Methylation of L1RE1, RARB, and RASSF1 function as possible biomarkers for the differential diagnosis of lung cancer.

BACKGROUND: Lung cancer is the major cause of cancer-related deaths worldwide. Differential diagnosis can be difficult, especially when only small samples are available. Epigenetic changes are frequently tissue-specific events in carcinogenesis and hence may serve as diagnostic biomarkers. MATERIAL AND METHODS: 138 representative formalin-fixed, paraffin-embedded (FFPE) tissues (116 lung cancer cases and 22 benign controls) were used for targeted DNA methylation analysis via pyrosequencing of ten literature-derived methylation markers (APC, CDH1, CDKN2A, EFEMP1, FHIT, L1RE1, MGMT, PTEN, RARB, and RASSF1). Methylation levels were analyzed with the Classification and Regression Tree Algorithm (CART), Conditional Interference Trees (ctree) and ROC. Validation was performed with additional 27 lung cancer cases and 38 benign controls. TCGA data for 282 lung cancer cases was included in the analysis. RESULTS: CART and ctree analysis identified the combination of L1RE1 and RARB as well as L1RE1 and RASSF1 as independent methylation markers with high discriminative power between tumor and benign tissue (for each combination, 91% specificity and 100% sensitivity). L1RE1 methylation associated significantly with tumor type and grade (p<0.001) with highest methylation in the control group. The opposite was found for RARB (p<0.001). RASSF1 methylation increased with tumor type and grade (p<0.001) with strongest methylation in neuroendocrine tumors (NET). CONCLUSION: Hypomethylation of L1RE1 is frequent in tumors compared to benign controls and associates with higher grade, whereas increasing methylation of RARB is an independent marker for tumors and higher grade. RASSF1 hypermethylation was frequent in tumors and most prominent in NET making it an auxiliary marker for separation of NSCLC and NET. L1RE1 in combination with either RARB or RASSF1 could function as biomarkers for separating lung cancer and non-cancerous tissue and could be useful for samples of limited size such as biopsies.

Reviewing the current evidence supporting early B-cells as the cellular origin of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly malignant skin cancer characterized by early metastases and poor survival. Although MCC is a rare malignancy, its incidence is rapidly increasing in the U.S. and Europe. The discovery of the Merkel cell polyomavirus (MCPyV) has enormously impacted our understanding of its etiopathogenesis and biology. MCCs are characterized by trilinear differentiation, comprising the expression of neuroendocrine, epithelial and B-lymphoid lineage markers. To date, it is generally accepted that the initial assumption of MCC originating from Merkel cells (MCs) is unlikely. This is owed to their post-mitotic character, absence of MCPyV in MCs and discrepant protein expression pattern in comparison to MCC. Evidence from mouse models suggests that epidermal/dermal stem cells might be of cellular origin in MCC. The recently formulated hypothesis of MCC originating from early B-cells is based on morphology, the consistent expression of early B-cell lineage markers and the finding of clonal immunoglobulin chain rearrangement in MCC cells. In this review we elaborate on the cellular ancestry of MCC, the identification of which could pave the way for novel and more effective therapeutic regimens.

[Merkel cell carcinoma: cutaneous manifestation of a highly malignant pre-/pro-B cell neoplasia? : Novel concept about the cellular origin of Merkel cell carcinoma]. / Merkelzellkarzinom: kutane Manifestation einer hochmalignen Prä-/pro-B-Zell-Neoplasie? : Neues Konzept zum zellulären Ursprung des Merkelzellkarzinoms.

Merkel cell carcinoma (MCC) is a relatively rare but highly malignant non-melanoma skin cancer of the elderly and immunosuppressed patients. The discovery of the Merkel cell polyomavirus (MCPyV) in 2008 significantly impacted the understanding of the etiopathogenesis of MCC. MCPyV is clonally integrated into the MCC genome and approximately 80% of MCC are MCPyV-positive. Recent results of clinical trials using blockade of the PD-1 immune modulatory pathway are promising for the future treatment of MCC. Despite this major progress of the past few years, the cellular origin of MCC still remains obscure. Based on histomorphology, gene expression profiling, and molecular analyses, we have recently hypothesized that MCC originates from pre­/pro-B cells. Here we review putative cells of MCC, including Merkel cells, (epi­)dermal stem cells, and pro­/pre-B cells. In the present work, the focus is on the concept of pre­/pro-B cells as the cellular origin of MCC, which might also impact the understanding of other human small cell malignancies of unknown cellular origin, such as small cell carcinomas of the lung and other anatomical locations. In addition, this concept might pave the way for novel treatment options, especially for advanced MCC.

