DLL3在胃肠胰神经内分泌肿瘤中的表达及其潜在诊断价值-动脉网

Exploring the expression of DLL3 in gastroenteropancreatic neuroendocrine neoplasms and its potential diagnostic value

Nature 等信源发布 2025-01-26 17:41

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Abstract

摘要

Delta-like protein (DLL3) is a novel therapeutic target. DLL3 expression in gastroenteropancreatic neuroendocrine tumors (GEP-NECs) is poorly understood, complicating the distinction between well-differentiated neuroendocrine tumors G3 (NET G3) and poorly differentiated NEC. DLL3 immunohistochemistry (IHC) was performed on 248 primary GEP-NECs, correlating with clinicopathological parameters, NE markers, PD-L1, Ki67 index, and prognosis.

Delta样蛋白（DLL3）是一种新型的治疗靶点。DLL3在胃肠胰神经内分泌肿瘤（GEP-NEC）中的表达知之甚少，使高分化神经内分泌肿瘤G3（NET G3）和低分化NEC之间的区别变得复杂。对248例原发性GEP NEC进行DLL3免疫组织化学（IHC），与临床病理参数，NE标志物，PD-L1，Ki67指数和预后相关。

Achaete-scute complex-like 1 (ASCL1) IHC was performed on some GEP-NECs. DLL3 IHC was conducted on 36 GEP-NETs, 29 gastric adenocarcinomas (GACs), and metastatic tumors (9 lymph node metastases and 19 distant metastases). DLL3 expression rates were 54.8% in GEP-NECs at the primary site, associated with small cell neuroendocrine carcinoma (SCNEC) (.

在一些GEP NEC上进行了Achaete-scute复合物样1（ASCL1）IHC。DLL3 IHC在36个GEP NET，29个胃腺癌（GAC）和转移性肿瘤（9个淋巴结转移和19个远处转移）上进行。DLL3在原发部位的GEP NEC中的表达率为54.8%，与小细胞神经内分泌癌（SCNEC）相关。

< 0.001), chemotherapy before baseline (

<0.001），基线前化疗(

= 0.015), and at least two NE markers (

=0.015），和至少两个NE标记(

= 0.048). DLL3 expression in metastatic GEP-NECs was similar to that of primary tumors. Expression rates in NET G1, NET G2, NET G3, and GACs were 0%, 0%, 15.8%, and 0%, respectively, highlighting DLL3 as a powerful tool for identifying poorly differentiated NEC. DLL3 expression was related to ASCL1 in GEP-NECs, especially in SCNEC.

DLL3在转移性GEP NEC中的表达与原发性肿瘤相似。NET G1，NET G2，NET G3和GAC中的表达率分别为0%，0%，15.8%和0%，突出显示DLL3是鉴定低分化NEC的有力工具。DLL3表达与GEP-NEC中的ASCL1有关，特别是在SCNEC中。

It was not correlated with progression-free survival (PFS) or overall survival(OS), regardless of cutoff value (1%, 50%, 75%). In conclusion, DLL3 targeted therapy may offer potential for the treatment of poorly differentiated NEC of the digestive system, although further studies are needed to validate its efficacy..

无论临界值（1%，50%，75%）如何，它与无进展生存期（PFS）或总生存期（OS）无关。总之，DLL3靶向治疗可能为治疗消化系统低分化NEC提供潜力，尽管需要进一步的研究来验证其疗效。。

Introduction

简介

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) are highly heterogeneous tumors that constituting approximately 65% of all neuroendocrine neoplasms and rank as the second most common gastrointestinal cancer

胃肠胰神经内分泌肿瘤（GEP-NENs）是高度异质性的肿瘤，约占所有神经内分泌肿瘤的65%，是第二大最常见的胃肠道肿瘤

. Improved imaging techniques and enhanced awareness have contributed to an increased incidence from 1.1/100,000 in 1973 to 6.9/100,000 in 2012

改进的成像技术和提高的意识使发病率从1973年的1.1/100000增加到2012年的6.9/100000

. According to the 2019 WHO grading system, GEP-NENs are categorized as neuroendocrine tumors (NETs), neuroendocrine carcinomas (NECs), or mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs) based on histological differentiation, mitotic count, and the Ki-67 proliferation index. Well-differentiated NETs are further stratified into Grade1 (G1), Grade2 (G2), and Grade3 (G3) subtypes, whereas NECs with poorly differentiated NECs are classified as small cell NEC (SCNEC) and large cell NEC (LCNEC).

根据2019年世界卫生组织分级系统，GEP-NENs根据组织学分化，有丝分裂计数和Ki-67增殖指数分为神经内分泌肿瘤（NETs），神经内分泌癌（NECs）或混合神经内分泌非神经内分泌肿瘤（MiNENs）。分化良好的NETs进一步分为1级（G1），2级（G2）和3级（G3）亚型，而具有低分化NEC的NEC被分类为小细胞NEC（SCNEC）和大细胞NEC（LCNEC）。

. NECs are often non-functional, highly aggressive, and frequently diagnosed late with distant metastasis, resulting in dismal 5-year survival rates below 5%

。NEC通常无功能，具有高度侵袭性，并且经常被诊断为晚期远处转移，导致5年生存率低于5%

Owing to the advanced stage and poor prognosis of GEP-NECs, treatments primarily aim to extend survival and enhance quality of life. Within the neuroendocrine spectrum, GEP-NECs share similar molecular and transcriptional profiles with small cell lung carcinoma (SCLC). Therefore, GEP-NECs treatment was similar to that for SCLC.

由于GEP NEC的晚期和预后不良，治疗主要旨在延长生存期和提高生活质量。在神经内分泌谱中，GEP-NEC与小细胞肺癌（SCLC）具有相似的分子和转录谱。因此，GEP NEC治疗与SCLC相似。

Systemic chemotherapy, typically platinum-based regimens such as etoposide plus cisplatin (EC) and irinotecan plus cisplatin (IC), is the first-line of treatment for metastatic GEP-NECs. Notably, a Ki-67 index > 55% reliably predicts the reactivity of platinum-based chemotherapy.

全身化疗，通常是基于铂的方案，例如依托泊苷加顺铂（EC）和伊立替康加顺铂（IC），是转移性GEP NEC的一线治疗方法。。

. Although some studies have suggested efficacy for agents such as 5-fluorouracil, irinotecan, and oxaliplatin, there are still no standardized second-line regimens. Ongoing trials, such as the one using 5-fluorouracil + leucovorin + irinotecan offer promise (NCT03387592)

尽管一些研究表明5-氟尿嘧啶，伊立替康和奥沙利铂等药物有效，但仍没有标准化的二线治疗方案。正在进行的试验，例如使用5-氟尿嘧啶++亚叶酸钙++伊立替康的试验提供了希望（NCT03387592）

. Advances in molecular detection technology and basic experiments have led researchers to investigate the molecular pathways and interactions in neuroendocrine neoplasms, offering potential targets for GEP-NECs. For instance, sunitinib, a multitarget tyrosine kinase inhibitor primarily inhibiting vascular endothelial growth factor receptor and platelet-derived growth factor receptor, elicited positive responses in 11 of 20 patients with GEP-NECs in a phase II trial encompassing 6 NET G3 and 20 NEC cases.

分子检测技术和基础实验的进步使研究人员研究了神经内分泌肿瘤中的分子途径和相互作用，为GEP NEC提供了潜在的靶点。例如，舒尼替尼是一种多靶点酪氨酸激酶抑制剂，主要抑制血管内皮生长因子受体和血小板衍生生长因子受体，在包括6例NET G3和20例NEC病例的II期试验中，在20例GEP NEC患者中，有11例引起阳性反应。

. Phase II and III clinical trials testing single- or multi-drug combinations targeting various pathways, including XPO1 (NCT02250885), PARP (NCT04209595), and HDAC (NCT05076786), are currently ongoing for GEP-NECs. Among them, the negative regulator of the Notch pathway, DLL3, has drawn significant attention as a potential therapeutic target for neuroendocrine neoplasms.

