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.

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.

Germline copy number variations associated with breast cancer susceptibility in a Japanese population.

Although copy number variations (CNVs) are expected to affect various diseases, little is known about the association between CNVs and breast cancer susceptibility. Therefore, we investigated this relation. Array comparative genomic hybridization was performed to search for candidate CNVs related to breast cancer susceptibility. Subsequent quantitative real-time polymerase chain reaction was carried out for confirmation. We found seven CNV markers associated with breast cancer risk. The means of the relative copy numbers of patients with a history of breast cancer and women in the control group were 0.8 and 1.8 for Hs06535529_cn on 1p36.12 (P < 0.0001), 2.9 and 2.2 for Hs03103056_cn on 3q26.1 (P < 0.0001), 1.2 and 1.8 for Hs03899300_cn on 15q26.3 (P < 0.0001), 1.0 and 1.5 for Hs03908783_cn on 15q26.3 (P < 0.0001), and 1.1 and 1.7 for Hs03898338_cn on 15q26.3 (P < 0.0001), respectively. Interestingly, nine or more copies of Hs04093415_cn on 22q12.3 were found only in 8/193 (4.1 %) patients with a history of breast cancer and in none of the controls (P = 0.0081). Similarly, 12 or more copies of Hs040908898_cn on 22q12.3 were found only in 7/193 (3.6 %) patients with a history of breast cancer and in none of the controls (P = 0.016). A combination of two CNVs resulted in 80.3 % sensitivity, 80.6 % specificity, 82.4 % positive predictive value, and 78.3 % negative predictive value for the prediction of breast cancer susceptibility. These findings may lead to a new means of risk assessment for breast cancer. Confirmatory studies using independent data sets are needed to support our findings.

[DNA polymorphisms].

Genetic variations, including single nucleotide polymorphisms (SNP), variable numbers of repetitive sequences such as microsatellite polymorphisms, and small insertion-deletion polymorphisms (INDELs), have been reportedly associated with various diseases. SNP is just a single base change in a DNA sequence, with the usual alternative of two possible nucleotides at a given position. Microsatellite polymorphisms are variations in the number of short nucleotide repeats observed at microsatellite loci. INDELs are small insertions and deletions ranging from 1 to 10,000 bp in length. Another variation in the human genome is that of genomic structural variants, including copy number variations (CNVs). The CNVs involve gains or losses of several to hundreds of kilobases of genomic DNA among phenotypically normal individuals and at least 291,801 CNV regions have been identified. Recent studies have described the associations of CNVs with various common disorders, especially with mental illness. In order to make an extensive public catalog of human genetic variations, including SNPs and structural variants, and their haplotype contexts, the 1,000 Genomes Project has been performed with international collaboration using the genomes of about 2,500 unidentified people from about 25 populations around the world with next-generation sequencing technologies. This resource will support genome-wide association studies and other medical research studies. In this review, we focus on HapMap and the association between the various genetic variations and diseases.

DNA copy number aberrations associated with lymphovascular invasion in upper urinary tract urothelial carcinoma.

Recent studies have reported that lymphovascular invasion (LVI) is a predictor of patient prognosis in upper urinary tract urothelial carcinoma (UUTUC). DNA copy number aberrations (DCNAs) identified by array-based comparative genomic hybridization (aCGH) had not previously been examined in UUTUC. We therefore examined DCNAs in UUTUC and compared them with DCNAs in LVI. We applied aCGH technology using DNA chips spotted with 4,030 BAC clones to 32 UUTUC patients. Frequent copy number gains were detected on chromosomal regions 8p23.1 and 20q13.12, whereas frequent copy number losses were detected on chromosomal regions 13q21.1, 17p13.1, 6q16.3, and 17p11.2. DCNAs occurred more frequently in tumors with LVI than in those without it (P = 0.0002), and this parameter was more closely associated with LVI than with the tumor grade or pT stage. Disease-specific survival rate was higher in tumors without LVI than in those with it (P = 0.0120); however, tumor grade and stage were not significant prognostic factors of patient outcome. These data support our hypothesis that tumors with LVI have more genetic alterations in terms of total numbers of DCNAs than those without, and provide proof that aggressive adjuvant therapy should be considered for UUTUC patients with LVI.

Luminal A and luminal B (HER2 negative) subtypes of breast cancer consist of a mixture of tumors with different genotype.

