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Here, we report two cases showing tumor-like white matter lesions; one case was diagnosed as having inflammatory disease, and the other was diagnosed as having astrocytoma. Their outcomes were completely distinct despite similar pathology. Prior to biopsy, perfusion computed tomography (CT) and magnetic resonance imaging (MRI) were conducted. The two mass-forming lesions were distinct in edema level and vascularity patterns on CT and MRI. However, pathological examination of brain biopsy specimens revealed commonalities, including (1) proliferation of glial cells, (2) perivascular lymphocytic infiltration, and (3) appearance of numerous macrophages. Although atypical astrocytes proliferated in both cases, nuclear atypia was more distinct in case 2 than in case 1. The immunohistochemical results were the same for both cases: isocitrate dehydrogenase 1 (IDH1) R132H mutation was negative, and alpha thalassaemia mental retardation X-linked (ATRX) was retained. Faint immunoreactivity for p53 was observed in a few glial cells, and Ki-67 immunoreactive cells were markedly reduced in numbers (< 1%). Inflammatory reactions were evident in both cases: T cells dominantly infiltrated over B cells in the perivascular area in case 1, whereas both T and B cells infiltrated in case 2. Molecular analysis revealed wild-type IDH1 and IDH2 in both cases. However, a telomerase reverse transcriptase (TERT) sequence mutation was detected in case 2 but not in case 1. Eventually, case 1 was diagnosed as having inflammatory lesions, whereas case 2 was diagnosed as having diffuse astrocytoma associated with inflammatory reactions. The prognosis was favorable for case 1, whereas case 2 died 10 months following biopsy. These data indicated the diagnostic value of molecular analysis, for example, a TERT mutation, in association with the radiological findings. Although in case 2, histopathological evidence did not suggest high-grade glioma, the case met the new diagnostic criteria: "diffuse astrocytic glioma, IDH wild-type, with molecular features of glioblastoma, World Health Organization (WHO) grade IV," according to cIMPACT-NOW, update 3. Thus, interdisciplinary approaches are essential for accurate diagnosis of newly categorized white matter diseases.

The case of 70-year-old man with mantle cell lymphoma (MCL) carrying t(11;14) translocation that relapsed as nodal lymphoma combining MCL and classic Hodgkin lymphoma (cHL) 9 years after autologous peripheral blood stem cell transplant (auto-PBSCT) is reported. Lymph nodes contained two separate areas of MCL and cHL-like components. Hodgkin and Reed-Sternberg (HRS)-like cells were accompanied by a prominent histiocyte background. HRS-like cells were CD5- , CD15+ , CD20- , CD30+ , PAX5+ , Bob.1- , Oct2- and EBER+ . The MCL component expressed cyclin D1 and SOX11, whereas cyclin D1 and SOX11 expressions were reduced and lost, respectively, in HRS-like cells. Polymerase chain reaction results showed a single clonal rearrangement of the IGH gene in MCL and cHL-like components. CCND1 break apart fluorescence in situ hybridization showed split signals in both MCL and HRS-like cells, suggesting that MCL and cHL-like components were clonally related. Acquisition of p53 expression and Epstein-Barr virus (EBV)-positivity was seen in HRS-like cells. The patient died of disease progression with elevated hepatobiliary enzymes. The autopsy showed both MCL and cHL-like components around the bile ducts, splenic white pulp and bone marrow. The two components were phenotypically distinct, but genetically related, suggesting that transformation of MCL to HRS-like cells during the course of MCL in association with EBV infection.

The patient was a 74-year-old woman with rheumatoid arthritis who developed ataxia. MRI revealed T2-hyperintense lesions predominantly in the left middle cerebellar peduncle. Punctate or linear Gd enhancement was also observed on T1-weighted images. A brain biopsy was conducted and the pathology revealed a mild demyelinated lesion. Polymerase chain reaction (PCR) of biopsied brain tissues revealed the presence of JC virus (JCV) DNA, but JCV-infected oligodendroglia-like cells were not apparent on immunohistochemistry. Sensitive in-situ hybridization, however, detected three JCV-positive cells and the infiltration of CD4+ and CD8+ T cells and plasma cells was also observed. Immunosuppressants were tapered off and mirtazapine and mefloquine administered, resulting in a favorable outcome.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by opportunistic infection of JC polyomavirus (JCV). Today, increased attention has been focused on PML development in multiple sclerosis (MS) patients under disease-modifying therapies (DMT). Although in the acquired immunodeficiency syndrome (AIDS) era, PML was thought to be a rapidly progressive disease with poor prognosis, drug-associated PML is relatively slow in progress, and a favorable outcome may be expected with early diagnosis. However, early PML diagnosis on magnetic resonance imaging (MRI) is frequently difficult, and JCV DNA copy number in cerebrospinal fluid (CSF) is usually low. To facilitate early PML diagnosis on MRI, the pre-mortem images were compared with neuropathology of the post-mortem brain, and underlying pathology corresponding to the MRI findings was evaluated. As a result, PML lesions of the autopsied brain were divided into three parts, based on the disease extension patterns: (A) Progressive white matter lesion in the right frontoparietal lobe including the precentral gyrus. Huge demyelinated lesions were formed with fusions of numerous small lesions. (B) Central lesion including deep gray matters, such as the putamen and thalamus. The left thalamic lesion was contiguous with the pontine tegmentum. (C) Infratentorial lesion of brainstem and cerebellum. Demyelination in the pontine basilar region and in cerebellar white matter was contiguous via middle cerebellar peduncles (MCPs). In addition, (D) satellite lesions were scattered all over the brain. These observations indicate that PML lesions likely evolve with three steps in a tract-dependent manner: (1) initiation; (2) extension/expansion of demyelinating lesions; and (3) fusion. Understanding of the PML disease evolution patterns would enable confident early diagnosis on MRI, which is essential for favorable prognosis with good functional outcome.

