Lack of significant associations between single nucleotide polymorphisms in LPAL2-LPA genetic region and all cancer incidence and mortality in Japanese population: The Japan public health center-based prospective study.

BACKGROUND: High lipoprotein (a) level is an established cardiovascular risk, but its association with non-cardiovascular diseases, especially cancer, is controversial. Serum lipoprotein (a) levels vary widely by genetic backgrounds and are largely determined by the genetic variations of apolipoprotein (a) gene, LPA. In this study, we investigate the association between SNPs in LPA region and cancer incidence and mortality in Japanese. METHODS: A genetic cohort study was conducted utilizing the data from 9923 participants in the Japan Public Health Center-based Prospective Study (JPHC Study). Twenty-five SNPs in the LPAL2-LPA region were selected from the genome-wide genotyped data. Cox regression analysis adjusted for the covariates and competing risks of death from other causes, were used to estimate the relative risk (hazard ratios (HR) with 95% confidence intervals (CI)) of overall and site-specific cancer incidence and mortality, for each SNP. RESULTS: No significant association was found between SNPs in the LPAL2-LPA region and cancer incidence or mortality (overall/site-specific cancer). In men, however, HRs for stomach cancer incidence of 18SNPs were estimated higher than 1.5 (e.g., 2.15 for rs13202636, model free, 95%CI: 1.28-3.62) and those for stomach cancer mortality of 2SNPs (rs9365171, rs1367211) were estimated 2.13 (recessive, 95%CI:1.04-4.37) and 1.61 (additive, 95%CI: 1.00-2.59). Additionally, the minor allele for SNP rs3798220 showed increased death risk from colorectal cancer (CRC) in men (HR: 3.29, 95% CI:1.59 - 6.81) and decreased CRC incidence risk in women (HR: 0.46, 95%CI: 0.22-0.94). Minor allele carrier of any of 4SNPs could have risk of prostate cancer incidence (e.g., rs9365171 dominant, HR: 1.71, 95%CI: 1.06-2.77). CONCLUSIONS: None of the 25 SNPs in the LPAL2-LPA region was found to be significantly associated with cancer incidence or mortality. Considering the possible association between SNPs in LPAL2-LPA region and colorectal, prostate and stomach cancer incidence or mortality, further analysis using different cohorts is warranted.

Development of a new histological identification method of human sinoatrial node suitable for immunohistochemical study.

Histological identification of the human sinoatrial node (SAN) remains a challenge. Conventional identification methods, such as Lev's method, have certain limitations. The aim of our study was to develop a new histological identification method that could properly identify the sinoatrial node, applicable to the immunohistochemical study of intra-nodal structures. Thirty-nine human autopsied hearts were included in this study. The cases included 23 men and 16 women ranging in age from 20 to 99 years. The sinoatrial area from eight control samples was cut in the vertical section using the conventional Lev's method. In our new method, called the "En face one-block method," the sinoatrial node was cut in "En face" at the junction of the right border of the right appendage and superior vena cava, placed in one long cassette, and serially cut using a microtome. Immunostaining was performed using primary antibodies against CD31, podoplanin (D2-40), S-100, and other proteins. The average area of the SAN on the slide glass in our new method was 32.2 mm2, which was significantly larger than that (3.59 mm2) of the control samples by Lev's method. The SAN area was positively correlated with age (r = 0.357; p = 0.026), especially in women (r = 0.626; p = 0.0095). The SAN group had significantly lower percentage of CD31-positive blood capillaries, higher percentage of podoplanin-positive lymphatic channels, and S-100-positive peripheral nerves. We successfully developed a novel cutting method applicable to immunohistochemical studies, with which we could provide a bird's-eye view of the sinoatrial nodes.

Gross Cystic Disease Fluid Protein-15 (GCDFP-15) Expression Characterizes Breast Mucinous Carcinomas in Older Women.

The predominant histological subtype of breast mucinous carcinoma in older women is type B (hypercellular type), and, in younger women, it is type A (hypocellular type). The characteristics of mucinous carcinomas of the same histological subtype may differ between older and younger women. This study aims to systematically clarify the pathological/immunohistochemical features of mucinous carcinomas. A total of 21 surgical cases of mucinous carcinoma (type A/B: 9/12 cases) in the older group (≥65 years) and 16 cases (type A/B: 14/2 cases) in the younger group (≤55 years) (n = 37) were included. Gross cystic disease fluid protein-15 (GCDFP-15) and eight other markers were used for immunostaining. The GCDFP-15-positive rate in the older group was high regardless of the histological subtype (type A, 77.8%; type B, 91.7%). The GCDFP-15 positivity in the older group was significantly higher than that in the younger group (p < 0.001 for Allred score). Among type A, GCDFP-15 positivity was significantly higher in the older group than in the younger group (p = 0.042 for the Allred score and p = 0.007 for the positivity rate). The present results suggest that GCDFP-15 expression characterizes mucinous carcinomas in older women.

