Distinct Distribution of Immunocytes in a Retropharyngeal Lymphadenopathy Associated with Kawasaki Disease: A Case Study Compared with Tonsillitis.

OBJECTIVES: Retropharyngeal lesions are often associated with Kawasaki disease (KD). A 4-year-old male first presented a peritonsillar and retropharyngeal abscess-like lesion. Surgical tonsillectomy was performed to avoid a risk of mediastinal abscess, but he fulfilled the diagnostic criteria of KD after the operation. This prompted us to perform a histological study on the KD tonsils. METHODS: The histopathology of the KD tonsil specimens were compared with hypertrophic tonsils obtained from 4 patients with chronic tonsillitis unrelated to KD assessed by the immunostainings. RESULTS: KD tonsils showed small lymphatic follicles and neutrophil infiltration in the peritonsillar muscle layer, with no evidence of vasculitis or abscess formation. The KD tonsils exclusively showed (1) predominant activated CD4+ T cells in the perifollicular interstitium, (2) sparse scattering of CD68+ monocytes/macrophages in the lymphatic follicles, and (3) polyclonal carcinoembryonic antigen-positive cells in the lymphatic follicles and venules with the high endothelial cells. CONCLUSIONS: The uniquely distributed immunocytes suggest the inflammatory process of KD involving the pathogen-associated molecules.

Analysis of centromere signal patterns in breast cancer cells with chromosomal instability using image cytometry combined with centromere fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with centromeric probes is a method used to detect chromosomal instability (CIN), a hallmark of most cancers. However, no studies thus far have investigated the relationship between centromeric FISH signals and the cell cycle in cancer cells. In this study, the chromosome content in each cell cycle phase was evaluated with respect to the number of centromeric FISH signals in two breast cancer cell lines and eight surgically resected breast cancer specimens using image cytometry. Variations in chromosome number were detected at each phase of the cell cycle but were not associated with proliferative capacity in the cell lines. Furthermore, the chromosome doubling frequency differed in each cell line and clinical specimen. These results reveal two aspects of centromeric FISH signal variation in breast cancers that exhibit CIN, and suggest that chromosome doubling is a remarkable occurrence that may increase the heterogeneity of tumors.

Copy number aberrations using multicolour fluorescence in situ hybridization (FISH) for prognostication in non-muscle-invasive bladder cancer (NIMBC).

OBJECTIVE: To investigate if detection of copy number aberrations of chromosomes 3, 7, 9p21, and 17 using multicolour fluorescence in situ hybridization (FISH) predicts patient outcome in non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: In all, 118 bladder wash samples were prospectively collected from patients who underwent transurethral resection of bladder tumour (median age 50.5 years, male/female: 91/27, tumour grade 1/2/3: 18/52/42, stage pTis/Ta/T1: 8/62/42) from 2007 to 2010. The 118 samples were analysed using the UroVysion® kit to detect the copy numbers of chromosomes 3, 7, 9p21, and 17. The variant fraction (VF; the sum of the non-modal copy number fraction of each chromosome) was defined as abnormal when the percentage was ≥16%. The percentage deletion of 9p21 (fraction of null or one copy number of the 9p21 locus) was defined as abnormal when the percentage was ≥12%. Maffezzini risk criteria were also analysed in our cohorts. RESULTS: There was recurrence in 57 (48.3%) patients and disease progression in 12 (10.1%), with a median follow-up of 35.7 months. Multivariate analysis showed that the percentage 9p21 loss (>12%) was an independent prognostic factor for recurrence (P < 0.001, odds ratio [OR] 3.24, 95% confidence interval [CI] 1.85-5.62). For disease progression, tumour grade, positive urine cytology, concurrent carcinoma in situ, and a mean VF of >16% were significant prognostic factors in univariate analysis. In multivariate analysis, a mean VF of >16% was a prognostic factor for disease progression (P = 0.048, OR 6.07, 95% CI 1.02-57.45). CONCLUSIONS: Multicolour-FISH analysis using a commercially available kit could be a powerful tool not only for diagnosis, but also for prognostication in patients with NMIBC.

Analysis of bronchoalveolar lavage fluid in a mouse model of bronchial asthma and H1N1 2009 infection.

