Andrzej Kram

Expression of ras oncogene p21 protein in relation to regional spread of human breast carcinomas.

The oncogenes most frequently detected in human tumors belong to the ras gene family (Ha‐ras, Ki‐ras, and N‐ras). These genes encode a group of closely related 21,000 dalton proteins termed p21. An immunohistochemical study of ras p21 expression was carried out on paraffin sections of 54 human breast carcinomas using monoclonal antibodies to p21. The control group consisted of ten cases of benign fibrocystic disease. The p21 expression was significantly higher in cancer cells than in epithelial cells of control specimens. No correlations, however, were observed between oncogene product expression and tumor size, histologic type, or grade. As a group, tumors with axillary lymph node metastases expressed higher levels of ras p21 than nonmetastasizing tumors. However, because of the significant overlap in individual p21 values, it is unlikely that the immunohistochemical assay for p21 could be used to predict the behavior of mammary carcinomas.

Mutational analysis of CDKN2A gene in a group of 390 larynx cancer patients

CDKN2A gene belongs to the genes involved in cell cycle regulation. When is absent or inactivated by mutation or promoter hypermethylation a cell may undertake an uncontrolled proliferation. Inactivation of CDKN2A gene is observed in many human malignancies, including larynx cancer. In this study we investigated mutations in exon 1 and exon 2 of CDKN2A gene in a large group of 390 laryngeal cancers. We found 40 different alterations (17%) and nearly half of them was not described previously. Out of these alterations two transversions in codon 108: c.322G>C (Asp108His) and c.322G>T (Asp108Tyr) as well as a G>A transition in codon 110 (Trp110X) were found more frequently (altogether: 7 cases in codon 108 and 10 cases in codon 110). This result, concerning the location of these codons in the ankyrin repeat structures, may suggest that these two codons may be critical hot-spots in larynx carcinogenesis.

A rare mutation in a rare tumor-SMARCB1-deficient malignant glomus tumor

Malignant glomus tumor (MGT), also known as glomangiosarcoma, is a rare soft tissue neoplasm (1 per 5,000 of all soft tissue neoplasms) which stems from modified epithelioid smooth muscle cells of the glomus body. At present there is no uniform treatment for MGT. The genomic landscape of soft tissue sarcoma remains only partially known. According to the 2013 WHO classification, over 50 histological subtypes of soft tissue sarcomas (STS) exist which can be distinguished based on morphology and immunohistochemical profile. Recently, Mosquera et al. found NOTCH gene family rearrangements and novel fusion genes MIR143-NOTCH2 and MIR143-NOTCH1 to be likely drivers of benign and MGTs (Mosquera et al., 2013). We report a 37-year-old patient with a MGT close to critical neurovascular structures in the neck area who has been treated by multiple surgical procedures and simultaneous-integrated boost intensity-modulated radiation therapy (Supporting Information). Facing a possible disease recurrence, we searched for underlying genomic alterations of this patient’s tumor to suggest viable treatment options. According to National Comprehensive Cancer Network guidelines for STS, we started by Sanger sequencing of BRAF, NRAS, KRAS, KIT, PDGFRA, TSC1 as well as TP53 mutation hotspots. No molecular defects were revealed in the screened genes. Thus, although the lack of TP53 alterations in the tumor could suggest a favorable outcome (Hoadley et al., 2014), we failed to obtain any therapeutic clues. Guided by the findings of Mosquera et al. we also performed fluorescence in situ hybrydization (FISH) analysis using NOTCH1 and NOTCH2 break-apart probes but did not find any rearrangements or amplifications. Finally, we performed target panel sequencing of a fresh frozen tumor and matched blood samples using COSMIC database-derived gene panel. We identified 24 somatic non-synonymous point mutations and 123 small insertions and deletions (INDELs), out of which two were located in protein coding regions. Notably, the identified alterations included a stop gain mutation C>T in exon 6 of SMARCB1 (Q244*) and a frameshift truncation in exon 9 of the same gene resulting in c.1148delC. These findings were validated with Sanger sequencing of both regions (Fig. 1). SMARCB1 Q244* is located in the highly conserved Repeat 1 region that is crucial for histone deacetylase (HDAC)-dependent transcriptional repression of Cyclin D1 promoter. C.1148delC is located in C terminal region proven to be instrumental in complete transcriptional repression as cells with mutations spanning that region presented about 50% of repression activity (Zhang et al., 2002). In order to further validate the functional effect of these mutations in the studied tumor, we performed immunohistochemical (IHC) staining that confirmed the loss of expression of the gene product of SMARCB1, as well as the tumor specific Cyclin D1 protein overexpression (Supporting Information Fig. 2). SMARCB1, known as hSNF5, INI1, BAF47, is a highly conserved core subunit of the SWF/SNF complex responsible for cell differentiation regulation, cell cycle control, and apoptosis (Roberts and Orkin, 2004). SMARCB1 inactivating mutations were initially found to be associated with malignant rhabdoid tumors (Versteege et al., 1998). Subsequently, germline and somatic SMARCB1 alterations have been reported in other tumor types, most commonly CNS, kidney, and soft tissue tumors (Agaimy, 2014). Surprisingly, SMARCB1 alterations dominate in rare tumor cases and have prevalent rhabdoid cell phenotype. Rarity of SMARCB1 defects is illustrated by the fact that whereas SWI/SNF complex mutations are suspected to be involved in about 20% of tumors (Kadoch et al., 2013) genomic profiling of over 3,000 tumors studied by TCGA showed no SMARCB1 alteration driven cases.

