May Britt Kalvenes

Importance of P-Cadherin, β-Catenin, and Wnt5a/Frizzled for Progression of Melanocytic Tumors and Prognosis in Cutaneous Melanoma

Purpose: It has been proposed that melanoma cells shift from E-cadherin to N-cadherin expression during tumor development, and recent gene profiling has shown increased expression of Wnt5a/Frizzled in aggressive melanomas possibly by interactions with β-catenin. We therefore wanted to investigate the role of cadherin subtypes, β-catenin, and Wnt5a/Frizzled in melanocytic tumors, with focus on prognosis in nodular melanomas. Experimental Design: The immunohistochemical expression of E-cadherin, N-cadherin, P-cadherin, β-catenin, and Wnt5a/Frizzled was examined using tissue microarrays of 312 melanocytic tumors. Results: Cytoplasmic expression of P-cadherin was associated with increasing tumor thickness (P = 0.005) and level of invasion (P = 0.019), whereas membranous staining was associated with thinner (P = 0.012) and more superficial (P = 0.018) tumors. Increased cytoplasmic P-cadherin was associated with reduced survival (P = 0.047). Lack of nuclear β-catenin expression was related to increased tumor thickness (P = 0.002) and poor patient survival in univariate (P = 0.0072) and multivariate (P = 0.004) analyses. Membranous expression of N-cadherin was significantly increased from primary tumors to metastatic lesions, whereas E-cadherin staining tended to be decreased. Wnt5a and its receptor Frizzled were highly coexpressed, and nuclear expression of both markers was significantly reduced from benign nevi to melanomas, with a shift from nuclear to cytoplasmic expression in malignant tumors. In addition, Wnt5a expression was significantly associated with nuclear β-catenin expression. Conclusions: Alterations in the expression and subcellular localization of cell adhesion markers are important in the development and progression of melanocytic tumors, and strong cytoplasmic P-cadherin expression and loss of nuclear β-catenin staining were associated with aggressive melanoma behavior and reduced patient survival.

Altered antibody pattern to Epstein-Barr virus but not to other herpesviruses in multiple sclerosis: a population based case-control study from western Norway

OBJECTIVE The prevalence of anti-EBV antibodies was studied in a group of 144 patients with multiple sclerosis and 170 age, sex, and area matched controls from the county of Hordaland, western Norway. The prevalence of three other herpesviruses, herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV), were also included. METHODS Antibodies to various virus antigens were determined by enzyme linked immunosorbent assay (ELISA) and indirect immunfluorescence (IIF) in serum samples from 144 patients with multiple sclerosis and 170 controls. RESULTS All of the 144 patients with multiple sclerosis had IgG antibodies to EBV compared with 162 of 170 controls (p=0.008). The frequency of IgG antibodies to EBV capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA) was significantly higher in patients with multiple sclerosis compared with the controls (p<0.000001, p=0.01, and p<0.0001 respectively). The presence of antibodies was independent of the initial course of the disease and the disease activity at the time of blood sampling. The prevalence of IgG antibodies to HSV, CMV, and VZV did not differ between cases and controls. CONCLUSION The results suggest a role for EBV in the aetiology of multiple sclerosis.

Loss of PTEN expression is associated with metastatic disease in patients with endometrial carcinoma.

The PTEN tumor suppressor gene frequently is involved in endometrial carcinoma. Loss of heterozygosity and mutations reportedly are common, although the biologic importance of these changes remain largely unknown. The objective of this study was to assess the pattern of PTEN expression by immunohistochemistry in a large series of patients with endometrial carcinoma.

