Low Natural Killer Cell Count Research Articles

The Prognostic Value of Dynamic Change of NK-Cell Counts in Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Key words: Diffuse large B-cell lymphoma, Natural killer cell count, prognosis. Introduction Natural killer (NK) cell counts at diagnosis have been reported to be associated with poor outcomes, but the dynamic change and prognostic value of NK cell counts after treatment in diffuse large B-cell lymphoma (DLBCL)remain unknown. NK cell count was test by flow cytometry at diagnosis, interim and the end of treatment. Receiver operating characteristic (ROC) curve was used to determine the best cutoff value for survival analysis. Overall survival (OS) and progression-free survival (PFS) were analyzed by the method of Kaplan-Meier and Cox proportional hazard regression model. A total of 175 DLBCL patients were analyzed and 45 of them were divided into the low NK cell counts with the cut-off value was 94.5/ul. Patients with low NK cell counts were associated with B symptoms (p < 0.001), high LDH (p=0.002), advanced stage (p < 0.001) and high IPI scores (p=0.001). Survival analysis showed patients with low NK cell counts at diagnosis showed more inferior OS and PFS than those with high NK cell counts (p < 0.001 and p < 0.001). There were 60 patients showed low NK cell counts at the interim, consist of 31patients with low and 29 with high NK cell counts at diagnosis and 65 patients had low NK cell counts at the end of the treatment, consist of 31patients with low and 34 with high NK cell counts at diagnosis. But NK cell count at the interim or end of treatment had no impact on the PFS (p = 0.415 and p = 0.011) and OS (p = 0.537 and p = 0.836) of DLBCL patients. Multivariable analysis revealed that NK cell counts was an independent prognostic factor OS [relative risk (RR);95% confidence interval (CI),0.157-0.984, p = 0.046] and PFS (RR,0.335;95%CI,0.191-0.588, p < 0.001） in all DLBCL patients independent of IPI. In conclusion, low NK cell counts at diagnosis is a simple and effective adverse prognostic factor in DLBCL patients treated with R-CHOP. However, the NK cell counts at the interim and the end of the treatment showed no impact on outcome.

AFM24 in combination with autologous NK cells (SNK01) in patients with advanced/metastatic epidermal growth factor receptor (EGFR) expressing solid tumors: Initial results from the phase 1 dose-escalation study.

26 Background: AFM24 is a novel tetravalent bispecific IgG1-scFv fusion antibody (scFv-IgAb) binding specifically to EGFR on target cells and CD16A on NK cells, mediating specific lysis of EGFR+ tumor cells via NK cell-mediated antibody-dependent cellular cytotoxicity. SNK01 cell therapy is an NK cell-derived adoptive cellular immunotherapy technology that can produce large scale ex vivo expanded and activated autologous NK cells (1,000–10,000-fold expansion of NK cells with >95% cell purity), even in patients with low primary NK cell counts. The antitumor activity of SNK01 may be enhanced when combined with AFM24. Methods: This is a proof-of-concept phase 1, 3+3 dose escalation study to determine the maximum tolerated dose and/or recommended phase 2 dose of AFM24 in combination with SNK01 (NCT05099549). Secondary endpoints are safety/tolerability, preliminary efficacy and pharmacokinetics (PK). Patients with advanced or metastatic, histologically or cytologically confirmed EGFR+ solid tumors (≥1% of positive tumor cells) refractory to standard-of-care therapies were enrolled. AFM24 starting dose was 160 mg followed by 480 mg. AFM24 is administered once weekly, concomitantly with 4.0 x109 SNK01 cells once weekly until disease progression, intolerable toxicity, patient withdrawal. Cycles are 28 days and CTs are at screening, cycle 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of January 27, 2023, 6 patients (5 microsatellite stable colorectal and 1 head and neck cancer) received combination therapy, 3 in each cohort. Median (range) age was 59 (41–64), mean number (range) of prior therapies was 5 (3–7). ECOG performance status was 0 for 3 patients and 1 for the other 3. No dose limiting toxicities or unexpected toxicities were observed. Preliminary results suggest consistent safety events with both drugs in monotherapy. The most frequent treatment-emergent adverse events related to AFM24 was Grade 1/2 infusion related reactions occurring in 5/6 patients. There were no Grade ≥3 events related to AFM24 or SNK01. In the low dose cohort (160 mg), 2 patients had stable disease for 106 days (15 weeks) and 100 days (14 weeks), respectively. At 480 mg, 1 patient with SD had 2% tumor burden reduction and 2 subsequent carcinoembryonic antigen reductions. The patient proceeded to Cycle 3. No difference in PK data was observed in comparison to AFM24 alone. Enrollment continues at 480 mg. Conclusions: In this first study combining a bispecific NK cell engager with autologous NK cell infusions; two dose cohorts have completed, and escalation has proceeded. Early PK data are consistent with AFM24 monotherapy with no new safety signals, suggesting that the combination of AFM24 and SNK01 cells is tolerated and warrants further investigation. Clinical trial information: NCT05099549 .

