Open the Technical Black Box of Tumor Mutational Burden (TMB): Factors Affecting Harmonization and Standardization of Panel-Based TMB.

As tumor mutational burden (TMB) has been approved as a predictive biomarker for immune checkpoint inhibitors (ICIs), next-generation sequencing (NGS) TMB panels are being increasingly used clinically. However, only a few of them have been validated in clinical trials or authorized by administration. The harmonization and standardization of TMB panels are thus essential for clinical implementation. In this review, preanalytic, sequencing, bioinformatics and interpretative factors are summarized to provide a comprehensive picture of how the different factors affect the estimation of panel-based TMB. Among the factors, poor DNA quality, improper formalin fixation and residual germline variants after filtration may overestimate TMB, while low tumor purity may decrease the sensitivity of the TMB panel. In addition, a small panel size leads to more variability when comparing with true TMB values detected by whole-exome sequencing (WES). A panel covering a genomic region of more than 1Mb is more stable for harmonization and standardization. Because the TMB estimate reflects the sum of effects from multiple factors, deliberation based on laboratory and specimen quality, as well as clinical information, is essential for decision making.

Genetic and Functional Effects of Adiponectin in Type 2 Diabetes Mellitus Development.

Diabetes mellitus (DM) is a common chronic metabolic disease, and the C57BLKsJ-db/db mice are good animal models for type 2 diabetes mellitus (T2DM). In this study, Western blotting and immunohistochemistry (IHC) were employed to examine the protein expression of adiponectin in the liver tissues of T2DM mice with different disease courses (4, 16, and 32 weeks). Adiponectin expression reduced in the liver tissues of T2DM mice in different disease courses. The genotypic and allelic frequencies of the adiponectin gene rs1063538 and rs2241766 single nucleotide polymorphisms (SNPs) in a Taiwanese population (570 T2DM patients and 1700 controls) were investigated. Based on the genetic distribution of the rs2241766 locus, the distribution frequency of the T allele in the T2DM group (72.8%) was higher than in the control group (68.8%). Individuals carrying the G allele had a 0.82-fold greater risk of developing T2DM than individuals carrying the T allele. Differences were evident in the genotypic and allelic distributions (p < 0.05). Enzyme-linked immunosorbent assay (ELISA) was used to measure changes in serum adiponectin protein concentrations in the healthy population and in patients with T2DM. Serum adiponectin concentration in patients with T2DM was lower than in the control group. In summary, adiponectin was determined to be a T2DM susceptibility gene and may be involved in T2DM progression.

Analytical validation of human epidermal growth factor receptor 2 immunohistochemistry by the use of the A0485 antibody versus the 4B5 antibody and breast versus gastric scoring guidelines in ovarian clear cell carcinoma.

AIMS: The aim of this study was to evaluate human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) in ovarian clear cell carcinoma (OCCC) by using two antibodies and two scoring guidelines in correlation with HER2 amplification and clinicopathological features. METHODS AND RESULTS: A tissue microarray was constructed by use of a total of 71 OCCC cases for IHC (the A0485 antibody and the 4B5 antibody) and dual-colour silver in-situ hybridisation (DISH). Two pathologists independently scored the IHC according to the 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) breast cancer guidelines (breast guidelines) and the 2016 ASCO/CAP gastro-oesophageal adenocarcinoma guidelines (gastric guidelines). IHC concordances between A0485 and 4B5 were 87.3-93.0%. Three to 16 (4.2-22.5%) cases had an IHC score of 2+/3+ with frequent basolateral/lateral membranous staining. The 4B5 antibody yielded fewer IHC 2+ cases than the A0485 antibody (n = 2-6 versus n = 5-12). Five (7.0%) cases had HER2 amplification as determined with DISH. Cases with papillary-predominant growth patterns were significantly more likely to have HER2 amplification (P = 0.0051). In predicting DISH results, IHC scored according to the gastric guidelines yielded 100%/100% sensitivity and 83.3-95.5%/98.2-100% specificity, and IHC scored according to the breast guidelines yielded 60-80%/33.3-66.7% sensitivity and 95.5-100%/100% specificity (including/excluding IHC 2+ cases). One case had intratumoral heterogeneity, with discordant results between primary and metastatic tumour specimens. CONCLUSION: We demonstrated HER2 amplification in 7% of OCCC cases, and the molecular change is significantly associated with papillary-predominant growth patterns. In predicting HER2 amplification, a combination of 4B5 IHC and gastric guidelines provides the best sensitivity and fewer equivocal (IHC 2+) cases. Given the intratumoral heterogeneity, assessment of HER2 status on whole tissue sections and on both primary and metastatic tumour specimens is recommended.

