1. Involvement of cyclin D3 in liver metastasis of colorectal cancer, revealed by genome-wide copy-number analysis
Laboratory Investigation (2005) 85, doi: /labinvest ; published online 27 June 2005

2. ABSTRACT
subtractive comparative genomic hybridization (CGH) for 20 CRC patients
Analysis of 11 CRC cell lines using array-based CGH (CGH-array)

3. Quantitative RT-PCR showed that CCND3 was significantly upregulated in liver-metastatic lesions compared with primary lesions
immunohistochemical analysis of 120 primary CRC tumors

4. Comparative Genomic Hybridization (CGH)
比較基因體雜交法1992
CGH is a molecular cytogenetic method of screening a tumor for genetic changes.

5. CGH原理 如果胚胎細胞DNA 與正常細胞基因等量，雜交後對應的染色體區域便呈黃色。

6. Conventional CGH in 20 primary tumors.

7. Regions with the most frequent copy-number
gains in primary tumors were at 13q (70%), 20q (65%), 8q (55%), 20p (40%), 7p and 7q (25% each), and 1q (20%).
losses in primary tumors were seen at 18q (55%), 18p and 8p (50% each), 1p (40%), 17p (35%), 4q (30%), 4p (25%), and 3p (35%)

8. Conventional CGH in liver metastases of the same tumors

9. Common regions for the most frequent copy-number
gains in metastatic tumors were at 8q (70%), 20q and 13q (60% each), 20p (45%), 7p (40%), 7q (35%), Xq (25%), 1q (20%), and 6p (15%).
losses in metastatic tumors were seen at 8p (60%), 18p and 18q (55% each), 14q (30%), 17p (25%), 4p and 22q (20% each), 4q (15%), and 1p (15%).

11. subtractive (CGH)

12. subtractive CGH結果
our direct comparisons of 20 paired samples by means of subtractive CGH analysis identified gain in 6p as a metastasis-specific change,
but we found no significant differences at chromosomes 7, 8, 13, 18, or 20, where major chromosomal aberrations occurred in both primary and metastatic tumors

13. CGH之重要性 CGH 可以針對整個染色體進行全面性的比對，解析度最好可高達1M bp
其解析度雖較FISH 差，但卻能同時偵測23 對染色體的異常，不似FISH只能偵測某特定片段
但在偵測染色體的著絲點（centromere ）、端粒點
（telomere）、及X、Y 染色體較不準確。

14. CGH-Array Analysis 比較基因體雜交微矩陣分析法（Microarray-based comparative
　genomic hybridization），簡稱Array-CGH。
此技術為CGH 改進後發展的新技術，
將基因晶片（Gene-chip）的概念應用在CGH 的分析上

15. 1992 年9 月，Mel Simon發表利用細菌人工染色體（bacterial artificial chromosome，BAC）進行基因大片段的選殖（cloning）技術
此種選殖技術可插入約 kb 的DNA，且選殖效率高而穩定，隨著微矩陣技術的成熟，細菌人工染色體(BAC）也被廣泛的使用於微矩陣中
以細菌人工染色體製備的微矩陣，稱為基因晶片，可以取代玻璃片上準備雜合的體染色體（metaphase
genomic）
使雜交後的影像解析度可以大大提高

16. CGH-Array重要性 以BAC 代替的基因晶片，其解析度可達100-200k basepair，較統的CGH 的解析度更高
且將染色體序列整合成基因晶片更是加快電腦分析的速度。
Array-CGH 與CGH 最大的不同是在玻片上準備雜合的染色體以BAC 代替metaphase
genomic ，其他步驟皆相同。

17. CGH-Array結果
Among the 19 genes within 6p that were spotted on the MCG Cancer Array-800, CCND3, located at 6p21.1 (log 2 ratio=2.25/2.28), was the only one amplified, and in only two of the cell lines (COLO201 and COLO205).
With this approach, we detected high-level amplification of CCND3 (6p21.1) in two cell lines (COLO201 and COLO205),

19. FISH證實 Amplification of CCND3 was confirmed by FISH analysis:
BAC RP11-720D9, which contains the CCND3 gene, generated a remarkably increase in FISH signals in COLO201 ;
a similar result was obtained with COLO205 (data not shown). In the three metastatic tumor samples,

20. Fluorescence In Situ Hybridization (FISH)
螢光原位雜交法
利用標記有螢光的探針（probe）與分裂中期（metaphase）的染色體進行雜合
（hybridization），可偵測目標染色體中特定片段的缺失（deletion）或放大
（amplification）。

21. FISH重要性 FISH 的解析度最小可達10k basepair 的長度，因為有許多的遺傳缺陷不能單靠條紋染色技術就可確定

22. Real-Time Quantitative RT-PCR
real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments revealed CCND3 as the most probable target of additional amplification during metastasis
showed that CCND3 was significantly upregulated in liver-metastatic lesions compared with primary lesions

24. Results
The results implied involvement of cyclin D3 in liver metastasis of CRC
Our experiments also confirmed the power of subtractive CGH and CGH-array analysis for identifying cancer-related genes.