Visualization of intradermal blood vessel structures by dual-wavelength photoacoustic microscopy and characterization of three-dimensional construction of livedo-racemosa in cutaneous polyarteritis nodosa.

BACKGROUND: Photoacoustic microscopy is expected to have clinical applications as a noninvasive and three-dimensional (3D) method of observing intradermal structures. OBJECTIVE: Investigate the applicability of a photoacoustic microscope equipped with two types of pulsed lasers that can simultaneously recognize hemoglobin and melanin. METHODS: 16 skin lesions including erythema, pigmented lesions, vitiligo and purpura, were analyzed to visualize 3D structure of melanin granule distribution and dermal blood vessels. 13 cases of livedo racemosa in cutaneous polyarteritis nodosa (cPN) were further analyzed to visualize the 3D structure of dermal blood vessels in detail. Vascular structure was also analyzed in the biopsy specimens obtained from tender indurated erythema of cPN by CD34 immunostaining. RESULTS: Hemoglobin-recognition signal clearly visualized the 3D structure of dermal blood vessels and melanin-recognition signal was consistently reduced in vitiligo. In livedo racemosa, the hemoglobin-recognition signal revealed a relatively thick and large reticular structure in the deeper layers that became denser and finer toward the upper layers. The numerical analysis revealed that the number of dermal blood vessels was 1.29-fold higher (p<0.05) in the deeper region of the lesion than that of normal skin. The CD34 immunohistochemical analysis in tender indurated erythema revealed an increased number of dermal vessels compared with normal skin in 88.9% (8/9) of the cases, suggesting that vascular network remodeling had occurred in cPN. CONCLUSION: The photoacoustic system has an advantage in noninvasively detecting dermal blood vessel structures that are difficult to recognize by two-dimensional histopathology specimen examination and is worth evaluating in various skin diseases.

DNA methylation alterations of ADCY5, MICAL2, and PLEKHG2 during the developmental stage of cryptogenic hepatocellular carcinoma.

AIM: The aim of this study was to clarify the significance of DNA methylation alterations of cryptogenic hepatocellular carcinomas (HCCs). METHODS: Using the Infinium assay, we performed genome-wide DNA methylation analysis of 250 liver tissue samples, including noncancerous liver tissue (U-N) and corresponding cancerous tissue (U-T) from patients with cryptogenic HCC without a history of excessive alcohol use and hepatitis virus infection, and whose U-N samples showed no remarkable histological features (no microscopic evidence of simple steatosis, any form of hepatitis including nonalcoholic steatohepatitis, or liver cirrhosis). RESULTS: We identified 3272 probes that showed significant differences of DNA methylation levels between U-N and normal liver tissue samples from patients without HCC, indicating that a distinct DNA methylation profile had already been established at the precancerous U-N stage. U-Ns have a DNA methylation profile differing from that of noncancerous liver tissue of patients with nonalcoholic steatohepatitis-related, viral hepatitis-related, and alcoholic liver disease-related HCCs. Such DNA methylation alterations in U-Ns were inherited by U-Ts. The U-Ns showed DNA methylation alteration of ADCY5, resulting in alteration of its mRNA expression, whereas noncancerous liver tissue of patients with nonalcoholic steatohepatitis-, viral hepatitis-, or alcoholic liver disease-related HCCs did not. DNA methylation levels of MICAL2 and PLEKHG2 in U-Ts were correlated with larger tumor diameter and portal vein involvement, respectively. CONCLUSIONS: U-N-specific DNA hypermethylation of ADCY5 may have significance, even from the precancerous stage in liver showing no remarkable histological features. DNA hypomethylation of MICAL2 and PLEKHG2 may determine the clinicopathological features of cryptogenic HCC.

Phospholipid isotope tracing reveals ß-catenin-driven suppression of phosphatidylcholine metabolism in hepatocellular carcinoma.

