Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4-ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

A mouse model for EML4-ALK-positive lung cancer.

EML4-ALK is a fusion-type protein tyrosine kinase that is generated in human non-small-cell lung cancer (NSCLC) as a result of a recurrent chromosome inversion, inv (2)(p21p23). Although mouse 3T3 fibroblasts expressing human EML4-ALK form transformed foci in culture and s.c. tumors in nude mice, it has remained unclear whether this fusion protein plays an essential role in the carcinogenesis of NSCLC. To address this issue, we have now established transgenic mouse lines that express EML4-ALK specifically in lung alveolar epithelial cells. All of the transgenic mice examined developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth, confirming the potent oncogenic activity of the fusion kinase. Although such tumors underwent progressive enlargement in control animals, oral administration of a small-molecule inhibitor of the kinase activity of ALK resulted in their rapid disappearance. Similarly, whereas i.v. injection of 3T3 cells expressing EML4-ALK induced lethal respiratory failure in recipient nude mice, administration of the ALK inhibitor effectively cleared the tumor burden and improved the survival of such animals. These data together reinforce the pivotal role of EML4-ALK in the pathogenesis of NSCLC in humans, and they provide experimental support for the treatment of this intractable cancer with ALK inhibitors.

Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts.

PURPOSE: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2)(p21p23) in the genome of non-small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes. EXPERIMENTAL DESIGN: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs. RESULTS: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens. CONCLUSIONS: These data reinforce the importance of accurate diagnosis of EML4-ALK-positive tumors for the optimization of treatment strategies.

[Hypersensitity pneumonitis in a greenhouse rose grower].

A 54-year-old woman presented with chronic cough. She had been engaged in growing roses in plastic greenhouses. Chest CT scan showed bilateral diffuse ground-glass attenuation. Bronchoalveolar lavage fluid demonstrated the increase of total cell counts with predominant lymphocyte cells, and transbronchial lung biopsy specimen revealed lymphocyte alveolitis with granuloma. Precipitating antibody against the extract of the Penicillium species obtained in the greenhouses was detected by the double immunodiffusion test, which led to a diagnosis of hypersensitivity pneumonitis caused by Penicillium species. This case suggests the risk of hypersensitivity pneumonitis caused by fungi in closed spaces with high temperature and humidity such as greenhouses.

Preventive effects of edaravone, a free radical scavenger, on lipopolysaccharide-induced lung injury in mice.

BACKGROUND AND OBJECTIVE: Reactive oxygen species (ROS) play an important role in the pathogenesis of acute lung injury (ALI) and pulmonary fibrosis. It was hypothesized that edaravone, a free radical scavenger, would be able to attenuate LPS-induced lung injury in mice by decreasing oxidative stress. METHODS: For the in vivo experiments, lung injury was induced in female BALB/c mice by the intranasal instillation of LPS. Edaravone was given by intraperitoneal administration 1 h before the LPS challenge. For the in vitro experiments, MH-S cells (murine alveolar macrophage cell line) were exposed to edaravone, followed by stimulation with LPS. RESULTS: In the LPS-induced ALI mouse model, the administration of edaravone attenuated cellular infiltration into and the concentrations of albumin, IL-6, tumour necrosis factor-alpha, keratinocyte-derived chemokine and macrophage inflammatory protein-2 in BAL fluid. In addition, the in vitro studies showed that the elevated IL-6 secretion from MH-S cells in response to LPS was significantly attenuated by co-incubation with edaravone. CONCLUSIONS: In an experimental murine model, a free radical scavenger may prevent ALI via repression of pro-inflammatory cytokine production by lung macrophages.

Mouse microRNA profiles determined with a new and sensitive cloning method.

MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77,436 Small-RNA species was sequenced, with 11,776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner.

Identification of a constitutively active mutant of JAK3 by retroviral expression screening.

To identify transforming genes in acute myeloid leukemia (AML) we here constructed a retroviral cDNA expression library from an AML patient, and then used this library to infect a mouse cell line 32Dcl3-mCAT. cDNA inserts of the cell clones which proliferated in the presence of granulocyte colony-stimulating factor were derived from JAK3 encoding a JAK3 mutant with a valine-to-alanine substitution at codon 674 and two additional amino acid substitutions. The transforming activity of JAK3(V674A) was confirmed by its introduction into 32Dcl3-mCAT. Sequencing of the original JAK3 cDNA derived from the patient, however, failed to detect the V674A mutation.

Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.

Biphenotypic acute leukemia (BAL) is a relatively rare subtype of acute leukemia characterized by the presence of both myeloid and lymphoid cell surface antigens. We have now screened for transforming genes in BAL blasts with the use of the focus formation assay with a retroviral cDNA expression library constructed from malignant blasts isolated from a BAL patient. Some of the retroviral inserts recovered from transformed foci were found to encode wild-type purinergic receptor P2Y, G protein coupled, 8 (P2RY8). The oncogenic potential of P2RY8 was confirmed with the in vitro focus formation assay as well as with an in vivo tumorigenicity assay in nude mice. A variety of luciferase-based reporter assays revealed that P2RY8 increased both the trans-activation activities of CREB and Elk-1 as well as the transcriptional activities of the serum response element and enhancer-promoter fragments of the c-Fos and c-Myc genes. Quantitation of P2RY8 mRNA in CD34(+) cells of bone marrow showed that P2RY8 expression is frequently increased in leukemia patients, especially in those with refractory disease. Our data thus reveal an abundant expression of P2RY8 in leukemic cells and its unexpected role in the pathogenesis of acute leukemia.

Pulmonary nocardiosis with bilateral diffuse granular lung shadows in a patient with subcutaneous panniculitic T-cell lymphoma.

A 40-year-old woman undergoing prednisolone and cyclosporine therapy for subcutaneous panniculitic T-cell lymphoma complained of a cough for a few weeks. A chest X-ray revealed bilateral diffuse granular shadows. Additionally, the patient was discovered to have multiple subcutaneous abscesses. Gram-stained smears of sputum and pus from the abscess showing branched gram-positive rods led to a diagnosis of pulmonary nocardiosis with dissemination to the lungs and subcutaneous tissues. Combination therapy consisting of sulfamethoxazole/trimethoprim and panipenem/betamipron produced rapid improvement of radiographic abnormalities. It is suggested that pulmonary nocardiosis should be considered in the differential diagnosis of diffuse granular shadows on chest X-rays, especially in immunocompromised patients.

Screening for genetic abnormalities involved in ovarian carcinogenesis using retroviral expression libraries.

The purpose of this study was to screen for genes involved in ovarian carcinogenesis in an attempt to develop an effective molecular-targeted therapy for ovarian cancer. We constructed retroviral expression libraries for the human ovarian cancer cell lines SHIN-3 and TYK-CPr, and performed a focus formation assay with 3T3 cells. As a result, proteasome subunit beta-type 2 (PSMB2), ubiquitin-specific protease 14 (USP14), and keratin 8 (KRT8) were identified from SHIN-3, and polymerase II RNA subunit (POLR2E), chaperonin containing T-complex polypeptide 1 subunit 4 (CCT4), glia maturation factor beta (GMFB), and neuroblastoma ras viral oncogene homolog (NRAS) from TYK-CPr. NRAS gene analysis revealed a CAA --> AAA substitution at codon 61, resulting in a Glu --> Lys change at position 61. When the mutant NRAS was introduced into fibroblasts for its expression, many transformed foci were generated, confirming the transforming ability of the mutant NRAS.

Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer.

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening.

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.