David J. Kwiatkowski

Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology

Objective: To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Participants: Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Evidence: Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Consensus Process: Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). Conclusions: The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products hamartin and tuberin, respectively. Here, we show that hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.

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.

Mutational Analysis in a Cohort of 224 Tuberous Sclerosis Patients Indicates Increased Severity of TSC2, Compared with TSC1, Disease in Multiple Organs

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.

Reversal of learning deficits in a Tsc2 +/− mouse model of tuberous sclerosis

Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2+/− mice) show deficits in learning and memory. Cognitive deficits in Tsc2+/− mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.

Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs.

IMPORTANCE Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments and enroll patients into clinical trials. OBJECTIVES To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival. DESIGN, SETTING, AND PARTICIPANTS From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival. INTERVENTIONS Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies. MAIN OUTCOMES AND MEASURES Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival. RESULTS From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity score-adjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006). CONCLUSIONS AND RELEVANCE Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01014286.

Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin

Gelsolin, an 82 kDa actin-binding protein, has potent actin filament-severing activity in vitro. To investigate the in vivo function of gelsolin, transgenic gelsolin-null (Gsn-) mice were generated and found to have normal embryonic development and longevity. However, platelet shape changes are decreased in Gsn- mice, causing prolonged bleeding times. Neutrophil migration in vivo into peritoneal exudates and in vitro is delayed. Gsn- dermal fibroblasts have excessive actin stress fibers and migrate more slowly than wild-type fibroblasts, but have increased contractility in vitro. These observations establish the requirement of gelsolin for rapid motile responses in cell types involved in stress responses such as hemostasis, inflammation, and wound healing. Neither gelsolin nor other proteins with similar actin filament-severing activity are expressed in early embryonic cells, indicating that this mechanism of actin filament dynamics is not essential for motility during early embryogenesis.

Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR

Tuberous sclerosis (TSC) is a familial tumor syndrome due to mutations in TSC1 or TSC2, in which progression to malignancy is rare. Primary Tsc2(-/-) murine embryo fibroblast cultures display early senescence with overexpression of p21CIP1/WAF1 that is rescued by loss of TP53. Tsc2(-/-)TP53(-/-) cells, as well as tumors from Tsc2(+/-) mice, display an mTOR-activation signature with constitutive activation of S6K, which is reverted by treatment with rapamycin. Rapamycin also reverts a growth advantage of Tsc2(-/-)TP53(-/-) cells. Tsc1/Tsc2 does not bind directly to mTOR, however, nor does it directly influence mTOR kinase activity or cellular phosphatase activity. There is a marked reduction in Akt activation in Tsc2(-/-)TP53(-/-) and Tsc1(-/-) cells in response to serum and PDGF, along with a reduction in cell ruffling. PDGFRalpha and PDGFRbeta expression is markedly reduced in both the cell lines and Tsc mouse renal cystadenomas, and ectopic expression of PDGFRbeta in Tsc2-null cells restores Akt phosphorylation in response to serum, PDGF, EGF, and insulin. This activation of mTOR along with downregulation of PDGFR PI3K-Akt signaling in cells lacking Tsc1 or Tsc2 may explain why these genes are rarely involved in human cancer. This is in contrast to PTEN, which is a negative upstream regulator of this pathway.

Rapid turnover of actin in dendritic spines and its regulation by activity.

Dendritic spines are motile structures that contain high concentrations of filamentous actin. Using hippocampal neurons expressing fluorescent actin and the method of fluorescence recovery after photobleaching, we found that 85 ± 2% of actin in the spine was dynamic, with a turnover time of 44.2 ± 4.0 s. The rapid turnover is not compatible with current models invoking a large population of stable filaments and static coupling of filaments to postsynaptic components. Low-frequency stimulation known to induce long-term depression in these neurons stabilized nearly half the dynamic actin in the spine. This effect depended on the activation of N-methyl-d-aspartate (NMDA) receptors and the influx of calcium. In neurons from mice lacking gelsolin, a calcium-dependent actin-binding protein, activity-dependent stabilization of actin was impaired. Our studies provide new information on the kinetics of actin turnover in spines, its regulation by neural activity and the mechanisms involved in this regulation.