Molekulare Charakterisierung von seltenen benignen und malignen aberranten Differenzierungen der Harnblase und Identifizierung möglicher Zielstrukturen für eine personalisierte Therapie

Maurer, Angela; Pradel, Gabriele (Thesis advisor); Knüchel-Clarke, Ruth (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Urinary bladder carcinoma predominantly show urothelial differentiation. Rarer squamous or glandular differentiated tumors of the urinary bladder are usually not considered in clinical trials due to the smaller number of cases. Furthermore, little is known about molecular alterations and possible developmental relationships of these tumors. Therefore, as part of this thesis, these rare tumors were further investigated in two projects using different molecular methods. In the first project a FFPE cohort of predominantly squamous differentiated urothelial carcinoma (MIX) and pure squamous cell carcinoma (PECA) of the urinary bladder was examined by immunohistochemistry (IHC) for the expression of seven markers (AR, ER-alpha, CD117, PD-1, PD-L1, EGFR, HER2) as well as for the loss of expression of four DNA mismatch repair (MMR) proteins (MSH2, MSH6, MLH1, PMS2) to identify potential therapeutically relevant targets in squamous differentiated bladder carcinoma (SQ-BLCA). Some cases with PD-L1 expression or MMR loss could be identified, which might benefit from immune checkpoint inhibitor therapy. Furthermore, high EGFR expression (95% positive, 61% overexpression) was shown for SQ-BLCA (n = 125), and therefore EGFR-inhibitior therapy was further investigated as a possible therapeutic option for SQ-BLCA. SCaBER (a squamous differentiated bladder carcinoma cell line) showed high sensitivity (GI50 = 0, 28 μM) towards EGFR inhibition with erlotinib and gefitinib in cell culture experiments. Combination treatments of SCaBER with one EGFR-inhibitor (erlotinib, gefitinib) and one chemotherapeutic agent (cisplatin, gemcitabine) each, which were also performed, showed predominantly synergistic effects for the combinations and concentrations tested (calculation of the drug effect of the combination treatments was performed using the Chou-Talaly method). For the combination of erlotinib and cisplatin, a concentration-dependent antagonism was observed for erlotinib concentrations below the GI50 of erlotinib (0,03 - 0, 26 μM). The high sensitivity of SCaBER to EGFR inhibition indicates that SQ-BLCA may respond well and possibly even better than other bladder cancer to EGFR-inhibitor therapy. However, possible antagonistic effects of EGFR inhibition with chemotherapeutic agents (especially cisplatin) should be considered in therapy or study planning (e.g. dose, timing, sequence). In the second project, a FFPE cohort of rare glandular bladder carcinomas (primary adenocarcinoma of the urinary bladder (BAC), n = 12; adenocarcinoma of the urachus (UAC), n=13; glandular differentiated urothelial carcinoma (UCg), n=11) and some glandular preinvasive precursor lesions (Cystitis glandularis (CG), n=3; intestinal metaplasia (IM), n = 1) was analyzed for genomic alterations in 21 genes using NGS and SNapShot analysis. IHC of SWI/SNF complex and MMR proteins was also performed. Comparison of the obtained results with publicly available data for muscle-invasive bladder carcinoma (BLCA) and colorectal adenocarcinoma (CORAD) was aimed at identifying similarities and differences in tumorigenesis, as some cases of glandular bladder carcinomas histologically resemble CORAD rather than BLCA. TP53 alterations were the most frequently detected alteration in both BAC, UAC, and UCg, whereas more "urothelial" TERT-promoter mutations were predominantly detected in UCg and more "colorectal" SMAD4 alterations were detected only in BAC and UCg. The low frequency of APC changes in all three entities compared with CORAD was striking. Furthermore, some potentially therapeutically relevant oncogenic alterations in BRAF, KRAS, NRAS and PIK3CA were detected. Activating FGFR3 mutations were not detected in any of the samples tested. IHC showed no case with MMR loss, but loss of expression of a SWI/SNF protein was detected in three cases. When comparing the changes in BAC, UAC, and UCg with BLCA and CORAD, UCg shows a distinctly "urothelial" pattern of aberrations, whereas BAC and UAC show both "urothelial" and "colorectal" changes. UAC and BAC thus form a separate group between CORAD and BLCA with similar alteration profiles and have more in common with each other than with BLCA or CORAD. In the three CG cases which were also sequenced, no pathological changes were detected in the 21 genes examined. However, the IM sample studied showed a TERT promoter mutation as well as a mutation of the FBXW7 gene, indicating a possible precancerous character of this lesion. In summary, this thesis has successfully used retrospective multicenter FFPE cohorts of rare urinary bladder carcinoma with two different methods to identify possible approaches for therapies as well as correlations in their development. For SQ-BLCA, EGFR was identified as a potential therapeutically relevant target for both MIX and PECA, with promising in vitro data. For glandular bladder tumors as well as glandular preinvasive precursors, first molecular data was collected (UCg) or existing data was supplemented (BAC, UAC, CG, IM), more similarities between BAC and UAC than previously described were revealed, and some potentially therapeutically relevant alterations were identified.