Rossana Capizzuto's research while affiliated with Istituto Clinico Humanitas IRCCS and other places

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide is a curative treatment for many hematological malignancies, yet a majority of patients still suffers from recurrent infections. Post-transplant infusion of memory T cells could potentially enhance immunological protection without increasing the risk of eliciting acute graft-versus-host disease, which is mainly induced by naïve T cells. Here, we performed longitudinal analysis of the lymphocyte compartment in 19 haplo-HSCT patients previously enrolled in a phase II prospective clinical trial (ClinicalTrials.gov Identifier: NCT04687982), in which they received post-transplant CD45RA-depleted donor lymphocyte infusions (DLI). T cell receptor sequencing analysis showed that, surprisingly, CD45RA-depleted DLI do not increase T cell clonal diversity, but lead to prominent expansion of a selected number of infused memory T cell clones, suggestive of recruitment of these cells in the immune response. Pathogen-specific memory T cells, including cytomegalovirus (CMV)-specific cells, engrafted and were able to persist for at least one month. Deep immunophenotyping revealed strong polyfunctional effector CMV-specific T cell responses in the majority of patients, with their expansion correlating with the frequency of CMV-specific cells in the donor. These findings provide a rationale behind the suggested improved protection against viral infections for patients receiving CD45RA-depleted DLI.

Background : Allogeneic stem cell transplantation from haploidentical donor using post-transplantation cyclophosphamide has been employed to cure hematological diseases. Due to slow immunological reconstitution, there is an increased incidence of viral infection. Objective : The aim of our study was to prospectively evaluate the efficacy and the feasibility of a CD45RA+ depleted donor lymphocytes infusion (DLI) in terms of reduction of viral infection early after haploidentical transplantation. Study design : This a prospective single center study. We enrolled 23 patients, of whom 19 were evaluable. GVHD prophylaxis were the same for all patients. The primary end point was 100-day cumulative incidence of viral infections. Results : The primary endpoint was met, since the 100-day cumulative incidence of viral infection was 32%. The median time from transplantation to first CD45RA+ depleted DLI was 55 days (range 46-63). 28% of patients had CMV reactivation, no patients reactivated HHV6; 1 patient developed BK virus related haemorrhagic cystitis. Most of the patients received the planned 3 infusions. Only 1 patient developed grade 2 acute GVHD and 2 patients moderate chronic GVHD. All evaluable patients were off immunosuppressive therapy at last follow-up. With a median follow-up was 12 months (range 3-23), the 1-y OS and PFS was 79% and 75%, respectively; the 100-day and 1-y NRM were 5% and 12%, respectively. Conclusion : CD45RA+ depleted DLI are feasible in patients treated with haploidentical transplantation. The toxic profile is good with a low risk to develop GVHD both acute and chronic.

Background Despite the introduction of several effective multidrug combination in the treatment landscape of multiple myeloma (MM), high dose chemotherapy (HDC) is still an important phase in the treatment of MM. On the other hand, chemotherapy used to mobilize stem cell (SC) do not contribute in disease control or long term survival Aims To analyze the efficacy of granulocyte colony stimulating factor (GCSF) alone to mobilize SC, in a consecutive series of multiple myeloma patients candidate to HDC. Methods From January 2016 to January 2019, 61 consecutive MM patients underwent GCSF alone stem cell mobilization in our institution. Every patients received GCSF at the dose of 10 mcg/kg/day for 5 days. Peripheral blood circulating CD34+/mcl were checked from day +4. Plerixafor was administered on demand when CD34+ cells were less than 20/mcl at day +4 or +5, or when CD34 harvested was less than 2 × 10^6/kg after the first or second apheresis. The stem cell harvest were performed processing 2.5 − 3 times total blood volume. After collection, the patients received single or double course of melphalan 200 mg/m ² or 1 course of melphalan 140 mg/m ² according to local guideline. The minimal CD34+ number to perform a single HDC was 2x106/kg. Results Patient characteristics are listed in Table 1. All patients but 2 (3%) achieved the target to perform the planned HCD. Median total CD34+ × 10 ⁶ /kg harvested was 8.6. The proportion of patients able to achieve the goal with one apheresis was 50%. Only 1 patient underwent a third apheresis. Nine patients (14%) received plerixafor, 7 on day +4 and 2 on day +5. Median blood CD34+/mcl on day+4 and +5 was 33.5 (15–98), and 63 (10–171), respectively. Median total blood leucocyte counts (10^3/mm^3) on day 4+ and on day +5 was 41310 and 49250, respectively. Median CD34+ × 10 ⁶ /kg in the first and second apheresis was 6.4 (0,8–16) and 4.7 (1.5–8), respectively. Median total leucocyte count (10 ⁶ ) in the graft on first and second apheresis was respectively 71280 and 49250. After HDC, all patients but one experienced engraftment. Summary/Conclusion GCSF alone stem cell mobilization is an effective and safe approach after induction in MM patients candidate to HDC. The main advantages are the possibility to predict peak CD34+ level (4–5 days), to schedule apheresis in an outpatient program and to avoid unnecessary toxicity with chemotherapy. image

