Single-Cell DNA Sequencing Reveals an Evolutionary Pattern of CHIP in Transplant Eligible Multiple Myeloma Patients Enrica Borsi, Ilaria Vigliotta, Andrea Poletti, Gaia Mazzocchetti, Vincenza Solli, Luca Zazzeroni, Marina Martello, Silvia Armuzzi, Barbara Taurisano, Ajsi Kanapari, Ignazia Pistis, Elena Zamagni, Lucia Pantani, Serena Rocchi, Katia Mancuso, Paola Tacchetti, Ilaria Rizzello, Simonetta Rizzi, Elisa Dan, Barbara Sinigaglia, Michele Cavo, Carolina Terragna Cells, 2024 Clonal hematopoiesis of indeterminate potential (CHIP) refers to the phenomenon where a hematopoietic stem cell acquires fitness-increasing mutation(s), resulting in its clonal expansion. CHIP is frequently observed in multiple myeloma (MM) patients, and it is associated with a worse outcome. High-throughput amplicon-based single-cell DNA sequencing was performed on circulating CD34+ cells collected from twelve MM patients before autologous stem cell transplantation (ASCT). Moreover, in four MM patients, longitudinal samples either before or post-ASCT were collected. Single-cell sequencing and data analysis were assessed using the MissionBio Tapestri® platform, with a targeted panel of 20 leukemia-associated genes. We detected CHIP pathogenic mutations in 6/12 patients (50%) at the time of transplant. The most frequently mutated genes were TET2, EZH2, KIT, DNMT3A, and ASXL1. In two patients, we observed co-occurring mutations involving an epigenetic modifier (i.e., DNMT3A) and/or a gene involved in splicing machinery (i.e., SF3B1) and/or a tyrosine kinase receptor (i.e., KIT) in the same clone. Longitudinal analysis of paired samples revealed a positive selection of mutant high-fitness clones over time, regardless of their affinity with a major or minor sub-clone. Copy number analysis of the panel of all genes did not show any numerical alterations present in stem cell compartment. Moreover, we observed a tendency of CHIP-positive patients to achieve a suboptimal response to therapy compared to those without. A sub-clone dynamic of high-fitness mutations over time was confirmed.
Long-Term Outcome After Adoptive Immunotherapy With Natural Killer Cells: Alloreactive NK Cell Dose Still Matters Sarah Parisi, Loredana Ruggeri, Elisa Dan, Simonetta Rizzi, Barbara Sinigaglia, Darina Ocadlikova, Andrea Bontadini, Valeria Giudice, Elena Urbani, Sara Ciardelli, Chiara Sartor, Gianluca Cristiano, Jacopo Nanni, Letizia Zannoni, Gabriella Chirumbolo, Mario Arpinati, Russell E. Lewis, Francesca Bonifazi, Giovanni Marconi, Giovanni Martinelli, Cristina Papayannidis, Stefania Paolini, Andrea Velardi, Michele Cavo, Roberto M. Lemoli, Antonio Curti Frontiers in Immunology, 2022 Recently, many reports were published supporting the clinical use of adoptively transferred natural killer (NK) cells as a therapeutic tool against cancer, including acute myeloid leukemia (AML). Our group demonstrated promising clinical response using adoptive immunotherapy with donor-derived alloreactive KIR-ligand-mismatched NK cells in AML patients. Moreover, the antileukemic effect was correlated with the dose of infused alloreactive NK cells (“functional NK cell dose”). Herein, we update the results of our previous study on a cohort of adult AML patients (median age at enrollment 64) in first morphological complete remission (CR), not eligible for allogeneic stem cell transplantation. After an extended median follow-up of 55.5 months, 8/16 evaluable patients (50%) are still off-therapy and alive disease-free. Overall survival (OS) and disease-free survival (DFS) are related with the dose of infused alloreactive NK cells (≥2 × 105/kg).
