斑点追踪分层分析及miRNA早期诊断并预测蒽环类药物心脏毒性的实验和临床研究

Anthracycline remains a commonly used chemotherapeutic agent in the treatment of a wide spectrum of hematological malignancies and solid tumors. However, their efficacy is undermined by potential life-threatening cardiotoxicity. It has been reported that more than 50% patients treated with anthracycline had subclinical left ventricular dysfunction during the first year. Thus, an non-invasive, sensitive and reliable method in detecting cardiac function is of vital importance during anthracycline administration to balance the cardiac risk and the potential cancer treatment benefits. In our previous studies, we demonstrated that strain values analyzed by speckle tracking echocardiography decreased significantly after anthracycline exposure, even though LVEF remained stable and within normal range. In animal models, we found that strain value decrease after doxorubicin treatment was in accordance with histological lesions. Cardiotoxicity induced by anthracycline has a regional pattern and the subendocardial region of myocardium is most sensitive to anthracycline, which has been improved by cardiac magnetic resonance. Therefore, our hypothesis is that early cardiac injury caused by anthracycline could be investigated by multi-layer analysis based on speckle tracking echocardiography. Furthermore, during chronic doxorubicin treatment, consistent dose-dependent response of several miRNAs were observed in rat hearts. The introduction of reliable early miRNA biomarkers may help to assess drug-induced cardiotoxicity. Based on the data above, we intend to build cardiac toxicity rabbit models using different dosage of anthracycline：① to analyze the correlation between speckle tracking multi-layer and pathological examination; ② to build networks of regulated miRNAs and to evaluation the correlation between histopathological examinations and the levels of miRNAs. Furthermore, we follow up the patients with lymphoma treated with anthracycline: ① to determine the cut-off values of the parameters of speckle tracking multi-layer analysis in detecting cardiac scar burden analyzed by cardiac magnetic resonance; ② to analyze the cut-off values of the parameters of speckle tracking multi-layer analysis in predicting early cardiotoxicity induced by anthracycline; ③ to detect the cut-off values of the plasma miRNAs levels in predicting early cardiotoxicity induced by anthracycline. The purpose of our study is to find out non-invasive, reliable and sensitive echocardiographic parameters and plasma biomarkers for early detection and prediction anthracycline-induced cardiac toxicity and to be helpful to target patients at high risk of cardiotoxicity, who could benefit from closer monitoring or earlier initiation of cardioprotective therapy.

蒽环类治疗第一年亚临床心功能损害发病率达50%，且难以逆转，早期检出意义重大。前期发现，斑点追踪显像（STI）可先于LVEF识别药物导致的左室功能异常，且与心肌病理改变一致。由于蒽环类药物的心肌损伤早期主要累及心内膜下心肌，STI分层分析有望早期发现局部心功能改变。另外，蒽环类药物诱导特异性心肌miRNA表达变化，且与病理改变一致，有望通过分析血浆miRNA表达水平监测早期心脏毒性。本研究拟建立蒽环类心脏毒性动物模型：1、分析STI分层分析参数与心肌病理相关性；2、筛选心肌及血浆中miRNA表达谱及其表达水平变化与心肌病理的相关性。随访蒽环类化疗患者，分析：1、STI参数识别心肌疤痕的敏感、特异性；2、STI参数对心脏毒性的预测阈值及监测时间点；3、根据动物实验结果，筛选并确定血浆miRNA表达水平变化及其对心脏毒性的预测阈值及监测时间点；4、STI与miRNA对预测心脏毒性的权重。

蒽环类治疗第一年亚临床心功能损害发病率达50%，且难以逆转，早期检出意义重大。我们的研究建立了蒽环类药物心脏毒性的大鼠模型。动物研究发现，在LVEF下降前，斑点追踪分层分析参数可以发现隐匿性心脏毒性的发生，且RS，CS-ENDO下降的程度与病理改变和肌钙蛋白升高一致。另外，基因芯片提示，蒽环类药物处理后，大鼠血浆多个miRNA表达发生改变。通过基因库比对及筛选，初步认为miR-122-5p，miR-19b-3p，miR-20a-5p，miR-223-3p，miR-92a-3p表达降低，血浆miR-208a-5p表达增高可能可以提示化疗后的心肌损伤。在临床研究中，我们入选了化疗后患者，与正常对照组进行比较。研究发现，化疗后即使LVEF无变化，斑点追踪分层应变参数GLS, GCS, CS-ENDO, LS-EPI, LS-MID,LS-ENDO均出现了显著的下降，提示斑点追踪分层应变参数可能可以早于LVEF发现药物所致的隐匿性心脏损害。我们的进一步研究针对初发NHL化疗患者进行系列随访，在化疗前，化疗第3周期，第6周期结束后及化疗后半年进行系列随访。研究发现，尽管常规超声心动图参数未发生变化，与化疗前比较，第3周期结束后，斑点追踪参数GLS, GCS及CS心内膜-外膜梯度均明显下降，超敏肌钙蛋白明显上升，第三周期结束后GLS的下降率及超敏肌钙蛋白上升可以预测随访期间心脏毒性的发生。这两项参数重复性良好，敏感性特异性高。我们同时确定了这两项参数的预测截值及监测时间点，具有很强的临床实际应用价值。另外，根据动物实验的结果，我们初步筛选了miR-19b-3p，miR-20a-5p，及miR-208a-5p作为候选miRNA。第3周期结束后，血浆miR-19b-3p，miR-20a-5p表达降低，血浆miR-208a-5p表达增高，这些miRNA有望成为心脏损害新的无创检测手段。.综上所述，本课题通过动物实验和临床研究，对蒽环类药物的心脏毒性反应早期监测总结出一套无创，敏感，相对成熟的指标。研究发现，基于斑点追踪的应变参数，血清超敏肌钙蛋白及血浆miRNA可以帮助早期诊断药物所致的隐匿性心脏毒性，敏感性特异性良好。.本研究严格按照申请书的内容实施了研究计划，顺利完成既定目标。