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: In patients with acute leukemia (AL) at high risk of relapse, because of unfavorable cytogenetics, molecular markers and disease status, the most powerful therapy is hematopoietic stem cell transplantation. In HLA-haploidentical stem cell transplantation we showed adoptive immunotherapy with naturally occurring T regulatory cells (nTregs) followed by conventional T cells (Tcons) prevented graft-versus-host-disease (GvHD) (Di Ianni et al., Blood 2011) and was associated with low transplant-related mortality (TRM) and relapse rate (Martelli et al., Blood 2014) in patients with AL conditioned with single-dose TBI. However, patients that are heavily pretreated (e.g., because of multiple induction chemotherapy cycles, previous autologous or allogeneic transplant, previous irradiation, infections, etc.) and elderly patients are not expected to withstand a high-intensity TBI-based conditioning regimen. For these patients with an indication to transplant we, therefore, designed a chemotherapy only-based conditioning regimen. We enrolled 24 patients with high risk AL (19 AML and 5 ALL, median age: 46, range 21-64). Twenty-two patients were transplanted in CR and 2 in relapse. For 5 patients this was the second transplant. Patients received a conditioning regimen that included thiotepa (10 mg/Kg on days -10 and -9), fludarabine (200 mg/sqm from day -10 to -7) and melphalan (80-120 mg/sqm on day -5). Fiftheen patients also received ATG (6 mg/kg on days -24 and -23). Seven patients also received cyclophosphamide (20 mg/Kg on days -8 and -7) (and no ATG). Two patients received only Treosulfan (36 gr/sqm from day -8 to -6), fludarabine and thiotepa. On day -4 patients were received an infusion of 2x10e6/kg nTregs (93% ± 8 SD FoxP3+ cells). On day 0 they received CD34+ cells (mean 9.5x10e6/kg ±3.1 SD) and Tcons (1x10e6/kg). No post-transplant pharmacologic immune suppressive GvHD prophylaxis was given. Twenty-two of the 24 patients engrafted. CD4+ and CD8+ cell counts reached 200/µL on days 56 (range 30-100) and 35 (range 20-120) respectively with a wide T-cell repertoire as analyzed by Spectratyping. Eight of the 22 engrafted patients (36%) developed ≥ grade II acute GvHD. Three of the 8 patients died, 3 are alive with cGvHD and 2 are currently under immunosuppressive therapy. The incidence of TRM was 32% (7/22). Only one patient (in relapse at the time of transplant) relapsed. At a median follow-up of 24 months (range 5-44), 62% of the patients are alive and leukemia-free. The mechanisms underlying Treg suppression of GVHD with no loss of GVL activity were clarified by our murine transplant models. NSG mice received human myeloid or lymphoblastic leukemia and HLA mismatched Tregs/Tcons. Mice that received leukemia and Tcons (without Tregs) cleared leukemia but died of GvHD. Human T cells harvested from their bone marrow, liver and gut displayed a CD8+ phenotype and mediated alloreactivity against human leukemia. Mice treated with Tregs alone died of leukemia. No human T cells were found in their bone morrow. Human CD4+ T cells were instead recovered in the liver and gut. They had retained their regulatory function as they inhibited mixed lymphocyte reaction. Mice that received human leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. Human T cells harvested from liver and gut were composed of CD4+/FOXP3- T cells (60%) and CD8+ T cells (40%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against human leukemia. In contrast, human T cells harvested from bone marrow were still predominantly CD8+ and still displayed potent alloreactivity against human leukemia. In conclusion, peripheral blood Tregs used for adoptive immunotherapy were predominantly CD45RO+ (and negative for the bone marrow homing receptor CXCR4) and, when infused in mice, migrated to GvHD target organs (i.e., liver and gut) where they blocked Tcons and prevented GvHD. However, they were unable to migrate to the bone marrow, thereby allowing Tcons to exert unopposed alloantigen recognition and leukemia killing. These results demonstrate haploidentical transplantation with a chemotherapy-based conditioning regimen and Treg/Tcon immunotherapy is not only feasible in fragile patients but also exerts a powerful anti-leukemic effect. Apparently, the Treg/Tcon immunotherapy plays a key role in the GvL effect. Disclosures Pierini: Stanford University: Patents & Royalties.

