Relation Between CD8+T Lymphocyte Infiltration and Efficacy of Neoadjuvant Chemotherapy for Triple-negative Breast Cancer
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摘要:目的
探讨三阴性乳腺癌组织CD8+T淋巴细胞浸润（CD8+Tils）的特点与患者预后的关系。
方法回顾性分析术前行新辅助化疗的126例三阴性乳腺癌患者的临床病理资料，采用免疫组织化学法分析CD8+Tils与临床病理特征的关系；Kaplan-Meier法绘制生存曲线，Cox风险比例回归模型分析患者无病生存时间（DFS）的预后影响因素。
结果高密度CD8+Tils浸润与年龄 < 60岁、病理高分级、临床高分期显著相关（P < 0.05）。CD8+Tils高密度浸润患者术后pCR率较低密度组高（66.7% vs. 19.8%, P=0.000）。高密度组中位DFS显著长于CD8+Tils低密度组（49 vs. 25月, P < 0.05）。多因素分析显示病理高分级、肿瘤直径 > 2 cm、淋巴结转移、脉管侵犯、CD8+Tils低密度浸润均为预后不良影响因素（P < 0.05），CD8+Tils为独立预后因素。
结论CD8+Tils有可能是三阴性乳腺癌患者独立预后指标，高密度浸润患者术后pCR率高、DFS长、远期疗效更优。
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关键词:
- 三阴性乳腺癌 /
- CD8+T淋巴细胞浸润 /
- 新辅助化疗
Abstract:ObjectiveTo investigate the relation between the characteristics of CD8+T lymphocyte infiltration and the prognosis of triple-negative breast cancer patients.
MethodsWe retrospectively analyzed the clinicopathological data of 126 patients with triple-negative breast cancer undergoing preoperative neoadjuvant chemotherapy. Immunohistochemical staining was used to analyze the relation between CD8+T lymphocyte infiltration and clinicopathological characteristics. Kaplan-Meier method was used to draw the survival curve, and Cox risk ratio regression model was used to analyze the prognostic factors affecting disease-free survival time (DFS).
ResultsHigh-density CD8+Tils was associated with age < 60 years old, high pathological grade and high clinical stage (P < 0.05). The pCR rate of high-density CD8+Tils group was higher than that of the low-density group (66.7% vs. 19.8%, P=0.000). The median DFS of the high-density group was significantly longer than that of the low-density group (49 vs. 25 months, P < 0.05). Multivariate analysis showed that high pathological grade, tumor diameter > 2 cm, lymph node metastasis, vascular invasion and CD8+Tils low-density infiltration were factors for poor prognosis (P < 0.05), and CD8+Tils was an independent prognostic factor.
ConclusionCD8+Tils may be an independent prognostic indicator for triple-negative breast cancer. The patients with high-density infiltration have high postoperative pCR rate, long DFS and better long-term efficacy.
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0 引言
