Dynamic Interplay in Tumor Ecosystems: Communication between Hepatoma Cells and Fibroblasts.

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 ß1, 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 α6ß4 and α6ß1 were upregulated in HLE, while α5ß1 and αVß1 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.

Genomic instability in giant cell tumor of bone. A study of 52 cases using DNA ploidy, relocalization FISH, and array-CGH analysis.

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.

Aberrant Expression of Syndecan-1 in Cervical Cancers.

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.

Lymphoepithelioma-like carcinoma of the breast: not Epstein-Barr virus-, but human papilloma virus-positive.

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.

Placental protein 13 (galectin-13) has decreased placental expression but increased shedding and maternal serum concentrations in patients presenting with preterm pre-eclampsia and HELLP syndrome.

Placental protein 13 (PP13) is a galectin expressed by the syncytiotrophoblast. Women who subsequently develop preterm pre-eclampsia have low first trimester maternal serum PP13 concentrations. This study revealed that third trimester maternal serum PP13 concentration increased with gestational age in normal pregnancies (p < 0.0001), and it was significantly higher in women presenting with preterm pre-eclampsia (p = 0.02) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome (p = 0.01) than in preterm controls. Conversely, placental PP13 mRNA (p = 0.03) and protein, as well as cytoplasmic PP13 staining of the syncytiotrophoblast (p < 0.05) was decreased in these pathological pregnancies compared to controls. No differences in placental expression and serum concentrations of PP13 were found at term between patients with pre-eclampsia and control women. In contrast, the immunoreactivity of the syncytiotrophoblast microvillous membrane was stronger in both term and preterm pre-eclampsia and HELLP syndrome than in controls. Moreover, large syncytial cytoplasm protrusions, membrane blebs and shed microparticles strongly stained for PP13 in pre-eclampsia and HELLP syndrome. In conclusion, parallel to its decreased placental expression, an augmented membrane shedding of PP13 contributes to the increased third trimester maternal serum PP13 concentrations in women with preterm pre-eclampsia and HELLP syndrome.

Deletion analysis of tumor and urinary DNA to detect bladder cancer: urine supernatant versus urine sediment.

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.

Persistent long-term human herpesvirus 6 (HHV-6) infection in a patient with langerhans cell histiocytosis.

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.

Stromal syndecan-1 expression is an adverse prognostic factor in oral carcinomas.

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.

Activation of villous trophoblastic p38 and ERK1/2 signaling pathways in preterm preeclampsia and HELLP syndrome.

Preterm preeclampsia is associated with the failure of trophoblast invasion, placental hypoxic/ischemic injury and the release of toxic substances, which promote the terminal pathway of preeclampsia. In term preeclampsia, factors yet unknown trigger the placenta to induce the terminal pathway. The contribution of the villous trophoblast to these pathologic events has not been fully elucidated. Here we aimed to study how stress and signaling pathways influence trophoblastic functions in various subforms of preeclampsia. Tissue microarrays (TMAs) were constructed from placentas obtained from pregnant women in the following groups: 1-2) preterm preeclampsia with (n = 8) or without (n = 7) HELLP syndrome; 3) late-onset preeclampsia (n = 8); 4-5) preterm (n = 5) and term (n = 9) controls. TMA slides were stained for phosphorylated Akt-1, ERK1/2, JNK, and p38 kinases, and trophoblastic immunostainings were semi-quantitatively evaluated. BeWo cells were kept in various stress conditions, and the expression of FLT1, GCM1, LEP, and PGF was profiled by qRT-PCR, while Akt-1, ERK1/2, JNK, and p38 kinase activities were measured with phospho-kinase immunoassays. We found that: 1) Placental LEP and FLT1 expression was up-regulated in preterm preeclampsia with or without HELLP syndrome compared to controls; 2) Mean pp38 immunoscore was higher in preterm preeclampsia, especially in cases with HELLP syndrome, than in controls. 3) Mean pERK1/2 immunoscore was higher in preterm preeclampsia with HELLP syndrome than in controls. 4) In BeWo cells, ischemia up-regulated LEP expression, and it increased JNK and decreased ERK1/2 activity. 5) Hypoxia up-regulated FLT1 and down-regulated PGF expression, and it increased ERK1/2, JNK and p38 activity. 6) IL-1ß treatment down-regulated PGF expression, and it increased JNK and p38 activity. 7) The p38 signaling pathway had the most impact on LEP, FLT1 and PGF expression. In conclusion, hypoxic and ischemic stress, along with unknown factors, activates trophoblastic p38 signaling, which has a key role in villous trophoblastic functional changes in preterm preeclampsia. The activation of ERK1/2 signaling may induce additional trophoblastic functional changes in HELLP syndrome, while distinct mechanisms may promote late-onset preeclampsia.

Detection of Chlamydia pneumoniae in liver transplant patients with chronic allograft rejection.

