Prognostic value of PD­L1 expression and CD68 macrophages in tumor nest of patients with primary gastric cancer.

The programmed death receptor 1/programmed death receptor ligand 1 axis (PD-1/PD-L1) is involved in tumor immune escape and is a potential prognostic biomarker and anti-tumor immunotherapy target in patients with gastric cancer (GC). However, the results of studies obtained in recent years have been inconsistent. The present study aimed to determine the possible predictive significance of PD-L1 in conjunction with three proteins linked with PD-L1 regulation in patients with primary GC. In the present study, the PD-L1, human epidermal growth factor receptor 2 (HER2), cluster of differentiation (CD)133 and microphage-associated CD68 expression levels were identified by multiplexed immunohistochemistry and assessed by automated pathological analysis system in 93 GC tumors and neighboring normal tissues arrayed on the same tissue microarray. All four proteins were statistically analyzed in relation to the clinicopathological characteristics. The expression levels of HER2, CD133 and CD68 were considerably higher in cancer tissues compared with neighboring normal tissues (P<0.05), however, the reverse trend was detected for PD-L1 expression (P=0.0577), particularly in tumor nest (TN; P<0.05). There was no significant correlation between the HER2 and CD133 expression levels and clinicopathological factors. However, significant relationships were found between PD-L1 expression and the TNM stage, pathological differentiation and survival status of patients (P<0.05). Moreover, survival time was prolonged in individuals with elevated PD-L1 expression in TN and GC tissues, but no significant correlation was identified (P=0.0881). The CD68 expression level in tumor stroma, but not in TN, was significantly correlated with poor pathological differentiation in patients with GC (P<0.05). However, PD-L1+CD68+ macrophages were strongly related to lower tumor size (diameter <5 cm), early TNM stage (stage I+II), good pathological differentiation and overall survival in TN (P<0.05). In conclusion, PD-L1+CD68+ macrophage infiltration in TN might be a potential indicator of prognosis in patients with primary GC and merits further investigation.

Cytokine levels in patients with non-M3 myeloid leukemia are key indicators of how well the disease responds to chemotherapy.

Acute myeloid leukemia (AML) is a malignant hematological neoplastic disease. Autocrine or paracrine cytokines released by leukemic cells regulate the proliferation of AML cells. It is uncertain whether cytokines can indicate whether patients with AML are in remission with chemotherapy. The goal of this study was to evaluate the levels of Th1/Th2/Th17 cytokines in AML patients before and after chemotherapy to determine whether the cytokine levels could predict disease remission after chemotherapy. It was found that the levels of IL-5, IL-6, IL-8, IL-10, TNF-α, TNF-ß, IL-17F, and IL-22 were significantly increased at the time of AML diagnosis in patients who achieved remission after two chemotherapy treatments (P < 0.05). After chemotherapy, the cytokine levels were reduced in patients with remission, while the levels of IL-6 and IL-8 were raised in patients without remission (P < 0.05). A comparison of cytokine levels before and after chemotherapy in patients who achieved remission showed areas under the curve (AUCs) of 0.69 for both IL-6 and IL-8. In addition, a comparison of the remission and non-remission groups after chemotherapy showed an AUC of 0.77 for IL-6. We then calculated the cutoff value using receiver operating characteristic curves. Values of IL-6 < 9.99 and IL-8 < 8.46 at the time of diagnosis were predictive of chemotherapy success and remission, while IL-6 > 14.89 at diagnosis suggested that chemotherapy would not be successful and remission would not be achieved. Multifactorial analysis showed that age, Neu, IL-6, and IL-8 were independent risk factors for AML prognosis, and IL-6 (OR = 5.48, P = 0.0038) was superior to age (OR = 3.36, P = 0.0379), Neu (OR = 0.28, P = 0.0308), IL-8 (OR = 0.0421, P = 0.0421). In conclusion, IL-6 levels were found to be predictive of the likelihood of remission.

Expression of Th1/2/17 Cytokines in CML with or without Pulmonary Bacterial and Fungal Coinfection.

