Constitutional Mismatch Repair Deficiency Syndrome
Constitutional Mismatch Repair Deficiency Syndrome
An early and definite diagnosis of CMMRD in a paediatric and young adult cancer patient is desired for several reasons. (1) Although the true cancer risk of CMMRD is currently not known (and may be overestimated since we have to rely on a potentially biased cohort of patients), the available data from the published patients with this rare disorder suggest that it is extraordinarily high. CMMRD patients who survive their first malignancy or in whom a premalignancy has been removed have a high risk of developing a second often different (pre-) malignancy. To improve the prognosis, surveillance should be offered at least for bowel cancer and brain tumours as recently proposed by the European consortium. (2) Early and definite diagnosis may also be a prerequisite in the future to adjust treatment to the underlying MMR defect and the high risk of a secondary malignancy. Currently, no information is available regarding the optimal treatment for CMMRD patients. But careful attention should be given to a possibly reduced efficacy and increased cytotoxicity of certain chemotherapeutic agents due to constitutionally impaired mutation repair. Since MMR-deficient cells are profoundly resistant to O methylators such as temozolomide, the risk of therapy failure may be increased in CMMRD patients treated with this drug. Furthermore, these drugs may increase the risk of second primary tumours by accelerating the rate of unrepaired mutations. (3) Siblings of a CMMRD patient have a 25% risk of having inherited the same genotype and, hence, equally a high risk for childhood cancer. (4) The parents of CMMRD patients and 50% of their siblings as well as other more distantly related family members are heterozygous for the MMR mutation(s) and, therefore, have an increased risk for LS-associated tumours in adulthood. A definite molecular diagnosis is needed to offer the families of CMMRD patients appropriate counselling and discuss with them the options of predictive testing as well as prenatal/preimplantation diagnostics if this is desired.
It might be considered desirable to establish a diagnosis before the development of the first malignancy in a biallelic MMR gene mutation carrier. Currently, however, this seems to be only achievable for siblings of a tumour patient with a molecularly confirmed diagnosis. The non-neoplastic features of CMMRD may be too subtle and unspecific to raise the suspicion in a child without a malignancy if there is no family history of CMMRD or diagnosis of LS in the family. Therefore, it was decided at the first meeting of the C4CMMRD consortium that, at the time being, the group will only propose clinical criteria that should raise the suspicion of CMMRD when present in a cancer patient. Questions that were discussed among the group were
Ad A): The spectrum of malignancies in CMMRD is very broad (see Table 2). Therefore, any malignancy in a paediatric (young adult) patient could be a CMMRD-associated one. Nevertheless, the index of suspicion should be higher in tumour entities that are overrepresented in CMMRD patients compared with their proportion in all tumours of the general population. Taking this into account, we developed a scoring system for the suspected diagnosis of CMMRD. Tumours assigned three points are highly specific for CMMRD syndrome. A diagnosis of CMMRD should be suspected in patients with these tumour entities no matter whether they show additional (non-neoplastic) features of CMMRD or not. Malignancies assigned two points are overrepresented in CMMRD but less specific. Additional features or tumours that add up to three points need to be present in patients with these malignancies to suspect CMMRD. All other malignancies are assigned one point, and additional tumours or features strongly pointing into the direction of CMMRD need to be present to raise a suspicion that should entail further diagnostic steps.
Colorectal or other cancers of the LS spectrum are extremely rare below the age of 25 years even in LS and FAP. Because they are highly suggestive for CMMRD, which phenotypically presents in several cases as a very early onset form of LS, three points are assigned to these tumours.
Multiple bowel adenomas are a frequent finding in CMMRD, which hence shows clinical overlap also with (attenuated) FAP. Herkert et al proposed that in the absence of proven APC or MUTYH germline mutations, patients with childhood-onset adenomatous polyposis should be considered for MMR gene mutation testing, especially when they have features of NF1. In CMMRD, both the adenoma formation and the adenoma carcinoma transition may be accelerated due to the greatly enhanced mutation rate in neoplastic and non-neoplastic tissue. In agreement with this notion, most (35/52) of the CMMRD patients with adenomas showed high-grade dysplasia in at least one of them or had synchronous bowel cancer. Therefore, CMMRD syndrome should be considered as a differential diagnosis also in a patient under the age of 25 years (for considerations concerning the age limit of 25 years, see ad (B)) with (i) multiple adenomas if a heterozygous APC mutation or biallelic MUTYH mutations are absent (exclusion of POLD1 and POLE hotspot mutations may also be considered) or (ii) in a patient with a single adenoma with high-grade dysplasia. Consequently, multiple bowel adenomas and absence of an APC/MUTYH mutation and/or one adenoma with high-grade dysplasia under the age of 25 have a score of three points.
