Era of Comprehensive Cancer Genome Analyses
Authors: Yasushi Yatabe, Roman K. Thomas
Journal: Journal of Clinical Oncology (2014)

The dramatic responses seen in patients with advanced solid tumors bearing kinase mutations who receive kinase inhibitors have shifted the therapeutic paradigm to approaches that are based on the molecular characteristics of a given cancer. As an example, testing for somatic genetic alterations affecting genes encoding the epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) has become a diagnostic routine in lung cancer. Unfortunately, however, acquired resistance to the initial kinase inhibitor will eventually develop during the course of treatment, thus necessitating drugs that can overcome resistance. Accordingly, several next-generation inhibitors of EGFR and ALK have been developed, with impressive early signs of clinical activity. Thus, in addition to the initial genomic diagnosis, precise determination of the actual mechanism of resistance is a key requirement to provide optimal clinical care to patients throughout the different lines of therapy. Although EGFR mutations and ALK rearrangements have already reached the general clinical routine, several additional pairs of cancer mutations and response to new therapeutic drugs are currently in clinical development (eg, ROS1, BRAF, FGFR2, RET, and NTRK1). As a consequence, the requirements for comprehensive cancer genomic diagnosis are growing at a dramatic pace, with the number of genes that need to be tested increasing steadily. In addition, the major types of therapeutically relevant genome alterations in cancer (eg, gene mutations, amplifications or deletions, and rearrangements or fusions) pose entirely different challenges to their detection. Finally, in contrast to these demands, most diagnostic samples in patients with advanced non–small-cell lung cancer are small, with minimal tumor content, and it is often difficult to obtain larger tumor tissue because of tumor location and patient comorbidity. The technical approaches to address this complexity can be grossly subdivided into three generations of development (Table 1): one, conventional Sanger sequencing (for mutation detection) plus microscopy coupled with fluorescence in situ hybridization (FISH; for detection of amplifications, rearrangements, or fusions); two, multiplex panel testing for mutation detection in addition to FISH; and three, comprehensive capture-based massively parallel sequencing. Although the advantage of Sanger sequencing is in the low associated costs, this technique typically lacks the sensitivity for identification of mutations in heterogeneous cancer specimens (eg, in those with low tumor content) and is therefore not ideally suited for clinical application. Several approaches for simultaneous (ie, multiplex) detection of cancer gene mutations have been developed over the past several years, with the goal of providing a comprehensive view of the mutational pattern in a given tumor. These approaches use various underlying analytic methods, ranging from mass-spectrometric genotyping over real-time polymerase chain reaction (PCR) to multiplex PCR-based massively parallel next-generation sequencing. All of these approaches have in common enhanced analytic sensitivity for mutations occurring at low frequencies in heterogeneous cancer specimens and a broad coverage of therapeutically relevant mutations. In contrast to sequencing-based methods, genotyping has the inherent disadvantage that individual mutations must be prespecified; that is, rare mutations affecting the same residue in the protein, but which are not part of the genotyping panel, will be missed. In addition, these secondgeneration approaches all have in common that cancer gene amplifications or rearrangements or gene fusions still require detection by FISH. In contrast, third-generation cancer genome analysis methods similarly use massively parallel next-generation sequencing, but they involve hybrid capture-based shotgun sequencing of cancer genome sequencing libraries prepared from formalin-fixed tissue. The advantage of such an approach lies in the possibility of covering all sites of recurrent genome alterations coupled with high sensitivity and the capability of reliably identifying all three types of cancer genome alterations (mutations, amplifications or deletions, and rearrangements or fusions) in a single assay. Thus, capture-based massively