GEP NEC目前正在进行II期和III期临床试验，测试针对各种途径的单药或多药组合，包括XPO1（NCT02250885），PARP（NCT04209595）和HDAC（NCT05076786）。其中，Notch通路的负调节因子DLL3作为神经内分泌肿瘤的潜在治疗靶点引起了人们的极大关注。

The Notch pathway, known for its highly conserved signaling cascade, is a critical factor in cellular transformation, including cell proliferation, epithelial-mesenchymal transition, neuroendocrine cell differentiation, chemoresistance, and immune microenvironment modulation

Notch通路以其高度保守的信号级联反应而闻名，是细胞转化的关键因素，包括细胞增殖、上皮-间质转化、神经内分泌细胞分化、化学抗性和免疫微环境调节

. As an inhibitory ligand of the Notch pathway

.作为Notch途径的抑制性配体

, DLL3 interacts with various Notch receptors (Notch1-4) to facilitate malignant transformation. DLL3 expression is absent or minimal in normal cells and is predominantly localized within the Golgi apparatus and cytoplasmic vesicles. Conversely, in malignant NENs, DLL3 translocates to the cell membrane surface, inhibiting the CIS notch pathway.

，DLL3与各种Notch受体（Notch1-4）相互作用以促进恶性转化。DLL3表达在正常细胞中不存在或很少，主要位于高尔基体和细胞质囊泡内。相反，在恶性NEN中，DLL3易位至细胞膜表面，抑制顺式notch途径。

ASCL1

, an intrinsic transcription factor in normal cells, regulates DLL3 expression, guiding neuroendocrine cell differentiation and initiating SCLC

，一种正常细胞中的内在转录因子，调节DLL3的表达，指导神经内分泌细胞分化并启动SCLC

ASCL1

activation increases DLL3 expression, enhancing the inhibition of Notch1 signaling

激活增加DLL3表达，增强对Notch1信号的抑制

. Both the inhibition of Notch1 and the upregulation of

Notch1的抑制和Notch1的上调

ASCL1

contribute to NEN development

为NEN发展做出贡献

. The efficacy of DLL3-targeted therapy has been confirmed in SCLC. Rova-T, a classic antibody-drug conjugate (ADC) in a phase I clinical trial (NCT01901653), has shown efficacy against recurrent or refractory SCLC

DLL3靶向治疗的疗效已在SCLC中得到证实。

. However, severe adverse reactions have halted further clinical investigations of Rova-T. Next-generation ADCs aim to enhance tumor cell uptake by refining drug linkers to mitigate toxicity and optimize pharmacokinetics for improved clinical utility

然而，严重的不良反应停止了Rova-T的进一步临床研究。下一代ADC旨在通过改进药物接头来减轻毒性并优化药代动力学以提高临床效用，从而增强肿瘤细胞的摄取

. Tarlatamab, a bispecific T cell engager (TCE) targeting DLL3 on tumor cells and CD3 on T cells, has demonstrated promising results in a phase I trial, with a 13% objective response rate (ORR) and 71% of patients with SCLC experiencing relief for ≥ 6 months

Tarlatamab是一种靶向肿瘤细胞上DLL3和T细胞上CD3的双特异性T细胞参与者（TCE），在I期试验中显示出有希望的结果，客观缓解率（ORR）为13%，71%的SCLC患者缓解≥6个月

. Other candidate drugs, such as HPN328 and AMG 119, have also shown beneficial antitumor responses in clinical and preclinical stages

其他候选药物，如HPN328和AMG 119，在临床和临床前阶段也显示出有益的抗肿瘤反应

. Patients with DLL3-expressing SCLC or NEC are currently enrolled in the first human Phase I trial of BI 764,532 (TCE) (NCT04429087). Although DLL3-targeted therapies are designed for tumors expressing DLL3, the immunohistochemical positivity for DLL3 expression on tumor cells was not a required criterion for patient enrollment in the trials.

。尽管针对DLL3的疗法是针对表达DLL3的肿瘤而设计的，但DLL3在肿瘤细胞上表达的免疫组织化学阳性并不是患者入选试验的必要标准。

Therefore, DLL3 immunohistochemistry does not have a predictive role in determining eligibility for the trials..

因此，DLL3免疫组织化学在确定试验资格方面没有预测作用。。

Although DLL3 expression was initially identified in SCLC, in vitro investigations have revealed its diverse oncogenic role; elevated expression promotes aggressive behavior through Snail overexpression

尽管最初在SCLC中鉴定出DLL3表达，但体外研究表明其具有多种致癌作用；高表达通过Snail过表达促进攻击行为

. Moreover, DLL3 is highly expressed not only in SCLC but also in lung LCNEC. In a retrospective study of pulmonary LCNEC, over 74% (70/94) of patients expressed DLL3

此外，DLL3不仅在SCLC中高表达，而且在肺LCNEC中也高表达。在一项肺LCNEC的回顾性研究中，超过74%（70/94）的患者表达DLL3

. DLL3 expression extends beyond lung cancer to various invasive malignancies, including prostate cancer, bladder SCNEC, malignant melanoma, glioblastoma, and medullary thyroid carcinoma

DLL3的表达超越了肺癌，扩展到各种侵袭性恶性肿瘤，包括前列腺癌，膀胱SCNEC，恶性黑色素瘤，胶质母细胞瘤和甲状腺髓样癌

, suggesting its potential as a biomarker for neuroendocrine-origin malignancies. However, DLL3 expression in GEP-NECs is poorly understood. This study aimed to investigate DLL3 expression in GEP-NECs and analyze its clinicopathological correlation and the relationship between DLL3 expression and patient prognosis..

，表明其作为神经内分泌源性恶性肿瘤的生物标志物的潜力。然而，对GEP NEC中DLL3的表达知之甚少。本研究旨在研究DLL3在GEP NEC中的表达，并分析其临床病理相关性以及DLL3表达与患者预后的关系。。

Methods

方法

Patient selection

患者选择

All available information in this retrospective study was sourced from the Peking University Cancer Hospital. Basic patient information spanning 2010 to 2023, including age, sex, primary tumor site, histological classification, TNM stage, chemotherapy prior to baseline, and some aspects of prognosis, were primarily collected from medical records.

这项回顾性研究中的所有可用信息均来自北京大学肿瘤医院。主要从医疗记录中收集2010年至2023年的基本患者信息，包括年龄，性别，原发肿瘤部位，组织学分类，TNM分期，基线前化疗以及预后的某些方面。

Telephone follow-up was specifically used to obtain detailed prognostic information, which may not have been fully documented in the medical records or required updates beyond what was originally recorded. TNM staging refer to the American Joint Committee on Cancer (AJCC) 8th edition.

电话随访专门用于获得详细的预后信息，这些信息可能尚未在医疗记录中得到充分记录，也可能没有超出最初记录的更新。TNM分期是指美国癌症联合委员会（AJCC）第8版。

.After re-examination by two experienced pathologists, it was ensured that each case met the WHO definition of neuroendocrine tumors: the shape was organ-like, trabecular and palisade-like, the mitotic count and Ki67 index strictly adhered to the standards of different grades, and expressed at least one neuroendocrine marker (Syn, CgA, CD56).

经过两位经验丰富的病理学家的重新检查，确保每个病例均符合WHO对神经内分泌肿瘤的定义：形状为器官样，小梁状和栅栏状，有丝分裂计数和Ki67指数严格遵守不同等级的标准，并表达至少一种神经内分泌标志物（Syn，CgA，CD56）。

Cases that did not meet these conditions were excluded from the study. Cases that lacked sufficient tissue samples for further staining were also excluded. The study protocol received approval from the Medical Ethics Committee of Peking University Cancer Hospital (approval number 2023KT29.), and all patients provided informed consent before tissue sample utilization..

不符合这些条件的病例被排除在研究之外。还排除了缺乏足够组织样本进行进一步染色的病例。该研究方案已获得北京大学肿瘤医院医学伦理委员会的批准（批准号2023KT29），所有患者在使用组织样本之前均已获得知情同意。。

Pathological material

病理材料

The sample composition of this experiment is summarized in Supplementary Fig. 1. We obtained 248 tumor tissue samples from primary GEP-NECs (including surgery and biopsy samples), 19 distant metastatic GEP-NECs samples (all were liver metastases) and 9 lymph node metastases. Additionally, 36 GEP-NETs (8 G1, 9 G2, 19 G3) samples and 29 GACs samples were also collected.

