Kyungsoo Jung

Comprehensive analysis of RET and ROS1 rearrangement in lung adenocarcinoma

The success of crizotinib in ALK-positive patients has elicited efforts to find new oncogenic fusions in lung cancer. These efforts have led to the discovery of novel oncogenic fusion genes such as ROS1 and RET. However, the molecular and clinicopathologic characteristics associated with RET or ROS1 fusion, compared with ALK fusion-positive lung cancer, remain unclear. We accordingly analyzed the clinicopathologic characteristics of RET- and ROS1-fusion-positive lung adenocarcinomas. We further performed immunohistochemistry and fluorescence in situ hybridization analysis (FISH) in 15 cases of RET and 9 cases of ROS1 fusion tumors by identified NanoString’s nCounter screening. RET fusion-positive patients were younger in age, never-smokers, and in early T stage; ROS1 fusion-positive patients had a higher number of never-smokers compared with patients with quintuple-negative (EGFR−/KRAS−/ALK−/ROS1−/RET−) lung adenocarcinoma. Histologically, RET and ROS1 fusion tumors share the solid signet-ring cell and mucinous cribriform pattern, as previously mentioned in the histology of ALK fusion tumors. Therefore, it can be presumed that fusion gene-associated lung adenocarcinomas share similar histologic features. In immunohistochemistry, the majority of 15 RET and 9 ROS1 fusion-positive cases showed positivity of more than moderate intensity and cytoplasmic staining for RET and ROS1 proteins, respectively. In FISH, the majority of RET and ROS1 rearrangement showed two signal patterns such as one fusion signal and two separated green and orange signals (1F1G1O) and an isolated 3′ green signal pattern (1F1G). Our study has provided not only characteristics of fusion gene-associated histologic features but also a proposal for a future screening strategy that will enable clinicians to select cases needed to be checked for ROS1 and RET rearrangements based on clinicohistologic features.

MET is a potential target for use in combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

MET, a cell surface receptor for hepatocyte growth factor, is involved in the development of triple‐negative/basal‐like breast cancer (TNBC/BLBC). However, its utility as a therapeutic target in this subtype of breast cancer is poorly understood. To evaluate MET fully as a potential therapeutic target for TNBC/BLBC, we investigated the relationship between MET expression and clinical outcomes of patients with breast cancer and the functional effect of MET inhibition. Using automated immunohistochemistry (Ventana), we analyzed MET expression in 924 breast cancer patients with relevant clinicopathologic parameters. BLBC showed the strongest relationship with MET expression (57.5%, p < 0.001). High expression of MET in breast cancer resulted in poor overall survival (p = 0.001) and disease‐free survival (DFS, p = 0.010). MET expression was relatively high in TNBC cell lines, and the silencing of MET via small interfering RNA reduced cell proliferation and migration. We observed reduced TNBC cell viability after treatment with the MET inhibitor PHA‐665752. In the most drug‐resistant cell line, MDA‐MB‐468, which showed elevated epidermal growth factor receptor (EGFR) expression, silencing of EGFR resulted in increased sensitivity to PHA‐665752 treatment. We confirmed that PHA‐665752 synergizes with the EGFR inhibitor erlotinib to decrease the viability of MDA‐MB‐468 cells. TNBC patients coexpressing MET and EGFR showed significantly worse DFS than that in patients expressing EGFR alone (p = 0.021). Our findings strongly suggest that MET may be a therapeutic target in TNBC and that the combined therapy targeting MET and EGFR may be beneficial for the treatment of TNBC/BLBC patients.

HER2 as a novel therapeutic target for cervical cancer

Surgery and radiation are the current standard treatments for cervical cancer. However, there is no effective therapy for metastatic or recurrent cases, necessitating the identification of therapeutic targets. In order to create preclinical models for screening potential therapeutic targets, we established 14 patient-derived xenograft (PDX) models of cervical cancers using subrenal implantation methods. Serially passaged PDX tumors retained the histopathologic and genomic features of the original tumors. Among the 9 molecularly profiled cervical cancer patient samples, a HER2-amplified tumor was detected by array comparative genomic hybridization and targeted next-generation sequencing. We confirmed HER2 overexpression in the tumor and serially passaged PDX. Co-administration of trastuzumab and lapatinib in the HER2-overexpressed PDX significantly inhibited tumor growth compared to the control. Thus, we established histopathologically and genomically homologous PDX models of cervical cancer using subrenal implantation. Furthermore, we propose HER2 inhibitor-based therapy for HER2-amplified cervical cancer refractory to conventional therapy.

