Motonao Nakao

Is lobular endocervical glandular hyperplasia a cancerous precursor of minimal deviation adenocarcinoma?: a comparative molecular-genetic and immunohistochemical study.

Although lobular endocervical glandular hyperplasia (LEGH) was originally described as a distinct hyperplastic glandular lesion of the uterine cervix, recent studies have raised a question that LEGH may be a cancerous precursor of minimal deviation adenocarcinoma (MDA) and other mucinous adenocarcinomas (MACs) of the uterine cervix. In the present study, we studied LEGH, MDA, and MAC by using molecular-genetic and immunohistochemical methods for chromosomal imbalance, microsatellite instability, human papillomavirus (HPV) infection, and gastric pyloric-type mucin secretion to clarify their relationship. Comparative genomic hybridization revealed recurrent chromosomal imbalances, that is, gains of chromosome 3q and a loss of 1p, which were common to MDA and MAC, in 3 of 14 LEGHs analyzed (21%). LEGHs with chromosomal imbalances showed a degree of cellular atypia in the hyperplastic glandular epithelium. Dual-color fluorescence in situ hybridization confirmed a gain of chromosome 3 fragment in these cervical glandular lesions. HPV in situ hybridization revealed that high-risk HPV (types 16 and 18) was positive in over 80% of MACs, but negative in all LEGHs and MDAs examined. Microsatellite instability was rarely detected in these cervical glandular lesions. Our present study results demonstrated a molecular-genetic link between LEGH and cervical mucinous glandular malignancies including MDA and MAC, and are thought to support the hypothesis that a proportion of LEGHs are cancerous precursors of MDA and/or MAC.

A copy number gain of the 6p arm is linked with advanced hepatocellular carcinoma: an array-based comparative genomic hybridization study.

In accordance with cancer progression, genomic aberrations accumulate in cancer cells in a stepwise fashion. However, whether there are genomic changes linked with tumour progression remains unclarified. The purpose of this study is to elucidate the relationship between genomic alterations and clinical stages in hepatocellular carcinoma (HCC). A technology of array‐based CGH using DNA chips spotted with 1440 BAC clones was applied to 42 surgically removed HCCs to examine the DNA copy number aberrations. A frequent copy number gain was detected on chromosomal regions 1q, 8q and Xq. In particular, gains of 1q42.12, 1q43 and 8q24.3 were detected in more than 65% of tumours. A frequent copy number loss was detected on chromosomal regions 1p, 4q, 6q, 8p and 17p. Losses of 8p21 and 17p13 were detected in more than 55% of HCCs. However, the DNA copy number gains of clones on 6p and 8q24.12 were more frequent in stage III/IV tumours than in stage I/II tumours (p < 0.001). In particular, the gain of the whole 6p was virtually limited to advanced‐staged HCCs. The gain of the whole 6p is suggested to be a genomic marker for the late stages in HCCs. These observations therefore support the concept of genomic staging in HCC. Copyright

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

BackgroundCell 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.MethodsArray-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.ResultsThe 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.ConclusionsEstablished 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.

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.

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.

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.

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.

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.

The development of a mini-array for estimating the disease state of gastric adenocarcinoma by array CGH

BackgroundThe treatment strategy usually depends on the disease state in the individual patient. However, it is difficult to estimate the disease state before treatment in many patients. The purpose of this study was to develop a BAC (bacterial artificial chromosome) mini-array allowing for the estimation of node metastasis, liver metastasis, peritoneal dissemination and the depth of tumor invasion in gastric cancers.MethodsInitially, the DNA copy number aberrations (DCNAs) were analyzed by array-based comparative genomic hybridization (aCGH) in 83 gastric adenocarcinomas as a training-sample set. Next, two independent analytical methods were applied to the aCGH data to identify the BAC clones with DNA copy number aberrations that were linked with the disease states. One of the methods, a decision-tree model classifier, identified 6, 4, 4, 4, and 7 clones for estimating lymph node metastasis, liver metastasis, peritoneal dissemination, depth of tumor invasion, and histological type, respectively. In the other method, a clone-by-clone comparison of the frequency of the DNA copy number aberrations selected 26 clones to estimate the disease states.ResultsBy spotting these 50 clones together with 26 frequently or rarely involved clones and 62 reference clones, a mini-array was made to estimate the above parameters, and the diagnostic performance of the mini-array was evaluated for an independent set of 30 gastric cancers (blinded – sample set). In comparison to the clinicopathological features, the overall accuracy was 66.7% for node metastasis, 86.7% for liver metastasis, 86.7% for peritoneal dissemination, and 96.7% for depth of tumor invasion. The intratumoral heterogeneity barely affected the diagnostic performance of the mini-array.ConclusionThese results suggest that the mini-array makes it possible to determine an optimal treatment for each of the patients with gastric adenocarcinoma.