Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

Comparative Analysis of miRNA and EMT Markers in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignant condition in the world. Micro RNAs (miRNAs) as well as epithelial to mesenchymal transition (EMT) play an important role in the pathogenesis of CRC. We performed a comparative analysis of the expression of selected miRNA genes and EMT markers in bioptic samples from patients (n = 45) with primary CRC or metastatic (m)CRC to the regional lymph node using reverse transcription-quantitative PCR and IHC staining. Results: Out of all miRNA analyzed, the miR-17 expression was most significantly different and associated with lower risk of CRC spread to the lymph node. In addition, significant relationships were found between the tumor side localization and several miRNAs expressions (miR-9, miR-29b, miR-19a, miR-19b, miR-21, miR-106a, miR-20a and miR-17). In addition, of the examined EMT markers, only VEGFA expression correlated with tumor progression (tumor grade G2). In the examined set of patient samples and their matched healthy tissue, several specific molecular markers (miRNAs associated with EMT and tumor progression) were identified with a promising prognostic potential. Their further examination in larger patient cohorts is planned to validate the present data.

Ovarian Combined Low-grade Serous and Mesonephric-like Adenocarcinoma: Further Evidence for A Mullerian Origin of Mesonephric-like Adenocarcinoma.

Mesonephric-like adenocarcinomas are rare neoplasms occurring in the uterine corpus and ovary which bear a close morphologic resemblance to cervical mesonephric adenocarcinomas. They also have a similar immunophenotype and harbor similar molecular abnormalities to mesonephric adenocarcinomas and it is debated whether they are truly of mesonephric origin or represent Mullerian neoplasms closely mimicking mesonephric adenocarcinomas. We report an unusual case with bilateral ovarian serous borderline tumors and extraovarian low-grade serous carcinoma (invasive implants). In one ovary, there was a component of mesonephric-like adenocarcinoma. The immunophenotypes of the serous and the mesonephric-like components were distinct and as expected for the individual tumor types (serous component diffusely positive with WT1 and estrogen receptor and negative with GATA3, TTF1 and CD10; mesonephric-like component WT1 and estrogen receptor negative and GATA3, TTF1, and CD10 positive; both components diffusely positive with PAX8 and exhibiting "wild-type" p53 immunoreactivity). In all components (bilateral serous borderline tumors, low-grade serous carcinoma and mesonephric-like adenocarcinoma), an identical KRAS mutation was detected (NM_004985.4): c.35G>A, p.(G12D) proving a clonal association between the serous and mesonephric-like components and excluding a collision neoplasm. This represents the second reported case of a combined ovarian low-grade serous tumor and mesonephric-like adenocarcinoma; in the previously reported case, an identical NRAS mutation was present in both components. These 2 cases provide evidence that ovarian mesonephric-like adenocarcinomas have, at least in some cases, a Mullerian origin and differentiate along mesonephric lines. We present additional evidence for this by reviewing associated findings in published and unpublished ovarian mesonephric-like adenocarcinomas; 8 of 11 of these neoplasms contained other Mullerian lesions in the same ovary, mainly endometriosis and adenomas/adenofibromas.

[Testing of mutations in BRCA1 and BRCA2 genes in tumor tissues - possibilities and limitations]. / Vysetrení mutací genu BRCA1 a BRCA2 v nádorových tkáních - moznosti a limitace.

