Peripheral immunological features of COVID-19 patients in Taizhou, China: A retrospective study.

BACKGROUND: Abnormal peripheral immunological features are associated with the progression of coronavirus disease 2019 (COVID-19). METHODS: Clinical and laboratory data were retrieved in a cohort of 146 laboratory-confirmed COVID-19 patients. Potential risk factors for the development of severe COVID-19 were evaluated. RESULTS: On admission, lymphocytes, CD3+, CD4+ and CD8+ T cells, eosinophils, and albumin and pre-albumin were dramatically lower, whereas neutrophils, and interleukin (IL)-10, C-reactive protein (CRP), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were significantly higher in severe cases. By the second week after discharge, all variables improved to normal levels. Covariate logistic regression results showed that the CD8+ cell count and CRP level were independent risk factors for severe COVID-19. CONCLUSION: Lower peripheral immune cell subsets in patients with severe disease recovered to normal levels as early as the second week after discharge. CD8+ T cell counts and CRP levels on admission are independent predictive factors for severe COVID-19.

Intergrating conventional MRI, texture analysis of dynamic contrast-enhanced MRI, and susceptibility weighted imaging for glioma grading.

BACKGROUND: The application of conventional magnetic resonance imaging (MRI) in glioma grading is limited and non-specific. PURPOSE: To investigate the application values of MRI, texture analysis (TA) of dynamic contrast-enhanced MRI (DCE-MRI) and intratumoral susceptibility signal (ITSS) on susceptibility weighted imaging (SWI), alone and in combination, for glioma grading. MATERIAL AND METHODS: Fifty-two patients with pathologically confirmed gliomas who underwent DCE-MRI and SWI were enrolled in this retrospective study. Conventional MRIs were evaluated by the VASARI scoring system. TA of DCE-MRI-derived parameters and the degree of ITSS were compared between low-grade gliomas (LGGs) and high-grade gliomas (HGGs). The diagnostic ability of each parameter and their combination for glioma grading were analyzed. RESULTS: Significant statistical differences in VASARI features were observed between LGGs and HGGs ( P < 0.05), of which the enhancement quality had the highest area under the curve (AUC) (0.873) with 93.3% sensitivity and 80% specificity. The TA of DCE-MRI derived parameters were significantly different between LGGs and HGGs ( P < 0.05), of which the uniformity of Ktrans had the highest AUC (0.917) with 93.3% sensitivity and 90% specificity. The degree of ITSS was significantly different between LGGs and HGGs ( P < 0.001). The AUC of the ITSS was 0.925 with 93.3% sensitivity and 90% specificity. The best discriminative power was obtained from a combination of enhancement quality, Ktrans- uniformity, and ITSS, resulting in 96.7% sensitivity, 100.0% specificity, and AUC of 0.993. CONCLUSION: Combining conventional MRI, TA of DCE-MRI, and ITSS on SWI may help to improve the differentiation between LGGs and HGGs.

Activation of the cardiac proteasome promotes angiotension II-induced hypertrophy by down-regulation of ATRAP.

Proteasomal degradation is critical to maintaining cardiac function and is altered in various diseases. Angiotensin II (Ang II) acts as a growth factor to induce cardiac growth. Here we aimed to test whether proteasome is involved in the development of Ang II-induced cardiac hypertrophy and dissect its molecular mechanisms. Cardiac hypertrophy was induced by Ang II infusion (1000 ng/kg/min) using mini-osmotic pumps. Starting 1 day before implantation, the mice were injected with the proteasome inhibitor bortezomib (BTZ, 50 µg/kg, 3 times per week) or with vehicle. After 14 days, the pool of ubiquitinated proteins was reduced but the protein expression of proteasome subunits (including ß1i, ß2i and ß5/ß5i) was markedly up-regulated in left ventricular hypertrophy versus control, which was accompanied by a significant increase in proteasome activities. Furthermore, Ang II-treated mice showed a significant increase in blood pressure but decrease in cardiac contractile function, and significant left ventricular hypertrophy, fibrosis and inflammation, which were all attenuated in BTZ-treated mice. Mechanistically, these beneficial effects were associated with the inhibition of degradation of angiotensin II type 1 receptor-associated protein (ATRAP) and inactivation of AT1R-mediated p38 MAPK and STAT3 signaling pathways. In conclusion, our data indicate that the activation of proteasome is required for the Ang II-induced cardiac hypertrophy, and suggest that the inhibition of proteasome activity by BTZ could be a potential therapeutic strategy for the treatment of cardiac hypertrophy and other heart diseases.

