Clinicopathological variables of included study candidates

FFPE tissue blocks of normal oral mucosa (n = 10) obtained during therapeutic or surgical extractions were included as a control group.

Immunohistochemistry

From each FFPE tissue block selected, 3 ?m thick sections were made on poly-l-lysine (0.1% [w/v] in H2O) (Sigma-Aldrich, Missouri, USA) coated slides. Sections were deparaffinized and rehydrated with xylene and serial dilutions of ethanol to distilled water. Tissue sections were immersed in EDTA buffer at a pH of 9 (EZ 2, Biogenex, Fremont, USA, ready to use), and heat-induced epitome retrieval was done using autoclave method at 120?C, 12?15 psi for 15 min. For each sample, anti-p63 antibody (Clone: 4A4) (Biogenex, Fremont, USA, mouse IgG, ready to use) was used as the primary antibody for 45 min incubation at room temperature in a humidity chamber. The antigen?antibody binding was detected with labeled anti-mouse polymer-horseradish peroxidase detection system and 3, 3'-diaminobenzidine + chromogen (Biogenex, Fremont, USA). Tissue sections were briefly immersed in hematoxylin for counterstaining. In all cases, staining of dysplastic epithelial cells served as positive internal controls for anti-p63 antibody, and the antigenic potential of the tissue blocks was confirmed by applying pan-cytokeratin cocktail (Biogenex, Fremont, USA, mouse IgG, ready to use) as primary antibody on the subsequent sections. For negative control, the primary antibody was replaced by mouse-negative control (nonimmune serum in phosphate-buffered saline with 0.09% sodium azide).

Quantitative assessment of p63 expression

The p63-stained slides were initially analyzed at low magnification (original magnification ? 100) to select cancer islands which were defined as cancer tissue without fibroblasts and vasculature. Five HPFs (original magnification ? 400) were selected in tumor proper area for each case of experimental group and in the epithelium of the control group, and the percentage of immune-reactive dysplastic cells was calculated by counting the dysplastic epithelial cells using manual tag function in the selected HPFs using Image Pro Express ver. 6.0 (Media Cybernetics Inc. Rockville, Maryland, USA) analysis software. The percentage of immune-reactive dysplastic cells for each case was calculated using the following formula.

Percentage of p63 immune-reactive dysplastic cells = Total no. of p63-positive dysplastic epithelial cells/Total no. of dysplastic epithelial cells ? 100%

The percentage p63 expression for each sample was calculated, and the results were tabulated against the corresponding data on the survival status of the respective study candidate [Table 2].

Clinicopathological parameters of the study candidates along with their survival status

Results

Statistical analysis

The data were statistically analyzed using GraphPad Prism 5.03 for Windows (GraphPad Software Inc., La Jolla, CA, USA). P <0>

p63 expression in normal oral mucosa

Normal human oral mucous epithelium had a basal and parabasal pattern of p63 expression. The labeling was only nuclear, with nuclei showing an intense staining, stronger in the basal layer with respect to the parabasal layer (with nuclei of the parabasal layer showing only a faint staining). In general, keratinocytes of suprabasal layers were not immunolabeled by anti-p63 antibody although a slight expression of p63 was recorded in some areas [Figure 1]d. Thus, normal epithelium included a mean of 20.86% (range: 9.26%?36.59%) of stained cells.

Percentage p63 expression and mean Anneroth scores of various grades of oral squamous cell carcinoma

Similarly, a statistically significant correlation (P?= 0.013) was obtained between MAS and the Broders' histological grading of the tumor; the MAS was lower in well-differentiated tumors (Grade I) when compared to that of the poorly differentiated counterparts (Grade III) [Table 3].

To analyze the prognostic significance of p63, the study candidates were subclassified into three subgroups based on their percentage p63 [removed]subgroup x [n?= 5]: <50 class="i" xss=removed>n?= 9]: 50%?75% p63 expression; subgroup z [n?= 13]: >75% p63 expression).

In addition, the prognostic applicability of Broders' classification (Grade I SCC [n?= 10]; Grade II SCC [n?= 10]; and Grade III SCC [n?= 07]) and Anneroth's multifactorial grading system (MAS: ?2.5 [n?= 14]; MAS: 2.6?4.0 [n?= 13]).

