Erzsébet Csernák

Human papillomavirus in head and neck cancer: Molecular biology and clinicopathological correlations

Human papillomaviruses are known to cause cancers of the cervix and other anogenital tract sites. Epidemiologic and molecular pathology studies have also suggested that HPV infection may be associated with cancers of the head and neck. Modes of transmission of HPV infection in the head and neck region have not been fully resolved; however, perinatal transmission and an association between sexual behavior and risk for HPV-positive cancers have been presented.Among the HPV types infecting the mucosa, high-risk, intermediate-risk and low-risk genotypes are defined, depending on their presence in carcinoma or precursor lesions. The phylogenic groups of HPVs also showed a definite correlation with the morphology of head and neck tumors. The groups A6, A7, and A9 include viruses that are frequently demonstrated in basaloid and verrucosus squamous cell carcinomas known to associate with HPV infection. Integration of HPV DNA into the host cell genome occurs early in cancer development and is an important event in malignant transformation.There is a trend for patients with HPV-positive tumors to be nondrinkers or light drinkers, the majority of these patients are females, and the median age is lower than in the case of HPV-negative tumors, but this latter difference was not always statistically significant. In the Kaplan-Meier survival model, the HPV-positive verrucous and basaloid squamous cell carcinomas showed better survival rates than the HPV-negative typical squamous cell carcinomas. An increased radiocurability of HPV-positive head and neck squamous cell carcinoma (HNSCC) has also been demonstrated.

DETECTION OF N-MYC GENE AMPLIFICATION IN NEUROBLASTOMA BY COMPARATIVE, IN SITU,AND REAL-TIME POLYMERASE CHAIN REACTION

We have used semiquantitative comparative and real-time quantitative polymerase chain reactions (PCR) to detect n-myc gene-amplification in 20 frozen neuroblastoma biopsies and IMR 32 cell line to predict biological behavior of the tumors. Two primer pairs were used for the semiquantitative method to co-amplify a 520-bp fragment of the beta-globin gene--used as a single copy reference standard--and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analyses were performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labeled fluorescent probe pair to detect the product. Calibration curve, set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was used for quantitative analysis. The semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, whereas quantitative LightCycler analysis was able to detect even 2-fold amplification. Differentiated neuroblastomas seldom show n-myc amplification. In spite of this, we have found two partly differentiated tumor samples that contained n-myc amplification. In these cases in situ PCRs were performed to examine the tumor heterogeneity. We used biotinated ATP labeling and the same primer pair as for the LightCycler analysis. In both cases differentiated cells did not show n-myc gene amplification, whereas considerable amplification was detected in the neuroblasts.

DNA content heterogeneity in neuroblastoma analyzed by means of image cytometry and its potential significance

The aim of this study was to determine the frequency and significance of the tumor DNA content heterogeneity in 33 previously untreated human neuroblastomas. We used image cytometry to selectively analyze neuroblasts by excluding karyorrhectic or stromal cells from cytometric measurements. DNA content heterogeneity with more than one clonal subpopulation on DNA histogram was found in 8 of 33 cases. Of these 8 cases, 4 showed MYCN amplification. Double labeling fluorescent in situ hybridization with probes for the centromeric region of chromosome 2 and MYCN gene was used to confirm the DNA content heterogeneity. DNA content heterogeneity was associated with poorer prognosis in this study (P<0.05). There was a significant correlation between euploidy (di- and tetraploidy) and worse prognosis, but only when heterogeneous neuroblastomas with euploid cell population were assigned to euploid tumors (P=0.006). Our results may explain the conflicting data in the literature regarding ploidy and suggest that DNA content heterogeneity and the presence of a euploid population may predict worse prognosis in neuroblastoma patients.

Application of Targeted Next-generation Sequencing, Truseq Custom Amplicon Assay for Molecular Pathology Diagnostics on Formalin-fixed and Paraffin-embedded Samples

The implementation of targeted therapies revolutionized oncology. As the number of new oncogenic driver mutations, which provide molecular targets for prediction of effective and selective therapies, is increasing, the implementation of fast and reliable methods by molecular pathology labs is very important. Here we report our results with TruSeq Custom Amplicon assay performed on formalin-fixed and paraffin-embedded material. The oligo capture probes targeted the hotspot regions of 10 well-known oncogenes linked to clinical diagnosis and treatment of lung and colorectal adenocarcinomas, melanomas, and gastrointestinal stromal tumors. Fifteen previously genotyped formalin-fixed and paraffin-embedded DNA samples from different tumor types were selected for massively parallel sequencing. A bioinformatics pipeline was developed to identify high-quality variants and remove sequence artifacts. With the exception of 1 sample, which was of lower quality than the others, relevant mutations corresponding to tumor types could be reliable detected by the developed bioinformatical pipeline. This study indicates that the application of TruSeq Custom Amplicon assay is a promising tool in molecular pathology diagnostics, but it is important to standardize sample processing (including fixation, isolation procedure, sample selection based on quality assessment, and rigorous variant calling) to achieve the highest success rate and avoid false results.

