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BackgroundDe 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.MethodsAn 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.ResultsSignificantly 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).ConclusionsAlthough 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.

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.

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 maintanence (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.

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

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.

To assess kinetics of plasmatic cytokines during radiation therapy (RT) for locally advanced and early-stage non-small cell lung cancer (NSCLC). This prospective study was conducted on 15 early-stage NSCLC underwent to extreme hypofractionated regimen (52 Gy in 8 fractions) with stereotactic body RT (SBRT), and 13 locally advanced NSCLC underwent to radical moderated hypofractionated regimen (60 Gy in 25 fractions) with intensity modulated RT (IMRT). For patients undergoing SBRT, peripheral blood samples were collected on the first day of SBRT (TFd), the last day (TLd) and 45 days (T45d) after the end of SBRT. For patients undergoing IMRT, blood samples were collected at: TFd, 2 weeks (T2w), 4 weeks (T4w), TLd, and T45d. The following cytokines were measured: IL-1, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17A, EGF, FGF-2, INF-γ, MIP-1α, MIP-1β, TGF-α, TNF-α, and VEGF. Cytokine levels measured in different RT time and compared. No difference in baseline levels of cytokines was documented between patient radiation approaches (except for MIP-1α). For SBRT patients, a mean reduction of IL-10 and IL-17 plasma level was documented between TLd and TFd, respectively (p

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.

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.

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.

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.

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.

We investigated EBV viremia in matched serum and peripheral blood mononuclear cells (PBMCs) from one of the largest Italian cohort of Undifferentiated Carcinoma of Nasopharyngeal Type (UCNT) patients (N=34). By using a LMP-1 real-time PCR assay, we found that EBV DNA detection rate was 74% (median 8417 copies/ml) and 24% (median 164 copies/10(6)cells) on serum and PBMCs, respectively. Significantly higher serum EBV DNA levels were detected in patients with advanced UCNT (nodal stage N2 versus N0-1 and N3 versus N0-1, P=0.03 and 0.018; overall stage IV versus I-II, P=0.03). During the follow-up, there was also a statistically significant difference of EBV DNA viral load between patients with and without clinical relapse (P=0.008). We concluded that serum EBV DNA reflects the biological activity of the UCNT and may be a prognostic factor also in a low-incidence region.

The new generation assay Versant HCV RNA 3.0v (Bayer Diagnostics) was evaluated to quantify hepatitis C virus (HCV) RNA levels in liver biopsy specimens from patients with HCV and human immunodeficiency virus (HIV) coinfection. A total of 25 liver biopsies and sera collected at the time of liver biopsy were used. The efficiency of HCV RNA recovery from spiked samples was between 38.6 and 50.7%, and reproducible measurements of viral load were observed (the intra- and interrun coefficients of variation were 0.5 to 13% and 3.5 to 24.7%, respectively), with good specificity and sensitivity. Linearity was evaluated in the range of 96,154 to 769 IU/μg by using a serially diluted high-titer sample. Coinfected patients had high HCV RNA viral loads in serum and liver (498,471 IU/ml and 231,495 IU/μg, respectively), and both levels were correlated ( r = 0.63; P < 0.01). The amount of hepatic HCV RNA was significantly higher among patients with genotype 1 than among patients with genotype 3 ( P < 0.01). The virological end-of-treatment response in the serum was associated with a lower pretreatment intrahepatic HCV viral load ( P = 0.03). The new version of b-DNA is a sensitive, specific, and reproducible method for quantitating HCV RNA in the liver. Given its positive analytical performance, the assay will be used to evaluate the HCV RNA levels in the serum and liver during follow-up of patients treated with an anti-HCV therapeutic regimen.

