br Results Cyclin D overexpression
Results: Cyclin D1 overexpression was found in 28.7% of the cases of OSCC. It was significantly and positively
associated with the following clinicopathological parameters: low tumor diﬀerentiation degree (p = 0.030), invasive morphology (p = 0.045), and proliferative phenotype according to tumor cell ki-67 expression (p = 0.018).
Conclusions: Cyclin D1 overexpression is an event of oral carcinogenesis associated with clinicopathological parameters classically associated with a poor prognosis in patients with OSCC.
& Miller, 1995). Numerous studies have focused on the prognostic value of emerging molecular biomarkers (Bettendorf, Piﬀkò, & Bànkfalvi, 2004; Monteiro Diniz-Freitas, Warnakulasuriya, Garcia-Caballero, Forteza-Vila et al., 2018; Monteiro, Diniz-Freitas, Warnakulasuriya, Garcia-Caballero, Forteza et al., 2018; Silva et al., 2011), and our group has proposed immunohistochemical biomarkers that may improve prediction of the prognosis of individual patients (González-Moles,
Cyclin D1 protein is encoded by the CCND1 gene in chromosome band 11q13 and promotes Meropenem progression during the G1-S phase (Sherr & Roberts, 2004). It has also been attributed with numerous emerging functions, including the regulation of cell migration and mi-tochondrial metabolism and the inhibition of cell diﬀerentiation and DNA repair (Pestell, 2013). Gene amplification is the main mechanism of cyclin D1 overexpression in OSCC, showing a two-fold higher rate
Corresponding author at: Oral Medicine Department, School of Dentistry, University of Granada, Paseo de Cartuja s/n, 18071, Granada, Spain.
E-mail addresses: firstname.lastname@example.org (P. Ramos-García), email@example.com (M.Á. González-Moles), firstname.lastname@example.org (L. González-Ruiz), email@example.com (Á. Ayén), firstname.lastname@example.org (I. Ruiz-Ávila), email@example.com (M. Bravo), firstname.lastname@example.org (J.A. Gil-Montoya).
than observed in other human cancers (Ramos-García, Gil-Montoya et al., 2017; Ramos-García, Ruiz-Ávila et al., 2017). However, nu-merous molecular alterations have also been implicated in cyclin D1 dysregulation and oncogenic activation, including chromosomal trans-locations, mutations, polymorphisms, and the activation of pathways involved in human carcinogenesis (MAPK, PI3K, Wnt, NF-κβ) (González-Moles, Plaza-Campillo et al., 2014; González-Moles, Ruiz-Ávila et al., 2014; Ramos-García, Gil-Montoya et al., 2017; Ramos-García, Ruiz-Ávila et al., 2017). Cyclin D1 overexpression is frequently associated with T and N status, advanced clinical stage, high histolo-gical grade, reduced survival, and lack of response to treatment, among other poor prognostic factors (Ramos-García, Gil-Montoya et al., 2017; Ramos-García, Ruiz-Ávila et al., 2017), and it has been described as one of the markers with greatest potential prognostic value in oral cancer patients (Ramos-Garcia et al., 2018).
With this background, the objective of this study of patients with OSCC was to evaluate the clinicopathological significance of cyclin D1 overexpression as measured by an semi-automatized im-munohistochemical technique.
2. Material & methods
We conducted a retrospective study of 54 patients aged between 45 and 87 yrs (63.0 ± 12.0 yrs) with 68 OSCCs under treatment in the Hospital Complex of Jaen (Spain); 40 patients (74.1%) were males (Table 1). The criteria for inclusion in the study were the following: 1. Having oral squamous cell carcinoma. 2. Availability of hospital clinical history. 3. Availability of a paraﬃned block of the tumor. The exclusion criteria were: 1. Absence of a clinical history or relevant data of the history. 2. Absence of paraﬃn block or suﬃcient tissue material for performing the immunohistochemical technique. 3. Deterioration of the tissue material necessary for the realization of the im-munohistochemistry technique. All participants had signed informed consent to the preservation and subsequent use of their biological samples for research. The study has been submitted to the ethics committee CEIM /CEI Provincial de Granada (Registration No. CD12018). After approval of the study by the ethics committee of the hospital, we reviewed the hospital records of the patients and gathered data on the clinicopathological characteristics of their lesions.
An immunohistochemical study of cyclin D1 and Ki-67 proteins was conducted in the Pathology Department Granada University Hospital Complex (Spain). Peroxidase-antiperoxidase and avidin-biotin techni-ques were applied in five 4-μm sections from each paraﬃn block (tumor), utilizing automated Autostainer Link equipment (Dako, Carpinteria, CA, USA) and EnVision™FLEX reagents (K8002; Dako) ac-cording to the manufacturer’s instructions. This system permits de-waxing and rehydration followed by heat-induced epitope recovery. The reproducibility of the process was ensured by loading the whole coverslip, guaranteeing the identical heating of all sections in each cycle. We used rabbit monoclonal anti-human cyclin D1 antibody (Clone EP12) (Dako) and primary Mib-1 antibody against ki-67 (Dako), recommended by the manufacturer for this automated system. Counterstaining was done using the EnVision™ Flex Hematoxylin system (K8008; Dako), which stains in light nuclear blue, followed by perma-nent mounting of the samples in DPX. For the negative control, the primary antibody was replaced by saline phosphate buﬀer. Tissue from an OSCC of known cyclin D1 and Ki67 expression served as positive control. No control group was used for cyclin D1 expression, given the well-documented and consistent negativity for cyclin D1 of healthy oral epithelia (Das, Khare, Singh, & Sharma, 2011; Goto, Kawano, Kobayashi, Sakai, & Yanagisawa, 2002; Kuo, Lin, Hahn, Cheng, & Chiang, 1999; Mishra & Das, 2009; Mishra, Nagini, & Rana, 2015; Wang et al., 2006; Wong et al., 2003; Zhang et al., 2015). Slides were digi-talized using a Philips IntelliSite Ultra-Fast Scanner (Philips Digital Pathology Solutions, Best, The Netherlands), and the tumor expression of markers was evaluated in four randomly selected tumor fields of 0.191 mm2 (equivalent to 40x magnification) with the Philips IMS viewer (Philips Digital Pathology Solutions), which oﬀers high magni-fication, definition, and reproducibility. The expression in each field was measured with a semi-automated cell count technique (Ramos-Garcia et al., 2018; Ramos-García et al., 2018), using Adobe Photoshop