Direct Tumor Irradiation Potentiates Adoptive NK Cell Targeting Against Parental and Stemlike Cancer in Human Liver Cancer Models.

PURPOSE: Radiation therapy (RT) has been shown to effectively induce the expression of intercellular adhesion molecule-1 (ICAM-1), which is recognized by lymphocyte function-associated antigen 1 (LFA-1) expressed on natural killer (NK) cells. However, the potential synergistic antitumor immune response of tumor irradiation and administered NK cells has not been explored in intractable human liver cancers. Furthermore, NK cell targeting against both parental and cancer stemness has never been investigated. METHODS AND MATERIALS: Highly activated ex vivo NK cells were administered into the human liver tumor-bearing mice. Tumor direct RT was optimized according to tumor bearing site. HepG2 and Hep3B ICAM-1 knockout cells were generated using CRISPR/CAS9. Stemness tumor spheres were generated. NK cell cytolysis against parental and tumor sphere was evaluated using flow cytometry and real-time cytotoxicity assay. RESULTS: A combination of adoptive NK cell therapy with RT significantly improved therapeutic efficacy over monotherapies against subcutaneous, orthotopic, and metastatic human liver tumor models. Direct tumor irradiation potentiated NK cell recognition and conjugation against liver cancer through the LFA-1/ICAM-1 axis. Suppression of immune synapse formation on NK cells using high-affinity LFA-1 inhibitors or ICAM-1 knockout liver cancer induced "outside-in" signal blocking in NK cells, resulting in failure to eliminate liver tumor despite the combination therapy. NK cells effectively recognized and targeted triple-high epithelial cell adhesion molecule+CD133+CD24+ liver cancer expressing upregulated ICAM-1 in the irradiated tumor microenvironment, which led to prevention of the initiation of metastasis, improving survival in a metastatic model. In addition, the LFA-1/ICAM-1 axis interruption between NK cells and stemness liver tumor spheres significantly diminished NK cell cytolysis. Consistent with our preclinical data, the LFA-1/ICAM-1 axis correlated with survival outcomes in patients with metastatic cancer from the The Cancer Genome Atlas databases. CONCLUSIONS: NK cells in combination with tumor irradiation can provide synergistic therapeutic effects for NK cell recognition and elimination against both parental and stemlike liver cancer through LFA-1/ICAM-1.

Efficacy of natural killer cell therapy combined with chemoradiotherapy in murine models of head and neck squamous cell carcinoma.

BACKGROUND AIMS: Natural killer (NK) cell-based cancer immunotherapy is effective when combined with other treatment modalities such as irradiation and chemotherapy. NK cell's antitumor function to treat solid tumor, including head and neck squamous cell carcinoma (HNSCC), has been targeted recently. This study assessed NK cell recruitment in response to chemoradiation therapy (CRT) in HNSCC. METHODS: Ex vivo expansion of NK cell, flow cytometry, cell viability assay, cytotoxicity assay, immunohistochemistry, and animal model were performed. RESULTS: Mouse NK cells were recruited to the tumor site by CRT in a nude mouse model. Furthermore, expanded and activated human NK cells (eNKs) were recruited to the tumor site in response to CRT, and CRT enhanced the anti-tumor activity of eNK in an NOD/SCID IL-2Rγnull mouse model. Various HNSCC cancer cell lines exhibited different NK cell ligand activation patterns in response to CRT that correlated with NK cell-mediated cytotoxicity. CONCLUSIONS: Identifying the activation patterns of NK cell ligands during CRT might improve patient selection for adjuvant NK cell immunotherapy combined with CRT. This is the first study to investigate the NK cell's antitumor function and recruitment with CRT in HNSCC mouse model.

Intensified NK cell therapy in combination with low-dose chemoradiotherapy against human colorectal cancer.

