Oral Oncology xxx (2014) xxx–xxx

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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology

Review

Genomic DNA copy number alterations from precursor oral lesions to oral squamous cell carcinoma Iman Salahshourifar a, Vui King Vincent-Chong a, Thomas George Kallarakkal a,b, Rosnah Binti Zain a,b,⇑ a b

Oral Cancer Research and Coordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia Department of Oral-Maxillofacial Surgical and Medical Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e

i n f o

Article history: Received 1 October 2013 Received in revised form 30 January 2014 Accepted 5 February 2014 Available online xxxx Keywords: Copy number alterations Oral lesions Oral cancer Oral squamous cell carcinoma Array comparative genomic hybridization

a b s t r a c t Oral cancer is a multifactorial disease in which both environmental and genetic factors contribute to the aetiopathogenesis. Oral cancer is the sixth most common cancer worldwide with a higher incidence among Melanesian and South Asian countries. More than 90% of oral cancers are oral squamous cell carcinoma (OSCC). The present study aimed to determine common genomic copy number alterations (CNAs) and their frequency by including 12 studies that have been conducted on OSCCs using array comparative genomic hybridization (aCGH). In addition, we reviewed the literature dealing with CNAs that drive oral precursor lesions to the invasive tumors. Results showed a sequential accumulation of genetic changes from oral precursor lesions to invasive tumors. With the disease progression, accumulation of genetic changes increases in terms of frequency, type and size of the abnormalities, even on different regions of the same chromosome. Gains in 3q (36.5%), 5p (23%), 7p (21%), 8q (47%), 11q (45%), 20q (31%) and losses in 3p (37%), 8p (18%), 9p (10%) and 18q (11%) were the most common observations among those studies. However, losses are less frequent than gains but it appears that they might be the primary clonal events in causing oral cancer. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Oral cancer is the sixth most common cancer worldwide and more than 90% of oral cancers are histopathologically squamous cell carcinoma [1,2]. The highest incidence of oral cancer has been seen in some Melanesian and South Asian countries [3,4]. Males have shown significantly higher rate of oral cancer in the most countries around the world [3,4]. While in contrast, Malaysian Indian females have shown a remarkable higher incidence of oral cancer according to the second report of the national cancer registry in Malaysia, 2003 [5]. Genetic factors play an important role in the aetiology of oral squamous cell carcinoma (OSCC). However, environmental risk factors would be the major triggers of the disease. Therefore, comprehensive research on the lifestyle habits of ethnic groups would

add to the growing body of knowledge in the aetiology of the disease. It is well documented that OSCC may be transformed from the clinically diagnosed oral potentially malignant disorders (OPMDs) which had been earlier termed precancer and premalignant lesions [6]. OPMDs refer to all clinical presentations that carry a risk of oral cancer [6]. In support of the revised terminology, this paper uses OPMDs to mean premalignant and precancerous lesions that were stated in the literature. We aimed at systematically reviewing the common genomic copy number alterations (CNAs) and their frequency on 12 studies that have been conducted on OSCCs using array comparative genomic hybridization (aCGH). In addition, we reviewed the literature dealing with the causes and genetic changes that drive these OPMDs to malignant tumors.

Clinical perspective of OPMDs Abbreviations: LGD, low grade dysplasia; HGD, high grade dysplasia; CIS, carcinoma in situ; OSCC, oral squamous cell carcinoma. ⇑ Corresponding author at: Oral Cancer Research and Coordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: +60 (03) 7967 4896; fax: +60 (03) 7954 7301. E-mail address: [email protected] (R.B. Zain).

Majority of the OSCCs are preceded by OPMDs that manifest genetic alterations. All OPMDs may not transform into OSCC but they belong to a family of disorders characterized by certain morphological alterations amongst which some have an increased potential for malignant transformation. Presence of an OPMD is an

http://dx.doi.org/10.1016/j.oraloncology.2014.02.005 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Salahshourifar I et al. Genomic DNA copy number alterations from precursor oral lesions to oral squamous cell carcinoma. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.02.005