该实验的样品组成总结在补充图1中。我们从原发性GEP NEC（包括手术和活检样本）获得248个肿瘤组织样本，19个远处转移性GEP NEC样本（均为肝转移）和9个淋巴结转移。此外，还收集了36个GEP NET（8个G1、9个G2、19个G3）样品和29个GAC样品。

Each sample underwent formalin fixation and paraffin embedding (FFPE). Some cases had immunohistochemical staining results for three neuroendocrine markers, Ki67, and PD-L1, when included in the study. For cases lacking any markers, additional immunohistochemical staining was carried out on available tumor tissues.

每个样品均经过福尔马林固定和石蜡包埋（FFPE）。当纳入研究时，一些病例对三种神经内分泌标志物Ki67和PD-L1进行了免疫组织化学染色。对于缺乏任何标记的病例，对可用的肿瘤组织进行了额外的免疫组织化学染色。

The assessment of neuroendocrine marker results follows the 5th edition WHO criteria.

神经内分泌标志物结果的评估遵循第5版WHO标准。

, the Ki67 index was evaluated as a percentage, and the expression of PD-L1 was evaluated by CPS (22C3, Agilent DaKo, Denmark, 1:50). PD-L1 expression was defined as the ratio of the number of immune-related cells (tumor cells, lymphocytes, macrophages) expressing PD-L1 to the number of all tumor cells, with CPS ≥ 1 defined as positive..

，以百分比评估Ki67指数，并通过CPS（22C3，安捷伦达科，丹麦，1:50）评估PD-L1的表达。PD-L1表达定义为表达PD-L1的免疫相关细胞（肿瘤细胞，淋巴细胞，巨噬细胞）数量与所有肿瘤细胞数量的比率，其中CPS≥1定义为阳性。。

Immunohistochemical staining

免疫组织化学染色

FFPE specimens were sectioned into 4-µm-thick slices using a rotary microtome, followed by incubation in EDTA solution (pH = 8.4) at 95 °C for 36 min. A DLL3 antibody (clone SP347, Roche, ready-to-use) was used for staining with Ventana, whereas ASCL1 antibody (24B72D11.1, BD Biosciences, 1:100) was stained with Leica Bond III.

使用旋转切片机将FFPE样品切成4μm厚的切片，然后在EDTA溶液（pH=8.4）中于95℃温育36分钟。使用DLL3抗体（克隆SP347，Roche，即用型）用Ventana染色，而ASCL1抗体（24B72D11.1，BD Biosciences，1:100）用Leica Bond III染色。

The color reaction was achieved using diaminobenzidine (ZSGB-BIO, Beijing, China). Staining outcomes were evaluated using an optical microscope with the 20× or 40× magnification. Each slide was reviewed by two experienced pathologists. Only when both pathologists agree on the result was it adopted, in cases of disagreement, the specimens were re-evaluated and discussed to achieve a consensus.

使用二氨基联苯胺（ZSGB-BIO，Beijing，China）实现显色反应。使用放大20倍或40倍的光学显微镜评估染色结果。每张幻灯片都由两位经验丰富的病理学家进行了审查。只有当两位病理学家都同意结果时，才能采用，如果存在分歧，则对标本进行重新评估和讨论以达成共识。

DLL3 and ASCL1 results were binarily categorized. For DLL3, positive staining was defined as a reaction in ≥ 1% of tumor cells, regardless of intensity, based on criteria established by other studies.

DLL3和ASCL1结果被二元分类。对于DLL3，根据其他研究建立的标准，阳性染色被定义为≥1%的肿瘤细胞中的反应，无论强度如何。

. Any punctate, cytoplasmic and/or membranous staining was considered positive, as previously described

如前所述，任何点状，细胞质和/或膜状染色均被认为是阳性的

. DLL3-high was characterized by ≥ 50% positive tumor cells

DLL3高的特征是肿瘤细胞阳性率≥50%

and ≥ 75% positive tumor cells

和≥75%的阳性肿瘤细胞

. For ASCL1, tumor cell positivity was categorized as 0, 1, 2, or 3 (negative, faint, moderate, or strong, respectively). We adopted semi-quantitative algorithms with H-score, which was calculated by multiplying the percentage of positive tumor cells (%) by the corresponding staining intensity. An H-score ≥ 50 is defined as positive.

对于ASCL1，肿瘤细胞阳性分为0、1、2或3（分别为阴性，微弱，中度或强）。我们采用了具有H评分的半定量算法，该算法是通过将阳性肿瘤细胞的百分比（%）乘以相应的染色强度来计算的。H评分≥50被定义为阳性。

Statistical analysis

统计分析

We conducted all analyses using SPSS software (IBM Corp., Armonk, NY, USA, version 25). The correlation between DLL3 expression and clinicopathological features was evaluated using the chi-squared test or Fisher’s exact test. Median progression-free survival (PFS) and overall survival (OS) were determined using Kaplan–Meier analysis, and survival differences were assessed using log-rank tests based on DLL3 expression status.

我们使用SPSS软件（IBM Corp.，Armonk，NY，USA，版本25）进行了所有分析。使用卡方检验或Fisher精确检验评估DLL3表达与临床病理特征之间的相关性。使用Kaplan-Meier分析确定中位无进展生存期（PFS）和总生存期（OS），并使用基于DLL3表达状态的对数秩检验评估生存差异。

The statistical significance level was set at .

统计显着性水平设定为。

< 0.05.

< 0.05。

Results

结果

Patient profile

患者简介

Table

表

provides a summary of the baseline characteristics of patients diagnosed with GEP-NECs. Among the 248 primary GEP-NECs cases, the age range was 23 to 85 years, with a median of 62 years. The study included 182 (73.4%) males and 66 (26.6%) females. The total number of SCNEC, LCNEC, and MiNENs cases were 138 (55.7%), 101 (40.7%), and 9 (3.6%), respectively.

提供了诊断为GEP NEC的患者的基线特征的摘要。在248例原发性GEP NEC病例中，年龄范围为23至85岁，中位数为62岁。该研究包括182名（73.4%）男性和66名（26.6%）女性。SCNEC，LCNEC和MiNENs病例总数分别为138（55.7%），101（40.7%）和9（3.6%）。

Gastric NECs accounted for the largest proportion (148, 59.7%) in this cohort, followed by the esophagus (51, 20.6%), and pancreas (24, 9.7%). T1-stage patients accounted for 3.2% (8/248), while T2-T4-stage patients accounted for 43.1% (107/248). Patients with stage N0 and N1-N3 accounted for 11.7% (29/248) and 33.1% (82/248), respectively; 40.7% (101/248) of patients with M stage were M0 stage, and 5.2% (13/248) were M1 stage.

胃NEC在该队列中占最大比例（148，59.7%），其次是食道（51，20.6%）和胰腺（24，9.7%）。T1期患者占3.2%（8/248），T2-T4期患者占43.1%（107/248）。N0期和N1-N3期患者分别占11.7%（29/248）和33.1%（82/248）；M期患者中40.7%（101/248）为M0期，5.2%（13/248）为M1期。

Additionally, 64 (25.8%) patients received chemotherapy before baseline. The rates of Ki67 proliferation index in different intervals 25–50%, 51–75%, and 76–100% were 11.7% (29/248), 39.5% (98/248), and 35.1% (87/248), respectively. In 75.4% (187/248) of cases, at least 2 NE markers were positive, while in 12.5% (31/248) of cases < 2 NE markers were positive.

此外，64名（25.8%）患者在基线前接受了化疗。Ki67增殖指数在25-50%，51-75%和76-100%的不同区间分别为11.7%（29/248），39.5%（98/248）和35.1%（87/248）。在75.4%（187/248）的病例中，至少有2个NE标志物呈阳性，而在12.5%（31/248）的病例中，2个NE标志物呈阳性。

A positive reaction of PD-L1 was shown in 6.0% (15/248) of cases..

6.0%（15/248）的病例显示PD-L1阳性反应。。

Table 1 Baseline characteristics of patients with GEP-NECs.

