title: Computational analysis of p63(+) nuclei distribution pattern by graph theoretic approach in an oral pre-cancer (sub-mucous fibrosis).
abstract: BACKGROUND
  Oral submucous fibrosis (OSF) is a pre-cancerous condition with features of chronic, inflammatory and progressive sub-epithelial fibrotic disorder of the buccal mucosa. In this study, malignant potentiality of OSF has been assessed by quantification of immunohistochemical expression of epithelial prime regulator-p63 molecule in correlation to its malignant (oral squamous cell carcinoma [OSCC] and normal counterpart [normal oral mucosa [NOM]). Attributes of spatial extent and distribution of p63(+) expression in the epithelium have been investigated. Further, a correlated assessment of histopathological attributes inferred from H&E staining and their mathematical counterparts (molecular pathology of p63) have been proposed. The suggested analytical framework envisaged standardization of the immunohistochemistry evaluation procedure for the molecular marker, using computer-aided image analysis, toward enhancing its prognostic value.
  
  
  METHODS
  In histopathologically confirmed OSF, OSCC and NOM tissue sections, p63(+) nuclei were localized and segmented by identifying regional maxima in plateau-like intensity spatial profiles of nuclei. The clustered nuclei were localized and segmented by identifying concave points in the morphometry and by marker-controlled watersheds. Voronoi tessellations were constructed around nuclei centroids and mean values of spatial-relation metrics such as tessellation area, tessellation perimeter, roundness factor and disorder of the area were extracted. Morphology and extent of expression are characterized by area, diameter, perimeter, compactness, eccentricity and density, fraction of p63(+) expression and expression distance of p63(+) nuclei.
  
  
  RESULTS
  Correlative framework between histopathological features characterizing malignant potentiality and their quantitative p63 counterparts was developed. Statistical analyses of mathematical trends were evaluated between different biologically relevant combinations: (i) NOM to oral submucous fibrosis without dysplasia (OSFWT) (ii) NOM to oral submucous fibrosis with dysplasia (OSFWD) (iii) OSFWT-OSFWD (iv) OSFWD-OSCC. Significant histopathogical correlates and their corroborative mathematical features, inferred from p63 staining, were also investigated into.
  
  
  CONCLUSIONS
  Quantitative assessment and correlative analysis identified mathematical features related to hyperplasia, cellular stratification, differentiation and maturation, shape and size, nuclear crowding and nucleocytoplasmic ratio. It is envisaged that this approach for analyzing the p63 expression and its distribution pattern may help to establish it as a quantitative bio-marker to predict the malignant potentiality and progression. The proposed work would be a value addition to the gold standard by incorporating an observer-independent framework for the associated molecular pathology.
authors: School of Medical Science and Technology, Kharagpur, India.
authors: Swarnendu
authors: S
authors: Bag
authors: School of Medical Science and Technology, Kharagpur, India.
authors: Sailesh
authors: S
authors: Conjeti
authors: School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India.
authors: Raunak Kumar
authors: RK
authors: Das
authors: Department of Oral and Maxillofacial Pathology, Guru Nanak Institute of Dental Sciences and Research, Kolkata, West Bengal, India.
authors: Mousami
authors: M
authors: Pal
authors: School of Medical Science and Technology, Kharagpur, India.
authors: Anji
authors: A
authors: Anura
authors: Department of Oral and Maxillofacial Pathology, Guru Nanak Institute of Dental Sciences and Research, Kolkata, West Bengal, India.
authors: Ranjan Rashmi
authors: RR
authors: Paul
authors: Department of Electrical and Electronics Communication Engineering, Indian Institute of Technology, Kharagpur, West Bengal, India.
authors: Ajoy Kumar
authors: AK
authors: Ray
authors: Department of Biochemistry, University of Calcutta, Kolkata, West Bengal, India.
authors: Sanghamitra
authors: S
authors: Sengupta
authors: School of Medical Science and Technology, Kharagpur, India.
authors: Jyotirmoy
authors: J
authors: Chatterjee
keywords: Dysplasia; graph theory; oral squamous cell carcinoma; oral submucous fibrosis; p63; quantitative immunohistochemistry