establishment of CMMRD diagnosis.

C.i) Cancer Type and Age of Presentation

In order to assess criterion for clinical diagnosis, the working group reviewed most common primary malignancy sites, as well as typical age of first presentation. Atypical cancer site and range of age of diagnosis was also assessed to determine the full spectrum of CMMRD.

Literature review of several larger published CMMRD cohorts that outline primary cancer site, as well as unpublished data from the IRRD consortium and the C4CCMMRD database are summarized in Table 4. It reveals that 86-100% of individuals with CMMRD have reported a cancer diagnosis. [START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Durno | The gastrointestinal phenotype of germline biallelic mismatch repair gene mutations[END_REF][START_REF] Wimmer | Constitutional mismatch repair-deficiency syndrome: have we so far seen only the tip of an iceberg?[END_REF] This may be a result of ascertainment bias, because cancer is the most common criterion leading to the diagnosis of CMMRD in index cases. Penetrance may also be dependent on timing of the publication as new cancers are likely to develop over time. In fact, one publication reported that at the time of recruitment, 19 of 23 (82.6%) individuals had a history of cancer, but by the time of publication, 22 individuals were affected (95.6%). [START_REF] Bakry | Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium[END_REF] Wimmer et al (2014) outlined the most common CMMRD tumors and age of first diagnosis to include; WHO grade III/IV glioma <25 yrs, NHL of T-cell lineage < 18 yrs, GI adenocarcinoma <25 yrs, or GI adenomatous polyposis < 18 yrs. [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF] These represent 60-84% of first diagnosis in CMMRD as outlined in Table 4. [START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Durno | The gastrointestinal phenotype of germline biallelic mismatch repair gene mutations[END_REF][START_REF] Wimmer | Constitutional mismatch repair-deficiency syndrome: have we so far seen only the tip of an iceberg?[END_REF] We acknowledge there may be an overlap of cohorts within the two large consortiums; the IRRD consortium and C4CMMRD database, therefore, we used recent, unpublished information from these two databases only to examine the common cancers outlined above. By expanding the criteria to all subtypes of brain, cerebral and hematological malignancies, an additional 18.75% (18/96) of the IRRD cases and 34% (31/90) from the C4CMMRD database would be included as primary malignancies. The contribution of GI cancers as the primary diagnosis was also assessed, and accounted for 15% (15/96) of IRRD cases and 18% (16/90) of C4CMMRD cases, most often reported as colorectum or small bowel cancer. Other studies have reported adenomatous polyposis as the first diagnosis, often diagnosed under age 18. [START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Bakry | Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium[END_REF][START_REF] Aronson | Gastrointestinal Findings in the Largest Series of Patients With Hereditary Biallelic Mismatch Repair Deficiency Syndrome: Report from the International Consortium[END_REF] Based on expertise and literature review, this working group defined new "hallmark tumors" in CMMRD as: (a) glioma or CNS embryonal tumors <25 yrs, (b) hematological cancer (excluding Hodgkin's lymphoma) < 18 yrs, (c) gastrointestinal adenocarcinoma <25 yrs, and (d) gastrointestinal adenomatous polyposis of more than 10 adenomas < 18 yrs (after ruling out polyposis conditions). Using this definition of hallmark cancers, it would account for 76-96% of primary diagnoses reported in Table 4.