Background and Aims: Activating mutations in the CTNNB1 gene encoding ß-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). HCC with CTNNB1 mutations show profound alterations in lipid metabolism including increases in fatty acid oxidation and transformation of the phospholipidome, but it is unclear how these changes arise and whether they contribute to the oncogenic program in HCC. Methods: We employed untargeted lipidomics and targeted isotope tracing to quantify phospholipid production fluxes in an inducible human liver cell line expressing mutant ß-catenin, as well as in transgenic zebrafish with activated ß-catenin-driven HCC. Results: In both models, activated ß-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid flux analysis in human cells revealed a large reduction in phosphatidylcholine (PC) production rates as assayed by choline tracer incorporation. We developed isotope tracing lipid flux analysis for zebrafish and observed similar reductions in phosphatidylcholine synthesis flux accomplished by sex-specific mechanisms. Conclusions: The integration of isotope tracing with lipid abundances highlights specific lipid class transformations downstream of ß-catenin signaling in HCC and suggests future HCC-specific lipid metabolic targets.

Clinicopathological significance of androgen receptor expression in extramammary Paget disease: An analysis of 92 patients.

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm arising in apocrine gland-rich areas. Although - like normal apocrine glands - EMPD frequently expresses androgen receptor (AR), the clinical significance of AR expression remains unclear. The present study investigated the clinicopathological impact of AR expression in EMPD. Immunohistochemistry for AR was performed in a retrospective cohort of 92 EMPD patients with 108 EMPD lesions, including 102 primary lesions, five lymph node [LN] metastases and one local recurrence. The total AR staining score was calculated as staining intensity score (IS 0-3) × positive-cell percentage score (PS 1-4). Expression levels were graded as Grade 1 (scores 0 and 1), Grade 2 (scores 2-4), and Grade 3 (scores 6-12). Higher expression grade was correlated with tumor thickness (P = 0.011), LN metastasis (P = 0.008), and higher EMPD stage (P = 0.023). Grade 1 EMPDs did not invade into the dermis and did not generate metastatic and/or recurrent lesions, whereas only Grade 2 or 3 EMPDs did so. AR expression in invasive components was significantly higher (P = 0.023) than in non-invasive components remaining within the epidermis. AR expression was further elevated in metastatic and/or recurrent lesions relative to locally invasive lesions (P = 0.014). These results clearly indicate that increased AR expression is associated with malignant progression of EMPD and that androgen blockade might be an effective therapy. Furthermore, AR expression assessed by immunohistochemistry may have potential for prediction of LN metastasis and local recurrence in EMPD.

DNA methylation status of the SPHK1 and LTB genes underlies the clinicopathological diversity of non-alcoholic steatohepatitis-related hepatocellular carcinomas.

PURPOSE: This study was performed to identify the DNA methylation profiles underlying the clinicopathological diversity of non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinomas (HCCs).  METHODS: Genome-wide DNA methylation analysis of 88 liver tissue samples was performed using the Infinium assay. RESULTS: Principal component analysis revealed that distinct DNA methylation profiles differing from such profiles in normal control liver tissue had already been established in non-cancerous liver tissue showing NASH, which is considered to be a precancerous condition. Hierarchical clustering separated 26 NASH-related HCCs into Cluster I (n = 8) and Cluster II (n = 18). Such epigenetic clustering was significantly correlated with histopathological diversity, i.e. poorer tumor differentiation, tumor steatosis and development of a scirrhous HCC component. Significant differences in DNA methylation levels between the two clusters were accumulated in molecular pathways participating in cell adhesion and cytoskeletal remodeling, as well as cell proliferation and apoptosis. Among tumor-related genes characterizing Clusters I and II, differences in the levels of DNA methylation and mRNA expression for the SPHK1, INHBA, LTB and PDE3B genes were correlated with poorer tumor differentiation. 5-Aza-2'-deoxycytidine treatment of HCC cells revealed epigenetic regulation of the SPHK1 and LTB genes. Knockdown experiments showed that SPHK1 promotes cell proliferation, represses apoptosis and enhances migration, whereas LTB enhances migration of HCC cells. DNA hypomethylation resulting in increased expression of SPHK1 and LTB in poorly differentiated HCCs may underlie the aggressive phenotype of such HCCs. CONCLUSION: These data indicate that DNA methylation profiles may determine the clinicopathological heterogeneity of NASH-related HCCs via alterations of tumor-related gene expression.

Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis.