Molecular breakdown: a comprehensive view of anaplastic lymphoma kinase (ALK)‐rearranged non‐small cell lung cancer

Most anaplastic lymphoma kinase (ALK)‐rearranged non‐small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK‐rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule‐associated protein‐like 4 (EML4)–ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK‐rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4–ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p < 0.05). In vitro experiments revealed that EML4–ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non‐homologous end‐joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4–ALK variants, new ALK somatic mutations, and novel ALK‐fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright

MET Exon 14 Skipping Mutations in Lung Adenocarcinoma: Clinicopathologic Implications and Prognostic Values

Introduction Response to mesenchymal‐epithelial transition (MET) inhibitors in NSCLC with mesenchymal‐epithelial transition gene (MET) exon 14 skipping (METex14) has fueled molecular screening efforts and the search for optimal therapies. However, further work is needed to refine the clinicopathologic and prognostic implications of METex14 skipping. Methods Among 795 East Asian patients who underwent a surgical procedure for NSCLC, we screened 45 patients with quintuple‐negative (EGFR‐negative/KRAS‐negative/anaplastic lymphoma kinase gene [ALK]‐negative/ROS1‐negative/ret proto‐oncogene [RET]‐negative) lung adenocarcinomas by using reverse‐transcriptase polymerase chain reaction and found 17 patients (37.8%) with METex14 skipping. We also investigated the effect of small interfering RNA (siRNA) targeting skipping junction in cells with METex14 skipping. Results The median age of the 17 patients was 73 years. The acinar subtype was predominant (52.9%), followed by the solid subtype (35.3%). MET immunohistochemistry demonstrated 100% sensitivity and 70.4% specificity. Multivariate analyses showed that patients with METex14 skipping had a higher recurrence rate than those with ALK fusion (versus METex14 skipping) (hazard ratio = 0.283, 95% confidence interval: 0.119–0.670) in stage I to IIIA disease; however, the differences in overall survival were not significant after adjustment for pathologic stage (p = 0.669). Meanwhile, siRNA decreased MET‐driven signaling pathways in Hs746T cells, and combined treatment with siRNA and crizotinib inhibited cell proliferation in crizotinib‐resistant H596 cells. Conclusions The prevalence of METex14 skipping was quite high in East Asian patients without other driver mutations in lung adenocarcinomas. METex14 skipping was associated with old age, the acinar or solid histologic subtype, and high MET immunohistochemical expression. The prognosis of patients with METex14 skipping was similar to that of patients with major driver mutations. siRNA targeting the junction of METex14 skipping could inhibit MET‐driven signaling pathways in cells with METex14 skipping.

LYN expression predicts the response to dasatinib in a subpopulation of lung adenocarcinoma patients

Therapies targeting SRC family kinases (SFKs) have shown efficacy in treating non-small cell lung cancer (NSCLC). However, recent clinical trials have found that the SFK inhibitor dasatinib is ineffective in some patient cohorts. Regardless, dasatinib treatment may benefit some NSCLC patient subgroups. Here, we investigated whether expression of LYN, a member of the SFK family, is associated with patient survival, the efficacy of dasatinib, and/or NSCLC cell viability. LYN expression was associated with poor overall survival in a multivariate analysis, and this association was strongest in non-smoker female patients with adenocarcinoma (ADC). In lung ADC cells, LYN expression enhanced cell proliferation, migration, and invasion. Dasatinib inhibited LYN activity and decreased cell viability in LYN-positive ADC cell lines and xenografts. Additionally, we identified the SFKs SRC and YES as candidate dasatinib targets in LYN-negative ADC cell lines. Our findings suggest that LYN is a useful prognostic marker and a selective target of dasatinib therapy in the lung ADC subpopulation especially in female non-smokers with lung ADC.

Integrin β3 Inhibition Enhances the Antitumor Activity of ALK Inhibitor in ALK-Rearranged NSCLC

Purpose: Anaplastic lymphoma kinase (ALK)-positive cancers are sensitive to small-molecule ALK kinase inhibitors, but most cases experience failure following treatment. Hence, additional drug targets and combination therapeutic treatments are needed. We investigated gene expression that is regulated by the expression of ALK and explored its roles in cancer progression and therapeutic implication. Experimental Design: We screened ALK-rearranged non–small cell lung cancer (NSCLC) cases using immunohistochemistry and fluorescence in situ hybridization and then conducted multiplex gene expression analysis. We also performed a clinicopathologic analysis to validate the findings. Additional cellular experiments, including inhibition and migration assays, and in vivo lung cancer model studies were performed. Results: Among patients with ALK-rearranged NSCLC, integrin β3 (ITGB3) was one of the overexpressed genes in comparison with that in ALK-negative NSCLC (P = 0.0003). ALK and integrin β3 expression were positively correlated, and we discovered that high integrin β3 mRNA expression was associated with metastasis and more advanced tumor stages (P < 0.005; P < 0.05). Furthermore, we found that inhibition of both ALK and integrin β3 led to increased drug sensitivity in vitro and in vivo (both P < 0.05). Conclusions: We discovered a positive correlation between ALK and integrin β3 expression levels in ALK-rearranged NSCLC. Our findings suggest that high integrin β3 expression in ALK-rearranged NSCLC is associated with tumor progression and a worse prognosis. This finding demonstrates the prognostic value of integrin β3 and provides a rationale for combination treatment with ALK and integrin β3 inhibitors in patients with ALK-rearranged NSCLC. Clin Cancer Res; 24(17); 4162–74. ©2018 AACR.

