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2020-1-14 17:51| 发布者: admin| 查看: 280| 评论: 0

摘要: 【肾上腺皮质肿瘤的组织化学诊断】The use of histochemistry in benign ACTs is limited. In the setting of primary aldosteronism, Luxol fast blue (see Fig. 8) can be used to identify spironolactone bodies ...


The use of histochemistry in benign ACTs is limited. In the setting of primary aldosteronism, Luxol fast blue (see Fig. 8) can be used to identify spironolactone bodies. Histochemistry has gained popularity with the introduction of the reticulin algorithm because the algorithm is now regarded as a simple and reproducible method that can be used to separate ACAs from ACCs (see Figs. 19 and 20); however, clinicians should recognize pitfalls and pearls in the interpretation of reticulin findings. Although validation of the reticulin algorithm in large series of pediatric ACCs is currently lacking, this algorithmic approach has been shown to aid in making the diagnosis of conventional as well as oncocytic and myxoid variants. The backbone of the reticulin algorithm is the reticulin histochemical stain (most commonly Gordon-Sweet-Silver). Assessment of this staining in ACTs evaluates both quantitative (loss of continuity in reticulin fibers) and qualitative (abnormal reticulin network with irregular thickness of fibers, frayed appearance, or pericellular pattern leading to a meshlike appearance) changes. Qualitative alterations in some ACTs may be the source of underestimation of malignancy because clinicians may encounter ACCs rich in areas of meshlike pericellular reticulin staining that can be mistaken for an unaltered reticulin framework. By being aware of this potential pitfall, additional careful examination of the reticulin histochemistry can solve this quandary because ACCs tend to show variable degrees of reticulin disruption even in the presence of qualitative changes. Although the quantitative alterations are most helpful, ACAs with areas of degeneration (eg, hemorrhage, extensive postbiopsy changes) can also show variable disruption of the reticulin framework. Furthermore, examples of very focal loss of reticulin staining and foci of incomplete or complete pericellular pattern have also been described in some ACAs. Therefore, altered reticulin framework should be taken into consideration along with other histologic findings as well as biomarker profiling when evaluating an ACT.

组织化学在良性病变中的应用是有限的。在原发性醛固酮增多症的背景下,Luxol快速蓝(见图8)可用于识别螺内酯体。随着reticulin算法的引入,组织化学变得越来越流行,因为该算法现在被认为是一种简单且可重复的方法,可用于区别ACAs和ACCs(见图19和20)。然而,临床医生在解释网织纤维结果时应该认识到陷阱和提示。尽管目前还缺乏在大量儿童ACCs中对reticulin算法的验证,但这种算法方法已被证明有助于诊断经典型/普通型以及嗜酸细胞型和黏液样亚型。reticulin算法的核心是网织纤维组织化学染色(最常见的Gordon-Sweet -Silver法)。在ACTs中对这种染色的评估同时进行了定量(网织纤维的连续性丧失)和定性(纤维厚度不规则、磨损外观或导致网状外观的细胞周围模式的异常网织纤维网)改变。一些ACTs的定性改变可能是低估恶性肿瘤的原因,因为临床医生可能会遇到富含网状细胞周围网状纤维染色区域的ACCs,这些区域可能被误认为是未改变的网织纤维框架。通过意识到这一潜在的陷阱,对网织纤维组织化学的额外仔细检查可以解决这一难题,因为即使在有质变的情况下,ACCs也倾向于显示不同程度的网织纤维破坏。虽然数量上的改变是最有帮助的,伴变性区域的ACAs(如出血,广泛的活检后改变)也可以显示不同程度的网织纤维框架破坏。此外,在一些ACAs中还描述了网织纤维染色的非常局灶性丢失和不完全或完全细胞周围模式的局灶性。因此,在评估一个ACT时,网织纤维框架的改变应结合其他组织学表现及生物标记物来综合评价。


Immunohistochemistry has become an integral component of the diagnostic work-up of ACTs. Biomarkers are routinely used to address various clinical needs, including confirmation of the adrenocortical origin, distinction of functional tumors, supporting the diagnosis of malignancy, providing prognostic and theranostic information in ACCs, and facilitating the screening process for germline pathogenesis. Relevant immunohistochemical biomarkers are summarized in Box 1.



