Penile squamous cell carcinoma (SCC) is a rare disease characterized by dismal prognosis in many patients. While in some cases the diagnosis is made at an advanced stage with early metastatic disease and death, in other cases the tumour persists as a localized lesion. Given the limited knowledge about penile SCC, there is a strong need to identify molecular markers distinguishing the carcinomas with metastatic potential from less aggressive tumours.
Sequencing libraries were constructed by KAPA HyperPlus kit with UMI adapters (Roche) and 300 ng of genomic DNA isolated from 112 FFPE SCC samples. The target was captured by KAPA HyperCapture custom panel consisting of 359 genes (1 163 kbp; which includes common tumour-related genes together with genes mutated in SCC; MSK-Impact) and sequenced by NextSeq (Illumina). The pipeline for biostatistical mutation analysis was constructed and optimized in CLC Genomics Workbench (Qiagen). The pipeline additionally includes tools for Tumour Mutation Burden (TMB) calculation, Microsatellite Instability (MSI) detection and Copy Number Variation (CVN) analysis.
Initial sequencing shows high target specificity (75-80 % of the UMI reads); target average coverage of 200-400x (depending on the overall quality of the sample) and avg. UMI group size of 3-5 reads. The achieved sensitivity enabled reliable TMB calculation (900-950 kbp of CDS with coverage > 100x) and MSI detection for each sample. However, reliable CNV analysis was possible in only approx. 50% of the samples due to the inferior binding ratio of AT/GC rich areas in older samples. The final analyses are in progress.
We managed to optimize high sensitivity DNA NGS analysis for the rare 112 penile SCC cases collected between 2003-2021. First NGS results indicate successful mutation, TMB, and MSI analyses in most samples, contrary to CNV analysis, which is more sample quality sensitive. Results should provide penile SCC genomic landscape overview, which will serve for further markers selection for reliable differentiation between carcinomas with metastatic potential from less aggressive tumours, further stratifying patient care. Supported by Ministry of Health of the Czech Republic (NU21J-03-00019).
The authors.
Ministry of Health of the Czech Republic (NU21J-03-00019).
All authors have declared no conflicts of interest.