表1 GEP NEC患者的基线特征。

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Relationship between DLL3 expression and clinicopathological features

DLL3表达与临床病理特征的关系

DLL3 staining was positive in the cytomembrane, cytoplasm, and punctate, in which cytoplasmic and membranous staining was diffuse, whereas intermittent perinuclear staining was punctate. However, we identified four cases in which nuclear positivity was observed alongside cytoplasmic and membranous staining, a pattern not previously reported in other studies.

DLL3染色在细胞膜，细胞质和点状中呈阳性，其中细胞质和膜染色呈弥漫性，而间歇性核周染色呈点状。然而，我们确定了四例在细胞质和膜染色的同时观察到核阳性的病例，这种模式以前在其他研究中没有报道过。

The nuclear positivity in these cases may represent a nonspecific or incidental reaction. Representative images are presented in Fig. .

在这些情况下，核阳性可能代表非特异性或偶然反应。代表性图像如图所示。

a. Moreover, DLL3 has a heterogeneous expression pattern in GEP-NECs. Correlations between DLL3 expression and clinicopathological features are presented in Table

a、此外，DLL3在GEP NEC中具有异质表达模式。DLL3表达与临床病理特征之间的相关性见表

. Positive expression was observed in 68.1% of SCNEC (94/138) compared to 38.6% of LCNEC (39/101) and 33.3% (3/9) of MiNENs (

在68.1%的SCNEC（94/138）中观察到阳性表达，而LCNEC（39/101）和MiNENs的阳性表达率分别为38.6%和33.3%（3/9）(

< 0.001). Of note, in MiNENs, the neuroendocrine component exhibited positive DLL3 expression, while the adenocarcinoma component showed negative expression (Fig.

<0.001）。值得注意的是，在MiNENs中，神经内分泌成分表现出DLL3阳性表达，而腺癌成分表现出阴性表达（图）。

b). The expression rate of DLL3 was highest in the esophagus (68.6%, 35/51), followed by the colorectum (53.8%, 7/13), stomach (52.7%, 78/148), pancreas (41.7%, 10/24), and small intestine (25.0%, 2/8). DLL3 showed significant expression differences among different T-stage cases (T1 100.0% vs. T2-T4 53.3%, .

b）。DLL3在食管中的表达率最高（68.6%，35/51），其次是结直肠（53.8%，7/13），胃（52.7%，78/148），胰腺（41.7%，10/24）和小肠（25.0%，2/8）。DLL3在不同T期病例中显示出显着的表达差异（T1 100.0%比T2-T4 53.3%。

= 0.009). Patients who underwent chemotherapy prior to baseline demonstrated a higher prevalence of DLL3 expression compared to individuals who did not undergo previous therapy (67.2% vs. 50.5%,

在基线之前接受化疗的患者与未接受过既往治疗的患者相比，DLL3表达的患病率更高（67.2%比50.5%，

= 0.015). In the group with < 2 NE positive markers, the expression rate of DLL3 was 38.7%, significantly lower than that in the group with ≥ 2 NE positive markers (56.7%,

==0.015）。在2个NE阳性标记物组中，DLL3的表达率为38.7%，显着低于≥2个NE阳性标记物组（56.7%，

= 0.048). No associations were observed between DLL3 expression and sex, N stage, M stage, Ki67 index, or PD-L1 expression.

= 0.048）。在DLL3表达与性别，N期，M期，Ki67指数或PD-L1表达之间未观察到关联。

Fig. 1

图1

(

一

) DLL3 staining was positive in the cytomembrane, cytoplasm, and nucleus, in which cytoplasmic and membranous staining was diffuse, whereas intermittent perinuclear staining was punctate. The second picture also showed the heterogeneity of DLL3 staining. (

)DLL3染色在细胞膜，细胞质和细胞核中呈阳性，其中细胞质和膜染色呈弥漫性，而间歇性核周染色呈点状。第二张图片还显示了DLL3染色的异质性。（笑声）(

b类

) In MiNENs, the neuroendocrine component exhibited positive DLL3 expression, whereas the adenocarcinoma component showed negative. (

)在MiNENs中，神经内分泌成分显示DLL3阳性表达，而腺癌成分显示阴性。（笑声）(

c级

) The staining of DLL3 was positive in NET G3, while negative in NET G1, NET G2, and GACs. NEC Neuroendocrine carcinoma. NET Neuroendocrine tumor. GAC Gastric adenocarcinoma.

)。NEC神经内分泌癌。净神经内分泌肿瘤。GAC胃腺癌。

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Table 2 Correlation of DLL3 expression with clinicopathological characteristics.

表2 DLL3表达与临床病理特征的相关性。

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DLL3 expression in metastatic tumors

DLL3在转移性肿瘤中的表达

The expression of DLL3 in metastatic tumors is shown in Supplementary Fig. 2. The staining pattern of DLL3 in metastatic lesions was similar to that in primary tumors. In lymph node metastasis and distant metastases, the positive rate of DLL3 was 44.4% and 52.6%, respectively. Chi-square analysis showed no significant difference in the expression of DLL3 between primary tumor, lymph node metastases, and distant metastases (.

DLL3在转移性肿瘤中的表达如补充图2所示。DLL3在转移性病变中的染色模式与原发性肿瘤相似。在淋巴结转移和远处转移中，DLL3的阳性率分别为44.4%和52.6%。卡方分析显示DLL3在原发肿瘤，淋巴结转移和远处转移之间的表达无显着差异（。

= 0.818) (Fig.

=0.818）（图。

a).

a）。

Fig. 2

图2

(

一

) The expression rate of DLL3 in primary tumors and metastatic tumors. The expression rate of DLL3 between ASCL1 negative proup and ASCL1 positive group in GEP-NECs (

)DLL3在原发性肿瘤和转移性肿瘤中的表达率。GEP NEC中ASCL1阴性proup和ASCL1阳性组之间DLL3的表达率(

b类

), GEP-SCNEC (

（SCNEC）

c级

), and GEP-LCNEC (

（GEP-LCNEC）

). GEP-NECs Gastroenteropancreatic neuroendocrine carcinomas. GEP-SCNEC Gastroenteropancreatic small cell neuroendocrine carcinoma, GEP-LCNEC Gastroenteropancreatic large cell neuroendocrine carcinoma.

)。GEP NECs胃肠胰神经内分泌癌。GEP-SCNEC胃肠胰小细胞神经内分泌癌，GEP-LCNEC胃肠胰大细胞神经内分泌癌。

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Explore the differential diagnosis value of DLL3

探讨DLL3的鉴别诊断价值

To explore the expression of DLL3 in well-differentiated GEP-NETs, we stained 36 cases of GEP-NETs. Among the GEP-NETs cases, none of the eight cases of NET G1 and nine cases of NET G2 exhibited positive DLL3 staining, while 3 out of 19 (15.8%) cases of NET G3 were positive for DLL3 staining. The DLL3 expression was also heterogeneous, and all of them were positive in the cytoplasm and membrane (Fig. .

为了探索DLL3在分化良好的GEP-NETs中的表达，我们对36例GEP-NETs进行了染色。。DLL3的表达也是异质的，它们在细胞质和膜中均为阳性（图）。

c). Since the morphological overlap between NET G3 and NECs or poorly differentiated adenocarcinoma and NECs often puts pathologists in a dilemma with respect to differential diagnosis, we further assessed the value of DLL3 in differential diagnosis. Therefore, we performed DLL3 staining on additional GACs with different degrees of differentiation.

c）。由于NET G3和NEC或低分化腺癌和NEC之间的形态学重叠经常使病理学家在鉴别诊断方面陷入困境，因此我们进一步评估了DLL3在鉴别诊断中的价值。因此，我们对具有不同分化程度的其他GAC进行了DLL3染色。

The results show that no positive DLL3 staining was detected in 29 GACs cases, regardless of the degree of differentiation (Fig. .