Effects of Casein Kinase 2 Alpha 1 Gene Expression on Mice Liver Susceptible to Type 2 Diabetes Mellitus and Obesity.

Diabetes mellitus (DM) is a chronic disease found worldwide. Notably, BKS.Cg- Dock7m +/+ Leprdb/JNarl mice are useful animal models for studying type 2 diabetes mellitus (T2DM). In this study, we investigated casein kinase 2 alpha 1 (CSNK2A1) gene and protein expression in the liver tissues of mice at different ages (4, 16, and 32 weeks) using real-time quantitative polymerase chain reactions, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay. Our data paved the way for exploring BKS.Cg- Dock7m +/+ Leprdb/JNarl in the mouse model by demonstrating a significant increase in gene and protein expression in T2DM (+Leprdb/+Leprdb) mouse liver when compared to control (+Dock7m/+Dock7m) mouse liver. We also observed that CSNK2A1 protein level in the serum of T2DM patient group was higher than that of the control group, although the data was not statistically significant. Based on our findings, we can now understand the role of CSNK2A1 gene upregulation when encountering T2DM pathologies.

Loss of Response Gene to Complement 32 (RGC-32) in Diabetic Mouse Retina Is Involved in Retinopathy Development.

Diabetic retinopathy (DR) is a severe and recurrent microvascular complication in diabetes. The multifunctional response gene to complement 32 (RGC-32) is involved in the regulation of cell cycle, proliferation, and apoptosis. To investigate the role of RGC-32 in the development of DR, we used human retinal microvascular endothelial cells under high-glucose conditions and type 2 diabetes (T2D) mice (+Leprdb/ + Leprdb, db/db). The results showed that RGC-32 expression increased moderately in human retinal endothelial cells under hyperglycemic conditions. Histopathology and RGC-32 expression showed no significant changes between T2D and control mice retina at 16 and 24 weeks of age. However, RGC-32 expression was significantly decreased in T2D mouse retina compared to the control group at 32 weeks of age, which develop features of the early clinical stages of DR, namely reduced retinal thickness and increased ganglion cell death. Moreover, immunohistochemistry showed that RGC-32 was predominantly expressed in the photoreceptor inner segments of control mice, while the expression was dramatically lowered in the T2D retinas. Furthermore, we found that the level of anti-apoptotic protein Bcl-2 was decreased (approximately 2-fold) with a concomitant increase in cleaved caspase-3 (approximately 3-fold) in T2D retina compared to control. In summary, RGC-32 may lose its expression in T2D retina with features of DR, suggesting that it plays a critical role in DR pathogenesis.

Study of sex-biased differences in genomic profiles in East Asian hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is characterized by a notable sex disparity in incidence and tumor aggressiveness. Revealing differences in genetic landscapes between male and female HCCs may expand the understanding of sexual disparities mechanisms and assist the development of precision medicine. Although reports on the sex disparity of HCC are accumulated, studies focusing on sex-related biomarkers among Asian populations remain limited. Here, we conducted a comprehensive genomic profiling analysis to explore differences between male and female patients within a cohort of 195 Taiwanese HCC patients. We did not detect any sex-biased genomic alterations. However, when our investigation extended to the TCGA dataset, we found higher frequencies of gene copy gains in CCNE2 and mutations in CTNNB1 and TP53 among male patients. Besides, we further evaluated the associations between genomic alterations and patients' prognosis by sex. The results showed that female patients harboring tumors with STAT3 gain and alterations in the JAK-STAT pathway displayed a poor prognosis. These two factors remained independently associated with unfavorable prognosis even after adjusting for the patient's age and stage characteristics (Hazard ratio = 10.434, 95% CI 3.331-32.677, P < 0.001; Hazard ratio = 2.547, 95% CI 1.195-5.432, P = 0.016, respectively). In summary, this study provides valuable insights into understanding sex disparity in HCC in the East Asian population. Validation through larger cohorts and extensive sequencing efforts is warranted.

