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This text is based on the recommendations accepted by the 4th Hungarian Consensus Conference on Breast Cancer, modified on the basis of the international consultation and conference within the frames of the Central-Eastern European Academy of Oncology. The recommendations cover non-operative, intraoperative and postoperative diagnostics, determination of prognostic and predictive markers and the content of cytology and histology reports. Furthermore, they address some specific issues such as the current status of multigene molecular markers, the role of pathologists in clinical trials and prerequisites for their involvement, and some remarks about the future., Competing Interests: Author BJ was employed by the company Medserv Kft. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cserni, Francz, Járay, Kálmán, Kovács, Krenács, Tóth, Udvarhelyi, Vass, Vörös, Krivokuca, Kajo, Kajová Macháleková and Kulka.)

Giant cell tumour of bone (GCTB) is a rare and intriguing primary bone neoplasm. Worrisome clinical features are its local destructive behaviour, its high tendency to recur after surgical therapy and its ability to create so-called benign lung metastases (lung 'plugs'). GCTB displays a complex and difficult-to-understand cell biological behaviour because of its heterogenous morphology. Recently, a driver mutation in histone H3.3 was found. This mutation is highly conserved in GCTB but can also be detected in glioblastoma. Denosumab was recently introduced as an extra option of medical treatment next to traditional surgical and in rare cases, radiotherapy. Despite these new insights, many 'old' questions about the key features of GCTB remain unanswered, such as the presence of telomeric associations (TAs), the reactivation of hTERT, and its slight genomic instability. This review summarises the recent relevant literature of histone H3.3 in relation to the GCTB-specific G34W mutation and pays specific attention to the G34W mutation in relation to the development of TAs, genomic instability, and the characteristic morphology of GCTB. As pieces of an etiogenetic puzzle, this review tries fitting all these molecular features and the unique H3.3 G34W mutation together in GCTB.

Modulated electro-hyperthermia (mEHT) is a selective cancer treatment used in human oncology complementing other therapies. During mEHT, a focused electromagnetic field (EMF) is generated within the tumor inducing cell death by thermal and nonthermal effects. Here we investigated molecular changes elicited by mEHT using multiplex methods in an aggressive, therapy-resistant triple negative breast cancer (TNBC) model. 4T1/4T07 isografts inoculated orthotopically into female BALB/c mice were treated with mEHT three to five times. mEHT induced the upregulation of the stress-related Hsp70 and cleaved caspase-3 proteins, resulting in effective inhibition of tumor growth and proliferation. Several acute stress response proteins, including protease inhibitors, coagulation and heat shock factors, and complement family members, were among the most upregulated treatment-related genes/proteins as revealed by next-generation sequencing (NGS), Nanostring and mass spectrometry (MS). pathway analysis demonstrated that several of these proteins belong to the response to stimulus pathway. Cell culture treatments confirmed that the source of these proteins was the tumor cells. The heat-shock factor inhibitor KRIBB11 reduced mEHT-induced complement factor 4 (C4) mRNA increase. In conclusion, mEHT monotherapy induced tumor growth inhibition and a complex stress response. Inhibition of this stress response is likely to enhance the effectiveness of mEHT and other cancer treatments.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vancsik, Máthé, Horváth, Várallyaly, Benedek, Bergmann, Krenács, Benyó and Balogh.)

Methotrexate (MTX) is a commonly used antimetabolite, which inhibits folate and DNA synthesis to be effective in the treatment of various malignancies. However, MTX therapy is hindered by the lack of target tumor selectivity. We have designed, synthesized and evaluated a novel glucose-methotrexate conjugate (GLU-MTX) both in vitro and in vivo, in which a cleavable linkage allows intracellular MTX release after selective uptake through glucose transporter-1 (GLUT1). GLU-MTX inhibited the growth of colorectal (DLD-1), breast (MCF-7) and lung (A427) adenocarcinomas, squamous cell carcinoma (SCC-25), osteosarcoma (MG63) cell lines, but not in WI-38 healthy fibroblasts. In tumor cells, GLU-MTX uptake increased 17-fold compared to unconjugated MTX. 4,6-O-ethylidene-α-D-glucose (EDG), a GLUT1 inhibitor, significantly interfered with GLU-MTX induced growth inhibition, suggesting a glucose-mediated drug uptake. Glu-MTX also caused significant tumor growth delay in vivo in breast cancer-bearing mice. These results show that our GLUT-MTX conjugate can be selectively uptake by a range of tumor cells to cause their significant growth inhibition in vitro, which was also confirmed in a breast cancer model in vivo. GLUT1 inhibitor EDG interfered with these effects verifying the selective drug uptake. Accordingly, GLU-MTX offers a considerable tumor selectivity and may offer cancer growth inhibition at reduced toxicity.

Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21 waf1 positive tumor cells suggested that p21 waf1 -mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.

There have been some relevant changes in the diagnosis and treatment of breast cancer to implement the updating of the 2016 recommendations made during the 3rd national consensus conference on the disease. Following a wide interdisciplinary consultation, the present recommendations have been finalized after their public discussion at the 4th Hungarian Breast Cancer Consensus Conference. The recommendations cover non-operative, intraoperative and postoperative diagnostics, the determination of prognostic and predictive markers and the content of the cytology and histology reports. Furthermore, it touches some special issues such as the current status of multigene molecular markers, the role of pathologists in clinical trials and prerequisites for their involvement, some relevant points about the future. The most important changes include the integration of the TNM 8th edition, the WHO classification of breast tumors 5th edition, the ASCO/CAP HER2 assessment guidelines from 2018, and the Yokohama terminology for cytology reporting; a more detailed text on tumor-infiltrating lymphocytes and size determination after neoadjuvant therapy and a broader discussion of molecular tests.

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p < 0.001) compared to normal samples, which was confirmed by real-time PCR and in situ hybridization. LINC00152 promoter hypomethylation detected in colorectal cancer (p < 0.01) was strongly correlated with increased LINC00152 expression (r=-0.90). Silencing of LINC00152 significantly suppressed cell growth, induced apoptosis and decreased cyclin D1 expression (p < 0.05). Whole transcriptome analysis of LINC00152-silenced cells revealed significant down-regulation of oncogenic and metastasis promoting genes (e.g. YES proto-oncogene 1, PORCN porcupine O-acyltransferase), and up-regulation of tumour suppressor genes (e.g. DKK1 dickkopf WNT signalling pathway inhibitor 1, PERP p53 apoptosis effector) (adjusted p < 0.05). Pathway analysis confirmed the LINC00152-related activation of oncogenic molecular pathways including those driven by PI3K/Akt, Ras, WNT, TP53, Notch and ErbB. Our results suggest that promoter hypomethylation related overexpression of LINC00152 can contribute to the pathogenesis of colorectal cancer by facilitating cell progression through the up-regulation of several oncogenic and metastasis promoting pathway elements.

