Brasanac D, Boricic I, Todorovic V, Tomanovic N, Radojevic S
Institute of Pathology, School of Medicine, University of Belgrade, Dr Subotica 1/II, 11000 Belgrade, Serbia and Montenegro.
Br J Dermatol. 2005 Dec;153(6):1166-75. doi: 10.1111/j.1365-2133.2005.06898.x.
Actinic keratosis (AK) has been defined as a precancerous lesion or an early phase in the evolution of squamous cell carcinoma (SCC) and histological changes seen in the individual cells of an AK are indistinguishable from those seen in SCC, which invade the dermis. Cyclin A is an increasingly utilized proliferation marker that has functions in both S phase (DNA replication) and initiation of mitosis, whereas alterations of beta-catenin, the molecule involved in cell-cell adhesion and in signalling transduction, could promote invasive and proliferative capacities of malignant tumours.
To determine cyclin A and beta-catenin expression pattern in cutaneous SCC and in in situ lesions classified as keratinocytic intraepidermal neoplasia (KIN) and, using traditional terms, as AK and Bowen's disease (BD), and to analyse it in relation to SCC differentiation, diameter and thickness.
Immunohistochemical staining was performed on 110 formalin-fixed paraffin-embedded tissue samples with the streptavidin-biotin technique using antibodies to cyclin A and beta-catenin. On histological examination, 53 lesions were diagnosed as AK, 16 as BD and 41 as SCC-11 well differentiated (WD), 16 moderately differentiated (MD) and 14 poorly differentiated (PD). Using KIN classification, 22 lesions were KIN1, 23 were KIN2 and 24 were KIN3. For cyclin A, distribution and labelling index (LI), and for beta-catenin, level of membranous staining and presence of aberrant (nuclear/cytoplasmic) localization were examined.
Diffuse cyclin A presence was observed more frequently in BD than in AK (P < 0.0001) or SCC (P = 0.0002), and in SCC-PD compared with SCC-WD (P < 0.0001) or SCC-MD (P = 0.0003). Differences between KIN3 and KIN2, as well as KIN3 and KIN1 lesions, were statistically significant (P < 0.0001), and the same result appeared when KIN1 and KIN2 cases were grouped and compared with those of KIN3 (P < 0.0001). Cyclin A LI was significantly lower (P < 0.05) in AK than in BD or SCC, but no difference between BD and SCC was found, and LI in BD was even higher than in SCC-WD or SCC-MD, while analysis regarding SCC differentiation and KIN classification revealed the same correlation as for the cyclin A distribution. Reduced or absent beta-catenin membranous staining was found in 90 cases (81.8%), more often in SCC than in AK (P = 0.03) or in AK and BD grouped together (P = 0.02). There was no statistical difference between SCCs of various level of differentiation, or between different KIN grades. Diffuse loss of membranous beta-catenin staining showed 36 lesions (32.7%), more frequently SCC than AK (P = 0.003) or AK and BD grouped (P = 0.006), as well as SCC-PD compared with SCC-WD (P = 0.01) and SCC-MD (P = 0.03), whereas all KIN comparisons remained nonsignificant. Aberrant beta-catenin cellular localization demonstrated 28 lesions (25.5%), most often in the basal or peripheral parts and in the lesions with diffuse beta-catenin loss (P = 0.009), but revealed no correlation with the histological type, SCC level of differentiation or KIN grades. Diffuse loss of membranous beta-catenin staining was found to be significantly more frequent in SCC thicker than 4 mm (P = 0.03), while all other comparisons between cyclin A or beta-catenin with the tumour size remained nonsignificant. Cyclin A LI was higher in cases with diffuse loss of membranous staining (P = 0.001) or with aberrant cellular localization of beta-catenin (P = 0.002).
Cyclin A LI showed greater difference between AK and BD than between BD and SCC, suggesting that increased proliferation (measured by cyclin A LI) characterizes progression of in situ lesions from AK to BD, whereas reduced beta-catenin expression separates more clearly SCC from the in situ lesions. Diffuse pattern of loss of membranous beta-catenin staining correlated better with the type of lesion, SCC differentiation and tumour size than reduced expression in general or aberrant cellular localization of beta-catenin. KIN classification does not seem to be supported by our findings, except when KIN1 and KIN2 lesions (in situ, partial thickness) are grouped.