确认肾上腺皮质起源的生物标志物:类固醇生成因子-1(SF-1,最特异), Melan-A, calretinin, alpha-inhibin, syn;






Several locoregional (eg, renal cell carcinoma, pheochromocytoma, epithelioid perivascular epithelioid cell neoplasms, sarcoma) and metastatic neoplasms (eg, melanoma, hepatocellular carcinoma) can simulate ACTs. Therefore, the confirmation of cortical origin should be considered in all ACTs, especially in the absence of adrenocortical hormone excess. Failure to confirm adrenocortical origin resulting in misdiagnoses has clearly been shown as being one of the major lessons from consultations practices. To confirm adrenocortical origin, steroidogenic factor-1 (SF-1) (Fig. 21), a transcription factor characteristic of steroidogenic tissues, is the most specific diagnostic biomarker. Negativity for SF-1 in sarcomatoid components of ACCs as well as in ACTs with suboptimal tissue fixation are important pitfalls in the interpretation of SF-1 staining.

一些局部区域性肿瘤(如肾细胞癌、嗜铬细胞瘤、上皮样血管周上皮样细胞肿瘤、肉瘤)和转移性肿瘤(如黑色素瘤、肝细胞癌)可以类似于ACT。因此,在所有考虑的 ACTs中,尤其是在没有肾上腺皮质激素过量的情况下,应确认是皮质来源。未能确认肾上腺皮质起源导致误诊已是实践中一个主要的教训。为了确认肾上腺皮质起源,类固醇生成因子-1(SF-1)(图21)是一种具有类固醇生成组织特征的转录因子,是最特异的诊断性生物标记物。在ACCs的肉瘤样成分中SF-1阴性,以及组织固定不良的ACTs是解释SF-1染色的重要陷阱。

Fig. 21. Confirmation of adrenocortical origin. Several biomarkers have been proposed to confirm adrenal cortical origin, including synaptophysin, alpha-inhibin, Melan-A, and calretinin. However, among these, SF-1 stands out as the best biomarker to confirm cortical origin. Shown is diffuse nuclear SF-1 staining in an adrenocortical carcinoma. The left side of the composite photomicrograph illustrates SF-1 positivity in an adrenocortical carcinoma (low magnification). The right side of this composite photomicrograph represents high magnification of SF-1 expression in the same tumor.

图21 肾上腺皮质起源的确认:一些生物标记物已经被提出来证实肾上腺皮质的起源,包括syn,alpha-inhibin,Melan-A和calretinin。然而,其中,SF-1是确认皮质起源的最佳生物标记物。图示肾上腺皮质癌弥漫性SF-1核染色。左侧显示肾上腺皮质癌(低倍镜)SF-1阳性,右侧为高倍镜下同一肿瘤中SF-1的表达。

Once adrenocortical origin has been established, several biomarkers can be used to aid in distinguishing ACA from ACC, especially with challenging surgical specimens and particularly in the evaluation of core biopsy material. Mete and colleagues reported that juxtanuclear insulinlike growth factor 2 (IGF-2) (Fig. 22) staining optimized at 1:3000 to 1:6000 dilutions was the most useful diagnostic biomarker of adult ACCs because this pattern of staining was absent in ACAs. The juxtanuclear Golgi pattern is thought to reflect impairment in translation and processing of the IGF-2 molecule in the Golgi apparatus, which results in IGF-2 overexpression.


Fig. 22. IGF-2 immunohistochemistry. Juxtanuclear IGF-2 immunostaining is a feature of adrenocortical carcinoma. Shown are conventional (upper) and oncocytic (lower) adrenocortical carcinoma with juxtanuclear IGF-2 staining.

图22 IGF-2免疫组化:IGF-2免疫染色是肾上腺皮质癌的一个特征。经典型/普通型(上)和嗜酸细胞型(下)肾上腺皮质癌中靠近细胞核IGF-2染色。

Abnormal p53 immunoexpression is a wellknown marker of malignancy in several organs. Although pediatric ACCs tend to show more frequent TP53 alterations, overexpression (more frequent) (Fig. 23) or global loss as a result of TP53 gene mutation has been seen in about 20% to 25% of adult ACCs. Aberrant expression has been associated with high-grade proliferative features and tumor aggressiveness.


Fig. 23. p53 immunohistochemistry. Shown is p53 overexpression in an adrenocortical carcinoma.

图23 p53免疫组织化学:图示p53在肾上腺皮质癌中的过表达。

Diffuse nuclear and cytoplasmic beta-catenin expression (Fig. 24) reflecting the activation of the Wnt pathway can be a feature of ACCs. This finding alone should not warrant a diagnosis of malignancy because ACAs can also harbor CTNNB1 mutations and show activation of the Wnt pathway. In addition, patients with familial adenomatous polyposis (FAP) can also manifest with ACTs, including ACAs. In contrast, there is a general consensus that ACCs with diffuse cytoplasmic and nuclear beta-catenin reactivity are more frequently associated with a poor prognosis.