Circulating E3 ligases are novel and sensitive biomarkers for diagnosis of acute myocardial infarction.

Ubiquitin ligase (E3) is a decisive element of the ubiquitin-proteasome system (UPS), which is the main pathway for intracellular protein turnover. Recently, circulating E3 ligases have been increasingly considered as cancer biomarkers. In the present study, we aimed to determine if cardiac-specific E3 ligases in circulation can serve as novel predictors for early diagnosis of acute myocardial infarction (AMI). By screening and verifying their tissue expression patterns with microarray and real-time PCR analysis, six of 261 E3 ligases, including cardiac-specific Rnf207 and cardiac- and muscle-enriched Fbxo32/atrogin-1, Trim54/MuRF3, Trim63/MuRF1, Kbtbd10/KLHL41, Asb11 and Asb2 in mouse heart, were selected for the present study. In the AMI rats, the levels of five E3 ligases including Rnf207, Fbxo32, Trim54, Trim63 and Kbtbd10 in the plasma were significantly increased compared with control animals. Especially, the plasma levels of Rnf207 was markedly increased at 1 h, peaked at 3 h and decreased at 6-24 h after ligation. Further evaluation of E3 ligases in AMI patients confirmed that plasma Rnf207 level increased significantly compared with that in healthy people and patients without AMI, and showed a similar time course to that in AMI rats. Simultaneously, plasma level of cardiac troponin I (cTnI) was measured by ELISA assays. Finally, receiver operating characteristic (ROC) curve analysis indicated that Rnf207 showed a similar sensitivity and specificity to the classic biomarker troponin I for diagnosis of AMI. Increased cardiac-specific E3 ligase Rnf207 in plasma may be a novel and sensitive biomarkers for AMI in humans.

AGGF1 protects from myocardial ischemia/reperfusion injury by regulating myocardial apoptosis and angiogenesis.

Angiogenic factor with G patch and FHA domains 1 (AGGF1) is a newly identified proangiogenic protein, which plays an important role in vascular disease and angiogenesis. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. This study investigated whether AGGF1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Wild-type (WT) C57BL/6 J mice were treated at 30 min prior to I/R injury with anti-AGGF1 neutralizing antibody (3 mg/kg) or recombinant human AGGF1 (rhAGGF1, 0.25 mg/kg). After I/R injury, the infarct size, the number of TUNEL-positive cardiomyocytes, Bax/Bcl2 ratio, inflammatory cytokine expression and angiogenesis were markedly increased as compared with sham control. Treatment of WT mice with anti-AGGF1 neutralizing antibody resulted in exaggeration of myocardial I/R injury but reducing angiogenesis. In contrast, administration of rhAGGF1 markedly reversed these effects. Furthermore, anti-AGGF1- or rhAGGF1-mediated effects on I/R-induced cardiac apoptosis, inflammation and angiogenesis were dose dependent. In addition, the protective effects of AGGF1 on cardiomyocyte apoptosis and inflammation were confirmed in cultured cardiomyocytes after I/R. Finally, these effects were associated with activation of ERK1/2, Stat3 and HIF-1α/VEGF pathways and inhibition of activation of NF-κB, p53 and JNK1/2 pathways. In conclusion, we report the first in vivo and in vitro evidence that AGGF1 reduces myocardial apoptosis and inflammation and enhances angiogenesis, leading to decreased infarct size after I/R injury. These results may provide a novel therapeutic approach for ischemic heart diseases.

Prognostic risk stratification value of MACC1 expression in patients with gastric cancer.

