Genetic diagnosis and therapeutic strategies for rare cancer = 유전체 분석을 통한 희귀암의 진단 및 치료 연구

다국어 초록 (Multilingual Abstract)

Rare cancer refers to cancer that occurs in less than 6 patients per 100,000 people, and as the incidence of rare cancer increases every year, 25% of cancer deaths are due to rare cancer. However, due to a lack of research, diagnosis and treatment are not performed properly. Until now, diagnosis has been performed using various methods, but diagnosis through genetic analysis is more important in that it can provide a malignant mechanism or treatment target.
In this study, a panel for the diagnosis and treatment of rare cancers and a panel for research on the mechanism of cancer development were designed and targeted gene sequencing was conducted. To investigate the oncogenic fusion genes in ambiguous tumors, we performed targeted RNA sequencing in 165 tumor samples and identified a total of 34 types of fusion genes. Eight novel fusion genes were also identified, and we confirmed the oncogenicity of novel fusion gene, PTPRG-RAF1 and the effect of small molecules in RAF1 fusion expressing cell lines. In addition, the effect of gene therapy was also verified using short interfering RNA (siRNA) for NUT carcinoma with BRD4-NUTM1 fusion gene, which is known to have no standard treatment yet. After siRNA treatment, it was confirmed that the BRD4-NUTM1 RNA expression in the BRD4-NUT expressing cell line decreased, and the cell viability also decreased. Afterwards, using xenograft mouse models with siRNA treatment, and the tendency of tumor size to decrease was confirmed.
In addition, target gene sequencing was performed using another panel to find the mechanism of malignancy in neurofibromatosis type 1 (NF1) which was not diagnosed as cancer but had a lifetime risk of malignancy of about 10% for NF1 patients. We performed targeted sequencing in 91 NF1 patients, genotypes and phenotypes were compared to find out what driver mutations are the cause of cancer, while RNA expression is confirmed through transcriptome sequencing to find differences between benign and malignant tumors.
This study demonstrated the utility of targeted sequencing as a theragnostic tool and suggested the possibility in the treatment of rare cancer patients as part of precision medicine.

국문 초록 (Abstract)

희귀암은 10만명당 6명 이하의 환자가 발생하는 암종을 말하며, 희귀암의 발생이 매년 증가함에 따라 암 사망자의 25%가 희귀암으로 사망하고 있다. 그러나 연구가 부족하여 진단과 치료가 제대로 이루어지지 않고 있는 실정이다. 희귀암 연구에 있어서 명확한 진단은 반드시 선제적으로 이루어져야 하는 과제이며, 정확한 진단은 향후 정확한 치료 연구에 영향을 줄 뿐 아니라, 환자에게 있어서도 적절한 치료 기회를 제공할 수 있다는 점에서 중요하다. 현재까지 여러 방법을 통해 진단을 시행하고 있으나 유전체 분석을 통한 진단은 악성화 기전 또는 치료 타깃을 제공할 수 있다는 점에서 더 중요하다.
본 연구에서는 희귀암의 진단과 치료를 위한 패널과 암의 발생 기전 연구를 위한 패널을 각각 디자인하여 목표 유전자 시퀀싱(Targeted gene sequencing)을 진행하였다. 이를 통해 진단이 불명확한 고형암에서 암유전자(oncogene)으로 작용하는 융합유전자를 찾았고, 기존에 보고된 적 없는 PTPRG-RAF1 융합유전자를 선정하여 융합유전자가 종양성 영향을 지니는지, 이를 표적으로 하는 약물로 치료 효과를 볼 수 있는지 확인하였다. 추가적으로 치료 약물이 아직까지 없다고 알려진 BRD4-NUTM1 융합유전자 유래 암종에 대해서도 치료 방안을 찾기 위해 짧은 간섭 RNA(siRNA)를 이용하여 유전자 치료 효과에 대해서도 검증해보았다.
추가적으로 암으로 판정되지 않으나 악성화의 전생애위험도(lifetime risk)가 10% 정도인 신경섬유종증 1형 환자를 대상으로 악성화의 기전을 찾기 위해 또 다른 패널을 사용하여 목표 유전자 시퀀싱을 진행하였다. 신경섬유종 유래 양성 종양 조직과 악성 종양 조직을 이용하여 암의 원인이 되는 드라이버 변이(driver mutation)이 무엇인지 찾기 위해 유전형과 표현형을 비교하는 한편, 전사체 분석을 통해 유전자 발현의 정도를 확인하여 악성 종양 특이적으로 발현이 높은 유전자를 확인함으로써 치료 가능성에 대해 모색해 보았다.
결론적으로, 본 연구에선 차세대 염기서열 분석을 통한 암의 유전체 분석이 희귀암에서 암의 진단 뿐만 아니라 환자 맞춤형 치료 전략 수립에 있어서 필수적으로 이루어져야 함을 제시한다.

