Biological Predictors of De Novo Tumors in Solid Organ Transplanted Patients During Oncological Surveillance: Potential Role of Circulating TERT mRNA.

BACKGROUND: De novo tumors are a major cause of morbidity and mortality after long-term solid organ transplantation. Chronic immunosuppression strongly affects solid organ transplanted (SOT) patients' immune system by promoting immune evasion strategies and reactivations of viruses with oncogenic potential, ultimately leading to cancer onset. In this scenario, an oncological Surveillance Protocol integrated with biobanking of peripheral blood samples and evaluation of immunovirological and molecular parameters was activated for SOT patients at CRO-IRCCS Aviano, with the aim of identifying suitable biomarkers of cancer development. METHODS: An exploratory longitudinal study was designed based on two serial peripheral blood samples collected at least three months apart. Forty nine SOT patients were selected and stratified by tumor onset during follow-up. Spontaneous T-cell responses to EBV, CMV and tumor associated antigens, EBV-DNA and CMV-DNA loads, and circulating TERT mRNA levels were investigated. RESULTS: Significantly higher levels of circulating TERT mRNA were observed 3.5-23.5 months before and close to the diagnosis of cancer as compared to tumor-free patients. Plasmatic TERT mRNA levels >97.73 copies/mL at baseline were significantly associated with the risk of developing de novo tumors (HR=4.0, 95%C.I. = 1.4-11.5, p=0.01). In particular, the risk significantly increased by 4% with every ten-unit increment in TERT mRNA (HR=1.04, 95%C.I. = 1.01-1.07, p=0.01). CONCLUSIONS: Although obtained in an exploratory study, our data support the importance of identifying early biomarkers of tumor onset in SOT patients useful to modulate the pace of surveillance visits.

The dose-response relationship between tobacco smoking and the risk of lymphomas: a case-control study.

BACKGROUND: Previous studies have provided limited support to the association between tobacco smoking and lymphomas with weak evidence of a dose-response relationship. METHODS: We investigated the relationship between tobacco smoking and risk of non-Hodgkin lymphomas (NHL) and Hodgkin lymphomas (HL) through logistic regression spline models. Data were derived from an Italian hospital-based case-control study (1999-2014), which enrolled 571 NHLs, 188 HLs, and 1004 cancer-free controls. Smoking habits and other lifestyle factors were assessed through a validated questionnaire. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression, adjusting for potential confounders. RESULTS: Compared to never smokers, people smoking ≥15 cigarettes/day showed increased risks of both NHL (OR = 1.42, 95% CI: 1.02, 1.97) and HL (OR = 2.47, 95% CI: 1.25, 4.87); the risk was particularly elevated for follicular NHL (OR = 2.43; 95% CI:1.31-4.51) and mixed cellularity HL (OR = 5.60, 95% CI: 1.31, 23.97). No excess risk emerged for former smokers or people smoking <15 cigarettes/day. Spline analyses showed a positive dose-response relationship with significant increases in NHL and HL risks starting from 15 and 21 cigarettes/day, respectively, with the most evident effects for follicular NHL and mixed cellularity HL. Smoking duration was significantly associated with the HL risk only (OR = 2.15, 95% CI: 1.16, 3.99). CONCLUSIONS: These findings support a role of tobacco smoking in the etiology of both NHL and HL, providing evidence of a direct association of risk with smoking intensity.

Hepatitis B and C viruses and risk of non-Hodgkin lymphoma: a case-control study in Italy.