Modulated electro-hyperthermia (mEHT) is a complementary antitumor therapy applying capacitive radiofrequency at 13.56 MHz. Here we tested the efficiency of mEHT treatment in a BALB/c mouse isograft model using the firefly luciferase-transfected triple-negative breast cancer cell line, 4T1. Tumors inoculated orthotopically were treated twice using a novel ergonomic pole electrode and an improved mEHT device (LabEHY 200) at 0.7 ± 0.3 W for 30 min. Tumors were treated one, two, or three times every 48 h. Tumor growth was followed by IVIS, caliper, and ultrasound. Tumor destruction histology and molecular changes using immunohistochemistry and RT-qPCR were also revealed. In vivo, mEHT treatment transitionally elevated Hsp70 expression in surviving cells indicating heat shock-related cell stress, while IVIS fluorescence showed a significant reduction of viable tumor cell numbers. Treated tumor centers displayed significant microscopic tumor damage with prominent signs of apoptosis, and major upregulation of cleaved/activated caspase-3-positive tumor cells. Serial sampling demonstrated substantial elevation of heat shock (Hsp70) response twelve hours after the treatment which was exhausted by twenty-four hours after treatment. Heat shock inhibitors Quercetin or KRIBB11 could synergistically amplify mEHT-induced tumor apoptosis in vitro. In conclusion, modulated electro-hyperthermia exerted a protective heat shock response as a clear sign of tumor cell stress. Exhaustion of the HSR manifested in caspase-dependent apoptotic tumor cell death and tissue damage of triple-negative breast cancer after mEHT monotherapy. Inhibiting the HSR synergistically increased the effect of mEHT. This finding has great translational potential.

Analogously to the natural selective forces in ecosystems, therapies impose selective pressure on cancer cells within tumors. Some tumor cells can adapt to this stress and are able to form resistant subpopulations, parallel with enrichment of cancer stem cell properties in the residual tumor masses. However, these therapy-resistant cells are unlikely to be sufficient for the fast tumor repopulation and regrowth by themselves. The dynamic and coordinated plasticity of residual tumor cells is essential both for the conversion of their regulatory network and for the stromal microenvironment to produce cancer supporting signals. In this nursing tissue "niche", cancer-associated fibroblasts are known to play crucial roles in developing therapy resistance and survival of residual stem-like cells. As paracrine messengers, extracellular vesicles carrying a wide range of signaling molecules with oncogenic potential, can support the escape of some tumor cells from their deadly fate. Here, we briefly overview how extracellular vesicle signaling between fibroblasts and cancer cells including cancer progenitor/stem cells may contribute to the progression, therapy resistance and recurrence of malignant tumors.

Introduction: The anti-cancer properties of high-dose intravenous ascorbic acid have been demonstrated in various malignancies. In our recent study, we tested topically applied ascorbic acid to treat basal cell carcinoma (BCC), and achieved a good clinical response., Aim: Based on these results, we decided to examine the efficacy and tolerability of high-dose intravenous ascorbic acid (IVA) for locally advanced BCC., Material and Methods: In this pilot study, patients diagnosed with locally advanced BCC who were not amenable to radiation, surgical or local therapy (no other treatment option was available at the time) received intravenous ascorbic acid (1-1.8 g/kg), in an outpatient setting, 1-3 times per week for a mean duration of 42 ±23.6 weeks. This therapy was generally well tolerated., Results: Among 4 patients who had a total of 165 (mean: 41 ±51, range: 1-114) skin lesions, 3 patients achieved stable disease and one had progressive disease. There was substantial variability in individual tumor response to therapy. With the aid of two-photon microscopy and second harmonic generation imaging techniques, alterations in collagen structure were observed between tumor nests during IVA therapy., Conclusions: Our results suggest that IVA is well tolerated in a small group of patients with extensive BCCs. However, in the era of smoothened (Smo) receptor inhibitors, it may only be considered as an adjuvant therapy in treatment-resistant cases., Competing Interests: Dr. Szipőcs holds shares in R&D Ultrafast Lasers Ltd. The remaining authors declare no conflict of interest., (Copyright: © 2019 Termedia Sp. z o. o.)

We aimed to analyze the expression of cell-cycle regulation markers - minichromosome maintenance protein 2 (MCM2), Ki-67, Cyclin-A and phosphohistone-H3 (PHH3) - in pre-treatment core-biopsy samples of breast carcinomas in correlation with known predictive and prognostic factors. Totally 52 core biopsy samples obtained prior to neoadjuvant therapy were analyzed. Immunohistochemistry was performed to analyze the expression of MCM2, Ki-67, Cyclin A and PHH3, which were correlated with the following clinicopathological parameters: clinical TNM, tumor grade, biological subtype, the presence of tumor infiltrating lymphocytes (TIL), pathological tumor response rate to the neoadjuvant therapy and patient survival. All investigated markers showed higher expression in high grade and in triple negative tumors (p < 0.01 and p < 0.05, respectively). Hormone receptor negative tumors showed significantly higher expression of Ki-67 (p < 0.01), MCM2 (p < 0.01) and Cyclin A (p < 0.01) than hormone receptor positive ones. Tumors with increased TIL showed significantly higher Ki-67 expression (p = 0.04). Pattern analysis suggested that novel cell-cycle marker-based subgrouping reveals predictive and prognostic potential. Tumors with high MCM2, Cyclin A or PHH3 expression showed significantly higher rate of pathological complete remission. Tumors with early relapse (progression-free survival ≤2 years) and shortened overall survival also show a higher rate of proliferation. Our cell cycle marker (Ki-67, MCM2, Cyclin A, PHH3) based testing could identify tumors with worse prognosis, but with a favorable response to primary systemic therapy. The pattern of cell-cycle activity could also be useful for predicting early relapse, but our findings need to be further substantiated in larger patient cohorts.

(1) Background: Successful treatment of acute kidney injury (AKI)-induced chronic kidney disease (CKD) is unresolved. We aimed to characterize the time-course of changes after contralateral nephrectomy (Nx) in a model of unilateral ischemic AKI-induced CKD with good translational utility. (2) Methods: Severe (30 min) left renal ischemia-reperfusion injury (IRI) or sham operation (S) was performed in male Naval Medical Research Institute (NMRI) mice followed by Nx or S one week later. Expression of proinflammatory, oxidative stress, injury and fibrotic markers was evaluated by RT-qPCR. (3) Results: Upon Nx, the injured kidney hardly functioned for three days, but it gradually regained function until day 14 to 21, as demonstrated by the plasma urea. Functional recovery led to a drastic reduction in inflammatory infiltration by macrophages and by decreases in macrophage chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) mRNA and most injury markers. However, without Nx, a marked upregulation of proinflammatory (TNF-α, IL-6, MCP-1 and complement-3 (C3)); oxidative stress (nuclear factor erythroid 2-related factor 2, NRF2) and fibrosis (collagen-1a1 (Col1a1) and fibronectin-1 (FN1)) genes perpetuated, and the injured kidney became completely fibrotic. Contralateral Nx delayed the development of renal failure up to 20 weeks. (4) Conclusion: Our results suggest that macrophage activation is involved in postischemic renal fibrosis, and it is drastically suppressed by contralateral nephrectomy ameliorating progression.