Background Primary central nervous system lymphoma (PCNSL), a relatively rare brain tumor, bears a dire prognosis. On occasion, rapid progression of the tumor makes immediate diagnosis and initiation of therapy imperative. To achieve swift diagnosis, we adopt flow cytometry (FCM) in addition to conventional histopathology. The aim of this study is to reveal utility and drawbacks of FCM diagnosis for PCNSL. Methods Patients with suspected PCNSL on neuroradiological findings and received both FCM and histological diagnosis between August 2015 and April 2020 were retrospectively enrolled into the study. Tumor samples were collected by craniotomy with either of endoscope or microscope. The patients’ electronic medical records were investigated, and histological findings, results of FCM, and other clinical data were evaluated. Results Twenty seven patients met the eligibility criteria. Twenty three patients (11 males and 12 females) were diagnosed with PCNSL by histological confirmation, and 22 cases were B-cell type lymphoma and 1 was T-cell type. Median age at diagnosis was 65. FCM analysis showed lymphoma pattern in 20 cases, but in the other 3 lymphoma cases (FCM discordant: FCM-D) and 4 non-lymphomatous tumor cases, FCM results did not show lymphoma pattern (sensitivity: 86.4%, specificity: 100%). Analysis of FCM-D cases showed infiltration of T lymphocytes or astrocytes into the tumor tissue, indicating tumor microenvironmental reaction, were observed, and it is assumed that those reactions deceived FCM diagnosis. Conclusions Despite some disadvantages, diagnosis of PCNSL by FCM is rapid and reliable.

Background: Solitary fibrous tumors (SFTs) are rare and can be misdiagnosed because of their various radiological appearances. Purpose: To clarify the characteristic MRI findings of SFTs by analyzing their radiological-pathological correlation. Material and Methods: Nine consecutive patients with SFT who underwent magnetic resonance imaging (MRI) prior to surgery were analyzed. Eight patients underwent contrast-enhanced MRI, and three underwent dynamic MRI. Radiological-pathological correlation analysis, co-occurrence matrix, run-length matrix, and histogram analysis were performed to assess the relationship between pathological findings T1- and T2-weighted images (T1-WI and T2-WI). Results: All nine lesions ranged in size from 20 to 36 mm. Seven lesions were located in the superior portion of the retrobulbar space found outside of the muscle cone, and two lesions in the inferior portion were located within it. No significant correlation was observed between the amount of collagenous tissue and the qualitative evaluation of the signal on T1-WI and T2-WI. Kurtosis on T2-WI was significantly correlated with the amount of collagenous tissue ('ρ' = –0.97, 'p' < 0.0001) and endothelial cells ('ρ' = –0.49, 'p' = 0.0479). Conclusion: Kurtosis in the histogram analysis on T2WI showed a strong correlation with the amount of collagenous tissue.

We present the case of a 52-year-old woman with right hemiparesis due to a mass lesion in the left parietal white matter and corpus callosum. The lesion was hyperintense on diffusion weighted image and homogenously enhanced with gadolinium on magnetic resonance imaging, and was radiologically indistinguishable with lymphoma. Following progressive aggravation of symptoms, craniotomy for biopsy of the lesion was performed, and it was revealed that the patient had anti-myelin oligodendrocyte glycoprotein-associated disease by histopathological and serological diagnosis. Initial treatment with steroid dramatically improved the symptoms, but they exacerbated again. Then, through cerebrospinal fluid examination, it was revealed that the patient had B-cell lymphoma.

A 70-year-old woman with a human T-cell leukemia virus type 1 infection without any focal neurological symptoms showed age-related atherosclerotic changes in the white matter without any suspicious signal changes suggestive of progressive multifocal leukoencephalopathy (PML) based on the findings of MRI. Viral polymerase chain reaction (PCR) revealed 6,700 copies/mL of the JC virus genome in the cerebrospinal fluid (CSF). An immuno-pathological examination of the autopsied brain revealed JC virus capsid proteins, and in situ hybridization confirmed a JC virus infection, indicating that an active infection begins at the radiologically indistinguishable phase of PML. An early JC virus infection is probably associated with small, scattered demyelinating lesions around the cortico-medullary area of the cortex.

Although progressive multifocal leukoencephalopathy (PML) is known as a fatal disease, some recent cases have shown favorable prognosis with early diagnosis. Therefore, detection of early PML lesions using magnetic resonance imaging (MRI) is important. PML lesions are divided into 4 groups based on distinct developing patterns: A)cerebral lesion, B)central lesion including deep gray matter, C)infratentorial lesion of the brain stem and cerebellum, and D)punctate lesions in the deep white matter. These lesions develop in 3 steps: 1)initiation of a small demyelinating lesion, 2)extension/expansion, and 3)fusion. It is likely that the viruses first reach the brain via the bloodstream and form small demyelinating foci (initiation). Second, the demyelinating foci spread along nerve fibers or expand at the sites (extension/expansion). Finally, the foci fuse with one another to form larger demyelinating lesions (fusion). Understanding the spreading patterns of the virus could help early MRI diagnosis of PML, which is important for favorable prognosis. (Received May 7, 2020; Accepted May 11, 2020; Published September 1, 2020).

In the present study, we analyzed the inflammatory profiles of brain tissues obtained from patients with progressive multifocal leukoencephalopathy (PML) due to John Cunningham (JC) virus infection to identify potential prognostic factors. The study included seven patients (two men, five women) who had been pathologically diagnosed with PML, and all of whom were HIV negative. Fixed brain samples were analyzed via hematoxylin and eosin (HE) staining and Kluver–Barrera (KB) staining. We then performed immunohistochemistry (IHC) specific to JC virus capsid proteins (VP1 and VP2/3) and lymphocyte surface markers (CD4, CD8, CD138, and PD-1). The mean age at onset was 53.4, while the mean duration until biopsy/autopsy was 4.7 months. Four patients were included in the good prognosis (GP) group, while three were included in the poor prognosis (PP) group. Pathological analysis revealed a significantly larger number of CD4-positive T-cell infiltrations (P = .029) in the GP group, along with a preserved CD4:CD8 ratio. Larger numbers of CD138-positive plasma cells were also observed in the GP group (P = .029) than in the PP group. Linear regression analyses revealed a significant association between the numbers of CD138-positive plasma cells and PD-1-positive cells (R2 = 0.80). Viral loads in the cerebrospinal fluid, a controlled inflammatory response mediated by CD4- and CD8-positive T cells, and plasma cells are associated with PML prognosis. Our findings further indicate that regulatory plasma cells may regulate inflammatory T-cell activity via a PD-1/PD-L1 immuno-checkpoint pathway, thereby protecting the uninfected brain from excessive immune-mediated damage during an active JC virus infection.

