Fine-needle biopsy of thyroid nodules and the contribution of molecular analysis of BRAF and RAS mutations.

BACKGROUND: Thyroid cancer is the most common endocrine malignancy. There is a significant overdiagnosis of thyroid carcinomas that would never clinically manifest, with consequent unnecessary surgical treatment. The fine-needle biopsy and subsequent cytologic examination is of crucial importance in the differential diagnosis of thyroid nodules. On the other hand, a significant portion of the results are indeterminate. OBJECTIVE: To assess the relationship of BRAF/RAS mutations in biopsy specimens to histological characteristics of thyroid nodules in individuals who undergone fine-needle biopsy and surgery. METHODS: This cross-sectional study involves 170 subjects with indeterminate cytology analyzed for BRAF/RAS mutations. RESULTS: Of all 170 patients with indeterminate cytological finding, 103 were indicated for surgery. Of these, 31 were BRAF and 25 RAS positive. Thyroid cancer was diagnosed in 59 patients, while 44 patients had non-malignant thyroid lesions. The BRAF V600E mutation was detected in 30 patients, and the RAS (K-RAS, N-RAS, and H-RAS) mutation in 13 patients with thyroid cancer. In all BRAF-positive nodules, thyroid cancer was histologically confirmed. This means a 100 % positive predictive value of BRAF testing in our study. CONCLUSION: Stratification of thyroid lesions with uncertain results of fine-needle cytology using genetic markers can help to deliver more tailored medical treatment (Tab. 6, Ref. 19).

Case Report: The Role of Molecular Analysis of the MUTYH Gene in Asymptomatic Individuals.

MUTYH-associated polyposis (MAP) is a rare hereditary condition caused by the biallelic mutation in the MUTYH gene encoding MUTYH glycosylase. This enzyme is a key member of the base excision repair (BER) pathway responsible for the repair of DNA lesions formed by reactive oxygen species (ROS). We report two cases of MAP. In case 1, a 67-year-old woman who presented with a personal history of colorectal and endometrial cancer and a family history of cancer syndromes underwent multigene panel testing that revealed a germline homozygous (biallelic) pathogenic variant c.1187G > A (p.Gly396Asp) in the MUTYH gene. Subsequent sequencing analysis performed in the offspring of the proband identified all three asymptomatic offspring as carriers of this pathogenic variant. In case 2, a 40-year-old woman with a strong family history of colorectal cancer [the proband's sister was a carrier of the pathogenic variant c.536A > G (p.Tyr179Cys) of the MUTYH gene] and renal cancer underwent sequencing analysis of the MUTYH gene. The pathogenic heterozygous (monoallelic) variant c.536A > G (p.Tyr179Cys) of the MUTYH gene was identified in the proband. We found another pathogenic variant of the MUTYH gene-heterozygous (monoallelic) mutation c.1187G > A (p.Gly396Asp) in the genome of the proband's husband. Molecular analysis of their offspring revealed that they are compound heterozygotes for MUTYH pathogenic variants c.536A > G (p.Tyr179Cys)/c.1187G > A (p.Gly396Asp). This paper shows the importance of genetic testing of asymptomatic relatives of the proband to ensure an early surveillance and management of individuals positive for pathogenic variant (s) in the MUTYH gene.

[Molecular pathology of endometrial carcinoma - a review]. / Molekulová patológia endometriálneho karcinómu - prehlad.

Endometrial carcinoma (EC) is the most common malignancy of the female genital tract in developed countries. According to its histomorphologic characteristics EC is divided into endometroid and serous carcinoma; among less common subtypes there are clear cell, mucinous, neuroendocrine and undifferentiated carcinoma and carcinosarcoma. Endometroid and serous EC were essential for the so-called dual classification of EC (type I and type II), which considered mainly epidemiological, clinical and endocrine characteristics. It was shown that part of the high-grade serous carcinomas (type II) can develop from the endometroid EC by a multiplication of genomic changes and there are also EC, in which both basic types are overlapping. Today it is known that clinical and histological presentation of the EC reflects the genetic and epigenetic alterations affecting mainly PTEN, PIK3CA, KRAS, CTNNB1 and TP53 genes, or leading to microsatellite instability. However, these changes are variably present in both types of EC; therefore dual division of EC has appeared very rigid. The novel classifications should represent an integrated system which also incorporates the results of the gene expression analyses and multiparallel DNA sequencing. Based on these findings EC were divided into four molecular categories: a) POLE/ultra mutated; b) hyper mutated microsatellite instable; c) "copy number low" d) "copy number high" serous-like carcinoma. This division better reflects the biological characteristics of each EC and represents a base for the individual therapy.

Comprehensive genetic characterization of hereditary breast/ovarian cancer families from Slovakia.