High-grade gliomas, including glioblastoma, gliosarcoma, anaplastic astrocytoma, oligodendroglioma and others, are rare tumours in childhood and adolescence. According to the data of the French Brain Tumour Data Bank and the German childhood cancer register (http://www.kinderkrebsregister.de), they represent 15% of all brain/CNS tumours in childhood and adolescence. Hence, assuming that a quarter of all tumours in childhood are brain/CNS tumours, high-grade gliomas represent less than 5% of all tumours in childhood/adolescence. In contrast, 26% of all malignancies so far seen in CMMRD patients are high-grade gliomas. Equally, CNS-PNET constituting 3.5% of all so far reported malignancies in CMMRD (vs 0.5% of all childhood tumours in the general population) are overrepresented in CMMRD. Among the haematological tumours, NHL are overrepresented in CMMRD. NHL constitute ~5–7% of all childhood tumours but 14% of all CMMRD-associated malignancies. Of note, at least 65% (20/31) of the CMMRD-associated NHL were from the T-cell lineage and at least 42% (13/31) were T-cell lymphoblastic lymphomas. This is in contrast to the distribution in the general population where ~65% are from the B-cell lineage and more than 50% are mature B-NHL. Hence, NHL of the T-cell lineage mainly account for the overrepresentation of NHL in CMMRD. Taken together, T-NHL, high-grade gliomas and CNS-PNET are considered malignancies typical for CMMRD and are assigned two points in the scoring system.
Ad B): The age at diagnosis of the first malignancy ranges from 0.4 to 39 years in CMMRD patients (Table 1). However, the vast majority, that is, 120 of the 146 CMMRD patients, were younger than 18 years of age when their first tumour was diagnosed. Only 17 patients were between 18 and 25 years when they developed their first tumour, which was a CRC in 11 patients, a glioblastoma in 1 patient and an oligodendroglioma in 1 patients. The remaining four patients had colorectal adenomas when they were young adults. Only four patients were older than 25 years when they were diagnosed with their first tumour; in all four cases a CRC. These four patients carried at least one allele a likely hypomorphic MSH2, MSH6 and PMS2 mutation, respectively, and, therefore, may genetically as well as clinically represent an intermediate phenotype between CMMRD and LS. Based on the observations in the patients reported so far, we set the age limit at diagnosis of the first tumour at 25 years for patients with CRC, colorectal adenomas and malignant gliomas. For all other patients with malignancies, the age limit is <18 years (see Table 5).
Ad C): Non-neoplastic features are weighted according to their specificity for CMMRD and their frequency in the general population with one point or two points.
Presence of (segmental) NF1 signs, primarily multiple (≥6) CALMs and freckling, is so far the most commonly reported non-neoplastic feature associated with CMMRD, and it has been stressed already in several reports that presence of one or more of these signs should raise the suspicion of CMMRD in any paediatric cancer patient with the exception of children/young adults with clearly NF1-associated malignancies, such as a peripheral nerve sheath tumour (MPNST) or a juvenile myelomonocytic leukaemia (JMML) or with a parent who is also diagnosed with NF1. Hence, this highly specific feature is weighted two points.
Although many CMMRD patients came to attention through the presence of NF1 signs, our analysis of the reported cases clearly indicates that limiting suspicion to patients showing classical NF1 signs will miss a number of patients. In several CMMRD patients, the number of CALMs has been reported to be below six, which is the necessary number to be diagnostic for NF1. Furthermore, several CMMRD patients do not show classical NF1-associated CALMs but have different kinds of skin hyperpigmentation and hypopigmentation. As an isolated finding these alterations are frequent in the general population. A study assessing the frequency of CALMs among school children aged 4–11 years showed that ~20% (146/732) had one CALM. However, only 4.1% (30/732) and 1.2% (9/732) had two and three CALMs, respectively. Merks and colleagues report a slightly lower frequency of solitary and multiple CALM in 13% and 3.3% of school children, respectively. Hence, the likelihood of having a childhood malignancy and two or more CALMs (hyperpigmented macules) by pure chance is very low. The same is likely the case for hypopigmented macules. Therefore, the presence of two or more skin areas of hyperpigmentation or hypopigmentation with a minimum diameter of 1 cm should be another feature that should raise the suspicion of CMMRD. This feature is also weighted two points.
