CD27/CD70 pathway activation in primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder.

Primary cutaneous CD4+ small or medium T-cell lymphoproliferative disorder (PCSM-LPD) is a clonal T-cell proliferation disease confined to the skin. PCSM-LPD shares expression of T follicular helper (Tfh) cell markers with various mature T-cell lymphomas. However, the benign presentation of PCSM-LPD contrasts the clinical behavior of other Tfh-lymphomas. The aim of our study was to delineate the molecular similarities and differences between PCSM-LPD and other Tfh-derived lymphomas to explain the clinical behavior and unravel possible pathological mechanisms. We performed targeted next-generation sequencing of 19 genes recurrently mutated in T-cell neoplasms in n = 17 PCSM-LPD with high and in n = 21 PCSM-LPD with low tumor cell content. Furthermore, gene expression profiling was used to identify genes potentially expressed in the PD1-positive (PD1+) neoplastic cells. Expression of some of these genes was confirmed in situ using multistain immunofluorescence. We found that PCSM-LPD rarely harbored mutations recurrently detected in other T-cell neoplasms. PCSM-LPD is characterized by the invariable expression of the T-cell-receptor-associated LCK protein. CD70 and its ligand CD27 are co-expressed on PD1+ PCSM-LPD cells, suggestive of autoactivation of the CD70 pathway. In conclusion, PCSM-LPD differs from disseminated lymphomas of Tfh origin by their mutation profile. Activation of CD70 signaling also found in cutaneous T-cell lymphoma represents a potential driver of neoplastic proliferation of this benign neoplasia of Tfh. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Interpreting whole genome and exome sequencing data of individual gastric cancer samples.

BACKGROUND: Gastric cancer is the fourth most common cancer and the second leading cause of cancer death worldwide. In order to understand the genetic background, we sequenced the whole exome and the whole genome of one microsatellite stable as well as one microsatellite unstable tumor and the matched healthy tissue on two different NGS platforms. We here aimed to provide a comparative approach for individual clinical tumor sequencing and annotation using different sequencing technologies and mutation calling algorithms. RESULTS: We applied a population-based whole genome resource as a novel pathway-based filter for interpretation of genomic alterations from single nucleotide variations (SNV), indels, and large structural variations. In addition to a comparison with tumor genome database resources and a filtering approach using data from the 1000 Genomes Project, we performed pyrosequencing analysis and immunohistochemistry in a large cohort of 428 independent gastric cancer cases. CONCLUSION: We here provide an example comparing the usefulness and potential pitfalls of different technologies for a clinical interpretation of genomic sequence data of individual gastric cancer samples. Using different filtering approaches, we identified a multitude of novel potentially damaging mutations and could show a validated association between a mutation in GNAS and gastric cancer.

Lysyl oxidase like-4 monoclonal antibody demonstrates therapeutic effect against head and neck squamous cell carcinoma cells and xenografts.

A new member of the lysyl oxidase (LOX) family, lysyl oxidase-like 4 (LOXL4), is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. A monoclonal antibody (mAb) derived from fusion of Balb/c mouse splenocytes immunized with LOXL4 specific peptide was used to evaluate its therapeutic efficacy in 15 HNSCC cell lines associated with LOXL4 overexpression. For xenograft experiments 41 severe combined immunodeficient (SCID) mice were used to analyze LOXL4-mAb mediated tumor regression. Cell viability was analyzed using cytotoxicity-, and clonogenic-assays. Significant suppression of tumor cell growth was observed in 12 out of 15 (80%) tumor cell lines after 48 hr exposure to the mAb (LD50 of 15 µg/ml to 45 µg/ml). The effect induced by the antibody could be blocked by pre-incubation of the antibody with the peptide used for immunization of the mice and antibody generation, indicating that the effect of the antibody is specific. In mice inoculated with HNSCC cells, i.v. injections of the LOXL4-mAb resulted within 70 days in extensive tumor destruction in all treated animals whereas no tumor regression occurred in control animals. In mice pre-immunized i.v. with LOXL4-mAb and subsequently injected with HNSCC cells, tumor development was considerably delayed in contrast to non LOXL4-mAb pre-immunized animals. These results demonstrate that the LOXL4-mAb has potent antitumor activity and suggest its suitability as a therapeutic immune agent applicable to HNSCC exhibiting tumor specific upregulation of LOXL4.