BACKGROUND: Hepatitis C virus (HCV) has been consistently associated to non-Hodgkin lymphoma (NHL); conversely, few studies have evaluated a comprehensive serological panel of hepatitis B virus (HBV) in NHL etiology. METHODS: We conducted a case-control study in Italy in 1999-2014, enrolling 571 incident, histologically confirmed NHLs and 1004 cancer-free matched controls. Study subjects provided serum for HCV and HBV testing and for HCV RNA. Odds ratios (ORs) and corresponding 95 % confidence intervals (CIs) were estimated by logistic regression, adjusting for potential confounders. RESULTS: Circulating HCV RNA was detected in 63 (11.1 %) NHL cases and 35 (3.5 %) controls (OR = 3.51, 95 % CI: 2.25-5.47). Chronic HBV infection (i.e., positive to HBV surface antigen - HBsAg(+)) was found in 3.7 % of cases and 1.7 % of controls (OR = 1.95, 95 % CI: 1.00-3.81); a significantly elevated OR was observed for B-cell NHL (2.11, 95 % CI: 1.07-4.15). People with serological evidence of past HCV or HBV infection, vaccination against HBV, or detectable antibodies against HBV core antigen (anti-HBc(+)) alone were not at increased NHL risk. CONCLUSIONS: Our results support a role of chronic HCV infection in NHL in Italy and suggest an involvement of HBV infection. Associations were clearest for B-cell NHL and diffuse large B-cell lymphoma. Prevention and treatment of HCV and HBV infection may diminish NHL incidence, notably in areas with high prevalence of hepatitis viruses infection.

Cell-free DNA as a prognostic marker in stage I non-small-cell lung cancer patients undergoing stereotactic body radiotherapy.

OBJECTIVE: To evaluate whether plasma cell-free DNA (cfDNA) was related to clinical outcome in inoperable stage I non-small cell lung cancer (NSCLC) patients undergoing stereotactic body radiotherapy (SBRT). MATERIALS AND METHODS: Plasma cfDNA was assessed at baseline, before the last day and 45 days after the end of SBRT, in 22 NSCLC patients. Twenty-two healthy controls were also evaluated. RESULTS: Plasma cfDNA was higher in patients than in controls. An association with unfavourable disease-free survival was found for continuous baseline cfDNA increments (HR = 5.9, 95%CI: 1.7-19.8, p = 0.04). CONCLUSION: Plasma cfDNA may be a promising prognostic biomarker in high-risk NSCLC patients.

Plasma biomarkers of clinical response during chemotherapy plus combination antiretroviral therapy (cART) in HIV+ patients with advanced Kaposi sarcoma.

This study aimed to evaluate plasma concentration of selected cancer-associated inflammatory and immune-modulated cytokines in HIV+ patients with advanced Kaposi sarcoma (KS), and to explore candidate biomarkers capable of predicting clinical outcome in response to chemotherapy (CT) plus combination antiretroviral therapy (cART).Thirty-seven plasma cytokines/chemokines were assessed by Luminex technology in 27 consecutive HIV+ KS patients, followed-up during CT and cART of maintenance (m-cART). Associations between plasma concentration of biomarkers and patient clinical response to m-cART were evaluated by means of Hazard Ratios (HRs) and corresponding 95% Confidence Intervals (CIs).Plasma baseline concentration of Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor (HGF) and endoglin were found to be associated with m-cART clinical response (HR:1.56, 95%CI:1.09-2.22, p = 0.01; HR:0.32, 95% CI:0.10-0.99, p = 0.05; HR:0.72, 95% CI:0.54-0.96, p = 0.03, respectively). The multivariate analysis confirmed the associations of baseline plasma G-CSF and HGF concentration with m-cART clinical complete remission response (HR:1.78, 95% CI:1.15-2.74, p = 0.009; HR:0.19, 95% CI:0.04-0.95, p = 0.04). Our exploratory study suggested that plasma G-CSF, HGF and endoglin may be novel predictors of clinical response during m-cART in HIV+ KS patients. Nonetheless, these findings should be further validated in an independent population study.

γ-Herpesvirus load as surrogate marker of early death in HIV-1 lymphoma patients submitted to high dose chemotherapy and autologous peripheral blood stem cell transplantation.

