Gieri Cathomas

Arterial Neovascularization and Inflammation in Vulnerable Patients Early and Late Signs of Symptomatic Atherosclerosis

Background—Atherosclerosis is complicated by cardiovascular events such as myocardial infarction, stroke, or peripheral arterial occlusive disease. Inflammation and pathological neovascularization are thought to precipitate plaque rupture or erosion, both causes of arterial thrombosis and cardiovascular events. We tested the hypothesis that arterial inflammation and angiogenic events are increased throughout the arterial tree in vulnerable patients, ie, in patients who suffered from cardiovascular events, compared with patients who never suffered from complications of atherosclerosis. Methods and Results—In a postmortem study, we quantified the inflammatory infiltrate and microvascular network in the arterial wall of iliac, carotid, and renal arteries. Tissue microarray technology was adapted to investigate full-thickness arterial sectors. We compared 22 patients with symptomatic atherosclerosis with 27 patients who never had suffered from any cardiovascular event. The absolute intimal macrophage content was 2- to 4-fold higher in vulnerable patients at all 3 arterial sites analyzed (P<0.05). Patients with symptomatic atherosclerosis had a denser network of vasa vasorum than patients with asymptomatic disease (33±2 versus 25±2 adventitial microvessels per 1 mm2; P=0.008). Hyperplasia of vasa vasorum was an early and macrophage infiltration was a late sign of symptomatic atherosclerosis. Conclusions—High intimal macrophage content and a hyperplastic network of vasa vasorum characterize vulnerable patients suffering from symptomatic atherosclerosis. These changes are uniformly present in different arterial beds and support the concept of symptomatic atherosclerosis as a panarterial disease.

Transmission of Human Herpesvirus 8 Infection from Renal-Transplant Donors to Recipients

BACKGROUND Human herpesvirus 8 (HHV-8) has been detected in all forms of Kaposis sarcoma, including transplantation-associated Kaposis sarcoma. To investigate the possibility of transmission of HHV-8 through allografts, we measured the seroprevalence of HHV-8 before and after renal transplantation. METHODS Using an enzyme-linked immunosorbent assay with the recombinant HHV-8 protein orf 65.2, we analyzed serum samples from 220 renal-transplant recipients for the presence of antibodies to HHV-8 on the day of transplantation and one year later. Positive results were confirmed by an indirect immunofluorescence assay that detects antibodies to latent antigen and by Western blotting. Follow-up lasted at least four years. RESULTS The seroprevalence of HHV-8 in graft recipients increased from 6.4 percent on the day of transplantation to 17.7 percent one year after transplantation. Seroconversion occurred within the first year after transplantation in 25 patients, and Kaposis sarcoma developed in 2 of them within 26 months after transplantation. Sequential serum samples were obtained from 10 of the patients with seroconversion, and in 8 of these patients, IgM antibodies to HHV-8 appeared within three months after transplantation. In the case of six patients who seroconverted, serum samples from the donors were available, and five (83 percent) tested positive for HHV-8. In a control group of eight patients who were seronegative at the time of transplantation and who received allografts from HHV-8-negative donors, none seroconverted within the year after transplantation. CONCLUSIONS HHV-8 is transmitted through renal allografts and is a risk factor for transplantation-associated Kaposis sarcoma.

β-Catenin Mutations Are Frequent in Human Hepatocellular Carcinomas Associated with Hepatitis C Virus Infection

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Hepatitis B virus and hepatitis C virus infections, exposure to aflatoxin, and excessive intake of alcohol have been identified as major risk factors. However, the molecular mechanisms underlying their development are still poorly understood. Recently, β-catenin, one of the key components of the Wnt signaling pathway, has been found to be mutated in about 20% of HCCs, suggesting a role of the Wnt pathway in their development. In this study, we examined β-catenin and APC mutations in 22 HCCs associated with HCV infection, using single-strand conformation polymorphism (SSCP) followed by direct DNA sequencing. β-Catenin mutations were found in nine (41%) cases, but no APC mutations were found. β-Catenin immunohistochemistry revealed nuclear accumulation of β-catenin protein in all nine tumors with a β-catenin mutation and two additional tumors without a mutation. These results suggest that activation of the Wnt signaling pathway by β-catenin mutation contributes significantly to the hepatocellular carcinogenesis associated with HCV infection.