BACKGROUND: In recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH. RESULTS: Normal liver tissue (NLT), non-cancerous liver tissue showing histological findings compatible with non-alcoholic fatty liver from patients without HCC (NAFL-O), non-cancerous liver tissue showing NASH from patients without HCC (NASH-O), non-cancerous liver tissue showing non-alcoholic fatty liver from patients with HCC (NAFL-W), non-cancerous liver tissue showing NASH from patients with HCC (NASH-W) and NASH-related HCC were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4050 CpG sites showed alterations of DNA methylation in NASH-W samples relative to NLT samples. Such alterations at the precancerous NASH stage were inherited by or strengthened in HCC samples. Receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among them, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage. The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, and for 3 of them sufficient sensitivity and specificity were successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, NAFL-W and NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort. CONCLUSIONS: After a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.

Intraoperative laparoscopic detection of sentinel lymph nodes with indocyanine green and superparamagnetic iron oxide in a swine gallbladder cancer model.

Mapping of sentinel lymph nodes (SLNs) can enable less invasive surgery. However, mapping is challenging for cancers of difficult-to-access visceral organs, such as the gallbladder, because the standard method using radioisotopes (RIs) requires preoperative tracer injection. Indocyanine green (ICG) and superparamagnetic iron oxide (SPIO) have also been used as alternative tracers. In this study, we modified a previously reported magnetic probe for laparoscopic use and evaluated the feasibility of detecting SLNs of the gallbladder using a laparoscopic dual tracer method by injecting ICG and SPIO into five swine and one cancer-bearing swine. The laparoscopic probe identified SPIO nanoparticles in the nodes of 4/5 swine in situ, the magnetic field counts were 2.5-15.9 µT, and fluorescence was detected in SLNs in all five swine. ICG showed a visual lymph flow map, and SPIO more accurately identified each SLN with a measurable magnetic field quite similar to the RI. We then developed an advanced gallbladder cancer model with lymph node metastasis using recombination activating gene 2-knockout swine. We identified an SLN in the laparoscopic investigation, and the magnetic field count was 3.5 µT. The SLN was histologically determined to be one of the two metastatic lymph nodes. In conclusion, detecting the SLNs of gallbladder cancer in situ using a dual tracer laparoscopic technique with ICG and SPIO was feasible in a swine model.

Aberrant DNA methylation results in altered gene expression in non-alcoholic steatohepatitis-related hepatocellular carcinomas.

PURPOSE: The aim of this study was to investigate DNA methylation alterations in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinomas (HCCs). METHODS: Genome-wide DNA methylation analysis was performed using the Infinium Human Methylation 450 K BeadChip, and levels of mRNA expression were analyzed by quantitative reverse transcription-PCR. RESULTS: Compared to 36 samples of normal control liver tissue (C), DNA methylation alterations were observed on 19,281 probes in 22 samples of cancerous tissue (T) obtained from patients showing histological features compatible with NASH in their non-cancerous liver tissue (N). Among those probes, 1396 were located within CpG islands or their shores and shelves, designed around the transcription start sites of 726 genes. In representative genes, such as DCAF4L2, CKLF, TRIM4, PRC1, UBE2C and TUBA1B, both DNA hypomethylation and mRNA overexpression were observed in T samples relative to C samples, and the levels of DNA methylation and mRNA expression were inversely correlated with each other. DNA hypomethylation occurred even in N samples at the precancerous NASH stage, and this was inherited by or further strengthened in T samples. DNA hypomethylation of DCAF4L2, CKLF and UBE2C was observed in both NASH-related and viral hepatitis-related HCCs, whereas that of TRIM4, PRC1 and TUBA1B occurred in a NASH-related HCC-specific manner. DNA hypomethylation and/or mRNA overexpression of these genes was frequently associated with the necroinflammatory grade of NASH and was correlated with poorer tumor differentiation. CONCLUSION: DNA methylation alterations may occur under the necroinflammatory conditions characteristic of NASH and participate in NASH-related hepatocarcinogenesis through aberrant expression of tumor-related genes.

Establishment of a model of sentinel lymph node metastasis using immunodeficient swine.

