Preliminary evaluation of two bathing methods for the management of Malassezia overgrowth in dogs with atopic dermatitis.

BACKGROUND: Antifungal shampoos are widely used for canine Malassezia dermatitis. Few studies have evaluated effective bathing methods for atopic dogs with Malassezia overgrowth. OBJECTIVES: To evaluate the efficacy of an emollient bathing product (AFLOAT VET) and 2% miconazole/2% chlorhexidine shampoo (2% MIC/CHX) in atopic dogs, and to evaluate the influence on skin barrier function of both products in healthy dogs. ANIMALS: Sixteen atopic dogs with secondary Malassezia overgrowth and 11 healthy dogs. METHODS AND MATERIALS: This study was a randomized, single-blinded trial. The dogs were randomly treated with either emollient bathing or 2% MIC/CHX, twice weekly for four weeks. Clinical assessment used the Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04), pruritus Visual Analog Scale (pVAS), and cytological evaluation of yeast numbers at Day (D)0, D14 and D28. Skin barrier function was determined by measuring transepidermal water loss (TEWL) after a single bathing procedure with each product in the healthy dogs. RESULTS: The pVAS scores and yeast counts were significantly reduced on D28 compared with D0 in both groups (P < 0.05). CADESI-04 was significantly decreased on D28 in the emollient bathing group (P = 0.003). There were no significant differences in each endpoint score between the groups. In healthy dogs, TEWL was significantly increased after bathing in both groups (P < 0.01). CONCLUSION: An emollient bathing product can be effective for Malassezia overgrowth in dogs with atopic dermatitis. Bathing with shampoo products might affect skin barrier function even when using an emollient product.

Contradictory intrahepatic immune responses activated in high-load hepatitis C virus livers compared with low-load livers.

We found a HLA class II histocompatibility antigen gene, DQ alpha 1 chain (HLA-DQA1), that was expressed more than 9-fold higher in high-load hepatitis C virus (HCV) livers than low-load HCV livers using transcriptomics of chronic HCV-infected livers. To further investigate this finding, we examined which cells were positive for HLA-DQA1 and what liver immune responses were different between HCV-high and -low livers. HLA-DQA1-positive cells were significantly increased in the HCV-high group, and most positive cells were identified as non-parenchymal sinusoid cells and lymphocytic infiltrates in the portal area. Parenchymal hepatocytes were negative for HLA-DQA1. HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were positive for CD20 (B cells) or CD3 (T cells). mRNA levels of antigen-presenting cell (APC) markers such as CD68 and CD11c were significantly upregulated in the HCV-high group and were correlated with HLA-DQA mRNA levels. CD8B mRNA (CD8+ T cells) was upregulated in both HCV-positive livers compared with HCV-negative livers, whereas CD154 mRNA (CD4+ T helper cell) was upregulated in the HCV-high group compared with the HCV-low group. The immune regulatory molecules FOXP3 mRNA (regulatory T cell, T reg) and programmed cell death ligand-1 (PD-L1) mRNA were significantly increased in the HCV-high group. HCV-high livers had two molecular immune responses: increased APC numbers and adaptive immunity and the induction of immune tolerance. The local hepatic imbalance of contradictory immune responses might be responsible for high HCV loads.

Expression of von Hippel-Lindau tumor suppressor protein (pVHL) characteristic of tongue cancer and proliferative lesions in tongue epithelium.

BACKGROUND: Patients with tongue cancer frequently show loss of heterozygosity (LOH) of the von Hippel-Lindau (VHL) tumor suppressor gene. However, expression of VHL protein (pVHL) in tongue cancer has rarely been investigated and remains largely unknown. We performed immunohistochemical staining of pVHL in tongue tissues and dysplasia, and examined the association with LOH and its clinical significance. METHODS: Immunohistochemical staining of pVHL in formalin-fixed, paraffin-embedded sections of cancerous and other tissues from 19 tongue cancer patients showed positivity for LOH of VHL in four samples, negativity in four samples, and was non-informative in 11 samples. The staining pattern of pVHL was also compared with those of cytokeratin (CK) 13 and CK17. RESULTS: In normal tongue tissues, pVHL staining was localized to the cytoplasm of cells in the basal layer and the area of the spinous layer adjacent to the basal layer of stratified squamous epithelium. Positive staining for pVHL was observed in the cytoplasm of cancer cells from all 19 tongue cancer patients. No differences as a result of the presence or absence of LOH were found. Notably, cytoplasm of poorly differentiated invasive cancer cells was less intensely stained than that of well and moderately differentiated invasive cancer cells. pVHL staining was also evident in epithelial dysplasia lesions with pVHL-positive cells expanding from the basal layer to the middle of the spinous layer. However, no CK13 staining was noted in regions of the epithelium, which were positive for pVHL. In contrast, regions with positive staining for CK17 closely coincided with those positive for pVHL. CONCLUSIONS: Positive staining for pVHL was observed in cancerous areas but not in normal tissues. pVHL expression was also detected in lesions of epithelial dysplasia. These findings suggest that pVHL may be a useful marker for proliferative lesions.

