Tumor Driver Mutations Research Articles

Genetic Background May Contribute to PAM50 Gene Expression Breast Cancer Subtype Assignments

Recent advances in genome wide transcriptional analysis have provided greater insights into the etiology and heterogeneity of breast cancer. Molecular signatures have been developed that stratify the conventional estrogen receptor positive or negative categories into subtypes that are associated with differing clinical outcomes. It is thought that the expression patterns of the molecular subtypes primarily reflect cell-of-origin or tumor driver mutations. In this study however, using a genetically engineered mouse mammary tumor model we demonstrate that the PAM50 subtype signature of tumors driven by a common oncogenic event can be significantly influenced by the genetic background on which the tumor arises. These results have important implications for interpretation of “snapshot” expression profiles, as well as suggesting that incorporation of genetic background effects may allow investigation into phenotypes not initially anticipated in individual mouse models of cancer.

Identification of Somatic Genomic Alterations in Circulating Tumors Cells: Another Step Forward in Non–Small-Cell Lung Cancer?

The last decade has witnessed substantial progress in defining the molecular determinants of non–small-cell lung cancer (NSCLC) and demonstrating how these can be exploited in the clinic. In 2004, the identification of somatic mutations in the epidermal growth factor receptor (EGFR) gene provided the first insight into a clinically relevant NSCLC driver oncogene. EGFR mutations in NSCLC are transforming, enhance the activity of the kinase domain of EGFR, and increase the affinity for EGFR tyrosine kinase inhibitors (TKIs). In EGFR-mutated NSCLCs (ie, tumors with classic EGFR exon 19 deletions or the L858R mutation, which are found in approximately 15% of all NSCLCs), clinical and radiographic responses are achieved in most patients with the use of two commercially available reversible EGFR TKIs: gefitinib and erlotinib. These EGFR TKIs improve outcomes when compared with cytotoxic chemotherapies, and the evidence-based use of these drugs, as recommended by the American Society of Clinical Oncology and other practice and regulatory agencies, is now restricted to EGFR-mutated NSCLCs in the first-line treatment of advanced tumors. The significant palliative benefits that are offered by matching a tumor driver mutation with an appropriate inhibitor paved the way for the genomic characterization of NSCLCs and the development of novel TKIs that can target these changes. The deciphering of NSCLCs using next-generation sequencing (through whole-genome, exome, and transcriptome techniques) has identified numerous driver oncogenic events (ie, activating mutations or rearrangements) involving targetable kinases, including ROS1, ERBB2, BRAF, and RET, among others. The separation of NSCLCs into distinct, actionable subtypes is mostly clear in lung adenocarcinomas of never-smokers (Figure 1), in which almost all tumors have a mutually exclusive driver oncogene for which TKIs are either clinically available or in earlyto late-stage development. Amid the exciting series of discoveries that have occurred during the last decade, one of the most remarkable has been the story of how anaplastic lymphoma kinase (ALK) gene alterations became a biomarker and therapeutic target in NSCLC. ALK is a transmembrane tyrosine kinase that had been previously implicated in the pathogenesis of anaplastic large cell lymphoma and neuroblastoma. In 2007, a Japanese group from Jichi Medical University identified novel fusion oncogenes involving the kinase domain of ALK in patients with NSCLC. ALK rearrangements, either inversions or translocations, characterize the genomic changes observed in NSCLC. The most frequent event is an inversion in the short arm of chromosome 2 that results in the fusion of the echinoderm microtubule-associated protein-like 4 (EML4) with ALK, leading to the production of an EML4-ALK fusion tyrosine kinase. Several EML4-ALK variants have now been described, with EML4-ALK E13;A20 and E20;A20 being the most common. Few other tumor types harbor these EML4-ALK rearrangements, and it remains unclear why NSCLCs can acquire these somatic changes. Interestingly, some clinical and pathologic features are associated with an increased incidence of tumors containing ALK rearrangements. These include never or light smoking history ( 15 pack-years), young age, and adenocarcinoma histology with signet rings. The reported prevalence of ALK rearrangements in NSCLC is approximately 5%; however, in neversmokers or light smokers with lung adenocarcinoma, the prevalence may be as high as 20%. Therefore, as many as 10,000 new cases of ALK-rearranged NSCLC are expected in the United States this year. Other/unknown

Abstract 3927: Evaluating new therapies in Gastrointestinal Stromal Tumor using in vivo molecular optical imaging.