High levels of NK Cells in graft are associated with reduced febrile neutropenia after haploidentical peripheral blood stem cell transplantation.

To investigate the impact of natural killer (NK) cells in the graft on the outcome following haploidentical peripheral blood stem cell transplantation (haplo-PBSCT). We retrospectively collected data from patients who had undergone haplo-PBSCT at our centre from January 2019 to November 2021. The percentage of NK cells in stem cell grafts was detected by flow cytometry. Based on the median (range) count of NK cells (1.8 [0.4-6.0] × 108/kg), patients were separated into high and low NK cell count groups. Data from 96 patients were analysed. Patients were evenly distributed (48 in each group) into high and low NK cell count groups. There was no significant difference in neutrophil and platelet recovery between the two groups. However, the rates of febrile neutropenia, bacterial infection, and invasive fungal disease (IFD) were significantly reduced in the high compared with the low NK cell count group. There was no significant difference in rates of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections between groups. There was no significant difference between groups in grades II and above acute graft versus host disease (GVHD), progression-free survival (PFS), or overall survival (OS). A high number of NK cells in the graft may reduce febrile neutropenia, IFD and bacterial infection following haplo-PBSCT but it has no significant effect on aGVHD, PFS, or OS.

Prognostic impact of peripheral natural killer cells in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal non-Hodgkin lymphoma with a poor prognosis. We aimed to evaluate the prognostic impact of circulating NK cells in PCNSL. Patients diagnosed with PCNSL who were treated at our institution between December 2018 and December 2019 were retrospectively screened. Patient variables including age, sex, Karnofsky performance status, diagnostic methods, location of lesions, lactate dehydrogenase, cerebrospinal fluids (CSF), and vitreous fluids involvement or not were documented. NK cell count and NK cell proportion (NK cell count/lymphocyte count) in the peripheral blood were evaluated by flow cytometry. Some patients underwent two consecutive NK cell tests before and three weeks after chemotherapy (before the next chemotherapy). The fold change in NK cell proportion and NK cell counts were calculated. CD56-positive NK cells in tumor tissue were assessed by immunohistochemistry. NK cell cytotoxicity assay was performed using flow cytometry. A total of 161 patients with PCNSL were included in this study. The median NK cell count of all NK cell tests was 197.73/μL (range 13.11-1889.90 cells/μL). The median proportion of NK cells was 14.11% (range 1.68-45.15%) for all. Responders had a higher median NK cell count (p<0.0001) and NK cell proportion (p<0.0001) than non-responders. Furthermore, Responders had a higher median fold change in NK cell proportion than non-responders (p=0.019) or patients in complete remission/partial remission (p<0.0001). A higher median fold change in NK cell count was observed in responders than in non-responders (p=0.0224) or patients in complete remission/partial remission (p=0.0002). For newly diagnosed PCNSL, patients with a high NK cell count (>165 cells/μL) appeared to have a longer median overall survival than those with a low NK cell count (p=0.0054). A high fold change in the proportion of NK cells (>0.1957; p=0.0367) or NK cell count (>0.1045; p=0.0356) was associated with longer progression-free survival. Circulating NK cells from newly-diagnosed PCNSL demonstrated an impaired cytotoxicity capacity compared to those from patients with PCNSL in complete remission or healthy donors. Our study indicated that circulating NK cells had some impact on the outcome of PCNSL.

Lenalidomide maintenance fails to overcome the unfavourable prognosis of low NK-cell counts in rituximab-chemotherapy responsive elderly DLBCL patients: A LYSA group study.

Low baseline NK-cell counts (NKCCs) in patients with diffuse large B-cell lymphoma (DLBCL) are associated with a poor prognosis. The REMARC phase III trial (NCT01122472) showed that lenalidomide maintenance prolonged PFS in rituximab-chemotherapy responders. We conducted a REMARC ancillary study analysing the impact of lenalidomide maintenance on the prognostic value of low NKCCs. Blood samples from 335 elderly French patients enrolled in the REMARC trial were analysed by flow cytometry to obtain NKCCs at diagnosis (n = 220), at randomization (n = 186) and/or six months after randomization (n = 184). Baseline NKCCs < 100 cells/μl were associated with shorter PFS and OS (HRs=[2.2 (1.4, 3.3), p < 0.001] and [2.8 (1.7, 4.5), p < 0.001], respectively), independently of aaIPI. In a competing risk analysis, low NKCCs at baseline were associated with a higher risk of relapse/progression (p=0.0025), but not of death without progression (p=0.33). Lenalidomide did not affect the prognosis value of low baseline NKCCs (p =0.6349). Similar results were obtained for low NKCCs at randomization. Our results demonstrate that low NKCCs at baseline and post rituximab-chemotherapy are robust prognostic factors in DLBCL and reveal that lenalidomide has no impact on this parameter. Other therapeutic strategies aiming at improving NK-cell function could improve outcomes in DLBCL.