Transmembrane serine protease TMPRSS2 activates hepatitis C virus infection.

UNLABELLED: The human liver reacts to hepatitis C virus (HCV) with a balanced response consisting of host anti- and proviral activities. To explore these subtle host responses, we used oligonucleotide microarrays to investigate the differential gene expression between two groups of liver samples with high and low HCV loads (>100-fold difference). We identified and validated 26 genes that were up-regulated in livers with high HCV loads, including transmembrane protease serine 2 (TMPRSS2). Trypsin inhibitors inhibited the infection of Huh7-25-CD81 cells with cell-culture-derived HCV (HCVcc) of Japanese fulminant hepatitis 1 isolate at the postbinding and entry step, and trypsin enhanced HCVcc infection at an early stage of infection. Several major transmembrane serine proteases, in particular, furin and hepsin, were detected in Huh7-25-CD81 cells, but TMPRSS2 was not. Huh7-25-CD81 cell clones stably expressing TMPRSS2- WT (wild type) and inactive TMPRSS2-mutant genes showed positive and negative enhancement of their susceptibility to HCVcc infection, respectively. The enhanced susceptibility of TMPRSS2-WT Huh7-25-CD81 cells was confirmed by knockdown of TMPRSS2 using small interfering RNA. The cell-surface protease activity of TMPRSS2-WT cells was markedly active in the cleavage of QAR and QGR, corresponding to amino acid residues at P3 to P1. CONCLUSION: The cell-surface activity of a trypsin-like serine protease, such as TMPRSS2, activates HCV infection at the postbinding and entry stage. Host transmembrane serine proteases may be involved in the sensitivity, persistence, and pathogenesis of HCV infection and be possible targets for antiviral therapy.

TMPRSS2 is an activating protease for respiratory parainfluenza viruses.

Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.

CLEC4M-positive and CD81-negative Huh7 cells are not susceptible to JFH-1 HCVcc infection but mediate transinfection.

C-type lectin domain family 4, member M (CLEC4M), a trans-membrane protein specifically expressed in liver sinusoidal endothelial cells, is considered a candidate receptor for hepatotropism of hepatitis C virus (HCV). CLEC4M was previously reported to capture artificial HCVpp (pseudoparticle) and transmit it to hepatocytes (transinfection) via CLEC4M-positive cells. It is still not known whether CLEC4M acts as a receptor for HCVcc (cell-culture-produced HCV) transinfection or whether CLEC4M is an entry receptor for HCVcc. Initially, we established stably CLEC4M-positive and HCV-replication-permissive cell lines by introducing a CLEC4M expression vector into Huh7-25 cells (Huh7-25-CLEC4M) by transfection. Huh7-25 is a mutant cell line that is resistant to JFH-1 HCVcc due to the lack of expression of CD81 but permissive for replication of JFH1 HCV RNA. When Huh7-25-CLEC4M cells were infected with HCVcc and cultured for 6 days, none were positive for infection. Next, to examine whether CLEC4M functions as a receptor for transinfection, Huh7-25-CLEC4M cells were inoculated with HCVcc and thereafter co-cultured with Huh7-it cells, which are susceptible to HCV infection. The amount of HCV RNA was increased in Huh7-it cells co-cultured with Huh7-25-CLEC4M cells, and the transinfection was inhibited in the presence of anti-CLEC4M antibody during inoculation. Thus, CLEC4M cannot substitute for CD81 as an entry receptor for JFH-1 HCVcc. It just mediates transinfection without internalization of HCVcc. CD81 is still crucial for HCV entry into hepatocytes, and CLEC4M in liver sinusoidal endothelial cells may be responsible for hepatotropism of HCV infection by trapping circulating HCV to transmit it to adjacent hepatocytes.

