Dasatinib cessation after deep molecular response exceeding 2 years and natural killer cell transition during dasatinib consolidation

Tyrosine kinase inhibitors (TKI) improve the prognosis of patients with chronic myelogenous leukemia (CML) by inducing substantial deep molecular responses (DMR); some patients have successfully discontinued TKI therapy after maintaining DMR for ≥1 year. In this cessation study, we investigated the optimal conditions for dasatinib discontinuation in patients who maintained DMR for ≥2 years. This study included 54 patients with CML who were enrolled in a D‐STOP multicenter prospective trial, had achieved DMR, and had discontinued dasatinib after 2‐year consolidation. Peripheral lymphocyte profiles were analyzed by flow cytometry. The estimated 12‐month treatment‐free survival (TFS) was 62.9% (95% confidence interval: 48.5%‐74.2%). During dasatinib consolidation, the percentage of total lymphocytes and numbers of CD3⁻ CD56⁺ natural killer (NK) cells, CD16⁺ CD56⁺ NK cells and CD56⁺ CD57⁺ NK‐large granular lymphocytes (LGL) were significantly higher in patients with molecular relapse after discontinuation but remained unchanged in patients without molecular relapse for >7 months. At the end of consolidation, patients whose total lymphocytes comprised <41% CD3⁻ CD56⁺ NK cells, <35% CD16⁺ CD56⁺ NK cells, or <27% CD56⁺ CD57⁺ NK‐LGL cells had higher TFS relative to other patients (77% vs 18%; P < .0008; 76% vs 10%; P < .0001; 84% vs 46%; P = .0059, respectively). The increase in the number of these NK cells occurred only during dasatinib consolidation. In patients with DMR, dasatinib discontinuation after 2‐year consolidation can lead to high TFS. This outcome depends significantly on a smaller increase in NK cells during dasatinib consolidation.     

Final 3-year Results of the Dasatinib Discontinuation Trial in Patients With Chronic Myeloid Leukemia Who Received Dasatinib as a Second-line Treatment

Introduction

We previously reported an interim analysis of the DADI (dasatinib discontinuation) trial. The results showed that 48% of patients with chronic myeloid leukemia in the chronic phase who maintained a deep molecular response (DMR) for ≥ 1 year could discontinue second- or subsequent-line dasatinib treatment safely at a median follow-up of 20 months. However, the results from longer follow-up periods would be much more useful from a clinical perspective.

Programmed cell death 1‐expressing CD56‐negative natural killer (NK) cell expansion is a hallmark of chronic NK cell activation during dasatinib treatment

Dasatinib treatment markedly increases the number of large granular lymphocytes including natural killer (NK) cells in a proportion of Ph⁺ leukemia patients, which associates with a better prognosis. In‐depth immune profiling of NK cells can predict therapeutic response in these patients. In the present study, we showed that CD56‐negative (CD56^neg) NK cells increased exclusively in cytomegalovirus‐seropositive (CMV⁺) patients treated with dasatinib. The increase longitudinally paralleled with progressive differentiation of CD56^dim NK cells during dasatinib therapy driven by CMV reactivation as shown by principal component analysis on 19 NK cell markers. The CD56^neg NK cells showed downregulation of NK‐activating receptors, upregulation of PD‐1, and lower cytotoxicity and cytokine production, indicating that these cells are anergic and dysfunctional as seen in chronic infections with HIV‐1 or hepatitis C virus. Moreover, cytolytic activity of CD56^dim and CD56^neg NK cells against leukemia cells was partially restored by nivolumab in proportion to the frequency of PD‐1⁺ NK cells. The proportion of patients who achieved deep molecular responses at 2 years was significantly higher in dasatinib‐treated patients with ≥3% CD56^neg NK cells than in those with fewer CD56^neg NK cells (54.5% vs 15.8%, P = .0419). These findings suggest that CD56^neg NK cells may be an exhausted population induced by chronic activation through CMV reactivation during dasatinib therapy. Expansion of CD56^neg NK cells is a hallmark of chronic NK cell activation in patients treated with dasatinib and may predict a better clinical outcome. Furthermore, PD‐1 blockade may enhance anti‐leukemia responses of such NK cells.     