Alterations of RB1, p53 and Wnt pathways in hepatocellular carcinomas associated with hepatitis C, hepatitis B and alcoholic liver cirrhosis

Major etiologic factors associated with human hepatocellular carcinomas (HCCs) include infection with hepatitis C (HCV) and hepatitis B virus (HBV), excess alcohol intake and aflatoxin B1 exposure. While the G→T p53 mutation at codon 249 has been identified as a genetic hallmark of HCC caused by aflatoxin B1, the genetic profile associated with other etiologic factors appears to be less distinctive. In our study, we screened HCCs resulting from HCV infection (51 cases), HBV infection (26 cases) or excess alcohol intake (23 cases) for alterations in genes involved in the RB1 pathway (p16INK4a, p15INK4b, RB1, CDK4 and cyclin D1), the p53 pathway (p53, p14ARF and MDM2) and the Wnt pathway (β‐catenin, APC). Alterations of the RB1 pathway, mainly p16INK4a methylation, loss of RB1 expression and cyclin D1 amplification, were most common (69–100% of cases). There was a significant correlation between loss of RB1 expression and RB1 methylation. All 24 HCCs with RB1 promoter methylation lacked RB1 expression, while none of the 67 cases with RB1 expression exhibited RB1 methylation (p < 0.0001), suggesting that promoter methylation is a major mechanism of loss of RB1 expression in HCCs. Alterations of the p53 pathway consisted mostly of p53 mutations or p14ARF promoter methylation (20–48%). Mutations of the p53 gene were found at a similar frequency (13–15%) in all etiologic groups, without any consistent base change or hot spot. Mutations of β‐catenin were found in 13–31% of cases, while no APC mutations were detected in any of the HCCs analyzed. With the exception of only 3 of 39 cases (8%), cyclin D1 amplification and β‐catenin mutations were mutually exclusive, supporting the view that cyclin D1 is a target of the Wnt signaling pathway. Overall, the RB1, p53 and Wnt pathways were commonly affected in HCCs of different etiology, probably reflecting common pathogenetic mechanisms, i.e., chronic liver injury and cirrhosis, but tumors associated with alcoholism had more frequent alterations in the RB1 and p53 pathways than those caused by HCV infection.

Cytomegalovirus (CMV)—Specific T Cell Immunity after Renal Transplantation Mediates Protection from CMV Disease by Limiting the Systemic Virus Load

The role of cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) and T helper cells (Th) in controlling CMV infection, as detected by antigenemia assay and polymerase chain reaction (PCR) in blood leukocytes, and CMV disease was investigated in 20 renal transplant recipients. Within 3 months after transplant, CMV-specific CTL and Th responses were demonstrable in 11 (55%) and 15 (75%) patients, respectively; CMV infection was detected by antigenemia and PCR in 19 (95%) patients each. During the month of first CMV detection, there was an inverse correlation between CTL response and antigenemia at >/=20 positive cells/105 leukocytes (P=.007) but no association with lower antigenemia levels or PCR positivity. CMV disease developed in 7 (35%) patients and was associated with high-level antigenemia but was inversely correlated with detection of CTLs (P=.04). After renal transplantation, CMV-specific CTLs limit the systemic virus load as reflected by antigenemia levels and thereby mediate protection from CMV disease.

HER2 gene status in primary breast cancers and matched distant metastases

IntroductionThe status of the gene encoding human EGF-like receptor 2 (HER2) is an important prognostic and predictive marker in breast cancer. Only breast cancers with HER2 amplification respond to the targeted therapy with trastuzumab. It is controversial to what degree the primary tumour is representative of distant metastases in terms of HER2 status. Discrepancies in HER2 status between primary tumours and distant metastases have been described, but their reasons remain unclear. Here, we compared HER2 status on cytological specimens of distant metastases with the result from the primary carcinomas, and explored the prevalence of and the reasons for discrepant results.MethodsHER2 status was determined by fluorescence in situ hybridisation. HER2 gene amplification was defined as a HER2/chromosome 17 signal ratio of 2 or more. HER2 results from cytological specimens of matched distant metastases were compared with the results from the corresponding primary tumours (n = 105 patients). In addition, lymph node metastases were analysed in 31 of these patients.ResultsHER2 amplification was found in 20% of distant metastases. HER2 status was discordant between the primary tumour and distant metastasis in 7.6% of the 105 patients. Re-evaluation revealed that in five patients (4.7%), discrepancies were due to interpretational difficulties. In two of these patients, focal amplification had initially been overlooked as a result of heterogeneity in the primary tumours or in the metastases, respectively. A further three patients had borderline amplification with a ratio close to 2. Discrepancy remained unexplained in three patients (2.9%).ConclusionHER2 gene status remains highly conserved as breast cancers metastasise. However, discrepant results do occur because of interpretational difficulties and heterogeneity of HER2 amplification. Cytological specimens from distant metastases are well suited for HER2 fluorescence in situ hybridisation analysis.