Development of targeted cancer therapy is accompanied by a search for markers allowing prediction of response to the particular treatment. Recently, the interest is focused, among other neoplasms, also on the therapy of ovarian cancer using new inhibitors of poly (ADP-ribose) polymerase (PARP) proteins, nuclear enzymes involved in the repair of single-stranded DNA breaks. The greatest benefit from the administration of PARP inhibitors have patients with a deleterious or potentially deleterious germ-line or somatic mutation of BRCA1 or BRCA2, two genes responsible for repair of double stranded DNA breaks. There has been described a wide spectrum of mutations of BRCA 1/2, from point substitutions to large deletions, including sometimes even several exons of the gene.Unlike the testing of germ-line mutations provided for many years by the medical geneticists, somatic mutations in the tumor tissue have not been routinely tested so far. Detection of BRCA1/2 mutations in the tumor is significantly different from testing of germ-line mutations. In comparison with the analysis of DNA isolated from blood samples, testing of DNA isolated from the FFPE tissue encounters challenges based on heterogeneous representation of tumor cells in the tissue samples, on the presence of multiple neoplastic clones and on the infiltration of tissue by the non-neoplastic elements, as well as difficulties caused by variable proportion of apoptotic and necrotic cells deteriorating the overall quality of isolated DNA.Regarding the testing methods, NGS appears to be the optimal choice because of its complexity, speed of implementation into routine diagnostics as well as sensitivity for detection of a BRCA 1/2 mutations. When introduced into everyday laboratory practice, the functioning quality control system is of utmost importance. Provided there is a high probability of detection of the so far unreported variations in BRCA 1/2 genes, an introduction of a shared database of somatic variants diagnosed in the Czech Republic would be of enormous benefit.

Hypersensitivity to chemoradiation in FANCA carrier with cervical carcinoma-A case report and review of the literature.

OBJECTIVE: Compared to Fanconi anemia (FA) patients with homozygous defective two-alleles inheritance, there is a scarce or no evidence on one defective allele FANCA carriers, with respect to their cancer incidence, clinical and in vitro radiosensitivity and chemosensitivity. On that account, we report a case of a 30-year old FANCA mutation carrier woman with uterine cervix adenocarcinoma who was treated with chemoradiotherapy, in which unexpected acute toxicity and fatal late morbidity occured. METHODS: We also report the results of an in vitro test for radiosensitivity, immunohistochemical examination with FANCA staining and human papillomavirus genotypization, and a review of the literature for FA carrier patients with respect to cancer incidence, clinical and in vitro response to chemo/radiotherapy, options of early heterozygosity detection, and methods of in vitro prediction of hypersensitivity to oncologic treatment. CONCLUSION: Although there are no standard guidelines for management of FA carriers with malignancies and reports about chemo- or radiosensitivity in this population are scarce; patients with FA-A heterozygosity may have a high rate of complications from chemo/radiotherapy. Up to now, an optimum method for the prediction of radiosensitivity and the best parameter has not been found. Clinical radioresponsiveness is unpredictable in FA carriers and there is a pressing need of new rapid and predictive in vitro assays of radiation responses. Until then, the treatment of FA carriers with malignancies should be individualized, with respect to potential hypersensitivity to ionizing radiation or cross-linking agents.

Prognostic Factors for Survival in Glioblastoma: A Retrospective Analysis Focused on the Role of Hemoglobin.

Background: Although several prognostic factors for survival have been identified in glioblastoma, there are numerous other potential markers (such as hemoglobin) whose role has not yet been confirmed. The aim of this study was to evaluate a wide range of potential prognostic factors, including HIF-1α and hemoglobin levels, for survival in glioblastoma. A secondary aim was to determine whether hemoglobin levels were associated with HIF-1α expression. Methods: A retrospective study of 136 patients treated for glioblastoma at our institution between 2012 and 2021 was performed. Cox univariate and multivariate analyses were carried out. Kaplan-Meier survival curves were generated. In addition, bivariate non-parametric correlation analyses were performed for key variables. Results: Median survival was 11.9 months (range: 0-119.4). According to the univariate analysis, 13 variables were significantly associated with survival: age, performance status, extent of surgery, tumor depth, tumor size, epilepsy, postoperative chemoradiotherapy, IDH mutations, CD44, HIF-1α, HIF-1ß, vimentin, and PDFGR. According to the multivariate regression analysis, only four variables remained significantly associated with survival: age, extent of surgery, epilepsy, and HIF-1α expression. No significant association was observed between hemoglobin levels (low <120 g/L in females or <140 g/L in males vs. high ≥120 or ≥140 g/L) and survival or HIF-1α/HIF-1ß expression. Conclusions: In this retrospective study of patients with glioblastoma, four variables-age, extent of surgery, HIF-1α expression, and epilepsy-were significant prognostic factors for survival. Hemoglobin levels were not significantly associated with survival or HIF-1α expression. Although hypoxia is a well-recognized component of the glioblastoma microenvironment, more research is needed to understand the pathogenesis of onset tumor hypoxia and treatment implication.

