Giovanni Riva

Severe pancytopenia and hemophagocytosis after HHV-8 primary infection in a renal transplant patient successfully treated with foscarnet

We report the occurrence of human herpesvirus (HHV)-8 primary infection in an adult male kidney recipient. Four months after transplantation, the patient developed visceral Kaposi sarcoma, and 1 month later he presented with progressive and severe peripheral cytopenia, in the presence of a normocellular or hypercellular bone marrow (BM) with hemophagocytosis. HHV-8 was the sole pathogen detected by polymerase chain reaction either in the serum or in the BM. HHV-8 latent nuclear antigen was detected in immature progenitor cells from the BM. Immunosuppressive therapy was reduced, and the patient was treated with foscarnet for 2 weeks, leading to a dramatic normalization of blood cell counts, concomitantly with the disappearance of HHV-8 viremia. At the end of antiviral therapy, the patient received chemotherapy, and Kaposi sarcoma regressed in 2 months. Severe peripheral cytopenia may be a posttransplant complication after HHV-8 infection, for which treatment with foscarnet seems appropriate.

Treatment of herpesvirus associated primary effusion lymphoma with intracavity cidofovir.

Primary effusion lymphoma (PEL) is a B-cell non-Hodgkin lymphoma, involving the serous cavities, which is invariably associated with Kaposi sarcoma-associated herpesvirus (KSHV)/ human herpesvirus-8 (HHV-8) and often with Epstein–Barr virus (EBV) infections. The disease accounts for less than 3% of AIDS-associated lymphomas, as recognized by the WHO, but a few cases have also been reported in transplant patients as well as in elderly human immunodeficiency virus (HIV)-negative men originating from areas endemic for HHV-8 infection, and at high risk for classic Kaposi sarcoma (KS). PEL has a poor prognosis, with a median survival of few months, in the majority of affected patients. In AIDS patients, combined chemotherapy, including high-dose methotrexate, may be attempted with the possibility to achieve complete remission, at least in some cases. On the contrary, in elderly and transplant patients, the use of chemotherapeutic regimens has been generally hampered by severe toxicity, and has revealed unsuccessful in the vast majority of the treated patients, raising the need for alternative treatment approaches. Cidofovir is an antiviral with a broad spectrum of activity against DNA viruses, and has been reported to be one of the most effective agents to inhibit HHV-8 replication in vitro. In addition to its antiviral activity, a potent antitumor activity has been recently attributed to cidofovir. Topical application of cidofovir resulted effective to treat both neoplastic genital lesions and recurrent laryngeal papillomatous lesions caused by human papillomavirus (HPV). In animal models, cidofovir has been found to be effective on virus-associated tumors, such as in nude mice with EBV-associated nasopharyngeal carcinoma, in which intratumoral injection of cidofovir induced in vivo regression of the tumors. The antitumor activity of cidofovir is not specifically related to the antiviral action of the compound. Consistent with this, cidofovir has also been shown to inhibit the growth of tumors, such as hemangiosarcomas, which are not associated with oncogenic viruses. In the current study, we tested whether cidofovir exerts an antitumor effect against two PEL cell lines in vitro, namely the HHV-8-positive BCBL-1 and the HHV-8-positive and EBVpositive HBL-6 cell lines. The EBV-negative and HHV-8negative RAMOS cell line was tested as control. Then, we investigated the effect of intracavity injections of cidofovir for the treatment of three elderly, HIV negative, patients with PEL. The BCBL-1, HBL-6 and the RAMOS cell lines were cultured in RPMI 1640 medium, supplemented with 15% of heat inactivated fetal calf serum (FBS), glutamine 1 mM and antibiotics, at 371C in a 5% CO2 humidified incubator. Cells were treated with cidofovir at 0.01, 0.1, 0.5, and 1 mM, for 3 and 6 days. At the end of the incubation period, cell count and viability were evaluated in triplicate, with Trypan blue dye exclusion assay. Apoptosis was studied by a combination of methods, including, morphology, propidium iodide staining (50 mg/ml) and analysis by flow cytometry (Becton and Dickinson Italia, Milano, Italy), the DNA fragmentation assay, and the in situ cell death detection kit (Boheringer Mannheim, Mannheim, Germany), which relies on the use of terminal deoxynucleotidyl transferase (TdT) that catalyses the polymerization of fluorescein-labeled nucleotides to free 30-hydroxyl residues of DNA fragments generated by endonucleases during apoptosis (TUNEL). Cidofovir caused a dose-dependent inhibition of BCBL-1 and HBL-6 cell proliferation and viability (Figure 1a–d), while in RAMOS cells, cidofovir inhibited proliferation but had no effect on viability (Figure 1e and f). Cidofovir induced a dose-dependent apoptosis in both the BCBL-1 and HBL-6 cell lines, after 3 and 6 days, as detected by flow cytometry analysis (Figure 2a). Apoptosis induced by cidofovir was confirmed by the observation of typical apoptotic features of cell morphology, by the characteristic ladder of fragmented genomic DNA on the DNA fragmentation assay, as well by the detection of fluorescein-labeled DNA strand breaks in apoptotic cells, by TUNEL assay (Figure 2b–d in supplementary information). On the contrary, RAMOS cells did not show any significant apoptosis up to 6-day treatment with cidofovir (Figure 2a and analysis of cell cycle distribution of RAMOS cells in supplementary information). In three patients, (pt. 1, pt. 2 and pt. 3) a diagnosis of HHV-8positive PEL, in Ann Arbor stage IV, was made, on the basis of morphologic, immunophenotypic and molecular analysis, as described. EBV coinfection was documented in pt. 1, while serology for HIV, hepatitis B and C viruses was negative in all three patients. In detail, pt. 1, a 96-year-old Italian man, was hospitalized for bilateral pleural effusion. No clinical evidence of KS was found. The patient was subjected to pleural drainage every 2 weeks for 4 months, without receiving any chemotherapy. Then, he was treated with two doses of intrapleura cidofovir, at 2.5 mg/kg, every 1 week. No recurrence of pleural effusion was observed after the second injection. PEL relapsed at the same site, as documented on standard radiographic examination, 10 months later. The patient refused cidofovir therapy and was subjected to pleural drainage. The patient died for heart failure. Pt. 2, a 70-year-old Italian man, was hospitalized for recurrent peritoneal effusions. The patient had a history of KS, for which he had received chemotherapy and radiotherapy 10 years before, with complete tumor regression. The patient was subjected to peritoneal drainages every 10 days for 2 months. Then, the patient received three doses of intraperitoneal cidofovir, at 5 mg/kg, every 1 week. No recurrence of peritoneal effusion was observed after the last cidofovir injection. PEL relapse was documented in the pleura 5 months later. The patient died for a cerebrovascular accident, with no peritoneal relapse. Pt. 3, a 77-year-old Italian man, was hospitalized for a 5-month history of bilateral pleural effusion requiring repeated pleural drainages. Chemotherapy was avoided because the patient had been suffering from heart failure. He was treated with three doses of intrapleura cidofovir, Received 31 May 2004; accepted 19 November 2004; Published online 20 January 2005 Correspondence: Dr M Luppi, Department of Oncology and Hematology, University of Modena and Reggio Emilia, Policlinico, Via del Pozzo 71, 41100 Modena, Italy; Fax: þ 39 059 4224549; E-mail: mluppi@unimore.it