Complex phenotypic heterogeneity of combined hepatocellular-cholangiocarcinoma with a homogenous TERT promoter mutation.

To clarify the mechanism underlying the development and poor prognosis of combined hepatocellular-cholangiocarcinoma (cHCC-CCA), we characterized liver cancer driver mutations and poor prognostic markers in both the HCC and intrahepatic CCA (iCCA) components of a cHCC-CCA tumor. The telomerase reverse transcriptase (TERT) promoter mutation C228T was quantified by digital polymerase chain reaction using DNA from multiple microdissected cancer components of a single cHCC-CCA nodule. The protein expression of cancer-related markers, including TERT, was examined by serial thin-section immunohistochemistry and double-staining immunofluorescence. TERT promoter mutation and TERT protein expression were detected in all cancer components but not in noncancer regions. TERT promoter mutation frequencies were similar among components; those of TERT protein-positive cancer cells were higher in iCCA and mixed components than in HCC. The frequencies of Ki67- and p53-positive cells were similarly higher in iCCA and mixed components than in HCC. However, double-positive cells for the three proteins were unexpectedly rare; single-positive cells dominated, indicating phenotypic microheterogeneity in cancer cells within a component. Interestingly, HCC and CCA marker protein immunohistochemistry suggested dedifferentiation of HCC and transdifferentiation from HCC to iCCA in HCC and iCCA components, respectively. Such phenotypic intercomponent heterogeneity and intracomponent microheterogeneity were detected in a tumor nodule of cHCC-CCA uniformly carrying the early HCC driver mutation. Moreover, poor prognostic markers were randomly expressed without a regular pattern, consistent with the poor prognosis.

Protein from the fraction remaining after RNA extraction is useful for proteomics but care must be exercised in its application.

Simultaneous isolation of mRNA and proteins from a single small biopsy specimen can be useful for integrated omics studies. Here, we have improved the method for extracting protein from the fraction remaining after RNA isolation by TRIzol reagent, for application in protein and proteome analyses. Protein yield was reduced by half, but the patterns developed on 2D gels were equivalent to conventional urea extractions. Thus, although quantitative profiles of individual proteins were different from conventionally-isolated samples, overall profiles were similar. Therefore, this particular protein source is useful for proteomics but care must be exercised in its application.

ß-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers.

The level of expression of housekeeping genes is in general considered stable, and a representative gene such as glyceraldehyde-3-phosphate dehydrogenase is commonly used as an internal control for quantitating mRNA. However, expression of housekeeping genes is not always constant under pathological conditions. To determine which genes would be most suitable as internal controls for quantitative gene expression studies in human liver diseases, we quantified 12 representative housekeeping genes in 27 non-cancerous liver tissues (normal, chronic hepatitis C with and without liver cirrhosis). We identified ß-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. We conclude that it is important to determine the most appropriate control gene for the particular condition to be analyzed.

A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report.

BACKGROUND: Von Hippel-Lindau disease (VHL) is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH) of the VHL gene is common in squamous cell carcinoma tissues of the tongue. CASE PRESENTATION: We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C) in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C) revealed a deletion of the mutant C allele in the carcinoma tissues. CONCLUSIONS: The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.

Direct molecular evidence for both multicentric and monoclonal carcinogenesis followed by transdifferentiation from hepatocellular carcinoma to cholangiocarcinoma in a case of metachronous liver cancer.

