Assessing minimal tumour cell requirements and DNA input for accurate molecular analysis of FFPE tissues using MALDI-TOF MS

Abstract

Aim Determine in FFPE core biopsies and cell blocks, the minimal tumour cell count and DNA input to accurately detect mutations using MALDI- TOF mass spectrometry (MS) platform and Sequenom panel profiling >200 mutations in EGFR, BRAF, KRAS, NRAS and cKIT. Methods Core biopsies and cell blocks were quantitated for tumour cells/hpf in cases with and without mutations. Mean tumour cell number/5hpf was quantified representing 20% of total cell block/core biopsy volume. Student t-test compared tumour cell numbers from samples with and without mutations. Fractional unextended primer (UEP) versus input DNA was recorded against dilutions of DNA. Results Mean tumour cell count was 98 (range 10–335) and 126/hpf (range 28–214) respectively and was not significantly different when compared with mutation negative cases. Dilution data showed that 0.91 to 1.00 of UEP used in all reactions containing DNA template (range 4–40 ng). 0.01 UEP used in assays with no DNA template. Allelic ratio 5%. Discussion MALDI-TOF MS platform accurately detects mutations to a level of 98 tumour cells/hpf (mean) of FFPE sections and is accurate up to a total input of DNA to 4 ng. This cut off is important for accurate diagnosis of molecular mutations in specimens with minimal tumour cells.