TERC telomerase subunit gene copy number in different disease stages of non-hodgkin lymphoma and in hepatitis C.

ABSTRACT Focal amplification of specific regions of the genome creates high copy number and expression of oncogenes in tumors. By applying fluorescence in situ hybridization (FISH) to leukocytes of hepatitis C (HCV) patients and non-Hodgkin lymphoma (NHL) patients, we estimated gene dosage of the TERC gene at 3q26.3. Higher TERC copy numbers were found in NHL at diagnosis compared to HCV patient groups. Higher TERC copy numbers were also observed in NHL patient at diagnosis and relapse compared to patients in remission. We believe that the TERC gene amplification is involved in the process of genetic instability leading to tumor genesis such as in NHL.

Telomere aggregates in hepatitis C patients.

Telomeres of tumor nuclei tend to form aggregates (TA). The aim of this study was to estimate the TA formation in leukocytes of patients with chronic hepatitis C (HCV) which is considered to be premalignant disease, in patients of HCV who eradicated the virus. PNA Telomere kit (Dako) was used to evaluate the TA formation with the utilization of 2D fluorescence microscopy. A higher rate of TA was found in both HCV groups as compared to controls. Our results indicate that HCV patients have some of the components that create the cascade of events leading to malignancies.

Presence of hepatitis C virus DNA sequences in the DNA of infected patients.

BACKGROUND: Hepatitis C virus (HCV) consists of a single positive RNA molecule. In the present study we investigated the possibility that HCV may undergo integration into the genomic DNA of infected cells. MATERIALS AND METHODS: HCV(+) patients (n = 51) and 21 HCV(-) controls were investigated for HCV integration. RNase treated DNA samples of mononuclear cells (MNC) and liver biopsies of the patients were screened by PCR and seminested PCR processes for detection of integration. Positive results were further investigated by means of Southern analysis of patient's DNA as well as sequencing of PCR products of patient's DNA. RESULTS: Positive PCR results were detected in 4/51 of the HCV(+) patients and in none of the control group. Southern analysis showed the presence of HCV sequence in a 23 kbp band of the patient which is much larger than the viral genome itself (9.646 kbp). Sequencing of cloned PCR products showed an identity of over 95.0% to HCV. CONCLUSIONS: As much as we are aware this is the first demonstration of the possible integration of HCV sequences into the DNA of HCV(+) patients.

Replication status as a marker for predisposition for lymphoma in patients with chronic hepatitis C with and without cryoglobulinemia.

OBJECTIVE: Essential mixed cryoglobulinemia (EMC) type II is associated with hepatitis C virus (HCV) in 90% of the patients with this disorder. A significant subset of these patients is at risk to develop non-Hodgkin lymphoma (NHL). The objective of this study was to examine whether the presence of EMC, a presumably premalignant step of lymphoproliferation, is associated with changes in the replication state of normal structural genes. MATERIALS AND METHODS: The study group included three subgroups: (1) seven patients with HCV without EMC; (2) eight patients with HCV associated with EMC. 3. Seven patients with follicular lymphoma; and (3) six healthy individuals served as control group. Monocolor fluorescent in situ hybridization (FISH) with probes to p53, RB-1, and 21q22 was applied to leukocytes nuclei for the evaluation of replication timing. RESULTS: Asynchronous replication (SD) rate was similar in patients with NHL and those with HCV associated with EMC and both are significantly higher when compared to patients with HCV without EMC and to normal controls (p < 0.01) for each comparison. This held true for all studied loci (21q22, RB-1, and p53). Patients infected by HCV (but without EMC) had a significantly higher rate of asynchronous pattern in comparison with healthy controls (p < 0.01). CONCLUSIONS: Patients with a "premalignant" clinical condition HCV with EMC already demonstrate asynchronous type of replication which is similar to patients who already have an established malignant disease (i.e., NHL). In the future, replication may be used to assess the risk of malignant transformation in patients with "benign" proliferation.

Extension of transplantation free time by lamivudine in patients with hepatitis B-induced decompensated cirrhosis.