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indicator of risk of likely future malignancies at any another clinically normally appearing mucosa in the oral cavity and not at the specific site [6]. Leukoplakia, erythroplakia, oral lichen planus (OLP), and oral submucous fibrosis (OSMF) are the more common OPMDs. These disorders can be widely distributed in the oral cavity where the floor of mouth and the ventrolateral aspect of the tongue are high risk sites to develop cancer [7], while cancers also occurs at other sites such as lips, cheek, alveolar and palatal mucosa. Leukoplakia is defined as a term to recognize white plaques of questionable risk having excluded other known diseases or disorders that carry no increased risk of cancer [6]. The estimated reported prevalence of leukoplakia worldwide is approximately 2% [8]. The risk of malignant transformation in an oral leukoplakia is dependent on certain factors such as presence of epithelial dysplasia, type and location of the lesion [8]. Erythroplakias is defined as a fiery red patch that cannot be categorized clinically or pathologically as any other definable disease. Majority of the erythroplakias undergo malignant transformation but there is a dearth of enough documented series to calculate a reliable malignant transformation rate [9]. Oral submucous fibrosis (OSMF), a well-recognized OPMD is characterized by fibrosis of the lining mucosa of the upper digestive tract involving the oral cavity, oropharynx and frequently the upper third of the oesophagus [6]. Studies from India and subsequently from Taiwan have estimated the rate of risk of malignant transformation in OSMF to be 7.6% and 1.9% respectively [10,11]. Oral lichen planus (OLP) is an inflammatory condition of unknown aetiology. OLP is primarily mediated by the T-lymphocytes that accumulate beneath the epithelium of the oral mucosa and increase the rate of differentiation of the stratified squamous epithelium [6]. The potential malignant nature of OLP has remained controversial. The principal drawback in studying the malignant transformation of OLP has been attributed to the absence of universally accepted criteria for diagnosis of OLP. This might result in false positive findings leading to inclusion of lesions that are similar to OLP and carry an inherent malignant potential such as erythroleukoplakia and proliferative verrucous leukoplakia [12]. The presence or absence of oral epithelial dysplasia (OED) in OPMDs will guide the clinician towards the type of management. However, accurate histopathological diagnosis and grading of epithelial dysplasia is extremely challenging. Evaluation of dysplasia is very subjective leading to considerable inter- and intra-observer variability [13]. The latest WHO classification has recommended an objective grading of OED that takes into account the levels of the involved epithelium [14]. OED has been subcategorized into mild (mild/ grade 1), moderate and severe and has been indicative of prognosis with the highest potential for malignant change being severe dysplasia and carcinoma-in situ being grouped with the malignancies [15]. However, subjective evaluations of classifications of dysplasia led to suggestions of only 2 categories namely high risk dysplasia or low risk dysplasia [15]. High risk dysplasia implies to an epithelium with moderate to severe dysplasia that carry a higher risk of transformation, while low risk dysplasia implies to an epithelium with questionable or mild dysplasia [15].

Causes of OPMDs Epithelial cells being the first shield of human body protection are the most exposed cells to environmental risk factors. Oral epithelial dysplasia, a precursor to OSCC often results from exposure to environmental risk factors. Smoking, alcohol drinking and betel quid chewing are proven risk factors of oral cancer that would be the main causes of oral epithelial dysplasias [16–19]. In India and Southeast Asia, betel quid chewing is found to be associated with increased risk of OSCC [11,16,20,21]. Betel quid chewers

and tobacco smokers have been shown a greater incidence of OPMDs [20]. In addition, betel quid chewers are more susceptible to develop OSMF, an OPMD with a transformation rate of 7.6% [11]. Evidence showed that these risk factors in a process known as field cancerization could alter the genomic profile of the field and cause OSCC [22,23]. In a study in UK, 92.9% and 85% of oral cavity and pharyngeal cancers among men and women respectively have been linked to fourteen lifestyle and environmental factors [24]. Of these, tobacco has been identified as the major contributor for all types of cancers, accounting for approximately 12% of all deaths above the age of 30 years and 71% of all lung cancer deaths in the world [25]. Oral leukoplakia is one of the most common OPMDs with a malignant transformation rate that ranges from 0.13% to 17.5% [26]. Presence of epithelial dysplasia is an important risk for malignant transformation [27]. Overall, a malignant transformation rate of 16% has been estimated in oral epithelial dysplasia [28]. It should be kept in mind that however people with the risk habits have higher potential to develop OPMDs and OSCC but those without risk habits may also develop OSCC [29,30]. It is wellknown that human papilloma virus (HPV) plays a major role in all cervical cancers through inactivation of p53 and Rb tumor suppressor genes [31]. Evidence has shown that HPV significantly contributes to the development of oropharyngeal cancer and also in some oral cavity cancers [32]. HPV infection is common but only HPV16 and 18 are involved in the majority of HPV related cancers [31,32]. In a study, 51.4% of OSCC samples were HPV positive versus 24.8% of healthy individuals (P < 0.001, OR = 4.3) [33]. High risk HPV 16 was found to be significantly associated with OSCC cases in that study. Apart from HPV, herpes simplex virus (HSV) and Epstein–Barr virus (EBV) that belong to the same family may play a role in the etiology of OSCC. EBV infection is very common, producing a latent and life-long persistent infection in 90% of the population worldwide but all of them may not develop cancer [34]. Epithelium of salivary glands and B-lymphocytes cells are two targets of EBV infection [35]. EBV infection has been implicated in several malignant disorders such as Burkitt’s lymphoma, nasopharyngeal carcinoma and gastric adenocarcinoma [36]. A high frequency of EBV infection has been detected among OSCC cases compared with OPMDs and controls [37–39]. There are two types of HSV namely HSV-1 and HSV-2. HSV-1 is primarily involved in oral and ocular infections while HSV-2 causes genitalinfections [40]. High prevalence of HSV infection has been detected among OSCC cases in developed countries [37]. Co-infections with oncogenic viruses such as HPV, EBV and HSV may serve as potential risk markers for the development of OSCC [37]. Significance of OPMDs in research It has always been difficult to predict the potential of an OPMD to undergo transformation based on the clinical and histological features. Furthermore, the assessment of these OPMDs is complex. A seven-year follow up study showed that one-third of the OPMDs transformed into invasive OSCC [41]. Understanding the genetic alterations that involve in disease progression would add to the growing body of knowledge on oral carcinogenesis. Hence, these genetic data would be very useful for clinicians to predict the malignancy transformation rate of OPMDs, the disease progression and the treatment planning. Genetic changes of OPMDs Risk of cancer significantly increases with age, therefore accumulation of genomic changes is in direct correlation with age