Description: Introduction Trans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect transplantation outcomes when the mother acts as donor for the child. Indeed, survival after T cell-depleted HLA haploidentical haematopoietic transplantation was improved using the mother as donor (vs all other family members) (Stern et al., Blood 112:2990, 2008; Kruchen et al., BMT 50:1367 2015). However, maternal donors were associated with increased incidence of GvHD and decreased survival after un-manipulated HLA haploidentical blood and marrow grafts (Wang Y et al., Blood 124:843, 2014). Patients and Methods A retrospective EBMT registry-based study was performed in a combined series of adult (n=333) and pediatric (n=105) patients with acute leukemia (AML=268, ALL=160, Mixed phenotype=10) who received transplant from one MHC mismatched family donor. Forty-four percent (233) of patients were in I or II complete remission (CR) at the time of transplant, 193 were in chemo-resistant relapse or in CR 〉 2. Median age was 32.7 (range: 0.67-70). Seventy-one percent of patients received ex-vivo T cell depleted transplants. Twenty-two patients were given bone marrow and peripheral blood stem cells, the others were given peripheral blood cells only. The mother was used as donor in 98 patients. Results Eighty patients developed acute GvHD ≥ grade II. At a median follow up of 58.7 months, 130 patients are alive without disease, 146 relapsed and 152 died of non-relapse mortality (NRM). Univariate analyses of factors influencing relapse-free survival (RFS) showed age, disease status at transplant, Karnofsky score and ex-vivo T cell depletion impacted significantly. As transplantation outcomes from family members other than mothers did not differ from one another, such transplants were combined for analyses. When compared with transplantation from all other family members (n=330), transplantation from mother donors (n=98) was associated with lower relapse incidence (RI) (28% vs 39%, P=0.06) and non-relapse mortality (NRM) (30% vs 40%, P=0.09) and, consequently, better relapse-free survival (RFS) (43% vs 21%, P 〈 0.001). Multivariate analyses showed transplantation from mother donors was an independent factor predicting better RFS (other donors vs mothers: HR: 1.42; CI: 1.01-2.00; P=0.04) and lower RI (other donors vs mothers: HR: 1.93; CI: 1.16-3.22; P = 0.01). In addition, transplantation in relapse (vs remission) predicted worse RFS (HR: 2.33, CI: 1.80-3.00, P 〈 0.001) such as ≥18 (vs 〈 18) adversely impacted on RFS (HR: 1.40, CI: 1.00-1.96, P=0.04). Discussion Our retrospective analyses in 438 HLA haploidentical hematopoietic transplants for acute leukemia patients (pediatric and adult) show that transplantation from mother donors, when compared with transplantation from any other family member, is an independent factor predicting better outcomes, i.e., better RFS and lower RI. Mothers should therefore be preferred when selecting an HLA haploidentical family donor. Further clinical and preclinical studies are needed to unveil the mysteries underlying mother-to-child immune interaction during pregnancy and its bearing on the reproductive success of the human species. Disclosures Ciceri: MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Description: Introduction and Aim: HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is increasingly offered to patients with high-risk acute myeloid (AML) or lymphoid leukemia (ALL). Unfortunately, graft manipulation employed to overcome the HLA barrier significantly delays immune reconstitution, posing the patients at risk of infections. Accordingly, non-relapse mortality after haplo-HSCT clearly extends beyond day 100 post-transplant. Over the years, different approaches have been investigated to speed-up immune reconstitution. In the absence of validated immune biomarkers, it is however difficult to evaluate the clinical impact of accelerated immune reconstitution. The aim of this EBMT retrospective study is to explore immune-cell counts early after haplo-HSCT as predictive of its overall outcome. Methods and Patients: Among AML and ALL patients in the EBMT database who underwent haplo-HSCT in the period 2001-2012, criteria for study entry were survival beyond day 100 and availability of differential immune-cell counts (CD3+, CD4+, CD8+ T cells, CD19+ B cells, CD16+/CD56+ NK cells) within this period. Accordingly, statistical analysis was landmarked at day 100. Of 259 patients meeting these criteria (age 2-70, median 33), 67 (26%) were children. The underlying disease was AML in 162 cases (63%), while ALL in the remaining (including 5 cases of bi-phenotypic leukemia). Fifty-two percent of patients were transplanted in CR1. The stem-cell source was G-CSF mobilized peripheral blood in all but one patient (〉99%) and 171 received TBI (66%). The graft was manipulated in 199 patients (78%), including CD34-selection (50%), ex vivo T-cell depletion (15%) or both (13%). Female-to-male transplants were 68 (26%), while 204 (79%) recipients were CMV seropositive. Sustained hematopoietic engraftment was reached in 246 patients (95%) Results: The estimated overall survival at 2yrs was43%. The estimated cumulative incidence of death due to relapse was 33%, while that of death due to other causes was 35% (51% of those were infections) The occurrence of grade III-IV GVHD and of chronic GVHD was 9% and 