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Research paper

Concurrently dilated lymphatic ducts within mucosa and

submucosa: early sign of cell dissemination and node metastasis?

Bin Jianng1*, Itzhak Avital

2*, Yin Jiang Ding

1, Aviram Nissan

3, Mladjan Protic

4,

Anton Bilchik5, Ciaran Mannion

6, Jeffrey Mason

7, Jinlian Wang8, Anahia Jewett

9,

Alexander Stojadinovic2¥

, Yan-gao Man2¥

1National medical Centre of Colorectal Disease, Third Affiliated Hospital of Nanjing

University of Chinese Medicine, Nanjing, Jiangsu, China

2Bon Secours Cancer Institute, Bon Secours Health System, Richmond VA, USA

3Department of Surgery, Hadassah Hebrew University Medical Center, Jerusalem, Israel

4Clinical Center of Vojvodina,

University of Novi Sad, Serbia

5University of California; Oncology Research Center, Los Angeles, USA

6Department of Pathology, Hancksack University Medical Center, Hancksack, NJ, USA

7Laboratory of Proteomics and Protein Sciences, Veterans Affairs Medical Center, Baltimore,

MD, USA

8Genetic & Genomics Department, Icahn Mount Sinai Medical School, New York, NY, USA

9Tumor Immunology Laboratory, Jonson Comprehensive Cancer Center, Los Angeles, CA,USA

* : They contribute equally to the project and are considered as the co-first author.

New Approaches combating Cancer & Aging 2014; 1: 55-71

¥: Corresponding author:

Alexander Stojadinovic, M.D. FACS. Professor of Surgery and Medicine

Medical Director, Bon Secours Cancer Institute, Bon Secours Health System, Virginia, USA

Phone: 804-221-7362 E-mail Address: stojadinovicmd2011@gmail.come

Yan-gao Man, MD., PhD

Director of Research Laboratory and international Collaboration

Bon Secours Cancer Institute, Bon Secours Health System, Virginia, USA

Editor-in-chief, Journal of Cancer (http://www.jcancer.org Impact Factor: 2.639)

Phone: 301-879-0816 Email address:yangao_man@bshsi.org or yanmann@aol.com

This is an open-access article distributed under the terms of the International Standard Serial

Number (2372-7837) and the International Union for Difficult-to-treat-Diseases

(www/iudd.org). Reproduction is permitted for personal, noncommercial use, provided that the

article is in whole, unmodified, and properly cited.

Received: 2014.12-11; Accepted: 2014.12-24; Published: 2014.12-31

Abstract

Background: Lymph node metastasis is one of the most important

prognostic factors in cancer, but can be difficult to detect. Our current

study tested a novel hypothesis that intravasation of tumor cells at the

primary site may stimulate lymphatic endothelial cell proliferation or

disrupt the normal pathway of dietary transport and fluid homeostasis

resulting in concurrently dilated lymphatic ducts in the mucosa and

submucosa.

Materials and Methods: The frequency of concurrently dilated

lymphatic ducts in the mucosa and submucosa and disseminated cells

within dilated lymphatic ducts in 100 primary colorectal cancer (CRC)

patients with known node status were compared using a panel of

epithelial and endothelial markers.

Results: Our study showed (1) node positive cases had a significantly

higher (p<0.005) frequency of concurrent dilated lymphatic ducts than

their morphologically similar node negative counterparts, (2) within the

mucosa, concurrently dilated lymphatic ducts with disseminated tumor

科研文章

粘膜和粘膜下层内同时扩张的淋巴管：

细胞游离和淋巴结转移的早期征兆？

江滨 1*,Itzhak Avital2*, 丁义江 1,Aviram Nissan3, Mladjan

Protic4, Anton Bilchik5 ,Ciaran Mannion6, Jeffrey Mason7王

金莲 8, Anahid Jewett9, Alexander Stojadinovic2¥ 满延高 2¥

1中国国家结直肠疾病中心, 南京中医大学第三附属医院,

南京, 江苏 2美国Bon Secours癌症研究院与医疗系统, 维吉尼亚 3以色列Hadassah Hebrew大学医学中心外科系,耶路萨冷 4塞比亚 Vojvodina临床中心,Novi Sad, 大学，Novi Sad,

5美国加利弗尼亚大学肿瘤研究院, 洛杉矶 6美国新泽西Hackensack大学医学中心病理学系,汉克萨克 7美国退伍军人医学中心，蛋白质组学及蛋白质科学实验室, 巴尔

底摩 8美国Icahn Mount Sinai医学院遗传和基因组学系,纽约 9美国加利弗尼亚Jonson综合癌症中心肿瘤免疫实验室.

*: 此两作者贡献均等，两者皆视为第一作者

新法抗癌抗衰 2014年第1期第55至71页

¥: 通讯作者:

Alexander Stojadinovic, 医学博士, 医学与外科教授

医学主任, Bon Secours癌症研究所与医疗系统 , . 维吉尼亚

电话: 804- 221-7364 电子邮件：stojadinovicmd2011@gmail.com

满延高，医学博士，哲学博士

国际合作与实验室主任

美国Bon Secours医疗系统癌症研究所, 维吉尼亚

[癌症杂志]主编 (http://www.jcancer.org 影响因子: 2.639);