The therapeutic potential of adoptive natural Killer (NK) cells immunotherapy in combination with chemoradiotherapy, the main treatment modality for colorectal cancer (CRC), has not yet been explored. Here, we aimed to investigate the efficacy of NK cells to potentiate primary tumor control and improve survival outcomes, especially in combination with low-dose chemoradiotherapy. Ex vivo activated NK cells (> 90% purity) from healthy donors were obtained. NK cells were administered intravenously to the CRC-bearing mice and intensified in vivo in combination with low-dose 5-fluorouracil (0.5 mg/kg or 1 mg/Kg) and irradiated tumors with low doses (2 Gy or 4 Gy). Real-time NK cell cytotoxicity demonstrated a synergistic killing effect of a combination of low-dose chemoradiotherapy, mainly through NKp30 and NKG2D, showing a decrease in NK cell degranulation after blocking NKG2D and NKp30. In vivo tumor characteristics after combination treatment showed decreased CD112, CD155, MICA, and MICB expression. Under the combination strategy, 70% of the mice had free lung metastasis and 90% without secondary gross tumors, indicating suppressed distant metastasis to lung and axillary regions. This combination therapy resulted in significantly synergistic antitumor activity against primary solid tumors compared to chemoradiotherapy only. Furthermore, the intensified NK cell administration showed significantly better primary tumor control and survival outcomes than the non-intensified NK cell administration in a human colorectal HT-29 model treated with low-dose chemoradiotherapy. Optimized NK cell therapy combined with low-dose chemoradiotherapy can provide effective therapeutic potential for intractable cold human colorectal cancer.

The Updated World Health Organization Classification Better Predicts Survival in Patients With Endocervical Adenocarcinoma (KROG 20-07).

PURPOSE: The 2020 World Health Organization classification divided endocervical adenocarcinoma (ADC) into human papillomavirus-associated (HPVA) and human papillomavirus-independent (HPVI) ADCs. This multi-institutional study aimed to investigate the clinical features and prognosis of patients with endocervical ADC based on the updated World Health Organization classification. METHODS AND MATERIALS: We retrospectively reviewed the 365 patients with endocervical ADC who underwent radical hysterectomy from 7 institutions. Tumor characteristics, patterns of failure, and survival outcomes were compared between HPVA and HPVI ADCs. RESULTS: Two hundred seventy-five (75.3%) and 90 (24.7%) patients had HPVA and HPVI ADC diagnoses, respectively. In all cases, the 5-year disease-free survival (DFS) and overall survival (OS) rates were 58.2% and 71.3%, respectively. HPVI ADC showed higher rates of local recurrence (25.6% vs 10.9%) and distant metastasis (33.3% vs 17.5%) than HPVA ADC. Multivariate survival analysis revealed that HPVI ADC showed significantly worse DFS (hazard ratio [HR], 1.919; 95% confidence interval [CI], 1.324-2.781; P < .001), distant metastasis-free survival (HR, 2.100; 95% CI, 1.397-3.156; P < .001), and OS (HR, 2.481; 95% CI, 1.586-3.881; P < .001) than HPVA ADC. Patients with gastric- and serous-type HPVI ADC had significantly worse OS than those with other HPVI ADCs (P = .020). Similarly, invasive stratified mucin-producing-type HPVA ADC showed significantly worse OS than other HPVA ADCs (P < .001). CONCLUSIONS: We demonstrated that HPVI ADC exhibited inferior DFS and OS and higher rates of local and distant recurrence compared with HPVA ADC. Gastric- and serous-type HPVI ADCs and invasive stratified mucin-producing-type HPVA ADC showed worse OS than other types of HPVI and HPVA ADCs, respectively. Our observation of significant differences in prognoses according to the histologic types needs to be validated in larger cohorts of patients with endocervical ADC.

Randomized multicenter phase II trial of prophylactic irradiation of para-aortic lymph nodes in advanced cervical cancer according to tumor hypoxia: Korean Radiation Oncology Group (KROG 07-01) study.

We conducted a prospective phase II study on whether extended-field irradiation (EFI) confers survival benefits depending on hypoxic markers in locally advanced uterine cervical cancer (LAUCC). RNA-seq was performed to identify immune and hypoxic gene signatures. A total of 288 patients were randomized to either EFI or pelvic radiotherapy (PRT). All patients completed chemoradiotherapy. Overall, significantly higher 5-year para-aortic recurrence free survival (PARFS) rate occurred in EFI (97.6%) than in PRT group (87.2%), with marginal tendency to improve disease-free survival (DFS; 78% vs 70%, P = .066). Subgroup analyses were performed based on carbonic anhydrase 9 (CA9)-only positive, CA9/hypoxia-inducible factor (HIF) double positive and CA9 negative. In the CA9-only positive, EFI successfully increased 5-year PARFS (100% vs 76.4%, P = .010), resulting in significantly improved long-term DFS (85.7% vs 54.7%, P = .023) compared to the PRT, while there was no such benefit of EFI in the CA9/HIFs double positive. RNA-seq analysis identified distinct immunehigh subgroup with negative correlation with hypoxia gene signatures (R = -.37, P < .01), which showed a higher 5-year DFS than the immunelow (P = .032). Hypoxia-related genes were upregulated in the CA9/HIFs double positive compared to CA9 negative (P < .05). Only 17.4% of patients in CA9-negative group showed immunelow signatures, while 40.0% of patients in the double-positive group exhibited immunelow signatures. In conclusion, EFI improved PARFS significantly in all patients, but therapeutic efficacy of EFI in terms of improved DFS was solely observed in CA9-only positive LAUCC, and not in CA9/HIFs double-positive subgroup. RNA-seq analysis suggested that hypoxia-induced immunosuppression may be related to treatment resistance in LAUCC.