The patients with increased p63 [removed]subgroup z) had poorer survival rates than the patients with comparatively lesser p63 [removed]subgroup x, subgroup y). Among participants of subgroup x (05/27), the survival proportion was 100.00 after 949 days whereas data of participants of subgroup y (9/27) showed a survival proportion of 77.78 after 926 days of follow-up. Whereas in participants with the highest p63 expression, subgroup z (13/27), the survival proportion after 820 days of follow-up was 23.07. The statistical comparison of the survival curves was done by log-rank (Mantel-Cox) test which showed statistical significance (P?= 0.0049) between the survival curves of patients of subgroups x, y, and z, respectively [Figure 2].

Table 4: Study candidates tabulated based on mean survival (days)

Similarly, when the X ? SEM of MAS of study participants classified based on survival period following diagnosis, it was compared; although there was an increase in the MAS of patients with <479>479 days of survival in all the three histological grades of the neoplasm (Grade I, II, III SCCs), statistical significance was obtained only for well-differentiated neoplasms [Table 4].

Discussion

The p63 proteins are important in the formation of the oral mucosa, and in normal oral mucosa, there is a balance between the six proteins belonging to the p63 family. In contrast, an imbalance in levels between them is seen in SCCs, in the same area.[12],[13] Although numerous studies have preferably used semi-quantitative analysis and quick scoring methods for grading immunoperoxidase expression in immunohistochemistry,[12],[14] our primary intent was to exactly quantify the p63 expression of every HPF assessed. Hence, quantitative assessment was done using manual tag function of Image Pro Express ver. 6.0 (Media Cybernetics Inc., Rockville, MD, USA) analysis software. Although, being a comparatively more time-consuming process than semi-quantitative analysis, the results could be stipulated to the exact percentage of immunoperoxidase expression in each HPF included, as compared to results expressed in ?range? in semi-quantitative methods. This method can be preferred at centers with a limited access to automated quantification facilities.

Cancer arises in a multistep process resulting from the sequential accumulation of genetic and epigenetic defects and the clonal expansion of selected cell populations.[15] The p53 gene, first described in 1979, was the first tumor suppressor gene to be identified. It was originally believed to be an oncogene ? a cell-cycle accelerator ? but genetic and functional data obtained 10 years after its discovery showed it to be a tumor suppressor.[16] In 1997?1998, two additional members of the p53 family, namely, p63 and p73 which had a close structural homology with their predecessor were discovered.[17]

The complexity of the study of p63 is due to the existence of multiple isoforms (six known isoforms, namely, TA-p63?, TA-p63 ?, TA-p63 ?, ?N-p63?, ?N-p63 ?, and ?N-p63 ?) with opposing functions.[18] The multiple numbers of antibodies that are needed to be employed distinguish between these isoforms have made the ability to analyze the expression of p63 difficult in human tumors. Many studies have found that p63 is overexpressed in human tumors while other studies have shown a loss of expression of p63.[6]

The present study was intended to evaluate whether the amount of p63 [removed]expressed as percentage expression) could be related to any of the histological grading which is generally used to define the aggressiveness of the tumor such as the Broders' classification and Anneroth's multifactorial grading system, which takes into consideration various morphological and histological parameters previously mentioned. Furthermore, the multifactorial grading system is considered to have greater significance in predicting the growth capacity and outcome of the tumor.[19] Although the multifactorial grading of invasive sites/front has shown highly significant prognostic value,[20],[21] since the intent was to use incisional biopsy tissue, parameters of Anneroth's multifactorial grading system were preferred over Bryne's multifactorial grading system,[21] since the grading criteria of the latter were not applicable for most of the incisional biopsies included in the study since they contained only the tumor tissue.[20]

Interestingly, the survival curves showed a statistically significant correlation (P?= 0.0003) between the study samples when categorized based on their MAS [Figure 2]. Hence, the Anneroth's multifactorial grading system can be preferred for initial assessment of prognosis and the aggressiveness of the tumor when incisional biopsy tissue alone is available for the pathologist. Moreover, we advocate the use of Anneroth's multifactorial grading system for routine histopathological reporting, over Broders' system, since the survival curves showed no prognostic significance (P?= 0.1016) [Figure 2]. This finding was in consensus with the results of various previously conducted studies.[19],[20],[21],[22],[23]