Brain malformations such as agenesis of corpus callosum and non-therapy-induced cavernoma as well as pilomatricomas/epitheliomas of the Malherbe are rare in the general population. But they seem to have a higher incidence in CMMRD patients (see sections 'Premalignancies and non-malignant tumours in CMMRD patients' and 'Non-neoplastic features of CMMRD'). Therefore, these features should also raise the suspicion of CMMRD when present in a childhood cancer patient and are, thus, weighted depending on their frequency in the general population 1–2 points. The frequency of brain malformations in CMMRD patients still needs to be evaluated prospectively. As long as they have not been shown to be more frequent, we do not advocate performing cranial MRI unless required for other clinical reasons to test for these features only in order to confirm the diagnosis CMMRD in a cancer patient. The brain malformations add one point to the score of a cancer patient. Multiple pilomatricomas, which are extremely rare and always indicate an underlying genetic defect, add two points. A single pilomatricoma adds only one point.
Consanguinity of the parents and/or homozygosity for one (founder) mutation is observed in 46 of the 91 so far reported families with CMMRD patients. However, consanguineous marriages are common in several ethnic and religious groups of the European population. Therefore, we decided that consanguinity should add one point and raise the suspicion of CMMRD in a patient with a paediatric cancer that is overrepresented in CMMRD syndrome and in any other paediatric cancer patient who has one of the other features.
Due to the impaired class switch recombination, reduced IgG2/4 and/or IgA levels could be observed in several CMMRD patients. But this feature is neither very specific for CMMRD nor particularly rare in the general population. Hence, it is also assigned one point.
Other criteria for suspecting CMMRD in a child with a malignancy are (i) a sibling with a malignancy and (ii) the molecular diagnosis of LS or an LS-associated tumour before the age of 60 years in a first-degree, second-degree or third-degree relative of the patient. When assigning points to these criteria, it was taken into consideration that typical CMMRD tumours in a sibling should raise a higher index of suspicion (two points) than any other tumour in a sibling (one point). Equally, a definite diagnosis of LS in the family is assigned two points, whereas an LS-associated tumour before the age of 60 years in a first-degree, second-degree or third-degree relative of the patient is scored only one point. A LS-associated tumour in a relative that has been shown to be microsatellite stable, and hence, is very likely a sporadic tumour should not be taken into account.
According to the three-point scoring system summarised in Table 5, CMMRD syndrome should be suspected in an individual who reaches a score of minimum three points. The points should be counted by adding the points assigned to the malignancy/malignancies and those assigned to additional features. When using this system, it has to be kept in mind that several of these features listed in Table 5 may be present also in a patient with a different (childhood) cancer predisposition syndrome, like Fanconi anaemia, ataxia telangiectasia, Bloom syndrome, Peutz–Jeghers syndrome, tuberous sclerosis and others. These syndromes are usually associated with characteristic anomalies that are not listed in Table 5. Therefore, patients with signs specific for another cancer predisposition syndrome except for NF1 should first be tested for the other cancer predisposition syndrome. Patients who have clearly NF1-associated malignancies such as JMML or MPNST and signs reminiscent of NF1 should be first tested for a NF1 mutation. There is also an overlap of CMMRD with Li Fraumeni syndrome (LFS). The tumour spectrum of the latter encompasses brain tumours and, to a lesser extent, haematological neoplasms. In families fulfilling Chompret's criteria, a TP53 germline mutation should be ruled out first.
Patients reaching a score of three points should be further analysed to confirm or refute the suspected diagnosis of CMMRD syndrome. The diagnostic steps in these patients largely follow the protocols developed for LS, which involves analysis of microsatellite instability (MSI) and/or immunohistochemical (IHC) staining of the four MMR proteins.
IHC analysis to assess the expression loss of the affected MMR protein can be effectively employed in all solid tumours of CMMRD patients and has the advantage to guide subsequent mutation analysis in the four MMR genes. In general, biallelic truncating mutations in PMS2 or MSH6 will result in isolated loss of these proteins, whereas mutations in MLH1 or MSH2 will lead to concurrent loss of MLH1/PMS2 or MSH2/MSH6, respectively, since MLH1 and MSH2 are the obligatory partners in the formation of MLH1/PMS2 and MSH2/MSH6 heterodimers. Of note, in the case of an underlying missense mutation, IHC may show normal expression of the affected MMR gene, which may be a possible pitfall when using IHC analysis to confirm suspected CMMRD. In contrast to LS where expression loss is observed only in neoplastic cells, in CMMRD patients IHC detects expression loss of one (or two) of the MMR proteins in both neoplastic and non-neoplastic tissues. Hence, negative IHC staining in neoplastic and surrounding normal cells should not be interpreted as a failure of proper staining and care should be taken to use a (on slide) staining control from a different individual. In principle, expression loss of one of the MMR genes can also be demonstrated in blood lymphocytes of CMMRD patients, as shown by western blotting but possibly also by IHC staining on cytospin preparations.
