Tibor Görögh

p16INK4a overexpression predicts translational active human papillomavirus infection in tonsillar cancer.

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin‐dependent kinases p16INK4a. In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type‐specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence‐labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT‐PCR were performed, and for p16INK4a detection, immunohistochemistry was performed. With 21/39 (53%) HPV‐positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16INK4a staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16INK4a expression was of statistical significance (p = 0.02). Kaplan‐Meier estimations revealed better survival outcome for patients with HPV‐positive tumors with detectable E6/E7 mRNA and p16INK4a overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16INK4a staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16INK4a as a surrogate marker with the potential to impact the standard of care of HPV DNA‐positive head and neck carcinomas.

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in patients with head‐and‐neck squamous‐cell carcinoma

Human papillomaviruses (HPVs) have been recognized as an essential pathogenic factor in anogenital cancer. HPV DNA has also been found in a subgroup of head‐and‐neck squamous‐cell carcinomas (HNSCCs), and a causative role of the virus in the development of these tumors has been suggested by the concomitant inactivation of the tumor‐suppressor protein pRb. Using 4 second‐generation ELISAs, we found antibodies against at least 1 of the oncoproteins E6 and E7 of the high‐risk HPV types 16 and 18 in 11 of 92 sera (12%) taken from HNSCC patients at or near diagnosis, in 1 of 52 sera (2%) taken from HNSCC patients >6 months after diagnosis and in 10 of 288 sera (3.5%) taken from sex‐ and age‐matched healthy control individuals of the normal population. In 11 of the 12 seropositive HNSCC cases, antibodies were directed against HPV16 proteins. In patients, the HPV16 antibodies were mostly of high titer, and in 6 cases, antibodies against both HPV16 oncoproteins were present. Seven of the 8 HPV16 antibody–positive sera from the control group were of low titer, and none of the 10 antibody‐positive sera reacted with both oncoproteins of the same HPV type. The HPV type of the antigens detected by the antibodies in HNSCC patients correlated well with that of the HPV DNA found in the tumor. Of 19 patients known to have HPV16 DNA–positive tumors, 7 (37%) also had HPV16 E6 and/or E7 antibodies. Our finding suggests that the antibodies were formed in an immune response against HPV E6 and E7 proteins expressed in the HNSCC and thus strongly supports the concept of a biologically active role of HPV in the development of a subgroup of HNSCC. Int. J. Cancer 85:815–818, 2000.

HPV DNA, E6*I-mRNA expression and p16INK4A immunohistochemistry in head and neck cancer – How valid is p16INK4A as surrogate marker?

It has been proposed that p16(INK4A) qualifies as a surrogate marker for viral oncogene activity in head and neck cancer (HNSCC). By analyzing 78 HNSCC we sought to validate the accuracy of p16(INK4A) as a reliable marker of active HPV infections in HNSCC. To this end we determined HPV DNA (HPVD) and E6*I mRNA (HPVR) expression status and correlated these results with p16(INK4A) staining. In tonsillar SCC 12/20 were HPVD+ and 12/12 of these showed active HPV infections whereas in non-tonsillar SCC 10/58 were HPVD+ and 5/10 showed active HPV infections. Thus, we prove about 8% of non-tonsillar SCC to be also correlated with HPV-associated carcinogenesis. Strikingly, 3/14 (21.4%) of tonsillar and non-tonsillar HPVD+/HPVR+ cases did not show p16(INK4A) overexpression and these cases would have been missed when applying initial p16(INK4A) staining only. However, in 13 cases negative for HPV, DNA p16(INK4A) was overexpressed. In conclusion, our data confirm tonsillar SCC to be predominantly but not only associated with active HPV infections. Furthermore, our data show that p16(INK4A) overexpression is not evident in a subgroup of HNSCC with active HPV infection. Definitive HPV data should therefore be utilized in diagnostics and treatment modalities of HPV positive and HPV negative HNSCC patients, resulting in a paradigm shift regarding these obviously different tumor entities.

Metalloproteinases and their inhibitors: influence on tumor invasiveness and metastasis formation in head and neck squamous cell carcinomas.