Our objective was to develop an electromagnetic tumor therapy device in a consortial cooperation between Semmelweis University and Oncotherm Ltd., to provide data and contribute to the development of the next generation of devices through preclinical, clinical and developmental modules via in vivo, in vitro studies, and patient treatments. Our numerous preclinical studies support the efficacy of mEHT. Clinical treatments were performed in 181 patients with inoperable and/or oligometastatic solid tumors. The protocols were developed, an international guideline was completed, and the planned steps of device development were realized. By optimizing previous selective RF techniques based on recent research findings, we can provide the most modern evidence-based treatment in the future.

Modulated electro-hyperthermia (mEHT) is a non-invasive treatment modality of cancer where electric field generated by 13.56 MHz radiofrequency can selectively accumulate in malignant tumors compared to adjacent normal tissues. This effect is based on the metabolic shift in cancer cells which upregulates glycolysis even under oxygenated conditions (Warburg effect), resulting in elevated lactate and ion concentration. The concomitant increased permittivity can induce dielectric polarization and rotational friction of dipole molecules resulting in elevated core temperature, which can be controlled at 42 °C with the treating instrument. Complementary application of loco-regional mEHT can improve the efficiency of chemo-, radio- and recently molecular targeted therapies based on increasing local perfusion and xenobiotic concentration, resolving tumor hypoxia and improved immune surveillance supported by high-fever range hyperthermia. We earlier showed that mEHT has its own tumor inhibiting/destructing effect, however, its mechanism had not been clarified. In this project we have investigated the molecular mechanism of action of mEHT treatment using in vitro and in vivo models of colorectal adenocarcinoma.

The poor prognosis of head and neck squamous cell carcinoma (HNSCC) is partly due to the lack of reliable predictive markers. Connexin 43 (Cx43) protein and its cell-communication channels have been assigned tumor suppressor functions while the anti-apoptotic Bcl-2 (B-cell lymphoma-2) protein has been associated with negative prognostic significance in cancer. This study aimed to test the role of Cx43 protein on Bcl-2 expression, tumor progression and response to taxane-based treatment in HNSCC. Human papillomavirus (HPV) negative HNSCC cell lines were tested for paclitaxel sensitivity through measuring apoptosis induction, cell viability and changes in Cx43 and Bcl-2 levels using flow cytometry, cell viability assay, immunocytochemistry and western blot. Inhibition of Cx43 expression using siRNA increased Bcl-2 protein levels in SCC25 (tongue squamous cell carcinoma) cells, while forced Cx43 expression reduced Bcl-2 levels and supported paclitaxel cytotoxicity in FaDu (hypopharynx squamous cell carcinoma) cells. In vitro results were in line with protein expression and clinicopathological features tested in tissue microarray samples of HNSCC patients. Our data demonstrate that elevated Cx43 and reduced Bcl-2 levels may indicate HNSCC sensitivity to taxane-based treatments. On the contrary, silencing of the Cx43 gene GJA1 (gap junction protein alpha-1) can result in increased Bcl-2 expression and reduced paclitaxel efficiency. Clinical tumor-based analysis also confirmed the inverse correlation between Cx43 and Bcl-2 expression.

Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21 waf1 and p27 kip . Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication.

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 μm (mean±S.D.: 1.17 ± 0.34 μm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro , HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.

Mutations of the multifunctional protein calreticulin (CALR) are recognised as one of the main driver alterations involved in the pathogenesis of Philadelphia negative myeloproliferative neoplasms (Ph - MPN) and also represent a major diagnostic criterion in the most recent World Health Organization classification of myeloid neoplasms. Nowadays, quantitative assessment of the driver mutations is gaining importance, as recent studies demonstrated the clinical relevance of the mutation load reflecting the size of the mutant clone. Here, we performed for the first time a manual and automated quantitative assessment of the CALR mutation load at protein level using CAL2, a recently developed CALR mutation specific monoclonal antibody, on a cohort of 117 patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) and compared the CALR protein mutation loads with the CALR mutation load values established by a molecular assay. Eighteen different CALR mutations were detected in the cohort of the 91 CALR mutant cases. Mutation loads of the CALR mutations were between 13% and 94% with mean value in PMF cases significantly higher than ET cases (49.94 vs 41.09; t-test, p=0.004). Cases without CALR mutation (n=26) showed no or only minimal labelling with the CAL2 antibody, while all 18 different types of CALR mutations were associated with CAL2 labelling. The CALR mutation load showed a significant correlation (p=0.03) with the occurrence of major thrombotic events, with higher mutation load in patients presenting with these complications. We report a 100% concordance between the mutation status determined by immunohistochemistry and the CALR molecular assay, and we extend the applicability of this approach to 16 rare CALR mutations previously not analysed at protein level., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Carcinomas are complex structures composed of hierarchically organized distinct cell populations such as cancer stem cells and non-stem (bulk) cancer cells. Their genetic/epigenetic makeup and the dynamic interplay between the malignant cell populations and their stromal fibroblasts are important determinants of metastatic tumor invasion. Important mediators of these interactions are the small, membrane-enclosed extracellular vesicles, in particular exosomes. Both cancer cell and fibroblast-derived exosomes carry a set of regulatory molecules, including proteins and different species of RNA, which cooperatively support metastatic tumor spread. Here, we briefly overview potential links between cancer stem cells and the exosome-mediated fibroblast-enriched metastatic niche formation to discuss their role in the promotion of tumor growth and metastatic expansion in breast carcinoma models., Competing Interests: The authors declare no competing interests.

Colorectal sessile serrated adenomas (SSA) are hypothesized to be precursor lesions of an alternative, serrated pathway of colorectal cancer, abundant in genes with aberrant promoter DNA hypermethylation. In our present pilot study, we explored DNA methylation profiles and examined selected gene mutations in SSA. Biopsy samples from patients undergoing screening colonoscopy were obtained during endoscopic examination. After DNA isolation and quality analysis, SSAs (n = 4) and healthy controls (n = 5) were chosen for further analysis. DNA methylation status of 96 candidate genes was screened by q(RT)PCR using Methyl-Profiler PCR array system. Amplicons for 12 gene mutations were sequenced by GS Junior Instrument using ligated and barcoded adaptors. Analysis of DNA methylation revealed 9 hypermethylated genes in both normal and SSA samples. 12 genes (CALCA, DKK2, GALR2, OPCML, PCDH10, SFRP1, SFRP2, SLIT3, SST, TAC1, VIM, WIF1) were hypermethylated in all SSAs and 2 additional genes (BNC1 and PDLIM4) were hypermethylated in 3 out of 4 SSAs, but in none of the normal samples. 2 SSAs exhibited BRAF mutation and synchronous MLH1 hypermethylation and were microsatellite instable by immunohistochemical analysis. Our combined mutation and DNA methylation analysis revealed that there is a common DNA methylation signature present in pre-neoplastic SSAs. This study advocates for the use of DNA methylation as a potential biomarker for the detection of SSA; however, further investigation is needed to better characterize the molecular background of these newly recognized colorectal lesions.