Tissue contexture determines the pattern and density of tumor-infiltrating immune cells in HPV-associated squamous cell carcinomas of oropharynx and uterine cervix.

The profile of the antitumor immune response is an important factor determining patient clinical outcome. However, the influence of the tissue contexture on the composition of the tumor microenvironments of virally induced tumors is not clearly understood. Therefore, we analyzed the immune landscape of two HPV-associated malignancies: oropharyngeal squamous cell carcinoma (OPSCC) and squamous cell carcinoma of uterine cervix (CESC). We employed multiplex immunohistochemistry and immunofluorescence to evaluate the density and spatial distribution of immune cells in retrospective cohorts of OPSCC and CESC patients. This approach was complemented by transcriptomic analysis of purified primary tumor cells and in silico analysis of publicly available RNA sequencing data. Transcriptomic analysis showed similar immune profiles in OPSCC and CESC samples. Interestingly, immunostaining of OPSCC tissues revealed high densities of immune cells in both tumor stroma and tumor epithelium, whereas CESC samples were mainly characterized by the lack of immune cells in the tumor epithelium. However, in contrast to other immune cell populations, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were abundant in both segments of CESC samples and CESC-derived tumor cells expressed markedly higher levels of the PMN-MDSC chemoattractants CXCL1, CXCL5, and CXCL6 than OPSCC tumor cells. Taken together, despite their having the same etiologic agent, the immune infiltration pattern significantly differs between OPSCC and CESC, with a noticeable shift toward prominent MDSC infiltration in the latter. Our data thus present a rationale for a diverse approach to targeted therapy in patients with HPV-associated tumors of different tissue origins.

Prognostic value of blood cell count-derived ratios in BRAF-mutated metastatic melanoma.

BACKGROUND: The treatment and prognosis of metastatic melanoma have changed during the last decade to include immunotherapy or targeted therapy as standard therapeutic options for BRAF-mutated melanoma. However, predictive and/or prognostic markers are lacking, especially in clinical situations where several options are available. The aim of this study was to determine the association of pre-therapeutic blood cell count-derived ratios (BCDR) with survival in patients with BRAF-mutated metastatic melanoma. METHODS: We evaluated the prognostic role of BCDR in therapy-naïve patients with BRAF-mutated metastatic melanoma treated with immune checkpoint inhibitors or targeted therapy. The impact of BCDR on survival was analysed using univariate and multivariate Cox proportional hazard models. RESULTS: We enrolled 46 patients treated with BRAF inhibitors and 20 patients who received anti-PD-1 checkpoint inhibitors. The median progression-free survival (PFS) and overall survival (OS) were 8.3 and 18.2 months, respectively, with no statistical difference between groups. The objective response rate was 39% (30% in the anti-PD-1 and 44% in the targeted therapy groups). Baseline BCDR values were associated with improved PFS and OS in the immunotherapy group. Only the platelet-to-lymphocyte ratio (PLR) was associated with OS and PFS in the targeted therapy group. Independent prognostic indicators for PFS were lactate dehydrogenase, PLR and the lymphocyte-to-monocyte ratio (LMR) and those for OS were LMR, toxicity and the number of initial metastases. CONCLUSION: BCDR had a substantial prognostic value in patients with BRAF-mutated metastatic melanoma treated with immune checkpoint inhibitors. However, a prognostic role for BCDR seemed less apparent in patients treated with targeted therapies.

DNA hypermethylation of CADM1, PAX5, WT1, RARß, and PAX6 genes in oropharyngeal cancer associated with human papillomavirus.

