Munehiro Enomoto

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.

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.

High-resolution analysis of chromosome copy number alterations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified, with single nucleotide polymorphism-typing microarrays

Angioimmunoblastic T-cell lymphoma (AILT) and peripheral T-cell lymphoma, unspecified (PTCL-u) are relatively frequent subtypes of T- or natural killer cell lymphoma. To characterize the structural anomalies of chromosomes associated with these disorders, we here determined chromosome copy number alterations (CNAs) and loss of heterozygosity (LOH) at >55 000 single nucleotide polymorphism loci for clinical specimens of AILT (n=40) or PTCL-u (n=33). Recurrent copy number gain common to both conditions was detected on chromosomes 8, 9 and 19, whereas common LOH was most frequent for a region of chromosome 2. AILT- or PTCL-u-specific CNAs or LOH were also identified at 21 regions, some spanning only a few hundred base pairs. We also identified prognosis-related CNAs or LOH by several approaches, including Coxs proportional hazard analysis. Among the genes that mapped to such loci, a poor prognosis was linked to overexpression of CARMA1 at 7p22 and of MYCBP2 at 13q22, with both genes being localized within regions of frequent copy number gain. For a frequent LOH region at 2q34, we also identified IKAROS family zinc-finger 2 cDNAs encoding truncated proteins. Our data indicate that AILT and PTCL-u consist of heterogeneous subgroups with distinct transforming genetic alterations.

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.

MicroRNA expression profiles of human leukemias.

1 Taksin AL, Legrand O, Raffoux E, de Revel T, Thomas X, Contentin N et al. High efficacy and safety profile of fractionated doses of Mylotarg as induction therapy in patients with relapsed acute myeloblastic leukemia: a prospective study of the alfa group. Leukemia 2007; 21: 66–71. 2 Mrozek K, Bloomfield CD. Chromosome aberrations, gene mutations and expression changes, and prognosis in adult acute myeloid leukemia. Hematology 2006 Education program book. American Society of Hematology: Washington, DC, 2006, 169–177. 3 Garrido SM, Bryant E, Appelbaum FR. Allogeneic stem cell transplantation for relapsed and refractory acute myeloid leukemia patients with 11q23 abnormalities. Leuk Res 2000; 24: 481–486. 4 Larson RA, Sivers EL, Stadtmauer EA, Lowenberg B, Estey EH, Dombret H et al. Final report on the efficacy and safety of gemtuzumab ozogamicin (Mylotarg) in patients with CD33 positive acute myeloid leukemia in first recurrence. Cancer 2001; 104: 1442–1452. 5 Muñoz L, Nomdedéu JF, Villamor N, Guardia R, Colomer D, Ribera JM et al. Acute myeloid leukemia with MLL rearrangements: clinicobiological features, prognostic impact and value of flow cytometry in the detection of residual leukemic cells. Leukemia 2003; 17: 76–82.

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.

Analysis of chromosome copy number in leukemic cells by different microarray platforms

Changes in the copy number of chromosomes, or copy number alterations (CNAs), are frequently apparent in the genome of cancer cells and may result in the amplification of oncogenes or deletion of tumor suppressor genes. Such CNAs range in size from an entire chromosome to several kilobase pairs, with many of the smaller changes being undetectable by conventional bacterial artificial chromosome-based comparative genomic hybridization (CGH), which has a resolution of several hundred kilobase pairs. This limitation has recently been overcome by the adaptation of high-density oligonucleotide microarrays that were originally developed for typing of single nucleotide polymorphisms (SNPs) to the evaluation of CNAs. Sophisticated software for such analysis, including dChip (http://biosun1.harvard.edu/complab/dchip) and CNAG (http://www.genome.umin.jp/CNAG.html), is now available online. Although high-density SNP-typing arrays allow determination of CNAs at a resolution of o100 kbp, it is not a simple task to link such data to changes in the number of the corresponding genes. The HGU133 Plus 2.0 (HGU133P2) microarray manufactured by Affymetrix (USA) was designed to quantitate the abundance of 447 000 human transcripts and has been widely used for gene expression profiling. Most of the probe sequences on the array are targeted to exons corresponding to the 30 untranslated region of each transcript. Although, as far as we are aware, there have been no reports on the use of this microarray for CNA assessment or CGH, we reasoned that HGU133P2 may be a useful platform for evaluation of CNAs because (1) cDNA sequences are one of the most updated and comprehensive information for human genome, and (2) copy number values could be calculated directly for each gene. Furthermore, it would be straightforward to compare the copy number and expression level of a given gene through separate hybridization of genomic DNA and mRNA to the array. For this purpose, we modified a protocol for labeling and hybridizing genomic DNA, provided by Affymetrix (Supplementary Information). To examine the fidelity of CNA analysis with the HGU133P2 array, we first isolated genomic DNA from mononuclear cells (MNCs) of peripheral blood from a healthy male volunteer (karyotype: 46,XY) and a healthy female volunteer (karyotype: 46,XX). Genomic DNA was also isolated from a human leukemia cell line (KCL22) with trisomy 8 plus t(9;22) and from transformed human B cells with a karyotype of 48,XXXX or of 47,XY,þ 21 (both from Coriell Cell Repositories, http://ccr.coriell.org). The DNA was fragmented by treatment with DNase I, end-labeled with biotin-conjugated deoxynucleotides and subjected to hybridization with the HGU133P2 array. We first chose 26 354 probe sets that gave reliable signals F those that received the ‘Present’ call from the GeneChip operating software (GCOS, Affymetrix) and had a signal intensity of X1.0 F in the control experiment with the normal male genome. This group contained 960 probe sets that mapped to the X chromosome. The signal intensities of these X chromosome-specific probe sets in the experiment with the normal female genome (46,XX) were then normalized by those of the corresponding probe sets in the data for 46,XY. The mean7s.d. value of these XX/XY ratios for the X chromosome-specific probe sets was 2.0771.19 (Figure 1a) and thus matched the predicted value of 2. The intensities of these probe sets for B cells with the