title: Fractal analysis of nuclear histology integrates tumor and stromal features into a single prognostic factor of the oral cancer microenvironment.
abstract: BACKGROUND
  The lack of prognostic biomarkers in oral squamous cell carcinoma (OSCC) has hampered treatment decision making and survival in OSCC remains poor. Histopathological features are used for prognostication in OSCC and, although useful for predicting risk, manual assessment of histopathology is subjective and labour intensive. In this study, we propose a method that integrates multiple histopathological features of the tumor microenvironment into a single, digital pathology-based biomarker using nuclear fractal dimension (nFD) analysis.
  
  
  METHODS
  One hundred and seven consecutive OSCC patients diagnosed between 1998 and 2006 in Calgary, Canada were included in the study. nFD scores were generated from DAPI-stained images of tissue microarray (TMA) cores. Ki67 protein expression was measured in the tumor using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA®). Lymphocytic infiltration (LI) was measured in the stroma from haematoxylin-eosin (H&E)-stained TMA slides by a pathologist.
  
  
  RESULTS
  Twenty-five (23.4%) and 82 (76.6%) patients were classified as high and low nFD, respectively. nFD was significantly associated with pathological tumor-stage (pT-stage; P = 0.01) and radiation treatment (RT; P = 0.01). High nFD of the total tumor microenvironment (stroma plus tumor) was significantly associated with improved disease-specific survival (DSS; P = 0.002). No association with DSS was observed when nFD of either the tumor or the stroma was measured separately. pT-stage (P = 0.01), pathological node status (pN-status; P = 0.02) and RT (P = 0.03) were also significantly associated with DSS. In multivariate analysis, nFD remained significantly associated with DSS [HR 0.12 (95% CI 0.02-0.89, P = 0.04)] in a model adjusted for pT-stage, pN-status and RT. We also found that high nFD was significantly associated with high tumor proliferation (P < 0.0001) and high LI (P < 0.0001), factors that we and others have shown to be associated with improved survival in OSCC.
  
  
  CONCLUSIONS
  We provide evidence that nFD analysis integrates known prognostic factors from the tumor microenvironment, such as proliferation and immune infiltration, into a single digital pathology-based biomarker. Prospective validation of our results could establish nFD as a valuable tool for clinical decision making in OSCC.
authors.affiliation: Department of Oncology, University of Calgary, Calgary, Canada. pbose@ucalgary.ca.|Current Address: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada. pbose@ucalgary.ca.
authors.forename: Pinaki
authors.initials: P
authors.lastname: Bose
authors.affiliation: Department of Cancer Epidemiology and Prevention Research, CancerControl Alberta, Alberta Health Services, Calgary, Alberta, T2N 2T9, Canada. nigel.brockton@albertahealthservices.ca.
authors.forename: Nigel T
authors.initials: NT
authors.lastname: Brockton
authors.affiliation: Department of Anatomic Pathology, Calgary Laboratory Services, Rockyview General Hospital, Calgary, Alberta, T2V 1P9, Canada. Kelly.Guggisberg@cls.ab.ca.
authors.forename: Kelly
authors.initials: K
authors.lastname: Guggisberg
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery, University of Calgary, Calgary, Alberta, T2N 4Z6, Canada. scnakone@ucalgary.ca.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Nakoneshny
authors.affiliation: Functional Tissue Imaging Unit, Translational Laboratories, Tom Baker Cancer Centre, Calgary, Alberta, T2N 4N2, Canada. Elizabeth.Kornaga@albertahealthservices.ca.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kornaga
authors.affiliation: Immunology and Inflammation Research, Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, Connecticut, 06877, USA. alexander.klimowicz@boehringer-ingelheim.com.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Klimowicz
authors.affiliation: Department of Physics, San Diego State University, San Diego, California, 92182-1233, USA. mtambasco@mail.sdsu.edu.
authors.forename: Mauro
authors.initials: M
authors.lastname: Tambasco
authors.affiliation: Department of Surgery, Division of Otolaryngology-Head and Neck Surgery, University of Calgary, Calgary, Alberta, T2N 4Z6, Canada. jdort@ucalgary.ca.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Dort
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000276:immunology / Q000401:mortality / Q000473:pathology*; D002467:Cell Nucleus / Q000473:pathology*; D018572:Disease-Free Survival; D005260:Female; D017709:Fractals; D006801:Humans; D053208:Kaplan-Meier Estimate; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000276:immunology / Q000401:mortality / Q000473:pathology*; D015999:Multivariate Analysis; D011379:Prognosis; D011446:Prospective Studies; D059016:Tumor Microenvironment