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摘要:
背景 以肝细胞肝癌(hepatocellular carcinoma，HCC)为主的肝肿瘤在中国人死亡危险因素中排第5位，肝细胞肝癌的5年生存率仅为12.5%，对确诊肝癌的患者进行分级治疗显得尤为重要。目前仍然没有被广泛接受可以在术前对肝细胞肝癌患者的微血管浸润(microvascular invasion，MVI)进行精准预测的方法。 目的 探讨血浆循环核酸(circulating free DNA，cfDNA)在术前预测肝细胞肝癌微血管浸润风险中的作用。 方法 连续收集解放军总医院第一医学中心肝胆外科2019年3 - 10月61例肝肿瘤患者术前外周血标本，根据术后病理结果选取其中43例诊断为肝细胞肝癌和6例良性肿瘤的患者进行基因组测序，并进行了染色体不稳定分析，即超灵敏染色体非整倍体检测(ultrasensitive-chromosomal aneuploidy detector，UCAD)得到染色体不稳定性(chromosome instability，CIN)评分，该评分是对比患者cfDNA与人类正常基因组的测序结果，通过数学方法计算出患者循环核酸不稳定性的程度。分析CIN对于诊断HCC、预测MVI及术后复发的价值。 结果 对患者的CIN评分进行log10转换后，HCC患者的CIN评分高于良性病变患者，差异有统计学意义(3.152±0.421 vs 2.282±0.099，P=0.032)。血浆CIN评分 ≥ 1 697.5预测MVI阳性的敏感度为76.9%，特异性为86.7%，优于甲胎蛋白以及现有的预测模型。所有患者中位随访时间5个月，14例血浆CIN评分 ≥ 1 697.5的HCC患者中有5例在半年内复发(35.7%)，而29例CIN评分较低(＜1 697.5)的患者仅有2例(5.71%)复发(P=0.019)。 结论 cfDNA染色体不稳定性评分 ≥ 1 697.5可在术前预测肝癌患者发生MVI的风险，优于现有的预测指标，并且提示术后早期复发的风险。 Abstract:Background Liver cancer, mainly as hepatocellular carcinoma (HCC), ranks the fifth among the risk factors for death in Chinese, and the five-year survival rate of HCC is only 12.5%. Therefore, it is particularly important to carry out treatment according tumor stage. Currently, there is still no widely accepted method to accurately predict microvascular invasion in patients with HCC before surgery. Objective To investigate the role of circulating free DNA (cfDNA) in predicting the risk of microvascular invasion (MVI) and prognosis of hepatocellular carcinoma (HCC). Methods Preoperative peripheral blood samples were collected from 61 patients with liver tumors from March to October in 2019 in the Department of Hepatobiliary Surgery of the First Medical Center of Chinese PLA General Hospital. According to the postoperative pathologic results, 43 patients were diagnosed with hepatocellular carcinoma and 6 cases with benign tumors. These blood specimens were genome sequenced, then their chromosome instability was analyzed. UCAD (Ultrasensitive-Chromosomal Aneuploidy Detector) was applied to get chromosome instability (chromosome instability, CIN) score, which made a comparison between the cfDNA of the patient and the sequencing results of the normal human genome, and calculated the degree of circulating nucleic acid instability of the patients through mathematical methods. Results After log conversion, the CIN score of HCC patients was significantly higher than that of the benign lesions (3.152 ± 0.421 vs 2.282 ± 0.099, P=0.032). The sensitivity and specificity of plasma CIN score ≥ 1697.5 to predict MVI positive result was 76.9% and 86.7%, respectively, which was better than AFP and the existing prediction model. Among the 43 HCC patients, tumor recurred within half a year in 5 (35.7%) patients with high CIN score (≥ 1 697.5), while only 2 (5.71%) in patients with low CIN score (＜1 697.5) (P=0.019). Conclusion cfDNA chromosomal instability score ≥ 1 697.5 can predict the risk of MVI in patients with liver cancer before surgery and suggest the risk of early postoperative recurrence, which is better than the existing prediction indicator. -