MSI tests for small misalignments, that is, insertion–deletion loops (IDL), which arise during DNA replication and remain uncorrected in the absence of a functional MMR system. It takes advantage of the fact that repeated-sequence motifs consisting of units of one, two or a few more nucleotides, that is, microsatellites, are frequent targets of IDL errors that result in shortening or lengthening of these sequence motifs, a phenomenon termed MSI. In tissues derived from clonally proliferating MMR-deficient cells, that is, neoplastic cells, MSI is easily detected by PCR amplification and fragment analysis of a set of microsatellite markers. MSI analysis following the current protocols for LS uses a panel of 5–6 dinucleotide and/or mononucleotide repeat markers. This approach is a reliable tool to diagnose MMR deficiency in gastrointestinal and other LS-associated tumours of CMMRD patients. However, standard MSI analysis as applied in LS frequently fails to show MSI in brain tumours and other malignancies. Currently, the reasons for this observation are unknown, but it has been shown that more subtle shifts (shortening or lengthening) of microsatellite alleles can be observed also in brain tumours and may be indicative of CMMRD. Furthermore, it might be worth testing whether a different panel of microsatellite markers would be more sensitive in brain and other tumours of CMMRD patients.
In principle, MSI can be observed also in DNA from normal, that is, non-neoplastic, cells of CMMRD patients. However, because altered microsatellite alleles are present only in a small proportion of the cells from normal tissue, so far most approaches used technically demanding single-molecule analyses (eg, small-pool PCR) to show MSI in normal tissue, that is, germline MSI (gMSI). Recently, a much more simple method to detect gMSI was presented. This assay relies on the analysis of 'stutter' peaks typically associated with microsatellite PCR products. When quantified by a novel publicly available software application, the relative peak height of the 'stutter' peaks of selected dinucleotide microsatellites significantly increases in DNA of CMMRD patients compared with normal controls as has been confirmed also in a larger cohort of samples (Bodo et al., in preparation). 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, if this assay shows in a larger cohort of samples that it is a reliable, simple and rapid tool to detect CMMRD at least in patients with PMS2, MLH1 or MSH2 mutations, it would be a good screening tool and an alternative to IHC in cases where appropriate tissue is not available.
Taken together, both IHC staining of the MMR genes and MSI analysis are diagnostic methods to substantiate the suspected diagnosis. Since IHC will also guide target-gene mutation analysis and has been shown to render reliable results in most solid tumours, it is considered the preferred method. But as outlined, both methods have potential pitfalls and may fail to confirm the suspected diagnosis. Therefore, we recommend combining both assays if needed. The final confirmation of the diagnosis CMMRD should come from the determination of the causative biallelic mutations in the patient.
According to the recommendations of national and international human genetic societies and the legislation of most European countries, genetic counselling must be offered to the patients and/or their parents prior to performing mutation analysis in the affected child. Patients and/or their parents should be informed by a team of paediatric oncologists and medical geneticists about the suspected diagnosis if this is substantiated by MSI and/or IHC analysis. Considering the burden of this syndrome, psychological support should systematically be proposed to families. The family has to be informed of potential therapeutic implications of the test result and also of the high risk for a second malignancy in a patient with a positive test result. Genetic counselling must also include information on the potential 25% recurrence risk in a sibling and on the risks for LS-associated cancer in possible heterozygous mutation carriers, particularly both parents.
With the informed consent of the patient and/or the parents, mutation analysis will be initiated. Reliable and robust comprehensive analysis exists for all four MMR genes now including the historically difficult PMS2 gene. However, when analysing PMS2, which is the affected gene in more than 50% of CMMRD patients, special care has to be taken to avoid pitfalls that arise from high prevalence of hybrid alleles that result from sequence exchange of the functional gene with its pseudogene PMS2CL.
Preferentially, targeted gene mutation analysis is performed. However, in cases where tumour tissue is not available for IHC analysis or the results are inconclusive, mutation analysis of all four MMR genes can be considered. It is expected that with the implementation of next-generation sequencing techniques this may be possible at reasonable costs in many laboratories in the near future.
