研究目的

1. 了解卵巢恶性生殖细胞肿瘤（MOGCT）患者不同临床阶段循环肿瘤DNA（ctDNA）状态，探究ctDNA与临床指标及预后之间的关系。

2. 探究 MOGCT 患者不同临床阶段循环肿瘤细胞（CTC）数量变化情况，分析CTC与临床特征及预后之间的关联。

3. 探究MOGCT患者CTC及播散肿瘤细胞（DTC）体外培养条件，建立个体化MOGCT 细胞系，开展药物敏感性测试并与患者临床用药疗效进行相关性分析。

研究方法

1. 收集患者肿瘤组织和不同时间节点的外周血，对肿瘤组织进行WES检测，采用基于肿瘤突变信息的个体化ctDNA检测方法，检测外周血ctDNA状态并计算MTM值，分析其与预后的关系。

2. 收集患者不同时间节点的外周血，采用LABYRINTH-K01系统进行CTC富集分离并计数，分析CTC数量和临床特征及预后的相关性。

3. 收集患者腹水或腹腔冲洗液以及计数剩余的外周血，采用LABYRINTH-CE01系统收集DTC和CTC进行体外培养，成功建立的细胞系进行化疗药物剂量-反应实验，获得药敏数据，与临床用药疗效对比分析。

研究结果

1. 共14例患者进行ctDNA检测。ctDNA总体基线阳性率92.9%。治疗结束3个月时ctDNA状态和预后相关，ctDNA阳性患者较阴性患者无进展生存期（PFS）显著缩短（P=0.044），治疗结束后任一点ctDNA阳性患者PFS与持续阴性患者相比也呈缩短趋势（P=0.15）。治疗和随访期间ctDNA变化可以反映患者临床治疗效果并提示复发。

2. 共39例MOGCT患者采集到基线外周血、28例患者采集到治疗结束和（或）随访期间外周血进行CTC检测。MOGCT患者基线CTC数量与mIGCCCG分层相关，高危组患者基线CTC数量显著高于中低危组（7.75 vs. 2.2，P=0.044）。治疗后CTC清除率和预后相关，治疗结束3个月时，CTC下降率不足70%的患者与超过70%的患者相比 PFS 显著缩短，复发风险升高（P=0.016，HR=13.06）。

3. 纳入33例患者的35例腹水/腹腔冲洗液样本进行DTC富集分离和体外培养，28例实现体外存活，15例体外扩增细胞量达到10^7及以上，最大增殖代数19代，最长培养时间129天。纳入15例外周血样本进行CTC培养，3例实现短期扩增，最大传代数4代，最长培养时间48天。使用个体化细胞系进行药物剂量-反应实验，10代之内的细胞对药物的敏感性特征稳定，同一患者来源的DTC和CTC对同种药物的敏感性也无统计学差异（P>0.05）。选取患者015和020进行体外细胞药物敏感性和临床用药疗效的相关性分析。临床上患者015比020对药物反应更加敏感，体外实验中，015细胞顺铂的半抑制浓度比020更低（12.94μM vs. 33.08μM），同等顺铂浓度条件下015细胞存活率更低（P=0.0126），表明015的细胞系对顺铂更敏感，与临床疗效相一致。016细胞系药敏实验数据可以部分解释患者016对化疗的响应情况，进行疗效预测评估。

研究结论

1. 本研究首次实现MOGCT患者的ctDNA和CTC检测，证实了基于肿瘤突变信息的个体化ctDNA检测和非抗原依赖性CTC检测方法的可行性。

2. MOGCT患者治疗后ctDNA状态和预后相关，ctDNA的动态变化可以辅助疗效评估并监测复发。

3. MOGCT患者外周血基线CTC数量与mIGCCCG分层相关，治疗后CTC清除率和预后相关。

4. 采用非抗原依赖性CTC富集技术对MOGCT患者腹水/腹腔冲洗液中的肿瘤细胞进行捕获，经过体外培养可以建立个体化细胞系，体外细胞对化疗药物的敏感性和供体患者临床用药疗效相一致，可对患者用药疗效进行预测评估。

Objectives

1. To investigate circulating tumor DNA (ctDNA) status at different clinical stages in malignant ovarian germ cell tumor (MOGCT) patients and explore its relationship with clinical indicators and prognosis.

