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.

The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion-type tyrosine kinase is an oncoprotein found in 4 to 5% of non-small-cell lung cancers, and clinical trials of specific inhibitors of ALK for the treatment of such tumors are currently under way. Here, we report the discovery of two secondary mutations within the kinase domain of EML4-ALK in tumor cells isolated from a patient during the relapse phase of treatment with an ALK inhibitor. Each mutation developed independently in subclones of the tumor and conferred marked resistance to two different ALK inhibitors. (Funded by the Ministry of Health, Labor, and Welfare of Japan, and others.).

Purpose: EML4-ALK is a transforming fusion tyrosine kinase, several isoforms of which have been identified in lung cancer. Immunohistochemical detection of EML4-ALK has proved difficult, however, likely as a result of low transcriptional activity conferred by the promoter-enhancer region of EML4. The sensitivity of EML4-ALK detection by immunohistochemistry should be increased adequately. Experimental Design: We developed an intercalated antibody-enhanced polymer (iAEP) method that incorporates an intercalating antibody between the primary antibody to ALK and the dextran polymer-based detection reagents. Results: Our iAEP method discriminated between tumors positive or negative for EML4-ALK in a test set of specimens. Four tumors were also found to be positive for ALK in an archive of lung adenocarcinoma (n = 130) and another 4 among fresh cases analyzed in a diagnostic laboratory. These 8 tumors were found to include1with EML4-ALK variant1,1with variant 2, 3 with variant 3, and 2 with previously unidentified variants (designated variants 6 and 7). Inverse reverse transcription-PCR analysis revealed that the remaining tumor harbored a novel fusion in which intron 24 of KIF5B was ligated to intron 19 of ALK. Multiplex reverse transcription-PCR analysis of additional archival tumor specimens identified another case of lung adenocarcinoma positive for KIF5B-ALK. Conclusions: The iAEP method should prove suitable for immunohistochemical screening of tumors positive for ALK or ALK fusion proteins among pathologic archives. Coupling of PCR-based detection to the iAEP method should further facilitate the rapid identification of novel ALK fusion genes such as KIF5B-ALK.Gene fusion is a major mechanism of carcinogenesis in hematologic malignancies and sarcomas (1). Identification of the BCR-ABL fusion kinase, which is generated as a result of the balanced chromosome anomaly t(9;22)(q34;q11) in chronic myelogenous leukemia (2), has thus been followed by the discovery of many fusion-type oncogenes (3). In contrast, it has remained unclear whether such translocationdependent fusion-type oncogenes also play a major role in the pathogenesis of epithelial tumors. Recently, however, almost 50% of prostate cancer cases have been suggested to harbor gene fusions involving ETS transcription factor loci (4), and we have discovered a recurrent chromosome translocation, inv(2)(p21p23), in non -small cell lung cancer (NSCLC) that results in the production of an EML4-ALK fusion-type protein tyrosine kinase (PTK;.Forced expression of EML4-ALK in lung epithelial cells induced the rapid development of hundreds of lung cancer nodules in mice, and peroral administration of inhibitors of the PTK activity of EML4-ALK was shown to clear such tumors from the lungs, demonstrating the pivotal role of EML4-ALK in the pathogenesis of NSCLC positive for this fusion kinase (9). This latter observation also supports the clinical application of ALK

The effect of motor activity on the left fronto-central region of the human brain was analyzed spatially and temporally by using noninvasive near-infrared light (NIR) topography. The changes in oxygenation states caused by motor activity were measured using intensity-modulated NIR spectroscopy at ten measurement positions on the head surface. The subject randomly performed unilateral finger opposition for 30 s as motor stimulation. When the subject performed contralateral (right) finger movement, significant increases in both oxygenated hemoglobin (oxy-Hb) and total hemoglobin (total-Hb) and decreases in deoxygenated hemoglobin (deoxy-Hb) were observed in a particular area. By mapping the static topograms of the changes of each Hb and comparing them with an anatomical image of MRI, it was found that the particular area was located on the motor cortex along the central sulcus. By mapping the dynamic topograms of the changes of total-Hb, which reflect the cerebral blood volume, and analyzing the spatiotemporal hemodynamic changes associated with the brain activity, it was found that the regional change in cerebral blood volume in the primary motor area overlaps the global change around the motor cortex. These results demonstrate that NIR topography can be used to effectively observe the human brain activity.