A cell-based functional assay that accurately links genotype to phenotype in familial HLH

AbstractFamilial forms of the severe immunoregulatory disease hemophagocytic lymphohistiocytosis (HLH) arise from biallelic mutations in the PRF1, UNC13D, STXBP2, and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding biallelic mutations in patients with HLH symptoms and reduced natural killer (NK)–cell cytotoxicity. However, patients often have a low NK-cell count or receive immunosuppressive therapies that may render the NK-cell cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances in which carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11, and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of, otherwise cryptic, cases of FHL or HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of patients with HLH or FHL who inherit mutations of undetermined pathogenicity.

Blood Immune Cell Biomarkers in Lung Cancer Patients Undergoing Treatment with a Combination of Chemotherapy and Immune Checkpoint Blockade.

Simple SummaryTumor cells can evade destruction via immune cells by expressing coinhibitory membrane molecules, which can suppress tumor-specific T cells. Immune checkpoint inhibitor therapies act by blocking these inhibitory pathways via monoclonal antibodies. Although this type of immunotherapy has shown promising results for advanced cancers of different entities during recent years, an important challenge is to identify the baseline characteristics of patients who will mostly benefit from such treatment. Blood biomarkers have limitations to reflect the tumor microenvironment but are easier to handle than markers in tumor lesions. The aim of our study was to identify blood cell parameters correlating with patients’ survival in 90 patients with non-small cell lung cancer undergoing immune checkpoint inhibitor therapy combined with chemotherapy. We found that patients with a neutrophil-to-lymphocyte ratio ≥6.1, a percentage of HLA-DRlow monocytes ≥22%, a frequency of slan+ non-classical monocytes <0.25%, and/or of dendritic cells ≤0.14% of leukocytes had a poorer prognosis. Long-term survivors were patients without any of the risk factors investigated. Our results implicate that blood neutrophil counts, special types of monocytes, and the number of blood dendritic cells might be useful predictive biomarkers for cancer patients’ survival.Although immune checkpoint inhibitor (ICI) therapies have improved the treatment of patients with advanced non-small cell lung cancer (NSCLC), several patients do not achieve durable clinical responses. Biomarkers for the prediction of therapy responses are urgently needed. To identify blood cell parameters correlating with patients’ survival, immune cells from 90 patients with NSCLC undergoing a combination of ICI and chemotherapy were prospectively monitored. At the time point of the first and third antibody administration, complete leukocyte blood count, the percentage of HLA-DRlow monocytes, the percentage of 6-Sulfo LacNAc (slan)+CD16+ non-classical monocytes, and the number of circulating dendritic cell (DC) subtypes, as well as T-, B-, and NK cells were determined by multi-color flow cytometry in peripheral blood. The prognostic value of the immune cell parameters investigated was evaluated by patients’ survival analysis, with progression-free survival (PFS) as the main criterion. A total of 67 patients (74.4%) showed a partial remission or a stable disease, and 35% of patients even survived 12 months and longer. Patients with a neutrophil-to-lymphocyte ratio (NLR) ≥6.1, a frequency of HLA-DRlow monocytes ≥22%, of slan+ non-classical monocytes <0.25% of leukocytes, and/or a sum of myeloid DC (MDC) and plasmacytoid DC (PDC) ≤0.14% of leukocytes had a poorer prognosis. The hazard ratio for PFS was 2.097 (1.208–3.640) for the NLR, 1.964 (1.046–3.688) for HLA-DRlow monocytes, 3.202 (1.712–5.99) for slan+ non-classical monocytes, and 2.596 (1.478–4.56) for the MDC/PDC sum. Patients without any of the four risk factors showed the best PFS. Furthermore, low NK cell counts correlated with shorter PFS (cutoff 200 cells/µL). Female patients had lower baseline NK cell counts and a shorter PFS. Our study confirms the usefulness of blood immune cells as biomarkers for clinical response and survival in NSCLC patients undergoing a combined ICI/chemotherapy.

Peripheral blood natural killer cells in sarcoidosis are associated with early cardiac involvement

To evaluate the distribution of circulating immune cell subsets in peripheral blood of patients with sarcoidosis and investigate if there is an association with an underlying cardiac involvement. Eighty-five newly diagnosed treatment-naïve patients with sarcoidosis (50 women) were included in the study. All patients underwent a thorough cardiac investigation, including cardiac magnetic resonance imaging (CMR). Of all patients, 19 (23.53%) had myocardial involvement, and the NK subpopulation in these patients in peripheral blood was significantly decreased compared to patients without (n=63, p=0.001 and p=0.003 respectively). The absolute number of NKT cells (CD3+CD16/56+ ) in patients with cardiac involvement was highly correlated with T2map increased values in MRI (r=-686, p=0.041) showing that low NKT cell count correlates with the inflammatory process of the heart. No difference in CD19, CD3, CD4, CD8 and CD3- NK cell counts was found between groups. Lung severity was not found to correlate with the number of NK cells. We found that low NK cell count in peripheral blood of patients with sarcoidosis is associated with cardiac involvement, and the number of NK-T cells correlates with CMR findings indicative of myocardial inflammation. This finding might have a potential clinical application in detecting clinically silent cardiac involvement in sarcoidosis and may also suggest potential targets for therapeutic interventions.