Dried blood spot is the feasible matrix for detection of some but not all hepatitis B virus markers of infection.

OBJECTIVE: Use of dried blood spots (DBS) for detection of hepatitis B virus (HBV) markers of infection has the potential to facilitate diagnosis of HBV infection especially in resource-limited countries. The aim of this study was to evaluate the feasibility of DBS for detection of various markers of HBV infections. RESULTS: Fifty-four DBS samples were engineered from well-characterized plasma samples. All DBS samples were tested for HBsAg, total anti-HBc and HBV DNA, 20 of 54 samples were also tested for HBeAg using commercially available assays. HBsAg was detected in 24 of 25 (96%), HBV DNA in 22 of 25 (88%), total anti-HBc in all 9 (100%), and HBeAg in all 7 (100%) DBS samples. The average difference in HBV DNA levels between DBS eluates and corresponding plasma samples was 2.7 log10 IU/mL. Fifteen DBS eluates positive for HBV DNA were sequenced and all of them belonged to HBV genotype A. Thirteen samples which were negative for all HBV markers showed HBeAg false positivity. Therefore, DBS is a reliable sample matrix for detection of HBsAg, total anti-HBc and HBV DNA, but not HBeAg. Further feasibility studies of DBS for diagnostic purposes and epidemiologic studies are warranted.

Development of an intervention system for linkage-to-care and follow-up for hepatitis B and C virus carriers.

BACKGROUND: Poor connections in the cascade of viral hepatitis care have been discussed around the world. In 2011 in Japan, 500,000 to 1.25 million hepatitis B and C virus carriers needed to consult with hepatologists, so linkage-to-care (LTC) needs to be promoted. Therefore, in this study, to improve LTC and care-seeking behaviors, we attempted to establish a community-based intervention system and evaluate its effectiveness by analyzing behavior modifications. METHODS: In a model city, Okazaki (population: 387,887 as of 2019), LTC was encouraged among HBV and HCV carriers by annually mailed brochures, and their care-seeking behaviors were followed up through questionnaires for 8 years (2012-2019). Their behavior modifications and demographic characteristics were analyzed anonymously in cooperation with community health workers, hepatologists, and researchers. RESULTS: Through regional HBsAg and anti-HCV screening, 333 HBV and 208 HCV carriers were identified. Before the intervention, only 34.7% (25/72) of HBV- and 34.3% (24/70) of HCV-positive individuals had consulted with hepatologists. However, in 2019, after the intervention, these proportions increased to 79.8% (91/114) and 91.2% (52/57), respectively. Access to outpatient care and treatment uptake also continuously improved. However, individuals over 70 years of age were significantly less likely to engage in care-seeking behaviors (p < 0.05), and significantly fewer HCV-positive females received treatment (p = 0.03). CONCLUSIONS: A paper-based reiterative intervention encouraging LTC and follow-up successfully improved the care-seeking behaviors of hepatitis virus-positive individuals and enabled their behavior modifications to be monitored. Further trials are required to advance the system by age- and gender-specific interventions.

Simultaneous analyses of urinary eicosanoids and related mediators identified tetranor-prostaglandin E metabolite as a novel biomarker of diabetic nephropathy.

Diabetic nephropathy is a major complication of diabetes mellitus, and thus novel biomarkers are desired to evaluate the presence and progression of diabetic nephropathy. In this study, we sought to identify possible metabolites related to diabetic nephropathy among urinary eicosanoids and related mediators. Using liquid chromatogram-tandem mass spectrometry, we optimized the lipid extraction from urine using the Monospin C18 as a solid-phase extraction cartridge and measured the urinary lipid mediators in 111 subjects with type 2 diabetes mellitus as well as 33 healthy subjects. We observed that 14 metabolites differed significantly among the clinical stages of nephropathy. Among them, levels of tetranor-prostaglandin E metabolite (tetranor-PGEM), an arachidonic acid metabolite, were significantly higher in subjects with stage 1 nephropathy than in healthy subjects and increased with the progression of nephropathy. We also observed that levels of maresin-1, a docosahexaenoic acid metabolite, and leukotriene B4-ethanolamide, an arachidonoyl ethanolamide metabolite, were significantly lower in subjects with stage 3-4 nephropathy than in healthy subjects and those with stage 1-2 nephropathy. Finally, using a comprehensive analysis of urinary eicosanoids and related mediators, we concluded that tetranor-PGEM was capable of discriminating clinical stages of nephropathy and thus useful as a novel biomarker for diabetic nephropathy.

ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvß6 (ß6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin ß6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin ß6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin ß6 as a possible therapeutic target or diagnostic marker candidate.

Proteome analysis demonstrates involvement of endoplasmic reticulum stress response in human myocardium with subclinical left ventricular diastolic dysfunction.

AIM: Heart failure is increasing in Japan, in particular that with preserved ejection fraction (HFpEF) prevalent in older-aged patients. The purpose of this study was to investigate the pathophysiology during the early stage of left ventricular (LV) diastolic dysfunction by the quantitative proteome analysis of human myocardium. METHODS: Among 331 post-mortem autopsy patients, we selected 23 patients (aged 79 ± 9.6 years) with echocardiographic data and without major comorbidities, except hypertension. Cryopreserved autopsy tissue of the LV myocardium was subjected to proteome analysis. LV diastolic function was evaluated by echocardiographic data. Thirteen patients were classified into the impaired diastolic function (IDF) group, and 10 the normal cardiac function group. We performed comparative proteome analysis between the IDF and normal groups by isobaric tags for relative and absolute quantitation (iTRAQ) using nano-liquid chromatography-tandem mass spectrometry. RESULTS: The iTRAQ-based proteome analysis revealed 57 differentially expressed proteins in the IDF group. Molecular network analysis of differentially expressed proteins indicated that endoplasmic reticulum (ER) stress was a potentially important event. Furthermore, the expressions of proteins associated with the ER stress response, such as glucose-regulated protein 78 kDa, inositol-requiring kinase 1α and spliced X-box binding protein 1, were significantly decreased in the IDF group. CONCLUSIONS: This study suggested that reduced ER stress responses were involved during the early stage of LV diastolic dysfunction. Geriatr Gerontol Int ••; ••: ••-•• Geriatr Gerontol Int 2021; 21: 577-583.

A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice.

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20S637A protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes.

B7-H3 expression in upper tract urothelial carcinoma associates with adverse clinicopathological features and poor survival.

B7-H3, a member of the B7 superfamily, is an immune checkpoint molecule. An association between B7-H3 expression and poor survival has been reported in many types of cancer. However, its prognostic value in patients with upper tract urothelial carcinoma (UTUC) has not yet been reported. The aim of this study was to examine the clinical significance of tumor B7-H3 expression in UTUC. B7-H3 positivity was observed in 36 of 271 cases (13 %) by immunohistochemistry and was significantly associated with several adverse clinicopathological features such as tumor grade, tumor stage, and lymph node metastasis. In addition, B7-H3 positivity was significantly associated with shorter metastasis-free survival and cancer-specific survival. We also found that B7-H3/programmed cell death ligand-1 (PD-L1) co-positivity was significantly associated with worse prognosis. These results suggest the utility of B7-H3 positivity and B7-H3/PD-L1 co-positivity as novel prognostic biomarkers in UTUC, and the potential usefulness of B7-H3 targeted therapy for patients with UTUC, the effect of which may be enhanced by combination with programmed cell death-1 /PD-L1 blockade.

Urinary autotaxin concentrations are associated with kidney injury.

BACKGROUND: While basic researches have shown the involvement of the autotaxin-lysophosphatidic acid (ATX-LPA) axis in the pathogenesis of kidney diseases, no clinical studies have revealed the association between urinary ATX concentrations and kidney disease yet. We investigate the clinical characteristics in relation to the urinary ATX concentrations and the potential association between urinary ATX concentrations and various kidney diseases. METHODS: We measured the urinary ATX concentrations in residual urine samples after routine clinical testing from a total of 326 subjects with various kidney diseases and healthy subjects. We compared the urinary ATX concentrations in relation to clinical parameters and urinary biomarkers, and investigated their association with various kidney diseases. RESULTS: The urinary ATX concentrations were associated with the gender, eGFR, presence/absence of hematuria, serum ATX, urinary concentrations of total protein (TP), microalbumin, N-acetyl-ß-D-glucosaminidase (NAG), α1-microglobulin (α1-MG), and transforming growth factor-ß. Multiple regression analyses identified urinary α1-MG, age, urinary TP, NAG, and hematuria as being significantly associated with the urinary ATX concentrations. Urinary ATX concentrations were higher in subjects with membranous nephropathy and systemic lupus erythematosus than in the control subjects. CONCLUSIONS: Urinary ATX might be associated with pathological conditions of the kidney associated with kidney injury.