Background: The presence of human leukocyte antigen donor-specific antibodies (DSAs) increases the risk of graft failure in T-cell-replete haploidentical hematopoietic stem cell transplantation (haplo-HSCT) CASE REPORT: A 49-year-old female with high-risk acute myeloid leukemia in first complete remission received a haplo-HSCT from her daughter. Pretransplant recipient screening examination showed high DSAs levels against unshared class I leukocyte antigens. Results: The patient underwent a desensitization program consisting of plasma exchange (PEX), polyvalent intravenous (IV) immunoglobulins, and IV tacrolimus and mycophenolate mofetil (MMF). This protocol resulted in the disappearance of the DSA anti HLA B41. Engraftment was prompt with stable full donor chimerism. Conclusions: This case report suggests that the adopted scheme is safe for reducing DSA levels and facilitating donor engraftment in patients scheduled for haplo-HSCT.

Background and objectives: Several transplantation outcomes have been shown to be associated with the infused bone marrow cell dose/kg of the recipient’s body weight. The donor bone marrow density is directly related to the infused cell dose. The aim of the present study was to identify donor-related variables that are associated with high donor bone marrow density. Materials and methods: We retrospectively analysed the predictive factors of high marrow density in 65 consecutive HLA-haploidentical bone marrow donors har- vested at our centre between 2009 and 2013. Results: Body mass index (BMI) and peripheral white blood cell (WBC) count were directly associated with bone marrow density (regression coefficient b = 5 33 and b = 2 93, respectively; P < 0 01). The likelihood of obtaining a collection with a high density was first predicted using BMI (BMI ≥30, mean density = 25 8 TNC/ ml 9 10^6). Second, donors with a BMI <30 were split into two groups according to peripheral WBC count (WBC <8 9 10^3/mm^3: mean density = 18 4 TNC/ ml 9 10^6; WBC ≥8 9 10^3/mm^3: mean density = 23 1 TNC/ml 9 10^6). We also observed that the density of the first collected bag directly correlated with the overall density (R^2 = 0 69, P < 0 01). Conclusion: The donor-related features BMI and WBC count affect the cell quan- tity obtainable with the harvest and should be taken into account when choosing the donor. Key words: bone marrow density, bone marrow donors, donor selection, haploidentical transplantation, predictive collection tree.

We explored the efficacy of the IGEV regimen (ifosfamide, gemcitabine, vinorelbine and prednisone) combined with a fixed dose of lenograstim (263 mug/day) to mobilize peripheral blood stem cells (PBSCs) in 90 Hodgkin's lymphoma patients. The median total CD34+ cells/mul peak, colony-forming units granulocyte-macrophage and white blood cells for all individual collection sets were 85/mul, 12 x 10(4)/kg and 20 700/mul, respectively. An adequate number of CD34+ cells (more than 3 x 10(6) or 6 x 10(6) CD34+ cells/kg depending on whether single or tandem high-dose chemotherapy was used) were collected in 89 out of 90 (98.7%) mobilized patients, whereas the only failure reached 2.3 x 10(6) CD34+ cells/kg. The median CD34+ cell collections were 11 x 10(6)/kg (range 2.3-39 x 10(6)/kg) and 10 x 10(6)/kg (range 6-22.0 x 10(6)/kg) with a median of 1 and 2 leukaphereses for patients eligible for single high-dose treatment and for candidates for tandem transplant, respectively. Target yields were reached in 71.43 and 49.09% and additionally in 17.14 and 43.64% of cases after the first and second apheresis procedures, respectively. Hematological and non-hematological side effects were acceptable, and no toxic deaths occurred. Thirty-four patients received a single and 47 received tandem transplantation with rapid engraftment. These results confirm that the IGEV regimen with lenograstim support can be used successfully and safely to mobilize PBSCs.

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.