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Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Immunotherapy targeting inhibitory pathways to unleash the antileukemia T-cell response is a promising strategy for the treatment of leukemia, but we must first understand the underlying molecular mechanisms. Eomesodermin (Eomes) and T-bet are both T-box transcription factors that regulate CD8+ T-cell responses in a context-specific manner. Here, we examined the role of these transcription factors in CD8+ T-cell immunity in AML patients. We report that the frequency of Eomes+T-betlow CD8+ T cells increased in newly diagnosed AML. This cell subset produced fewer cytokines and displayed reduced killing capacity, whereas depletion of Eomes by siRNA reversed these functional defects. Furthermore, Eomes bound the promoter of T-cell immunoglobulin and ITIM domain (TIGIT) and positively regulated the expression of this inhibitory receptor on patient-derived T cells. A high frequency of Eomes+T-betlow CD8+ T cells was associated with poor response to induction chemotherapy and shorter overall survival in AML patients. These findings have significant clinical implications as they not only identify a predictive and prognostic biomarker for AML, but they also provide an important target for effective leukemia therapeutics.Significance:These findings reveal that a high frequency of Eomes+T-betlow CD8+ T cells predicts poor clinical outcome in AML and that targeting Eomes may provide a therapeutic benefit against AML.

Supplemental Table 1: Analysis of potential confounding factors; Supplemental Table 2: Multivariate Cox proportional hazard analysis; Supplemental Figure 1: The expression of CXCR5 and TCF1; Supplemental Figure 2: Comparing Fraction III CD8 T cells vs. overall survival; Supplemental Figure 3: The expression of CD39.

TIGIT expression associates with up-regulation of multiple inhibitory receptors.

Legends for supplemental figures

TIGIT+ CD8+ T cells from AML patients retain capacity of proliferation and cytotoxicity.

Purpose: T-cell immunoglobulin and immunoreceptor tyrosine–based inhibitory motif (ITIM) domain (TIGIT) is a recently identified T-cell coinhibitory receptor. In this study, we aimed to determine the clinical impact of TIGIT in patients with acute myelogenous leukemia (AML) and dissect the role of TIGIT in the pathogenesis of leukemia progression.Experimental Design: TIGIT expression on T cells from peripheral blood collected from patients with AML was examined by flow cytometry. The correlation of TIGIT expression to clinical outcomes, including rate of complete remission and relapse post-allogeneic stem cell transplantation (alloSCT) in AML patients, was analyzed. Phenotypic and functional study (cytokine release, proliferation, killing, and apoptosis) of TIGIT-expressing T cells were performed. Using siRNA to silence TIGIT, we further elucidated the regulatory role of TIGIT in the T-cell immune response by dissecting the effect of TIGIT knockdown on cytokine release and apoptosis of T cells from AML patients.Results: TIGIT expression on CD8+ T cells is elevated in AML patients and high-TIGIT correlates with primary refractory disease and leukemia relapse post-alloSCT. TIGIT+ CD8+ T cells display phenotypic features of exhaustion and exhibit functional impairment manifested by low production of cytokines and high susceptibility to apoptosis. Importantly, their functional defects are reversed by TIGIT knockdown.Conclusions: TIGIT contributes to functional T-cell impairment and associates with poor clinical outcome in AML. Our study suggests that blockade of TIGIT to restore T-cell function and antitumor immunity may represent a novel effective leukemia therapeutic. Clin Cancer Res; 22(12); 3057–66. ©2016 AACR.

High-dose cytarabine (HiDAC) consolidation for acute myeloid leukemia (AML) induces transient profound myelosuppression and potential morbidity/mortality. PrE0901 was a phase I multi-center trial evaluating the safety/toxicity of eltrombopag in AML patients receiving HiDAC consolidation. We used a standard 3 + 3 design employing a unique dose-escalation/de-escalation strategy. One hundred four patients were screened, 54 declined participation, 35 were deemed medically ineligible, and 14 were treated on study. Three patients were treated in cohorts 1-4 and two were treated in cohort 5. Eltrombopag + HiDAC was well-tolerated and no dose-limiting toxicities were observed. Median time to platelet recovery of all patients treated was 22.5 (range 16-43) days. Observationally, eltrombopag 150 mg once daily starting on day 3 of consolidation demonstrated the fastest and most consistent platelet recovery (median 19 days). Further investigation is needed to define the optimal role, dose, and schedule of eltrombopag in the treatment of chemotherapy associated myelosuppression.