光化性角化病（AK）被定义为癌前病变或鳞状细胞癌（SCC）演变的早期阶段，AK单个细胞中所见的组织学变化与侵袭真皮的SCC中所见变化无法区分。细胞周期蛋白A是一种越来越常用的增殖标志物，在S期（DNA复制）和有丝分裂起始中均起作用，而参与细胞间黏附和信号转导的β-连环蛋白的改变可促进恶性肿瘤的侵袭和增殖能力。
确定细胞周期蛋白A和β-连环蛋白在皮肤SCC以及分类为角质形成细胞表皮内瘤变（KIN）、传统上称为AK和鲍恩病（BD）的原位病变中的表达模式，并分析其与SCC分化、直径和厚度的关系。
采用链霉亲和素-生物素技术，使用针对细胞周期蛋白A和β-连环蛋白的抗体，对110份福尔马林固定石蜡包埋组织样本进行免疫组织化学染色。组织学检查中，53个病变诊断为AK，16个为BD，41个为SCC - 11个高分化（WD）、16个中分化（MD）和14个低分化（PD）。采用KIN分类，22个病变为KIN1，23个为KIN2，24个为KIN3。检测细胞周期蛋白A的分布和标记指数（LI），以及β-连环蛋白的膜染色水平和异常（核/细胞质）定位情况。
BD中细胞周期蛋白A弥漫性存在的情况比AK（P < 0.0001）或SCC（P = 0.0002）更常见，SCC - PD中比SCC - WD（P < 0.0001）或SCC - MD（P = 0.0003）更常见。KIN3与KIN2以及KIN3与KIN1病变之间的差异具有统计学意义（P < 0.0001），当将KIN1和KIN2病例分组与KIN3病例比较时也出现相同结果（P < 0.0001）。AK中细胞周期蛋白A LI显著低于BD或SCC（P < 0.05），但BD与SCC之间未发现差异，BD中的LI甚至高于SCC - WD或SCC - MD，而关于SCC分化和KIN分类的分析显示与细胞周期蛋白A分布具有相同的相关性。90例（81.8%）发现β-连环蛋白膜染色减少或缺失，SCC中比AK中更常见（P = 0.03），或在AK和BD合并组中更常见（P = 0.02）。不同分化程度的SCC之间或不同KIN分级之间无统计学差异。36个病变（32.7%）出现β-连环蛋白膜染色弥漫性缺失，SCC中比AK中更常见（P = 0.003）或在AK和BD合并组中更常见（P = 0.006），SCC - PD中比SCC - WD（P = 0.01）和SCC - MD（P = 0.03）更常见，而所有KIN比较均无统计学意义。28个病变（25.5%）出现β-连环蛋白异常细胞定位，最常出现在基底或周边部分以及β-连环蛋白弥漫性缺失的病变中（P = 0.009），但与组织学类型、SCC分化程度或KIN分级无相关性。发现β-连环蛋白膜染色弥漫性缺失在厚度大于4 mm的SCC中明显更常见（P = 0.03），而细胞周期蛋白A或β-连环蛋白与肿瘤大小的所有其他比较均无统计学意义。膜染色弥漫性缺失的病例中细胞周期蛋白A LI更高（P = 0.001），β-连环蛋白细胞异常定位的病例中也更高（P = 0.002）。
细胞周期蛋白A LI在AK和BD之间的差异大于BD和SCC之间的差异，表明增殖增加（通过细胞周期蛋白A LI测量）是原位病变从AK进展到BD的特征，而β-连环蛋白表达降低更清楚地将SCC与原位病变区分开来。β-连环蛋白膜染色弥漫性缺失模式与病变类型、SCC分化和肿瘤大小的相关性比β-连环蛋白总体表达降低或异常细胞定位更好。除了将KIN1和KIN2病变（原位、部分厚度）分组外，我们的发现似乎不支持KIN分类。