Background: Tyrosine kinase inhibitors (TKIs) are the standard therapy for patients with chronic myeloid leukemia (CML). While their use greatly increases patient survival rates and can lead to normal life expectancy, bacterial infections in the lungs continue to play a significant role in determining patient outcomes. Methods: In this study, the medical records of 272 CML and 53 healthy adults were analyzed. Information on age, sex, body temperature, procalcitonin (PCT), C-reactive protein (CRP), and cytokine levels were collected from patients. Since the data belonged to a nonstate distribution, we used the Mann-Whitney U test to examine differences between groups. Cut-off values were analyzed by receiver operating characteristic (ROC) curves. Results: No significant differences in the Th1/2/17 levels were observed in relation to TKI treatment. Further analysis showed that the levels of the interleukins IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-22, IL-12p70, IL-17A, IL-17F, and IL-1ß, interferon (IFN-γ), and tumor necrosis factors (TNF α and ß) were higher in patients with pulmonary bacterial infections compared with uninfected patients. IL-6, IL-8, and IL-10 levels in CML patients with bacterial and fungal coinfection were higher than those in patients without infection. The areas under the ROC curves (AUCs) were found to be 0.73 for IL-5, 0.84 for IL-6, 0.82 for IL-8, 0,71 for IL-10, and 0.84 for TNF-α. AUC values were higher for patients with pulmonary bacterial infection, especially IL-6 (AUC = 0.84, cut-off = 13.78 pg/ml) and IL-8 (AUC = 0.82, cut-off = 14.35 pg/ml), which were significantly better than those for CRP (AUC = 0.80, cut-off = 6.18 mg/l), PCT (AUC = 0.71, cut-off = 0.25 ng/ml), and body temperature (AUC = 0.68, cut-off = 36.8°C). In addition, according to the cut-off values, we found that 83.33% of patients with pulmonary bacterial infections had IL-6 ≥ 13.78 pg/ml, while when IL-6, IL-8, and IL-10 levels simultaneously exceeded the cut-off values, the probability of pulmonary bacterial infection was 93.55%. Conclusions: TKI treatment did not appear to affect cytokine expression in CML patients. However, CML patients with pulmonary bacterial infection had significantly higher levels of Th1/2/17 cytokines. In particular, abnormally elevated IL-6, IL-8, and IL-10 levels were associated with a pulmonary bacterial infection in patients with CML.

Cytokines help suggest aplastic anemia with pulmonary bacterial or co-fungal infection.

Although aplastic anemia (AA) does not come under the category of blood malignant diseases, the infection that frequently occurs in this bone marrow failure can make it worse. Pulmonary infection is the most prevalent but limiting clinical diagnosis. To find biomarkers predicting bacterial or bacterial-combined fungal infections in the lungs, we reviewed 287 AA medical records including 151 without any infection, 87 with pure pulmonary bacterial infection, and 49 with bacterial and fungal infection were reviewed. There were substantial changes in IL-17F, IL-17A, IFN-γ, IL-6, IL-8, and IL-10 levels between the non-infected and lung bacterial infection groups (P < 0.05). Further, a significant variation in IL-17A, TNF-ß, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-22, and IL-12p70, between the uninfected group and the pulmonary bacterial and fungal infection group (P < 0.05) was observed. The results further revealed significant differences in TNF-ß, IL-12p70, IL-6, IL-8, and IL-10 between the pulmonary bacterial infection group and the fungal infection group (P < 0.05). Moreover, by calculating ROC and cut-off values, we determined that IL-6 (AUC = 0.98, Cut-off = 14.28 pg/ml, P = 0.0000) had a significant advantage than other cytokines, body temperature (AUC = 0.61, P = 0.0050), PCT (AUC = 0.57, P = 0.0592), and CRP (AUC = 0.60, P = 0.0147) in the detection of lungs bacterial infections. In addition, IL-6 (AUC = 1.00, Cut-off = 51.50 pg/ml, P = 0.000) and IL-8 (AUC = 0.87, Cut-off = 60.53 pg/ml, P = 0.0000) showed stronger advantages than other cytokines, body temperature (AUC = 0.60, P = 0.0324), PCT (AUC = 0.72, Cut-off = 0.63 ng/ml, P = 0.0000) and CRP (AUC = 0.79, Cut-off = 5.79 mg/l, P = 0.0000) in distinguishing bacteria from fungi. This may suggest that IL-8 may play a role in differentiating co-infected bacteria and fungi. Such advantages are repeated in severe aplastic anemia (SAA) and very severe aplastic anemia (VSAA).In conclusion, aberrant IL-6 elevations in AA patients may predict the likelihood of bacterial lung infection. The concurrent increase of IL-6 and IL-8, on the other hand, should signal bacterial and fungal infections in patients.These findings may help to suggest bacterial or fungal co-infection in patients with AA (Focus on VSAA and SAA).

Interferon-induced protein 16 expression in colorectal cancer and its correlation with proliferation and immune signature markers.

