Neoadjuvant Durvalumab Alone or Combined with Novel Immuno-Oncology Agents in Resectable Lung Cancer: The Phase II NeoCOAST Platform Trial.

Neoadjuvant chemoimmunotherapy improves pathologic complete response rate and event-free survival in patients with resectable non-small cell lung cancer (NSCLC) versus chemotherapy alone. NeoCOAST was the first randomized, multidrug platform trial to examine novel neoadjuvant immuno-oncology combinations for patients with resectable NSCLC, using major pathologic response (MPR) rate as the primary endpoint. Eighty-three patients received a single cycle of treatment: 26 received durvalumab (anti-PD-L1) monotherapy, 21 received durvalumab plus oleclumab (anti-CD73), 20 received durvalumab plus monalizumab (anti-NKG2A), and 16 received durvalumab plus danvatirsen (anti-STAT3 antisense oligonucleotide). MPR rates were higher for patients in the combination arms versus durvalumab alone. Safety profiles for the combinations were similar to those of durvalumab alone. Multiplatform immune profiling suggested that improved MPR rates in the durvalumab plus oleclumab and durvalumab plus monalizumab arms were associated with enhanced effector immune infiltration of tumors, interferon responses and markers of tertiary lymphoid structure formation, and systemic functional immune cell activation. SIGNIFICANCE: A neoadjuvant platform trial can rapidly generate clinical and translational data using candidate surrogate endpoints like MPR. In NeoCOAST, patients with resectable NSCLC had improved MPR rates after durvalumab plus oleclumab or monalizumab versus durvalumab alone and tumoral transcriptomic signatures indicative of augmented immune cell activation and function. See related commentary by Cooper and Yu, p. 2306. This article is featured in Selected Articles from This Issue, p. 2293.

A Randomized Phase II Study of MEDI0680 in Combination with Durvalumab versus Nivolumab Monotherapy in Patients with Advanced or Metastatic Clear-cell Renal Cell Carcinoma.

PURPOSE: MEDI0680 is a humanized anti-programmed cell death-1 (PD-1) antibody, and durvalumab is an anti-PD-L1 antibody. Combining treatment using these antibodies may improve efficacy versus blockade of PD-1 alone. This phase II study evaluated antitumor activity and safety of MEDI0680 plus durvalumab versus nivolumab monotherapy in immunotherapy-naïve patients with advanced clear-cell renal cell carcinoma who received at least one prior line of antiangiogenic therapy. PATIENTS AND METHODS: Patients received either MEDI0680 (20 mg/kg) with durvalumab (750 mg) or nivolumab (240 mg), all intravenous, every 2 weeks. The primary endpoint was investigator-assessed objective response rate (ORR). Secondary endpoints included best overall response, progression-free survival (PFS), safety, overall survival (OS), and immunogenicity. Exploratory endpoints included changes in circulating tumor DNA (ctDNA), baseline tumor mutational burden, and tumor-infiltrated immune cell profiles. RESULTS: Sixty-three patients were randomized (combination, n = 42; nivolumab, n = 21). ORR was 16.7% [7/42; 95% confidence interval (CI), 7.0-31.4] with combination treatment and 23.8% (5/21; 95% CI, 8.2-47.2) with nivolumab. Median PFS was 3.6 months in both arms; median OS was not reached in either arm. Because of adverse events, 23.8% of patients discontinued MEDI0680 and durvalumab and 14.3% of patients discontinued nivolumab. In the combination arm, reduction in ctDNA fraction was associated with longer PFS. ctDNA mutational analysis did not demonstrate an association with response in either arm. Tumor-infiltrated immune profiles showed an association between immune cell activation and response in the combination arm. CONCLUSIONS: MEDI0680 combined with durvalumab was safe and tolerable; however, it did not improve efficacy versus nivolumab monotherapy.

Phase 1b study of pegylated arginine deiminase (ADI-PEG 20) plus Pembrolizumab in advanced solid cancers.

