Yuki Togashi

RET, ROS1 and ALK fusions in lung cancer

Through an integrated molecular- and histopathology-based screening system, we performed a screening for fusions of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1, receptor tyrosine kinase (ROS1) in 1,529 lung cancers and identified 44 ALK-fusion–positive and 13 ROS1-fusion–positive adenocarcinomas, including for unidentified fusion partners for ROS1. In addition, we discovered previously unidentified kinase fusions that may be promising for molecular-targeted therapy, kinesin family member 5B (KIF5B)-ret proto-oncogene (RET) and coiled-coil domain containing 6 (CCDC6)-RET, in 14 adenocarcinomas. A multivariate analysis of 1,116 adenocarcinomas containing these 71 kinase-fusion–positive adenocarcinomas identified four independent factors that are indicators of poor prognosis: age ≥50 years, male sex, high pathological stage and negative kinase-fusion status.

KIF5B-ALK, a Novel Fusion Oncokinase Identified by an Immunohistochemistry-based Diagnostic System for ALK-positive Lung Cancer

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 include 1 with EML4-ALK variant 1, 1 with 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.

EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers.

Introduction: Very recently, we have found a novel fusion product between the echinoderm microtubule-associated protein-like4 (EML4) and the anaplastic lymphoma kinase (ALK) in non-small cell lung cancers (NSCLCs). Tumors featuring EML4-ALK fusion constitute one subtype of NSCLC that might be highly sensitive to ALK inhibitors. Herein, we present results of a first large scale study of EML4-ALK fusion in lung cancers. Methods: Using reverse transcription-polymerase chain reaction for EML4-ALK fusion mRNA, we investigated 149 lung adenocarcinomas, 48 squamous cell carcinomas, 3 large-cell neuroendocrine carcinomas, and 21 small-cell carcinomas. For EML4-ALK-positive cancers, we further investigated the presence of ALK fusion proteins by immunohistochemistry. Results: Five of 149 adenocarcinomas (3.4%) showed EML4-ALK fusion mRNA, this being totally lacking in carcinomas of other types (0/72). In all the fusion-positive cases, ALK fusion protein could be detected in the cytoplasm immunohistochemically. The five fusion cases featured two EML4-ALK variant 1 fusions and three variant 2 fusions. Histologically, both variant 1 cases were mixed type adenocarcinomas, showing papillary with bronchioloalveolar components. Interestingly, all three variant 2 cases were acinar adenocarcinomas, the link being statistically significant (p = 0.00018). None of the five fusion-positive cases demonstrated any mutations of EGFR or KRAS, pointing to a mutually exclusive relationship (p = 0.014). There was no association with smoking habits. Conclusions: In the present first investigation of EML4-ALK fusion in a large study of lung cancers (5/221), we found an interesting histotype-genotype relationship. Furthermore, we could detect the fusion protein by immunohistochemistry, pointing to possible clinical applications.

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.

EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset

A subset of lung cancers harbors a small inversion within chromosome 2p, giving rise to a transforming fusion gene, EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene), which encodes an activated tyrosine kinase. We have earlier examined the presence of EML4-ALK by multiplex reverse transcription-polymerase chain reaction in 363 specimens of lung cancer, identifying 11 adenocarcinoma cases featuring the fusion gene. In this study, we clinicopathologically examined the characteristics of the EML4-ALK-positive cases, including the mutation status of EGFR, KRAS, and TP53, and whether they were of thyroid transcription factor-1 (TTF-1) cell lineage or not. Of 11 patients, 4 (36%) with EML4-ALK-positive lung adenocarcinomas who were below 50 years of age were affected by these diseases, as compared with 12 of 242 patients (5.0%) with EML4-ALK-negative lung adenocarcinomas (P=0.00038). EML4-ALK-positive lung adenocarcinomas were characterized by less-differentiated grade (P=0.0082) and acinar-predominant structure (P<0.0001) in histology. Furthermore, the presence of EML4-ALK appears to be mutually exclusive for EGFR and KRAS mutations (P=0.00018), whereas coexisting with TP53 mutations at a low frequency (1/11=9.1%), and correlating with non- or light smoking (P=0.040), in line with the TTF-1 immunoreactivity. Thus, EML4-ALK-positive tumors may form a distinct entity among lung adenocarcinomas, characterized by young onset, acinar histology, no or rare mutations in EGFR, KRAS, and TP53, and a TTF-1 cell lineage, all in agreement with the prevalence in non- or light smokers.

