RT-PCR assay to detect FGFR3::TACC3 fusions in formalin-fixed, paraffin-embedded glioblastoma samples

Abstract

Background

One targeted treatment option for isocitrate dehydrogenase (IDH)-wild-type glioblastoma focuses on tumors with fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions. FGFR3::TACC3 fusion detection can be challenging, as targeted RNA next-generation sequencing (NGS) is not routinely performed, and immunohistochemistry is an imperfect surrogate marker. Fusion status can be determined using reverse transcription polymerase chain reaction (RT-PCR) on fresh frozen (FF) material, but sometimes only formalin-fixed, paraffin-embedded (FFPE) tissue is available.

Aim

To develop an RT-PCR assay to determine FGFR3::TACC3 status in FFPE glioblastoma samples.

Methods

Twelve tissue microarrays with 353 historical glioblastoma samples were immunohistochemically stained for FGFR3. Samples with overexpression of FGFR3 (n = 13) were subjected to FGFR3::TACC3 RT-PCR on FFPE, using 5 primer sets for the detection of 5 common fusion variants. Fusion-negative samples were additionally analyzed with NGS (n = 6), FGFR3 Fluorescence In Situ Hybridization (n = 6), and RNA sequencing (n = 5).

Results

Using RT-PCR on FFPE material of the 13 samples with FGFR3 overexpression, we detected an FGFR3::TACC3 fusion in 7 samples, covering 3 different fusion variants. For 5 of these FF was available, and the presence of the fusion was confirmed through RT-PCR on FF. With RNA sequencing, 1 additional sample was found to harbor an FGFR3::TACC3 fusion (variant not covered by current RT-PCR for FFPE). The frequency of FGFR3::TACC3 fusion in this cohort was 9/353 (2.5%).

Conclusions

RT-PCR for FGFR3::TACC3 fusions can successfully be performed on FFPE material, with a specificity of 100% and (due to limited primer sets) a sensitivity of 83.3%. This assay allows for the identification of potential targeted treatment options when only formalin-fixed tissue is available.

Successful (targeted) therapy options for isocitrate dehydrogenase (IDH)-wild-type glioblastoma (GBM) are greatly desired.¹ Rapid advances in tumor genotyping are creating opportunities for the identification of targeted treatments from which at least a subset of patients might benefit. One such approach focuses on targeting gene fusions involving fibroblast growth factor receptor 3 (FGFR3) and the coiled-coil domain of the transforming acidic coiled-coil-containing protein 3 (TACC3). This is the most prevalent identified gene fusion in adult gliomas, occurring in 3.0%–8.3% of IDH-wild-type gliomas.^2–5FGFR3::TACC3 fusion occurs mostly in IDH-wild-type GBM,⁶ including in histologically lower-grade IDH-wild-type diffuse astrocytomas with molecular features of GBM.⁷ Interestingly, a better survival rate has been reported for GBM cases positive for FGFR3::TACC3 fusion compared to cases without this fusion.^6,8,9

The biology of FGFR::TACC fusion proteins is not completely understood,¹⁰ but it is hypothesized that the FGFR3 fusion leads to loss of the microRNA (miR)-99a binding site, resulting in FGFR3 overexpression.¹¹ The oncogenic effects may then be caused by accumulation of the proteins in the nucleus and direct phosphorylation of substrates that are essential for mitosis while concurrent activation of growth-promoting pathways allows the cells to remain viable.¹² More recently, it was suggested that the FGFR3::TACC3 fusion is oncogenic due to the activation of oxidative phosphorylation and mitochondrial metabolism.¹³ In addition, FGFR3::TACC3 fusion protein might be able to induce mitotic segregation defects leading to aneuploidy, when localized at the mitotic spindle poles.^5,14

Two phase I trials of Erdafitinib (JNJ-42756493), an oral pan-FGFR inhibitor, have independently shown the response of 3 patients (n = 2 in 1 trial, n = 1 in the other trial) with FGFR3::TACC3 fusion-positive GBM.^2,13,15 Wang et al. described 1 case in which a patient with FGFR3::TACC3 fusion-positive, TERT promoter mutant GBM was treated with Anlotinib (multi-target tyrosine kinase inhibitor) and temozolomide, resulting in a partial response that was maintained for more than 17 months.¹⁶ There are several ongoing trials that include patients with glioblastoma, IDH-wild-type, to test treatment targeting FGFR-signaling (not all limited to FGFR fusion-positive tumors).¹⁷