The survival curves showed a statistically significant correlation (P?= 0.0049) when the study candidates were classified based on their percentage p63 expression [Figure 2]. Studies conducted by Lo Muzio et al. in 2005,[14] Gu et al. in 2008,[24] and Loljung et al. in 2014[12] found a significant correlation of p63 expression and patient survival in OSCCs, and Cho et al. in 2003[25] and Moergel et al. in 2010[26] associated increased p63 expression with radiation resistance whereas, on the other hand, the present study results are at odds with the findings of the studies conducted by Bortoluzzi et al. in 2004[27] and Monteiro et al. in 2016.[28]

Data from the p63 field currently demonstrate that p63 can act as a tumor suppressor or as an oncogene. The data are most consistent with supporting the idea that the ?Np63 isoforms have oncogenic activities while the TAp63 isoforms have tumor suppressive activities.[5],[6],[7] In addition, discovery of the interactions of p63 in NOTCH pathway,[8] beta-catenin signaling pathway,[29] and control of growth signaling pathways involving cyclin kinase inhibitor, p21 and p57,[8] have validated the existence of a correlation between p63 expression and the invasive behavior of various tumors.

Flores, based on an extensive review of studies conducted on p63, put forth a hypothesis that the downregulation or loss of TAp63 and/or overexpression of ?Np63 may lead to inhibition of the functions of TAp63, p53, and TAp73 which, in turn, will result in the development of an invasive and metastatic tumor. Furthermore, it was evident that mutant p53 could bind to TAp63 and TAp73, which would inhibit their function leading to the development of an invasive and metastatic tumor.[6]

Summary and Conclusion

In summary, we have shown expression of p63 to correlate with survival in OSCCs, where high expression was seen in tumors with poorer survival after treatment. Although the results of the present study have shown considerable promising evidence for the applicability of p63 as a prognostic marker for OSCCs, the completeness of the follow-up is crucial in any study of survival. Hence, it is intended to extend the follow-up for a longer period (5?10 years) and also accommodating additional cases, which we intend, will aid toward validating the applicability of p63 as a prognostic marker for OSCCs. Furthermore, the usage and importance of Anneroth's multifactorial grading system over Broders' grading system in routine histopathological reporting for incisional biopsies of OSCCs is stressed.

Conflict of Interest

There are no conflicts of interest.

References

1. peight PM, Palmer S, Moles DR, Downer MC, Smith DH, Henriksson M, et al. The cost-effectiveness of screening for oral cancer in primary care. Health Technol Assess 2006;10:1-144, iii-iv.
2. ?Balasubramaniam AM, Sriraman R, Sindhuja P, Mohideen K, Parameswar RA, Muhamed Haris KT. Autofluorescence based diagnostic techniques for oral cancer. J Pharm Bioallied Sci 2015;7 Suppl 2:S374-7.
3. anaka T, Tanaka M, Tanaka T. Oral carcinogenesis and oral cancer chemoprevention: A review. Patholog Res Int 2011;2011:431246.
4. ukuda M, Kusama K, Sakashita H. Molecular insights into the proliferation and progression mechanisms of the oral cancer: Strategies for the effective and personalized therapy. Jpn Dent Sci Rev 2012;48:23-41.
5. raziano V, De Laurenzi V. Role of p63 in cancer development. Biochim Biophys Acta 2011;1816:57-66.
6. lores ER.?The roles of p63 in cancer. Cell Cycle 2007; 6: 30-4
7. uo X, Mills AA.?p63, cellular senescence and tumor development. Cell Cycle 2007; 6: 305-11
8. otto GP.?Crosstalk of Notch with p53 and p63 in cancer growth control. Nat Rev Cancer 2009; 9: 587-95
<li x


|?Figure.1p63 expression in (a) Grade I oral squamous cell carcinoma, (b) Grade II oral squamous cell carcinoma, (c) Grade III oral squamous cell carcinoma, (d) Normal oral mucosa (immunoperoxidase, original magnification ?400)






|?Figure.2Kaplan?Meier survival curves based on various criteria of classification