Autologous stem cell transplantation (ASCT) is a feasible procedure for human immunodeficiency virus-1 (HIV-1) lymphoma patients, whose underlying disease and intrinsic HIV-1- and ASCT-associated immunodeficiency might increase the risk for γ-herpesvirus load persistence and/or reactivation. We evaluated this hypothesis by investigating the levels of Epstein-Barr virus (EBV)- and Kaposi sarcoma-associated herpesvirus (KSHV)-DNA levels in the peripheral blood of 22 HIV-1-associated lymphoma patients during ASCT, highlighting their relationship with γ-herpesvirus lymphoma status, immunological parameters, and clinical events. EBV-DNA was detected in the pre-treatment plasma and peripheral blood mononuclear cells (PBMCs) of 12 (median 12,135 copies/mL) and 18 patients (median 417 copies/10(6) PBMCs), respectively; the values in the two compartments were correlated (r = 0.77, p = 0.0001). Only EBV-positive lymphomas showed detectable levels of plasma EBV-DNA. After debulking chemotherapy, plasma EBV-DNA was associated with lymphoma chemosensitivity (p = 0.03) and a significant higher mortality risk by multivariate Cox analysis adjusted for EBV-lymphoma status (HR, 10.46, 95% CI, 1.11-98.32, p = 0.04). After infusion, EBV-DNA was detectable in five EBV-positive lymphoma patients who died within six months. KSHV-DNA load was positive in only one patient, who died from primary effusion lymphoma. Fluctuations in levels of KSHV-DNA reflected the patient's therapy and evolution of his underlying lymphoma. Other γ-herpesvirus-associated malignancies, such as multicentric Castleman disease and Kaposi sarcoma, or end-organ complications after salvage treatment were not found. Overall, these findings suggest a prognostic and predictive value of EBV-DNA and KSHV-DNA, the monitoring of which could be a simple, complementary tool for the management of γ-herpesvirus-positive lymphomas in HIV-1 patients submitted to ASCT.

Autograft HIV-DNA load predicts HIV-1 peripheral reservoir after stem cell transplantation for AIDS-related lymphoma patients.

Autologous stem cell transplantation (ASCT) is a widely used procedure for AIDS-related lymphomas, and it represents an opportunity to evaluate strategies curing HIV-1 infection. The association of autograft HIV-DNA load with peripheral blood HIV-1 reservoir before ASCT and its contribution in predicting HIV-1 reservoir size and stability during combination antiretroviral therapy (cART) after transplantation are unknown. Aiming to obtain information suggesting new functional cure strategies by ASCT, we retrospectively evaluated HIV-DNA load in autograft and in peripheral blood before and after transplantation in 13 cART-treated HIV-1 relapse/refractoring lymphoma patients. Among them seven discontinued cART after autograft infusion. HIV-DNA was evaluated by a sensitive quantitative real-time polymerase chain reaction (PCR). After debulking chemotherapy/mobilization, the autograft HIV-1 reservoir was higher than and not associated with the peripheral HIV-1 reservoir at baseline [median 215 HIV-DNA copies/10(6) autograft mononuclear cells, range 13-706 vs. 82 HIV-DNA copies/10(6) peripheral blood mononuclear cells (PBMCs), range 13-479, p = 0.03]. After high dose chemotherapy and autograft infusion, HIV-DNA levels reached a plateau between month 6 and 12 of follow-up. No association was found between peripheral HIV-DNA levels at baseline and after infusion in both cART interrupting and not interrupting patients. Only in the last subgroup, a stable significant linear association between autograft and peripheral blood HIV-1 reservoir emerged from month 1 (R(2) = 0.84, p = 0.01) to month 12 follow-up (R(2) = 0.99, p = 0.0005). In summary, autograft HIV-1 reservoir size could be influenced by the mobilization phase and predicts posttransplant peripheral HIV-1 reservoir size in patients on continuous cART. These findings could promote new research on strategies reducing the HIV-1 reservoir by using the ASCT procedure.

Multiplex analysis of blood cytokines as a prognostic tool in HIV related non-Hodgkin lymphoma patients: a potential role of interleukin-7.

