The Pan-Cytokeratin Staining Intensity and Fractal Computational Analysis of Breast Tumor Malignant Growth Patterns Prognosticate the Occurrence of Distant Metastasis

Abstract

Improved prognosis of breast cancer outcome could prolong patient survival by reliable identification of patients at high risk of metastasis occurrence which could benefit from more aggressive treatments. Based on such clinical need, we prognostically evaluated the malignant cells in breast tumors, as the obvious potential source of unexploited prognostic information. The patient group was homogeneous, without any systemic treatments or lymph node spread, with smaller tumor size (pT1/2) and a long follow-up. Epithelial cells were labeled with AE1/AE3 pan-cytokeratin antibody cocktail and comprehensively analyzed. Monofractal and multifractal analyses were applied for quantification of distribution, shape, complexity and texture of malignant cell clusters, while mean pixel intensity and total area were measures of the pan-cytokeratin immunostaining intensity. The results surprisingly indicate that simple binary images and monofractal analysis provided better prognostic information then grayscale images and multifractal analysis. The key findings were that shapes and distribution of malignant cell clusters (by binary fractal dimension; AUC = 0.29), their contour shapes (by outline fractal dimension; AUC = 0.31) and intensity of the pan-cytokeratin immunostaining (by mean pixel intensity; AUC = 0.30) offered significant performance in metastasis risk prognostication. The results reveal an association between the lower pan-cytokeratin staining intensity and the high metastasis risk. Another interesting result was that multivariate analysis could confirm the prognostic independence only for fractal but not for immunostaining intensity features. The obtained results reveal several novel and unexpected findings highlighting the independent prognostic efficacy of malignant cell cluster distribution and contour shapes in breast tumors.

Small Cell Lung Cancer Therapeutic Responses Through Fractal Measurements: From Radiology to Mitochondrial Biology

“…The fractal dimension box counting method has been capable of differentiating between malignant and normal tissue in a number of neoplastic studies involving hepatocellular carcinoma [57], endometrioid endometrial adenocarcinoma [58], oral squamous cell carcinoma [59], breast cancer [60,61], prostate adenocarcinoma [62], and renal cell carcinoma [63]. However, it was not until more recently that FD was utilized alongside LC as an additional parameter of fractal geometry to quantify colorectal adenocarcinomas [64,65], breast cancer [66], and cervical cancer [67].…”

Analysis of Spatial Distribution and Prognostic Value of Different Pan Cytokeratin Immunostaining Intensities in Breast Tumor Tissue Sections

“…Immunostaining of keratin reveals the shapes, sizes, and distribution of malignant cell patches in cancers caused by the neoplastic transformation of epithelial cells and thus presents a paramount prognostic potential. While the cytokeratin staining of malignant nests [ 16 ] and their prognostic value in breast tumors [ 17 , 18 , 19 , 20 , 21 ] have been established, this is the first systemic analysis of the pan cytokeratin immunostaining based on its intensity. We revealed the spatial distribution and prognostic value for each of the produced seven narrow ranges of pan cytokeratin immunostaining intensities.…”

“…Our previous report indicated that the low intensity of pan cytokeratin immunostaining is associated with a higher metastasis risk [ 18 ]. The separation of immunostaining intensities allowed us to extend the previous study by providing a detailed insight into the prognostic performance of pan cytokeratin.…”

“…Malignant epithelial cells are usually organized in tumor nests of variable sizes and shapes scattered throughout stroma which contains mostly fibroblasts, some immune cells, and few epithelial buds [ 17 ]. We have previously found that low pan cytokeratin staining intensity is associated with a poor breast cancer outcome [ 18 ].…”

“…Intrigued by our previous finding that low pan cytokeratin staining intensity is associated with a poor outcome [ 18 ], we set out to perform a detailed computational investigation of the spatial distribution and prognostic value of the discrete pan cytokeratin staining intensities.…”