结果表明，无论分化程度如何，在29例GACs病例中均未检测到阳性DLL3染色（图）。

c). DLL3 may serve as a useful differential diagnostic tool with a sensitivity of 54.8% and a specificity of 84.2% when differentiating NECs from NET G3, and a sensitivity of 54.8% and a specificity of 100.0% when differentiating NECs from GACs, with a cutoff value of 1% positive tumor cells. However, with the threshold raised to 50% positive tumor cells, the sensitivity declined to 31.9%, while the specificity increased to 94.7% when identifying NECs and NET G3 and to 100.0% when identifying NECs and GACs, reflecting the stricter standard (Table .

c）。DLL3可以作为一种有用的鉴别诊断工具，当区分NEC和NET G3时，灵敏度为54.8%，特异性为84.2%，当区分NEC和GAC时，灵敏度为54.8%，特异性为100.0%，截止值为1%阳性肿瘤细胞。然而，随着阈值提高到50%阳性肿瘤细胞，敏感性下降到31.9%，而当鉴定NEC和NET G3时，特异性增加到94.7%，当鉴定NEC和GAC时，特异性增加到100.0%，反映了更严格的标准（表。

Table 3 Evaluation of DLL3 as a diagnostic marker for GEP-NECs.

表3 DLL3作为GEP NEC诊断标志物的评估。

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ASCL1 expression

ASCL1表达

Almost all positive reactions for ASCL1 were located to the nucleus, with only one case showing an unusual punctate positivity around the nucleus (Fig.

几乎所有ASCL1阳性反应都位于细胞核，只有一例在细胞核周围表现出异常的点状阳性（图）。

a). The expression status of 111 samples of ASCL1 and DLL3 is shown in Fig.

a）。111个ASCL1和DLL3样品的表达状态如图所示。

b. In addition, it was observed that in samples with co-expression of DLL3 and ASCL1, tumor cells expressing ASCL1 also exhibited DLL3 expression, demonstrating a spatial consistency. (Fig.

b、此外，观察到在具有DLL3和ASCL1共表达的样品中，表达ASCL1的肿瘤细胞也表现出DLL3表达，证明了空间一致性。（图。

c). Similarly, its expression was also heterogeneous inside the tumor. Overall, ASCL1 expression was detected in 14.4% (16/111) of GEP-NECs cases. Interestingly, DLL3 expression rate differed between the ASCL1 positive and ASCL1 negative groups (

c）。同样，其表达在肿瘤内也是异质的。。有趣的是，ASCL1阳性组和ASCL1阴性组的DLL3表达率不同(

= 0.002) (Fig.

=0.002）（图。

b). In the ASCL1 positive group, the DLL3 positive rate was 87.5% (14/16), whereas 47.4% (45/95) showed positive DLL3 staining in the ASCL1 negative group. Similarly, DLL3 expression was higher in the ASCL1 positive group among GEP-SCNECs (100.0% vs. 56.8%,

b）。在ASCL1阳性组中，DLL3阳性率为87.5%（14/16），而在ASCL1阴性组中，DLL3阳性率为47.4%（45/95）。同样，在GEP SCNEC中，ASCL1阳性组的DLL3表达更高（100.0%比56.8%，

= 0.02) (Fig.

=0.02）（图。

c). However, the differential expression of DLL3 was not significant in GEP-LCNECs (33.3% vs. 37.8%,

c）。然而，DLL3的差异表达在GEP LCNEC中并不显着（33.3%对37.8%，

= 0.687) (Fig.

=0.687）（图。

d).

d）。

Fig. 3

图3

(

一

) The positive reactions for ASCL1 were located in the nucleus, with only one case showing an unusual dot-like positivity around the nucleus. (

)ASCL1的阳性反应位于细胞核内，只有一例在细胞核周围显示出异常的点状阳性。（笑声）(

b类

) The expression of ASCL1 and DLL3 in 111 cases. (

)111例中ASCL1和DLL3的表达。（笑声）(

c级

) In the samples with co-expression of DLL3 and ASCL1, the spatial distribution of tumor cells expressed by DLL3 and ASCL1 was consistent.

)在DLL3和ASCL1共表达的样品中，DLL3和ASCL1表达的肿瘤细胞的空间分布是一致的。

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Follow-up and prognosis

随访和预后

The prognosis information of 199 patients was obtained. The median follow-up time was 19.7 months (range: 0.8–140.4 months). Survival analysis was conducted for different cutoff values of DLL3 expression (

获得了199例患者的预后信息。中位随访时间为19.7个月（范围：0.8-140.4个月）。对DLL3表达的不同临界值进行生存分析(

= 199) (Fig.

=199）（图。

). In the DLL3 positive group (with the 1% cutoff value), the median PFS and median OS were 12.3 months (95% CI: 7.9–16.6) and 24.4 months (95% CI: 20.2–28.6), respectively, while in the DLL3 negative group, the median PFS and median OS were 13.4 months (95% CI: 10.5–16.3) and 25.9 months (95% CI: 18.9–32.9), respectively.

)。DLL3阳性组（临界值为1%）的中位PFS和中位OS分别为12.3个月（95%CI:7.9-16.6）和24.4个月（95%CI:20.2-28.6），而DLL3阴性组的中位PFS和中位OS分别为13.4个月（95%CI:10.5-16.3）和25.9个月（95%CI:18.9-32.9）。

The log-rank test showed that there was no significant difference in the prognosis between the DLL3 negative group and the DLL3 positive group (Fig. .

。

a). Moreover, no significant difference in survival was observed between high and low DLL3 expression groups, regardless of whether the cutoff value defined by high DLL3 expression was 50% or 75% (Fig.

a）。此外，无论高DLL3表达定义的临界值是50%还是75%，在高DLL3表达组和低DLL3表达组之间均未观察到存活率的显着差异（图）。

b and c).

b和c）。

Fig. 4

图4

Kaplan-Meier survival curves for progression-free survival (PFS) and overall survival (OS) of GEP-NECs patients (

GEP NECs患者无进展生存期（PFS）和总生存期（OS）的Kaplan-Meier生存曲线(

= 199) according to DLL3 status with the cut-off value 1%(a), 50%(b), and 75%(c).

根据DLL3状态，截止值为1%（a），50%（b）和75%（c）。

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Discussion

讨论

High levels of DLL3 expression are found in carcinoid subtypes of pulmonary NETs that exhibit significant dendritic cell infiltration, suggesting its potential as a viable target for focused intervention. Previous studies have reported a 65% DLL3 expression rate in 37 patients with LCNEC

在肺NETs的类癌亚型中发现高水平的DLL3表达，其表现出显着的树突细胞浸润，表明其作为集中干预的可行靶标的潜力。先前的研究报道，37例LCNEC患者的DLL3表达率为65%

. Analysis of clinical trial groups revealed DLL3 expression in over 70% of SCLC cases

临床试验组的分析显示，超过70%的SCLC病例中DLL3表达

. A high expression rate of DLL3 was also observed in patients with castration-resistant neuroendocrine prostate cancer

在去势抵抗性神经内分泌前列腺癌患者中也观察到DLL3的高表达率

. A previous study reported a DLL3 expression rate of 76.9% in 13 cases of gastrointestinal-pancreatic NECs

先前的一项研究报道，在13例胃肠道胰腺NEC中，DLL3的表达率为76.9%

. We first assessed DLL3 positivity in a large sample of GEP-NECs (

。我们首先在大量GEP NEC样本中评估了DLL3阳性(

= 248) and found a positivity rate of 54.8%. Our study revealed different DLL3 expression rates between SCNEC and LCNEC, with SCNEC exhibiting a higher positivity rate than LCNEC. Regarding primary site distribution, the esophagus exhibited a high DLL3 expression rate, which was largely attributed to the high proportion of SCNEC in esophageal NECs.

= 248），阳性率为54.8%。我们的研究揭示了SCNEC和LCNEC之间不同的DLL3表达率，其中SCNEC表现出比LCNEC更高的阳性率。关于原发部位分布，食管表现出高DLL3表达率，这主要归因于食管NEC中SCNEC的高比例。

Similarly, this cohort showed a high expression rate of DLL3 in T1 stage patients, partly because SCNEC had an absolute advantage in the number of T1-stage patients (7/8). A single-cell transcriptome sequencing study of SCLC tumor cells demonstrated that chemotherapy resulted in decreased expression of therapeutic target genes, including .