Although several antibodies are available for immunohistochemical detection of Ki-67, even the most commonly used MIB-1 has not been validated yet. Our aim was to compare 5 commercially available antibodies for detection of Ki-67 in terms of agreement and their ability in predicting prognosis of breast cancer. Tissue microarrays were constructed from 378 breast cancer patients' representative formalin-fixed, paraffin-embedded tumor blocks. Five antibodies were used to detect Ki-67 expression: MIB-1 using chromogenic detection and immunofluorescent-labeled MIB-1, SP-6, 30-9, poly, and B56. Semiquantitative assessment was performed by 2 pathologists independently on digitized slides. To compare the 5 antibodies, intraclass correlation and concordance correlation coefficient were used. All the antibodies but immunofluorescent-labeled MIB-1 (at 20% and 30% thresholds, P=.993 and P=.342, respectively) and B56 (at 30% threshold, P=.288) separated high- and low-risk patient groups. However, there were a significant difference (P values for all comparisons≤.005) and a moderate concordance (intraclass correlation, 0.645) between their Ki-67 labeling index scores. The highest concordance was found between MIB-1 and poly (concordance correlation coefficient=0.785) antibodies. None of the antibodies except Ki-67 labeling index as detected by poly (P=.031) at 20% threshold and lymph node status (P<.001) were significantly linked to disease-free survival in multivariate analysis. At 30% threshold, this was reduced to lymph node status (P<.001) alone. Our results showed that there are considerable differences between the different Ki-67 antibodies in their capacity to detect proliferating tumor cells and to separate low- and high-risk breast cancer patient groups., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nuclear estrogen receptor (ER), progesterone receptor (PR) and Ki-67 protein positive tumor cell fractions are semiquantitatively assessed in breast cancer for prognostic and predictive purposes. These biomarkers are usually revealed using immunoperoxidase methods resulting in diverse signal intensity and frequent inhomogeneity in tumor cell nuclei, which are routinely scored and interpreted by a pathologist during conventional light-microscopic examination. In the last decade digital pathology-based whole slide scanning and image analysis algorithms have shown tremendous development to support pathologists in this diagnostic process, which can directly influence patient selection for targeted- and chemotherapy. We have developed an image analysis algorithm optimized for whole slide quantification of nuclear immunostaining signals of ER, PR, and Ki-67 proteins in breast cancers. In this study, we tested the consistency and reliability of this system both in a series of brightfield and DAPI stained fluorescent samples. Our method allows the separation of overlapping cells and signals, reliable detection of vesicular nuclei and background compensation, especially in FISH stained slides. Detection accuracy and the processing speeds were validated in routinely immunostained breast cancer samples of varying reaction intensities and image qualities. Our technique supported automated nuclear signal detection with excellent efficacy: Precision Rate/Positive Predictive Value was 90.23 ± 4.29%, while Recall Rate/Sensitivity was 88.23 ± 4.84%. These factors and average counting speed of our algorithm were compared with two other open source applications (QuPath and CellProfiler) and resulted in 6-7% higher Recall Rate, while 4- to 30-fold higher processing speed. In conclusion, our image analysis algorithm can reliably detect and count nuclear signals in digital whole slides or any selected large areas i.e. hot spots, thus can support pathologists in assessing clinically important nuclear biomarkers with less intra- and interlaboratory bias inherent of empirical scoring. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

Background: Metastasis is a leading cause of death among cancer patients. In the tumor microenvironment, altered levels of extracellular matrix proteins, such as collagens, can facilitate the first steps of cancer cell metastasis, including invasion into surrounding tissue and intravasation into the blood stream. However, the degree of misexpression of collagen genes in tumors remains understudied, even though this knowledge could greatly facilitate the development of cancer treatment options aimed at preventing metastasis., Methods: We systematically evaluate the expression of all 44 collagen genes in breast cancer and assess whether their misexpression provides clinical prognostic significance. We use immunohistochemistry on 150 ductal breast cancers and 361 cervical cancers and study DNA methylation in various epithelial cancers., Results: In breast cancer, various tests show that COL4A1 and COL4A2 overexpression and COL17A1 ( BP180 , BPAG2 ) underexpression provide independent prognostic strength (HR = 1.25, 95% CI = 1.17-1.34, p  = 3.03 × 10 -10 ; HR = 1.18, 95% CI = 1.11-1.25, p  = 8.11 × 10 -10 ; HR = 0.86, 95% CI = 0.81-0.92, p  = 4.57 × 10 -6 ; respectively). Immunohistochemistry on ductal breast cancers confirmed that the COL17A1 protein product, collagen XVII, is underexpressed. This strongly correlates with advanced stage, increased invasion, and postmenopausal status. In contrast, immunohistochemistry on cervical tumors showed that collagen XVII is overexpressed in cervical cancer and this is associated with increased local dissemination. Interestingly, consistent with the opposed direction of misexpression in these cancers, the COL17A1 promoter is hypermethylated in breast cancer and hypomethylated in cervical cancer. We also find that the COL17A1 promoter is hypomethylated in head and neck squamous cell carcinoma, lung squamous cell carcinoma, and lung adenocarcinoma, in all of which collagen XVII overexpression has previously been shown., Conclusions: Paradoxically, collagen XVII is underexpressed in breast cancer and overexpressed in cervical and other epithelial cancers. However, the COL17A1 promoter methylation status accurately predicts both the direction of misexpression and the increased invasive nature for five out of five epithelial cancers. This implies that aberrant epigenetic control is a key driver of COL17A1 gene misexpression and tumor cell invasion. These findings have significant clinical implications, suggesting that the COL17A1 promoter methylation status can be used to predict patient outcome. Moreover, epigenetic targeting of COL17A1 could represent a novel strategy to prevent metastasis in patients.

There have been relevant changes in the diagnosis and treatment of breast cancer to implement the updating of the 2010 recommendations made during the 2nd national consensus conference on the disease. Following a wide interdisciplinary consultation, the present recommendations have been finalized after their public discussion at the 3rd Hungarian Consensus Conference on Breast Cancer. The recommendations cover non-operative and intraoperative diagnostics, the work-up of operative specimens, the determination of prognostic and predictive markers and the content of the cytology and histology reports. Furthermore, it touches some special issues such as the current status of multigene molecular markers, the role of pathologists in clinical trials and prerequisites for their involvement, some relevant points about the future.