BACKGROUND: Recent studies suggest a powerful prognostic value for blood cytokine levels in different diseases. Non-Hodgkin lymphoma (NHL) still represents one of the main causes of death in the HIV setting, with a wide variation in outcome and survival among patients. We measured blood concentrations of 11 cytokines from HIV-NHL patients at diagnosis and correlated these with the patient outcome to evaluate the prognostic value. METHODS: Luminex technology was used to simultaneously measure serum levels of interleukin IL-2/5/6/7/8/10/13/15, INF-γ, TNF-α and VEGF. Eighty-one consecutive HIV-NHL patients, at diagnosis, were studied. Hazard Ratios (HRs) and corresponding 95% confidence intervals (CIs) of disease-free survival (DFS) and overall survival (OS) were computed according to cytokine levels. HRs were also calculated for continuous variation of IL-7. RESULTS: In the multivariate analysis, statistically significant associations to both DFS and OS were found for IL-7 serum levels ≥ 3.2 pg/mL (HR=5.55, 95%CI:2.38-12.95; HR=3.53, 95%CI:1.60-7.77, respectively), IL-8 ≥ 18 pg/mL (HR=2.69, 95%CI:1.15-6.30; HR=2.35, 95%CI:1.01-5.51, respectively) and IL-10 ≥ 13 pg/mL (HR=2.82, 95%CI: 1.19-6.71; HR=2.98, 95%CI:1.21-7.30, respectively). When the multivariate analyses were mutually adjusted for INF-γ, IL-7, IL-8, IL-10 and IL-15, serum IL-7 ≥ 3.2 pg/mL emerged as factor independently associated to increased risk of DFS (HR=3.63, 95%CI:1.47-8.93) and OS (HR=3.97, 95%CI:1.49-10.57). CONCLUSIONS: IL-7, measured at NHL diagnosis, was the only cytokine strongly and independently associated to both DFS and OS. The multiplex analysis of different blood cytokines' concentration might be useful in defining additive predictive markers in HIV-NHL management and ascertainment of their outcome.

Assessment of immunovirological features in HIV related non-Hodgkin lymphoma patients and their impact on outcome.

BACKGROUND: Despite the era of highly active antiretroviral therapy, non-Hodgkin lymphoma (NHL) remains one of the main causes of death in HIV-infected patients, with a wide variation on the outcome. OBJECTIVES: We investigated immunological status and EBV, HHV8, HIV viral load in a group of HIV-infected patients at diagnosis of NHL to evaluate their prognostic significance. STUDY DESIGN: Eighty-one consecutive HIV+ NHL patients were studied. CD4 and CD8 cell counts, HHV8 DNA, EBV DNA, HIV RNA and HIV DNA were assessed at diagnosis and at 3 months after chemotherapy initiation. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of disease free survival (DFS) and overall survival (OS) were computed according to CD4 and CD8 cell counts, EBV DNA, HIV RNA and HIV DNA. HRs were, thereafter, computed also for continuous variation of CD4, CD8 cell counts and EBV DNA. RESULTS: In the multivariate analysis, CD4<160 and CD8<590 cell/µl and EBV DNA≥300 c/ml were independently associated to DFS (HR=2.98; 95%CI: 1.26-7.03; HR=2.65, 95%CI: 1.13-6.19; HR=4.01; 95%CI: 1.81-8.91) and OS (HR=3.32; 95%CI: 1.41-7.83; HR=4.62, 95%CI: 1.91-11.19; HR=3.11, 95%CI: 1.42-6.80). HRs for DFS and OS decreased continuously with increasing CD4 and CD8 cell counts, while they increased continuously with increasing EBV DNA levels. CONCLUSIONS: The association with survival of low CD4 and CD8 cell counts and detectable EBV viremia, measured at lymphoma's diagnosis, identified three independent prognostic biomarkers that might help in the management of NHL HIV+ patients, offering complementary information in the ascertainment of their outcome.