Metabolic and immunological phenotype of rare lipomatoses: Dercum\u2019s disease and Roch-Leri mesosomatic lipomatosis

ContextDercum’s disease (DD) and Roch-Leri mesosomatic lipomatosis (LMS) are rare and poorly characterized diseases. The clinical presentation combines multiple lipomas, painful in DD in contrast with LMS, without lipoatrophy.ObjectiveTo identify any specific metabolic and immune phenotype of DD and LMS.Design and patientsThis monocentric retrospective study included 46 patients: 9 DD, 11 LMS, 18 lean and 8 obese controls. Metabolic and immunohematological characteristics of each group were compared.ResultsThe median age of the patients was similar in the 3 groups (31 years). The number of women, and of basophils, and CD3+, CD4+ and CD8+ T lymphocytes was significantly higher in the DD versus the LMS group, without any difference of the metabolic parameters. Weight, BMI, blood pressure, gamma-GT, leptin, fasting insulin and C-peptide levels, fat mass percentage, and intra/total abdominal fat ratio were significantly higher in each lipomatosis group compared with the lean group. Compared with the lean group, the DD group had significantly higher fasting blood glucose, LDL-cholesterol, platelets, leukocytes, basophils, and a lower NK cell count, whereas the LMS group had a significantly lower rate of CD3, CD4, and CD8 lymphocytes. Compared with the obese controls, basophils remained higher in DD and T lymphocytes subpopulations lower in LMS groups.ConclusionDD and LMS show a common background of obesity and metabolic phenotype, but a distinct immunohematological profile characterized by a higher number of basophils in DD patients, an inflammatory profile that could contribute to pain. T lymphocyte depletion was present in LMS. These findings could offer specific therapeutic opportunities, especially for painful DD.

Extensive Immunosuppression in the Acute Myeloid Leukemia (AML) Bone Marrow and Discovery of NK Cells As Biomarker of Superior Survival

BackgroundThe immune system has been harnessed in the treatment of AML patients by eliciting the graft-versus-leukemia response after hematopoietic stem cell transplant (HSCT). Recently, several other immunotherapy modalities such as immune checkpoint inhibitors, leukemia antigen-targeted antibodies, and adoptive cell therapy have been studied with hope for less toxic and more effective alternatives to conventional chemotherapeutic regimes. Yet, little is known on the clinical significance of the pretreatment immune microenvironment. Here, we characterized the immunologic landscape of the AML bone marrow (BM) with a novel multiplex immunohistochemistry (mIHC) method to identify prognostic immune biomarkers.MethodsDiagnostic-phase BM biopsies of AML patients (n=69) treated in the Helsinki University Hospital during 2005-2017 and non-leukemic controls (n=14) were collected. Using hematopathologic expertise, tissue microarrays (TMA) were constructed from duplicate BM spots and analyzed using mIHC combining simultaneously up to six out of 25 examined lymphoid, myeloid, and immune checkpoint marker. We complemented mIHC with machine learning-based image analysis. Immune cell subsets were compared with Mann-Whitney U test, and p-values adjusted with the Benjamini-Hochberg procedure (q-values). By combining mIHC findings with extensive diagnostic-phase demographic, peripheral blood and BM aspirate laboratory test, and treatment data, we developed a risk stratification model predicting relapse-free survival (RFS) using bootstrapped L1-penalized elastic net Cox regression. We validated results with diagnostic-phase AML BM aspirates (n=84) analyzed with flow cytometry (FC) in the centralized university hospital clinical laboratory (2005-2016).ResultsUnsupervised hierarchical clustering of integrated immune profiles separated AML patients distinctly from control subjects. In AML patients, immune cell subsets and phenotypes associated with anti-cancer immunity were decreased (e.g. lower granzyme B and CD57 expression in CD3+CD4+ and CD3+CD8+ T cells), while features of immunosuppression were accentuated (e.g. higher proportion of FOXP3+ CD4+ T cells and lower expression of class I HLA-ABC in all BM cells).Higher levels of T cell exhaustion markers PD1+ in both CD4+ and CD8+ T cells (15.7% vs. 1.7% and 13.2% vs. 2.0%, q<0.001 for both), and CTLA4+ (2.0% vs. 0.9%, q=0.02) in CD4+ T cells were noted in AML BM.Lower level of CD68+/pSTAT1+cMAF- (0.033% vs. 5.9%, q<0.001) and higher CD68+/pSTAT1-cMAF+ (24.8% vs. 1.7%, q<0.001), e.g. M1 and M2-polarized macrophages, were observed in AML vs. control BM, thus shifting the M1/M2 ratio from 3.5 in control to 0.0013 in AML BM. In addition, higher amount of CD11b+CD33+HLADR-, e.g. myeloid-derived suppressor cells (MDSC), were observed in AML BM (1.0% vs. 0.038% of all cells, q<0.001).The most predictive features from the L1-penalized regression analysis were selected with multivariate Cox regression. High patient age and low amount of CD3-CD56+ (NK) cells were significantly associated with worse RFS both in univariate and multivariate setting after adjusting for induction therapy protocol (AUC 0.84 and R2 0.45, p<0.001 log-rank test). Interestingly, the model predicted also overall survival (OS) (AUC 0.86 and R2 0.38, p<0.001). The model was categorized into even-sized low, intermediate (hazard ratio (HR) 2.5, 95%CI [1.1-5.6]) and high-risk groups (HR 8.3, 95%CI [3.7-18.8]) to predict RFS (Fig 1).In the independent validation cohort, CD45+CD2+CD3- (NK) cell count was categorized into even-sized groups based on tertiles. Low and intermediate NK cell count (<0.75% and 0.75-2.0% of all cells, respectively) predicted worse RFS (HR 3.3 p=0.005 and HR 2.2 p=0.05) and OS (HR 2.6 p=0.03 and HR 2.3 p=0.05) after adjusting for induction therapy protocol and HSCT.ConclusionTissue cytometrics of TMA slides with mIHC and automated image analysis enable fast and objective single-cell phenotyping of large patient cohorts. The AML BM is characterized by myeloid and lymphoid lineage immunosuppression. Results of immune cell analyses with both mIHC and FC suggest low NK cell count as biomarker of poor RFS and OS. Other treatment response and survival endpoints remain to be analyzed and complemented with extensive flow cytometric, cytogenetic and molecular genetic data. [Display omitted] DisclosuresPorkka:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Blood CD3-(CD56 or 16)+ natural killer cell distributions are heterogeneous in healthy adults and suppressed by azathioprine in patients with ANCA-associated vasculitides

