Higher Natural Killer Cell Research Articles

Prognostic significance of early nk cell recovery in pediatric t-cell replete allogeneic hematopoietic stem cell transplantation.

Early immune reconstitution, particularly of natural killer (NK) cells with strong graft-versus-leukemia effects, is crucial for the prognosis of patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). This retrospective study examined 122 pediatric patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) who underwent T-cell replete allo-HSCT at the Children's Hospital of Soochow University from 2019 to 2021. Peripheral blood lymphocyte counts on days 30 and 60 post-transplant were analyzed, focusing on NK cell recovery and its impact on overall survival (OS), relapse-free survival (RFS), non-relapse mortality (NRM), relapse, graft-versus-host disease, and CMV reactivation. Patients were categorized into high and low NK cell groups by the median NK cell counts at day 30 (NK30) and day 60 (NK60) after HSCT. Patients with high NK30 and NK60 levels had significantly better 3-year OS rates compared to their respective low NK count groups (86.8% ± 4.7% versus 67.6% ± 7.0%, P = 0.046; 95.7% ± 3.0% versus 63.7% ± 8.7%, P < 0.001, respectively). NK60 was found to be an independent predictor for 3-year OS and RFS in multivariate analysis. Early NK reconstitution was also related to lower NRM rates. However, this correlation disappeared in patients with ALL and those undergoing haploidentical transplantation. In addition, A positive association between early T cell and NK cell reconstitution was found in our study. Higher NK cell counts on days 60 can predict superior OS, RFS and lower NRM in pediatric AML allo-HSCT patients.

Effect of preoperative natural killer cell on postoperative pulmonary complications in patients of lung cancer − A single-center retrospective cohort study

BackgroundThe effect of preoperative natural killer (NK) cell abnormalities on postoperative pulmonary complications (PPCs) after thoracoscopic radical resection of lung cancer is still unclear. The main purpose of this study was to investigate the relationship between the preoperative NK cell ratio and PPCs. MethodsThe patients who underwent thoracoscopic radical resection for lung cancer were divided into a normal group and an abnormal group according to whether the proportion of preoperative NK cells was within the reference range. The main outcome was the incidence of PPCs during postoperative hospitalization. The demographic and perioperative data were collected. Propensity score matching was used to exclude systematic bias. Univariate logistic regression was used to test the relationship between the preoperative NK cell ratio and the incidence of PPCs. The restrictive cubic spline curve was used to analyze the dose–effect relationship between the preoperative NK cell ratio and the incidence of PPCs. ResultsA total of 4161 patients were included. After establishing a matching cohort, 910 patients were included in the statistical analysis. The incidence of PPCs in the abnormal group was greater than that in the normal group (55.2% vs. 31.6%). The incidence of PPCs first decreased and then increased with increasing NK cell ratio. The proportion of patients with Grade 3 or higher PPCs in the normal group was lower than that in the abnormal group [108 (23.7%) vs. 223 (49%)]. The indwelling time of the thoracic drainage tube in the abnormal group was longer than that in the normal group [3 (3, 4) vs. 3 (3, 5)]. A preoperative abnormal NK cell ratio constituted a risk factor for PPCs in each subgroup. ConclusionLung cancer patients with an abnormal proportion of peripheral blood NK cells before surgery were more likely to develop PPCs, their disease degree was more severe, and they had a prolonged duration of chest tube indwelling. Compared with those with abnormally high NK cell ratios, those with abnormally low NK cell ratios had more pronounced PPCs.

Density of tumor-infiltrating NK and Treg cells is associated with 5 years progression-free and overall survival in resected lung adenocarcinoma