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

Changes in the immune responses against human herpesvirus-8 in the disease course of posttransplant kaposi sarcoma

In nine patients with posttransplant Kaposi sarcoma (KS) T-cell responses to human herpesvirus (HHV)-8 latent and lytic antigens, as detected by enzyme-linked-immunospot (Elispot) assay, were absent at disease onset. Virus-specific T-cell responses were detected in six renal recipients at remission after a reduction of calcineurin inhibitors (CIs), and in two HHV-8 seropositive renal recipients without KS. In two liver recipients undergoing switch from CIs to sirolimus (SRL), normalization of the T-cell repertoire and recovery of both HHV-8-specific effector and memory T lymphocytes were associated with complete KS remission. In a renal recipient undergoing SRL conversion, the early recovery of HHV-8-specific effector but not of memory T lymphocytes, was associated only with partial remission. Neither rejection nor changes in graft function were observed after SRL conversion. HHV-8-specific T-cell responses are required to achieve posttransplant KS remission, and may be restored under SRL, while maintaining effective immunosuppression.

How I treat HHV8/KSHV-related diseases in posttransplant patients

Posttransplantation human herpesvirus-8 (HHV8)/Kaposi sarcoma herpesvirus (KSHV) primary infection and/or reactivations are associated with uncommon and sometimes fatal, neoplastic, and non-neoplastic diseases. HHV8-related clinical manifestations notably range from Kaposi sarcoma (KS) to either primary effusion lymphoma or multicentric Castleman disease B-cell malignancies, and from polyclonal HHV8-positive plasmacytic lymphoproliferative disorders to bone marrow failure and peripheral cytopenias, associated or not with hemophagocytic syndromes, and to acute hepatitis syndromes. We reviewed the patient series reported in the literature and summarized clinical management aspects, in terms of diagnosis, follow-up, and treatment. We described typical clinical presentations and histopathologic diagnostic features of these diseases, and we discussed the role of HHV8-specific serologic, molecular, and immunologic assays, particularly focusing on recent data from HHV8-specific T-cell monitoring in posttransplantation KS patients. We finally discussed actual therapeutic options, namely, the reduction or discontinuation of immunosuppressive therapy or the switch from calcineurin inhibitors to mTOR inhibitors, as alternatives to antineoplastic chemotherapy, along with the use of antiherpesvirus agents as prophylactic or therapeutic measures, and treatment with rituximab in posttrans-plantation multicentric Castleman disease patients and non-neoplastic HHV8-associated syndromes.