Lymph node metastasis occurs via the migration of cancer cells through the lymphatic system. Sentinel lymph node (SLN) biopsy is a common diagnostic strategy. SLNs have been studied using healthy rodents and large animals without metastasis. Here we used immunodeficient swine to establish a model of lymph node metastasis. We used RAG2-knockout immunodeficient swine. A431 human epithelial carcinoma cells expressing green fluorescent protein were injected subcutaneously into the posterior sides of the auricle, forelimb and hindlimb of knockout swine. Indigo carmine dye was injected subcutaneously 8 weeks after tumour cell transplantation. SLNs were extracted, observed using a stereoscopic fluorescence microscope and analysed histologically using haematoxylin and eosin staining, and immunohistochemistry. Lymphoid follicles were found in wild-type swine, and a few aggregated lymphocytes and immature lymphoid follicles were observed in knockout swine. Fluorescence in the lymph nodes indicated metastasis of tumour cells to the lymph nodes. Tumour cells replaced lymph node architectures, showed high-grade nuclear atypia and formed irregular tumour nests. Our model may be useful for the preclinical validation of diagnostic methods and minimally invasive treatment of metastatic cancer.

Disseminated histoplasmosis from a calcified lung nodule after long-term corticosteroid therapy in an elderly Japanese patient: A case report.

RATIONALE: Histoplasmosis occurs most commonly in Northern and Central America and Southeast Asia. Increased international travel in Japan has led to a few annual reports of imported histoplasmosis. Healed sites of histoplasmosis lung infection may remain as nodules and are often accompanied by calcification. Previous studies in endemic areas supported the hypothesis that new infection/reinfection, rather than reactivation, is the main etiology of symptomatic histoplasmosis. No previous reports have presented clinical evidence of reactivation. PATIENT CONCERNS: An 83-year-old Japanese man was hospitalized with general fatigue and high fever. He had been treated with prednisolone at 13 mg/d for 7 years because of an eczematous skin disease. He had a history of travel to Los Angeles, Egypt, and Malaysia 10 to 15 years prior to admission. Five years earlier, computed tomography (CT) identified a solitary calcified nodule in the left lingual lung segment. The nodule size remained unchanged throughout a 5-year observation period. Upon admission, his respiratory condition remained stable while breathing room air. CT revealed small, randomly distributed nodular shadows in the bilateral lungs, in addition to the solitary nodule. DIAGNOSIS: Disseminated histoplasmosis, based on fungal staining and cultures of autopsy specimens. INTERVENTIONS: The patient's fever continued despite several days of treatment with meropenem, minocycline, and micafungin. Although he refused bone marrow aspiration, isoniazid, rifampicin, ethambutol, and prednisolone were administered for a tentative diagnosis of miliary tuberculosis. OUTCOMES: His fever persisted, and a laboratory examination indicated severe thrombocytopenia with disseminated intravascular coagulation. He died on day 43 postadmission. During autopsy, the fungal burden was noted to be higher in the calcified nodule than in the disseminated nodules of the lung, suggesting a pathogenesis involving endogenous reactivation of the nodule and subsequent hematogenous and lymphatic spread. LESSONS: Physicians should consider histoplasmosis in patients with calcified nodules because the infection may reactivate during long-term corticosteroid therapy.

Magnetically Promoted Rapid Immunofluorescence Staining for Frozen Tissue Sections.

Current immunohistochemistry methods for diagnosing abnormal cells, such as cancer cells, require multiple steps and can be relatively slow compared with intraoperative frozen hematoxylin and eosin staining, and are therefore rarely used for intraoperative examination. Thus, there is a need for novel rapid detection methods. We previously demonstrated that functionalized fluorescent ferrite beads (FF beads) magnetically promoted rapid immunoreactions. The aim of this study was to improve the magnetically promoted rapid immunoreaction method using antibody-coated FF beads and a magnet subjected to a magnetic field. Using frozen sections of xenograft samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also determined the optimum magnetic force for the antibody reaction (from 7.79 × 10-15 N to 3.35 × 10-15 N) and washing (4.78 × 10-16 N), which are important steps in this technique. Furthermore, we stained paraffin-embedded tissue arrays and frozen sections of metastatic breast cancer lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the utility of this system in clinical specimens. Under optimal conditions, this ultra-rapid immunostaining method may provide an ancillary method for pathological diagnosis during surgery. (J Histochem Cytochem 58:XXX-XXX, 2010).