。SCLC肿瘤细胞的单细胞转录组测序研究表明，化疗导致治疗靶基因的表达降低，包括。

DLL3

DLL3级

. However, our results showed that DLL3 has a higher expression rate in patients receiving chemotherapy before baseline treatment, which prompts future research to focus on whether chemotherapy affects the expression of the

然而，我们的研究结果表明，DLL3在基线治疗前接受化疗的患者中具有较高的表达率，这促使未来的研究集中于化疗是否影响DLL3的表达

DLL3

DLL3级

gene.

基因。

Several studies have adopted various definitions for DLL3-high expression. When defined as staining in ≥ 50% of tumor cells, the DLL3-high expression rates in SCLC range from 32% to 79.5%

一些研究采用了DLL3高表达的各种定义。当定义为在≥50%的肿瘤细胞中染色时，DLL3在SCLC中的高表达率在32%至79.5%之间

. With a cutoff value of 75%, the positive rate of high expression was 70% in another study on SCLC

在另一项关于SCLC的研究中，高表达的阳性率为70%，截止值为75%

. Conversely, high expression rates of DLL3 in lung LCNEC are relatively low at 54%

相反，DLL3在肺LCNEC中的高表达率相对较低，为54%

. In this study, we applied 50% and 75% cutoff values to define DLL3-high expression in GEP-NECs, exploring the DLL3-high expression rate for the first time. Although these cutoff values have been frequently used in small cell lung cancer (SCLC), their application to GEP-NECs has not been previously reported.

。尽管这些临界值经常用于小细胞肺癌（SCLC），但它们在GEP NEC中的应用尚未见报道。

Our results showed a 31.8% expression rate of DLL3 with a cutoff value of 50% and a 25.4% expression rate of DLL3 with a cutoff value of 75%. Since GEP-NECs contains SCNEC and LCNEC, we analyzed the high expression rates in different subtypes (Supplementary Table 1). Irrespective of the cutoff value, the expression rate of DLL3 in SCNEC is always higher than that in LCNEC.

我们的结果显示DLL3的表达率为31.8%，截止值为50%，DLL3的表达率为25.4%，截止值为75%。由于GEP-NEC含有SCNEC和LCNEC，我们分析了不同亚型的高表达率（补充表1）。无论截止值如何，DLL3在SCNEC中的表达率始终高于LCNEC中的表达率。

Furthermore, the expression rate of DLL3 in the digestive system was lower than that in high-grade neuroendocrine tumors of the lung, regardless of the cutoff value. Recent studies in SCLC indicate significant improvements in ORR and PFS in patients with high expression of DLL3 (defined as positive staining in ≥ 50% of tumor cells); the high expression group demonstrated significant benefits in confirmed objective response (35% versus 0%) and disease control (90% versus 60%) compared to the low expression group of DLL3.

此外，无论临界值如何，消化系统中DLL3的表达率均低于肺部高级神经内分泌肿瘤。最近在SCLC中的研究表明，DLL3高表达患者的ORR和PFS显着改善（定义为≥50%肿瘤细胞的阳性染色）；与DLL3的低表达组相比，高表达组在确认的客观反应（35%对0%）和疾病控制（90%对60%）方面显示出显着的益处。

. Additionally, in another phase II clinical trial, the ORR was 14.3% in the DLL3 high-expression group, with high expression defined as positive staining in ≥ 75% of tumor cells

此外，在另一项II期临床试验中，DLL3高表达组的ORR为14.3%，高表达定义为≥75%的肿瘤细胞呈阳性染色

. In phase I/II studies of multiple cancer types, a higher remission rate was observed with high DLL3 expression

在多种癌症类型的I/II期研究中，观察到DLL3高表达的缓解率更高

. However, some patients with DLL3 expression in < 50% of tumor cells also exhibit stable disease for a prolonged duration

然而，一些DLL3在50%以下肿瘤细胞中表达的患者也表现出长期稳定的疾病

. Hence, in the following clinical phases, the assessment should focus on evaluating the efficacy in patients exhibiting any measurable DLL3 levels.

。

The field of immuno-oncology (IO) has revolutionized the landscape of cancer therapy, actively influencing patient outcomes

免疫肿瘤学（IO）领域彻底改变了癌症治疗的格局，积极影响患者的预后

. PD-L1 has been identified as the first biomarker for anti-PD-1 therapy and is included in the prescribing information for pembrolizumab. Currently, other immune therapy efficacy-related markers include tumor mutational burden (TMB), mismatch repair system deficiency (dMMR), high microsatellite instability (MSI-H), neoantigens, mutations in antigen presentation pathways, and circulating tumor DNA (ctDNA) as indicators for selecting patients who may benefit from immunotherapy.

PD-L1已被确定为抗PD-1治疗的第一个生物标志物，并包含在派姆单抗的处方信息中。目前，其他免疫治疗功效相关标志物包括肿瘤突变负荷（TMB），错配修复系统缺陷（dMMR），高微卫星不稳定性（MSI-H），新抗原，抗原呈递途径突变和循环肿瘤DNA（ctDNA）作为选择可能受益于免疫治疗的患者的指标。

. In a study on pancreatic ductal adenocarcinoma, high DLL3 expression was positively correlated with PD-L1/2 expression

在一项关于胰腺导管腺癌的研究中，高DLL3表达与PD-L1/2表达呈正相关

. Another study in SCLC also observed cases with high DLL3 expression and negative NOTCH1 expression to have a higher PD-L1 expression rate, revealing a favorable prognosis in such SCLC patients

另一项针对小细胞肺癌的研究还观察到DLL3高表达和NOTCH1阴性表达的病例具有较高的PD-L1表达率，这表明此类小细胞肺癌患者的预后良好

. However, our study did not find any correlation between DLL3 and PD-L1 expression in GEP-NECs. Although no significant correlation was observed, in our study cohort, 9 out of 40 (22.5%) patients co-expressed DLL3 and PD-L1, suggesting the feasibility of combined DLL3-targeted therapy and immunotherapy in some patients and providing a theoretical basis for developing new treatment strategies for patients with GEP-NEC, which requires further clinical validation..

然而，我们的研究没有发现GEP NEC中DLL3和PD-L1表达之间有任何相关性。虽然没有观察到显着的相关性，但在我们的研究队列中，40名患者中有9名（22.5%）共表达DLL3和PD-L1，这表明DLL3靶向治疗和免疫治疗联合治疗在某些患者中的可行性，并为开发GEP-NEC患者的新治疗策略提供了理论基础，这需要进一步的临床验证。。

In recent studies, four subtypes of SCLC have been identified based on the unique expression patterns of four key transcripts: SCLC-A, SCLC-N, SCLC-P, and SCLC-Y

在最近的研究中，基于四个关键转录本的独特表达模式，已经鉴定出四种SCLC亚型：SCLC-A，SCLC-N，SCLC-P和SCLC-Y

. Baine et al. confirmed higher DLL3 expression in ASCL1-high SCLC compared to other groups

Baine等人证实，与其他组相比，ASCL1高SCLC中DLL3的表达更高

. Wang et al. classified NECs into five subtypes (ASCL1, NEUROD1, HNF4A, POU2F3, and YAP1) across various tumor sites (including GEP)

Wang等人在不同的肿瘤部位（包括GEP）将NEC分为五种亚型（ASCL1，NEUROD1，HNF4A，POU2F3和YAP1）

. Although our study did not further classify subtypes, we first verified the expression of ASCL1 and DLL3 in 111 available tumor tissues, showing a significantly higher DLL3 expression rate in the ASCL1-positive group (likely corresponding to the A subtype) compared to the ASCL1-negative group (likely corresponding to non-A subtypes) at the protein level, confirming a significant correlation.

尽管我们的研究没有进一步对亚型进行分类，但我们首先验证了111个可用肿瘤组织中ASCL1和DLL3的表达，与ASCL1阴性组（可能对应于非a亚型）相比，ASCL1阳性组（可能对应于a亚型）的DLL3表达率显着更高，证实了显着的相关性。

However, this correlation was only evident in small cell GEP-NECs and was not seen in large cell or mixed types, which was quite different from that observed with pulmonary neuroendocrine tumors. The correlation of DLL3 and ASCL1 expression was observed in both SCLC and LCNEC of the lung.