2. To analyze circulating tumor cell (CTC) counts across different clinical stages and assess their association with clinical characteristics and prognosis.

3. To optimize in vitro culture conditions for DTCs from MOGCT patients and establish individualized cell lines. To analyze the correlation between the drug sensitivity test of patient-derived cell lines and clinical treatment efficacy.

Methods

1. Tumor tissues and peripheral blood samples were collected. Whole-exome sequencing (WES) was performed on tumor tissues and blood samples to identify somatic variants for ctDNA detection. The ctDNA status and MTM values were analyzed for their correlations with clinical characteristics and prognosis.

2. Peripheral blood samples from MOGCT patients at different timepoints were processed using the LABYRINTH-K01 system for CTC enrichment and enumeration. CTC counts were correlated with clinical features and prognosis.

3. DTC were enriched using the LABYRINTH-CE01 system from ascites or peritoneal washings for in vitro culture. Residual peripheral blood samples were used for CTC enrichment, isolation, and in vitro culture. Established cell lines were subjected to drug sensitivity tests with chemotherapy agents. Drug sensitivity results were then correlated with clinical outcomes.

Results

1. Fourteen patients were included for ctDNA analysis. The baseline ctDNA positivity rate was 92.9%. ctDNA-positive patients had significantly shorter PFS compared to ctDNA negative patients (P = 0.044). Additionally, ctDNA positivity at any time point after treatment showed a trend toward shorter PFS compared to continuously ctDNA-negative patients (P = 0.15). Changes in ctDNA levels during treatment and follow-up could reflect clinical treatment efficacy and detect disease relapse.

2. Peripheral blood samples for CTC detection were collected from 39 patients at baseline and from 28 patients after treatment and during follow-up. In MOGCT patients, the baseline CTC count is associated with modified International Germ Cell Cancer Collaborative Group (mIGCCCG) risk stratification, with poor-risk group patients having a significantly higher CTC count compared to the good- and intermediate-risk group (7.75 vs. 2.2, P=0.044). The clearance rate of CTC before and after treatment was correlated with prognosis. Three months after treatment, patients with a CTC reduction rate of less than 70% had significantly shorter PFS and increased risk of recurrence compared to those with a reduction rate over 70% (P = 0.016, HR = 13.06).

3. A total of 35 ascites or peritoneal washing samples from 33 patients were used for DTC enrichment, isolation, and in vitro culture. DTCs were successfully cultured in vitro in 28 samples, with 15 samples achieving cell amplification over 10⁷ cells. The maximum proliferation generation was 19 passages, and the maximum culture duration was 129 days. Short-term CTC expansion occurred in 3/15 blood samples, with a maximum proliferation generation of 4 passages and a longest culture duration of 48 days. Drug sensitivity remained stable within 10 passages. DTCs and CTCs derived from the same patient showed consistent sensitivity profiles to cisplatin, with no significant differences observed (P > 0.05). Patient-derived cell lines mirrored clinical drug sensitivity. Cell lines derived from patient 015 demonstrated a lower cisplatin IC50 (12.94μM vs. 33.08μM) and significantly reduced viability at equivalent cisplatin concentrations (P = 0.0126), consistent with its superior clinical response compared to patient 020. Drug sensitivity data of patient-derived cell line from patient 016 explained the patient’s clinical response to chemotherapy regimens and provided predictive evaluations of therapeutic efficacy.

Conclusion

1. This study is the first to implement ctDNA and CTC detection in MOGCT patients, validating the feasibility of personalized ctDNA monitoring based on tumor mutational profiles and label-independent CTC enrichment methodologies.

2. Post-treatment ctDNA status correlates with prognosis in MOGCT patients, and longitudinal ctDNA dynamics may aid in treatment response assessment and recurrence monitoring.

3. In MOGCT patients, peripheral blood baseline CTC counts show significant association with mIGCCCG risk stratification, while post-treatment CTC clearance predicts survival outcomes.

4. In MOGCT patients, DTCs isolated by the label-free CTC enrichment method from ascites and peritoneal washings can be cultured in vitro to establish personalized cell lines. Drug sensitivity profiles of patient-derived cell lines correlate with clinical treatment efficacy, supporting their utility for predictive therapeutic evaluation.