Summary Decitabine is a DNA‐hypomethylating agent that has been widely applied for the treatment of acute myeloid leukaemia (AML) patients who are elderly or unfit for intensive therapy. Although effective, the complete response rate to decitabine is only around 30% and the overall survival remains poor. Emerging data support that regulation of DNA methylation is critical to control immune cell development, differentiation and activation. We hypothesize that defining how decitabine influences the immune responses in AML will facilitate the development of novel immune‐based leukaemia therapeutics. Here, we performed phenotypic and functional immune analysis on clinical samples from AML patients receiving decitabine treatment and demonstrated a significant impact of decitabine on the immune system. T‐cell expression of inhibitory molecules was upregulated and the ability of CD8 T cells to produce cytokines was decreased upon decitabine treatment. Importantly, in an unbiased comprehensive analysis, we identified a unique immune signature containing a cluster of key immune markers that clearly separate patients who achieved complete remission after decitabine from those who failed to do so. Therefore, this immune signature has a strong predictive value for clinical response. Collectively, our study suggests that immune‐based analyses may predict clinical response to decitabine and provide a therapeutic strategy to improve the treatment of AML.

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Immunotherapy targeting inhibitory pathways to unleash the antileukemia T-cell response is a promising strategy for the treatment of leukemia, but we must first understand the underlying molecular mechanisms. Eomesodermin (Eomes) and T-bet are both T-box transcription factors that regulate CD8+ T-cell responses in a context-specific manner. Here, we examined the role of these transcription factors in CD8+ T-cell immunity in AML patients. We report that the frequency of Eomes+T-betlow CD8+ T cells increased in newly diagnosed AML. This cell subset produced fewer cytokines and displayed reduced killing capacity, whereas depletion of Eomes by siRNA reversed these functional defects. Furthermore, Eomes bound the promoter of T-cell immunoglobulin and ITIM domain (TIGIT) and positively regulated the expression of this inhibitory receptor on patient-derived T cells. A high frequency of Eomes+T-betlow CD8+ T cells was associated with poor response to induction chemotherapy and shorter overall survival in AML patients. These findings have significant clinical implications as they not only identify a predictive and prognostic biomarker for AML, but they also provide an important target for effective leukemia therapeutics. Significance: These findings reveal that a high frequency of Eomes+T-betlow CD8+ T cells predicts poor clinical outcome in AML and that targeting Eomes may provide a therapeutic benefit against AML.

We analyzed late fatal infections (LFIs) in allogeneic stem cell transplantation (HCT) recipients reported to the Center for International Blood and Marrow Transplant Research. We analyzed the incidence, infection types, and risk factors contributing to LFI in 10,336 adult and 5088 pediatric subjects surviving for ≥2 years after first HCT without relapse. Among 2245 adult and 377 pediatric patients who died, infections were a primary or contributory cause of death in 687 (31%) and 110 (29%), respectively. At 12 years post-HCT, the cumulative incidence of LFIs was 6.4% (95% confidence interval [CI], 5.8% to 7.0%) in adults, compared with 1.8% (95% CI, 1.4% to 2.3%) in pediatric subjects; P < .001). In adults, the 2 most significant risks for developing LFI were increasing age (20 to 39, 40 to 54, and ≥55 years versus 18 to 19 years) with hazard ratios (HRs) of 3.12 (95% CI, 1.33 to 7.32), 3.86 (95% CI, 1.66 to 8.95), and 5.49 (95% CI, 2.32 to 12.99) and a history of chronic graft-versus-host disease GVHD (cGVHD) with ongoing immunosuppression at 2 years post-HCT compared with no history of GVHD with (HR, 3.87; 95% CI, 2.59 to 5.78). In pediatric subjects, the 3 most significant risks for developing LFI were a history of cGVHD with ongoing immunosuppression (HR, 9.49; 95% CI, 4.39 to 20.51) or without ongoing immunosuppression (HR, 2.7; 95% CI, 1.05 to 7.43) at 2 years post-HCT compared with no history of GVHD, diagnosis of inherited abnormalities of erythrocyte function compared with diagnosis of acute myelogenous leukemia (HR, 2.30; 95% CI, 1.19 to 4.42), and age >10 years (HR, 1.92; 95% CI, 1.15 to 3.2). This study emphasizes the importance of continued vigilance for late infections after HCT and institution of support strategies aimed at decreasing the risk of cGVHD. ispartof: BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION vol:25 issue:2 pages:362-368 ispartof: location:United States status: published