Large granular lymphocytosis during dasatinib therapy

Dasatinib is a second generation tyrosine kinase inhibitor (TKI) approved for clinical use in patients with imatinib-resistant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph⁺ ALL). Large granular lymphocytes (LGLs) are medium to large cells with eccentric nuclei and abundant cytoplasm with coarse azurophilic granules. LGL lymphocytosis is caused by a proliferation of cytotoxic (CD8⁺) T cells and/or NK cells. In a proportion of CML and Ph⁺ ALL patients, there is a significant expansion of LGLs during dasatinib therapy. LGL lymphocytosis is seen in some cases with fevers, colitis, and pleural effusions (PE), suggesting an aberrant immune response mediated by these LGLs. LGLs may participate in the elimination of the residual leukemic cells, and LGL clonal expansion is associated with excellent, long-lasting therapy responses in dasatinib-treated patients. For a more comprehensive analysis, we analyzed the morphologic, phenotypic, clinical, and functional features of the LGL subsets amplified in vivo during dasatinib therapy.     

Prognostic value of CD57<Superscript>+</Superscript> T lymphocytes in the peripheral blood of patients with advanced gastric cancer

Background  Natural killer (NK)-like T cells comprising CD56⁺ T cells and CD57⁺ T cells belong to a subset of CD1d-independent NKT cells playing an important role in regulating immune responses. Although NK-like T cells are reported to increase in patients with advanced gastric carcinomas, it remains unknown how NK-like T cells are involved in disease progression in gastric cancer patients. Methods  The proportions of Th1 cells (interferon [IFN]-γ-producing CD4⁺ T cells), Th2 cells (IL-4-producing CD4⁺ T cells), and NK-like T cells (CD56⁺ T cells and CD57⁺ T cells) in the peripheral blood of 48 gastric cancer patients and 20 healthy controls were measured by two-color flow cytometry analysis and by intracellular cytokine analysis to investigate an association of these immune cells with the survival rate of gastric cancer patients. Results  Univariate analysis showed that Th1 cells and CD57⁺ T cells, as well as some clinicopathological factors, significantly influenced the survival rate. CD57-high (≧18%) patients survived for a significantly shorter period after surgery compared to CD57-low patients (P = 0.046; Kaplan-Meier, log-rank test) in the stage III–IV patients, but not in the stage I–II patients. Further, multivariate analysis showed that lymphatic invasion was a statistically significant independent risk factor in all the gastric cancer patients, but the proportion of CD57⁺ T cells as well as depth of tumor were statistically significant independent risk factors in patients with advanced carcinomas (stage III–IV). Conclusion  An increased proportion (≧18%) of CD57⁺ T cells in the peripheral blood of patients with advanced gastric carcinomas could indicate a poor prognosis.     

Expression of CD56 is an unfavorable prognostic factor for acute promyelocytic leukemia with higher initial white blood cell counts

Expression of CD56 has recently been introduced as one of the adverse prognostic factors in acute promyelocytic leukemia (APL). However, the clinical significance of CD56 antigen in APL has not been well elucidated. We assessed the clinical significance of CD56 antigen in 239 APL patients prospectively treated with all‐trans retinoic acid and chemotherapy according to the Japan Adult Leukemia Study Group APL97 protocol. All patients were prospectively treated by the Japan Adult Leukemia Study Group APL97 protocol. The median follow‐up period was 8.5 years. Positive CD56 expression was found in 23 APL patients (9.6%). Expression of CD56 was significantly associated with lower platelet count (P = 0.04), severe disseminated intravascular coagulation (P = 0.04), and coexpression of CD2 (P = 0.03), CD7 (P = 0.04), CD34 (P < 0.01) and/or human leukocyte antigen‐DR (P < 0.01). Complete remission rate and overall survival were not different between the two groups. However, cumulative incidence of relapse and event‐free survival (EFS) showed an inferior trend in CD56⁺ APL (P = 0.08 and P = 0.08, respectively). Among patients with initial white blood cell counts of 3.0 × 10⁹/L or more, EFS and cumulative incidence of relapse in CD56⁺ APL were significantly worse (30.8% vs 63.6%, P = 0.008, and 53.8% vs 28.9%, P = 0.03, respectively), and in multivariate analysis, CD56 expression was an unfavorable prognostic factor for EFS (P = 0.04). In conclusion, for APL with higher initial white blood cell counts, CD56 expression should be regarded as an unfavorable prognostic factor.     