While the majority of CMMRD cases present with a hallmark tumor, there is a broader spectrum of less common primary and metachronous malignancies. These include embryonal tumours (e.g. neuroblastoma, retinoblastoma, Wilms tumour), germ cell tumours (i.e. yolk sac tumour), sarcomas (e.g. osteosarcoma, dermatofibrosarcoma protuberans, rhabdomyosarcoma), ganglioneuroma, melanoma, urinary tract (bladder, kidney, ureter), prostate, breast, gynecological (endometrial, ovarian), and other GI (ampullary, gastric) cancers. [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF][START_REF] Baris | Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Poley | Biallelic germline mutations of mismatch-repair genes: a possible cause for multiple pediatric malignancies[END_REF][START_REF] Bakry | Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium[END_REF][START_REF] Wimmer | Constitutional mismatch repair-deficiency syndrome: have we so far seen only the tip of an iceberg?[END_REF][START_REF] Aronson | Gastrointestinal Findings in the Largest Series of Patients With Hereditary Biallelic Mismatch Repair Deficiency Syndrome: Report from the International Consortium[END_REF][START_REF] Bush | Delineating a new feature of constitutional mismatch repair deficiency (CMMRD) syndrome: breast cancer[END_REF][START_REF] Ramchander | Homozygous germ-line mutation of the PMS2 mismatch repair gene: a unique case report of constitutional mismatch repair deficiency[END_REF][START_REF] Gilpin | An Unusual IHC result: Is Late Onset Biallelic Mismatch Repair Deficiency (BMMRD) the explanation?[END_REF] Older age of onset of primary cancer has been noted in CMMRD and data for the larger studies are summarized in Table 4 [START_REF] Gilpin | An Unusual IHC result: Is Late Onset Biallelic Mismatch Repair Deficiency (BMMRD) the explanation?[END_REF] There are also atypical cancer sites diagnosed at older ages as the first malignancy in 2 reported CMMRD cases who presented with breast cancer over age 25. [START_REF] Hackman | A human compound heterozygote for two MLH1 missense mutations[END_REF][START_REF] Bush | Delineating a new feature of constitutional mismatch repair deficiency (CMMRD) syndrome: breast cancer[END_REF] Older ages of onset of first cancer appears to be more commonly associated with epithelial cancers, although older onset lymphoma (diagnosed at age 33) has also been reported. [START_REF] Ebner | Late-Onset Biallelic Mismatch Repair Deficiency Syndrome[END_REF] Genotype-phenotype correlations may explain some of this older onset cases. For example Li et al. (2015) described a founder homozygous PV of PMS2 in Inuit families displaying an attenuated phenotype with a delayed average age of onset of cancer associated to a residual PMS2 expression. [START_REF] Li | A homozygous PMS2 founder mutation with an attenuated constitutional mismatch repair deficiency phenotype[END_REF] These clinically unusual cases highlight the spectrum of CMMRD presentation and the challenges with establishing a diagnosis based on clinical presentation alone.

C.ii) Benign manifestations

Individuals with CMMRD often have features that are found in NF1, including café-au-lait macules (CALM), neurofibromas and axillary freckling. Only a minority of individuals with CMMRD meet the NIH diagnostic criteria for NF1, although 18.5% (n=27/146) have more than one NF1 feature and between 62-95% have CALM, albeit not all reach the threshold of >6 CALM. [START_REF] Wimmer | Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD)[END_REF][START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF][START_REF] Wimmer | Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1[END_REF] Conversely, CALM are common in the general population, often present at childbirth but can continue to grow and develop with age. The incidence, varying with age and race/ethnicity, is reported in 0.3% to 1.8% of White individuals between birth to childhood years, and in 18% to 27% within that age range in individuals who identify as Black. [START_REF]Cafe au Lait Macules and Associated Genetic Syndromes[END_REF] In addition to CMMRD and NF1, CALM can be associated with other hereditary conditions including Legius syndrome (SPRED1 gene), Noonan syndrome (occurring with multiple lentigines), other RASopathies, and

McCune-Albright syndrome (often mosaic and occurring with skeletal anomalies). Hypochromic spots, not present in NF1, are have been described in 16% (Lavoine et al) to 29% (unpublished C4CMMRD data) in CMMRD patients who have undergone a dermatological examination. [START_REF] Lavoine | Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort[END_REF] In case of suspicion of CMMRD, a complete and careful skin examination must be performed and may provide elements in favor of the diagnosis.

