*4.2. Assessment of mRNA by qRT-PCR*

Tumor specimens were assessed by qRT-PCR as previously described [43]. In short, RNA was extracted from a single 10 μm curl of FFPE tissue and processed according to a commercially available bead-based extraction method (Xtract kit; Stratifyer Molecular Pathology GmbH, Cologne, Germany). RNA was eluted with 100 μL of elution buffer. DNA was digested, and RNA eluates were then stored at −80 ◦C until use.

The mRNA levels of *CXCL9*, *PD1*, *PD-L1*, *KRT5*, *KRT20*, *KI67* and the reference genes *Calmodulin2* (*CALM2*) and *Beta-2 microglobulin* (*B2 M*) were determined by a one-step qRT-PCR using the SuperScript III RT-qPCR system (Invitrogen, Waltham, MA, USA) and gene specific primer-probe combinations (Stratifyer). Each patient sample or control was analyzed in duplicate in an ABI Step One PCR System (ThermoFisher, Darmstadt, Germany) according to the manufacturers' instructions. Gene expression was quantified with a modification of the method by Schmittgen and Livak by calculating 40-ΔCt, whereas ΔCt was calculated as the difference in Ct between the test gene and the mean of the reference genes [38,44].