Safety analysis of ex vivo-expanded canine natural killer cells in a xenogeneic mouse model of graft-versus-host disease.

Canine natural killer (NK) cells are large, granular lymphocytes that are neither B lymphocytes nor T lymphocytes. However, it has been reported that canine NK cells share some of the phenotypic characteristics of T lymphocytes, such as CD3 and CD5. Studies are needed to assess the safety of canine NK cells for immunotherapy, especially because the safety of using allogeneic NK cells as an immunotherapy for dogs has yet to be shown. In this study, the safety of cultured canine NK cells was assessed using a xenogeneic mouse model of graft-versus-host disease (GVHD). Mice were injected with either canine peripheral blood mononuclear cells (PBMCs) or cultured NK cells for 2 or 3 weeks. Data were then collected on changes in mice body weights, disease severity scores, and survival rates. Histopathological and immunohistochemical evaluations were also performed. All mice injected with canine PBMCs died within 45 days after injection. Severe clinical signs were caused by GVHD. The histopathological and immunohistochemical evaluations showed that mice injected with canine PBMCs had multiple lesions, including necrosis in their lungs, livers, kidneys, and stomachs, and the injected cells were present around the lesions. By contrast, no mice injected with cultured NK cells without removing the CD3+ TCR- cells exhibited any clinical abnormalities. Moreover, they all survived the 90-day experimental period without exhibiting any histopathological changes. Accordingly, the results of this study suggest that canine NK cells do not cause significant side effects such as GVHD and allogeneic NK cells can safely be used for cancer immunotherapy in dogs.

Gliomatosis cerebri having a poor performance status without recurrence after radiotherapy: a single institutional experience.

OBJECTIVE: Radiotherapy (RT) has been advocated for gliomatosis cerebri (GC) to delay tumor progression. However, patients with GC may experience poor performance status without recurrence after RT. Herein, we reviewed the responses of patients with GC treated with RT. MATERIALS AND METHODS: Seventeen patients with diffuse radiologic infiltration of more than two cerebral lobes and diagnosed as having grade II (N=12) or III (N=5) glioma were treated with a mean radiation dose of 51.8 Gy (range, 42-59.4 Gy). The mean radiation volume to a mean marginal dose of 42.8 Gy (range, 36-45 Gy) was 693.4 cc (range, 316.5-1279.6 cc), which was 45.3% of the mean whole brain volume. We reviewed the clinical prognostic factors related to progression-free survival (PFS) and overall survival (OS) in all patients and described characteristics of patients with poor performance (Eastern Cooperative Oncology Group performance scale 3-4) without recurrence. RESULTS: In all patients, the median PFS was 12.3 months, and oligodendroglial pathology (p=0.002) and non-enhanced tumor (p=0.002) were associated with an improved PFS. The median OS was 39.3 months, and young age and adjuvant chemotherapy were associated with improved OS (p=0.022 and 0.002, respectively). Based on multivariate analysis, adjuvant chemotherapy was significantly associated with a longer OS (p=0.012; hazard ratio=0.099; 95% CI, 0.016-0.596). Five patients (29.4%) showed poor performance without recurrence. The median age of these 5 patients was 65 years (range, 60-75 years). The mean radiation volume to a mean marginal dose of 41 Gy (range, 36-45 Gy) was 539.9 cc (range, 255.5-983.1 cc). The pathologic diagnosis was grade II glioma in 2 patients and grade III in 3 patients. The median OS was 10.0 months (range, 8.2-45.9 months). CONCLUSIONS: Some GC patients, especially the elderly, might have a poor performance status without recurrence after RT of a larger radiation field.

Microarray analysis of gene expression in lung cancer cell lines treated by fractionated irradiation.