Other clinical features reported in CMMRD patients include agenesis of the corpus callosum, grey matter heterotopia [START_REF] Baas | Agenesis of the corpus callosum and gray matter heterotopia in three patients with constitutional mismatch repair deficiency syndrome[END_REF] , venous anomalies [START_REF] Shiran | Multiple Brain Developmental Venous Anomalies as a Marker for Constitutional Mismatch Repair Deficiency Syndrome[END_REF] , multiple pilomatrixoma [START_REF] Chmara | Multiple pilomatricomas with somatic CTNNB1 mutations in children with constitutive mismatch repair deficiency[END_REF] , paediatric systemic lupus erythematosus [START_REF] Toledano | Paediatric systemic lupus erythematosus as a manifestation of constitutional mismatch repair deficiency[END_REF] , intracranial tuber-like lesions and renal angiomyolipoma. [START_REF] Shapira Rootman | The great mimicker: Phenotypic overlap between constitutional mismatch repair deficiency and Tuberous Sclerosis complex[END_REF] Decreased IgA and/or IgG2/4 levels 1 have also been reported, however, this was not substantiated in a recent study that could not consistently identify clinical or routine immunological laboratory parameters suggestive of primary immunodeficiency in 15 unrelated CMMRD patients. [START_REF] Tesch | No Overt Clinical Immunodeficiency Despite Immune Biological Abnormalities in Patients With Constitutional Mismatch Repair Deficiency[END_REF] These abnormalities have no clinical impacts in terms of immunodeficiency for the vast majority of patients. [START_REF] Tesch | No Overt Clinical Immunodeficiency Despite Immune Biological Abnormalities in Patients With Constitutional Mismatch Repair Deficiency[END_REF] While these manifestations have been observed, they are not routinely investigated at the time of CMMRD diagnosis. However, examinations of these benign manifestations can be of value, especially when diagnosis in doubtful, as they may provide arguments in favor of the diagnosis.

CMMRD is a legitimate differential diagnosis in children suspected to have sporadic NF1 in whom no NF1 or SPRED1 mutation is identified, as testing for CMMRD in these children would allow for cancer surveillance before their first malignancy. However, given that counseling and testing for CMMRD in these children is associated with potential harm and the estimated prevalence of CMMRD in these children is only 0.39%, the C4CMMRD consortium (Suerink et al 2018) developed criteria to preselect those children where CMMRD is more likely. The testing criteria outlines that an individual be tested if they fulfill the prerequisites (a) the presence of at least one diagnostic NF1 feature including at least two CALMs (b) absence of NF1 and SPRED1 germline mutations, (c) absence of NF1 signs in both parents. In addition the individual should have one or more features indicative of CMMRD in the family including: parental consanguinity, Lynch syndrome in one or both parents, siblings with NF1 features or childhood malignancy, LS-related cancer diagnosed before age 60 in first-degree or second-degree relative; or personally: atypical CALMs (irregular borders and/or pigmentation), hypopigmented skin patches, > 1 pilomatricoma, agenesis of the corpus callosum, non-therapy-induced cavernoma or multiple developmental vascular abnormalities. [START_REF] Suerink | Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy[END_REF]

D. Multi-gene panel testing to search for differential diagnoses

The differential diagnosis of CMMRD includes a wide range of other hereditary tumor conditions. Lynch syndrome and CMMRD are both caused by mismatch repair gene mutations, and while they phenotypically differ, atypical cases can mimic each other. There have been rare cases of LS with glioblastoma or GI cancers diagnosed in childhood or young adulthood, and CMMRD cases presenting with adult-onset cancer. [START_REF] Fernandez-Rozadilla | Early Colorectal Cancers Provide New Evidence for a Lynch Syndrome-to-CMMRD Phenotypic Continuum[END_REF][START_REF] Yang | Lynch syndrome-associated ultra-hypermutated pediatric glioblastoma mimicking a constitutional mismatch repair deficiency syndrome[END_REF][START_REF] Heath | Childhood cancers in families with and without Lynch syndrome[END_REF] Polyposis conditions can also mimic CMMRD including individuals with specific POLE variants (i.e. p.V411L or p.A456P) who present with childhood hypermutant brain and colorectal cancers. [START_REF] Wimmer | A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency[END_REF][START_REF] Lindsay | Germline POLE mutation in a child with hypermutated medulloblastoma and features of constitutional mismatch repair deficiency[END_REF] Conversely, individuals with CMMRD have also presented with adenomatous polyposis (i.e. 10-100 adenomas) mimicking conditions such as Familial Adenomatous Polyposis (FAP or attenuated FAP) and

MUTYH-Associated Polyposis (MAP).