Interferon-induced protein 16 (IFI16) is important for innate immune recognition of foreign/damaged DNA. Abnormal IFI16 expression is closely related to the occurrence of multiple malignant tumours, but its expression pattern in colorectal cancer (CRC) remains unclear. The present study aimed to investigated IFI16 expression and association with cell proliferation in CRC tissues and adjacent normal tissues. A multiplex immunofluorescence panel of antibodies against IFI16, Ki-67 and phosphorylated (p)-ERK1/2 was applied to assess a tissue microarray (TMA). The TMA included 77 CRC samples and 74 normal adjacent tissue samples which were collected from The First People's Hospital of Yunnan Province (Kunming, China) (3 paracancerous tissues were lost because of repeated cutting). Immunohistochemistry was used to detect CD8+ tumour-infiltrating lymphocyte (TIL) abundance and programmed death-ligand 1 (PD-L1) expression in cancer tissues. The present study demonstrated that IFI16 localized to the nucleus of CRC cells. Although IFI16 was weakly expressed in normal mucosal epithelial cells, absent to strong expression was detectable in different patients with CRC. Typically, IFI16 was not co-localized with Ki-67 within CRC cells. The multiplex immunofluorescence data demonstrated that the proportion of IFI16-/Ki-67+ cells from CRC tissues was 57.13%; however, that of IFI16+/Ki-67+ cells was 1.50%. The IFI16-/Ki-67+ phenotype was significantly positively associated with the tumor-node-metastasis stage and was marginally significantly correlated with lymph node metastasis. p-ERK1/2 protein was primarily localized to the cytoplasm and cell membrane of CRC cells and sometimes to the nucleus. Although, IFI16 demonstrated a strong correlation with p-ERK1/2, IFI16 did not co-localize with p-ERK1/2 and the proportion of IFI16 and p-ERK1/2 double-negative CRC cells was 84.95%. IFI16 expression displayed no significant association with CD8+ TILs or PD-L1. However, a strong positive correlation between CD8+ TILs and PD-L1 was observed. High CD8+ TIL infiltration in CRC tissue was associated with lower lymph node metastasis and tumor-node-metastasis stage. In summary, the results of the present study provided a novel insight for the role of IFI16 in CRC occurrence via the regulation of cancer cell proliferation.

Application of the chemokine-chemokine receptor axis increases the tumor-targeted migration ability of cytokine-induced killer cells in patients with colorectal cancer.

Cytokine-induced killer (CIK) cells are a group of heterogeneous immune cells which can be isolated from human peripheral blood mononuclear cells and have demonstrated therapeutic benefit both in hematologic malignancies and solid tumors, including colorectal cancer. However, poor tumor-targeted migration has limited the clinical efficacy of CIK cell treatment. The chemokine-chemokine receptor (CK-CKR) axis serves a role in the tumor-directed trafficking capacity of immune cells. Investigating the relationship between CKR profiles on the surface of CIK cells and chemokine expression levels in the tumor microenvironment may improve CIK cell therapy. In the present study, the spectrum of chemokine expression levels in tumor tissues from patients with colorectal cancer (CRC) and CKR expression profiles in CIK cells obtained from the same individuals with CRC were investigated. The results showed that chemokine expression levels in tumor tissues exhibited variability and cell line heterogeneity. However, the expression levels of a number of chemokines were similar in different CRC donors and cell lines. Expression levels of CXCLL10, CXCL11 and CCL3 were significantly higher in most tumor tissues compared with adjacent normal tissues and highly expressed in most CRC cell lines. In accordance with chemokine expression levels, CKR profiles on the surface of CIK cells also showed donor-to-donor variability. However, concordant expression profiles of CKRs were identified in different patients with CRC. CXCR3 and CXCR4 were highly expressed on the surface of CIK cells through the culture process. Importantly, the expression levels of all CKRs, especially CCR4, CXCR4 and CXCR3, were notably decreased during the course of CIK cell expansion. The changing trend of CKR profiles were not correlated with the chemokine expression profiles in CRC tissues (CCL3, CXCL12 and CXCL10/CXCL11 were highly expressed in CRC tissue). Re-stimulating CIK cells using chemokines (CCL21 and CXCL11) at the proper time point increased corresponding CKR expression levels on the surface of CIK cells and enhance tumor-targeted trafficking in vitro. These results demonstrated that modification of the CK-CKR axis using exogenous recombinant chemokines at the proper time point enhanced CIK cell trafficking ability and improved CIK antitumor effects.

