Hans Peter Dienes

Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV‐DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV‐DNA and expression of p16 and EGFR were analyzed. The 5‐year disease‐free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty‐eight percent of the cases contained oncogenic HPV‐DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV‐DNA (p < 0.001). Univariate analysis of the 5‐year DFS revealed a significantly better outcome for patients with p16‐positive tumors (84% vs. 49%, p = 0.009). EGFR‐negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5‐year DFS of 93% for p16+/EGFR− tumors vs. 39% for p16−/EGFR+ tumors (p = 0.003) and to a 5‐year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5‐fold increased risk for relapse in patients with p16‐negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV‐positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus.

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Histomorphologic Tumor Regression and Lymph Node Metastases Determine Prognosis Following Neoadjuvant Radiochemotherapy for Esophageal Cancer: Implications for Response Classification

Objective:We sought to quantitatively and objectively evaluate histomorphologic tumor regression and establish a relevant prognostic regression classification system for esophageal cancer patients receiving neoadjuvant radiochemotherapy. Patients and Methods:Eighty-five consecutive patients with localized esophageal cancers (cT2-4, Nx, M0) received standardized neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil, 36 Gy). Seventy-four (87%) patients were resected by transthoracic en bloc esophagectomy and 2-field lymphadenectomy. The entire tumor beds of the resected specimens were evaluated histomorphologically, and regression was categorized into grades I to IV based on the percentage of vital residual tumor cells (VRTCs). A major response was achieved when specimens contained either less than 10% VRTCs (grade III) or a pathologic complete remission (grade IV). Results:Complete resections (R0) were performed in 66 of 74 (89%) patients with 3-year survival rates of 54% ± 7.05% for R0-resected cases and 0% for patients with incomplete resections ortumor progression during neoadjuvant therapy (P < 0.01). Minor histopathologic response was present in 44 (59.5%) and major histopathologic response in 30 (40.5%) tumors. Significantly different 3-year survival rates (38.8% ± 8.1% for minor versus 70.7 ± 10.1% for major response) were observed. Univariate survival analysis identified histomorphologic tumor regression (P < 0.004) and lymph node category (P < 0.01) as significant prognostic factors. Pathologic T category (P < 0.08), histologic type (P = 0.15), or grading (P = 0.33) had no significant impact on survival. Cox regression analysis identified dichotomized regression grades (minor and major histomorphologic regression, P < 0.028) and lymph node status (ypN0 and ypN1, P < 0.036) as significant independent prognostic parameters. A 2-parameter regression classification system that includes histomorphologic regression (major versus minor) and nodal status (ypN0 versus ypN1) was established (P < 0.001). Conclusions:Histomorphologic tumor regression and lymph node status (ypN) were significant prognostic parameters for patients with complete resections (R0) following neoadjuvant radiochemotherapy for esophageal cancer. A regression classification based on 2 parameters could lead to improved objective evaluation of the effectiveness of treatment protocols, accuracy of staging and restaging modalities, and molecular response prediction.

Peginterferon plus Adefovir versus Either Drug Alone for Hepatitis Delta

BACKGROUND Chronic infection with hepatitis B virus and hepatitis delta virus (HDV) results in the most severe form of viral hepatitis. There is no currently approved treatment. We investigated the safety and efficacy of 48 weeks of treatment with peginterferon alfa-2a plus adefovir dipivoxil, peginterferon alfa-2a alone, and adefovir dipivoxil alone. METHODS We conducted a randomized trial in which 31 patients with HDV infection received treatment with 180 μg of peginterferon alfa-2a weekly plus 10 mg of adefovir daily, 29 received 180 μg of peginterferon alfa-2a weekly plus placebo, and 30 received 10 mg of adefovir alone weekly for 48 weeks. Follow-up was conducted for an additional 24 weeks. Efficacy end points included clearance of HDV RNA, normalization of alanine aminotransferase levels, and a decline in levels of hepatitis B surface antigen (HBsAg). RESULTS The primary end point--normalization of alanine aminotransferase levels and clearance of HDV RNA at week 48--was achieved in two patients in the group receiving peginterferon alfa-2a plus adefovir and two patients in the group receiving peginterferon alfa-2a plus placebo but in none of the patients in the group receiving adefovir alone. At week 48, the test for HDV RNA was negative in 23% of patients in the first group, 24% of patients in the second, and none of those in the third (P = 0.006 for the comparison of the first and third groups; P = 0.004 for the comparison of the second and third). The efficacy of peginterferon alfa-2a was sustained for 24 weeks after treatment, with 28% of the patients receiving peginterferon alfa-2a plus adefovir or peginterferon alfa-2a alone having negative results on HDV-RNA tests; none of the patients receiving adefovir alone had negative results. A decline in HBsAg levels of more than 1 log(10) IU per milliliter from baseline to week 48 was observed in 10 patients in the first group, 2 in the second, and none in the third (P<0.001 for the comparison of the first and third groups and P = 0.01 for the comparison of the first and second). CONCLUSIONS Treatment with peginterferon alfa-2a for 48 weeks, with or without adefovir, resulted in sustained HDV RNA clearance in about one quarter of patients with HDV infection. (Current Controlled Trials number, ISRCTN83587695.).