BACKGROUND: Liver transplantation for hepatitis B virus (HBV)-induced cirrhosis carries a high risk of graft reinfection and poor prognosis. Active viral replication is considered a contraindication for transplantation in most centers. Lamivudine, a new nucleoside analog, is a potent inhibitor of HBV replication that has been used safely for pretransplantation suppression of HBV replication. METHODS: We report the pattern of response to lamivudine treatment in three consecutive patients with decompensated cirrhosis due to the replicative phase of chronic HBV infection. RESULTS: In addition to virological and biochemical response, impressive clinical improvement was noted in all three patients, with disappearance of the ascites and marked improvement of synthetic liver function tests. One patient converted to anti-hepatitis B surface and is free of symptoms 20 months after initiation of treatment. The other two patients experienced significant clinical improvement for 8 to 9 months and were removed from the waiting list for transplantation. However, progressive liver disease recurred in both patients--one underwent liver transplantation and the other is a candidate for the procedure. CONCLUSION: The administration of lamivudine for pretransplantation HBV suppression was associated with impressive clinical and biochemical improvement. Lamivudine may extend the transplantation free time in such patients. The mechanism of this desirable effect should be explored.

Comparative genomic hybridization in polycythemia vera and essential thrombocytosis patients.

Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal chronic myeloproliferative disorders originating from a multipotent stem cell. Bone marrow examinations reveal chromosomal abnormalities in 15-43% of PV patients and 5% of ET patients, but no specific recurring abnormality has been found to date. We aimed to find cytogenetic aberrations in PV and ET by comparative genomic hybridization (CGH), a relatively new molecular cytogenetic technique. In this study, CGH analysis was performed on peripheral blood leukocytes of 12 PV patients and 8 ET patients. One patient (8.3%) with PV had an abnormal karyotype with a deletion in 7q11.2 and one patient with ET (12.5%) had a gain in 18p. Peripheral blood analysis by CGH revealed a low frequency of cytogenetic abnormalities in PV and ET patients. However, using CGH we were able to detect two cytogenetic aberrations that were not reported previously in these disorders.

Analysis of chromosomal aberrations in large hepatocellular carcinomas by comparative genomic hybridization.

Hepatocellular carcinoma (HCC) is a very common and highly malignant tumor, associated mainly with chronic viral hepatitis, cirrhosis of any cause, aflatoxin exposure and ethanol consumption. The aim of this study was to map genomic aberrations in HCC by a recently developed technique: comparative genomic hybridization (CGH). We applied CGH on 17 liver specimens, of which seven were HCCs. The rest were benign liver tumors, cirrhotic and normal livers, and other liver malignancies. Our study included mainly large tumors (mean size 10.5 cm) unrelated to viral hepatitis or cirrhosis. Our CGH analysis detected genomic imbalances in 42% of HCCs. The common aberrations included DNA gains of 1q, 9p, and 8q and DNA losses of 17p, 13q, 9q, 4q, and 11q. Also, we detected trisomies 8, 9, 18 and 21, which have not been reported previously. Gains and losses of DNA found in this study probably involve oncogenes and tumor suppressor genes that play a role in the puzzle of hepatocarcinogenesis. This study also suggests a possible link between the size of the tumor and the burden of genetic changes.

Bone marrow involvement, in intensively treated patients with intermediate grade non-Hodgkin's lymphoma, is a risk factor for granulocytopenia and fever.

Intensive combination chemotherapy administered to patients with non-Hodgkin's lymphoma (NHL) is inevitably associated with neutropenia and fever. However, the subset of patients who are at increased risk for myelotoxicity has not been clearly identified as yet. We evaluated 58 patients with NHL in order to verify whether bone marrow involvement (BMI) at diagnosis is a risk factor for neutropenia and infection following intensive chemotherapy. Patients with bone marrow involvement had significantly lower neutrophil counts both at diagnosis and following chemotherapy. An higher percentage of patients with BMI developed neutropenia (< 1000/mm3) following chemotherapy. Furthermore events of febrile neutropenia were more frequently encountered in patients with BMI. We conclude that BMI in patients with NHL is a risk factor for chemotherapy induced neutropenia and fever. The role of colony stimulating factors, administered in an attempt to prevent this complication, should be explored.

Seroconversion of hepatitis B during chemotherapy for malignant lymphoma.

We report a patient with chronic hepatitis B infection who developed lymphoma and was treated with concomitant cytotoxic and antiviral therapy. In contrast to the expected life threatening fulminant hepatitis that is often reported in this clinical setting, in our patient normalization of liver function tests with temporary loss of viral replication markers were seen. The implications of this rare clinical and serological course are discussed.

Association between alopecia and response to aggressive chemotherapy in patients with Hodgkin's disease.