Surgical resection of pulmonary adenocarcinoma is considered to be curative but progression-free survival (PFS) has remained highly variable. Antitumor immune response may be important, however, the prognostic significance of tumor-infiltrating natural killer (NK) and regulatory T (Treg) lymphocytes is uncertain. Resected pulmonary adenocarcinoma tissues (n = 115) were studied by immunohistochemical detection of NKp46 and FoxP3 positivity to identify NK and Treg cells, respectively. Association of cell densities with clinicopathological features and progression-free survival (PFS) as well as overall survival (OS) were analyzed with a follow-up time of 60 months. Both types of immune cells were accumulated predominantly in tumor stroma. NK cell density showed association with female gender, non-smoking and KRAS wild-type status. According to Kaplan-Meier analysis, PFS and OS proved to be longer in patients with high NK or Treg cell densities (p = 0.0293 and p = 0.0375 for PFS, p = 0.0310 and p = 0.0448 for OS, respectively). Evaluating the prognostic effect of the combination of NK and Treg cell density values revealed that PFS and OS were significantly longer in NKhigh/Treghigh cases compared to the other groups combined (p = 0.0223 and p = 0.0325, respectively). Multivariate Cox regression analysis indicated that high NK cell density was independent predictor of longer PFS while high NK and high Treg cell densities both proved significant predictors of longer OS. The NKhigh/Treghigh combination also proved to be an independent prognostic factor for both PFS and OS. In conclusion, NK and Treg cells can be components of the innate and adaptive immune response at action against progression of pulmonary adenocarcinoma.

Circulating Immune Cells Predict Prognosis and Clinical Response to Chemotherapy in Cholangiocarcinoma.

The immune system is linked to the prognosis and response to treatment of patients with cancer. However, the clinical implication of peripheral blood immune cells in cholangiocarcinoma (CCA) remains vague. Thus, we aimed to assess whether peripheral circulating immune cells could be used as an indicator for prognosis and chemotherapeutic efficacy in CCA. The distributions of immune subsets were analyzed in peripheral blood samples from 141 patients with CCA and 131 healthy volunteers by using flow cytometry. The variation in the subset distribution in the two groups and the relationship between clinicopathological features and the subpopulations were investigated. Meanwhile, we assessed the implications of lymphocyte subsets as predictors of chemotherapy outcomes and overall survival (OS). The proportion of total lymphocytes decreased, while the percentages of activated T cells as well as CD4+CD25+ regulatory T cells (Tregs) increased in CCA. Notably, lymphocyte proportion decreased in patients with regional lymph node (N) (p=0.016) and distant metastasis (M) (p= 0.001). Furthermore, our study showed that peripheral blood lymphocyte subsets were significantly correlated with chemotherapy efficacy, with increased proportions of CD3+ cells (p=0.021) and CD4+ cells (p=0.016) in the effective group. Finally, the Kaplan-Meier analysis indicated that patients with high natural killer (NK) cell proportion might have prolonged OS (p = 0.028). The relationship between circulating immune cells with prognosis and chemotherapy response in patients with CCA highlights their potential application as an indicator of CCA prognosis and stratification of chemotherapy response.

Role of High Endometrial Natural Killer Cell Concentration in Patients with Recurrent Miscarriage

Role of High Endometrial Natural Killer Cell Concentration in Patients with Recurrent Miscarriage

Abstract 1241: Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE)

Abstract With an annual incidence rate exceeding 660,000 cases, and a death toll surpassing 325,000 per year, head and neck cancer (HNC) ranks as the seventh most common cancer in the world. Surgery, radiation and chemotherapy are used to treat HNC patients with modest and variable clinical success. However, these treatments prove less effective for human papillomavirus negative (HPV-) HNC patients, a subset of HNC patients with markedly worse prognosis. While significant advancements have been made in cancer immunotherapy over the past decade, its success also remains elusive for HNC due to factors such as the hypoxic solid tumor microenvironment (TME). To address the critical need for an improved therapeutic intervention, we leveraged the ability of natural killer (NK) cells in killing cancer cells without prior sensitization by developing a novel tri-specific killer engager (TriKE) that can direct NK cell killing of tumor within the hypoxic solid TME. The TriKE is composed of three domains: a humanized nanobody arm binding the activating receptor CD16 on NK cells, an interleukin (IL)-15 moiety that can drive expansion of NK cells, and a nanobody arm binding B7H3, a protein which high expression can be negatively correlated with overall survival of HPV- HNC patients. B7H3 is a prime target candidate because it is highly expressed on HPV- HNC cells, but minimally expressed on healthy tissues. In vitro testing using HPV- HNC patient blood samples revealed that B7H3 TriKE enhances activation (measured by NK cell degranulation and interferon-gamma production) and expansion of NK cells from these patients at levels equivalent to those observed in healthy controls. Furthermore, B7H3 TriKE is efficacious in its ability to drive high NK cell cytotoxicity in both acute (&amp;lt;48-hours) and prolonged (7-days) hypoxic (1% oxygen) models of HNC. Under acute hypoxia, B7H3 TriKE induces significantly more killing of targets by NK cells compared to IL-15 treatment, where NK cell cytotoxicity is impaired. In addition, B7H3 TriKE can boost the killing efficacy of NK cells exposed to prolonged hypoxia, surpassing limitations seen with IL-15 treatment. These findings strongly suggest that the B7H3 TriKE can bypass hypoxic suppression of NK effector functions in the solid TME. In vivo studies using immunocompromised mice engrafted with HPV- HNC cells revealed that B7H3 TriKE treatment significantly extends the survival of mice, compared to IL-15 treatment. Moreover, NK cells persisted in the blood of B7H3 TriKE-treated mice 28 days post-NK cell injection, highlighting the promising clinical translation of this immunotherapy. More in-depth characterization of NK cells from HPV- HNC patients are underway but altogether, these robust pre-clinical data present a novel avenue for the management of HNC for these patients. We plan to translate results from these studies to clinical trials in fall 2024. Citation Format: Melissa Khaw, Nicholas A. Zorko, Carly Selleck, Laura Bendzick, Zachary Davis, Peter Hinderlie, Madison Shackelford, Ann Lu, James Lim, Naomi Fujioka, Margaret MacMillan, John Wagner, Martin Felices, Jeffrey S. Miller. Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1241.

