Keiko Nagata

Association of Merkel cell polyomavirus infection with clinicopathological differences in Merkel cell carcinoma.

Merkel cell polyomavirus is a novel polyomavirus that is monoclonally integrated into genomes of up to 80% of human Merkel cell carcinomas. Merkel cell polyomavirus-positive Merkel cell carcinomas showed less metastatic tendency and better prognosis according to some reports, whereas others disagree. In this study, we analyzed clinicopathological characteristics of 20 Merkel cell polyomavirus-positive and 6 Merkel cell polyomavirus-negative Merkel cell carcinoma cases, in which we already reported the association of Merkel cell polyomavirus infection with statistically significant morphological differences. Immunohistochemical expressions of cell cycle-related proteins, mutations of the TP53 tumor-suppressor gene (exons 4-9) and p14ARF promoter methylation status as well as detailed clinical data were analyzed and compared between Merkel cell polyomavirus-positive and Merkel cell polyomavirus-negative cases. Merkel cell polyomavirus-positive Merkel cell carcinomas showed better prognosis with one spontaneous regression case and significantly higher expression of retinoblastoma protein (P = .0003) and less p53 expression (P = .0005) compared to Merkel cell polyomavirus-negative Merkel cell carcinomas. No significant differences were found in expressions of p63, MDM2, p14ARF or MIB-1 index, and p14ARF promoter methylation status. Interestingly, frequency of TP53 non-ultraviolet signature mutation was significantly higher in Merkel cell polyomavirus-negative Merkel cell carcinomas than in Merkel cell polyomavirus-positive Merkel cell carcinomas (P = .036), whereas no significant difference was detected in TP53 ultraviolet signature mutations between two groups. These results suggest that Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas likely develop through different tumorigenic pathways and that the presence or absence of Merkel cell polyomavirus in the tumor is still an important factor that affects survival in patients with Merkel cell carcinoma.

Increased theophylline metabolism in the menstrual phase of healthy women

BACKGROUND We previously reported on a Japanese woman with premenstrual asthma whose serum theophylline concentration was lower before the onset of her menstrual period. We evaluated the clearance of theophylline in each phase of her menstrual cycle and found an increase in clearance in the premenstrual and menstrual phases. OBJECTIVE This study was designed to investigate whether changes in theophylline clearance may occur with the menstrual cycle in nonasthmatic women. METHODS Aminophylline, 250 mg, was infused intravenously for 45 minutes, and the clearance of theophylline was calculated in seven healthy, ovulatory women on days 1 or 2, 10, and 20 or 25 of the menstrual cycle. The serum concentration of theophylline was determined with an enzyme immunoassay (homogeneous). RESULTS Theophylline clearance was significantly higher and its half-life was significantly shorter in the menstrual phase than in the follicular phase. CONCLUSION The metabolism of theophylline, and consequently, its clearance were increased in healthy women around the onset of menses. Clinicians must therefore consider the menstrual variation in theophylline metabolism when prescribing this drug to female patients.

Interleukin-1 loop model for pathogenesis of Langerhans cell histiocytosis.

We propose Langerhans cell histiocytosis (LCH) is an inflammatory process that is prolonged by mutations. We hypothesize that Merkel cell polyomavirus (MCPyV) infection triggers an interleukin-1 (IL-1) activation loop that underlies the pathogenesis of LCH. Langerhans cells (LCs) are antigen presenting cells in the skin. When LCs encounter exogenous antigens, they migrate from the epidermis into draining lymphoid tissues to initiate T-cell activity. It has been proposed that LC migration-related factors, including E-cadherin, matrix metalloproteinase, and Notch ligand induce LCH activity. We found that the tyrosine phosphatase SHP-1, which binds IL-1 receptor-associated kinase 1, is expressed at a significantly higher level in LCH affecting multiple organ systems (MS-LCH) than in LCH affecting a single organ system (SS-LCH). IL-1 stimulates T helper 17 cells and their signature cytokine IL-17 had been a matter of controversy. We detected higher levels of IL-17A receptor expression in MS-LCH than in SS-LCH and proposed an IL-17 endocrine model that could settle the controversy. IL-1 is the first cytokine secreted in response to sensitizers and promotes LC migration from sentinel tissues. Myeloid differentiation primary response 88 (MyD88), downstream of the IL-1 receptor, has functions in both RAS signaling and inflammation, leading to human cell transformation. In 2010, an activating mutation in the B-rapidly accelerated fibrosarcoma gene (BRAF) V600E was found in LCH. This BRAF mutation induces phosphorylation of the extracellular signal-regulated kinase (ERK) that may play an important role with MyD88 in LCH pathogenesis. However, phosphorylated ERK (pERK) is rapidly dephosphorylated by dual specificity phosphatase 6 (DUSP6), and limited proliferation is predicted in BRAF mutant cells. MyD88 binds pERK via its D-domain, thereby preventing pERK–DUSP6 interaction and maintaining ERK in an active, phosphorylated state. We detected MCPyV-DNA in the peripheral blood cells of two out of three patients with LCH in high-risk organs but not in those of patients with LCH in non–high-risk organs (0/12; P = .029). MCPyV infection can trigger precursor LCH cells with BRAF mutation to produce IL-1; the IL-1 loop is amplified in all LCH subclasses. Our model indicates both BRAF mutation and IL-1 loop regulation as potential therapeutic targets.