Background: Pegylated arginine deiminase (ADI-PEG 20) is a metabolism-based strategy that depletes arginine, resulting in tumoral stress and cytotoxicity. Preclinically, ADI-PEG 20 modulates T-cell activity and enhances the therapeutic efficacy of programmed death-1 (PD-1) inhibition. Methods: A phase 1b study, including a dose-escalation cohort and an expansion cohort, was undertaken to explore the effects of ADI-PEG 20 in combination with pembrolizumab, an anti-PD-1 antibody, for safety, pharmacodynamics, and response. CD3 levels and programmed death-ligand 1 (PD-L1) expression were assessed in paired biopsies collected prior to and after ADI-PEG 20 treatment but before pembrolizumab. Results: Twenty-five patients, nine in the dose-escalation cohort and sixteen in the expansion cohort, were recruited. Treatment was feasible with adverse events consistent with those known for each agent, except for Grade 3/4 neutropenia which was higher than expected, occurring in 10/25 (40%) patients. Mean arginine levels were suppressed for 1-3 weeks, but increased gradually. CD3+ T cells increased in 10/12 (83.3%) subjects following ADI-PEG 20 treatment, including in three partial responders (p = .02). PD-L1 expression was low and increased in 3/10 (30%) of subjects. Partial responses occurred in 6/25 (24%) heavily pretreated patients, in both argininosuccinate synthetase 1 proficient and deficient subjects. Conclusions: The immunometabolic combination was safe with the caveat that the incidence of neutropenia might be increased compared with either agent alone. ADI-PEG 20 treatment increased T cell infiltration in the low PD-L1 tumor microenvironment. The recommended phase 2 doses are 36 mg/m2 weekly for ADI-PEG 20 and 200 mg every 3 weeks for pembrolizumab.

Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma.

Immune checkpoint therapy is being tested in the neoadjuvant setting for patients with localized urothelial carcinoma1,2, with one study reporting data in cisplatin-ineligible patients who received anti-PD-L1 monotherapy2. The study reported that patients with bulky tumors, a known high-risk feature defined as greater than clinical T2 disease, had fewer responses, with pathological complete response rate of 17%2. Here we report on the first pilot combination neoadjuvant trial ( NCT02812420 ) with anti-PD-L1 (durvalumab) plus anti-CTLA-4 (tremelimumab) in cisplatin-ineligible patients, with all tumors identified as having high-risk features (n = 28). High-risk features were defined by bulky tumors, variant histology, lymphovascular invasion, hydronephrosis and/or high-grade upper tract disease3-5. The primary endpoint was safety and we observed 6 of 28 patients (21%) with grade ≥3 immune-related adverse events, consisting of asymptomatic laboratory abnormalities (n = 4), hepatitis and colitis (n = 2). We also observed pathological complete response of 37.5% and downstaging to pT1 or less in 58% of patients who completed surgery (n = 24). In summary, we provide initial safety, efficacy and biomarker data with neoadjuvant combination anti-PD-L1 plus anti-CTLA-4, which warrants further development for patients with localized urothelial carcinoma, especially cisplatin-ineligible patients with high-risk features who do not currently have an established standard-of-care neoadjuvant treatment.

ARID1A mutation plus CXCL13 expression act as combinatorial biomarkers to predict responses to immune checkpoint therapy in mUCC.