BackgroundCytotoxic Natural Killer (NK) cells are increasingly recognized as a powerful tool to induce targeted cell death in cancer and autoimmune diseases. Still, basic blood NK cell parameters are poorly defined. The aims of this study were 1) to establish reference values of NK cell counts and percentages in healthy adults; 2) to describe these parameters in the prototype autoimmune disease group ANCA-associated vasculitis (AAV); and 3) to investigate whether NK cell counts and percentages may be used as activity biomarkers in the care of AAV patients, as suggested by a preceding study.MethodsCD3-(CD56 or 16)+ NK cell counts and percentages were determined in 120 healthy adults. Lymphocyte subset and clinical data from two German vasculitis centers were analyzed retrospectively (in total 407 measurements, including 201/49/157 measurements from 64/16/39 patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), respectively).ResultsCD3-(CD56 or 16)+ NK cell counts and percentages in healthy adults were highly variable, not Gaussian distributed and independent of age and sex. NK cell percentages ranged from 1.9 to 37.9% of lymphocytes, and were significantly more dispersed in AAV (0.3 to 57.6%), while the median percentage was not different between AAV and healthy donors. In contrast, median NK cell counts were significantly lower in AAV compared to healthy donors. Sub-group analyses revealed that NK cell counts were low independent of AAV entity and disease activity. Azathioprine therapy was associated with significantly lower NK cell counts and percentages compared to non-azathioprine therapies. In 13.6% of azathioprine-treated patients, percentages were </= 1% which may be interpreted as temporary NK cell deficiency. NK cell counts and percentages could not separate active from inactive AAV.ConclusionsNK cell counts and percentages in blood are heterogeneous and can presently not be recommended as biomarker in clinical care of AAV patients. Azathioprine treatment was associated with significantly low NK cells. These findings may be relevant for the development of drugs that aim at exploiting NK cell cytotoxicity and may help to identify patients at risk to develop malignant or infectious co-morbidities.

Association of Epstein-Barr virus infection with peripheral immune parameters and clinical outcome in advanced nasopharyngeal carcinoma

The purpose of this study was to investigate the association of Epstein-Barr virus (EBV) with peripheral blood immune cell counts and clinical outcomes in advanced nasopharyngeal carcinoma (NPC) patients. In a retrospective design, 146 patients with NPC at stage IV were enrolled in this study. The association of EBV status with peripheral blood immune cell counts, distant metastases, and long-term survival in patients with advanced NPC were determined. Eighty-seven (59.6%) of all patients were positive for EBV. Compared with patients with normal NK cell count, patients with lower NK cell count showed a significantly lower EBV viral load (median: 614.0 vs. 2190.0 copies/mL, P = 0.024). EBV-positive patients showed a significantly higher incidence of liver metastasis than EBV-negative patients (32.6% vs. 23.7%, P = 0.021). Multi-variant regression analysis showed that EBV infection was independently associated with liver metastasis (OR: 2.33, P = 0.043). EBV positive patients showed a significantly worse PFS (P = 0.001) and OS (P = 0.001) than EBV negative patients. Multivariate Cox regression analysis revealed that EBV infection was independently associated with a worse PFS (HR: 1.94, P = 0.003), and OS (HR: 2.12, P = 0.014) in advanced NPC. In conclusion, EBV infection is associated with a high risk of liver metastasis and is also an independent negative predictor for PFS and OS in patients with advanced NPC. EBV infection is associated with lower CD8% and higher NK%, while lower NK cell count is associated with lower EBV viral load.