Acute myeloid leukemia (AML) relapse after allogeneic stem cell transplant (alloSCT) remains a major therapeutic challenge. While patients with longer remission after initial alloSCT are recommended to receive cell therapy (CT) such as 2ndalloSCT or donor lymphocyte infusion (DLI), survival for patients who relapse within 6 months of alloSCT has been dismal. We evaluated the outcomes of AML relapse after alloSCT to assess the impact of different treatments on long-term survival. One hundred and seventy-two patients with AML underwent alloSCT at the Penn State Cancer Institute from January 2014 to August 2019. Sixty-nine patients relapsed (median age, 60 years; range, 10–75). Of these, 4 patients underwent 2ndalloSCT, and 26 received DLI. One-year overall survival (OS) in all cases was 20.3% (95% CI: 11.8–30.4%). Patients with ECOG performance status (PS) 0–2 at relapse showed a better 1-year OS than those with PS 3–4. Median OS for patients who received chemotherapy only or chemotherapy with CT was 74 or 173.5 days, respectively (p

Chimerism testing by short tandem repeats (STRs) is used to monitor engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). Generally, STR alleles are stable and transferred from parent to child or from donor to recipient. However, 3 cases did not follow this norm. Additional work-up with help from forensic literature solved these mysteries. In case 1, the patient received HSCT from his son. The son shared STR alleles in 22/23 loci except Penta E, which was explained by repeat expansion in the son. In case 2, the patient had been in remission for 14 years after HSCT for lymphoma and developed repeat expansion in CSF1PO in granulocytes. In case 3, a pre-HSCT patient demonstrated 3 alleles, with 2 peaks taller than the third, in the FGA locus (chromosome 4). A combination of a triallelic variant and leukemia-associated trisomy 4 explained the finding. STR number variants are rare and clinically inconsequential but can overlap malignancy-associated, clinically significant changes.

To improve the outcome of relapsed/refractory acute myeloid leukemia (AML), a randomized phase II trial of three novel regimens was conducted. Ninety patients were enrolled and were in first relapse or were refractory to induction/re-induction chemotherapy. They were randomized to the following regimens: carboplatin-topotecan (CT), each by continuous infusion for five days; alvocidib (formerly flavopiridol), cytarabine, and mitoxantrone (FLAM) in a timed sequential regimen; or sirolimus combined with mitoxantrone, etoposide, and cytarabine (S-MEC). The primary objective was attainment of a complete remission (CR). A Simon two-stage design was used for each of the three arms. The median age of the patients in the FLAM arm was older at 62 years compared to 55 years for the CT arm and the S-MEC arm. The overall response was 14% in the CT arm (5/35, 90% CI 7%−35%), 28% in the FLAM arm (10/36, 90% CI, 16%−43%), and 16% in the S-MEC arm (3/19, 90% CI, 4%−36%). There were nine treatment-related deaths, seven of which occurred in the FLAM arm with four of these in elderly patients. We conclude that the FLAM regimen had an encouraging response rate and should be considered for further clinical development but should be used with caution in elderly patients.