Recently, an increasing incidence of HPV-induced oropharyngeal squamous cell carcinoma (OPSCC) has been observed. Moreover, locoregionally advanced stages require a combined modal approach, and the prognosis is poor. Therefore, it is essential to find early diagnostic and prognostic biomarkers. DNA methylation changes play a crucial role in the process of carcinogenesis and are often investigated as promising biomarkers in many types of cancer. For analysis of DNA methylation levels of selected tumour suppressor genes in HPV-positive and HPV-negative samples (including primary tumours and corresponding metastases of metastasizing OPSCCs, primary tumours of non-metastasizing OPSCCs, and control samples), methylation-specific MLPA and methylation-specific high-resolution melting analyses were used. A significant difference in methylation between OPSCCs and the control group was observed in WT1, PAX6 (P < 0.01) and CADM1, RARß (P < 0.05) genes. CADM1 and WT1 hypermethylation was detected mostly in HPV-positive samples; all but one HPV-negative samples were unmethylated. Moreover, hypermethylation of PAX5 gene was observed in metastases compared with control samples and was also associated with shorter overall survival of all patients (P < 0.05). Associations described herein between promoter methylation of selected genes and clinicopathological data could benefit OPSCC patients in the future by improvement in screening, early detection, and prognosis of the disease.

Calcifying pseudoneoplasm of neuroaxis (CAPNON): a comprehensive immunohistochemical and morphological characterization of five cases.

Calcifying pseudoneoplasm of neuroaxis (CAPNON) is a rare lesion of the central nervous system with uncertain histogenesis. We further explored phenotypic spectrum of the entity with respect to possible histogenesis. We collected 5 cases of CAPNONs, performed a detailed morphological assessment, and performed an extensive immunohistochemical analysis (EMA, progesterone receptors, MUC4, SSTR2A, cytokeratin AE1/3, cytokeratin 18, GFAP, neurofilaments, desmin, nestin, synaptophysin, S100 protein, SOX10, CD56, Podoplanin, SATB2, ERG, CD45, and CD163) to elucidate the histogenesis. Furthermore, we performed NGS analysis of one case. The clinical course was benign in all cases. All lesions showed extensively calcified matrix in multilobular arrangement, with a palisade of osteoblast-like cells. Characteristic fibrohyaline matrix was notable in 4/5 cases, while one case was myxoid with rod-like calcifications. Metaplastic lamellar bone was present in 4/5 cases and psammoma bodies were present in 2/5 cases. In 4/5 cases, areas of entrapped glial tissue were present. Expression of EMA was focally present in 3/5 cases, SSTR2A and nestin in 2/5 cases, and progesterone receptor in 2/5 cases in rare cells. We did not observe concomitant expression of EMA, SSTR2A, and progesterone receptor in the same cellular subsets. In one case, NGS showed multiple chromosomal alterations and missense mutation in PIK3CA, attributable to the admixed meningothelial population compatible with meningioma. In another case, biphasic proliferation with myoepithelial phenotype was present. The lesions showed no lineage-specific immunoprofile. Additional pathology was identified in two cases, furthermore suggestive of a possible reactive origin of the lesion.

Role of N-Cadherin in Epithelial-to-Mesenchymal Transition and Chemosensitivity of Colon Carcinoma Cells.

(1) Background: N-cadherin expression, epithelial-to-mesenchymal transition (EMT) and aggressive biological phenotype of tumor cells are linked although the underlying mechanisms are not entirely clear. (2) Methods: In this study, we used two different in vitro cell models with varying N-cadherin expression (stabilized lines and primocultures) and investigated their select biological features including the degree of their chemoresistance both in vitro as well as in vivo. (3) Results: We report that although enforced N-cadherin expression changes select morphological and behavioral characteristics of exposed cells, it fails to successfully reprogram cells to the aggressive, chemoresistant phenotype both in vitro as well as in vivo as verified by implantation of those cells into athymic mice. Conversely, primocultures of patient-colonic cells with naturally high levels of N-cadherin expression show fully aggressive and chemoresistant phenotype pertinent to EMT (in vitro and in vivo), with a potential to develop new mutations and in the presence of dysregulated regulatory pathways as represented by investigated miRNA profiles. (4) Conclusions: The presented results bring new facts concerning the functional axis of N-cadherin expression and related biological features of colon cancer cells and highlight colon cancer primocultures as a useful model for such studies.