Blood natural killer cells during treatment in recurrent ovarian cancer

Objective Recent research indicated favorable prognostic impact of intratumoral natural killer (NK) cells in ovarian carcinoma (OC). The role of NK cells during chemotherapy in OC is unknown. We investigated impact of NK cells in OC patients treated with palliative chemotherapy. Methods Participants receiving palliative chemotherapy for recurrent OC (N = 72) had prospectively blood samples at baseline and before cycle 2. NK cell counts were quantified by flow cytometry. NK cell activity was measured by the NK Vue® assay, estimating interferon-gamma production. Overall survival (OS) was the primary endpoint. Cutoffs were predefined, NK numbers (≥184 × 106 cells/L vs. <184 × 106 cells/L) and NK activity (<200 pg/mL vs. ≥200 pg/mL). Results Median OS in patients with low vs. high NK cell count at baseline was 7.1 months vs. 15.6 months (p = .028), respectively, and before cycle 2 was 5.7 vs. 17.3 months, p < .001, respectively. The difference in restricted mean survival (ΔRMST) was 5.7 months (95% CI: 3.3–8.0) at cycle 2 vs. 2.5 months (95% CI: −0.6 to 5.6) at baseline, showing a significant difference with no overlap of confidence intervals. In multivariate analyses, low NK cell count remained significant with a hazard ratio (HR)=2.83, 95% CI: 1.53–5.22, p = .001 (baseline) and HR = 3.34, 95% CI: 1.67–6.71, p = .001 (before cycle 2). Patients with both low NK count and NK activity at baseline (N = 20) had median OS 6.5 months vs. 11.5 months in patients with either high activity, high count or both (p = .007). In parallel, patients with both low NK activity and count at cycle 2 (N = 18) had a median survival of 4.0 months vs. 15.4 months (p < .001). Conclusions A low blood NK cell count in recurrent metastatic ovarian cancer during chemotherapy is associated with unfavorable prognostic impact. Early increase in survival difference based on NK cell status suggests an association between NK cell count and treatment benefit.

Correlation between emotional regulation and peripheral lymphocyte counts in colorectal cancer patients.

BackgroundColorectal cancer is one of the most common cancers worldwide. Psychological morbidity has an important impact on quality of life and major clinical outcomes. Several data have shown that the immune system may be a key player on the relation between psychological features and cancer outcomes. Natural Killer (NK) cells have been shown to be influenced by psychological factors. The aim of this investigation was to assess the impact of anxiety, depression, and anger state, trait, and expression on the immune response, particularly, their effect on NK cells and CD8+ T cells in surgical colorectal cancer patients.MethodsWe studied 54 surgical colorectal cancer patients and assessed patients pre-surgically, post-surgically, and 12 months after surgery (follow-up). We applied the Hospital Anxiety and Depression Scale and the State-Trait Anger Expression Inventory and measured peripheral T cells, CD8+ T cells, and NK cells. We did a cross-sectional analysis as well as a longitudinal assessment of the variables during the follow-up period.ResultsPre-surgical assessment: Trait anger, angry reaction, and anger-out had a significant negative correlation with NK cells. The lymphocytes values were unaffected by the presence of clinical anxiety or depression. Post-surgical assessment: Patients without clinical anxiety had higher levels of T cells. Angry reaction was negatively correlated with NK cells. Lymphocytes values were unaffected by the presence of clinical depression. Follow-up assessment: Patients without clinical depression had higher T cell counts. Trait anger and angry reaction were negatively correlated with the levels of NK cells. The lymphocytes values were unaffected by the presence of clinical anxiety. Longitudinal assessment: Angry-temperament, anger expression, and anger-in reduced significantly from the first to the second assessment. Anxiety, state anger, and trait anger significantly diminished from the pre-surgical to the follow-up assessment. Depression levels did not alter during the follow-up period. The lymphocyte count, and particularly T cells and CD8+ T cells, was significantly higher in the follow-up when compared with the pre-surgical assessment.ConclusionOur study suggests the existence of a relation between psychological response and immune response in colorectal cancer patients. We identified the importance of emotional regulation as a potential modulator for NK cell counts. Higher values of propensity to experience anger states and express them outwards seem to be associated with lower NK cell counts.

A Phase 1, Open-Label, Multi-Center, Dose Escalation and Dose Expansion Study of NKTR-255 As a Single Agent in Relapsed or Refractory Hematologic Malignancies and in Combination with Daratumumab As a Salvage Regimen for Multiple Myeloma

A Phase 1, Open-Label, Multi-Center, Dose Escalation and Dose Expansion Study of NKTR-255 As a Single Agent in Relapsed or Refractory Hematologic Malignancies and in Combination with Daratumumab As a Salvage Regimen for Multiple Myeloma

Effect of Plasma-Derived Exosomes of Refractory/Relapsed or Responsive Patients with Diffuse Large B-Cell Lymphoma on Natural Killer Cells Functions.