BACKGROUND: Bronchial asthma is known as a risk factor of admission to the intensive care unit. However, the mechanism by which pandemic 2009 H1N1 (A(H1N1)pdm09) infection increases the severity of symptoms in patients with bronchial asthma is unknown; therefore, we aimed at determining this mechanism. METHODS: Inflammatory cell levels in the bronchoalveolar lavage (BAL) fluid from the non-asthma/mock, non-asthma/A(H1N1)pdm09, asthma/mock, and asthma/A(H1N1)pdm09 groups were determined using BALB/c mice. Cell infiltration levels, cytokine levels, and viral titers were compared among the groups. RESULTS: Neutrophil, monocyte, interleukin (IL)-5, IL-6, IL-10, IL-13, and tumor necrosis factor (TNF)-α levels were significantly higher in the BAL fluid from the non-asthma/A(H1N1)pdm09 and asthma/A(H1N1)pdm09 groups than in the mock groups (p<0.05 for neutrophils and monocytes; p<0.01 for the rest). The number of eosinophils and CD8(+) lymphocytes and the level of transforming growth factor beta 1 (TGF-ß1) in BAL fluid in the asthma/A(H1N1)pdm09 group were significantly higher among all groups (p<0.05 for eosinophils and CD8(+) lymphocytes; p<0.01 for TGF-ß1). The levels of IL-6, IL-10, IL-13, and TNF-α were significantly higher in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group (p<0.05 for IL-6 and IL-10; p<0.01 for IL-13 and TNF-α). The level of IFN-γ in the asthma/A(H1N1)pdm09 group was significantly lower than that in the non-asthma/A(H1N1)pdm09 group (p<0.05). The viral titers in the BAL fluids were higher in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group (p<0.05). Histopathological examination showed more severe infiltration of inflammatory cells and destruction of lung tissue in the asthma/A(H1N1)pdm09 group than in the non-asthma/A(H1N1)pdm09 group. CONCLUSIONS: Severe pulmonary inflammation induced by elevated levels of cytokines, combined with increased viral replication due to decreased IFN-γ levels, may contribute to worsening respiratory symptoms in patients with bronchial asthma and A(H1N1)pdm09 infection.

Breast cancer cell lines carry cell line-specific genomic alterations that are distinct from aberrations in breast cancer tissues: comparison of the CGH profiles between cancer cell lines and primary cancer tissues.

BACKGROUND: Cell lines are commonly used in various kinds of biomedical research in the world. However, it remains uncertain whether genomic alterations existing in primary tumor tissues are represented in cell lines and whether cell lines carry cell line-specific genomic alterations. This study was performed to answer these questions. METHODS: Array-based comparative genomic hybridization (CGH) was employed with 4030 bacterial artificial chromosomes (BACs) that cover the genome at 1.0 megabase resolution to analyze DNA copy number aberrations (DCNAs) in 35 primary breast tumors and 24 breast cancer cell lines. DCNAs were compared between these two groups. A tissue microdissection technique was applied to primary tumor tissues to reduce the contamination of samples by normal tissue components. RESULTS: The average number of BAC clones with DCNAs was 1832 (45.3% of spotted clones) and 971 (24.9%) for cell lines and primary tumor tissues, respectively. Gains of 1q and 8q and losses of 8p, 11q, 16q and 17p were detected in >50% of primary cancer tissues. These aberrations were also frequently detected in cell lines. In addition to these alterations, the cell lines showed recurrent genomic alterations including gains of 5p14-15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28 that were barely detected in tumor tissue specimens. These are considered to be cell line-specific DCNAs. The frequency of the HER2 amplification was high in both cell lines and tumor tissues, but it was statistically different between cell lines and primary tumors (P = 0.012); 41.3 +/- 29.9% for the cell lines and 15.9 +/- 18.6% for the tissue specimens. CONCLUSIONS: Established cell lines carry cell lines-specific DCNAs together with recurrent aberrations detected in primary tumor tissues. It must therefore be emphasized that cell lines do not always represent the genotypes of parental tumor tissues.

Influence of cytological stains on comparative genomic hybridization analysis for DNA extracted from cytological smears.

