miRNA-34a와 miRNA-449a의 후생유전학적 변화로 인한 epithelial-mesenchymal transition과 세리티닙에 대한 획득 내성 기전 규명

Dept. of Medicine/박사Treatment with ALK tyrosine kinase inhibitors often elicits profound initial antitumor responses in ALK fusion-positive patients with lung adenocarcinoma. However, patients invariably develop acquired resistance to ALK inhibitors. Although the recent functional genetic studies characterized the mechanism of resistance to ALK inhibition, epigenetic mechanism of acquired resistance is poorly understood. Here we report histone H3 lysine 27 acetylation (H3K27ac) and microRNAs involved in acquired resistance to ceritinib, a second generation ALK inhibitor. Epithelial-to-mesenchymal transition with AXL activation was found in multiple in vitro and in vivo ALK-rearranged lung cancer models with acquired resistance to ceritinib. Genome-wide analysis identified that miR-34a and miR-449a are decreased with loss of H3K27ac and AXL and mesenchymal genes are increased in the process of acquired resistance. Pharmacological inhibition of AXL or ectopic expression of miR-34a or miR-449a restored sensitivity to ceritinib in resistant cells. We also found histone deacetylase inhibitor, panobinostat, synergistically induced anti-proliferative effects with ceritinib in resistant cells. Our findings demonstrate that H3K27ac remodeling is a crucial event in ceritinib resistance and inhibition of both ALK and HDAC could prevent or overcome acquired resistance to ALK inhibitors in individuals with ALK-rearranged lung cancer. EML4-ALK 양성 비소세포폐암 환자에서 ALK 타이로신 키나아제 억제제 치료 시 초기에는 우수한 반응을 보이나, 결국 약제 내성을 획득하게 된다. 비록 ALK 억제제에 대한 획득 내성 연구들이 진행된 바 있지만, 현재까지 후성유전학적 기전에 대한 연구는 전무하다. 본 연구에서는 세리티닙이라는 2세대 ALK 억제제에 대한 획득 기전 규명을 위해 RNA-seq, MBD-seq, ChIP-seq 통합 분석을 통해 내성 획득 세포의 전사체, DNA 메틸화, 히스톤 아세틸화 등 후성 유전체 통합 분석을 실행하였다. ALK 양성 내성 세포주에서 모세포주에 비해 H3K27ac 부위에 탈 아세틸화가 두드러지게 나타나는 것을 확인하였고, AXL 발현 증가와 상피-간엽전환 (epithelial-to-mesenchymal transition)이 발생하는 것을 확인하였다. H3K27 아세틸화 감소와 함께 mRNA 발현이 감소하는 유전자들을 선별한 결과, miR-34a와 miR-449a의 H3K27 탈아세틸화로 인하여 miR-34a와 miR-449a의 발현이 감소하고, 이에 따라 이들이 하위로 조절하는 AXL 과 mesenchymal 유전자들의 발현이 증가하는 기전을 규명하였다. 또한 AXL을 억제하거나 miR-34a 와 miR-449a를 외부에서 주입시켜 주었을 때 세리티닙에 대해 다시 민감성을 획득하였다. 히스톤 탈 아세틸화 효소 억제제인 Panobinostat 에 의한 miR-34a 와 miR-449a의 탈 아세틸화 및 발현 조절이 가능한지 확인하기 위해 Panobinostat 처리 후 q-PCR, RNA-seq, ChIP-seq 분석을 수행하였고, Panobinostat 이 내성 세포주의 H3K27ac 부위에서 아세틸화를 증가시키는 것을 볼 수 있었다. Panobinostat을 세리티닙과 함께 내성세포주에 처리하였을 경우 병용효과를 확인할 수 있었고, G1 arrest 유도 단백질로 알려진 CDK inhibitor protein(CIP/KIP family)인 p21의 발현과, 세포사멸 (Apoptosis) 관련 단백질인 cleaved PARP와 cleaved caspase 3의 발현을 증가시킨 것을 확인하였다. 또한 miR-34a 및 miR-449 가 표적으로 하는 AXL 및 MET 등의 단백질 및 N-cadherin의 발현은 감소시켰고, 감소된 E-cadherin의 발현을 다시 증가시켰다. 상기 결과들을 통해 H3K27ac 리모델링으로 인한 miR-34a와 miR-449a 발현 감소가 세리티닙의 획득 내성에 중요한 역할을 하며, 이를 극복하기 위한 새로운 치료 방법으로서 히스톤 탈 아세틸화 효소 억제제와 세리티닙의 병용 요법에 대한 전략을 제시해준다.ope