It is essential to carefully select patients who are eligible for targeted treatments.^6,9 Thus, it is important to identify FGFR3::TACC3 fusions in a specific and sensitive manner. However, the detection can be challenging because of the structural heterogeneity of the FGFR3::TACC3 fusions, with 15 distinct breakpoints described.¹⁸ Ideally, fusion detection should be performed using RNA-based techniques, to confirm whether the case meets the genomic criteria for trial inclusion.

Although it has been suggested that FGFR3::TACC3 fusion-positive GBM cases can have a specific morphology (tumor cells with monomorphous ovoid nuclei, nuclear palisading, a fine network of capillary vessels, micro-calcifications, and desmoplasia¹⁹), a recent case series showed that morphology alone is not reliable for identification of cases that are suspect for FGFR3::TACC3 fusion.²⁰ For screening purposes, it is possible to stain accumulated FGFR3::TACC3 proteins through immunohistochemistry (IHC) with an antibody against the N-terminal of FGFR3.¹² In normal brain tissue or glioma without FGFR3 aberration, this staining should be negative due to the suppression of FGFR3 protein expression by miRNA 99a.⁴ Staining for FGFR3 can be diffuse cytoplasmic, sometimes nuclear and/or membranous.¹¹ The sensitivity of FGFR3 IHC to detect FGFR3::TACC3 fusion was reported to be 100% and the specificity 88% for samples with moderate-to-strong staining intensity.¹¹

Several options are available to test for the presence of FGFR3::TACC3 fusions. A real-time (reverse transcription) polymerase chain reaction (RT-PCR) assay has been developed previously to detect all FGFR3::TACC3 variants using a single primer set,² but this can only be used for fresh frozen (FF) samples. Targeted RNA sequencing would be useable in this context; however, many labs do not have access to this technique. Next-generation sequencing (NGS) is not suitable for FGFR3::TACC3 fusion detection, as targeted DNA NGS panels that are used for mutation detection do not cover intronic regions to detect the breakpoints and the change in coverage caused by the duplication is too small to allow for reliable detection. Fluorescence In Situ Hybridization (FISH) is a technique that can be performed on FFPE material for fusion detection, but this is not suitable for the detection of FGFR3::TACC3 fusion due to the close proximity of FGFR3 and TACC3 on chromosome 4.²

While formalin-fixation and paraffin embedding (FFPE) is the most commonly used manner of tissue preservation in pathology, many molecular techniques perform suboptimal on FFPE samples. This is mainly due to DNA fragmentation. This fragmentation occurs significantly less in FF material, which is why FF samples are preferred for many molecular techniques. However, FF samples are often not available. In this study, we examine the sensitivity and specificity of an RT-PCR assay designed to detect 5 FGFR3::TACC3 fusion variants in FFPE GBM samples. The present study was designed to investigate the feasibility of RT-PCR as a tool for FGFR3::TACC3 fusion detection in FFPE samples.

Materials and Methods

Tissue Samples

In this study, we analyzed 12 tissue microarrays (TMAs; University Medical Center, Utrecht) with a total of 353 historical glioblastoma samples, constructed from FFPE tissue blocks. The samples dated from 2005 to 2014. IDH1 immunohistochemical staining was performed on the TMA’s: 94.2% of samples were negative for IDH1 staining. IDH mutation analysis was not routinely investigated; therefore, IDH1 mutation status was not confirmed at DNA level and IDH2 mutation status of the samples is unknown. It is estimated that >94% of samples would now be diagnosed as “glioblastoma, IDH-wildtype” and a few cases as “high-grade astrocytoma, IDH-mutant” using the current diagnostic criteria. However, this should not impede the aim of the study. The TMAs contained 3 cores (0.6 mm) from different areas of each tumor. Snap-frozen samples were collected with informed consent of the patients (METC 09-420 and 16-342).

