Slavko Gašparov

HER2 in Gastric Cancer: An Immunohistochemical Study on Tissue Microarrays and the Coressponding Whole-Tissue Sections with a Supplemental Fish Study

Since focal HER2 expression is an issue in GC, TMA construction from the paraffin-embedded surgically-obtained tissue may not reflect its real status. The aim of this study was to assess the HER2 status in tissue microarrays (TMAs) and the corresponding whole sections using HercepTest immunohistochemistry (IHC), and to correlate it and to assess the concordance of HER2 IHC and fluorescence in situ hybridization (FISH) in TMAs. Concordance of the HER2 expression status for 302 cases of gastric cancer using 9 paired TMAs was evaluated using a 2-mm core size and 305 corresponding whole sections. Concordance of the IHC and FISH HER2 status was compared. In addition,, the HER2 status was compared to clinicopathological characteristics and patients’ survival. Using the whole-section approach, HER2 over-expression was found in 25.2xa0% (HER2 3+ 6.6xa0%, HER2 2+ 18.7xa0%) of tumours. The overall concordance of IHC between the cores and the whole section was 84.9xa0%; 15.1xa0% of the tumours showed HER2 amplification. The overall concordance of IHC and FISH on cores was 75.7xa0%. The level of amplification correlated with the IHC score. Relationship between the intestinal and papillary types and tumour grade was observed for tumours with over-expression and amplification, whereas tumour location was related only to over-expression. There was a statistically significant difference in the overall survival of the patients, which was related to HER2 amplification. In conclusion, good concordance of the IHC HER2 results between tissue cores in TMA and whole sections, and excellent concordance of the IHC and FISH results on tissue cores was found. At least a part of the observed IHC HER2 heterogeneity could very likely be explained by fixation artifacts. With adequate fixation, a higher concordance of IHC HER2 between the cores and the whole sections can be expected. The TMA approach could enable an easier analysis of more than one representative tumour block.

Expression of Secreted Frizzled-Related Protein 1 and 3, T-cell Factor 1 and Lymphoid Enhancer Factor 1 in Clear Cell Renal Cell Carcinoma

Frequency and mortality of renal cell carcinoma (RCC) are increasing for decades. However, the molecular background of RCC tumorigenesis is still poorly understood. In current study we investigated the expression of TCF/LEF and SFRP family members (SFRP1 and SFRP3) to gain a better understanding of biological signaling pathways responsible for epidemiology and clinical parameters of clear cell RCC (cRCC). Thirty-six pairs of paraffin-embedded clear cRCC and adjacent nontumoral tissues samples using immunohistochemistry (IHC) were analyzed and compared with corresponding clinicopathological parameters. Immunohistochemistry indicated statistically significant decreased SFRP3 expression in tumor tissues but no consistency in SFRP1 expression in analyzed normal and tumor tissue. The TCF1 expression level was significantly weaker in normal tissue compared to tumor samples while LEF1 protein levels were significantly weaker in tumor tissue. To our knowledge, this is the first report on analysis of the expression of transcription factors TCF1 and LEF1 in clear cell renal cell carcinoma and their comparison with Wnt signal pathway antagonists belonging to SFRP family.

Bcl-2 and MALT1 genes are not involved in the oncogenesis of uterine tumors resembling ovarian sex cord tumors

Uterine tumors resembling ovarian sex cord tumors (UTROSCT) are rare entities. They were described by Clement and Scully in 1976 who classified them into groups I and II. Group I comprises typical endometrial stromal neoplasms with focal areas resembling ovarian sex cord elements and group II are predominantly or completely composed of ovarian sex cord-like elements. We report a case of UTROSCT type II with cytogenetic analysis. The tumor occurred in a 76-year-old woman who presented with vaginal bleeding. The tumor was lobulated, firm, yellow and histologically composed of sex cord-like elements. Tumor cells expressed vimentin, CD10, CD99 and alpha-actin. Cytogenetic analysis in a previously reported case detected translocation t(4;18)(q21.1;q21.3) in the majority of cells. Bcl-2 and MALT1 genes are located at or near the translocation breakpoints, and the aim of this study was to determine whether these genes were involved in chromosomal translocation or tumorigenesis. We did not find IgH translocation or the most common MALT translocations. Bcl-2 was also not involved in this oncogenesis.

Unlocking cancer glycomes from histopathological formalin-fixed and paraffin-embedded (FFPE) tissue microdissections

N- and O-glycans are attractive clinical biomarkers as glycosylation changes in response to diseases. The limited availability of defined clinical specimens impedes glyco-biomarker identification and validation in large patient cohorts. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are the common form of sample preservation in clinical pathology, but qualitative and quantitative N- and O-glycomics of such samples has not been feasible to date. Here, we report a highly sensitive and glycan isomer selective method for simultaneous N- and O-glycomics from histopathological slides. As few as 2000 cells isolated from FFPE tissue sections by laser capture microdissection were sufficient for in-depth histopathology-glycomics using porous graphitized carbon nanoLC ESI-MS/MS. N- and O-glycan profiles were similar between unstained and hematoxylin and eosin stained FFPE samples but differed slightly compared with fresh tissue. This method provides the key to unlock glyco-biomarker information from FFPE histopathological tissues archived in pathology laboratories worldwide.