电话： 301-879-0816; 电子邮件: yanmann@aol.com; yman@jcancer.org

本刊 为网上杂志,国际标准序列号为:2373-2806. 本刊为国际抗疑

难杂症联盟(www.iudd.org) 的学术刊物. 在保证如实完整反映本刊

所发论文的前提下,任何个人与非商业团体可免费下载任一文章

的全文或章节。

收稿：2014-10-1。接受：2014-10-20。发表：2014-10-22

摘 要

背景： 淋巴结转移是癌症临床预后最重要的因

素之一，但可能很难检测到。我们目前的研究旨

在检验一新假说：肿瘤细胞在病发病灶中入侵淋

巴管可能刺激淋巴管内皮细胞增殖或扰 乱正常

的淋巴液回流，因此导致在的粘膜和粘膜下层的

淋巴管异常扩张。

材料和方法：应用上皮细胞和内皮细胞标记

物，对100例已知淋巴结状况的原发性结肠直肠

癌（CRC）粘膜和粘膜下层内同时发生的淋巴管

扩张和菅中含扩散肿瘤细胞的频率，进行了对比

研究。

结果：我们研究揭示：（1）淋巴结阳性病例中

同时扩张之淋巴菅的频率显著高于（ P值 <

0.005）其形态学相似的淋巴结阴性病例； （2）

在粘膜层中，同时扩张并含扩散肿瘤细胞的淋巴

New Approaches combating Cancer & Aging 2014; Vol 1:55-71

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cells were exclusively seen in node positive cases and in only one node

negative case with confirmed metastasis, (3) concurrently dilated

lymphatic ducts with disseminated cells were interconnected with a

similar pattern extending to the entire primary tumor in consecutive

sections, and (4) most concurrently dilated lymphatic ducts with or

without disseminated cells are hard to detect in H&E stained or in

superficial sections, but they are easily seen in double immuno-stained

or deeper sections with epithelial and endothelial cell markers.

Conclusions: The presence or absence of concurrently dilated

lymphatic ducts in a single tissue section may accurately reflect the

presence or absence of disseminated cells within the entire tumor,

which may benefit early differentiation between node positive and node

negative CRC.

Keywords: Colorectal cancer; Lymph node metastasis; Early

Detection; Lymphatic duct; D2-40

Introduction

Cancer cell metastasis to lymph nodes is an indicator of

colorectal cancer (CRC) metastasis and is also one of the most

important prognostic factors. Before lymph node metastasis develop, a

vast majority of CRC can be cured by surgical resection alone; and,

greater than 75% of patients with node-negative CRC survive 5-years

or longer after resection [1-3]. After lymph node metastasis, the

treatment will be far more difficult and expensive, and only about 10%

of the CRC patients with distant disease spread survive 5-years or

longer [1-3].

Early detection of lymph node metastasis, however, can be

technically challenging for three main reasons. First, it has been

consistently documented that up to 25% of early stage (stage I/II), node

negative CRC patients develop systemic metastasis [4-11], which

suggests that metastasis could potentially occur at a much earlier stage

in the natural history of disease than is currently defined or assumed.

Consequently, an accurate assessment of nodal status may require a

fundamental change of the concept of cancer metastasis and the

emergence of new strategies and methodologies aimed at optimizing

staging accuracy. Second, the total number of the regional lymph

nodes draining a given primary CRC can be numerous, yet the

disseminated cells could metastasize to any of these nodes. Thus, the

12-node minimum has been established for an accurate assessment of

the nodal status in resected tumor specimens [4], which is a time-

consuming and resource intense process, particularly when adjunctive

measure must be utilized to achieve that quality benchmark. Third,

tumor cells metastasized to a lymph node(s) could lodge in any location

of a given node that is usually about 0.5-2.0rcm in diameter or larger,

in which thousands of 5-µm sections can be derived from a given node.

菅仅见于淋巴结阳性病例及一被证明有转移的淋巴结阴性病例；（3）在连续切片中，同时扩张并含扩散肿瘤细胞的淋巴菅在整个癌巢中以相似方式，互相连通；(4）绝大多数同时扩张的淋巴菅（无论其是否含扩散肿瘤细胞）都较难在苏木素-伊红染色切片或表面的切片中检出，但是它们却很容易在用上皮细胞和内皮细胞标记物双重免疫组化染色的切片或较深层的切片中被查出。

结论：在单一的组织切片中存在或缺乏同时扩

张的淋巴管，可能正确地反映了在整个癌瘤组

织内是否存在癌瘤细胞的淋巴结转移，它也可

能有利于早期鉴别有无淋巴结转移的原发性结

直肠癌病人。

关键词: 大肠癌 ；淋巴结转移 ；早期的检测；

淋巴导管 ；D2-40

导 言

癌细胞转移到淋巴结是结肠直肠癌（CRC）

转移的一个征象，并且是最重要的诊断因素之

一。在淋巴结转移发生之前，绝大多数结直肠

癌可用单纯外科切除手术治愈，而且手术后多

于 75%的无淋巴结转移的病人可存活五年或更

久【1-3】。在淋巴结发生转移后，其治疗则远

为困难，花费更多，并且仅有 10%的伴有远距

离部位扩散的结肠直肠癌病人能存活五年或更

久【1-3】。

然而，早期的淋巴结检测在技术上是有挑

战的，主要理由有三：首先，一贯的文献表

明，有多达 25%的早期（I 期/II 期）无淋巴结

转移结肠直肠癌病人发生了系统性转移 【4-

11】，这就证实在该病的自然历史中，这种转

移能在比后来人们界定或接受时期更早的时期

潜在的发生。继之，一个淋巴结状态的正确评

估，可能需要癌症转移概念的重要改变，出现

新的战略观点，以及形态学家在优化时期准确

性方面的协助。其次，所有能导流原发性结肠

直肠癌全部区域内的淋巴结数量众多，且散布

的癌细胞应能转移到任何一个这些淋巴结内。

因此，最少应有 12 个淋巴结可作为癌瘤切片标

本中淋巴结状态的正确评估之用 【4】，这是

一个消耗时间和资源紧张的过程，特别是当必

须利用附加措施来达到质量水准基点时，更是

如此。第三，转移到淋巴结的癌细胞应能寄宿

在该淋巴结的任一部位，此淋巴结的直径通常

约 0.5 至 2rcm 或更大，其中可从一个淋巴结内

切成成上千片 5 微米厚的切片。因此，仅检测

New Approaches combating Cancer & Aging 2014; Vol 1:55-71

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Hence, the examination of one or a few sections of each node is very

unlikely to be able to accurately determine the node status, while the

examination of all sections of all nodes would be technically

impracticable [12-14]. Early detection of surrogate indicators of nodal

metastasis at the primary tumor site could be even more difficult due to

its significantly larger size, significantly more cell types, and the lack

of effective biomarkers [15-18].