18% (7% extensive), respectively. As expected, overall survival was better in children (62% vs 36%, P=0.002 by Log-rank), who clearly had a lower incidence of death due to causes other than relapse compared with adults (10% vs 37%, P=0.0001). Negative prognostic factors for overall survival were any disease state other than CR1 at time of transplantation (P=0.002) and CMV seropositivity (P=0.009). Type of leukemia, TBI or graft manipulation had no effect on the outcome. By day 100 post-transplant, patients reached the following median immune-cell counts: 100 CD3+ T cells (range 0-2576), 30 CD4+ T cells (0-1714), 48 CD8+ T cells (0-1880), 276 CD16+/CD56+ NK cells (18-3581), 21 CD19+ B cells (0-790). Importantly, CD3+ counts above the first quartile (1Q) of the entire data set (29 cells per microL) were significantly associated with a better overall survival (P=0.0005 by Log rank) and a lower incidence of death due to causes other than relapse (P=0.002 by Gray test). The same held true for CD8+ counts (1Q: 15 cells per microL; P=0.003 on overall survival; P=0.0004 on death due to other causes). CD4+ counts also showed similar correlations, but at higher values (above the median). None of the other immune-cell counts analyzed correlated with clinical outcome. Strikingly, when challenged in multivariate analysis taking into account age category, CMV seropositivity, graft manipulation and CR1 status at transplant, CD3+ and CD8+ counts above the 1Q adjusted to fit optimal cut-off points were still significantly associated with a better overall survival (P=0.006 and P=0.015, respectively), but only CD8+ values associated with a lesser risk of death due to causes other than relapse (P=0.026). Conversely, similarly adjusted median CD4+ counts failed to show any association. Conclusions: Contrary to what is generally accepted, these results indicate that an accelerated CD8+, but not CD4+, T cell reconstitution associates with a more favorable clinical outcome after haplo-HSCT, likely due to its protective role against opportunistic viral infections. Moreover, they suggest that yet to be validated CD8+ cut-off points, rather than the commonly used arbitrary value of 200 CD4+ T cells per microL, should be considered as surrogate biomarkers in clinical trials. Disclosures Bonini: MolMed S.p.A: Consultancy.

Description: Posttransplant relapse is still a major cause of treatment failure in high-risk acute leukemia. Evidence from experimental bone marrow transplantation (BMT) showed coinfusion of conventional T lymphocytes (Tcons) with regulatory T lymphocytes (Tregs) suppressed lethal Graft-versus-Host Disease (GvHD) without impairing the Graft-versus-Leukemia (GvL) effect (Edinger et al., Nat Med. 2003;9:1144-1150). In HLA haploidentical transplantation for acute leukemia we demonstrated that donor-derived natural Tregs, coinfused with Tcons, protected recipients against GvHD (Di Ianni et al., Blood 2011, 117:3921-3928) and largely prevented posttransplant leukemia relapse, as only 5% of evaluable patients relapsed (Martelli et al., Blood 2014, 124:638-44). The mechanism by which the GvL effect is maintained in the absence of GvHD is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 and preferentially localize to the bone marrow while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010;184:4317-4326). Since peripheral blood Tregs to be used for adoptive immunotherapy are CD45RO+, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the bone marrow combined with regulated T cell alloreactivity at the periphery. Immunodeficient NSG mice (a total of 40 for each experimental group) received human myeloid or lymphoblastic leukemia (3x106) and HLA mismatched Tregs/Tcons (3x106). Mice that received leukemia and Tcons (without Tregs) cleared leukemia but died of GvHD. Human T cells harvested from their BM, spleen and liver were predominantly (90%) CD8+ and displayed potent alloreactivity (by 51-chromium release at an E:T ratio of 5:1) against human leukemia (lysis = 50% ± 15), autologous to leukemia PHA blasts (lysis = 60% ± 10) and mouse Con A blasts (lysis = 40% ± 8). Mice that received human leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. Human T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+/FOXP3- T cells (60%). Despite their FOXP3 negativity, purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction (by 50% at a Tcon/Treg ratio of 1:2). Purified CD8+ T cells displayed no alloreactivity against human leukemia, PHA blasts and mouse Con A blasts. In contrast, human T cells harvested from BM were still predominantly CD8+ and still displayed potent alloreactivity against human leukemia and autologous to leukemia PHA blasts and mouse ConA blasts, suggesting Tcons had retained their alloantigen recognition against human and mouse MHC. Finally, in mice treated with Tregs alone, human T cells were recovered only in the spleen and liver, they displayed a CD4+/FOXP3- phenotype and inhibited mixed lymphocyte reaction. No human T cells were found in the bone morrow, thus showing human CD45RO+ Tregs do not home to the bone marrow. In conclusion, Treg/Tcon adoptive immunotherapy mediates GvL effect in the absence of GvHD because Tcons that home to the bone marrow exert unopposed alloantigen recognition, while Tcons that home to the periphery are blocked by Tregs. Disclosures No relevant conflicts of interest to declare.

