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Abstract In cancer genome analysis, identifying pathogenic alterations and assessing their effects on oncogenic processes is important. Although whole exome sequencing (WES) can effectively detect such changes, driver alterations could not be identified in 27.8% of the cases, according to a previous study. The objectives of the present study were to evaluate the utility of whole genome sequencing (WGS) and clarify its differences with WES in terms of driver alteration detection. For this purpose, WGS analysis was conducted on 177 driverless WES samples, selected from 5,480 fresh frozen samples derived from 5,140 Japanese patients with cancer. These samples were selected as primary tumor, both WES and transcriptome profiling were performed, estimated tumor content of ≥ 30%, and no driver alterations were identified by WES. WGS identified driver and likely driver alterations in 68.4 and 22.6% of the samples, respectively. The most frequent alteration type was oncogene amplification, followed by tumor suppressor gene deletion and small variants located outside the coding region. In the remaining 9.0% of samples, no such signals were identified; therefore, further investigations are required. The current study clearly demonstrated the role and utility of WGS in identifying genomic alterations that contribute to tumorigenesis.

Abstract Heat shock proteins (HSPs) are molecular chaperones whose primary function is cytoprotection, supporting cell survival under (sub) lethal conditions. They have been implicated in various diseases such as inflammatory diseases and cancer due to their cytoprotective and immunomodulatory effects, and their biological mechanisms have been studied. Central family members include, HSP27, which is induced by various stimuli such as heat shock, hypoxia, hyperoxia, ultraviolet exposure, and nutritional deficiency, and HSP70, which is homeostatically expressed in many organs such as the gastrointestinal tract and has anti-cell death and anti-inflammatory effects. In this study, HSP27 and HSP70 were investigated during thrombus formation and dissolution in a deep vein thrombosis model by immunohistochemistry to determine their involvement in this process and whether their expression could be used as a forensic marker. In the process of thrombus formation and lysis, HSP27 and HSP70 were found to be expressed by immunohistochemical analysis. The role of inhibitors of HSP27 and HSP70 in the pathogenesis of thrombosis in mice was also investigated. When HSP27 or HSP70 inhibitors were administered, thrombi were significantly smaller than in the control group on day 5 after inferior vena cava ligation, indicating pro-thrombotic effects HSP27 and HSP70. If HSP27- or HSP70-positive cells were clearly visible and easily identifiable in the thrombus sections, the thrombus was presumed to be more than 10 days old. Thus, the detection of intrathrombotic HSP27 and HSP70 could forensically provide useful information for the estimation of thrombus ages. Collectively, our study implied that both HSP27 and HSP70 might be molecular targets for thrombus therapy and that the detection of HSP-related molecules such as HSP27 and HSP70 could be useful for the determination of thrombus ages.

Abstract We investigated the dynamics of the gene expression of M1 and M2 macrophage markers during skin wound healing in mice. Expression of M1-macrophage markers, such as Il12a, Tnf, Il6, Il1b, and Nos2 was upregulated after wounding and peaked at 1 or 3 days after injury, and that of M2-macrophage markers such as Mrc1, Cd163, Ccl17, Arg, and Tgfb1, peaked at 6 days after injury. Consistent with these findings, using triple-color immunofluorescence analysis revealed that F4/80+CD80+ M1 macrophages were more abundant than F4/80+CD206+ M2 macrophages on day 3 in mouse wound specimens, and that M2 macrophages were prominently detected in day 6 wounds. For application in forensic practice, we examined macrophage polarization using human wound specimens. The average ratios of CD68+iNOS+ M1 macrophages to CD68+CD163+ M2 macrophages (M1/M2 ratios) were greater than 2.5 for the wounds aged 2–5 days. Out of 11 wounds aged 1–5 days, five samples had the M1/M2 ratios of > 3.0. These observations propose that the M1/M2 ratios of 3.0 would indicate a wound age of 1–5 days as the forensic opinion. This study showed that M1 and M2 macrophages in human skin wound might be a promising marker for wound age determination.

Abstract The differences in genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma remain unclear. We developed a customized, targeted gene sequencing panel for efficient and sensitive identification of germline variants, including whole-gene deletion types for cancer-related drug-metabolizing enzyme genes in lung adenocarcinoma and squamous cell carcinoma. The minor allele frequencies of the variants, confirmed as clinically significant in the Japanese population, did not differ significantly from those of normal participants listed in the public database. Genotype analysis comparing lung adenocarcinoma (n = 559) and squamous cell carcinoma (n = 151) indicated that the variants of DPYD (rs190771411, Fisher’s exact test, P = 0.045; rs200562975, P = 0.045) and ALDH2 (rs568781254, P = 0.032) were associated with an increased risk of squamous cell carcinoma compared to adenocarcinoma. Conversely, whole-gene deletion of CYP2A6 was associated with adenocarcinoma but not squamous cell carcinoma. Notably, whole-gene deletion of CYP2A6 was confirmed in 22 patients with lung adenocarcinoma but not in any patients with squamous cell carcinoma. Most patients with whole-gene deletion of CYP2A6 were female non-smokers. The discovery of a whole-gene deletion of CYP2A6 in patients with lung adenocarcinoma may have an important role in clinical practice and advance our understanding of CYP2A6 germline variants and their association with carcinogenesis or their susceptibility to lung adenocarcinoma.