The fundamental functions of normal colonic lymphatic ducts

include: (a) immune surveillance, (b) maintenance of tissue fluid

homeostasis; and, (c) transport of dietary fat and fat-soluble vitamins

[19, 20]. The wall of a normal lymphatic duct consists of a single

layer of elongated endothelial cells often containing small pores, and

surrounded by a discontinuous basement membrane [21-23], which

allow tumor cell intravasation. Aberrant lymph drainage has been

linked to the development and progression of a number of benign and

malignant conditions, including inflammatory bowel disease,

pancreatitis, and CRC metastasis [24-27]. A recent study reported that,

of 192 CRC patients that had undergone surgical resection, 42 (22%)

had extended operation due to aberrant lymphatic drainage outside the

standard resection limits; patients with extended operation had a

significantly higher frequency of node positivity than patients with

standard oncologic resection, 62% versus 43%, respectively [28].

Together, these findings suggest tumor cell metastasis to local lymph

node may significantly impact the drainage pathway or the structural

feature of the associated lymphatic ducts. Thus, our current study

attempted to test a novel hypothesis that intravasation of tumor cells at

the primary site may either stimulate lymphatic endothelial cell

proliferation or disrupt the normal pathway of dietary transport and

fluid homeostasis, which results in concurrently dilated lymphatic ducts

in the mucosa and submucosa. Consequently, concurrently dilated

lymphatic ducts may represent an early sign of tumor cell

dissemination to regional or distant nodes.

Materials and Methods

Unstained consecutive and de-indentified tissue sections from

100 stage- and age-matched CRC patients with and without positive

lymph nodes were retrieved from the Achieves of the National Medical

Center of Colorectal Disease, The Third Affiliated Hospital of Nanjing

University of Chinese Medicine, Nanjing, China under an Institutional

Review Board (IRB)-approved protocol (#2008001). The first and last

sections from each case were stained with hematoxylin and eosin

(H&E) for morphological classification using established criteria. The

remaining sections were used for immunohistochemical (IHC) and

morphological assessment. After preliminary IHC and morphological

assessment, 8-cases are excluded due to the lack of a sufficient amount

of the normal tissue component or tissue falling from the slides.

淋巴结的一片或少量切片，很可能是不能精确

地确定此淋巴结的真实状态，而要检测所有淋

巴结的全部切片在技术上应是不现实【12-

14】。而在原始癌瘤部位早期检测淋巴结转

移，则因体积明显太大，细胞类型明显太多，

以及缺乏有效的生物标记物，则甚至更为困难

【15-18】。

正常结肠淋巴管的基本机能包括：（1）免

疫监视；（2）维持组织液体的内环境稳定；

以及（3）食物营养料脂肪和水溶性维生素的

转运【19-20】。正常淋巴管壁由一层长梭形

内皮细胞构成，管壁通常含有若干小孔，并由

一层不连续的基膜所环绕【21-23】，这就允

许癌瘤细胞的侵入。异常的淋巴导流可联系到

无数良性和恶性肿癌的发生和进展，包括感染

性结肠炎，胰腺炎，以及结肠直肠癌转移等

【24-27】。最近一项研究报告表明，在 192

例接受手术切除的结直肠癌病人中，其中有 42

例（占 22%）因为在标准切除范围外存在异常

的淋巴导流，而需扩大手术切除范围。需扩大

手术范围病人淋巴结阳性的频率显著高于无需

扩大手术的一病例，62%比 43%【28】。综合

起来，这些发现证实，癌瘤细胞转移到局部淋

巴结，可能明显地影响淋巴导管的导流通道或

结构特征。因此，我们的研究试图检测一个新

的假说： 即原始癌瘤部位癌瘤细胞的侵入，可

能会刺激淋巴管内皮细胞的增生，或是破坏食

物营养素的正常通道和液体的内环境稳定，这

就导致粘膜和粘膜下层内的淋巴管同时发生扩

张。因此，同时扩张的淋巴管可能代表癌瘤细

胞散播到区域部位或远离部位淋巴结的一个早

期信号。

材料和方法

一百例匿名、年龄和分期匹配、淋巴结阳

性与阴性结直肠癌病人的未染色连续组织切片

来自中国南京市南京中医药大学附属第三医院

的全国结肠直肠疾病医学中心。 实验由肛肠中

心之伦理与审核委员会批准(批号为 2008001).

每例病人的首个和最后一个切片用苏木素-伊

红（H&E）染色，按已确定标准作形态学分

类。病人的其余切片，用作形态学和免疫组织

化学（IHC）评估。经初步的形态学和免疫组

化评估后，八个病例因缺乏有效量的正常组织

成份或组织从切片上脱落丢失而被 排除。

New Approaches combating Cancer & Aging 2014; Vol 1:55-71

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Double IHC was utilized to assess the distribution of lymphatic

ducts within the mucosa and submucosa that are associated with

morphologically distinct muscularis mucosa (MM) with the following

monoclonal antibodies: (1) anti-cytokeratins (CK) AE1/AE3, which

react with all epithelium-derived cells; (2) anti-CK-19 (clone: clone:

RCK108, Dako, Carpinteria, CA, USA), which is thought to be a

cancer stem cell marker for most epithelium-derived malignancies,

including CRC [29,30]; and (3) anti-D2-40 (clone: D2-40; Signet,

Dedham, MA), which labels all lymphatic ducts.

A dilated lymphatic duct was defined as an open tube-like

structure with (1) a distinct lumen larger than the combined size of at

least 3-4 white blood cells, and (2) morphologically distinct

endothelial cells that shows strong D2-40-positivity. Concurrently

dilated lymphatic ducts in the mucosa and submucosa were defined as

the presence of dilated lymphatic ducts within the mucosa and

submucosa of the same microscopic field. To compare the frequencies

of concurrently dilated lymphatic ducts between node negative and

node positive cases, all structures with distinct mucosa and submucosa

clearly segregated by the MM were digitally photographed. The digital

images were independently reviewed on a computer screen at400-800%

magnification by at least two investigators, blinded to the nodal status

of the patients. The absolute number of concurrently dilated lymphatic

ducts within the mucosa and submucosa that are associated with

morphologically distinct MM in each case was counted, added, and

separately averaged between node positive and negative cases. The

averaged number between node positive and negative cases was

statistically compared using the Pearson’s Chi-square test. Statistical

significance was defined as p < 0.05.