Expression of keratin 13, 14 and 19 in oral squamous cell carcinomas from Sudanese snuff dippers: Lack of association with human papillomavirus infection

In stratified squamous epithelia, altered expression of keratins (Ks) is one possible marker of malignant potential. In the epithelium of the uterine cervix, presence of human papillomaviruses (HPVs) is increasingly regarded as a marker of risk for cervical cancer. However, a similar role in oral cancer and precancer remains controversial. To address these questions, formalin‐fixed, paraffin‐embedded oral carcinomas from Sudanese snuff dippers (n=14) and oral carcinomas from Sudanese (n=14), Swedish (n=19) and Norwegian (n=41) non‐snuff dippers were examined by immunohistochemistry for expression of K types 13, 14 and 19 using monoclonal antibodies. HPV infection was searched for in all the carcinomas by in situ hybridization (ISH) using the cocktail HPV OmniProbe and the ViraType probe. Carcinomas from Sudanese (snuff dippers/non‐snuff dippers) were also examined for HPV infection by polymerase chain reaction (PCR) using the general HPV primers GP5+/GP6+. For the oral carcinomas from snuff dippers, moderate to intense expression of K13 (71%; 10/14), K14 (86%; 12/14) and K19 (93%; 13/14) was found. For the oral carcinomas from non‐snuff dippers, weak to moderate expression of K13 (64%; 47/74), K14 (43%; 32/74) and K19 (45%; 33/74) was found. HPV DNA was not detected in any of the carcinomas from three countries when examined by ISH. The Sudanese (from snuff dippers/non‐snuff dippers) oral carcinomas were also negative for HPV DNA with the PCR. The present study shows that (i) there is a high level of expression of K13, K14 and K19 in oral carcinomas from snuff dippers compared to those from non‐snuff dippers, (ii) this high level of expression may arise from dysregulation of keratinocyte proliferation and maturation caused by damaging effects of snuff, (iii) the HPV genome is not found in Sudanese (snuff dippers/non‐snuff dippers), Swedish or Norwegian oral carcinomas, and (iv) this may suggest that these viruses do not play a prominent role in the aetiology of oral carcinomas from these countries.

BRAF-V600E expression in primary nodular melanoma is associated with aggressive tumour features and reduced survival

Background:Around 50% of primary melanomas harbour BRAF mutations, but their prognostic impact has not been clear. Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry. Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma.Methods:In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by immunohistochemistry using tissue microarray sections of paraffin-embedded archival tissue. Mutation status was assessed by real-time PCR in cases with sufficient tumour tissue (n=191).Results:Positive BRAF-V600E expression was present in 86 (35%) of the cases, and was significantly associated with increased tumour thickness, presence of tumour ulceration and reduced survival. Further, BRAF-V600E expression was an independent prognostic factor by multivariate analysis, whereas BRAF mutation status was not significant. There was 88% concordance between BRAF-V600E expression and mutation status.Conclusions:Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma.

Raised levels of antibodies to human viruses at the clinical onset of autoimmune chronic active hepatitis

Summary. Patients with autoimmune chronic active hepatitis (AICAH) often have very high titres of antibodies to rubella and/or measles virus. In the present study a young girl at the clinical onset of AICAH exhibited very high titres of antibodies against influenza viruses A and B, parainfluenza viruses, rubella virus and varicella‐zoster virus. The titres normalized over 2 months except for rubella and varicella‐zoster antibodies. Strong reactivities were seen against the rubella structural proteins E1, E2 and C in Western blot but IgM antibodies were not demonstrated. Total IgG was increased with normal ratios of subclasses. The IgG1 was the dominant antibody to E1 and E2, while IgG4 dominated the anti‐C response. There was no significant shift in subclass reactivities over one year from onset. The polymerase chain reaction (PCR), using a nested primer set, was negative for rubella virus RNA in a liver biopsy obtained at the clinical onset and in peripheral blood mononuclear cells (PBMC) 1 year later. Co‐cultivation experiments using PBMC and permissive cell lines were also negative for rubella virus. Hence, in the very early phase of AICAH there may be a transiently enhanced antibody response to various unrelated viruses.