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

Gap juctions are transmembrane communication channels known to be involved in the control of cell proliferation by mediating the exchange of ions and small molecules between cells. Gap junctions are composed of connexon hemichannels made up of 6 connexin proteins, which abnormal expression and functions have been linked to tumor progression and poorer prognosis. Here, we studied the prognostic impact of the most prevalent connexin isotype, connexin 43 (Cx43) in head and neck squamous cell carcinomas (HNSCC). Tissue microarrays made from tumor samples of 90 HNSCC patients were immunostained for Cx43 and cell cycle regulation-related biomarkers including p53, Ki67, p16, aurora A, geminin, and p21 proteins. Scoring and histopathologic evaluation were performed in digital slides. A 4-tier scoring distinguishing the percentage of positively stained tumor cells was used including score 1: <5%, score 2: 6% to 20%, score 3: 21% to 60%, and score 4: >60%. For statistics, Kaplan-Meier curves with log-rank tests, Cox-regression, and Pearson χ/Fisher exact tests were used.A significant positive correlation was found between Cx43 expression and disease-specific survival of patients (P=0.004). The rate of p21 protein-positive tumor cells also proved to be a significant positive prognostic marker (P=0.014). Cx43 levels also showed a significant positive correlation with p53 expression (P=0.036). However, there was no statistical association between Cx43 levels and the rest of the markers tested neither with T, N, or M stage.In conclusion, our data suggest that reduced Cx43 expression and low p21 protein levels may have a significant negative impact on HNSCC prognosis.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 μm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

Background: Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) loss of function is frequently detected in advanced colorectal cancer. Its detection is thought to have prognostic significance and it is being considered to predict responsiveness to anti-EGFR therapy. Unfortunately, while immunohistochemical assessment of PTEN expression is widespread, it lacks standardization and the results are hardly comparable across the available publications., Methods: Retrospectively collected, formalin-fixed and paraffin-embedded colorectal tumor tissue samples from 55 patients were combined into tissue microarray (TMA) blocks. We used three different PTEN antibodies to determine the frequency, intensity and intracellular pattern of PTEN immunohistochemical labeling: Neomarkers, Dako and CellSignaling. We evaluated the aforementioned parameters in selected regions of colorectal cancers and in their lymph node metastases by using three scoring methods that take into consideration both staining frequency and intensity (H1-H3-score). We also evaluated intracellular localization., Results: The Dako and CellSignaling antibodies stained predominantly cytoplasms, while the Neomarkers antibody specifically stained cell nuclei. PTEN H-scores were significantly lower in all tumor areas as compared to the normal colonic mucosa based on staining with the DAKO and CellSignaling antibodies. Intratumoral regional differences or differences between matching tumors and metastases were not detected with any of the antibodies. Neither Dako, neither CellSignaling, nor the Neomarkers antibodies revealed a significant correlation between PTEN expression and pT, Dukes/MAC and clinical stage. KRAS status, histological grade correlated with PTEN H-scores based on staining with the Neomarkers antibody. PTEN H-scores did not correlate with MMR status. PTEN H-scores did not show any correlation with relapse-free survival based on staining with either antibody., Conclusions: While PTEN expression decreased in colorectal cancer according to two antibodies, neither of the three applied PTEN antibodies could justify significant correlation with clinicopathological data, nor had prognostic value. Thus, we might conclude that immunohistochemical PTEN investigation remains a challenge requiring more standardized evaluation on larger number of cases to clarify its utility as a prognostic and predictive tool in CRC. The standardization of immunohistochemical method is key in the evaluation process, which is further discussed.

We aimed to analyze to what extent expression of four cell cycle regulation markers-minichromosome maintenance protein (MCM2), Ki-67, cyclin A, and phosphohistone-H3 (PHH3)-predict response to primary systemic therapy in terms of pathological complete remission (pCR). In search of an accurate and reproducible scoring method, we compared computer-assisted (CA) and routine visual assessment (VA) of immunoreactivity. We included 57 patients with breast cancer in the study. The cell cycle markers were detected using immunohistochemistry on pre-therapy core biopsy samples. Parallel CA (validated by manual labeling) and standard VA were performed and compared for diagnostic agreement and predictive value for pCR. CA and VA results were dichotomized based on receiver operating characteristic analysis defined optimal cut-off values. "High" was defined by staining scores above the optimal cut-off, while "low" had staining scores below the optimal cut-off. The CA method resulted in significantly lower values for Ki-67 and MCM2 compared to VA (mean difference, -3.939 and -4.323). Diagnostic agreement was highest for cyclin A and PHH3 (-0.586 and -0.666, respectively). Regardless of the method (CA/VA) used, all tested markers were predictive of pCR. Optimal cut-off-based dichotomization improved diagnostic agreement between the CA and VA methods for every marker, in particular for MCM2 (κ = 1, p < 0.000). Cyclin A displayed excellent agreement (κ = 0.925; p < 0.000), while Ki-67 and PHH3 showed good agreement (κ = 0.789, p < 0.000 and κ = 0.794, p < 0.000, respectively). We found all cell cycle markers (Ki-67, MCM2, cyclin A, and PHH3) predictive of pCR. Diagnostic agreement between CA and VA was better at lower staining scores but improved after optimal cut-off-based dichotomization.

The incidence of head and neck squamous cell carcinomas is still growing, and the long-term prognosis of advanced disease remains poor. Only a fraction of head and neck cancers are sensitive to the EGFR-inhibitor cetuximab, which is the only registered targeted therapy available today. In several cancers, gene copy number alterations of MET and PIK3CA have been found to be prognostic and predictive for therapy response. The aim of this study was to systematically analyze in head and neck cancers the pathological characteristics and prognostic significance of copy number changes of MET and PIK3CA genes. MET and PIK3CA copy numbers were analyzed by fluorescence in situ hybridization in tumor samples of 152 patients. Expression of EGFR, p16, and Ki67 was studied by immunohistochemistry. High polysomy of PIK3CA (chromosome 3) was found in 20 % of cases and amplification in 4.5 %. Regarding MET, 35 % of cases showed low or high polysomy of the gene (chromosome 7), while no intra-chromosomal amplification of MET was detected. PIK3CA copy number gain (high polysomy or amplification) was significantly associated with shorter disease-specific survival, larger tumor volume, and lower p16 expression. MET copy number gain (low or high polysomy) in tumors was significantly associated with shorter disease-specific survival and lower level of EGFR. PIK3CA and MET may play an important role in oncogenesis of certain specific subtypes of head and neck cancer. There is an urgent need for the development of novel targeted therapies against these tumors associated with poor prognosis.