To compare the frequency of disseminated cells in concurrently

dilated lymphatic ducts in both the mucosa and submucosa, 5 node

positive cases (in which 16 of 18, 12 of 12, 11 of 13, 10 of 10, and 6 of

6 lymph node examined were positive) and 5 age-, type-, size-, and

differentiation-matched node negative cases (in which all 17, 12, 11, 11,

and 11 lymphatic ducts examined were negative) were selected. A total

of 80-100 consecutive sections at 5-7 µm thickness were cut from each

paraffin-embedded tissue block, and a set of 8-10 of discontinuous

sections from each case were subjected to IHC staining with epithelial

and lymphatic endothelial cell phenotypic marker D2-40 and immune

cell-related markers. The frequencies of concurrently dilated lymphatic

ducts with disseminated tumor cells between node negative and node

positive cases were statistically compared with the Pearson’s Chi-

square test. Statistical significance was defined as p < 0.05.

应用双重免疫组化方法（IHC），来评估与形态清晰的粘膜肌（MM）相联的粘膜和粘膜下层内的淋巴管的分布。 所应用单克隆抗体有：（1）抗胞质角蛋白（CK）AE1-AE3 抗体，它能与所有上皮样起源的细胞发生反应；（ 2 ） 抗 胞 质 角 质 蛋 白 -19 抗 体 （ 克 隆 RCK108，DAKO，美国加州 Carpinteria），它被认可为绝大部分起源于上皮癌瘤（包括结肠直肠癌）的干细胞标记物；以及（3）抗 D2-40 抗体（克隆：D2-40；Signet，美国马萨诸塞州 Dedham），它能标记所有的淋巴管。

扩张的淋巴管的定义是：一个开放的管样

结构，且具有二个特征：（1）一个明显的管

腔，其大小至少要大于 3-4 个白细胞联合体

积；以及（2）在形态学上有明显的内皮，并

表现明显的 D2-40 阳性。在粘膜和粘膜下层同

时扩张的淋巴管的定义为：在同一显微镜视野

下在粘膜和粘膜下层内所存在的扩张的淋巴

管。为了对比在有无淋巴结转移病例之间伴发

淋巴管扩张的发生频率，所有的由清晰粘膜肌

分隔的粘膜和粘膜下层的结构，都作了数位相

机拍照。所有的数位相机图片，都经过至少两

位事先对淋巴结转移状态盲目无知的研究工作

者，在电脑银屏 400-800 倍放大条件下单独地

观察检测. 在有无淋巴结转移的病例之间，对

所有与清晰固有膜相连的粘膜和粘膜下层内扩

张淋巴管的绝对数量，作计数记录 .累加以及

平均值计算。对有无淋巴结转移之间的平均

值，使用 Pearson 氏 Chi-sguare 测算法作了统计

学上的对比，统计学差异的标准界定为 P 值

<0.05。

为了对比在粘膜和粘膜下层内伴发的扩张

淋巴管内扩散癌细胞的频率，选用了五例淋巴

结阳性病人（其中分别有 16/18、12/12、11/ 13,

10/10、和 6/6 淋巴结被检测为阳性），和五例

年令、类型、大小和分化，分期匹配淋巴结阴

性病人（其中分别有 17、12、11、11 和 11 个

淋巴管被检测为阴性）。总数有 80-100 个厚度

为 5-7 微米的连续切片，切自石蜡包埋的组织

块，每个病例有一套 8-10 张不连续的切片，应

用对上皮细胞和淋巴管内皮细胞遗传表现型标

记物 D2-40 和免疫细胞相关标记物，作免疫组

织化学染色。在有无淋巴结转移病人之间，对

内含分散癌细胞的伴发扩张淋巴管的频率，应

用 Pearson 氏 Chi-square 测算法，作了统计学上

的对比，统计学差异界定为 P值<0.05。

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To assess the specificity of the immunostaining, various

negative controls were used, including: (1) the substitution of the

primary antibody with the same isotype or pre-immune serum of the

antibody; and, (2) the omission of the secondary antibody. The

immunostaining procedure was repeated at least twice using the same

protocol and under the same conditions. Immunostained sections were

independently evaluated by two investigators, blinded to the nodal

status of the patient. A given cell was considered immunoreactive if

distinct immunoreactivity was consistently seen in sections with a

given antibody, whereas all negative controls lacked distinct

immunostaining.

Results

The density, size, and distribution of lymphatic ducts in morphologically

similar tissue components and locations among cases differed substantially.

The most noticeable difference between node negative and node positive

cases is the size of the duct lumen. As shown in Figure 1, a vast majority of

the lymphatic ducts within the mucosa and submucosa of node negative cases

generally lack a distinct lumen (A-D). In contrast, about 1/3 of node positive

cases harbored variable numbers of lymphatic ducts with a large open lumen

in both the mucosa and submucosa or crossing the MM (E-H). A total of 189

concurrently dilated lymphatic ducts within the mucosa and submucosa were

detected in 37 node positive cases, compared to 22 in 55 node negative cases.

The frequency of concurrently dilated lymphatic ducts was

significantly higher in node positive than in node negative cases (p<

0.0001) (Table 1).

A vast majority of the dilated lymphatic ducts harbored no disseminated

cells. As shown in Figure 2, the superficial section (A-B) from a node

positive CRC harbors over 10 concurrently dilated lymphatic ducts within the

mucosa and submucosa, while none contains disseminated tumor cells.

However, in the deeper sections (C-H), variable numbers of disseminated

tumor cells were found in some lymphatic ducts within the mucosa. It is

interesting to note that the superficial (A-B) and the last section (G-H) are at a

distance of about 500µm (about 100 5-µm sections), while the pattern of

concurrently dilated lymphatic ducts is very similar between these two

sections.