Mucorales -specific T cells emerge in the course of invasive mucormycosis and may be used as a surrogate diagnostic marker in high-risk patients

Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4(+) or CD8(+) subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Prevalence of Human Herpesvirus-6 Chromosomal Integration (CIHHV-6) in Italian Solid Organ and Allogeneic Stem Cell Transplant Patients

The unique phenomenon of human herpesvirus‐6 (HHV‐6) chromosomal integration (CIHHV‐6) may account for clinical drawbacks in transplant setting, being misinterpreted as active infection and leading to unnecessary and potentially harmful treatments. We have investigated the prevalence of CIHHV‐6 in 205 consecutive solid organ (SO) and allogeneic stem cell transplant (alloSCT) Italian patients. Fifty‐two (38.5%) of 135 solid organ transplant (SOT) and 16 (22.8%) of 70 alloSCT patients resulted positive for plasma HHV‐6 DNA by real‐time polymerase chain reaction. Seven SOT and three alloSCT patients presented HHV‐6‐related diseases, requiring antivirals. Two further patients (0.9%) were identified, presenting high HHV‐6 loads. The quantification of HHV‐6 on hair follicles disclosed the integrated state, allowing the discontinuation of antivirals. Before starting specific treatments, CIHHV‐6 should be excluded in transplant patients with HHV‐6 viremia by the comparison of HHV‐6 loads on different fluids and tissues. Pretransplantation screening of donors and recipients may further prevent the misdiagnosis of CIHHV‐6.

Emergence of BCR-ABL–specific cytotoxic T cells in the bone marrow of patients with Ph + acute lymphoblastic leukemia during long-term imatinib mesylate treatment

Imatinib mesylate has been demonstrated to allow the emergence of T cells directed against chronic myeloid leukemia cells. A total of 10 Philadelphia chromosome-positive acute lymphoblastic leukemia patients receiving high-dose imatinib mesylate maintenance underwent long-term immunological monitoring (range, 2-65 months) of (p190)BCR-ABL-specific T cells in the bone marrow and peripheral blood. (p190)BCR-ABL-specific T lymphocytes were detected in all patients, more frequently in bone marrow than in peripheral blood samples (67% vs 25%, P < .01) and resulted significantly associated with lower minimal residual disease values (P < .001), whereas absent at leukemia relapse. Specific T cells were mainly effector memory CD8(+) and CD4(+) T cells, producing interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (median percentage of positive cells: 3.34, 3.04, and 3.58, respectively). Cytotoxic subsets able to lyse BCR-ABL-positive leukemia blasts also were detectable. Whether these autologous (p190)BCR-ABL-specific T cells may be detectable under other tyrosine-kinase inhibitors, expanded ex vivo, and exploited for immunotherapy remains to be addressed.

Cytarabine-related lung infiltrates on high resolution computerized tomography: a possible complication with benign outcome in leukemic patients

Potentially fatal lung toxicity occurs in 12–20% of leukemic patients treated with cytarabine especially at intermediate to high doses, usually presenting as noncardiogenic pulmonary edema (NCPE). Anecdotally the association between cytarabine and the onset of bronchiolitis obliterans organizing pneumonia (BOOP) has been reported. We describe here three cases of patients affected by acute myeloid leukemia (AML) treated with chemotherapeutic regimens including high dose cytarabine, who developed early onset of fever, mild dyspnea, moderate hypoxemia on arterial blood gas analysis and lung infiltrates documented by high-resolution computerized tomography (HRCT), with a more indolent behaviour and a benign clinical outcome, compared with similar cases previously reported in the literature. Our cases widen the spectrum of clinical features of cytarabine-related toxicity in leukemic patients.