Frequent recurrence is a major issue in liver cancer and histological heterogeneity frequently occurs in this cancer type. However, it has remained elusive whether such cancers are multicentric or monoclonal. To elucidate the clonal evolution of hepatocellular carcinoma (HCC) recurrence and combined hepatocellular-cholangiocarcinoma (cHCC-CCA) development, the somatic mutation frequency and signatures in a patient with triple occurrence of liver cancer every three years were examined, with samples designated as #1HCC, #2HCC and #3cHCC-CCA, respectively. A total of four tumor regions, including HCC (#3HCC) and intrahepatic CCA (#3iCCA) components of #3cHCC-CCA, and three nontumor regions (#1N, #2N and #3N) were precisely dissected from formalin-fixed paraffin-embedded tissues of each surgical specimen. DNA was extracted and subjected to tumor-specific somatic mutation determination. Of note, five nonsynonymous single-nucleotide variants (SNVs), namely those of KMT2D, TP53, DNMT3A, PKHD1 and TLR4, were identified in #3cHCC-CCA. All five SNVs were detected in both #3HCC and #3iCCA and #2HCC but not in #1HCC. The telomerase reverse transcriptase (TERT) promoter mutation C228T, but not C250T, was observed in all tumors. Digital PCR of C228T also indicated the presence of the TERT promoter mutation C228T in nontumorous liver tissues (#1N, #2N and #3N) at a frequency of 0.11-0.83% compared with normal liver and blood samples. These results suggest the following phylogenetic evolution of three metachronous liver cancers: #1HCC was not related to #2HCC, #3HCC and #3iCCA; both #3HCC and #3iCCA arose from #2HCC. From the above, three novel findings were deduced: i) Both multicentric occurrence and intrahepatic metastasis may be involved in liver cancer in a three-year interval; ii) transdifferentiation from HCC to iCCA is a possible pathogenic mechanism of cHCC-CCA; and iii) a nontumorous, noncirrhotic liver may contain a preneoplastic region with a cancer driver mutation in the TERT promoter.

Phylogenetic analysis of combined lobular and ductal carcinoma of the breast.

Breast cancer manifests in diverse forms, with particular reference to various cell types harboring different mutations and gene expression profiles. To elucidate the clonal relationship between cancer cells in tumors composed of both ductal and lobular phenotypes, two combined lobular and ductal carcinoma (CLDC) cases were analyzed, including one mixed ductal­lobular carcinoma (MDL) lesion, by direct sequencing of the mitochondrial DNA D­loop, digital PCR targeting of chromosomes 1q and 16q, as well as next­generation sequencing. DNA was extracted from formalin­fixed paraffin­embedded tissue sections of different histological types, including invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, lobular carcinoma in situ, flat epithelial atypia, non­neoplastic mammary gland and extramammary organs, using laser­assisted microdissection. Mutations detected by the comprehensive cancer panel were validated by SYBR green allele­specific quantitative PCR (RRM1, AKT1, PIK3CA, RALGDS, EGFR, TP53, IL21R, DPYD, SGK1, CDH1, TIMP3 and KMT2C). CLDC, which shared the basic genetic alterations of 1q gain or 16q loss, progresses to invasive lobular or ductual carcinoma with the accumulation of further mutations. Cancer cells contained in an MDL lesion shared closely related genetic alterations, suggesting that these cells have the same origin, despite different histological features, namely 'lobular' or 'ductal'. By contrast, multiple lesions located away from the main tumor, diagnosed as CLDC (excluding an MDL lesion) were not always identical with different genetic alterations, despite being diagnosed as ductal carcinoma in situ. Thus, MDL should be defined as a distinct category separate from CLDC, whose components of 'lobular' and 'ductal' may have the same cellular origin.

2',5'-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro.

We found the 2',5'-oligoadenylate synthetase-like (OASL) gene to be significantly elevated by high virus loads in human liver infected with hepatitis C virus (HCV). Here, we determined whether OASL inhibited HCV replication using an in vitro system. We constructed three expression vectors of OASL to produce isoform a (OASLa), isoform b (OASLb), and the C-terminal ubiquitin-like domain of isoform a (Ub). When Huh7 JFH-1 HCV replicon cells were separately transfected with these three vectors, colony formation of HCV-replicating cells was inhibited by 95%, 94%, and 65%, respectively. Both OASLa and OASLb were also inhibitory for cells as well as the virus because colony formation of OASL-producing cells was reduced to 41% and 8%, respectively. Stable Huh7 clones producing each of the three OASLs were established and assessed for their inhibition of HCV replication using luciferase reporter gene-containing JFH-1 replicon RNA. HCV replication was inhibited by 50-90% in several stable OASL clones. Association analysis in six Ub clones expressing different levels of Ub mRNA showed that the degree of inhibition of HCV replication was significantly associated with the amount of Ub present. In conclusion, OASL possesses two domains with HCV inhibitory activity. The N-terminal OAS-homology domain without OAS activity is inhibitory for cell growth as well as HCV replication, whereas C-terminal Ub is inhibitory only for HCV replication. Therefore, OASLa, a major isoform of this molecule induced in human liver, may mediate anti-HCV activity through two different domains.