A 37-year-old woman with systemic lupus erythematosus presented with gait disturbance and cognitive dysfunction. Brain magnetic resonance imaging (MRI) revealed small, punctate, T2-/fluid-attenuated inversion recovery-hyperintense and T1-hypointense lesions without gadolinium enhancement, which is atypical for progressive multifocal leukoencephalopathy (PML). On a pathological examination of biopsied brain tissues, JC virus-infected cells were hardly detected via immunohistochemistry but were certainly detected via in situ hybridization, conclusively verifying the PML diagnosis. After tapering off the immunosuppressant and mefloquine administration, the MRI findings revealed gradual improvement, and she has been stable for over 18 months. A punctate MRI pattern is not specific to natalizumab-associated PML but may be a ubiquitous early sign useful for the early diagnosis of PML.

Patient 1 was a 59-year-old woman receiving prednisolone for idiopathic hypereosinophilia. Brain MRI of patient 1 disclosed slight gadolinium enhancement at lesions, indicating inflammation. Patient 2 was a 32-year-old woman with systemic lupus erythematosus under immunosuppressive therapy. Brain biopsy of patient 2 showed balanced infiltration of CD8+ and CD4+ T lymphocytes at the sites of lesions. Both subjects were diagnosed as having progressive multifocal leukoencephalopathy (PML) shortly after the onset of neurological symptoms and were treated with a combination of mefloquine, mirtazapine, and risperidone. Both patients remain alive with improved neurological symptoms even after long-term follow-up (24 months in patient 1 and 45 months in patient 2). Although the prognosis of PML is very poor, our findings suggest that pharmacotherapy may be effective for patients with well-controlled immune reactions against the JC virus.

BACKGROUND: Chemotherapy with high-dose methotrexate (HD-MTX) followed by whole brain radiotherapy (WBRT) is a conventional approach to treat primary central nervous system lymphomas (PCNSL), but some tumors relapse early leading to unfavorable outcome. Several biomarkers have been identified as prognostic factors in PCNSL, however, the correlation of both clinical factors including those related to MTX metabolism and B-cell differentiation and oncogenic biomarkers with response to and outcome by therapy is yet unclear. METHODS: We investigated 32 immunocompetent patients (19 males, 13 females) with PCNSL (all diffuse large B-cell type) treated with HD-MTX based therapy with or without WBRT since 2000 in our institution. Paraffin-embedded formalin-fixed tumor tissue sections were stained immunohistochemically with antibodies against following factors: B-cell differentiation markers (CD10, Bcl-6, Mum-1, CD138); MTX metabolism-related (MRP family, LRP, DHFR); cell cycle-related (p27KIP1, MIB-1); drug resistance-related (MGMT, MLH1, MSH2, MSH6, PMS2); and oncogenes (Myc, Bcl-2). Correlation between positivity of these factors and clinical outcomes were evaluated using logrank test and cox regression analysis. RESULTS: Among these factors, complete response to HD-MTX was significantly associated with longer progression-free survival (PFS)(P = 0.0012), while Bcl-6 expression as well as histological subtype (non-germinal center B-cell, non-GCB) was closely correlated with shorter PFS. Age (>60) (P = 0.006) and MSH2 expression (P = 0.017) were found to be better predictor for overall survival (OS), but in multivariate analysis, they were no longer significant. Other factors involved in MTX metabolism, DNA repair enzymes, and oncogenes did not affect outcome. CONCLUSIONS: Non-GCB subtype and Bcl-6 expression may be associated with worse outcome in patients with PCNSL treated with HD-MTX, while MTX-metabolism related factors did not influence prognosis. Further investigation is needed to assess Bcl-6 as a potential prognostic factor in PCNSL. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Some cancer cells lengthen their telomeres by alternative lengthening of telomeres (ALT); these are referred to as ALT cancer cells and do not express telomerase. The ALT mechanism involves the elongation of telomeric DNA repeats by homologous recombination. In interphase nuclei of ALT cancer cells, giant telomeres can be specifically observed by fluorescencein situhybridization (FISH) to detect telomeric repeats, and they are co-localized with promyelocytic-leukemia nuclear-bodies (PML-NBs). However, it how large telomeres specific to ALT and how they form a structural relation with PML-NBs. We refer to giant telomeres specific to interphase ALT cancer cells giant-telomeric nuclear-bodies (GT-NBs). To quantitatively define GT-NBs, we performed telomeric FISH of both interphase nuclei and metaphase chromosomes and analyzed telomere sizes by integrated FISH signals. The distributions of telomere sizes in telomerase-positive cells were similar in interphase nuclei and chromosomes. However, the distribution of telomere sizes in ALT cancer cells differed between interphase nuclei and chromosomes. Giant telomeres that were larger than those at chromosomal ends were only observed in interphase nuclei of ALT cancer cells. Accordingly, GT-NBs could be quantitatively defined as larger than the maximum size of telomeres at chromosomal ends. Furthermore, ALT cancer cells demonstrated fewer telomeric signals in interphase nuclei than in chromosomes. These findings indicate that GT-NBs could be formed by the aggregation of two or more telomeres at chromosomal ends. Furthermore, super-resolution microscopy showed that GT-NBs contain one or two PML-NBs. GT-NBs are considered aggregates of telomeres and could contain multiple sites of homologous recombination accompanied by PML-NBs. These findings may contribute to the development of therapeutic approaches for ALT cancer. (251 words)

The study aim to demonstrate the therapeutic tissue depth of photodynamic therapy (PDT) using the photosensitizer talaporfin sodium and semiconductor laser for malignant glioma from an autopsy finding. Three patients diagnosed with glioblastoma by pre-operative imaging (1 newly diagnosed patient and 2 patients with recurrence) were treated with intra-operative additional PDT and adjuvant therapy such as post-operative radiotherapy or chemotherapy. All three patients died of brain stem dysfunction owing to cerebrospinal fluid dissemination or direct invasion of the tumor cells from 13, 18, or 20 months after PDT. Antemortem magnetic resonance images demonstrated no tumor recurrence in the site of PDT, and autopsy was performed for the pathological analysis. Macroscopic observation demonstrated no tumor recurrence in two patients, but one patient demonstrated tumor recurrence in the therapeutic depth of PDT. Microscopic analysis demonstrated histopathological changes reaching depths of 9, 11, and 18 mm (mean: 12.7 mm) from the surface of the cavity of tumor resection, suggesting the therapeutic tissue depth of PDT to be in this range. This region demonstrated glial scarring with infiltration of T lymphocytes and macrophages, with slight degeneration of small vessel walls. However, viable tumor tissues were observed beyond or around the therapeutic tissue depth of PDT in two patients. PDT for glioblastoma prevented early local recurrence, which suggests the possibility that activation of the immune mechanisms was involved. The therapeutic tissue depth was suggested to be 9–18 mm from the surface of the cavity of tumor resection; however, the viable tumor tissues were demonstrated beyond this therapeutic range.