Radioimmunoprecipitation and immunoblot studies of antibodies to rubella virus in patients with chronic liver disease

SummaryPatients with autoimmune chronic active hepatitis (AICAH) and some other chronic liver disorders often have very high titres of rubella HI antibodies. In the present study sera from 46 patients with chronic liver disease and controls were examined for rubella antibodies using radioimmunoprecipitation assay (RIPA) and Western blot. RIPA appeared to be more suitable than Western blot for the study of the individual antibody specificities provided that proteins (possibly actin) interfering with the resolution of the E2 glycoprotein band are identified. It was shown that patients with high rubella HI titres reacted strongly against the E1 glycoprotein and in general also against the core protein (C). Reactivity to the E2 glycoprotein was detected with all sera from patients with chronic liver disease but varied more in strength. Three patients with post-acute rubella showed very faint E2 reactivity, but strong E1 and C reactivities. Patients with primary biliary cirrhosis had normal HI titres and showed no increase in reactivity in RIPA. The present findings show that patients with chronic liver disease and high rubella HI antibody titres exhibit an enhanced specific antibody response to rubella virus structural proteins.

Study of transcription in measles virus‐infected Vero cells using cDNA probes prepared from poly(A)RNA from uninfected and infected cells

From af primary plasmid cDNA library prepared from measles virus‐infected Vero cell poly(A)RNA, 435 clones selected at random were used to examine the sensitivity and specificity of cDNA probes derived from total poly(A)RNA from uninfected and infected Vero cells. The correlation between the abundance level of a particular species in the cDNA probe and the hybridization signal strength generated by the corresponding cDNA clone on a filter was reliably determined only when at least three independently prepared filters were examined. Variation in the amount of target plasmid was the most important cause of spurious signals. Variation in cDNA insert length did not disturb the signal strength within certain limits. cDNA species with abundance levels down to 0.08–0.01% were able to produce a hybridization signal above background. Unspecific cross‐hybridization was shown to define the sensitivity limit of mixed cDNA probes. Despite the many false signals present at different stages, cDNA probes provided valuable information: the cDNA probes were used to monitor relative RNA expression levels and to clone five different measles virus transcripts and 2 host cell transcripts more abundantly expressed in infected cells. The abundance levels of the measles virus nucleocapsid, phosphoprotein, matrix, fusion protein and haemagglutinin genes were 1.5%, 1.5%, 1%, 0.75% and 0.5%, respectively, of the total cDNA library.

Human papillomavirus infection in progressive and non‐progressive cervical intraepithelial neoplasia

Human papillomavirus (HPV) infection is common in cervical intraepithelial neoplasia (CIN) and is widely held to be responsible for its progression to grade 3. This thesis is examined here. Comparison of the level of HPV changes in 133 lesions that had not progressed to that in those from 197 women with histologically proven CIN 3 failed to reveal significant differences in their level of HPV infection on cytology, histology or in situ hybridization. However, in both these groups, some of the cases that did not show HPV positivity on in situ hybridization with probes reacting with the common HPV types did show evidence of HPV DNA using a general primer‐mediated polymerase chain reaction. This may indicate low‐copy number infections or non‐productive infections. Such reactions were more frequent in the women with progressive lesions, and it is probable that they may also have been at greater risk of cervical infection in general. The present findings suggest that a further factor, a co‐carcinogen, may be involved in progression to CIN 3, HPV being a common forerunner, providing a proliferative environment and thus favoring such an event.

Prognostic impact and concordance of TERT promoter mutation and protein expression in matched primary and metastatic cutaneous melanoma

Background:TERT promoter mutations are frequent in melanoma. Here we analysed the concordance and prognostic impact of TERT mutation and telomerase reverse transcriptase (TERT) protein expression in a large melanoma series.Methods:In 194 primary nodular melanomas with 72 matched loco-regional metastases, TERT promoter mutation status was assessed by Sanger sequencing and TERT protein expression by immunohistochemistry.Results:TERT mutations were found in 68% of primary melanomas and 64% of metastases, and the mutation status was discordant between primary tumour and metastasis in 24% of the cases. 6 of the 10 cases with discordant and wild-type metastases were also TERT wild type when re-tested in other intra-tumour regions, whereas 4 cases were mutation positive. TERT-mutated tumours tended to be thicker, have a higher mitotic count and higher patient age than TERT wild-type cases, but there was no significant association with reduced survival. TERT protein expression did not correlate with mutation status, but showed a similar discordancy between the primary and first metastatic lesion, and was significantly associated with reduced survival.Conclusions:TERT promoter mutations showed inter- and intra-tumoural discordancy, whereas only expression of TERT protein was associated with reduced patient survival.