BACKGROUND: The St Gallen International Expert Consensus 2011 has proposed a new classification system for breast cancer. The purpose of this study was to elucidate the relationship between the breast cancer subtypes determined by the new classification system and genomic characteristics. METHODS: Invasive breast cancers (n = 363) were immunohistochemically classified as follows: 111 (30.6%) as luminal A, 95 (26.2%) as luminal B (HER2 negative), 69 (19.0%) as luminal B (HER2 positive), 41 (11.3%) as HER2, and 47 (12.9%) as basal-like subtypes. RESULTS: The high expression of Ki-67 antigen was detected in 236 tumors; no cases of luminal A subtype showed high expression of the Ki-67 antigen, but more than 85% of tumors of the other subtypes showed high expression. In addition, DNA ploidy and chromosomal instability (CIN) were assessed using imaging cytometry and FISH, respectively. In this series, 336 (92.6%) tumors consisted of 129 diploid/CIN- and 207 aneuploid/CIN + tumors. Diploid/CIN- and aneuploid/CIN+ features were detected in 64.9% and 27.9% of luminal A, 41.1% and 49.5% of luminal B (HER2-), 11.6% and 81.2% of luminal B (HER2+), 4.9% and 90.2% of HER2, and 17.0% and 76.6% of basal-like subtypes, respectively. Unlike the luminal B (HER2+), HER2 and basal-like subtypes, the luminal A and luminal B (HER2-) subtypes were heterogeneous in terms of DNA ploidy and CIN. CONCLUSIONS: It is reasonable to propose that the luminal A and luminal B (HER2-) subtypes should be further divided into two subgroups, diploid/CIN- and aneuploid/CIN+, based on their underlying genomic status.

Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems.

Cell-penetrating peptides have gained attention owing to their promise in noninvasive delivery systems. Among the identified cell-penetrating peptides, the TAT peptide has been preferentially used for transduction into cells of diverse origins. However, this activity is nonselective between neoplastic and non-neoplastic cells. Here we describe artificial cell-penetrating peptides that are selectively and efficiently incorporated into human tumour cells, according to their lineage. Ten representative tumour lineage-homing cell-penetrating peptides were obtained by screening of a random peptide library constructed using messenger RNA display technology, and some of the isolates were further modified by amino-acid substitution. Their advantageous tumour cell-targeting ability is corroborated in an in vivo mouse model for imaging and growth suppression of metastatic xenoplant tumours. These cell-penetrating peptides are potentially useful for the efficient targeting of human neoplasms in a tumour origin-dependent manner, and provide a framework for the development of peptide-based anti-tumour technologies.

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.

Adenoma of the nipple, focusing on the contrast-enhanced magnetic resonance imaging findings: report of a case.

A woman in her 50s was referred to our hospital for an investigation of a right breast tumor. The tumor was palpated below the nipple, but there was no erosion or nipple discharge. Mammography showed a well-defined high-density tumor, measuring 2 cm in diameter, without calcification, and ultrasonography showed a low-echoic mass with a fluid component with posterior echo enhancement and a lateral shadow. Contrast-enhanced magnetic resonance imaging (CE-MRI) demonstrated a 1.3 × 0.8 cm solid component and a gradually increasing time-intensity curve. We performed lumpectomy and the pathological findings were adenoma of the nipple. The pattern of the time-intensity curve might be attributed to moderate fibrosis of the tumor. Contrast-enhanced MRI is therefore considered to be very useful in the diagnosis of breast disease because it can show the nature and extent of the breast lesion; however, we should be aware that various patterns have been observed on CE-MRI for adenoma of the nipple.

Centrosome amplification as a putative prognostic biomarker for the classification of urothelial carcinomas.