Please cite this article in press as: Salahshourifar I et al. Genomic DNA copy number alterations from precursor oral lesions to oral squamous cell carcinoma. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.02.005

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and lifestyle habits. Evidence has shown that OSCC in younger patients who are non-smokers had less copy number changes than both old and young patients who were smokers [42]. Different OPMDs, dependent on the site, size and presence/absence of dysplasia, might have variable competencies to transform into a malignancy. Accumulation of multiple genetic changes in OPMDs, most likely due to exposure to risk factors over a period of time, has a direct relationship with malignancy transformation. Genomic gains and losses are more frequent among OSCC (65.2%) than OPMD (40%) samples, indicating the disease progression [43]. It may be noted that OPMDs may not progress to a malignancy but discovery of biomarkers that may be expressed during the transition phase are critical to predict the disease progression. Concurrent with the disease progression, an increase in the accumulation of genetic changes in terms of frequency, type and extent of the abnormalities occur (Fig. 1). Despite similarities in the initial clonal genetic changes between OPMDs and OSCCs, there is a clear pattern of genetic change that drives the disease from low grade dysplasia to an invasive form [44]. A study showed that the size of initial abnormality and its frequency increase with the disease progression [43]. In contrast to OSCC, there are few investigations and there is a research gap on the genetic profiles of OPMDs. Low grade dysplasia (LGD) associated CNAs Non-dysplastic normal appearing cells (oral distant fields (ODFs) which are located at a distance from OPMDs have shown lower copy number changes (37%) as compared with their matched

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OPMDs (58%) [23] In addition, the type of recurrent copy number alterations was almost similar between ODFs and OPMDs with a higher frequency among OPMDs [23]. Of these, gain in 1p36.32pter, 7p22.2-pter, 8q24.3-qter, 11p15.5-pter, 16p13.3-pter and 20q13.33-qter were recurrent changes among both ODFs and OPMDs. In that study, 9p21.3 deletion was detected only among dysplastic OPMDs (4 of 8). High resolution customized array-CGH on the short arm of chromosome 3 lead to the identification of 3p25.1-p25.3 deletion as the only alteration in 13% of non-progressive LGDs (2 of 15) [43]. Progressive LGDs and HGDs almost exhibited similar frequencies of deletion (between 56% and 78%) in six recurrent regions on 3p including 3p25.3-p26.1, 3p25.1-p25.3, 3p24.1, 3p21.31-p22.3, 3p14.2 and 3p14.1, while OSCCs showed more than 90% of loss in these regions [43]. It is evident that genomic alterations increase sequentially in terms of number and extent from progressive LGDs to OSCCs [43–46]. A remarkable increase of whole chromosome arm changes was detected along with disease progression in comparison with segmental copy number changes [44]. In that study, 19% of non-progressive LGDs (4 of 21), 55% of progressive LGDs (5 of 9), 56% of HGDs (18 of 32), 71% of OSCCs (17 of 24) exhibited whole chromosome arm alterations. Therefore, identification of segmental 3p losses might be used as a biomarker to monitor the progress of the tumor, while whole 3p arm deletion demonstrates an advanced stage of tumor. Deletions are less frequent but it seems that clonal deletion of tumor suppressor genes might be the initial events in causing oral cancer [43,47–49]. Deletions at 3p14.2 and 9p21 might be the initial clonal abnormalities in low grade dysplasia and even in some histologically normal epithelial cells [48]. Deletion at 3p has been

Fig. 1. Putative sequence of genetic alterations that drive OSCC from normal appearing epithelial cells. Whole arm represents several chromosomal alterations on that specific arm and those highlighted in bold and underline have shown higher frequency. Group I: clinically and histologically normal labial mucosa may manifest subtle genetic changes. Group II: different OPMDs display varied histological appearances from hyperplasia to low grade dysplasia that may have some genomic instability. Group III: OPMDs with severe histopathological changes such as high grade dysplasia or carcinoma in situ with accumulation of genetic changes. Group IV: clinical appearance of histologically proven OSCC as an ulcer or proliferative growth inside the oral cavity Group V: advanced stage of OSCC with lymph node metastasis.

