Search

Tumors are intricate ecosystems where cancer cells and non-malignant stromal cells, including cancer-associated fibroblasts (CAFs), engage in complex communication. In this study, we investigated the interaction between poorly (HLE) and well-differentiated (HuH7) hepatoma cells and LX2 fibroblasts. We explored various communication channels, including soluble factors, metabolites, extracellular vesicles (EVs), and miRNAs. Co-culture with HLE cells induced LX2 to produce higher levels of laminin β1, type IV collagen, and CD44, with pronounced syndecan-1 shedding. Conversely, in HuH7/LX2 co-culture, fibronectin, thrombospondin-1, type IV collagen, and cell surface syndecan-1 were dominant matrix components. Integrins α6β4 and α6β1 were upregulated in HLE, while α5β1 and αVβ1 were increased in HuH7. HLE-stimulated LX2 produced excess MMP-2 and 9, whereas HuH7-stimulated LX2 produced excess MMP-1. LX2 activated MAPK and Wnt signaling in hepatoma cells, and conversely, hepatoma-derived EVs upregulated MAPK and Wnt in LX2 cells. LX2-derived EVs induced over tenfold upregulation of SPOCK1/testican-1 in hepatoma EV cargo. We also identified liver cancer-specific miRNAs in hepatoma EVs, with potential implications for early diagnosis. In summary, our study reveals tumor type-dependent communication between hepatoma cells and fibroblasts, shedding light on potential implications for tumor progression. However, the clinical relevance of liver cancer-specific miRNAs requires further investigation.

Syndecan-1, is a transmembrane heparan/chondroitin sulfate proteoglycan necessary for cell-cell and cell-matrix interactions. Its decreased level on the cell surface correlates with poor prognosis in several tumor types. Aberrant stromal localization of syndecan-1 is also considered an unfavorable prognostic factor in various human malignancies. In the presented work the question was addressed if changes in syndecan-1 expression are related to the prognosis of cervical cancer. Immunohistochemistry for syndecan-1 extracellular domain was performed on surgical specimens of primary cervical cancer. To follow the communication between tumor cells and stromal fibroblasts, their mono-and co-cultures were studied, detecting the expression of syndecan-1, smooth muscle actin, vimentin, and desmin. Immunohistochemistry of tumorous specimens revealed that while cell surface syndecan-1 expression was reduced on cancer cells, it appeared on the surface of tumor-associated fibroblasts. Until year 7, the cohort with high cell surface syndecan-1 expression had significantly longer survival. No difference in the same time-period could be detected when stromal syndecan-1 expression was analyzed. In vitro analysis revealed, that tumor cells can induce syndecan-1 expression on fibroblast, and fibroblasts showed that fibroblast-like cells are built by two cell types: (a) syndecan-1 positive, cytokeratin negative real fibroblasts, and (b) syndecan-1 and cytokeratin positive epithelial-mesenchymal transformed tumor cells. Syndecan-1 on the surface of cancer cells appears to be a positive prognostic marker. Although syndecan-1 positive fibroblasts promote tumor cell proliferation in vitro, we failed to detect their cancer promoting effect in vivo.

Topotecan (TpT) is a major inhibitory compound of topoisomerase (topo) I that plays important roles in gene transcription and cell division. We have previously reported that heparin and heparan sulfate (HS) might be transported to the cell nucleus and they can interact with topoisomerase I. We hypothesized that heparin and HS might interfere with the action of TpT. To test this hypothesis we isolated topoisomerase I containing cell nuclear protein fractions from normal liver, liver cancer tissues, and hepatoma cell lines. The enzymatic activity of these extracts was measured in the presence of heparin, liver HS, and liver cancer HS. In addition, topo I activity, cell viability, and apoptosis of HepG2 and Hep3B cells were investigated after heparin and TpT treatments. Liver cancer HS inhibited topo I activity in vitro. Heparin treatment abrogated topo I enzyme activity in Hep3B cells, but not in HepG2 cells, where the basal activity was higher. Heparin protected the two hepatoma cell lines from TpT actions and decreased the rate of TpT induced S phase block and cell death. These results suggest that heparin and HS might interfere with the function of TpT in liver and liver cancer.

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.

