Lucian R. Chirieac

Somatic mutations affect key pathways in lung adenocarcinoma

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Non-small cell lung cancer.

Most patients with non-small cell lung cancer (NSCLC) are diagnosed with advanced cancer. These guidelines only include information about stage IV NSCLC. Patients with widespread metastatic disease (stage IV) are candidates for systemic therapy, clinical trials, and/or palliative treatment. The goal is to identify patients with metastatic disease before initiating aggressive treatment, thus sparing these patients from unnecessary futile treatment. If metastatic disease is discovered during surgery, then extensive surgery is often aborted. Decisions about treatment should be based on multidisciplinary discussion.

Clinical Features and Outcome of Patients With Non–Small-Cell Lung Cancer Who Harbor EML4-ALK

PURPOSE The EML4-ALK fusion oncogene represents a novel molecular target in a small subset of non-small-cell lung cancers (NSCLC). To aid in identification and treatment of these patients, we examined the clinical characteristics and treatment outcomes of patients who had NSCLC with and without EML4-ALK. PATIENTS AND METHODS Patients with NSCLC were selected for genetic screening on the basis of two or more of the following characteristics: female sex, Asian ethnicity, never/light smoking history, and adenocarcinoma histology. EML4-ALK was identified by using fluorescent in situ hybridization for ALK rearrangements and was confirmed by immunohistochemistry for ALK expression. EGFR and KRAS mutations were determined by DNA sequencing. RESULTS Of 141 tumors screened, 19 (13%) were EML4-ALK mutant, 31 (22%) were EGFR mutant, and 91 (65%) were wild type (WT/WT) for both ALK and EGFR. Compared with the EGFR mutant and WT/WT cohorts, patients with EML4-ALK mutant tumors were significantly younger (P < .001 and P = .005) and were more likely to be men (P = .036 and P = .039). Patients with EML4-ALK-positive tumors, like patients who harbored EGFR mutations, also were more likely to be never/light smokers compared with patients in the WT/WT cohort (P < .001). Eighteen of the 19 EML4-ALK tumors were adenocarcinomas, predominantly the signet ring cell subtype. Among patients with metastatic disease, EML4-ALK positivity was associated with resistance to EGFR tyrosine kinase inhibitors (TKIs). Patients in the EML4-ALK cohort and the WT/WT cohort showed similar response rates to platinum-based combination chemotherapy and no difference in overall survival. CONCLUSION EML4-ALK defines a molecular subset of NSCLC with distinct clinical characteristics. Patients who harbor this mutation do not benefit from EGFR TKIs and should be directed to trials of ALK-targeted agents.

Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers.

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-α catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-α mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-α H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan–PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography–computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

Characterizing the cancer genome in lung adenocarcinoma

Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in ∼12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.

BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz–Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas

Lineage-survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development. Here we show that a peak of genomic amplification on chromosome 3q26.33 found in squamous cell carcinomas (SCCs) of the lung and esophagus contains the transcription factor gene SOX2, which is mutated in hereditary human esophageal malformations, is necessary for normal esophageal squamous development, promotes differentiation and proliferation of basal tracheal cells and cooperates in induction of pluripotent stem cells. SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 here cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These characteristics identify SOX2 as a lineage-survival oncogene in lung and esophageal SCC.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

Unique Clinicopathologic Features Characterize ALK-Rearranged Lung Adenocarcinoma in the Western Population

Purpose: The anaplastic large cell kinase gene (ALK) is rearranged in ∼5% of lung adenocarcinomas within the Asian population. We evaluated the incidence and the characteristics of ALK-rearranged lung adenocarcinomas within the western population and the optimal diagnostic modality to detect ALK rearrangements in routine clinical practice. Experimental Design: We tested 358 lung adenocarcinomas from three institutions for ALK rearrangements by fluorescent in situ hybridization (FISH) and immunohistochemistry with and without tyramide amplification. The clinicopathologic characteristics of tumors with and without ALK rearrangements were compared. Results: We identified 20 (5.6%) lung adenocarcinomas with ALK rearrangements within our cohort of western patients. ALK rearrangement was associated with younger age (P = 0.0002), never smoking (P < 0.0001), advanced clinical stage (P = 0.0001), and a solid histology with signet-ring cells (P < 0.0001). ALK rearrangement was identified by FISH in 95% of cases and immunohistochemistry with and without tyramide amplification in 80% and 40% of cases, respectively, but neither FISH nor immunohistochemistry alone detected all cases with ALK rearrangement on initial screening. None of the ALK-rearranged tumors harbored coexisting EGFR mutations. Conclusions: Lung adenocarcinomas with ALK rearrangements are uncommon in the western population and represent a distinct entity of carcinomas with unique characteristics. For suspected cases, dual diagnostic testing, with FISH and immunohistochemistry, should be considered to accurately identify lung adenocarcinomas with ALK rearrangement. (Clin Cancer Res 2009;15(16):5216–23)