Denisa Ilencikova

Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium ‘Care for CMMRD’ (C4CMMRD)

Constitutional mismatch repair deficiency (CMMRD) syndrome is a distinct childhood cancer predisposition syndrome that results from biallelic germline mutations in one of the four MMR genes, MLH1, MSH2, MSH6 or PMS2. The tumour spectrum is very broad, including mainly haematological, brain and intestinal tract tumours. Patients show a variety of non-malignant features that are indicative of CMMRD. However, currently no criteria that should entail diagnostic evaluation of CMMRD exist. We present a three-point scoring system for the suspected diagnosis CMMRD in a paediatric/young adult cancer patient. Tumours highly specific for CMMRD syndrome are assigned three points, malignancies overrepresented in CMMRD two points and all other malignancies one point. According to their specificity for CMMRD and their frequency in the general population, additional features are weighted with 1–2 points. They include multiple hyperpigmented and hypopigmented skin areas, brain malformations, pilomatricomas, a second childhood malignancy, a Lynch syndrome (LS)-associated tumour in a relative and parental consanguinity. According to the scoring system, CMMRD should be suspected in any cancer patient who reaches a minimum of three points by adding the points of the malignancy and the additional features. The diagnostic steps to confirm or refute the suspected diagnosis are outlined. We expect that application of the suggested strategy for CMMRD diagnosis will increase the number of patients being identified at the time when they develop their first tumour. This will allow adjustment of the treatment modalities, offering surveillance strategies for second malignancies and appropriate counselling of the entire family.

Human MSH6 Deficiency Is Associated with Impaired Antibody Maturation

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.

Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents

BACKGROUND & AIMS Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors in repetitive DNA sequences. We investigated whether these features could be used to identify patients with CMMRD. METHODS We examined MSI by PCR analysis and tolerance to methylating or thiopurine agents (functional characteristics of MMR-deficient tumor cells) in lymphoblastoid cells (LCs) from 3 patients with CMMRD and 5 individuals with MMR-proficient LCs (controls). Using these assays, we defined experimental parameters that allowed discrimination of a series of 14 patients with CMMRD from 52 controls (training set). We then used the same parameters to assess 23 patients with clinical but not genetic features of CMMRD. RESULTS In the training set, we identified parameters, based on MSI and LC tolerance to methylation, that detected patients with CMMRD vs controls with 100% sensitivity and 100% specificity. Among 23 patients suspected of having CMMRD, 6 had MSI and LC tolerance to methylation (CMMRD highly probable), 15 had neither MSI nor LC tolerance to methylation (unlikely to have CMMRD), and 2 were considered doubtful for CMMRD based on having only 1 of the 2 features. CONCLUSION The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients.

High-grade brain tumors in siblings with biallelic MSH6 mutations†‡

Biallelic germline mutations of Constitutional mismatch repair‐deficiency syndrome (CMMR‐D) genes, MLH1, MSH2, MSH6, and PMS2 are characterized by increased risk of childhood malignancy. We report a case with CMMR‐D caused by novel homozygous MSH6 mutations leading to gliomatosis cerebri and T‐ALL in an 11‐year‐old female and glioblastoma multiforme in her 10‐year‐old brother, both with rapid progression of the diseases. A literature review on brain tumors in CMMR‐D families shows that they are treatment‐resistant and lead to early death. Identification of patients with CMMR‐D is critical, and specific cancer screening programs with early surgery are recommended. Pediatr Blood Cancer 2011; 57: 1067–1070.

Evaluation of 2-year experience with EGFR mutation analysis of small diagnostic samples.

Mutation analysis of the epidermal growth factor receptor (EGFR) gene is an essential part of the diagnostic algorithm in patients with metastatic or recurrent non–small cell lung cancer (NSCLC). Small biopsies or cytology specimens represent >80% of the available diagnostic material. EGFR mutation analyses were realized on 835 samples (675 cytology specimens, 151 formalin-fixed paraffin-embedded blocks, 5 tumors, and 4 pleural effusions). EGFR mutation analysis was performed by high-resolution melting analysis in combination with mutant-enriched polymerase chain reaction and sequencing analysis. Because of increased risk of inaccuracy in histology diagnosis of small specimens, all subtypes of NSCLC were analyzed. EGFR mutations were detected in 83 cases (10%). EGFR mutation testing failed in 5% (42/835) and was associated with poor cellularity, low percentage of tumor cells, and bad quality of DNA. Although 281 samples were evaluated as insufficient material (poor cellularity and/or unrepresentative tumor content), mutation rates were 7%. Although only adenocarcinomas or NSCLC-not otherwise specified are recommended for EGFR mutation testing, EGFR mutations in 11% of the large cell carcinomas and 4% of the squamous cell carcinomas were observed. Our results indicate that defined algorithm for EGFR testing of small diagnostic samples is sensitive, fast, and suitable even for samples with poor cellularity. The results of this testing should be evaluated depending on tumor content and DNA quality for each sample individually. At the conclusion of our results, we recommend to realize EGFR mutation analysis of small diagnostic samples regardless of the histologic subtypes of NSCLC.