Identification of an intermediate signature that marks the initial phases of the colorectal adenoma-carcinoma transition.

The colorectal adenoma-carcinoma sequence describes the stepwise progression from normal to dysplastic epithelium and then to carcinoma. Only a small proportion of colorectal adenomas (CRAs) progress to colorectal carcinomas (CRCs). Endoscopic intervention is currently being used on patients with high grade dysplasia CRAs, with diameters of >1 cm, or villous components of >25% who are at higher risk than other CRA sufferers. During the process, biopsy samples are taken for conventional histological diagnosis, but poor pathomorphological sensitivity and specificity greatly limit the diagnostic accuracy. Unfortunately, there are no reliable molecular criteria available that can predict the potential development of CRA to CRC. Gene expression profiles of normal colorectal mucosa (NOR), CRA and different Dukes' stages of CRC biopsy specimens, which represent the gradual progress of the CRA to CRC sequence, were determined by Affymetrix technology. Representative regulated genes were further analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Intersectional analyses of discriminative expression signatures of CRC vs. CRA and CRA vs. NOR allowed the identification of an intermediate signature of 463 probe sets (psets) that mark the NOR--> CRA-->CRC progression. This signature represents a reservoir of candidate markers for the early diagnosis of higher-risk CRA, thus allowing for timely therapeutic intervention and more selective treatment. A further 279 CRC-specific psets pointing to the malignant transition from CRA to CRC were identified and these could represent potential therapeutic targets for CRC. The reliability of the results was further confirmed by qRT-PCR and IHC analyses of the 4-gene sets randomly selected from the 463 psets.

[Study on the relationship between HBV infection and polymorphism of CIITA G-944C gene among Jinuo, Dai and Aini minority populations in Yunnan province].

OBJECTIVE: To explore the relationship between HBV infection and the genotypes and allele frequencies of CIITA G-944C gene polymorphism in three minority populations (Jinuo, Dai and Aini population) in Xishuangbanna district, Yunnan province. METHODS: Polymerase chain reaction and sequencing method were used to study the genotypes and allele frequencies distributions of CIITA G-944C gene polymorphism in those three populations. Relationship between the genotypes distribution and HBV infection results were also analyzed. RESULTS: The rates on HBV infection and HBsAg carrier status in Aini minority population were 89.2% and 16.3%, which were significantly higher than in Jinuo (27.9% and 3.9%, χ(2) = 135.196 and 10.361, P = 0.000 and 0.001) and Dai population (44.9% and 6.6%, χ(2) = 96.783 and 8.748, P = 0.000 and 0.003) while among Aini population it was significantly different with the other two minority populations. The CC genotype and C allele frequencies were more distributed in Aini population than in the other two minority populations. In contrast, the GG genotype and G allele frequencies were lower than the other two minority populations, with χ(2) rates between Aini and Jinuo population were 11.841 and 12.208 and the P as 0.003 and 0.000 respectively while the χ(2) rates between Aini and Dai population were 23.902 and 20.220 with P value as 0.000 and 0.000. The genotypes frequencies of CIITA G-944C was significantly different in the infected individuals (IF) group and health control (HC) group in Jinuo population (χ(2) = 6.150 and 4.911, P = 0.046 and 0.027). When compared with HBsAg(+) group and HBsAg(-) group, the genotypes and allele frequencies were different in Aini population and the total three minority populations (χ(2) rates in Jinuo minority were 8.650 and 5.034 with P values as 0.013 and 0.025). However, the χ(2) rates in the whole population were 13.047 and 9.416 with P values as 0.001 and 0.002, respectively. The distribution of CC genotype and C allele gene in HBsAg(+) group was increasing. Data from non-condition logistic regression analysis and adjusting for confounding factors, the HBsAg(+) group had a significantly increase of HBsAg(-) group under the C allele Recessive Model (P = 0.000; OR = 2.964; 95%CI: 1.609 - 5.460). CONCLUSION: The genotypes and allele frequencies distribution of CIITA G-944C were different in the three ethnic populations. Polymorphism of this gene was closely associated with HBsAg carrier. The CC genotype patients were more easily to become HBsAg carrier.

[Screening of differentially expressed genes from patients with Bardet-Biedl syndrome by gene chip analysis].