Immune checkpoint therapy (ICT) can produce durable antitumor responses in metastatic urothelial carcinoma (mUCC); however, the responses are not universal. Despite multiple approvals of ICT in mUCC, we lack predictive biomarkers to guide patient selection. The identification of biomarkers may require interrogation of both the tumor mutational status and the immune microenvironment. Through multi-platform immuno-genomic analyses of baseline tumor tissues, we identified the mutation of AT-rich interactive domain-containing protein 1A (ARID1A) in tumor cells and expression of immune cytokine CXCL13 in the baseline tumor tissues as two predictors of clinical responses in a discovery cohort (n = 31). Further, reverse translational studies revealed that CXCL13-/- tumor-bearing mice were resistant to ICT, whereas ARID1A knockdown enhanced sensitivity to ICT in a murine model of bladder cancer. Next, we tested the clinical relevance of ARID1A mutation and baseline CXCL13 expression in two independent confirmatory cohorts (CheckMate275 and IMvigor210). We found that ARID1A mutation and expression of CXCL13 in the baseline tumor tissues correlated with improved overall survival (OS) in both confirmatory cohorts (CheckMate275, CXCL13 data, n = 217; ARID1A data, n = 139, and IMvigor210, CXCL13 data, n = 348; ARID1A data, n = 275). We then interrogated CXCL13 expression plus ARID1A mutation as a combination biomarker in predicting response to ICT in CheckMate275 and IMvigor210. Combination of the two biomarkers in baseline tumor tissues suggested improved OS compared to either single biomarker. Cumulatively, this study revealed that the combination of CXCL13 plus ARID1A may improve prediction capability for patients receiving ICT.

Comprehensive Molecular Characterization Identifies Distinct Genomic and Immune Hallmarks of Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.

Neoantigen responses, immune correlates, and favorable outcomes after ipilimumab treatment of patients with prostate cancer.

Tumors with high mutational burden (TMB) tend to be responsive to immune checkpoint blockade (ICB) because there are neoantigens available for targeting by reinvigorated T cells, whereas those with low TMB demonstrate limited clinical responses. To determine whether antigen-specific T cell responses can be elicited after treatment with ICB in cancers that have a low TMB, we conducted a clinical trial with ipilimumab in 30 patients with metastatic castration-resistant prostate cancer. We identified two distinct cohorts by survival and progression times: "favorable" (n = 9) and "unfavorable" (n = 10). Patients in the favorable cohort had high intratumoral CD8 T cell density and IFN-γ response gene signature and/or antigen-specific T cell responses. Two patients with a relatively low TMB had T cell responses against unique neoantigens. Moreover, six of nine patients in the favorable group are still alive at the time of analysis, with survival ranging from 33 to 54 months after treatment. All 10 patients in the unfavorable cohort have succumbed to their disease and had survival ranging from 0.6 to 10.3 months. Collectively, our data indicate that immunological correlates associated with effector T cell responses are observed in patients with metastatic prostate cancer who benefit from ICB.

B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Window-of-opportunity clinical trial of pembrolizumab in patients with recurrent glioblastoma reveals predominance of immune-suppressive macrophages.

BACKGROUND: We sought to ascertain the immune effector function of pembrolizumab within the glioblastoma (GBM) microenvironment during the therapeutic window. METHODS: In an open-label, single-center, single-arm phase II "window-of-opportunity" trial in 15 patients with recurrent (operable) GBM receiving up to 2 pembrolizumab doses before surgery and every 3 weeks afterward until disease progression or unacceptable toxicities occurred, immune responses were evaluated within the tumor. RESULTS: No treatment-related deaths occurred. Overall median follow-up time was 50 months. Of 14 patients monitored, 10 had progressive disease, 3 had a partial response, and 1 had stable disease. Median progression-free survival (PFS) was 4.5 months (95% CI: 2.27, 6.83), and the 6-month PFS rate was 40%. Median overall survival (OS) was 20 months, with an estimated 1-year OS rate of 63%. GBM patients' recurrent tumors contained few T cells that demonstrated a paucity of immune activation markers, but the tumor microenvironment was markedly enriched for CD68+ macrophages. CONCLUSIONS: Immune analyses indicated that pembrolizumab anti-programmed cell death 1 (PD-1) monotherapy alone can't induce effector immunologic response in most GBM patients, probably owing to a scarcity of T cells within the tumor microenvironment and a CD68+ macrophage preponderance.

Immune profiling of human tumors identifies CD73 as a combinatorial target in glioblastoma.

Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a subset of patients with specific tumor types1,2. Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73hi macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73-/- mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies.

High OX-40 expression in the tumor immune infiltrate is a favorable prognostic factor of overall survival in non-small cell lung cancer.

