ALBERT

Description: Key Points Haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents high-risk acute leukemia relapse. The GVL effect is separated from GVHD even across major HLA barriers.

Description: Patients undergoing full haplotype-mismatched hematopoietic transplantations may experience severe intractable invasive fungal infections. To verify whether an imbalanced production of T-helper 1 (TH1) and TH2 cytokines may be responsible for susceptibility to fungal infections, C3H/HeJ (H-2k) recipient mice were lethally irradiated, received transplantations with T-cell–depleted allogeneic bone marrow (BM) cells from mice ofH-2d haplotype, and were infected withCandida albicans. At different time-points after transplantation, mice were assessed for pattern of TH cytokine production and susceptibility to infection. The results show that a long-term, donor-type chimerism was achieved as early as 2 weeks after BM transplantation (BMT), at the time when high-level production of TH2 cytokines (interleukin-4 [IL-4] and IL-10) and impaired production of TH1 cytokines (interferon-γ [IFN-γ] and IL-12] were observed. At this time, mice were highly susceptible to both disseminated and mucosal infections, as indicated by decreased survival, uncontrolled fungal growth, and failure to develop protective TH1 immunity. However, a predominant production of TH1 cytokines was observed by week 5 after BMT, at the time when mice developed donor-type protective TH1 responses and were resistant to infections. Therapeutic ablation of IL-4 or IL-10 greatly increased resistance to candidiasis. These results indicate that a dysregulated production of TH cytokines occurs in mice undergoing T-cell–depleted allogeneic BMT. The transient predominant production of TH2 cytokines over that of IL-12 impaired the ability of mice to develop antifungal TH1 resistance, an activity that could be efficiently restored upon treatment with TH2 cytokine antagonists.

Description: Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)–haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when coinfused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

Description: Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity 〉90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

Description: Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+ cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+ cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+ leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+ subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+ cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+ fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34− cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Description: Nucleoplasmin (NPM) mutations have recently been described as a new kind of mutation in AML. In the presented work we have analyzed 977 AML cases from different prognostic subgroups. Fifteen different mutations (type A:n=245 (78.3%), B:n=21 (6.7%), D: n=28 (11.4%), I:n=3 (1%), J:n=4 (1.3%), K:n=2, G,H, L, M, N, O, P, Q, R: n=1, each (0.3%) were detected in 313 cases. No mutation was found in inv(16) (n=89), t(8;21) (n=98), t(15;17) (n=17), t(11q23)/MLL (n=8), inv(3) (n=8), other reciprocal translocations (n=3), and complex aberrant karyotypes (n=59). In the normal karyotype group the incidence was 299/603 (49.6%) and in all others 13/90 (14.4%). In the latter cohort cases with NPM+ were: − Y (1 case), +4 alone (1 case), +4 and +8 (2 cases), +4 with multiple trisomies (1 case),+8 (1 case), multiple other trisomies (1 case), del(5q) (1 case), − 7 alone (2 cases), del(9q) (2 cases), unbalanced translocation (1 case). Data for evaluation of prognosis were available in 401 AML patients with normal karyotype treated within the AMLCG99 study. Results were calculated in relation to MLL-PTD, FLT3-LM, FLT3-TKD, NRAS, KIT, and CEBPA mutations and correlated to further clinical characteristics. NPM-mutated cases were frequently associated with FLT3 mutations but rarely with other mutations. The NPM-mutated group had a higher CR rate (70.5% vs. 54.7%, p=0.003), a trend to longer OS (median 1012 vs. 549 days, p=0.0762), and significantly longer EFS (median 428 vs. 336 days; p=0.0121). There was a clear favourable impact of NPM+/FLT3-LM- on OS and EFS (264/395 cases; 66.8%). This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM+/FLT3-LM+ double positive pts. was similar to NPM-/FLT3-LM+ cases. We further analysed stability for the NPM mutation at diagnosis and relapse and found stability in 14/15 paired samples. One case with AML M5 lost the NPM mutation at relapse. This case had also a shift from normal karyotype to 46,XX,del(7)(q22),der(7)t(7;21)(p11;q?) and a complete shift of immunophenotype indicating a therapy-related AML. In addition, we have analyzed the applicability of NPM mutations as targets for minimal residual disease (MRD) detection. In total, 82 samples of 22 selected cases were analyzed by use of quantitative real time PCR for the three most common mutation types. Fifteen of these cases had a type A, 3 a type B, and 4 a type D mutation. Samples at diagnosis had an NPM/ABL ratio of 267.6 (range: 54.9 to 3174.7) while each 10 NPM negative cases had a median ratio of 0.03 (assay for A), 0.0001 (assay for B), and 0.002 (assay for D), showing that these assays were nearly specific for the respective mutation. Using limited dilution series of NPM+ in NPM- cDNA a median sensitivity of 1:100.000 could be shown. In both analyzed cases with relapse these were early detectable by increasing NPM levels. Thus, NPM mutations can be used for highly sensitive PCR-based MRD detection in AML, especially if other markers are lacking, i.e. in normal karyotype. In conclusion, our data showa nearly exclusive association of NPM+ to the normal karyotype,a good prognosis of NPM+/FLT3-LM- in normal karyotype.an intermediate prognosis of NPM+/FLT3-LM+ in normal karyotypeNPM as a new marker for PCR-based MRD-detection.