Abstract P113: Associations of Immune Cell Subsets With Coronary Artery Calcium Incidence and Progression in the Multi-Ethnic Study of Atherosclerosis

Introduction: Coronary artery calcium (CAC) and its progression strongly associates with incident cardiovascular disease (CVD). Although immune dysregulation and related inflammation are important causes of coronary artery disease, few data exist on associations between specific immune cell subsets and CAC presence and progression. Hypothesis: We hypothesized innate &amp; adaptive immune cell subsets are associated with longitudinal CAC changes Methods: In the Multi-Ethnic Study of Atherosclerosis (MESA), we examined associations of immune cell subsets (measured at baseline in peripheral blood by flow cytometry) with incident CAC and CAC progression. Of 975 participants sampled who had CAC and immune cell subsets measured at baseline (2000-02), 378 had CAC 0 at baseline and were used to examine incident CAC from MESA exam 2 (2002-04) through 5 (2010-12); the remaining 597 participants were included to analyze CAC progression from exam 1-5. Incident CAC analyses (N=378) used Weighted Cox regressions to determine hazard ratios of incident CAC, adjusted for age, gender, race, MESA site, education, smoking status, blood pressure, body-mass index, diabetes status, cholesterol, HDL cholesterol, statin use, and antihypertensive medication. CAC progression analyses among individuals with nonzero CAC at baseline (N=597) used weighted multivariable-adjusted linear mixed models to investigate associations of baseline immune cell subsets with CAC progression, with Benjamini-Hochberg correction for multiple comparisons. Results: Among 378 participants with CAC 0 and immune cell subsets measured at baseline, 149 had incident CAC between MESA examination 2-5. Of 19 subsets measured, natural killer (NK) cells (CD3-CD16+CD56+) were associated with an elevated risk of incident CAC (hazard ratio (HR) 1.26 per standard deviation (SD) higher NK cells; 95% confidence interval (CI) 1.03-1.56, p=0.03). Two CD4+ T cell subsets, Th1 (HR 0.81, 95% CI 0.66-0.99, p=0.04) and Th2 (HR 0.80, 95% CI 0.64-0.99, p=0.04) were associated with a borderline lower risk for incident CAC. In analyses of CAC progression for participants with nonzero CAC at baseline (N=597), CD19+ B-cells were associated with CAC progression (beta-coefficient=53.1 Agatston units per SD higher B-cells proportion, 95% CI 2.0-104.2, p=0.04), while intermediate monocytes (CD14+CD16+) (beta = -71.6 Agatston units per SD higher, 95% CI -134.9 - -8.3, p=0.03) and higher T-regulatory cells (CD4+CD25+CD127-) (beta = -71.6 Agatston units per SD higher, 95% CI -119.1 - -4.8, p=0.03) were negatively associated with CAC progression. Conclusions: Among individuals with CAC 0 at baseline, NK cells were associated with incident CAC. In individuals with nonzero CAC at baseline, T-regulatory cells and intermediate monocytes were negatively associated with CAC progression, whereas B-cells were positively associated with CAC progression.