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.

[The molecular diagnostics of viruses]. / Vírusok molekuláris diagnosztikája.

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.

Heparin and liver heparan sulfate can rescue hepatoma cells from topotecan action.

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.

Changes of placental syndecan-1 expression in preeclampsia and HELLP syndrome.

Preeclampsia is characterized by maternal systemic anti-angiogenic and pro-inflammatory states. Syndecan-1 is a cell surface proteoglycan expressed by the syncytiotrophoblast, which plays an important role in angiogenesis and resolution of inflammation. Our aim was to examine placental syndecan-1 expression in preeclampsia with or without hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome. Placentas were obtained from women in the following groups: (1) late-onset preeclampsia (n = 8); (2) early-onset preeclampsia without (n = 7) and (3) with HELLP syndrome (n = 8); (4) preterm controls (n = 5); and (5) term controls (n = 9). Tissue microarrays (TMAs) were constructed from paraffin-embedded placentas. TMA slides were immunostained for syndecan-1 and evaluated using microscopy, virtual microscopy, and semi-automated image analysis. Maternal sera from patients with preeclampsia (n = 49) and controls (n = 32) were immunoassayed for syndecan-1. BeWo cells were treated with Forskolin or Latrunculin B or kept in ischemic conditions. SDC1 expression and syndecan-1 production were investigated with qRT-PCR, confocal microscopy, and immunoassays. Syndecan-1 was localized to the syncytiotrophoblast apical membrane in normal placentas. Syndecan-1 immunoscores were higher in late-onset preeclampsia (p = 0.0001) and early-onset preeclampsia with or without HELLP syndrome (p = 0.02 for both) than in controls. Maternal serum syndecan-1 concentration was lower in preeclampsia (median, 673 ng/ml; interquartile range, 459-1,161 ng/ml) than in controls (1,158 ng/ml; 622-1,480 ng/ml). SDC1 expression and syndecan-1 immunostainings in BeWo cells and syndecan-1 concentrations in supernatants increased during cell differentiation. Disruption of the actin cytoskeleton with Latrunculin B decreased syndecan-1 release, while ischemic conditions increased it. Syncytiotrophoblastic syndecan-1 expression depends on the differentiation of villous trophoblasts, and trophoblastic syndecan-1 release is decreased in preeclampsia and HELLP syndrome. This phenomenon may be related to the disturbed syncytiotrophoblastic cortical actin cytoskeleton and associated with maternal anti-angiogenic and pro-inflammatory states in these syndromes.

Syndecan-1 enhances proliferation, migration and metastasis of HT-1080 cells in cooperation with syndecan-2.

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 activated targets of mTOR signaling pathway are characteristic for PDGFRA mutant and wild-type rather than KIT mutant GISTs.

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.

PP13, maternal ABO blood groups and the risk assessment of pregnancy complications.

BACKGROUND: Placental Protein 13 (PP13), an early biomarker of preeclampsia, is a placenta-specific galectin that binds beta-galactosides, building-blocks of ABO blood-group antigens, possibly affecting its bioavailability in blood. METHODS AND FINDINGS: We studied PP13-binding to erythrocytes, maternal blood-group effect on serum PP13 and its performance as a predictor of preeclampsia and intrauterine growth restriction (IUGR). Datasets of maternal serum PP13 in Caucasian (n = 1078) and Hispanic (n = 242) women were analyzed according to blood groups. In vivo, in vitro and in silico PP13-binding to ABO blood-group antigens and erythrocytes were studied by PP13-immunostainings of placental tissue-microarrays, flow-cytometry of erythrocyte-bound PP13, and model-building of PP13--blood-group H antigen complex, respectively. Women with blood group AB had the lowest serum PP13 in the first trimester, while those with blood group B had the highest PP13 throughout pregnancy. In accordance, PP13-binding was the strongest to blood-group AB erythrocytes and weakest to blood-group B erythrocytes. PP13-staining of maternal and fetal erythrocytes was revealed, and a plausible molecular model of PP13 complexed with blood-group H antigen was built. Adjustment of PP13 MoMs to maternal ABO blood group improved the prediction accuracy of first trimester maternal serum PP13 MoMs for preeclampsia and IUGR. CONCLUSIONS: ABO blood group can alter PP13-bioavailability in blood, and it may also be a key determinant for other lectins' bioavailability in the circulation. The adjustment of PP13 MoMs to ABO blood group improves the predictive accuracy of this test.

Clinical significance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas.

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.

Keratin-positive gastrointestinal stromal tumor of the stomach mimicking gastric carcinoma: diagnosis confirmed by c-kit mutation analysis.

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.

Prognostic significance of high-risk HPV status in advanced cervical cancers and pelvic lymph nodes.

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 presence of human papillomavirus 16 in neural structures and vascular endothelial cells.

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.