The significance of epidermal growth factor receptor (EGFR) signaling is well studied in a number of different tumors, but limited data is available with regard to head and neck squamous cell carcinoma (HNSCC). Since anti-EGFR therapies are currently under investigation in these malignancies as well, comprehensive information about the alteration of EGFR in HNSCC is necessary to design the most appropriate therapeutic protocols. We examined retrospectively the gene copy number of EGFR by FISH and the protein expression by immunohistochemistry using different epitope-specific antibodies in paraffin-embedded primary tumors of five different regions, from 71 HNSCC patients who had not been treated with anti-EGFR therapy. In seven cases corresponding lymph node metastases were also available for comparative analyses. We also determined the mutational status of tyrosine kinase (TK) domain (exon 19 and 21) and the extracellular deletion mutation (vIII) of EGFR, the KRAS mutation at codon 12 and the presence of HPV infection. Eight of the 71 cases (11.3%) showed EGFR gene amplification (most of them localized into the hypopharyngeal region) and the increased gene copy number (amplification+polysomy) was 43.7%. Despite pronounced intratumoral heterogeneity of EGFR protein expression being found, the high EGFR expression correlated with poor prognosis. On the other hand, the phosphorylation of EGFR was associated with prolonged survival. No mutations in the TK domain of EGFR were found in any of the HNSCC patients and only two cases were KRAS mutant at codon 12. We detected vIII deletion mutation of EGFR in 21% of the samples, but there was no statistically significant correlation between the presence of vIII mutant form and patient survival. EGFR vIII mutation was, however, associated with increased gene copy number. Fourteen of 71 cases (19.7%) were HPV-positive and the incidence of infection showed a decreasing tendency from the oral cavity towards the larynx. Interestingly, in contrast to previous findings, we could not observe improved survival in HPV-positive patients compared to non-infected patients, most probably due to the fact that the majority of these HNSCC patients were smokers and alcohol consumers. In conclusion, we found that increased EGFR protein levels and gene copy numbers (not gene amplification alone) have prognostic significance in the investigated HNSCC patient population. However, the relatively high incidence of the EGFR-vIII mutant form warrants careful therapeutic decision-making when choosing between different anti-EGFR treatment options., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The therapy for gastrointestinal stromal tumors (GISTs) has been revolutionized by tyrosin kinase inhibitors. Clinicopathologic studies have been conducted to assess therapeutical responses in cases with KIT and platelet-derived growth factor receptor α (PDGFRA) gene mutations. Cell culture data suggest that Akt/mammalian target of rapamycin (mTOR) kinase signaling may be important in GIST. The aim of our study was to determine the activity of the mTOR pathway in a larger series of GISTs (108 different cases) with different exon mutation types. The KIT and/or PDGFRA mutation status of 108 GIST patients was analyzed by direct DNA sequencing. Immunohistochemistry was performed on tissue microarrays using antibodies for phospho-p70S6 kinase, phospho-4EBP1, and phospho-S6, which are downstream target proteins of mTOR. DNA sequencing identified 73 cases with mutations of KIT and 12 cases with PDGFRA mutations. Wild-type receptors were present in 23 cases. KIT exon mutations were accompanied by the activation of the mTOR pathway in 28 of 73 (38.4%) cases, whereas PDGFRA mutant GISTs showed activation in 10 of 12 (83.3%) cases. Wild-type cases were accompanied by mTOR activation in 17 of 23 (73.9%) cases. Our results indicate that the activation of the mTOR pathway is not a general hallmark of GIST with KIT mutations. However, mTOR signaling seems to be activated in PDGFRA mutants and in wild-type cases, which suggests that mTOR or upstream mTOR inhibitors may be therapeutically useful in primary resistant GISTs and confirms earlier data that mTOR is a crucial survival pathway in resistant GISTs.

Genetic instability in relation to clinical behavior was studied in 52 cases of giant cell tumor of bone (GCTB). Ploidy was determined in the mononuclear cell population by using native cell smears and image cytometry. A relocalization technique allowed fluorescent in situ hybridization (FISH) analysis of CD68-negative neoplastic cells for numerical changes of chromosomes X, 3, 4, 6, 11, and telomeric association on 11p. Genome-wide alterations were tested using array comparative genomic hybridization (array-CGH) on magnetically separated CD68-negative tumor cells. CTNNB1, TP53, and BCL2 protein expression was also analyzed in formol-paraffin sections to see if their pathways are involved in the development of chromosomal instability. CD68-positive histiocytes showed no significant numerical chromosome and telomeric alterations. Based on ploidy values and clinical outcome, we could distinguish five groups as follows: diploid nonrecurrent (n = 20), tetraploid nonrecurrent (n = 6), diploid recurrent (n = 5), tetraploid and/or aneuploid recurrent (n = 14), and malignant cases (n = 7). Random individual-cell aneusomy was significantly (P < 0.001) more frequent in the recurrent groups (36.01 +/- 11.94%) than in the benign nonrecurrent cases (10.65 +/- 3.66%). The diploid recurrent group showed significantly (P < 0.001) increased balanced aneusomy compared with the diploid nonrecurrent group and the tetraploid nonrecurrent group represented eusomic polysomy. Array-CGH and FISH showed clonal aberrations almost exclusively in the malignant group. None of the protein markers tested showed significant correlation with elevated aneuploidy/polysomy (P = 0.56). Our results show that ploidy determination combined with FISH analysis may help predicting recurrence potential of GCTB and suggest that chromosomal abnormalities superimposed on telomeric associations could be responsible for an aggressive clinical course.