Combination of NK and Other Immune Markers at Early Phase Stratify the Risk of Sepsis Patients: A Retrospective Study.

Immune dysfunction plays a pivotal role in sepsis pathogenesis. Previous studies have revealed the crucial role of T cells and human leukocyte antigen-DR (HLA-DR) in sepsis. However, the function of natural killer (NK) cells remains unclear. This study aimed to investigate whether NK cells are associated with sepsis prognosis. In addition, we aimed to explore the interrelation and influence between NK and other immunological features in patients with sepsis. This retrospective, observational study included patients with sepsis from two hospitals in mainland China. The clinical characteristics and immune results during the early phase were collected. Patients were classified according to the level of immune cells to analyze the relationship between immunological features and 28-day mortality. A total of 984 patients were included in this study. Non-survivors were older and had lower levels of lymphocytes, monocytes, NK cells, HLA-DR, and T cells. Patients were classified into eight groups according to their levels of NK cells, HLA-DR, and T cells. Only patients with decreased NK and T cell counts showed a significant increase in 28-day mortality. An increase in CD8+ T cells was correlated with the alleviation of 28-day mortality only among patients with high NK cell levels. This study provides novel insights into the association between NK cells and 28-day mortality as well as the interrelation between NK cells and other immune cells in sepsis. The relationship between CD8+ T cells and 28-day mortality in sepsis is dependent on NK cell count.

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.

Pan-cancer analysis of natural killer (NK) cell infiltration in human malignancies: Molecular features and clinical implications.

2563 Background: NK cells are part of the innate immune system that are not antigen-specific, but can be redirected to targets of interest using multiple strategies. Advantages of NK cells over T cells include the use of allogeneic off-the-shelf products and lower risk of cytokine release syndrome. Numerous NK-specific immunotherapies are under development for the treatment of cancer, although none are yet FDA-approved. Here, we conducted a pan-cancer analysis of NK cell abundance in &gt;90,000 tumor samples across 45 cancer types using the Caris Precision Oncology Alliance (POA) database. Features of prostate cancer (PCa, n=3365) and renal cell carcinoma (RCC, n=1106) were explored in depth. Methods: DNA (targeted/whole-exome) and RNA (whole-transcriptome) sequencing was performed for patient tumors (N=90,916) submitted to Caris Life Sciences (Phoenix, AZ). NK cell fractions were inferred from RNA-seq data using quanTIseq (Finotello, 2019). Real-world overall survival (OS) was determined from insurance claims, and Kaplan-Meier estimates were calculated. Statistical significance was determined using X2 and Mann-Whitney U tests with corrections for multiple hypothesis testing where appropriate. Results: Median NK cell fractions ranged from 7-9% (medulloblastoma and gliomas) to 2% (thyroid and thymic cancers), with intermediate levels observed for PCa (4.6%) and RCC (3.1%). High (&gt; median) NK cell fractions were associated with improved OS (hazard ratios, 0.28–0.84, p&lt;0.05) for 28 of 45 cancer types, while 16 of 45 including low-grade glioma, GIST, and thyroid had HR &lt;1.0 (0.264-0.997) with 95% CI crossing 1.0. Improved OS was notable in PCa (HR 0.46, 95% CI 0.38–0.56, p&lt;0.0001) and RCC (HR 0.52; 95% CI 0.39–0.70, p&lt;0.0001). Unexpectedly, tumors with higher NK infiltrates were less frequently PD-L1+ (SP142) by IHC in PCa (2.8% vs 5.4%, p=0.08) and RCC (12.4% vs 30.8%, p&lt;0.0001) ( i.e. an inverse relationship), despite a lack of correlation of NK infiltration with dMMR/MSI status or tumor mutation burden (TMB) in both cancers. Conversely, increased NK infiltration was associated with a 1.4- to 2.1–fold increased mRNA expression of immunomodulatory receptors LAG3 and TIGIT in both PCa (p&lt;0.0001) and RCC (p&lt;0.0001), but a 1.4–fold decrease in B7-H3 in RCC (p&lt;0.0001). Conclusions: High NK cell infiltration is associated with improved OS in numerous cancer types, including many with inadequate therapeutic options. Our findings suggest broad deployment of NK engagers and CAR-NK products in a range of malignancies. The positive correlation between NK cells and LAG3/TIGIT suggest that combination approaches may be warranted. The inverse relationship between NK infiltrates and PD-L1, and lack of association with standard immune therapy markers (MSI status, TMB), indicates the potential use of NK cell approaches in situations where other immunotherapies are ineffective.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

A new natural killer cell-specific gene signature predicting recurrence in colorectal cancer patients.