Association between rs1229984 in ADH1B and cancer prevalence in a Japanese population.

Alcohol consumption is an established risk factor for cancer, but little is known regarding the effect of genetic polymorphisms in alcohol metabolism genes on alcohol-related cancer risk in the Japanese population. Associations between the ADH1B gene (alcohol dehydrogenase 1B), single nucleotide polymorphism (SNP) rs1229984 and cancer have been extensively studied yet evidence is inconsistent. This population-based case-control study primarily aimed to clarify any association between SNP rs1229984 in both overall and specific cancer risk in a Japanese population. The functional non-synonymous SNP rs1229984 (Arg48His) was genotyped using DNA samples from 1,359 consecutive autopsy cases registered in The Japanese Single Nucleotide Polymorphisms for Geriatric Research database. Medical and pathological record data from this database were used to categorise cases and controls. Results included 1,359 participants, 816 cases and 543 controls. Multinomial logistic regression analyses showed no significant association between rs1229984 presence and overall cancer risk in both dominant and recessive genetic inheritance models [Arg/Arg+Arg/His vs. His/His: Adjusted odds ratio (OR)=0.66 (95% CI=0.39-1.13; P=0.129), Arg/Arg vs. Arg/His+His/His: OR=0.95 (95% CI=0.75-1.20; P=0.657)]. However, results showed those homozygous for rs1229984 (genotype His/His) were at significantly decreased odds of lung cancer than other genotypes [recessive model: OR=0.64 (95% CI=0.44-0.93; P=0.020]. In conclusion, there was no significant association between rs1229984 and odds of overall or specific cancers except in lung cancer where His/His genotype decreased odds. To the best of our knowledge, the association between His/His and decreased odds of lung cancer is a novel finding. These findings require further validation in larger studies.

Association of CYP2A6 gene deletion with cancers in Japanese elderly: an autopsy study.

BACKGROUND: CYP2A6 is an enzyme involved in oxidation of a number of environmental chemicals, including nicotine, pro-carcinogenic nitrosamines and polycyclic aromatic hydrocarbons (PAHs). The whole gene deletion of CYP2A6 (CYP2A6*4) is prevalent in East Asian population. Whether or not CYP2A6*4 associates with cancer is still controversial. METHODS: We undertook an association study to determine whether deletion of CYP2A6 gene associates with total cancer and major cancer types employing data of consecutive autopsy cases registered in the Japanese single-nucleotide polymorphisms for geriatric research (JG-SNP) database. The presence of cancer were inspected at the time of autopsy and pathologically confirmed. Genotyping for CYP2A6 wild type (W) and deletion (D) was done by allele specific RT-PCR method. RESULTS: Among 1373 subjects, 826 subjects (60.2%) were cancer positive and 547 subjects (39.8%) were cancer negative. The genotype frequency in the whole study group for WW, WD and DD were 65.0, 30.6 and 4.4%, respectively, which obeyed the Hardy-Weinberg equilibrium (p = 0.20). Total cancer presence, as well as major cancers including gastric, lung, colorectal, and blood cancers did not show any positive association with CYP2A6 deletion. When male and female were separately analyzed, CYP2A6 deletion associated with decreased gastric cancer risk in female (OR = 0.49, 95%CI: 0.25-0.95, p = 0.021, after adjustment for age, smoking and drinking). When smoker and non-smoker were separately analyzed, CYP2A6 deletion associated with decreased total cancer in female nonsmokers (OR = 0.67, 95%CI: 0.45-0.99, p = 0.041 after adjustment). On the other hand, CYP2A6 deletion associated increase blood cancers in smokers (OR = 2.05, 95%CI: 1.19-3.53, p = 0.01 after adjustment). CONCLUSION: The CYP2A6 deletion may not grossly affect total cancer. It may associate with individual cancers in sex and smoking dependent manner. Further studies with larger sample size are warranted to confirm our results.

Analysis of urinary sphingolipids using liquid chromatography-tandem mass spectrometry in diabetic nephropathy.

