Supplementary Material for: Cell-Free DNA and Clinical Characteristics in Patients with Small Intestinal or Pancreatic Neuroendocrine Tumors

<b><i>Introduction:</i></b> Neuroendocrine tumors (NETs) are rare and characterized by a heterogeneous clinical course and an unmet need for better prognostic markers. Plasma cell-free DNA (cfDNA) has prognostic value in other malignancies but is not previously investigated in NETs. We studied cfDNA levels in patients with mainly low-grade small intestinal NET ­(siNET) or pancreatic NET (pNET) and evaluated the prognostic potential of cfDNA. <b><i>Materials and Methods:</i></b> We included 70 NET patients, siNET (<i>n</i> = 50) and pNET (<i>n</i> = 20). Plasma cfDNA levels were determined by droplet digital PCR for the <i>beta-2-microglobulin</i> gene every 6 months during a period of 3 years, including in a subgroup of 19 patients during peptide receptor radionuclide therapy (PRRT) therapy. <b><i>Results:</i></b> cfDNA levels were higher in both siNET and pNET compared to a previously established healthy cohort (<i>p</i> &lt; 0.0001). ­cfDNA levels did not predict overall survival (crude hazard ratio [HR] 0.95 [0.57–1.58], <i>p</i> = 0.837, adjusted for smoking status HR 0.77 [0.51–1.17], <i>p</i> = 0.22). The impact of cfDNA level on progression-free survival showed different trends in siNET and pNET. There was no effect of PRRT treatment on cfDNA levels and no difference in cfDNA levels between patients with and without progressive disease after PRRT (ANOVA <i>p</i> = 0.66). cfDNA levels were significantly higher in never-smokers and previous smokers than in current smokers (<i>p</i> = 0.029). <b><i>Discussion/Conclusion:</i></b> cfDNA levels are higher in NET patients than in healthy controls; however, there was no association with prognosis, and cfDNA levels were unaffected by PRRT. Our observations suggest that cfDNA levels are not associated with the disease course in low-grade NET in contrast to other malignancies.

**<i>引言：</i>** 神经内分泌肿瘤（Neuroendocrine tumors, NETs）属于罕见疾病，其临床病程具有高度异质性，且目前仍存在对更优质预后标志物的未被满足的临床需求。血浆游离脱氧核糖核酸（plasma cell-free DNA, cfDNA）在其他恶性肿瘤中已被证实具有预后价值，但此前尚未在神经内分泌肿瘤中开展相关研究。本研究针对以低级别小肠神经内分泌肿瘤（small intestinal NET, siNET）或胰腺神经内分泌肿瘤（pancreatic NET, pNET）为主的患者，检测了其血浆cfDNA水平，并评估了cfDNA的预后潜力。**<i>材料与方法：</i>** 本研究共纳入70例神经内分泌肿瘤患者，其中小肠神经内分泌肿瘤患者50例（<i>n</i> = 50），胰腺神经内分泌肿瘤患者20例（<i>n</i> = 20）。采用液滴数字聚合酶链反应（droplet digital PCR）检测针对β2-微球蛋白（beta-2-microglobulin）基因的血浆cfDNA水平，随访周期为3年，每6个月检测一次；其中19例接受肽受体放射性核素治疗（peptide receptor radionuclide therapy, PRRT）的患者亚组也纳入了该检测流程。**<i>结果：</i>** 与既往建立的健康对照队列相比，小肠神经内分泌肿瘤和胰腺神经内分泌肿瘤患者的血浆cfDNA水平均显著升高（<i>p</i> < 0.0001）。cfDNA水平无法预测患者总体生存率（粗风险比[HR] 0.95 [95%置信区间 0.57–1.58]，<i>p</i> = 0.837；校正吸烟状态后风险比为0.77 [95%置信区间 0.51–1.17]，<i>p</i> = 0.22）。cfDNA水平对无进展生存期的影响在小肠神经内分泌肿瘤和胰腺神经内分泌肿瘤中呈现出不同的趋势。肽受体放射性核素治疗对患者cfDNA水平无显著影响，且接受治疗后出现疾病进展与未出现疾病进展的患者之间，cfDNA水平亦无显著差异（方差分析[ANOVA] <i>p</i> = 0.66）。从不吸烟者和既往吸烟者的cfDNA水平显著高于当前吸烟者（<i>p</i> = 0.029）。**<i>讨论/结论：</i>** 神经内分泌肿瘤患者的血浆cfDNA水平高于健康对照人群，但该水平与患者预后无关联，且不受肽受体放射性核素治疗的影响。本研究结果提示，与其他恶性肿瘤不同，低级别神经内分泌肿瘤患者的血浆cfDNA水平与疾病病程并无关联。