Please cite this article in press as: Salahshourifar I et al. Genomic DNA copy number alterations from precursor oral lesions to oral squamous cell carcinoma. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.02.005

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detected in 31% of 35 dysplastic samples [46]. FHIT (Fragile Histidine Triad) on 3p14.2 is a tumor suppressor gene that regulates apoptosis and controls the cell cycle. Loss of heterozygosity (LOH) of this gene was detected frequently in epithelial tumors, especially with a higher frequency among those that are exposed to the environmental risk factors such as tobacco [50]. CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A) on 9p21 is a tumor suppressor gene that encodes the p16 and p14 proteins as the result of alternative reading frame. These proteins regulate two critical cell cycle pathways namely p53 and RB1 [51]. Oral dysplastic lesions with a deletion at 3p14.2 with or without a deletion at 9p21 have been known as high risk lesions [47], with relatively a high transformation rate [45,48]. Dysplastic lesions with 3p deletion and/or 9p deletion have shown a 22.6-fold increased transformation risk [47]. High risk lesions with LOH at the 4q/17p have showed progression rate of 63.1% in five years compared with low risk lesions that retained 3p and 9p [47]. The higher progression rate among these lesions might be attributed to the p53 mutation. A study on the genetic imbalances of 3p, 9p, 11q, and 17p using STR markers revealed that allelic imbalance at 9p was most frequent in oral leukoplakia [49]. In addition, oral leukoplakia lesions with LOH at 9p21 and 3p14 had a higher transformation rate [48], which would be in line with dysplastic features of the leukoplakia. Classification of OLP as a premalignant oral lesion has remained controversial despite the inability to detect LOH at 3p14.2, 9p21, and 17p13 regions, providing an molecular evidence for non- or less-malignant nature of OLPs [52].

High grade dysplasia (HGD) associated CNAs In addition to changes at 3p and 9p, transition from low to high grade dysplasias is strongly associated with multiple recurrent genetic changes. Alterations that tend to occur in association with higher grades of dysplasia and OSCC are almost similar and gains in 3q26-qter, 8q22.3, 8q11-q21, 8q24-qter, 11q13, 11q13.2-q13.4 have been detected frequently among HGDs [23,43,46,53]. It may be noted that inconsistency of chromosomal breakpoints might be attributed to the variations in the array platforms that have been used. Gains in 8q22-q23 were detected frequently among mild dysplasias and OSCCs, suggesting a very early clonal event in tumourigenesis [46], while gains encompassing the whole 8q arm were frequently detected in invasive OSCCs [44]. High resolution aCGH on 8q21-q24 revealed recurrent gains including 8q22, 8q24 and 8q21-q24 [54]. 8q24 is the hotspot region that harbors the MYC oncogene but 8q22 might enclose a novel oncogene as well [54]. Despite deletion at 3p14 among progressing LGDs, 75% of HGDs had recurrent alterations at 3p25.3-p26.1, 3p25.1-p25.3, 3p24.1, 3p21.31-p22.3, 3p14.2, and 3p14.1 [45]. This study also revealed a similar frequency of 3p alterations among HGDs and OSCCs but the size of the alterations were bigger and extended to the whole 3p arm among OSCCs as compared to HGDs [45]. Inactivation of CDKN2A occurs as an initial event but p53 (17p13.1) inactivation occurs later that subsequently increases the accumulation of genetic changes and aneuploidies [55]. Consistently, the p53 mutations were mainly found during transformation from LGD to HGD in the neoplastic progression of Barrett’s esophagus [56]. It has been well-known that mutation in p53, a key guardian gene in controlling of cell cycle, apoptosis and DNA repair, accounts for >50% of all cancer types [57]. Overexpression of p53 was detected among OPMDs as compared to OSCCs [58], while in contrast a higher mutation rate of p53 was detected among OSCCs but not in dysplastic matched oral samples [59,60]. This shows that down-regulation of p53 among OSCCs as compared to OPMDs could be the result of mutations that inactivate the p53

gene which subsequently leads to the loss of genomic integrity, resulting in an increase of genomic mutations. p53 inactivation might be induced by non-viral risk factors such as tobacco, alcohol [60,61] or HPV [31]. OSCCs with a mutation in the p53 gene have shown coamplification of CCND1 (11q13) and EGFR (7p11.2) as well [62,63], suggesting that they may play a role downstream of p53 in transition to the advanced stages of the disease. Clinical perspective of OSCC Despite advances in diagnostic technologies, the survival rate of OSCC has still remained unchanged over past three decades [3]. However early detection of OSCC can have a critical impact on management and therapy of the disease. Most of the cases present with late stage disease as the symptoms of OSCC are not clearly understood by the high risk groups and the general population [3]. OSCC primarily occurs in middle-aged and elderly population. However, a disturbing trend has been observed with a significant number of these cases being documented in younger adults in the recent years [1]. Similarly a disparity that used to exist with respect to the incidence of OSCC between males and females has dramatically reduced over the past fifty years. This may be attributable to a greater number of women being exposed to potential risk factors such as tobacco and alcohol [1]. OSCC in its early stages may manifest as a white patch (leukoplakia), red patch (erythroplakia) or a mixed red and white lesion (erythroleukoplakia) and is usually subtle and asymptomatic. With progression surface mucosal ulceration or an exophytic mass may develop. Ulcerated lesions are characterized by raised and rolled margins while exophytic masses may have a fungating or papillary surface. With advanced stage disease overt symptoms such as pain, loosening of teeth, dysphagia, trismus, parasthesia and neck masses usually manifest. In European and US populations, 40% of OSCCs involve the tongue where the posterior lateral border and the ventral surfaces are usually affected followed by the floor of the mouth. Among the Asian population OSCC of the buccal mucosa is more common which may be explained by the prevalence of betel quid/tobacco chewing habits [1,3,64]. Evolution and final outcome of the tumour is dependent on several factors that include anatomic site and disease staging to name a few. A higher metastatic rate for tumours is governed by the vascular and lymphatic networks of the affected anatomic site. Clinical staging of OSCC based on the TNM system has a significant influence on the outcome as the five-year survival rates are significantly lower in patients with stage IV disease in contrast to patients with stage I disease [65]. OSCC associated CNAs We aimed to determine common CNAs and their frequency among studies that have been conducted on OSCC samples using array comparative genomic hybridization (aCGH). We searched the Medline public database through PubMed using terms of array-CGH/copy number variations/copy number alterations/genomic profile plus oral squamous cell carcinoma/oral cancer. We included only studies that have been conducted on OSCC tumors using array-CGH. We excluded studies that have been conducted on cell lines, SNP and customized arrays and duplicated samples. In addition, the tumor sites were restricted to only oral cavity consisting of tong, gum, hard palate, soft palate, floor of mouth and buccal mucosa. Finally, twelve studies met our inclusion criteria (Table 1). CNAs have been categorized for each chromosome and the frequency of gains and losses were calculated (Table 2) and function of each gene in tumorigenesis is summarized