There is also a rare case presenting with > 30 juvenile polyps typically associated with Juvenile Polyposis Syndrome (JPS). [START_REF] Levi | The gastrointestinal manifestation of constitutional mismatch repair deficiency syndrome: from a single adenoma to polyposis-like phenotype and early onset cancer[END_REF] Early-onset malignancies can also overlap with Li-Fraumeni syndrome, caused by germline TP53 mutations, which increases the risk for hematological, sarcoma, brain and GI cancers presenting at early ages. [START_REF] Nguyen | MSI detection and its pitfalls in CMMRD syndrome in a family with a bi-allelic MLH1 mutation[END_REF] There has also been overlap with conditions that have CALM, skin manifestation such as NF1 and Legius syndromes, [START_REF] Wimmer | Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1[END_REF][START_REF] Suerink | Constitutional mismatch repair deficiency in a healthy child: On the spot diagnosis?[END_REF] and tuberous sclerosis [START_REF] Shapira Rootman | The great mimicker: Phenotypic overlap between constitutional mismatch repair deficiency and Tuberous Sclerosis complex[END_REF] . Parallel to MMR analysis, other syndromes should be searched through multi-gene panel testing encompassing hereditary colorectal and polyposis conditions, childhood malignancy conditions (i.e. TP53), or syndromes based on skin findings (i.e. NF1, Legius, tuberous sclerosis) according to clinical presentation.

Other important diagnostic scenarios.

Full Siblings

If a proband meets diagnostic or likely diagnostic criteria, our expert panel recommends CMMRD surveillance for full siblings until CMMRD is ruled out by at least one of the following methods (Note: LS diagnosis may still be possible):

• From criteria 1, 2 or 5: if siblings did not inherit one of the familial variants as identified in the index case.

• If sibling(s) reaches age 30 years with no history of colorectal polyps or CMMRD related cancer

• If sibling has negative ancillary testing (when index case has proven positive ancillary test)

Consideration for EPCAM

To date, there is one known case of homozygous EPCAM 3`UTR deletions, unpublished and followed by the IRRD consortium. This 5-year old has colorectal polyposis, small bowel adenomas and 1 CALM. There has also been a 9-year old child reported to have compound heterogyzous MSH2 mutation and EPCAM 3'UTR deletion (in trans), with 70-80 colorectal adenomas, and two synchronous colorectal cancers. [START_REF] Hh | Colorectal cancer in a 9year-old due to combined EPCAM and MSH2 germline mutations: case report of a unique genotype and immunophenotype[END_REF] To date, there have been no reports of biallelic EPCAM variants with deletions extending into MSH2, and while rare, one might expect this to resemble the phenotype of biallelic MSH2.

At this time, there is not enough clinical information to determine the impact on biallelic EPCAM 3' UTR deletions on cancer risks and whether it is isolated to the GI tract, or if it extends to the other hallmark cancers of CMMRD. This may be also dependent on the involvement of the MSH2 gene, as homozygous deletions that extend into MSH2 would be suspected to cause CMMRD. The working group recommend CMMRD surveillance for biallelic EPCAM 3' UTR deletions or individuals with EPCAM/MSH2, until further information becomes available.

Consideration for MSH3

There have been few reports of individuals with biallelic MSH3 described as having adult onset adenomatous polyposis of the colon. [START_REF] Adam | Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis[END_REF] Thyroid and small bowel adenomas, and astrocytoma have also been reported, all occurring in young adulthood. [START_REF] Adam | Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis[END_REF] It is unclear whether individuals with biallelic MSH3 require the same intensive screening as CMMRD patients, and at this time our expert panel concluded that insufficient evidence exists at this time to determine if cancer risk would be similar to CMMRD or FAP, and does not recommend CMMRD surveillance at this time.

As information continues to evolve on biallelic EPCAM and MSH3, as well as the expanding spectrum of CMMRD, the diagnostic criteria and surveillance recommendations should be assessed periodically.

Summary

This is the first consensus for diagnostic criteria for CMMRD which was achieved by a working group of international experts, including members of the IRRD and the C4CMMRD consortia who are involved in the management of most CMMRD individuals. The six diagnostic criteria described allows for clear surveillance recommendations and familial genetic counseling.

CMMRD is a condition with a variable clinical presentation encompassing a broad spectrum of cancers that may mimic other hereditary conditions. This working group defined a set of hallmark cancers, while recognizing there is variability in presentation of cancer site and age of onset. Benign manifestations are also broad and include features that overlap the phenotype of NF1, tuberous sclerosis, juvenile and adenomatous polyposis. Molecular testing is unable to conclusively identify biallelic pathogenic MMR gene variants in up to 40% of patients. The diagnostic criteria outlined is essential given the complexity of the syndrome.

