Gábor Sobel

Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.

RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for β globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives’ yields for various size real-time PCR amplicons. Longer amplicons (above ∼225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.

Changes of Cell Adhesion and Extracellular Matrix (ECM) Components in Cervical Intraepithelial Neoplasia

Cell-cell and cell-extracellular matrix interaction is crucial in tumor progression. Tight junction (TJ) proteins as occludin and claudins (CLDNs) play important role in this process together with several extracellular matrix components, as syndecan. Our previous work suggested significant changes in the expression of claudins even in the early stages of cervical carcinogenesis. The aim of our present work was to study the expression of occludin and syndecan-1, as compared to CLDNs, in early phases of cervical carcinogenesis. Paraffin sections of 50 samples were studied by immunohistochemistry, including cervical intraepithelial neoplasias (CIN-I-II-III), in situ carcinomas (CIS) and normal cervical samples. Occludin and CLDN-2 were found colocalized in the basal layer, while syndecan-1 and CLDN-1, -4 and -7 were coexpressed in the parabasal and intermedier layers in normal epithelia. Intensity of occludin staining decreased in CIN/CIS lesions, although it was more extended towards the upper epithelial layers with inverse relation with grades, as seen in the case of CLDN -2 expression. CLDN -1, -2, -4 and -7 were detected in the entire epithelium in CIN, showing decrease in CIS. The progression of CIN was associated with reduced syndecan-1 expression, in contrast to CLDN -1, -4 and -7 which increased toward CIS. The obtained data suggest that significant changes occur in the composition of cell adhesion complexes even in early stages of cervical carcinogenesis. The pattern of expression is characteristic for the alteration, the changes in the different components, however, are not parallel with each other.

Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression.

BackgroundComparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors.Preliminary densitometry analysis of laminin-1, α-smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts.MethodsIn vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored.ResultsWhile normal fibroblasts produced components of interstitial matrix and TGF-β1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α6β4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells.ConclusionsOur results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix.

Prenatal Diagnosis of a Giant Congenital Primary Cerebral Hemangiopericytoma

Congenital primary intracraniai hemangiopericytomas are exceptionally rare tumors. We present a case of a fetus, with the prenatal sonogram at 33 weeks of gestation revealing a large cerebral tumor. Because of the enlarged head, a cesarean section was performed. The tumor was confirmed by postnatal ultrasound, magnetic resonance imaging (MRI) and biopsy. Elevated intracraniai pressure and hemorrhage led to death on the 11th day. Autopsy revealed a 10×9 cm large inhomogeneous tumor located centrally, mainly in the posterior fossa. Histology showed a hypercellular and hypervascular tumor with extended necrosis and high mitotic rate. The tumor cells were positive for vimentin and CD34 antigens and negative for several neurological markers, desmin and CD31. The diagnosis of a congenital primary cerebral hemangiopericytoma was confirmed.

Pleomorphic rhabdomyosarcoma of the uterus in a postmenopausal patient

Pure rhabdomyosarcomas occurring in the adult uterus are very rare, with poor prognosis. We present a case of a 67-year-old woman with postmenopausal vaginal bleeding caused by pleomorphic rhabdomyosarcoma of the uterus, treated with hysterectomy, bilateral salpingooophorectomy, pelvic/paraaortic lymphadenectomy and partial sigmoidectomy. Postoperative chemotherapy (Doxorubicin) was given according to protocol. Followup examinations one year after surgery revealed no abnormalities or tumor recurrence. The rarity of this histological entity makes the presented case worthy of publication.(Pathology Oncology Research Vol 12, No 2, 102–104)

Hepatitis viruses and hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is among the most frequent malignancies worldwide. Hepatitis viruses, such as the hepatitis B virus (HBV) and hepatitis C virus (HCV) are undoubtedly listed in the etiology of HCC. Studies show that, in the near future, viral hepatitis will carry increasing weight in the etiology of HCC. This review briefly discusses the known carcinogenic effects of HBV and HCV in the light of experimental and human studies. The data show that viral proteins may directly interfere with gene products responsible for cell proliferation and cell growth. Many other signal transduction cascades may be affected as well. Direct integration of HBV viral sequences into the host genome increases the genomic instability. The genomic imbalance allows the development and survival of malignant clones bearing defected genomic information. HBV and HCV infection induces indirect and direct mechanisms through cellular damage, increased regeneration and cell proliferation, therefore enhancing the development of HCC.