The timing of plerixafor addition to G-Csf and chemotherapy affects immunological recovery after autologous stem cell transplant in multiple myeloma Giulia Tolomelli, Katia Mancuso, Paola Tacchetti, Francesca Patriarca, Monica Galli, Lucia Pantani, Beatrice Zannetti, Maria Rosa Motta, Simonetta Rizzi, Elisa Dan, Barbara Sinigaglia, Valeria Giudice, Andrea Olmo, Mario Arpinati, Gabriella Chirumbolo, Renato Fanin, Russell E. Lewis, Laura Paris, Francesca Bonifazi, Michele Cavo, Antonio Curti, Roberto M. Lemoli Bone Marrow Transplantation, 2020 Plerixafor inhibits CXCR4, thus inducing the mobilization of hematopoietic stem/progenitor cells in lymphoma and multiple myeloma (MM) patients eligible for autologous stem cell transplantation (ASCT). However, the kinetics of plerixafor-induced mobilization of lymphocyte subsets is poorly known. Here, we evaluated the graft content, the engraftment, and the immunological reconstitution of MM patients receiving plerixafor. Thirty-seven patients undergoing one or tandem ASCT were enrolled. After mobilization with cyclophosphamide plus G-CSF, plerixafor was added at hematological recovery regardless of CD34 + cell count. We evaluated the number of CD34 + , CD34 + /CD38 − , CD3 + , CD4 + , CD8 + , CD19 + , CD56 + /CD3 − , CD4 + /CD25 + /FOXP3 + , and CD138 + /CD38 + cells on each apheresis. Hematological and immunological recovery were determined at 30 days, 3, 6, 9, and 12 months after ASCT. Overall, 34/37 patients mobilized a median of 10.1 × 10 6 CD34 + cells/Kg (IQ 7.7–13.4). Patients with <20/µL CD34 + cells at plerixafor administration (18/33) had a significantly higher CD34 + cell fold increase, but not a higher absolute number, than 16/33 patients with ≥20/µL CD34 + cells. A similar CD34 + and immune graft composition was reported. A higher number of CD3 + and CD8 + cells/µL was observed at 3 months after first ASCT ( p < 0.05) in the group with ≥20 CD34 + cells/µL. Thus, in MM patients, the timing of plerixafor administration influences immunological recovery.
Intrabone transplant provides full stemness of cord blood stem cells with fast hematopoietic recovery and low GVHD rate: results from a prospective study Francesca Bonifazi, Elisa Dan, Myriam Labopin, Mariarosaria Sessa, Viviana Guadagnuolo, Martina Ferioli, Simonetta Rizzi, Sabrina De Carolis, Barbara Sinigaglia, Maria Rosa Motta, Andrea Bontadini, Valeria Giudice, Giovanni Martinelli, Mario Arpinati, Michele Cavo, Massimiliano Bonafé, Gianluca Storci Bone Marrow Transplantation, 2019 Umbilical Cord Blood (UCB) represents a valid option for patients with hematopoietic malignancies lacking an HLA matched donor. To overcome the limitation of the low stem cell dose of UCB, the intrabone (IB) route has been proposed. We report the results of a prospective study on a poor-prognosis cohort of 23 patients receiving intrabone single UCB transplant (Clinicaltrials.gov NCT00886522). Cumulative incidence of hematological recovery at day 90 was 82 ± 9% (ANC > 0.5 × 109/L) and 70 ± 10% (platelet > 50 × 109/L) and correlated with CD34 + cells in the graft. NRM was 20 ± 9%. No severe aGVHD and only one extensive cGVHD occurred, with fast immune reconstitution. To test the hypothesis that the direct IB injection could affect the expression of stem cells regulatory pathways, CD34 + cells from BM aspirates at day + 10, + 20, + 30, processed in hypoxic conditions mimicking the BM-microenvironment (7%pO2), were studied for the expression of c-Mpl, Notch1 and CXCR4. We found that the expression of c-Mpl in CD34 + cells at day + 10 significantly correlated with hematological recovery. In conclusion, IB-UCB transplant success is associated with low incidence of GVHD and high-speed platelet recovery; intrabone route may preserve full hematopoietic stemness by direct delivery of UCB stem cells into the hypoxic HSC niche.