In routine practice, gastrointestinal stromal tumor (GIST) can usually be identified with relative ease on the basis of a rather simple immunohistochemical panel besides its characteristic morphology. Still, serious differential diagnostic problems may arise because of the heterogeneity of these tumors in both morphologic appearance and clinical behavior. In our case, we present a metastatic, ulcerative, hemorrhagic GIST with epithelioid appearance, which displayed diffuse pan cytokeratin (AE1/AE3) positivity beside CD117 expression. As carcinomas may also be CD117-positive, definitive diagnosis was confirmed by the detection of a hexanucleotide deletion in the exon 11 of c-kit. This case demonstrates that although gastric carcinoma more commonly ulcerates or causes hemorrhage than GIST, keratin-positive GIST should also be considered from a differential diagnostic point of view. In these cases, c-kit mutation analysis may be necessary.

Lymphoepithelioma-like carcinoma of the breast is a rare tumor, with fewer than 20 cases documented in the literature. None of the published cases was Epstein-Barr virus positive, and our case was also Epstein-Barr virus negative. However, in our case, human papilloma virus (HPV) types 18 and 33 DNA could be demonstrated within the tumor tissue. Many years previously, the patient underwent hysterectomy for cervical carcinoma in situ which showed the presence of HPV-33. To the best of our knowledge, this is the first report on lymphoepithelioma-like carcinoma of the breast where high-risk HPV infection may be suggested as an etiological factor in a patient with a previous history of cervical carcinoma in situ.

The accumulation of genetic alterations plays a role in the evolution of bladder cancer. These changes can be detected in the urine by DNA analysis of the cells exfoliated from the bladder wall enabling us to detect bladder cancer. The urine supernatant, besides the urine sediment, contains DNA, however in a much smaller amount. The origin of DNA in these two fractions is probably different. Our aim was to evaluate which fraction (supernatant or sediment) provides more reliable results in detecting tumors. We analyzed blood, urine and tumor samples taken from 80 individuals (44 patients with bladder cancer, 20 control patients and 16 healthy volunteers) by using 12 microsatellite markers mapped on 6 chromosomes. Microsatellite alterations were detected in the urine sediment and supernatant in 86% of the cancer cases. Urine sediment alone had a sensitivity of 68%, while urine supernatant alone indicated aberrations in 80% of the tumors. In the superficial (Ta/T1) cases, a considerable difference in sensitivity, 84 vs. 67%, was found between the two fractions in favor of urine supernatant. We also detected deletions in the control groups, although in a much lower proportion. Loss of the 16q24 chromosomal region showed a significant correlation with tumor stage (p=0.02). Microsatellite analysis of the urine is an efficient and noninvasive molecular method to detect bladder cancer. The analysis of free DNA in the urine supernatant provides a higher detection rate. The marker on the chromosomal region 16q24 is suggested to have a prognostic value.

Langerhans cell histiocytosis (eosinophilic granuloma) was first diagnosed in the adolescence of a male patient presented. Several years later persisting human herpesvirus 6 (HHV-6) infection was recognized. The HHV-6 infection could be verified retrospectively in his historical histological samples; the continuous presence of HHV-6 could be established through 17 years of disease course. The patient was operated several times during this period for painful relapses, and developed diabetes insipidus. At variable time points during the clinical course, Varicella zoster (VZV), Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) infections were temporarily detected from blood samples and biopsy specimens. HHV-6 was the only virus continuously identified throughout the entire follow-up period. Antiviral therapy effectively cleared EBV and HHV-8, but HHV-6 remained detectable throughout the disease course. Since DNA sequences of HHV-6 could be detected in the pathologic histiocytes of eosinophilic granuloma, and from other samples taken later on, it is suggested that long-term HHV-6 infection may be associated with development or progression of Langerhans cell histiocytosis.