AIMS/INTRODUCTION: Sphingolipids, such as ceramides and sphingosine, are involved in the pathogenesis of diabetes; however, the modulation of urinary sphingolipids in diabetic nephropathy has not been fully elucidated. Therefore, we aimed to develop a simultaneous measurement system for urinary sphingolipids using liquid chromatography-tandem mass spectrometry and to elucidate the modulation of urinary sphingolipids in diabetic nephropathy. MATERIALS AND METHODS: We established a simultaneous measurement system for the urinary sphingosine, dihydrosphingosine, and six ceramide species (Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/18:1, Cer d18:1/20:0, Cer d18:1/22:0 and Cer d18:1/24:0), and we examined the urinary sphingolipids in 64 type 2 diabetes patients and 15 control participants. RESULTS: The established measurement system for the urinary sphingolipids showed good precision for Cer d18:1/16:0, Cer d18:1/20:0, Cer d18:1/22:0 and Cer d18:1/24:0. We observed that the urinary levels of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/20:0, Cer d18:1/22:0 and Cer d18:1/24:0 were elevated in patients with stage 3 of diabetic nephropathy, and were correlated with urinary biomarkers, such as albumin and N-acetyl-ß-d-glucosaminidase, and sediment score. CONCLUSIONS: Our method is useful for the measurement of ceramide in urine specimens, and urinary ceramides might be associated with the pathological condition of diabetic nephropathy, such as renal tubular injury.

Clinicopathological characteristics of gastric cancer with carbohydrate antigen 19-9 expression occurring in elderly individuals: An autopsy study.

The clinicopathological significance of carbohydrate antigen 19-9 (CA19-9) in gastric cancer (GC) remains obscure. Therefore, the current study aimed to clarify the clinicopathological value of CA19-9 in GC utilizing autopsy cases. We examined the expression of CA19-9 and mucin core proteins in GC immunohistochemically, and analyzed serum CA19-9 levels and clinicopathological variables or complications. We also investigated whether fucosyltransferases 2 and 3 (FUT2/3) allelic variants influence CA19-9 expression in GC. Compared to GC cases with negative CA19-9 expression (tCA19-9-N), those with positive CA19-9 expression (tCA19-9-P) demonstrated significant differences in characteristic features such as lymph node and distant organ metastases, lymphatic and venous permeation, and higher Tumor, Node, Metastasis (TNM) stages. Moreover, compared to GC cases with low serum CA19-9 levels (sCA19-9-L), those with high serum CA19-9 levels (sCA19-9-H) were related to venous permeation, higher proportion of lymph node and distant organ metastases, and higher TNM stages. Both tCA19-9-P GC and sCA19-9-H GC cases were significantly associated with coagulation abnormalities. sCA19-9-H GC cases correlated significantly with MUC1 and MUC5AC expression. FUT2/3 genotypes were not associated with CA19-9 expression in GC. These results suggest that CA19-9 can predict the risk of lymph node and distant metastases as well as of coagulation abnormalities.

Fumarate accumulation involved in renal diabetic fibrosis in Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is a spontaneous animal model of type 2 diabetes and early stage of diabetic nephropathy. However, the pathophysiological mechanisms contributing to the progression of diabetic nephropathy in GK rats remain unclear. Kidneys from 15-week old male diabetic GK/Jcl rats and age-matched Wistar rats, which have the same genetic background as GK rats, were used. Proteomic analyses of GK and Wistar kidneys were performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Differentially expressed proteins in GK rats were subjected to pathway analysis, and expression levels of hypoxia inducible factor 1α (HIF-1α) and transforming growth factor-ß1 (TGF-ß1), and fumarate accumulation in GK kidneys were examined. Azan staining and immunohistochemical staining of α-smooth muscle actin were performed in relation to fibrosis in GK kidneys. Proteomic analysis using 2D-DIGE, analysis of fumarate content, and expression analysis of HIF-1α, TGF-ß1, and α-smooth muscle actin of GK rat's kidney, suggested the mechanism of fibrosis characterized as two stages in diabetic nephropathy of GK rats. Abnormalities of glucose metabolism such as elevated levels of 2-oxoglutarate dehydrogenase and reduction of fumarate hydratase caused the accumulation of fumarate followed by the upregulation of HIF-1α and TGF-ß1 leading to fibrosis in diabetic nephropathy. Alterations in proteins involved in the tricarboxylic acid cycle are associated with fibrosis through fumarate accumulation in diabetic nephropathy of GK rats.

