Abstract 627: Detection of chromosomal rearrangements in clinical tissue samples by chromosome conformation capture
Authors: Gao Xiaobin , Wang Jianhui , Wang Jinglan , Tumwine Lynnette , Sklar Jeffrey 
Published: August 2015

Background

The detection of recurrent and specific chromosomal rearrangements has become an important element in the diagnostic evaluation of tumors and in decisions pertaining to their therapy. Several detection methods applicable to routine clinical samples currently exist, including in situ hybridization (FISH), RT-PCR, and direct DNA sequence analysis–all of which are associated with various drawbacks. An approach that has not been extensively explored for the detection of chromosomal rearrangements in clinical samples is the analysis of changes in chromatin conformation due to alterations of chromosomal structure. However, the extensive formalin fixation and paraffin embedding that these samples routinely undergo would appear to pose special challenges for analysis of chromatin in such tissues.

Design

To investigate the practicality and potential value of chromatin conformation assays for detecting chromosomal rearrangements in formalin-fixed and paraffin-embedded (FFPE) samples, we have analyzed fixed and embedded cell lines and FFPE biopsy specimens containing two different types of chromosomal translocations: the t(8;14)(q24;q32) found in Burkitt's lymphoma and the t(14;18)(q32q21) found in follicular B cell lymphoma. These two translocations place DNA of the IGH locus near that of oncogenes MYC and BCL-2, respectively. The strong enhancers of IGH induce the overexpression of the two oncogenes, presumably by looping of chromatin to permit interactions with the oncogene promoters. Utilizing primers designed to be complementary to DNA within IGH enhancers and MYC or BCL-2 promoters, we performed chromosome conformation capture (3C) to interrogate the possible chromatin interactions between the IGH enhancer and the two promoters in FFPE samples.

Results

Since conventional 3C procedures worked poorly with FFPE cell lines, probably because of heavy fixation (16 hours, 10% buffered formalin), we modified the procedures by partially de-crosslinking the specimens using heat and mild alkali. The 3C procedure modified in this manner can efficiently and specifically detect the t(8; 14) and t(14; 18) in FFPE cell lines and, at a lower sensitivity, in clinical biopsy specimens. In follicular lymphoma samples, the detection of t(14; 18) achieved high specificity with an analytic sensitivity of at least 10% tumor cells within a volume of ∼1000 total cells. The sensitivity with the Burkitt's samples was lower, probably because of differences in preservation of the tissue and chromatin, as suggested by results of FISH.

Conclusions

We report a modified 3C workflow that successfully detected chromosome rearrangements in clinical samples. In addition to diagnostic applications, this method, with possible further improvements, may permit study of chromatin interactions in clinical samples to increase understanding of epigenetic and other basic genomic features in many forms of cancer.

Citation Format: Xiaobin Gao, Jianhui Wang, Jinglan Wang, Lynnette Tumwine, Jeffrey Sklar. Detection of chromosomal rearrangements in clinical tissue samples by chromosome conformation capture. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2015-627