Serum interferon gamma level predicts recurrence in hepatocellular carcinoma patients after curative treatments

Host immunity may have important role in the prognosis of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the correlation between circulating immune regulators and clinical outcome in patients with HCC. Sixty‐three HCC patients were prospectively enrolled. Serum levels of interleukin‐10 (IL‐10), transforming growth factor‐beta (TGF‐β), interferon‐gamma (IFN‐γ) and interferon gamma‐inducible protein 10 (IP‐10) were measured, as well as the prevalence of regulatory T cells (Treg), NK⁺ T cells, invariant natural killer T cells (iNKT), programmed cell death‐1 (PD‐1)⁺CD8⁺ T cells, T helper 17 cells (Th17), CD69⁺ and CD45RO⁺ T cells in peripheral blood mononuclear cells (PBMC). Correlation between these immune regulators and clinical outcome were analyzed. A low serum IFN‐γ level (<50 pg/mL) was significantly associated tumor stage (BCLC stage B: 61.25% vs. stage A: 25%, p = 0.010) and tumor size (>5 cm: 53.8% vs. <5 cm: 25%, p = 0.047). Recurrence‐free survival was evaluated in 48 patients receiving curative treatment of HCC. By multivariate analysis, BCLC stage [hazard ratio (HR) = 32.180, p < 0.001], tumor size (HR = 15.373, p = 0.005), AST (HR = 3.796, p = 0.011) and IFN‐γ (HR = 0.354, p = 0.018) levels were independent factors associated with recurrence‐free survival. In conclusion, serum IFN‐γ level correlates with tumor stage and tumor size in HCC patients. Patients with lower baseline IFN‐γ levels have a higher risk of tumor recurrence after curative treatment. IFN‐γ may reflect host anti‐tumor immunity and may be a potential marker of HCC recurrence after curative treatment.     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

The immune response‐related mutational signatures and driver genes in non‐small‐cell lung cancer

Immune checkpoint blockade (ICB) therapy has achieved remarkable clinical benefit in non‐small‐cell lung cancer (NSCLC), but our understanding of biomarkers that predict the response to ICB remain obscure. Here we integrated somatic mutational profile and clinicopathologic information from 113 NSCLC patients treated by ICB (CTLA‐4/PD‐1). High tumor mutation burden (TMB) and neoantigen burden were identified significantly associated with improved efficacy in NSCLC immunotherapy. Furthermore, we identified apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) mutational signature was markedly associated with responding of ICB therapy (log‐rank test, P = .001; odds ratio (OR), 0.18 [95% CI, 0.06‐0.50], P < .001). The association with progression‐free survival remained statistically significant after controlling for age, sex, histological type, smoking, PD‐L1 expression, hypermutation, smoking signature and mismatch repair (MMR) (HR, 0.30 [95% CI, 0.12‐0.75], P = .010). Combined high TMB with APOBEC signature preferably predict immunotherapy responders in NSCLC cohort. The CIBERSORT algorithm revealed that high APOBEC mutational activity samples were associated with increased infiltration of CD4 memory activated T cells, CD8⁺ T cells and natural killer (NK) cells, but reduced infiltration of regulatory T cells. Besides, individual genes mutation of IFNGR1 or VTCN1 were only found in responders; however, the PTEN mutation was only found in non‐responders (Fisher's exact test, all P < .05). These findings may be applicable for guiding immunotherapy for patients with NSCLC.     