Lymph Node Size and Metastatic Infiltration in Colon Cancer

Background: Detection of metastatic lymph nodes in colon cancer is essential for determining stage and adjuvant treatment modalities. Lymph node size has been used as one possible criterion for nodal metastasis. Although enlarged regional lymph nodes are generally interpreted as metastases, few data are available that correlate lymph node size with metastatic infiltration in colon cancer.Methods: In a prospective morphometric study, the regional lymph nodes from 30 colon specimens from consecutive patients with primary colon cancer were analyzed. The lymph nodes were counted and the largest diameter of each lymph node was measured and analyzed for metastatic involvement by histological examination.Results: A total of 698 lymph nodes were present in the 30 specimens examined for this study. A mean number of 23 (range, 19–39) lymph nodes was found in each specimen. Of these nodes, 566 (81%) were tumor-free and 132 (19%) contained metastases. The mean diameter of the lymph nodes free of metastases was 3.9 mm, whereas those infiltrated by metastases averaged 5.9 mm in diameter (P< 0.0001). Of the tumor-free lymph nodes, 528 (93%) measured < 5 mm in diameter, whereas 70 (53%) lymph nodes containing metastases measured < 5 mm in diameter.Conclusions: Lymph node size is not a reliable indicator for lymph node metastasis in colon cancer. A careful histological search for small lymph node metastasis in the specimen should be undertaken to avoid false-negative node staging.

MUC1 and Nuclear β-Catenin Are Coexpressed at the Invasion Front of Colorectal Carcinomas and Are Both Correlated with Tumor Prognosis

Purpose: Overexpression of MUC1 and cytosolic interaction of the mucin with β-catenin are claimed to be involved in colorectal carcinogenesis. In vitro data published recently suggest that MUC1 overexpression results in an increase of steady state levels of nuclear β-catenin. We tried to elucidate the coexpression of both molecules in colorectal cancer to demonstrate possible correlations with clinical, pathological, and prognostic data. Experimental Design: An immunohistochemical double staining study was performed to characterize the expression and subcellular distribution of MUC1 and β-catenin in a series of 205 patients with colorectal carcinoma. The results were correlated with clinicopathological variables as well as overall survival. Results: MUC1 was strongly expressed in the tumor center and at the invasion front in ∼50% of the cases. Similar results were obtained with regard to nuclear accumulation of β-catenin at the invasive tumor parts. MUC1 protein expression in the tumor center correlated significantly with a low grade of differentiation, and nuclear β-catenin in the tumor periphery was more frequent in carcinomas of the left colon and rectum. Overexpression of MUC1 and β-catenin, as well as their nuclear coexpression at the invasion front correlated with a worse overall survival in an univariate analysis. However, only pathological tumor-node-metastasis staging and MUC1 at the invasion front revealed as independent prognostic factors. Conclusions: These results suggest that MUC1 and β-catenin are coexpressed at the invasion front of colorectal carcinomas and that this feature is associated with an accelerated course of disease and worse prognosis.