MET in gastric cancer--discarding a 10% cutoff rule.

AIMS: We aimed to develop a putative predictive biomarker score for future hepatocyte growth factor receptor (MET)-targeted therapy of gastric cancer (GC). METHODS AND RESULTS: MET expression and MET amplification were analysed by immunohistochemistry (IHC) and chromogenic in-situ hybridization (CISH) in 470 GC patients. Immunostaining was documented with the HistoScore. The percentage area of MET-amplified tumour cell clones was assessed by virtual microscopy. The expression of MET was heterogeneous in primary and metastatic GC. Immunostaining intensity (MET-IHC 2+/3+) correlated with MET amplification and a positive MET status was defined by a combination of MET-IHC 2+ or 3+ with MET amplification, or MET-IHC 3+ without MET amplification. The prognostic significance of the MET status was independent from the percentage area of positive tumour cells (e.g. <10 versus ≥10%). MET-positive GCs were microsatellite stable and of KRAS/PIK3CA wild-type. MET-positive GCs had a very poor prognosis, with a median survival of 5.4 months and a hazard ratio of 2.126. CONCLUSIONS: A combination of immunohistochemistry and CISH is suitable to assess MET status. If MET status is used as a predictive biomarker, prospective studies should pay specific attention to adequate tissue sampling, should ignore cutoff values for tumour areas, may consider the KRAS and PIK3CA genotype as negative predictive markers and should carry out the analysis expeditiously.

The role of the antileukoprotease SLPI in smoking-induced human papillomavirus-independent head and neck squamous cell carcinomas.

Recently, we showed that increased SLPI levels prevent human papillomavirus (HPV) infections and metastasis in smoking-induced, non-HPV-driven head and neck squamous cell carcinoma (HNSCC). Here, we focus on the role of SLPI in non-HPV-driven HNSCC, investigating tumor tissue and non-neoplastic mucosa from the same patients and from non-HNSCC patients. Gene and protein expression of SLPI and gene expression of annexin 2 (a SLPI receptor), nicotine receptor (α7AChR) and arylhydrocarbon receptor (AhR) were analyzed in HNSCC patients (20 smokers; 16 nonsmokers). SLPI-results were correlated with the patients' HPV status. Non-neoplastic mucosa of HNSCC patients and normal mucosa from non-HNSCC individuals (18 smokers; 20 nonsmokers) was analyzed for the same parameters. Tissue of the inferior turbinate (n = 10) was incubated with nicotine for analysis of the same genes. SLPI gene expression in tumor tissue was 109.26 ± 23.08 times higher in smokers versus nonsmokers. Non-neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49 ± 1.89-fold non-HNSCC; 18.02 ± 3.93-fold HNSCC patients). Annexin 2 gene expression was also increased in smokers. SLPI data were corroborated by immunohistochemistry. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r(2) = 0.15, p < 0.001) was shown ex vivo. Nicotine and smoking increased α7AChR and AhR gene expression. Five patients, showing no/low SLPI expression, were HPV16-positive. A significant correlation between smoking and SLPI expression in tumors and to our knowledge for the first time in mucosa of HNSCC and non-HNSCC patients was established. Together with the finding that all patients with HPV infection showed no/low SLPI expression, these data support our intriguing hypothesis that smoking induced upregulated SLPI prevents HPV infections.

MDM2 amplification is rare in gastric cancer.