Detection of apoptosis and matrical degeneration within the intervertebral discs of rats due to passive cigarette smoking.

Although low-back pain is considered to be associated with cigarette smoking, the influence of cigarette smoking on the intervertebral discs (IVD) has not been confirmed. We established a rat model of passive cigarette smoking-induced IVD degeneration, and investigated the cytohistological changes in the IVD and the accompanying changes in gene expression. IVD from rats exposed to 8 weeks of passive cigarette smoking were stained with Elastica van Gieson, and exhibited marked destruction of the supportive structure of the reticular matrix in the nucleus pulposus (NP). Positive signals on safranin O, alcian blue, type II collagen and aggrecan staining were decreased in the destroyed structure. Safranin O and type II collagen signals were also decreased in the cartilage end-plate (CEP) after 4- and 8-weeks of cigarette smoking. In the CEP, the potential for apoptosis was increased significantly, as demonstrated by staining for single-strand DNA. However, there were no signs of apoptosis in the NP or annulus fibrosus cells. Based on these findings, we hypothesized that passive cigarette smoking-induced stress stimuli first affect the CEP through blood flow due to the histological proximity, thereby stimulating chondrocyte apoptosis and reduction of the extracellular matrix (ECM). This leads to reduction of the ECM in the NP, destroying the NP matrix, which can then progress to IVD degeneration.

Caution for simple sequence repeat number variation in the mitochondrial DNA D-loop to determine cancer-specific variants.

The mitochondrial DNA (mtDNA) displacement loop (D-loop) is often altered in various cancer types, including with regard to simple sequence repeat number variation (SSRNV), which includes the C-tract and CA-tract. However, because of mitochondrial heteroplasmy and slippage errors by the Taq DNA polymerase used in polymerase chain reaction (PCR) analysis, it is difficult to precisely evaluate mtDNA D-loop SSRNV experimentally. In this study, to precisely determine cancer-specific variants in mtDNA SSRNV, various microscopic portions of cancerous tissues and normal control tissues were obtained from a patient with breast cancer, followed by laser-capture microdissection of formalin-fixed paraffin-embedded specimens. Regions containing (CA)7 repeats (positions 514-523) and (C)8 repeats (positions 303-315) of the mitochondria DNA D-loop were amplified and sequenced. Variant signals of mtDNA SSRs of (CA)7 and (C)8 were observed in normal and cancerous tissues, with the content of minor alleles (CA)6 and (C)7/(C)9 differing among samples. These results were confirmed by PCR using various primers and proofreading DNA polymerases. PCR of genomic SSRs of (CA)7 in the NAALD2 gene and (C)8 in the BMP6 gene showed a simple repeat in all samples that was different from the observed mtDNA SSRNV. The present study suggests a reliable procedure for determining cancer-specific variants in mtDNA SSRNV: Using a proofreading DNA polymerase for PCR, the background of slippage by PCR is determined by PCR of the same genomic sequence as the target. Due to the varied heteroplasmy level of mtDNA SSRNV among normal tissues, the second background of polymorphic variations should be determined by several normal tissue DNA as PCR templates. Finally, the cancer-specific variant, including its variation frequency, is determined by subtracting the two background signals from the variant signals in cancer. However, care must be taken, as normal heteroplasmy drifts observed in mtDNA SSRNV may complicate such estimations.

Allelic loss analysis of tumor suppressor genes regardless of heterozygosity: von Hippel-Lindau gene loss in renal cell carcinoma.

PURPOSE: VHL, the von Hippel-Lindau tumor suppressor gene, has no microsatellites, but rather single nucleotide polymorphisms inside the gene. However, their low heterozygosity is unfavorable for loss of heterozygosity analysis. We examined whether our modified single nucleotide polymorphism genotyping method would be useful for allelic loss analysis of the VHL gene in heterozygous and homozygous genotypes of sporadic renal cell carcinoma. MATERIALS AND METHODS: Genomic DNA was extracted from tumor and nontumor tissues in 35 cases of sporadic renal cell carcinoma. The single nucleotide polymorphism (rs1642742), G or A containing region of the VHL gene was amplified from sample DNA and subjected to primer extension reaction with fluorescent dideoxynucleotide triphosphate. Template directed incorporation of fluorescent dideoxyguanosine triphosphate or dideoxyadenosine triphosphate was quantitatively analyzed and the A/G (G/A) signal ratio was compared between tumor and nontumor tissues. RESULTS: We confirmed quantitative template directed incorporation of dideoxyguanosine triphosphate or dideoxyadenosine triphosphate using model templates with various ratios of DNA from the 2 genotypes AA and GG. In 20 heterozygous cases of renal cell carcinoma the A/G signal ratio was significantly differentiated between tumor and nontumor in 9 loss of heterozygosity positive cases but not in 11 loss of heterozygosity negative cases. A total of 15 homozygous renal cell carcinoma cases were tested by adding homozygous control DNA of a different genotype before analysis. Eight of the 15 cases showed a significantly lower signal ratio in tumor than in nontumor, whereas the other 7 showed no significant difference. CONCLUSIONS: Our modified single nucleotide polymorphism genotyping is broadly applicable to allelic loss analysis of tumor suppressor genes in heterozygous and homozygous tumors.