Collection and preservation of frozen microorganisms.

The storage of the different microorganisms over long periods is necessary to ensure reproducible results and continuity in research and in biomedical processes and also for commercial purposes. Effective storage means that a microorganism is maintained in a viable state free of contamination or genetic drift and must be easily restored without genotypic or phenotypic alterations to its original characteristics and properties. To this end, different techniques have been described and advances in cryopreservation technology have led to methods that allow low-temperature maintenance of a variety of cell types, minimizing the risks of genetic change and are now recommended for long-term storage of most microorganisms.This chapter summarizes the most important steps and components in the process of low- and -ultra-low temperatures freezing of bacteria, parasites, yeasts and fungi, viruses, and recombinant microorganisms.

Multicenter comparative study of Epstein-Barr virus DNA quantification for virological monitoring in transplanted patients.

BACKGROUND: EBV-related post-transplant lymphoproliferative diseases are usually accompanied by increased EBV DNA in peripheral blood. Monitoring EBV DNAemia is the basis for weighing decisions regarding initiation of pre-emptive or anti-EBV-related tumor therapy. However, the definition of clinically relevant cut-off values is hampered by the lack of standardization in EBV DNA testing. OBJECTIVES: To estimate inter-laboratory variability and to evaluate the impact of different matrices in EBV DNA load determination in Italian laboratories involved in monitoring of virus infections in transplanted patients. STUDY DESIGN: Two different proficiency panels were distributed among seven centers: the first contained cell-associated and cell-free EBVs; the second was prepared by spiking both cell-associated and cell-free EBVs in EBV DNA-negative whole blood from EBV seropositive healthy donors. Samples were extracted and amplified with different methods. Intra-laboratory and inter-laboratory variabilities was evaluated. RESULTS: 337 EBV DNA determinations were performed. Sensitivity was 100% for both panels, specificity was 100% for the first and 74% for the second panel, where whole blood was utilized as the matrix. Discrepant results in the second panel were restricted to samples containing low copy numbers. Quantification fell within ±0.5 log in 73% of the determinations. Values for cell-associated samples tended to be more heterogeneous than those obtained from cell-free samples. Good overall linearity was observed for each sample type; inter-laboratory variability ranged from 4.71% to 12.86%. CONCLUSIONS: The results of this multicenter study indicate that EBV DNAemia may be reliably quantified by different laboratories using a variety of commercial and in-house molecular assays.

Prevalence of antibodies against Kaposi's sarcoma associated herpes virus (KSHV) complement inhibitory protein (KCP) in KSHV-related diseases and their correlation with clinical parameters.

Kaposi's sarcoma-associated herpes virus (KSHV) encodes its own inhibitor of the complement system, designated KSHV complement control protein (KCP). Previously, we detected anti-KCP antibodies in a small group of 22 patients suffering from Kaposi's sarcoma (KS) and KSHV-related lymphoproliferative diseases (Vaccine, 25:8102-9). Anti-KCP antibodies were more prevalent in individuals suffering from KSHV-related lymphomas than KS and also in those with high titer of antibodies against lytic KSHV antigens. Herein we analyze anti-KCP antibodies in 175 individuals originating from three different groups from northern Sweden or Italy, which included patients suffering from classical or HIV-associated KS, Multicentric Castleman's Disease, KSHV-associated solid lymphoma, pleural effusion lymphoma and healthy individuals with detectable KSHV immune response. Our current study confirmed previous observations concerning antibody prevalence but we also analyzed correlations between anti-KCP antibodies and classical KS evolution, clinical stage and viral load in body fluids. Furthermore, we show that patient's anti-KCP antibodies are able to decrease the ability of KCP to inhibit complement. This fact combined with results of statistical analysis suggests that KCP inactivation by specific antibodies may influence progression of classical KS.

Association between Epstein-Barr virus infection and risk for development of pregnancy-associated breast cancer: joint effect with vitamin D?