Frequent appearance of club cell (Clara cell)-like cells as a histological marker for ALK-positive lung adenocarcinoma.

Anaplastic lymphoma kinase-rearranged (ALK+ ) lung cancers show characteristic histological features, such as solid signet ring cell patterns and mucinous cribriform patterns; however, these features are not always observed in ALK+ lung cancers. We noticed that club cell (Clara cell)-like cells (CLCs) were frequently present in the papillary portion of ALK+ lung adenocarcinomas. In this study, we investigated the importance of CLCs in papillary patterns of ALK+ lung cancers. We compared the histological features of 18 ALK+ cases with 62 control cases (22 epidermal growth factor receptor-positive (EGFR+ ) and 40 ALK- and EGFR-negative (ALK- /EGFR- ) cases). The present study analyzed presence of papillary pattern, proportion of papillary pattern area, presence of micropapillary pattern, frequency of CLCs and lengths of snout. The frequency of CLCs in ALK+ cases was significantly higher than that in EGFR+ cases and ALK- /EGFR- cases. Micropapillary pattern was more frequently observed in ALK+ cases than that in ALK- /EGFR- cases (P < 0.001). The present study indicated that the high frequency of CLCs in papillary patterns was significantly associated with ALK+ cases. When solid signet ring cell patterns and mucinous cribriform patterns are absent, the high frequency of CLCs in papillary adenocarcinoma could be a useful histological marker for ALK+ lung cancers.

Optimization of trypsin treatment condition utilizing immunohistochemistry for chromogenic in situ hybridization.

Chromogenic in situ hybridization (CISH) is a molecular technique used to visualize specific genes. Both heat treatment and protease treatment play important roles for the success of CISH on formalin-fixed paraffin-embedded (FFPE) tissue sections. In contrast to heat treatment, the optimal condition of protease treatment may vary depending on each sample. Because trypsin has a substrate specificity to cleave lysine and arginine, we hypothesized that trypsin could effectively degrade histones rich in lysine and arginine and that the removal of histones from DNA following heat treatment could improve CISH results. We selected 21 patients with lung adenocarcinoma previously known to be positive or negative for anaplastic lymphoma kinase (ALK) gene rearrangement and used FFPE tissue sections collected from these patients. Then, we assessed histone degradation among the following protease treatments; trypsin, pepsin, and proteinase K, and compared the ALK CISH results with results obtained using commercially available kits and these protease treatments. The results showed that trypsin effectively degraded histones. Additionally, compared with the other treatments, ALK CISH with trypsin treatment showed the most evaluable cells and the smallest standard deviation. Our study suggests that the degradation of histones by trypsin subsequent to heat treatment might improve CISH results.

Age-associated proteomic alterations in human aortic media.

AIM: Human vascular senescence, which mainly occurs in media, is not completely understood. Here, we used proteomic approaches to investigate age-associated changes in human aortic media with the goal of understanding the molecular mechanisms underlying vascular senescence. METHOD: Cryopreserved autopsy samples of aortic media from older-aged (aged 70-100 years, n = 25), middle-aged (aged 49-68 years, n = 24), and young (aged 21-39 years, n = 12) subjects were collected. We used two proteomic techniques, two-dimensional differential gel electrophoresis and isobaric tags for relative and absolute quantitation, and we subjected differentially-expressed proteins among age groups to immunohistochemical analyses. RESULTS: Proteomic analyses showed that the expression of lactadherin, which produces medin, was elevated in aortic media of older-aged individuals. Immunohistochemical and Congo red staining showed that lactadherin and apolipoprotein E were deposited, and that amyloidosis was enhanced in older-aged aortic media. Furthermore, the markers of oxidative damage (8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal) were significantly elevated in aortic media of middle-aged or older-aged individuals. The immunohistochemical expression of anti-oxidant proteins (thioredoxin and extracellular superoxide dismutase) was also high in middle-aged and older-aged groups. Oxidative damage might induce the disruption of smooth muscle cells, resulting in the decrement of α-actin, a highly-expressed protein in smooth muscle cells, and matrix remodeling, in which several proteins associated with extracellular matrix were altered with aging. CONCLUSIONS: Proteomic approaches showed that the elevated expression of lactadherin might contribute to amyloid deposition, enhancement of oxidative stress, induction of antioxidant proteins and matrix remodeling in older-aged aortic media. Geriatr Gerontol Int 2019; 19: 1054-1062.