The MDM2 proto-oncogene (MDM2) is a primary negative regulator of p53. The latter is frequently mutated in gastric cancer (GC). In the present study, we aimed to validate gene amplification, protein expression, and the putative tumor biological function of MDM2 in a well-characterized Western GC cohort. MDM2 amplification and protein expression were studied in a cohort of 327 GCs by fluorescence in situ hybridization (FISH) and immunohistochemistry. Gene amplification and protein expression were correlated with diverse clinicopathological patient characteristics including patient outcome. Immunohistochemically, 97 GCs (29.7%) were categorized as MDM2 positive and 230 GCs (70.3%) as negative. An amplification of MDM2 was found in 11 (3.4%) cases without evidence of intratumoral heterogeneity. Nine of these eleven (81.8%) cases showed MDM2 protein expression. MDM2 amplification correlated significantly with MDM2 protein expression (p < 0.001). On a case-by-case analysis, MDM2-amplified cases showed varied histological phenotypes and were most commonly microsatellite stable; EBV, HER2, and MET negative; and FGFR2 positive. A single case harbored both, MDM2 amplification and TP53 mutation. MDM2 amplification and MDM2 expression, respectively, did not correlate with overall or tumor-specific survival. Our targeted analysis of MDM2 in a well-characterized cohort of GC patients showed that MDM2 amplification is rare, of no specific histological phenotype, and may not be always mutually exclusive with TP53 mutations. Given the low number of cases, currently, no diagnostic or therapeutic recommendation related to MDM2 amplification can be given for GC of Western origin.

Absence of BRAF mutation in pediatric and adolescent germ cell tumors indicate biological differences to adult tumors.

The V600E mutation of the BRAF gene has been reported to be associated with poor prognosis of germ cell tumors in adult patients. We analyzed the mutational status of the BRAF and KRAS gene as well as MLH1 and MSH6 expression as surrogate markers for microsatellite instability in 70 pediatric germ cell tumors. Neither BRAF and KRAS mutations nor loss of MLH1 and MSH6 expression were found. Our data provide further evidence for patient age related biological differences in germ cell tumors and demonstrate that prognostic biomarkers cannot necessarily be transferred from one age group to the other.

FGFR2 overexpression and compromised survival in diffuse-type gastric cancer in a large central European cohort.

The significance of fibroblast growth factor receptor 2 (FGFR2) in gastric cancer (GC) has been studied predominantly in Asian patient cohorts. Data on White patients are scarce. Here, we aimed to independently validate the expression and putative tumor biological significance of FGFR2 in a large non-Asian GC cohort. Immunohistochemistry (IHC) was performed on large-area tissue sections from 493 patients with GC and evaluated using the HScore. GCs with moderate and strong FGFR2 expression were studied for Fgfr2 amplification using chromogenic in situ hybridization (CISH). Median overall survival was determined using the Kaplan-Meier method. The majority [240 (99.1%)] of FGFR2-positive GCs showed a variable combination of staining intensities with marked intratumoral heterogeneity, including weak [198 (40.2%) cases], moderate [145 (29.4%)], and strong [108 (21.9%)] staining in diverse combinations. 250 (50.9%) GCs expressed no FGFR2. Fgfr2 gene amplification was found in 40% of selected cases with high protein expression and was also heterogeneous at the cell level. FGFR2 protein expression did not correlate with patient survival in the entire cohort However, using different cutoff values, a negative correlation between FGFR2-expression and patient outcome was found for diffuse-type GC. FGFR2 expression was associated with a lower tumor grade and intestinal phenotype (p≤0.0001). FGFR2-positive diffuse-type GCs classify a small subset of patients with a poor tumor specific survival (5.29±1.3 vs. 14.67±1.9 months; p = 0.004).

Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine.

BACKGROUND: Cancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC. METHODS: The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3-10 tumor samples/patient) of the discovery cohort. RESULTS: In total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53-91% were not present in each patient's sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs. CONCLUSIONS: Neutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

p53 immunostaining cannot be used to predict TP53 mutations in gastric cancer: results from a large Central European cohort.