Table 1. Frequency of concurrently dilated lymphatic ducts in node positive and negative cases

表 1.淋巴结阳性和阴性病例内同时扩张淋巴管的频率

Node status

淋巴结状态

Number of cases

病例数量

Number of concurrently dilated ducts

扩张淋巴管的数量

P

P 值

Positive (阳性) 37 189 <0.0001

Negative (阴性) 55 22

为评估免疫学染色的特异性，应用了各种阴

性对照组，其中包括：（1）以同型性替代物或

免疫前的血清抗体取代抗体，以及 (2)省略第二

抗体。免疫学染色处理至少重复二次，且使用

同样的方案和在同样条件下进行。免疫学染色

切片经二名事先对病人的淋巴结转移状态盲目

无知的研究人员逐个评估，若在特定的抗体染

色的切片中都一致见到明显的免疫学反应，而

所有阴性对照组的切片应缺乏这种明显的免疫

染色特性，则该切片细胞可看作是具有免疫反

应性的细胞。

结 果

在所有病例中，在形态学上具有相似组织

成分和部位内的淋巴管的密度、大小和分布，

都明显的不同。在淋巴结阳性和阴性病人之

间，最明显的差别是淋巴管腔的大小。如图 1

所示，在淋巴结阴性病人粘膜和粘膜下层内的

绝大多数淋巴管，都缺乏明显的管腔（A-D 小

图），相反，在 1/3 有淋巴结阳性病人粘膜和

粘膜下层内的淋巴管或穿过粘膜肌的淋巴管，

都具有较大的开放的管腔（E-H 小图）。在 37

例淋巴结阳性病人粘膜和粘膜下层内，共检测

出 189 个同时扩张的淋巴管，而在 55 例淋巴结

阴性病人中，则其检出总数为 22 个。因此，在

淋巴结阳性病人中同时扩张淋巴管的频率明显

高于淋巴结阴性病人（P<0.0001）（见表 1）。

绝大多数扩张的淋巴管内，无隐藏有分散

的癌细胞。如图 2 所示，在淋巴结阳性病人的

表面切片（A-B 小图）的粘膜和粘膜下层内，

可见 10 个同时扩张的淋巴管，但其中不含有分

散的癌细胞。但是，在较深部切片（C-H 小

图）中，某些淋巴管内可见相当数量的分散的

癌细胞。非常有意义的是，这些表面切片（A-B

小图）和最后切片（G-H 小图）的距离约为

500 微米（约为 100 个 5 微米厚切片），而在

这两种切片之间同时扩张淋巴管的结构，却很

相似。

结果

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Figure 1. Different features of lymphatic ducts in node negative and positive cases

Human normal appearing colonic tissue sections distant from tumor were immunostained with

lymphatic endothelial cell marker D2-40 (brown). Black circles identify low magnification

views of the structures in B, D, F, and H, respectively. Black and blue arrows identify

lymphatic ducts within the mucosa and submucosa, respectively. Stars identify the MM. Note

that nearly all lymphatic ducts of node negative cases lack a distinct lumen (A-D). In contrast,

node positive cases harbor variable numbers of concurrently dilated lymphatic ducts with a

distinct open lumen, and some of them span the mucosa and the MM (red circle). A, C, E, and

G: 80X. B, D, F, and H. A higher (400X) of A, C, E, and G, respectively.

图 1. 淋巴结阳性和阴性病例中淋巴管的不同特征

远离癌巢貌似正常的人体结直肠组织切片，免疫组化染淋巴内

皮细胞标记物 D2-40（棕色）。黑色环状物分别表示在 B、D、

F 和 H 内结构的低倍镜观，黑色和兰色箭头分别表示在粘膜和

粘膜下层内鉴定的淋巴管，星形物表示粘膜肌层。注意：淋巴

结阴性病例中几乎所有的淋巴管都缺乏明显的管腔（A-D）。

相反，淋巴结阳性病例隐藏相当数量同时扩张的淋巴管，它们

具有明显的开放管腔，其中有些淋巴管横跨粘膜和粘膜肌层

（红色环状物）。A、C、E 和 G：放大 80 倍，B、D、F 和

H：分别为 A、C、E和 G 的高倍放大（400 倍）。

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Figure 2. Disseminated cells within dilated lymphatic ducts of mucosa in deeper sections

A set of four discontineous human CRC tissue sections from a node positive case were double

immunostained for D2-40 (brown) plus cytokeratins AE1/3 (red) (A-F) or triple immunostained for D2-40 (blue), Ki-67 (black), and leukocyte common antigen (LCA; red). Circles identifies the low magnification

view of the structure in B, D, F, and H. Black and blue arrows identify lymphatic ducts within the mucosa

and submucosa, respectively. Red arrows identify lymphatic ducts with disseminated cells. Stars identify the MM. Note that the superficial section (A-B) harbor over 10 open lumen lymphatic ducts within the

mucosa and submucosa, while none contain disseminated cells. However, in the deeper section (C-H),

several lymphatic ducts with disseminated disseminated cells are seen within the mucosa. The superficial section (A-B) and the last section (G-H) are at a distance of about 500µm (over 100 5-µm sections), while

the pattern of concurrently dilated lymphatic ducts is very similar. A, C, E, and G: 100X. B, D, F, and H: a

higher (300X) magnificantion of A, C,E, and G, respectively

图 2.在较深部切片扩张淋巴管内的散布癌细胞 淋巴结阳性结肠直肠癌病例的四张非连续组织切片，经 D2-40(棕 色）和角质蛋白 AE1/3(红色)（A-F）双重免疫组化染色，或经 D2-40(蓝色)、Ki-67（黑色）和白细胞共同抗原（LCA;红色）三重免疫组化染色。圆圈表明 B、D、F 和 H 结构的低倍观。黑色和蓝色箭头分别表明粘膜和粘膜下层内的淋巴管，红色表明具有散布癌细胞的淋巴管，星形物表明粘膜肌层。注意：表面切片（A-B）粘膜和粘膜下层内隐藏有 10 个以上有开口的淋巴管，但并不包含散布的癌细胞。然而，在较深部切片(C-H)粘膜层内，可见若干淋巴管内含散布的癌细胞。此表面切片（A-B）和最末切片（G-H）的距离约 500 微米(100 片以上厚度 5 微米切片距离)，而其中伴发的扩张淋巴管的方式都很相似。A、C、E 和 G：放大 100 倍，B、D、F 和 H：分别为 A、C、E 和 G 的高倍放大（300 倍）。

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Disseminated tumor cells were also seen within dilated lymphatic ducts of the submucosa distant from tumor. As shown in Figure 3, the superficial section (A-B) harbor a number of concurrently dilated lymphatic dusts with no disseminated cells. Again, some ducts in the deeper sections (C-H) contain variable numbers of disseminated tumor cells. It is interesting to note that disseminated tumor cells within the lymphatic ducts are often physically associated with LCA positive cells (G-H), suggesting that immune cells are very likely to significantly contribute to intravasation and circulation of tumor cells.

