We describe the case of a girl who presented numerous CALMs, bilateral axillar freckling and a plexiform neurofibroma of the right thigh (figure 1A–C), suggesting NF1,13 but no molecular screening for NF1 was performed at the time of diagnosis. No history of tumour or NF1 phenotype in relatives has been reported. She had been treated for scoliosis since the age of 4. She had an ophthalmological screening at the age of 6 which did not find neither Lisch nodules nor visual alteration, but no brain MRI was performed during childhood.

Skin features of patient 1: (A) axillar frecklings, (B) plexiform neurofibroma of the right thigh, (C) numerous café-au-lait macules.

At the age of 16, she was diagnosed with a right posterior parieto-occipital brain tumour revealed by headaches accompanied by hypoesthesia of the left upper limb. Brain MRI showed two synchronous contiguous lesions with a contrast enhancement, peripheral oedema and infiltration towards the corpus callosum. Multiple developmental venous brain anomalies were also reported but no focal area of signal intensity on T2/FLAIR was observed. A partial excision of the occipital part of the tumour was performed allowing for pathological diagnosis and somatic molecular analyses. The histopathology examination of the tissue biopsy revealed a glioblastoma with a few giant or multinucleated cells. IHC staining demonstrated expression of OLIG2, ATRX and P53 in the tumour cells. The occurrence of a high-grade glioma, a typical CMMRD-associated paediatric tumour, raised doubts about the NF1 diagnosis. Thus, immunostaining for MMR protein expression was performed and showed PMS2 loss in normal and tumour cells. Whole-tumour-exome sequencing revealed a typical pattern for CMMRD associated tumour14 with an ultra-high tumour burden (229 mut/Mb; above the threshold of 100 mut/Mb defined for ultra-hypermutator phenotype), a somatic POLE driver PV (c.857C>G p.Pro286Arg), multiple PVs in genes such as NF1 (two variants: c.2033dup p.(Ile679Aspfs*21) and c.532G>T p.(Glu178*)), PMS2 (two variants: c.695G>T p.(Gly232Val) and c.2275+1G>A), TP53, ATR and FANCA (table 1). Neither IDH1 p.Arg132His somatic mutation nor histone H3K27M or H3G34R/V somatic mutation was found. No microsatellite instability (MSI) (2.4%, ie, 202/7940) was reported according to MSI sensor (https://github.com/ding-lab/msisensor).

Molecular analysis of patient 1: germline and somatic alterations identified by RNA sequencing and whole exome sequencing of blood and tumour (glioblastoma, percentage of tumour cells: 80%)

In view of the patient’s phenotypic and tumour characteristics, constitutional genetic analyses were performed after genetic counselling. Germline genetic analysis revealed two different variants (compound heterozygous) in the PMS2 gene identified by NGS on two independent blood samples: one (c.2275+1G>A) classified as pathogenic inherited from the mother and the second (c.695G>T,p.(Gly232Val)), initially classified as VUS, inherited from the father. In addition, the germline genetic analysis also identified a PV in exon 18 of the NF1 gene (NM_001042492.3): c.2033dup, p.(Ile679Aspfs*21) (variant allele frequency (VAF): ~20% in a blood sample, 3% in a skin sample and 9% in a saliva sample) confirming the diagnosis of a mosaic NF1 associated with CMMRD in the patient (online supplemental figure S1). Cultured fibroblasts were enriched with cells with the NF1 PV (VAF=50% by Sanger sequencing).

Since one PMS2 variant was classified as VUS, we performed additional ancillary testing in order to confirm the diagnosis of CMMRD. Functional assays demonstrated that the cells of the patient displayed methylation tolerance and ex vivo MSI,11 confirming the diagnosis of CMMRD, although analyses showed clear MSI only after prolonged culture. The patient was included in the study of Gallon et al,12 her cMSI score confirmed the diagnosis of CMMRD in this patient, although she has one of the lowest cMSI score among the PMS2-associated CMMRD samples, probably due to the PMS2 missense variant causing their CMMRD syndrome. Based on these additional analyses, the c.695G>T p.(Gly232Val) PMS2 variant was finally reclassified as likely pathogenic (class 4), according to the ACMG-AMP criteria.15

After the partial surgery, the patient received adjuvant treatment combining radiotherapy to the right occipital lobe (54 Gy at the rate of 1.8 Gy per session) and immune checkpoint inhibition therapy with anti-PD1 as per the NIVOGLIO protocol (NCT04267146), without temozolomide. She had a good clinical and radiological response with a significant decrease of the residual brain tumour and, as per protocol, immunotherapy was discontinued after 1 year of treatment. She is in continuous remission 1.5 years after immunotherapy withdrawal and 2.5 years after the initial diagnosis. The identification of the CMMRD tumour predisposition syndrome enabled appropriate oncological surveillance to be proposed to this patient and her parents. Upper gastrointestinal endoscopy was normal. Colonoscopy at the age of 17 revealed a single 3 mm sessile polyp from the middle rectum, which has been excised. The pathological examination was in favour of a low-grade adenoma.

Among a series of 22 patients, we identified 2 additional patients with a NF1 mosaicism in blood DNA: c.4751dup, p.(Lys1585Glnfs*37) with VAF ~4% (100/2560 reads) for patient 2 and c.184_185dup, p.(Leu62Phefs*2) with VAF ~16% (191/1167 reads) for patient 3 (online supplemental figure S1). Patient 2 has previously been published in the C4CMMRD report on brain tumours.3 He was diagnosed with CMMRD after occurrence of T lymphoblastic lymphoma, with identification of PMS2 biallelic germline PV (c.2007–2A>G, p.?). Subsequently, during childhood, he developed a brain anaplastic astrocytoma grade III (IDH wild type with angiocentric features) with a loss of PMS2 expression by immunostaining and a high mutation load (rate=184 coding SNV/Mb). He is still alive with stable disease after a recurrence of his brain tumour treated with a further focal radiation therapy 2.5 years ago. Clinically, he presents multiple CALMs with hypopigmented skin lesions but no neurofibroma or other NF1-associated features.

Patient 3 is the brother of a boy who developed an acute leukaemia in the first years of life and a subsequent colonic adenocarcinoma, from which he died. Both brothers are carriers of compound heterozygous PMS2 PVs (c.1020_1021del p.(Arg341Alafs*23) and exons 13–15 deletion). CMMRD was identified in patient 3 prior to the occurrence of an acute B-lymphoblastic leukaemia (as young teenager) treated according to the French CAALL protocol. He is in complete remission 2 years after the end of the treatment. He presents with thoracic CALMs with segmental distribution exclusively on the right thoracic side and disseminated hypopigmented skin lesions. Like his brother, he also has multiple pilomatricomas, some of which have been surgically removed.