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摘要：
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摘要：目的 探讨肝细胞癌（hepatocellular carcinoma，HCC）相关三级淋巴样结构（tertiary lymphoid
structure，TLS）的免疫细胞组成。方法 以2016年4月至2019年10月北京大学第一医院收治的112例原
发性HCC患者为研究对象，收集其手术切除及HCC穿刺活检样本。对所有患者的肿瘤标本进行苏木
精-伊红（hematoxylin-eosin，HE）染色。采用免疫组织化学法对TLS阳性的标本进行CD3、CD20、
PD1、CD45RO、CD21和CD68染色。采用IPP（Image Pro Plus）软件计算TLS中阳性细胞占TLS的面
积百分比，比较其在不同HCC分期患者及不同部位（瘤内、瘤旁）的差异。结果 112例HCC患者中
共21例患者标本TLS阳性。TLS阳性组和TLS阴性组中早期HCC患者（CNLCⅠ～Ⅱ期）比例分别为
76.2%（16/21）和49.5%（45/91），差异有统计学意义（χ
2
= 4.919，P = 0.027）。TLS阳性早期HCC
患者，瘤内TLS的CD3+ [（69.19 ± 16.43）% vs（53.25 ± 14.48）%]、CD20+
[（63.63 ± 17.72）%
vs（44.44 ± 16.63）%]和CD21+
[（68.48 ± 12.78）% vs（11.75 ± 7.20）%]比例显著高于瘤旁TLS，而
PD1+
比例[（6.94 ± 3.64）% vs（20.56 ± 8.47）%]显著低于瘤旁，差异有统计学意义（P均＜ 0.05），
CD45RO+
[（44.56 ± 19.67）% vs（41.00 ± 16.07）%]和CD68+
[（10.25 ± 6.13）% vs（8.13 ±
5.78）%]比例差异无统计学意义（P均＞ 0.05）。16例早期HCC患者（CNLCⅠ～Ⅱ期）中，与自
身瘤旁TLS相比，瘤内TLS CD3+
、CD20+
和CD21+
比例升高例数分别为11例、15例和16例，PD1+
比
例下降14例，CD45RO+
（升高8例，下降8例）和CD68+
比例（升高7例，下降9例）升高与下降例数
相似。TLS阳性早期HCC患者瘤内TLS的CD3+
[（69.19 ± 16.43）% vs（35.60 ± 15.21）%]、CD20+
[（63.63 ± 17.72）% vs（32.80 ± 13.85）%]和CD21+
[（68.48 ± 12.78）% vs（10.00 ± 6.71）%]比例
显著高于晚期HCC患者（P均＜ 0.05），PD1+
[（6.94 ± 3.64）% vs（9.60 ± 3.78）%]、CD45RO+
[（44.56 ± 19.67）% vs（48.20 ± 13.94）%]和CD68+
[（10.25 ± 6.13）% vs（7.40 ± 3.65）%]比例差
异无统计学意义（P均＞ 0.05）。TLS阳性早期HCC患者瘤旁TLS的CD3+
[（53.25 ± 14.48）% vs
（19.80 ± 5.07）%]和CD20+
[（44.44 ± 14.63）% vs（21.60 ± 5.46）%]比例显著高于晚期HCC患者（P
均＜ 0.05），CD21+
[（11.75 ± 7.20）% vs（11.80 ± 3.90）%]、PD1+
[（20.56 ± 8.47）% vs（22.00 ±
9.49）%]、CD45RO+
[（41.00 ± 16.07）% vs（38.60 ± 9.99）%]和CD68+
[（8.13 ± 5.78）% vs（6.80 ±
4.15）%]比例差异无统计学意义（P均＞ 0.05）。结论 早期HCC患者瘤内T细胞、B细胞和树突状细胞
比例高于瘤旁TLS，也高于晚期HCC患者瘤内TLS，且其中耗竭型淋巴细胞减少，记忆型淋巴细胞和
巨噬细胞比例无变化。提示早期HCC患者瘤内TLS含有更多的活化淋巴细胞和树突状细胞，可能是其
发挥抗肿瘤免疫应答作用、延缓疾病进展和延长患者生存期的原因。
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Abstract: Objective To investigate the composition of tertiary lymphoid structure (TLS) of hepatocellular
carcinoma (HCC). Methods A total of 112 patients with primary HCC admitted to Peking University
First Hospital from April 2016 to October 2019 were selected as subjects. Surgical resection and liver
cancer biopsy samples were collected. Tumor specimens of all patients were stained by hematoxylin-eosin
(HE). The TLS positive specimens were stained with CD3, CD20, PD1, CD45RO, CD21 and CD68 by
immunohistochemistry. IPP (Image Pro Plus) software was used to calculate the area percentage of positive
cells in TLS, and the differences among patients with different HCC stages and different sites (intratumoral
and paratumoral) were compared. Results There were 21 patients out of 112 HCC patients whose TLS
were positive. The ratio of patients with early stage HCC (CNLCⅠ～Ⅱ stage) in TLS positive group and
TLS negative group were 76.2% (16/21) and 49.5% (45/91), respectively, the difference was statistically