Figure 3. Disseminated cells within dilated lymphatic ducts of submucosa in deeper sections.

A set of four discontineous normal appearing colonic tissue sections from a node positive case were double

immunostained for D2-40 (brown) and CK AE1/3 (red) (A-F), or CK AE1/3 (brown) and LCA (red). Circles

identifies the low magnification view of structure in B, D, F, and H. Black and blue arrows identify lymphatic

ducts within the mucosa and submucosa, respectively. Red arrows identify lymphatic ducts with disseminated cells.

Stars identify the MM. Note that the superficial section (A-B) harbor several concurrently dilated lymphatic ducts

within the mucosa and submucosa, while none contains disseminated cells. In the deeper sections (C-H), varible

numbers of disseminated are seen. It is interesting to note that disseminated cells within the lymphatic duct are

physically associated with LCA positive cells. A, C, E, and G: 100X. B, D, F, and H: a higher (300X)

magnificantion of A, C, E, and G, respectively.

散布的癌细胞也见于远离癌瘤的粘膜下层的扩

张淋巴管内。如图 3 所示，表面切片(A-B)内

隐藏有相当数量的扩张淋巴管，其管腔内并无

散布的癌细胞。再者，在深部切片（C-H）某

些淋巴管内却含有相当数量的散布癌细胞。有

意义的是：在淋巴管内散布的癌细胞，在生理

学上通常都与白细胞共同抗原（LCA）阳性细

胞相连接，表明这些免疫细胞很可能与癌细胞

的侵入和循环具有明显的关系。

图 3. 在较深部切片粘膜下层扩散淋巴管内散布的癌细胞 淋巴结阳性结肠直肠癌病例的四张非连续组织切片，经 D2-40(棕色)和 CK AE1/3（红色；A-F），或经 CK AE1/3（棕色）和白细胞共同抗原（LCA,红色）双重免疫组化染色。圆圈指示 B、D、F 和H 结构的低倍观，黑色和蓝色箭头分别指示粘膜和粘膜下层内的淋巴管，红色箭头指示淋巴管内包含散布的癌细胞，星形物指示粘膜肌层。注意：表面切片（A-B）粘膜和粘膜下层内隐藏若干同时扩张的淋巴管，但管内未含分散的癌细胞。而在较深部切片（C-H），其中可见相当数量的分散癌细胞。有意义的是，这些淋巴管内分散的癌细胞，在生理学上都与和白细胞共同抗原阳性细胞相连。A、C、E 和 G：放大 100 倍，B、D、F 和 H：分别为 A、C、E 和 G 的高倍放大（300 倍）。

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The number of disseminated cells within a given dilated lymphatic duct was generally small, ranged from a single to about 15 cells. Disseminated cells were seen in either the mucosa or the submucosa, or in both locations. As shown in Figure 4, each of 8 sections harbors concurrently dilated lymphatic ducts within the mucosa and submucosa, while the dilated lymphatic ducts within the mucosa of the first 3-superficial sections harbor no disseminated cells (A-F). In the 5-deeper sections (G-P), one of the dilated ducts on the upleft of the section starts to show disseminated cells, which persist in the same duct of all 5-deeper sections. It is interesting to note that concurrently dilated lymphatic ducts within the mucosa and submucosa in these discontineous sections appear to be interconnected or in direct physical contitunity although the number and lumen size of the ducts, and disseminated cells differed among the discontineous tissue sections.

在扩张淋巴管内散布癌细胞的数量，一般较少，从单个细胞至约 15 个细胞。分散的癌细胞可见于粘膜层或粘膜下层，或两者部位都有。如图 4 所示，8 个切片中每个切片粘膜层和粘膜下层内都隐藏有伴发的扩张淋巴管，而在最初 3 个表面切片粘膜的扩张淋巴管内，却无隐藏有扩散 的癌细胞（A-F）。在 5 个较深部切片中(G-P)，于切片的左上方有一个扩张的淋巴管内开始显有扩散的癌细胞，并一直出现在所有 5 个深部切片的同样的淋巴管内。有意义的是，尽管在这些不同切片中，其扩张淋巴管的数量和管腔大小以及分散癌细胞数量有所不同，但在这些不同切片中粘膜和粘膜下层内同时扩张的淋巴管似乎是相互联系的，或是直接相通的。

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Figure 4. Changeable status of dilated lymphatic ducts in multi-consecutive sections

A set of 8 discontineous CRC tissue sections from a node positive case were immunostained for D2-40 and other markers. Circles identifies the low magnification view of structure in B, D, F, H, J, L, N, and P.

Black and blue arrows identify lymphatic ducts within the mucosa and submucosa, respectively. Red

arrows identify lymphatic ducts with disseminated cells. Stars identify the MM. Note that each of 8 sections harbors concurrently dilated lymphatic ducts within the mucosa and submucosa, while the dilated

lymphatic ducts within the mucosal of the first 3-superficial sections harbor no disseminated cells (A-F). In

the 5-deeper sections (G-P), one of the dilated ducts on the left starts to show disseminated cells, which persist in the same duct of all 5-sections. A, C, E, G, I, K, M, and O: 100X. B, D, F, H, J, L, N, and P: a

higher (300X) magnificantion of A, C, E, G, I, K, M, and O, respectively.