Natural killer cell cytotoxicity is enhanced by very low doses of rIL-2 and rIFN-α in patients with renal cell carcinoma

Very low doses of recombinant interleukin-2 (rIL-2) and interferon-α (rIFN-α) induce, in patients with advanced renal cell carcinoma (RCC) clinical response rate and median survival time comparable to other protocols, other than immunological response in terms of expansion of NK cells and cT lymphocytes. The aim of this pilot study was to verify whether very low dose immunotherapy can enhance NK cell cytotoxicity against tumoral target cells. Eight patients with advanced and 13 patients with localised disease received 4-week cycles of rIL-2 (total dose per week 7 MIU/m², s.c.) and rIFN-α (total dose per week 3.6 MUI/m², i.m.) according to the scheme proposed by Buzio et al. Neutrophils, monocytes, eosinophils, NK cells (CD56+bright, CD56+dimmer, CD3-CD56 +), NK-T cells (CD3+CD56+), Th-lymphocytes, cT-lymphocytes, HLA-DR+ and CD25+  lymphocytes and NK cell cytotoxicity were evaluated before and after cycle. The treatment led to the significant expansion of eosinophils (P < 0.001), NK cells (P < 0.001), CD56+bright (P < 0.001), CD56+dimmer (P < 0.001), Th-lymphocytes (P = 0.001), cT-lymphocytes (P = 0.014), HLA-DR+ (P = 0.007) and CD25+(P = 0.002) cells. Neutrophils significantly decreased (P = 0.001), whereas no significant effect was observed on monocytes (P = 0.22) or NK-T cells (P = 0.20). Patients with localised disease responded significantly better to treatment than metastatic patients in terms of the expansion of CD56+bright (P = 0.038), DR+ (P = 0.021), CD25+ (P = 0.006) and Th-lymphocytes (P = 0.014). The NK cell cytotoxicity was significantly increased by the immunotherapy in the whole population (P = 0.021) and similarly in the two groups of patients (P = 0.860); a reverse relation, even if not significant, was seen between the variation of NK-T cells and NK cells cytotoxicity (r = −0.39; P = 0.074).     

Comparison of tumor‐infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2⁺, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8⁺ and CD4⁺ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2⁺ and TN breast cancers have a lower percentage of TILs and CD8⁺ and CD4⁺ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.     

A distinct distribution of natural killer cell subgroups in human tissues and blood

Natural killer (NK) cells have been subdivided according to their CD16/ CD56 expression into at least 2 subgroups. We examined the distribution of these NK subgroups in humans. In the blood of normal individuals, CD16⁺⁺/ CD56⁺/ CD3^– NK cells predominate, constituting more than 90% of all NK cells. In contrast, decidua is infiltrated almost exclusively by CD16^+/–/ CD56⁺/ CD3^– NK cells (>90%), a fact so far seen in context with decidua being an immunoprivileged tissue. However, this NK subgroup can also be detected in the blood, where it comprises about 10% of NK cells. We have found that normal (colon) as well as neoplastic (ovarian and urothelial carcinoma) tissues are also predominantly infiltrated by this CD16^+/– NK subgroup. Lymphatic fluid draining solid tissues contains CD16^+/– NK cells exclusively, with absolute numbers of NK cells being very low. No predominating NK subgroup was seen in ascites. CD16^+/– NK cells, when tested against the target cell lines K562 and JAR, revealed a cytotoxic spectrum different from CD16⁺⁺ NK cells and from T cells. A change in the CD16/ CD56/ CD3 phenotype was not seen in either subgroup in long-term cultures containing IL-2 (1,000 U/ ml). Our data indicate that the decidua is not the only solid tissue infiltrated by CD16^+/– NK cells. Other normal and malignant tissues were also infiltrated predominantly by this NK cell subgroup. We suggest that CD16^+/– NK cells represent a functionally distinct NK subgroup involved in the surveillance of solid tissues. Int. J. Cancer 78:533–538, 1998. © 1998 Wiley-Liss, Inc.     