An efficent antitumor and antiviral cellular immune response requires optimal interferon-gamma (IFN-gamma) secretion and perforin expression in CD8(+) T cells. The aim of this study was to define whether CD4(+) and CD8(+) T cells from patients with undifferentiated carcinoma of nasopharyngeal type (UCNT), a tumor regularly associated with the Epstein-Barr virus (EBV), have abnormal phenotype profiles, cytokine production, perforin and CD3-zeta expressions. Our data showed that CD4 and CD8 subset distribution was not grossly altered in the peripheral blood of UCNT patients, while tumor biopsies contained an increased proportion of CD8(+) T cells. The analysis of the CD4(+) subset showed a defect in interleukin-2 (IL-2) production and a moderate increase of IL-10 production, a situation consistent with a Th1/Th2 imbalance. We have also demonstrated that CD8(+) lymphocytes from UCNT patients had a marked impairment of IFN-gamma secretion and perforin expression. This impairment was not related to the presence of detectable EBV DNA in the plasma. In UCNT patients, the blockade of the perforin pathway and of IFN-gamma production may constitute important mechanisms for immune escape by the tumor and for impaired control of EBV replication.

Viral load is an important marker of activity of viral diseases for a number of viruses. We wished to evaluate whether the viral load of human herpesvirus 8 (HHV-8) in peripheral blood was a consistent feature of Kaposi's sarcoma (KS) patients and whether the viral load correlated with human immunodeficiency virus (HIV) RNA levels, CD4 counts, and/or the HHV-8 seroreactivity. Fifty-four consecutive plasma samples from 14 patients with KS were evaluated for HHV-8 viral load by quantitative real-time PCR. Samples were analyzed at the start of highly active antiretroviral therapy (HAART) and at different intervals during treatments. The median HHV-8 DNA load before HAART treatment was 8,998 (ranging from 170 to 40,100) copies/ml and 12,270 (ranging from 40 to 142,575) copies/ml during HAART. There were both increasing and decreasing trends. There was an association between HHV-8 DNA and HIV RNA viral loads (odds ratio [OR] = 5.40; 95% confidence interval [95% CI], 1.54 to 18.98) and between HHV-8 viral load and CD4 cell counts (OR = 7.24; 95% CI, 1.30 to 40.35). High HHV-8 viral load was also correlated with the titers of antibodies to the lytic HHV-8 antigen detected with immunofluorescence ( P < 0.01), but not with antibodies to the latent HHV-8 antigen. In conclusion, we found that HHV-8 viremia in KS is associated with HIV viral load, CD4 cell counts, and lytic HHV-8 serological reactivity. HHV-8 viral load monitored by real time PCR might be useful for determination HHV-8 viral load during the follow-up of KS patients.

The mode of transmission of human herpesvirus 8 (HHV8) was investigated in two seroepidemiological studies of Swedish women who completed a questionnaire about sexual behavior. Seropositivity for HHV8 antibodies, measured using an indirect immunofluorescence assay, was linked to a high number (>10) of sexual partners (P < 0.004). It also correlated strongly with a history of other sexually transmitted diseases (STD; P < 0.0001), in particular with a history of Chlamydia trachomatis infection and condyloma acuminata. There was appreciable HHV8 seropositivity already among virginal or monogamous women (9%). In summary, HHV8 transmission to women in Sweden may occur nonsexually. When sexual transmission occurs, it appears to be associated with high risk-taking sexual behavior.

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. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2790–2)

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.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.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.In the multivariate analysis, CD4160 and CD8590 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.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.

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.

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.

Objective. Aim of our study was to evaluate the kinetics of HIV-1 DNA levels (HIV DNA) and its clinical role in HIV-related lymphoma patients undergoing autologous stem cell transplantation (ASCT). Materials and methods. HIV DNA was measured by real-time PCR in the peripheral blood mononuclear cells (PBMCs) of 22 patients, 16 with non-Hodgkin’s lymphoma and 6 with Hodgkin’s disease. Results: Baseline HIV DNA levels were associated with HIV-1 RNA levels (HIV RNA) (r = 0.56, p = 0.02), but not with CD4 counts (r = - 0.10, p = 0.68).The viremia was undetectable for all the follow-up time post-ASCT, while HIV DNA could be evaluated in a high percentage of patients before ASCT and during the entire follow-up (median, 89.5%). At 3 months and at 1 year from transplantation, the median HIV DNA levels were 113 copies/106 PBMCs (baseline vs. 3 months, p > 0.05) and 66 copies/106 PBMCs (baseline vs. 1 year, p > 0.05), respectively. Moreover, baseline HIV DNA levels were significantly different between alive and deceased patients (p = 0.03), and the overall survival (OS) analysis showed that patients with higher HIV DNA levels at baseline had an increased and nearly significant risk of dying if compared to patients with lower levels (HR, 8.33, 95% CI, 0.99-70.06, p = 0.05). Conclusions: Our study demonstrated the association between increased HIV DNA levels at baseline and overall survival after ASCT, in one of the largest cohorts of HIV-lymphoma patients, treated with salvage therapy.