We recently conducted a randomized double-blind study in which we demonstrated that moderate/severe chronic graft-versus-host disease (cGVHD) but not cGVHD-free survival was reduced in patients receiving anti-T lymphocyte globulin (ATLG) versus placebo. In a companion study we performed immunophenotypic analysis to determine the impact of ATLG on immune reconstitution (IR) and to correlate IR with clinical outcomes. The randomized study (n = 254) included patients (aged 18 to 65 years) who underwent myeloablative transplants for acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia from HLA-matched unrelated donors. Ninety-one patients consented for the companion IR study (ATLG = 44, placebo = 47). Blood samples were collected on days 30, 100, 180, and 360 after hematopoietic cell transplantation (HCT), and multiparameter flow cytometry was performed in a blinded fashion. Reconstitution of CD3+ and CD4+ T cells was delayed up to 6 months post-HCT in the ATLG arm, whereas absolute regulatory T cell (Treg) (CD4+25+127-) numbers were lower only in the first 100 days. Analysis of the CD4+ Treg and conventional T cells (Tconv) (CD4+25–127+) compartments showed a profound absence of naive Tregs and Tconv in the first 100 days post-HCT, with very slow recovery for 1 year. B cell and natural killer cell recovery were similar in each arm. Higher absolute counts of CD3+, CD4+, CD8+ T, Tregs, and Tconv were associated with improved overall survival, progression-free survival, and nonrelapse mortality but not moderate/severe cGVHD. Although ATLG delays CD3+ and CD4+ T cell recovery post-transplant, it has a relative Treg sparing effect after the early post-HCT period, with possible implications for protection from cGVHD. ATLG severely compromises the generation of naive CD4+ cells (Treg and Tconv), potentially affecting the diversity of the TCR repertoire and T cell responses against malignancy and infection.

Unlike unrelated donor registries, transplant centers lack uniform approaches to related donor assessment and deferral. To test whether related donors are at increased risk for donation-related toxicities, we conducted a prospective observational trial of 11,942 related and unrelated donors aged 18-60 years. Bone marrow (BM) was collected at 37 transplant and 78 National Marrow Donor Program centers, and peripheral blood stem cells (PBSC) were collected at 42 transplant and 87 unrelated donor centers in North America. Possible presence of medical comorbidities was verified prior to donation, and standardized pain and toxicity measures were assessed pre-donation, peri-donation, and one year following. Multivariate analyses showed similar experiences for BM collection in related and unrelated donors; however, related stem cell donors had increased risk of moderate [odds ratios (ORs) 1.42; P

Purpose Despite the achievement of complete remission with chemotherapy in patients with acute myeloid leukemia (AML), relapse is common and the majority of patients will die of their disease. Patients who achieve a remission after refractory or relapsed disease as well as elderly patients have a very high rate of relapse even if they achieve a complete remission. A phase 3 randomized ECOG-ACRIN-led intergroup study was conducted to determine whether post-remission therapy with the farnesyl transferase inhibitor, tipifarnib (R115777), improved the disease-free survival (DFS) of adult patients with AML in complete remission (CR), at high risk for relapse. Patients and methods Adult patients with AML in remission after salvage therapy and/or over age 60 in first remission were enrolled in this study. They were randomly assigned to treatment with tipifarnib or observation (control). The primary objective was to compare the disease-free survival (DFS) between the two arms based on intention to treat, which includes all randomized patients. Results One hundred and forty-four patients were enrolled on the study. Median DFS was 8.9 vs 5.3 months, for tipifarnib vs observation (one-sided p = 0.026) and did not cross the pre-specified boundary to call the study positive. For the 134 eligible patients, median DFS was 10.8 vs 5.3 months for those randomized to tipifarnib vs observation (one-sided p = 0.008). Moreover in an ad hoc evaluation of all women (n = 71) median DFS was 12.1 vs 3.9 months for tipifarnib vs observation (one-sided p = 0.0004) while median OS was 26.5 vs 8.4 months respectively (one-sided p = 0.001). Conclusion This study was not able to demonstrate a benefit to tipifarnib as maintenance therapy in patients with AML in remission. While subsets of patients may indeed benefit, additional studies would be needed to elucidate that benefit which is unlikely given that other seemingly better options have since become available.