Preliminary stop of the TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC) trial.

The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial was stopped preliminary, due to lagging inclusions. This study aimed to evaluate the treatment efficacy and clinical applicability of imiquimod 5% cream in high-grade cervical intraepithelial neoplasia (CIN). The lagging inclusions were mainly due to a strong patient preference for either of the two treatment modalities. This prompted us to initiate a new study on the same subject, with a non-randomized, open-label design: the 'TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC)-3' study. Original TOPIC-trial: Medical Ethics Committee approval number METC13231; ClinicalTrials.gov Identifier: NCT02329171, 22 December 2014. TOPIC-3 study: Medical Ethics Committee approval number METC162025; ClinicalTrials.gov Identifier: NCT02917746, 16 September 2016.

TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC trial): study protocol for a randomized controlled trial.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical cancer. Whereas not all high grade CIN lesions progress to cervical cancer, the natural history and risk of progression of individual lesions remain unpredictable. Therefore, high-grade CIN is currently treated by surgical excision: large loop excision of the transformation zone (LLETZ). This procedure has potential complications, such as acute haemorrhage, prolonged bleeding, infection and preterm birth in subsequent pregnancies. These complications could be prevented by development of a non-invasive treatment modality, such as topical imiquimod treatment. The primary study objective is to investigate the efficacy of topical imiquimod 5% cream for the treatment of high-grade CIN and to develop a biomarker profile to predict clinical response to imiquimod treatment. Secondary study objectives are to assess treatment side-effects, disease recurrence and quality of life during and after different treatment modalities. METHODS/DESIGN: The study design is a randomized controlled trial. One hundred forty women with a histological diagnosis of high-grade CIN (CIN 2-3) will be randomized into two arms: imiquimod treatment during 16 weeks (experimental arm) or immediate LLETZ (standard care arm). Treatment efficacy will be evaluated by colposcopy with diagnostic biopsies at 20 weeks for the experimental arm. Successful imiquimod treatment is defined as regression to CIN 1 or less, successful LLETZ treatment is defined as PAP 1 after 6 months. Disease recurrence will be evaluated by cytology at 6, 12 and 24 months after treatment. Side-effects will be evaluated using a standardized report form. Quality of life will be evaluated using validated questionnaires at baseline, 20 weeks and 1 year after treatment. Biomarkers, reflecting both host and viral factors in the pathophysiology of CIN, will be tested at baseline with the aim of developing a predictive biomarker profile for the clinical response to imiquimod treatment. DISCUSSION: Treatment of high-grade CIN lesions with imiquimod in a selected patient population may diminish complications as a result of surgical intervention. More knowledge on treatment efficacy, side effects and long-term recurrence rates after treatment is necessary. TRIAL REGISTRATION: EU Clinical Trials Register EU-CTR2013-001260-34 . Registered 18 March 2013. Medical Ethical Committee approval number: NL44336.068.13 (Medical Ethical Committee Maastricht University Hospital, University of Maastricht). Affiliation: Maastricht University Hospital. Registration number ClinicalTrials.gov: NCT02329171.

Downregulation of MTSS1 expression is an independent prognosticator in squamous cell carcinoma of the lung.

BACKGROUND: The metastasis suppressor 1 (MTSS1) is a newly discovered protein putatively involved in tumour progression and metastasis. MATERIAL AND METHODS: Immunohistochemical expression of MTSS1 was analysed in 264 non-small-cell lung carcinomas (NSCLCs). RESULTS: The metastasis suppressor 1 was significantly overexpressed in NSCLC compared with normal lung (P=0.01). Within NSCLC, MTSS1 expression was inversely correlated with pT-stage (P=0.019) and histological grading (P<0.001). NSCLC with MTSS1 downregulation (<20%) showed a significantly worse outcome (P=0.007). This proved to be an independent prognostic factor in squamous cell carcinomas (SCCs; P=0.041), especially in early cancer stages (P=0.006). CONCLUSION: The metastasis suppressor 1 downregulation could thus serve as a stratifying marker for adjuvant therapy in early-stage SCC of the lung.