KLC1-ALK: A Novel Fusion in Lung Cancer Identified Using a Formalin-Fixed Paraffin-Embedded Tissue Only

The promising results of anaplastic lymphoma kinase (ALK) inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE), and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5′-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

Identification of anaplastic lymphoma kinase fusions in renal cancer: large-scale immunohistochemical screening by the intercalated antibody-enhanced polymer method.

Several promising molecular‐targeted drugs are used for advanced renal cancers. However, complete remission is rarely achieved, because none of the drugs targets a key molecule that is specific to the cancer, or is associated with “oncogene addiction” (dependence on one or a few oncogenes for cell survival) of renal cancer. Recently, an anaplastic lymphoma kinase (ALK) fusion, vinculin‐ALK, has been reported in pediatric renal cell carcinoma (RCC) cases who have a history of sickle cell trait. In this context, ALK inhibitor therapy would constitute a therapeutic advance, as has previously been demonstrated with lung cancer, inflammatory myofibroblastic tumors, and anaplastic large cell lymphomas.

Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma

ALK-positive large B-cell lymphoma is a rare subtype of lymphoma, and most cases follow an aggressive clinical course with a poor prognosis. We examined an ALK-positive large B-cell lymphoma case showing an anti-ALK immunohistochemistry pattern distinct from those of 2 known ALK fusions, CLTC-ALK and NPM-ALK, for the presence of a novel ALK fusion; this led to the identification of SQSTM1-ALK. SQSTM1 is an ubiquitin binding protein that is associated with oxidative stress, cell signaling, and autophagy. We showed transforming activities of SQSTM1-ALK with a focus formation assay and an in vivo tumorigenicity assay using 3T3 fibroblasts infected with a recombinant retrovirus encoding SQSTM1-ALK. ALK-inhibitor therapies are promising for treating ALK-positive large B-cell lymphoma, especially for refractory cases. SQSTM1-ALK may be a rare fusion, but our data provide novel biological insights and serve as a key for the accurate diagnosis of this rare lymphoma.

Pulmonary Inflammatory Myofibroblastic Tumor Expressing a Novel Fusion, PPFIBP1–ALK: Reappraisal of Anti-ALK Immunohistochemistry as a Tool for Novel ALK Fusion Identification

Purpose: The anaplastic lymphoma kinase (ALK) inhibitor crizotinib has been used in patients with lung cancer or inflammatory myofibroblastic tumor (IMT), both types harboring ALK fusions. However, detection of some ALK fusions is problematic with conventional anti-ALK immunohistochemistry because of their low expression. By using sensitive immunohistochemistry, therefore, we reassessed “ALK-negative” IMT cases defined with conventional immunohistochemistry (approximately 50% of all examined cases). Experimental Design: Two cases of ALK-negative IMT defined with conventional anti-ALK immunohistochemistry were further analyzed with sensitive immunohistochemistry [the intercalated antibody-enhanced polymer (iAEP) method]. Results: The two “ALK-negative” IMTs were found positive for anti-ALK immunohistochemistry with the iAEP method. 5′-rapid amplification of cDNA ends identified a novel partner of ALK fusion, protein-tyrosine phosphatase, receptor-type, F polypeptide-interacting protein-binding protein 1 (PPFIBP1) in one case. The presence of PPFIBP1–ALK fusion was confirmed with reverse transcriptase PCR, genomic PCR, and FISH. We confirmed the transforming activities of PPFIBP1–ALK with a focus formation assay and an in vivo tumorigenicity assay by using 3T3 fibroblasts infected with a recombinant retrovirus encoding PPFIBP1–ALK. Surprisingly, the fusion was also detected by FISH in the other case. Conclusions: Sensitive immunohistochemical methods such as iAEP will broaden the potential value of immunohistochemistry. The current ALK positivity rate in IMT should be reassessed with a more highly sensitive method such as iAEP to accurately identify those patients who might benefit from ALK-inhibitor therapies. Novel ALK fusions are being identified in various tumors in addition to IMT, and thus a reassessment of other “ALK-negative” cancers may be required in the forthcoming era of ALK-inhibitor therapy. Clin Cancer Res; 17(10); 3341–8. ©2011 AACR.