Recent studies have reported that centrosome amplification is closely related to chromosomal instability and patient prognosis in human malignancies. The purpose of this study was to elucidate the relationship between centrosome amplification and genomic alterations in urothelial carcinomas. Centrosomes were evaluated by immunohistochemistry using anti-γ-tubulin antibody. Array-based comparative genomic hybridization technology using DNA chips spotted with 4030 bacterial artificial chromosome clones was applied to 70 urothelial carcinomas to examine DNA copy number aberrations. Studying aberrations in the number of chromosomes 7, 9, and 17 using fluorescence in situ hybridization allowed the estimation of the degree of chromosomal instability. DNA copy number gains at 20p12.2, 5p15.2, 5p15.31, and 17q25.3 and losses at 17p12, 8p22, 2q37.3, 5q31.1, and 2q37.3 were more frequent in tumors with centrosome amplification than in those without it. The total numbers of DNA copy number aberrations and frequency of chromosomal instability were also larger in tumors with centrosome amplification than in those without it (P = .0263 and P < .0001, respectively). These parameters were more closely associated with centrosome amplification than with the subjectively assigned tumor grade (P = .0405 and P = .0020, respectively). Thus, these data suggest that centrosome amplification may have great potential as a biomarker for improved objective classification of urothelial carcinoma and estimation of prognosis.

Loss of 3p26.3 is an independent prognostic factor in patients with oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a common malignancy worldwide and the prognosis for patients with advanced-stage OSCC is particularly poor. To identify DNA copy number aberrations and candidate genes associated with a poor or favorable outcome, we analyzed the genome profiles of OSCC tumors by array-based comparative genomic hybrid-ization (A-CGH). This technique uses DNA microarray technology to detect genomic copy number variations at a higher resolution level than chromosome-based CGH. Fifty patients with primary OSCCs were included in the study. Of these 50 patients, 37 were treated surgically and 13 were treated without surgery and had received irradiation and/or chemotherapy. All samples were analyzed by A-CGH. Gains were detected frequently (>50%) at chromosomal regions 5p15.33, 7p22.3, 8q21.1-24.3, 9q34.3, 11q13, 16p13.3 and 20q13.3. Losses were frequently detected at 3p22, 3p14 and 4q35.2. High-level gains were recurrently (>10%) detected at each of 5p15, 7p22, 7p11, 8q24, 11q13, 11q22 and 22q11. Gains of 2p25.1, 11p15, 16p13.3, 16q24.3 and 20q13.3 were inversely correlated with nodal metastasis. In 37 of the 50 OSCC patients treated with surgery, gains of 8q12.1-24.22 and losses of 3p26.2-3 were associated with disease-specific survival (p<0.01). Loss of a 0.2 Mb chromosomal region in 3p26.3 was associated with a poor prognostic outcome in the Kaplan-Meier analysis (p<0.01 by the log-rank test). Multivariate analysis revealed that loss of 3p26.3 is an independent prognostic factor (p<0.01) of OSCC. Loss of a 0.2 Mb chromosomal region in 3p26.3 including the CHL1 (cell adhesion molecule with homology to L1CAM1) gene was identified as a novel potential marker for predicting the prognosis of patients with OSCC.

DNA copy number aberrations associated with the clinicopathological features of colorectal cancers: Identification of genomic biomarkers by array-based comparative genomic hybridization.

The aim of the present study was to investigate the chromosomal aberrations that are linked with the crucial clinicopathological features of colorectal cancer (CRC) and its prognosis by array-based comparative genomic hybridization (CGH). Fresh-frozen tumor tissues of 94 cases of CRC were analyzed by using bacterial artificial chromosome (BAC) CGH slides spotted with 4030 human BAC clones, which covered the whole range of the human genome at an average interval of 0.83 mega base pairs. DNA copy number aberrations (DCNAs) were identified in association with clinicopathological features: a gain of 8q24.3 and losses of 9q33.1 and 20p12.2 were associated with lymph node metastasis, gain of 8q24.3 and loss of 9q33.1 with disease stage, gain of 8q21.11 and loss of 10q21.3 with lymphovascular invasion and losses of 3p25.1, 10p15.3, 12q15 and 17p13.1 for venous invasion. These aberrations can be regarded as genomic biomarkers to predict the clinical outcome of patients with CRC, and are expected to serve to individualize the treatment of CRC patients.

BCL2L10 protein regulates apoptosis/proliferation through differential pathways in gastric cancer cells.