Rapid and Efficient Detection of EGFR Mutations in Problematic Cytologic Specimens by High-Resolution Melting Analysis

AbstractBackground and Objective: Chemotherapy for advanced non-small-cell lung cancer (NSCLC) remains marginally effective, with a 5-year overall survival rate of approximately 5%. Recently, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib was approved in Slovakia for the treatment of metastatic NSCLC. Gefitinib is a selective EGFR inhibitor that binds to the adenosine triphosphate binding pocket of the kinase domain and blocks downstream signaling pathways. Mutations of the EGFR gene, particularly an in-frame 15 bp deletion (delE746_A750) in exon 19 and the L858R mutation in exon 21, correlate with enhanced clinical responsiveness to EGFR tyrosine kinase inhibitors. However, the detection of these mutations and thereby prediction of the therapy outcome is sometimes unreliable due to the low sensitivity of direct sequencing if the proportion of tumor cells in the tissue is less than 25%. Therefore we decided to test the applicability of other methods, particularly high-resolution melting analysis (HRMA), for detection of these mutations in clinical samples. Methods: We analyzed 53 archival cytologic specimens for the presence of EGFR mutations, using the HRMA method. Results were verified by direct sequencing. For samples containing less than 25% tumor cells, we used mutant-enriched PCR before sequencing. We also performed a titration assay to establish the lower limit of the proportion of tumor cells for detection of EGFR mutations. Results: EGFR mutations were detected in 13 cases (24%). In-frame deletions in exon 19 were detected in eight cases (15%) and the L858R mutation in exon 21 was detected in five cases (9%). The positive results of the HRMA were confirmed by direct sequencing only in five of 13 cases. In the remaining eight positive samples, HRMA results were confirmed by sequencing analysis after mutant-DNA enrichment. The titration assay established that the lower limit for detection of EGFR mutations by HMRA was 1% tumor cells in the clinical sample. Conclusion: Our results indicated that HRMA in combination with mutant-enriched PCR represents a sensitive method for detection of EGFR mutations from cytologic specimens. When properly executed, this protocol allows identification of EGFR mutations in specimens containing a minimal percentage of tumor cells.

New Mutations Associated with Rasopathies in a Central European Population and Genotype–Phenotype Correlations

We performed the genetic analysis of Rasopathy syndromes in patients from Central European by direct sequencing followed by next generation sequencing of genes associated with Rasopathies. All 51 patients harboured the typical features of Rasopathy syndromes. Thirty‐five mutations were identified in the examined patients (22 in PTPN11, two in SOS1, one in RIT1, one in SHOC2, two in HRAS, three in BRAF, two in MAP2K1 and two in the NF1 gene). Two of them (p.Gly392Glu in the BRAF gene and p.Gln164Lys in the MAP2K1 gene) were novel with a potentially pathogenic effect on the structure of these proteins.

Thirty-Nine Novel Neurofibromatosis 1 (NF1) Gene Mutations Identified in Slovak Patients

We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had café‐au‐lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1.

High Yield of Pathogenic Germline Mutations Causative or Likely Causative of the Cancer Phenotype in Selected Children with Cancer

Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer. Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: (1) intellectual disability and/or congenital anomalies, (2) multiple malignancies, (3) family history of cancer, or (4) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome. Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 (n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy (EP300-based Rubinstein–Taybi syndrome, ARID1A-based Coffin–Siris syndrome, ACTB-based Baraitser–Winter syndrome, and EZH2-based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2, which are possibly involved in genetic cancer predisposition. Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594–603. ©2018 AACR.

Novel SCN1A variants in Dravet syndrome and evaluating a wide approach of patient selection.

Voltage-gated sodium channels are essential for generation and propagation of the action potential mainly in nerve and muscle cells. Causative variants in SCN1A gene which codes the main, pore-forming subunit of the channel expressed in central nervous system are associated predominantly with Dravet syndrome (DS), as well as with generalized epilepsy with febrile seizures plus (GEFS+) making it one of the most significant epilepsy gene. Our goal was to determine whether SCN1A screening is relevant in patients with a broad range of epileptic syndromes. 52 patients diagnosed with DS, generalized epilepsy with GEFS+ or similar types of epileptic syndromes were included. Sequencing of the protein coding parts of the gene complemented with MLPA analysis was carried out. One already described nonsense variant, four novel protein truncating variants and a deletion encompassing the whole SCN1A gene were revealed, all in heterozygous state. All identified variants were found in DS patients with 85.7% sensitivity, thus supporting the role of profound SCN1A gene variants in etiology of DS phenotype. No causative variants were identified in any of non-DS epileptic patients in our cohort, suggesting a minor, but not irrelevant role for SCN1A in patients with other types of childhood epilepsy.