Transglutaminase 2 is upregulated in primary hepatocellular carcinoma with early recurrence as determined by proteomic profiles.

The mechanism of early recurrence of hepato-cellular carcinoma (HCC) is not well understood. To examine whether early intrahepatic metastasis of HCC can be determined by the reliable molecular characteristics of the primary HCC, we focused on early-stage tumors of primary and solitary HCC cases. Proteomic differences were investigated between two groups, 11 early (recurrence within 12 months) and 10 late (no recurrence within 48 months) HCC cases, using two-dimensional fluorescence difference gel electrophoresis. Overall, 10 upregulated and 9 downregulated proteins were identified from a total of 1623 protein spots detected in early recurrent HCC. Cluster analysis using the 19 proteins successfully divided the 21 HCC samples exactly into the two above groups. A multifunctional protein, transglutaminase 2 (TGM2), was upregulated in the early recurrence group. Immunohistochemistry revealed the frequent observation of TGM2-positive HCC cells in the early group, with a tendency of TGM2-positive staining in HCC cells adjacent to fibrous stroma. To examine whether two major TGM2-associated pathways, epithelial-mesenchymal transition (EMT) and integrin signaling, were activated in the early recurrence group of HCC, downstream molecules of TGM2 were measured. The mRNA level of EMT-related genes was highly positively correlated with TGM2 mRNA. However, E-cadherin (CDH1) mRNA and protein were not downregulated in correlation with TGM2 expression. The phosphorylation of FAK and Akt and the downregulation of PTEN were not associated with the quantity of TGM2. Therefore, TGM2 might contribute to early HCC recurrence through signaling pathways not related to EMT and integrin signaling. The proteomics of strictly classified HCCs would be useful for characterizing pro-metastatic HCC and for developing a new therapeutic target for treatment of metastasis.

Particular gene upregulation and p53 heterogeneous expression in TP53-mutated maxillary carcinoma.

It has been demonstrated that tumor protein p53 (TP53) mutation in maxillary squamous cell carcinoma, is more treatment-resistant compared with the carcinoma without TP53 mutation. However, the association between TP53 mutation and treatment resistance remains unclear. As a first step in understanding the biological differences between tumors with and without TP53 mutation, a comprehensive gene expression analysis of maxillary squamous cell carcinoma with or without TP53 mutation was performed. A total of 42 genes were identified to be differentially expressed by >4-fold. Quantification of their mRNA using quantitative polymerase chain reaction indicated 18 genes with high expression and three genes with low expression in TP53 mutated tumors vs. TP53 wild-type tumors. The 18 genes included eight cell adhesion (DSC3, GRHL1, EPPK1, PROM2, ANXA8, DSP, JUP, and KRT6B) and four cell growth inhibition (SFN, CLCA2, SAMD9 and TP63) genes. Among these genes, DSC3, SFN, and CSTA, whose expression was markedly increased, also demonstrated high protein expression in immunohistochemical staining of TP53 mutated tumors. The TP53 mutated tumors demonstrated high nuclear staining of the TP53 protein only in tumor cells at the tumor margins adjacent to the stroma, whereas the tumor interior was negative for TP53. However, all tumor cells of TP53 wild-type tumors exhibited positive nuclear staining for the TP53 protein. The combined findings suggest that TP53 mutated tumors possess a phenotype opposite to that associated with cancer progression and malignant transformation, and exhibit tumor cell heterogeneity between the tumor interior and margins.

Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation.

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: 'microgenomics'. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.