Low-Dose Anti-T Lymphoglobulin as Prophylaxis for Graft-versus-Host Disease in Unrelated Donor Transplantations for Acute Leukemias and Myelodysplastic Syndromes Francesca Bonifazi, Jacopo Olivieri, Mariarosaria Sessa, Elisa Dan, Barbara Sinigaglia, Simonetta Rizzi, Maria Rosa Motta, Andrea Bontadini, Francesca Ulbar, Valeria Giudice, Cristina Papayannidis, Antonio Curti, Angela Chiereghin, Tiziana Lazzarotto, Michele Cavo, Mario Arpinati Biology of Blood and Marrow Transplantation, 2018 Chronic graft-versus-host disease (cGVHD) is a major complication after stem cell transplantation (HSCT). Several randomized studies already demonstrated that anti-T lymphoglobulin (ATLG) is effective in preventing GVHD after myeloablative unrelated and HLA-identical sibling transplants. However, the issue of doses and the potential increase of relapses still remain unsolved. Here we report data on 190 patients with acute leukemia and myelodysplastic syndrome who underwent an unrelated HSCT with low-dose ATLG (15 to 30 mg/kg) given at an earlier timing (days -6 to -2). HSCT was performed from HLA 10/10 (n = 62, 33%), 9/10 (n = 91, 48%), 8/10 (n = 30, 16%), and <8/10 (n = 7, 4%) identical unrelated donor. Peripheral blood was the stem cell source in 42% (n = 80). Median follow-up was 51 months. Grades II to IV and III to IV acute GVHD were 26% and 9%, respectively, and 2-year overall and moderate to severe cGVHD were 23% and 14%, respectively. The 3-year incidences of relapse and nonrelapse mortality were 26% and 18%, respectively. The rates of 3-year overall survival (OS), disease-free survival (DFS), and GVHD-free and relapse-free survival (GRFS) were 60%, 56% and 44%, respectively. Factors such as younger donor, good performance status, and early disease were associated with better outcome in terms of OS, DFS, and GRFS. Our data indicate that doses of ATLG lower that those used in randomized clinical trials can be used for GVHD prevention, even in the adult setting, without clear increases in relapse and infections; these findings need to be further validated by a prospective randomized study.
Reinfusion of highly purified CD133+ bone marrow-derived stem/progenitor cells in patients with end-stage liver disease: A phase I clinical trial Pietro Andreone, Lucia Catani, Cristina Margini, Lucia Brodosi, Stefania Lorenzini, Daria Sollazzo, Benedetta Nicolini, Rosaria Giordano, Tiziana Montemurro, Simonetta Rizzi, Elisa Dan, Valeria Giudice, Mariele Viganò, Andrea Casadei, Francesco G. Foschi, Deborah Malvi, Mauro Bernardi, Fabio Conti, Roberto M. Lemoli Digestive and Liver Disease, 2015 BACKGROUND Bone marrow stem/progenitor cells seem to be effective in liver regeneration after tissue injury. AIM To evaluate the feasibility and safety of the mobilization and reinfusion of CD133+ stem/progenitor cells in patients with end-stage liver disease. METHODS Autologous CD133+ stem/progenitor cells, mobilized with granulocyte-colony stimulating factor, were collected by leukapheresis and reinfused at increasing doses through the hepatic artery starting from 5×10(4)/kg up to 1×10(6)/kg. RESULTS 16 subjects with Model for End-stage Liver Disease (MELD) score between 17 and 25 were enrolled, 14 mobilized an adequate number of CD133+ stem/progenitor cells and 12 were reinfused. No severe adverse events related to the procedure were reported. MELD score significantly worsened during mobilization in Child Turcotte Pugh-C patients. A significant improvement of liver function was observed 2 months after reinfusion (MELD 19.5 vs. 16; P=0.045). Overall, 5 patients underwent liver transplantation within 12 months from reinfusion and 2 died because of progressive liver failure. CONCLUSIONS CD133+ stem/progenitor cells reinfusion in patients with end-stage liver disease is feasible and safe. A worsening of liver function was observed during mobilization in Child Turcotte Pugh-C patients. The temporary improvement of MELD score after reinfusion suggests that stem cells therapy may be a "bridge to transplant" approach for these patients.