Suggestions of the European Consortium 'Care for CMMRD'
Diagnostic Criteria That Should Raise the Suspicion of CMMRD Syndrome in a Cancer Patient
An early and definite diagnosis of CMMRD in a paediatric and young adult cancer patient is desired for several reasons. (1) Although the true cancer risk of CMMRD is currently not known (and may be overestimated since we have to rely on a potentially biased cohort of patients), the available data from the published patients with this rare disorder suggest that it is extraordinarily high. CMMRD patients who survive their first malignancy or in whom a premalignancy has been removed have a high risk of developing a second often different (pre-) malignancy. To improve the prognosis, surveillance should be offered at least for bowel cancer and brain tumours as recently proposed by the European consortium. (2) Early and definite diagnosis may also be a prerequisite in the future to adjust treatment to the underlying MMR defect and the high risk of a secondary malignancy. Currently, no information is available regarding the optimal treatment for CMMRD patients. But careful attention should be given to a possibly reduced efficacy and increased cytotoxicity of certain chemotherapeutic agents due to constitutionally impaired mutation repair. Since MMR-deficient cells are profoundly resistant to O methylators such as temozolomide, the risk of therapy failure may be increased in CMMRD patients treated with this drug. Furthermore, these drugs may increase the risk of second primary tumours by accelerating the rate of unrepaired mutations. (3) Siblings of a CMMRD patient have a 25% risk of having inherited the same genotype and, hence, equally a high risk for childhood cancer. (4) The parents of CMMRD patients and 50% of their siblings as well as other more distantly related family members are heterozygous for the MMR mutation(s) and, therefore, have an increased risk for LS-associated tumours in adulthood. A definite molecular diagnosis is needed to offer the families of CMMRD patients appropriate counselling and discuss with them the options of predictive testing as well as prenatal/preimplantation diagnostics if this is desired.
It might be considered desirable to establish a diagnosis before the development of the first malignancy in a biallelic MMR gene mutation carrier. Currently, however, this seems to be only achievable for siblings of a tumour patient with a molecularly confirmed diagnosis. The non-neoplastic features of CMMRD may be too subtle and unspecific to raise the suspicion in a child without a malignancy if there is no family history of CMMRD or diagnosis of LS in the family. Therefore, it was decided at the first meeting of the C4CMMRD consortium that, at the time being, the group will only propose clinical criteria that should raise the suspicion of CMMRD when present in a cancer patient. Questions that were discussed among the group were
Which tumour entities should raise a suspicion?
What is the age limit for a patient with a specific tumour entity to be suspected?
Which non-neoplastic feature should be considered a diagnostic criterion?
Ad A): The spectrum of malignancies in CMMRD is very broad (see Table 2). Therefore, any malignancy in a paediatric (young adult) patient could be a CMMRD-associated one. Nevertheless, the index of suspicion should be higher in tumour entities that are overrepresented in CMMRD patients compared with their proportion in all tumours of the general population. Taking this into account, we developed a scoring system for the suspected diagnosis of CMMRD. Tumours assigned three points are highly specific for CMMRD syndrome. A diagnosis of CMMRD should be suspected in patients with these tumour entities no matter whether they show additional (non-neoplastic) features of CMMRD or not. Malignancies assigned two points are overrepresented in CMMRD but less specific. Additional features or tumours that add up to three points need to be present in patients with these malignancies to suspect CMMRD. All other malignancies are assigned one point, and additional tumours or features strongly pointing into the direction of CMMRD need to be present to raise a suspicion that should entail further diagnostic steps.
Colorectal or other cancers of the LS spectrum are extremely rare below the age of 25 years even in LS and FAP. Because they are highly suggestive for CMMRD, which phenotypically presents in several cases as a very early onset form of LS, three points are assigned to these tumours.
Multiple bowel adenomas are a frequent finding in CMMRD, which hence shows clinical overlap also with (attenuated) FAP. Herkert et al proposed that in the absence of proven APC or MUTYH germline mutations, patients with childhood-onset adenomatous polyposis should be considered for MMR gene mutation testing, especially when they have features of NF1. In CMMRD, both the adenoma formation and the adenoma carcinoma transition may be accelerated due to the greatly enhanced mutation rate in neoplastic and non-neoplastic tissue. In agreement with this notion, most (35/52) of the CMMRD patients with adenomas showed high-grade dysplasia in at least one of them or had synchronous bowel cancer. Therefore, CMMRD syndrome should be considered as a differential diagnosis also in a patient under the age of 25 years (for considerations concerning the age limit of 25 years, see ad (B)) with (i) multiple adenomas if a heterozygous APC mutation or biallelic MUTYH mutations are absent (exclusion of POLD1 and POLE hotspot mutations may also be considered) or (ii) in a patient with a single adenoma with high-grade dysplasia. Consequently, multiple bowel adenomas and absence of an APC/MUTYH mutation and/or one adenoma with high-grade dysplasia under the age of 25 have a score of three points.