图 4.在多个连续切片中扩张淋巴管的易变性状态 淋巴结阳性结肠直肠癌病例的 8 张非连续组织切片，经 D2-40 和其它标记物的免疫组化染色。圆圈表明 B、D、F、H、J、L、N和 P 结构的低倍观，黑色和蓝色箭头分别表明粘膜和粘膜下层内的淋巴管，红色箭头表明淋巴管内扩散的癌细胞，星形物表明粘膜肌层。注意：8 个切片中每个切片粘膜和粘膜下层内都隐藏有同时扩张的淋巴管，但在最初 3 个表面切片粘膜扩张淋巴管内，却未见有隐藏扩散的癌细胞（A-F），而在 5 个较深部位切片中（G-P），左侧的一个扩张淋巴管开始出现扩散的癌细胞，它们存在于所有这 5 个切片的同一个淋巴管内。A、C、E、G、I、K、M 和O：放大 100 倍， B、D、F、H、J、L、N 和 P：分别为 A、C、E、G、I、K、M 和 O 的高倍放大（300 倍）。

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In one case in which 11 of 13 nodes examined were positive, however, the number of disseminated cells within a given lymphatic duct reached hundreds or more. Figure 5 shows a set of 10 discontinuous sections from this node positive case, which are stained with different markers. It can be seen again that concurrently dilated lymphatic ducts within the mucosa and submucosa of all sections (that spanned the distance of over 50 consecutive sections) appear to be interconnected or in direct physical continuity although the number and lumen size of the ducts differ among the discontinuous sections (A-H). Some of the dilated lymphatic ducts with disseminated cells penetrate the MM and reach deep to the submucosa (I-M). These disseminated cells showed strong expression of CK-19 in an overall CK-19 negative background (D-F). It is interesting to note that concurrently dilated lymphatic ducts are not only seen within the mucosa and submucosa adjacent to the tumor tissues, but also seen within the mucosa and submucosa distant from (at the normal colonic wall opposite to the tumor site) (A-H; squares). In addition, two normal appearing CK-negative epithelial structures harbor budding CK-19 positive cell clusters adjacent to dilated lymphatic ducts (I-K).

在一个病例中，受检的 13 个淋巴结有11 个为阳性，但其淋巴管内扩散的癌细胞的数量竟达到几百个或更多。图 5 示这个病例的一组 10 张非连续切片，经不同标记物染色处理。我们可见到，尽管在这些非连续切片中，淋巴管的数量和管腔大小不同，但在所有切片（切片横跨约 50 个以上连续切片的距离）粘膜和粘膜下层内同时扩张的淋巴管，似乎是相互联系的，或者是直接相通的（A-H）。具有扩散的癌细胞的某些扩张的淋巴管，可以穿过粘膜肌而到达粘膜下层深部（I-M）。虽然肿瘤组织内的细胞普遍缺乏 CK-18 表达，所有扩张淋巴管内的细胞是CK-19 强阳性。有意义的是，这些同时扩张的淋巴管，不仅见于邻近癌瘤组织的粘膜和粘膜下层，也见于远离部位的粘膜和粘膜下层（位于癌瘤部位对面的正常结肠壁内）（A-H；长方形区）。此外，有 2 个貌似正常， CK-阴性的上皮样结构，却隐藏有枝芽状的 CK-19 阳性细胞簇群，其位置邻近于扩张的淋巴管处（I-K）。

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Figure 5. Inter-connections of dilated lymphatic ducts among different tissue components

A set of 10 discontineous CRC tissue sections from a node positive case were immunostained for different

markers. Squares in A, C, E, G, I, K, M, and O identify low magnification views of the structures in B, D,

F, H, J, L, N, and P. Square and circle in Q identify low magnification views of structures in R and S,

respectively. Square and circle in T identify low magnification views of structures in U and V, respectively.

Black and green arrows dilated lymphatic ducts within the mucosa and submucosa, respectively. Stars in

R, S, U, and V identify MM. Curved lines identify dilated lymphatic ducts with disseminated cells crossing

the mucosa, MM, and the submucosa tissue components. Note that concurrently dilated lymphatic ducts

within the mucosa and submucosa are seen not only in normal appearing epithelial tissue adjacent to tumor,

but also in normal epithelial tissue at the opposite wall of the tumor site. A, C, E, G, I, K, M, O, Q, and T:

80X. B, D, F, H, J, L, N, P, R, S, U, and V: a higher (300X) of A, C, E, G, I, K, M, O, Q, and T,

respectively.

图 5. 不同组织成分中扩张淋巴管间的相互联系

淋巴结阳性结肠直肠癌病人的一套 10 张不连续切片，经不同标记免疫组化染色处理。A、C、E、G、I、K、M 和 O 内的长方形区，分别表示 B、D、F、H、J、L、N 和 P 内结构的低倍观。在 Q 内的长方形和圆圈，分别表示 R 和 S 内结构的低倍观。在 T 内的长方形和圆圈，分别表示 U 和 V 内结构的低倍观。黑色和绿色箭头分别表示粘膜和粘膜下层内的扩张淋巴管，R、S、U 和 V 内的星形物表示粘膜肌层，曲线表示横跨粘膜、粘膜肌和粘膜下层组织成分的扩张淋巴管及其内含的扩散癌细胞。注意：在粘膜和粘膜下层内扩张的淋巴管，不仅见于邻近癌瘤组织貌似正常的上皮组织内，而且也见于癌瘤组织对面的正常上皮组织壁上。A、C、E、G、I、K、M、O、Q 和 T：放大 80 倍，B、D、F、H、J、L、N、P、R、S、U 和 V:分别代表 A、C、E、G、I、K、M、O、Q 和 T 的高倍放大（300 倍）。

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All five node positive cases with concurrently dilated lymphatic ducts

containing the CK-19 positive disseminated cells within the mucosa (as those

shown in Figures 2, 4, and 5), whereas none of the 5-node negative cases

harbored similar ducts with disseminated cells (Table 2).

