Pathology – Research and Practice 212 (2016) 426–436 Contents lists available at ScienceDirect Pathology – Research and Practice jou rn al hom epage: www.elsevier.com/locate/prp Original article Differential immunohistochemical expression profiles of perlecan-binding growth factors in epithelial dysplasia, carcinoma in situ, and squamous cell carcinoma of the oral mucosa Mayumi Hasegawa a,b , Jun Cheng a , Satoshi Maruyama c , Manabu Yamazaki a , Tatsuya Abé a,c , Hamzah Babkair a , Chikara Saito b , Takashi Saku a,c,∗ a Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan b Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan c Oral Pathology Section, Department of Surgical Pathology, Niigata University Hospital, Niigata, Japan a r t i c l e i n f o Article history: Received 7 October 2015 Received in revised form 15 January 2016 Accepted 14 February 2016 Keywords: Perlecan Perlecan-binding growth factors Oral squamous cell carcinoma Carcinoma in situ Epithelial dysplasia Cell proliferating zone a b s t r a c t The intercellular deposit of perlecan, a basement-membrane type heparan sulfate proteoglycan, is con- sidered to function as a growth factor reservoir and is enhanced in oral epithelial dysplasia and carcinoma in situ (CIS). However, it remains unknown which types of growth factors function in these perlecan- enriched epithelial conditions. The aim of this study was to determine immunohistochemically which growth factors were associated with perlecan in normal oral epithelia and in different epithelial lesions from dysplasia and CIS to squamous cell carcinoma (SCC). Eighty-one surgical tissue specimens of oral SCC containing different precancerous stages, along with ten of normal mucosa, were examined by immuno- histochemistry for growth factors. In normal epithelia, perlecan and growth factors were not definitely expressed. In epithelial dysplasia, VEGF, SHH, KGF, Flt-1, and Flk-1were localized in the lower half of rete ridges (in concordance with perlecan, 33–100%), in which Ki-67 positive cells were densely packed. In CIS, perlecan and those growth factors/receptors were more strongly expressed in the cell proliferating zone (63–100%). In SCC, perlecan and KGF disappeared from carcinoma cells but emerged in the stromal space (65–100%), while VEGF, SHH, and VEGF receptors remained positive in SCC cells (0%). Immuno- fluorescence showed that the four growth factors were shown to be produced by three oral SCC cell lines and that their signals were partially overlapped with perlecan signals. The results indicate that per- lecan and its binding growth factors are differentially expressed and function in specific manners before (dysplasia/CIS) and after (SCC) invasion of dysplasia/carcinoma cells. © 2016 Elsevier GmbH. All rights reserved. 1. Introduction It remains a challenge to make objective histopathological diag- noses of oral borderline malignancies from epithelial dysplasia and carcinoma in situ (CIS) to microinvasive squamous cell carcinomas (SCC) only on hematoxylin and eosin (HE) stained sections, as the conventional grading systems are too heavily dependent on the subjectivity of pathologists, which leads to considerable disagree- ment [1–3]. Recently, we have proposed that the characteristic ∗ Corresponding author at: Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan. E-mail address: tsaku@dent.niigata-u.ac.jp (T. Saku). two-phase appearance, which results from a sharp and contrastive layering of the upper keratinized cell layer and the lower half basaloid cells, is recognized in some particular histological types of epithelial dysplasia or CIS [4–12], and it could be an impor- tant histopathological hallmark of potentially malignant epithelial lesions even on HE sections. In the lower half of the two-phase epithelial dysplasia, composed of basaloid cells which are immuno- histochemically positive for Ki-67 [5,6] as well as podoplanin [12,13], there are enriched intercellular deposits of extracellular matrix (ECM) molecules such as perlecan, a basement-membrane type heparan sulfate proteoglycan [14–18]. In addition, the basaloid cells in the lower half showed simultaneous loss of E-cadherin and nuclear translocation of -catenin from the cell membrane, which indicates that those basaloid cells form a cell proliferating center in the lower half [6]. http://dx.doi.org/10.1016/j.prp.2016.02.016 0344-0338/© 2016 Elsevier GmbH. All rights reserved.