A novel interstitial deletion of KAL1 in a Japanese family with Kallmann syndrome

AbstractWe identified a novel interstitial deletion that spanned from exons 5 to 10 of KAL1 in two Japanese brothers with X-linked Kallmann syndrome (KS; MIM no. 308700). Both brothers had hypogonadism, unilateral renal agenesis, and disturbance of the sense of smell, but they had no other neurological manifestations, including mental disturbance. Their mother was confirmed to be an asymptomatic carrier, by use of a comparative multiplex polymerase chain reaction (PCR) analysis. The present patients are further examples of patients with KS without mental disturbance caused by a mutation confined to KAL1.

Comparison of Akt/mTOR/4E-BP1 pathway signal activation and mutations of PIK3CA in Merkel cell polyomavirus-positive and Merkel cell polyomavirus-negative carcinomas.

Merkel cell polyomavirus (MCPyV) integrates monoclonally into the genomes of approximately 80% of Merkel cell carcinomas (MCCs), affecting their clinicopathological features. The molecular mechanisms underlying MCC development after MCPyV infection remain unclear. We investigated the association of MCPyV infection with activation of the Akt/mammalian target of rapamycin (mTOR)/4E-binding protein 1 (4E-BP1) signaling pathway in MCCs to elucidate the role of these signal transductions and to identify molecular targets for treatment. We analyzed the molecular and pathological characteristics of 41 MCPyV-positive and 27 MCPyV-negative MCCs. Expression of mTOR, TSC1, and TSC2 messenger RNA was significantly higher in MCPyV-negative MCCs, and Akt (T308) phosphorylation also was significantly higher (92% vs 66%; P = .019), whereas 4E-BP1 (S65 and T70) phosphorylation was common in both MCC types (92%-100%). The expression rates of most other tested signals were high (60%-100%) and not significantly correlated with MCPyV large T antigen expression. PIK3CA mutations were observed more frequently in MCPyV-positive MCCs (6/36 [17%] vs 2/20 [10%]). These results suggest that protein expression (activation) of most Akt/mTOR/4E-BP1 pathway signals was not significantly different in MCPyV-positive and MCPyV-negative MCCs, although these 2 types may differ in tumorigenesis, and MCPyV-negative MCCs showed significantly more frequent p-Akt (T308) activation. Therefore, certain Akt/mTOR/4E-BP1 pathway signals could be novel therapeutic targets for MCC regardless of MCPyV infection status.

Merkel cell polyomavirus (MCPyV) strains in Japanese merkel cell carcinomas (MCC) are distinct from Caucasian type MCPyVs: genetic variability and phylogeny of MCPyV genomes obtained from Japanese MCPyV-infected MCCs.

Abstract Most of merkel cell carcinomas (MCC), a rare, aggressive skin cancer with neuroendocrine features, harbor merkel cell polyomavirus (MCPyV). Seroepidemiological studies suggested high prevalence of MCPyV in the human population. More than ten sequence data on MCPyV strains in Japan have been available, whereas most sequence data were detected from patients living in Europe or European ancestry. Analysis of nine almost complete and 19 partial sequences from two oncogenes, small T antigen(ST) and large T antigen(LT) genomes obtained from 32 Japanese MCPyV-infected MCC revealed that each Japanese MCPyV-infected MCC harbored a specific MCPyV strain with some synonymous or, silent mutations and stop codons or deletions, but functional domains of T antigen had no amino acid changes. All stop codons were localized after retinoblastoma protein-binding domain. These Japanese MCPyV strains were very closely interrelated to themselves and a consensus sequence of Japanese strain was generated. Phylogenetic analysis of our nine sequences and 70 other sequences for ST and LT gene registered in GenBank indicated that Japanese or Asian MCPyV strains formed distinct clades from Caucasian clade, and phylogenetic tree of our nine and 75 other sequences for ST gene formed characteristic three clades and showed that all Japanese or Asian strains were included in the dominant clade. These suggested the possibility of geographically related genotypes of MCPyV. The genomic characterization of MCPyV variants will provide an important database and insights for illuminating their evolutional and biological differences.