[KRAS mutation testing in therapeutic algorithm for treatment of metastatic colorectal carcinoma]. / Vysetrení mutacního statutu genu KRAS jako soucást algoritmu lécby metastatického kolorektálního karcinomu.

Targeted therapy has become an integral part of treatment procedures of malignant tumors. Colorectal carcinomas are frequently targeted with monoclonal anti-EGFR antibodies (cetuximab and panitumumab). Activating somatic mutations in codons 12 and 13 of the exon 2 of KRAS gene are considered negative predictive factors of response to anti-EGFR therapy in patients with metastatic colorectal cancer. In the Czech Republic, evaluation of mutational status of KRAS gene is performed in several referral laboratories. In 2009, these laboratories performed 2580 tests of the KRAS mutational status--out of these, 60.2% cases reported non-mutated, wild-type KRAS. In one of the referral laboratories, we demonstrate the logistics of KRAS testing procedure. Stratification of patients with metastatic colorectal tumors based on their KRAS mutational status has evolved to a standard procedure. Laboratories performing these methods shall therefore adhere to the recommendations of the professional and accredited societies.

MicroRNA Deregulation in Papillary Thyroid Cancer and its Relationship With BRAF V600E Mutation.

BACKGROUND: MicroRNAs (miRNAs) are non-coding regulatory molecules 18-25 nucleotides in length that act as post-transcriptional regulators of gene expression. MiRNAs affect various biological processes including carcinogenesis. Deregulation of miRNAa expression has been described in a variety of tumors including papillary thyroid carcinoma (PTC). The aim of the present study was to investigate the role of selected miRNAs in PTC and find associations between miRNA expression and the BRAF (V600E) mutation. MATERIALS AND METHODS: The study group comprised a total of 62 patients with surgically treated PTC. The control group consisted of 30 patients with nodular goitre that were surgically treated in the same time period. The expression status of miR-146b, miR-181a, miR-187, miR-221 and miR-222 was determined using quantitative real-time PCR. BRAF mutation analysis was performed by PCR with reverse hybridization. RESULTS: MiR-146b, miR-181a, miR-187, miR-221 and miR-222 were up-regulated in PTC compared to normal thyroid gland tissue of the same patient. MiR-146b, miR-187, miR-221 and miR-222 were also up-regulated in PTC compared to nodular goitre. The recurrent tumors were statistically significantly associated with up-regulation of miR-221. The mutation V600E of BRAF gene was significantly associated with up-regulation of miR-146b and with down-regulation of miR-187. CONCLUSION: Over-expression of selected miRNAs in PTC compared to normal thyroid gland tissue and nodular goitre was found. Moreover, miR-221 may serve as a prognostic marker as its over-expression was significantly associated with recurrent tumors.

SMARCA4-Deficient Carcinoma of Uterine Cervix Resembling SCCOHT-Case Report.

Small cell carcinoma of hypercalcemic type (SCCOHT) is a rare gynaecological neoplasm, originating mostly in the ovaries. Cervical origin of this very aggressive malignancy with unknown histogenesis is an extremely rare condition, without published management recommendations. Alterations in SMARCA4 gene are supposed to play the major role in SCCOHT oncogenesis and their identification is crucial for the diagnosis. Adequate genetic counselling of the patients and their families seems to be of great importance. Optimal management and treatment approaches are not known yet but may extremely influence the prognosis of young female patients that suffer from this very resistant disease. Nowadays, a translational research seems to be the key for the further diagnostic and treatment strategies of SCCOHT. The purpose of the case report is to provide practical information and useful recommendations on the diagnosis, management, and treatment of SMARCA4-deficient carcinoma of the uterine cervix resembling SCCOHT.