Please cite this article in press as: Salahshourifar I et al. Genomic DNA copy number alterations from precursor oral lesions to oral squamous cell carcinoma. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.02.005

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Table 1 List of 12 studies. Studies

OSCC samples

Platform

Site

Vincent et al. [101] Chen et al. [84] O’Regan et al. [42] Sparano et al. [96] Freier et al. [97] Ambatipudi et al. [98] Yoshioka et al. [83] Snijders et al. [62] Noutomi et al. [46] Sugahara et al. [99] Cha et al. [53] Uchida et al. [100]

46 60 20 21 40 60 25 89 35 54 7 50

SurePrint G3 Human 1 M aCGH, Agilent GenoSensor Array 300, Vysis GenoSensor Array 300, Vysis 4134 BAC clones (Ultra GAPS; Corning, NY) DNA microarrays (matrix-CGH) 105 K Oligo aCGH, Agilent 44 k oligo aCGH, Agilent 2 K aCGH, UCSF CGH 44 K Oligo aCGH, Agilent 44 K Oligo aCGH, Agilent MAC array Karyo 4 K, Macrogen

Tongue, BM, FOM, HP, GUM BM and non BM Tongue, FOM, Soft palate, Gum Site has not been mentioned Soft palate, FOM, Mandible, maxilla Tongue and gingivobuccal complex Tongue, BM, Gum, FOM BM, FOM, Gum, Tongue, Tongue, gingiva, buccal region, FOM, and palate Tongue, Gingiva, Palate, BM, FOM Site has not been mentioned FOM, BM, Maxillary gingiva, Mandibular gingiva, Tongue

BM, Buccal mucosa; FOM, floor of mouth; HP, hard palate.

(Supplementary Table 1). It revealed that gains in 3q, 5p, 7p, 8q, 11q and 20q and losses in 3p, 8p and 18q were the most common reported CNAs (Fig. 2). Consistently, gain in 8q was the first most common reported aberration with the highest frequency (47%) among all OSCCs (Table 2). It appeared that 8q24 (PTK2, LY6K, MYC genes) and 8q22 (LRP12) might be the hotspot regions that harbor causative genes in the oral cancer tumorigenesis [54,66]. Gain in 11q13 was the second most common reported aberration (Table 2). This region harbors genes that play a role in tumor progression, invasion and metastasis. Overexpression of CCND1 on this region has been detected in the clinical samples with high grade dysplasia and OSCC and is associated with overall survival of the patients [43,67,68]. In addition, transgenic mice with CCND1 over-expression exhibited more susceptibility to develop oral epithelial dysplasia [69]. CCND1 is a proto-oncogene that controls G1/S transition in the cell cycle and shows amplification in a variety of cancers [70]. This gene perhaps plays a major role in tumorigenesis along with other cancer-related genes such as EMS1, ORAOV1 on this region in a complex pattern [71]. It appeared that ORAOV1 may involves in invasion and metastasis events [72]. Amplification of 7p11.2 has been detected as a frequent finding among OSCCs that subsequently would result in overexpression of EGFR [73]. Despite lack of remarkable difference in the EGFR expression between low grade dysplasia and normal epithelial cells, it is highly expressed in cases with high grade dysplasia and is associated with poor prognosis [73,74]. Furthermore, OPMDs with higher copy number of EGFR have shown a higher rate of transformation than those with normal copy number [75]. Gain and subsequently overexpression of both EGFR and CCND1 was found to be significantly associated with the progression of OSCC [76]. This evidence is consistent with the finding of coamplification of EGFR and CCND1 genes in high risk OPMDs [43]. It may be observed that alterations in gene expression usually resulted from genomic copy number changes that occur earlier in the disease progression [77]. Gain in 3q which contains several cancer related genes was the second most common reported aberration as well (Table 2). TP63 gene, a tumor protein which is located on 3q26 is highly expressed in transition from epithelial dysplasia to OSCC and it is associated with poor prognosis [78]. However, TP63 normally is expressed in epithelial tissues but cells with highly expression of TP63 exhibited severe dysplastic features [79,80]. Overexpression of TP63 and PIK3CA might be the consequence of gain in 3q, as higher copy number is associated with increased risk of disease progression. However due to the function of several isoforms, the oncogenic or suppressor role of TP63 has not been clearly known but it plays a pivotal role in the regulation of cell cycle, apoptosis, metastasis and tumorigenesis in interaction with p53 [80,81]. Losses in 3p, 8p and 9p were the most common observations, respectively (Table 2). However, as mentioned above, lost in 3p

