Hepatoid tenosynovial giant cell tumor - a rare morphologic variant case report.

We report a morphologically rare type of tenosynovial giant cell tumor (TSGCT), localized type, occurring in a 49-year-old man. Imaging examination revealed multiple nodular lesions around the right knee joint. The largest one extended to both intra- and extra-osseous region of the right distal femur. Histologically, the tumor consisted of epithelioid mononuclear cells and they looked like to have abundant eosinophilic cytoplasm reminiscent of hepatic tissues. In some areas, however, typical histologic features of TSGCT were observed. Electron microscopy revealed that the eosinophilic cytoplasm-like substance was intercellular fibrinous material surrounding the mononuclear tumor cells. Immunohistochemically, mononuclear tumor cells and multinucleate giant cells were positive for CD68 (Kp1) and some of the mononuclear tumor cells were also positive for desmin. Finally, we made the diagnosis of hepatoid TSGCT.

Diverse fusion patterns and heterogeneous clinicopathologic features of renal cell carcinoma with t(6;11) translocation.

Renal cell carcinoma (RCC) with t(6;11) translocation, involving the transcription factor EB (TFEB) and Alpha, also known as MALATI, (TFEB RCC), is extremely rare, with only 20 cases reported to date. It may be frequently misdiagnosed because of a lack of established characteristics. TFEB RCCs are predominantly seen in younger patients and are generally indolent, with only 2 cases of metastasis. Genetic analysis has been limited, showing break points upstream of TFEB exon 3, yielding only a single transcript. We examined 3 new adult Japanese TFEB RCC cases by means of precise clinicopathologic, immunohistochemical, cytogenetic, and molecular analyses and compared them with 200 ordinary RCCs. A 57-year-old man was the oldest patient with TFEB RCC at the time of this study. Although the tumor had histology typical of translocation RCC, its fusion points were different between the genomic and transcript coordinates. A 37-year-old man had an aggressive course resulting in death. The tumor had 2 variants of messenger ribonucleic acid. A 47-year-old man showed borderline histologic and immunohistochemical features between TFEB RCC and chromophobe-type RCC. The tumor had a fusion point in TFEB exon 4, downstream of the wild-type ATG in exon 3. Nuclear expression of the TFEB protein was detected, and a Western blotting analysis identified a protein similar in size to the wild-type TFEB protein. Immunohistochemistry is useful for the diagnosis of these tumors, and TFEB RCCs have heterogeneous clinicopathologic features and more diverse fusion patterns than previously thought, requiring attention to polymerase chain reaction experiments for diagnosis. Our study will contribute to the correct diagnosis of TFEB RCC.

Applications of nucleic acid chaperone activity of CspA and its homologues.

In Escherichia coli, the cold shock response is exerted upon temperature change from 37 to 15 degrees C and is characterized by induction of several cold shock proteins including its major cold shock protein, CspA. E. coli CspA family consists of nine members, CspA to CspI. CspA and some of its homologues play a critical role in cold acclimation of cells as RNA chaperones by destabilizing secondary structures in RNAs. Here, we showed that the nucleic acid melting activity of Csp proteins can be used to facilitate reactions, such as RT-PCR or RNA cleavage reactions by endoribonucleases, which are hindered by presence of secondary structures in the DNA/RNA substrate used. The low substrate specificity of Csps together with their compatibility with various enzymes and their stability and activity over a broad temperature range makes them ideal candidates to be used for a variety of processes.

Adult Xp11 translocation renal cell carcinoma diagnosed by cytogenetics and immunohistochemistry.

PURPOSE: To determine the incidence of Xp11 translocation renal cell carcinoma (RCC) in adult patients using cytogenetics and immunohistochemstry. EXPERIMENTAL DESIGN: Cytogenetic studies were prospectively done using tumor samples from 443 consecutive adult Japanese patients (ages 15-89 years) who underwent nephrectomy for RCC. TFE3 immunohistochemistry was done for cases in which cytogenetic results were not obtained. Clinicopathologic characteristics of Xp11 translocation RCC were examined. RESULTS: Mitotic cells suitable for cytogenetic analysis were obtained in 244 tumor samples (55%); among these, we identified 4 cases (1.6%) of Xp11 translocation RCC. TFE3 immunohistochemistry identified 3 positive cases (1.5%) among the remaining 199 cases. The median age of the 7 patients was 41 years (range, 15-59 years), and 15% of RCC patients (4 of 26) who were younger than ages 45 years had this type of RCC. Of the four Xp11 translocation RCC patients whose karyotypes were determined, two had an ASPL-TFE3 gene fusion. Of these 2, 1 had pulmonary metastasis at presentation, and the other developed liver metastasis 12 months after nephrectomy and died of the disease. The remaining two patients had PRCC-TFE3 and PSF-TFE3 gene fusions, respectively. Both had nodal involvement but remained disease free for 3 and 5 years, respectively, after surgical resection of lymph node metastases. Of the 3 immunohistochemically diagnosed patients, 1 had nodal metastases at presentation and died 9 months after surgery. CONCLUSIONS: This is the first report to determine the incidence of Xp11 translocation RCC in adult patients. We found that this disease is relatively common in young adults.

