A subset of small cell lung cancer with low neuroendocrine expression and good prognosis: a comparison study of surgical and inoperable cases with biopsy.

Patients with small cell lung carcinoma (SCLC) rarely demonstrate long-term survival. We previously reported that gene expression profiling identified a subset of SCLC with good prognosis in surgical cases. To find an easier way to routinely identify SCLC belonging to this subset, we conducted the present study with a hypothesis that neuroendocrine (NE) or basaloid (BA) phenotypes may influence prognosis. To confirm the subset, we used an array platform to analyze fresh samples. Because inoperable cases may differ from surgical cases, we enrolled 51 biopsy cases and 43 resected samples. To evaluate NE and BA phenotypes, we used NE (synaptophysin, chromogranin A, and CD56) and BA (p63 and CK34ßE12) markers. To varying extents, expression profiling based on the array platform duplicated the subsets. For NE phenotypes, 77% of surgical cases and 100% of biopsy cases were positive for at least 1 marker. For BA phenotypes, only 19% of surgical cases were positive for at least 1 marker, whereas there were no positive biopsy cases. Cases undergoing surgery were categorized based on NE and BA immunoreactivity; 58% into NE+BA-, 19% into NE+BA+, 23% into NE-BA-, and 0 into NE-BA+ groups. NE- patients (n = 10) demonstrated a significantly better prognosis (P = .0306) than their NE+ counterparts (n = 33), whereas no survival difference was evident between the BA+ and BA- groups. Multivariate analyses showed that NE positivity was an independent prognostic factor. In conclusion, the SCLC subset with good prognosis is identified by low NE marker expression, which was found only in surgical cases.

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.

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.

BACKGROUND: 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. METHODS: Anti-ALK immunohistochemistry was used to screen 355 tumor tissues, using the intercalated antibody-enhanced polymer (iAEP) method. The cohort consisted of 255 clear cell RCCs, 32 papillary RCCs, 34 chromophobe RCCs, 6 collecting duct carcinomas, 10 unclassified RCCs, 6 sarcomatoid RCCs, and 12 other tumors. RESULTS: Two patients (36- and 53-year-old females) were positive for ALK as determined by iAEP immunohistochemistry. Using 5'- rapid amplification of complementary DNA ends, we detected TPM3-ALK and EML4-ALK in these tumors. The results of this study were confirmed by fluorescence in situ hybridization assays. The 2 ALK-positive RCCs were unclassified (mixed features of papillary, mucinous cribriform, and solid patterns with rhabdoid cells) and papillary subtype. They comprised 2.3% of non-clear cell RCCs (2 of 88) and 3.7% of non-clear cell and nonchromophobe RCCs (2 of 54). CONCLUSIONS: The results of this study indicate that ALK fusions also exist in adult RCC cases without uncommon backgrounds. These findings confirm the potential of ALK inhibitor therapy for selected cases of RCC.

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.

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.

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 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.

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.