Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas.

BACKGROUND: Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK. RESULTS: In this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5' end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3' end of the ALK signal. Two patients showed a deletion of the 3'ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested. CONCLUSIONS: In a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.

Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer.

Chromosomal inversions within chromosome 2p, resulting in fusions between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes, are a recent focus of treatment options for non-small cell lung cancer. Thirteen EML4-ALK fusion variants have been identified, affecting eight EML4 exons. We have developed an exon scanning approach using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify known and potential variants involving the first 22 EML4 exons. A total of 55 formalin-fixed, paraffin-embedded lung cancer tumors were screened, of which 5 (9%) were positive for EML4-ALK fusions. Four positive cases harbored known fusion variants: variant 3a, 3b, or both in three cases and variant 1 in one case. The fifth positive specimen harbored two novel variants, designated 8a and 8b, involving exon 17 of EML4. Fluorescence in situ hybridization confirmed the presence of EML4-ALK fusions in three of the four RT-PCR-positive specimens with sufficient tissue for examination, and also confirmed absence of fusions in all 19 RT-PCR-negative specimens tested. Immunohistochemistry analysis confirmed ALK protein expression in the sample containing the novel 8a and 8b variants. This RT-PCR-based exon scanning approach avoids the limitations of screening only for previously identified EML4-ALK fusions and provides a simple molecular assay for fusion detection in a clinical diagnostics setting.

Somatic mutations of signaling genes in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths, with non-small-cell lung cancer (NSCLC) accounting for approximately 85% of cases. A significant proportion of NSCLC cases are not diagnosed until a late stage, when aggressive treatments are required but often prolong survival only modestly. Recent advances in molecular characterization of NSCLC have enabled identification of numerous cell growth and proliferation pathways that are disrupted in these tumors. This knowledge has provided insight into the mechanisms of tumor development in various histologic subtypes of NSCLC and has pointed the way toward targeted treatment strategies. In this review, we highlight literature findings of somatic mutations in genes involved in cell growth and proliferation that are commonly found in the various subtypes of NSCLC, and we discuss how these findings may relate to treatment strategies.

P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti.

This study describes the expression patterns of P-type Na(+)/K(+)-ATPase and V-type H(+)-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene, both ATPases are expressed in all of the osmoregulatory tissues of larvae (midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach, Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na(+)/K(+)-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na(+)/K(+)-ATPase, apical V-type H(+)-ATPase) and ion-transporting (V-type H(+)-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations, whereas the adult stomach has uniform expression of basolateral P-type Na(+)/K(+)-ATPase and apical V-type H(+)-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H(+)-ATPase (apically and cytoplasmically) whereas P-type Na(+)/K(+)-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na(+)/K(+)-ATPase (basal) and V-type H(+)-ATPase (apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na(+)/K(+)-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na(+)/K(+)-ATPase and V-type H(+)-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.

Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti.

Transport across insect epithelia is thought to depend on the activity of a vacuolar-type proton ATPase (V-ATPase) that energizes ion transport through a secondary proton/cation exchanger. Although several of the subunits of the V-ATPase have been cloned, the molecular identity of the exchanger has not been elucidated. Here, we present the identification of sodium/proton exchanger isoform 3 (NHE3) from yellow fever mosquito, Aedes aegypti (AeNHE3). AeNHE3 localizes to the basal plasma membrane of Malpighian tubule, midgut and the ion-transporting sector of gastric caeca. Midgut expression of NHE3 shows a different pattern of enrichment between larval and adult stages, implicating it in the maintenance of regional pH in the midgut during the life cycle. In all tissues examined, NHE3 predominantly localizes to the basal membrane. In addition the limited expression in intracellular vesicles in the median Malpighian tubules may reflect a potential functional versatility of NHE3 in a tissue-specific manner. The localization of V-ATPase and NHE3, and exclusion of Na+/K+-ATPase from the distal ion-transporting sector of caeca, indicate that the role of NHE3 in ion and pH regulation is intricately associated with functions of V-ATPase. The AeNHE3 complements yeast mutants deficient in yeast NHEs, NHA1 and NHX1. To further examine the functional property of AeNHE3, we expressed it in NHE-deficient fibroblast cells. AeNHE3 expressing cells were capable of recovering intracellular pH following an acid load. The recovery was independent of the large cytoplasmic region of AeNHE3, implying this domain to be dispensable for NHE3 ion transport function. 22Na+ uptake studies indicated that AeNHE3 is relatively insensitive to amiloride and EIPA and is capable of Na+ transport in the absence of the cytoplasmic tail. Thus, the core domain containing the transmembrane regions of NHE3 is sufficient for pH recovery and ion transport. The present data facilitate refinement of the prevailing models of insect epithelial transport by incorporating basal amiloride-insensitive NHE3 as a critical mediator of transepithelial ion and fluid transport and likely in the maintenance of intracellular pH.

Sindbis virus induces transport processes and alters expression of innate immunity pathway genes in the midgut of the disease vector, Aedes aegypti.

The midgut of hematophagous insects is the initial site of infection by arthropod-borne viruses (arboviruses) and plays a crucial role in vector competence. To further understand processes that occur in the midgut in response to infection by an arbovirus, DNA microarrays were used to analyze gene expression changes following infection by the alphavirus, Sindbis (MRE16 Malaysian strain). Midgut transcription profiles from mosquitoes fed blood containing 10(8)pfu/ml of virus were compared with those from mosquitoes ingesting blood meals having no virus. Transcription profiles from both experimental groups were analyzed at 1, 4, and 8 days post-feeding. Among the many transcription changes observed by microarray analysis, the most dramatic involved three genes that had 25-40-fold increases in transcript levels in virus infected mosquitoes at 4 days post-infection. These genes were synaptic vesicle protein-2 (SV2), potassium-dependent sodium/calcium exchanger (NCKX), and a homologue of Caenorhabditis elegans Unc-93, a putative component of a two-pore potassium channel. We speculate that these changes represent changes in vesicle transport processes. In addition to these observations, transcript changes were observed in infected mosquitoes that suggested involvement of Toll and c-jun amino terminal kinase immune cascades as a response to viral infection.

Blood meal induces global changes in midgut gene expression in the disease vector, Aedes aegypti.

Blood feeding is an essential developmental process for many arthropods and plays a significant role in disease transmission. Understanding physiological responses in the midgut is important because it is the primary site of blood meal digestion and pathogenic infection. Processes that occur in the midgut in response to a blood meal have been studied but are poorly understood. Here, we use cDNA microarrays to examine midgut gene expression on a global level in response to blood feeding to assist in unraveling these processes. We have developed Aedes aegypti microarrays consisting of clones obtained from an expressed sequence tag project. Individual clones were amplified by polymerase chain reaction and printed onto glass slides. These microarrays were used to study the effects of a blood meal on midgut gene expression over a 72-h time course. As a result, a number of genes involved in processes such as nutrient uptake and metabolism, cellular stress responses, ion balance, and PM formation, as well as a number of unknown genes were induced or repressed in response to a blood meal based on this microarray data.