Background: Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia., Methods: Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression., Results: A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups., Conclusions: These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

Objective: The majority of prostate cancers require androgen hormones for growth, and androgen ablation is an important part of the systemic treatment of advanced prostate cancer. Nevertheless, most of these cancers eventually relapse as they become less sensitive to androgen ablation and anti-androgen treatment. Elucidating the molecular events that are responsible for the conversion of androgen-sensitive cancers to androgen-refractory tumors may reveal new therapeutic opportunities., Methods: In the present study, we investigated nine androgen-sensitive and nine androgen-refractory prostate cancer samples to evaluate the expression levels of 10 selected proteins that have been implicated in oncogenesis and cancer progression., Results: Our immunohistochemical data show that three of the investigated proteins (i.e., minichromosome maintenance-2, methylguanine-DNA methyltransferase, and androgen receptor) are expressed at significantly different levels in the androgen-refractory cancer samples than in the androgen-sensitive tumors, whereas the expression levels of the seven other studied proteins (i.e., β-catenin, p27, p21, p16, Ki67, hypoxia-inducible factor 1 alpha, and geminin) are not significantly different regarding the two groups., Conclusions: Our data suggest that the increased expression of minichromosome maintenance-2 and decreased expression of methylguanine-DNA methyltransferase related to androgen receptor are indicative of the androgen-refractory stage in prostate cancer. Further studies are required to determine whether these expression changes play a causative role in the transition of androgen-sensitive to androgen-refractory prostate cancer.

Head and neck squamous cell carcinomas (HNSCC) show diverse clinicopathological features and are mostly linked with poor outcome. In this study, we tested if the expression of tumor growth, cell cycle and basement membrane anchorage related biomarkers allow prognostic and clinicopathological stratification of HNSCC. Archived HNSCC samples from 226 patients included into tissue microarrays (TMA) were tested using immunohistochemistry. Histopathological evaluation and the analysis of immunostaining for EGFR, Ki67, p53, p16(ink4) and Collagen XVII proteins were carried out in digital whole slides. Statistical evaluation was carried out using Pearson's Chi-square test and Kaplan-Meier survival analysis. In the tested cohort, hypopharyngeal cancers had the least favorable, and glottic cancers had the most favorable prognosis. High Ki67 positive tumor cell fractions were associated with significantly worse prognosis and elevated rate of lymph node metastasis. Both Ki67 and EGFR expression correlated significantly with the tumor localization. Ki67 index was the highest in the hypopharyngeal region and it proved to be the lowest in the glottic region. EGFR expression was the highest in the oral cavity and the lowest in the glottic region. The survival rate of patients with p16(ink4)-negative cancer was significantly lower than of those with p16(ink4)-positive disease. A significant inverse correlation was found between histological grade and the prognosis of HNSCC. Our data support that elevated Ki67 positive proliferating cell fractions contribute to the unfavorable prognosis of hypopharyngeal cancers, while glottic cancers have the most favorable prognosis because of the lowest Ki67 expression rate.

Chronic renal fibrosis is the final common pathway of end stage renal disease caused by glomerular or tubular pathologies. Genetic background has a strong influence on the progression of chronic renal fibrosis. We recently found that Rowett black hooded rats were resistant to renal fibrosis. We aimed to investigate the role of sustained inflammation and oxidative/nitrative stress in renal fibrosis progression using this new model. Our previous data suggested the involvement of podocytes, thus we investigated renal fibrosis initiated by doxorubicin-induced (5 mg/kg) podocyte damage. Doxorubicin induced progressive glomerular sclerosis followed by increasing proteinuria and reduced bodyweight gain in fibrosis-sensitive, Charles Dawley rats during an 8-week long observation period. In comparison, the fibrosis-resistant, Rowett black hooded rats had longer survival, milder proteinuria and reduced tubular damage as assessed by neutrophil gelatinase-associated lipocalin (NGAL) excretion, reduced loss of the slit diaphragm protein, nephrin, less glomerulosclerosis, tubulointerstitial fibrosis and matrix deposition assessed by periodic acid-Schiff, Picro-Sirius-red staining and fibronectin immunostaining. Less fibrosis was associated with reduced profibrotic transforming growth factor-beta, (TGF-β1) connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL-1a1) mRNA levels. Milder inflammation demonstrated by histology was confirmed by less monocyte chemotactic protein 1 (MCP-1) mRNA. As a consequence of less inflammation, less oxidative and nitrative stress was obvious by less neutrophil cytosolic factor 1 (p47phox) and NADPH oxidase-2 (p91phox) mRNA. Reduced oxidative enzyme expression was accompanied by less lipid peroxidation as demonstrated by 4-hydroxynonenal (HNE) and less protein nitrosylation demonstrated by nitrotyrosine (NT) immunohistochemistry and quantified by Western blot. Our results demonstrate that mediators of fibrosis, inflammation and oxidative/nitrative stress were suppressed in doxorubicin nephropathy in fibrosis-resistant Rowett black hooded rats underlying the importance of these pathomechanisms in the progression of renal fibrosis initiated by glomerular podocyte damage.

Background: Somatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line., Methods: Colonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion., Results: In case of normal aging SST mRNA expression did not alter, but decreased in cancer (p < 0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70% ± 0.79%) compared to CRC (0% ± 0%) (p < 0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2% ± 11.6%) compared to healthy young individuals (3.5% ± 1.9%) (p < 0.05)., Conclusions: In cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation.

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.

Background: Primary systemic treatment for ovarian cancer is surgery, followed by platinum based chemotherapy. Platinum resistant cancers progress/recur in approximately 25% of cases within six months. We aimed to identify clinically useful biomarkers of platinum resistance., Methods: A database of ovarian cancer transcriptomic datasets including treatment and response information was set up by mining the GEO and TCGA repositories. Receiver operator characteristics (ROC) analysis was performed in R for each gene and these were then ranked using their achieved area under the curve (AUC) values. The most significant candidates were selected and in vitro functionally evaluated in four epithelial ovarian cancer cell lines (SKOV-3-, CAOV-3, ES-2 and OVCAR-3), using gene silencing combined with drug treatment in viability and apoptosis assays. We collected 94 tumor samples and the strongest candidate was validated by IHC and qRT-PCR in these., Results: All together 1,452 eligible patients were identified. Based on the ROC analysis the eight most significant genes were JRK, CNOT8, RTF1, CCT3, NFAT2CIP, MEK1, FUBP1 and CSDE1. Silencing of MEK1, CSDE1, CNOT8 and RTF1, and pharmacological inhibition of MEK1 caused significant sensitization in the cell lines. Of the eight genes, JRK (p = 3.2E-05), MEK1 (p = 0.0078), FUBP1 (p = 0.014) and CNOT8 (p = 0.00022) also correlated to progression free survival. The correlation between the best biomarker candidate MEK1 and survival was validated in two independent cohorts by qRT-PCR (n = 34, HR = 5.8, p = 0.003) and IHC (n = 59, HR = 4.3, p = 0.033)., Conclusion: We identified MEK1 as a promising prognostic biomarker candidate correlated to response to platinum based chemotherapy in ovarian cancer.