Renal Choriocarcinoma Gestational or Germ Cell Origin

Choriocarcinoma is a rare, highly malignant trophoblastic tumor with gestational or, rarely, germ cell origin. Primary extragenital localization is extremely rare. This report describes a choriocarcinoma case clinically mimicking a primary renal cell carcinoma with multiplex pulmonary metastases. Differentiation from a sarcomatoid renal cell carcinoma with trophoblastic differentiation and identification of the exact origin, namely gestational or germ cell origin by molecular genetic methods is of great importance as it helps determine the prognosis and the most effective therapy of the disease. The Investigator Hexaplex ESS Kit was used for DNA polymorphism studies. This showed foreign alleles in the tumor DNA that confirmed the presence of paternal DNA and the gestational origin of the tumor.

Vastagbélrák-hajlamosító DNS-szekvencia-variációk tumormentes és vastagbél-daganatos populációban Magyarországon. Az egyedi daganathajlam becslése

: Genome-wide association studies (GWAS) using population-based designs have identified many genetic loci, at which common variants can influence the risk of developing the sporadic colon cancers. These are single nucleotide polymorphisms (SNPs) located on different chromosomes, close to genes involved in cancer developing process, and the SNPs modify their functions, and as a consequence the cancer risk is increased. Our aim was to provide frequency distributions data of variable (risk) allele of six independent SNPs in patients with colorectal cancers and in control Hungarian population, predicting the increased risk effect of sequence variant of SNPs. We also investigated the frequency distribution of tumor localization between right or left half of large bowel as well as the RAS mutation status. 47 non-tumorous patients and 47 patients with colorectal cancer were given oral mucosa cells or blood samples for SNP analysis. After DNA isolation, an LC480 (Roche) type PCR instrument, asymmetric LATE PCR method and melting point analysis were used for detection of sequence variations, by the assistance of two SNP specific primers, unlabeled specific probe and intercalating fluorescent dye. Genomic frequency distribution of variable alleles of SNPs predisposed to tumor development have been investigated in colorectal cancer carrier patients and the results have been compared with the same allele frequency distribution data obtained from the non-tumorous control patients and from CEU population stored in SNPnexus data base. The homozygous risk alleles of SNPs showed a 1.5-2.3-time increase in colorectal cancer carrier patients then in control and CEU patients, but the heterozygous risk allele distribution was identical in tumorous and control population. The frequency distribution of homozygous risk alleles of six SNPs was also investigated in the same time and some patients. Among 47 patients with colorectal cancer, in 3 patients carrying 3 SNPs with homozygous risk alleles, in additional 5 tumor samples two and 24 samples contain only one SNPs homozygous risk alleles, and in 15 patients, SNPs with homozygous risk alleles do not occur. In 47 control patients, only 3 samples contain two SNPs with homozygous risk alleles and 17 samples contain only one SNP with homozygous risk alleles. Significant differences of the tumorous and the control population can be seen detected. NRAS mutation was not found. Our results showed a real increased risk effect of several newly recognized low-penetrance colorectal cancer susceptibility genetic variants by influence of the regulation of neighboring genes, however, the degree of cancer risk is individual, and influenced by others environmental factors, such as dietary factors. Orv Hetil. 2018; 159(40): 1614-1623.

Mixed Ovarian Tumor Composed of Brenner Tumor and Adult-Type Granulosa Cell Tumor: A Case Report of a Very Rare Mixed Ovarian Tumor and a Review of the Literature:

A combination of ovarian tumors with the same histogenetic origin but different histologic subtype is relatively common, whereas a co-occurrence of tumors with different histogenetic origin is rare. We report a case of mixed ovarian tumor composed of Brenner tumor and adult-type granulosa cell tumor, a combination that to the best of our knowledge has not been reported in the literature until now.

The mutational frequency of BRAF and KRAS in low grade serous testicular neoplasms - a case series.

Low‐grade serous neoplasms of the testis are rare neoplasms that show striking morphological similarities with the better‐understood ovarian neoplasms. This study is to see if there are similar molecular abnormalities in these two tumours. The cell of origin, relationship with serous ovarian tumour and the pathogenesis of these neoplasms are not fully established.