High-grade gliomas, including glioblastoma, gliosarcoma, anaplastic astrocytoma, oligodendroglioma and others, are rare tumours in childhood and adolescence. According to the data of the French Brain Tumour Data Bank and the German childhood cancer register (http://www.kinderkrebsregister.de), they represent 15% of all brain/CNS tumours in childhood and adolescence. Hence, assuming that a quarter of all tumours in childhood are brain/CNS tumours, high-grade gliomas represent less than 5% of all tumours in childhood/adolescence. In contrast, 26% of all malignancies so far seen in CMMRD patients are high-grade gliomas. Equally, CNS-PNET constituting 3.5% of all so far reported malignancies in CMMRD (vs 0.5% of all childhood tumours in the general population) are overrepresented in CMMRD. Among the haematological tumours, NHL are overrepresented in CMMRD. NHL constitute ~5–7% of all childhood tumours but 14% of all CMMRD-associated malignancies. Of note, at least 65% (20/31) of the CMMRD-associated NHL were from the T-cell lineage and at least 42% (13/31) were T-cell lymphoblastic lymphomas. This is in contrast to the distribution in the general population where ~65% are from the B-cell lineage and more than 50% are mature B-NHL. Hence, NHL of the T-cell lineage mainly account for the overrepresentation of NHL in CMMRD. Taken together, T-NHL, high-grade gliomas and CNS-PNET are considered malignancies typical for CMMRD and are assigned two points in the scoring system.
Ad B): The age at diagnosis of the first malignancy ranges from 0.4 to 39 years in CMMRD patients (Table 1). However, the vast majority, that is, 120 of the 146 CMMRD patients, were younger than 18 years of age when their first tumour was diagnosed. Only 17 patients were between 18 and 25 years when they developed their first tumour, which was a CRC in 11 patients, a glioblastoma in 1 patient and an oligodendroglioma in 1 patients. The remaining four patients had colorectal adenomas when they were young adults. Only four patients were older than 25 years when they were diagnosed with their first tumour; in all four cases a CRC. These four patients carried at least one allele a likely hypomorphic MSH2, MSH6 and PMS2 mutation, respectively, and, therefore, may genetically as well as clinically represent an intermediate phenotype between CMMRD and LS. Based on the observations in the patients reported so far, we set the age limit at diagnosis of the first tumour at 25 years for patients with CRC, colorectal adenomas and malignant gliomas. For all other patients with malignancies, the age limit is <18 years (see Table 5).
Ad C): Non-neoplastic features are weighted according to their specificity for CMMRD and their frequency in the general population with one point or two points.
Presence of (segmental) NF1 signs, primarily multiple (≥6) CALMs and freckling, is so far the most commonly reported non-neoplastic feature associated with CMMRD, and it has been stressed already in several reports that presence of one or more of these signs should raise the suspicion of CMMRD in any paediatric cancer patient with the exception of children/young adults with clearly NF1-associated malignancies, such as a peripheral nerve sheath tumour (MPNST) or a juvenile myelomonocytic leukaemia (JMML) or with a parent who is also diagnosed with NF1. Hence, this highly specific feature is weighted two points.
Although many CMMRD patients came to attention through the presence of NF1 signs, our analysis of the reported cases clearly indicates that limiting suspicion to patients showing classical NF1 signs will miss a number of patients. In several CMMRD patients, the number of CALMs has been reported to be below six, which is the necessary number to be diagnostic for NF1. Furthermore, several CMMRD patients do not show classical NF1-associated CALMs but have different kinds of skin hyperpigmentation and hypopigmentation. As an isolated finding these alterations are frequent in the general population. A study assessing the frequency of CALMs among school children aged 4–11 years showed that ~20% (146/732) had one CALM. However, only 4.1% (30/732) and 1.2% (9/732) had two and three CALMs, respectively. Merks and colleagues report a slightly lower frequency of solitary and multiple CALM in 13% and 3.3% of school children, respectively. Hence, the likelihood of having a childhood malignancy and two or more CALMs (hyperpigmented macules) by pure chance is very low. The same is likely the case for hypopigmented macules. Therefore, the presence of two or more skin areas of hyperpigmentation or hypopigmentation with a minimum diameter of 1 cm should be another feature that should raise the suspicion of CMMRD. This feature is also weighted two points.
