Federica Sorà

Immune reconstitution after autologous peripheral blood progenitor cell transplantation: Effect of interleukin-15 on T-cell survival and effector functions

OBJECTIVEnThe aim of this study was to evaluate the occurrence of T-cell spontaneous apoptosis (A(spont)) and its modulation in vitro by the interleukin-2 receptor (IL-2R) gamma-chain (gammac)-signaling cytokine IL-15 in patients transplanted with autologous peripheral blood progenitor cells (PBPC) for hematologic malignancies.nnnMATERIALS AND METHODSnPatients were examined on days 30-60, 60-90, and 90-120 after PBPC infusion. Dissipation of mitochondrial transmembrane potential, a hallmark of T-cell apoptosis, has been detected using the fluorescent probe 3,3-dihexyloxacarbocyanine iodide, after short-term T-cell culture in the absence or presence of exogenous cytokines. Expression of Bcl-2 family members has been studied by flow cytometry and reverse transcriptase polymerase chain reaction. T-cell proliferative responses to recall antigens have been estimated in autologous mixed leukocyte cultures.nnnRESULTSnA(spont) was seen in 45% +/- 6% of CD4(+) and 55% +/- 6% of CD8(+) T cells cultured in the absence of cytokines. Of interest, IL-15 and, to a lesser extent, its structural cousin IL-2 counteracted T-cell A(spont) by inhibiting the processing of caspase-3 and up-regulating Bcl-2 mRNA and protein levels. Cell division tracking confirmed that IL-15 did not rescue T cells from A(spont) by promoting proliferation but rather acted as a genuine survival factor. Addition of a gammac-blocking antibody to cytokine-conditioned cultures abrogated both apoptosis inhibition and Bcl-2 induction by IL-15, suggesting involvement of the IL-2Rgammac signal transduction pathway. Whereas cytokine-unprimed posttransplant T cells mounted inadequate responses to recall antigens, T cells conditioned with IL-15 expanded vigorously, indicating restoration of antigen-specific proliferation.nnnCONCLUSIONSnT cells recovering after autologous PBPC transplantation are highly susceptible to spontaneous apoptosis in vitro. This phenomenon can be counteracted by the gammac-signaling cytokine IL-15. These findings suggest that IL-15 might be a promising immunomodulating agent to improve postgrafting T-cell function.

Immune reconstitution after autologous selectedperipheral blood progenitor cell transplantation:comparison of two CD34+ cell-selection systems

BACKGROUND: Selection of CD34+ PBPCs has been applied as a method of reducing graft contamination from neoplastic cells. This procedure seems to delay lymphocyte recovery, while myeloid engraftment is no different from that with unselected PBPC transplants.

Dasatinib restores full donor chimerism in a patient with imatinib-resistant Ph+ ALL relapsing after unrelated cord blood transplantation

The t(9;22) translocation, known as the Philadelphia (Ph) chromosome, represents the most adverse prognostic factor for adult acute lymphoblastic leukemia (ALL) and, until recently, conventional chemotherapy programs have not been effective in patients with this diagnosis [1], allogeneic stem cell transplantation (SCT) being therefore considered the only option for a cure [2]. Since imatinib was introduced as the first molecularly targeted therapy, the treatment for Phþ ALL has been rapidly changing [3], but unfortunately the emergence of resistance to imatinib remains a major problem in the treatment of Phþ leukemias [4,5]. The need for alternative approaches for imatinib-resistant Phþ leukemias has led to the development of a second generation of targeted therapies, such as AMN107 (nilotinib) and BMS354825 (dasatinib). The progression-free survival (PFS) achieved to date with dasatinib is clinically relevant, considering the very poor prognosis of this difficult-to-treat population, and may open a window of opportunity for many of these patients [6,7]. Here we describe the case of a Phþ ALL patient who received salvage therapy with dasatinib, achieving a durable molecular remission for more than 8 months, after failure of both imatinib-based treatment and subsequent allogeneic cord blood transplantation (CBT). A 37-year-old woman was referred to our hospital in May 2005 with a history of night-sweats and tiredness. The initial laboratory evaluation revealed haemoglobin 12.7 g/dl, WBC 25610/l, platelet count 150610/l, LDH 3500 UI/l, and 72% blast cells in the blood smear. A bone marrow aspirate showed 89% blast cells and the immunophenotype was consistent with a pre-B ALL. The karyotype was 46,XX,t(9;22) 7q7 in 100% of metaphases, and presence of the Bcr/Abl fusion gene was demonstrated using reverse transcriptase polymerase chain reaction (RT-PCR). She was treated with induction therapy according to GIMEMA LAL 0904 protocol, including prednisone, vincristine, daunoblastine, and asparaginase. Imatinib 800 mg per day was added to standard induction chemotherapy according to the presence of Ph chromosome. The patient achieved a hematologic and cytogenetic complete remission (CR) after induction, without any major complication, and in July 2005 she was started on maintenance treatment with imatinib 400 mg per day. She had no HLA-matched sibling and search for an unrelated donor was initiated. At more than 6 months after diagnosis, a bone marrow aspirate revealed 29% of blasts and cytogenetic response was lost (30% Phþ metaphases). Imatinib was escalated to 800 mg per day and prednisone and weekly vincristine were administered, but blast cell percentage increased to 38%; the patient underwent a standard salvage treatment (HAM), obtaining a hematologic CR, whereas her bone marrow remained positive for the t(9;22) translocation.