Monoclonality in Helicobacter pylori-positive gastric biopsies: an early detection of mucosa-associated lymphoid tissue lymphoma.

Mucosa-associated lymphoid tissue (MALT) is not present in healthy gastric mucosa, but it can develop in sites of long-persisting inflammation and is connected with the development of MALT lymphoma. A monoclonal lymphocyte population is one of the characteristics of such lymphomas. In this study we analyzed gastric biopsies (formalin fixed and paraffin embedded or frozen) in 93 patients with dyspepsia accompanied by Helicobacter pylori infection. We applied PCR and single-cell immunocytochemistry to detect the clonality of the gastric B-cell population. Immunocytochemistry performed on 33 frozen biopsies showed two samples with monoclonal pattern. PCR analysis of immunoglobulin heavy-chain (IgH) gene rearrangements revealed two monoclonal populations out of 161 biopsies from 60 patients. We conclude that PCR analysis was the most sensitive method, which gave us insight into the nature of the earliest stage of MALT lymphoma in gastric biopsies.

Is quadrant biopsy sufficient in men likely to have advanced prostate cancer? Comparison with extended biopsy

We hypothesized that quadrant prostate biopsy (QPB) provides sufficient first-line pathological evaluation of patients with presumed advanced prostate cancer (PC). The aim of this study was to investigate whether the reduction of core number in first-line PB from 6–12 to 4 in patients with presumed advanced PC leads to loss of clinically relevant information. We retrospectively studied 113 men that underwent PB, classified in two groups: “H” (high) and “L” (low likelihood of having advanced PC), according to PSA, digital rectal and transrectal ultrasound findings. Pathological results of 6–12-core PB and QPB were retrospectively compared for the presence of malignancy, percentage of positive cores, Gleason score (GS), and the presence of high-grade prostatic intraepithelial neoplasia (HGPIN). PC detection rate was not impaired in group H but dropped significantly in group L, and the percentage of positive cores was not significantly changed in group H (p=0.39), but decreased in group L (p=0.04), due to sampling scheme reduction. No HGPIN was missed with QPB in group H, while 2 HGPINs were missed in group L. No significant change in GS in either group was observed (p=0.12, p=0.13) due to reduction to QPB. We conclude that in patients with presumed advanced PC, reduction of the number of cores in PB may be an acceptable diagnostic strategy, but further studies are needed to analyze the impact of PB scheme reduction on other relevant pathological information obtained from PB.

Clinical Significance of VEGF-A and Microvessel Density in Diffuse Large B-Cell Lymphoma and Low-Grade Follicular Lymphoma

Angiogenesis is essential for the development, growth and progression of tumors. Although vascular endothelial growth factor (VEGF) is a well-known proangiogenic factor, its impact on lymphoma has not yet been fully clarified. The aim of this study was to evaluate VEGF-A -expression and microvessel density (MVD) in aggressive lymphoma such as diffuse large B-cell lymphoma (DLBCL), in indolent lymphomas such as low-grade follicular lymphoma (FL), and in lymph node reactive follicular hyperplasia (FH). In 80 prospective and retrospective cases (30 DLBCL, 30 FL and 20 FH), CD31 was analyzed by immunohistochemical staining assessing density of blood vessels, as well as the total number of CD31 positive endothelial cells. The results were compared with relevant clinical data. MVD was 85% in FH, followed by 60% in DLBCL and 43% in low-grade FL. VEGF-A was significantly higher in DLBCL than in low-grade FL and FH. A statistically significant association of MVD and VEGF-A with the International Prognostic Index (IPI) was found in DLBCL. High MVD and VEGF-A expression was observed in DLBCL patients with high IPI, while there was no statistically significant association between MVD and VEGF-A with the Follicular Lymphoma International Prognostic Index in low-grade FL. Our results suggested an important relationship between angiogenesis and high-grade lymphoma.

Is it possible to overcome antiapoptotic API2/MALT1 events in tumor B-cells by influencing Tregs in MALT lymphoma?

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) comprises approximately 50% of primary gastric lymphoma. Proliferation of tumor cells infected with Helicobacter pylori is facilitated by the presence of T cells activated by H. pylori antigens. Unlike the majority of MALT lymphomas, tumors bearing the t(11;18)(q21;q21) resulting in production of a chimeric protein API2/MALT1 are often resistant to H. pylori eradication therapy, and require more aggressive therapeutic approach including chemotherapy. The authors hypothesize that a subset of patients with translocation-positive MALT lymphoma might benefit from a novel therapeutic approach that would address intercellular communication pathways between various cell types in the tumor microenvironment. A subset of T cells called regulatory T cells (Tregs) are one of the major immunomodulators of antitumor response mechanisms. There are several potential tools that could have a substantial impact on this particular T cell population, such as interleukin (IL)-15, indoleamine 2,3-dioxigenase (IDO), anti-CD25 antibodies. Introducing some of these components into treatment protocols for patients with API2/MALT1 translocation-positive MALT lymphomas might also prove to be benefitial for other lymphomas with increased number of intratumoral Tregs.