Pitfalls and technical aspects during the research of intestinal anastomotic healing in rats.

BACKGROUND: Fundamental experimental research into intestinal anastomotic healing in rodent models will gain increasing interest in the future. METHODS: The aim of this study was to describe our 5-year experience with a standardized experimental setup of small and large bowel anastomoses in a rodent model and present a basic set of assessment tools investigating anastomotic healing. Anastomotic technique, perioperative complications such as anastomotic insufficiency (AI) and obstructive ileus were in the focus. RESULTS: During different studies with varying study patterns, 167 rat small bowel anastomoses and 120 colonic anastomoses were performed. Overall mortality was 3.6% in small bowel and 2.5% in colonic anastomoses, AI occurred in 2.9 and 4%, respectively. A postoperative obstructive ileus was seen in 3/167 small bowel anastomoses and none in the colonic group. CONCLUSION: When performing experimental intestinal anastomoses in a standardized operative setting and critically considering special perioperative issues, the incidence of relevant complications can be maintained at an adequately low level.

[Merkel cell polyomavirus in the pathogenesis of non-melanoma skin cancer]. / Das Merkel-Zell-Polyomavirus in der Pathogenese nichtmelanozytärer Hauttumoren.

Merkel cell carcinoma (MCC) is a very aggressive neuroendocrine carcinoma of the skin. The recently identified Merkel cell polyomavirus (MCPyV) is present in the majority of MCCs. MCPyV clonally integrates in the tumor DNA and tumor-specific viral mutations are detected within the large T-antigen. To elucidate a possible role of MCPyV in the pathogenesis of other non-melanoma skin cancers (NMSC), i.e. squamous cell carcinoma, Bowen's disease and basal cell carcinoma we tested a group of these tumors in immunosuppressed and immunocompetent patients. In addition we tested MCPyV-positive tumors for viral mutations within the large T-antigen. MCPyV DNA was significantly more frequently detected in the NMSC of the immunosuppressed patients (p<0.001). No tumor specific mutations were found within the large T-antigen. The presence of the virus in tumor cells was confirmed by FISH analysis. Although MCPyV is present in the tumor cells of squamous cell carcinoma, Bowen's disease and basal cell skin carcinoma, further investigations into the role of MCPyV in the pathogenesis of these tumors is needed.

Performance of a polymer-based DNA chip platform in detection and genotyping of human papillomavirus in clinical samples.

Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.

'Light-chain escape-multiple myeloma'-an escape phenomenon from plateau phase: report of the largest patient series using LC-monitoring.

PURPOSE: More intensive and novel therapy options in multiple myeloma (MM) hold the promise to improve treatment outcome. However, disease evolution, induced with long disease duration and extensive pretreatment, has resulted in changes in the biological behaviour of MM and unusual relapse emergence, such as of extramedullary (EM) disease or a shift in secretion from intact immunoglobulin (Ig) to free-light chains (FLCs) only. METHODS: We studied ten patients since 2004, thoroughly assessed relevant patient characteristics, prominent similarities, SFLC-changes, therapy response, mode and speed of progression, and the incidence of light-chain escape (LCE)-MM within our entire myeloma patient cohort. Serum FLCs (SFLCs) were determined via Freelite-assay (Dade-Behringer Nephelometer). RESULTS: This report summarizes the to date largest series of ten patients, whose MM appeared stable, as judged by conventional monitoring of intact Ig levels, but developed severe organ dysfunction as a consequence of initially undetected LC-progression. Median number of anti-MM cycles before LCE occurrence was six, including autologous and/or allogeneic stem cell transplants and novel drugs, predominantly thalidomide, in 4/10. Classic diagnostics, such as electrophoresis and quantitative Ig measurement proved futile to detect LC-progression, whereas SFLCs were reliable markers. The LCE-MM prevalence within 407 MM patients treated in our institution between 2004 and 2007 was 2.46%. CONCLUSIONS: Our report suggests that early detection of LCE-MM by means of serial SFLC measurements may prevent unnecessary complications, allows to detect unusual relapse manifestations in the era of intensive and biological therapy options and possibly also permits to improve treatment results in LCE-MM.

Induction of early murine cytomegalovirus infection by different reporter gene-associated recombinant viruses.