and 9p could be as an early events in the oral tumorigenesis but chromosomal instability extends to the whole arm of the chromosomes in advanced stages of the disease. Of course the genetic heterogeneity could be another possibility that should be kept in consideration. However, genome of OSCC patients undergoes a large number of genetic changes but balance chromosomal rearrangements are undetectable by aCGH. As recurrent isochromosome, translocation, aneuploidy and polysomy were found frequently among OSCCs [76,82]. Tumor metastasis associated CNAs A remarkable differences were found between the genomic profile of non-metastatic primary tumors and their paired lymph node metastatic tumors (LNMs) [83]. Gains in 7p11.2-p22.3, 8q11.22q24.3, 17q24.3-q25.3, with an average of 64%, 91% and 62% respectively, were detected more significantly among LNMs as compared with 10%, 31% and 8.5% in non-metastatic primary tumors [83]. Moreover, gain in several regions including 7p12 (EGFR), 11q13 (FGF3/FGF4, CCND1, EMS1), 17q11.2 (THRA) and 20q12 (AIB1) showed a significant association with LNM (p-value < 0.05) [84]. Of these, gain in 20q12 was found to be the most significant (p-value < 0.005). Lack of a significant difference between genomic profile of metastatic primary tumors and LNMs indicates that further CNA changes might not be needed for invasion to lymph node. Another evidence showed similar copy number changes between primary OSCC and paired metastatic tumors but significant gains in 3q26.3 (PIK3CA gene) significantly showed higher copy number changes within the metastatic cohort [85]. Despite lack of difference in copy number of 11q13 between primary and metastatic OSCC samples, the overexpression of FGF4 on this region showed a significant association with decreased survival rate [85]. Overexpression of TLN1 gene which is located on 9p13.3 has been suggested to be involved in metastasis [86]. Proliferation and apoptosis associated CNAs Tumoregenesis is a multistep process by which imbalance between proliferation and apoptosis play a critical role in tumoregenesis. This balance is controlled by several genes such as TP53, CDKN2A, CCND1, TGFBR2 and SMAD4 [87]. TP53, CDKN2A, CCND1 are acting in same pathway to inhibit cell cycle. TP53 plays a central role in this pathway to either arrest cell cycle or induce apoptosis [88]. Amplification of CCND1 at 11q13.3 was found to be significantly associated with increased cell proliferation [77]. EMS1 is another gene on this chromosomal region by which its amplification showed association with high proliferation in oral cancer [89]. CDKN2A on 9p21.3 is a negative regulator of cell pro-

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Table 2 Top most common reported chromosomal amplifications and deletions among 473 OSCC samples (12 studies).

Gain

Loss

Chromosomal region

Frequency

Frequency of aberrations for each chromosome n = 473 (%)

Studied candidate genes

Reference

3q27-q29 3q26 3q29 3q27.2-3q27.3 3q26 3q24-q29 3q24-q25 3q26-qte 3q25.31-q36.3 3q21.3-q29

46/60 9/20 11/20 10/21 10/40 36/60 2/89 29/35 7/7 14/25

36.5

TP63, TERC, ZASC1, EPHB3,BCL6, TM4SF1, SOX2, CLDN1, SCCRO, PIK3CA

[84] [42] [42] [96] [97] [98] [62] [46] [53] [83]

5p13 5p15.33, 5p13.1p13.2 5p15 5p15.33-p11 5p13.2 5p15 5p 5p13.1-p15 5p15.33

27/60 7/21

23

TERT, RAD1, SKP2, SEC6L1

[84] [96]

7p12.3-p12.1 7p11 7p22.3-p11.1 7p11.2 7p12-p11.2 7p21.1 7p22.3 7p12.3-p12.1 7p11.2-22.3

8/20 6/40 30/60 4/89 5/7 7/7 5/50 32/60 6/25

21

FSCN1, IL6, EGFR, GNA12

[42] [97] [98] [62] [53] [53] [100] [84] [83]

8q11.1-q24.4 8q24.12-q13 8q24.11-q24.13 8q24.23-q24.3 8q24 8q11.1-q24.4 8q22-23 8q 8q24.1-q24.2 8q21.1-24.3 8q11.1-q24.3