Presence of Chlamydia trachomatis DNA in the amniotic fluid in women with preterm prelabor rupture of membranes.

OBJECTIVE: The primary aim of this study was to assess the rate and load of amniotic fluid Chlamydia trachomatis DNA and their associations with intra-amniotic infection and intra-uterine inflammatory complications in women with preterm prelabor rupture of membranes (PPROM). The secondary aim was to assess the short-term morbidity of newborns from PPROM pregnancies complicated by amniotic fluid C. trachomatis DNA. METHODS: A retrospective study of 788 women with singleton pregnancies complicated by PPROM between 24 + 0 and 36 + 6 weeks of gestation was performed. Transabdominal amniocenteses were performed at the time of admission. C. trachomatis DNA in the amniotic fluid was assessed by real-time polymerase chain reaction using a commercial AmpliSens® C. trachomatis/Ureaplasma/Mycoplasma hominis-FRT kit, and the level of Ct DNA was quantified. RESULTS: Amniotic fluid C. trachomatis DNA complicated 2% (16/788) of the PPROM pregnancies and was present in very low loads (median 57 copies DNA/mL). In addition to amniotic fluid C. trachomatis DNA, other bacteria were detected in 62% (10/16) of the C. trachomatis DNA-complicated PPROM pregnancies. Amniotic fluid C. trachomatis DNA was associated with intra-amniotic infection, histologic chorioamnionitis (HCA), and funisitis in 31%, 47%, and 33%, respectively. The presence of C. trachomatis DNA accompanied by Ureaplasma species in the amniotic fluid was associated with a higher rate of HCA than the presence of amniotic fluid C. trachomatis DNA alone. The composite neonatal morbidity in newborns from PPROM pregnancies with amniotic fluid C. trachomatis DNA was 31%. CONCLUSION: The presence of C. trachomatis DNA in the amniotic fluid is a relatively rare condition in PPROM. Amniotic fluid C. trachomatis DNA in PPROM is not related to intensive intra-amniotic and intr-auterine inflammatory responses or adverse short-term neonatal outcomes.

Non-conventional mucosal lesions (serrated epithelial change, villous hypermucinous change) are frequent in patients with inflammatory bowel disease-results of molecular and immunohistochemical single institutional study.

Chronically inflamed mucosa in inflammatory bowel disease (IBD) is associated with an increased risk of cancer. Besides IBD-associated dysplasia, there are non-conventional mucosal changes that may act as potential precursors. The aim of the study was to retrospectively review samples from IBD patients focusing on detection of such lesions with evaluation of their immunohistochemical and molecular properties. Surgical specimens and/or endoscopical biopsy samples of IBD patients examined during a 10-year period were reviewed. Detected mucosal lesions were divided into three groups-group 1 (non-conventional or putative precursor lesions - PPLs) with serrated or villous hypermucinous morphology, group 2 (true serrated polyps fulfilling valid criteria), and group 3 (IBD-associated neoplasia). Lesions from all groups were analyzed with antibodies against MLH1, p53, and MGMT and by molecular testing of KRAS, NRAS, and BRAF mutation. Samples from 309 IBD patients were reviewed. A total of 88 mucosal lesions were found in 51 patients. Most common were lesions from group 1 with superficial serrated epithelial change seen in 41 samples (46.6%) and villous hypermucinous change in 6 (6.8%). Group 2 consisted of 15 true serrated polyps. Six conventional IBD-dysplasia cases and 11 carcinomas were seen in group 3. Six lesions from group 1 were associated with invasive carcinoma whereas two shared the same mutation in KRAS or BRAF. Lesions from group 1 were characterized by loss of MGMT expression in 44.6%, aberrant p53 expression, and by mutations in KRAS gene in 42.9% of cases. This study proves the existence of mucosal changes other than conventional IBD-dysplasia and extends the knowledge about their immunohistochemical and molecular properties and relation to carcinoma further supporting their potential role in IBD-related carcinogenesis.