SNP typing of aldehyde dehydrogenase2 gene with Cycleave ICAN.

A simple and non-expensive platform is critical to realize on-site SNP typing. In this study we typed an SNP existing at the 487th residue of human aldehyde dehydrogenase2 [wild: Glu (GAA); mutant: Lys (AAA)] using our unique isothermal DNA amplification method, ICAN and cycling probes. Both genotypes were identified by the naked eye using a non-expensive UV transilluminator as well as with real-time PCR apparatus or a fluorescence detector. Since ICAN does not need thermal cycling, a cost- and space-limiting factor when fabricating apparatus, the combination of ICAN and cycling probes will be able to realize affordable on-site SNP typing in the near future.

Investigation of the molecular mechanism of ICAN, a novel gene amplification method.

Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, thermostable RNaseH and a DNA polymerase with strand-displacing activity (H. Mukai et al. J. Biochemistry, in the preceding paper in this issue). Here we elucidated the mechanism of ICAN by analysing the nicking site of RNaseH, behaviour of chimeric primers and extension products. We found that the ICAN reaction was composed of two unique mechanisms, multi-priming and template-switching, that were responsible for the highly efficient amplifying capability of ICAN. The simultaneous occurrence of two types of reactions, one based on multi-priming and the other based on template-switching, is likely to drive the DNA amplification in ICAN.

Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe.

Unscheduled overexpression of human WAPL promotes chromosomal instability.

Previously, we have isolated and characterized a novel human gene termed human WAPL that has the characteristics of an oncogene in uterine cervical cancer. WAPL is inducible by human papillomavirus (HPV) E6 and E7 oncoproteins. On the other hand, recent studies have revealed that WAPL regulates sister chromatid resolution by controlling the association of cohesin and chromatin. However, the effects of WAPL overexpression on cervical carcinogenesis are still unclear. Here, we show that WAPL overexpression induces generation of multinucleated cells. In addition, WAPL-overexpressing cells demonstrated increases in chromatid breaks in comparison with control cells. These results were obtained even in HPV-negative cell lines. High frequent premature sister separation by disregulation of cohesin may lead to these results. Thus, our study suggests that unscheduled overexpression of WAPL disturbs mitosis and cytokinesis, and contributes to tumor progression by induction of chromosomal instability (CIN).

Fusion between CIC and DUX4 up-regulates PEA3 family genes in Ewing-like sarcomas with t(4;19)(q35;q13) translocation.

Ewing's family tumors (EFTs) are highly malignant tumors arising from bone and soft tissues that exhibit EWS-FLI1 or variant EWS-ETS gene fusions in more than 85% of the cases. Here we show that CIC, a human homolog of Drosophila capicua which encodes a high mobility group box transcription factor, is fused to a double homeodomain gene DUX4 as a result of a recurrent chromosomal translocation t(4;19)(q35;q13). This translocation was seen in two cases of soft tissue sarcoma diagnosed as Ewing-like sarcoma. CIC-DUX4 exhibits a transforming potential for NIH 3T3 fibroblasts, and as a consequence of fusion with a C-terminal fragment of DUX4, CIC acquires an enhanced transcriptional activity, suggesting that expression of its downstream targets might be deregulated. Gene expression analysis identified the ETS family genes, ERM/ETV5 and ETV1, as potential targets for the gene product of CIC-DUX4. Indeed, CIC-DUX4 directly binds the ERM promoter by recognizing a novel target sequence and significantly up-regulates its expression. This study clarifies the function of CIC and its role in tumorigenesis, as well as the importance of the PEA3 subclass of ETS family proteins in the development of EFTs arising through mechanisms different from those involving EWS-ETS chimeras. Moreover, the study identifies the role of DUX4 that is closely linked to facioscapulohumeral muscular dystrophy in transcriptional regulation.

Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis.

A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10(4) to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.

EWSR1 is fused to POU5F1 in a bone tumor with translocation t(6;22)(p21;q12).