急性早幼粒细胞白血病(acute promyelocytic leukemia, APL)是以t(15;17)(q24;q21)或PML-RARα融合基因阳性为特征的特殊类型急性髓系白血病(acute myeloid leukemia, AML)。典型APL骨髓早幼粒细胞≥30%非红系有核细胞，常规表达CD13、CD33和CD117，不表达CD34和HLA-DR。由于严重的出凝血障碍，APL患者早期死亡率高达9%~30%[1]。全反式维甲酸(all trans retinoic acid, ATRA)和亚砷酸(arsenic trioxide, ATO)在APL中的联合应用，使其长期生存率提高至90%以上[2]。及时明确诊断、尽早应用ATRA有利于减少患者早期死亡。现报道1例骨髓形态早幼粒细胞无明显升高，以原始粒细胞为主，且强表达CD34并伴add(21)(q22)的复杂核型APL，经ATRA联合去甲氧柔红霉素和阿糖胞苷(IA)方案诱导治疗后获形态学及遗传学完全缓解，并结合相关文献分析其临床特征，为此类APL临床诊断和治疗提供参考。
1 临床资料
患者，男，17岁。因“活动后胸闷、气短1周伴间断鼻腔出血3次”于2022年4月11日入院。查体：全身皮肤黏膜苍白，左上肢可见一处瘀斑(2 cm×2 cm)，不高出皮肤，压之不褪色，全身浅表淋巴结未触及明显肿大，胸骨无压痛，肝脾肋下未触及。血常规：WBC 0.57×109/L，RBC 2.60×1012/L，HGB 92 g/L，PLT 38×109/L，N 0.1×109/L。凝血指标：纤维蛋白原1.87 g/L，D-二聚体0.82 mg/L，纤维蛋白降解产物14.5 μg/ml，抗凝血酶Ⅲ 122.9%。骨髓细胞学：原始粒细胞61%，早幼粒细胞0.5%，明显核浆发育紊乱，胞质量中等，呈天蓝色，含少许嗜天青颗粒，偶见Auer小体，胞核多不规则，可见凹陷，折迭、肾形等，核质呈微细颗粒状，分布均匀，见图 1A。组织化学染色：过氧化物酶阳性。提示AML-M2骨髓象。免疫分型：CD34、CD117、cMPO、CD13、CD33阳性；HLA-DR、CD16、CD64、CD56、CD20、CD11b、cCD79a阴性，见图 1B。考虑AML-M2。染色体核型：46，XY，t(15;17)(q24;q21)，add(17)(p13)，add(21)(q22)[19]/46，XY[1])，见图 1C。融合基因：PML-RARα(bcr1)阳性，96.223%表达。荧光原位杂交(FISH)：51%的细胞为PML-RARα阳性细胞，见图 1D。髓系白血病相关基因：WT1 83.352%表达，GATA2基因突变频率6.58%，其余常见AML基因突变检测结果均为阴性。
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图 1 患者临床资料Figure 1 Clinical data of patientA: morphology of bone marrow cells on admission (Wright's staining×100); B: immunophenotyping showed that CD34 was highly expressed (black arrow); C: patient's chromosome karyotype: 46, XY, t(15; 17)(q24; q21), add(17)(p13), add(21)(q22)[19]/46, XY[1](black arrow); D: GLP PML-RARα probe revealed that 51% of the cells showed two yellow, one red, and one green signals (white arrow); E: 100% cells showed PML-RARα negative cells after ATRA combined with IA regimen (white arrow); F: chromosomes returned to normal karyotype.综合上述结果明确诊断为急性早幼粒细胞白血病(中危组)。随即给予ATRA+IA方案诱导治疗。2022年5月11日骨髓细胞学示治疗后完全缓解，染色体核型及融合基因转阴，白血病微小残留(minimal residual disease, MRD)检测阴性。同时予以腰椎穿刺+鞘内注射预防中枢神经系统白血病，脑脊液常规生化检查指标均正常。5月13日继续原方案巩固治疗。6月14日骨髓细胞学：完全缓解骨髓象；FISH显示PML-RARα(bcr1)融合基因阴性，见图 1E；染色体核型：46，XY[20]，见图 1F；WT1 0.071%；GATA2-L3751基因突变比例 < 0.001%；MRD检测阴性。
随后规律服用ATRA及复方黄黛片维持治疗，8月9日、10月11日复查示遗传学及分子生物学为持续缓解状态：染色体核型正常，PML-RARα(bcr1)无表达，MRD未见白血病残留细胞。后续依据《中国急性早幼粒细胞白血病诊疗指南》[3]继续规范治疗。治疗过程及结果见图 2。
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图 2 患者治疗过程与细胞表型改变Figure 2 Treatment process and phenotypic changesATRA: all-trans retinoic acid; ATO: arsenic trioxide; IDA: idarubicin; Ara-C: cytarabine; FCM: flow cytometry; FISH: fluorescence in stitu hybridization; M: myeloblast; P: promyelocytic; CR: complete remission.2 讨论
