46
그림21.도출 된cut-off value 로 내부 데이터에 대한OS의 카플란-마이어 곡선
그림22. 도출 된cut-off value 로 내부 데이터에 대한EFS의 카플란-마이어 곡선
48
표12. 도출 된cut-off value 에 대한internal validation 콕스 회귀분석 결과
Variable
OS
HR 95% CI P
myc3.regroup1 Reference
myc3.regroup2 2.2 1.1 – 4.3 0.022 *
5. COO 가 확인 된 환자군에 대한cut-off value재검증
예후를 예측하는데 영향을 미칠 것으로 알려져 있는 COO와IPI, 도출 된cut-off value 를 비교 분석 했다.비교분석을 위해COO 가 확인 되지 않은 환자를 제외 한180명을 대상으로 했다.40).
앞서 도출 된cut-off value 에 따라 음성군과 양성군으로 이분화 했다. 이를 콕스 회귀 분석 결과가 앞선 결과와 같이 양성군이 유의하게 예후가 나쁨을 보였다(HR, 2.1, 95%
CI, 1.06 – 4.2, p= 0.033.) .
COO 는 양성군이 음성군에 비해 유의하지 않게 예후가 나쁜 경향을 보였다(HR, 1.3,
95% CI, 0.65 – 2.7, p= 0.443).
IPI 지수3 미만인 모집을 음성군, 3 이상인 모집을 양성군으로 나누어 분석했다. 그 결
과 음성군에 비해 양성군이 양성군이 유의하게 예후가 나쁨을 보였다(HR, 2.7, 95% CI, 1.47 – 5.0, p = 0.001).
그 결과, COO 로 예측한 결과 보다MYC 단백의 발현의 강도와 비율이 예측에 적합하 다는 결과를 도출 할 수 있었다.
이로써Opal 다중 면역 형광 염색으로 정량화 하여cut-off value 를 강도3+ 이상, 양성
세포 비율60% 이상으로 하는 것이 판독 기준으로 제시 가능하다는 결론을 내렸다(표 13.).
50
표13. COO 가 확인된180명의 환자에 대한 도출 된 cut-off value, COO, IPI의 단변량 분석의 결과
Variable
OS
HR 95% CI P
myc3.group1 Reference
myc3.group2 2.1 1.06 – 4.2 0.033 *
COO 1 Reference
COO 2 1.3 0.65 – 2.7 0.443
IPI 1 Reference
IPI 2 2.7 1.47 – 5.0 0.001 **
결론
본 연구는MYC 단백 발현 강도를 객관적으로 정량함 으로써R-CHOP요법에 반응하 지 않는 나쁜 예후의DLBCL환자군을 예측하는데 의미가 있다.
MYC 단백발현의 강도가3+ 이상이며 양성세포비율이 60% 의cut-off value가 연구결
과 가장 명확한 예후를 예측할 수 있었다.
비록 현재까지MYC의 병태생리에 대해 폭 넓게 연구되어있지 않지만, 본 연구결과를
기반으로MYC 의 생물학적 역할에 대해 규명하여R-CHOP 에 반응하지 않는DLBCL
환자군을 예측하는 중요한 표지 인자로서 활용될 수 있으며 또한 적절한 치료법을 제 시하는데 기여할 수 있을 것 이다.
현재 임상진단기법으로 널리 활용되고 있는 면역화학염색 기법에도 도출된 cut-off value 가 적용 가능하다면 병리의사의 진단의 보조적 도구로 유용하게 사용 될 것 이다.
또한 이 연구는 AI 를 이용한 새로운 진단 기술을 개발하는데 기저 연구가 될 것 으로 사료된다.
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ABSTRACT
Introduction: A large B-cell lymphoma (DLBCL) is a common disease that accounts for approximately 40% of non-Hodgkin's lymphomas. DLBCL has improved compared to the past due to standard chemotherapy (r-CHOP; rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), but about 40% of patients still do not respond to treatment and eventually die. Previous studies to predict and define these patients and to develop new therapies have reported a number of associations between the expression of MYC protein and the poor prognosis of DLBCL. However, immunohistochemistry (IHC) used in previous studies has a variety of staining due to the diversity of MYC expression intensity. For this reason, it is difficult to apply it to the diagnosis due to the inconsistency between the readers and the cut-off value. In order to overcome these problems, it is required to carry out quantitative analysis with an experimental technique and scientific basis that can objectively quantify the intensity of expression. In this study, MYC protein expression cut-off value was derived from DLBCL prognosis prediction by quantitative image analysis using multiplexed immunofluorescence staining and statistical analysis combined with clinical indicators.
Methods: Between 2007 and 2012, paraffin tissues of DLBCL patients diagnosed at Asan Medical Center in Seoul were studied. Of these, TMAs were made in 192 patients except for patients who received treatment other than R-CHOP, DLBCL patients with primary central nervous system, and small amounts of tissue that could not be used for TMA (tissue microarray). TMA immunohistochemistry was performed by using Opal multiphoton staining method, in which each antibody was labeled with a different fluorescent material and quantitatively measured the fluorescence intensity. The antibody used was stained with CD20 and MYC to confirm the expression of MYC protein in tumor cells. In addition, CD3 staining was performed for analysis except T cell, which is a non-tumor cell. The stained slides were collected by a Vectra slide scanner (PerkinElmer, Waltham, Mass.) At 20 nm intervals. Quantified data was obtained with inForm ™ Tissue Finder ™ image analysis software (PerkinElmer, Waltham, Mass.). The expression intensity of MYC was divided by the number of quartiles (0+, 1+, 2+, 3+). The cut-off value was calculated by performing survival analysis between patient groups according to MYC protein expression intensity and positive cell ratio. The resulting cut-off value was internally compared to confirm the