FGFR3 N-Terminus Immunohistochemistry

TMA slides (4 μm) were stained with a mouse monoclonal antibody raised against the N-terminal region of FGFR3 (specifically against amino acids 15-124 of FGFR3 of human origin, SC-13121, Santa Cruz Biotechnology, Dallas, TX; dilution 1:200), using the Ventana Benchmark Ultra automated staining instrument (Ventana Medical Systems, Tucson, AZ), according to manufacturer instructions. Expression of FGFR3 was semiquantitatively scored by 1 neuropathologist (W.H.), based on staining intensity: 0 (negative, N), 1+ (low, L), 2+ (high, H), as described previously by Theelen et al.²¹ The highest score of the 3 TMA tissue cores from each sample was used to select cases for further analysis, to compensate for intratumoral heterogeneity. Of the samples that showed at least 1 TMA core with positive FGFR3 staining, corresponding whole-tumor FFPE slides were stained as well.

RNA Extraction and cDNA Synthesis

Total RNA was extracted from FF tissue using RNeasy Mini Kit (Qiagen, Hilden, Germany) and from FFPE tissue using RNeasy FFPE kit (Qiagen, Hilden, Germany) according to manufacturer instructions. For 2 samples (samples 7 [FFPE] and 14 [FFPE], analyzed at a later point in time), RNA was isolated using Maxwell RSC automated instrument according to manufacturer instructions. One to three micrograms of total RNA were retrotranscribed using Superscript III (Invitrogen), Oligo-dT15 (Promega), and Random primers (Promega) according to respective manufacturers’ instructions.

RT-PCR on FF

RT-PCR of FGFR3::TACC3 was performed on FF material by applying the protocol from Lasorella et al.¹² The PCR mix consisted of 2.5 μL PCR buffer, 1 μL dNTP (10 mM), 0.75 μL forward primer, 0.75 μL reverse primer (0.3 uM), 0.5 μL MgSO₄ (50 mM), 0.2 μL platinum Taq DNA polymerase (Thermo Fisher Scientific), 1 μL (50 ng) complementary DNA (cDNA), and up to 25 μL Milli-Q. The primers used for FF were FGFR3exon11-Forward: 5’-CGTGAAGATGCTGAAAGACGATG-3’ and TACC3exon14-Reverse: 5’-AAACGCTTGAAGAGGTCGGAG-3’. Amplification conditions were: 94°C for 3 minutes, 40 cycles “94°C—30 seconds, 58°C—30 seconds, 68°C—1 minute 40 seconds” and finally 68°C for 7 minutes. The RT-PCR assay was first tested on GBM-1123, a case known to be positive for the FGFR3::TACC3 fusion.⁵ Subsequently, GBM-1123 was used as a positive control.

RT-PCR on FFPE

The PCR protocol and reagents used for FFPE material were the same as for FF material. Except, instead of 1 μL cDNA, 10 μL cDNA was used as input for the PCR reaction, and different primers were used.

For FFPE samples, 5 primer sets were created for 5 of the most commonly observed fusion variants (Supplementary Table 2), covering 60% of previously reported FGFR3::TACC3 fusions in human gliomas.¹⁸ These covered the fusion variants: FGFR3ex17::TACC3ex11 (20%), 17-8 (14%), 17-10 (11%), 17-6 (9%), 18-5 (6%). Forward primers were created for FGFR3 and reverse primers were created to be reverse complimentary to different regions in the TACC3 gene. The PCR program was the same as for FF. The quality of the cDNA from FFPE tissue was verified through the presence of the reference genes GAPDH (100 base pairs [bp] amplified product) and β-actin (274 bp amplified product).

Synthetic DNA sequences were used as a positive control to compare FGFR3::TACC3 positive FFPE samples to the predicted amplicon lengths (Figure 1). After amplification by fusion-specific PCR, agarose gel electrophoresis was performed to confirm the presence of the product based on the expected length of the amplicon. Next, the PCR products were purified and subjected to Sanger sequencing. BLAST analysis was performed using FGFR3 (NM_000142) and TACC3 (NM_006342) reference sequences.

Figure 1.

Schematic representation of the primers used in the RT-PCR assay for FFPE material and the expected amplicon length of the product in base pairs (bp). For the exact sequences and references, see Supplementary Table 2.