Integrative Genomic and Transcriptomic Analyses of Tumor Suppressor Genes and Their Role on Tumor Microenvironment and Immunity in Lung Squamous Cell Carcinoma

Non-small-cell lung cancers (NSCLCs) are largely classified into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), which have different therapeutic options according to its molecular profiles and immune checkpoint expression, especially PD-L1, which is a suppressive factor in the tumor microenvironment. The tumor microenvironment can be altered by the genomic mutations on specific innate immune genes as well as tumor suppressor genes, so it is essential to comprehend the association between tumor microenvironment and tumor suppressor genes to discover the promising immunotherapeutic strategy to overcome the resistance of immune check point blockade. In this study, we aimed to analyze how the somatic mutations in tumor suppressor genes affect the tumor immune microenvironment through a comprehensive analysis of mutational profiling on the representative tumor suppressor genes (TP53, CDKN2A, PTEN, RB1, BRCA1, BRCA2) and immune gene expression in The Cancer Genome Atlas (TCGA) 155 lung squamous cell carcinoma (LUSC) and 196 lung adenocarcinoma (LUAD) samples. Several microenvironmental factors, such as the infiltrating immune and stromal cells, were suppressed by the mutated tumor suppressor genes in LUSC, unlike in the LUAD samples. In particular, infiltrating immune cells such as macrophage, neutrophil, and dendritic cells were significantly reduced in tumors with mutated tumor suppressor genes' group. In addition, the gene expressions for interleukin production and lymphocyte differentiation and PGC, C7, HGF, PLA2G2A, IL1RL1, CCR2, ALOX15B, CXCL11, FCN3 were significantly down-regulated, which were key immune genes for the cross-talk between LUSC microenvironment and tumor suppressors. Therefore, we generated evidence that TSG mutations in LUSC have an impact on tumor immune microenvironment, which suggests that TSG non-mutated patients will have the more inflamed tumors and are more likely to respond to immune checkpoint blockade therapy.ope

Psychosocial Impact of Cancer Patients on Their Family Members

PURPOSE: A population-based study was conducted in order to examine the characteristics of family members of cancer patients in comparison with the general population and also to evaluate the psychosocial impact of cancer patients on their family members. MATERIALS AND METHODS: From the Fourth Korea National Health and Nutrition Examination Surveys (KNHANES IV) (2007-2009) dataset, we identified 460 cancer patients and then selected family members of these patients who were aged 20 years or older (n=565). The control group was sampled from members of families without a cancer patient with matching for sex and age (n=2,260). Serial conditional logistic regression models were used for comparison of characteristics between family members of cancer patients and subjects in the control group. RESULTS: Family members of cancer patients were less employed (57.9% vs. 63.0%, p<0.001), more functionally limited (20.2% vs. 16.5%, p=0.032), and had lower self-rated health (p=0.023) compared with sex and age-matched control subjects. They also had a significantly higher level of stress (79.7% vs. 76.1%, p=0.008), history of depression (12.9% vs. 10.2%, p=0.035), and current depressive symptoms (5.5% vs. 3.5%, p=0.038). However, higher physical activity was reported in family members of cancer patients (13.6% vs. 9.6%, p=0.003) than in control subjects. The presence of a cancer patient in the family showed an association with current depressive symptoms (odds ratio, 1.62; 95% confidence interval, 1.05 to 2.48; p=0.028), however, the association was no longer significant after adjustment for household income, education level, and employment status (p=0.304). CONCLUSION: Family members of cancer patients are more susceptible to depression, probably due to adverse change in socioeconomic status. Use of multidisciplinary approaches for promotion of psychological health and well-being is essential.ope