APL患者PML-RARα融合基因转录产生的PML-RARα融合蛋白，通过募集并结合共抑制因子、组蛋白去乙酰化酶和DNA甲基化酶阻碍骨髓细胞分化基因的表达，以显性负向作用抑制髓系细胞分化[4]。ATRA及ATO通过参与分化相关转录基因激活、诱导细胞自噬及抑制和降解Pin1等，促进未成熟早幼粒细胞成熟和分化、诱导肿瘤细胞凋亡[2]，在APL治疗中显示出明显临床疗效。然而APL细胞组织因子和癌症促凝剂等促凝血因子的异常表达可通过激活凝血因子Ⅹ触发凝血级联反应，同时近期研究表明，APL患者组织型纤溶酶原复合物及其AnnexinA2受体表达增加，进而引起纤溶亢进。凝血及纤溶异常共同导致严重出凝血障碍，使相当一部分APL患者诱导治疗前死亡[5]。因此，及时明确诊断、尽早ATRA治疗至关重要。
细胞遗传学和基因检测作为APL诊断金标准具有明显时间滞后性，形态学及免疫表型可实现快速筛查。经典APL患者骨髓早幼粒细胞异常增多[6]，本例患者早幼粒细胞仅0.5%，原始粒细胞占61%，导致APL初步诊断存在困难。先前研究显示变异型APL新的融合基因可能导致细胞形态发生改变，如细颗粒型APL形态学有时可类似AML-M2或AML-M5[7]，但其形态的不典型性在PML-RARα(bcr1)APL中尚未见报道。CD34为未成熟细胞标志，超过90%APL细胞呈阴性[7]。Ceni等研究表明CD34仅在1/24 APL样本中表达，多参数流式细胞术分析显示，利用CD34、HLA-DR联合SSC平均荧光强度区分APL和其他类型AML准确性约91.2%[8]。Tran等利用CD117、CD13、CD56、CD64、MPO构建的APL筛查模型敏感度约91.7%，特异性约93.1%[9]。流式细胞术提供了快速简便的APL初步诊断手段。然而，Verma等曾报道一例CD34及HLA-DR呈阳性而被误诊为AML(MPO阳性)的APL[10]。APL快速筛查中应警惕非典型形态学表现及异常免疫表型的存在。本例患者骨髓原始粒细胞61%，早幼粒细胞仅0.5%，免疫分型约22.6%有核细胞CD34阳性。根据骨髓细胞形态学及免疫分型极易与AML-M2混淆，染色体分析示t(15;17)(q24;q21)并伴add(17)(p13)及add(21)(q22)，PML-RARα融合基因阳性，明确诊断为APL。
位于21q22染色体的AML1/RUNX1基因参与调控造血干细胞向成熟血细胞分化及p53和转化生长因子-β信号通路相关靶基因的表达[11]，常与AML-M2发生相关。Ishii等报道过1例染色体核型为46，XY，add(21)(q22)的M2型AML，其原始粒细胞59.6%，早幼粒细胞18.4%，可见Auer小体，高表达CD34、CD13、CD33、HLA-DR[12]。21q22异常是否与类似AML-M2的细胞形态及免疫表型相关暂未明确。除此之外，add(21)(q22)也被报道伴发于急性淋巴细胞白血病[13]、B细胞淋巴瘤[14]等。本病例形态学及免疫表型的不典型性是否可考虑与伴发add(21)(q22)有关，还有待进一步明确。该病例反映了APL骨髓细胞形态学及免疫表型的复杂性，单一或快速检查手段无法及时做出准确诊断，需进一步综合染色体及FISH等各方面信息以明确诊断。
Competing interests: The authors declare that they have no competing interests.作者贡献：孔天东：论文撰写陈露：文献搜集及试验数据整理统计段方方、王留晏、周寒丽：病例资料筛选及随访登记赵晓丽：部分随访登记刘萌萌：实验实施刘丹娜：数据统计分析及论文审校 -
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图 1 高低密度CD8+Tils组pCR率对比
Figure 1 Comparison of pCR rates between high- and low- density CD8+Tils groups
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图 2 三阴性乳腺癌组织高低密度组CD8+Tils的表达
Figure 2 Expression of CD8+Tils in high- and low- density groups of triple negative breast cancer tissue
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图 3 CD8+Tils浸润密度对三阴性乳腺癌患者DFS的影响
Figure 3 Effects of CD8+TiLs infiltration densities on DFS of TNBC patients
表 1 CD8+淋巴细胞高密度浸润与入组患者临床病理资料关系(n(%))
Table 1 Relation between high-density infiltration of CD8+ lymphocytes and clinicopathological data of involved patients(n(%))
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表 2 不同临床病理特征生存风险的单因素及多因素分析
Table 2 Univariate and multivariate analyses of survival hazards for different clinicopathological features
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