BACKGROUND: The prognostic significance of metastasis-associated in colon cancer-1 (MACC1) has been explored in a variety of malignancies. However, its clinical relevance in patients with gastric cancer (GC) is limited, also remains controversial. METHOD: In this study, we retrospectively evaluated the prognostic value of lesion MACC1 expression in 347 GC patients. Lesion MACC1 expression was analyzed with immunohistochemistry and grouped as MACC1low (n = 172) and MACC1high (n = 175) cases. RESULTS: Data revealed that the degree of MACC1 expression is not related to patient sex, age and disease stage (all p > 0.05). Survival analysis showed that only post-operation advanced pT (p = 0.018), pN (p < 0.001), pM (p = 0.001) and AJCC stages (p < 0.001) are significantly associated with shorter survival, while no obvious difference was observed between MACC1low and MACC1high cases (p = 0.158). However, we found that survival for female (p = 0.032), older (p = 0.028), and early disease stage (pT stage I + II, p = 0.033) patients with MACC1high are remarkably worse than those with MACC1low. CONCLUSION: In summary, our findings revealed that, though MACC1 expression is not associated with the survival of the whole cohort, the prognostic risk stratification value of lesion MACC1 expression in subgroups of patients with gastric cancer should be noted.

Dynamic changes of soluble HLA-G and cytokine plasma levels in cervical cancer patients: potential role in cancer progression and immunotherapy.

PURPOSE: Chronic inflammation has been proven to be an important factor in carcinogenesis. Cytokines are the central mediators in the inflammatory microenvironment, and their release may be influenced by soluble HLA-G (sHLA-G). The aim of this study was to monitor the dynamic process of these soluble factors in patients with cervical cancer at Taizhou Hospital of Zhejiang Province, trying to understand their relationship with diagnosis, treatment, and prognosis. METHODS: We quantified plasma levels of sHLA-G and 12 cytokines using ELISA and flow cytometry, respectively, in the peripheral blood of patients with cervical cancer divided into three groups: preoperation, postoperation and clinical relapse. Healthy women were used as the control group. Data were analysed by non-parametric tests, receiver-operating characteristic (ROC) curves, and Kaplan-Meier plotter (log-rank test). RESULTS: In this study, our findings showed that preoperation plasma levels of sHLA-G and the cytokines IL-6, IL-10, and IFN-γ in cervical cancer patients had a good discriminatory effect between cervical cancer patients and healthy women. It should be noted that plasma levels of sHLA-G, IL-6, and IL-10 were significantly decreased within 30 days after radical hysterectomy (P < 0.05). A positive correlation was observed between IL-6 and IL-10, IL-8 and IL-17 levels preoperatively. In contrast, sHLA-G levels were negatively correlated with IL-10 but not with other cytokines. An increased survival rate in patients with cervical cancer was associated with IL-5 < 1.70 pg/mL, IL-17 < 2.30 pg/mL, and IFN-α < 2.26 pg/mL preoperatively. In addition, our findings showed that the levels of cytokines IL-6, IL-8, IL-12p70, IL-17, and IFN-γ may be related to 5-year relapse rates and/or the metastasis of cervical cancer. CONCLUSION: The current findings enhance our understanding of the dynamic process (preoperation, postoperation and clinical relapse) of sHLA-G and these cytokines in the plasma of patients with cervical cancer from diagnosis to prognosis. These biomarkers may play a potential therapeutic target role of such dynamic changes in the immunotherapy for cervical cancer.

Impact of Sample Processing and Storage Conditions on RNA Quality of Fresh-Frozen Cancer Tissues.

Background: A biobank is a central resource that supports basic and clinical research. RNA quality of fresh-frozen tissue specimens in the biobank is highly associated with the success of downstream applications. Therefore, it is very important to evaluate the impact of tissue processing and storage conditions on RNA quality. Methods: A total of 238 surgically removed tissue specimens, including esophagus, lung, liver, stomach, colon, and rectal cancer, were used to evaluate RNA quality. Two tissue homogenization methods, manual and TissueLyser, were compared and the impacts of temperature fluctuation, tissue types, storage period, and clinicopathological parameters on RNA quality were analyzed. Results: RNA integrity was not influenced by tissue homogenization methods and tissue types. However, RNA integrity number (RIN) values were significantly correlated with temperature fluctuation. When the power of a -80°C freezer was cut off, RNA integrity of frozen tissues was not significantly affected until the temperature increased to 0°C. When the temperature rose to room temperature and remained for 4 hours, RNA integrity was almost completely destroyed. In addition, various cancer tissues with short-term storage at -80°C (<5 years) or high tumor differentiation had higher RINs. Conclusions: Tissue processing and storage conditions affected RNA quality of fresh-frozen cancer tissues. It is necessary to keep storage temperature stable and keep specimens at ultralow temperatures during homogenization. Also, for a biobank containing multiple types of cancer tissue samples, it is better to store them in liquid nitrogen if the storage duration is more than 5 years.