Abstract Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors with an estimated annual incidence of ∼6,000 new cases in the United States. The majority (∼75%) of GISTs possess gain-of-function mutations in KIT exons 9, 11, 13 or 17, causing constitutive activation of the kinase receptor, with smaller subsets possessing either gain-of-function mutations in PDGFRA (exons 12, 14 or 18) or BRAF (exon 15). Management of GIST has been transformed by the identification of these tumor driver mutations leading to unprecedented disease control of advanced GIST with the introduction of imatinib mesylate (IM). Despite the efficacy of IM, most patients experience primary and/or secondary resistance within 2 years of treatment. Therefore additional therapies are needed and methods to optimize screening of novel approaches are warranted. Efficacy of treatment is typically assessed using Response Evaluation Criteria In Solid Tumors (RECIST) guidelines. Using RECIST, patients with advanced disease have equivalent survival when they have stable disease as when they have complete or partial responses. The Choi criteria define treatment response as a >10% decrease in tumor size or a >15% decrease in tumor density of tumor lesions assessed on CT scans. Response by Choi criteria has been shown to correlate better with FDG-PET than RECIST response, but can only be used with contrast enhanced CT imaging. RECIST and Choi both require a period of time on therapy before changes indicative of response can be observed. None of these criteria however informs us directly about cell death. Here we evaluate the use of molecular imaging technology using near-infrared (NIR) imaging probes in combination with three-dimensional fluorescence molecular tomography (FMT) for assessing therapeutic response and ultimately optimizing our understanding of the biologic effects of these agents. We determine the potential of two NIR probes (PSVueTM794 and KcapQ647) for detecting apoptosis and compare this to tumor size measured by MRI in response to IM treatment in GIST-T1 xenografts. PSVueTM794, used as a marker of apoptosis, binds to phosphatidylserine residues exposed on the surface of apoptotic cells and, in addition, binds to negatively-charged necrotic regions found in various tumors. KcapQ647, a cell penetrating activatable probe was used to detect apoptosis-associated caspase acitivity. Our studies revealed statistically significant increases in apoptotic activity due to IM treatment (compared to control) using both the PSVue TM794 and KcapQ647 probes (p=1.41e-04, 0.002 respectively) as early as 24 hours post IM treatment. These findings were confirmed by IHC. We believe that this methodology will allow for faster and more effective screening of novel therapies in preclinical GIST models. Citation Format: Lori A. Rink, Harvey Hensley, Karthik Devarajan, James R. Johnson, David Piwinica-Worms, Andrew K. Godwin, Margaret von Mehren. Evaluating new therapies in Gastrointestinal Stromal Tumor using in vivo molecular optical imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3927. doi:10.1158/1538-7445.AM2013-3927

Abstract PR1: Oncogenic driver mutations shape the nature of immune responses in tumors.

Abstract The composition of tumor-associated leukocytes and their pro- or anti-tumor polarization are heterogeneous among different tumor types. In addition, the abundance of leukocyte subtypes can vary even among tumors of the same type. Understanding what determines this heterogeneity is important since the immune cell contexture of tumors can serve as a strong predictor of clinical outcomes and response to anti-cancer therapy. Given that tumorigenesis is driven by mutations in various types of proto-oncogenes and tumor-suppressor genes, we postulated that the identity of driver mutation could shape the leukocytic composition of tumors. To test this, we used the established mouse models for liver tumorigenesis induced by hydrodynamic transfection of c-MYC (MYC) or myr-AKT1+N-RasV12 (AKT+RAS) oncogenes. The resulting somatic integration and long-term overexpression of MYC or AKT+RAS in the liver leads to development of hepatoblastoma and hepatocellular carcinoma, respectively. We profiled liver-infiltrated CD45+ leukocytes in these two models by staining liver single-cell suspensions with lineage-specific leukocyte markers, followed by polychromatic FACS analysis. A massive increase in the CD11b+/F4/80-/Ly6Ghi/Ly6Cmed fraction of CD45+ leukocytes was detected in the AKT+RAS, but not in the MYC model. Compared to the vehicle-injected controls, AKT+RAS livers exhibited a 3-fold increase in this leukocyte population both in pre-neoplastic stages and in tumors. Overexpression of either oncogenes alone did not trigger such increase, suggesting that CD11b+/F4/80-/Ly6Ghi/Ly6Cmed response resulted from cooperation between AKT and RAS. Consistent with the FACS results, IHC staining of liver tissue sections with the neutrophil marker 7/4 antigen revealed an abundant positive staining both in pre-neoplastic livers and in tumors in the AKT+RAS model, while only a small infiltration of 7/4 antigen-positive cells was observed the MYC model only at tumor stage. To address if CD11b+/F4/80-/Ly6Ghi/Ly6Cmed play a role in tumorigenesis, we treated mice with the rat monoclonal Gr-1 antibody to systemically deplete Ly6G/Ly6C-expressing cells. Treatment with Gr-1 starting on day -1 before oncogene transfection ablated CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells from the livers and showed a tendency to increase the tumor burden in AKT+RAS mice for nearly 50%, suggesting that these cells act in early stages to suppress AKT+RAS-induced tumorigenesis. In contrast, Gr-1 treatment starting on day 4 after oncogene transfection no longer had an effect on AKT+RAS-induced tumorigenesis. Nevertheless, the same protocol significantly extended survival of mice and reduced tumor burden in the MYC model, suggesting a tumor-promoting role of CD11b+/F4/80-/Ly6Ghi/Ly6Cmed in MYC-induced tumorigenesis. In conclusion, our results demonstrate that the leukocytic composition and the nature of immune responses during tumorigenesis are shaped, at least in part, by tumor-driving mutations. We are currently elucidating the mechanism by which CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells promote or suppress tumorigenesis driven by MYC and AKT+RAS. This abstract is also presented as Poster A46. Citation Format: Vladislava Juric, Brian Ruffell, J. Michael Bishop. Oncogenic driver mutations shape the nature of immune responses in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR1.