Objective The purpose of this study was to investigate effect of plasma-derived exosomes of refractory/relapsed or responsive diffuse large B-cell lymphoma (DLBCL) patients on natural killer (NK) cell functions. Materials and Methods In this cross-sectional and experimental study, NK cells were purified from responsive patients (n=10) or refractory/relapsed patients (n=12) and healthy donors (n=12). NK cells were treated with plasma-derived exosomes of responsive or refractory/relapsed patients. We examined the expression levels of hsa-miR-155-5p, hsa- let-7g-5p, INPP5D (SHIP-1) and SOCS-1 in NK cells quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Percentages of NK cells expressing CD69, NKG2D and CD16, NK cell cytotoxicity and NK cell proliferation (using flow-cytometry) as well as interferon-gamma (IFN-γ) level in the supernatant of NK cells using ELISA were also investigated. Results We observed an increased level of hsa-miR-155-5p and a decreased level of SOCS-1 in NK cells treated with exosomes compared to untreated NK cell in healthy donors and DLBCL patients. An increase in hsa-miR-155-5p level was associated with an increased level of IFN-γ in healthy donors. The decreased levels of hsa-let-7g-5p were observed in NK cells treated with exosomes in comparison with untreated NK cells in DLBCL patients (P<0.05). There was no significant difference in the percentage of CD69+NK cells and NKG2D+ NK cells in the absence or presence of exosomes of DLBCL patients in each group. Furthermore, we observed significant reduction of NK cell proliferation in DLBCL patients and healthy donors in the presence of exosomes of refractory/relapsed patients (P<0.05). A significant decrease was observed in cytotoxicity of NK cell in patients with DLBCL treated with exosomes of responsive patients. ConclusionOur findings demonstrated adverse effect of plasma-derived exosomes of DLBCL patients on some functions of NK cell. It was also determined that low NK cell count might be associated with impaired response to R-CHOP and an increased recurrence risk of cancer.

Prognostic Impact of Natural Killer Cell Count in Follicular Lymphoma and Diffuse Large B-cell Lymphoma Patients Treated with Immunochemotherapy.

Natural killer (NK) cells are key effector cells for anti-CD20 monoclonal antibodies (mAb), such as obinutuzumab and rituximab. We assessed whether low pretreatment NK-cell count (NKCC) in peripheral blood or tumor tissue was associated with worse outcome in patients receiving antibody-based therapy. Baseline peripheral blood NKCC was assessed by flow cytometry (CD3-CD56+ and/or CD16+ cells) in 1,064 of 1,202 patients with follicular lymphoma treated with obinutuzumab or rituximab plus chemotherapy in the phase III GALLIUM trial (NCT01332968) and 1,287 of 1,418 patients with diffuse large B-cell lymphoma (DLBCL) treated with obinutuzumab or rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (G-CHOP or R-CHOP) in the phase III GOYA trial (NCT01287741). The prognostic value of tumor NK-cell gene expression, as assessed by whole-transcriptome gene expression using TruSeq RNA sequencing, was also analyzed. The association of baseline variables, such as treatment arm, was evaluated using multivariate Cox regression models using a stepwise approach. In this exploratory analysis, low baseline peripheral blood NKCC was associated with shorter progression-free survival (PFS) in both follicular lymphoma [hazard ratio (HR), 1.48; 95% confidence interval (CI), 1.02-2.14; P = 0.04] and DLBCL (HR, 1.36; 95% CI, 1.01-1.83; P = 0.04), and overall survival in follicular lymphoma (HR, 2.20; 95% CI, 1.26-3.86; P = 0.0058). Low tumor NK-cell gene expression was associated with shorter PFS in G-CHOP-treated patients with DLBCL (HR, 1.95; 95% CI, 1.22-3.15; P < 0.01). These findings indicate that the number of NK cells in peripheral blood may affect the outcome of patients with B-cell non-Hodgkin lymphoma receiving anti-CD20-based immunochemotherapy.

Increased heat shock protein 70 (Hsp70) serum levels and low NK cell counts after radiotherapy \u2013 potential markers for predicting breast cancer recurrence?