It is generally thought that skilled behavior in human beings results from a functional hierarchy of the motor control system, within which reusable motor primitives are flexibly integrated into various sensori-motor sequence patterns. The underlying neural mechanisms governing the way in which continuous sensori-motor flows are segmented into primitives and the way in which series of primitives are integrated into various behavior sequences have, however, not yet been clarified. In earlier studies, this functional hierarchy has been realized through the use of explicit hierarchical structure, with local modules representing motor primitives in the lower level and a higher module representing sequences of primitives switched via additional mechanisms such as gate-selecting. When sequences contain similarities and overlap, however, a conflict arises in such earlier models between generalization and segmentation, induced by this separated modular structure. To address this issue, we propose a different type of neural network model. The current model neither makes use of separate local modules to represent primitives nor introduces explicit hierarchical structure. Rather than forcing architectural hierarchy onto the system, functional hierarchy emerges through a form of self-organization that is based on two distinct types of neurons, each with different time properties (“multiple timescales”). Through the introduction of multiple timescales, continuous sequences of behavior are segmented into reusable primitives, and the primitives, in turn, are flexibly integrated into novel sequences. In experiments, the proposed network model, coordinating the physical body of a humanoid robot through high-dimensional sensori-motor control, also successfully situated itself within a physical environment. Our results suggest that it is not only the spatial connections between neurons but also the timescales of neural activity that act as important mechanisms leading to functional hierarchy in neural systems.

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.

Since its publication in 2007, the Tokyo Guidelines for the management of acute cholangitis and cholecystitis (TG07) have been widely adopted. The validation of TG07 conducted in terms of clinical practice has shown that the diagnostic criteria for acute cholecystitis are highly reliable but that the definition of definite diagnosis is ambiguous. Discussion by the Tokyo Guidelines Revision Committee concluded that acute cholecystitis should be suspected when Murphy's sign, local inflammatory findings in the gallbladder such as right upper quadrant abdominal pain and tenderness, and fever and systemic inflammatory reaction findings detected by blood tests are present but that definite diagnosis of acute cholecystitis can be made only on the basis of the imaging of ultrasonography, computed tomography or scintigraphy (HIDA scan). These proposed diagnostic criteria provided better specificity and accuracy rates than the TG07 diagnostic criteria. As for the severity assessment criteria in TG07, there is evidence that TG07 resulted in clarification of the concept of severe acute cholecystitis. Furthermore, there is evidence that severity assessment in TG07 has led to a reduction in the mean duration of hospital stay. As for the factors used to establish a moderate grade of acute cholecystitis, such as leukocytosis, ALP, old age, diabetes, being Electronic supplementary material The online version of this article

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 smallmolecule 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.transgenic mouse ͉ surfactant protein C ͉ molecular targeted therapy L ung cancer remains the leading cause of cancer deaths, with almost 1.3 million people dying annually from this condition worldwide (www.who.int/cancer/en). Although a variety of chemotherapeutic regimens have been developed to treat this intractable disease, their efficacy is limited and depends on cancer subtype. Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer cases and is less sensitive to cytotoxic drugs than is small cell lung cancer. Unless tumor cells are surgically resected at an early clinical stage, individuals with NSCLC can expect a median survival time of less than 1 year (1).A subset of individuals with NSCLC (mostly nonsmokers, young females, and those of Asian ethnicity) have been shown to harbor mutations in the epidermal growth factor receptor (EGFR) gene (2-4). Such mutations result in constitutive activation of the EGFR tyrosine kinase, the oncogenic potential of which has been demonstrated in a transgenic mouse system (5). Small-molecule drugs that specifically inhibit the catalytic activity of EGFR have been found to exhibit clinical efficacy in the treatment of NSCLC patients, especially in those with an activated EGFR (6, 7).We recently developed a system for the construction of retroviral cDNA libraries from small quantities of clinical specimens (8-10), and we applied this technology to NSCLC to screen for oncogenes that might be potential drug targets (11). With the use of a focus-formation assay performed with mouse 3T3 fibroblasts, we identified a fusion-type oncogene, EML4-ALK, in an NSCLC specimen of a smoker (12). A small inversion within t...