Loss of heterozygosity at 6p21 and HLA class I expression in esophageal squamous cell carcinomas in China

Background and objective: The loss or downregulation of human leukocyte antigen ( HLA-Ⅰ) has been proposed to contribute to immune evasion by cancer cells. Since the human leukocyte antigen (HLA-Ⅰ) complex is located at 6p21.3, loss of heterozygosity of this region may alter HLA class Ⅰ tumor phenotypes. The aim of this study was to analysis loss of heterozygosity (LOH) of chromosome 6p in ESCC samples and correlate this with HLA class I expression. Materials and methods: A total of 87 formalin-fixed, paraffin-embeded and frozen-fresh of ECSS lesions were collected. HLA-Ⅰand antigen-processing machinery component expression was investigated by immunohistochemistry with anti-HLA classⅠ monoclonal antibody and a panel of 49 ESCCs with downregulated HLA class Ⅰ expression wereselected for LOH studies using 3 microsatellite markers located at 6p21.3 (D6S105,D6S265,D6S273). Results: HLA-Ⅰantigen,TAP1 and LMP were lost or down-regulated in 57.5, 29.8 and 47.0% of the ESCC lesions, respectively. In 23/49(46.9%) of the ESCCs, allelic loss for at least one locus at 6p21.3 was found. Conclusions: Our data show that downregulation of HLA class I expression is correlated with loss of heterozygosity regions at 6p21.3 in ESCC.     

Expression of classical human leukocyte antigen class I antigens,HLA‐E and HLA‐G,is adversely prognostic in pancreatic cancer patients

The expression of classical human leukocyte antigen class I antigens (HLA‐I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA‐I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA‐I and non–classical HLA‐I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA‐I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune‐related genes to characterize PDAC tumor microenvironments. Lower expression of HLA‐I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA‐E and HLA‐G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA‐I antigens was significantly correlated with shorter survival. Higher HLA‐I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD‐L1 and PD‐L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA‐I in three PDAC cell lines. It also upregulated surface expression of HLA‐E, HLA‐G and immune checkpoint molecules, including PD‐L1 and PD‐L2. These results suggest that the higher expression of HLA‐I, HLA‐E and HLA‐G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA‐I expression on PDAC cells.     

The prognostic role of classical and nonclassical MHC class I expression in endometrial cancer

The aim of this study was to investigate classical MHC class I and nonclassical MHC (human leukocyte antigen‐G [HLA‐G]) expression in a large cohort of patients with endometrial cancer, to determine the prognostic value of these cell surface markers and their relation with clinicopathological variables. Tissue microarrays containing epithelial endometrial carcinoma tissue from 554 patients were stained for classical and nonclassical MHC class I using the following monoclonal antibodies: 4H84 (anti‐HLA‐G), β2‐m (anti‐beta‐2‐microglobulin) and HC‐10 (MHC class I antigen heavy chain). Expression data were linked to known clinicopathological characteristics and survival. HLA‐G upregulation and MHC class I downregulation in neoplastic cells was observed in 40% and 48%, respectively. Nonendometrioid tumor type, advanced stage disease (FIGO stage ≥II) and poorly or undifferentiated tumors were associated with MHC class I downregulation. Absence of HLA‐G expression was independently associated with MHC class I downregulation. In univariate analysis, MHC class I downregulation was a predictor of worse disease‐specific survival. Prognostic unfavorable tumor characteristics were correlated with downregulation of MHC class I expression in endometrial cancer cells. Furthermore, downregulated MHC class I has a negative impact on disease‐specific survival, observed in a large cohort of patients with endometrial cancer. As there seems to be a relation between classical and nonclassical MHC class I molecules (HLA‐G), further research is warranted to unravel this regulatory mechanism.     

