Comparative genomic hybridization (CGH) had been developed as a molecular cytognetic technique that compensates difficulties in conventional cytogenetics and fluorescence in situ hybridization (FISH), allowing the entire genome to be scanned, in a single step, for copy number aberrations in chromosomal material.⁵⁾

In this technique, the genomic DNAs of test and reference samples are isolated, and labeled in red and green fluorescent dyes, respectively. Each of labeled DNAs is subject to competitive hybridization to metaphase chromosomes from normal cells. The ratio of red and green fluorescent signals is measured along the longitudinal axis of each chromosome. Hybridization of repetitive sequences is blocked by adding Cot-1 DNA. The chromosomal regions involved in deletion or amplification in test DNA appear red or green, respectively, but the chromosomal regions equally represented in test and reference DNAs appear yellow.

The use of CGH for analysis of solid tumors has revealed a number of recurrent chromosomes copy number aberrations including amplifications that had not been detected previously by any existing techniques so far. For example, CGH could reveal frequent amplifications at chromosome 3q26-27 and 20q13 in various tumors, and first led to the identification of target genes, PIK3CA at 3q26 amplification in ovarian cancer⁶⁾, and ZNF217 at 20q13 amplification in breast cancer.⁷⁾

We have also extensively performed CGH analysis in over 900 cases in various types of solid tumors together with some types of hematological malignancies. Consequently, we have constructed the CGH database compiling those results through the internet (http://www.cghtmd.jp/).

Through our CGH analysis, we detected a number of novel and nonrandom amplifications in various tumors and could identify target genes within those amplicons. Among those, we detected distinct amplification at 8p23.1 in malignant fibrous histiocytoma (MFH) by CGH and isolated a novel gene designated MASL1 (MFH-amplified sequences with leucine-rich tandem repeats 1) within this narrowly defined region.⁸⁾

Recently, MASL1 has been reported to be translocated in immunoblastic B-cell lymphoma cell line OCI-LY8.⁹⁾ Furthermore, cIAP1, a member of the IAP (antiapoptotic) gene family, was identified as a target for the 11q22 amplification in esophageal squamous cell carcinomas (ESCs) and cervical squamous cell carcinomas (CSCCs).¹⁰⁾ Immunohistochemical analysis of primary CSCCs from patients treated only with radiotherapy demonstrated that patients with high levels of nuclear cIAP1 staining showed significantly poorer prognosis.¹¹⁾

We also detected frequent amplifications at 17q23 in neuroblastoma (NB) cell lines and a phosphatase, PPM1D was a target molecule upregulated due to gene amplification. A significant correlation between higher expression of PPM1D and poorer clinical outcome was ascertained.¹²⁾ PPM1D was also a target for 17q amplification in uterine endometrial cancer.¹³⁾ Furthermore, frequent 5p12-13 amplification was detected in both small cell lung cancer (SCLC) and non-small cell lung cancers (NSCLC), and S-Phase kinase associated protein 2 (SKP2) was identified as the target.¹⁴⁾ Interestingly, downregulation of SKP2 appeared suppressed DNA synthesis, and induction of apoptosis in lung-cancer cells,¹⁵⁾ as well as inhibition of invasion and migration of NSCLC cells.¹⁶⁾

Taken together, those results strongly suggest that targeting molecules within novel amplifications detected by CGH could represent a promising new therapeutic approach for intractable cancer.