POU5F1(OCT3/4) is a sequence-specific transcription factor that is essential for keeping germ cells and embryonic stem cells in an immature and pluripotent status. In this article, we report that POU5F1 was fused to EWSR1 in a case of undifferentiated sarcoma derived from pelvic bone with chromosomal translocation t(6;22)(p21;q12). The EWSR1-POU5F1 chimera consists of exons 1-6 of EWSR1 and exons 2-5 and a part of exon 1 of POU5F1. The predicted amino acid sequence indicates that the chimera is composed of the N-terminal QSY domain of EWS that functions as a transcriptional activation domain and the C-terminal POU DNA-binding domains derived from POU5F1. The t(6;22) tumor does not belong to any known categories of bone and soft-tissue tumors (BSTs). It is suggested that EWS-POU5F1 may act as an oncogenic transcription factor and that its expression may contribute to undifferentiated and immature phenotypes of BST.

[Evaluation of the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA by the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN)].

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA, combined with luminescence detection by a probe hybridization. In the performance tests, this system was able to detect 10 to 100 copies of C. trachomatis/N. gonorrhoeae DNA for only 3.5 hours, and was highly specific to C. trachomatis/N. gonorrhoeae without any cross-reaction to C. pneumoniae, N. lactamica, N. sicca or N. meningitidis. When we tested 60 clinical samples of urine and cervical swabs, the interpretive results were completely consistent with those obtained by Roche PCR system. Of 13 positive samples by the ICAN and PCR systems for C. trachomatis, four were negative by EIA method(IDEIA Chlamydia). These results indicate that the ICAN system is an efficient and sensitive system to simultaneously detect C. trachomatis/N. gonorrhoeae DNA.

Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions.

In this study, we describe the cytological and cytogenetic features of six Epstein-Barr virus (EBV)-infected natural killer (NK) cell clones. Three cell clones, SNK-1, -3 and -6, were derived from patients with nasal T/NK-cell lymphomas; two cell clones, SNK-5 and -10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK-11, was from a patient with hydroa vacciniforme (HV)-like eruptions. An analysis of the number of EBV-terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK-cell lymphomas but also those isolated from CAEBV and HV-like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T-cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV-positive NK cells, which proliferated in the diseased individuals. The results showed that EBV-positive NK cells in malignant and non-malignant lymphoproliferative diseases shared common cytological and cytogenetic features.

Seasonal change of persistent organic pollutant concentrations in air at Niigata area, Japan.

The concentrations of persistent organic pollutants (POPs), such as HCB, alpha-, beta-, gamma- and delta-HCH, trans- and cis-chlordane (t-CHL, c-CHL), DDE, DDD and DDT, in ambient air have been measured at five sampling points in Niigata area, Japan (Niigata, Maki, Tsubame, Jouzo and Yahiko) during the period from September 1999 to November 2001. HCB, alpha-HCH, t-CHL and c-CHL showed higher concentrations than the other chemicals in all locations. All the POPs except t-CHL and c-CHL collected at urban sites of the Niigata Plain was almost the same in their concentration levels. Higher concentrations of t-CHL and c-CHL in residential areas should be attributed to the past usage of the chemical as a termiticide. At Yahiko (remote site), most of the POPs showed lower concentrations than those measured at the other sampling sites, although alpha-HCH and gamma-HCH were comparable with the concentrations found at the other sampling sites. All POPs except alpha-HCH and gamma-HCH tend to decrease 41-80% in their concentrations from 2000 to 2001. The lower POPs concentrations in winter and the higher POPs concentrations in summer at every sampling point can be partly explained by temperature differences. Applying the equation of the logarithm of the POP partial pressure in air versus reciprocal temperature (lnPa=m/T+b) to our data, linear relations were observed. HCB gave a poor linearity and the smallest slope, while beta-HCH, t-CHL and c-CHL gave good linearities and large slopes in the equation. The results suggest that HCB level is influenced by not only the emission from terrestrial sources but the global-scale background pollution. A peculiar observation is that beta-HCH concentration measured in our study showed large temperature dependence, indicating there could be a source of contamination in the surrounding areas.

Evaluation of activated carbon fiber filter for sampling of organochlorine pesticides in environmental water samples.