Multi-target fluorescence in situ hybridization in bladder washings for prediction of recurrent bladder cancer

The objective of this study was to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for predicting recurrence of urothelial carcinoma (UC) after transurethral resection. One hundred and thirty‐eight patients (median age 68.5 years) with a history of UC were eligible for this prospective study. FISH was applied to cytospin specimens prepared from bladder washings taken during a negative control cystoscopy. The multi‐target FISH test UroVysion® (Abbott/Vysis) containing probes to the centromeres of chromosomes 3, 7, 17 and the 9p21 locus was used. UC recurrence was defined as a positive biopsy during follow‐up. The median follow‐up time was 19.2 (4–52) months. FISH was positive in 50 (36%) patients and negative in 88 (64%) patients. A recurrence occurred in 39% of the patients with a positive FISH test and in 21% of patients with a negative FISH test. FISH positivity according to manufacturers criteria, at the time of a negative cystoscopy, was not significantly associated with the risk of recurrence (p = 0.12). However, the sensitivity of the FISH test to predict recurrence was significantly improved by considering specimens with rare (≤10) tetraploid cells as negative (p < 0.006). In addition, presence of 9p21 deletion was significantly associated with recurrence (p < 0.01). Notably, positive standard cytology was an independent factor for subsequent recurrence in this study (p < 0.001). Taken together, multi‐target FISH may help to stratify the risk of recurrence of UC at the time of a negative follow‐up cystoscopy. Defining the optimal threshold for FISH positivity requires consideration of tetraploid pattern and 9p21 deletion. Our results also emphasize the paramount importance of conventional cytology for UC surveillance.

Cytomegalovirus infection and graft rejection in renal transplantation.

Background. Cytomegalovirus (CMV) infection and CMV disease have been associated with acute and chronic graft rejection. The introduction of the sensitive CMV antigenemia pp65 assay for detection of CMV infection allowed us to study the time course of CMV infection and acute rejection and the long-term outcome in renal transplant recipients with and without a CMV risk constellation. Methods. Prospective single center study including 48 renal transplant recipients at risk for CMV infection (donor and/or recipient CMV seropositive) and a control group of 36 CMV seronegative recipients of CMV seronegative kidney donors. Evidence of CMV infection was monitored by the CMV antigenemia pp65 assay every 1 to 2 weeks and compared with the occurrence of acute rejection in the posttransplant period and graft function at 5 years. Results. CMV infection developed in 83% (40/48) of patients of the CMV risk group within 4 months posttransplant. A total of 18 of patients experienced an acute rejection episode (control group 16/36;P =0.65). In 12/18 CMV infection followed rejection and in three patients antigenemia preceded the diagnosis of rejection. In three patients CMV antigenemia remained negative. Five-year follow up: Patient survival (44/48 vs. 31/36;P =0.48), graft survival (38/48 vs. 27/36;P =0.79), number of patients with at least one acute rejection episode: CMV risk group: 42.1%, control group 51% (P =0.46), serum creatinine: CMV risk group:130±66 &mgr;mol/liter, control group: 126±37 &mgr;mol/liter (P =0.56), proteinuria: CMV risk group: 0.02±0.02 g/mmol creatinine, control group: 0.02±0.02 g/mmol creatinine (P =1.0). Conclusion. CMV infection within 4 months posttransplant, as defined by a positive antigenemia assay was not found to be a risk factor for acute graft rejection or chronic graft dysfunction at 5 years.

Oral hairy leukoplakia in a HIV-negative renal transplant patient: a marker for immunosuppression?

We report the case of a 58-year-old renal transplant patient who developed oral hairy leukoplakia. Examination for HIV-1 and HIV-2 infection was negative. Biopsy of the lateral tongue showed ballooned prickle cells and electron microscopy revealed herpes-type viruses. In situ hybridization and examinations with the Southern blot technique yielded Epstein-Barr virus. Serology for Epstein-Barr virus was reactive. Immunological investigation of the patient showed a marked decrease of T-helper and T-suppressor cells as the result of immunosuppressive regimen. Oral hairy leukoplakia may be a marker for severe immunosuppression but is not necessarily associated with HIV infection.

Effect of buffered formalin on amplification of DNA from paraffin wax embedded small biopsies using real-time PCR

Background: The isolation of good quality DNA from routinely fixed and processed biopsy samples is crucial for the success of subsequent molecular analysis. Aims: To compare the amount of β actin DNA extracted from upper gastrointestinal tract biopsies fixed in buffered and unbuffered formalin. Methods: Amounts of β actin DNA extracted from forceps biopsies of the upper gastrointestinal tract fixed in unbuffered (n  =  22) and buffered formalin (n  =  16) were estimated by quantitative real-time polymerase chain reaction. Results: The yield of β actin DNA was significantly higher in biopsies fixed in buffered formalin than in those fixed in unbuffered formalin (median 2.8 × 104 and 5.3 × 102 DNA molecules, respectively; p < 0.005). Furthermore, fixation in buffered formalin led to a more reproducible DNA extraction, as indicated by the coefficient of variation (1.0 and 2.2, respectively). Conclusions: This study indicates that tissue samples should be fixed in buffered formalin to facilitate the use of molecular pathology analysis in routine biopsy material.