The reason for and consequences of BCL2L10 down-regulation in gastric carcinoma are poorly understood. Our aim was to investigate the function of the protein BCL2L10 in gastric carcinoma. We investigated BCL2L10 expression using quantitative real-time PCR and immunoblotting. The methylation status of the BCL2L10 gene promoter was examined by bisulphite sequencing in fresh gastric normal and carcinoma tissues. We studied apoptosis and proliferation regulation in gastric cancer cell lines using flow cytometry, fluorescence staining, murine xenografting and immunoblotting. Pathway inhibitors were applied to confirm the major pathways involved in apoptosis or proliferation regulation. We observed significant correlations between lower BCL2L10 expression and CpG island hypermethylation of the BCL2L10 gene promoter in gastric carcinoma, apoptosis induced by over-expressed BCL2L10 through mitochondrial pathways, and proliferation accelerated by BCL2L10 siRNA via the PI3K-Akt signalling pathway in gastric cancer cell lines. The pro-apoptotic effect of BCL2L10 and growth promotion by BCL2L10 siRNA in gastric cancer cells suggest that it may be a tumour suppressor.

Loss of BCL2L10 protein expression as prognostic predictor for poor clinical outcome in gastric carcinoma.

AIMS: BCL2L10 protein is an apoptosis-related member of the Bcl-2 protein family. The clinical significance of its expression in gastric carcinoma is poorly understood. The aim was to investigate BCL2L10 expression and its clinical and prognostic significance in gastric carcinoma patients. METHODS AND RESULTS: Immunohistochemistry, real-time polymerase chain reaction (PCR) and immunoblotting all revealed extensive loss of BCL2L10 expression in gastric cancer cells. The scaled BCL2L10 expression data was categorized into three groups (groups 0-2) to facilitate statistical analysis. A significant correlation was observed between the lower BCL2L10 expression group and shorter disease-free survival (P=1.956×10(-18)). Multivariate regression analysis showed that loss of BCL2L10 protein expression [P=4.883×10(-8), hazard ratio (HR)=0.252] is an independent prognostic predictor of gastric carcinoma. The receiver operator characteristic (ROC) curve showed that the area for BCL2L10 protein was 0.817 (P=8.331×10(-14)), indicating that loss of BCL2L10 protein expression is an excellent prognostic predictor of gastric carcinoma. CONCLUSIONS: Loss of BCL2L10 protein expression predicts poor clinical outcome in gastric carcinoma.

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.

The loss of 8p23.3 is a novel marker for predicting progression and recurrence of bladder tumors without muscle invasion.

There are few reliable markers to distinguish tumors with aggressive characteristics from others at the time of initial diagnosis in non-muscle-invasive bladder cancer. The purpose of this study was to identify a genomic marker that allows the prediction of prognosis for non-muscle-invasive bladder cancers. We screened the genome-wide copy number in 41 patients with non-muscle-invasive urothelial carcinoma of the bladder by array-based comparative genomic hybridization using arrays spotted with 4,030 bacterial artificial chromosome clones. A loss of 8p23.3 (clone 923) was correlated significantly with a higher histological grade (P = 0.0026) and advanced pathological stage (P = 0.0148). Both recurrence-free and progression-free survival rates were lower in patients with tumors without 8p23.3, compared with those with 8p23.3 (P = 0.0146 and 0.0473, respectively; log-rank test). These data suggest that the loss of 8p23.3 is a novel genomic marker allowing estimation of biological characteristics of non-muscle-invasive bladder cancer.

Connexin 26 expression correlates with less aggressive phenotype of intestinal type-gastric carcinomas.

Connexin 26 (Cx26), one of the gap junction-forming family members, is more controversial than other members, as a tumor suppressor. Here, we assessed Cx26 expression in gastric carcinoma, which has not been investigated before, and its clinical significance including survival analyses. Cx26 expression was assessed in 205 tissue samples from gastric carcinoma by immunohistochemistry. Of 205 gastric carcinoma cases, 79 (38.5%) were positive for Cx26 with mainly cytoplasmic localization compared to sporadic membranous staining in normal epithelium, and the expression levels were confirmed by Western blotting and real-time PCR. Negative associations were revealed between Cx26 expression and most clinicopathologic features (all P<0.05). Notably, high Cx26 expression was associated with histological intestinal-type (P=0.017) and early stage of gastric carcinoma. The multivariate regression analysis revealed that positive Cx26 expression was an independent prognostic predictor of intestinal-type GC (P=0.023, HR=2.019). Our findings suggest that aberrant expression of Cx26 in cytoplasm plays a tumor-suppressor role in gastric carcinoma and is an independent biomarker for favorable prognosis in intestinal-type gastric carcinoma.

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.