Paola Terranova

Daclizumab as useful treatment in refractory acute GVHD: a paediatric experience

GVHD remains a serious complication after allogeneic SCT. We describe 13 paediatric patients treated with daclizumab for refractory acute GVHD (aGVHD). After 30 days of daclizumab administration, all patients with cutaneous aGVHD reached complete response. Among patients with gastrointestinal disease, 50 and 30% had complete and partial response, respectively, whereas 11 and 55% of patients with hepatic aGVHD achieved CR and PR, respectively. Overall, complete (46%) and partial (46%) responses were demonstrated in 92% of our patients, whereas the remaining patients (8%) were nonresponders. No life-threatening infectious episodes were recorded within 100 days from transplant in this selected group of paediatric patients. Overall 46% of patients were alive at a median of 461 days from SCT, but 50% of them developed chronic GVHD. In our experience, daclizumab proved to be a useful and safe treatment for refractory and steroid-resistant/dependent aGVHD, in particular for cutaneous and low-moderate intestinal involvement.

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children.

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 105 PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 105 PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached ⩾20 000 copies per 105 PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as ⩾20 000 geq per 105 PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.

Analysis of the antigen specific T cell repertoires in HIV infection

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Impaired immune response to Candida albicans in cells from Fanconi anemia patients

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. Several studies show alterations of the immunological status of FA patients including defects in peripheral blood lymphocyte subsets, serum immunoglobulin levels, and inflammatory cytokines. However scanty information is available on the response of FA cells to specific infectious antigens. In this work we examined the response of FA cells to different immunological stimuli and found a defective response of IL-1β, TNF-α and IL-17 to Candida albicans stimulation thus pointing to a potentially impaired response to fungal infections of FA patients.

Clinical features and therapeutic challenges of cytopenias belonging to alps and alps-related (ARS) phenotype

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Sirolimus as a rescue therapy in children with immune thrombocytopenia refractory to mycophenolate mofetil

To the Editor: Immune thrombocytopenia (ITP) represents the most common bleeding disorder in children characterized by the immune-mediated destructionof platelets and their precursorssecondary toproliferation and differentiation of autoreactive B-cells due to an abnormal T-cell response. Most cases in children have a self-limited course; however, the disease can become persistent or chronic. The standard first-line treatment are steroids/intravenous immunoglobulins usually given with platelets count <20–30 3 10/L or in patients with significant bleeding symptoms regardless of platelet count. Second/further-line therapies include splenectomy, rituximab, thrombopoietin receptor agonists, and immunosuppressant drugs; however, the choice is often individualized and based on the clinician’s experience or guidelines due to the lack of validated data. Mycophenolate mofetil (MMF) was shown to be effective in patients with ITP; however, some patients do not respond, especially those with primary ITP. Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that has been showed to be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and other primary or secondary autoimmune cytopenias. A prospective multi-institutional trial suggested that sirolimus was effective and safe in refractory cytopenias; however, little data were reported on patients with primary ITP. Herein, we report the results of a retrospective data review performed on ITP patients treated with sirolimus at the IRCCS Istituto Giannina Gaslini, Genoa (16 patients), and at University of Catania (3 patients). We included children with primary ITP or ITP secondary to an ALPS-related syndrome (ARS), which was defined as the presence of cytopenia and at least one absolute or primary additional criterion for ALPS. Patients with definitive/probable ALPS and with multilineage cytopenias were excluded. According to the ITP International Working Group criteria, responses to sirolimus were classified as a complete response (CR), which was defined as a platelet count 100 3 10/L; a partial response (PR), which was defined as a platelet count 30 3 10/L and at least a 2-fold increase from the baseline count; or no response, which was defined as a platelet count <30 3 10/L or less than a 2fold increase from the baseline. Recurrence was defined by a platelet count <30 3 10/L or by the appearance of bleeding symptoms after sirolimus withdrawal in responding patients. Sirolimus was given at an initial dose of 2-2.5 mg/m/day. Serum levels were monitored to keep drug levels at 5–15 ng/mL. The response and toxicity were assessed by periodical clinical/biochemical follow-up. Between 2005 and 2016, 19 patients (9 males, 10 females) with primary ITP (n510) or ITP secondary to ARS (n59) were treated with sirolimus. Median age was 9.8 years (range: 1.8–20), and 17 of the 19 (89%) patients had previously failed MMF treatment. The median time between diagnosis and sirolimus therapy was 22 months (range: 6– 106). Thirteen patients (68%) achieved a response that was complete and partial in 10 (53%) and 3 (16%). Of the 17 patients who had previously failed MMF therapy, 11 (65%) responded to sirolimus rescue. The median time between starting sirolimus and achieving a PR or CR was 50 days (range: 14–135) and 158 days (range: 14–579), respectively. The patient’s clinical characteristics, previous treatments, response, and toxicity are reported in Table 1. Five/Ten patients (50%) and 8/9 (89%) with primary ITP and with ARS-ITP, responded to the treatment, respectively (P50.06). One patient with ARS relapsed during tapering but recovered after dose adjustment. Sirolimus was given for a median of 15 months (range: 1–71). The median follow-up time was 21 months (range: 4–71). Treatment was well tolerated, and no adverse effects were reported. To the best of our knowledge, this is the largest cohort of ITP patients treated with sirolimus, and the results show that the drug is safe and efficacious. Patients were treated with the same homogeneous approach and most were had previously received MMF. In previous reports, this drug given to adults and children with ITP was successful in about 50%-60% of patients; however, the patients who did not respond required further-line therapies. In this study, most of the children who responded (85%) had previously failed to respond to MMF treatment demonstrating that sirolimus is an efficient rescue agent in these setting of patients. In addition, the treatment sequence of MMF followed by sirolimus, successfully used in ALPS patients, can also be applied in patients with primary or secondary ITP. Sirolimus is a mTOR inhibitor that targets the PI3K/AKT/mTOR pathway thus modulating Band T-lymphocyte proliferation and, more specifically, abnormal T-cells. Contrary to MMF, it also promotes the proliferation of regulatory T-cells. Little data are available on the use of sirolimus in ITP. The only prospective study for autoimmune multilineage cytopenias also included adults and patients with ALPS and thus showed very limited data for children with primary ITP. The largest cohort of children with ITP reported so far included 12 patients and showed that sirolimus was a safe steroid-sparing agent. However, these patients were previously treated with different drugs, and sirolimus was mostly coadministered with steroids. In our homogeneous