Murine cytomegalovirus (MCMV) has provided useful models for acute, chronic and latent CMV infection because of its similarities in structure and biology with human CMV. We report the induction of acute MCMV hepatitis with different bacterial artificial chromosome (BAC)-cloned virus constructs [MCMV-SEAP which includes the gene for secreted alkaline phosphatase (SEAP) under Rous sarcoma virus (RSV)-promoter control, MCMV-GFP which includes the gene for enhanced green fluorescent protein (eGFP) under HCMV-ie promoter control, MCMV-HBs includes the gene for hepatitis B surface antigen (HBsAg) under simian virus (SV)40-promoter control and the DeltaMC95.21 virus in which the m152 gene was deleted and substituted by the reporter gene lacZ] in order to elucidate the histopathological changes together with different reporter-gene products in the liver tissue and the effect of the deletion of a certain gene. All the virus constructs induced a similar mild acute hepatitis which had its climax from days 3 to 5 post-infection in immunocompetent mice. In situ, the reporter-gene products beta-galactosidase and secreted alkaline phosphatase could be visualized in relation to the inflammatory changes. The composition of the invading cell populations did not change even in the absence of the m152 gene. Additionally discrete inflammatory changes were seen in kidney and serosa while the other organs were not involved. This model helps us understand the immunological and histopathological mechanisms of the CMV-induced hepatitis, which plays an important role especially in the immunocompromised patient. The morphological changes can be analysed while the respective reporter gene product is expressed by the virus construct.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted.

Tumours ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis.

Specific induction of pp125 focal adhesion kinase in human breast cancer.

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT-PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51+/-0.84 (mean+/-standard deviation), which was strongly induced to 2.91 (+/-1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0+/-0.63 vs 2.27+/-0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation.

Epstein-Barr virus in gastric carcinomas and gastric stump carcinomas: a late event in gastric carcinogenesis.

BACKGROUND: To determine at what stage during gastric carcinogenesis Epstein-Barr virus (EBV) enters the gastric epithelial cells, the presence of EBV was investigated in two pathogenetically related but distinct forms of adenocarcinoma of the stomach-gastric carcinoma of the intact stomach (GCIS) and gastric stump carcinoma (GSC)-and their presumed precursor lesions. PATIENTS AND METHODS: Eleven patients with EBV positive GCIS and eight patients with EBV positive GSC, demonstrated by the highly sensitive EBV encoded RNA 1/2 (EBER1/2) RNA in situ hybridisation (RISH) technique, were studied. Paraffin wax embedded tissue available from preoperative gastric biopsies and tumour adjacent tissue from the resection specimens containing normal gastric mucosa, inflamed gastric mucosa, and preneoplastic lesions (intestinal metaplasia and dysplasia) was investigated by EBER1/2 RISH, in addition to EBV nuclear antigen 1 (EBNA-1) and latent membrane protein 1 (LMP-1) immunohistochemistry (IHC). RESULTS: In both GCIS and GSC and their precursor lesions EBER1/2 transcripts were restricted to the carcinoma cells. In addition, positivity of EBNA-1 IHC was also restricted to the tumour cells. IHC for LMP-1 was negative in all cases tested. CONCLUSIONS: The absence of EBER1/2 transcripts in preneoplastic gastric lesions (intestinal metaplasia and dysplasia) and their presence in two distinct types of gastric carcinoma strongly suggest that EBV can only infect neoplastic gastric cells and thus is a late event in gastric carcinogenesis.

Increased apoptosis in gastric mucosa adjacent to intestinal metaplasia.

BACKGROUND: The biological processes involved in the development of gastric mucosal atrophy and intestinal metaplasia are still incompletely understood. Reports testing the hypothesis that apoptosis leads to atrophy have yielded conflicting results. The availability of new antibodies for the detection of apoptotic cells in tissue sections has facilitated the analysis of the role of apoptosis in the gastritis-atrophy-intestinal metaplasia sequence. METHODS: Archival material from 40 gastric resection specimens with normal mucosa (n = 5), chronic active gastritis (n = 17), or intestinal metaplasia (n = 18) was studied. Immunohistochemistry was performed using antibodies directed against cleaved cytokeratin 18 and active caspase 3. Slides were scored on a 0-3 scale for the presence of apoptotic cells. RESULTS: Normal gastric mucosa contained low numbers of apoptotic cells at the surface epithelium (mean score, 0.20). This number was significantly increased in cases with chronic gastritis (mean score, 1.06) and in those with intestinal metaplasia (mean score, 2.56). Within the intestinal metaplasia cases, 44 different foci of intestinal metaplasia were identified. In 39 of these 44 areas, concentrations of apoptotic cells were seen immediately adjacent to the foci of intestinal metaplasia, but not in the metaplastic epithelium itself. CONCLUSIONS: Apoptosis is uncommon in normal gastric mucosa. Chronic inflammation and intestinal metaplasia are associated with increased apoptosis, but occur mainly at the mucosal surface and not in the deeper layers. These findings do not support the concept that apoptosis underlies the loss of gastric glands and leads to atrophy, but the observed concentration of apoptotic epithelial cells adjacent to foci of intestinal metaplasia could be related to heterogeneity of epithelial damage, causing apoptosis, to which intestinal metaplasia is a response.