25/46 37/60 11/20 13/21 17/40 44/60 32/35 NS/20 7/7 10/50 17/25

47

PRKDC, YWHAZ, LRP12, RAD21, EXT1, MYC, NDRG1, PTK2, LY6K

[101] [84] [42] [96] [97] [98] [46] [99] [53] [100] [83]

11q11 11q23.3-q25 11q13 11q13 11q13 11q13 11q13.3 11q13.5 11q13 11q 11q13.3 11q13.1 11q13.1-q13.3

24/46 26/46 41/60 12/20 15/40 28/60 10/89 2/89 17/35 3/20 21/50 7/50 11/25

45

CCND1, ORAOV1, FGF19, FGF4, FGF3, FADD, PPFIA1, EMS1, SHANK2, TPCN2, OCIM, BIRC3

[101] [101] [84] [42] [97] [98] [62] [62] [46] [99] [100] [100] [83]

20q11.21-q13.33 20q13 20q12 20q 20q13.33 20q13 20q 20q 20q13.3 20q11.21-q13.33

24/46 27/60 27/60 NS/20 7/21 8/40 24/35 NS/20 5/50 12/25

31

AIB1, BCL2L1, MMP9, SNAI1, BMP7

[101] [84] [84]] [42] [96] [97] [46] [99] [100] [83]

3p14.2 3p12-p13, 3p14.2, 3p24.3 3p24.2-p24.3 3p21-3p12 3p14.2 3p14-p21 3p

32/60 NS/20

37

FANCD2, VHL, TIMP4, PPARG, XPC, RARB,TGFBR2, MLH1,DLEC1, CSRNP1, CTNNB1, RASSF1, WNT5A, FHIT

[84] [42]

12/40 30/60 2/89 16/35 NS/20 7/7 5/50

10/21 15/40 33/60 22/35 NS/20

[97] [98] [62] [46] [99] [53] [100]

[96] [97] [98] [46] [99]

Please cite this article in press as: Salahshourifar I et al. Genomic DNA copy number alterations from precursor oral lesions to oral squamous cell carcinoma. Oral Oncol (2014), http://dx.doi.org/10.1016/j.oraloncology.2014.02.005

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I. Salahshourifar et al. / Oral Oncology xxx (2014) xxx–xxx Table 2 (continued) Chromosomal region

Frequency

Frequency of aberrations for each chromosome n = 473 (%)

Studied candidate genes

Reference

3p21.3 3p14 3p22, 3p14, 3p26.3 3p11.1-3p26.3

7/7 7/7 >25/50

8p23.2 8p32 8p 8p 8p11.23-p23.2

11/21 11/40 50/60 NS/20 10/25

18

PCM1, CSMD1, LZTS1

[96] [97] [98] [99] [83]

9p21.3 9p21 9p 9p21.3

37/60 5/20 NS/20 5/10

10

CDKN2A, MTAP, CDKN2B

[84] [42] [99] [83]

18q 18q21-q23 18q11.2-q23 18q22-qter 18q

8/21 10/40 6/10 28/35 NS/20

11

SMAD4, BCL2, SERPINB5, MALT1

[96] [97] [83] [46] [99]

[53] [53] [100]

17/25

[83]

Notice that not specified (NS) samples were excluded to estimate the pooled frequency of aberrations per each chromosome which are highlighted in bold.

among OSCC patients with betel quid chewing [84]. However, copy number alterations of many cancers have been well-studied but the causative mechanism that is mediated by the environmental risk factors is unknown and needs a large cohort of samples to achieve the statistical significance.

A step before cancer, concluding remarks and outlook

Fig. 2. Replicated and recurrent amplifications and deletions among 12 aCGH studies on primary OSCC tumors. Genomic alterations that were reported >2 times are included in the histogram. Data were extracted from references in Table 2.

liferation, hence its low expression result in unlimited proliferation and advanced stage of disease [90]. Loss in this chromosomal region has been frequently detected among OSCCs (Table 2). Apoptosis is negative regulator of proliferation in which many genes including SOX2 (3q26.33), PTK2 (8q24.3), BIRC3 (11q22.2), CSRNP1 (3p22.2), BCL (18q21.332) play a role in this event (Supplementary Table 1). Risk habit associated CNAs Cigarette smoking, alcohol drinking and betel quid chewing are well-known risk habits associated with OSCC. It has been revealed that the cigarette smoking could induce certain copy number changes that might be mediated by the genomic instability [91]. However, multiple evidence studies on from lung cancer support the role of environmental risk factors in causing specific genomic changes [91,92], but there is a paucity of research for OSCC. Evidence has shown differential pattern of DNA copy number alterations between OSCC patients with and without betel quid chewing [84]. It was observed that losses were more frequently identified than gains among a cohort of OSCC patients who had chewed betel quid. Loss of 3p14.2 (FHIT) and 10q21.3 (EGR2) and gains in 19q13.32 (GLTSCR2) and 7q11.23 (CYLN2) were the most statistically significant findings