Tumor‐infiltrating M2 macrophage in pretreatment biopsy sample predicts response to chemotherapy and survival in esophageal cancer

The association between the tumor microenvironment (TME) and treatment response or survival has been a recent focus in several types of cancer. However, most study materials are resected specimens that were completely modified by prior chemotherapy; therefore, the unmodified host immune condition has not yet been clarified. The aim of the present study was to evaluate the relationship between TME assessed in pre–therapeutic biopsy samples and chemoresistance in esophageal cancer (EC). A total of 86 endoscopic biopsy samples from EC patients who received neoadjuvant chemotherapy (NAC) prior to surgery were evaluated for the number of intratumoral CD4⁺ lymphocytes (with/without Foxp3 expression), CD8⁺ lymphocytes (with/without PD‐1 expression), monocytes (CD14⁺) and macrophages (CD86⁺, CD163⁺ and CD206⁺) by multiplex immunohistochemistry (IHC). The number of tumor‐infiltrating CD206⁺ macrophages I significantly correlated with cT, cM, cStage and neutrophil/lymphocyte ratio (NLR), whereas the number of lymphocytes (including expression of Foxp3 and PD‐1) was not associated with clinico‐pathological features. The high infiltration of CD163⁺ or CD206⁺ macrophages was significantly associated with poor pathological response to NAC (P = 0.0057 and 0.0196, respectively). Expression of arginase‐1 in CD163⁺ macrophages tended to be higher in non–responders (29.4% vs 18.2%, P = 0.17). In addition, patients with high infiltration of M2 macrophages exhibited unfavorable overall survival compared to those without high infiltration of M2 macrophages (5‐year overall survival 57.2% vs 71.0%, P = 0.0498). Thus, a comprehensive analysis of TME using multiplex IHC revealed that M2 macrophage infiltration would be useful in predicting the response to NAC and long‐term survival in EC patients.     

Programmed death ligand‐1 is associated with tumor infiltrating lymphocytes and poorer survival in urothelial cell carcinoma of the bladder

Drugs blocking programmed death ligand‐1 (PD‐L1) have shown unprecedented activity in metastatic and unresectable bladder cancer. The purpose of the present study was to investigate the expression, clinical significance and association of PD‐L1 with tumor‐infiltrating lymphocytes (TIL) in resectable urothelial cell carcinoma of the bladder (UCB). In this retrospective study, 248 UCB patients who received radical cystectomy or transurethral resection were examined. Immunohistochemistry was used to evaluate PD‐L1 expression and stromal CD8⁺ TIL, Th1 orientation T cell (T‐bet⁺) and PD‐1⁺ TIL densities within the intratumoral regions and associated stromal regions. Of the 248 specimens, 23% showed PD‐L1 expression in tumor cells and 55% in tumor‐infiltrating immune cells. CD8⁺ TIL, T‐bet⁺ TIL and PD‐1⁺ TIL were distributed throughout the tumor tissues and were more frequently distributed in stromal regions than in intratumoral regions. PD‐L1⁺ tumor cells and PD‐L1⁺ immune cells were positively associated with aggressive clinical features (all P < .05). Both PD‐L1⁺ tumor cells and PD‐L1⁺ immune cells were associated with poorer recurrence‐free and overall survival (all P < .05). Multivariate analysis showed that PD‐L1⁺ immune cells were an independent prognostic factor for overall (P = .001) and recurrence‐free survival (P = .024). Notably, high stromal CD8⁺ TIL and PD‐1⁺ TIL density were associated with poorer overall survival (P = .031 and P = .001, respectively). In the stroma, CD8⁺ TIL density has strong positive association with PD‐L1⁺ immune cells and PD‐1⁺ TIL density (all P < .0001). These results suggested that an exhausted immune state occurred in the tumor stroma in UCB. Further clinical development of immune‐checkpoint inhibitors may be effective for resectable patients with UCB.     

Long‐term prognostic significance of interleukin‐17‐producing T cells in patients with non‐small cell lung cancer