Immunoglobulin expressions are only associated with MCPyV-positive Merkel cell carcinomas but not with MCPyV-negative ones: comparison of prognosis.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer, often associated with Merkel cell polyomavirus (MCPyV). Recently, immunoglobulin (Ig) expression was reported in MCC, thereby suggesting that B cells might be their cellular ancestors. We tested 30 MCCs (20 MCPyV-positive and 10 MCPyV-negative) using immunohistochemistry for the expressions of IgG, IgA, IgM, Ig&kgr;, Ig&lgr;, terminal desoxynucleotidyl transferase, paired box gene 5 (PAX5), octamer transcription factor-2 (Oct-2), and sex-determining region Y-box 11 (SOX11). We performed in situ hybridization for Ig&kgr;-mRNA or Ig&lgr;-mRNA and Ig heavy chain (IgH) gene rearrangement (IgH-R) analyses. The expressions of PAX5, TdT, Oct-2, and SOX11 were not significantly different between MCPyV-positive and MCPyV-negative MCCs. At least 1 of IgG, IgA, IgM, or Ig&kgr; was expressed in MCPyV-positive (14/20, 70%) and none in MCPyV-negative MCCs (P=0.0003). There was a higher tendency for Ig&kgr;-mRNA expression (7/19, using in situ hybridization) and IgH-R (10/20, using polymerase chain reaction) in MCPyV-positive than in MCPyV-negative MCCs (0/10 and 2/10, respectively), thus suggesting a different Ig production pattern and pathogenesis between the 2 types of MCC. Ig expression or IgH-R in MCPyV-positive MCCs might be associated with MCPyV gene integration or expression in cancer cells but do not necessarily suggest a B-cell origin for MCCs. IgH expression or IgH-R nonsignificantly correlated with improved prognosis. However, these might be important factors that influence the survival of neoplastic cells and might allow the development of novel therapies for patients with MCPyV-positive MCCs.

A new in situ hybridization and immunohistochemistry with a novel antibody to detect small T-antigen expressions of Merkel cell polyomavirus (MCPyV)

BackgroundApproximately 80% of Merkel cell carcinomas (MCCs) harbor Merkel cell polyomavirus (MCPyV) which monoclonally integrates into the genome and has prognostic significance. The presence or absence of MCPyV is usually diagnosed using CM2B4 immunohistochemistry (IHC) for MCPyV-large T antigen (LT) protein. However, this method poses a risk of misdiagnosis.MethodsIn this study, we determined MCPyV infection in MCCs using real-time PCR for MCPyV-LT DNA and prepared 16 cases of MCPyV-DNA-positive and -negative groups. Diagnostic sensitivity and specificity of conventional PCR for MCPyV-small T antigen (MCPyV-ST), IHC using a newly developed polyclonal antibody (ST-1) for MCPyV-ST protein (MCPyV-ST) (aa: 164–177), and in situ hybridization (ISH) as well as real-time PCR for MCPyV-ST mRNA were compared against CM2B4-IHC for sensitivity (0.94, 15/16) and specificity (0.94, 15/16).ResultsThe followings are the respective sensitivity and specificity results from examinations for MCPyV-ST gene: conventional PCR for the MCPyV-ST (0.94, 1.0), ST-1-IHC (0.69, 1.0), real-time PCR for ST mRNA (1.0, no data), ST mRNA ISH (0.94, 1.0). Each of the MCPyV-pseudonegative (1/16) and -pseudopositive (1/16) diagnoses evaluated using CM2B4-IHC were accurately corrected by examinations for MCPyV-ST or its expression as well as real-time PCR for MCPyV-LT. Sensitivity of CM2B4-IHC (0.94) was superior to that of ST-1-IHC (0.69) but equal to that of ST mRNA-ISH (0.94). Specificities of ST-1-IHC (1.0) and ST mRNA-ISH (1.0) were superior to that of CM2B4-IHC (0.94).ConclusionsTherefore, combined application of ST mRNA-ISH and ST-IHC as well as CM2B4-IHC is recommended and will contribute to the diagnostic accuracy for MCPyV infection in MCCs.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9966295741144834

The evolution of Epstein-Barr virus inferred from the conservation and mutation of the virus glycoprotein gp350/220 gene

To study variations of Epstein-Barr virus (EBV), we analyzed the gp350/220 gene for several cell lines and Japanese wild isolates using direct sequencing. The N-terminal region was highly conserved in all EBVs except for Jijoye/P3HR-1 and a few isolates. The variation of the region coincided with EBV types A and B (also referred to as types 1 and 2) and were, respectively, designated as the types a and b. The type A/a was detected in most Japanese cell lines and wild isolates, and was classified as China1 type with latent membrane protein (LMP) 1 gene. The type B/b was detected in only a few wild isolates with the Med and China2 types. The C-terminus had more diversity than the N-terminus and lacked the divergence between types A/a and B/b. The phylogenetic analyses of the gp350/220 and LMP1 genes may suggest a mode of EBV evolution into types A/a and B/b and then to LMP1 subtypes.

Presence of Epstein–Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves’ disease patients and in healthy individuals

Abstract Graves’ disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein–Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves’ disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves’ disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves’ disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves’ disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves’ disease.