Real-World Experience of Nivolumab in Non-small Cell Lung Cancer in Korea

Purpose: The introduction of immune checkpoint inhibitors represents a major advance in the treatment of lung cancer, allowing sustained recovery in a significant proportion of patients. Nivolumab is a monoclonal anti-programmed death cell protein 1 antibody licensed for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) after prior chemotherapy. In this study, we describe the demographic and clinical outcomes of patients with advanced NSCLC treated with nivolumab in the Korean expanded access program. Materials and methods: Previously treated patients with advanced nonsquamous and squamous NSCLC patients received nivolumab at 3 mg/kg every 2 weeks up to 36 months. Efficacy data including investigator-assessed tumor response, progression data, survival, and safety data were collected. Results: Two hundred ninety-nine patients were treated across 36 Korean centers. The objective response rate and disease control rate were 18% and 49%, respectively; the median progression-free survival was 2.1 months (95% confidence interval [CI], 1.87 to 3.45), and the overall survival (OS) was 13.2 months (95% CI, 10.6 to 18.9). Patients with smoking history and patients who experienced immune-related adverse events showed a prolonged OS. Cox regression analysis identified smoking history, presence of immune-related adverse events as positive factors associated with OS, while liver metastasis was a negative factor associated with OS. The safety profile was generally comparable to previously reported data. Conclusion: This real-world analysis supports the use of nivolumab for pretreated NSCLC patients, including those with an older age.ope

Combatting acquired resistance to osimertinib in EGFR-mutant lung cancer

The discovery of activating mutations in epidermal growth factor receptor (EGFR) in non-small-cell lung cancer transformed the care and prognosis of patients and heralded the era of 'personalized medicine' in thoracic oncology. Osimertinib, a third-generation EGFR inhibitor, has been established as the preferred EGFR inhibitor for newly diagnosed patients which urged the need to develop treatment options for patients progressing on first-line osimertinib. However, acquired resistance invariably emerges and numerous efforts have been attempted to delay or overcome acquired resistance. In this article, we thoroughly reviewed the current understanding of osimertinib resistance mechanisms and explored the established and emerging treatment options. Newer treatment strategies targeting EGFR-dependent or -independent resistance mechanisms, novel approaches using bispecific antibodies and antibody-drug conjugates will be discussed. Moreover, what to do with brain only progression, and how to incorporate immunotherapy in EGFR-mutant lung cancer will be discussed. Lastly, future perspectives on the ongoing clinical trials and combination of front-line therapy will be introduced.ope

Prognostic Value of CD200R1 mRNA Expression in Head and Neck Squamous Cell Carcinoma

Immune system dysfunction is associated with head and neck squamous cell carcinoma (HNSCC) development and progression and immune checkpoint inhibitors have demonstrated substantial survival benefits in platinum-refractory HNSCC; therefore, we examined the prognostic value of immune-related gene (IRG) expression in HNSCC. We analyzed the expression of 82 IRGs in 71 patients with HNSCC enrolled in a feasibility study for a prospective HNSCC biomarker-driven umbrella trial (Korean Cancer Study Group TRIUMPH study, NCT03292250). CD200R1 was identified as an independent prognostic factor and validated in GEO and TCGA database. CD2000R1 mRNA expression was found to be an independent favorable prognostic factor in patients with HNSCC. Moreover, CD200R1 was found to affect genes and pathways associated with the immune response, while seven differentially expressed genes (CD8A, DOK2, CX3CR1, TYROBP, CXCL9, CD300LF, IFNG) were associated with CD200R1 expression. Samples with higher CD200R1 expression displayed higher tumor-infiltrating immune cell counts both in silico and in histological analysis. These findings will help in the development of more accurate prognostic tools and suggest CD200R1 modulation as a HNSCC immunotherapy.ope

Advances in the management of non-small-cell lung cancer harbouring EGFR exon 20 insertion mutations

Epidermal growth factor receptor (EGFR) mutation is one of the key oncogenic mutations in non-small-cell lung cancer with adenocarcinoma histology. Exon 19 deletions and exon 21 L858R substitutions account for 90%, while EGFR exon 20 insertions constitute 4–10% of EGFR mutations and are the third most prevalent activating EGFR mutations. EGFR exon 20 insertions are associated with decreased sensitivity to EGFR tyrosine kinase inhibitors and, until recently, effective targeted therapy against these tumours remained an unmet clinical need and chemotherapy was the only treatment of choice available. The approval of amivantamab and mobocertinib for patients who have progressed after chemotherapy represents an important step forward in the management of these patients. Here in this review, we summarize the epidemiology, structure and the tumour microenvironment of EGFR exon 20 insertion and also review the systemic treatments, including targeted therapies and ongoing clinical trials in EGFR exon 20 insertion mutations, as well as detection methods for EGFR exon 20 insertion. Lastly, resistant mechanisms and future directions are addressed. © The Author(s), 2023.ope