The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which underlie NK cell sensitivity. Here, a novel CRC-specific NK cell gene signature that infers NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. In contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the signature includes genes representative of NK cells of different maturation states, activation status and anatomical origin. Our signature also accurately discriminates murine NK cells, demonstrating the applicability of this geneset when mining datasets generated from preclinical studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies.

Chemical screening identifies novel small molecule activators of natural killer cell cytotoxicity against cancer cells.

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.

The Association of Circulating CD14++CD16+ Monocytes, Natural Killer Cells and Regulatory T Cells Subpopulations With Phenotypes of Cardiovascular Disease in a Cohort of Peritoneal Dialysis Patients

The altered expression of immune cells including monocyte subsets, natural killer (NK) cells and CD4+CD25+ regulatory T cells (Tregs) in end-stage kidney disease, affect the modulation of inflammation and immunity with significant clinical implications. The aim of this study was to investigate the profile of specific immune cells subpopulations and their correlations with phenotypes of established cardiovascular disease (CVD), including coronary artery disease (CAD) and heart failure (HF) in peritoneal dialysis (PD) patients.Materials and Methods: 29 stable PD patients and 13 healthy volunteers were enrolled. Demographic, laboratory, bioimpedance measurements, lung ultrasound and echocardiography data were collected. The peripheral blood immune cell subsets analysis was performed using flow cytometry.Results: PD patients compared to normal controls had lower total lymphocytes (22.3 ± 6.28 vs. 31.3 ± 5.54%, p = <0.001) and B-lymphocytes (6.39 ± 3.75 vs. 9.72 ± 3.63%, p = 0.01) as well as higher CD14++CD16+ monocytes numbers (9.28 ± 6.36 vs. 4.75 ± 2.75%, p = 0.0002). PD patients with prevalent CAD had NK cells levels elevated above median values (85.7 vs. 40.9%, p = 0.04) and lower B cells counts (3.85 ± 2.46 vs. 7.2 ± 3.77%, p = 0.03). Patients with increased NK cells (>15.4%) had 3.8 times higher risk of CAD comparing with patients with lower NK cell levels (95% CI, 1.86 – 77.87; p = 0.034). B cells were inversely associated with the presence of CAD (increase of B-lymphocyte by 1% was associated with 30% less risk for presence of CAD (95% CI, −0.71 – 0.01; p = 0.05). Overhydrated patients had lower lymphocytes counts (18.3 ± 4.29% vs. 24.7 ± 6.18%, p = 0.006) and increased NK cells [20.5% (14.3, 23.6) vs. 13.21% (6.23, 19.2), p = 0.04)]. In multiple logistic regression analysis the CRP (OR 1.43; 95% CI, 1.00 – 2.05; p = 0.04)] and lymphocytes counts (OR 0.79; 95% CI, 0.63–0.99; p = 0.04)] were associated with the presence of lung comets. Patients with higher NK cells (>15.4%, n = 15) were more likely to be rapid transporters (D/P creatinine 0.76 ± 0.1 vs. 0.69 ± 0.08, p = 0.04). Patients displaying higher Tregs (>1.79%) were older (70.8 ± 10.7 years vs. 57.7 ± 14.7years, p = 0.011) and had higher nPCR (0.83 ± 0.14 vs. 0.91 ± 0.17, p = 0.09).Conclusion: Future research is required to evaluate the role of immune cells subsets as potential tools to identify patients at the highest risk for complications and guide interventions.

Prognostic value of natural killer cell activity for patients with HER2 + advanced gastric cancer treated with first-line fluoropyrimidine-platinum doublet plus trastuzumab.