[BEX2 regulates cell cycle through the interaction with INI1/hSNF5].

BEX2 (Brain expressed X-linked protein 2), a 13 kDa protein, is highly expressed in brain and testis. It is reported that the protein expression of BEX2 dramatically alters during the embryo development, but little is known about its function. By means of yeast two-hybrid screening, we isolated that INI1/hSNF5 was a binding partner for BEX2, a key component of SWI/SNF chromosome remolding complex. GST Pull-down experiment interaction is physical and specific. Further analysis using truncated mutations demonstrated that the two partner for BEX2, a key component of SWI/SNF chromosome remolding complex. GST Pull-down experiment confirmed that BEX2 can interact with INI1/hSNF5 directly and specifically. Truncated mutations analysis further demonstrated that the two conserved reverse repeats sequences within INI1/hSNF5 were necessary for the interaction. Sub-cellular localization showed that both BEX2 and INI1/hSNF5 mainly localized in cell nucleus, which indicated that the interaction may be involved in the regulation of gene expression. Our experiments also showed that co-overexpressing of the two proteins affected cell cycle by increasing the cells in S phase, indicating that BEX2 could regulate cell cycle by interacting with INI1/hSNF5.

Prognostic significance of the immune checkpoint HLA-G/ILT-4 in the survival of patients with gastric cancer.

OBJECTIVE: Human leukocyte antigen-G (HLA-G) and its receptors, including immunoglobulin-like transcripts (ILT)-2 and ILT-4, are closely associated with cancer development and clinical outcomes of patients. However, the clinical significance of HLA-G and ILT-2/-4 in gastric cancer (GC) is limited. METHODS: In this study, the percentage of HLA-G-, ILT-2 and ILT-4 positive tumor cells in 127 GC lesion suspensions of tumor cells gated for epithelialcelladhesionmolecule(EpCAM) was determined using multicolor flow cytometry and their clinical significance was evaluated. RESULTS: Our data showed that the median percentages of HLA-G-, ILT-2, and ILT-4 expressing GC cells were 18.0%, 67.80%, and 1.42%, respectively, and co-expression of HLA-G/ILT-2, HLA-G/ILT-4, and ILT-2/ILT-4 was 16.9%, 1.42%, and 1.70%, respectively. Kaplan-Meier survival results revealed that besides post-operation N status (p = 0.006), M status (p = 0.001), and AJCC clinical stage (p < 0.001), only high percentage of ILT-4+ GC cells was a significant factor for worse survival of patients with GC (overall survival [OS]: 42.9 months vs. 84.5 months; p = 0.031). However, among female patients with GC (n = 31), high percentage of either HLA-G+ (OS: 18.5 months vs. 89.3 months; p = 0.001) or ILT-4+ (OS: 17.9 months vs. 85.8 months; p = 0.002) GC cells was markedly associated with a poor prognosis. CONCLUSION: Our findings revealed that among HLA-G, ILT-2, and ILT-4, only a high percentage of ILT-4+ GC cells was significantly related to poor prognosis in the entire cohort of patients with GC. However, high percentage of HLA-G+ and ILT-4+ GC cells is associated with poor clinical outcome among female patients with GC.

Pre-operative neutrophil-to-lymphocyte ratio is an independent prognostic factor in patients with gastric cancer.