We report four cases of high-grade astrocytoma with a BRAF V600E mutation, ATRX inactivation, and CDKN2A/B homozygous deletion. Children to young adults aged 3–46 presented with a well demarcated contrast-enhancing mass in the supratentorial area. Pathological examination revealed packed growth of short spindle to round polygonal cells including some pleomorphic cells. The tumors had less ability to infiltrate into the adjacent brain parenchyma and presented a circumscribed growth pattern. Mitosis was readily found, accompanied by focal necrosis and/or microvascular proliferation. Tumors were histologically similar in part to pleomorphic xanthoastrocytoma (PXA) or anaplastic PXA, but did not fit criteria for either neoplasm. A BRAF V600E mutation and homozygous deletion of CDKN2A/B were observed, which is similar to the genetic features of PXA or epithelioid glioblastoma, but the additional loss of ATRX nuclear immunoreactivity and absence of TERT promoter mutation were unusual findings, indicating a novel genetic profile. Despite their malignant histological features, all patients had a favorable clinical course and remained alive for 6 months to 28 years under standard medical treatment for malignant glioma. In summary, high grade astrocytomas with BRAF V600E, ATRX, and CDKN2A/B alternations had unique clinicopathological features and may be a novel subset of high grade glioma.

A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti‐EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild‐type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O 6‐methylguanine‐DNA methyltransferase (MGMT) was detected by methylation‐specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post‐treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by reactivation of the asymptomatic persistent pathogen human polyomavirus JC (JC virus). The pathology of affected brain tissues demonstrates oligodendroglia-like cells with viral inclusions in their enlarged nuclei, a diagnostic hallmark of this disease. Today, the pathological features of this disease are expanding, partly due to an unsteady balance between viral virulence and host immunity. Intranuclear viral inclusions were initially thought to be amphophilic materials comprising the entire enlarged nucleus, based on HE staining (full inclusions). Howevewr, recent immunohistochemical analyses detected the presence of intranuclear viral inclusions in dots (dot-shaped inclusions). The dot-shaped inclusions reflect clustered progeny virions at punctuated subnuclear domains called promyelocytic leukemia nuclear bodies, and are indicative of early-stage viral infection or suppressed viral proliferation. Second, the JC virus is usually reactivated in patients with impaired immunity, and therefore the inflammatory reactions are poor. However, the causes of immunosuppression are divergent, as seen with the frequent use of immunosuppressive drugs, including natalizumab. Therefore, the degree of host immunity is variable; some patients show marked anti-viral inflammatory reactions and a good prognosis, indicating that a strong resistance against viral infection remains. Recovery of the immune system may also induce paradoxical clinical worsening, known as immune reconstitution inflammatory syndrome, the mechanism of which has not been clarified. The virus-host interactions have increased in complexity, and the pathology of PML is diverging. In this review, the pathology of PML will be described, with a focus on the intranuclear target of JC virus infection and host inflammatory reactions.

Introduction Removal of midline paraventricular gliomas is difficult because of their deep localization and invasive character, requiring biopsy for pathologic diagnosis. This study aimed to assess the pathologic findings and clinical course of midline paraventricular gliomas diagnosed using a neuroendoscope. Methods This study was performed as a retrospective investigation using a neuroendoscope of 26 patients whose tumors were diagnosed as midline paraventricular gliomas. The main loci of the lesions were the thalamus (11 patients), tectum (6 patients), and other areas (9 patients). Of these 26 patients, 21 (81%) had accompanying obstructive hydrocephalus. Surgery was performed via the lateral ventricle using a flexible scope. For patients with obstructive hydrocephalus, we added endoscopic third ventriculostomy, septostomy, and/or plasty of the foramen of Monro. Pathologic diagnosis was determined according to hematoxylin-eosin staining and immunohistochemistry using anti-GFAP, anti-Ki-67, anti-H3-K27M, and anti-IDH1-R132H antibodies. Results The pathologic diagnoses were grade I (5 patients), grade II (3 patients), grade III (6 patients), and grade IV (4 patients) gliomas. Six patients were diagnosed as having high-grade glioma, which was difficult to distinguish between grade III and grade IV. Two patients were undiagnosable. H3-K27M was strongly positive in 8 of 15 patients with high-grade glioma. All patients with high-grade gliomas died or received best supportive care within 2 years after surgery. Conclusions Neuroendoscopic surgery is useful for midline paraventricular gliomas in terms of the treatment of obstructive hydrocephalus, as well as pathologic diagnosis and genetic analysis, which are required under the World Health Organization 2016 classification.

One-third of lung malignancies demonstrate a proneural/neuroendocrine phenotype or type of differentiation. However, it has not been clearly elucidated how proneural/neuroendocrine differentiation is controlled in lung cancers. We recently demonstrated that the POU3F2 gene plays a significant role in proneural/neuroendocrine differentiation of lung cancers. Because class III POU genes (POU3F1, POU3F2, POU3F3, and POU3F4) and class IV POU genes (POU4F1, POU4F2, and POU4F3) share similar properties in neural development, we analyzed the association between class III/IV POU genes and a proneural/neuroendocrine phenotype in lung cancers using seven small cell lung cancer (SCLC) cell lines and twelve non-SCLC (NSCLC) cell lines. Class III/IV POU gene expression was generally restricted to SCLC cells. However, the forced expression of class III/IV POU genes in the NSCLC cell lines induced the expression of neuroendocrine-specific markers (neural call adhesion molecule 1, synaptophysin, and chromogranin A) and proneural transcription factors (achaete-scute homolog-like 1, NeuroD1, and thyroid transcription factor 1) in various degrees. Furthermore, each class III/IV POU gene induced other class III/IV POU genes, suggesting the mutual induction of class III/IV POU genes. These findings suggest that the expression of class III/IV POU genes is important for the proneural/neuroendocrine differentiation of lung cancer cells.