Genomic and tumour microenvironmental biomarkers of immune checkpoint inhibitor response in advanced Taiwanese melanoma.

Objective: Genomic biomarkers predicting immune checkpoint inhibitor (ICI) treatment outcomes for Asian metastatic melanoma have been rarely reported. This study presents data on next-generation sequencing (NGS) and tumour microenvironment biomarkers in 33 cases. Methods: Thirty-three patients with advanced melanoma, who underwent ICI treatment at the Chang Gung Memorial Hospital in Taiwan, were recruited. The study evaluated clinical outcomes, including response rate, disease control rate, progression-free survival (PFS) rate and overall survival (OS) rate. Archived tissue samples from 33 cases were subjected to NGS by ACTOnco, and ACTTME was employed in 25 cases. Results: The most prevalent driver mutations were BRAF mutations (24.2%), followed by NRAS (15.2%), KIT (12.1%), KRAS (9.1%) and NF1 (9.1%) mutations. Acral/mucosal melanomas exhibited distinct mutation patterns compared to non-acral melanomas. Tumour mutational burden estimated using ACTOnco was not associated with ICI efficacy. Notably, genetic alterations in the p53 pathway (CDKNA2 loss, MDM2 gain/amplification and TP53 mutation) accounted for 36.4% and were significantly associated with unfavourable PFS (median PFS 2.7 months vs. 3.9 months, P = 0.0394). Moreover, 26 genes were identified as differentially expressed genes that were upregulated in patients with clinical benefits compared to those without benefits. Four genes, GZMH, GZMK, AIM2 and CTLA4, were found to be associated with both PFS and OS. Conclusion: Genetic alterations in the p53 pathway may be critical in Asian patients with melanoma undergoing ICI treatment. Further investigation is required to explore this mechanism and validate these findings.

Interpretation According to Clone-Specific PD-L1 Cutoffs Reveals Better Concordance in Muscle-Invasive Urothelial Carcinoma.

Because immune checkpoint inhibitors have been approved for treating advanced urothelial carcinoma (UC), programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assays have been widely used as companion or complementary diagnostic tests for predicting treatment outcomes. Because different clones, scoring algorithms, and cutoffs have been used for interpretation, this study investigated the variation, correlation, and concordance of four validated PD-L1 clones (SP142, SP263, 22C3, and 28-8) and proposed a practical solution for the harmonization of PD-L1 IHC. A tissue microarray, including 46 muscle-invasive UCs, was constructed for PD-L1 testing with the four clones. Tumor cell (TC) and immune cell (IC) expression was analyzed. SP142 had significantly low TC expression, whereas SP263, 22C3, and 28-8 exhibited a moderate correlation (rho ≥ 0.6), with almost perfect concordance (intraclass correlation coefficient > 0.8) in TC expression. Fair to moderate correlation and concordance were observed in IC expression in most pairwise comparisons of clones. Substantial concordance (kappa > 0.6) was noted when high PD-L1 expression was defined by applying clone-specific cutoffs to each clone. Our findings imply that a universal cutoff value is not feasible for UC; we propose that PD-L1 IHC assays for UC should be interpreted according to a clone-specific scoring algorithm and cutoff value.

Good staining quality ensuring the reproducibility of Ki67 assessment.

AIMS: Although Ki67 labelling index (LI) is a prognostic and predictive marker in breast cancer, its accuracy and reproducibility must be validated before its clinical application. We aimed to evaluate the agreement of Ki67 LI in clinical practice in Taiwan. METHODS: We conducted a Ki67 immunohistochemistry (IHC) proficiency test. The participants performed the Ki67 IHC test and measured the Ki67 LI of 10 cases of breast cancer tissue on a microarray slide. The staining quality was centrally reviewed based on the Ki67 staining of the tonsil surface epithelium. RESULTS: Ki67 staining and counting methods are diverse in Taiwan. The reproducibility of Ki67 LI was poor to good (intraclass correlation coefficient: 0.581, 95% CI 0.354 to 0.802). The reproducibility and agreement in the high staining quality group were significantly higher than those in the low staining quality group. The majority of the Ki67 LIs derived from the low staining quality group were underestimated. Different counting methods did not reveal significant differences when determining Ki67 LI with microarray sections. CONCLUSIONS: We suggest using the surface epithelium of the tonsil as external control and achieving optimal staining results that consist of a high positive parabasal layer, a low positive intermediate layer and a negative superficial layer. Good Ki67 staining quality can minimise the staining variations among different laboratories, and it is essential for the reproducibility of Ki67 LI.

