Fabio Forghieri

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.

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.

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.

Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Diagnosis of invasive aspergillosis by tracking Aspergillus-specific T cells in hematologic patients with pulmonary infiltrates.

Invasive aspergillosis (IA) is a leading cause of infection-related mortality in hematologic patients, with death rate ranging from 50 to 90%.1 The reason for this extremely poor outcome is because of the difficulties in a timely and undoubted diagnosis, which still relies on a very high degree of suspicion. The current diagnostic tools are limited by invasiveness, slowness, relative insensitiveness, lack of standardization and unpredictable kinetics.2 The most widely studied test, Galctomannan antigenemia (GM), has been demonstrated to be highly variable in performance, with sensitivity ranging between 29 and 100%, and is affected by several factors related either to the fungus or to the host.2 Furthermore, the detection of Aspergillus DNA through polymerase chain reaction (PCR) is still hampered by the difficulties in understanding the fungal DNA release and kinetics other than by technical barriers.2 For all these reasons, establishing an early diagnosis of IA remains a challenge.1, 2

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.

Pain and emotional distress in leukemia patients at diagnosis.

The few available studies on leukemia patients have so far ddressed only the issue of physical pain and emotional distress in erminally ill patients [1,2] The prevalence of pain during the last months of life of Italian cancer patients was reported to be 83% n lymphoma and leukemia patients, according to what reported y their caregivers [3]. This result is much higher compared with ther studies [4] reporting a prevalence of 5% in leukemia [4] and 8% in lymphoma patients [5]. We used the Edmonton Symptoms Assessment System (ESAS) o investigate the frequency of physical pain and both ESAS and ospital Anxiety and Depression Scale (HADS) to assess emoional distress in 53 patients (36 male, 17 female; median age 50 ears-range 32–72) with acute leukemia (40 myeloid and 13 lymhoblastic), at diagnosis (T0), during the neutropenia phase (T15), nd at discharge, as well as in 76 patients with solid cancers (47 ale, 29 female; median age 65 years-range 42–83), as control roup. The diagnosis of depression and/or anxiety was made with score of eight of 21 or more in the HADS questionnaire [6]. In he ESAS analysis, depression and anxiety were considered present ith a score of two of 10 or more [6]. During the induction chemotherapy phase, mild pain (from 1 to of the Numerical Rating Scale-NRS of the ESAS) was reported in 9.5%, 27%, and 16.6%, while moderate (from 4 to 6) to severe pain from 7 to 10) was reported in 11.6%, 20.4% and 5.5% of the cases, at 0, T15 and discharge, respectively. According to the HADS score, epression was reported in 21%, 36% and 33%, at T0, T15 and disharge, respectively, while anxiety in 30%, 40%, 36% of the cases, t the same time intervals, respectively. Depression was reported n 32% while anxiety in 38% of all questionnaires, respectively, colected at all time intervals, during all clinical phases. According to he ESAS score, depression was reported in 45%, 38.6% and 27.7%, t T0, T15 and discharge, while anxiety in 46.5%, 40.4%, 41.6% of he cases, at the same time intervals, respectively. ESAS showed sensitivity of 57% and 74%, and a specificity of 67% and 67% for epression and anxiety, respectively, when all questionnaires were onsidered. In solid cancer patients, mild to moderate to severe ain was reported in 38% of the cases, at diagnosis. HADS score as positive for depression in 25% while for anxiety in 19.7% of he cases. ESAS score was positive for depression in 28.9% while or anxiety in 40.7% of the cases, showing a sensitivity of 63% and 6.6% and a specificity of 82% and 70% for depression and anxiety, espectively. Symptom severity appeared similar or even higher n leukemia compared with solid cancer patients, at diagnosis, as eported for patients referred to palliative care (Table 1) [2,4]. We suggest that, contrary to what is generally accepted, eukemia patients may suffer from physical pain and emotional disress since the time of diagnosis. Further studies are warranted to onfirm and extend these findings.

Pathogenetic Mechanisms of Hepatitis C Virus-Induced B-Cell Lymphomagenesis

Hepatitis C virus (HCV) infection is probably the most common chronic viral infection and affects an estimated 180 million people worldwide, accounting for 3% of the global population. Although the liver is considered to be the primary target, extrahepatic manifestations are well recognized among patients with chronic HCV infection. Epidemiological studies have clearly demonstrated a correlation between chronic HCV infection and occurrence of B-cell non-Hodgkins lymphomas (B-NHL). The clinical evidence that antiviral therapy has a significant role in the treatment at least of some HCV-associated lymphoproliferative disorders, especially indolent B-NHL, further supports the existence of an etiopathogenetic link. However, the mechanisms exploited by HCV to induce B-cell lymphoproliferation have so far not completely clarified. It is conceivable that different biological mechanisms, namely, chronic antigen stimulation, high-affinity interaction between HCV-E2 protein and its cellular receptors, direct HCV infection of B-cells, and “hit and run” transforming events, may be combined themselves and cooperate in a multifactorial model of HCV-associated lymphomagenesis.

Variability of Voriconazole Trough Levels in Haematological Patients: Influence of Comedications with cytochrome P450(CYP) Inhibitors and/or with CYP Inhibitors plus CYP Inducers.

Voriconazole plasma exposure greatly varies among haematological patients. The purpose of this study was to identify the magnitude of influence of comedications with CYP inhibitors and/or with CYP inhibitors plus CYP inducers on voriconazole trough level (Cmin). Voriconazole Cmin was retrospectively assessed among haematological patients who underwent therapeutic drug monitoring (TDM). Univariate and multivariate linear mixed‐effect regression analyses were performed to identify the independent predictors of normalized Cmin. Of the 83 included patients, 35 had comedications with CYP inhibitors (omeprazole or pantoprazole) and 21 with CYP inhibitors (omeprazole or pantoprazole) plus CYP inducers (methylprednisolone, dexamethasone, phenobarbital, rifampin or carbamazepine). Median Cmin value (n = 199) was 2.4 mg/L with a wide range of distribution (<0.2–13.5 mg/L). Median (IQR) normalized voriconazole Cmin value was significantly higher in the presence of CYP inhibitors (4.20 mg/L, 3.23–5.51 mg/L) than either in the absence of interacting cotreatments (2.55 mg/L, 1.54–3.47 mg/L) or in the presence of CYP inhibitors plus CYP inducers (2.16 mg/L, 1.19–3.09 mg/L). The presence of CYP inhibitors was highly significantly associated with Cmin >5.5 mg/L (OR: 23.22, 95% CI: 3.01–179.09, p = 0.003). No significant association emerged when CYP inhibitors were coadministered with CYP inducers (OR: 3.53, 95% CI: 0.36–34.95, p = 0.280). The amount of expected Cmin increase was significantly influenced by both the type and the dose of the administered proton pump inhibitor. The study highlights that the benefit from TDM of voriconazole may be maximal in those patients who are cotreated with CYP inhibitors and/or with CYP inhibitors plus CYP inducers, especially when receiving proton pump inhibitors (PPIs) at very high dosages intravenously.