Selective Inhibition of ADAM28 Suppresses Lung Carcinoma Cell Growth and Metastasis.

ADAM28 (a disintegrin and metalloproteinase 28) is overexpressed by carcinoma cells in non-small cell lung carcinomas (NSCLC) and plays an important role in cancer cell proliferation and metastasis by reactivation of insulin-like growth factor-1 (IGF-1) and escaping from von Willebrand factor (VWF)-induced apoptosis through digestion of IGF-binding protein-3 and VWF, respectively. To aim for new target therapy of NSCLC patients, we developed human neutralizing antibodies 211-12 and 211-14 against ADAM28, which showed IC50 values of 62.4 and 37.5 nmol/L, respectively. Antibody 211-14 recognized the junctional region between cysteine-rich domain and secreted-specific domain and showed a KD value of 94.7 pmol/L for the epitope-containing peptide. This antibody detected monkey and human secreted-form ADAM28s, although it was not reactive with mouse membrane-anchored ADAM28m. Antibody 211-14 effectively inhibited IGF-1-stimulated cell proliferation of lung adenocarcinoma cell lines with ADAM28 expression, including PC-9 cells, and promoted VWF-induced cell death in these cell lines. In lung metastasis models, antibody 211-14 significantly reduced tumor growth and metastases of PC-9 cells and prolonged survivals in the antibody-treated mice compared with the control IgG-treated ones. Combination therapy of the antibody and docetaxel was more effective than that of bevacizumab and docetaxel and showed further elongation of survival time compared with monotherapy. No adverse effects were observed even after administration of 10-fold more than effective dose of anti-ADAM28 antibody to normal mice. Our data demonstrate that antibody 211-14 is a neutralizing antibody specific to ADAM28s and suggest that this antibody may be a useful treatment remedy for NSCLC patients. Mol Cancer Ther; 17(11); 2427-38. ©2018 AACR.

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses.

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.

Genome-wide DNA methylation analysis during non-alcoholic steatohepatitis-related multistage hepatocarcinogenesis: comparison with hepatitis virus-related carcinogenesis.

The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples using the Illumina Infinium HumanMethylation450 BeadChip. After Bonferroni correction, 3331 probes showed significant DNA methylation alterations in 113 samples of non-cancerous liver tissue showing NASH (NASH-N) as compared with 55 samples of normal liver tissue (NLT). Principal component analysis using the 3331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection (viral-N). Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95. Jonckheere-Terptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without hepatocellular carcinoma (HCC) were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in 22 samples of NASH-related HCC (NASH-T) themselves. NASH- and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N samples and 37 samples of HCC associated with HBV or HCV infection, were observed in tumor-related genes, such as WHSC1, and were frequently associated with mRNA expression abnormalities. These data suggested that NASH-specific DNA methylation alterations may participate in NASH-related multistage hepatocarcinogenesis.

Prevalence of Patent Foramen Ovale in the Japanese Population- Autopsy Study.

BACKGROUND: Patent foramen ovale (PFO) can cause ischemic stroke because of paradoxical embolism. Autopsy studies have shown that the prevalence of PFO is 25% in whites or blacks. However, there is a paucity of data on the prevalence of PFO in Asians. The aim of this study was to clarify the prevalence of PFO in the Japanese population. METHODS AND RESULTS: We reviewed 52,717 autopsy reports, which were collected and edited by the Japanese Society of Pathology from 2009 to 2012. Next, we inspected consecutive 103 formalin-fixed specimens that had already been examined by certified pathologists from 2009 to 2013 to find PFO and atrial septal aneurysm (ASA). ASA was defined as ≥10 mm protrusion of the septum into the left or the right atrium. In the database of the Japanese Society of Pathology, the incidence of PFO was 0.08% (43/52,717). Inspection of heart specimens disclosed that the prevalence of PFO was 13.6% (14/103). None of the PFO cases was reported at the original autopsy. PFO was more frequently found in the subjects with ASA (50%) than in those without ASA (9.7%) (P=0.004). CONCLUSIONS: PFO is under-reported in autopsy reports. Re-evaluation of heart specimens disclosed that the prevalence of PFO was 13.6%. The prevalence was lower than reported in the past.