Human papillomavirus (HPV) is known as a strictly epitheliotropic pathogen. Our results raised the possibility that HPV 16 is present in neural cells and in the vascular endothelium. By in situ hybridization, we have detected HPV 16 E6 ORF sequence in small blood vessels and peripheral nerves adjacent to oral and cervical cancers. The same structures have clearly shown immunohistochemical reactivity for the E6 oncoprotein. These results were verified by PCR applied to E6 and L1 ORFs following microscopic laser dissection of the immunohistochemically positive nerves and vessels. These observations suggest that HPV 16 DNA and protein are present in neurons and endothelial cells in the vicinity of HPV-associated tumors. The HPV 16 genome presumably exists in a non-replicating form in the neurons and constitutively produces high levels of E6 and E7 proteins with an unknown neuropathological outcome.

Syndecan-1, a transmembrane proteoglycan, may exert anti-proliferative effects and promote cell growth by binding various growth factors. Malignant epithelial cells often down-regulate their own syndecan-1 production, and are capable of inducing an aberrant syndecan-1 expression in stromal fibroid cells. Decreased tumor cell syndecan-1 levels in human head and neck squamous cell carcinomas indicate poor prognosis, however, no correlation between stromal syndecan-1 expression and clinical parameters has previously been established. By means of immunohistochemistry, we observed a decrease in tumor cell syndecan-1 reactivity in 33/39 oral carcinoma cases, the degree of which, however, correlated only weakly with the clinical outcome (p = 0.097). Conversely, stromal syndecan-1 positivity proved to be a significant risk factor of recurrence (Cox analysis, p = 0.03) and tumor-specific death (p = 0.023) within a 24-month period after operation. Taken together, stromal expression of syndecan-1 is a reliable factor of adverse prognosis in oral carcinomas.

Objective: In this study, we investigated the presence of high-risk (HR) HPV types most prevalent in the Hungarian population in surgically removed cervical cancers and pelvic lymph nodes. The aim of our work was to determine the prognostic significance of HPV status in the lymph nodes draining the tumor., Methods: Primary tumor specimens from 150 patients and 900 lymph node samples (six per case) were studied. Fifty-six/150 were early (FIGO IA-IB) stage, while 94/150 were advanced (FIGO IIA-IIIB) stage cancers. Beside histopathological evaluation, DNA extracted from the tissue samples was subjected to nested PCR to detect characteristic type-specific sequences of HPVs 16, 18 and 33. Moreover, clinicopathological data were collected for an average 48-month postoperative follow-up period for the purposes of statistical analysis., Results: The presence of HR-HPV types in the lymph nodes shows no correlation with disease-free survival, whereas the presence of lymph node metastases significantly decreases life expectancy (P = 0.002). Lymph nodes with metastases more frequently carry HR-HPV than nodes with no evidence of tumorous infiltration (65% versus 36%, P < 0.001); however, a high number of metastases surrounding HR-HPV-positive tumors were found negative for the viruses (42/120)., Conclusions: HR-HPV status of pelvic lymph nodes draining cervical cancers has no noticeable influence on the life expectancy of the patients. HR-HPV-positive tumor cells do not necessarily have a selective advantage in forming metastases. Presumably, a number of alterations in cellular genes rather than the presence of papillomavirus DNA may have a decisive role in the progression of cervical cancers.

The purpose of this study was to collect data about the incidence of high-risk HPV (16, 18, 33) types in paraffin embedded cervical bioptic material, including LSIL, HSIL and cervical cancers using immunohistochemistry and nested PCR methods. In our study were included randomly selected 10 LSIL, 18 HSIL and 30 cervical cancer cases. We analyzed the expression of HPV in this specimens with immunohistochemistry used DAKO K1H8 antibody and CHEMICON Mab HPV 16, 16 antibody using LSAB method and Tiramin amplification method, and nested PCR for HPV 16, 18 and 33. In LSIL cases three, in HSIL cases eight and in carcinoma 20 cases were positive for HPV 16 or 18 for immunohistochemistry or PCR. Although this proportion in lower than those reported in the literature, our work signals the existence of the infection in our country and presents a relatively cheap diagnostic method.