In lung tumors, the association between carcinoids and high-grade neuroendocrine tumors (HGNETs) is controversial. To understand the phenotypic similarities/differences between lung carcinoids and HGNETs, we comparatively investigated the expression of three kinds of developing neural transcription factors (DNTFs: BRN2, TTF1 and ASCL1) and multiple endocrine neoplasia type 1 (MEN1) as well as RB1 and P53 using 18 carcinoids and 16 HGNETs. The DNTFs were expressed in 10 of the 18 carcinoids and in all the HGNETs, while normal neuroendocrine cells, which are considered the major cell origin of lung carcinoids and small cell carcinomas, did not express DNTFs. Both the DNTF(-) and DNTF(+) carcinoids contained typical and atypical carcinoids. All the DNTF(-) carcinoids examined were formed in the bronchial wall. All the MEN1(-) carcinoids examined were classified into the DNTF(-) carcinoids, while all the HGNETs expressed MEN1. This finding suggests that DNTF(-) MEN1(-) carcinoids are unlikely to be precursors of HGNETs. Although the status of RB1 and P53 between carcinoids and HGNETs were apparently different, the DNTF(+) carcinoids of two male patients and one female patient revealed morphologies resembling HGNET cells and relatively high Ki67 indices. Further investigation of DNTF expression in carcinoids might provide important clues to understand the association between carcinoids and HGNETs.

Lobular carcinoma in situ (LCIS) clinically constitutes a risk factor for the subsequent development of either invasive lobular carcinoma (ILC) or invasive ductal carcinoma (IDC). In order to approach the possibility of this common precursor of both ILC and IDC, we investigated combined lobular and ductal carcinomas. Thirty-two cases of lobular carcinoma were picked up out of 773 cases of operated breast carcinomas. The histopathological detailed re-examination using immunostain of E-cadherin and β-catenin revealed a rather high frequency of combined lobular carcinomas than previous reports. Clinicopathologically, combined lobular carcinomas were younger and smaller than pure lobular carcinomas, and the cytological atypia was relatively low. These results suggested that combined lobular carcinomas could be detected in the earlier stage of breast cancer. Furthermore, the lobular and ductal components of combined carcinomas coexisted in the neighborhood and were distributed contiguously. The immunohistochemical phenotypes of both components were accorded in most combined cases. A genetic analysis using methylation-specific PCR on the HUMARA gene demonstrated that the same allele was inactivated in both lobular and ductal components in all detectable cases of combined carcinoma. Therefore, it is reasonable to assume that both lobular and ductal components of combined carcinomas are clonal and derived from the LCIS as the common precursor lesion, which may contradict the conventional concept that the lobular and ductal carcinomas arise from distinct differentiation pathways.

BRN2 is a developmental neural cell-specific POU domain transcription factor and is crucial for cell lineage determination. We investigated the importance of BRN2 in the expression of the lineage-specific transcription factors (achaete-scute homolog-like 1 (ASCL1) and NeuroD1 (ND1)) and neural/neuroendocrine marker molecules (neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYP) and chromogranin A (CHGA)) in small cell lung cancer (SCLC) using cultured lung cancer cells. All examined SCLC cell lines expressed BRN2, as well as ASCL1, ND1, NCAM1, SYP and CHGA. The expression levels of ASCL1, ND1, NCAM1, SYP and CHGA considerably decreased when BRN2 was knocked down in SCLC cells, and the addition of a BRN2 transgene into non-SCLC (NSCLC) cells induced the expression of ASCL1, ND1, NCAM1, SYP and CHGA. However, the BRN2 gene was not activated by the forced expression of ASCL1 or ND1 in NSCLC cells. The knockdown of BRN2 caused significant growth retardation with decrease of S to G2 phase population and mitotic cell rates and unaltered Ki-67-labeled or apoptotic cell rates in SCLC cells, indicating increase of G1 phase population. These findings suggest that BRN2 is a higher level regulator than ASCL1 and ND1 and BRN2 might be involved in aggressiveness of SCLC.

A 53-year-old man was admitted for sub-acute progressive dementia and Gerstmann syndrome. MRI demonstrated lesions in the white matter involving the left parietal lobe, accompanied by speckled or faint linear peripheral enhancement. Brain biopsy revealed JC virus infection in oligodendrocytes and balanced infiltration of CD8+ and CD4+ T lymphocytes. We diagnosed progressive multifocal leukoencephalopathy (PML) with controlled inflammation. The finding of CD4/CD8 T cells in the infected PML brain suggested therapeutically valuable immune system involvement, which we decided to preserve by withholding corticosteroids. We treated the patient with risperidone, cytarabine and mefloquine to suppress virus replication, but not with the corticosteroid that is conventionally used in inflammatory PML cases. The patient was discharged three months after admission, and one year later, his score on the Mini-Mental State Examination had recovered to 26/30, from 5/30 on admission.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by reactivation of the JC virus associated with impaired host immunity. However, PML may occur even without an evident cause of immunosuppression. In such cases, JC virus DNA in cerebrospinal fluid (CSF) may not be detectable with polymerase chain reactions (PCR), and a brain biopsy may be performed. Pathology may exhibit marked inflammatory reactions around the JC virus-infected cells, although typical intranuclear viral inclusions would rarely be seen. Atypical inflammation in patients with PML likely an indication of the host's immune-response against the virus. this is usually observed in those with relatively-mild immunosuppression with favorable prognosis. Here, we describe cases of PML that exhibited inflammatory reactions; a recent topic, PML with immune reconstruction inflammatory syndrome (IRIS), will also be discussed.