Thyroid fine-needle aspiration cytology in Taiwan: a nationwide survey and literature update.

In Taiwan, thyroid fine-needle aspiration cytology is easily accessible and reliable for evaluating thyroid nodules. The sonographic pattern plays a major role and is the deciding factor for aspiration. We conducted a nationwide survey in 2017 and it revealed that 31% of laboratories had adopted The Bethesda System for Reporting Thyroid Cytopathology. There was a relatively high unsatisfactory rate (24.04%) and low rates of indeterminate diagnoses, including atypia of undetermined significance/follicular lesions of undetermined significance: 4.87%, and follicular neoplasm/suspicious for a follicular neoplasm: 0.35%. Moreover, the risks of malignancy in benign, atypia of undetermined significance, and suspicious for a follicular neoplasm were relatively high. These may reflect strict diagnostic criteria for indeterminate categories and better patient selection for surgery. Improvements in specimen sampling and continuing education programs are crucial. Newly-developed thyroid cytology technologies, such as immunocytochemistry, molecular testing, and computerized cytomorphometry, may further facilitate cytology diagnoses.

Diagnostic Agreement for High-Grade Urothelial Cell Carcinoma in Atypical Urine Cytology: A Nationwide Survey Reveals a Tendency for Overestimation in Specimens with an N/C Ratio Approaching 0.5.

In the Paris System (TPS), standardized cytomorphological criteria and diagnostic categories were proposed for reporting urine cytology. To evaluate the diagnostic agreement and interobserver concordance for assessing TPS criteria, the Taiwan Society of Clinical Cytology organized an online survey with 10 atypical urine cytology cases. A total of 137 participants completed the survey. The mean agreement of diagnosis was 51.2%, ranging from 34.3% to 83.2% for each case. For 60% (6/10) of cases, the agreement was <50%. The interobserver concordance of diagnosis and cytological criteria assessment showed poor agreement. The nuclear-to-cytoplasmic (N/C) ratio had the highest kappa value of 0.386, indicating a significantly higher interobserver concordance and reproducibility than the other three TPS criteria. The correct rate of assessing the N/C ratio increased as the N/C ratio increased (correlation coefficient: 0.891, p < 0.01). Three cases with an N/C ratio near 0.5 were overestimated. Poor interobserver concordance of diagnosis and TPS criteria was revealed. Compared with other cytological features, the N/C ratio assessment was quantitative and more reproducible, but a tendency to overestimate cells was noted when the N/C ratio was approximately 0.5. Continuing education programs should emphasize the accurate assessment of N/C ratio to improve the application of TPS.

Impact of the COVID-19 pandemic on cytology practice: An international survey in the Asia-Pacific region.

BACKGROUND: The purpose of the current study was to examine the impact of coronavirus disease 2019 (COVID-19) on various aspects of cytology practice in the Asia-Pacific region. METHODS: An online questionnaire was distributed to cytopathology laboratories in 24 Asia-Pacific countries to explore the impact of restrictive measures on access to health care, use of general and personal protective equipment (PPE), and changes in cytology workflow and workload from February to April 2020. RESULTS: A total of 167 cytopathology laboratories from 24 countries responded to the survey; the majority reported that restrictive measures that limited the accessibility of health care services had been implemented in their cities and/or countries (80.8%) and their hospitals (83.8%). The respondents noted that COVID-19 had an impact on the cytologic workflow as well as the workload. Approximately one-half of the participants reported the implementation of new biosafety protocols (54.5%) as well as improvements in laboratory facilities (47.3%). Rearrangement or redeployment of the workforce was reported in 53.3% and 34.1% of laboratories, respectively. The majority of the respondents reported a significant reduction (>10%) in caseload associated with both gynecological (82.0%) and nongynecological specimens (78.4%). Most laboratories reported no significant change in the malignancy rates of both gynecological (67.7%) and nongynecological specimens (58.7%) compared with the same period in 2019. CONCLUSIONS: The results of the survey demonstrated that the COVID-19 pandemic resulted in a significant reduction in the number of cytology specimens examined along with the need to implement new biosafety protocols. These findings underscore the need for the worldwide standardization of biosafety protocols and cytology practice.