INTRODUCTION: OX-40 co-stimulatory signaling plays a role in mounting anti-tumor immune responses and clinical trials targeting this pathway are ongoing. However, the association of with OX-40 protein expression with clinical outcomes and pathological features in non-small cell lung cancer (NSCLC) are largely unknown. METHODS: Surgically-resected stage I-III NSCLC specimens (N = 100) were stained by immunohistochemistry (IHC) for the following immune markers: OX-40, PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FOXP3, granzyme B, and ICOS. Immune-related markers mRNA expression were also assessed. We evaluated the association of OX-40 levels with major clinicopathologic variables, including molecular driver mutations. RESULTS: OX-40 IHC expression was observed in all tested tumors, predominantly localized in the membrane of the tumor immune infiltrate, and was not associated with a specific clinicopathologic or molecular subtype. High OX-40 expression levels measured by IHC median score were associated with better overall survival (OS) (p = 0.002), independent of CD3/CD8, PD-L1, and ICOS expression. High OX-40 IHC score was associated with increased expression of immune-related genes such as CD3, IFN-gamma, ICOS, CD8, CXCL9, CXCL10, CCL5, granzyme K. CONCLUSIONS: High OX-40 IHC expression in the tumor immune infiltrate is associated with favorable prognosis and increased levels of immune-related genes including IFN-gamma in patients with surgically resected stage I-III NSCLC. Its prognostic utility is independent of PD-L1 and other common markers of immune activation. High OX-40 expression potentially identifies a unique subgroup of NSCLC that may benefit from co-stimulation with OX-40 agonist antibodies and potentially enhance the efficacy of existing immune checkpoint therapies.

Autoimmune antibodies correlate with immune checkpoint therapy-induced toxicities.

Immune checkpoint (IC) therapy provides substantial benefits to cancer patients but can also cause distinctive toxicities termed immune-related adverse events (irAEs). Biomarkers to predict toxicities will be necessary to improve management of patients receiving IC therapy. We relied on serological analysis of recombinant cDNA expression libraries to evaluate plasma samples from patients treated with IC therapy and identified autoantibodies, both in pretreatment and on-treatment samples prior to the development of irAEs, which correlate with the development of immune-related hypophysitis (anti-GNAL and anti-ITM2B autoantibodies) and pneumonitis (anti-CD74 autoantibody). We developed an enzyme-linked immunosorbent assay and tested additional patient samples to confirm our initial findings. Collectively, our data suggest that autoantibodies may correlate with irAEs related to IC therapy, and specific autoantibodies may be detected early for the management of irAEs.

Blockade of CTLA-4 and PD-1 Enhances Adoptive T-cell Therapy Efficacy in an ICOS-Mediated Manner.

Adoptive transfer of tumor-reactive T cells (ACT) has led to modest clinical benefit in the treatment of solid tumors. Failures with this therapy are primarily due to inadequate infiltration and poor function of adoptively transferred cells in the tumor microenvironment. To improve the efficacy of ACT, we combined ACT with dual blockade of CTLA-4 and PD-1. Treatment with anti-CTLA-4 plus anti-PD-1 compared with monotherapy resulted in durable antitumor responses, enhanced effector function of ACT, utilizing PMEL-1 transgenic (Tg+) CD8+ T cells, and improved survival. Using PMEL-1ICOS-/- mice, we showed that deletion of the inducible T-cell costimulator (ICOS) receptor abolished the therapeutic benefits, with selective downregulation of Eomesodermin (Eomes), interferon gamma (IFNγ), and perforin. Higher expression of IFNγ and Eomes was noted in human ICOShi CD8+ T cells compared with ICOSlow counterparts. Together, our data provide direct evidence that ACT combined with immune-checkpoint therapy confers durable antitumor responses, which largely depended on CD8+ T-cell-intrinsic expression of ICOS. Our study provides a foundation of testing combinatorial therapy of ACT of CD8 T cells and dual blocking of CTLA-4 and PD-1 in patients with melanoma.