Frontline Dasatinib Treatment in a “Real-Life” Cohort of Patients Older than 65 Years with Chronic Myeloid Leukemia

Dasatinib (DAS) has been licensed for the frontline treatment in chronic myeloid leukemia (CML). However, very few data are available regarding its efficacy and toxicity in elderly patients with CML outside clinical trials. To address this issue, we set out a “real-life” cohort of 65 chronic phase CML patients older than 65 years (median age 75.1 years) treated frontline with DAS in 26 Italian centers from June 2012 to June 2015, focusing our attention on toxicity and efficacy data. One third of patients (20/65: 30.7%) had 3 or more comorbidities and required concomitant therapies; according to Sokal classification, 3 patients (4.6%) were low risk, 39 (60.0%) intermediate risk, and 20 (30.8%) high risk, whereas 3 (4.6%) were not classifiable. DAS starting dose was 100 mg once a day in 54 patients (83.0%), whereas 11 patients (17.0%) received less than 100 mg/day. Grade 3/4 hematologic and extrahematologic toxicities were reported in 8 (12.3%) and 12 (18.5%) patients, respectively. Overall, 10 patients (15.4%) permanently discontinued DAS because of toxicities. Pleural effusions (all WHO grades) occurred in 12 patients (18.5%) and in 5 of them occurred during the first 3 months. DAS treatment induced in 60/65 patients (92.3%) a complete cytogenetic response and in 50/65 (76.9%) also a major molecular response. These findings show that DAS might play an important role in the frontline treatment of CML patients >65 years old, proving efficacy and having a favorable safety profile also in elderly subjects with comorbidities.

Prevalence, characteristics and management of occult hepatitis B virus infection in patients with chronic lymphocytic leukemia: a single center experience.

Several reports have emphasized the risk of hepatitis B virus (HBV) reactivation in patients with lymphoproliferative disorders undergoing cytotoxic treatment. To determine the prevalence of occult B infection (OBI) in a population with chronic lymphocytic leukemia (CLL) and management with universal prophylaxis (UP) in all patients undergoing chemoimmunotherapy or targeted prophylaxis (TP) in patients experiencing seroreversion during therapy, we analyzed 397 patients with CLL from our database. The prevalence of OBI in our patients with CLL was 8.6% (34 patients). When comparing patients with OBI/CLL with those with CLL, we did not find any statistical difference among clinical–biological parameters and time dependent endpoints except for a lower peripheral blood lymphocyte count in the OBI/CLL group (p = 0.036). From 2000 to 2010 careful follow-up and TP were adopted; two out of 10 patients (20%) showed seroreversion. From June 2010 we adopted UP during and 12 months after immunosuppressive treatment in all patients with CLL with OBI; no evidence of seroreversion was detected.

Kinetics of peg-filgrastim after high-dose chemotherapy and autologous peripheral blood stem cell transplantation

Peg-filgrastim is a form of G-CSF with a sustained duration of action due to self-limited clearance. We administered 6u2009mg peg-filgrastim to 18 autograft recipients on day +1 after transplantation for hematologic malignancies. Plasma samples were collected at baseline and during transplantation. Hematopoietic recovery and clinical outcomes were compared to the historical data of 54 patients not receiving G-CSF. Patients receiving peg-filgrastim achieved a serum level of 115u2009000u2009pg/ml on day +2, 24u2009h after drug administration. Drug level maintained a plateau until day +8 and, after day +10, declined concomitantly with myeloid recovery. Patients experienced prompt neutrophil recovery: days +9 and +10 to 500 and 1000 neutrophils per microliter, and 4 days with an absolute neutrophil count <100 cells per microliter. Duration of antibiotic therapy was significantly shortened, but we did not observe significant differences in other end points. In conclusion, peg-filgrastim was well tolerated and efficacious, and hastened myeloid recovery.

Clonal gammopathies and allogeneic stem cell transplantation.

recentlypublishedinyourjournal.Inthepaper,theauthorsclaimedthat there was an increase of clonal gammopathiesfollowing alemtuzumab-based reduced-intensity condition-ing hematopoietic stem cell transplantation. The authorsconcludethattherewasnodifferenceinthenumberofviralinfections (that is, CMV and HBV) in patients developingclonal gammopathies. The majorcausative role forthedevelopment of clonal gammopathies after organ trans-plantation is thought to be EBV as supported by severalreports in the literature.