Morphological, immunohistochemical and molecular features of inflammatory bowel disease associated colorectal carcinoma and associated mucosal lesions - Single institution experience.

BACKGROUND: Patients with inflammatory bowel disease (IBD) - ulcerative colitis (UC) and Crohn's disease (CD) have an elevated risk of developing colorectal carcinoma (CRC). Major risk factor in IBD patients is the continuous chronic inflammation leading to development of dysplasia and carcinoma. Nevertheless, other types of non-conventional but suspicious mucosal changes serrated change/dysplasia, NOS and villous hypermucinous change, have also been reported in IBD patients. Preneoplastic potential of these lesions is still not well elucidated. AIMS: The aim of this study was identification of IBD-associated CRCs focusing on finding related precursor lesions in the surgical specimen or in archival biopsy samples followed by a detailed morphological, immunohistochemical and molecular evaluation. For the purpose of the study the mucosal lesions were divided into conventional IBD-associated dysplasia and non-conventional lesions that were merged under a provisory term of putative preneoplastic lesions (PPL). METHODS: A total of 309 consecutive IBD colectomy specimens diagnosed during a 10-year period were reviewed. Detailed morphological evaluation, immunohistochemical analysis of mismatch repair (MMR) proteins, p53 and O6-methylguanine DNA methyltransferase (MGMT) expression and molecular analysis for KRAS, NRAS and BRAF gene mutation were performed in the retrieved CRC cases as well as in the detected dysplasia and PPLs of these patients. RESULTS: We identified 11 cases of morphologically heterogenous IBD-associated CRCs, occurring in 5 males and 6 females, aged 26-79 years (mean 44 years). A total of 22 mucosal lesions were revealed in 8 CRC patients comprising conventional IBD-associated dysplasia (4 lesions), PPLs as serrated change/dysplasia NOS (11 lesions), villous hypermucinous change (5 lesions), and two true serrated lesions (one sessile serrated adenoma and one traditional serrated adenoma). More than one type of lesion was found in 6 patients. Seven CRC cases harbored mutation of KRAS/NRAS and one case of BRAF. Two patients with KRAS-mutated CRC showed the same mutation in PPL in the same specimen (one serrated change NOS and one TSA with high-grade dysplasia). Similarly, one BRAF-mutated carcinoma case presented the same mutation in serrated change/dysplasia, NOS in the same specimen. Of the CRCs, two showed deficient MMR system profile, six presented with loss of MGMT expression, and six showed aberrant p53 expression. PPLs showed deficient MGMT expression (14 cases) and aberrant p53 (10 cases) as well. CONCLUSION: IBD-associated CRCs are very heterogeneous entities. Besides conventional IBD-related dysplasia, other types of mucosal lesions may be associated with long lasting IBD and CRC e.g. villous hypermucinous change and serrated change/dysplasia, NOS. Since these lesions share certain genetic or immunohistochemical changes with the related CRC, a suspicion is raised that these lesions may also have preneoplastic potential. Awareness of these changes is necessary to prevent their missing and under-reporting, and further studies of these lesions should be carried out.

Tumor-infiltrating B cells affect the progression of oropharyngeal squamous cell carcinoma via cell-to-cell interactions with CD8+ T cells.