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.

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, p0.05). 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.

The quantitative evaluation of human herpesvirus 8 (HHV8) DNA is not well described in the clinical management of HHV8-related lymphoproliferative diseases.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.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.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, p0.001 and r=0.73, p0.001, 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).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.

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.

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.

The effects of CD34 mobilization with chemotherapy and G-CSF administration were evaluated in 13 HIV-positive patients with relapsed lymphomas and low CD4 counts. After mobilization, CD4+ cells increased significantly while HIV-RNA and integrated HIV-DNA showed no increases. G-CSF led to an increase of CD4+ cells with limited effects on HIV replication.

A population-based serosurvey of Human Herpesvirus type 8 (HHV8) in Västerbotten County, an area of Northern Sweden with high incidence of Kaposi's Sarcoma, was conducted. Serum samples from an age- and sex-stratified random sample of 520 subjects (260 men and 260 women) participating in a population-based biobanking project were tested for antibodies against HHV8, using a sensitive indirect immunofluorescence assay to latent and lytic HHV8 antigens. Buffy coat DNA was also analyzed for viral DNA using real time PCR assay. HHV8 DNA was not detectable in any one of the buffy coat samples. Eighty-four subjects (16.2%) were HHV8 seropositive, 14.4% for the lytic HHV8 antigen, and 1.7% for the latent HHV8 antigen. HHV8 seroprevalences were not associated significantly with sex or age. HHV8 seropositivity was more common among smokers (OR: 1.95, 95% CI: 1.02-3.75), but was less common among consumers of wine and spirits (OR: 0.44, 95% CI: 0.25-0.77 and OR: 0.50, 95% CI: 0.26-0.95, respectively). In summary, HHV8 has an intermediate high and stable seroprevalence rate in Northern Sweden, but environmental determinants that can explain the viral distribution were not found.

Purpose: Cytokines such as IL-10 and IL-18 seem to be involved in the inflammatory response of undifferentiated carcinoma of nasopharyngeal type (UCNT). The aim of this study was to evaluate the correlation between functional single nucleotide polymorphisms (SNPs) in the promoter region of IL-10 and IL-18 genes and the virological and clinical characteristics in a large case series of Caucasian patients suffering from UCNT, a tumor regularly associated with the Epstein Barr Virus (EBV). Methods: Eighty-nine patients with histologically confirmed UCNT and 130 healthy donors were included in our study. DNA was examined for the polymorphisms of IL-10 gene at positions –1082, −819, −592 by direct sequencing and IL-18 gene at position −607 and −137 by allele –specific PCR. EBV DNA serum viremia was evaluated by QC-PCR. Results: The distributions of the IL-10 and IL-18 genetic variants were not different between UCNT patients and healthy controls. The frequency of IL-10 –1082G allele, which is associated with high IL-10 expression, showed a nearly statistically significant increase in UCNT patients EBV DNA-negative as compared to healthy controls (OR=3.3 95% CI: 1.2–9.8). Subjects with C/C or C/G combined IL-18 genotypes showed an increased risk of being with Stages III-IV (OR=2.1 95% CI: 1.2–6.6). Conclusion: This study was performed to improve the definition of the pathogenetic factors implicated in UCNT by addressing the correlation between cytokine polymorphisms and clinical parameters. This is the first study investigating the possible role of the IL-18 and IL-10 polymorphisms in the development and outcome of UCNT. In our genetic analysis there is no evidence for involvement of IL-10 promoter polymorphisms alone in the genetic predisposition to this tumor. On the other hand, IL18 genetic variants may represent a genetic risk factor for tumor aggressiveness.