Differentially expressed genes in association with in vitro invasiveness of human epithelioid sarcoma.

AIMS: Differential display reverse transcription polymerase chain reaction (RT-PCR) was performed to identify genes associated with the invasive potential of human epithelioid sarcoma. METHODS: Two different clonal subpopulations, GRU-1A and GRU-1B, derived from the same human epithelioid sarcoma cell line GRU-1 and known to differ greatly in their invasive potential were compared by means of mRNA fingerprinting. RESULTS: Using a set of 10 arbitrary upstream primers and nine anchored oligo-dT primers, 22 candidate gene fragments were identified; differential expression was confirmed in four of these fragments by northern blot analysis. At the mRNA level, apoferritin light chain was predominantly expressed by the highly invasive cell line GRU-1A. In contrast, the mitochondrial gene M1, encoding cytochrome c oxidase I, and the TI-227H gene were expressed more strongly by the low invasive cell line GRU-1B. Furthermore, a novel gene fragment was identified and cloned that was preferentially expressed in the low invasive cell line GRU-1B, and therefore might have an inhibitory role in invasion. Consequently, this gene fragment was designated as expressed in low invasive sarcoma cells (ELISC-1). CONCLUSIONS: A novel gene fragment (ELISC-1) and three known genes were identified as potential regulators of tumour invasiveness. Cloning of the entire sequence of ELISC-1 and subsequent investigations are required to establish its biological role.

Distinct chromosomal aberrations in Epstein-Barr virus-carrying gastric carcinomas tested by comparative genomic hybridization.

BACKGROUND & AIMS: Approximately 10% of gastric adenocarcinomas carry the human pathogenic Epstein-Barr virus (EBV). The role of EBV in the pathogenesis of these carcinomas remains to be established. METHODS: To obtain a comprehensive overview of chromosomal aberrations in EBV-carrying and EBV-negative gastric carcinomas we performed comparative genomic hybridization (CGH) on 44 gastric carcinomas, 10 EBV-positive, and 34 EBV-negative. Additionally, DNA flow cytometry was done. RESULTS: Loss of chromosome 4p (P < 0.001) and of 11p (P < 0.02) was exclusively restricted to EBV-carrying gastric carcinomas. In addition, loss of 18q (P < 0.02) was significantly more frequent in EBV-carrying gastric carcinomas. The latter involves loci, which have already been linked to gastric carcinogenesis such as the DCC and SMAD4 gene. In contrast, the losses on chromosome 4 and 11 do not yet harbor a gene related to gastric carcinogenesis. No significant correlation was found between DNA-ploidy and the EBV-status. A number of chromosomal aberrations were found at comparable frequencies in both groups, i.e., losses of chromosome 17, 12q, and loss of 1p. Interestingly, gains of 13q (10/34) and 3q (5/34) and loss of 1q (5/34) were solely observed in EBV-negative gastric carcinomas. CONCLUSIONS: These data indicate that EBV-carrying and EBV-negative gastric carcinomas have different pathogenetic pathways in which EBV might play a crucial role.

Unique transcription pattern of Epstein-Barr virus (EBV) in EBV-carrying gastric adenocarcinomas: expression of the transforming BARF1 gene.

