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Background We screened resected tumor tissues from patients with lung cancer for EGFR mutations, ALK rearrangements, and rearranged during transfection (RET) gene variants (including RET rearrangements and the Kinesin Family Member 5B (KIF5B)-RET fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome. Method We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using RET break-apart and KIF5B-RET SY translocation probes) and a commercial RET transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining. Results Among the 581 specimens, 51 (8.8%) specimens harbored RET rearrangements, including 12 cases (2.1%) carrying a KIF5B-RET fusion gene. Surprisingly, RET expression was lower in KIF5B-RET fusion gene-positive than in RET wild-type specimens. We detected activating EGFR mutations in 11 (21.6%) of the 51 RET variant-positive specimens. Among the KIF5B-RET fusion gene-positive specimens, p-ERK expression was significantly lower in the EGFR mutation subgroup showing RET expression than in the EGFR mutation subgroup that did not express RET. Similarly, the RET rearrangement group showed significant variation in the expression level of p-AKT (P = 0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the RET rearrangement group regardless of RET expression. Conclusion This result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring RET variants.

Background: The KIF5B-RET rearrangement is detected with the frequency of 1 similar to 2 % in 'triple marker'-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational changes are known to be mutually exclusive, but the co-existence of ALK rearrangement with activating mutations of EGFR is rarely found. Methods: We examined the KIF5B-RET fusion gene in frozen tissues from 154 surgically resected lung tumors using RT-PCR with direct sequencing and the mutation status of EGFR and KRAS genes using PNA clamping. We tested KIF5B-RET translocation in Formalin Fixed Paraffin Embedded using fluorescence in situ hybridization. We also measured RET mRNA and protein expression by RT-PCR and immunohistochemistry, respectively. Results: The existence of KIF5B-RET fusion gene was identified in 9 patients. The mean age was 67.2 and M: F ratio 4: 5. Of 9 patients, 3 patients harbored wild type of EGFR and KRAS gene. However, KIF5B-RET fusion gene coincided with EGFR or KRAS mutation in 6 patients. These six pts were also positive for both RET break-apart probes (23.9 %) and KIF5B-RET fusion (44.4 %). However, there were no correlations between RET mRNA and protein expression in the KIF5B-RET-positive patients. The median disease free survival and overall survival were 23.9 months and 29.5 months, respectively. Conclusions: Taken together, our data suggest one-step screening platform for KIF5B-RET as well as EGFR, K-RAS, ALK oncogenic mutations be necessary for lung adenocarcinoma patients because EGFR or KRAS mutation are not infrequently found in KIF5B-RET-positive patients.

e20610 Background: RET rearrangements have been identified in 1-2% of lung adenocarcinomas. The most common fusion is the KIF5B-RET, the function and roles of the RET fusion oncogene, and its downstream signaling molecules remain unclear. Methods: We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from lung adenocarcinoma patients and investigated them using FISH with RET break-apart and KIF5B-RET SY translocation probes. NanoString’s nCounter technology was used to assay RETtranscripts. We evaluated the protein expressions of RET and RET-related signaling molecules, including p-AKT and p-ERK, using TMA-based IHC staining. Results: Using FISH, we identified 51 cases (8.8%) of RET variants and 10 cases (1.7%) of KIF5B-RET fusion genes among the 581 cases. RET protein expression was lower in the group harboring KIF5B-RET fusion gene than that in the group harboring a wild type RET gene. We found the activating EGFR mutations in 11 (21.6%) cases of 51 RET variants. For the group with KIF5B-RET fusion gene, the expression of p-ERK was significantly lower in EGFR mutation subgroup with presence of RET protein compared to EGFR mutation subgroup with absence of RET protein. For the group with RET rearrangement, there were significant differences in the expression level of p-AKT (P = 0.028) and, p-ERK protein expression was remarkably increased, especially in cases with no RET protein expression. Conclusions: Taken together, the expression of p-ERK protein was meaningfully increased in the RET variants group regardless of RET protein expression. This result suggests that RET inhibitors combined with ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring the RET variants.

e20014Background: KIF5B-RET gene rearrangement occurs in about 1% of lung adenocarcinomas. In recent, the targeted agents to inhibit RET phosphorylation have been examined in several clinical studies. However, little was known about how this fusion gene drive lung cancer. Methods: We established transformed HEK293T cells with KIF5B-RET fusion gene using Lenti virus system. In transformed HEK293T cells, we identified transcription factors which are activated using qRT-PCR array and then, checked mRNA, protein, expressions of FOXA2. We performed immunohistochemistry to evaluate the expressions of FOXA2 protein in tumor tissues obtained from 38 lung adenocarcinoma patients who were screened by FISH, RT-PCR, and Nanostring analysis. Results: In the qRT-PCR array, the transcription factors of which the relative expression increased more than 1.5 in KIF5B-RET transformed HEK293T cells were MYF5, STAT5A, ESR1 and FOXA2. We confirmed the increased expressions of FOXA2 mRNA and protein in transformed HEK293T cells...

e18528 Background: KIF5B-RET fusion genes are formed when part of the RET gene fuses with part of the KIF5B gene. This fusion gene induces the proliferation and differentiation of tumor cells by tr...

7537 Background: The KIF5B-RET rearrangement is detected with the frequency of 1~2% in ‘triple marker’-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational chang...