As proliferation-driven neoplasms, an accurate assessment of cell proliferation biomarkers in ACTs is a crucial clinical task for pathologists assessing these tumors. Several biomarkers have been used to do this, such as Ki67, phosphohistone-H3 (PHH3), p53, BUB1B, HURP, and NEK2. PHH3 (Fig. 25) can be considered to distinguish mitotic figures from apoptotic or cells with crush artifact; thus, it can assist mitotic count to enable accurate mitotic tumor grading. 


Fig. 24. Beta-catenin immunohistochemistry. Beta-catenin reactivity can be variable in adrenocortical neoplasms. Not all adrenocortical carcinomas manifest with nuclear and cytoplasmic beta-catenin expression; some adrenocortical carcinomas display variable nuclear staining (left). Diffuse nuclear and cytoplasmic beta-catenin expression in adrenocortical carcinoma (right). Diffuse nuclear and cytoplasmic beta-catenin reactivity alone does not warrant a diagnosis of malignancy; however, adrenocortical carcinomas with diffuse cytoplasmic and nuclear beta -catenin reactivity are more frequently associated with a poor prognosis.

图24 β-catenin免疫组化:在肾上腺皮质肿瘤中β-catenin反应不一。并非所有的肾上腺皮质癌都有核和胞质β-catenin表达;一些肾上腺皮质癌显示多少不等核染色(左)。肾上腺皮质癌(右)弥漫性核和胞质β-catenin表达。仅弥漫性核和胞质β-catenin表达不能保证可以诊断为恶性肿瘤;然而,具有弥漫性细胞质和核β-catenin表达的肾上腺皮质癌更常与预后不良相关。

Fig. 25. PHH3 immunohistochemistry. PHH3 can be used to facilitat counting mitotic figures, because it also allows distinction of mitotic figures from apoptotic bodies and cells with crush artifact. Shown is an atypical mitotic figure using PHH3.

图25 PHH3免疫组化:PHH3可用于核分裂象的计数,因为它可区分核分裂象与凋亡小体和有挤压伪影的细胞。图中可见一个核分裂象表达PHH3。

Ki67 is one of the most important biomarkers and is readily available in almost all pathology laboratories. Most ACCs show a Ki67 proliferation exceeding 5% (Fig. 26). The MIB1 antibody (anti–human Ki67 monoclonal antibody) is considered the gold standard for this assessment. The management of patients with ACCs requires the knowledge of an accurate Ki67 labeling index along with other tumor characteristics. Several pitfalls and pearls exist in the assessment of the Ki67 proliferation index. Because ACCs are well known to display intratumoral proliferative heterogeneity (see Fig. 26), the first step in obtaining an accurate and meaningful Ki67 labeling index is the selection of the right tumor block based on high mitotic density seen in hematoxylin-eosin–stained sections. Tumor blocks with poor tissue fixation can result in impaired detection of nuclear antigen. If there is any concern regarding tissue fixation, multiple blocks should be assessed. Although the antibody and staining methods used can vary from one laboratory to another, visual assessment is no longer an acceptable option for the analysis of the Ki67 in ACCs. Manual counting or automated image analysis nuclear algorithms from hot spots of nuclear labeling (preferably 1000-2000 tumor cells) should be assessed in ACCs (Fig. 27). Some studies have classified ACCs into 3 groups: low-risk (grade 1), intermediate-risk (grade 2), and high-risk (grade 3) categories based on the Ki-67 index with different cutoffs (<10%, 10%–19%, ≥20%; or <20%, 20%–50%, >50%). Overall, Ki67 labeling index is considered an important factor in prognosticating ACCs as well as in determining the need for adjuvant therapies. A recent French series of pediatric ACCs also highlighted the impact of Ki67 because pediatric ACCs with poor prognosis had 2 of the following parameters: Ki67 greater than 15%, mitotic activity greater than 15 per 20 high-power fields, vascular invasion, tumor necrosis, and adrenal capsule invasion.