Expression of tolerogenic HLA-G molecules in cancer prevents antitumor responses

In this paper, we focus our attention on the relevance of HLA-G in cancer in the light of our recent advances on the expression and immunological function of HLA-G. Regarding HLA-G function, we recently showed that in addition to its direct inhibitory effects on T, APC and NK function, HLA-G induces suppressor cells via two distinct processes: (i) either by cell differentiation of naïve T cells into lasting suppressor T cells or (ii) by rapid transfer of HLA-G from APC or tumor cells to T or NK cells converting them into temporary HLA-G-positive suppressor cells. Regarding HLA-G expression, we described that tumor-microenvironment factors such as hypoxia, IDO and, TNF-α regulate the expression of HLA-G by tumor cells in a way that favors tumor escape from NK lysis. These findings reinforce the role of HLA-G as one mechanism of tumor-driven immune evasion and provide potential targets for testing novel anticancer treatment strategies.     

Expression of SAMHD1 and its mutation on prognosis of colon cancer

The expression of sterile α motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) and its mutation play a key role in the prognosis of colon cancer. The aim of the present study was to investigate the mechanism and the role of SAMHD1 in colon cancer. Microarray data from 187 patients with colon cancer and 45 adjacent normal tissue obtained from the Gene Expression Omnibus (GEO) were analyzed. A protein-protein interaction (PPI) network was constructed to identify key genes associated with colon cancer prognosis. Cox proportional hazard regression and survival analyses were performed to identify the potential for SAMHD1 to serve as a prognostic biomarker. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed to assess the expression levels and distribution of SAMHD1 in tissues and cells. Western blotting (WB) and Cell Counting Kit-8 (CCK-8) assays were used to identify the proliferation and apoptotic effects of SAMHD1 on HT-29 (Cas9-SAMHD1) cell lines. A total of 6,905 consistently differentially expressed genes were identified in the GEO database. Through the PPI network, SAMHD1 was found to be associated with Kirsten rat sarcoma virus (KRAS). SAMHD1 expression was negatively associated with KRAS. Proportional hazards regression and survival analyses demonstrated that low expression of SAMHD1 was associated with increased patient mortality. IHC and IF results demonstrated that SAMHD1 expression in patients with colon cancer was decreased compared with controls (both P<0.05). CCK-8 and WB results showed that proliferation was significantly promoted, and the expression levels of apoptosis-related proteins were significantly inhibited in the D137N and D311A groups as a result of a mutation in the deoxynucleoside triphosphohydrolase (dNTPase) site (both P<0.05 vs. wild-type). Proliferation was inhibited and apoptosis-related protein expression levels were promoted in the wild-type (WT) and D137N groups following 20 µg/ml 5-fluorouracil (5-FU) treatment (both P<0.05). WB and CCK-8 results showed cell proliferation was promoted and cell apoptosis-related protein expression was inhibited in the D137N group following treatment with 20 µg/ml 5-FU (all P<0.05) compared with the WT group. In conclusion, SAMHD1 expression was low in colon cancer. The dNTPase function of SAMHD1 may inhibit colon cancer cell proliferation and may enhance apoptosis. In addition, first-line chemotherapy with 5-FU has a time-dependent effect, which may provide novel options for clinical treatment of colon cancer.     

HLA class I and class II expression of pulmonary adenocarcinoma cells and the influence of interferon gamma

Background: The clinico-biological significance of HLA (both class I antigen and class II one) expressed on tumor cells still remains controversial. Methods: Tumor cells were freshly separated from 33 surgical specimens of pulmonary adenocarcinoma. The tumor cells were incubated for 24 h in the presence or absence of IFN-γ (130 International Units/ml). After incubation, the cells were cytocentrifuged onto glass slides and immunostained with either an anti-HLA class I (A, B, C) monoclonal antibody or anti-HLA class II (DR) one. Results: In 22 of 33 cases (66.7%), the HLA class I were individually expressed by more than 60% of tumor cells while so were the HLA class II in 15 (45.4%). No significant correlation was observed between the HLA class I expression and the HLA class II one. The proportion of HLA class I-positive tumor cells correlated with neither the grade of histological differentiation nor the stage of disease. In contrast, the proportion of HLA class II-positive tumor cells correlated with both the grade of histological differentiation and the stage. In most cases, IFN-γ was found to increase the proportion of class II-positive tumor cells as well as that of class I-positive cells. Conclusions: The above findings thus suggested that the HLA class II expression might therefore represent a manifestation of cellular differentiation and that IFN-γ may, as a result, have the potential to differentiate cancer cells.     

Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer

Patients with squamous cell carcinoma of the head and neck (SCCHN) have depressed antitumour immunity. The presence of CD4+CD25+ (Treg) cells in these patients might be, in part, responsible for downregulation of antitumour immune responses. To evaluate the frequency and characteristics of Treg in the peripheral circulation of patients with SCCHN, we used multicolour flow cytometry. Expression of CCR7, CD62L, zeta chain and Annexin V binding to Treg and non-Treg CD4+ lymphocyte populations were evaluated. Treg were confirmed to be Foxp3+ and GITR+. The Treg frequency was significantly elevated in patients with active disease and those with no evidence of disease (NED) following curative therapies. Both Treg and non-Treg CD4+ T cells in patients were significantly enriched in CCR7- and CD62L- cell subsets. Although Treg in patients contained a higher proportion of double negative (CCR7-CD62L-) cells, the majority of Tregs were CCR7-CD62L+. The proportion of Annexin V+CD4+ T cells was higher in patients (P<0.00005) than normal controls (NC), and Treg were significantly more sensitive to apoptosis than non-Treg in patients and NC. Expression of zeta was reduced in all subsets of CD4+ T cells obtained from patients vs NC. The data suggest that Treg in patients with SCCHN largely contain T cells with the 'effector' phenotype, which bind Annexin V and have low zeta expression, consistent with their activation state and a rapid turnover in the peripheral circulation.     

CD19+CD24hiCD38hiBregs involved in downregulate helper T cells and upregulate regulatory T cells in gastric cancer

Regulatory B cells (Bregs) play a critical role in inflammation and autoimmune disease. We characterized the role of Bregs in the progression of gastric cancer. We detected an increase in Bregs producing IL-10 both in peripheral blood mononuclear cells (PBMCs) and in gastric tumors. Multicolor flow cytometry analysis revealed that a subset of CD19⁺CD24^hiCD38^hi B cells produces IL-10. Functional studies indicated that increased Bregs do not inhibit the proliferation of CD3⁺T cells or CD4⁺ helper T cells (Th cells). However, Bregs do suppress the secretion of IFN-γ and TNF-α by CD4⁺Th cells. CD19⁺CD24^hiCD38^hiBregs were also found to correlate positively with CD4⁺FoxP3⁺ regulatory T cells (Tregs). Neutralization experiments showed that Bregs convert CD4⁺CD25⁻ effector T cells to CD4⁺FoxP3⁺Tregs via TGF-β1. Collectively, these findings demonstrate that increased Bregs play a immunosuppressive role in gastric cancer by inhibiting T cells cytokines as well as conversion to Tregs. These results may provide new clues about the underlying mechanisms of immune escape in gastric cancer.     

β-asarone suppresses HCT116 colon cancer cell proliferation and liver metastasis in part by activating the innate immune system

Studies have revealed that β-asarone exerts a powerful inhibitory effect on the proliferation of human cancer cells. The authors'' previous study demonstrated that β-asarone could induce LoVo colon cancer cell apoptosis in vitro and in vivo, indicating its anticancer properties. The present study aimed to determine the antineoplastic effect of β-asarone in HCT116 colon cancer cells. An in vitro proliferation assay using a real time cell analyzer demonstrated that β-asarone effectively decreased HCT116 cell proliferation in a dose-dependent manner. Bioinformatics analysis revealed that differentially expressed genes following β-asarone inhibition were involved in the ‘cell cycle’, ‘cell division’, ‘cell proliferation’ and ‘apoptosis’. Subsequently, a xenograft assay evidenced the inhibitory effect of β-asarone on the growth of HCT116 tumors in vivo. Further detection of immune-associated cytokines and cells suggested that β-asarone might be involved in the antitumor immune response by stimulating granulocyte-colony stimulating factor and increasing the number of macrophage cells in the spleen. Additionally, a murine model of splenic-transplantation verified the strong suppressive role of β-asarone in colon cancer liver metastasis in vivo. Taken together, the results of the current study revealed that β-asarone decreased HCT116 colon cancer cell proliferation and liver metastasis potentially by activating the innate immune system, supporting the multi-system regulation theory and providing a basis for further mechanistic studies on colon cancer.     

