Monica Maccaferri

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.

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.

Chronic eosinophilic leukaemia with ETV6-NTRK3 fusion transcript in an elderly patient affected with pancreatic carcinoma

To the Editor: An 82-yr-old woman with previous history of breast cancer, surgically treated in 1992, and arterial hypertension underwent distal pancreatectomy and splenectomy in July 2008 for a poorly differentiated adenocarcinoma of the pancreatic tail with regional lymph nodes and liver metastases. She was subsequently started on palliative chemotherapeutic treatment, consisting of gemcitabine 1000 mg ⁄m days 1 and 8 every 3 wk. On February 2009, having completed eight cycles, the chemotherapy was withdrawn because hepatic metastases increased in size and number. Until March 2009, the complete blood count was unrevealing (WBC count 7.2 · 10 ⁄L with a normal differential count, Hb 10.2 g ⁄dL, Plt count 300 · 10 ⁄L). One month later, the patient was admitted because of low-grade fever, bone pain and marked fatigue. The laboratory investigations revealed leukocytosis with marked eosinophilia and thrombocytopenia (WBC count 73 · 10 ⁄L with eosinophils 78%, Hb 11.4 g ⁄dL, Plt count 69 · 10 ⁄L). The diagnostic work-up excluded either allergic reactions or parasitic infestations or autoimmune disorders or T-cell populations with an abnormal phenotype (1, 2), raising the hypothesis of an eosinophilic leukaemoid reaction secondary to the metastatic pancreatic cancer (3). However, the morphological examination of peripheral blood (PB) smear showed eosinophilia accompanied with circulating myeloid immature precursors and blasts (3%) (Fig. 1A), while bone marrow (BM) trephine biopsy documented marked proliferation of eosinophil granulocytopoiesis, with a blast cell count < 20% (Fig. 1B). Moreover, moderate to severe BM fibrosis was documented on Gomori methenamine silver staining (not shown). These morphological pictures were consistent with a myeloproliferative neoplasm with eosinophilia (MNE) (4). Unfortunately, serum tryptase level was not measured. Extensive FISH and molecular studies on both PB and BM samples failed to detect PDGFRA, PDGFRB and FGFR1 rearrangements (4), but the clonality of the myeloid population was confirmed by further cytogenetic and FISH studies. In particular, conventional G-banding showed 46, XX, )7, +8 karyotype (Fig. 2A), while whole chromosome painting and metaphase FISH examinations performed with specific probes showed that ETV6 gene, located on 12p13, was cryptically rearranged with 15q25 (Fig. 2B,C). A submicroscopic t(12;15)(p13;q25) resulting in the fusion gene ETV6-NTRK3 on der(15) was thus documented. Moreover, RNA isolated from the BM sample was subjected to reverse transcriptase–PCR using primers designed to amplify either the 731-bp solid cancer ETV6-NTRK3 variant (5) or the 601-bp acute myeloid leukaemia (AML) ETV6-NTRK3 variant (6). These latter molecular analyses demonstrated the 731and 1265-bp products, respectively, as expected when the solid cancer-associated fusion transcript is detected (Fig. 2D). Both FISH and molecular examinations, performed as described (7) on formalin-fixed paraffinembedded pancreatic carcinoma sections, were negative for ETV6-NTRK3 fusion transcript, whereas amplification of ETV6 gene was documented (not shown).

Mucorales-Specific T Cells in Patients with Hematologic Malignancies

Background Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. Methods and Findings By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. Conclusions Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

May the indirect effects of CIHHV-6 in transplant patients be exerted through the reactivation of the viral replicative machinery?