Brain malformations such as agenesis of corpus callosum and non-therapy-induced cavernoma as well as pilomatricomas/epitheliomas of the Malherbe are rare in the general population. But they seem to have a higher incidence in CMMRD patients (see sections 'Premalignancies and non-malignant tumours in CMMRD patients' and 'Non-neoplastic features of CMMRD'). Therefore, these features should also raise the suspicion of CMMRD when present in a childhood cancer patient and are, thus, weighted depending on their frequency in the general population 1–2 points. The frequency of brain malformations in CMMRD patients still needs to be evaluated prospectively. As long as they have not been shown to be more frequent, we do not advocate performing cranial MRI unless required for other clinical reasons to test for these features only in order to confirm the diagnosis CMMRD in a cancer patient. The brain malformations add one point to the score of a cancer patient. Multiple pilomatricomas, which are extremely rare and always indicate an underlying genetic defect, add two points. A single pilomatricoma adds only one point.
Consanguinity of the parents and/or homozygosity for one (founder) mutation is observed in 46 of the 91 so far reported families with CMMRD patients. However, consanguineous marriages are common in several ethnic and religious groups of the European population. Therefore, we decided that consanguinity should add one point and raise the suspicion of CMMRD in a patient with a paediatric cancer that is overrepresented in CMMRD syndrome and in any other paediatric cancer patient who has one of the other features.
Due to the impaired class switch recombination, reduced IgG2/4 and/or IgA levels could be observed in several CMMRD patients. But this feature is neither very specific for CMMRD nor particularly rare in the general population. Hence, it is also assigned one point.
Other criteria for suspecting CMMRD in a child with a malignancy are (i) a sibling with a malignancy and (ii) the molecular diagnosis of LS or an LS-associated tumour before the age of 60 years in a first-degree, second-degree or third-degree relative of the patient. When assigning points to these criteria, it was taken into consideration that typical CMMRD tumours in a sibling should raise a higher index of suspicion (two points) than any other tumour in a sibling (one point). Equally, a definite diagnosis of LS in the family is assigned two points, whereas an LS-associated tumour before the age of 60 years in a first-degree, second-degree or third-degree relative of the patient is scored only one point. A LS-associated tumour in a relative that has been shown to be microsatellite stable, and hence, is very likely a sporadic tumour should not be taken into account.
According to the three-point scoring system summarised in Table 5, CMMRD syndrome should be suspected in an individual who reaches a score of minimum three points. The points should be counted by adding the points assigned to the malignancy/malignancies and those assigned to additional features. When using this system, it has to be kept in mind that several of these features listed in Table 5 may be present also in a patient with a different (childhood) cancer predisposition syndrome, like Fanconi anaemia, ataxia telangiectasia, Bloom syndrome, Peutz–Jeghers syndrome, tuberous sclerosis and others. These syndromes are usually associated with characteristic anomalies that are not listed in Table 5. Therefore, patients with signs specific for another cancer predisposition syndrome except for NF1 should first be tested for the other cancer predisposition syndrome. Patients who have clearly NF1-associated malignancies such as JMML or MPNST and signs reminiscent of NF1 should be first tested for a NF1 mutation. There is also an overlap of CMMRD with Li Fraumeni syndrome (LFS). The tumour spectrum of the latter encompasses brain tumours and, to a lesser extent, haematological neoplasms. In families fulfilling Chompret's criteria, a TP53 germline mutation should be ruled out first.
Diagnostic Steps to Substantiate the Diagnosis CMMRD
Patients reaching a score of three points should be further analysed to confirm or refute the suspected diagnosis of CMMRD syndrome. The diagnostic steps in these patients largely follow the protocols developed for LS, which involves analysis of microsatellite instability (MSI) and/or immunohistochemical (IHC) staining of the four MMR proteins.
IHC analysis to assess the expression loss of the affected MMR protein can be effectively employed in all solid tumours of CMMRD patients and has the advantage to guide subsequent mutation analysis in the four MMR genes. In general, biallelic truncating mutations in PMS2 or MSH6 will result in isolated loss of these proteins, whereas mutations in MLH1 or MSH2 will lead to concurrent loss of MLH1/PMS2 or MSH2/MSH6, respectively, since MLH1 and MSH2 are the obligatory partners in the formation of MLH1/PMS2 and MSH2/MSH6 heterodimers. Of note, in the case of an underlying missense mutation, IHC may show normal expression of the affected MMR gene, which may be a possible pitfall when using IHC analysis to confirm suspected CMMRD. In contrast to LS where expression loss is observed only in neoplastic cells, in CMMRD patients IHC detects expression loss of one (or two) of the MMR proteins in both neoplastic and non-neoplastic tissues. Hence, negative IHC staining in neoplastic and surrounding normal cells should not be interpreted as a failure of proper staining and care should be taken to use a (on slide) staining control from a different individual. In principle, expression loss of one of the MMR genes can also be demonstrated in blood lymphocytes of CMMRD patients, as shown by western blotting but possibly also by IHC staining on cytospin preparations.
