Definition of parameters and thresholds to detect MYCN amplification in retinoblastomas

Retinoblastoma is a malignant childhood neoplasm where MYCN amplification defines a subset of tumors with worse prognosis. FISH (fluorescence in situ hybridization) represents a fast and reliable method to measure gene copy numbers in various tumors but has not yet been systematically evaluated in retinoblastoma. In this study, we define criteria for FISH detection of MYCN amplification in a systematic unbiased approach by using a well characterized series of 44 clinical retinoblastoma samples. We (i) determined potential measurements and parameters by a comprehensive literature review, (ii) analyzed a retrospective cohort of samples with known MYCN amplification, (iii) determined statistically measurements and cut-offs, which allow reliable detection of amplified tumors, and (iv) applied these criteria to a prospective cohort. We demonstrate that average gene copy number (AVGCN) of MYCN/cell, MYCN/CEN2 ratio, and MYCN-CEN2 difference reveal the lowest statistical variance in amplified samples, if at least 50 cells were counted. The combination of these three parameters and cut-offs, namely AVGCN ≥ 10, MYCN/CEN2 ratio ≥ 3, and MYCN-CEN2 difference ≥ 8, allowed a reliable distinction between amplified and non-amplified cases. The prevalence of MYCN-amplified cases was 4/33 (12.1%) among prospective clinical samples indicating a higher percentage of positive tumors than previously reported. Our data provide the first evidence for well-grounded MYCN FISH criteria in retinoblastoma.