In the present study, we investigated the influence of cytological stains in analyzing DNA extracted from cytological slides by comparative genomic hybridization (CGH). Multiple imprint cytological slides were prepared for fresh-frozen breast cancer tissue samples and the slides were stained by three staining methods for each sample. Under microscopic observation, cancer cells were selectively microdissected from the slides and forwarded to DNA extraction, whole genome amplification, and CGH analysis. CGH was successfully performed for all methylgreen-stained and May-Grunwald-Giemsa (MGG)-stained cytological smear slides, but for two Papanicolaou (PAP)-stained slides. The number of chromosomal imbalances detected were 5-10 in methylgreen-stained slides and 5-9 in MGG-stained slides. The chromosomal imbalances resemble each other between methylgreen-stained and MGG-stained slides. The present study indicates that the MGG stain is preferred to the PAP stain for the purpose of cytogenetical analysis by CGH for DNA extracted from cytological smear slides.

Biological characteristics in bladder cancer depend on the type of genetic instability.

PURPOSE: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. EXPERIMENTAL DESIGN: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry. RESULTS: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. CONCLUSIONS: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability.

In vivo 3D analysis of systemic effects after local heavy-ion beam irradiation in an animal model.

Radiotherapy is widely used in cancer treatment. In addition to inducing effects in the irradiated area, irradiation may induce effects on tissues close to and distant from the irradiated area. Japanese medaka, Oryzias latipes, is a small teleost fish and a model organism for evaluating the environmental effects of radiation. In this study, we applied low-energy carbon-ion (26.7 MeV/u) irradiation to adult medaka to a depth of approximately 2.2 mm from the body surface using an irradiation system at the National Institutes for Quantum and Radiological Science and Technology. We histologically evaluated the systemic alterations induced by irradiation using serial sections of the whole body, and conducted a heart rate analysis. Tissues from the irradiated side showed signs of serious injury that corresponded with the radiation dose. A 3D reconstruction analysis of the kidney sections showed reductions in the kidney volume and blood cell mass along the irradiated area, reflecting the precise localization of the injuries caused by carbon-beam irradiation. Capillary aneurysms were observed in the gill in both ventrally and dorsally irradiated fish, suggesting systemic irradiation effects. The present study provides an in vivo model for further investigation of the effects of irradiation beyond the locally irradiated area.

A novel technology allowing immunohistochemical staining of a tissue section with 50 different antibodies in a single experiment.

Immunohistochemical (IHC) examination is frequently necessary for a histological differential diagnosis of tumors. To simplify IHC examination, we have developed a novel device called a "multiplex-immunostain chip (MI chip)." The chip is a panel of antibodies contained in a silicon rubber plate that consists of 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows IHC staining of a tissue section with 50 different antibodies in a single experiment, reducing the time, effort, and expense of IHC analysis. In addition, it enables pathologists to compare expression of multiple antigens on a tissue section simply by changing microscopic fields on a single slide. These features are unique to the MI chip technology. The method requires no expensive instruments. This device can be used in various applications in differential diagnosis of tumors and the field of cell biology.

Multicolor FISH and cytometric analyses allow classification of urothelial carcinomas into two subtypes, low- and high-grade tumors.

The biological behaviors of urothelial carcinomas are linked roughly to histological grade, but there are many cases whose precise classification is difficult. In addition, it remains unclear whether there are differences in the types of genetic changes between low- and high-grade urothelial carcinomas. Fifty-seven biopsy specimens of urothelial carcinomas and 9 control urothelial specimens were examined by four-color FISH with centromere-specific probes for chromosomes 3, 7, and 17, and a locus-specific probe for 9p21 that covers p16INK4a and p15INK4b. Nuclear DNA ploidy was determined with laser scanning cytometry (LSC). FISH and LSC data allowed us to classify urothelial cancers into two groups. Tumors in one group showed minimal intercellular variation in centromere copy numbers and DNA diploidy, and these tumors were histologically comparable to low-grade (grades I and II) tumors. In contrast, tumors in the other group showed a large intercellular variation in centromere copy numbers and DNA aneuploidy, suggesting chromosomal instability. These tumors were all high-grade (grades II and III) tumors. Numerical abnormalities of 9p21 signals were detected in all cancers; this variation in 9p21 signals was also associated with histological grade. The present findings suggest that the pathogenetic pathways are different between low- and high-grade carcinomas. High-grade tumors are characterized by chromosomal instability, whereas low-grade carcinomas are not.

