Role of Technological Advances in Identifying Genomic Rearrangements

The advantages of newer methods to study breast cancer genome have resulted in a greater understanding of the patterns of genomic instability and the underlying gene rearrangements observed within breast cancer (Table 2). Some of these methods include FISH, break-apart FISH, array CGH, polymerase chain reaction (PCR)-based techniques, whole-exome sequencing, whole-genome sequencing (WGS), RNA-seq, single primer enrichment technology (SPET), and anchored multiplex PCR (AMP).

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(To view a larger version of Table 2, click here.)

FISH is a powerful tool for detecting specific genomic alterations and has gained popularity due to its clinical application in identifying ERBB2 amplifications in breast cancer,17,45 BCR-ABL fusions in leukemia,46 and EML4-ALK fusions in lung cancer.47 However, since it uses predesigned gene probes, a major drawback is the inability to detect novel alterations. Further, even with known, locus-specific, high-resolution probes, cytogenetic techniques are currently limited in the ability to differentiate between duplications, inversions, or deletions occurring in small regions of a few hundred kilo-bases or less.48Therefore, standard FISH methods for clinical evaluation may significantly underestimate the prevalence of pathogenic gene rearrangements in solid tumors (Table 2). For example, FISH relies on use of gene-specific probes. Common receptor tyrosine kinase (RTK) fusions with known, recurrent gene partners can be identified using this method. However, fusions with an unknown partner can lead to false negative results. Break-apart FISH describes a technique where two fluorescent probes are designed to recognize one gene. Break-apart FISH can detect the presence of novel fusions with the known gene. In the presence of a rearrangement involving a second gene, the two probes are no longer found in proximity, ie, they break apart. While this technique can establish the presence of a fusion affecting the designated gene, it will not identify novel gene partners, the location of breakpoints in the gene partner, or the functionality of the fusion. Therefore, other methods such as reverse transcriptase-PCR (RT-PCR) and RNA-seq are needed to confirm the presence of such gene fusions. Fusions that occur within the same chromosomal band or with intronic breakpoints are beyond the resolution of cytogenetic techniques. PCR-based assays can be used to detect such intra-band and intron events only when breakpoints are known.

The development of more efficient high-throughput sequencing methods and data analysis pipelines has made the identification of pathogenic rearrangement events across all malignancies more affordable and more accessible. WGS and RNA-seq provide an unbiased view of the genome and expressed transcripts, respectively (Table 2). A subset of these next-generation sequencing methods is designed to capture rearrangement events in particular, eg, intron capture and RNA-Seq. Using WGS and RNA-seq, Stephens et al36 showed that multiple rearrangements are present in many breast cancers, with >50% of them occurring within coding regions.36 These rearrangements can lead to deregulation of gene expression and/or the formation of fusion transcripts, resulting in novel fusion proteins.49