Supplementary Material for: ctDNA in Neuroendocrine Carcinoma of Gastroenteropancreatic Origin or of Unknown Primary: The CIRCAN-NEC Pilot Study

Introduction: Gastroenteropancreatic neuroendocrine carcinomas (GEPNEC) are characterized by a heterogeneous molecular profile and a poor prognosis. Circulating tumour DNA (ctDNA) analysis may be useful for NEC management. This study aimed at describing ctDNA mutations, to assess their predictive value for response to chemotherapies, and their change according to disease progression. Methods: The CIRCAN-NEC study included patients with GEPNEC or NEC from an unknown primary, scheduled to begin first- or second-line chemotherapy. Blood samples were collected prior to chemotherapy initiation, at first evaluation, and during disease progression. ctDNA was sequenced by next-generation sequencing (NGS). Molecular response was defined as a decrease of at least 30% of the mutant allele fraction. Results: All 24 patients included received platinum-etoposide first-line chemotherapy; 19 received a FOLFIRI-based post-first-line regimen. Twenty-two patients had at least one driver mutation: TP53 (n = 21), RB1 (n = 2), KRAS (n = 4), and BRAF (n = 3). Ten (42%) had an “adenocarcinoma-like” profile. Five of 6 patients with matching ctDNA/tissue NGS harboured at least one concordant mutation (44% concordance at the gene level). The concordance rate between ctDNA mutation/immunohistochemistry profile was 64% (7/11) for TP53/p53+ and 14% (1/7) for RB1/pRb−. In this pilot study including few patients by subgroups, patients with KRAS (HR = 3.60, 95% CI [1.06–12.04]) and BRAF (HR = 4.25, 95% CI [1.11–16.40]) mutations had shorter progression-free survival (PFS) under platinum-etoposide, while the 2 patients with RB1 mutations had shorter PFS under FOLFIRI-based chemotherapy. Twenty-eight periods of treatment were assessed: 10 patients had a molecular response (7/10 had a morphological response), which was associated with longer PFS (HR = 0.37, 95% CI [0.15; 0.91]). Conclusion: This pilot study shows a high sensitivity of ctDNA assessment, which is encouraging for the future management of GEPNEC (tumour molecular diagnosis and evaluation of disease progression).引言：胃肠胰神经内分泌癌（Gastroenteropancreatic neuroendocrine carcinomas, GEPNEC）以分子特征异质性显著、预后较差为核心特征。循环肿瘤DNA（circulating tumour DNA, ctDNA）分析对神经内分泌癌（neuroendocrine carcinomas, NEC）的临床管理具有潜在应用价值。本研究旨在阐明ctDNA的突变谱特征，评估其对化疗应答的预测效能，并分析其随疾病进展的动态变化。方法：CIRCAN-NEC研究纳入拟接受一线或二线化疗的GEPNEC或原发灶不明NEC患者。分别于化疗起始前、首次疗效评估阶段及疾病进展时采集血液样本。采用下一代测序（next-generation sequencing, NGS）技术对ctDNA进行测序。分子应答定义为突变等位基因分数至少降低30%。结果：所有纳入的24例患者均接受了依托泊苷联合铂类的一线化疗；其中19例后续接受了以FOLFIRI为基础的二线治疗方案。22例患者检出至少1种驱动基因突变：TP53突变21例、RB1突变2例、KRAS突变4例、BRAF突变3例。10例（42%）呈现"腺癌样"分子特征。在6例可匹配ctDNA与组织NGS结果的患者中，5例检出至少1种一致性突变，基因水平一致率为44%。ctDNA突变与免疫组化结果的一致率方面，TP53/p53阳性的一致率为64%（7/11），RB1/pRb阴性的一致率为14%（1/7）。在这项亚组患者数量有限的先导性研究中，携带KRAS（HR=3.60，95%置信区间CI[1.06–12.04]）或BRAF（HR=4.25，95%CI[1.11–16.40]）突变的患者在接受依托泊苷联合铂类化疗时，无进展生存期（progression-free survival, PFS）更短；而2例携带RB1突变的患者在接受FOLFIRI为基础的化疗时PFS更短。共评估28个治疗周期：10例患者出现分子应答，其中7/10同时出现形态学应答；分子应答与更长的PFS相关（HR=0.37，95%CI[0.15–0.91]）。结论：本先导性研究证实ctDNA检测具有较高的灵敏度，这为GEPNEC的后续管理——包括肿瘤分子诊断与疾病进展评估——提供了积极的研究前景。