5370 - Evaluation of different diagnostic methods for identification of FGFR alteration in advanced urothelial carcinomas: Proficiency Results based on multiple RNA extraction kits and mutation detection methods

Background

Pan FGFR inhibitors such as Erdafitinib are approved in patients with advanced urothelial carcinoma (aUC) harboring FGFR alterations. Based on the necessity of sensitive and reproducible identification of these alterations in diagnostic routines this study aimed to compare different RNA isolation techniques and the QIAGEN therascreen® FGFR RGQ RT-PCR Kit with the SNaPshot mutational analysis.

Methods

Nucleic acids were extracted from 47 UC patients using the QIAGEN RNeasy® DSP FFPE Kit, STRATIFYER Xtract or Maxwell RNA (Promega) isolation kit. Mutations of the FGFR3 gene were detected using the SNaPshot method as well as via the QIAGEN therascreen® FGFR RGQ RT-PCR Kit. Intrinsic molecular subtypes were assessed using immunohistochemistry (IHC) of GATA3, FOXA1, CK20, CK5 and CD44 as well as RT-qPCR for gene expression of CK20 and CK5.

Results

All three different RNA isolation kits showed comparable amount of extracted RNA (Median 290, 177 and 226 ng/ul). Six hotspot FGFR3 mutations were identified with 100% concordance. The main difference were the necessary working hours: per 10 samples, 4 vs. 4.5 vs 8 hours were needed for Stratifyer, Maxwell or QIAGEN RNA isolation. In addition, of 47 analyzed samples 100% concordance between QIAGEN therascreen® FGFR RGQ RT-PCR Kit and SNapShot analysis were achieved with 18 mutations identified (14 S249C, 2 R248C and 2 Y375). Immunohistochemical basal marker profile (CD44 and CK5) as well as high CK5 expression was observed in 28 (6/28 FGFR3-altered). Luminal marker profile (IHC markers: GATA3, FOXA1 and CK20 as well as expression of CK20) was detected in 37 samples (5/37 with FGFR3 alteration, n.s.).

Conclusions

Different RNA extraction methods presented with comparable amount of nucleic acids with variant working hours. Moreover, a 100% concordance between the well established SNaPshot analysis compared to the QIAGEN therascreen® FGFR RGQ RT-PCR Kit for FGFR3 mutations was observed. So far, the frequency of FGFR3 mutations in aUC was not related to the type of UC as defined by marker profils.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

BRIDGE consortium.

Funding

Janssen.

Disclosure

R. Wirtz: Officer / Board of Directors: Stratifyer. M. Eckstein: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Janssen. A. Hartmann: Advisory / Consultancy: Janssen. All other authors have declared no conflicts of interest.