Circulating tumor cells for differential diagnosis of adnexal mass

Circulating Tumor Cells for Differential Diagnosis of Adnexal Mass Dong Hoon Suh 1, Jiyoon Bu 2, Yoon-Tae Kang 2, Kidong Kim 1, Jae Hong No 1, Yong-Beom Kim 1, Young-Ho Cho 2 1 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital 2 Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST)

Differential diagnosis of uterine adnexal mass Mercado C et al. Gynecol Oncol 2010 sensitivity specificity CA-125 75% 80% OVA1 test 93% 43% ROMA 93% 90% RMI 79% 90% Liao XY et al. J Cancer Res Ther 2014 Ueland FR et al. Obstet gynecol 2011 Ortiz-Munoz B Tumor Biol 2014 Kaijser J et al. Hum Reprod Update 2014 There is no method accurate enough for differentiating between malignant and benign tumor of adnexal mass.

Circulating tumor cells [Circulating biomarkers in tumor metastasis] Masuda T et al. Mol Oncol 2016 [Image of a single CTC isolated in breast cancer patient] De Luca F et al. Oncotarget 2016

Circulating tumor cells in previous studies In breast cancer: CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies. Bulfoni M et al. Breast Cancer Res 2016 In colorectal cancer: CTC analysis represent a potential complementary tool for prediction of colorectal cancer patients outcome. de Albuquerque A et al. J Transl Med 2012 In prostate cancer: Poorer survival outcomes were observed in vimentin- and Ki67-positive CTC patients. Lindsay CR et al. BMC Cancer 2016

Background 기존 CTC 선별 platforms CellSearch (FDA approved) 상피세포의 surface marker (EpCAM) 발현기반 상피세포특성을가진암종에서만유효, 변형된 CTC 경우선별오류발생 활성유지어려워배양과추가특성분석어려움. 기타 : ISET, Caltech, NDDK 세포크기만으로선별하는기술은백혈구가많이섞이는단점 KAIST platform CTC 복합적인물리적특성기반 크기, 변형성, 밀도 배양하여물리적특성과항암제반응특성분석가능 RBC CTC WBC Tapered Slit Array

Objective 새로운혈중암세포검출 platform 의자궁부속기종양의감별진단검사로서의성능평가 자궁부속기종양환자에서혈중암세포의양적, 질적특성파악 임상병리학적특성과의연관성규명

Method 검체의확보및이동 수술 난소종양환자 채혈 항암치료

환자발굴 수술이필요한난소종양환자 ( 담당교수 ) 초음파상악성감별이요구되어, CT, CA125, HE4 시행, r/o malig 인경우 r/o ovarian cancer 로 w/u 상 ovarian cancer 로 primary debulking or NAC 예정 환자동의 동의서서명후연구대상자번호부여 수술전 / 중 검체채취 수술전 ( 혈액검체 B1) : 마취직후 incision 전 15cc (10cc+5cc) 채혈및연구간호사연락 수술중 ( 혈액검체 B2) : 종양측난소정맥 (2.1) 15cc 와 omentum 정맥 15cc (mass 있는경우가능한 mass 근처, 2.2) 수술중 ( 조직검체 ): 원발병소 (OT), 정상난소 (ON), omental tumor (MT), 정상 omentum (MN) 1x1x1cm 수술후혈액 수술후 ( 혈액검체 B3): 10cc ( 연구간호사가환자퇴원시 CA125, HE4 채혈과동일날연구채혈예약오더넣기 ) 악성인경우 1주기항암제 prechemo lab 채혈시 CA125, HE4 와함께외래채혈 양성인경우 postop 1mo 에외래방문 수술후 ( 혈액검체 B4): 10cc ( 연구간호사가 CA125, HE4 채혈오더확인후같은날로연구채혈예약오더넣기 ) cycle 3-4 CA125, HE4 채혈시함께외래채혈

IF staining Fluorescent images of the cells from metastatic cancer patients, scale bar=10μm

Cytopathologic criteria 1. 강한 CK+, DAPI+, and CD45 2. 세포질대비핵의크기 3. 세포의형태 (Irregular shape, etc) 및크기 (>12um)

Results Sample N (%) Positive/negative CTC number, mean (range) B1 (preop. Peripheral) 76 (98.7)* 42(55.3)/34(44.7) 1.59 (0-23) B2 (intraop.) B2.1 (intraop. Ovarian v.) 38 (49.4) 19(50)/19(50) 0.97 (0-7) B2.2 (intraop. Omental v.) 15 (19.5) 7(46.7)/8(53.3) 0.67 (0-4) B3 (postop. <1mo) 51 (66.2) 25(49)/26(51) 0.75 (0-4) B4 (after chemotx) 6 (25.0) 4(66.7)/2(33.3) 1.25 (0-3) *1 명 neoadjuvant CTX 중 among 24 patients with cancer