OBJECTIVE: To evaluate the prognostic significance of peripheral lymphocyte count and its derived inflammatory markers, such as neutrophil-to-lymphocyte ratio (NLR), in a cohort of patients with gastric cancer (GC). METHODS: In this retrospective study, the clinical characteristics and follow-up information, both pre- and post-operative within one week of laboratory findings, of 338 patients with GC who underwent radical gastrectomy were retrieved, and their prognostic significance was evaluated. RESULTS: Both lower pre- and post-operative lymphocyte counts and higher NLR and SSI were significantly related to advanced tumour (pT) and disease stages (American Joint Committee on Cancer [AJCC]) in patients with GC. Log-rank survival analysis showed that, in addition to traditional pT, pN, pM, and AJCC stages, both lower pre- (p = 0.041) and post-operative (p = 0.002) lymphocyte counts and higher NLR (ppre < 0.001 and ppost = 0.008) and SSI (ppre = 0.014 and ppost = 0.145) were associated with worse survival. Cox proportional hazards analysis revealed that pre-operative NLR (p = 0.018; hazard ratio = 1.778) was an independent predictor of prognosis in patients with GC. Moreover, when the pre-operative NLR was divided into NLRlow and NLRhigh, NLRhigh showed stratified prognostic value for patient sex (male, p = 0.001; female, p = 0.044), age (younger, p = 0.005; older, p = 0.005), and AJCC stage III (p = 0.007). CONCLUSION: Pre-operative NLR is an independent prognostic factor for patients with GC and has stratified prognostic value for patients with AJCC stage III GC.

Prognostic Significance of Immune Checkpoints HLA-G/ILT-2/4 and PD-L1 in Colorectal Cancer.

Immune checkpoint inhibitors (ICIs) have become a promising area of research for cancer treatment. In addition to the well-known ICIs targeting PD-1/PD-L1, HLA-G/ILT-2/-4 is relatively new immune checkpoint that has been evaluated in early clinical trials in patients with advanced solid tumors. In this study, the expression of HLA-G (n=157), ILT-2/4 (n=82), and PD-L1 (n=70) in epithelial cell adhesion molecule (EpCAM)-positive colorectal cancer (CRC) cells was analyzed by multicolor flow cytometry, and the prognostic significance of these molecules was evaluated. In EpCAM+ CRC cells, the median percentages of HLA-G, ILT-2, ILT-4, and PD-L1 were 14.90%, 67.70%, 8.55% and 80.30%, respectively. In addition, a positive correlation was observed between them (all p<0.001). Higher levels of these immune checkpoint proteins are associated with lymph node metastasis. In addition to the AJCC stage (p=0.001), Kaplan-Meier survival analysis showed that higher levels of HLA-G (p=0.041), ILT-2 (p=0.060), ILT-4 (p<0.001), PD-L1 (p=0.012), HLA-GILT4 (p<0.001) and ILT-2ILT-4 (p<0.001) were significantly associated with shorter survival of CRC patients. When CRC patients were stratified by early and advanced AJCC stages, HLA-G levels were only related to the survival among CRC patients with early disease stage (p=0.024), while ILT-4 levels were significant for both CRC patients with early (p=0.001) and advanced (p=0.020) disease stages. Multivariate cox regression analysis revealed that advanced AJCC stage (HR=2.435; p=0.005) and higher ILT-4 levels (HR=2.198; p=0.063) were independent risk factors for poor outcomes in patients with CRC. In summary, among the immune checkpoints, HLA-G/ILT-2/4 and PD-L1, ILT-4 is the most significant prognostic indicator of CRC. This finding indicated that a combination of immunotherapy strategies, such as ILT-4 blockade, could improve the clinical outcomes in patients with cancer. Moreover, multicolor flow cytometry can be employed as a reliable and efficient, alternative to immunohistochemistry, for evaluating the immune checkpoint proteins expressed in tumor lesions.

Intratumor Heterogeneity of HLA-G Expression in Cancer Lesions.

Signaling pathway between human leukocyte antigen (HLA)-G and immune inhibitory receptors immunoglobulin-like transcript (ILT)-2/4 has been acknowledged as one of immune checkpoints, and as a potential target for cancer immunotherapy. Like other immune checkpoints, inter- and even intratumor heterogeneity of HLA-G could render a rather complexity for HLA-G-target immunotherapy. However, little information for intratumor heterogeneity of HLA-G is available. In this study, HLA-G expression in a serial section of colorectal cancer (CRC) lesions from three CRC patients (each sample with serial section of 50 slides, 10 randomized slides for each antibody), three different locations within a same sample (five CRC), and three case-matched blocks that each includes 36 esophageal cancer samples, were evaluated with immunohistochemistry using anti-HLA-G antibodies (mAbs 4H84, MEM-G/1 and MEM-G/2 probing for all denatured HLA-G isoforms, 5A6G7, and 2A12 probing for denatured HLA-G5 and HLA-G6 isoforms). Our results revealed that, in addition to the frequently observed inter-tumor heterogeneity, intratumor heterogeneous expression of HLA-G is common in different areas within a tumor in CRC and esophageal cancer samples included in this study. Moreover, percentage of HLA-G expression probed with different anti-HLA-G antibodies also varies dramatically within a tumor. Given HLA-G has been considered as an important immune checkpoint, intratumor heterogeneity of HLA-G expression, and different specificity of anti-HLA-G antibodies being used among studies, interpretation and clinical significance of HLA-G expression in cancers should be with caution.