Immunologic Correlates of Pathologic Complete Response to Preoperative Immunotherapy in Hepatocellular Carcinoma.

In hepatocellular carcinoma (HCC), surgical resection is associated with high recurrence rate, and no effective adjuvant therapy currently exists. We initiated a pilot randomized trial of perioperative immunotherapy with nivolumab and ipilimumab for resectable HCC. Here, we provide an illustrative report of a case that achieved a complete response and report immunologic correlates of this complete pathologic response to perioperative immunotherapy. Clinical response was correlated with an increase in CD8+ T-cell infiltration, with an increase in two effector T-cell clusters. This study is ongoing, and the final results may contribute to a paradigm shift in the perioperative treatment of HCC, leading to the incorporation of immunotherapy in the curative setting.

Characterization and Comparison of GITR Expression in Solid Tumors.

PURPOSE: Determine the differential effect of a FcγR-binding, mIgG2a anti-GITR antibody in mouse tumor models, and characterize the tumor microenvironment for the frequency of GITR expression in T-cell subsets from seven different human solid tumors.Experimental Design: For mouse experiments, wild-type C57BL/6 mice were subcutaneously injected with MC38 cells or B16 cells, and BALB/c mice were injected with CT26 cells. Mice were treated with the anti-mouse GITR agonist antibody 21B6, and tumor burden and survival were monitored. GITR expression was evaluated at the single-cell level using flow cytometry (FC). A total of 213 samples were evaluated for GITR expression by IHC, 63 by FC, and 170 by both in seven human solid tumors: advanced hepatocellular carcinoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, pancreatic carcinoma, head and neck carcinoma, melanoma, and ovarian carcinoma. RESULTS: The therapeutic benefit of 21B6 was greatest in CT26 followed by MC38, and was least in the B16 tumor model. The frequency of CD8 T cells and effector CD4 T cells within the immune infiltrate correlated with response to treatment with GITR antibody. Analysis of clinical tumor samples showed that NSCLC, renal cell carcinoma, and melanoma had the highest proportions of GITR-expressing cells and highest per-cell density of GITR expression on CD4+ Foxp3+ T regulatory cells. IHC and FC data showed similar trends with a good correlation between both techniques. CONCLUSIONS: Human tumor data suggest that NSCLC, renal cell carcinoma, and melanoma should be the tumor subtypes prioritized for anti-GITR therapy development.

Comparison of immune infiltrates in melanoma and pancreatic cancer highlights VISTA as a potential target in pancreatic cancer.

Immune checkpoint therapy (ICT) has transformed cancer treatment in recent years; however, treatment response is not uniform across tumor types. The tumor immune microenvironment plays a critical role in determining response to ICT; therefore, understanding the differential immune infiltration between ICT-sensitive and ICT-resistant tumor types will help to develop effective treatment strategies. We performed a comprehensive analysis of the immune tumor microenvironment of an ICT-sensitive tumor (melanoma, n = 44) and an ICT-resistant tumor (pancreatic cancer, n = 67). We found that a pancreatic tumor has minimal to moderate infiltration of CD3, CD4, and CD8 T cells; however, the immune infiltrates are predominantly present in the stromal area of the tumor and are excluded from tumoral area compared with melanoma, where the immune infiltrates are primarily present in the tumoral area. Metastatic pancreatic ductal adenocarcinomas (PDACs) had a lower infiltration of total T cells compared with resectable primary PDACs, suggesting that metastatic PDACs have poor immunogenicity. Further, a significantly higher number of CD68+ macrophages and VISTA+ cells (also known as V-domain immunoglobulin suppressor of T cell activation) were found in the pancreatic stromal area compared with melanoma. We identified VISTA as a potent inhibitory checkpoint that is predominantly expressed on CD68+ macrophages on PDACs. These data suggest that VISTA may be a relevant immunotherapy target for effective treatment of patients with pancreatic cancer.