图 1 染色体拷贝数变异覆盖率图示(A：HCC患者；B：良性肿瘤患者)
Figure 1. Chromosome copy number coverage chart (A: HCC patients; B: Benign tumor patients)
图 2 49例患者的染色体不稳定性评分及ROC曲线
A：HCC患者及良性肿瘤患者CIN评分比较；B：CIN评分为788.8时预测HCC的能力
Figure 2. Results of CIN scores and ROC curves in 49 patients
A: Comparison of CIN score between the HCC patients and the benign tumor patients; B: Ability to predict HCC with CIN score of 788.8
图 3 CIN评分与微血管侵犯的相关性
A：不同MVI程度患者以及良性肿瘤患者CIN平均值；B：CIN评分 ≥ 1 679.5预测MVI；C：CIN评分 ≥ 1 679.5预测MVI 2
Figure 3. Correlation between CIN scores and MVI
A: Mean CIN of patients with different degrees of MVI and benign tumors; B: CIN score ≥ 1 679.5 in predicton of MVI; C: CIN score ≥ 1 679.5 in predicton of MVI 2
图 4 CIN高评分(≥ 1 679.5)与术后复发的相关性
A：HCC患者的CIN评分与术后随访，其中黑色线代表CIN高评分，蓝色系代表CIN低评分，红色线代表肿瘤复发，绿色线代表未复发；B：HCC患者术后无病生存曲线，红色代表CIN低评分，蓝色代表CIN高评分
Figure 4. Correlation between CIN ≥ 1 679.5 and postoperative recurrence
A: CIN score and postoperative follow-up of HCC patients, in which the black line represents the CIN high score, the blue line represents the CIN low score, the red line represents tumor recurrence, and the green line represents no recurrence; B: Postoperative disease-free survival curve for HCC patients, with red representing low CIN score and blue representing high CIN score
表 1 HCC患者各项临床指标与CIN评分的关系(n)
Table 1. Relationship between clinical indicators and CIN score in the HCC patients (n)
Characteristic cfDNA CIN Positive Negative χ2 P Age 0.044 0.834 ≥57 yrs 11 6 ＜ 57 yrs 16 10 Gender − 0.635 Male 23 15 Female 4 1 Tumor size − 0.001 ≥ 5 cm 16 1 3 - 5 cm 7 6 ＜ 3 cm 4 9 AFP − 0.033 ≥ 200 ng/μL 10 1 ＜ 200 ng/μL 17 15 MVI − 0.139 2 4 0 1 7 2 0 16 14 HBV cps − 0.719 ＞1×103 7 3 ＜1×103 20 13 Count of lesions − 0.695 Multiple 5 2 Single 22 14 − calculated by Fisher’s exact test. 
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表 2 各项检查指标与MVI的关系
Table 2. Relationship between each index and MVI
Variable Predict MVI (M1+M2) Predict M2 P OR 95% CI P OR 95% CI cfDNA CIN 0.007 2.09 1.27，3.76 0.074 1.88 0.96，4.12 CTC 0.155 3.46 0.94，54.18 0.997 0.00 − AFP 0.075 1.17 0.99，1.39 0.151 1.20 0.94，1.59 ALP 0.066 1.02 1.00，1.05 0.059 1.04 1.00，1.10 BIL 0.589 1.03 0.93，1.13 0.233 1.08 0.94，1.20 ALB 0.820 1.01 0.90，1.15 0.349 1.12 0.92，1.50 AST 0.997 1.00 0.96，1.03 0.921 1.00 0.92，1.00 ALT 0.781 1.00 0.96，1.02 0.548 0.98 0.91，1.00 GGT 0.719 1.00 0.99，1.01 0.391 1.01 0.99，1.00 HBV＞
1 000 cps0.638 1.00 NA，1.00 0.722 1.00 NA，1.00 Size 0.188 1.13 0.95，1.37 0.325 1.12 0.86，1.40 Age＞57 yrs 0.147 0.95 0.88，1.01 0.522 0.97 0.85，1.10 Gender (male) 0.615 1.64 0.20，11.24 0.399 2.92 0.13，30.00
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