Different classes of antibiotics exhibit disparate negative impacts on the therapeutic efficacy of immune checkpoint inhibitors in advanced non-small cell lung cancer patients

It has been reported that antibiotics (ATBs) have adverse effect on the efficacy of treatment with immune checkpoint inhibitors (ICIs) in cancer patients. Since different classes of ATBs have different antibacterial spectrum, we aimed to study whether all ATBs had similar or different negative effects on the clinical outcomes of ICIs in patients with advanced non-small cell lung cancer (NSCLC). Patients with advanced NSCLC who received ICIs were included in this retrospective study and grouped by the class of ATBs they had used around the ICIs treatment time. The overall survival (OS) and the progression free survival (PFS) of patients among these groups were compared using Kaplan-Meier method and Cox proportional hazards model. A total of 148 eligible patients were enrolled, and 80 patients used ATBs. The results indicated that quinolones had no significant negative consequence on the clinical outcomes, while β-lactams significantly shortened the OS and PFS of patients. Furthermore, patients exposed to the combination of β-lactams and quinolones suffered the worst OS and PFS. Moreover, the subgroup analysis of β-lactams revealed that only penicillins, but not carbapenems and cephalosporins, markedly reduced both OS and PFS. In addition to the class of ATBs used, the time frame of ATBs used also affected the clinical outcomes of ICIs therapy. Patients receiving ATBs within 60 days prior to and 30 days after the initiation of ICI treatment had significantly shorter OS and PFS compared with those who did not use ATBs. This study demonstrated that different classes of ATBs had disparate negative impacts on the clinical outcomes, and the use of β-lactams, especially penicillins, should be avoided in advanced NSCLC patients who are receiving or scheduled to receive ICIs within 60 days.     

Identification of a ternary protein-complex as a therapeutic target for K-Ras-dependent colon cancer

A cancer phenotype is driven by several proteins and targeting a cluster of functionally interdependent molecules should be more effective for therapeutic intervention. This is specifically important for Ras-dependent cancer, as mutated (MT) Ras is non-druggable and targeting its interaction with effectors may be essential for therapeutic intervention. Here, we report that a protein-complex activated by the Ras effector p38γ MAPK is a novel therapeutic target for K-Ras-dependent colon cancer. Unbiased proteomic screening and immune-precipitation analyses identified p38γ interaction with heat shock protein 90 (Hsp90) and K-Ras in K-Ras MT, but not wild-type (WT), colon cancer cells, indicating a role of this complex in Ras-dependent growth. Further experiments showed that this complex requires p38γ and Hsp90 activity to maintain MT, but not WT, K-Ras protein expression. Additional studies demonstrated that this complex is activated by p38γ-induced Hsp90 phosphorylation at S595, which is important for MT K-Ras stability and for K-Ras dependent growth. Of most important, pharmacologically inhibition of Hsp90 or p38γ activity disrupts the complex, decreases K-Ras expression, and selectively inhibits the growth of K-Ras MT colon cancer in vitro and in vivo. These results demonstrated that the p38γ-activated ternary complex is a novel therapeutic target for K-Ras-dependent colon cancer.     

Up‐regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration‐resistant prostate cancer

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor‐induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome‐wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration‐resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin‐2 (IL‐2) and transforming growth factor‐β (TGF‐β) pathways. Studies revealed that the levels of expression of genes responsible for T‐cell proliferation (C‐FOS, C‐JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4⁺CD25^highCD127^– Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.     

Expression of human leukocyte antigen class I and β2‐microglobulin in colorectal cancer and its prognostic impact