significant (χ
2
= 4.919, P = 0.027). For HCC patients in early stage with TLS positive, the percentages of CD3+
[(69.19 ± 16.43)% vs (53.25 ± 14.48)%], CD20+
[(63.63 ± 17.72)% vs (44.44 ± 16.63)%] and CD21+
[(68.48 ±
12.78)% vs (11.75 ± 7.20)%] of intratumoral TLS were significantly higher than those of paratumoral TLS,
the proportion of PD1+
[(6.94 ± 3.64)% vs (20.56 ± 8.47)%] was lower than that of paratumoral TLS, the
differences were statistically significant (all P ＜ 0.05). There were no statistically significant differences in
the percentages of CD45RO+
[(44.56 ± 19.67)% vs (41.00 ± 16.07)%] and CD68+
[(10.25 ± 6.13)% vs (8.13 ±
5.78)%] (all P ＞ 0.05). For the 16 patients with early HCC (CNLCⅠ～ II), compared with paraneoplastic
TLS of themselves, the number of patients whose percentages of CD3+
, CD20+
and CD21+
of intratumoral
TLS increased were 11 cases, 15 cases and 16 cases, respectively, the number of patients whose CD45RO+
(increased: 8 cases, decreased: 8 cases) and CD68+
(increased: 7 cases, decreased: 9 cases) ratio increased is
similar to those CD45RO+
and CD68+
ratio decreased. The percentages of CD3+
[(69.19 ± 16.43)% vs (35.60 ±
15.21)%], CD20+
[(63.63 ± 17.72)% vs (32.80 ± 13.85)%] and CD21+
[(68.48 ± 12.78)% vs (10.00 ± 6.71)%]
of early stage HCC patients with TLS positive were significantly higher than those of advanced HCC patients
with TLS positive (all P ＜ 0.05). There were no significant differences in the percentages of PD1+
[(6.94 ±
3.64)% vs (9.60 ± 3.78)%], CD45RO+
[(44.56 ± 19.67)% vs (48.20 ± 13.94)%] and CD68+
[(10.25 ± 6.13) % vs
(7.40 ± 3.65)%] (all P ＞ 0.05). The percentages of CD3+
[(53.25 ± 14.48)% vs (19.80 ± 5.07)%] and CD20+
[(44.44 ± 14.63)% vs (21.60 ± 5.46)%] in paratumoral TLS of HCC patients in early stage with TLS positive
were significantly higher than those of advanced HCC patients with TLS positive (all P ＜ 0.05). There were
no significant differences in the percentages of CD21+
[(11.75 ± 7.20)% vs (11.80 ± 3.90)%], PD1+
[(20.56 ±
8.47)% vs (22.00 ± 9.49)%], CD45RO+
[(41.00 ± 16.07)% vs (38.60 ± 9.99)%] and CD68+
[(8.13 ± 5.78)% vs
(6.80 ± 4.15)%] (all P ＞ 0.05). Conclusions The proportion of T cells, B cells and dendritic cells of intratumoral
TLS of patients with early stage HCC were higher than those of paratumoral TLS, and also higher than those of
intratumoral TLS of patients with advanced HCC, and the proportion of depletion lymphocytes decreased, while
the proportion of memory lymphocytes and macrophages didn’t change. It is suggested that TLS in HCC patients
in early stage contains more activated lymphocytes and dendritic cells, which may be the reason for its anti-tumor
immune response, delaying the progression of the disease and prolonging the survival of patients.
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