Mask wearing in pre-symptomatic patients prevents SARS-CoV-2 transmission: An epidemiological analysis.

OBJECTIVES: Pandemic COVID-19 has become a seriously public health priority worldwide. Comprehensive strategies including travel restrictions and mask-wearing have been implemented to mitigate the virus circulation. However, detail information on community transmission is unavailable yet. METHODS: From January 23 to March 1, 2020, 127 patients (median age: 46 years; range: 11-80) with 71 male and 56 female, were confirmed to be infected with the SARS-CoV-2 in Taizhou, Zhejiang, China. Epidemiological trajectory and clinical features of these COVID-19 cases were retrospectively retrieved from electronic medical records and valid individual questionnaire. RESULTS: The disease onset was between January 9 to February 14, 2020. Among them, 64 patients are local residents, and 63 patients were back home from Wuhan from January 10 to 24, 2020 before travel restriction. 197 local residents had definite close-contact with 41 pre-symptomatic patients back from Wuhan. 123 and 74 of them contact with mask-wearing or with no mask-wearing pre-symptomatic patients back from Wuhan, respectively. Data showed that incidence of COVID-19 was significantly higher for local residents close-contact with no mask-wearing Wuhan returned pre-symptomatic patients (19.0% vs. 8.1%, p < 0.001). Among 57 close-contact individuals, 21 sequential local COVID-19 patients originated from a pre-symptomatic Wuhan returned couple, indicated dense gathering in congested spaces is a high risk for SARS-CoV-2 transmission. CONCLUSIONS: Our findings provided valuable details of pre-symptomatic patient mask-wearing and restriction of mass gathering in congested spaces particularly, are important interventions to mitigate the SARS-CoV-2 transmission.

Genetic variability and oncogenic risk association of human papillomavirus type 58 E6 and E7 genes in Taizhou area, China.

It is well recognized that the association of human papillomavirus (HPV) and cervical carcinogenesis is based on the presence of HPV DNA sequence. The E6 and E7 oncoproteins encoded by high-risk HPV types play a key role in carcinogenesis. HPV58 type accounts for a larger share of cervical disease in China, whereas data on HPV58 genetic variability in China is limited. We aimed to evaluate the diversity of HPV58 genetic variants by sequencing the entire E6 and E7 genes. Phylogenetic trees were constructed by Maximum likelihood method by MEGA 5.05 software. In this study, the overall HPV infection rate was 22.6% (2891/12780) in Southeast China and the prevalence of HPV58 infection rate was 2.6% (335/12780). 26 nucleotides substitutions were observed in E6 and E7 genes with 10 novel substitutions and 17 non-synonymous substitutions. We obtained 25 distinct variation patterns which the accession GenBank numbers as MH348918-MH348942. All of HPV58 variants belong to lineage A, while no lineage B, C and D were detected in Taizhou area, Southeast China. The sublineage A1, A2, and A3 variants were found in 136 (68.3%), 39 (19.6%), and 24 (12.1%) of HPV58 isolates, respectively. The sublineage A3 variants with T20I/G63S substitutions at E7 oncoprotein carried a significantly higher risk for high-grade cervical intraepithelial neoplasia (CIN2 or worse, CIN2+) when compared with other HPV58 variants (odds ratio = 4.41, P < 0.05). Nevertheless, there was no association between HPV58 (sub) lineages and cervical lesions. These data provide the critical characteristics of HPV58 variants to assist further investigation of carcinogenic association and the development of next generation vaccines and diagnostic assays in China.

Biobanking of Fresh-Frozen Gastric Cancer Tissues: Impact of Long-Term Storage and Clinicopathological Variables on RNA Quality.