OBJECTIVE: To investigate the differential gene expression profile from patients with Bardet-Biedl syndrome (BBS) by oligonucleotide microarray technique. METHODS: Total RNA of 3 probands with BBS and 4 healthy siblings were isolated from peripheral blood mononuclear cells and reverse-transcribed to cDNAs. Then the cDNAs were subjected for microarray screening with Affymetrix U133 Plus 2.0 array. Genechip scanner was applied to screen the hybridization signals. Genes differentially expressed between the BBS probands and controls were identified by using GCOS1.4 software with the standard of two-fold change (P<0.05) of expression. RESULTS: Fifteen genes were up-regulated 2 or more fold and another 15 genes were down-regulated 2 or more fold in the BBS patients, among them 12 genes were related to signaling pathway and cell cycle by Gene Ontology (GO) analysis. CONCLUSION: The differentially expressed genes identified may correlate with the function or structure of cilia. Their roles in the BBS genesis need to be further studied.

Novel hepatitis B virus subgenotype in the southern Yunnan Province of China.

Hepatitis B virus (HBV) infection is highly prevalent in China. To identify the genotypes of HBV in the southern Yunnan Province of China, full-length HBV genomes were extracted from 1 Dai and 4 Hani HBV carriers and linked with the pMD T-18 vector. For each patient, 3-10 clones were sequenced directly and a consensus sequence was created. Genotypic and serotypic analysis revealed 4 HBV/B (2 B2 with adw2 and 2 new subgenotypes with ayw1) and 1 HBV/C (C1 with adrq+) genotypes. The divergences of the entire genome sequences of the new subgenotype were 0-0.9% and 2.99-6.48% between other known HBV/B. Divergences in other coding regions revealed that it was more similar to B3 and B4 in the precore/core gene (2.02 and 2.09%, respectively), and similar to B3 and B5 in the preS1/S2/S gene (2.24 and 2.78%, respectively). Phylogenetic trees using the precore/core and X genes both revealed a new clad separating from the major trunk of genotype B with a 99% bootstrap value. These results show that the 2 consensus isolates are a mosaic of B3-B5, which we designated to subgenotype B6. Considering the geographical distances, the relationship between B6 and other HBV/B subgenotypes (B3-B5) and HBV evolution needs to be further studied.

FOXL2 gene mutations and blepharophimosis-ptosis-epicanthus inversus syndrome (BPES): a novel mutation detected in a Chinese family and a statistic model for summarizing previous reported records.

Previous studies found that the forkhead transcription factor 2 (FOXL2) gene mutations are responsible for both types of blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) but have not established any systematic statistic model for the complex and even contradictory results about genotype-phenotype correlations between them. This study is aimed to find possible mutations of FOXL2 gene in a Chinese family with type II BPES by using DNA sequencing and to further clarify genotype-phenotype correlations between FOXL2 mutations and BPES by using a systematic statistical method, namely Multifactor Dimensionality Reduction (MDR). A novel mutation (g.933_965dup) which could result in an expansion of the polyalanine (polyAla) tract was detected in all patients of this family. MDR analysis for intragenic mutations of FOXL2 gene reported in previous BPES studies indicated that the mutations which led to much stronger disturbance of amino acid sequence were responsible for more type I BPES, while other kinds of mutation were responsible for more type II BPES. In conclusion, the present study found a novel FOXL2 gene mutation in a Chinese BPES family and a new general genotype-phenotype correlation tendency between FOXL2 intragenic mutations and BPES, both of which expanded the knowledge about FOXL2 gene and BPES.

Virologic characteristics of hepatitis B virus in patients infected via maternal-fetal transmission.

AIM: To determine whether HBV with the same characteristics causes dissimilar mutations in different hosts. METHODS: Full-length HBV genome was amplified and linked with pMD T18 vector. Positive clones were selected by double-restriction endonuclease digestion (EcoRI and HindIII) and PCR. Twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (S)], and the phylogenetic and mutational analysis was performed using BioEditor, Clustal X and MEGA software. Potential immune epitopes were determined by the Stabilized Matrix Method (SMM), SMM-Align Method and Emini Surface Accessibility Prediction. RESULTS: All of the 27 sequences were genotype C, the divergence between the mother and son was 0%-0.8%. Compared with another 50 complete sequences of genotype C, the mother and her son each had 13 specific nucleotides that differed from the other genotype C isolates. AA 1-11 deletion in preS1 was the dominant mutation in the mother (14/18). The 1762T/1764A double mutation existed in all clones of the mother, 3 of them were also coupled with G1896A mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential HLA class I epitopes and one B cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the P gene. CONCLUSION: The HBV strain in the son came from his mother, and discrepant mutation occurred in the mother and her son during infection.