Background: Chlamydia pneumoniae is one of the possible pathogenetic factors of atherosclerotic processes. Foam cell arteriopathy is a generally accepted pathologic feature of chronic liver allograft rejection and has several similarities to the early lesions of atherosclerosis. The aim of the authors' study was to show any existing correlation between the occurrence of Chlamydia pneumoniae and the presence of foam cell arteriopathy in transplanted livers with chronic rejection., Methods: Ten liver samples from patients with chronic liver rejection including foam cell arteriopathy and 10 liver samples from healthy individuals were analyzed for the presence of Chlamydia pneumoniae by specific immunohistochemistry and polymerase chain reaction (PCR). Liver samples from two transplant patients with chronic liver rejection without any evidence of foam cell arteriopathy and nine patients with acute liver allograft rejection were also investigated by PCR., Results: In all 10 rejected liver samples, Chlamydia pneumoniae was detected by PCR, whereas only one of the healthy control samples and one of the samples with acute rejection were found to be positive. Immunohistochemistry showed similar results. The positive signals of Chlamydia pneumoniae were localized mainly in the hepatocytes, sinusoidal and perisinusoidal cells, and the cells of portal tracts, whereas most of the altered hepatic arteries showed no or very weak positivity., Conclusions: The results strongly suggest an association between the occurrence of Chlamydia pneumoniae and the presence of foam cell arteriopathy in transplanted livers.

For many years data of cancer research indicated that viruses can cause cancer. Virus infections induce cancer by different mechanisms. To predict the significance of a viral DNA fragment in human cells we have to be aware of the changes the particular virus is able to induce there.However, no matter which mechanisms of viral carcinogenesis are utilized, generally other factors (environmental, chemical, immunodeficiency, etc.) are also needed to induce invasive cancer in human. Before the introduction of nucleic acid based detection technique virus identification was a long and cumbersome process. This has been eliminated by the invention of recombinant gene technology and polymerase chain reaction. Virus nucleic acid can be detected without amplification using Southern, Northern and in situ hybridization. Techniques for target (polymerase chain reaction)or signal (hybrid capture, tyramine) amplification improved the sensitivity of detection. In the meantime, for the successful use of the arsenal of new methods we have to consider the characteristic feature of molecular virus research. A major achievement of molecular virus detection is that it proved the pathological significance of viruses in human cancers even in those where this was not expected. Hopefully these informations will increase the effort for elimination of oncogene virus infections.

Objective: The purpose of this study was to collect data about the incidence of high-risk HPV (16, 18, 33) types in in situ cervical cancers, and to evaluate the reliability of the morphological signs of HPV infection by comparing the presence of these signs to the PCR-proven HPV virus infection., Methods: Fifty patients who underwent conisation at the Department of Obstetrics and Gynecology of Semmelweis University, Budapest, Hungary because of in situ cervical cancer were examined retrospectively for the presence of HPV infection by the PCR technique. The direct and indirect morphological signs of HPV infection identified in the histological and cytological samples were compared to the actual results of virus DNA amplification by PCR in the identical histological sections. The evaluation of the cytological smears and the histological sections was accomplished independently by two different pathologists., Results: E6 open reading frame of HPV 16, 18 or 33 was detected by PCR in 56% (28 cases) of the histological sections of the 50 examined patients with in situ cancer. In 92% (26 patients) of the 28 HPV positive patients one HPV type was detected, while in one of the remaining two cases two HPV types (16/33), or all three types could be detected. The direct morphological signs for HPV infection proved to be 75% sensitive and 50% specific when compared to the results of PCR. Their predictive value for HPV infection was 65%. For the indirect HPV signs the sensitivity was 64% and specificity 31%. The predictive value, prognosticating the presence of HPV 16, 18, 33 infection was 54% in the same sections. Using significance analysis no significant relationship (p = 0.7728) could be detected between the positivity of indirect signs and the presence of HPV 16, 18, 33 infection, while in case of direct signs the relationship was almost significant (p = 0.0675). The joint testing of the direct and indirect signs did not improve the results (p = 0.1338). During the review of the cytological smears the specificity of the cytology in predicting true HPV infections was found to be 68% and sensitivity was 20%. The predictive value was only 50%. A significance analysis was not accomplished by this diagnostic method because of the missing data (see text)., Conclusion: The method of Nawa et al. seems to be a reliable approach for the detection of HPV DNA in paraffin-embedded material. The three main types of HPV (16, 18, 33) are probably represented in lower percentages in CIN III in Hungary, but a larger survey is needed to obtain reliable data. The direct and indirect morphological signs of HPV infection failed to show a significant relationship with the PCR proven presence of HPV 16, 18, 33.