Downregulation of human leukocyte antigen (HLA) class I has been postulated to be a mechanism of adaptive immune escape in various tumors, especially microsatellite instability–high (MSI‐H) colorectal cancer (CRC). In this study, we aimed to investigate HLA class I and β2‐microglobulin (β2M) expression in MSI‐H and microsatellite‐stable (MSS) CRCs and determine its prognostic impact. The representative areas from the tumor center (TC) and tumor periphery (TP) from 300 CRCs, including 161 MSI‐H and 139 MSS cases, were selected to construct a tissue microarray. Immunohistochemistry (IHC) for HLA A/B/C, β2M, CD3, and CD8 was performed. Reduced HLA A/B/C expression was detected in 113 (70.2%) MSI‐H and 54 (38.8%) MSS cases, while reduced β2M expression was observed in 69 (42.9%) MSI‐H and 17 (12.2%) MSS cases. Although reduced β2M expression was associated with higher pathological tumor (pT) stage in MSI‐H CRC with borderline significance, no association was found between HLA A/B/C and β2M expression and survival. Interestingly, reduced HLA A/B/C expression in MSS was associated with higher stage, and reduced HLA A/B/C and β2M expression was an independent prognostic factor in multivariate analysis. In conclusion, reduced HLA A/B/C and β2M expression was frequently observed in immunotherapy‐naive MSI‐H CRC, suggesting the possibility of primary resistance to immune checkpoint inhibitor. Interestingly, downregulation of HLA A/B/C and β2M was associated with poor prognosis in MSS cancers. Overall, IHC for HLA A/B/C and β2M might be a feasible predictive or prognostic tool in CRC.     

CCL17 and CCL22 chemokines within tumor microenvironment are related to accumulation of Foxp3+ regulatory T cells in gastric cancer

It has been reported that an increased population of regulatory T cells (Tregs) is one of the reasons for impaired anti-tumor immunity. Recently, Foxp3 has been reported as a reliable marker of Tregs. The authors investigated the frequency of Foxp3(+) Tregs within CD4(+) cells in TILs, regional lymph nodes and PBLs of gastric cancer patients (n = 45). Furthermore, to elucidate the mechanisms behind Treg accumulation within tumors, they evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3(+) Tregs in gastric cancer. CD4(+)CD25(+)Foxp3(+) Tregs as a percentage of CD4(+) cells were counted by flow cytometry and evaluated by immunohistochemistry. Moreover, an in vitro migration assay using Tregs derived from gastric cancers was performed in the presence of CCL17 or CCL22. As a result, the frequency of Foxp3(+) Tregs in TILs was significantly higher than that in normal gastric mucosa (12.4% +/- 7.5% vs. 4.1% +/- 5.3%, p < 0.01). Importantly, the increase in Tregs in TILs occurred to the same extent in early and advanced disease. Furthermore, the frequency of CCL17(+) or CCL22(+) cells among CD14(+) cells within tumors was significantly higher than that of normal gastric mucosa, and there was a significant correlation between the frequency of CCL17(+) or CCL22(+) cells and Foxp3(+) Tregs in TILs. In addition, the in vitro migration assay indicated that Tregs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3(+) Tregs, with such an observation occurring in early gastric cancer.     

High casein kinase 1 epsilon levels are correlated with better prognosis in subsets of patients with breast cancer

Reliable biological markers that predict breast cancer (BC) outcomes after multidisciplinary therapy have not been fully elucidated. We investigated the association between casein kinase 1 epsilon (CK1ε) and the risk of recurrence in patients with BC. Using 168 available tumor samples from patients with BC treated with surgery +/− chemo(radio)therapy, we scored the CK1ε expression as high (≥1.5) or low (<1.5) using an immunohistochemical method. Kaplan-Meier analysis was performed to assess the risk of relapse, and Cox proportional hazards analyses were utilized to evaluate the effect of CK1ε expression on this risk. The median age at diagnosis was 60 years (range 35-96). A total of 58% of the patients underwent breast conservation surgery, while 42% underwent mastectomy. Adjuvant chemotherapy and radiation therapy were administered in 101 (60%) and 137 cases (82%), respectively. Relapse was observed in 24 patients (14%). Multivariate analysis found high expression of CK1ε to be associated with a statistically significant higher disease-free survival (DFS) in BC patients with wild-type p53 (Hazard ratio [HR] = 0.33; 95% CI, 0.12-0.91; P = 0.018) or poor histological differentiation ([HR] = 0.34; 95% CI, 0.12-0.94; P = 0.039) or in those without adjuvant chemotherapy ([HR] = 0.11; 95% CI, 0.01-0.97; P = 0.006). Our data indicate that CK1ε expression is associated with DFS in BC patients with wild-type p53 or poor histological differentiation or in those without adjuvant chemotherapy and thus may serve as a predictor of recurrence in these subsets of patients.