Helicobacter pylori infecting strains may include colony subtypes with different cytotoxin-associated gene (cag) genotypes. We sought to determine whether the cag heterogeneity of infecting strains is related to the clinical outcome of infection. Gastric biopsies for culture and histologic study were taken from 19 patients infected with cagA-positive strains (6 with duodenal ulcer, 8 with atrophic gastritis, and 5 with nonatrophic gastritis). For each biopsy, DNA was extracted from 10 single colonies and from a sweep of colonies. Polymerase chain reaction (PCR) for cagA and cagE (both located in the right half of cag) and virB11 (located in the left half of cag) was performed. Random amplified polymorphic DNA PCR (RAPD-PCR) and sequencing of glmM PCR product were performed to verify strain identity of colonies with different cag genotypes. In all patients, PCR from sweeps were positive for cagA, showing that all specimens contained cagA-positive H. pylori subtypes. In 11 patients, PCR products from all colonies were positive for cagA, cagE, and virB11, but in 8 patients, PCR products from varying numbers of colonies were negative for 1 or more cag genes. RAPD-PCR and sequencing of glmM PCR product confirmed the strain identities of colonies with different cag genotypes. We detected cag deletions in 6 of 8, 2 of 5, and 0 of 6 patients with atrophic gastritis, nonatrophic gastritis, and duodenal ulcer, respectively (P = .02). In conclusion, changes in cag genotype in single colony isolates from subjects infected with cagA-positive H. pylori strains are more common in atrophic than in nonatrophic gastritis or duodenal ulcer. These findings are consistent with host-induced (acid secretion?) adaptive changes in cag genotype during infection.

Sexual history has consistently been found to be a risk factor for the development of prostate cancer. An association between prostate cancer and herpes simplex virus type 2 (HSV-2) or Kaposi sarcoma-associated herpesvirus/human herpesvirus type 8 (HHV-8) infections has also been reported. Linkage of data on a cohort of 20,243 healthy Finnish men identified 165 cases of prostate cancer that were diagnosed up to 24 years after donation of a serum sample. Two control subjects were matched by age, sex, and municipality of residence to each case patient. Serum levels of immunoglobulin G against HSV-2 and HHV-8 were determined. Neither HSV-2 infection (odds ratio [OR], 0.93 [95% confidence interval {CI}, 0.44-1.96]) nor HHV-8 infection (OR, 0.74 [95% CI, 0.19-2.88]) was associated with prostate cancer.

Background. Primary effusion lymphoma (PEL) represents a peculiar lymphoma infected with human herpesvirus 8 (HHV-8) and occurs predominantly in human immunodeficiency virus (HIV)-infected patients. The aim of the present study was to evaluate the immunologic and virological parameters, including HHV-8 viremia, of 5 HIV-infected patients with PEL whose disease was diagnosed and treated at our institute. Methods. Five patients were enrolled in the study. Biological parameters, such as latent and lytic HHV-8 antigen levels, plasma HHV-8 load, Epstein-Barr virus plasma DNA load, HIV-1 load, and CD4 cell count, were assessed before treatment, during therapy, and at follow-up. Results. Four patients were treated with chemotherapy and highly active antiretroviral therapy (HAART), and 1 was treated with HAART alone; 3 of 5 patients reached complete remission. HHV-8 could be detected before the initiation of therapy in plasma from all patients analyzed. HHV-8 levels decreased after therapy in 4 patients. During the whole observation period, plasma HHV-8 load showed a statistically significant inverse correlation with CD4 cell count but no significant correlation with HIV load and response to therapy. Conclusions. Our analysis demonstrates that HHV-8 can be detected in the plasma at the onset of PEL; its prognostic role needs to be explored. CD4 cell count seems to be the most important indicator of progression of PEL.