We aimed to evaluate the prognostic value of natural killer (NK) cell activity for patients with HER2 + advanced gastric cancer (AGC) treated with first-line fluoropyrimidine-platinum doublet plus trastuzumab. Forty-one patients with HER2 + AGC who received fluoropyrimidine-platinum doublet plus trastuzumab as first-line treatment were prospectively enrolled. NK cell activity was evaluated using the NK Vue®. The median age was 63.5years, and 31 patients (75.6%) were male. Patients with low baseline NK cell activity (≤ median, n = 21) were associated worse progression-free survival (PFS) and overall survival (OS) compared with patients with high baseline NK cell activity (> median, n = 20) with a median PFS of 4.21 vs. 9.53months (P < 0.001), and median OS of 8.15months vs. 17.82months (P = 0.025), respectively. In the multivariate analysis, low baseline NK cell activity was independently associated with poor PFS (HR 4.35, P = 0.007). NK cell activity recovered to a normal range in nine patients (47.4%) with a low baseline NK cell activity (n = 19) after two cycles of treatment. The median PFS and OS among patients with recovered NK cell activity were significantly better than that among patients with persistently low NK cell activity (PFS, P = 0.038; OS, P = 0.003). Our results demonstrated the prognostic value of baseline NK cell activity for patients with HER2 + AGC treated with fluoropyrimidine-platinum doublet plus trastuzumab. The association between treatment outcomes and dynamic changes in NK cell activity suggests that NK cell treatment may improve treatment outcomes, especially for patients with low baseline NK cell activity.

3 Cases of Cancer Patients Whose Natural Killer Cell Activity Improved with Traditional Korean Medicine Treatment: A Case Series

Objectives: High natural killer cell activity (NKA) is associated with a clinically favorable status in patients with cancer. This study explores whether traditional Korean medicine (TKM) improves NKA in patients with cancer.Methods: We report three clinical cases involving patients with cancer who underwent NKA tests while they received TKM treatment, including acupuncture, moxibustion, wild ginseng pharmacopuncture, and Korean herbal medicines. We analyzed the treatment process and NKA test results of each patient.Results: The NKA of all three patients presented with an increasing tendency during the treatment process. Patient 1, who had been diagnosed with breast cancer, received inpatient treatment 3 times between September 16, 2017 and February 27, 2019. The NKA increased from 7.2 pg/mL to 571.7 pg/mL. Patient 2, who had thyroid cancer, was hospitalized 4 times between July 23, 2019 and August 27, 2020. The NKA improved from 317.4 pg/mL to 2000 pg/mL. Patient 3, who had liver cancer, received inpatient treatment 4 times between May 27, 2016 and January 10, 2017. The NKA increased from 22.2 pg/mL to 459.5 pg/mL. The chief complaints of these patients also were alleviated.Conclusions: TKM may be an effective treatment for accelerating NKA. Further research is needed to establish evidence that TKM stimulates NKA.

Demonstration of a Single-Parameter, Rapid Flow Cytometry Assay in Assessing Natural Killer Cell Cytotoxic Function

Assessing cytotoxic function of immune effector cells, such as natural killer (NK) cells, is necessary to determine their potency. Various cytotoxicity assays measuring target cell death have been reported, however these assays typically feature a number of limitations that curb their broad applicability. Quantitative assays of NK cell killing traditionally measure the release of chromium or a fluorescent dye from labelled target cells. These assays typically show low reproducibility and often underestimate cell death for many cell types. Recent strategies aimed at ensuring manufacture of high NK cell doses commonly include NK cell co-culture with antigen presenting cells as feeder cells for their expansion. Such expansion platforms result in a final NK cell product with a high content of dead or dying feeder cells. This end product also complicates the accurate measurement of target cell death in NK cell cytotoxicity assays. Here we report a rapid flow cytometry assay for quantifying target cell killing after single-color labeling of the target cells prior to co-incubation with NK cells. In this assay, target cells are first labeled with calcein-AM (CAM), a green fluorescent dye. CAM-labeled target cells are then co-incubated with feeder cell expanded-NK cells in 96-well plates using various NK cell-to-target ratios. After 4 hours of NK cell-target cell co-incubation, a fixed volume of the cell suspension was analyzed using a flow cytometer capable of reading 96-well plates. The live, CAM-labeled target cells were gated and quantitated for all the treatment conditions to determine the number of CAM-labeled target cells present per fixed volume.Using this assay, we observed highly consistent cytotoxicity results against both suspension and adherent cell targets. The accuracy of this assay was also demonstrated by NK cell dose-dependent responses. Importantly, the presence of dead feeder cells in the NK cell suspension does not interfere with the analysis of this assay. In addition, the gating strategy is simple, consistent, and the analysis between treatment groups does not require cumbersome calculations or compensation that could introduce variability. Assessment of target cell death was also shown to be more reliable and consistent when compared with the measurement of CAM release using a microplate reader. The latter assay has previously been shown to be highly variable with inconsistent results depending on target cell and CAM dilution. Our flow cytometry assay consistently identifies live cells, avoiding errors due to variability in dye intake and dye release detection. DisclosuresNo relevant conflicts of interest to declare.

