RASSF1A and KRAS Expression in Oral Leukoplakia with Dysplasia and in Squamous Cell Carcinoma

Research Article

J Dent & Oral Disord. 2021; 7(1): 1154.

RASSF1A and KRAS Expression in Oral Leukoplakia with Dysplasia and in Squamous Cell Carcinoma

Costa V1, El-Achkar VNR1, Ribeiro MP1, Tristao SS1, Kowalski LP2, Pinto CAL3, Carvalho YR1, da Cruz-Perez DE4 and Kaminagakura E1*

1Department of Biosciences and Oral Diagnosis, Sao Paulo State University (Unesp), Brazil

2Department of Head and Neck Surgery, University of Sao Paulo Medical School, Brazil

3Department of Anatomic Pathology, AC Camargo Cancer Center, Brazil

4Department of Clinical and Preventive Dentistry, Pernambuco Federal University (UFPE), Brazil

*Corresponding author: Kaminagakura E, Department of Biosciences and Oral Diagnosis, Sao Paulo State University (Unesp), Brazil

Received: January 21, 2021; Accepted: February 17, 2021; Published: February 24, 2021


The aim of this study is to investigate the role of RASSF1A and KRAS protein immunoexpression in Oral Leukoplakia with Epithelial Dysplasia (OLD) and in Oral Squamous Cell Carcinoma (OSCC). Immunohistochemical staining for RASSF1A and KRAS was performed and a semiquantitative analysis was applied to samples of the Control Group (CG, n=20), OLD (n=39), and OSCC (n=100). No significant difference was observed between RASSF1A immunoexpression and OLP and OSCC groups (p>0.05). KRAS expression was higher in OSCC than in OLP and CG (p<0.05). No association was observed between RASSF1A or KRAS expression and alcohol/tobacco use or clinicopathological features (p>0.05) in the OSCC group. Also, patients with OSCC who presented KRAS overexpression had a worse disease-free survival rate (p=0.04). RASSF1A expression was similar in OLD and OSCC groups, suggesting that it plays a critical role in the early stage of OSCC. KRAS expression was higher in OSCC when compared with normal and dysplastic tissues, showing that KRAS expression increases with malignant progression.

Keywords: Oral leukoplakia; Oral cancer; Oral squamous cell carcinoma; Oncoprotein


Oral leukoplakia is recognized as a white plaque of questionable risk, diagnosed excluding other known disorders or diseases that carry no increased risk for cancer [1]. It is considered as the most common potentially malignant oral disorder encountered in clinical practice [2,3]. Its malignant transformation is reported in about 1% of cases, resulting in 20 per 100,000 new cases of Oral Squamous Cell Carcinoma (OSCC) per year [2].

Squamous Cell Carcinoma (SCC) is the most common malignant neoplasm of the oral cavity. Alcohol abuse and tobacco use are the major risk factors for oral cancer [4]. They increase the Reactive Oxygen Species (ROS) causing oxidant/antioxidant imbalance. This is accompanied by lipid peroxidation, oxidative DNA damage, damage to macro- and micro-molecules of cells, and disturbances of antioxidant defense, which can initiate the malignant process [5].

Mutant KRAS (Kirsten Murine Sarcoma Virus) elevates intracellular Reactive Oxygen Species (ROS) levels and leads to oxidative DNA damage. Ras Association Domain Family 1 Isoform A (RASSF1A) is known to play a role as a Ras effector. The suppressive effect of RASSF1A on ROS production is triggered by activated K-RAS. RASSF1A attenuates KRAS-triggered oxidative DNA damage and chromosomal damage [6].

Despite advances in diagnostic methods, the malignant transformation predictor of oral leukoplakia into OSCC is still based on conventional histopathological examination [3]. In recent years, the interest in genes that predispose to the carcinogenesis of oral leukoplakia has grown. Therefore, the aims of this study are to investigate the pattern of RASSF1A and KRAS immunoexpression in Oral Leukoplakia with Epithelial Dysplasia (OLD) and compare to OSCC and to epithelial without dysplasia (control group/CG), and to associate the results with clinicopathological features.

Materials and Methods

The Research Ethical Committee of the University of Sao Paulo State approved this study (CAAE 34246314.9.1001.0077). Previously, calibrated examiners performed all the following analyzes independently and the Kappa test was used to determine agreement between them (VC, MPR and EK).

A retrospective analysis from 1990 to 2007 was performed and 100 samples with diagnosis of OSCC were obtained from the files of the Department of Pathology, AC. Camargo Cancer Center, Sao Paulo, Brazil. During a period from 1992 to 2016, 39 samples with diagnosis of OLD and 20 samples of epithelial tissue from fibroma without epithelial dysplasia or inflammation (control group/CG) were obtained from the files of the Department of Biosciences and Oral Diagnosis, São Paulo State University, São José dos Campos and from Department of Clinical and Preventive Dentistry, Pernambuco Federal University, Brazil. Clinicopathological features, variables and follow-ups were collected from patients’ medical history charts. Cases of OSCC were staged according to the AJCC cancer staging manual [7]. Histopathological diagnoses and histological grades were reviewed and were classified according to El-Naggar et al., 2017 [4].

Tissue Microarray (TMA)

For OSCC samples, TMA was built following methods similar to those reported earlier by Kaminagakura et al., 2011 [8].

Immuno HistoChemistry (IHC)

The methods used herein are similar to those reported earlier by Costa et al., 2019 [9]. The sections were incubated with primary antibodies against RASSF1A (1:200, clone NBP1-89411, Novus Biologicals, CO, EUA) and KRAS (1:100, clone ab55391, Abcam, MA, USA) at 4°C overnight.

A semiquantitative analysis using an Olympus CX31 light microscope (Olympus, New York, Ny, USA) with a 10x/0.25 objective was performed. For IHC analysis, less than 30% of the cytoplasmic immunostained positive cells were considered underexpressed, and ≥30% were considered overexpressed according to the modified criteria proposed by Cao et al., 2013 [10] and Elsabah et al., 2013 [11].

Statistical analysis

Statistical analyses were performed using the SPSS program, version 23.0 (SSPInk, IL, USA). The association between qualitative variables was evaluated by X2 test or Fisher’s exact test, as appropriate. For OSCC, the Kaplan-Meier estimator of the survival function was considered for survival analysis. Only results with p<0.05 were considered statistically significant.


The clinicopathological data and immunoexpression status are summarized in (Table 1).