BACKGROUND: To identify differentially expressed genes between parent and radioresistant lung cancer cell lines established by fractionated irradiation. MATERIALS AND METHODS: Lung cancer cell lines (A549, NCI-H1650) were irradiated with several fractionation schemes. Clonogenic assays were used to identify radioresistant cell lines. We compared the gene expression profiles on a cDNA microarray. RESULTS: Four established cell (A549-2G, A549-5G, H1650-2G and H1650-5G) were shown to be radioresistant (p≤0.05). Seventy-two genes were commonly altered in A549-G and 655 genes in H1650-G, compared to their parental cells. Genes in the wingless-type MMTV integration site family (WNT) signaling pathway were the ones most frequently altered in both A549-G and H1650-G cells. Those involved in inflammation; integrin, platelet-derived growth factor (PDGF), interleukin, transforming growth factor-beta (TGFB), epidermal growth factor receptor (EGFR) signaling, were commonly altered in radioresistant H1650 sublines. CONCLUSION: The major gene expression changes during irradiation are related to WNT signaling pathway.

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture.

BACKGROUND AIMS: Interleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture. METHODS: IL-2/IL-15-activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry. RESULTS: Compared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the "first week" group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the "first week" group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of "first week" NK cells that received IL-21 treatment for an additional 2 days compared with the "first week" NK cells (P < 0.05). CONCLUSIONS: These data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.

A phase III concurrent chemoradiotherapy trial with cisplatin and paclitaxel or docetaxel or gemcitabine in unresectable non-small cell lung cancer: KASLC 0401.

PURPOSE: Concurrent chemoradiotherapy (CCRT) is recommended for the management of patients with unresectable non-small cell lung cancer (NSCLC). This prospective study aimed to compare the efficacy of concurrently delivered cisplatin doublets with paclitaxel, or docetaxel, or gemcitabine. METHODS: The main eligibility criteria consisted of previously untreated stage IIIB NSCLC. The subjects were randomized into three arms: paclitaxel 45 mg/m(2)/week (TP), docetaxel 20 mg/m(2)/week (DP), and gemcitabine 350 mg/m(2)/week (GP) in addition to cisplatin 20 mg/m(2)/week. Three-dimensional conformal radiotherapy was given once daily, weekly 5 fractions and the total prescription dose was 60-66 Gy. The primary endpoint was response rate, and the secondary endpoints were survival and toxicity. RESULTS: A total of 101 patients were recruited into this trial of whom 93 (TP: 33, DP: 29, GP: 31) patients were treated with CCRT from March 2005 to July 2007. Similar response rates were observed across arms: TP: 63.6 %, DP: 72.4 %, GP: 61.3 % (p = 0.679). There was no statistically significant difference of median survival (TP: 27.3, DP: 27.6, GP: 16.5 months, p = 0.771). In subgroup analysis, a survival benefit of consolidation chemotherapy was not seen, but leucopenia (63.2 %) and neutropenia (68.4 %) more than grade 3 were significantly high in DP arm. The grade ≥3 radiation esophagitis was more frequent in the GP arm (22.6 %, p = 0.163). CONCLUSIONS: Among the three arms, no statistically significant difference in response rate, survival, and toxicity was observed. However, clinically significant radiation toxicity was more frequent in the GP arm.

Overnight storage of blood in ACD tubes at 4{degrees}C increases NK cell fraction in peripheral blood mononuclear cells.

A considerable variabilility in the effects of sample handling on NK cytotoxicity has been observed. Using flow cytometry, NK cytotoxicity assays and lymphocyte subset analysis of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMCs) isolated from whole blood stored under various conditions were performed. The NK cytotoxicity of samples in heparin tubes stored overnight at 4 and 22°C, as well as at 22°C in acid citrate dextrose (ACD) tubes, was lower than that of a fresh sample. However, the NK cytotoxicity of samples in an ACD tube stored at 4°C was similar to that of a fresh sample. Based on lymphocyte subset analysis, samples in an ACD tube stored at 4°C showed a lower percentage of CD3+ T cells and a higher percentage of CD16/56+ NK cells compared to samples stored under other conditions. The NK cytotoxicity of fresh samples and samples in ACD tubes stored in a Styrofoam cooler box did not differ significantly; however, the differences were inconsistent. Overnight storage of peripheral blood in ACD tubes at 4°C is optimum for retention of NK cytotoxicity, the level of which is similar to that of fresh blood. This may be associated with an increased NK-cell fraction in Ficoll-Hypaque-separated PBMCs after overnight storage.

Ex vivo expansion of natural killer cells using cryopreserved irradiated feeder cells.