BACKGROUND: Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, a better stratification of patients and the development of novel therapeutic protocols are crucially needed. The importance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC. METHODS: We utilized transcriptomic analysis of immune response-related genes in 18 OPSCC samples with respect to human papillomavirus (HPV) status. The density and localization of CD20+, CD8+ and DC-LAMP+ cells were subsequently analyzed in 72 tissue sections of primary OPSCC samples in relation to patients' prognosis. The immunohistochemical approach was supplemented by flow cytometry-based analysis of phenotype and functionality of TIL-Bs in freshly resected primary OPSCC tissues. RESULTS: We observed significantly higher expression of B cell-related genes and higher densities of CD20+ B cells in HPV-associated OPSCC samples. Interestingly, CD20+ TIL-Bs and CD8+ T cells formed non-organized aggregates with interacting cells within the tumor tissue. The densities of both intraepithelial CD20+ B cells and B cell/CD8+ T cell interactions showed prognostic significance, which surpassed HPV positivity and CD8+ TIL density in stratification of OPSCC patients. High density of TIL-Bs was associated with an activated B cell phenotype, high CXCL9 production and high levels of tumor-infiltrating CD8+ T cells. Importantly, the abundance of direct B cell/CD8+ T cell interactions positively correlated with the frequency of HPV16-specific CD8+ T cells, whereas the absence of B cells in tumor-derived cell cultures markedly reduced CD8+ T cell survival. CONCLUSIONS: Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can predict patients with excellent prognosis, who would benefit from less invasive treatment. We propose that in extensively infiltrated tumors, TIL-Bs might recruit CD8+ T cells via CXCL9 and due to a highly activated phenotype contribute by secondary costimulation to the maintenance of CD8+ T cells in the tumor microenvironment.

High-risk human papillomavirus infection and p16INK4a protein expression in laryngeal lesions.

A total of 88 samples of laryngeal lesions (23 vocal cord nodules (VCNs), 23 papillomas (PAs), 18 dysplasias (DYs), and 24 carcinomas (CAs)) were analyzed for p16INK4a protein (p16) expression by immunohistochemistry and for high-risk human papillomavirus (HR-HPV) infection using chromogene in situ hybridization (CISH) and polymerase chain reaction (PCR). The series comprised 62 males and 26 females, aged 1-87 years (median 55 years). p16 expression was detected in 2 of 23 (9%) VCNs, 18 of 23 (78%) PAs, 9 of 18 (50%) DYs, and 14 of 24 (58%) CAs. Using CISH, HR-HPV DNA was detected in 3 of 23 (13%) VCNs, in 19 of 23 (83%) PAs, in 12 of 18 (67%) DYs, and in 14 of 24 (58%) CAs. HR-HPV DNA was found in six of nine (67%) PAs by PCR. A statistically significant difference in p16 expression and HR-HPV DNA presence detected by CISH was observed between VCNs and PAs (p<0.000001). The sensitivity and specificity of p16 expression for HR-HPV DNA presence detected by CISH was 0.612 and 0.773, respectively. Our study confirms a potential role of HR-HPV infection not only in the pathogenesis of malignant, but also in benign laryngeal lesions.

Cervical human papillomavirus infection in women with preterm prelabor rupture of membranes.

OBJECTIVE: To evaluate the association between cervical human papillomavirus (HPV) infection at the time of admission and the presence of microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation (IAI) in women with preterm prelabor rupture of membranes (PPROM) and to determine the association between cervical HPV infection and short-term neonatal morbidity. METHODS: One hundred women with singleton pregnancies complicated by PPROM between the gestational ages of 24+0 and 36+6 weeks were included in the study. The presence of HPV DNA was evaluated in scraped cervical cells using polymerase chain reaction (PCR). Amniotic fluid samples were obtained by transabdominal amniocentesis. RESULTS: The rate of cervical HPV infection in women with PPROM was 24%. The rates of MIAC and IAI were not different between women with cervical HPV infection and those without cervical HPV infection [MIAC: with HPV: 21% (5/24) vs. without HPV: 22% (17/76), p = 1.00; IAI: with HPV: 21% (5/24) vs. without HPV: 18% (14/76), p = 0.77]. There were no differences in the selected aspects of short-term neonatal morbidity between women with and without cervical HPV infection. CONCLUSIONS: In women with PPROM, the presence of cervical HPV infection at the time of admission is not related to a higher risk of intra-amniotic infection-related and inflammatory complications or worse short-term neonatal outcomes.