ALBERT

Description: Key Points Wnt pathway is frequently mutated in CLL. Wnt pathway mutations can lead to pathway activation and enhanced CLL survival.

Description: Abstract 1892 Patients with advanced hematological malignancies remain at high risk for eventual disease progression following reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We hypothesized that vaccination with whole leukemia cells during the critical period of immune reconstitution early after transplant may enhance antitumor immunity and facilitate expansion of leukemia-reactive T cell responses. We tested this hypothesis in a prospective clinical trial, in which patients with advanced chronic lymphocytic leukemia (CLL) received up to 6 vaccine doses initiated between day 30–45 following RIC allo-HSCT. Each vaccine consisted of 1×107 irradiated autologous tumor cells admixed with 1×107 irradiated K562 bystander cells secreting GM-CSF (GM-K562). All patients received tacrolimus and mini-methotrexate as graft-versus-disease (GvHD) prophylaxis. Tacrolimus was maintained at therapeutic levels during the vaccination period without taper. Twenty-two patients were enrolled, all with advanced disease (median number of prior therapies 3; range 2–11). Many of the leukemias expressed markers associated with aggressive disease (e.g. unmutated IgVH - 68%) and displayed high-risk cytogenetic abnormalities (sole del(11q) - 41%; sole del(17p) - 23%; del(11q and 17p) - 18%). Greater than 50% (n=13) of patients had persistent marrow involvement (≥10%) at time of allo-HSCT. Eighteen of 22 subjects were vaccinated after allo-HSCT and received a median of 6 (range 1–6) vaccines. The remaining 4 patients were precluded from vaccination due to development of acute GvHD before day 45. Vaccines were generally well tolerated, but mild, transient injection site erythema was common. Only one grade 4 event (neutropenia) with a possible attribution to treatment occurred. We observed a similar incidence of grade II-IV aGvHD at 1 year in the 18 vaccinated patients (39%; 95% CI: 17–61%) and 42 control CLL patients that underwent RIC allo-HSCT at our institution from 2004–2009 (31%; 95%CI: 18–46%). At a median follow-up of 2.9 (range 1–4) years, the estimated 2-year rates of progression-free survival and overall survival of vaccinated study participants were 80% (95% CI: 54–92%) and 84% (95% CI: 58–95%). With these promising clinical results, we next focused on gaining insight into the mechanism that generated the observed clinical graft-versus-leukemia (GvL) responses. To delineate the specific contribution of vaccination to the overall GvL effect, we performed T cell assays to detect CLL-specific reactivity in serial pre- and post-HSCT samples obtained from vaccinated patients (n=9) who received median of 6 vaccines (range 3–6). In comparison, we examined T cell responses in study subjects (n=4) that developed aGvHD at a median of 44.5 days (range 26–56) after HSCT; and control CLL patients (n=4; no vaccine, no GvHD in the early post-transplant period) that were not enrolled in the study. Although early post-transplant vaccination had no impact on recovering absolute T cell numbers, reactivity of CD8+ T cells from the vaccinated patients was consistently directed against autologous tumor cells but not alloantigen bearing-recipient cells (PHA T cell blasts and fibroblasts) in IFNγ ELISpot assays. A peak response against autologous tumor cells was reached at day 60 after allo-HSCT (average 221 SFC/5×105 cells vs. 29 and 33 average SFC/5×105cells for PHA blasts and fibroblasts, respectively). CD8+ T cell clones were isolated from 4 vaccinated study subjects by limiting dilution and 17% (range 13–33%) reacted solely against CLL-associated antigens. In contrast, broad CD8+ T cell reactivity indicating an alloantigen response was observed in GvHD patients, while no increase in T cell reactivity against tumor-associated or alloantigens was seen in control patients. Tumor-reactive CD8+ T cells isolated from vaccinated patients secreted a broad profile of effector cytokines (GM-CSF, TNFα and IP10). Moreover, the amount of cytokines secreted by these CLL-specific CD8+ T cells steadily increased following early post-transplant vaccination, but not after allo-HSCT alone or in relation to GvHD. Our studies reveal that vaccination with autologous whole CLL/GM-K562 cells between days 30–100 after allo-HSCT is associated with induction of immunity against recipient CLL cells, and suggest that this is an effective strategy for promoting GvL following RIC allo-HSCT. Disclosures: Brown: Genzyme, Celgene: Research Funding; Calistoga, Celgene, Genentech, Pharmacyclics, Novartis, Avila: Consultancy. Cutler:Pfizer, inc: Research Funding; Astellas, Inc: Consultancy, Research Funding.

Description: Key Points Marrow CD8+ T-cell infiltrates may be a novel predictor of response to donor lymphocyte infusions in patients with relapsed CML. Reversal of T-cell exhaustion is tightly linked to effective antileukemia responses to donor lymphocyte infusions.