然而，这种相关性仅在小细胞GEP NEC中明显，在大细胞或混合型中未见，这与肺神经内分泌肿瘤观察到的差异很大。在肺的SCLC和LCNEC中均观察到DLL3和ASCL1表达的相关性。

. Nonetheless, these results suggest that patients with the ASCL1 subtype may benefit from DLL3-targeted therapy. Furthermore, the study by Wang et al. revealed that A and N types of GEP-NEC are classified as NE-high types

尽管如此，这些结果表明ASCL1亚型患者可能会从DLL3靶向治疗中受益。此外，Wang等人的研究表明，A型和N型GEP-NEC被归类为NE高型

. Similarly, in our cohort, we found that among the 13 ASCL1-positive cases that underwent NE staining in available tissues, 10 cases were positive for ≥ 2 NE markers. Additionally, in tumors positive for ≥ 2 NE markers, the expression rate of DLL3 was higher than that of the subgroup of < 2 NE markers, which is consistent with the results observed in pulmonary neuroendocrine carcinomas.

同样，在我们的队列中，我们发现在可用组织中进行NE染色的13例ASCL1阳性病例中，有10例对≥2个NE标记物呈阳性。此外，在≥2个NE标记阳性的肿瘤中，DLL3的表达率高于<2个NE标记的亚组，这与在肺神经内分泌癌中观察到的结果一致。

It has been found that a classic pathway in the development of SCLC involves tumor initiation under the premise of bi-allelic mutations in

已经发现，SCLC发展的经典途径涉及在双等位基因突变的前提下肿瘤的发生

RB1

RB1型

and

和

TP53

TP53型

, driven by NOTCH signaling inactivation

，由NOTCH信号失活驱动

. While the roles of the DLL3/Notch pathway and the loss of

虽然DLL3/Notch途径的作用和

RB1

RB1型

and

和

TP53

TP53型

mutations in the development of GEP-NECs have not been extensively studied, in high-grade neuroendocrine tumors of the digestive system,

GEP NEC发育中的突变尚未在消化系统的高级神经内分泌肿瘤中得到广泛研究，

TP53

TP53型

and

和

皇家银行

mutations are the most common genetic alterations, differing from the frequently occurring

突变是最常见的遗传变异，不同于频繁发生的突变

MEN1

男性1

ATRX/DAXX

, and

，以及

mTOR

pathway activation in NET

NET中的途径激活

. This may explain why DLL3 is negative in well-differentiated neuroendocrine tumors, but shows positive reactions in some high-grade neuroendocrine tumors, suggesting that part of digestive neuroendocrine tumors may share a similar carcinogenic pathway with SCLC. However, this speculation needs further confirmation in future studies..

这可以解释为什么DLL3在分化良好的神经内分泌肿瘤中呈阴性，但在某些高级神经内分泌肿瘤中呈阳性反应，这表明部分消化神经内分泌肿瘤可能与SCLC具有相似的致癌途径。然而，这种推测需要在未来的研究中进一步证实。。

DLL3 staining in all GACs cases showed negative results regardless of the degree of differentiation. Among the nine cases of MiNENs comprising a mixture of adenocarcinoma and NECs, no positive signal was detected in the adenocarcinoma component. Hence, DLL3 staining may serve as a tool to differentiate gastric NECs from GACs with 100% specificity, particularly in biopsy samples where limited tissue samples may hinder a definitive diagnosis.

无论分化程度如何，所有GAC病例的DLL3染色均显示阴性结果。。因此，DLL3染色可以作为区分胃NEC和GAC的工具，具有100%的特异性，特别是在活检样本中，有限的组织样本可能会阻碍明确的诊断。

Based on the classification established by the 2010 World Health Organization, NENs were divided into highly differentiated NETs (NET G1 and NET G2) and poorly differentiated NECs.

根据2010年世界卫生组织建立的分类，NEN分为高分化NET（净G1和净G2）和低分化NEC。

. Despite being highly proliferative, certain tumors progress slowly and have a favorable prognosis, while others follow an aggressive course. To address this heterogeneity, a subgroup termed NET G3 was introduced into the GEP-NENs classification system, representing an intermediate prognosis between NET G1/G2 and NEC.

尽管具有高度增殖性，但某些肿瘤进展缓慢，预后良好，而其他肿瘤则呈侵袭性。为了解决这种异质性，将称为NET G3的亚组引入GEP NENs分类系统，代表NET G1/G2和NEC之间的中间预后。

. Nevertheless, distinguishing NET G3 with a relatively favorable prognosis from poorly differentiated NECs remains challenging for pathologists. Currently, a comprehensive diagnostic approach combining clinical performance, morphology, immunohistochemistry, and molecular biomarkers is preferred. These biomarkers include RB, DAXX/ATRX, SSTR2, CgA, and p53.

然而，对于病理学家来说，区分预后相对较好的NET G3和分化较差的NEC仍然具有挑战性。目前，优选结合临床表现，形态学，免疫组织化学和分子生物标志物的综合诊断方法。这些生物标志物包括RB，DAXX/ATRX，SSTR2，CgA和p53。

. No large-sample studies have confirmed the value of DLL3 in distinguishing NET G3 from NEC. A small-sample study on GEP-NENs found no DLL3 expression in NET G3 (0/5)

。没有大样本研究证实DLL3在区分净G3和NEC方面的价值。对GEP-NENs的小样本研究发现，NET G3（0/5）中没有DLL3表达

. Chen et al. utilized various methods, including WES, FISH, qPCR, and IHC, to identify DLL3 expression in three out of eight GEP-NET G3 cases, with no abnormal DLL3 expression in NET G2

Chen等人利用各种方法，包括WES、FISH、qPCR和IHC，在八分之三的GEP-NET G3病例中鉴定DLL3的表达，而在NET G2中没有异常的DLL3表达

. Our study marks the first discovery of DLL3 protein expression in GEP-NET G3 with a relatively large sample size, suggesting the differential diagnostic value of DLL3,and calling for further research on DLL3-targeting agents in NET G3.

我们的研究标志着首次在样本量相对较大的GEP-NET G3中发现DLL3蛋白表达，表明DLL3具有鉴别诊断价值，并呼吁进一步研究NET G3中的DLL3靶向药物。

Additionally, there was no significant difference in DLL3 expression among primary tumors, lymph node metastases, and distant metastases. Therefore, DLL3 staining in metastases appears to reflect the condition of primary tumors, suggesting the feasibility of applying DLL3-targeted therapy in advanced patients with distant organ metastases..

此外，DLL3在原发性肿瘤，淋巴结转移和远处转移中的表达没有显着差异。因此，转移瘤中的DLL3染色似乎反映了原发性肿瘤的状况，表明在晚期远处器官转移患者中应用DLL3靶向治疗的可行性。。

In a study on DLL3 expression in SCLC without targeted therapy, patient prognosis was found to be unrelated to DLL3 expression status, with high or low expression (≥ 50% cutoff) showing no impact on survival

在一项未经靶向治疗的小细胞肺癌中DLL3表达的研究中，发现患者预后与DLL3表达状态无关，高表达或低表达（截止值≥50%）对生存率没有影响

. Similar findings were observed in the GEP-NECs cohort in our study. However, another study encompassing SCLC, carcinoid, and atypical carcinoid cases noted that DLL3-high expression (≥ 50% positive tumor cells) correlated with improved OS in SCLC (

在我们的研究中，在GEP NECs队列中观察到了类似的发现。然而，另一项涵盖SCLC，类癌和非典型类癌病例的研究指出，DLL3高表达（≥50%阳性肿瘤细胞）与SCLC中OS的改善相关(

= 0.049), without adjusting for age, tumor dimension, and stage

=0.049），

. Conversely, a small-sample cohort study of GEP-NENs (

相反，GEP NENs的小样本队列研究(

= 46) observed significantly better PFS and OS in the DLL3-negative group, largely because of the predominance of NET G1/G2/G3 cases in their study cohort

46）在DLL3阴性组中观察到明显更好的PFS和OS，主要是因为在他们的研究队列中净G1/G2/G3病例占优势

. This study was the largest GEP-NECs cohort to date and found that the expression of DLL3 has no relationship with the prognosis of patients with GEP-NECs.