We assessed the performance of a Real Time PCR assay to be used for EBV viremia evaluation in clinical specimens. Sensitivity and intra-/interassay reproducibility were evaluated by using DNA serial dilutions from the Namalwa cell line. EBV DNA was analyzed in serum samples from 39 patients (pts) with undifferentiated type nasopharyngeal carcinoma (UCNT), from 5 infectious mononucleosis (IM) pts and from 18 healthy donors. Results obtained by Real Time PCR were compared with those obtained by quantitative competitive (QC)-PCR assay.We thereafter measured the dynamics of EBV DNA load in 5 HIV-seropositive (HIV+) and 9 HIV-seronegative (HIV-, as controls) pts with lymphoma, treated with high-dose chemotherapy (HCT) followed by autologus stem-cell transplantation (ASCT). We found a sensitivity of 100% at 10 EBV copies. The Spearman correlation for both the intra- and the interassay reproducibility was statistically significant (r=0.99; p20 copies/reaction and >30% for EBV viral loads

Purpose: To describe the clinical features and outcome of HIV-associated primary effusion lymphoma (PEL) and to compare them with those of the other HIV-associated non-Hodgkin’s lymphomas (NHLs). Patients and Methods: From April 1987 to June 2002, 277 patients with HIV infection and systemic NHL were diagnosed and treated in our institution. Clinical features and outcome of PEL patients were compared with the features and outcomes of 162 patients belonging to the following histologic subtypes: plasmoblastic lymphoma of oral cavity (PBLOC, n = 11), immunoblastic lymphoma (IBL, n = 76), and centroblastic B-cell lymphoma (CBCL, n = 75). Results: Among the 277 NHL patients, PEL was diagnosed in 11 patients (4%). Eight of 11 patients were treated with a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)–like regimen. Complete remission was reached in 42% of patients, with a median survival time of 6 months. When the clinical features and outcome of 11 PEL patients were compared with the other three groups of patients affected by NHL, at the onset of the disease, no statistically significant differences were observed in demographic data, CD4 absolute number, HIV viremia plasma levels, and clinical characteristics. When we compared the outcome of PEL patients with the CBCL group, a statistically significant worse outcome was observed; however, the clinical outcome of PEL patients was not significantly different from the outcome observed in the other two groups (PBLOC and IBL groups). Conclusion: PEL is a rare HIV-associated NHL type occurring as a late manifestation of HIV infection with a poor clinical outcome and a shorter overall survival compared with CBCL patients.

The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene ( cag ) island may have different compositions in infecting strains. In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA -positive H . pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA -negative strains. Three loci in the cag island ( cagA , cagE , and virB11 ) and the conserved gene glmM ( ureC ) were investigated by PCR. The levels of anti- H . pylori and anti-CagA antibodies in patient sera were also measured. For subjects infected with cagA -negative strains, all sweep isolates were also negative for cagE and virB11 , suggesting the complete absence of the cag island. For subjects infected with cagA -positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island. cagA , cagE , and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies. The deletion of virB11 was always associated with the deletion of cagA and/or cagE . H . pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment. Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical. All of the patients had detectable antibodies against H . pylori , and 9 of 10 had anti-CagA antibodies. In conclusion, we show that a single infecting H . pylori strain may include variable proportions of colony subtypes with different cag genotypes. The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H . pylori infections.

The major antibody-reactive epitope of the small viral capsid antigen (sVCA) of human herpesvirus 8 (HHV-8) was defined by use of overlapping peptides. Strong IgG reactivity was found among approximately 50% of 44 human immunodeficiency virus-positive or -negative patients with Kaposi's sarcoma and 13 subjects who were seropositive by immunofluorescence assay (IFA) for the latent HHV-8 nuclear antigen. Only 1 of 106 subjects seronegative for both lytic and latent HHV-8 antigens and 10 of 81 subjects IFA-seropositive only for the lytic HHV-8 antigen had strong IgG reactivity to this epitope. Among 534 healthy Swedish women, only 1.3% were strongly seropositive. Comparison of the peptide-based and purified sVCA protein-based ELISAs found 55% sensitivity and 98% specificity. However, only 1 of 452 serum samples from healthy women was positive in both tests. In conclusion, the defined sVCA epitope was a specific, but not very sensitive, serologic marker of active HHV-8 infection. Such infection appears to be rare among Swedish women, even with sexual risk-taking behavior.