Mechanisms of endocrine secretory granule (SG) formation in thyroid C cells and medullary thyroid cancer (MTC) cells have not been fully elucidated. Here we directly demonstrated that PROX1, a developmental homeobox gene, is transcriptionally involved in SG formation in MTC, which is derived from C cells. Analyses using gene expression databases on web sites revealed that, among thyroid cancer cells, MTC cells specifically and highly express PROX1 as well as several SG-forming molecule genes. Immunohistochemical analyses showed that in vivo MTC and C cells expressed PROX1, although follicular thyroid cancer and papillary thyroid cancer cells, normal follicular cells did not. Knockdown of PROX1 in an MTC cells reduced SGs detected by electron microscopy, and decreased expression of SG-related genes (chromogranin A, chromogranin B, secretogranin II, secretogranin III, synaptophysin, and carboxypeptidase E). Conversely, the introduction of a PROX1 transgene into a papillary thyroid cancer and anaplastic thyroid cancer cells induced the expression of SG-related genes. Reporter assays using the promoter sequence of chromogranin A showed that PROX1 activates the chromogranin A gene in addition to the known regulatory mechanisms, which are mediated via the cAMP response element binding protein and the repressor element 1-silencing transcription factor. Furthermore, chromatin immunoprecipitation-PCR assays demonstrated that PROX1 binds to the transcriptional regulatory element of the chromogranin A gene. In conclusion, PROX1 is an important regulator of endocrine SG formation in MTC cells.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder caused by JC virus infection. JC virus was recently found to target promyelocytic leukemia nuclear bodies (PML-NBs), punctuate domains in the nuclei. Thus, the virus progenies cluster in dots as intranuclear inclusions (ie, as dot-shaped inclusions). In the present study, both the viral major and minor capsid proteins were expressed from polycistronic expression vectors with a powerful promoter, and formation into virus-like particles (VLPs) was examined by electron microscopy. When the upstream regulatory sequence including the agnogene (nt 275 to 490) was present, capsid protein expression was suppressed, but numerous VLPs were efficiently formed with restricted accumulation to PML-NBs. VLPs were uniform, and the cells were severely degraded. In contrast, when the 5′ terminus of the agnogene (nt 275 to 409; 135 bp) was deleted, capsid protein expression was markedly enhanced, but VLPs were more randomly produced in the nucleus outside of PML-NBs. VLPs were pleomorphic, and cell degradation was minimal. JC virus association with PML-NBs was confirmed in human brain tissues by structured illumination microscopy. PML-NBs were shaped in spherical shells, with viral capsid proteins circumscribing the surface. These findings indicate that PML-NBs are intranuclear locations for pathogenic JC virus proliferation. Either the agnogene or its product likely supports efficient progeny production at PML-NBs, leading to subsequent degeneration of host glial cells.

Progressive multifocal leukoencephalopathy (PML) is caused by reactivation of latently infected JCV when hosts' immune system is impaired by HIV infection, hematologic diseases, collagen diseases, immunemodulatory therapy and so on. PML was rare but HIV infection and Natalizumab have made it much more common while the prognosis is much better than other PML. PML patients present with various signs and symptoms including hemiparesis, dementia, aphasia, visual disturbance, cranial nerve paresis, cerebellar signs and bladder bowel disturbance. Brain MRI reveals characteristic demyelinating lesions in the CNS white matter and CSF mild increase of protein with or without mild mononuclear pleocytosis. Detection of JCV genome from CSF is crucial for the clinical diagnosis of PML. PML was once thought to be fatal but some HIV infected PML patients showed halting progression or even recovery after introduction of HAART. In addition, anti-malarial drug mefloquine was found to be effective. Recovery of immunity may provoke some inflammatory responses known as immune reconstruction inflammatory syndrome (IRIS) which requires high dose corticosteroid. In Japan, we are providing free test of CSF-JCV genome and organized a unique system for surveillance and clinical research of PML. Using this system we hope to improve diagnosis and therapy of PML in Japan.

Gastrointestinal stromal tumors (GISTs) show a high incidence of gain-of-function mutations of the c-kit gene, which encodes a receptor tyrosine kinase KIT. This mutation is seen independently of metastasis and/or recurrence of tumors; thus, the factors involved in tumor proliferation rate and malignancy are still not known. Some mesenchymal and epithelial tumors have been reported to co-express KIT and its ligand, stem cell factor (SCF), for autonomous proliferation by the autocrine mechanism. The purpose of this study is to clarify whether GIST cells produce SCF, despite mutated KIT with constitutive activation. Immunohistochemically, we examined the co-expression of KIT and SCF in 36 GIST cases. All cases were found to be KIT-positive, and of these, 21 cases, including four recurrent or metastatic GISTs, showed co-expression of SCF. MIB-1 labeling index was significantly higher, and the average tumor size was larger in SCF-positive cases. By confocal microscopy, KIT was expressed on the cellular membrane, around which SCF was distributed less densely. Western blot analysis revealed that the membrane-bound SCF of 31 kDa was found to be approximately 10 times more abundant than the soluble SCF of 18.5 kDa, suggesting continuous KIT activation. These results indicate that proliferation of GIST cells can be caused not only by the gain-of-function mutation of c-kit, but also by the autocrine mechanism of the SCF/KIT system. Thus, SCF expression would be a useful marker for tumor proliferation.

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder due to human polyomavirus JC infection in which there are viral inclusions in enlarged nuclei of infected oligodendrocytes. We report that the pathogenesis of this disease is associated with distinct subnuclear structures known as promyelocytic leukemia nuclear bodies (PML-NBs). Postmortem brain tissues from 5 patients with the disease were examined. Affected cells with enlarged nuclei contained distinct dot-like subnuclear PML-NBs that were immunopositive for PML protein and nuclear body protein Sp100. Major and minor viral capsid proteins and proliferating cell nuclear antigen, an essential component for DNA replication, colocalized with PML-NBs. By in situ hybridization, viral genomic DNA showed dot-like nuclear accumulation, and by electron microscopy, virus-like structures clustered in subnuclear domains, indicating that PML-NBs are the site of viral DNA replication and capsid assembly. Molecules involved in the ubiquitin proteosome pathway (i.e. ubiquitin and small ubiquitin-like modifier 1) did not accumulate in the nuclei with viral inclusions, indicating that cell degeneration may not be dependent on this pathway. When viral progeny production was advanced, PML-NBs were disrupted. These data suggest that: 1) PML-NBs allow for efficient viral propagation by providing scaffolds, 2) disruption of PML-NBs is independent of the ubiquitin-proteasome pathway, and 3) this disruption probably heralds oligodendrocyte degeneration and the resulting demyelination.

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to CNS. Despite low tendency of systemic dissemination, its prognosis is poor with median overall survival of 4 years when treated with standard of care high-dose methotrexate-based chemoradiotherapy. Despite majority of PCNSL is diffuse large B-cell lymphoma (DLBCL), its response to therapy and biological behavior differ from the systemic disease, necessitating detailed investigation of its pathogenesis and genetic alterations. Here we performed whole-exome sequencing (WES) on 41 PCNSL and paired normal specimens as well as RNA sequencing. Seven cases were classified as germinal center B-cell-like (GCB) subtype (17%), while 34 cases were non-GCB. Progression-free survival was significantly longer in cases with GCB subtype as in systemic DLBCL (p = 0.027). WES yielded sequencing data with a mean coverage of 158Χ with 98% of the targeted bases covered by ≥20 independent reads. We identified a total of 17,385 somatic mutations, of which 62% were nonsynonymous single nucleotide variations. The predominant nucleotide substitution was a C > T transition (57.8%), especially in the context of the sequence GCG. WES revealed a substantial extent of aberrant somatic hypermutation (aSHM) with a high frequency of nonsynonymous somatic mutations especially in PIM1 (90.2%) and BTG2 (65.9%). Genes mutated other than aSHM include MYD88 (85.4%), most of which being L265P activating mutation. These mutations were not associated with GCB/non-GCB subtypes. Notably, mutations in a number of genes related to the NF-κB signaling pathway were also prominent. Copy number aberration analysis identified gains in chromosomes 1q, 7, 12, and 18q, and a loss of 6q. These results suggest that MYD88 and PIM1 mutations may play an important role in PCNSL development regardless of GCB subtypes, and NF-κB pathway could be the therapeutic targets of this intractable disease.