Abstract P24: Evidenced-based natural therapies and immune-enhancing strategies to control, prevent, and treat co-morbidities COVID-19 and cancer

Abstract The purpose of this research is to provide evidence-based natural therapies and foods that will enhance the immune system to fight the virus SARS CoV-2 and cancer. Minority populations that include Black African Americans and Hispanics are experiencing higher mortality rates due to both socioeconomic and lifestyle factors in the US and globally. Successful lessons learned from controlling previous 1918 pandemic flu will be discussed. Hypotheses: We hypothesize that targeting lifestyle factors such as diet, exercise, and hydrothermal therapy that enhance the immune system can effectively control and reduce the risk to COVID-19 disease and cancer simultaneously. The target cells for the SARS CoV-2 virus contain the ACE2 and TMPRSS2 receptors which serve as portals of entry. They are expressed throughout the body including the nose, lungs, heart, bladder, pancreas, kidneys and brain found on the epithelial and vascular tissue cells of these organs. Covid-19 affects multiple organs in the body and disrupts the innate immune system, especially the natural killer cells, monocytes, and neutrophils. Studies have shown that countries where BCG was used have less than 10% of COVID-19 cases and countries that do not use the BCG vaccine such as the US and Italy had high cases. It was also seen that people who had higher natural killer cell activity had 100% survival rates. Enhancing the innate immune system through natural therapies has shown to enhance immune cells to fight COVID-19 as well as cancer and destroy it. Methodology: Our research is exploratory and is based on recently published literature on COVID-19 diseases. The following databases were searched: CINAHL, Web of Science, PubMed, EBSCOHost, Google Scholar, Academic Search Complete, AccessMedicine, MedlinePlus, Nutrition Care Manuel, JSTOR and Publons. Results: During the Spanish flu pandemic death rates of those who received the best medical care was 6.7% compared with those who received hydrotherapy at 1.34%. Hydrotherapy will increase the body temperature and stimulate the activity of the immune cells, which can kill the viruses. Increasing the body temperature to 39.5 degrees centigrade was shown to kill the virus. Following the hot water bath with cold treatment was shown to be even more effective. Many foods have been found to enhance the immune system and to fight the flu virus. Foods such as blueberries, garlic, and probiotics were shown to improve natural killer activity. Vitamin C, Vitamin D, Zinc, and selenium play a role in immunity. Foods rich in antioxidants such as kale, blueberries, red bell peppers, and all citrus fruits also help enhance the immune system. During the 1918 flu pandemic that received hydrotherapy and other natural remedies, out of the 120 students and staff exposed, there were 90 patients, none very sick, and there were no deaths. Natural remedies used in the Spanish flu pandemic and should be instituted to help enhance immunity and lessen the deadly impact of COVID-19 cancer patients. Citation Format: Padma P. T. Uppala, Sherri Isaak, Brian Wong, Kimberly Ferreira, Carlos Garberoglio, John Kelly. Evidenced-based natural therapies and immune-enhancing strategies to control, prevent, and treat co-morbidities COVID-19 and cancer [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P24.

Prognostic Value of Natural Killer Cells Besides Tumor-Infiltrating Lymphocytes in Breast Cancer Tissues

BackgroundEach subgroup of immune cells has a different prognostic role in breast cancer; however, the prognostic impact of tumor-infiltrating natural killer cells (TINKs) is still not well established. Our aim was to assess the prognostic impact of natural killer (NK) cells in breast carcinomas. Materials and MethodsNK cells infiltration were assessed by immunohistochemistry (IHC). Statistical analyses were performed to evaluate the correlation of NK cells with clinical-pathological features and outcome. ResultsCD56 IHC was realized in 126 patients. NK cells infiltration showed significant and positive association with tumor high Scarff-Bloom-Richardson (SBR) grade. NK cells were significantly associated with HER2-positive breast cancer and triple-negative breast cancer subtypes. Analyses showed significant and inverse correlation with progesterone and estrogen receptors expression status. High NK cells were significantly related to high Ki-67 labeling index. Our data showed that high NK cells infiltrate was significantly associated with tumor-infiltrating lymphocytes in breast cancer tissues.At a median follow-up of 5.5 years, high CD56 expression (≥ 5 cells/10 high power field) was associated significantly with a good overall survival and with good disease-free survival. ConclusionIn this study, we assessed the important prognostic role of TINKs in breast carcinomas, which seems to be evident despite its association with aggressive pathological features. Thus evaluation of NK cells can be standardized and integrated in daily routine.