Tables and Figures

Figure 1: # Ancillary testing is described in further detail above. Does not include tumor mutation burden and signature at this time.

Functional testing should be published with proven high sensitivity and specificity performed in an accredited (e.g. CAP inspected) laboratory authorised to give a clinically usable report. If discrepancy occurs among tests, multiple ancillary tests should be used to reach more conclusive decision.

> Hallmark CMMRD cancer: Glioma or CNS embryonal tumors <25 yrs, Hematological cancer (excluding Hodgkin's lymphoma) < 18 yrs, GI adenocarcinoma <25 yrs, or >10 adenomatous GI polyps < 18 yrs (after ruling out polyposis conditions).

+ C4CMMRD criteria outlined in Figure 1 ^ Individuals with two positive ancillary tests for CMMRD in the absence of the described phenotype, can be assessed on a caseby-case basis, but these are atypical CMMRD cases and additional assessment is required to determine surveillance.

(

  IRRD), the C4CMMRD (European consortium, Care for CMMRD) as well as other clinical and molecular geneticists, genetic counsellors, pediatric hematologist and oncologists, from Canada, United States, Austria, Israel and France. To validate some of the recommendations, the clinical working group used data from the IRRD consortium, based in Toronto Canada at the Hospital for Sick Children and from the C4CMMRD database, based in Villejuif, France at Gustave Roussy Cancer Campus. The IRRD consortium was established in 2007 to assess individuals suspected of having syndromes with replication repair deficiencies, including a large cohort of CMMRD patients (n=110), originating from 45 countries. Updates on surveillance and health status are obtained at regular intervals. The European Care for CMMRD (C4CMMRD) consortium was established in 2013. One of its objectives was to create a database dedicated to CMMRD patients. This database contains retrospective and prospective data, from 15 countries, with approximately 100 patients with confirmed CMMRD diagnosis. It collaborates with other national databases in European countries to address specific issues about CMMRD. Based on literature review and expertise from the panel, draft clinical criteria were established by the working group at an International Replication Repair Deficiency workshop held in Toronto on Oct 15-16, 2017. The draft clinical criteria from the working group were presented to all

  . Using unpublished data from the two largest consortiums (IRRD and C4CMMRD, respectively),11-15.6% of cases presented at age 18 or older, and 1-7% at age 25 or older. The oldest age at first malignancy reported in CMMRD was rectal cancer at age 40 in an individual with in trans P/LP PMS2 variants (c.2007-?_2589+?del (del 12-15)/ c.614A>C) who had confirmed loss of PMS2 protein in tumor and non-neoplastic tissue.

Wimmer et al (2014):Scoring system to determine germline testing eligibility for CMMRD 1 Indication for CMMRD testing in a cancer patient ≥3 points Malignancies/premalignancies: one is mandatory; if more than one is present in the patient, add the points:

	Carcinoma from the LS spectrum* at age <25 years	3 points
	Multiple bowel adenomas at age <25 years and absence of APC/MUTYH mutation(s) or a single high-grade
	dysplasia adenoma at age <25 years	3 points
	WHO grade III or IV glioma at age <25 years	2 points
	NHL of T-cell lineage or sPNET at age <18 years	2 points
	Any malignancy at age <18 years	1 point
	Additional features: optional; if more than one of the following is present, add the points
	Clinical sign of NF1 and/or ≥2 hyperpigmented and/or hypopigmented skin alterations Ø>1 cm	2 points
	Diagnosis of LS in a first-degree or second-degree relative	2 points
	Carcinoma from LS spectrum* before the age of 60 in 1 st , 2 nd or 3rd-degree relative	1 point
	A sibling with carcinoma from the LS spectrum*, high-grade glioma, sPNET or NHL	2 points
	A sibling with any type of childhood malignancy	1 point
	Multiple pilomatricomas in the patient	2 points
	One pilomatricoma in the patient	1 point
	Agenesis of the corpus callosum or non-therapy-induced cavernoma in the patient	1 point
	Consanguineous parents	1 point
	Deficiency/reduced levels of IgG2/4 and/or IgA	1 point
	*Colorectal, endometrial, small bowel, ureter, renal pelvis, biliary tract, stomach, bladder carcinoma. CMMRD,
	constitutional mismatch repair deficiency; LS, Lynch syndrome; NHL, non-Hodgkin's lymphomas; sPNET,
	supratentorial primitive neuroectodermal tumors