Abstract Heme oxygenase-1 (HO-1), an inducible stress-response protein, exerts anti-oxidant and anti-apoptotic effects. However, its significance in forensic diagnosis of acute ischemic heart diseases (AIHD) such as myocardial infarction (MI) is still unknown. We examined the immunohistochemical expression of HO-1 in the heart samples to discuss their forensic significance to determine acute cardiac ischemia. The heart samples were obtained from 23 AIHD cases and 33 non-AIHD cases as controls. HO-1 positive signals in cardiomyocyte nuclear were detected in 78.2% of AIHD cases, however, that were detected in only 24.2% control cases with statistical difference between AIHD and non-AIHD groups. In contrast to HO-1 protein expression, there was no significant difference in the appearance of myoglobin pallor regions and leukocyte infiltration in the hearts between AIHD and non-AIHD groups. From the viewpoints of forensic pathology, intracardiac HO-1 expression would be considered a valuable marker to diagnose AIHD as the cause of death.

In cancer genome analysis, identifying pathogenic alterations and assessing their effects on oncogenic processes is important. Although whole exome sequencing (WES) can effectively detect such changes, driver alterations could not be identified in 27.8% of the cases, according to a previous study. The objectives of the present study were to evaluate the utility of whole genome sequencing (WGS) and clarify its differences with WES in terms of driver alteration detection. For this purpose, WGS analysis was conducted on 177 driverless WES samples, selected from 5,480 fresh frozen samples derived from 5,140 Japanese patients with cancer. These samples were selected as primary tumor, both WES and transcriptome profiling were performed, estimated tumor content of ≥ 30%, and no driver alterations were identified by WES. WGS identified driver and likely driver alterations in 68.4 and 22.6% of the samples, respectively. The most frequent alteration type was oncogene amplification, followed by tumor suppressor gene deletion and small variants located outside the coding region. In the remaining 9.0% of samples, no such signals were identified; therefore, further investigations are required. The current study clearly demonstrated the role and utility of WGS in identifying genomic alterations that contribute to tumorigenesis. [ABSTRACT FROM AUTHOR]

Abstract Posttransplantation cyclophosphamide (PTCy) has become a popular option for haploidentical hematopoietic stem cell transplantation (HSCT). However, personalized methods to adjust immune intensity after PTCy for each patient’s condition have not been well studied. Here, we investigated the effects of reducing the dose of PTCy followed by α-galactosylceramide (α-GC), a ligand of iNKT cells, on the reciprocal balance between graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect. In a murine haploidentical HSCT model, insufficient GVHD prevention after reduced-dose PTCy was efficiently compensated for by multiple administrations of α-GC. The ligand treatment maintained the enhanced GVL effect after reduced-dose PTCy. Phenotypic analyses revealed that donor-derived B cells presented the ligand and induced preferential skewing to the NKT2 phenotype rather than the NKT1 phenotype, which was followed by the early recovery of all T cell subsets, especially CD4+Foxp3+ regulatory T cells. These studies indicate that α-GC administration soon after reduced-dose PTCy restores GVHD-preventing activity and maintains the GVL effect, which is enhanced by reducing the dose of PTCy. Our results provide important information for the development of a novel strategy to optimize PTCy-based transplantation, particularly in patients with a potential relapse risk.

Heat shock proteins (HSPs) are molecular chaperones whose primary function is cytoprotection, supporting cell survival under (sub) lethal conditions. They have been implicated in various diseases such as inflammatory diseases and cancer due to their cytoprotective and immunomodulatory effects, and their biological mechanisms have been studied. Central family members include, HSP27, which is induced by various stimuli such as heat shock, hypoxia, hyperoxia, ultraviolet exposure, and nutritional deficiency, and HSP70, which is homeostatically expressed in many organs such as the gastrointestinal tract and has anti-cell death and anti-inflammatory effects. In this study, HSP27 and HSP70 were investigated during thrombus formation and dissolution in a deep vein thrombosis model by immunohistochemistry to determine their involvement in this process and whether their expression could be used as a forensic marker. In the process of thrombus formation and lysis, HSP27 and HSP70 were found to be expressed by immunohistochemical analysis. The role of inhibitors of HSP27 and HSP70 in the pathogenesis of thrombosis in mice was also investigated. When HSP27 or HSP70 inhibitors were administered, thrombi were significantly smaller than in the control group on day 5 after inferior vena cava ligation, indicating pro-thrombotic effects HSP27 and HSP70. If HSP27- or HSP70-positive cells were clearly visible and easily identifiable in the thrombus sections, the thrombus was presumed to be more than 10 days old. Thus, the detection of intrathrombotic HSP27 and HSP70 could forensically provide useful information for the estimation of thrombus ages. Collectively, our study implied that both HSP27 and HSP70 might be molecular targets for thrombus therapy and that the detection of HSP-related molecules such as HSP27 and HSP70 could be useful for the determination of thrombus ages. [ABSTRACT FROM AUTHOR]