We read with great interest the article by Lee et al. (1) about the clinical significance of the human herpesvirus 6 integrated in host chromosome (CIHHV-6) in liver transplant patients. The authors show that CIHHV-6 may be associated with higher rates of allograft rejection and opportunistic infections, previously attributed only to active HHV-6 infection, possibly as a result of viral immunomodulation (1, 2). Unfortunately, no data have so far been reported on how such an immunomodulatory activity may be exerted, in particular whether it may be mediated by the activation of the replicative machinery of the integrated genome. Of note, it has recently been demonstrated that CIHHV-6 may actively replicate upon chemical stimuli and infect permissive cells, at least in vitro (3). We would like to discuss the issue of the pathogenicity of CIHHV-6 by adding new experimental data and by extending the clinical follow-up of a previously reported patient with the variant A CIHHV-6, undergoing an allogeneic stem-cell transplantation (allo-SCT) from an unrelated donor for a high-risk myelodysplastic syndrome (4). At day 275 posttransplant, the patient underwent a bone marrow (BM) biopsy because of severe leukopenia and increasing HHV-6A DNA plasma values. Graft-versus-host disease manifestations were absent and either peripheral or BM blood donor chimerisms resulted almost 100% (Fig. 1A). The reduction of HHV-6 loads would be expected with improving donor chimerism in alloSCT patients, with such a CIHHV-6 donor/recipient combination (5). The biopsy demonstrated a reduced BM cellularity without signs of myelodysplasia. After antiviral treatment, leukocytes recovered and HHV-6 loads reduced. A second similar episode was recorded at day 342 and again resolved with the administration of antiviral therapy. Long-term BM cultures (LTBMCs) were performed with the BM-derived cells from the patient, either alone (LTBMC1) or further allogeneically stimulated with BM-derived cells from another histoincompatible subject (LTBMC2) (6). Either the human leukocyte antigen-unrelated SCT donor or the histoincompatible subject was HHV-6B seropositive. After 48 hr, nonadherent cells have been removed, and immunohistochemical analysis with a mouse monoclonal antibody against the HHV-6A early protein (gp41/38) (Advanced Biotechnologies) was performed, as described (4), on ethanolfixed cells either at days 10, 30, and 50 of LTBMC1 or at days 17, 32, and 57 of LTBMC2. The gp41/38 antibody highlighted cytoplasmic reactivity in some BM adherent cells either at day 10 of LTBMC1 or at day 32 of LTBMC2, confirming their productive infection with HHV-6A (Fig. 1B, C). Unfortunately, the same analysis on formalin-fixed paraffin-embedded BM biopsy specimens resulted negative, possibly due to the antiviral treatment, reducing HHV-6 gene expression, in vivo. These results demonstrate that CIHHV-6 may produce proteins, at least in ex vivo culture, possibly as a result of the allogeneic reaction between the patient’s cells and those of either his human leukocyte antigen-unrelated SCT donor or the histoincompatible subject. It has been reported that human cytomegalovirus, a beta herpesvirus closely related to HHV-6, may reactivate from latency in peripheral blood mononuclear cells after allogeneic stimulation (6). In our case, the possibility that the protein expression comes from a superinfecting episomal copy is very unlikely because (a) HHV-6A latency is unlikely to involve the formation of viral episomes (3) and (b) either the SCT donor or the histoincompatible subject was latently infected with the HHV-6B. These findings cast doubt on a possible CIHHV-6 reactivation as culprit in the patient’s two episodes of leukopenia with increased HHV-6 loads, at day 263 and 342 posttransplant, respectively, also when considering that both episodes resolved after the administration of antivirals. In conclusion, Lee’s, Arbuckle’s, and our data challenge the belief that CIHHV-6 could be an inert genome and only represents a confounding element in the diagnosis of HHV-6 active infection (1, 3, 7). Alternatively, as it may be supposed that HHV-6 may exert its pathogenic potential through either full replication or the sole expression of viral proteins, we believe that transplant clinicians should be alerted to the possibility of HHV-6-associated diseases/indirect effects in their CIHHV-6 patients and also consider the administration of antivirals in those patients with signs and symptoms consistent with viral infection, when all the other causes have been extensively and repeatedly ruled out. The observation by Lee et al. (1) that the only CIHHV-6 patient undergoing valganciclovir prophylaxis developed neither rejection nor opportunistic infections may spur investigations on whether the administration of antiviral prophylaxis with an HHV-6 active agent may reduce the occurrence of HHV-6 indirect effects in patients with CIHHV-6. Further in vivo studies are needed to clearly define the replicative potential of CIHHV-6.

Long-term molecular remission with persistence of BCR-ABL1-specific cytotoxic T cells following imatinib withdrawal in an elderly patient with Philadelphia-positive ALL.

least 2 weeks thereafter. Treatment with bendamustine and alemtuzumab was not to be interrupted for CMV viraemia, but was to be held if there was evidence of CMV infection (i.e. fever) until such evidence resolved. Summary treatment outcomes and baseline characteristics of the nine patients enrolled are shown in Table I. Three subjects were enrolled in each cohort, at which point the trial was terminated due to slow accrual. One case (Patient 9) was not evaluable for response. Evaluable responses included one case of progressive disease, four cases of stable disease and three partial responses. Adverse events are summarized in Table II. There were five serious adverse events (myelosuppression, hypotension, infection and two cases of rash). A DLT of prolonged neutropenia occurred in one patient in the highest dosing cohort (C). In summary, this phase I dose escalation study demonstrates that patients with high risk CLL can be safely treated with bendamustine in combination with alemtuzumab without excessive toxicity or CMV reactivation. More data are needed to determine the efficacy of this combination in less heavily pre-treated patients.

Organising pneumonia mimicking invasive fungal disease in patients with leukaemia

Clinical charts from 63 consecutive highly immunocompromised haematologic patients presenting with pulmonary nodular lesions on CT scan, classified as either probable or possible invasive fungal disease (IFD) according to the revised EORTC/MSG classification, were retrospectively studied. Histopathological analysis of lung tissues, available for 23 patients, demonstrated proven IFD in 17 cases (14 invasive aspergillosis and 3 invasive zygomycosis), diffuse alveolar damage in one and organising pneumonia (OP) in five cases. In the OP cases, three of which have been defined as probable IFD according to EORTC/MSG classification, extensive immunohistochemical, molecular and immunological analyses for fungi were negative. Our case descriptions extend the notion that OP may be encountered as a distinct histopathological entity in pulmonary nodular lesions in patients with leukaemia with probable/possible IFD.

Interferon-alpha may restore sensitivity to tyrosine-kinase inhibitors in Philadelphia chromosome positive acute lymphoblastic leukaemia with F317L mutation

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The importance of cytogenetic and molecular analyses in eosinophilia-associated myeloproliferative neoplasms: an unusual case with normal karyotype and TNIP1- PDGFRB rearrangement and overview of PDGFRB partner genes

Fusion genes derived from the platelet-derived growth factor receptor beta (PDGFRB) or alpha (PDGFRA) play an important role in the pathogenesis of eosinophilia-associated myeloproliferative neopla...