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to the central nervous system (CNS), and majority of PCNSL is pathologically classified as diffuse large B-cell lymphoma (DLBCL). We have now performed whole-exome sequencing for 41 tumor tissues of DLBCL-type PCNSL and paired normal specimens and also RNA-sequencing for 30 tumors, revealing a very high frequency of nonsynonymous somatic mutations in PIM1 (100 %), BTG2 (92.7 %), and MYD88 (85.4 %). Many genes in the NF-κB pathway are concurrently mutated within the same tumors. Further, focal deletion or somatic mutations in the HLA genes are associated with poor prognosis. Copy number amplification and overexpression of genes at chromosome 7q35 were both found to predict short progression-free survival as well. Oncogenic mutations in GRB2 were also detected, the effects of which in cultured cells were attenuated by inhibitors of the downstream kinases MAP2K1 and MAP2K2. Individuals with tumors positive for MYD88 mutations also harbored the same mutations at a low frequency in peripheral blood mononuclear cells, suggesting that MYD88 mutation-positive precancerous cells originate outside of the CNS and develop into lymphoma after additional genetic hits that confer adaptation to the CNS environment.

The proportion of white matter in the brain has increased during evolution, and white matter comprises approximately half of the human brain. Its macroscopic as well as microscopic structures change during development, aging, and disease progression as well as following physical or mental training. Knowledge about the structural plasticity of the white matter may alter our cortex-oriented view of brain functions and expand our strategies for diagnosis and treatment, including rehabilitation, since the gray and white matter are complementary. Although the presence of white matter lesions is easy to detect with magnetic resonance imaging of the brain, their qualitative differentiation requires vast knowledge about the underlying processes. Examples from multiple ischemic lesions caused by different disease processes affecting the cerebral arteries are presented for comparison. It is worth considering "what matters more in the white matter" by taking into account the basic structures of the brain as well as their plasticity. Such "thinking inside of the brain" may further expand our understanding of the brain to improve our clinical interpretations and treatments.

Human polyomavirus JC (JCV) is a causative agent for progressive multifocal leukoencephalopathy, a fatal demyelinating disorder. The viruses form intranuclear viral inclusions in infected oligodendrocytes. The outer capsid of JCV is thought to be composed of 360 molecules of major capsid protein VP1, and minor capsid proteins VP2 and VP3 in an appropriate ratio. However, the regulatory mechanisms of gene expression for the capsid proteins, their nuclear transport, and formation of viral inclusions are not well understood. We have recently clarified the following regarding the mechanism underlying JCV virion assembly; (i) major and minor capsid proteins are synthesized from messenger RNAs, the expression ratio of which is determined by alternative splicing, (ii) messenger RNAs for the major and minor capsid proteins are polycistronic, and their translation occurs downstream of the regulatory protein, agnoprotein, (iii) major and minor capsid proteins are translocated to the nucleus in a cooperative manner and accumulate at the dot-shaped intranuclear structures called promyelocytic leukemia nuclear bodies (PML-NBs), (iv) efficient viral replication can occur at the PML-NBs, where capsid assembly is likely to be associated with viral DNA replication. PML-NBs are the sites for expression of important nuclear functions for the host cells. The finding that the target of JCV infection is the PML-NB may contribute greatly to our understanding of the mechanism underlying cellular degeneration, which occurs after the formation of intranuclear viral inclusions.

The human polyomavirus JC (JCV) replicates in the nuclei of infected cells. Here we report that JCV virions are efficiently assembled at nuclear domain 10 (ND10), which is also known as promyelocytic leukemia (PML) nuclear bodies. The major capsid protein VP1, the minor capsid proteins VP2 and VP3, and a regulatory protein called agnoprotein were coexpressed from a polycistronic expression vector in COS-7 cells. We found that VP1 accumulated to distinct subnuclear domains in the presence of VP2/VP3 and agnoprotein, while VP1 expressed alone was distributed both in the cytoplasm and in the nucleus. Mutation analysis revealed that discrete intranuclear accumulation of VP1 requires the presence of either VP2 or VP3. However, VP2 or VP3 expressed in the absence of VP1 showed diffuse, not discrete, nuclear localization. The C-terminal sequence of VP2/VP3 contains two basic regions, GPNKKKRRK (cluster 1) and KRRSRSSRS (cluster 2). The deletion of cluster 2 abolished the accumulation of VP1 to distinct subnuclear domains. Deletion of the C-terminal 34 residues of VP2/VP3, including both cluster 1 and cluster 2, caused VP1 to localize both in the cytoplasm and in the nucleus. Using immunoelectron microscopy of cells that coexpressed VP1, VP2/VP3, and agnoprotein, we detected the assembly of virus-like particles in discrete locations along the inner nuclear periphery. Both in oligodendrocytes of the human brain and in transfected cells, discrete nuclear domains for VP1 accumulation were identified as ND10, which contains the PML protein. These results indicate that major and minor capsid proteins cooperatively accumulate in ND10, where they are efficiently assembled into virions.