Ethnic difference of driver mutation frequencies and correlations between driver mutations and histologic subtypes in lung adenocarcinoma.

1552 Background: The frequencies of known driver mutation in lung adenocarcinoma from patients in the United States have been reported by the NCI’s Lung Cancer Mutation Consortium (LCMC), indicating driver mutations were detected in 54% (280/516) of tumors. In this report, mutations found: EGFR 17%, KRAS 22%, HER2 0.6%, PIK3CA 1.2%, BRAF 2%, MET amplification 0.6%, MAP2K1 0.4%, NRAS 0.4%, AKT 0%, ALK rearrangements 7%. However little is known about ethnic difference of driver mutation frequencies and correlations between driver mutations and histological subtypes in lung adenocarcinoma. Methods: Known driver mutations in tumors from 97 Japanese patients with lung adenocarcinoma who underwent surgical resection between 1999 and 2003 in National Cancer Center Hospital East were analyzed by next-generation sequencing and confirmed by Sanger sequencing. Correlations between driver mutations and histological subtypes were also assessed. Results: Driver mutations were detected in 72% of tumors. Mutations found: EGFR 57%, KRAS9%, HER2 2%, PIK3CA 2%, BRAF 1%, MET amplification 1%, MAP2K1 0%, NRAS 0%, AKT 0%. Due to the limitation of rearrangement detection by exon-sequencing, ALK rearrangements were not analyzed. Compared with the report by LCMC, the frequency of EGFR mutations was high and that of KRAS mutations was low in the present study. All mutations were mutually exclusive. The number of predominant histological subtypes of tumors harbored EGFR mutations were papillary 28, acinar 3, solid 5, lepidic 19. That with KRAS mutations showed papillary 2, acinar 2, solid 2, lepidic 3, and HER2 mutations showed papillary 1 and acinar 1. Two tumors harbored PIK3CA mutations showed both histological acinar pattern. Each of BRAF mutation and MET amplification showed lepidic and papillary pattern, respectively. Conclusions: It was suggested that there should be ethnic difference of driver mutation frequencies in lung adenocarcinoma between Asian and non-Asian patients, although the details of ethnic distribution included in LCMC study has not been opened. In addition, each driver mutations did not correspond to specific histological subtypes of lung adenocarcinoma.

Cell population genetics and deep sequencing: A novel approach for drivers discovery in hepatocellular carcinoma

We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues.Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all nine tumor and seven nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein coding mutation. Hence, by using a cell-population genetic definition,this approach identified three coding changes (CCNG1, P62,and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.

Abstract IA2: Cancer genomes and their implications for basic and applied research

Abstract Human cancer is, in essence, a genetic disease. Though this pivotal fact has been established over the past three decades, only recently has the compendium of genetic alterations in tumors been identified at a genome-wide scale. The basic landscapes of cancer genomes are becoming clear and the implications of this research are evident. These landscapes are populated by a few “mountains” – genes that are mutated at high frequency in specific tumor types – but are dominated by hills, representing genes that are mutated in only a relatively small fraction of the tumors. The vast majority of genetic alterations are point mutations, and most of these are passengers, i.e., provide no selective growth advantage to the tumor cell. Moreover, virtually all of the driver mutations in common solid tumors are in tumor suppressor genes rather than in oncogenes. All the bona fide cancer genes identified to date affect one or more of a dozen pathways that are central to neoplasia. Appreciation of this landscape has profound implications for both understanding the disease and managing patients with cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA2.

Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data

We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues. Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all 9 tumor and 7 nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein-coding mutation. Hence, by using a cell-population genetic definition, this approach identified three coding changes (CCNG1, P62, and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.

Identification of driver mutations in tumor specimens from 1,000 patients with lung adenocarcinoma: The NCI’s Lung Cancer Mutation Consortium (LCMC).

CRA7506 Background: The ability to detect driver mutations like EGFR and EML4-ALK in tumor specimens from patients with lung cancer and administer agents targeting those molecular lesions has revolutionized the management of adenocarcinoma of the lung. The availability of multiplexed assays to detect mutations permits the identification of multiple driver mutations from tumors at diagnosis. The number of molecular lesions and new agents to target them continues to grow. To exploit this, we created the LCMC to determine 10 driver mutations in tumors from 1,000 patients and to give the results to clinicians for care and entry onto targeted therapeutic trials based on these findings. Methods: The 14 member LCMC is prospectively enrolling patients to test tumors from patients with lung adenocarcinoma in CLIA laboratories for KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, MEK1, and NRAS using standard multiplexed assays and fluorescence in situ hybridization (FISH) for ALK rearrangements and MET amplifications. All are stage IIIB/IV, PS 0-2, have available tissue, and signed consent. Results: 830 patients have been registered with 50 enrolling monthly. We detected a driver mutation in 60% (252/422, 95% CI 55 to 65%) of tumors thus far. Mutations found: KRAS 107 (25%, 95% CI 21 to 30%), EGFR 98 (23%, 95% CI 19 to 27%), ALK rearrangements 14 (6%, 95% CI 4 to11%), BRAF 12 (3%, 95% CI 1 to 5%), PIK3CA 11 (3%, 95% CI 1 to 5%), MET amplifications 4 (2%, 95% CI 0.5 to 5%), HER2 3, (1%, 95% CI 0.1 to 2%), MEK1 2 (0.4%, 95% CI 0.1 to 2%), NRAS 1 (0.2%, 95% CI 0.01 to 1%), AKT1 0 (0%, 95% CI 0 to 1%). 95% of molecular lesions were mutually exclusive. Conclusions: We detected an actionable driver mutation in 60% of tumors from prospectively studied patients with lung adenocarcinoma. Results of EGFR mutation testing are given to treating physicians to select erlotinib as initial treatment per NCCN and ASCO guidelines. Patients with other driver mutations are offered participation in LCMC-linked trials of agents targeting the mutation identified, e.g. crizotinib with EML4-ALK. At half of LCMC sites, multiplexed testing for all mutations is now routine practice in their pathology departments. Supported by 1RC2CA148394-01.

Applications of the human p53 knock‐in ( Hupki ) mouse model for human carcinogen testing

Tumor-driving mutations in the TP53 gene occur frequently in human cancers. These inactivating mutations arise predominantly from a single-point mutation in the DNA-binding domain of this tumor suppressor gene (i.e., exons 4-9). The human p53 knock-in (Hupki) mouse model was constructed using gene-targeting technology to create a mouse strain that harbors human wild-type TP53 DNA sequences in both copies of the mouse TP53 gene. Replacement of exons 4-9 of the endogenous mouse TP53 alleles in the Hupki mouse with the homologous normal human TP53 gene sequences has offered a humanized replica of the TP53 gene in a murine genetic environment. The Hupki mouse model system has proven to be an invaluable research tool for studying the underlying mechanisms of human TP53 mutagenesis. The utility of the Hupki mouse model system for exploring carcinogen-induced TP53 mutagenesis has been demonstrated in both in vivo animal experiments and in vitro cell culture experiments. Here, we highlight applications of the Hupki mouse model system for investigating mutagenesis induced by a variety of environmental carcinogens, including sunlight ultraviolet radiation, benzo[a]pyrene (a tobacco smoke-derived carcinogen), 3-nitrobenzanthrone (an urban air pollutant), aristolochic acid (a component of Chinese herbal medicine), and aflatoxin B1 (a food contaminant). We summarize the salient findings of the respective studies and discuss their relevance to human cancer etiology.