Description: In HLA-haploidentical stem cell transplantation we showed adoptive immunotherapy with naturally occurring T regulatory cells (nTregs) followed by conventional T cells (Tcons) prevented acute and chronic graft-versus-host-disease (GvHD), favoured lymphoid reconstitution and immunity against pathogens (Di Ianni et al., Blood 2011). One major concern is whether a graft-versus-leukemia (GvL) effect is maintained since FoxP3+Tregs can also suppress immune response against tumour. However, we showed in immunodeficient NSG mice engrafted with human myeloid and lymphoblastic acute leukemia cells (7x106 iv), Treg (3x106)+Tcon(3x106) infusion rescued mice from leukemia death in the absence of GvHD. In contrast, controls died of leukemia (when not treated or infused with Tregs only) or of GvHD (when infused with Tcons only).Since September 2008, 45 consecutive patients (21 male; 24 female; median age 40, range 20-59) with high-risk acute leukemia ( 33 AML, 12 ALL) were transplanted. The conditioning regimen included TBI, thiotepa, fludarabine and cyclophosphamide (25 patients in the 1st clinical trial) or alemtuzumab/ATG (20 patients in the 2nd clinical trial). On day -4, patients received an infusion of freshly isolated donor Tregs (mean 2.5/kg ±1; Foxp3 81.01%±16.47; Helios/FoxP3 54%±8.4; CD127 11.72%±7.653) followed on day 0 by a megadose of purified CD34+ cells (9.7x106/kg ± 3.1) and Tcons (mean 1.1 /kg ±0.6; CD3 90.72%± 9.6; CD4 57.77% ± 8.85; CD8 31.21% ± 8.59). No post-transplant GvHD prophylaxis was given. 43 of the 45 patients achieved primary, sustained full-donor type engraftment. Only 6/43 evaluable patients developed ≥ grade II acute GvHD (3 alive, treated and now off-therapy). At a median follow-up of 28 months (range 4-58) no patient has developed chronic GvHD. CD4 and CD8 counts reached 100/µL medianly on days 40 (range 25-150) and 45 (range 18-100) and 200/µL on days 55 (range 45-160) and 60 (range 50-140). In KIR-ligand mismatched transplants (Ruggeri et al., Science 2002; Ruggeri et al., Blood 2007), donor versus recipient alloreactive NK cells were also preserved. At a median follow-up of 46 months (range 7-58) 23/43 patients are alive and disease free with a probability of DFS of 0.56. The cumulative incidence of TRM is 0.37. The cumulative incidence of relapse was 0.06 as only 2 AML patients transplanted from non-NK alloreactive donors have relapsed to date. This incidence is extremely low considering the high-risk patient population (9 with FLT-3/ITD; 8 with complex karyotypes; 4 with t(9:22); 2 PIF; 1 secondary AML; 1CR after second-line induction; 1 with CNS and skin localization at diagnosis; 19 ≥ 2CR). In conclusion, adoptive immunotherapy with Tregs and Tcons exerts a powerful GVT activity in both AML and ALL patients in the absence of GvHD. Disclosures: No relevant conflicts of interest to declare.

Description: Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.