Abstract PS5-26: Pd-l1 mRNA expression in plasma-derived exosomes predicts prognosis and response to anti-pd-l1 antibodies in unresectable locally advanced triple negative breast cancer (TNBC) treated with atezolizumab-paclitaxel

Abstract Background Despite modern therapies have improved outcomes of patients with non-TNBC cancers, patients diagnosed with unresectable locally advanced TNBC usually have poor outcome for its aggressive clinical behaviour. Over time for cancer management, predicting and monitoring response to treatments is crucial, better if through easy non-invasive procedures. Atezolizumab plus nanoparticle albumin-bound (nab)-Paclitaxel prolonged progression-free survival (PFS) and overall survival (OS) among patients with TNBC, as demonstrated by phase III IMpassion130 trial. Despite this, there is a dire need for biomarkers reflecting treatment response. In our study, we correlated plasma PD-L1 mRNA levels with prognosis and response to anti -PD-L1 antibodies in unresectable locally advanced TNBC in the era of Atezolizumab. Methods We assessed 30 consecutive patients with unresectable locally advanced TNBC treated with Atezolizumab plus nab-Paclitaxel. Blood samples were collected at time point 0 (at baseline) and after 2 months and we assessed the association between PD-L1 mRNA copies per ml in plasma-derived exosomes and response to treatment. Exosomal PD-L1 mRNA in plasma was determined using Bio-Rad QX100 digital droplet PCR system and exoRNeasy kit. Objective responses were defined following the RECIST criteria v.1.1 Results At baseline, patients with complete and partial response (CR+PR, n=11) displayed a significantly higher number of PD-L1 mRNA copies per ml compared to ones with stable or progressive disease (SD+PD, n=19) (mean value: 745.6±131.1 vs 124.7±34.4, p&amp;lt;0.001). In patients with CR and PR mean PD-L1 copies/ml were 745.6±131.1 and 175.4 at baseline vs 2 months, respectively (p=0.001).In patients with stable disease the mean ± s.e.m. values were 270±71.1 vs 217.5±17.3 copies per ml (p=0.614) while in progressive disease PD-L1 mRNA levels were 112.1±31.2 vs 454.3±46.2 copies per ml at baseline vs 2 months, respectively (p&amp;lt;0.001).Two months after the start of Atezolizumab plus nab-Paclitaxel decrease of PD-L1 mRNA copies per ml relative to pre-treatment PD-L1 mRNA copies per ml, was positively correlated with an objective response to treatment.Increase of PD-L1 mRNA copies per ml was significantly associated with worse and shorter OS: median OS in patients with increase of PD-L1 mRNA copies/ml was 5 months (range 2-7 months, 95%CI 1.1-6.1); on the other side median OS in patients with decrease of PD-L1 mRNA copies/ml was more than double (range 8-15 months) (p&amp;lt;0.001).Moreover, prolonged survival outcomes (p&amp;lt;0.001) were recorded in cases with low baseline circulating Lox1+ Myeloid-derived suppressor cells (MDSC) (r=0.62, p&amp;lt;0.001) and high Natural Killer cells (NK) (r=0.66, p&amp;lt;0.001): the NK-to-MDSC ratio (NMr) was significantly higher in patients with higher number of PD-L1 mRNA copies per ml at baseline (p=0.002). During PD-L1 blockade, 2 months after starting treatment, NK progressively raised in clinical benefit group while declined in non-responders (p=0.001) Conclusions Despite the study’s limitations, our data suggest exosomal PD-L1 is significantly associated with outcome and response to treatment. Citation Format: Lucrezia Raimondi, Laura Di Benedetto, Victoria Bitca, Gian Paolo Spinelli. Pd-l1 mRNA expression in plasma-derived exosomes predicts prognosis and response to anti-pd-l1 antibodies in unresectable locally advanced triple negative breast cancer (TNBC) treated with atezolizumab-paclitaxel [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-26.