Frequency assessment of BRAF mutation, KRas mutation, and RASSF1A methylation in nodular goitre based on fine-needle aspiration cytology specimens

INTRODUCTION Standard pre-operative diagnosis of nodular goitre is not always conclusive. The decision about nodular goitre surgery is increasingly based on molecular methods. The aim of the study was to determine BRAF T1799A mutation and KRas proto-oncogene mutation, and the analysis of RASSF1A promoter methylation level in cytological material obtained from FNAB specimens of thyroid nodules. MATERIAL AND METHODS The study population consisted of 85 women and 12 men. The study material was genomic DNA isolated from peripheral blood and thyroid bioptates. Pyrosequencing was used for the evaluation of RASSF1 methylation level. KRas mutation was investigated with Sanger sequencing. BRAF mutation was analysed by standard methods of real-time amplification detection (real-time PCR) with the use of specific starters surrounding the mutated site. RESULTS A significant positive correlation was demonstrated between mean methylation of four CpG islands of RASSF1A gene and thyroid tumour volume and its largest diameter (p < 0.05). KRas mutation was not detected in any of the 97 patients. In 7/85 subjects (8.2%) BRAF mutation was observed. In 6/7 patients with BRAF mutation, FNAB of thyroid nodules confirmed a benign nature of the lesions; the material was non-diagnostic in one patient, and papillary thyroid cancer was diagnosed on the basis of postoperative histopathology assessment. CONCLUSIONS The results of genetic tests reported in our study indicate that the presence of BRAF mutation or higher RASSF1A methylation levels in FNAB cytology specimens of benign lesions may be useful in the assessment of oncological risk, while the evaluation of KRas proto-oncogene mutation is not a valuable test in pre-operative diagnosis of nodular goitre.

Frequency assessment of BRAF mutation, KRas mutation, and RASSF1A methylation in nodular goitre based on fine-needle aspiration cytology specimens Ocena częstości występowania mutacji genów BRAF, KRas oraz.

INTRODUCTION Standard pre-operative diagnosis of nodular goitre is not always conclusive. The decision about nodular goitre surgery is increasingly based on molecular methods. The aim of the study was to determine BRAF T1799A mutation and KRas proto-oncogene mutation, and the analysis of RASSF1A promoter methylation level in cytological material obtained from FNAB specimens of thyroid nodules. MATERIAL AND METHODS The study population consisted of 85 women and 12 men. The study material was genomic DNA isolated from peripheral blood and thyroid bioptates. Pyrosequencing was used for the evaluation of RASSF1 methylation level. KRas mutation was investigated with Sanger sequencing. BRAF mutation was analysed by standard methods of real-time amplification detection (real-time PCR) with the use of specific starters surrounding the mutated site. RESULTS A significant positive correlation was demonstrated between mean methylation of four CpG islands of RASSF1A gene and thyroid tumour volume and its largest diameter (p < 0.05). KRas mutation was not detected in any of the 97 patients. In 7/85 subjects (8.2%) BRAF mutation was observed. In 6/7 patients with BRAF mutation, FNAB of thyroid nodules confirmed a benign nature of the lesions; the material was non-diagnostic in one patient, and papillary thyroid cancer was diagnosed on the basis of postoperative histopathology assessment. CONCLUSIONS The results of genetic tests reported in our study indicate that the presence of BRAF mutation or higher RASSF1A methylation levels in FNAB cytology specimens of benign lesions may be useful in the assessment of oncological risk, while the evaluation of KRas proto-oncogene mutation is not a valuable test in pre-operative diagnosis of nodular goitre.