Performance of a new HPV and biomarker assay in the management of hrHPV positive women: Subanalysis of the ongoing multicenter TRACE clinical trial (n > 6,000) to evaluate POU4F3 methylation as a potential biomarker of cervical precancer and cancer

The ongoing Triage and Risk Assessment of Cervical Precancer by Epigenetic Biomarker (TRACE) prospective, multicenter study aimed to provide a clinical evaluation of the CONFIDENCE™ assay, which comprises a human papillomavirus (HPV) DNA and a human epigenetic biomarker test. Between 2013 and 2015 over 6,000 women aged 18 or older were recruited in Hungary. Liquid‐based cytology (LBC), high‐risk HPV (hrHPV) DNA detection and single target host gene methylation test of the promoter sequence of the POU4F3 gene by quantitative methylation‐specific polymerase chain reaction (PCR) were performed from the same liquid‐based cytology sample. The current analysis is focused on the baseline cross‐sectional clinical results of 5,384 LBC samples collected from subjects aged 25 years or older. The performance of the CONFIDENCE HPV™ test was found to be comparable to the cobas® HPV test with good agreement. When applying the CONFIDENCE Marker™ test alone in hrHPV positives, it showed significantly higher sensitivity with matching specificity compared to LBC‐based triage. For CIN3+ histological endpoint in the age group of 25–65 and 30–65, the methylation test of POU4F3 achieved relative sensitivities of 1.74 (95% CI: 1.25–2.33) and 1.64 (95% CI: 1.08–2.27), respectively, after verification bias adjustment. On the basis of our findings, POU4F3 methylation as a triage test of hrHPV positives appears to be a noteworthy method. We can reasonably assume that its quantitative nature offers the potential for a more objective and discriminative risk assessment tool in the prevention and diagnostics of high‐grade cervical intraepithelial neoplasia (CIN) lesions and cervical cancer.

Performance of a New HPV and Biomarker Assay in Management of hrHPV Positive Women

The ongoing Triage and Risk Assessment of Cervical Precancer by Epigenetic Biomarker (TRACE) prospective, multicenter study aimed to provide a clinical evaluation of the CONFIDENCE™ assay, which comprises a human papillomavirus (HPV) DNA and a human epigenetic biomarker test. Between 2013 and 2015 over 6,000 women aged 18 or older were recruited in Hungary. Liquid‐based cytology (LBC), high‐risk HPV (hrHPV) DNA detection and single target host gene methylation test of the promoter sequence of the POU4F3 gene by quantitative methylation‐specific polymerase chain reaction (PCR) were performed from the same liquid‐based cytology sample. The current analysis is focused on the baseline cross‐sectional clinical results of 5,384 LBC samples collected from subjects aged 25 years or older. The performance of the CONFIDENCE HPV™ test was found to be comparable to the cobas® HPV test with good agreement. When applying the CONFIDENCE Marker™ test alone in hrHPV positives, it showed significantly higher sensitivity with matching specificity compared to LBC‐based triage. For CIN3+ histological endpoint in the age group of 25–65 and 30–65, the methylation test of POU4F3 achieved relative sensitivities of 1.74 (95% CI: 1.25–2.33) and 1.64 (95% CI: 1.08–2.27), respectively, after verification bias adjustment. On the basis of our findings, POU4F3 methylation as a triage test of hrHPV positives appears to be a noteworthy method. We can reasonably assume that its quantitative nature offers the potential for a more objective and discriminative risk assessment tool in the prevention and diagnostics of high‐grade cervical intraepithelial neoplasia (CIN) lesions and cervical cancer.

Women’s knowledge about cervical cancer

The human papillomavirus-infection is one of the most frequent sexually transmitted disease; it is detectable in nearly all cases of cervical cancer. Nowadays, the incidence of cervical cancer is unacceptable high. Aim: Our aim was to evaluate women’s knowledge about the human papillomaviruses and cervical cancer. We tried to determine the possible connection between the higher mortality rates, the low participation rate of screenings and the knowledge about cervical cancer. Methods: The related questionnaire contained 18 questions and was fi lled in by 422 women in certain cities and villages. The completed questionnaires were classifi ed by age, place of residence, qualifi cation, children in family, and human papillomavirus vaccination in anamnesis. Results: The results showed that almost half of the women had a basic knowledge, but the number of correct answers to functional questions (e.g. „How can one decrease the risk of infection?”) were less than it was expected according to the international literature. 56% of the women knew that the cervical cancer mainly caused by viruses, but only 17% of them named the right combination of the risk factors. The rate of correct answers was much lower in high school circles and 42% of the women knew that males can be infected by human papillomaviruses. Only 44% of them participate on cervical cancer screening once a year and 43% of them thought that cervical cancer and precancerous lesions do not mean serious risk and danger. 80% of the women knew that screening involves smear taking. The signifi cance of knowledge level differences between groups was estimated by χ²-probe. Conclusions: On the basis of the results, half of the women said to be familiar with the basic questions. In our opinion, it can be a benefi cial consequence of educational campaigns. Although there were several issues, which were implemented by not more than 20% of correct answers.

Dysregulation of microRNA Expression in Human Cervical Preneoplastic and Neoplastic Lesions

Data discussed in recent reviews demonstrated that dysregulation of microRNA (miRNA) expression profiles occurs during cervical carcinogenesis and characteristic up- or downregulation of certain miRNAs might be used as biomarkers. The majority of altered miRNAs, however were found to be inconsistent upon comparison with cancerous and normal cervical epithelia in the discussed studies due to several reasons. The results obtained in this present review suggest the need for further investigations on miRNAs on larger sample sizes in order to indicate sensitivity and specificity by means of well defined, “unified” methods. In addition, obtaining further data on the clinical course and outcome of patients in comparison to the dysregulation of miRNA expression profile could turn miRNAs into prognostic and/or progression markers. Inhibition of overexpressed miRNAs, as suggested by some authors, might even serve as target for cancer therapy.