Single-Cell DNA Sequencing Reveals an Evolutionary Pattern of CHIP in Transplant Eligible Multiple Myeloma Patients.

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.

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.

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 × 106 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.

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.

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.

Larger Size of Donor Alloreactive NK Cell Repertoire Correlates with Better Response to NK Cell Immunotherapy in Elderly Acute Myeloid Leukemia Patients.

PURPOSE: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. EXPERIMENTAL DESIGN: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53-73) received NK cells from haploidentical KIR-ligand-mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. RESULTS: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6-68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3-51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively;P= 0.03). CONCLUSIONS: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells.

Reinfusion of highly purified CD133+ bone marrow-derived stem/progenitor cells in patients with end-stage liver disease: A phase I clinical trial.

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.

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.

Positive selection and transplantation of autologous highly purified CD133(+) stem cells in resistant/relapsed chronic lymphocytic leukemia patients results in rapid hematopoietic reconstitution without an adequate leukemic cell purging.

We assessed the capacity of positively selected autologous CD133(+) hematopoietic stem cells (HSCs) to reconstitute lymphomyelopoiesis in chronic lymphocytic leukemia (CLL) patients receiving myeloablative chemotherapy. Ten resistant/relapsed CLL patients underwent HSC mobilization with chemotherapy and granulocyte-colony stimulating factor (G-CSF). Positive selection of circulating CD133(+) HSCs was performed by immunomagnetic technique. Highly purified HSCs were reinfused after busulphan/melphalan myeloablative treatment. A median number of 4.2 x 10(6) CD34(+) cells/kg and of 3.14 x 10(6) CD133(+) cells/kg were collected. Immunomagnetic selection resulted in the reinfusion of a median number of 2.45 x 10(6) CD133(+) cells/kg (median purity: 94.8%; median recovery: 84%) and 2.4 x 10(6) CD34(+) cells/kg (median purity: 93%; median recovery: 71%). HSC selection resulted in a median T cell and CD19(+)/CD5(+) cell depletion of 3.85 log and 2.8 log, respectively. At the molecular level, however, 7 of 8 valuable purified HSC fractions were contaminated by leukemic cells. All CLL patients showed rapid and sustained myeloid engraftment after reinfusion of purified CD133(+) cells. Immunologic reconstitution was comparable to that routinely observed in patients reinfused with unmanipulated leukapheresis products and no late infectious complications were observed. With a median follow-up of 28 months for transplanted patients, 5 patients are in clinical complete remission, 3 are in partial remission, and 1 is in progression. In conclusion, the reinfusion of highly purified CD133(+) HSCs allowed the rapid and sustained recovery of hematopoiesis after myeloablative treatment in resistant/relapsed CLL patients. However, the purging potential of positive selection of CD133(+) cells is not adequate to achieve tumor-free autografts.

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.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the efficacy of pegfilgrastim, in combination with salvage chemotherapy, in mobilizing CD34(+) stem cells into the peripheral blood of pretreated lymphoma patients. DESIGN AND METHODS: This was an open-label phase II study including 25 pretreated patients (Hodgkin's disease=4; aggressive non-Hodgkin's lymphoma=21). The primary end-point of the study was the successful mobilization of a target cell dose of 2x10(6) CD34(+) cells/kg in lymphoma patients receiving ifosfamide, epirubicin and etoposide (IEV) chemotherapy and a fixed dose (6 mg) of pegfilgrastim given as single subcutaneous injection. RESULTS: Following chemotherapy, all patients had grade 4 neutropenia that lasted a median of 1.5 days (1-3). Pegfilgrastim treatment was well tolerated and only 2/25 patients required pain-control medication. CD34+ cells were mobilized in all patients. The median (range) peak value of peripheral blood CD34+ cells after IEV chemotherapy and pegfilgrastim was 141x10(6)/L (12.8-386) and occurred almost invariably on day +14 (13-16). Twenty-three of the 25 patients underwent a single standard volume leukapheresis to collect a median of 8.7x10(6) CD34(+) cells/kg (1.78-17.3). Twenty four/25 patients (96%) reached the target cell dose of 2x10(6) CD34(+) cells/kg. High concentrations of circulating CD34+ cells (> 50x10(6)/L) were observed for several days after the achievement of the peak value. All the study patients were transplanted with their pegfilgrastim-mobilized CD34(+) cells and showed a rapid and sustained engraftment after high-dose chemotherapy. INTERPRETATION AND CONCLUSIONS: Our results show that pegfilgrastim as an adjunct to chemotherapy is a predictable and highly effective mobilization regimen in pretreated lymphoma patients.

High-dose therapy with autologous transplantation for aggressive non-Hodgkin's lymphoma: the Bologna experience.