Engrailed 1 overexpression as a potential prognostic marker in quintuple-negative breast cancer

ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by poor patient prognosis and for which no targeted therapies are currently available. TNBC can be further categorized as either basal-like (BLBC) or quintuple-negative breast cancer (QNBC). In the present study, we aimed to identify novel molecular therapeutic targets for TNBC by analyzing the mRNA expression of TNBC-related genes in publicly available microarray data sets. We found that Engrailed 1 (EN1) was significantly overexpressed in TNBC. Using breast cancer cell lines, we found that EN1 was more highly expressed in TNBC than in other breast cancer subtypes. EN1 expression was analyzed in 199 TNBC paraffin-embedded tissue samples by immunohistochemistry. EN1 protein expression was positively associated with reduced overall survival (OS) rate in patients with QNBC, but not those with BLBC. The importance of EN1 expression in QNBC cell viability and tumorigenicity was evaluated using the QNBC cell lines, HCC38 and HCC1395. Based on our data, EN1 may promote the proliferation, migration, and multinucleation of QNBC cells, likely via the transcriptional activation of HDAC8, UTP11L, and ZIC3. We also demonstrated that actinomycin D effectively inhibits EN1 activity in QNBC cells. The results of the present study suggest that EN1 activity is highly clinically relevant to the survival prognosis of patients with QNBC and EN1 is a promising potential therapeutic target for future QNBC treatment.

Phenylethanolamine N-methyltransferase downregulation is associated with malignant pheochromocytoma/paraganglioma

Malignant pheochromocytoma/paraganglioma (PCC/PGL) is defined by the presence of metastases at non-chromaffin sites, which makes it difficult to prospectively diagnose malignancy. Here, we performed array CGH (aCGH) and paired gene expression profiling of fresh, frozen PCC/PGL samples (n = 12), including three malignant tumors, to identify genes that distinguish benign from malignant tumors. Most PCC/PGL cases showed few copy number aberrations, regardless of malignancy status, but mRNA analysis revealed that 390 genes were differentially expressed in benign and malignant tumors. Expression of the enzyme, phenylethanolamine N-methyltransferase (PNMT), which catalyzes the methylation of norepinephrine to epinephrine, was significantly lower in malignant PCC/PGL as compared to benign samples. In 62 additional samples, we confirmed that PNMT mRNA and protein levels were decreased in malignant PCC/PGL using quantitative real-time polymerase chain reaction and immunohistochemistry. The present study demonstrates that PNMT downregulation correlates with malignancy in PCC/PGL and identifies PNMT as one of the most differentially expressed genes between malignant and benign tumors.

Abstract 4391: MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) are known as poor outcome subtypes with deficiency of targeted molecular therapy. Previous studies showed that MET may play a critical role in the development of TNBC and may be a rational therapeutic target. In order to evaluate MET as a potential therapeutic target for TNBC, we investigated genomic aberration and expression status of MET and evaluated efficacy of MET inhibitor, PHA-665752 in TNBC cell lines. MET protein expression was relatively high in TNBC cell lines. The depletion of MET using small interfering RNA (siRNA) led decreased cell proliferation and migration. PHA-665752 treatment resulted in decreased cell viability, and most of these cell lines were found to be moderately sensitive in their response to PHA-665752. The most resistant cell line, MDA-MB-468, showed exclusively high expression of EGFR, and then EGFR depletion using siRNA induced increased sensitivity to PHA-665752. In our cohort, BLBC was the subtype showing the highest relationship with MET expression among 5 subtypes (77/134, 57.5%, P<0.001). MET expression had a poorer OS (P=0.001) and DFS (P=0.010). Interestingly, TNBC patients with co-expression of MET and EGFR worsen the DFS significantly compared with EGFR expression only. Our findings highly suggest that MET can be a potential therapeutic target in TNBC, and the combined inhibition of MET and EGFR may benefit for the treatment of BLBC patients. Citation Format: Yu Jin Kim, Jong-Sun Choi, Ji-Young Song, Kyungsoo Jung, Ensel Oh, Young Kee Shin, Yoon-La Choi. MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2013-4391