목차 (Table of Contents)

• Table of Contents
• Abstract................................................................................................... vii
• Chapter 1. General background............................................................. 1
• 1. Rare cancer and ultra-precision medicine.......................................... 1
• 2. Purpose of study................................................................................... 2
• Chapter 2. Targeted RNA sequencing for genetic diagnosis and treatment of rare cancer........................................................................ 3
• 1. Introduction........................................................................................... 3
• 2. Materials and Methods ......................................................................... 5
• A. Sample collection............................................................................. 5
• B. Panel design..................................................................................... 6
• C. RNA extraction and targeted sequencing....................................... 7
• D. RT-PCR............................................................................................ 8
• E. Identification of novel fusions......................................................... 8
• F. Cell lines........................................................................................... 9
• G. MTS assay....................................................................................... 10
• H. Spheroid culture.............................................................................. 11
• I. Soft agar assay.................................................................................. 11
• J. Invasion assay.................................................................................. 12
• K. Drug screening................................................................................ 12
• L. Western blotting............................................................................... 13
• M. In vitro siRNAs assays targeting the junction region of the fusion gene....................................................................................................... 13
• N. qRT-PCR......................................................................................... 14
• O. Immunohistochemistry.................................................................... 14
• P. In vivo experiment........................................................................... 14
• Q. Cell-free RNA isolation and BRD4-NUTM1 transcript quantification in the plasma......................................................................................... 16
• 3. Result.................................................................................................... 21
• A. Development of CGMP panel.......................................................... 21
• B. Identification of fusions in diagnostically uncertain tumors......... 24
• C. The potential as diagnostic tool of fusion gene detection in ambiguous cancers............................................................................... 28
• D. Clinicopathologic features of novel fusions in clinical samples... 34
• E. Functional characterization of PTPRG-RAF1............................... 43
• F. Patient demographics and characterization of the partner fused with NUTM1.................................................................................................. 47
• G. Development of an siRNA targeting the junction region of the fusion gene....................................................................................................... 53
• H. In vivo efficacy of the siRNA targeting the junction region of the fusion gene............................................................................................ 56
• I. The potential of assaying the junction sequence for predicting and monitoring cancer treatment................................................................ 59
• 4. Discussion.............................................................................................. 62
• Chapter 3. Mechanism of Malignancy in Rare Cancer with Targeted sequencing.............................................................................................. 72
• 1. Introduction........................................................................................... 72
• 2. Materials and Methods ......................................................................... 73
• A. Sample collection............................................................................. 73
• B. Panel design...................................................................................... 74
• C. DNA extraction and targeted sequencing....................................... 74
• D. Multiplex Ligation dependent probe amplification (MLPA).......... 75
• E. Mutation calling................................................................................. 75
• F. Nanostring DSP GeoMX.................................................................... 76
• G. Data analysis..................................................................................... 76
• 3. Result.................................................................................................... 78
• A. Development of NF1 panel............................................................. 78
• B. Correlation between genotype and phenotype of NF1................. 81
• C. Comparison of RNA expression differences.................................. 89
• 4. Discussion............................................................................................ 97

참고문헌 (Reference)

1. Small-Molecule Targeting of BET Proteins in Cancer, French, C. A., 131: p. 21-58, , 2016