Millions of people are recurrently exposed to the risk habits but only a few among them develop OSCC. Over the past years, it has been well-known that oral cancer is a genetic disorder but the susceptibility to oral cancer is an issue that has not been clearly known. Many studies have been conducted on the genetic changes in the cancerous tissues leading to the identification of a long list of genes or regions while there is a research gap in searching the predisposing genetic risk factors of oral cancer. Despite the rarity of familial cases, having a higher risk of oral cancer among 1st degree relatives and higher incidence in certain ethnic groups favours the role of genetic makeup in the cause of oral cancer. There is evidence showed that gene expression alteration in cancer is associated with copy number changes [77], but single nucleotide variations (SNPs) are also able to alter the gene expression or the binding abilities. It may be noted that major variations in the pattern of expression would be expected from copy number alterations than SNPs. Most of the SNPs are located in the non-coding regions, hence their role to cause disease has largely remained unknown. Hence, focusing on the steps before copy numbers changes may result in detection of biomarkers for early diagnosis of OSCC. Unlike high penetrance mutations, genetic variations might have a modest effect to increase susceptibility to cancer through changing the expression levels or binding abilities. Therefore, searching for these genetic variations and their interaction with environmental risk factors should be a part of study on oral cancer. It is been well-known that an individual’s genetic makeup has a strong association with the risk of late onset complex disorders such as hypertension, heart diseases, diabetes and cancer. The advent of high-throughput technologies has resulted in the importance of modifier and low penetrance genes receiving too much attention in cancer [93,94]. As genome-wide association studies (GWAS) being successful to identify genetic variations that increase the susceptibility to some common cancers [93]. To the best of our knowledge, only one GWAS research has been conducted on oral

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cancer using a small number of samples [95]. Thus, as concluding remarks, high-throughput genotyping studies using large number of samples would result in identification of significant genes with low but remarkable effect in the OSCC etiology. Conflict of interest statement None declared. Acknowledgments This study was supported by Ministry of Higher Education High Impact Research Grant (H-18001-00-C000008). The authors are indebted to Dr. Mannil Thomas Abraham, Department of Oral & Maxillofacial Surgery, Hospital Tengku Ampuan Rahimah, Klang, Selangor, Malaysia and Dr. Siti Mazlipah Ismail Department of Oro-Maxillofacial Surgical and Medical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia for contributing cases for images and photomicrographs for this manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.oraloncology. 2014.02.005. References [1] Neville BW, Day TA. Oral cancer and precancerous lesions. CA Cancer J Clin 2002;52(4):195–215. [2] Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: implications for therapy. J Dent Res 2008;87(1):14–32. [3] Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45(4–5):309–16. [4] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61(2):69–90. [5] Lim GCC, Halimah Y. Second report of the national cancer registry. Cancer incidence in Malaysia 2003. Kuala Lumpur: National Cancer Registry; 2004. [6] Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36(10):575–80. [7] Jaber MA, Porter SR, Speight P, Eveson JW, Scully C. Oral epithelial dysplasia: clinical characteristics of western European residents. Oral Oncol 2003;39(6):589–96. [8] Petti S. Pooled estimate of world leukoplakia prevalence: a systematic review. Oral Oncol 2003;39(8):770–80. [9] van der Waal I. Potentially malignant disorders of the oral and oropharyngeal mucosa; terminology, classification and present concepts of management. Oral Oncol 2009;45(4–5):317–23. [10] Hsue SS, Wang WC, Chen CH, Lin CC, Chen YK, Lin LM. Malignant transformation in 1458 patients with potentially malignant oral mucosal disorders: a follow-up study based in a Taiwanese hospital. J Oral Pathol Med 2007;36(1):25–9. [11] Murti PR, Bhonsle RB, Pindborg JJ, Daftary DK, Gupta PC, Mehta FS. Malignant transformation rate in oral submucous fibrosis over a 17-year period. Community Dent Oral Epidemiol 1985;13(6):340–1. [12] Gonzalez-Moles MA, Scully C, Gil-Montoya JA. Oral lichen planus: controversies surrounding malignant transformation. Oral Dis 2008;14(3):229–43. [13] Warnakulasuriya S. Histological grading of oral epithelial dysplasia: revisited. J Pathol 2001;194(3):294–7. [14] Speight PM. Update on oral epithelial dysplasia and progression to cancer. Head Neck Pathol 2007;1(1):61–6. [15] Warnakulasuriya S, Reibel J, Bouquot J, Dabelsteen E. Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med 2008;37(3):127–33. [16] Zain RB, Gupta PC, Warnakulasuriya S, Shrestha P, Ikeda N, Axell T. Oral lesions associated with betel quid and tobacco chewing habits. Oral Dis 1997;3(3):204–5. [17] Thomas G, Hashibe M, Jacob BJ, Ramadas K, Mathew B, Sankaranarayanan R, et al. Risk factors for multiple oral premalignant lesions. Int J Cancer 2003;107(2):285–91. [18] Pentenero M, Broccoletti R, Carbone M, Conrotto D, Gandolfo S. The prevalence of oral mucosal lesions in adults from the Turin area. Oral Dis 2008;14(4):356–66. [19] Jaber MA, Porter SR, Gilthorpe MS, Bedi R, Scully C. Risk factors for oral epithelial dysplasia–the role of smoking and alcohol. Oral Oncol 1999;35(2):151–6.

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