A simple method for quantitative analyses of organic chlorine pesticides (OCPs) in environmental water samples such as rainwater, river water and seawater using activated carbon fiber filters (ACFF) is described. ACFF was used as adsorbent to collect the chemicals in water samples. The collection of OCPs was completed almost for one day by stirring the mixture of the sample and the ACFF chips at room temperature. The adsorbed OCPs on the ACFF could be extracted easily with toluene-ethanol (4:1) mixed solvent. The purified extract by a florisil column chromatograph was followed by the analysis using high-resolution gas chromatograph/high-resolution mass spectrometer. Recoveries of OCPs spiked to actual samples such as rainwater, river water and seawater samples were approximately more than 80%, and the coefficients of variations were within 10%. This method was applied to the actual samples and was confirmed to be applicable for monitoring sub-ng/l level OCPs in environmental water samples.

Pulmonary metastases from a low-grade endometrial stromal sarcoma confirmed by chromosome aberration and fluorescence in-situ hybridization approaches: a case of recurrence 13 years after hysterectomy.

Pulmonary metastasis from low-grade endometrial stromal sarcomas (ESSs) occasionally are found after long, disease-free periods, mostly as incidental histological or radiological discoveries. We describe a case of low-grade ESS presenting as nodular pulmonary metastases finally diagnosed by estrogen-receptor staining, cytogenetic and fluorescence in situ hybridization (FISH) analyses, and perusal of the histology of hysterectomy material. An abnormal nodule in the lung field was discovered by means of chest X-ray of a 47-year-old woman. She had been disease free for 13 years after hysterectomy for an alleged leiomyoma. A computed tomographic scan revealed nodules, with fluctuation in size over the 2-year period, in both lungs. Finally the lesion in the left lung was resected, and pulmonary endometriosis was suspected because of the lack of stromal cell nuclear atypia and positive immunohistochemical reactions for estrogen and progesterone receptors. However, a characteristic karyotype was identified cytogenetically: 46, XX, t(7;17)(p15;q11), the translocation of which, specific to ESS, was confirmed by FISH analysis. A final diagnosis of pulmonary metastases from an ESS could be made by reviewing the histology of the previous uterine tumor. In this case, metastatic lesions from an ESS showed a decrease as well as an increase in size, despite the malignant potential. Immunostaining for estrogen and progesterone receptors and cytogenetic and FISH analyses, together with clinical information on the past gynecological history, are valuable diagnostic keys.

[Development of the detection system for Mycobacterium tuberculosis DNA by using the isothermal DNA amplification method ICAN].

The isothermal and chimeric primer-initiated amplification of nucleic acids(ICAN) is a new isothermal DNA amplification method composed of exo- Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the detection system, combined the ICAN with luminescence detection by a probe hybridization, for Mycobacterium tuberculosis DNA targeting the IS6110 insertion element. We examined performance tests of the system. This system was able to detect one copy of Mycobacterium tuberculosis DNA for only 3.5 hours, and performance of the system was equivalent or better to the Roche PCR system. We also examined a detection system by using magnetic beads, which system could shorten detection time for 2.5 hours. It was shown that the ICAN system was an efficient and sensitive detection system for Mycobacterium tuberculosis DNA from mass samples.

Secretion of interleukin-6 and vascular endothelial growth factor by spindle cell sarcoma complicating Castleman's disease (so-called 'vascular neoplasia').

So-called 'vascular neoplasia' (VN) is a rare tumour of unknown origin that complicates hyaline vascular type Castleman's disease (CD). This paper reports a case of VN complicating CD of hyaline vascular type, in which neoplastic cells were shown to secrete interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). In this case, VN first occurred in the retroperitoneum of a 60-year-old male. The lesion showed typical morphology, with three distinct areas: (1) a lymph node-like area with regressively transformed lymph follicles showing hyaline vascular changes and with a hypervascular interfollicular region filled with slit-like vascular channels; (2) an area composed of spindle cell sarcoma; and (3) an area showing angiolipomatous hamartoma. A proportion of the cells in the spindle cell area showed severe pleomorphism. Subcutaneous recurrence after 8 months was composed purely of pleomorphic spindle cells. A karyotypic analysis of the recurrent tumour showed 47, XXY with some instability. Supernatant from primary culture contained high levels of IL-6 and VEGF, suggesting high secretion of these cytokines from neoplastic cells. Immunohistochemically, p53 overexpression was identified only in the pleomorphic spindle cells of the primary lesion and metastatic tumour. No features suggestive of vascular origin were shown on immunohistochemical or electron microscopic analysis of the neoplastic cells. Human herpesvirus type 8 was not detected by immunohistochemistry or PCR analysis. High levels of IL-6 and/or VEGF have been reported to play a role in CD. This is the first case report that clarifies the site of such cytokine production, showing the possibility of CD as a paraneoplastic phenomenon.