The clinical significance and implications of absence of alopecia and/or neutropenia in patients treated with chemotherapy for malignant disease is unknown. We hypothesized that there is a common mechanism of resistance to the effects and the adverse effects of anticancer treatment, which may have clinical implications. To evaluate this hypothesis, we conducted a retrospective analysis of the charts of 17 consecutive patients with Hodgkin's disease who received at least 4 courses of combination chemotherapy (ABVD or MOPP/ABV). Twelve patients underwent complete alopecia while 5 patients retained their hair or had only minimal hair loss. The two groups had similar pretreatment characteristics. Ten (83%) of the patients with alopecia achieved complete remission as compared with 2 (40%) of the patients who retained their hair (P = 0.11). Also, patients without alopecia had fewer episodes of neutropenia (0% vs 33%, P = 0.02), delays of scheduled treatments (0% vs 66%, P = 0.02) or number of courses with dose reductions (20% vs 41%, P = 0.39). These differences shows a clear trend which did not reach statistical significance due to the small number of patients. We suggest that, in intensively treated patients with Hodgkin's disease, the absence of alopecia may predict a poor response to treatment. Also, patients without alopecia probably have fewer episodes of leukopenia, deferral of treatment courses or number of courses with dose reductions. We hypothesize that there is a common mechanism of resistance to cytotoxic agents that may prevent apoptotic death in both normal and malignant cells.

Telomere aggregates in non-Hodgkin lymphoma patients at different disease stages.

Telomeres of tumor nuclei tend to form aggregates (TA). The same phenomenon was also observed in premalignant states. The aim of this study was to estimate TA formation in leukocytes of patients with non-Hodgkin lymphoma (NHL) at different disease stages (diagnosis, treatment, relapse, and remission). The peptide nucleic acid Telomere Kit was used to evaluate TA formation, using two-dimensional fluorescence microscopy. A higher rate of TA was found in all the NHL stages (including remission) than in the control group. Significantly higher TA formation was also observed in the relapse group, compared to the diagnosis group. It may be possible that patients with higher TA numbers are prone to relapse. From our previous results involving replication pattern, random aneuploidy rate, and (recently) TA formation, it can be concluded that the patients in remission are at higher risk of developing relapse than the normal population throughout their life span. The genetic instability parameters remain in the cells of these patients, who must continue to be monitored throughout their life.

Telomere length in Hepatitis C.

Telomeres are nucleoprotein structures located at the termini of chromosomes that protect the chromosomes from fusion and degradation. Hepatocyte cell-cycle turnover may be a primary mechanism of telomere shortening in hepatitis C virus (HCV) infection, inducing fibrosis and cellular senescence. HCV infection has been recognized as potential cause of B-cell lymphoma and hepatocellular carcinoma. The present study sought to assess relative telomere length in leukocytes from patients with chronic HCV infection, patients after eradication of HCV infection (in remission), and healthy controls. A novel method of manual evaluation was applied. Leukocytes derived from 22 patients with chronic HCV infection and age- and sex-matched patients in remission and healthy control subjects were subjected to a fluorescence-in-situ protocol (DAKO) to determine telomere fluorescence intensity and number. The relative, manual, analysis of telomere length was validated against findings on applied spectral imaging (ASI) in a random sample of study and control subjects. Leukocytes from patients with chronic HCV infection had shorter telomeres than leukocytes from patients in remission and healthy controls. On statistical analysis, more cells with low signal intensity on telomere FISH had shorter telomeres whereas more cells with high signal intensity had longer telomeres. The findings were corroborated by the ASI telomere software. Telomere shortening in leukocytes from patients with active HCV infection is probably due to the lower overall telomere level rather than higher cell cycle turnover. Manual evaluation is an accurate and valid method of assessing relative telomere length between patients with chronic HCV infection and healthy subjects.

Bcl-2 rearrangement in patients with chronic hepatitis C associated with essential mixed cryoglobulinemia type II.

Hepatitis C virus (HCV) infection is found in 80% to 90% of patients with essential mixed cryoglobulinemia (EMC) type II, which is associated with monoclonal IgMk produced by monoclonal B cells. It was investigated whether bcl-2 rearrangement is associated with the clonal B-cell proliferation of EMC induced by hepatitis C. The study groups were composed of 15 patients with HCV and EMC, 12 patients with HCV without EMC, and 7 patients with chronic liver disease (CLD) unrelated to HCV. Fluorescence in situ hybridization with probes was applied to JH and to bcl-2 to study whether JH/bcl-2 translocation was present in these patients. Thirteen of 15 (86%) of patients with HCV-related EMC had the JH/bcl-2 translocation, a significantly higher rate than in HCV patients without EMC (16%; P < .001). Bcl-2 rearrangement was not detected in the patients with CLD not related to HCV. The JH/bcl-2 translocation may constitute a pathogenetic link for the development of NHL in patients with HCV infection. (Blood. 2000;96:2910-2912)