Ki67是最重要的生物标志物之一,在几乎所有的病理实验室中都很容易获得。大多数ACCs显示Ki67增殖超过5%(图26)。MIB1抗体(抗人Ki67单克隆抗体)被认为是该评估的金标准。ACCs患者的治疗需要了解准确的Ki67标记指数和其他肿瘤特征。在Ki67增殖指数的评估中存在一些陷阱和提示。众所周知,因为ACCs存在肿瘤内增殖的异质性(见图26),获得准确和有意义的Ki67标记指数的第一步是根据苏HE染色切片中的高核分裂象密度选择合适的肿瘤块。组织固定不良的肿瘤块可导致核抗原检测受损。如果对组织固定有任何顾虑,应评估多个蜡块。尽管使用的抗体和染色方法因实验室而异,但视觉评估不再是分析ACCs中Ki67的可接受选择。人工计数或自动图像分析核标记热点(最好是1000~2000个肿瘤细胞)的核算法应在ACCs中进行评估(图27)。一些研究将ACCs分为3类:低风险(1级)、中风险(2级)和高风险(3级),分别基于Ki-67指数和不同的临界值(<10%、10%~19%、 ≥20%;或<20%、20%~50%、>50%)。总的来说,Ki67标记指数被认为是预测ACCs以及确定是否需要辅助治疗的重要因素。法国最近的一系列儿童ACCs也强调了Ki67的影响,因为预后不良的儿童ACCs有以下两个参数:Ki67大于15%,核分裂象>15个/20HPF,血管侵犯,肿瘤坏死,肾上腺包膜侵犯。

Fig. 26. Ki67 immunohistochemistry in adrenocortical neoplasms. Adrenocortical carcinomas are proliferation driven neoplasms; most adrenocortical carcinomas have a Ki67 labeling index that exceeds 5%; however, these tumors often show in tratumoral proliferative heterogeneity. Shown is Ki67 staining pattern in an adrenocortical carcinoma.

图26 肾上腺皮质肿瘤的Ki67免疫组化研究:肾上腺皮质癌是增殖驱动的肿瘤;大多数肾上腺皮质癌的Ki67增殖指数超过5%;然而,这些肿瘤往往表现为肿瘤间增殖异质性。图示肾上腺皮质癌的Ki67染色模式。

Fig. 27. Assessment of Ki67 immunohistochemistry. Accurate assessment of the Ki67 labeling index is of clinical significance because this information has prognostic and therapeutic implications. Visual assessment is no longer an acceptable option for the analysis of the Ki67 in adrenocortical carcinomas. Manual counting or automated image analysis nuclear algorithms from hot spots of nuclear labeling should be considered. Shown is the assessment of Ki67 using an automated image analysis nuclea algorithm.

图27 Ki67免疫组织化学评价:准确评估Ki67标记指数具有临床意义,因为该信息具有预后和治疗意义。视觉评估不再是肾上腺皮质癌中Ki67分析的可接受选择。人工计数或自动图像分析应考虑核标记热点的核算法。所示为使用自动图像分析核算法对Ki67的评估。



Fig. 28. Integrated practical approach to adult adrenocortical tumors with no extra-adrenal spread. Following careful macroscopic examination and an initial microscopic assessment that includes confirmation of adrenocortical origin of the lesion in question, evaluation of the reticulin framework is a critical step in the diagnostic approach to adrenocortical tumors. Suspicious features in the setting of a preserved reticulin framework include mitotically active tumors with fewer than 5 mitoses per 50 HPFs, presence of necrosis, adrenocortical tumors with suspicious but not unequivocal vascular invasion, and/or other invasive growth features. In these situations, IGF-2 immunohistochemistry is often useful. The additional immunohistochemical application of Ki67, p53, and beta-catenin as well as molecular testing can also be helpful in further classifying the challenging neoplasms. In addition to serving a diagnostic purpose, the use of Ki67, p53, and beta-catenin also has a prognostic role in the evaluation of adrenocortical carcinomas.

图28 成人肾上腺皮质肿瘤无肾上腺外扩散的综合实用方法:经过仔细的大体检查和初步的组织学评估,包括确定病变为肾上腺皮质起源,评估网织纤维框架是肾上腺皮质肿瘤诊断方法的关键步骤。保留网织纤维框架背景下的可疑特征包括少于5个核分裂象/50HPF、坏死,肾上腺皮质肿瘤伴可疑但不明确的血管侵犯和/或其他侵袭性生长特征。在这些情况下,IGF-2免疫组化通常是有用的。Ki67、p53和β-catenin的免疫组化应用以及分子检测也有助于进一步区分具有挑战性的肿瘤。除了用于诊断目的外,Ki67、p53和β-catenin在肾上腺皮质癌的预后评估中也有一定的作用。





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