Currently, feeder cells are γ-irradiated immediately before use for the ex vivo expansion of natural killer (NK) cells from human peripheral blood. Storing irradiated feeder cells by cryopreserving them in multiple vials would be more convenient than irradiating cells each time they are needed. We compared NK cell expansion using cryopreserved-irradiated feeder cells (cryopreserved group) and freshly-irradiated feeder cells (fresh group). To expand NK cells, peripheral blood mononuclear cells were isolated and co-cultured with-100 Gy-irradiated K562 leukemia cells that had been modified to express 4-1BB ligand and membrane-bound (mb) interleukin (IL)-15 (K562-mb15-41BBL cells) for three weeks in the presence of IL-2 and IL-15. Fresh and cryopreserved K562-mb15-41BBL feeder cells expressed similar levels of 4-1BB ligand, whereas membrane-bound IL-15 expression was lower in the cryopreserved cells than in the fresh cells. The NK cell expansion rate did not differ between the two groups (980-fold vs. 1058-fold, respectively), although the mean NK cell purity was higher in the fresh-group than in the cryopreserved-group at day 14 (94.1% vs. 92.5%, respectively) and day 21 (97.1% vs. 95.4%, respectively). The NK cells from the two feeder cell groups did not differ in cytotoxicity against various malignant cell lines at effector-to-target ratios of 4:1, 2:1, and 1:1, or in the expression pattern of NK cell receptors [cluster of differentiation (CD)-16, natural killer group-2, member D (NKG2D), CD69, NKp30, NKp44, NKp46, and CD158b] and level of interferon-γ secretion. Our results demonstrate that cryopreserved irradiated feeder cells can be used for the ex vivo expansion of human NK cells and provide a convenient improvement on current methods.

Respiratory motional effect on cone-beam CT in lung radiation surgery.

The cone-beam CT (CBCT), which is acquired using an on-board imager (OBI) attached to a linear accelerator, is used effectively in the verification of setup accuracy for lung radiation surgery. In this study, the respiratory organ motional effect on the CBCT was evaluated with a properly devised phantom system, and the level of possible error in conditions of a real clinical process was assessed. In a comparison study between the CBCT in static status and CBCT images acquired in 20 different motional cases, we confirmed that the image quality and information of CBCT were degraded, with an increase of motional ranges in the region of inhomogeneous structures. The 4D-CT MIP (50 approximately 55%) for the planning of lung radiation surgery and the 4D-CT MIP (full phase) were compared with CBCT in the various motional cases for the evaluation of the influence of the motional effect on CBCT in the process of the setup error correction. The average ratio of relative difference between plan CT: 4D-CT MIP (50% approximately 55%) and CBCT was 5.79% and between plan CT: 4D-CT MIP (50% approximately 55%) and 4D-CT MIP (full phase) was 42.95% in the phantom study. In the analysis of clinical cases of lung radiation surgery, the gross tumor volumes were compared in each CT image. The average ratio of relative difference between plan CT: 4D-CT MIP (50 approximately 55%) and CBCT was 10.72% and between plan CT: 4D-CT MIP (50 approximately 55%) and 4D-CT MIP (full phase) was 28.19%. These results showed that, although a respiratory organ motional effect on CBCT introduced variation in image quality, the error as a result of this variation could be estimated relatively low in the setup error correction for a gated-lung radiation surgery when the planning was performed in 4D-CT MIP (50 approximately 55%), which already included a related signal of motional effect.

Low hMLH1 expression prior to definitive chemoradiotherapy predicts poor prognosis in esophageal squamous cell carcinoma.

The present study evaluated the pretreatment expression patterns of hMLH1, MDM2, p53, and pRb protein to determine whether these could predict the outcome of definitive concurrent chemoradiotherapy (CCRT) in 51 patients with stage I-IVa esophageal squamous cell carcinoma. High immunoreactivies of hMLH1, MDM2, p53, and pRb were detected in 90.2%, 19.6%, 27.5%, and 66.7% of entire patients, respectively. High hMLH1 expression was found to favor earlier stage, less locoregional failure, and longer cause-specific survival, and all were with significance. However, the expressions of MDM2, p53, and pRb were not found to be clinically significant. Thirty-three patients with high hMLH1 and pRb expression tended to survive longer than four patients with low hMLH1 and pRb expression. We suggest that the expression of hMLH1 is a potential marker of tumor response and survival. Determinations of this protein expression might be useful for selecting esophageal squamous cell carcinoma patients for definitive CCRT.