这项研究是迄今为止最大的GEP NEC队列研究，发现DLL3的表达与GEP NEC患者的预后无关。

This study had some limitations. Being a single-center study with a modest sample size, future research should involve larger cohorts to validate the differences in DLL3 expression across primary tumor sites and further assess the diagnostic performance between NET G3 and NECs in the digestive system.

这项研究有一些局限性。作为样本量适中的单中心研究，未来的研究应该涉及更大的队列，以验证原发肿瘤部位DLL3表达的差异，并进一步评估NET G3和NEC在消化系统中的诊断性能。

Although cytoplasmic and/or membranous staining was considered positive, as reported by other studies.

尽管其他研究报道，细胞质和/或膜染色被认为是阳性的。

, the presence of nuclear positivity remains poorly understood. This discrepancy highlights a limitation in our study, as the relationship between DLL3 staining type (cytoplasmic, membranous, punctata and nuclear) and therapeutic response to DLL3-targeted therapy remains unclear. Further investigation is required to elucidate whether different DLL3 staining patterns are associated with varying clinical outcomes.

，对核阳性的存在仍然知之甚少。这种差异突出了我们研究的局限性，因为DLL3染色类型（细胞质，膜，点状和核）与对DLL3靶向治疗的治疗反应之间的关系尚不清楚。需要进一步研究以阐明不同的DLL3染色模式是否与不同的临床结果相关。

Moreover, the study’s retrospective nature leads to potential biases stemming from progression in clinical practices over time. This highlights the necessity for prospective studies to more effectively clarify the influence of DLL3 expression on patient prognosis..

此外，该研究的回顾性导致了随着时间的推移临床实践进展产生的潜在偏差。这突出了前瞻性研究的必要性，以更有效地阐明DLL3表达对患者预后的影响。。

Conclusion

结论

Our study confirmed DLL3 expression in GEP-NECs and found that DLL3 expression was related to the SCNEC subtype and chemotherapy. The DLL3 expression rate in NET G3 supports the application of DLL3-targeted therapy in high-grade NETs of the digestive system. DLL3 IHC was conducive for distinguishing between NET G3 and NECs, as well as GACs and NECs.

我们的研究证实了DLL3在GEP NEC中的表达，并发现DLL3表达与SCNEC亚型和化疗有关。NET G3中的DLL3表达率支持DLL3靶向治疗在消化系统高级NET中的应用。DLL3 IHC有助于区分净G3和NEC，以及GAC和NEC。

Finally, we confirmed the correlation between DLL3 and ASCL1 protein expression in GEP-NECs. Our study suggests that DLL3 expression is not a prognostic factor in patients with GEP-NECs..

最后，我们证实了GEP NEC中DLL3和ASCL1蛋白表达之间的相关性。我们的研究表明，DLL3表达不是GEP NEC患者的预后因素。。

Data availability

数据可用性

The datasets generated and/or analysed during the current study are not publicly available due patient privacy but are available from the corresponding author on reasonable request.

由于患者的隐私，当前研究期间生成和/或分析的数据集无法公开获得，但可根据合理要求从通讯作者处获得。

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Acknowledgements

致谢

I would like to express my sincere gratitude to my supervisor, Dr. Y. Sun, for her guidance and support throughout my research journey. I am also deeply grateful to my colleagues, who have provided assistance in data collection and proofreading of manuscript. Finally, thanks to the Neukio Biotherapeutics for providing the DLL3 antibody..

我要向我的导师Y.Sun博士表示衷心的感谢，感谢她在我整个研究过程中的指导和支持。我也非常感谢我的同事，他们在数据收集和稿件校对方面提供了帮助。。。

Funding

资金

This work was supported by Capital’s Funds for Health Improvement and Research (2024-2-1024), Beijing Natural Science Foundation (7232018), National Natural Foundation of China (92259302), Beijing Natural Science Foundation (Z200015), and horizontal scientific research project (NEUK300-03).

这项工作得到了首都健康改善与研究基金（2024-2-1024），北京自然科学基金（7232018），国家自然基金（92259302），北京自然科学基金（Z200015）和横向科学研究项目（NEUK300-03）。

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作者信息

Authors and Affiliations

作者和隶属关系

State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers, Beijing Key Laboratory of Carcinogenesis and Translational Research, Department of Pathology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Haidian District, Beijing, 100142, China

胃肠道癌症整体综合管理国家重点实验室，北京大学肿瘤医院病理科北京癌变与转化研究重点实验室，北京市海淀区阜成路52号，北京100142

L. Yin, R. Wang, K. Jiang, Y. Hu, X. Zhao & Y. Sun

五十、尹，R。王，K。江，Y。胡，X。赵和Y。太阳

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Pathology, Peking University Cancer Hospital and Institute, Beijing, 100142, China

北京大学肿瘤医院病理科致癌作用与转化研究重点实验室（教育部），北京100142

X. Ma

十、马萨诸塞州

Neukio Biotherapeutics, Shanghai, 200131, China

Neukio Biotherapeutics，上海，200131

L. Zhang, Z. Wang & T. Long

张，Z。王缩略图长

State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers, Beijing Key Laboratory of Carcinogenesis and Translational Research, Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute, Beijing, 100142, China

M. Lu

M、鲁

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Gastrointestinal Oncology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Haidian District, Beijing, 100142, China

北京大学肿瘤医院胃肠道肿瘤科癌变与转化研究重点实验室（教育部），北京市海淀区阜成路52号，北京100142

J. Li

J、李

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Contributions

捐款

M.L, J.L and Y.S privided the ideas. L.Y and R.W collected the date and followed up patients. X.M carried out immunohistochemical staining on the tissues. L.Z, Z.W, and T.L provided the DLL3 antibody. L.Y analyzed the data, made all the figures and tables, and wrote the manuscript. K.J, Y.H, X.Z and Y.S revised the manuscript and all the figures and tables.

M、 L、J.L和Y.S提出了这些想法。五十、 Y和R.W收集了日期并对患者进行了随访。十、 M对组织进行免疫组织化学染色。五十、 Z，Z.W和T.L提供了DLL3抗体。五十、 Y分析了数据，制作了所有的图表，并撰写了手稿。K、 J，Y.H，X.Z和Y.S修改了手稿和所有的图表。

Y.S completed the submission. Y.S provided funding acquisition. All authors read and approved the manuscript..

Y、 S完成了提交。Y、 S提供了资金收购。所有作者都阅读并批准了手稿。。

Corresponding authors

通讯作者

Correspondence to

通信对象

J. Li

J、李

或

Y. Sun

Y、太阳

Ethics declarations

道德宣言

Ethics declarations

道德宣言

Human samples and clinical data were collected and used in accordance with the principles of the Declaration of Helsinki and approved by the Ethics committee of Peking University Cancer Hospital. All participants provided written informed consent, approval number 2023KT29.

根据赫尔辛基宣言的原则收集和使用人体样本和临床数据，并经北京大学肿瘤医院伦理委员会批准。所有参与者均提供了书面知情同意书，批准号为2023KT29。

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Additional information

其他信息

Publisher’s note

出版商注释

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

Electronic supplementary material

电子补充材料

Below is the link to the electronic supplementary material.

以下是电子补充材料的链接。

Supplementary Material 1

补充材料1

Supplementary Material 2

补充材料2

Supplementary Material 3

补充材料3

Supplementary Material 4

补充材料4

Rights and permissions

权限和权限

Open Access

开放存取

This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

本文是根据知识共享署名非商业性NoDerivatives 4.0国际许可证授权的，该许可证允许以任何媒介或格式进行任何非商业性使用，共享，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出您是否修改了许可材料。

You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

根据本许可证，您无权共享源自本文或其部分的改编材料。本文中的图像或其他第三方材料包含在文章的知识共享许可证中，除非该材料的信用额度中另有说明。。

To view a copy of this licence, visit .

要查看此许可证的副本，请访问。

http://creativecommons.org/licenses/by-nc-nd/4.0/

Reprints and permissions

重印和许可

About this article

关于本文

Cite this article

引用本文

Yin, L., Wang, R., Ma, X.

尹，L.，王，R.，马，X。