BRCA1/2 mutations are not a common cause of malignant melanoma in the Polish population

The association of BRCA1/2 mutations with melanoma is not completely determined; the interpretation of variants of unknown significance is also problematic. To evaluate these issues we explored the molecular basis of melanoma risk by performing whole-exome sequencing on a cohort of 96 unrelated Polish early-onset melanoma patients and targeted sequencing of BRCA1/2 genes on additional 30 melanoma patients with familial aggregation of breast and other cancers. Sequencing was performed on peripheral blood. We evaluated MutationTaster, Polyphen2, SIFT, PROVEAN algorithms, analyzed segregation with cancer disease (in both families with identified BRCA2 variants) and in one family performed LOH (based on 2 primary tumors). We found neither pathogenic mutations nor variants of unknown significance within BRCA1. We identified two BRCA2 variants of unknown significance: c.9334G>A and c.4534 C>T. Disease allele frequency was evaluated by genotyping of 1230 consecutive melanoma cases, 5000 breast cancer patients, 3500 prostate cancers and 9900 controls. Both variants were found to be absent among unselected cancer patients and healthy controls. The MutationTaster, Polyphen2 and SIFT algorithms indicate that c.9334G>A is a damaging variant. Due to lack of tumour tissue LOH analysis could not be performed for this variant. The variant segregated with the disease. The c.4534 C>T variant did not segregate with disease, there was no LOH of the variant. The c.9334G>A variant, classified as a rare variant of unknown significance, on current evidence may predisposes to cancers of the breast, prostate and melanoma. Functional studies to describe how the DNA change affects the protein function and a large multi-center study to evaluate its penetrance are required.

Founder Mutations for Early Onset Melanoma as Revealed by Whole Exome Sequencing Suggests That This is Not Associated with the Increasing Incidence of Melanoma in Poland

Purpose Germline mutations within melanoma susceptibility genes are present only in minority of melanoma patients and it is expected that additional genes will be discovered with next generation sequence technology and whole-exome sequencing (WES). Materials and Methods Herein we performed WES on a cohort of 96 unrelated Polish patients with melanoma diagnosed under the age of 40 years who all screened negative for the presence of CDKN2A variants. A replication study using a set of 1,200 melanoma patient DNA samples and similarly large series of healthy controls was undertaken. Results We selected 21 potentially deleterious variants in 20 genes (VRK1, MYCT1, DNAH14, CASC3, MS4A12, PRC1, WWOX, CARD6, EXO5, CASC3, CASP8AP2, STK33, SAMD11, CNDP2, CPNE1, EFCAB6, CABLES1, LEKR1, NUDT17, and RRP15), which were identified by WES and confirmed by Sanger sequencing for an association study. Evaluation of the allele distribution among carriers and their relatives in available family trios revealed that these variants were unlikely to account for many familial cases of melanoma. Replication study revealed no statistically significant differences between cases and controls. Conclusion Although most of the changes seemed to be neutral we could not exclude an association between variants in VRK1, CREB3L3, EXO5, and STK33 with melanoma risk.

Ocena częstości występowania mutacji genów BRAF, KRas oraz metylacji genu RASSF1A w wolu guzkowym na podstawie badania materiału cytologicznego uzyskanego drogą biopsji aspiracyjnej cienkoigłowej

INTRODUCTION Standard pre-operative diagnosis of nodular goitre is not always conclusive. The decision about nodular goitre surgery is increasingly based on molecular methods. The aim of the study was to determine BRAF T1799A mutation and KRas proto-oncogene mutation, and the analysis of RASSF1A promoter methylation level in cytological material obtained from FNAB specimens of thyroid nodules. MATERIAL AND METHODS The study population consisted of 85 women and 12 men. The study material was genomic DNA isolated from peripheral blood and thyroid bioptates. Pyrosequencing was used for the evaluation of RASSF1 methylation level. KRas mutation was investigated with Sanger sequencing. BRAF mutation was analysed by standard methods of real-time amplification detection (real-time PCR) with the use of specific starters surrounding the mutated site. RESULTS A significant positive correlation was demonstrated between mean methylation of four CpG islands of RASSF1A gene and thyroid tumour volume and its largest diameter (p < 0.05). KRas mutation was not detected in any of the 97 patients. In 7/85 subjects (8.2%) BRAF mutation was observed. In 6/7 patients with BRAF mutation, FNAB of thyroid nodules confirmed a benign nature of the lesions; the material was non-diagnostic in one patient, and papillary thyroid cancer was diagnosed on the basis of postoperative histopathology assessment. CONCLUSIONS The results of genetic tests reported in our study indicate that the presence of BRAF mutation or higher RASSF1A methylation levels in FNAB cytology specimens of benign lesions may be useful in the assessment of oncological risk, while the evaluation of KRas proto-oncogene mutation is not a valuable test in pre-operative diagnosis of nodular goitre.