Cancer Genome Sequencing—An Interim Analysis

Cancer is a genomic disease associated with the accumulation of mutations (1). The prevailing models of tumorigenesis stress tumor progression as the result of sequential mutations in key cancer genes, each mutation driving a new round of clonal proliferation (2, 3). By cataloging all clonal mutations found in a variety of cancers, it is hoped that it will be possible to identify the key mutated genes of cancer and, in so doing, revolutionize basic and clinical cancer research (4). A series of studies have now been undertaken to annotate the cancer genomes of several different tumor types and it is timely to summarize the results and to evaluate the implications of these findings.

Early attempts at systematically identifying multiple somatic mutations within individual cancers yielded few prevalent clonal alterations (5–7). The relatively limited sequence coverage of these initial studies spurred on attempts at more comprehensive screens of the cancer genome. The first complete sequencing of all likely coding exons of a human cancer genome, conducted in breast and colon cancers (8, 9), concluded that these cancers contain a median of 84 and 76 clonal mutations, respectively, which alter protein function (Table 1). Although almost 10% of the 18,197 genes studied were detectably mutated in at least one specimen, each tumor displayed a unique and diverse profile of mutated genes, and other than those previously known (e.g., TP53 and APC), no new prevalently mutated genes were identified. The authors of this study proposed that the cancer genome landscape is composed of a handful of commonly mutated gene “mountains” but dominated by a vastly larger number of infrequently mutated gene “hills” (Fig. 1A), a view consistent with a large number of mutations, each providing a small fitness advantage, driving tumor progression (9). It is this clonal mutational heterogeneity, the authors concluded, which underlies the wide variation in tumor behavior and responsiveness to therapy.

Subsequent studies on diverse cancer types (Table 1) have generated the most extensive catalogs of somatic point mutations yet undertaken (10–14). The primary conclusion to be drawn from these studies is that most cancer types display substantial heterogeneity at the genetic level. Individual solid organ tumors harbor on average >50 nonsilent clonal mutations in the coding regions of different genes (colorectal ≈ breast >> pancreatic > glioblastoma multiforme), and although a few of these genes are mutated in a high proportion of tumors, the prevalence at which the majority are mutated among different tumors of the same cancer type is low. Additionally, these studies focused exclusively on identifying mutations in exons of known protein-coding genes. With the ever-increasing recognition that so-called “junk” DNA and intronic sequences contain functional elements, including regulatory regions and noncoding RNAs, the total number of clonal mutations with functional consequences cannot be easily estimated.

The characterization of the first hematopoietic cancer genome represents an important methodologic milestone in cancer genome sequencing—truly unbiased whole genome sequencing (15). Prior attempts at resequencing the tyrosine kinase gene family in acute myeloid leukemia (AML) yielded few mutations (16, 17). By exhaustively interrogating the cancer and paired normal genomes of a single AML patient using massively parallel sequencing, Ley and colleagues (15) identified 500 to 1,000 nonsynonymous somatic mutations throughout the cancer genome, of which 10 mutations were in protein-coding genes. Although the overall number of protein coding mutations is considerably fewer than that described for solid organ tumors, similar to the mutational diversity of other tumor types, the genes identified are mutated in only a small fraction of AML cases. Surprisingly, none of the 8 newly identified genes were found to be mutated in a further 187 cases of AML.

Although the absence of prevalently mutated genes is disappointing from a targeted therapeutic perspective, the major shortcoming of all the above studies is not in the complexity of the problem they are attempting to address but in the fact that, by design, they do not analyze the deeper heterogeneity within individual tumors. A primary tumor is itself genomically heterogeneous, with each cell having a unique mutational signature. This genetic variation within a cancer cell population reflects the history and dynamics of clonal evolution and, importantly, serves as a reservoir of genetic diversity from which therapy-resistant clones may arise (18). Analysis of disseminated single cells in minimal residual disease has shown that there is a high level of genomic heterogeneity within individual lesions as well as between primary tumors and metastatic cells (19, 20). Although current “next-generation” sequencers can detect multiple rare subclones with frequencies as low as 1 in 5,000 (21), analyzing rarer subclones awaits an, as yet, undeveloped future generation of DNA sequencing methodologies. It is this intraspecimen diversity, not detected by the cancer genome sequencing studies, that may be clinically most relevant and responsible for much of the diverse spectrum of clinical phenotypes. The question arises of whether there is, at a clinically meaningful level, a single consensus cancer genome per tumor.

We posit that a complete description of the cancer genome must capture the mutational heterogeneity within individual neoplasms as well as between different tumors. In addition to the clonal genomic landscape proposed, comprising mountains and hills of frequently and infrequently mutated genes within a given tumor type, additional mutational diversity exists within individual tumors themselves, with a large number of subclonal mutations being present in only one or a few cells (Fig. 1B). It is this mutational landscape, overlooked by the cancer genome sequencing studies to date, that is the basis for the wide variations in tumor behavior and responsiveness to therapy and represents the clinically most important features of the cancer genome.

New DNA sequencing technologies are undoubtedly important for guiding biological and medical research; however, many limitations exist. To accurately quantify the subclonal diversity of a single tumor, it may be necessary to sequence millions of individual genome equivalents per tumor. Present sequencing efforts only detect mutations that are present in the majority of the cells of a neoplasm. Until more advanced technologies become available or existing ones are retooled, a critical feature of the genomic landscape will continue to go unexplored.

The mutational heterogeneity of cancer, which has been recognized for many years (22) and now confirmed by DNA sequencing, underlies the complexity of the cancer genome landscape. Currently, the heterogeneity within individual tumor types alone confounds many of the original expectations of the cancer genome strategy. A future of multiple targeted therapies and patient stratification, based on a mutational signature of defined key genes for each cancer type, seems less hopeful than initially anticipated. Although ongoing studies will continue to uncover the remarkable heterogeneity inherent in tumorigenesis, without addressing the deeper complexity of each individual cancer genome, we may not significantly affect patient care.

L.A. Loeb: consultant/advisory board, Stratus, Inc. The other authors disclosed no potential conflicts of interest.

Grant support: NIH grants CA102029 and CA78885 (L.A. Loeb) and AG033485 (J.J. Salk) and National Cancer Institute/Health Research Board of Ireland Joint Research Fellowship (E.J. Fox).

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