Description: Transplantation of peripheral blood hematopoietic cells from HLA haplotype-mismatched family members is a therapeutic strategy for patients with high-risk acute leukemia who need transplantation and do not have matched donors. As T cell alloreactions cause lethal GvHD in mismatched transplants, only T cell-depleted hematopoietic grafts can be used. In adults, because of declining thymic function, immune-recovery originates from expansion of the mature T cells infused with the graft. In T cell depleted mismatched transplant immune recovery is hindered by the paucity of the starting T-cell population. Slow recovery of functional T cell immunity to pathogens is responsible for 35% infection-related mortality which remains the most pressing clinical issue. In murine MHC-haploidentical bone marrow transplant models we demonstrated donor-versus-recipient alloreactive NK cells ablate recipient-type lympho-hematopietic cells such as leukemic cells, the T cells that cause rejection and the antigen-presenting cells which trigger GvHD. Consequently mismatched T cell-replete transplants can be performed without GvHD (Ruggeri et al., Science 2002). Unexpectedly, in recent experiments, we observed alloreactive NK cells hastened immune-reconstitution. Pre-transplant infusion of alloreactive NK cells promoted brisk recovery of donor B and T cell precursors which matured correctly and of donor DCs. Rapidly reconstistuting DCs were crcuicial in protecting mice from infectious challenges. We next demonstrated:interaction between alloreactive NK cells and NK-susceptible recipient DCs alone was responsible for immune-rebuilding,NK conditioned mice remain receptive to accelerated immune rebuilding even when transplanted a week after NK conditioning, therefore the NK-DC interaction appears to release an as yet unknown immune-rebuilding factor which acts upon bone marrow and thymus microenvironements stably over time,quantitative PCR on bone marrow and thimus of NK conditioned mice shows several-fold increased expression of cytokines implicated in B, T and myeloid cell maturation, such as IL-7 and c-Kit ligand;the accelerated immune rebuilding effect can be reproduced by conditioning mice with the infusion of NK:DC co-culture supernatants. These observation prompted an analysis of infectious mortality in 178 acute leukemia patients who received haploidentical transplant at our Center. Transplantation from KIR ligand-mismatched (i.e., NK alloreactive) donors, in addition to controlling AML relapse, offers statistically significant protection from infectious mortality in AML and ALL patients. Studies are in progress to identify “immune rebuilding factor(s)”, produced in consequence of the interaction between donor alloreactive NK and recipient DCs, in the hope they might be exploited to boost immune-recovery and help reduce infection mortality after haploidentical hematopoietic transplantation.