Matrix metalloproteinases (MMPs) play an important role in tumor invasiveness. This study investigates the expression status of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in head and neck squamous cell carcinomas (HNSCC).

HPV status in patients with head and neck of carcinoma of unknown primary site: HPV, tobacco smoking, and outcome.

OBJECTIVES Infection with human papillomavirus (HPV) is linked to oropharyngeal cancer. This analysis investigated possible associations between HPV status, smoking history and survival outcome in patients with neck metastasis and carcinoma of unknown primary (CUP). MATERIALS AND METHODS Registries at the Universities of Hamburg and Kiel were searched for patients with CUP diagnosed from 2002 to 2011 who had formalin-fixed and paraffin-embedded metastatic lymph node samples available. All patients underwent routine diagnostic procedures to establish the primary site and received radiotherapy (60Gy using conventional fractionation) with or without concurrent cisplatin-based chemotherapy depending on disease extent. Genotyping was performed using polymerase chain reaction; p16([INK4a]) expression was assessed using immunohistochemistry. RESULTS Sixty-three patients were included; 23 (37%) had HPV DNA/p16+ samples and 40 (63%) were negative for either/both markers. A high proportion of patients had a history of tobacco smoking; significantly fewer patients with HPV+/p16+ samples were smokers than those who were negative for either/both markers (61% vs. 90%, respectively; p = 0.0067). There were no statistically significant differences between overall or recurrence-free survival in HPV+/p16+ patients vs. those negative for either/both markers. Overall survival appeared to be superior in patients with <10 pack-years smoking history and HPV+/p16+ disease. CONCLUSIONS This study, the largest to date investigating HPV status in head and neck CUP, identified HPV and p16 overexpression in over one-third of patients. Tobacco smoking history appeared to affect survival in HPV+/p16+ patients. Smoking status should be considered as a prognostic factor in patients with CUP, along with HPV DNA status.

The role of RANTES in nasal polyposis.

Background Characteristic infiltrates of eosinophils are a hallmark of nasal polyps (NPs). Several studies suggest that members of the CC chemokine family may be involved in this process. RANTES (regulated on activation, normal t-cell-expressed and secreted) is a member of the CC chemokine family with chemotactic activity on mainly eosinophils and T lymphocytes. Thus, RANTES is an interesting target for the recruitment of eosinophils and T lymphocytes into the nose. The degree of the tissue eosinophilia has been reported to correlate with the severeness of the symptomatology of the disease and the extension on the lower respiratory tract, as well as with the probability of the recurrence of NPs. Therefore, we hypothesized that high numbers of eosinophils correlate with high levels of RANTES and that associated atopic diseases modify this correlation. Methods Total RNA was extracted from NP homogenates, reverse transcribed and RANTES mRNA expression analyzed using semiquantitative reverse transcription polymerase chain reaction and Northern blot analysis. Histological studies divided NPs in an eosinophilic and low eosinophilic group. Additionally, RANTES protein concentration was measured in homogenates by a RANTES-specific enzyme-linked immunosorbent assay. Results This study has clearly shown that RANTES is expressed and secreted in NPs. The group with a high tissue eosinophilia had a significant higher RANTES gene expression and protein production than NPs without tissue eosinophilia. The isolated coincidence of acetylsalicyl acid intolerance with chronic hyperplastic sinusitis/NP additionally increased significantly the RANTES amounts in NPs. Conclusion Increased RANTES leads to increased tissue eosinophilia. Associated acetylsalicylic acid intolerance seems to enhance the amount of RANTES in NPs and might explain in part the more severe clinical course in those patients. Thus, RANTES appears to play an important role in mobilization of eosinophils into the local inflamed tissue.