Four molecular subgroups of gastric cancer (GC) have been proposed, ie, Epstein-Barr virus (EBV)-positive, microsatellite instable, chromosomal instable (CIN), and genomically stable GC. Based on the complex relationship between chromosomal instability and TP53 mutational status, we hypothesized that the typical clinicopathological characteristics caused by chromosomal instability are correlated with the p53 expression that is detected by immunohistochemistry. Four hundred sixty-seven whole-tissue sections of patients with therapy-naive GC were stained with anti-p53 antibody. The histoscore and staining pattern were analyzed for each slide. Different algorithms of immunohistochemistry evaluation were formed and correlated with clinicopathological characteristics. The algorithms were validated by assessing the mutational status of TP53 in 111 cases. Four hundred forty-two GCs were p53 positive, and 25 were negative, including 414 GCs with a homogeneous pattern and 53 GCs with a heterogeneous staining pattern. There was no correlation with overall or tumor-specific survival. In comparison with clinicopathological characteristics, the algorithm high versus low showed correlations with microsatellite instability, hepatocyte growth factor receptor (MET), and TP53 mutational status. The algorithm Q1/Q4 versus Q2/Q3 appeared to be correlated with the phenotype as per the Laurén classification, microsatellite instability, EBV status, and p53 expression pattern. The algorithm <90% = 0 and <50% = 3+ versus ≥90% = 0 or ≥50% = 3+ showed correlations with the EBV status, microsatellite instability, grading, and p53 expression pattern. The algorithm homogeneous versus heterogeneous did not correlate with any clinicopathological characteristic. Our results showed that the immunohistochemistry of p53, TP53 mutational status, and CIN subtype were connected. However, different algorithms for p53 immunohistochemical evaluation cannot be used to predict TP53 mutations in CIN GCs in individual cases.

SOX gene expression in human osteoarthritic cartilage.

OBJECTIVE: While the developmental role of the SOX transcription factors in fetal chondrocyte differentiation is well documented, much less is known about the expression of SOX family members in normal and osteoarthritic adult cartilage. Therefore, the aim of the present study was to present a thorough analysis of SOX gene expression in normal and osteoarthritic human adult cartilage. METHODS: RNA from normal and osteoarthritic knee cartilage from human adults was analyzed by gene expression profiling using GeneChip technology (Affymetrix) and quantitative real time PCR. RESULTS: Most members of the SOX transcription factor family showed no or very low expression levels in normal and osteoarthritic cartilage from adults. In contrast, SOX9 expression was fairly high in normal cartilage, amounting to approximately 20% of GAPDH levels. SOX9 transcript levels were substantially reduced in osteoarthritis. SOX6 levels were reduced, albeit starting from a low basis expression in normal tissue. CONCLUSION: The presented data indicate that the role of the SOX transcription factor family in adult human cartilage is most probably restricted to a few members, with SOX9 being the most prominent. Furthermore, the reduction of SOX9 and SOX6 transcript levels in osteoarthritic chondrocytes might be responsible for the loss of phenotypic stability of osteoarthritic chondrocytes.

miRNA-expression in tonsillar squamous cell carcinomas in relation to HPV infection and expression of the antileukoproteinase SLPI.