Table 1 : Example of Surveillance protocol for patients with CMMRD

 1 GYN, gynecologic; Q, every; U/S, ultrasound; VCE, visual capsule endoscopy. (adapted from Tabori et al Clin Cancer Res (2017)[START_REF] Tabori | Clinical Management and Tumor Surveillance Recommendations of Inherited Mismatch Repair Deficiency in Childhood. Clinical cancer research : an official journal of the American Association for[END_REF]

	Examination	Start age Frequency Tumors	Comment
	MRI brain		At	Q	6	Brain tumors	Should not be replaced
			diagnosis	months			with WBMRI
	WBMRI		6 years	Once	a	All tumors	Should	not	replace
				year			dedicated CNS imaging
	CBC		1 year	Q	6	Leukemia	May be considered
				months
	Abdominal U/S	1 year	Q	6	Lymphoma	May be considered
				months
	Upper		4 to 6	Once	a	Gastrointestinal	Upper	and	lower
	gastrointestinal	years	year		tumors	endoscopy, to increase
	endoscopy; VCE,					once polyps are found
	ileocolonoscopy
	GYN	exam,	20 years Once	a	Genitourinary	As per LS guidelines
	transvaginal U/S,		year		cancers
	pipelle curettage,
	urine cytology,

Table 2 : CMMRD Diagnostic criteria

 2 Biallelic-impacts same gene on both parental alleles (i.e. PMS2/PMS2), P-pathogenic (ACMG C5), LP-likely pathogenic (ACMG C4), VUS-(ACMG C3). Multi-gene panel testing is recommended to investigate overlapping conditions. Consider phenotype of individual to rule out overlapping syndromes. All families should be assessed in a specialized centre for diagnosis. ** Consanguinity further support a diagnosis of CMMRD due to a homozygous MMR gene mutation that is unidentifiable ^ In trans variants can be proven by testing parents, offspring or other relatives. If unavailable to confirm variants in trans, individual should fulfil criterion #3 ^^ If unavailable to confirm variants in trans, individual should fulfil criterion #6

	Criterion		Germline result*	Positive	Clinical Phenotype
			PMS2, MSH6, MSH2,	Ancillary testing #
			MLH1
			Biallelic pathogenic	Not required	Not required if under age 25 (if no
		1	variants (P/P)*, confirmed in trans^	unless unaffected >25 yrs, then one	malignancy over age 25, ancillary testing required)
				required #
			Biallelic P/LP or	One required #	Hallmark CMMRD cancer diagnosis >
			LP/LP* variants,	unless unaffected	or C4CMMRD criteria of 3 points +
	Definitive	2	confirmed in trans^^	by hallmark	(then 2 ancillary tests required)
	Diagnosis			cancer > , then 2
	(Strong			required
	evidence		Heterozygous P or LP	One required #	Hallmark CMMRD cancer diagnosis >
	of	3	variant (+/-VUS* or
	CMMRD)		likely benign variants)
			No P or LP MMR	Two required #	Hallmark CMMRD cancer diagnosis >
			variants (including
		4	VUS/VUS)** or
			No testing available
			(i.e. deceased proband)
			Biallelic P/LP* or	Not required	C4CMMRD criteria of 3 points +
	Likely	5	LP/LP variants
	Diagnosis		confirmed in trans^^
	(Moderate		Heterozygous P or LP	Two required #	a. C4CMMRD criteria of 3 points +
	evidence		variant or no testing		b. Individual < age 18 with NF1
	of	6 ^	available (i.e. deceased		features (i.e. no malignancy or
	CMMRD)		proband)		polyposis history)
					c. Malignancy under age 30

*

protuberans, rhabdomyosarcoma, renal pelvis carcinoma, myofibromatosis +: WHO grade III/IV glioma <25 yrs, NHL of T-cell lineage < 18 yrs, GI adenocarcinoma <25 yrs, or GI adenomatous polyposis < 18 yrs ~ Glioma or CNS embryonal tumors <25 yrs, Hematological cancer (excluding Hodgkin's lymphoma) < 18 yrs, GI adenocarcinoma <25 yrs, or >10 adenomatous GI polyps < 18 yrs (after ruling out polyposis conditions).