The BamHI-Z-encoded Epstein—Barr virus (EBV) replication activator (ZEBRA) is a key mediator of the switch from latency to productive cycle in EBV virus. Antibodies against ZEBRA are a marker of EBV reactivation and are regularly found among patients with infectious mononucleosis (IM) or nasopharyngeal carcinoma (NPC), but are only rarely found among healthy EBV-seropositive donors. In order to define the serologically reactive epitopes in the ZEBRA protein, we synthesized a set of overlapping peptides and tested them for reactivity with serum samples from EBV-seronegative persons, patients with NPC, IM, chronic fatigue syndrome, lymphoma or from healthy donors. Three major EBV-specific epitopes were found. These epitopes were further defined and optimized using substitution or truncation analogues of the peptides. Reactivity with epitope number 22 was found in 63% of NPC patients' sera, with < 2% of healthy donors' sera being positive. Serological reactivity with epitope number 19 was associated with IM (57% positive, 5% healthy donors positive). Serum antibodies against epitope 1 were found among healthy donors, but were significantly elevated among patients with NPC, IM or lymphomas. In conclusion, different serologically reactive epitopes in the ZEBRA protein associate with different EBV-associated diseases.

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.

Background: One of the major concern for ASCT in HIV-positive patients (pts) is that post-transplant immunosuppression might worsen the immunosuppression and enhance the HIV replication. Therefore we analyzed whether the immune system of HIV-positive pts might support an immune recovery similar to the HIV-negative pts who underwent the same ASCT program. Methods: The kinetics and the extent of immunereconstitution were assessed by measuring: TCR excision circles (TRECs), CD4, CD8, CD56, CD4, CD45RA, CD19 cells, and HIV pro and viraemia Results: 15 HIV-positive and 7 HIV-negative pts with relapsed DBCL underwent ASCT. Before the induction therapy TRECs/106 PBMC mean value was 962±2183 and 2948±5485 in HIV-positive and HIV-negative pts respectively. The nadir was reached before ASCT (150±11 vs 927±1842). At one year the mean value of TRECs/106 PBMC returned to the baseline in HIV-negative pts, while in HIV-positive pts largely overcame the baseline (2440±2799). Before the induction therapy, HIV-positive pts showed a significant lower CD4 (174±110 vs 386±200) CD56 (86±129 vs 144±92) and CD4/CD8 ratio in comparison with HIV-negative pts. CD4 count nadir was reached during aplastic period in both groups. No differences were present in the dynamics of CD4, CD45RA, CD8 and CD56 recovery between the two groups. At one year CD4 count returned to the baseline value in HIV-negative pts while in HIV-positive pts overcame 70% the baseline value. Before ASCT, all HIV-positive pts were on HAART and HIV-viraemia was Conclusions: The dynamics of the immunereconstitution was similar in HIV-positive and HIV-negative pts, but the tymic output unexpectedly seemed to be enhanced in HIV-positive pts. Supported by ISS grants.

3121 Background: HIV-PI inhibit the conversion of the active form of metalloproteinase-2 (MMP-2), thus preventing activation of proteases necessary for angiogenesis. In NPC, MMPs and VEGF are overexposed and their expression correlates with a poor prognosis. Methods: Since January 2002, we started the compassionate use of Indinavir for extensively pretreated pts. The aims of the study were to evaluate: feasibility and safety, serum and/or plasma level of the “anti-angiogenic surrogate markers” (MMP-2, MMP-3, MMP-9, VEGF and β FGF). Pts refractory to first-line, second-line conventional or to high dose chemotherapy (CT) plus ASCT and radiotherapy (RT) and pts who could not receive any intensive CT or RT were allowed to receive Indinavir, 800 mg tid po. alone or with concomitant Carboplatin (AUC 5) every 21 days. Toxicity according to WHO scale and biochemical tests for the “surrogate markers” were performed every 4, 8 weeks. Results: Six pts were enrolled. Five pts received therapy for a median time of 28 ...