The aim of this study was to determine if micro-(mi-)RNAs are involved in the previously reported inverse correlation between the antileukoproteinase SLPI, HPV, and smoking habit of head and neck squamous cells carcinoma (HNSCC) patients. HPV-status and SLPI-protein expression were determined in tonsillar SCC (TSCC; n=126). Differentially expressed miRNAs dependent on HPV-status and SLPI-expression were detected by microarray; possible binding-sites in SLPI- and HPVE6-mRNAs were determined in silico. Survival rates were estimated testing prognostic values of HPV-status, SLPI- and miRNA-expression. miRNA-array identified 24 up-regulated and 10 down-regulated miRNAs in HPV-positive versus HPV-negative TSCC (p<0.01; HPV-positivity: 42.1%). HPV-positivity resulted in two up-regulated miRNAs in SLPI-positive TSCC. Of 16 further miRNAs, eight miRNAs were up- and eight were down-regulated in SLPI-negative TSCC. RT-q-PCR-validation of the four most differentially expressed miRNAs showed that miR-363 is expressed strongest in SLPI-negative/HPV-positive TSSC. In silico-analysis of all differentially expressed miRNAs identified miR-363, miR-210, miR-130a, and miR-181a with possible binding sites in the HPV16-E6-mRNA, but none were predicted in the SLPI-mRNA. HPV-positivity, low SLPI-levels and high miR-363-levels are significantly associated with better survival rates. The data presented here show that miR-363 is associated with HPV-positive/SPLI-negative TSCC. The prognostic value of miR-363 suggests a role in the assumed inverse correlation of smoking and SPLI-expression in the mode of HPV-infections in tonsillar but possibly also other HNSCC.

Clinicopathologic Characteristics of Microsatellite Instable Gastric Carcinomas Revisited: Urgent Need for Standardization.

Microsatellite instable gastric cancer (MSI-GC) is a specific molecular subtype of GC. We studied the phenotypes, genotypes, and clinicopathologic characteristics of MSI-GC in a white GC cohort and compared our findings with an extended literature review. The study cohort consisted of 482 patients. Specimens were available from 452 cases and were used for immunostaining (MLH1, PMS2, MSH2, MSH6) and molecular biological analyses (BAT-25, BAT-26, NR-21, NR-24, NR-27; Epstein-Barr virus in situ hybridization). Thirty-four (7.5%) GCs were MSI. Loss of MLH1 and/or PMS2 was found in 30 (88%) MSI-GC, 3 (9%) showed loss of MSH2 and/or MSH6. One (3%) MSI-GC was identified only by molecular biological testing. A single case was heterogeneous and contained microsatellite-stable and instable tumor areas. Twenty-one (62%) MSI-GCs showed unusual histologic features. MSI-GC was not found in diffuse-type or Epstein-Barr virus-positive GC. MSI-GC was significantly more prevalent in elderly patients, distal stomach, and was associated with a significantly lower number of lymph node metastases and a significantly better overall and tumor-specific survival. MSI-GC constitutes a small but relevant subgroup of GC with distinct clinicopathologic characteristics. Our literature review illustrates the shortcomings of missing standardized testing algorithms with prevalences of MSI-GC ranging from 0% to 44.5%. Future studies should test the hypothesis that patients with MSI-GCs may not need adjuvant/perioperative chemotherapy. However, this will require a standardized, quality-controlled diagnostic algorithm of MSI for GC.

Complex APC germline mutation associated metaplasia and intraepithelial neoplasia (CAM-IEN) of the gallbladder.

Preneoplasic and neoplastic changes of the gallbladder of patients with a familial adenomatous polyposis (FAP) are rare, and very little is known about their incidence in patients with an attenuated FAP. We herein report on a unique case of a woman with an attenuated FAP who shows eight distinct, partially preneoplastic differentiation patterns within the gallbladder mucosa, which are: (1) regular gallbladder epithelium, (2) low grade biliary intraepithelial neoplasia, (3) papillary adenoma, (4) Paneth cell metaplasia, (5) goblet cell metaplasia, (6) pancreatic metaplasia, (7) pseudopyloric metaplasia, and (8) neuroendocrine differentiation. Moreover, this is the first case of a KRAS mutation in a gallbladder adenoma of a patient with an APC germline mutation, which is highly suggestive of an early event of malignant transformation. As a consequence of our findings, clinicians should draw special attention to the gallbladder of FAP patients, and a simultaneous protective cholecystectomy of FAP patients, which undergo colectomy and show conspicuous changes of the gallbladder mucosa, should be performed in these patients in order to eliminate the risk of a synchronous or metachronous gallbladder neoplasia.