Parvoviruses in Blood Donors and Transplant Patients, Italy

To the Editor: Parvoviruses (PARV) 4 and 5 are 2 genotypes of a novel human parvovirus, with 92% nucleotide identity, identified in the plasma sample of a patient screened for acute HIV infection and in samples of manufactured plasma pools (1,2). Recently, PARV4 and PARV5 were identified in blood samples from 3 of 26 cadavers from the United Kingdom, all of whom were positive for hepatitis C virus RNA and had a history of intravenous drug use (3). PARV4/5 were also found in bone marrow (BM) and lymphoid tissues from 17 of 24 HIV-positive cadavers from Scotland (4) and in BM aspirates from 16 of 35 Italian patients with AIDS (5). Little or no information is available about the epidemiology and clinical correlates of infection with these novel viruses. To provide insights into their pathogenic potential in vivo, we assessed the frequency of PARV4/5 viremia in healthy patients, transplant patients, and those with suspected viral disease. We performed a retrospective molecular study for the presence of PARV4/5 sequences in 4 groups of 417 Italian HIV-negative persons. Group 1 consisted of 100 blood donors recruited from the Transfusion Centre of Modena (northern Italy); group 2, 84 patients with hematologic diseases showing clinical signs of viral etiology but negative results for the most common viruses (herpesviruses, adenovirus, hepatitis virus, and coxsackie virus). For both of these groups, DNA was extracted for analysis from serum specimens and peripheral blood mononuclear cells (PBMCs). Groups 3 and 4 comprised recipients of kidney and allogeneic BM/peripheral blood stem cell (PBSC) transplants, for which DNA was extracted from serum specimens collected at 6 and 12 months, respectively, after transplantation. The nested PCR method was used to amplify a shared sequence of PARV4 and its variant PARV5 and was specific for the open reading frame 1. First step PCR was performed as previously described (2) with a sensitivity of 1–10 copies, on 1 μg PBMC DNA and on one fifth of DNA extracted from 0.25 mL of serum. Primers for second round PCR were PV4NS1Fn2 (5′-GTTGATGGYCCTGTGGTTAG-3′) and PV4NS1Rn2 (5′-CCTTTCATATTCAGTTCCTGTTCAC-3′). All positive results were confirmed by direct sequencing. We found 3 positive case-patients, including 2 renal transplant recipients and 1 patient with a suspected viral disease; none of the blood donors tested positive on single-round PCR. On nested PCR, 1 blood donor had positive results; the positivity rate did not increase in the other groups (Table). In the first 2 groups, PARV4/5 sequences were detected only in the serum samples, not in the PBMCs collected at the same time. These sequences suggest that PBMCs are not a major site of viral replication. Similar to B19 infection, which is rarely reactivated in the setting of BM/PBSC transplantation (6,7), none of the BM/PBSC transplant patients were PARV4/5 positive. The detection of PARV4/5 sequences in the serum collected at 12 months after transplantation was not associated with the occurrence of any symptoms in the 2 renal recipients. Of note, the available serum samples collected from both recipients before transplantation, and at 6 and 24 months after transplantation, were PARV4/5 negative, which suggests that asymptomatic PARV4/5 infection may transiently occur after solid organ transplantation or may be acquired throughout transfusion or transplantation. Similarly, the rate of B19 infection in solid organ transplant recipients is low (1.4%–1.8%), and most B19 DNA–positive patients remain asymptomatic (8,9). Table Analysis of 417 patients tested for parvoviruses 4/5 by PCR* PARV4/5 sequences were detected in the serum collected from 1 patient affected with Wegener granulomatosis. This patient was under long-term treatment with steroids, concomitant with the development of a clinical syndrome for which a viral cause was suspected, including fever, severe anemia, a histologic-examination–proven postinfectious glomerulonephritis, and eryhtroid hypoplasia, with dsyserythropoiesis and dismegakaryopoiesis on BM examination. Serologic and molecular tests for the most common viruses, including B19, were negative and the patient died of multiple organ failure 1 month later. Single-cell PCR performed on the DNA extracted from isolated BM erythroid and myeloid progenitors in the formalin-fixed, paraffin-embedded BM tissue biopsy specimens, collected 2 days before death and at autopsy, were PARV4/5 negative. While the PARV4/5 viremia, in the absence of other known viral agents, suggests a possible contribution of this novel parvovirus to the patient’s clinical syndrome, the absence of the virus in the BM cells suggests that its in vivo tropism may markedly differ from that of B19. In conclusion, although the frequency of PARV4/5 viremia is very low in the general Italian population, it is slightly higher in certain subgroups of iatrogenically immunosuppressed patients and it is not clear to which extent immunosuppression enhances viral reactivation and/or primary infection. Failure to detect PARV4/5 DNA in all but 4 study patients does not necessarily indicate a rarity of past viral exposure or infection in transplant patients or indeed in the general population. Further studies are needed to confirm a possible pathogenic role of PARV4/5 infection,