Objective:To clarify the clinical, neuropathologic, and virologic characteristics of progressive multifocal leukoencephalopathy (PML) and its immune reconstitution inflammatory syndrome (IRIS) in a patient with fingolimod-treated MS.Methods:A case study.Results:A 34-year-old patient with MS using fingolimod for 4 years had a gradual progression of right hemiparesis and aphasia with a new subcortical white matter lesion in the precentral gyrus by initial MRI. Blood tests were normal, except for lymphopenia (160 cells/μL). One month after the cessation of fingolimod, brain MRI depicted a diffusely exacerbated hyperintensity on fluid-attenuated inversion recovery and diffusion-weighed imaging in the white matter with punctate gadolinium enhancement, suggesting PML-IRIS. A very low level of JC virus (JCV)-DNA (15 copies/mL) was detected in the CSF as judged by quantitative PCR. Brain tissues were biopsied from the left frontal lesion, which showed some small demyelinated foci with predominant loss of myelin-associated glycoprotein with infiltrations of lymphocytes and macrophages, but clear viral inclusion was not observed with hematoxylin-eosin staining. JCV-DNA was uniquely detectable in an active inflammatory demyelinating lesion by in situ hybridization, possibly suggesting an early phase of PML. DNA extracted from the brain sample was positive for JCV-DNA (151 copies/cell). It took 3 months to normalize the blood lymphocyte count. The patient was treated with 1 g of IV methylprednisolone for 3 days and a weekly oral dose (375 mg) of mefloquine, and her symptoms gradually improved.Conclusion:Low CSF JCV-DNA and unfound viral inclusions initially made her diagnosis difficult. The clinical course of fingolimod-associated PML may be associated with mild immune reconstitution.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by reactivation of the asymptomatic persistent pathogen human polyomavirus JC (JC virus). The pathology of affected brain tissues demonstrates oligodendroglia-like cells with viral inclusions in their enlarged nuclei, a diagnostic hallmark of this disease. Today, the pathological features of this disease are expanding, partly due to an unsteady balance between viral virulence and host immunity. Intranuclear viral inclusions were initially thought to be amphophilic materials comprising the entire enlarged nucleus, based on HE staining (full inclusions). Howevewr, recent immunohistochemical analyses detected the presence of intranuclear viral inclusions in dots (dot-shaped inclusions). The dot-shaped inclusions reflect clustered progeny virions at punctuated subnuclear domains called promyelocytic leukemia nuclear bodies, and are indicative of early-stage viral infection or suppressed viral proliferation. Second, the JC virus is usually reactivated in patients with impaired immunity, and therefore the inflammatory reactions are poor. However, the causes of immunosuppression are divergent, as seen with the frequent use of immunosuppressive drugs, including natalizumab. Therefore, the degree of host immunity is variable; some patients show marked anti-viral inflammatory reactions and a good prognosis, indicating that a strong resistance against viral infection remains. Recovery of the immune system may also induce paradoxical clinical worsening, known as immune reconstitution inflammatory syndrome, the mechanism of which has not been clarified. The virus-host interactions have increased in complexity, and the pathology of PML is diverging. In this review, the pathology of PML will be described, with a focus on the intranuclear target of JC virus infection and host inflammatory reactions.

In progressive multifocal leukoencephalopathy, JC virus–infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed “promyelocytic leukemia nuclear bodies” (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 μm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.

One-third of lung malignancies demonstrate a proneural/neuroendocrine phenotype or type of differentiation. However, it has not been clearly elucidated how proneural/neuroendocrine differentiation is controlled in lung cancers. We recently demonstrated that the POU3F2 gene plays a significant role in proneural/neuroendocrine differentiation of lung cancers. Because class III POU genes (POU3F1, POU3F2, POU3F3, and POU3F4) and class IV POU genes (POU4F1, POU4F2, and POU4F3) share similar properties in neural development, we analyzed the association between class III/IV POU genes and a proneural/neuroendocrine phenotype in lung cancers using seven small cell lung cancer (SCLC) cell lines and twelve non-SCLC (NSCLC) cell lines. Class III/IV POU gene expression was generally restricted to SCLC cells. However, the forced expression of class III/IV POU genes in the NSCLC cell lines induced the expression of neuroendocrine-specific markers (neural call adhesion molecule 1, synaptophysin, and chromogranin A) and proneural transcription factors (achaete-scute homolog-like 1, NeuroD1, and thyroid transcription factor 1) in various degrees. Furthermore, each class III/IV POU gene induced other class III/IV POU genes, suggesting the mutual induction of class III/IV POU genes. These findings suggest that the expression of class III/IV POU genes is important for the proneural/neuroendocrine differentiation of lung cancer cells.

Human polyomavirus JC (JCV) can encode the three capsid proteins VP1, VP2, and VP3, downstream of the agnoprotein in the late region. JCV virions are identified in the nucleus of infected cells. In this study, we have elucidated unique features of JCV capsid formation by using a eukaryotic expression system. Structures of JCV polycistronic late RNAs (M1 to M4 and possibly M5 and M6) generated by alternative splicing were determined. VP1 would be synthesized from M2 RNA, and VP2 and VP3 would be synthesized from M1 RNA. The presence of the open reading frame of the agnoprotein or the leader sequence (nucleotides 275 to 409) can decrease the expression level of VP1. VP1 was efficiently transported to the nucleus in the presence of VP2 and VP3 but distributed both in the cytoplasm and in the nucleus in their absence. Mutation analysis indicated that inefficiency in nuclear transport of VP1 is due to the unique structure in the N-terminal sequence, KRKGERK. Within the nucleus, VP1 was localized discretely and identified as speckles in the presence of VP2 and VP3 but distributed diffusely in their absence. These results suggest that VP1 was efficiently transported to the nucleus and localized in the discrete subnuclear regions, possibly with VP2 and VP3. By electron microscopy, recombinant virus particles were identified in the nucleus, and their intranuclear distribution was consistent with distribution of speckles. This system provides a useful model with which to understand JCV capsid formation and the structures and functions of the JCV capsid proteins.

In lung tumors, the association between carcinoids and high-grade neuroendocrine tumors (HGNETs) is controversial. To understand the phenotypic similarities/differences between lung carcinoids and HGNETs, we comparatively investigated the expression of three kinds of developing neural transcription factors (DNTFs: BRN2, TTF1 and ASCL1) and multiple endocrine neoplasia type 1 (MEN1) as well as RB1 and P53 using 18 carcinoids and 16 HGNETs. The DNTFs were expressed in 10 of the 18 carcinoids and in all the HGNETs, while normal neuroendocrine cells, which are considered the major cell origin of lung carcinoids and small cell carcinomas, did not express DNTFs. Both the DNTF(-) and DNTF(+) carcinoids contained typical and atypical carcinoids. All the DNTF(-) carcinoids examined were formed in the bronchial wall. All the MEN1(-) carcinoids examined were classified into the DNTF(-) carcinoids, while all the HGNETs expressed MEN1. This finding suggests that DNTF(-) MEN1(-) carcinoids are unlikely to be precursors of HGNETs. Although the status of RB1 and P53 between carcinoids and HGNETs were apparently different, the DNTF(+) carcinoids of two male patients and one female patient revealed morphologies resembling HGNET cells and relatively high Ki67 indices. Further investigation of DNTF expression in carcinoids might provide important clues to understand the association between carcinoids and HGNETs.