In inflammatory bowel diseases the presence of free-circulating DNA (fcDNA) sequences in the sera is an established phenomenon, albeit its real biological function still remains unclear. In our study the immunobiologic effects of a single-dose, intravenously administered fcDNA of normal and colitic origin were assayed in DSS-colitic and control mice. In parallel with disease and histological activity evaluations changes of the TLR9 and inflammatory cytokine signaling gene expression profiles were assayed in isolated cells of the lamina propria. Intravenously administered colitis-derived fcDNA displayed a more prominent beneficial action regarding the clinical and histological severity of DSS-colitis than that of fcDNA of normal origin. Systemic administration of colitis-derived fcDNA significantly altered the expression of certain TLR9-related and proinflammatory cytokine genes in a clinically favorable manner. Presumably due to induction of severe colitis, the subsequent marked inflammatory environment may result changes in fcDNA with a potential to promote the downregulation of inflammation and improvement of tissue regeneration. Elucidating mechanisms of innate immune alterations by nucleic acids may provide further insight into the etiology of inflammatory bowel diseases, and develop the basis of novel nucleic acid-based immunotherapies.

As mammalian Target of Rapamycin (mTOR) plays role in protein synthesis and metabolism, mTOR pathway activation is involved in the pathogenesis of several types of tumors. Our aim was to elucidate its role in medulloblastoma in terms of prognosis and as a therapeutic target. Members of activated mTOR complex 1 (mTORC1) pathway, phospho-mTOR (p-mTOR) and phospho-S6 (p-S6) were examined by immunohistochemistry in formalin fixed paraffin embedded samples of 40 patients with medulloblastoma, and results were compared to clinical features and survival of patients. In proliferation assays, Daoy and UW228-2 medulloblastoma cell lines were tested by rapamycin, an mTORC1 inhibitor, and NVP-BEZ235, a dual mTOR and phosphatidylinositol 3-kinase (PI3K) inhibitor, each in monotherapy and in combination with cytostatic drugs (cisplatin, etoposide). Components of mTORC1 and mTORC2 complexes were also examined in these cell lines. Neither presence of p-mTOR (32.5 %) nor p-S6 (32.5 %) correlated with age, gender or histological subtype. In 22.5 % of cases simultaneous expression of p-mTOR and p-S6 was shown. Kaplan-Meier analysis showed inferior survival of patients expressing both marker proteins, but it was not statistically significant, probably due to low case number. UW228-2 cells had greater sensitivity to mTOR inhibitors, possibly due to its higher mTORC1 specific protein expression levels, compared to Daoy cells. In both cell lines antiproliferative effect of cytostatic drugs was significantly enhanced by mTOR inhibitors (p < 0.05). Based on our in vitro and clinicopathological studies mTOR inhibitors may have a role in the future treatment of a subset of patients with medulloblastoma.

Aim: We investigated tissue biomarkers in non-small cell lung cancer (NSCLC) to find indicators of brain metastasis and peritumoral brain edema., Patients and Methods: Fifty-two cases were studied out of which 26 had corresponding brain metastatic tissue. Clinicopathological characteristics of tumors were correlated with biomarkers of cell adhesion, cell growth, cell cycle and apoptosis regulation that were previously immunohistochemically studied but never analyzed separately according to histological subgroups, gender and smoking history., Results: Increased collagen XVII in adenocarcinoma (ADC) and increased caspase-9, CD44v6, and decreased cellular apoptosis susceptibility protein (CAS) and Ki-67 in squamous cell carcinoma (SCC) correlated significantly with brain metastasis. Increased β-catenin, E-cadherin and decreased caspase-9 expression in primary SCC, and decreased CD44v6 expression in brain metastatic SCC tissues showed a significant correlation with the extent of peritumoral brain edema. Positive correlation between smoking and biomarker expression could be observed in metastatic ADCs with p16 and caspase-8, while-negative correlation was found in SCC without brain metastasis with caspase-3, and in SCC with brain metastasis with p27., Conclusion: Our results highlight the importance of separate analysis of biomarker expression in histological subtypes of NSCLC., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Urogenital tract infection with Chlamydia trachomatis is a leading cause of sexually transmitted infections. There is currently no commercially available vaccine against C. trachomatis. The highly conserved plasmid of chlamydiae has been considered to be a virulence factor and the plasmid proteins have important roles in the Chlamydia-specific immune response. This study was designed to evaluate the efficacy of vaccination with plasmid proteins in the prevention of C. muridarum lung infection in a mouse model. C57BL/6N mice were immunised 3 times subcutaneously with recombinant pGP3 or pGP4 and infected with C. muridarum. Immunisation of the mice with recombinant pGP3 or pGP4 protein caused a significantly lower chlamydial burden in the lungs of the infected mice; the lower IFN-γ level indicated a reduced extent of inflammation. In vitro or in vivo neutralisation of C. muridarum with sera obtained from immunised mice did not reduce the number of viable C. muridarum in the lungs of mice. However, adoptive transfer of the CD4(+) spleen cells isolated from the immunised mice resulted in a significantly reduced bacterial burden. Our results indicate that it is not the pGP3- and pGP4-specific antibodies, but the CD4(+) cells that are responsible for the protective effect of the immune response to plasmid proteins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Early molecular detection of the colorectal dysplasia-carcinoma transition may augment the accuracy of diagnosis in case of biopsy orientation errors. The combination of high-throughput microarray-based biomarker screening with tissue microarray-based prospective protein biomarker expression analysis could represent an additional test in routine automated diagnostic procedures. Our aim was to test and select protein markers to identify protein expression profile alterations, focusing on the dysplasia-carcinoma transition in sporadic colorectal tumors. Dysplasia-carcinoma transition-specific transcript sets were previously identified using HGU133plus2 microarrays and Taqman RT-PCR cards. Here, 26 potential dysplasia-carcinoma transition-specific markers were tested by immunohistochemistry at the protein level using tissue microarrays in a total of 168 independent colonic biopsy samples. A set of 26 transcripts [including matrix metalloproteinase-3 (MMP3) and chemokine (C-X-C motif) ligand 1 (CXCL1)] has been determined recently, indicating a linear expression correlation with the adenoma-dysplasia-carcinoma sequence, thereby having the potential to discriminate between dysplasia and early malignancy. Currently, we find that high-grade dysplastic sessile adenomatous-stage and early-stage colorectal cancer conditions can be differentiated correctly by the stromal expression of MMP3 and CXCL1, respectively, on tissue microarray-based analysis. Furthermore, in cases of sporadic colorectal tumors, MMP3 protein expression in the lamina propria itself seems to be highly specific for the detection of tumorous transition. Our current and recent results indicate that appropriate antibody marker combinations are highly suitable for tissue microarray-based and digital microscopy-based, automated, high-capacity diagnostic application in tumorous colonic diseases.

