ACTH-secreting pituitary carcinoma with TP53, NF1, ATRX and PTEN mutations Case report and review of the literature

Pituitary carcinoma(PC) is a devastating disease with high mortality rate. The molecular background for the development of this rare tumor entity and the mechanism of metastasis are unknown. There are only a few case reports and singular larger case series considering only restricted number of sequenced genes by the PC [ 8 , 11 , 12 , 13 , 14 , 15 , 16 ]. From our case report and review of the literature we propose special improvements of diagnostic approach in case of PC suspicion by application of NGS for at least several genes found in PC biology ATRX, CDKN2A, CDKN2B, H-Ras, NF1, PTEN, SDHB, MSH2, and TP53 genes, as well as these involved in PitNET biology e.g., USP8 or USP48. This technique may be implemented into clinical practice to detect the genetic drivers for planning targeted therapies for PC as several identified gene mutations are potential targets for pharmacological therapy. Tumor tissue was fixed in 4% paraformaldehyde directly after surgical resection, dehydrated, embedded in paraffin, and then sectioned at 2 µm according to standard lab protocols. For all immunohistochemical stains paraffin-embedded tissue was deparaffinized, rehydrated. All immunohistochemical stainings were performed using automatic staining machines (Ventana BenchMark TX and Ventana Discovery Ultra), Roche Diagnostics, Mannheim, Germany). The following primary antibodies were used: ATRX(1:400, HPA001906, Atlas Antibodies, Bromma, Sweden), GH(1:1000, PA0704, Leica Biosystems, Buffalo Grove, IL, United States), Prolactin(1:1000, ab11301, Abcam, Cambridge, UK), TSH(1:10000, Epredia, Portsmouth, NH, United States), ACTH(1:500, RP045, Diagnostic BioSystems, Hanhgzhou, China), FSH(1:200, M3504, DAKO, Glostrup, Denmark), LH(1:300, M3502, DAKO, Glostrup, Denmark), Pit-1(1:200, HPA041646, Sigma-Aldrich, Taufkirchen, Germany), T-pit(1:1500, AMAb91409, Atlas Antibodies, Bromma, Sweden), TTF-1(1:50, M3575, DAKO, Glostrup, Denmark, Ki-67(1:750, 275R-15, Cell Marque, Rocklin, CA, United States), p53(1:800, M7001, DAKO, Glostrup, Denmark), chromogranin(1:800, M0869, DAKO, Glostrup, Denmark, synaptophysin (1:500, M7315, DAKO, Glostrup, Denmark), EGFRvIII (1:250, T170B620, Absolute Antibody, Oxford, Great Britain). The tissue was further sectioned 10 times at 10 µm according to standard lab protocol. HE stains were utilized for the selection of tumor area. Tumor tissue was then manually microdissected using a fine needle under an inverted microscope. The DNA was isolated using Maxwell RSC DNA FFPE Kit (AS1450, Promega). DNA panel sequencing was done using a self-customized targeted panel, manufactured by Qiagen (CDHS-21330Z-424). This panel targets the complete coding regions and splice-sites of six genes (ATRX, EGFR, NF1, NF2, PTEN, TP53), as well as mutation hotspots of further 14 genes (AKT, BRAF, CTNNB1, FGFR1, FGFR2, H3F3A, HIST1H3B, HIST1H3C, IDH1, IDH2, KRAS, PI3CA, PIK3R1, TERT-promoter). The library was constructed according to the manufacturer’s instructions. Sequencing was done on an Illumina MiniSeq sequencing system (paired-end, 2 × 151 bp, average coverage 500x). Data were analyzed with the Qiagen CLC Genomics workbench, using a self-customized workflow. Variants were annotated with information from the 1000 genome project, dbSNP, ClinVar and COSMIC. Only variants with an allele frequency ≥ 5% and a total target coverage of ≥40x were analyzed further. Variants not annotated by ClinVar were additionally analyzed with VarSome ( www.varsome.com ). Primers previously described by Sbiera et al. were used for both amplification and sequencing the specific regions of USP8 and USP48 genes [ 37 ]. A PCR reaction volume of 25 μl containing 40 ng of template DNA, 0.1 μM of each primer, 100 μM dNTPs (deoxyribonucleotide triphosphates), Dream Taq polymerase buffer, and 1.25 U Dream Taq DNA Polymerase was prepared and amplified after initial denaturation at 95 °C for 3 min followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 45 s, and elongation at 72 °C for 60 s. PCR products were sequenced using Mix2Seq Kit NightXpress and performed by Eurofinsgenomics.