References

1. peight PM, Palmer S, Moles DR, Downer MC, Smith DH, Henriksson M, et al. The cost-effectiveness of screening for oral cancer in primary care. Health Technol Assess 2006;10:1-144, iii-iv.
2. ?Balasubramaniam AM, Sriraman R, Sindhuja P, Mohideen K, Parameswar RA, Muhamed Haris KT. Autofluorescence based diagnostic techniques for oral cancer. J Pharm Bioallied Sci 2015;7 Suppl 2:S374-7.
3. anaka T, Tanaka M, Tanaka T. Oral carcinogenesis and oral cancer chemoprevention: A review. Patholog Res Int 2011;2011:431246.
4. ukuda M, Kusama K, Sakashita H. Molecular insights into the proliferation and progression mechanisms of the oral cancer: Strategies for the effective and personalized therapy. Jpn Dent Sci Rev 2012;48:23-41.
5. raziano V, De Laurenzi V. Role of p63 in cancer development. Biochim Biophys Acta 2011;1816:57-66.
6. lores ER.?The roles of p63 in cancer. Cell Cycle 2007; 6: 30-4
7. uo X, Mills AA.?p63, cellular senescence and tumor development. Cell Cycle 2007; 6: 305-11
8. otto GP.?Crosstalk of Notch with p53 and p63 in cancer growth control. Nat Rev Cancer 2009; 9: 587-95
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12. ?Loljung L, Coates PJ, Nekulova M, Laurell G, Wahlgren M, Wilms T, et al. High expression of p63 is correlated to poor prognosis in squamous cell carcinoma of the tongue. J Oral Pathol Med 2014;43:14-9.
13. ?Thurfjell N, Coates PJ, Uusitalo T, Mahani D, Dabelsteen E, Dahlqvist A. et al.?Complex p63 mRNA isoform expression patterns in squamous cell carcinoma of the head and neck. Int J Oncol 2004; 25: 27-35
14. Lo Muzio L, Santarelli A, Caltabiano R, Rubini C, Pieramici T, Trevisiol L, et al. p63 overexpression associates with poor prognosis in head and neck squamous cell carcinoma. Hum Pathol 2005;36:187-94.
15. ?Califano J, van der Riet P, Westra W, Nawroz H, Clayman G, Piantadosi S.?et al.?Genetic progression model for head and neck cancer: Implications for field cancerization. Cancer Res 1996; 56: 2488-92
16. ?Vogelstein B, Lane D, Levine AJ.?Surfing the p53 network. Nature 2000; 408: 307-10
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19. ?Anneroth G, Hansen LS. A methodologic study of histologic classification and grading of malignancy in oral squamous cell carcinoma. Scand J Dent Res 1984;92:448-68.
20. Bryne M, Koppang HS, Lilleng R, Stene T, Bang G, Dabelsteen E. New malignancy grading is a better prognostic indicator than Broders' grading in oral squamous cell carcinomas. J Oral Pathol Med 1989;18:432-7.
21. Bryne M, Koppang HS, Lilleng R, Kjaerheim A. Malignancy grading of the deep invasive margins of oral squamous cell carcinomas has high prognostic value. J Pathol 1992;166:375-81.
22. ?Jakobsson PA, Eneroth CM, Killander D, Moberger G, M?rtensson B. Histologic classification and grading of malignancy in carcinoma of the larynx. Acta Radiol Ther Phys Biol 1973;12:1-8.
23. ?Anneroth G, Hansen LS, Silverman S Jr. Malignancy grading in oral squamous cell carcinoma. I. Squamous cell carcinoma of the tongue and floor of mouth: Histologic grading in the clinical evaluation. J Oral Pathol 1986;15:162-8.
24. Gu X, Coates PJ, Boldrup L, Nylander K.?p63 contributes to cell invasion and migration in squamous cell carcinoma of the head and neck. Cancer Lett 2008; 263: 26-34
25. Cho NH, Kim YB, Park TK, Kim GE, Park K, Song KJ.?P63 and EGFR as prognostic predictors in stage IIB radiation-treated cervical squamous cell carcinoma. Gynecol Oncol 2003; 91: 346-53
26. ?Moergel M, Abt E, Stockinger M, Kunkel M.?Overexpression of p63 is associated with radiation resistance and prognosis in oral squamous cell carcinoma. Oral Oncol 2010; 46: 667-71
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28. ?Monteiro LS, Delgado ML, Ricardo S, Amaral BD, Salazar F, Pacheco J, et al. Prognostic significance of CD44v6, p63, podoplanin and MMP-9 in oral squamous cell carcinomas. Oral Dis 2016;22:303-12.
29. Koga F, Kawakami S, Kumagai J, Takizawa T, Ando N, Arai G. et al.?Impaired Delta Np63 expression associates with reduced beta-catenin and aggressive phenotypes of urothelial neoplasms. Br J Cancer 2003; 88: 740-7

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