Successful Second Unrelated Donor Hematopoietic Stem Cell Transplant in a Patient With Dyskeratosis Congenital After First Graft Rejection

Dyskeratosis congenita is a rare congenital telomeropathy characterized by cutaneous and nail dystrophy, oral leukoplakia, and bone marrow failure. Pulmonary fibrosis and cancers are late manifestations. Allogeneic hematopoietic stem cell transplant represents the only cure for those with bone marrow failure with this disease, but outcomes reported are overall poor, with organ toxicities, graft failure, and graft-versus-host disease as main issues. Although reduced intensity conditioning regimens seem to be related to better outcomes, a standard regimen for dyskeratosis congenita has never been defined. Here, we report a successful long-term outcome of an 8-year-old girl with dyskeratosis congenita who received 2 consecutive allogeneic hematopoietic stem cell transplants from different unrelated donors, because of rejection after the first one, both conditioned with fludarabine-based reduced intensity conditioning regimen. The second transplant was complicated by severe hemorrhagic cystitis and acute grade IV graft-versus-host disease in the early phase and mild chronic graft-versus-host disease and ureteral stenosis in the late phase. This experience confirms that dyskeratosis congenita is at high risk for transplant-related morbidity but that a fludarabine-based reduced intensity conditioning regimen is a safe and feasible option as a preparative regimen, as shown here in a second transplant after first graft rejection. To reduce the risk of graft-versus-host disease, more effective prophylaxis schedules should be chosen in cases of unrelated donor, and haploidentical hematopoietic stem cell transplant with in vitro α/ β + and CD19+ depletion should be considered.

Evaluation of Chimerism Dynamics after Allogeneic Hematopoietic Stem Cell Transplantation in Children with Nonmalignant Diseases

It is recognized that chimerism following hematopoietic stem cell transplantation (HSCT) is a dynamic process. The aims of this study were to describe the evolution of chimerism in children with nonmalignant diseases who underwent allogeneic HSCT, and to analyze the risk factors influencing chimerism status. A total of 101 HSCTs were performed in 85 patients with nonmalignant diseases. The donor was unrelated in 62.4% of HSCTs. Reduced-intensity conditioning (RIC) regimen was administered in 48.5% of patients. Acute graft-versus-host disease (aGVHD) occurred in 51.7% and chronic GVHD (cGVHD) in 39.7% of patients. Analysis of chimerism was performed through amplification of 9 specific short tandem repeats by polymerase chain reaction at engraftment and 1, 6, and 12 months after HSCT. Upon first evaluation, complete chimerism (CC) was detected in 34.7% and mixed chimerism (MC) in 55.4%, whereas graft failure occurred in 9.9% of patients. Severe aGVHD was associated with CC (P = .031). The last chimerism evaluation showed CC in 72.1%, stable MC in 12.8%, and progressive MC in 3.5%. CC was associated with a higher incidence of aGVHD (P = .016) and cGVHD (P = .022), whereas the RIC regimen was associated with graft failure (P = .026). One- and 3-year overall survival (OS) was 87.4% and 80.5%, respectively, with a lower OS at 3 years in patients with CC compared with those with MC (P = .008). aGVHD and cGVHD represent factors favoring CC, thus close, careful follow-up of chimerism is recommended in patients affected by nonmalignant disease.