BACKGROUND: Few studies have evaluated the role of the ubiquitous Epstein-Barr virus (EBV) infection, together with levels of the immunomodulator, vitamin D, in different breast cancer entities. We studied, prospectively, the association of EBV and vitamin D status with the risk of pregnancy-associated breast cancer (PABC), breast cancer diagnosed during pregnancy or 1 year post-partum, using a nested case-control study. METHODS: Serum vitamin D and antibodies to EBV were measured for 108 PABC cases of the Finnish Maternity Cohort, and 208 controls matched for date of birth, date of sampling and parity. The joint effect of vitamin D and EBV on the risk of PABC was evaluated. RESULTS: EBV seropositivity was generally not associated with the risk of PABC. Among individuals with sufficient (≥75 nmol/l) levels of vitamin D, we, however, found similar increased risk estimates for PABC associated with serum immunoglobulin G (IgG) antibodies to EBV early antigens [odds ratio (OR)=7.7, 95% (confidence interval) CI 1.4-42.3] and the viral reactivator protein, ZEBRA (OR=7.8, 95% CI 1.1-61.2). CONCLUSION: Immunological markers of EBV reactivation status among individuals with sufficient vitamin D levels were consistently associated with increased risk of the disease. This suggests that EBV reactivation may be an indicator of the progression of breast cancer occurring soon after pregnancy, while the virus probably is not the aetiological agent.

Immune recovery after autologous stem cell transplantation is not different for HIV-infected versus HIV-uninfected patients with relapsed or refractory lymphoma.

BACKGROUND: High-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT) are feasible and effective salvage treatments for human immunodeficiency virus (HIV)-related relapse or refractory lymphoma. Among the main concerns with ASCT in HIV-infected persons is the additional immune depletion caused by treatment, which could amplify the preexisting immune deficit. The aims of our study were to assess the impact of conventional chemotherapy before salvage treatment was administered, in this population, and to evaluate immune reconstitution dynamics during ASCT. METHODS: All 33 HIV-infected and HIV-uninfected patients who underwent comparable ASCT protocols at the National Cancer Institute (Aviano, Italy) who underwent 1 month of follow-up after transplantation were included in a prospective immunological study. Demographic, clinical, and immunovirological data were obtained before administration of induction therapy, during transplantation, and at 24 months of follow-up. RESULTS: Before HDC, no significant differences were observed in CD4(+) cell subsets and signal joint T cell receptor excision circles (sjTRECs), although HIV-infected persons had inverted ratios of CD4(+) cells to CD8(+) cells because they had higher CD8(+) T cell counts, compared with HIV-uninfected persons. After ASCT, this inversion was also observed in HIV-uninfected patients up to 24 months. CD4(+) cell subsets had similar recoveries, with a temporary setback in HIV-infected persons 3 months after reinfusion, together with an increase in infections. sjTRECs demonstrated similar dynamics in both populations and serve as a useful predictive marker of recovery of CD4(+) cell subsets. No significant changes emerged in HIV DNA levels during the follow-up period, with values at 24 months significantly lower than those at baseline. CONCLUSIONS: Our study demonstrated that ASCT in HIV-infected persons with lymphoma does not worsen the initial immune impairment and does not enhance viral replication or the peripheral HIV reservoir in the long term.

Predictive value of HIV type 1 DNA levels on overall survival in HIV-related lymphoma Patients treated with high-dose chemotherapy (HDC) plus autologous stem cell transplantation (ASCT).