Antizyme inhibitor 1 genetic polymorphisms associated with diabetic patients validated in the livers of diabetic mice.

Diabetes mellitus (DM) is a complex disease caused by absolute or relative insulin deficiency. The C57BLKsJ-db/db mouse model is a useful animal model for studying type 2 DM (T2DM). The present study investigated the association between an antizyme inhibitor 1 (AZIN1) gene polymorphism (rs1062048) and T2DM susceptibility in 2,270 Taiwanese individuals (570 patients with T2DM and 1,700 controls). Additionally, the present study investigated AZIN1 gene and protein expression in the liver tissues of mice in three age groups (4, 16 and 32 weeks) through reverse transcription-quantitative PCR, western blotting and immunohistochemistry. The data indicated that the genotype frequency distribution of the rs1062048 single-nucleotide polymorphism differed significantly between the patients with T2DM and controls (P<0.05). Furthermore, gene and protein expression levels of AZIN1 were significantly lower in early stage and late stage T2DM mouse liver samples than in control samples. Overall, the data suggested that AZIN1 expression is involved in T2DM development.

Increased Expression of Ecto-NOX Disulfide-thiol Exchanger 1 (ENOX1) in Diabetic Mice Retina and its Involvement in Diabetic Retinopathy Development.

BACKGROUND/AIM: Diabetic retinopathy (DR) is a type of retinal damage caused by a complication of diabetes and is a major cause of blindness in working-age adults. Ecto-NOX disulfide-thiol exchanger 1 (ENOX1) is a member of the ecto-NOX family involved in the plasma membrane electron transport pathway. This study aimed to investigate the role of ENOX1 in the development of DR. MATERIALS AND METHODS: Human retinal endothelial cells (HRECs) and human retinal pigment epithelial cells (HREpiCs) exposed to a high concentration (25 mM) of D-glucose and type 2 diabetes (T2D) mice (+Leprdb/+Leprdb, db/db) with retinopathy were used as models to determine the ENOX1 expression levels there. RESULTS: Our results showed that ENOX1 expression levels did not significantly change in both HRECs and HREpiCs under hyperglycemic conditions for 48 h. Nevertheless, ENOX1 expression increased significantly in T2D mouse retinas, particularly in the photoreceptor layer, compared to the control mouse retinas. CONCLUSION: Different retinal ENOX1 expression in T2D mice and control mice suggested that ENOX1 may be involved in DR development.

Cholesteryl Ester Transfer Protein Genetic Variants Associated with Risk for Type 2 Diabetes and Diabetic Kidney Disease in Taiwanese Population.

Cholesteryl ester transfer protein (CETP) plays an important role in lipid metabolism. Low levels of high-density lipoprotein cholesterol (HDL-C) increase the risk of type 2 diabetes (T2D). This study investigated CETP gene variants to assess the risk of T2D and specific complications of diabetic kidney disease (DKD) and diabetic retinopathy. Towards this, a total of 3023 Taiwanese individuals (1383 without T2D, 1640 with T2D) were enrolled in this study. T2D mice (+Leprdb/+Leprdb, db/db) were used to determine CETP expression in tissues. The A-alleles of rs3764261, rs4783961, and rs1800775 variants were found to be independently associated with 2.86, 1.71, and 0.91 mg/dL increase in HDL-C per allele, respectively. In addition, the A-allele of rs4783961 was significantly associated with a reduced T2D risk (odds ratio (OR), 0.82; 95% confidence interval (CI), 0.71‒0.96)), and the A-allele of rs1800775 was significantly related to a lowered DKD risk (OR, 0.78; 95% CI, 0.64‒0.96). CETP expression was significantly decreased in the T2D mice kidney compared to that in the control mice (T2D mice, 0.16 0.01 vs. control mice, 0.21 0.02; p = 0.02). These collective findings indicate that CETP variants in the promoter region may affect HDL-C levels. Taiwanese individuals possessing an allele associated with higher HDL-C levels had a lower risk of T2D and DKD.