Quantitative analysis of centromeric FISH spots during the cell cycle by image cytometry.

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G2 phase was larger than that in the G0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G2 phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.

Elucidation of proliferative capability of mononuclear tetraploid cells, emerging spontaneously from diploid cells, using image cytometry and fluorescence in situ hybridization.

OBJECTIVES: Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method. MATERIALS AND METHODS: In this study, two completely disomic cell lines were used, TIG-7, a fibroblast cell line and CAL-51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony-forming ability were examined. RESULTS: Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG-7 and CAL-51. Colonies of TIG-7 TCs were not observed, but were observed of CAL-51 TCs. CONCLUSIONS: Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG-7 TCs do not proliferate but that mononuclear CAL-51 TCs are able to.

A case of gastric adenocarcinoma with conspicuous binuclear cytologic features.

Binuclear cells have been occasionally observed in nonneoplastic and carcinoma cells. However, in clinical cases, few reports have analyzed and discussed the origins and features, including the proliferative capacity, of binuclear cells. We describe the case of a 75-year-old man with gastric cancer with microscopically prominent binuclear cells in the resected tissue and ascitic fluid. Image cytometry and chromosomal analysis were performed on cells isolated from the ascitic fluid. The DNA histogram pattern showed aneuploidy and the fluorescence in situ hybridization pattern of centromeres 7 and 11 was similar to that of most other mononuclear cancer cells. Furthermore, the binuclear cells showed low proliferative capability based on 5-bromo-2'-deoxyuridine incorporation. Our results demonstrated that the binuclear cells were derived from mononuclear aneuploid cells through incomplete cell division, and, in this case, may have impaired proliferative capacity.

Loss of 6q or 8p23 is associated with the total number of DNA copy number aberrations in adenoid cystic carcinoma.

We analyzed 10 adenoid cystic carcinomas (ACCs) of the salivary glands by array-based comparative genomic hybridization (a-CGH) using DNA chips spotted with 4,030 bacterial artificial chromosome clones. After the data smoothing procedure was applied, a total of 88 DNA copy number aberrations (DCNAs) were detected. The frequent (≥30%) DCNAs were loss of 6q23-27 and 8p23, and gains of 6p, 6q23, 8p23 and 22q13. High-level gains were detected on 12q15, including MDM2 in two cases. These two cases showed an immunohistochemically high-level (>50%) expression of MDM2 and a low-level expression of p53 (<20%). Furthermore, the total number of DCNAs was significantly greater in ACCs with loss of 6q compared to other ACCs, and in ACCs without the loss of 8p23 compared to other ACCs, respectively. Although limitations exist, a-CGH detected several candidate chromosomal imbalances associated with accumulation of DCNAs in ACCs.

DNA copy number aberrations associated with aneuploidy and chromosomal instability in breast cancers.

Biological characteristics of a tumor are primarily affected by its genomic alterations. It is thus important to ascertain whether there are genomic changes linked with DNA ploidy and/or chromosomal instability (CIN). In the present study, using fresh-frozen samples of 46 invasive breast cancers, laser scanning cytometry, array-based comparative genomic hybridization, and chromosome fluorescence in situ hybridization were performed to assess DNA ploidy, DNA copy number aberrations (DCNAs), and CIN status. Both ploidy and CIN status were examined in 36 tumors, resulting in 23 aneuploid/CIN+ tumors, 1 aneuploid/CIN-, 2 diploid/CIN+, and 10 diploid/CIN- tumors. Comparison of the aCGH data with the DNA ploidy and CIN status identified cytogenetically 11 characteristic breast cancers with distinctive DCNAs. The 11 tumors were classified into two types; one type is diploid/CIN- phenotype containing 4 DCNAs, and the other aneuploid/CIN+ phenotype containing 7 DCNAs. In 30 (65.2%) of the 36 breast cancers, the status of DNA ploidy and CIN depended on the type of DCNAs. Furthermore, the DNA ploidy phenotype depended on the dominant type of DCNAs even in tumors with a mixture of multiple DCNAs of one type and a single DCNA of the other type. Tumors with multiple DCNAs of both types represented aneuploidy and over three quarters of breast cancers carry at least one type of the DCNAs. These results suggested that, in breast cancers, the status of DNA ploidy and CIN was likely to determine at the beginning of carcinogenesis.