Table 2. Frequency of concurrently dilated lymphatic ducts with disseminated cells in node

positive and negative cases

表 2.在有无淋巴结转移病例中伴发的含有分散癌细胞的扩张淋巴管的频率

Node status 淋巴结状态

Number of cases 病例数量

Number of ducts with disseminated cells

含有分散癌细胞的淋巴管数量

P P 值

Positive (阳性) 5 45 <0.0001

Negative (阴性) 5 0

Discussion

In summary, our current study has detected the following unique alterations

that have not been previously reported: (1) the frequency of concurrently

dilated lymphatic ducts within the mucosa and submucosa distant from or

adjacent to the tumor is significantly higher (p<0.001) in node positive than

in node negative cases, (2) dilated lymphatic ducts with disseminated

tumor cells are exclusively present in the normal appearing mucosa of

node positive cases, (3) A vast majority of dilated lymphatic ducts

harbor no disseminated cells, but disseminated cells are consistently

detectable in some of the consecutive sections, and (4) in node positive

cases with a high number of positive nodes, concurrently dilated

lymphatic ducts within the mucosa and submucosa appear to be

interconnected or in direct physical continuity, and to present in the

entire primary tumor, including normal epithelial structures distant

from the in situ or invasive tissue component.

Together, these findings suggest that concurrently dilated

lymphatic ducts within the mucosa and submucosa represent an early

sign of tumor cell dissemination and node metastasis. Concurrently

dilated lymphatic ducts within the mucosa and submucosa are most

likely to be caused by disseminated tumor cells, which may either

stimulate proliferation of lymphatic endothelial cells or disrupt the

normal pathway of fluid homeostasis and dietary transport. Consistent

with our speculation are the facts that: (1) although most concurrently

dilated lymphatic ducts harbor no disseminated cells, disseminated cells

are consistently detectable in the consecutive sections, and (2) all node

positive cases with a high number of positive nodes harbor

concurrently dilated lymphatic ducts that appear to interconnected or in

direct physical continuity, and to present in the entire primary tumor.

Consequently, the presence of concurrently dilated lymphatic ducts

within the mucosa and submucosa is most likely to signify the presence

of disseminated tumor cells within the dilated ducts. In another words,

the presence or absence of concurrently dilated lymphatic ducts in a

single tissue section may accurately reflect the presence or absence of

所有 5 个淋巴结阳性病例的粘膜内全都有

同时扩张的淋巴管，内含 CK-19 阳性的扩散癌

细胞 （见图 2、4 和 5），而所有 5 个淋巴结

阴性病例的粘膜内， 全都缺乏 CK-19 阳性的

扩散癌细胞和同时扩张的淋巴管 （见表 2）。

讨 论 概括来看，本实验观察到下列独特、过去

未曾报道过的现象： （1）淋巴结阳性病例

中，其远离或邻近癌瘤组织的粘膜和粘膜下层

内，同时扩张淋巴管的频率，明显高于淋巴结

阴性病例（P 值<0.001）；(2) 在貌似正常的粘

膜组织内，仅淋巴结阳性病例含有扩散癌细胞

的扩张淋巴管；（3）绝大多数同时扩张的淋

巴管内不包含扩散的癌细胞，但是分散扩癌细

胞可以始终如一地见于某些连续切片中； 及

（4）在高数量淋巴结阳性病例中，其粘膜和

粘膜下层内同时扩张的淋巴管似乎是相互联系

的，或者是直接连通的，并且存在于整个原发

性癌瘤内，包括邻近或远离原位癌或侵袭癌的

正常上皮组织成分中。

综合起来，这些发现揭示，在粘膜和粘膜

下层内同时扩张的淋巴管可能是癌瘤细胞扩散

和淋巴结转移的一个早期征兆。在粘膜和粘膜

下层内伴发的淋巴管扩张，很可能是由于扩散

的癌细胞引致：侵入淋巴管的癌细胞刺激淋巴

管内皮细胞的增生，或者破坏了液体内环境稳

定和食物营养料转运的正常通道。与我们推测

相一致的事实有：（1）尽管大多数同时扩张

的淋巴管内未含有扩散的癌细胞，但是扩散的

癌细胞却始终如一地见于连续切片中；以及

（2）淋巴结阳性 病例含同时扩张淋巴管的频

率显著高于淋巴结阴性病例；而且, 同时扩张

的淋巴管似乎是相互联系的，或者是直接相通

的，并且存在于整个癌瘤组织内。因此，在粘

膜和粘膜下层内存在的同时扩张的淋巴管，很

可能表明扩张的淋巴管内存在扩散的癌细胞。

换言之，在单一组织切片中存在或缺乏同时扩

张的淋巴管，可能正确地反映了整个癌瘤组织

内存在或缺乏扩散的癌细胞。如此推测得以证

New Approaches combating Cancer & Aging 2014; Vol 1:55-71

http://naca.iudd.org/index.html Page69

disseminated cells within the entire tumor. If confirmed at a larger

number of cases, our findings would have significant clinical

applications. First, as such ducts with disseminated cells are easily

appreciable, examination of a given double immuno-stained sections

for D2-40 and Ck-19 (or CK AE1/AE3) may significantly facilitate the

early detection of CRC metastasis and differentiation between node

positive and negative CRC. Second, examination of the biopsy

samples of given CRC before the surgery may significantly benefit the

decision making of the operation extent.

Conflict of Interest Disclosure

The authors have declared that no competing interest exists.

Acknowledgements

This study was supported by the United States Military Cancer Institute

and Grant 2RO1CA090848–05A2 from the National Cancer Institute

and the California Oncology Research Institute (CORI), the Joyce E

and Ben B Eisenberg Foundation, the Hearst Foundation, the Davidow

Charitable Fund, the Rod Fasone Memorial Cancer Fund, Mrs. Ruth

Weil, the Sequoia Foundation for achievement in the arts and education,

and Mrs. Marguerite Perkins Mautner.

This study was also supported in part by grants 200601060,

201108004from science and technology bureau of Nanjing,

ZKX11004from Health bureau of Nanjing, LZ11105 from Jiangsu

province bureau of traditional Chinese medicine to Jiang Bin. This

study was also supported in part by grant 2008-02 from the US Military

Cancer Institute and Henry M. Jackson Foundation to Dr. Yan-gao Man.

The translation of this article from English to Chinese was made by Dr.

Shizhang Shang of Georgetown University Medical School,

Washington DC, USA, and typing was completed by Ms. Rui Gao of

Maryland, USA. Journal of Cancer greatly appreciates their assistance

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致 谢

本研究为美国军队癌症中心和全国癌症研究所

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