Germline mutations in the BRCA1/2 genes account for the majority of hereditary breast ovarian cancer (HBOC). Identification of causal mutations may have significant impact on clinical management of such families. Despite high mutation detection rate, many HBOC cases remain without identified cause. These cases warrant use of several analysis methods, such as those for large genomic rearrangements and DNA copy number changes, or analysis other genes, shown to be associated with increased HBOC risk. We assessed 585 Slovak HBOC for the presence of mutations in BRCA genes. Sequencing revealed mutations in 100 families, representing 17.1% (88 and 12% of mutations were located in BRCA1 and BRCA2, respectively). Four of the mutations, c.80+4del4, c.1938_1947del10 and c.1166delG in BRCA1 and c.6589delA in BRCA2 gene have been described only in Slovak population. Using MLPA analysis, we detected two large genomic rearrangements in three families, a deletion of exons 21 and 22, and a rare deletion of a whole BRCA1 gene. Twenty-seven different variants of uncertain clinical effect (four novel) and 14 distinct SNP BRCA1 haplotypes were detected. Their potential effect was considered using the prediction software packages Align-GVGD, Pmut and Polyphen. We observed that the best clinical criterion for the initiation of BRCA1 analysis is the presence of breast cancer at 40 years of age in the association with the presence of ovarian cancer diagnosed around the age of 50. Conversely, the best clinical criterion for starting with BRCA2 analysis is the presence of breast cancer diagnosed in older age (above 50), or the presence of breast cancer in conjunction with carcinomas at different sites e.g., prostate, colorectum, ovary and uterus. Finally we have seen that the analyses of other HBOC risk gene TP53 and specific mutation in CHEK2*c.1100delC in Slovak HBOC families were not efficient since no mutations were found in these genes.

Identification of a novel mutations BRCA1*c.80 + 3del4 and BRCA2*c.6589delA in Slovak HBOC families.

Mutations in the BRCA1 and BRCA2 genes account for the majority of hereditary breast ovarian cancer (HBOC) cases. However, after BRCA1 and BRCA2 screening still the most HBOC cases remain negative for any mutational event. Accordingly, in these cases raises the relevance to analyze the unusual BRCA1/2 variants of uncertain clinical significance. Complex RNA/cDNA analysis may constitute the solution and help to interpret the HBOC syndrome in the family. In our study we analyzed the novel, to our knowledge, not yet published mutations identified in Slovak HBOC families, c.80 + 3del4 (IVS2 + 3delAGTC) in BRCA1 gene and mutation c.6589delA (6817delA) in BRCA2 gene. To determine the effect of the BRCA1 mutation, we applied different approaches: segregation analysis of mutation with disease, presence in the set of unaffected controls and finally RNA/cDNA BRCA1 analysis. Novel BRCA2 mutation was determined performing direct sequencing analysis. In conclusion, considering the results from all used techniques we approved the mentioned mutations as seriously pathogenic and disease causing with clear effect on the onset of HBOC syndrome.

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers.

BACKGROUND: Depending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts. METHODS: The main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes. RESULTS: We found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region. CONCLUSION: LGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.

Multiplex SNaPshot genotyping for detecting loss of heterozygosity in the mismatch-repair genes MLH1 and MSH2 in microsatellite-unstable tumors.

BACKGROUND: In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously. METHODS: We used the Applied Biosystems SNaPshot Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the method's diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification. RESULTS: The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2). CONCLUSIONS: Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.

[The first molecular analysis of a Hungarian HNPCC family: a novel MSH2 germline mutation]. / Az elsó magyar hereditaer nem polypoticus colorectalis carcinoma család molekuláris vizsgálata: az MSH2 gén uj mutációja.

BACKGROUND: Hereditary nonpolyposis colorectal cancer is an inherited disease characterized by onset at an early age, an excess of synchronous and metachronous large bowel tumors and a variety of extracolorectal malignancies. Basal and squamous cell carcinomas of the skin are not customarily included in the tumor spectrum of the syndrome. The disease is caused by a germline mutation in one of the DNA mismatch repair genes, most commonly MSH2 or MLH1, and typically presents with microsatellite instability and frequent loss of mismatch repair protein expression in the tumor tissue. PATIENT: The case of a 62-year old woman who had a history of colon cancer at the age of 46 years, endometrial cancer at the age of 56 years, baso-squamous, and squamous cell cancer of the face at the ages of 53, 54, 62 and 58 years, respectively, and rectal cancer at 60 is reported. Her family fulfills the Amsterdam criteria for the diagnosis of hereditary nonpolyposis colorectal cancer. The baso-squamous cell, the squamous cell, the endometrial and the rectal cancers were assessed for the microsatellite instability status and the expression of the MSH2 and MLH1 mismatch repair proteins, and the p53 tumor suppressor protein by immunohistochemistry. Mutational screening using an automated capillary DNA sequencer was performed by the direct genomic sequencing of 17 fragments of the MSH2 gene, which covers promoter, all exons and flanking intronic regions. RESULTS: All cancers displayed microsatellite instability and were positive for the p53 protein. The immunohistochemical staining in the baso-squamous cell, the squamous cell, the rectal and endometrial cancers were negative for MSH2 and positive for MLH1 proteins. DNA sequencing analysis revealed a mutation c.2292G > A in exon 14 of the MSH2 gene, which is altering the 764. amino acid, the tryptophan to STOP codon (p.W764X). Thus the MSH2 protein is presumably truncated by 171 aminoacids. CONCLUSION: To the best of authors' knowledge, this is the first molecular characterization of a Hungarian hereditary nonpolyposis colorectal cancer family. According to the Human Mutation Database and International Collaborative Group of HNPCC Database, this mutation is novel, has not been reported previously. Cutaneous baso-squamous and squamous cell cancers may present as part of the HNPCC phenotype. Detection of the loss of mismatch repair protein expression and mismatch repair gene mutation mapping, represents a significant improvement of the diagnosis of this syndrome in Hungary. These examinations identify the mutation carriers who are at an increased risk of developing cancers.