Background and Aims: Sporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations., Materials and Methods: Colorectal biopsies from healthy children (n1 = 14), healthy adults (n2 = 10), adult adenomas (n3 = 10) and CRCs (n4 = 10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n1 = 6), adult (n2 = 41) samples and in CRC (n3 = 34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n1 = 6, n2 = 6, n3 = 6)., Results: Mitotic index (MI) was significantly higher (p<0.05) in intact juvenile (MI = 0.33±0.06) and CRC samples (MI = 0.42±0.10) compared to healthy adult samples (MI = 0.15±0.06). In contrast, apoptotic index (AI) was decreased in children (0.13±0.06) and significantly lower in cancer (0.06±0.03) compared to healthy adult samples (0.17±0.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC., Conclusion: Colorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium.

Background: Ischemia-reperfusion (I/R) is the main cause of acute kidney injury (AKI) in patients. We investigated renal microRNA (miRNA) expression profiles and the time course of changes in selected miRNA expressions after renal I/R to characterize the miRNA network activated during development and recovery from AKI., Methods and Results: One day after lethal (30 minutes) and sublethal (20 minutes) renal ischemia, AKI was verified by renal histology (tubular necrosis, regeneration), blood urea nitrogen (BUN) level, renal mRNA expression, and plasma concentration of neutrophil gelatinase-associated lipocalin (NGAL) in C57BL/6J mice. On the first day after 30-minute, lethal I/R miR-21, miR-17-5p, and miR-106a were elevated out of the 21 miRNAs successfully profiled on the Luminex multiplex assay. After 20-minute, sublethal I/R, renal miR-17-5p and miR-106a expressions were elevated on the first and second days of reperfusion, while miR-21 expression increased later and lasted longer. Renal miR-17-5p and miR-21 expressions correlated with each other. Renal function returned to normal on the fourth day after sublethal I/R., Conclusions: Our results demonstrate that besides miR-21, miR-17-5p, and miR-106a are additionally activated during the maintenance and recovery phases of renal I/R injury. Furthermore, a correlation between renal miR-17-5p and miR-21 expressions warrants further investigation of how they may influence each other and the outcome of renal ischemia-reperfusion injury.

Angiosarcomas constitute a heterogeneous group of highly malignant vascular tumors. Angiosarcoma of bone is rare and poorly characterized. For angiosarcoma of soft tissue, some pathways seem to be involved in tumor development. Our aim was to evaluate the role of these pathways in angiosarcoma of bone. We collected 37 primary angiosarcomas of bone and used 20 angiosarcomas of soft tissue for comparison. Immunohistochemistry was performed on constructed tissue microarrays to evaluate expression of CDKN2A, TP53, PTEN, BCL2, CDK4, MDM2, cyclin D1, β-catenin, transforming growth factor-β (TGF-β), CD105, phospho-Smad1, phospho-Smad2, hypoxia-inducible factor-1α, plasminogen activator inhibitor type 1 (PAI-1), VEGF, CD117 and glucose transporter--1. PIK3CA was screened for hotspot mutations in 19 angiosarcomas. In nearly 55% of the angiosarcoma of bone, the retinoblastoma (Rb) pathway was affected. Loss of CDKN2A expression was associated with a significantly worse prognosis. No overexpression of TP53 or MDM2 was found, suggesting that the TP53 pathway is not important in angiosarcoma of bone. Angiosarcoma of bone showed highly active TGF-β signaling with immunoreactivity for phospho-Smad2 and PAI-1. Although the phosphatidylinositol 3-kinase (PI3K)/Akt pathway seems to be active in both tumor groups, different mechanisms were involved: 41% of angiosarcoma of bone showed a decrease in expression of PTEN, whereas in angiosarcoma of soft tissue overexpression of KIT was found (90%). PIK3CA hotspot mutations were absent. In conclusion, the Rb pathway is involved in tumorigenesis of angiosarcoma of bone. The PI3K/Akt pathway is activated in both angiosarcoma of bone and soft tissue, however, with a different cause; PTEN expression is decreased in angiosarcoma of bone, whereas angiosarcomas of soft tissue show overexpression of KIT. Our findings support that angiosarcomas are a heterogeneous group of vascular malignancies. Both angiosarcoma of bone and soft tissue may benefit from therapeutic strategies targeting the PI3K/Akt pathway. However, interference with TGF-β signaling may be specifically relevant in angiosarcoma of bone.

The mesenchymal, clear cell, and dedifferentiated chondrosarcoma subtypes are extremely rare, together constituting 10% to 15% of all chondrosarcomas. Their poor prognosis and lack of efficacious treatment emphasizes the need to elucidate the pathways playing a pivotal role in these tumors. We constructed tissue microarrays containing 42 dedifferentiated, 23 clear cell, and 23 mesenchymal chondrosarcomas and performed immunohistochemistry to study the expression of growth plate-signaling molecules and molecules shown to be involved in conventional chondrosarcoma. We observed high expression of SOX-9 and FGFR-3, as well as aberrant cellular localization of heparan sulfate proteoglycans, in all subtypes. TGFβ signaling through p-SMAD2 and PAI-1 was highly active in all chondrosarcoma subtypes, which suggests that TGFβ inhibitors as a possible therapeutic strategy in rare chondrosarcoma subtypes. As in conventional chondrosarcoma, antiapoptotic proteins (Bcl-2, and/or Bcl-xl) were highly expressed in all subtypes. Inhibition with the BH-3 mimetic ABT-737 rendered dedifferentiated chondrosarcoma cell lines sensitive to doxorubicin or cisplatin. Our data indicate that antiapoptotic proteins may play an important role in chemoresistance, suggesting a promising role for targeting Bcl-2 family members in chondrosarcoma treatment, irrespective of the subtype., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The objective of the study was to examine proliferation and apoptosis associated gene expression in the whole sequence parathyroid lesions to reveal specific features of carcinoma. This study was based on surgically removed parathyroid tissues, gene expression analysis was performed both at gene and protein level. First, mRNA isolation was performed from deep-frozen tissue samples, and further apoptosis pathway-specific cDNA macroarray analysis was carried out. The results were validated with real-time PCR. Subsequently, protein expression was analyzed with immunhistochemistry on Tissue Micro Array multi-blocks derived from several paraffin-embedded samples. cDNA macroarrays revealed elevated expression of both pro-apoptotic (FAS receptor, TRAIL ligand, CASPASE8, and -4) and anti-apoptotic (cIAP1, APOLLON) genes in benign proliferative lesions compared to that in normal gland. TMA studies showed overexpression of KI67, P53, SURVIVIN and APOLLON protein and failure of expression of P27, BCL2, BAX, CHROMOGRANIN-A, SYNAPTOPHYSIN, CYCLIND1, FLIP, TRAIL, CK8, CK18, CK19 in parathyroid carcinoma was detected. These alterations in gene expression of the investigated products could be used in differentiation between beningn and malignant proliferative processes of the parathyroid gland. Authors conclude that a series of alterations in gene expression such as overexpression of APOLLON, P53, KI67 and suppression of P27, BCL2, BAX lead to uncontrolled cell proliferation, but still not leading to increased apoptotic activity in parathyroid carcinoma.