Recipient chimerism increase has been used to predict leukemia relapse in post-hematopoietic cell transplant (HCT) patients with conventional GVHD prophylaxis. However, the value of recipient chimerism increase in patients with post-transplant cyclophosphamide (PTCy) is not clear. We compared PTCy to conventional GVHD prophylaxis (non-PTCy) patients regarding engraftment kinetics and the clinical significance of the 2 chimerism parameters, increasing mixed chimerism (IMC) and degree of recipient chimerism increase at the first event (Δ increase). We studied both total and T-cell-specific chimerism. While leukemia relapse is manifested by an increase in total cell recipient chimerism, an increase in T-cell-specific recipient chimerism may be more impactful in predicting relapse because of the effect of increased T-cell-specific chimerism on the graft-versus-leukemia effect. A total of 220 patients (PTCy: 44, non-PTCy: 176) with AML, MDS, and ALL underwent HCT at our institution from January 2014 to September 2020 and were included in this study (Table). Chimerism was tested at least monthly for the first 3 months, followed by every 3 months up until 1-year post-HCT, and then every 6-12 months thereafter. Short tandem repeat or quantitative PCR were used when percent recipient chimerism was ≥5% and PTCy patients achieved complete donor chimerism (CC) in total cells earlier at a deeper level (>99%) as compared to non-PTCy patients. Deeper total cell CC (>99%) was achieved in 79.5% of PTCy vs. 51% of non-PTCy patients at day 250, while CC (>95%) was achieved in almost 90% of patients in both groups within 100 days (Figure 1A and B). In comparison, the percentage of PTCy patients achieving T-cell-specific CC was significantly higher at day 250 post-HCT: CC (>95%/>99%) was 79.7%/68.4% in PTCy patients vs. 56.1%/37.5% in non-PTCy patients (Figure 1C and D). To evaluate their impact in predicting relapse, IMC was stratified into no IMC, 1 IMC (≥1 nonconsecutive IMC), and 2 IMC (≥2 consecutive IMC), and degree of recipient chimerism increase at the first event (Δ increase) was stratified into Two IMC (total), 1 IMC (T-cell), and 2 IMC (T-cell) groups were associated with shorter DFS in non-PTCy patients but not in PTCy patients (Figure 2). One and 2 IMC groups (both total and T-cell) were associated with relapse risk in non-PTCy patients. Furthermore, 1 IMC (T-cell) in non-PTCy patients showed a strong association in relapse risk (HR 7.0 (95%CI 2.83-17.8) p Δ increase ≥1% (total and T-cell) and Δ increase ≥0.1% (T-cell) were associated with shorter DFS in non-PTCy patients, while only Δ increase ≥1% (T-cell) only showed a trend towards shorter DFS in PTCy patients (Figure 3). The Cox regression model showed Δ increase ≥1% in both total, and T-cell chimerism was associated with relapse risk in non-PTCy patients (HR 6.4 (95%CI 2.9-14.2) p This is one of the most extensive studies investigating engraftment kinetics and the association of total and T-cell recipient chimerism increase to predict leukemia relapse in PTCy and non-PTCy HCT recipients. We found that PTCy HCT recipients achieved deeper engraftment earlier as compared to non-PTCy recipients. In addition, the two chimerism parameters (IMC and Δ increase) are less reliable in predicting relapse in PTCy than non-PTCy recipients. However, other factors, such as disease type, conditioning regimen, and donor HLA disparity, may have affected engraftment kinetics and the significance of chimerism parameters. Further investigations are warranted to elucidate the impact of the engraftment kinetics and recipient chimerism increase to predict relapse, especially in the PTCy setting. Figure 1 Figure 1. Disclosures Rakszawski: SeaGen: Speakers Bureau. Naik: Takeda: Other: Virtual Advisory Board Member ; Sanofi: Other: Virtual Advisory Board Member ; Kite: Other: Virtual Advisory Board Member. Rybka: Spark Therapeutics: Consultancy; Merck: Consultancy. Claxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.