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The majority of cancers are diagnosed using excised biopsy specimens. These are graded, using a gold-standard histopathology protocol based on haemotoxylin and eosin ('H  +  E') chemical staining. However the grading is done by eye and if the same biopsy is graded by different practitioners, they typically only agree ~70% of the time. The resulting overtreatment problem constitutes a massive unmet need worldwide. Our new "Digistain" technology, uses mid-infrared imaging to map the fractional concentration of nucleic acids, i.e. the nuclear-to-cytoplasmic chemical ratio (NCR) across an unstained biopsy section. It allows a quantitative 'Digistain index' (DI) score, corresponding to the NCR, to be reproducibly extracted from an objective physical measurement of a cancer. Our objective here is to evaluate its potential for aiding cancer diagnosis for the first time. We correlate the DI scores with H  +  E grades in a double-blind clinical pilot trial. Two adjacent slices were taken from 75 breast cancer FFPE blocks; one was graded with the standard H  +  E protocol, and also used to define a 'region of interest' (RoI). Digistain was then used to acquire a DI value averaged over the corresponding RoI on the other (unstained) slice and the results were statistically analysed. We find the DI score correlates significantly (p  =  0.0007) with tumor grade in a way that promises to significantly reduce the inherent subjectivity and variability in biopsy grading. The NCR is elevated by increased mitotic activity because cells divide when they are younger and, on average, become smaller as the disease progresses. Also, extra DNA and RNA is generated as the nuclear transcription machinery goes awry and nuclear pleomorphism occurs. Both effects make the NCR a recognized biomarker for a wide range of tumors, so we expect Digistain will find application in a very wide range of cancers.

Mid-infrared imaging in breast cancer tissue: an objective measure of grading breast cancer biopsies