Patients with aggressive non-Hodgkin's lymphoma (NHL) who relapse after initial therapy have a poor prognosis and with standard dose salvage therapy the outlook remains poor. In this work we examine the patient characteristics and outcome of patients with aggressive NHL treated with HDT and autologous transplantation at our Institute from 1982 to 1999. A retrospective analysis was performed examining patient characteristics, prior chemotherapy regimens, pretransplant disease status, HDT regimen, source of stem cells, time for hematopietic recovery, complications of transplantation, response rates, overall survival (OS) and relapse-free survival (RFS). One hundred and thirty-four patients with aggressive NHL were treated with estimated 10-year OS and RFS rates of 50% and 66%, respectively. Disease status (sensitive vs. refractory) pre-HDT was the most powerful predictive parameter for OS and RFS, at both univariate and multivariate analysis. For the entire cohort, transplant-related mortality was only 3.5% without evidence of second malignancies. Our results confirm that HDT with autologous transplantation is associated with a durable RFS in a remarkable proportion of aggressive NHL patients with very low global early and late toxicity. Improved patient selection, transplant timing, ongoing improvements in supportive care, and selected phase III trials should increase outcomes further.

High-dose therapy with autologous transplantation for Hodgkin's disease: the Bologna experience.

BACKGROUND AND OBJECTIVES: In this work we examine the characteristics and outcome of patients with Hodgkin's disease (HD) treated with high-dose therapy (HDT) and autologous transplantation at our Institute between 1982 to 2000. DESIGN AND METHODS: A retrospective analysis was performed examining patients' characteristics, prior chemotherapy regimens, pre-transplant disease status, HDT regimen, source of stem cells, time for hematopoietic recovery, complications of transplantation, response rates, overall survival (OS) and relapse-free survival (RFS). RESULTS: Ninety-seven patients with HD were treated and had estimated 10-year OS and RFS rates of 32% and 60%, respectively. Disease status (sensitive vs. refractory) before HDT was the most powerful predictive parameter for OS and RFS in both univariate and multivariate analyses. The rate of transplant-related mortality in the whole cohort was only 1% whereas the rate of second malignancies was 3%. INTERPRETATION AND CONCLUSIONS: Our results confirm that HDT with autologous transplantation is associated with a durable RFS in a remarkable proportion of HD patients and that the procedure has a very low global early and late toxicity.

Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti-idiotype vaccination.

Circulating monocytes from multiple myeloma patients enrolled in a clinical study of anti-idiotype vaccination were labelled with clinical-grade anti-CD14 microbeads and positively selected with the CliniMACS instrument. Cells were then grown, according to good manufacturing practice guidelines, in fetal-calf-serum-free medium in cell culture bags and differentiated to dendritic cells (DC) with granulocyte-macrophage colony stimulating factor plus interleukin 4 (IL-4), followed by either tumour necrosis factor-alpha (TNF-alpha) or a cocktail of IL-1beta, IL-6, TNF-alpha and prostaglandin-E2. The CD14+ cell yield was increased from 17.6 +/- 6.5% to 93.8 +/- 6.3% (recovery 64.4 +/- 15.4%, viability > 97%). After cell culture, phenotypic analysis showed that 86.7 +/- 6.8% of the cells were DC: 2.27 +/- 0.9 x 108 DC/leukapheresis were obtained, which represented 20.7 +/- 4.6% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (28.6 +/- 3% of initial CD14+ cells) of DC than TNF-alpha alone, with secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic T cells and efficient presentation of tumour idiotype to autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of preloaded DC. The recovery of thawed, viable DC was 78 +/- 10%. Finally, interferon-alpha-2b was at least as efficient as IL-4 in inducing the differentiation of mature, functional DC from monocytes.

Human CD34(+) blood cells induce T-cell unresponsiveness to specific alloantigens only under costimulatory blockade.

OBJECTIVES: The immunogenic role of human CD34(+) cells in allogeneic hematopoietic stem cell transplantation is controversial. In this study we tested the role of CD40 and CTLA4 ligands on CD34(+) cell costimulation of HLA-mismatched lymphocytes. MATERIALS AND METHODS: An anti-CD40L monoclonal antibody (hu5C8) and/or CTLA4-Ig molecule were used in primary mixed lymphocyte culture (MLC) with irradiated CD34(+) blood cells and allogeneic responders. Then, secondary MLC, cytotoxic activity, and effector cytokine expression and production were measured. RESULTS: Each reagent was able to reduce anti-CD34(+) cell alloreactivity, but only the combination of the anti-CD40L monoclonal antibody and CTLA4-Ig induced greater than 90% inhibition of T-cell response in primary MLC and prevented generation of cytotoxic T cells when priming with purified CD34(+) cells. Importantly, responder cells activated by allogeneic CD34(+) cells in the presence of anti-CD40L monoclonal antibody and CTLA4-Ig entered a state of antigen-specific unresponsiveness while responding to third party antigen, tetanus toxoid, or phytohemagglutinin, and showed suppression of interferon-gamma and increase of interleukin-10 expression and release. Interestingly, addition of interleukin-2 in secondary MLC did not reverse T-cell anergy. CONCLUSIONS: The results demonstrate that human CD34(+) blood progenitors stimulate T-cell responses potently and can induce T-cell unresponsiveness only when both B7:CD28 and CD40:CD40L pathways are blocked, with an increase of interleukin-10-producing cells. Therefore, our data allow design of in vivo studies aimed at achieving T-cell tolerance across HLA barriers by using purified CD34(+) cells and costimulatory blockade.