The presence of interleukin (IL)‐17‐producing T cells has recently been reported in non‐small cell lung cancer (NSCLC) patients. However, the long‐term prognostic significance of these populations in NSCLC patients remains unknown. In the present study, we collected peripheral blood from 82 NSCLC patients and 22 normal healthy donors (NC). Percentages of IL‐17‐producing CD4⁺T (Th17), CD8⁺T (Tc17) and γδT cells (γδT17) were measured to determine their association with clinical outcomes and overall survival (OS) in NSCLC. All NSCLC patients were followed up until July 2018. Median follow‐up time was 13.5 months (range 1‐87 months). The 3‐ and 5‐year survival rate was 27% and 19.6%, respectively. We found that Th17 cells and γδT17 cells were significantly increased, whereas Tc17 cells were markedly decreased in patients with NSCLC compared with those in NC. In addition, Th17 cells were significantly positively associated with T helper type 1 cells (Th1), whereas γδT17 cells were significantly negatively associated with γδT ⁺ interferon (IFN)‐γ⁺ cells. High percentages of peripheral Tc17 cells were significantly associated with favorable 5‐year OS (P = .025), especially in patients with early TNM stage (P = .016). Furthermore, high percentages of peripheral Th17 cells were positively associated with favorable 5‐year OS in patients with late TNM stage (P = .002). However, no significant association was observed between γδT17 cells and OS, regardless of the TNM stage. In conclusion, our findings suggest that enhanced Th17 and reduced Tc17 cells in the peripheral blood could be a significant predictor of a favorable prognosis for NSCLC patients.     

A child case of CD34, CD33, HLA-DR, CD7, CD56 stem cell leukemia with thymic involvement

It has been reported that the CD56⁺/CD7⁺/CD3⁻ phenotype of natural killer (NK) cells develop from the CD34⁺/HLA-DR⁻ bone marrow (BM) mononuclear cell population in long-term BM culture (LTBMC). An HLA-DR⁻/CD33⁺/CD56⁺/CD16⁻ myeloid/natural killer cell acute leukemia has been described. We report here a 7-year-old boy who developed stem cell acute leukemia with superior vena cava syndrome secondary to thymic involvement. Surface marker analyses revealed that the leukemia cells showed CD34⁺/HLA-DR⁻/CD33⁻/CD7⁺/CD56⁺ phenotype. When stimulated with phorbol ester in vitro the leukemic cells morphologically differentiated to myeloid cells developing CD13, CD15 and CD56 antigens. Our results suggest that CD34⁺/HLA-DR⁻/CD7⁺/CD56⁺ stem cell leukemia may arise from transformation of a pluripotent precursor cell, which could differentiate to both myeloid and NK cell lineages.     

NK- and T-cell subsets in malignant mesothelioma patients: Baseline pattern and changes in the context of anti-CTLA-4 therapy

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK-cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti-CTLA-4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL-15 stimulation improved NK cell-mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56^bright and CD56^dim NK subsets and increased serum concentrations of the cytokines IL-10, IL-8 and TNF-α. After tremelimumab treatment, the ratio between the CD56^bright and CD56^dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM-3⁺CD8⁺ T-cell frequency, high DNAM-1⁺CD56^dim NK-cell frequency and high expression levels of NKp46 on the CD56^dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.     

NK depletion results in increased CCL22 secretion and Treg levels in Lewis Lung Carcinoma via the accumulation of CCL22‐secreting CD11b+CD11c+ cells

Tumor‐induced immune suppression involves the accumulation of suppressive infiltrates in the tumor microenvironment such as regulatory T‐cells (Tregs). Previous studies demonstrated that NK‐dependant increases in CCL22 secretion selectively recruit Tregs toward murine lungs bearing Lewis Lung Carcinoma (LLC). To extend the in vitro studies, the present studies utilized in vivo depletion of NK cells to ascertain the contribution of NK‐derived CCL22 toward total CCL22 and subsequent Treg levels in both normal and LLC‐bearing lungs. However, NK depletion had the unexpected effect of increasing both CCL22 secretion and Treg levels in the lungs of NK‐depleted LLC‐bearing mice. This was concurrent with an increase in tumor burden. Flow cytometry and a series of both immunomagnetic and FACS isolations were used to identify the CCL22‐producing cellular fractions in LLC‐bearing lungs. A novel CD11b⁺CD11c⁺ cell population was identified that accumulates in large numbers in NK‐depleted LLC‐bearing lung tissue. These CD11b⁺CD11c⁺ cells secreted large amounts of CCL22 that may overcompensate for the loss of NK‐derived CCL22 in the lungs of NK‐depleted LLC‐bearing mice. Taken together, these data suggest that NK cells play both a positive and negative role in the regulation of CCL22 secretion and, in turn, the recruitment of Tregs toward LLC‐bearing lungs.