Selective upregulation and amplification of the lysyl oxidase like‐4 (LOXL4) gene in head and neck squamous cell carcinoma

Members of the lysyl oxidase family (LOX) are copper and lysyl‐tyrosine quinone cofactor‐containing amine oxidases that are important for the assembly and maintenance of components of the extracellular matrix. Our previous results demonstrated that a novel member, LOXL4, is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. Results of the current study showed overexpression of the LOXL4 transcript in 74% (46 of 62) of invasive HNSCC tumours and 90% of both primary and metastatic HNSCC cell lines. Significant correlation was found between LOXL4 expression and local lymph node metastases versus primary tumour types (p < 0.01) and higher tumour stages (p < 0.01). Immunocytochemistry demonstrated cellular overexpression of the LOXL4 protein that correlated with the increased mRNA transcription in HNSCC cells. HNSCC cell lines displayed in significant subset of nuclei increased copies of the LOX4 gene locus on chromosome 10q24, demonstrated by fluorescence in situ hybridization (FISH). Extensive metaphase cytogenetic analysis was performed on UTSCC19A cells, identifying an isochromosome i(10)(q10). Taken together, these results highlight LOXL4 expression as a distinctive trait and suggest a functional role for LOXL4 in the molecular pathogenesis of invasive head and neck carcinomas. Copyright

Human papillomaviruses in lymph node neck metastases of head and neck cancers.

Conclusion The results of this study corroborate earlier findings that human papillomavirus (HPV)16 is the most prevalent type of HPV in squamous cell carcinomas of the head and neck (SCCHNs) and reinforce a possible influence of HPV on SCCHN progression by showing that the majority of HPV-positive patients harbor HPV16 (or HPV33) both in their primary tumors and in lymph node neck metastases (LNNMs). Objective HPVs are causally associated with carcinomas of the uterine cervix and have also been linked to a subset of SCCHNs. In order to further investigate the predicted causative role of HPV in SCCHNs, we analyzed pairs of primary tumors and LNNMs or LNNMs alone for the presence of HPV DNA using polymerase chain reaction (PCR). Material and methods DNA was extracted from fresh frozen tissue samples of primary tumors and the corresponding LNNMs of 18 patients and from LNNMs alone in 17 patients. For the detection and typing of HPV, PCR was performed using both type-specific and consensus primer pairs, followed by Southern hybridization and, in selected cases, sequencing of the PCR products. Results Of the 35 patients investigated, 22 (63%) were found to have HPV DNA in their tumors: HPV16 DNA in 21 cases and HPV33 in 1. The highest HPV prevalence was detected in tumors of Waldeyers tonsillar ring (8/9 patients; 89%). Of the 18 patients in whom primary tumors and LNNMs were analyzed, 7 (39%) were HPV-positive in both samples (HPV16, n=6; HPV33, n=1), in 3 (17%) the primary tumors were HPV-negative and the LNNMs HPV16-positive and in 1 (5.5%) the primary tumor contained HPV16 and the LNNM was negative. Interestingly, of the 7 patients in whom LNNMs had been detected only several months after diagnosis and treatment of the primary tumors, only 1 showed infection with HPV (HPV33).

Human papillomavirus infection in head and neck cancer: The role of the secretory leukocyte protease inhibitor

We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non-malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases.

Implications of galactocerebrosidase and galactosylcerebroside metabolism in cancer cells

Galactosylcerebroside is known to be overexpressed upon the cellular surface of a variety of cancers. In squamous cell carcinomas of the head and neck, one explanation for galactosylcerebroside accumulation has been identified as a transcriptional repression of the galactocerebrosidase gene. Galactocerebrosidase is the enzyme responsible for degrading galactosylcerebroside to ceramide. Ceramide is an important apoptosis activator, whereas galactosylcerebroside functions as an inhibitor. A shift of the ceramide metabolism balance in favor of glycosylated forms has been identified as a mechanism of drug resistance for several antineoplastic agents. Our review elaborates on possible explanations for galactocerebrosidase suppression and on other explanations for increased glycosphingolipid concentration within cancer cell membranes. Furthermore, conjecturable influences of a repressed galactocerebrosidase expression on tumor biology are to be explained. The inhibiting transcription factors YY1 and AP2 have been identified as potential galactocerebrosidase gene suppressors. The resulting accumulation of galactosylcerebroside promotes a reduction of cellular adhesion and inhibits apoptosis, leading to increased cellular growth, migration and prolonged cell survival contributing to carcinogenesis.