BackgroundBreast cancer is the most common invasive tumor in women worldwide and the second cause of cancer-related deaths. After breast conserving surgery the tumor bed gets irradiated. Radiation-induced tumor cell death has been found to be associated with the release of damage-associated molecular patterns (DAMPs) including free Hsp70 that can stimulate inflammatory immune responses. Therefore, Hsp70 serum levels as well as the composition of lymphocyte subpopulations have been measured in breast cancer patients during therapy and in the follow-up period as potential predictors for clinical outcome.MethodsThe serum of 40 breast cancer patients, who received a breast-conserving surgery and adjuvant radiotherapy (RT) was examined for soluble, free Hsp70 using the R&D Human HSP70 DuoSet and lipHsp70 ELISA. Lymphocyte subpopulations and total lymphocyte counts were analysed by multiparameter flow cytometry in the peripheral blood. Blood samples were collected before (t1), after 30 Gy (t2) and 60 Gy (t3), 6 weeks (t4), 6 months (t5) and 1 year (t6) after RT. Clinical responses were assessed regularly up to 5 years after RT.ResultsPatients who developed a contralateral recurrence or metastases within the first 2 years after RT had significantly higher serum Hsp70 values at the end of RT (t3; p = 0.03) up to 6 weeks after RT (t4; p = 0.007) compared to patients who either remained disease-free or developed a secondary endometrial carcinoma. Clinicopathological parameters such as age, tumor size, grading and TNM-stage of the resected tumors, adjuvant chemotherapy and irradiation dose did not affect serum Hsp70 levels. Elevated free Hsp70 levels might be indicative for a chronic inflammatory response which could support tumor recurrence. Lymphocyte subpopulation analysis revealed lower NK cell counts after RT in recurrence/metastases patients as compared to disease-free patients. In contrast, no significant changes were observed in the proportion of T and B cells.ConclusionLongitudinal elevated serum levels of free Hsp70 up to 6 weeks after RT and dropping NK cell counts might be predictive for an unfavourable prognosis in patients with breast cancer.

A nearly fatal primary Epstein-Barr virus infection associated with low NK-cell counts in a patient receiving azathioprine: a case report and review of literature

BackgroundSymptomatic primary Epstein-Barr virus infection is a usually self-limiting illness in adolescents. We present a case of an adolescent who had been receiving azathioprine for inflammatory bowel disease for four years and developed a life-threatening primary Epstein-Barr virus infection successfully treated with rituximab.Case presentationAn 11-year-old girl presented with chronic, bloody diarrhea. Endoscopic biopsies confirmed a diagnosis of chronic ulcerative colitis with features of Crohn’s disease. Azathioprine was initiated after one year due to active colitis. She responded well and remission was achieved. At the age of 16 years she developed a life-threatening Epstein-Barr virus infection including severe multiple organ failure and was critically ill for 4 weeks in the intensive care unit. Natural killer cells were virtually absent in the lymphocyte subset analysis. Azathioprine was stopped on admission. She was initially treated with corticosteroids, acyclovir and intravenous immunoglobulin. Approximately 30 days after admission, she developed signs of severe hepatitis and pneumonitis and received weekly rituximab infusions for 8 weeks. Primary immunodeficiency was excluded by whole exome sequencing in two independent laboratories. Persistent viremia stopped when the natural killer cell count started to rise, approximately 90 days after the cessation of azathioprine.ConclusionsWe found 17 comparable cases in the literature. None of the previous cases reported in the literature, who had been treated with azathioprine and developed either a severe or a fatal Epstein-Barr virus infection, underwent full genetic and prospective immunological workup to rule out known primary immunodeficiencies. Recently, azathioprine has been shown to cause rather specific immunosuppression, resulting in natural killer cell depletion. Our case demonstrates that slow recovery from azathioprine-induced natural killer cell depletion, 3 months after the stopping of azathioprine, coincided with the clearance of viremia and clinical recovery. Finally, our choice of treating the patient with rituximab, as previously used for patients with a severe immunosuppression and Epstein-Barr virus viremia, appeared to be successful in this case. We suggest testing for Epstein-Barr virus serology before starting azathioprine and measuring natural killer cell counts during the treatment to identify patients at risk of developing an unusually severe primary Epstein-Barr virus infection.

Low absolute NK cell counts in peripheral blood are associated with inferior survival in patients with mantle cell lymphoma.

Although risk stratification of mantle cell lymphoma (MCL) is most frequently performed using the simplified MCL International Prognostic Index (sMIPI), the identification of host-related factors and tumor microenvironment, including absolute monocyte counts (AMC) and peripheral blood T lymphocyte subsets, especially absolute natural killer cell counts (ANKC) has been suggested to be critical in the prediction of prognosis and the guidance of treatment. This study was aimed at investigating whether peripheral blood ANKC and AMC at diagnosis had an impact on MCL prognosis. A total of 92 newly diagnosed MCL patients was enrolled in this retrospective study. Flow cytometric analysis was conducted on fresh peripheral blood samples with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The median follow-up was 42 months (range, 2-144 months) and the median overall survival (OS) of all cases was 45 months. High AMC (> 0.6 × 109/L) was the parameter associated with inferior progression free survival (PFS) (P= 0.044) and poor OS (P= 0.028) while low ANKC (⩽ 0.1 × 109/L) was associated with unfavorable OS (P= 0.023) by univariable analysis. Multivariable analysis revealed that only low ANKC (⩽ 0.1 × 109/L) was statistically significant in worse OS (P= 0.009) independent of sMIPI. Low ANKC (⩽ 0.1 × 109/L) proved to be a significant predictor of inferior OS in patients with MCL.