Description: After hematopoietic transplantation invasive aspergillosis is one of the most lethal infections. Susceptibility to invasive aspergillosis may be due to GvHD and its prophylaxis and treatment in T cell-replete transplants, and to T-cell-depletion in haploidentical transplants. Studies in mice and humans show that adaptive T-helper type-1 immune effector mechanisms are involved in control of invasive aspergillosis (Hebart et al. Blood, 2002; Cenci et al. J Immunol, 2000). In vivo T-cell priming induced by DCs pulsed with A. fumigatus conidia protects bone marrow transplanted mice from invasive aspergillosis (Bozza et al. Blood, 2000). This study monitored recovery of anti-Aspergillus immune competence in recipients of T cell-replete matched transplants and of T cell-depleted matched or haploidentical transplants for acute leukaemia. Patients: 32 pediatric recipients of matched T-replete transplants from unrelated donors (n=21), unrelated cord blood (n=2), and matched siblings (n=9) (median age: 10.5 years; range: 0.5–24); 20 adult recipients of matched T cell-depleted transplants (median age: 43 years; range 18–65), and 46 adult recipients of haploidentical transplants (median age: 34 years; range: 9–64). In all we monitored recovery of CD4+ T-cells and Aspergillus-specific CD4+ T-cells (by LDA) monthly for 18 months after transplant. Total CD4+ T-cell counts were higher after T-replete matched than after T-depleted matched or haploidentical transplant. At 9 months, CD4+ cells were: 1332±337 in T-cell replete transplant recipients, 364±62 in T cell-depleted matched transplant recipients, and 218±186 in haploidentical transplant recipients (p=0.000). Incidence of acute GvHD 〉 grade II was 60% after T-replete transplantation, 0% after T cell-depleted matched and 9% after haploidentical transplantation. Aspergillus-specific T cells were first detected 15–18 months after T-replete matched transplantation (when immune suppressive GvHD prophylaxis/therapy was being withdrawn); 7–9 months after T cell-depleted matched transplantation and 9–12 months after haploidentical transplantation (p=0.000). Incidence of invasive aspergillosis was 21%, with a 10% mortality after T-replete transplants, 0% after T cell-depleted matched (p=0.000) and 7% with 4% mortality after haploidentical transplants (p=0.000). Although T cell counts were significantly higher after T-replete transplants their function appeared to be impaired by post-transplant immune suppression/GvHD. T-replete transplants were associated with a higher incidence of invasive aspergillosis and aspergillosis-related deaths. Specific Aspergillus immune competence recovered faster after T cell-depleted transplants, whether matched or haploidentical. These results show that T-cell depletion and no post-transplant immune suppression may provide a better pattern of immune recovery than T cell-replete transplantation and challenge the widely held belief that immune recovery after T cell-depleted transplants, particularly the haploidentical, is unduly delayed.

Description: The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P ≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

Description: Results of family haploidentical allogeneic stem cell transplantation (Haplo-SCT) have been reported from single-center studies as a curative treatment option for patients with high risk acute leukemias. We analysed 273 adult patients with de novo acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) receiving an Haplo-SCT transplanted from 1995 to 2002 in 75 EBMT centers. Overall, 170 AML patients underwent transplantation in CR1 (n=39), CR2 (n=34) or in advanced disease (n=97); median age was 38y (16–70). Overall, 103 ALL patients underwent transplantation in CR1 (n=31), CR2 (n=22) or in advanced disease (n=50); median age was 26y (16–56). Graft composition was based on CD34+ cell selection of PBSC, with median CD34+ cells x106/kg of 7.3 (1.1–45.5) in AML and 8.3 (1.14–30) in ALL. Conditioning regimen was TBI-based in 69% and 87% in AML and ALL respectively. Primary engraftment was documented in 87% AML and in 83% of ALL patients, with ANC 0.5 x109/L in a median of 13 days (8–35) and a 2y probability of PLT 〉 50 x109/L of 80+/− 5%. The use of TBI was significantly associated with engraftment (90% vs 71%) when adjusting for CD34 dose. The cumulative incidence of acute GvHD 〉=II was 16% in AML and 15% in ALL. With a median follow-up of 19 months (1–85), the estimated leukemia-free survival (LFS) at 2 years in AML was 39±9%, 33 ±10% and 4±3% and in ALL was 28±9%, 16±8, and 0% for CR1, CR2 and advanced patients respectively. Cumulative incidence using competing risks, for relapse (RI) at 2 y in AML was 8 ±8%, 9 ±9%, 25±8% for CR1, CR2 and advanced; RI in ALL was 27±16% and 41 ±20% in CR1 and CR2. The non-relapse mortality (NRM) at 2 y in AML was 52±18%, 56 ±19%, 69±8% for CR1, CR2 and advanced patients; NRM in ALL was 44±18% and 41±21% in CR1 and CR2. In a multivariate analysis for LFS, advanced disease status at transplant (RR 0.4), ALL (RR 0.5), recipient’s age 〉33 years (RR 0.4), and absence of TBI in conditioning (RR 0.6) were factors associated with lower LFS. Factors associated with higher transplant-mortality were advanced disease status (RR 2.01), absence of TBI (RR 1.6) and year of transplant