The kinetics and predictive value of HIV-1 DNA (HIV DNA) levels in relapsed or refractory HIV lymphoma patients, treated with high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT), were investigated. HIV DNA was measured by real-time PCR in the peripheral blood mononuclear cells (PBMCs) of 22 patients observed for a median follow-up of 31.0 months. At baseline, HIV DNA was found to be correlated with HIV-1 RNA (HIV RNA) (r = 0.56), but not with CD4(+) counts (r = -0.10). HIV RNA load was under control for the entire follow-up, while HIV DNA levels were almost always detectable (baseline levels vs. 1 year from ASCT levels, p > 0.05). Baseline HIV DNA levels were significantly different between alive and deceased patients (p = 0.03), and the overall survival (OS) analysis showed that for patients with higher HIV DNA levels at baseline there was a higher and nearly significant risk of death if compared to patients with lower levels (HR, 8.33, 95% CI, 0.99-70.06, p = 0.05). Our study demonstrated that high HIV DNA levels at baseline could predict overall survival after ASCT in one of the largest cohorts of HIV lymphoma patients treated with salvage therapy.

No risk of maternal EBV infection for childhood leukemia.

We performed a large nested case-control study within the Finnish and Icelandic maternity cohorts to verify/falsify the association of maternal EBV infection with an increased risk of acute lymphoblastic leukemia (ALL) in the offspring found in previous studies. All hematologic malignancies diagnosed among children born during 1983 to 2006 in Finland and 1997 to 2005 in Iceland were identified through national cancer registries. For each index mother of a leukemia case, three matched control mothers with cancer-free offspring were identified. First trimester sera from 561 ALL and 144 non-ALL index mothers and from 2,105 control mothers were analyzed for antibodies to EBV viral capsid antigen (IgG and IgM), early antigen (IgG) and ZEBRA protein (IgG). Conditional logistic regression-based estimates of odds ratios and 95% confidence intervals adjusted for birth order and sib-ship size were calculated. Overall, there was no evidence of increased risk of ALL associated to EBV viral capsid antigen IgM (odds ratio, 0.9; 95% confidence interval, 0.5-1.8). The early antigen and ZEBRA antibodies (EBV reactivation markers) were also not associated with risk. The data argue against a role of EBV in ALL.

Human herpesvirus 8 DNA quantification in matched plasma and PBMCs samples of patients with HHV8-related lymphoproliferative diseases.

BACKGROUND: The quantitative evaluation of human herpesvirus 8 (HHV8) DNA is not well described in the clinical management of HHV8-related lymphoproliferative diseases. OBJECTIVES: To evaluate and to compare HHV8 viral load in different blood compartments from patients with multicentric Castleman's disease (MCD), primary effusion lymphoma (PEL) and HHV8-associated solid lymphoma (SLY) and to establish which clinical sample would be preferable for HHV8 DNA testing. STUDY DESIGN: We assessed HHV8 DNA in plasma and peripheral blood mononuclear cells (PBMCs) paired samples from 7 PEL, 8 MCD, 2 SLY HIV+ patients at the diagnosis and during the course of the illness by using a real time PCR assay. RESULTS: HHV8 viremia was always detectable at diagnosis. HHV8 DNA levels were correlated in matched pairs of samples at diagnosis and during follow-up (Spearman correlation coefficient: r=0.83, p<0.001 and r=0.73, p<0.001, respectively). The performance characteristics of the PCR assay with both materials did not show disparity by the analysis of the receiver operating characteristic (ROC) curve (X(1)(2)=0.50; p=0.48). CONCLUSIONS: Plasma or PBMCs are both adequate samples for HHV8 DNA quantification and Real time PCR provides a reliable method to estimate viral replication in patients with HHV8-related lymphoproliferations, where HHV8 viral load is a consistent feature.

Antibodies against Kaposi sarcoma-associated herpes virus (KSHV) complement control protein (KCP) in infected individuals.

Kaposi sarcoma-associated herpesvirus (KSHV) is the most important etiopathological factor of Kaposi's sarcoma (KS) and some specific types of malignant lymphomas. One of the viral lytic genes encodes the KSHV complement control protein (KCP), which functionally mimics human complement inhibitors. Although this protein provides an advantage for evading the complement attack, it can serve as target for adaptive immune response. Herein, we identified anti-KCP IgG antibodies in patients with KS and KSHV-related lymphomas. KCP-specific antibodies were only detected in sera of those patients who had high titres of antibodies against lytic or latent KSHV antigens. Complement control protein domain 2 (CCP2) was found to be the most immunogenic part of the KCP protein. Furthermore, pre-incubation of KCP-expressing CHO cells with patient sera containing anti-KCP antibodies resulted in an increased complement deposition when incubated with human serum.