Varying Mutational Alterations in Multiple Primary Melanomas.

In melanoma, the mitogen-activated protein (MAP) kinase pathway plays a crucial oncogenic role. Recent studies identified additional genetic alterations, eg, TERT-promoter mutations. Up to 8% of melanoma patients present with multiple primary melanomas (MPMs). The pathogenesis is not fully understood, and data on the genetic diversity of MPMs are limited. To identify putative diagnostic and therapeutic consequences, we assessed the mutational status of the BRAF and NRAS genes and TERT promoter in patients with MPMs. The study cohort consisted of 96 patients with 237 malignant melanomas. The BRAF, NRAS, and TERT-promoter genotypes were assessed in all MPMs and were correlated with patients' clinicopathological characteristics. BRAF mutations were found in 84 melanomas (35.4%), NRAS mutations, in 33 (14.0%); and TERT-promoter mutations, in 112 (47.3%). Mutation patterns were concordant between first and subsequent primary tumors in 23.9% of patients and were discordant in 61.4% of patients. The genetic alterations were partially different in 14.7% of patients. By Cox regression analysis, only the NRAS mutation had a significant negative prognostic impact on time to progression to stage III (P = 0.016) and on distant metastasis-free survival (P = 0.032). In the majority of primary melanomas in patients with MPMs, BRAF, NRAS, and TERT-promoter genotypes were discordant. Thus, molecular testing for targeted therapy should be performed on metastatic tissue and not on primary tumors.

The human chondrosarcoma HCS-2/8 cell line is responsive to BMP-7, but not to IL-1beta.

Cultures of primary chondrocytes as in vitro model systems for studying the cellular behavior of chondrocytes are notoriously difficult to cultivate and propagate. One way to circumvent these problems appears to be the use of immortalized/immortal chondrocytic cell lines. In the present study, we were interested whether the chondrosarcoma derived HCS-2/8 cells are suitable for studying major cellular reaction pattern in response to key anabolic (BMP-7) and catabolic (IL-1beta) factors. Therefore, we used cDNA array and real-time PCR technology in order to evaluate gene expression triggered by stimulation with IL-1beta (0,1-100 ng/ml) and BMP-7 in confluent monolayer cultures. HCS-2/8 cells hardly responded to IL-1beta, but showed good responsiveness to BMP-7. We found 12 genes up- and 17 significantly down-regulated by BMP-7 (out of 340 investigated genes). Besides the expected activation of anabolic genes chondrocytic cells after BMP-stimulation try to neutralize activation of the BMP-signalling cascade by expressing intra- and extracellular BMP-antagonists. Chondrosarcoma derived cell lines are a potential substitute for primary articular chondrocytes promising consistent expression of a differentiated chondrocyte phenotype with sufficient proliferative capacity. However, as shown by this study one needs to carefully select the cell line depending on the effects which one intends to study. In this respect, HCS-2/8 cells are a validated tool for studying BMP-effects on chondrocytes, but not e.g. effects of interleukin-1.

Geographical and anatomical influences on human papillomavirus prevalence diversity in head and neck squamous cell carcinoma in Germany.