Glutathione transferase-A2 s112T polymorphism predicts survival, transplant-related mortality, busulfan and bilirubin blood levels after allogeneic stem cell transplantation F. Bonifazi, G. Storci, G. Bandini, E. Marasco, E. Dan, E. Zani, F. Albani, S. Bertoni, A. Bontadini, S. De Carolis, M. R. Sapienza, S. Rizzi, M. R. Motta, M. Ferioli, P. Garagnani, M. Cavo, V. Mantovani, M. Bonafe Haematologica, 2014 Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione homeostasis, with the aim of testing their impact on the clinical outcome of 185 busulfan-conditioned allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic factor for poorer survival (RR=2.388), for increased any time- and 100-day transplant-related mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan area under the concentration-time curve (1214.36±570.06 vs. 838.10±282.40 mMol*min) and higher post-transplant bilirubin serum levels (3.280±0.422 vs. 1.874+0.197 mg/dL). In vitro, busulfan elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein, solute carrier organic anion transporters, and even GSTA2. It is worthy of note that GSTA2 also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a patho-physiological model in which busulfan-induced inflammation leads to the impairment of post-transplant bilirubin metabolism.
Intensification of GVHD prophylaxis with low-dose ATG-F before allogeneic PBSC transplantation from HLA-identical siblings in adult patients with hematological malignancies: Results from a retrospective analysis F Bonifazi, G Bandini, M Arpinati, G Tolomelli, M Stanzani, M R Motta, S Rizzi, V Giudice, E Dan, E Massari, P Tazzari, A Bontadini, P Pagliaro, M Baccarani Bone Marrow Transplantation, 2012 Several studies have shown that chronic GVHD (cGVHD) is more frequent in patients receiving transplants from PBSC than in those receiving BM. In the setting of PBSC-unrelated transplants, the addition of anti-T-cell globulin (ATG) has shown a significant decrease in incidence/severity of cGVHD, without an increase in relapses or infections. However, no prospective data are yet available in the sibling setting. We retrospectively analyzed the effects of intensification of standard GVHD prophylaxis (CsA+MTX) by the addition of low-dose ATG in 245 patients receiving a transplant from HLA-identical sibling. From 1996 to 2001, patients received PBSC as the preferred source (group 2), and then ATG was added before transplant (group 3) because of a high cGVHD rate. Patients receiving BM in the same time period were analyzed as a control group (group 1). The incidence of grade III−IV acute GVHD and cGVHD was not significantly different in the three groups, but extensive cGVHD was highest in group 2 (38%) compared with group 3 (21%) or group 1 (28%; P=0.03). OS, TRM and time to relapse/progression were similar in the three groups. Our analysis shows that adding ATG to PBSC sibling allogeneic transplants can lower cGVHD, without an increase of relapse. Further prospective studies are needed to confirm these findings.
Phase II study of a single pegfilgrastim injection as an adjunct to chemotherapy to mobilize stem cells into peripheral blood of pretreated lymphoma patients Journal of Supportive Oncology, 2005
Phase II study of a single pegfilgrastim injection as an adjunct to chemotherapy to mobilize stem cells into the peripheral blood of pretreated lymphoma patients Haematologica, 2005
High-dose therapy with autologous transplantation for aggressive non-Hodgkin's lymphoma: The Bologna experience Pier Luigi Zinzani, Monica Tani, Annalisa Gabriele, Filippo Gherlinzoni, Antonello de Vivo, Paolo Ricci, Giuseppe Bandini, Roberto Massimo Lemoli, Maria Rosa Motta, Simonetta Rizzi, Valeria Guidice, Maurizio Zompatori, Vittorio Stefoni, Lapo Alinari, Gerardo Musuraca, Enrica Marchi, Simona Bassi, Roberto Conte, Stefano Pileri, Sante Tura, Michele Baccarani Leukemia and Lymphoma, 2004
High-dose therapy with autologous transplantation for Hodgkin's disease: The Bologna experience Haematologica, 2003
Allogeneic peripheral blood stem cell transplantation in patients with early-phase hematologic malignancy: A retrospective comparison of short-term outcome with bone marrow transplantation Haematologica, 1998
Immunologic and clinical modifications following low-dose subcutaneous administration of rIL-2 in non-Hodgkin's lymphoma patients after autologous bone marrow transplantation Bone Marrow Transplantation, 1996
Autologous bone marrow transplantation in late first complete remission improves outcome in acute myelogenous leukemia Leukemia, 1996
Stem cell factor (c-kit ligand) enhances the interleukin-9-dependent proliferation of human CD34+ and CD34+CD33-DR- cells Experimental Hematology, 1994
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