Supplementary Material for: Ki-67 Index of 55% Distinguishes Two Groups of Bronchopulmonary Pure and Composite Large Cell Neuroendocrine Carcinomas with Distinct Prognosis

<b><i>Background:</i></b> Little information is available concerning prognostic factors for bronchopulmonary large cell neuroendocrine carcinomas (BP-LCNECs) and even less is known about combined LCNECs (Co-LCNECs). We investigated whether an integrated morphological, immunohistochemical, and molecular approach could be used for their prognostic evaluation. <b><i>Methods:</i></b> Morphological (including combined features), proliferative (mitotic count/Ki-67 index), immunohistochemical (napsin A, p40, TTF-1, CD44, OTP, SSTR2A, SSTR5, mASH1, p53, RB1, and MDM2), and genomic (<i>TP53</i>, <i>RB1</i>, <i>ATM</i>, <i>JAK2</i>, <i>KRAS</i>, and <i>STK11</i>) findings were analyzed in BP-LCNECs from 5 Italian centers, and correlated with overall survival (OS). The Ki-67 index was expressed as the percentage of positive cells in hot spots as indicated in the WHO 2019 Digestive System Tumors and, for Co-LCNECs, the Ki-67 index was evaluated only in the LCNEC component. <b><i>Results:</i></b> A total of 111 LCNECs were distinguished into 70 pure LCNECs, 35 Co-LCNECs (27 with adenocarcinoma [ADC] and 8 with squamous cell carcinoma [SqCC]), and 6 LCNECs with only napsin A immunoreactivity. The Ki-67 index cutoff at 55% evaluated in the neuroendocrine component was the most powerful predictor of OS (log-rank <i>p</i> = 0.0001) in all LCNECs; 34 cases had a Ki-67 index &lt;55% (LCNEC-A) and 77 had a Ki-67 index ≥55% (LCNEC-B). Statistically significant differences in OS (log-rank <i>p</i> = 0.0001) were also observed between pure and Co-LCNECs. A significant difference in OS was found between pure LCNECs-A and Co-LCNECs-A (<i>p</i> &lt; 0.05) but not between pure LCNECs-B and Co-LCNECs-B. Co-LCNEC-ADC and LCNEC napsin A+ cases had longer OS than pure LCNEC and Co-LCNEC-SqCC cases (log-rank <i>p</i> = 0.0001). On multivariable analysis, tumor location, pure versus combined features, and napsin A, but no single gene mutation, were significantly associated with OS after adjustment for Ki-67 index and study center (<i>p</i> &lt; 0.05). <b><i>Conclusions:</i></b> The Ki-67 proliferation index and the morphological characterization of combined features in LCNECs seem to be important tools for predicting clinical outcome in BP-LCNECs.**背景：** 目前关于支气管肺大细胞神经内分泌癌（bronchopulmonary large cell neuroendocrine carcinomas, BP-LCNECs）的预后影响因素相关研究数据较为匮乏，而针对联合型大细胞神经内分泌癌（combined LCNECs, Co-LCNECs）的认知则更为有限。本研究旨在探讨整合形态学、免疫组织化学及分子生物学的综合分析方法，是否可用于此类肿瘤的预后评估。 **方法：** 本研究对来自5个意大利医学中心的BP-LCNEC患者样本开展分析，涵盖形态学特征（含联合分型特征）、增殖活性指标（核分裂计数/Ki-67指数）、免疫组织化学标志物（napsin A、p40、TTF-1、CD44、OTP、SSTR2A、SSTR5、mASH1、p53、RB1及MDM2）以及基因组学指标（*TP53*、*RB1*、*ATM*、*JAK2*、*KRAS*及*STK11*），并将上述指标与患者总生存期（overall survival, OS）进行相关性分析。Ki-67指数以《WHO消化系统肿瘤分类（2019版）》中规定的热点区域阳性细胞百分比表示；针对Co-LCNECs，仅在大细胞神经内分泌癌成分中评估Ki-67指数。 **结果：** 本研究共纳入111例LCNEC病例，其中70例为纯型大细胞神经内分泌癌，35例为联合型大细胞神经内分泌癌（27例伴腺癌[adenocarcinoma, ADC]成分，8例伴鳞状细胞癌[squamous cell carcinoma, SqCC]成分），另有6例仅表现为napsin A免疫反应阳性的LCNEC。在所有LCNEC病例中，以神经内分泌成分中Ki-67指数≥55%作为截断值，是预测总生存期最具效力的指标（log-rank检验，*p* = 0.0001）。据此可将病例分为两组：34例Ki-67指数<55%（命名为LCNEC-A组），77例Ki-67指数≥55%（命名为LCNEC-B组）。纯型与联合型LCNEC患者的总生存期亦存在显著统计学差异（log-rank检验，*p* = 0.0001）。纯型LCNEC-A组与联合型LCNEC-A组的总生存期存在显著差异（*p* < 0.05），但纯型LCNEC-B组与联合型LCNEC-B组之间未观察到类似差异。伴ADC成分的联合型LCNEC以及napsin A阳性LCNEC病例的总生存期长于纯型LCNEC及伴SqCC成分的联合型LCNEC病例（log-rank检验，*p* = 0.0001）。多变量分析结果显示，在校正Ki-67指数及研究中心因素后，肿瘤部位、纯型/联合型分型特征以及napsin A表达情况与总生存期显著相关（*p* < 0.05），而单基因突变则无此关联。 **结论：** Ki-67增殖指数以及LCNEC的联合分型特征，可作为预测支气管肺大细胞神经内分泌癌患者临床结局的重要辅助工具。