Anti-CTLA-4 Immunotherapy Does Not Deplete FOXP3+ Regulatory T Cells (Tregs) in Human Cancers.

PURPOSE: CTLA-4 was the first inhibitory immune checkpoint to be identified. Two mAbs, ipilimumab (IgG1) and tremelimumab (IgG2), which block the function of CTLA-4, have demonstrated durable clinical activity in a subset of patients with advanced solid malignancies by augmenting effector T-cell-mediated immune responses. Studies in mice suggest that anti-CTLA-4 mAbs may also selectively deplete intratumoral FOXP3+ regulatory T cells via an Fc-dependent mechanism. However, it is unclear whether the depletion of FOXP3+ cells occurs in patients with cancer treated with anti-CTLA-4 therapies. EXPERIMENTAL DESIGN: Quantitative IHC was used to evaluate the densities of intratumoral CD4+, CD8+, and FOXP3+ cells in stage-matched melanoma (n = 19), prostate cancer (n = 17), and bladder cancer (n = 9) samples treated with ipilimumab and in paired melanoma tumors (n = 18) treated with tremelimumab. These findings were corroborated with multiparametric mass cytometry analysis of tumor-infiltrating cells from paired fresh melanoma tumors (n = 5) treated with ipilimumab. RESULTS: Both ipilimumab and tremelimumab increase infiltration of intratumoral CD4+ and CD8+ cells without significantly changing or depleting FOXP3+ cells within the tumor microenvironment. CONCLUSIONS: Anti-CTLA-4 immunotherapy does not deplete FOXP3+ cells in human tumors, which suggests that their efficacy could be enhanced by modifying the Fc portions of the mAbs to enhance Fc-mediated depletion of intratumoral regulatory T cells.See related commentary by Quezada and Peggs, p. 1130.

The distribution of T-cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia.

BACKGROUND: Phenotypic characterization of immune cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) is lacking. METHODS: T-cell infiltration was quantified on BM biopsies from 13 patients with AML, and flow cytometry was performed on BM aspirates (BMAs) from 107 patients with AML who received treatment at The University of Texas MD Anderson Cancer Center. The authors evaluated the expression of inhibitory receptors (programmed cell death protein 1 [PD1], cytotoxic T-lymphocyte antigen 4 [CTLA4], lymphocyte-activation gene 3 [LAG3], T-cell immunoglobulin and mucin-domain containing-3 [TIM3]) and stimulatory receptors (glucocorticoid-induced tumor necrosis factor receptor-related protein [GITR], OX40, 41BB [a type 2 transmembrane glycoprotein receptor], inducible T-cell costimulatory [ICOS]) on T-cell subsets and the expression of their ligands (41BBL, B7-1, B7-2, ICOSL, PD-L1, PD-L2, and OX40L) on AML blasts. Expression of these markers was correlated with patient age, karyotype, baseline next-generation sequencing for 28 myeloid-associated genes (including P53), and DNA methylation proteins (DNA methyltransferase 3α, isocitrate dehydrogenase 1[IDH1], IDH2, Tet methylcytosine dioxygenase 2 [TET2], and Fms-related tyrosine kinase 3 [FLT3]). RESULTS: On histochemistry evaluation, the T-cell population in BM appeared to be preserved in patients who had AML compared with healthy donors. The proportion of T-regulatory cells (Tregs) in BMAs was higher in patients with AML than in healthy donors. PD1-positive/OX40-positive T cells were more frequent in AML BMAs, and a higher frequency of PD1-positive/cluster of differentiation 8 (CD8)-positive T cells coexpressed TIM3 or LAG3. PD1-positive/CD8-positive T cells were more frequent in BMAs from patients who had multiply relapsed AML than in BMAs from those who had first relapsed or newly diagnosed AML. Blasts in BMAs from patients who had TP53-mutated AML were more frequently positive for PD-L1. CONCLUSIONS: The preserved T-cell population, the increased frequency of regulatory T cells, and the expression of targetable immune receptors in AML BMAs suggest a role for T-cell-harnessing therapies in AML.