TGF-β1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis

Transforming growth factor-β1 (TGF-β1) is a powerful stimulus for collagen formation in vitro. To determine the in vivo effects of TGF-β1 on liver fibrogenesis, we generated transgenic mice overexpressing a fusion gene [C-reactive protein (CRP)/TGF-β1] consisting of the cDNA coding for an activated form of TGF-β1 under the control of the regulatory elements of the inducible human CRP gene promoter. Two transgenic lines were generated with liver-specific overexpression of mature TGF-β1. After induction of the acute phase response (15 h) with lipopolysaccharide (100 μg ip), plasma TGF-β1 levels reached >600 ng/ml in transgenic animals, which is >100 times above normal plasma levels. Basal plasma levels of uninduced transgenic animals were about two to five times above normal. As a consequence of hepatic TGF-β1 expression, we could demonstrate marked transient upregulation of procollagen I and procollagen III mRNA in the liver 15 h after the peak of TGF-β1 expression. Liver histology after repeated induction of transgene expression showed an activation of hepatic stellate cells in both transgenic lines. The fibrotic process was characterized by perisinusoidal deposition of collagen in a linear pattern. This transgenic mouse model gives in vivo evidence for the important role of TGF-β1 in stellate cell activation and liver fibrogenesis. Due to the ability to control the level of TGF-β1 expression, this model allows the study of the regulation and kinetics of collagen synthesis and fibrolysis as well as the degree of reversibility of liver fibrosis. The CRP/TGF-β1 transgenic mouse model may finally serve as a model for the testing of antifibrogenic agents.

High Specificity of Quantitative Excision Repair Cross- Complementing 1 Messenger RNA Expression for Prediction of Minor Histopathological Response to Neoadjuvant Radiochemotherapy in Esophageal Cancer

Purpose: The excision repair cross-complementing 1 (ERCC1) gene is coding for a nucleotide excision repair protein involved in the repair of radiation- and chemotherapy-induced DNA damage. We examined the potential of quantitative ERCC1 mRNA expression to predict minor or major histopathological response to neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil, and 36 Gy of radiation) followed by transthoracic en bloc esophagectomy in patients with locally advanced esophageal cancer (cT2–4, Nx, M0). Experimental Design: Tissue samples were collected by endoscopic biopsy before treatment. RNA was isolated from biopsies, and quantitative real-time reverse transcriptase PCR assays were performed to determine ERCC1 mRNA expression. Relative mRNA levels (tumor/normal ratios) were calculated as (ERCC1/β-actin in tumor)/(ERCC1/β-actin in paired normal tissue). ERCC1 expression levels were correlated with the objective histopathological response in resected specimens. Histomorphological regression was defined as major response when resected specimens contained <10% of residual vital tumor cells or in case a pathologically complete response was achieved. Results: Twelve of 36 tumors showed a major histopathological response, and 24 of 36 showed a minor histopathological response. Relative expression levels of ERCC1 of >1.09 were not associated with a major histopathological response (sensitivity, 62.5%; specificity, 100%) and 15 of 24 patients with minor histopathological response to the delivered neoadjuvant radiochemotherapy could be unequivocally identified. This association of dichotomized relative ERCC1 mRNA expression and histopathological response was statistically significant (P < 0.001). Conclusions: Relative expression levels of ERCC1 mRNA determined by quantitative real-time reverse transcriptase-PCR appear highly specific to predict minor response to our neoadjuvant radiochemotherapy protocol in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (42%) of patients.

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

Background In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. Methodology HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3′-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. Principal Findings The 3′-UTR of the collagen-1 and −4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. Conclusions Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.

Long‐term follow‐up of antimitochondrial antibody–positive autoimmune hepatitis

Antimitochondrial antibodies (AMAs) are the serological hallmark for primary biliary cirrhosis (PBC). When AMAs are detected in patients with chronic hepatitis, they negatively impact on the autoimmune hepatitis (AIH) scoring system. The purpose of this study was to determine if AMAs detected in the sera of patients with overt AIH have clinical or pathological significance. All patients with a clinicopathologic diagnosis of AIH from one center were reviewed. Only those meeting the criteria for probable or definite AIH according to the International Autoimmune Hepatitis Group were included. Patients found to be consistently AMA‐positive via enzyme‐linked immunosorbent assay (ELISA) for pyruvate dehydrogenase complex E2 subunit were reviewed in detail. Fifteen of 126 patients with typical features of AIH (pretreatment AIH score >10) had detectable AMAs in serum. None had any histologic features suggestive of PBC. None had detectable anti‐liver–kidney–microsomal antibodies. Of these 15 patients, all have remained persistently AMA‐positive via ELISA. All 15 patients have been followed long‐term, and their clinical course remained typical for AIH. No bile duct damage typical of PBC was seen on initial or follow‐up liver biopsies. Conclusion: Patients with overt AIH who test positive for AMAs at initial presentation and are treated with corticosteroid therapy have shown no clinical or histologic evidence of PBC despite the continued detection of AMAs over a follow‐up of up to 27 years. (HEPATOLOGY 2008;48:550–556.)