Targeted sequencing identifies genetic alterations that confer primary resistance to EGFR tyrosine kinase inhibitor (Korean Lung Cancer Consortium)

BACKGROUND: Non-small-cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations may exhibit primary resistance to EGFR tyrosine kinase inhibitor (TKI). We aimed to examine genomic alterations associated with de novo resistance to gefitinib in a prospective study of NSCLC patients. Patients and methods: One-hundred and fifty two patients with activating EGFR mutations were included in this study and 136 patients' tumor sample were available for targeted sequencing of genomic alterations in 22 genes using the Colon and Lung Cancer panel (Ampliseq, Life Technologies). Results: All 132 patients with EGFR mutation were treated with gefitinib for their treatment of advanced NSCLC. Twenty patients showed primary resistance to EGFR TKI, and were classified as non-responders. A total of 543 somatic single-nucleotide variants (498 missense, 13 nonsense) and 32 frameshift insertions/deletions, with a median of 3 mutations per sample. TP53 was most commonly mutated (47%) and mutations in SMAD4 was also common (19%), as well as DDR2 (16%), PIK3CA (15%), STK11 (14%), and BRAF (7%). Genomic mutations in the PI3K/Akt/mTOR pathway were commonly found in non-responders (45%) compared to responders (27%), and they had significantly shorter progression-free survival and overall survival compared to patients without mutations (2.1 vs. 12.8 months, P=0.04, 15.7 vs. not reached, P<0.001). FGFR 1-3 alterations, KRAS mutations and TP53 mutations were more commonly detected in non-responders compared to responders. Conclusions: Genomic mutations in the PI3K/Akt/mTOR pathway were commonly identified in non-responders and may confer resistance to EGFR TKI. Screening lung adenocarcinoma patients with clinical cancer gene test may aid in selecting out those who show primary resistance to EGFR TKI (NCT01697163).ope

Recommendations for the Use of Next-Generation Sequencing and the Molecular Tumor Board for Patients with Advanced Cancer: A Report from KSMO and KCSG Precision Medicine Networking Group

Next-generation sequencing (NGS) is becoming essential in the fields of precision oncology. With implementation of NGS in daily clinic, the needs for continued education, facilitated interpretation of NGS results and optimal treatment delivery based on NGS results have been addressed. Molecular tumor board (MTB) is multidisciplinary approach to keep pace with the growing knowledge of complex molecular alterations in patients with advanced solid cancer. Although guidelines for NGS use and MTB have been developed in western countries, there is limitation for reflection of Korea's public health environment and daily clinical practice. These recommendations provide a critical guidance from NGS panel testing to final treatment decision based on MTB discussion.ope

Brief Report: Heterogeneity of Acquired Resistance Mechanisms to Osimertinib and Savolitinib

Introduction: MET amplification is a frequently observed mechanism of resistance to osimertinib, and coinhibition strategy of MET and EGFR revealed promising results in recent clinical trials. Nevertheless, acquired resistance mechanisms to combined EGFR and MET inhibition are poorly understood. In this study, we investigated the mechanisms of acquired resistance to osimertinib and savolitinib by using pretreatment and post-treatment tissue analysis. Methods: Whole-exome sequencing was performed in EGFR-mutant, MET-amplified patients who received osimertinib and savolitinib using tissues obtained both before and after therapy. All patients achieved partial response or durable stable disease to osimertinib and savolitinib before developing acquired resistance. Results: After progression on osimertinib and savolitinib, whole-exome analysis revealed MET-dependent mechanisms of resistance, such as acquired MET p.D1246H mutation, MET p.Y1230C mutation, and MET copy number gain. As for MET-independent mechanisms, development of ERBB2 mutation and amplification and copy number gains in amplifications in CCNE, CCND1, CDK6, and EGFR were observed. Patient 2 harbored an acquired PIK3CA p.H1047R mutation in which resistance could be overcome with combination of PI3K inhibitor and osimertinib in the patient-derived xenograft model. Conclusions: Our study reveals that acquired resistance to savolitinib plus osimertinib can occur from both MET-dependent and MET-independent mechanisms.ope