Mieke Henfling

Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Detailed analysis of cell cycle kinetics upon proteasome inhibition

We have studied specific effects of proteasome inhibition on cell cycle progression. To this end, the protease inhibitors MG115, calpain inhibitor I, and calpain inhibitor II, which display differential inhibitory effects on proteasomes, were used. Cell kinetic studies using bromodeoxyuridine pulse labeling revealed a complete block of G1/S and metaphase transitions and a delayed progression through S phase in cell cultures treated with 54 microM of MG115. Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetics. Calpain inhibitor II and MG2M, which is a structural analogue of MG115, had no effect on the cell cycle. The inhibitory effect of MG115 treatment was reversible, because the cell cycle was immediately resumed when the MG115-containing culture medium was replaced by fresh culture medium. Because ubiquitinated proteins accumulated after MG115 treatment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked. By comparison of biochemical and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics.

Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

HPV‐related HNSCC generally have a better prognosis than HPV‐negative HNSCC. However, a subgroup of HPV‐positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16‐positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD‐SCC‐2, UM‐SCC‐047, UM‐SCC‐104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT‐PCR and DIPS‐PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration‐specific staining patterns and signals indicating transcriptional activity using FISH. APOT‐ and DIPS‐PCR identified integration‐derived fusion products in six cell lines and only episomal products for UM‐SCC‐104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16‐positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

CD44 and OTP Are Strong Prognostic Markers for Pulmonary Carcinoids

Purpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histologic classification in typical and atypical carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, that is, orthopedia homeobox (OTP), CD44, and rearranged during transfection (RET). Experimental Design: These genes were analyzed in 56 frozen carcinoids by quantitative real-time PCR (qRT-PCR). RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was conducted on 292 carcinoids. Results: Low mRNA expression levels of CD44 (P = 1.8e−5) and OTP (P = 0.00054), and high levels of RET (P = 0.025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP but not for RET. Within all carcinoids as well as atypical carcinoids, absence of CD44 protein was significantly associated with low 20-year survival (P = 0.00014 and 0.00013, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (P = 5.2−6). Multivariate analyses revealed that age at diagnosis, histopathology, stage, and cytoplasmic OTP immunoreactivity were independent predictors of prognosis. Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories. Clin Cancer Res; 19(8); 2197–207. ©2013 AACR.

MEN1 Gene Mutation and Reduced Expression Are Associated With Poor Prognosis in Pulmonary Carcinoids

CONTEXT MEN1 gene alterations have been implicated in lung carcinoids, but their effect on gene expression and disease outcome is unknown. OBJECTIVE Our objective was to analyze MEN1 gene and expression anomalies in lung neuroendocrine neoplasms and their correlations with clinicopathologic data and disease outcome. DESIGN We examined 74 lung neuroendocrine neoplasms including 58 carcinoids and 16 high-grade neuroendocrine carcinomas (HGNECs) for MEN1 mutations (n = 70) and allelic losses (n = 69), promoter hypermethylation (n = 65), and mRNA (n = 74) expression. Results were correlated with disease outcome. RESULTS MEN1 mutations were found in 7 of 55 (13%) carcinoids and in 1 HGNEC, mostly associated with loss of the second allele. MEN1 decreased expression levels correlated with the presence of mutations (P = .0060) and was also lower in HGNECs than carcinoids (P = .0024). MEN1 methylation was not associated with mRNA expression levels. Patients with carcinoids harboring MEN1 mutation and loss had shorter overall survival (P = .039 and P = .035, respectively) and low MEN1 mRNA levels correlated with distant metastasis (P = .00010) and shorter survival (P = .0071). In multivariate analysis, stage and MEN1 allelic loss were independent predictors of prognosis. CONCLUSION Thirteen percent of pulmonary carcinoids harbor MEN1 mutation associated with reduced mRNA expression and poor prognosis. Also in mutation-negative tumors, low MEN1 gene expression correlates with an adverse disease outcome. Hypermethylation was excluded as the underlying mechanism.

An exploration of pathways involved in lung carcinoid progression using gene expression profiling

Pulmonary carcinoids comprise a well-differentiated subset of neuroendocrine tumors usually associated with a favorable prognosis, but mechanisms underlying disease progression are poorly understood. In an explorative approach to identify pathways associated with progression, we compared gene expression profiles of tumors from five patients with a favorable and five with a poor disease outcome. Differentially expressed genes were validated using quantitative real-time PCR on 65 carcinoid tumors, in combination with survival analysis. One of the identified pathways was further examined using immunohistochemistry. As compared with other chromosomal locations, a significantly higher number of genes downregulated in carcinoids with a poor prognosis were located at chromosome 11q (P = 0.00017), a region known to be frequently lost in carcinoids. In addition, a number of upregulated genes were found involved in the mitotic spindle checkpoint, the chromosomal passenger complex (CPC), mitotic kinase CDC2 activity and the BRCA-Fanconi anemia pathway. At the individual gene level, BIRC5 (survivin), BUB1, CD44, IL20RA, KLK12 and OTP were independent predictors of patient outcome. For survivin, the number of positive nuclei was also related to poor prognosis within the group of carcinoids. Aurora B kinase and survivin, major components of the CPC, were particularly upregulated in high-grade carcinomas and may therefore comprise therapeutic targets for these tumors. To our knowledge, this is the first expression profiling study focusing specifically on pulmonary carcinoids and progression. We have identified novel pathways underlying malignant progression and validated several genes as being strong prognostic indicators, some of which could serve as putative therapeutic targets.

DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis

The cytokeratin 8/18 (CK8/18) cytoskeleton network is an early target for caspase cleavage during apoptosis. Recent reports suggest that the highly conserved and ubiquitous death effector domain containing DNA binding protein (DEDD) plays a role in the recruitment of procaspase-9 and -3 at this CK8/18 scaffold. DEDD interacts with both the CK8/18 intermediate filament network and procaspase-3 and –9. It is suggested that the CK8/18 fibrils may provide a scaffold for the proximity-induced autocleavage and activation of procaspase-9 in close association with caspase-3.We addressed this issue by investigating DEDD staining patterns in various cell lines and by correlating these expression patterns with the sensitivity of these cell lines for roscovitine-induced apoptosis. We showed that in some cell lines DEDD revealed a bright filamentous staining pattern in others DEDD staining was weak and diffusely distributed in the cytoplasm of the cells. The difference in staining patterns was irrespective of the phosphorylation status of the cytokeratin filaments. In cells showing a filamentous staining pattern, DEDD was strongly associated with the CK8/18 cytokeratin filaments as evidenced by double immunofluorescence and its resistance to extraction with Triton X-100. Subcellular fractionation indicates that DEDD co-purifies with CK18, which corroborates a strong association of DEDD and the cytokeratin network. DEDD was either mono- or diubiquinated. Cells showing a filamentous DEDD distribution are more apoptosis-prone as evidenced by the rapid appearance of M30 CytoDeath-positive cells after induction of apoptosis. The sensitivity towards apoptosis is irrespective of the procaspase-3 content of the cells. Our data support the notion that DEDD-mediated accumulation of procaspases at the cytokeratin scaffold leads to an increase in the local concentration, which renders cells more apoptosis-prone.

Upregulation of AKR1C1 and AKR1C3 expression in OPSCC with integrated HPV16 and HPV-negative tumors is an indicator of poor prognosis: AKR1C expression and prognosis in HPV16-positive and -negative OPSCC

Different studies have shown that HPV16‐positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome‐wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty‐three fresh‐frozen HPV‐16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo‐keto‐reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV‐positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non‐hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV‐positive (p ≤0.001) and ‐negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV‐negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.

Abstract 2380: Reduced CD44 and OTP gene expression are strong indicators of poor prognosis in pulmonary carcinoids.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histological classification in typical and atypical (AC) carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, i.e., OTP, CD44 and RET. Methods: These genes were analyzed in 56 frozen carcinoids by quantitative RT-PCR. RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was performed on 292 carcinoids. Results: Low mRNA expression levels of CD44 (p=1.3e-5) and OTP (p=0.012), and high levels of RET (p=0.0025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP, but not for RET. Within all carcinoids as well as ACs, absence of CD44 protein was significantly associated with low 20-year survival (p=8.6e-5 and p=0.00015, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (p=2.9e-5). Multivariate analysis revealed that CD44 and OTP immunostaining combined with histopathology is the optimal indicator for patient outcome. Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories. Citation Format: Dorian RA Swarts, Mieke Henfling, Leander van Neste, Robert J. van Suylen, Anne-Marie Dingemans, Winand NM Dinjens, Annick Haesevoets, Martina Rudelius, Erik Thunnissen, Marco Volante, Wim van Criekinge, Manon van Engeland, Frans CS Ramaekers, Ernst-Jan M. Speel. Reduced CD44 and OTP gene expression are strong indicators of poor prognosis in pulmonary carcinoids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2013-2380

Abstract 2255: Diagnostic and prognostic value of oncogenic human papillomavirus in patients with carcinoma of unknown primary of the neck

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective. To date, there is no consensus on the optimal diagnostic and therapeutic strategy for patients with carcinoma of unknown primary (CUP) of the neck. These tumors are heterogeneous in their clinical and biological characteristics, and a pre-operative prognostic marker is desirable to optimize therapy and improve outcome and survival. Human papillomavirus (HPV) is an etiologic factor in a subgroup of head and neck squamous cell carcinomas and has been identified as a significant prognostic biomarker. We sought to determine if HPV also may add relevant information on the origin and prognosis of these tumors. Material and method. 47 patients (mean age 58.7 years; 40 men, 7 women) presenting with CUP of the neck between 1994 and 2008 in Cologne were examined by standard diagnostic procedures including radiological imaging of the head, neck, thorax and abdomen, positron emission tomography (PET), panendoscopy, curettage of the nasopharynx, bilateral tonsillectomy and blind probes of the base of tongue. All patients were surgically treated with neck dissection of the diseased neck as well as adjuvant chemoradiation. The mean follow up time was 34 months. Formaldehyde-fixed, paraffin-embedded tissue specimens of the 47 metastases were examined retrospectively by means of p16INK4A immunohistochemistry, HPV-specific PCR and fluorescence in situ hybridization. Results were correlated with clinical follow-up data. Results. Oncogenic HPV was present in 12/47 (26%) metastases (10 with HPV 16), which showed also p16INK4A overexpression. In 42% of CUP patients the primary tumor was discovered during follow-up. A significant correlation between HPV positivity and later detection of the primary tumor in the oropharynx was found (p=0.038). Moreover, patients with a p16INK4A- (13/47) or HPV-positive tumor had a more favorable overall 5-year survival rate then p16INK4A- and HPV-negative tumors (69% vs 33%, p=0.05; 65% vs 37%, p=0.093; respectively). Conclusion. HPV is present in a quarter of neck metastases of CUP patients and the presence of oncogenic HPV and p16INK4A expression can serve to locate the primary tumor in the oropharynx. In addition, both biomarkers are indicators of a favorable prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2011-2255