Serum antibody response to lytic and latent Epstein-Barr virus antigens in undifferentiated nasopharyngeal carcinoma patients from an area of nonendemicity.

Epstein-Barr virus (EBV)-associated undifferentiated carcinoma of the nasopharyngeal type (UCNT) is highly prevalent in southeast China, where immunoglobulin A (IgA) antibodies to viral capsid antigen and early antigen (EA) represent important markers, routinely used to assist in diagnosing this malignancy. Our study aimed at determining the EBV serological profiles of 78 UCNT patients from Italy, an area of nonendemicity for this tumor, using different assays specific for both lytic and latent EBV antigens. Serum IgA against both EA and EBNA1 and IgG and IgA to the latent membrane protein 1 (LMP1), to EA, and to the EBV transactivator ZEBRA protein were assessed. These serological responses were then evaluated according to the clinicopathologic parameters at diagnosis. The sensitivities of the IgG assays were 37.7% for LMP1, 73.6% for EA, and 61.0% for ZEBRA. EA/EBNA1 IgA reactivity was 84.4%, and a high association (odds ratio [OR], 2.6; 95% confidence interval [CI], 1.7 to 4.0) with UCNT was observed. When EBV serological reactivities were analyzed according to the tumor, node, and metastasis staging system (TNM), a statistically significant association was found between N stage and IgG antibody rates for EA (OR, 3.6; 95% CI, 1.2 to 10.9) and ZEBRA (OR, 2.6; 95% CI, 1.2 to 5.5) and between M stage and IgG antibody rates for ZEBRA (OR, 7.1; 95% CI, 3.2 to 16.0) and LMP1 (OR, 14.0; 95% CI, 1.8 to 110.9). Our results show that no single serological marker allows the detection of all UCNT cases. EA/EBNA1 IgA represents a reliable marker for diagnosis, with a high predictive value also in areas where UCNT is not endemic, such as Italy. The analysis of serological results according to TNM classification is consistent with a progressive impairment of humoral immune response to EBV as the disease advances and may be used to improve the accuracy of diagnosis.

Activation of maternal Epstein-Barr virus infection and risk of acute leukemia in the offspring.

After identifying an association between maternal Epstein-Barr virus (EBV) reactivation and acute lymphoblastic leukemia (ALL), the authors analyzed a nested case-control study within Finnish and Icelandic maternity cohorts with 7 million years of follow-up to confirm EBV's role in ALL. Offspring of 550,000 mothers were followed up to age 15 years during 1975-1997 by national cancer registries to identify leukemia cases. Mothers of cases and three quarters of matched mothers of controls were identified by national population registers. First-trimester sera from mothers of 304 ALL cases and 39 non-ALL cases and from 943 mothers of controls were analyzed for antibodies to viral capsid antigen, early antigen, and EBV transactivator protein ZEBRA. Relative risk, estimated as odds ratio (95% confidence interval), was adjusted for birth order and sibship size. Combining early antigen and/or ZEBRA immunoglobulin G antibodies with the presence of viral capsid antigen immunoglobulin M antibodies did not increase the estimate for ALL risk for viral capsid antigen immunoglobulin M alone (odds ratio = 1.9, 95% confidence interval: 1.2, 3.0). Both ZEBRA immunoglobulin G antibodies and viral capsid antigen immunoglobulin M antibodies were associated with an increased risk of non-ALL in the offspring (odds ratio = 4.5, 95% confidence interval: 1.3, 16; odds ratio = 5.6, 95% confidence interval: 1.1, 29, respectively), suggesting EBV reactivation in the mothers of non-ALL cases. EBV reactivation may be associated with a proportion of childhood leukemia.