MSI tests for small misalignments, that is, insertion–deletion loops (IDL), which arise during DNA replication and remain uncorrected in the absence of a functional MMR system. It takes advantage of the fact that repeated-sequence motifs consisting of units of one, two or a few more nucleotides, that is, microsatellites, are frequent targets of IDL errors that result in shortening or lengthening of these sequence motifs, a phenomenon termed MSI. In tissues derived from clonally proliferating MMR-deficient cells, that is, neoplastic cells, MSI is easily detected by PCR amplification and fragment analysis of a set of microsatellite markers. MSI analysis following the current protocols for LS uses a panel of 5–6 dinucleotide and/or mononucleotide repeat markers. This approach is a reliable tool to diagnose MMR deficiency in gastrointestinal and other LS-associated tumours of CMMRD patients. However, standard MSI analysis as applied in LS frequently fails to show MSI in brain tumours and other malignancies. Currently, the reasons for this observation are unknown, but it has been shown that more subtle shifts (shortening or lengthening) of microsatellite alleles can be observed also in brain tumours and may be indicative of CMMRD. Furthermore, it might be worth testing whether a different panel of microsatellite markers would be more sensitive in brain and other tumours of CMMRD patients.
In principle, MSI can be observed also in DNA from normal, that is, non-neoplastic, cells of CMMRD patients. However, because altered microsatellite alleles are present only in a small proportion of the cells from normal tissue, so far most approaches used technically demanding single-molecule analyses (eg, small-pool PCR) to show MSI in normal tissue, that is, germline MSI (gMSI). Recently, a much more simple method to detect gMSI was presented. This assay relies on the analysis of 'stutter' peaks typically associated with microsatellite PCR products. When quantified by a novel publicly available software application, the relative peak height of the 'stutter' peaks of selected dinucleotide microsatellites significantly increases in DNA of CMMRD patients compared with normal controls as has been confirmed also in a larger cohort of samples (Bodo et al., in preparation). 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, if this assay shows in a larger cohort of samples that it is a reliable, simple and rapid tool to detect CMMRD at least in patients with PMS2, MLH1 or MSH2 mutations, it would be a good screening tool and an alternative to IHC in cases where appropriate tissue is not available.
Taken together, both IHC staining of the MMR genes and MSI analysis are diagnostic methods to substantiate the suspected diagnosis. Since IHC will also guide target-gene mutation analysis and has been shown to render reliable results in most solid tumours, it is considered the preferred method. But as outlined, both methods have potential pitfalls and may fail to confirm the suspected diagnosis. Therefore, we recommend combining both assays if needed. The final confirmation of the diagnosis CMMRD should come from the determination of the causative biallelic mutations in the patient.
Counselling and Genetic Testing
According to the recommendations of national and international human genetic societies and the legislation of most European countries, genetic counselling must be offered to the patients and/or their parents prior to performing mutation analysis in the affected child. Patients and/or their parents should be informed by a team of paediatric oncologists and medical geneticists about the suspected diagnosis if this is substantiated by MSI and/or IHC analysis. Considering the burden of this syndrome, psychological support should systematically be proposed to families. The family has to be informed of potential therapeutic implications of the test result and also of the high risk for a second malignancy in a patient with a positive test result. Genetic counselling must also include information on the potential 25% recurrence risk in a sibling and on the risks for LS-associated cancer in possible heterozygous mutation carriers, particularly both parents.
With the informed consent of the patient and/or the parents, mutation analysis will be initiated. Reliable and robust comprehensive analysis exists for all four MMR genes now including the historically difficult PMS2 gene. However, when analysing PMS2, which is the affected gene in more than 50% of CMMRD patients, special care has to be taken to avoid pitfalls that arise from high prevalence of hybrid alleles that result from sequence exchange of the functional gene with its pseudogene PMS2CL.
Preferentially, targeted gene mutation analysis is performed. However, in cases where tumour tissue is not available for IHC analysis or the results are inconclusive, mutation analysis of all four MMR genes can be considered. It is expected that with the implementation of next-generation sequencing techniques this may be possible at reasonable costs in many laboratories in the near future.