BACKGROUND: Molecular research is increasingly dependent on high-quality biobanked biospecimens. Preanalytical variables in tissue processing and preservation may influence the RNA quality and research results. Hence, the effect of long-term storage and clinicopathological parameters on RNA quality needs to be elucidated. METHODS: Ninety gastric cancer tissue samples were collected and fresh-frozen in a -80°C freezer for 12 years (2006-2017). The histology was assessed and RNA integrity number (RIN) was detected by an Agilent 2100 Bioanalyzer. The impact of storage duration on RNA integrity and histomorphology was analyzed. The difference between RIN values and clinical variables was analyzed. Correlations between pathological parameters such as tumor cell percentage, stroma percentage, necrosis extent, cellularity, and RIN were assessed, respectively. RESULTS: Long-term storage at -80°C for 12 years did not adversely affect RNA integrity and histomorphology. RNA integrity was also not influenced by tumor location, estimated blood loss, cold/warm ischemia time, and surgical approach. However, RIN values were significantly correlated with the tumor cell percentage and stroma percentage. Gastric cancer tissues with higher tumor cell percentage or lower stroma percentage had higher RIN values. CONCLUSIONS: RNA quality of fresh-frozen gastric cancer tissues is influenced by clinical and histological parameters. Standard tissue collection procedure and histological quality control remain essential for tissue biobanking.

Association of HLA-G 3' UTR polymorphism and expression with the progression of cervical lesions in human papillomavirus 18 infections.

BACKGROUND: Human leukocyte antigen (HLA)-G is an immune checkpoint molecule, which expression in cervical cancer cells enables them to escape immunosurveillance. To date, limited information has been published on the association of HLA-G genetic background in malignant cells with levels of HLA-G expression and the clinical outcome of patients. METHODS: We investigated the influence of the HLA-G 14 bp In/Del (rs66554220) and + 3142C/G (rs1063320) polymorphisms in 130 cases of HPV16 infection, 130 cases of HPV18 infection and 185 age-matched, unrelated, HPV-negative, and cytologically normal Chinese Han women. Case-matched cervical biopsy tissues were evaluated by immunohistochemistry. RESULTS: Our findings show that the frequency of alleles, 14 bp In (38.5% vs 29.2%, OR = 1.52, P < 0.05) and + 3142G (72.7% vs 57.0%, OR = 2.01, P < 0.05), were significantly increased in the HPV18-infected group compared with the control group. The HLA-G polymorphisms (alleles 14 bp In and + 3142G) are also associated with the progression of HPV18-related cervical lesions. Moreover, HLA-G expression increased from CIN1 to CIN2/3 lesions and was highest in patients with adenocarcinoma; however, a significant association between these characteristics and the HLA-G polymorphisms was not observed. CONCLUSION: Our results support that the HLA-G 14 bp In and + 3142G alleles are risk factors for HPV18 infections and influence the progression of HPV18-related cervical lesions. This suggests that HLA-G-driven immune mechanisms play an important role in cervical carcinogenesis.

Predictive value of different proportion of lesion HLA-G expression in colorectal cancer.

Differential expression of HLA-G has been observed among cancer types and tumors from individuals with the same type of cancer; however, its clinical significance is rather limited. In this study, expression and predictive relevance of HLA-G expression in 457 primary colorectal cancer (CRC, ncolon = 232, nrectal = 225) patients was investigated. Data showed 70.7% (323/457) of the CRC were HLA-G expression when the above 5% (HLA-GLow) was considered as positive, which wasn't associated with patient survival (p = 0.109). However, HLA-G expression above 55% (HLA-GHigh) was associated with a worse prognosis of CRC patients (p = 0.042). Furthermore, a shorter survival was found for the female (p = 0.042) and elder (p = 0.037) patients whose HLA-G expression was above HLA-GLow level. HLA-G expression above HLA-GHigh level showed a worse prognosis for female (p = 0.013), elder (p = 0.023), colon cancer (p = 0.016), advanced tumor burden (T3+4, p = 0.018), regional lymph node status (N1+2, p = 0.044), and advanced clinical stage patients (AJCC III+IV, p = 0.037). In conclusion, our results demonstrated for the first time that combination of differential lesion HLA-G expression notably improved the value of traditional survival prediction for CRC patients.