Results Median f/u: 2 months (0-9 months) Patient characteristics Characteristic Benign (n=40) Malignant (n=37)* p Age (yr) 44.9±11.6 52.6±13.0 0.008 Preoperative serum CA125 (U/ml) 187.7±443.4 1339.8±2382.8 0.006 Preoperative serum ROMA 10.9±15.5 46.5±39.8 <0.001 Preoperative RMI 692.8±1867.9 3404.3±7227.2 0.033 Laparoscopic operation 20 (50.0) 6 (16.7) 0.002 Tumor size (cm) 8.9±6.0 12.5±6.1 0.014 Presence of CTC 16 (40.0) 26 (72.2) 0.005 Number of CTC 1.4±3.7 1.8±1.7 0.606 Values are presented as number (%) or mean ± standard deviation *including 13 borderline malignant tumors B1 from preoperative peripheral blood Characteristic Non-cancer (n=53) Cancer (n=24) p Presence of CTC 23 (43.4) 19 (82.6) 0.002 Number of CTC 1.4±3.4 2.0±1.6 0.431

ROC curves for differential diagnosis: malignant vs. benign AUC P value 95% CI CTC_B1 0.641 0.035 0.5-0.8 CA125 0.603 0.124 0.5-0.7 ROMA 0.753 <0.001 0.6-0.9 RMI 0.576 0.257 0.4-0.7

ROC curves for differential diagnosis: cancer vs. benign AUC P value 95% CI CTC_B1 0.685 0.013 0.6-0.8 CA125 0.755 0.001 0.6-0.9 ROMA 0.804 <0.001 0.7-0.9 RMI 0.698 0.008 0.6-0.8

Effect of surgery Non-cancer (n=31) Cancer (n=20) Number (mean±sd) p Number (mean±sd) p CTC 0.135 0.016 Preop (B1) 1.68±4.21 2.20±1.54 Postop (B3) 0.55±0.72 1.05±1.28 ROMA 0.290 0.006 Preop 5.84±2.18 67.08±38.34 postop 4.96±2.61 30.57±21.13

Results in cancer Preoperative serum CA125, ROMA, and RMI score No association with CTC _B1. Stage, grade, tumor size, lymph node and omental metastasis, and serous histology No significant association with CTC_B1, B21, and B22. Notably, a significant association between substantial amount of ascites and CTC _B1 was found in 37 women with malignant tumor (p=0.036).

CTC in patients with malignancy (n=37) CTC_B1 (-) CTC_B1 (+) P FIGO stage >0.999 I-II 6 (60.0) 15 (57.7) III-IV 4 (40.0) 11 (42.3) Histology 0.709 Serous 5 (50.0) 16 (61.5) Non-serous 5 (50.0) 10 (38.5) Grade 0.657 I 3 (42.9) 5 (31.3) II-III 4 (57.1) 11 (68.8) Tumor size >0.999 11.8cm 5 (50.0) 13 (50.0) >11.8cm 5 (50.0) 13 (50.0) LN metastasis >0.999 Absent 4 (80.0) 15 (78.9) Present 1 (20.0) 4 (21.1) Omental metastasis 0.295 Absent 6 (100) 17 (73.9) Present 0 6 (26.1) Ascites 0.039 Absent 10 (100) 17 (65.4) Present 0 9 (34.6)

Conclusion Performance of CTC platform for differential diagnosis of adnexal tumor not more excellent compare to controls including ROMA Qualitative and quantitative Surgical reduction of tumor burden well reflected in cancer

Future direction Role as prognostic marker in cancer Association with EMT markers according to the presence of gross metastasis Vimentin/E-cadherin expression in CTC from B2.2 Association with ascites VEGF expression in CTC In vitro study with cultured CTCs

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CTC 연구환자수술장에서채취해야할검체요약 마취후팔에서정맥혈 15cc (B1) 종양유무와상관없이, 2 개의정맥 ( 난소정맥, 대망정맥 ) 과 2 곳의조직 ( 난소와대망 ) 에서각각최소 1 개의검체채취 - 요약표참고 Ovary tumor Omentum tumor 난소정맥 (15cc) 대망정맥 (15cc) 난소조직 (1x1cm) 대망조직 (1x1cm) Unilateral 있음 1 개 : Tumor 측 1 개 : Tumor 주변 2 개 :Tumor 와정상 2 개 :Tumor 와정상 Unilateral 없음 1 개 : Tumor 측 1 개 : 가능한정맥 2 개 :Tumor 와정상 1 개 : 정상 Bilateral, focal Bilateral, focal Bilateral, whole Bilateral, whole 있음 1개 : 더큰 tumor 측 1개 : Tumor 주변 2개 :Tumor 와정상 2개 :Tumor 와정상 없음 1개 : 더큰 tumor 측 1개 : 가능한정맥 2개 :Tumor 와정상 1개 : 정상 있음 1개 : 더큰 tumor 측 1개 : Tumor 주변 1개 : Tumor 2개 :Tumor 와정상 없음 1개 : 더큰 tumor 측 1개 : 가능한정맥 1개 : Tumor 1개 : 정상 검체생략이가능한경우 ( 아래 2 경우를제외, 모든경우는요약표에따라검체채취가이루어져야함 ) 요약표에따라채취를시도하였으나기술적으로불가능한경우 복강경수술의경우난소정맥과대망정맥채혈