Identification of DNA copy number aberrations associated with metastases of colorectal cancer using array CGH profiles.

It is important to estimate the biological characteristics of tumors, including the nodal status at the time of diagnosis for optimal treatment of individual cancer patients. Array-based comparative genomic hybridization (aCGH) was performed on 77 sporadic colorectal adenocarcinomas using a chip spotted with 4030 BAC clones. The nodal status was compared with an aCGH profiles depicted using a combination of decision-tree classifier and a Self-Organizing Map (SOM) analysis. Node metastasis was not detected in any of the 6 poorly differentiated adenocarcinomas with a 3q loss. A SOM analysis following the decision-tree classification of the aCGH data allowed for the differentiation in chromosomal regions between high- and low-level decreases in the DNA copy number. Node metastasis was detected in all 5 tumors with the high-level decrease in DNA copy number at Xp, irrespective of the histological type. Node metastasis was also found exclusively in 6 tumors with increase in DNA copy number at the chromosomal region between 11q13.3 and 11q22.3. Copy number aberrations linked to nodal metastasis were identified more collectively by the combination of the decision-tree classifier and a SOM analysis than by the conventional analysis method in aCGH analysis.

9p21 index as estimated by dual-color fluorescence in situ hybridization is useful to predict urothelial carcinoma recurrence in bladder washing cytology.

Recent studies have shown that chromosome 9p21 locus is frequently deleted in the early stages of urothelial carcinogenesis. To study the predictive value of the 9p21 aberrations in recurrence of urothelial carcinoma of the urinary bladder, we applied dual-color fluorescence in situ hybridization for 9p21 and chromosome 9 centromere to the bladder washing cytology samples that were obtained from the patients with urothelial carcinoma of the urinary bladder treated by transurethral resection. For the evaluation, the 9p21 index was defined as the ratio of the mean number of 9p21 signals per nucleus for that of the chromosome 9 centromere signals per nucleus in each of the bladder washing cytology samples. The 9p21 index values of the bladder washing cytology samples with no (G0) cytologic atypia were significantly higher than those of the bladder washing cytology samples with moderate (G2) (P < .01) and severe (G3) (P < .001) cytologic atypia, but the index values did not statistically differ from those of the bladder washing cytology samples with mild (G1) cytologic atypia. Recurrence-free survival in the patients with a low 9p21 index value (<0.9) was significantly poorer in comparison with the patients with a high 9p21 index value (>0.9). Furthermore, 2 patients of bladder washing cytology G1 with a low 9p21 index value recurred much sooner than the other patients of the bladder washing cytology G1 category. These findings indicate that a decreased 9p21 index value is associated with recurrence of urothelial carcinoma of the urinary bladder, and the 9p21 index may be useful as a marker to identify patients with elevated risk of recurrence of urothelial carcinoma of the urinary bladder.

The prognosis in spindle-cell sarcoma depends on the expression of cyclin-dependent kinase inhibitor p27(Kip1) and cyclin E.

The aim of the present study was to examine the prognostic significance of p27(Kip1) and cyclin E expression in patients with spindle-cell soft tissue sarcomas. In 46 cases of spindle-cell sarcoma including 17 pre-operative biopsy materials, the expression of p27(Kip1) and cyclin E was immunohistochemically examined. The expression of p27(Kip1) decreased in the nuclei of metastatic primary tumor cells (stage IV), whereas the expression of cyclin E increased in those lesions. On univariate analysis, when the expression of p27(Kip1) and cyclin E was analyzed together, patients with spindle-cell sarcoma exhibiting low expression of p27(Kip1) and high expression of cyclin E showed lower distant-metastasis-free survival (DMFS) and overall survival (OS) than those with other combinations of the two parameters (both P < 0.0001). Multivariate analysis revealed that patients with low p27(Kip1) and high cyclin E expression also showed a decrease in DMFS (P = 0.0007, relative risk = 21.3) and OS (P = 0.005, relative risk = 20.8). These results suggest that the combination analysis of p27(Kip1) and cyclin E expression even in biopsy specimens allows the prediction of the clinical behavior of spindle-cell sarcoma.