Fig. 2

Intratumoral heterogeneity and Copy number variation. A Non-synonymous mutations were unevenly distributed among patients and patient samples. Each row represents a patient sample and each column represents one non-synonymous mutation. B Copy number variation analyses showed marked intratumoral heterogeneity (maroon denotes amplification and dark blue deletion). C–J Case #5 yields homogeneous amplifications in MDM2 (all ten samples) and a heterogeneous amplification of CD274 (PD-L1; 2/10 samples including a single lymph node metastasis). MDM2 amplification was confirmed independently in all samples, i.e., primary tumor (C, F; non-neoplastic mucosa as a reference in D) and all lymph node metastases (E). Amplification of CD274 was associated with strong PD-L1 immunostaining only in a single sample (I) and only in a single lymph node metastasis. All other samples were immunonegative for PD-L1 (J). The PD-L1-positive tumor area (G) showed a phenotype, different from the remainder (H). Primary tumor (C); corresponding non-neoplastic mucosa (D); lymph node metastasis corresponding to sample G13390 (E, G, I) and a sample of the primary tumor without CD274 amplification (PD-L1-immunonegative; F, H, J). Fluorescence in situ hybridization (orange signal: MDM2, green signal: reference centromere; C–F); H&E staining (G; H) and anti-PD-L1-immunostaining (I, J). Original magnifications 1000-fold (C–F), 400-fold (G–J)