Approximately 10% of gastric adenocarcinomas worldwide are associated with human EBV. These carcinomas generally do not express the latent membrane protein 1 (LMP1), the major known EBV oncogene. Recently, another EBV gene [ie., BARF1 (BamHI A rightward open reading frame)] was shown to have transforming and immortalizing capacities. Therefore, in this study, we investigated the expression of BARF1 in EBV-carrying gastric adenocarcinomas in relation to the expression of other latent EBV transcripts. In the present study, 10 of 132 gastric adenocarcinomas tested positive for EBV using EBER1/2-RNA in situ hybridization. We demonstrate BARF1 gene transcription in nine EBV-carrying gastric adenocarcinomas (with sufficient RNA quality) using the BARF1-specific nucleic acid sequence-based amplification assay. In addition, we also detected other latent EBV transcripts (ie., BARF0-, LMP2A-, and Q/K-driven EBNA1 transcripts in these carcinomas using reverse transcription-PCR analysis. No expression of LMP1, EBNA2, and ZEBRA (either at transcription or protein level) was found. In addition, two cases were positive for BHRF1 transcripts, the viral bcl-2 homologue. Thus, together with BARF1 transcription, a unique and distinct EBV latency type has been found in EBV-associated gastric adenocarcinomas. Because BARF1 exerts immortalizing effects on human epithelial cells in vitro and EBV-carrying gastric adenocarcinomas lack the expression of LMP1, the BARF1 gene might act as the viral oncogene in EBV-carrying gastric carcinomas. The BARF1 gene offers an alternative way for EBV-mediated oncogenesis other than LMP1.

Retinoblastoma-protein (prb) expression and prognosis in squamous-cell carcinomas of the esophagus.

The retinoblastoma gene (RB) is a typical tumor-suppressor gene. Inactivation of RB has been shown in a variety of human cancers, including esophageal squamous-cell carcinomas. In the present study, samples of normal esophageal squamous epithelium (n = 10), severe squamous-cell dysplasias (n = 19), carcinomas in situ (n = 14), invasive squamous-cell carcinomas (n = 172), and 2 continuous esophageal-carcinoma cell lines were immunohistochemically analyzed for pRb expression. The specificity of immunostaining was tested by Western-blot analysis of pRb expression in the cell lines. In normal esophageal epithelium, nuclear pRb expression was restricted to the parabasal cell layer, whereas, in a considerable portion of severe dysplasias and carcinomas in situ, pRb over-expression was found. Among carcinomas, 161 of 172 cases showed pRb expression, as did the 2 esophageal-carcinoma cell lines, whereas 11 carcinomas were negative. Expression of pRb among carcinomas was not correlated with pT category, pN category or tumor grade. In the univariate survival analysis, patients with pRb-negative tumors showed lower 2-year and 5-year survival rates (27.3%/9.1%) than patients with pRb-positive tumors (42.8%/25.8%; not significant). In conclusion, pRb protein can be detected by immunohistochemistry in a high percentage of squamous-cell carcinomas of the esophagus and its precursor lesions. However, expression of the pRb protein has no significant impact on the prognosis. Int. J. Cancer (Pred. Oncol.) 84:618-622, 1999.

Expression of p21WAF1 predicts outcome of esophageal cancer patients treated by surgery alone or by combined therapy modalities.

The p21WAF1 protein is an important regulator of the cell cycle. Its expression and prognostic significance were investigated immunohistochemically in samples of normal esophageal squamous epithelium (n = 10), severe squamous cell dysplasia (n = 20), carcinoma in situ (n = 14), permanent esophageal squamous cell carcinoma cell lines (n = 3), and invasive squamous cell carcinomas treated either by potentially curative resection (n = 172) or by combined modality therapy (radiochemotherapy +/- surgery; n = 38). Whereas p21WAF1 expression in the normal epithelium was restricted to a few cells adjacent to the basal cell layer, p21WAF1 overexpression was frequently found in preneoplasias and invasive carcinomas. Expression of p21WAF1 in invasive carcinomas was not correlated with tumor differentiation, pT category, or pN category. Among carcinomas treated by potential curative resection, univariate (P = 0.0025) and multivariate (P = 0.0081) survival analysis showed significant correlation of strong p21WAF1 expression (> or =50% p21WAF1-positive tumor cells) with poor overall survival. Univariate survival analysis (P = 0.0006) revealed the same prognostic influence in the group of patients treated by combined modality therapy. We conclude that overexpression of p21WAF1 protein is a frequent event in preneoplasias and neoplasias of the esophagus. Immunohistochemical examination of p21WAF1 expression may provide important prognostic information for decision-making in the treatment of patients with esophageal cancer.