Description: Abstract 1903 Donor lymphocyte infusion (DLI) can provide curative treatment for relapsed hematologic malignancies following allogeneic hematopoietic stem cell transplant (HSCT). However, the precise mechanism by which DLI eliminates leukemia cells in vivo has not been established. We hypothesized that marrow-infiltrating immune populations play a critical role in DLI responses since marrow is the primary site of disease and a reservoir of high-avidity antigen-specific memory T cells that can recognize tumor antigens, therefore potentially mediating graft-versus-leukemia (GvL) responses. We performed immunohistochemical staining of immune cells in serial marrow biopsies collected before and after DLI from 29 patients with relapsed CML. Twenty-two patients achieved cytogenetic remission within twelve months (‘responders’) while 7 patients demonstrated persistent disease (‘non-responders’). While no significant changes in the numbers of circulating T cells were seen between patient groups following DLI, the median number of marrow-infiltrating CD8+ T cells increased 2-fold in responders but remained unchanged in non-responders (P=0.02), demonstrating that clinical response to DLI is associated with T cell responses at the site of disease that may not be apparent in the peripheral blood. To investigate whether immune cell infiltration of the marrow could predict DLI response, we compared pre-treatment samples from both patient groups. Responders exhibited significantly higher proportions of CD8+ T cells in pre-DLI marrow compared to non-responders (5% vs 2.5%; P=0.01). Because disease burden is a known risk factor for ineffectual DLI response, we evaluated the interaction between disease burden and pre-existing CD8+ T cell infiltrate through the clinical course of 8 patients with high (≥70%) pre-treatment marrow cellularity. Three of 8 had ≥5% CD8+ T cell marrow infiltrates, and all 3 subsequently achieved cytogenetic remission. In contrast, 5 of 8 patients had

Description: Abstract 2417 Marrow is a major site of disease development and progression for chronic lymphocytic leukemia (CLL), as well as a priming site for antigen-specific T cells and a reservoir for memory T cells. To determine the extent to which T cells in the marrow microenvironment have an altered phenotype and function in CLL, we analyzed the immunophenotypic characteristics of marrow-infiltrating T cells of 18 CLL patients compared to 11 normal donors. Chemotherapy-naïve CLL patients (n=7) possessed comparable quantities of marrow T cells compared to normal donors (median CD8+ T cells/μl = CLL 904 vs normal 1247; median CD4+ T cells/μl = CLL 1975 vs normal 1110). However, we identified several aberrant characteristics among T cells infiltrating the marrow of CLL patients. First, the ratio of CD8+ to regulatory T cells (CD4+CD25+FOXP3+) was depressed (median ratio CLL 14 vs normal 41), indicating more regulatory T cells per effector T cells in CLL. Second, compared to normal marrow T cells, CLL marrow contained proportionally fewer functional effector CD8+ T cells (CD27+CD28+)(median normal 57%, CLL 48%) and more immunosenescent cells (CD27-CD28-)(median normal 21%, CLL 30%). Third, the T cell differentiation state of CLL CD8+ T cells was skewed to favor a phenotype of increased terminal differentiation (CD45RA+CCR7-)(median CLL 55% vs normal 40%), and decreased naïve (CD45RA+CCR7+) cells (median CLL 21% vs normal 31%) compared to normal donors. These differences were further accentuated in CLL samples collected within 4 months from treatment with conventional chemotherapy (n=11). Finally, by immunohistochemical staining of CLL marrow biopsies, we observed marrow-infiltrating lymphocytes to express PD-1 (mean of infiltrating T cells, untreated CLL 12%, treated CLL 35%, present even 〉6 months after therapy), a marker associated both with immuno-activation and inhibition. While the majority of PD-1+ CD8 T cells of normal donors (n=5) and treated CLL patients (n=4) were differentiated towards effector memory (CD45RA-CCR7-) cells (median normal 46% vs untreated CLL 16%, p=0.07; treated CLL 61%), the PD-1+ T cells from untreated CLL patients (n=5) were terminally differentiated (CD45RA+CCR7+)(median normal 23% vs untreated CLL 65%, p=0.04; treated CLL 24%). These results indicate an exhausted rather than an activated T cell phenotype in untreated patients. Paired immunophenotypic analysis on blood and marrow from the same individuals (n=9) demonstrated an increased percentage and intensity of PD-1 expression on T cells from marrow compared to blood (percentage CD8+ T cells BM vs blood p = 0.05). Interestingly, PD-1 was also detected on CLL cells (n=16) but not normal B cells (median normal 0%, vs CLL 17%, p = 0.004). The ligand for PD-1, PD-L1, was detected in the marrow vasculature by immunohistochemical staining of biopsies, suggesting that the marrow microenvironment plays a role in the induction of PD-1 associated immunosuppression. Ligation of blood PD-L1 on CLL-T cells led to a 2-fold decrease in activation (measured as CD69 expression) of CD3/CD28 stimulated patient T cells. In summary, we identify several phenotypic and functional alterations within marrow-infiltrating T cells of CLL patients. We speculate these together may contribute to impaired priming of host immunity against the tumor. The PD-1 pathway appears to be activated in CLL, especially in the setting of chemotherapeutic treatment. Since anti-PD1 antibodies are now clinically available, it may be possible to target this pathway to improve anti-tumor responses. Disclosures: No relevant conflicts of interest to declare.