The increased knowledge regarding HPV-infections in head and neck squamous cell carcinoma (HNSCC) has unexpectedly contributed to several uncertainties related to i) prevalence diversities depending on tumour site and geographical origin of the patients, ii) proportion of HPV-driven tumours among HPV-DNA-positive cases, and iii) identification of patients with HPV-attributed survival benefit. To investigate this heterogeneity, we analysed 307 HNSCC cases (tonsillar, n=135; non-tonsillar, n=172) from eight health care centers mostly from Northern Germany and determined HPV-DNA/mRNA and p16INK4A-status and combined results with the patient outcome. Overall HPV-DNA prevalence rate was 23.5% (72/307); attributed to: 43.7% (59/135) and 7.6% (13/172) tonsillar and non-tonsillar cases, respectively. Among these, 96.6% tonsillar and 38.5% non-tonsillar SCC were HPV-mRNA-positive. Although the study cohort was composed of patients from regions of rather close proximity, prevalence rates showed diversities of up to 40% in HNSCC subsite analysis with the lowest prevalence for tonsillar SCC in metropolitan areas (22.2%) vs. 50.9% in rural areas. Survival analysis identified p16INK4A alone as strongest predictor, followed by HPV-DNA-status alone or in combination with p16INK4A. This survival benefit was shown for tonsillar and non-tonsillar cases. Smoking significantly correlated with HPV-status, however, it does not influence survival when stratified for HPV. In conclusion, the data emphasize the urge for further data on HPV-infection in HNSCC to, e.g. clarify to what extent survival benefits of p16INK4A-positive patients are truly attributed to HPV-infections.

Bone morphogenetic protein-mediating receptor-associated Smads as well as common Smad are expressed in human articular chondrocytes but not up-regulated or down-regulated in osteoarthritic cartilage.

Bone morphogenetic proteins (BMPs) are supposed to be important for cartilage matrix anabolism. In this study, we investigated whether the intracellular mediators of BMP activity, Smads 1, 4, 5, and 8, are expressed in normal human articular chondrocytes in vivo and in vitro and whether alterations in expression and distribution pattern are found in osteoarthritic cartilage or in vitro after stimulation with interleukin (IL)-1, because down-regulation of these mediators could be responsible for the decrease of anabolic activity in osteoarthritic cartilage. RNA was isolated from normal and osteoarthritic human knee cartilage and analyzed by (quantitative) polymerase chain reaction (PCR) technology. Articular chondrocytes were cultured in alginate beads and short-term high-density monolayer cultures with and without stimulation by IL-1. In addition, immunolocalization of the receptor-associated Smads (R-Smads) was performed on sections of normal and diseased articular cartilage. Reverse-transcription (RT)-PCR analysis showed a moderate expression of all Smads investigated in normal, early degenerative, and late stage osteoarthritic cartilage. Immunolocalization detected the R-Smads in most chondrocytes on the protein level in all specimen groups investigated. In vitro, the Smads were also expressed and partly up-regulated by Il-1beta in alginate bead culture. Of note, for Smad 1, two truncated splice variants were expressed by articular chondrocytes missing exon 4 as well as exons 3 and 4. Our study showed that BMP-receptor Smads 1, 5, and 8 as well as common Smad (C-Smad) 4 are expressed and present in human normal and osteoarthritic articular chondrocytes corroborating the importance of BMPs and BMP signaling for articular cartilage. This study is the first to describe splicing variants for Smad 1. Smads 1, 4, and 5 are up-regulated in vitro by Il-1beta, suggesting a linkage of the Il-1 and BMP-signaling pathways within the chondrocytes. None of the Smads were grossly up- or down-regulated in osteoarthritic chondrocytes, suggesting that differences in overall expression levels of the investigated Smad proteins are not relevant for metabolic activity of articular chondrocytes in vivo.

Quantification of mRNA expression levels in articular chondrocytes with PCR technologies.

Unlike any other technology in molecular biology, the polymerase chain reaction (PCR) has changed the technological armamentarium of molecular scientists working on cartilage, in terms of outstanding sensitivity and accuracy. Four approaches to determine mRNA expression levels by PCR amplification of specific cDNA sequences are currently in use and are discussed in this chapter: conventional PCR with end-point determination, conventional PCR in the logarithmic amplification phase, conventional PCR using internal competitive DNA fragments, and real-time PCR as offered by TaqMan technology and others. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels within cartilage and cultured chondrocytes, as in other tissues and cell types. This technology offers outstanding sensitivity and accuracy in terms of determination of the amount of cDNA molecules. However, this method cannot account for factors such as efficiency of RNA isolation and reverse transcription conditions. Thus, normalization of the acquired data is required, with all its limitations as described.