Immune Reconstitution following Transplantation of Autologous Peripheral CD34+ Cells

The recovery of lymphocyte count, CD4+ and CD8+ T-cell subsets, natural killer (NK) cells and CD19+ B cells has been evaluated during the first 4 months after the infusion of autologous CD34+ peripheral blood progenitor cells (PBPC; group A; 33 patients) or autologous unselected PBPC (group B; 36 patients) for hematological malignancies. Lymphocyte count promptly recovered in both patient cohorts, although the repopulation of CD3+ T cells occurred more rapidly in group B compared with group A. The count of CD4+ T lymphocytes remained <200/µl during the study period in patients transplanted with CD34+ PBPC, being significantly lower compared with group B (p = 0.0019 and p = 0.0035 on days 30 and 60, respectively). CD8+ T cells rapidly increased both in group A and B and CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T cells displayed an activated phenotype in both groups of patients, coexpressing the HLA-DR antigen throughout the study period. No differences in the repopulation kinetics of NK cells and CD19+ B cells were observed. Further investigations are encouraged to characterize T cell competence following transplantation of CD34+ PBPC.

Glutamine parenteral supplementation in stem cell transplant

Pytlik et al1 recently described the results of a randomized double-blind placebo-controlled study on alanyl-glutamine dipeptide administered in autologous transplant patients. What is really surprising is not only the lack of any effect of glutamine supplementation but the detrimental effect, which was statistically significant, on several end points of the study (survival, hospitalization, mucositis, opioid requirements, and relapse). If our first goal is first do no harm, the results of this study obviously do not go in this direction and this raises the question of a premature withdrawal of the study, considering also the significant increment in the cost of the transplant procedure. Although several adjustments were made by the authors in dissecting the patient population, excluding multiple sclerosis and multiple myeloma, these were not responsible for major differences in their results. Heterogeneity of the patient population as stated by the authors might be responsible for the lack of any effect. We would like to draw attention to the following points with regard to patient characteristics and the design of the above-mentioned study:

Low risk of hepatitis B virus reactivation in patients with resolved infection and chronic myeloid leukemia treated with tyrosine kinase inhibitors

The hepatitis B virus (HBV) infection is a crucial issue for the management of patients with hematological malignancies, who often receive treatments that suppress the immune system with a consequent increased risk of HBV reactivation. Thus, routine screening for the assessment of HBV serological status is mandatory, as effective antiviral therapies are available for the treatment of HBV infection. According to the current guidelines, the universal prophylaxis with high genetic barrier drugs, such as entecavir or tenofovir, has a well-established efficacy in the prevention of HBV hepatitis in HBV surface antigen (HBsAg) positive patients.[1] However, the best strategy for the management of HBsAg negative patients with resolved HBV infection who are HBV core antibodies (antiHBc) positive with or without hepatitis B surface antibodies (antiHBs) positivity is less clear. During and after a profound suppression of the immune system, serum HBsAg detectability may be observed in these patients, usually associated with a decline of antiHBs when present at baseline, before immunosuppression; this has been defined as ‘reverse seroconversion’, and may lead to HBV infection reactivation, defined as the presence of significant HBV-DNA and alanine aminotransferase (ALT) serum levels above the normal upper limit, and eventually to HBV hepatitis, defined as ALT serum levels exceeding three times the upper limit of normal or an increase of more than 100 IU/L compared with the baseline, prechemotherapy, values.[2–4] In patients with hematological malignancies and resolved HBV infection, this risk has been reported to be as high as 12% during conventional chemotherapy, but it can rise to 14–50% in patients treated with hematopoietic stem cell transplantation,[5] or in patients treated with monoclonal antilymphocyte B and T (anti-CD20 and anti-CD52) antibodies.[3,6] According to the current evidence-based guidelines of the American Gastroenterological Association,[7] the risk of HBV reverse seroconversion and reactivation in HBsAg negative/antiHBc positive patients treated with B-cells depleting agents (e.g. rituximab) is high (>10%); therefore, in these subjects antiviral prophylaxis with drugs presenting a high barrier to resistance is recommended. Conversely, this risk is considered moderate (1–10%) in patients treated with tumor necrosis factor alpha (TNF) inhibitors, with other cytokine or integrin inhibitors (e.g. vedolizumab), with high or moderate doses of daily corticosteroids for more than 4 weeks, with antracyclin derivatives, and with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib. In these cases, antiviral prophylaxis over monitoring is suggested, although the strength of recommendations is weak and derived from evidence of moderate quality due to the insufficient data published in these settings.[7,8] Indeed, few data about the occurrence of HBV reverse seroconversion in Western patients receiving TKIs for a long period are available. We conducted a retrospective study to investigate the prevalence of resolved HBV infection and the incidence of HBV reactivation in a cohort of patients affected by chronic myeloid leukemia (CML) treated with TKIs in our Institution during the last 15 years. At the time of CML diagnosis, screening for HBsAg, HBeAg, antiHBe, antiHBc IgG and IgM, antiHBs, and HBV-DNA was performed in all patients using commercially available kits (COBAS Ampli Prep/COBAS TaqMan HBV test, Roche Diagnostics, Switzerland). Patients with serum markers of resolved HBV infection (HBsAg negative, antiHBc positive with or without antiHBs, HBV-DNA negative) and antiHBs positive patients who had never been vaccinated and who had lost serum antiHBc were enrolled in the study. After the beginning of TKIs treatment, each patient underwent clinical evaluation at three-month intervals, or whenever necessary based on the course of the disease; HBV virological assessment and liver function tests, as well as routine