Fluorescence In Situ Hybridization

FGFR3 IHC positive, but FGFR3::TACC3 fusion-negative samples were analyzed with FISH for potential FGFR3 amplification. FFPE slides were hybridized with an IGH/FGFR3 (IGH, immunoglobulin heavy locus) translocation dual fusion FISH probe (Cytocell, Cambridge, UK). FFPE slides were prepared for FISH using protocol as described by Richardson et al.²² To determine FGFR3 gene copy numbers, 50 tumor cell nuclei per tumor were assessed on FGFR3 and IGH gene copy numbers at 100× magnification using a Leica DM5500 B microscope system with Leica application suite advanced fluorescence software (Leica Microsystems, Rijswijk, The Netherlands). An FGFR3-IGH ratio was calculated and defined as <1.5: normal copy numbers, 1.5–2.0: copy number gain, >2: gene amplification.²³

Next-Generation Sequencing

FGFR3 IHC positive, but FGFR3::TACC3 fusion-negative samples were also analyzed with NGS²⁴ to detect a possible mutation in FGFR3. The panel includes FGFR3 codon 248–277, 368–402, 632–653, 691–719, and 772-807. Other mutations/amplifications found were also reported (Supplementary Table 1).

RNA Sequencing

Total RNA was isolated from FF samples using Maxwell RSC automated instrument according to manufacturer instructions, aiming for a minimum concentration of 20 ng/µL. Library preparation, sequencing, and data analysis were performed as described in the article by J. Hehir-Kwa et al.²⁵ In short, RNAseq libraries were generated with 300 ng RNA using the KAPA RNA HyperPrep Kit with RiboErase (Roche) and subsequently sequenced on a NovaSeq 6000 system (2 × 150 bp) (Illumina). The RNA sequencing data were processed as per the GATK 4.0 best practices workflow for variant calling, using a wdl- and cromwell-based workflow (https://gatk.broadinstitute.org/hc/enus/sections/360007226651-Best-Practices-Workflows). This included performing quality control with Fastqc (version 0.11.5) to calculate the number of sequencing reads and the insert size, Picard (version 2.20.1) for RNA metrics output and MarkDuplicates. The raw sequencing reads were aligned using Star (version 2.7.0f) to Genome Reference Consortium Human Build 38 and gencode version 29. Gene fusion detection was performed using Star fusion (version 1.6.0).²⁶

Results

FGFR3 IHC Screening

TMA slides were screened for FGFR3 overexpression using FGFR3 IHC. Out of 353 samples, 308 were interpretable (with at least 1 evaluable tissue core) (Figure 2). Out of 308 glioblastoma samples, 13 samples (4.2%) showed low (1+, n = 10) or high (2+, n = 3) FGFR3 staining intensity (Table 1). An example of high- and low-intensity staining patterns is shown in Supplementary Figure 1. Expression of FGFR3 fusion protein was verified on whole FFPE tissue slides using the same N-terminal FGFR3 antibody. Of these, 4 showed homogeneous expression of the tumor tissue, whereas 9 showed intratumoral heterogeneity for FGFR3 staining with both positive and negative tumor areas. Both homogeneous and heterogeneous staining patterns varied in intensity. All cases positive for FGFR3 IHC on TMA were also positive on their corresponding whole slide, though in a few cases, staining intensity differed between TMA and whole slide, as exemplified in Table 1.

Figure 2:

Flow diagram showing sample selection and results. The cohort consisted of 353 historical glioblastoma samples in 12 tissue microarrays (TMAs). FGFR3 IHC was interpretable for 308 samples, of which 13 showed FGFR3 overexpression (1+ or 2+). Out of these 13 samples, 7 were positive for FGFR3::TACC3 fusion upon testing of FFPE material with RT-PCR. One additional sample with FGFR3::TACC3 fusion was found with RNA sequencing. The remaining samples were all negative for FGFR3 amplification (tested with fluorescence in situ hybridization [FISH]), and only 1 sample had an FGFR3 mutation upon testing with next-generation sequencing.

Table 1:

Overview of all 13 cases with positive IHC on TMAs

Case ID	Dx	FGFR3 IHC TMA	FGFR3 IHC whole slide	RT-PCR (FFPE)	RNAseq (FF)
1	GBM	1+	Heterogeneous, 1+	negative	No reads
2	GBM	1+	Heterogeneous, 1+	FGFR3ex17::TACC3ex10	—
3	GBM	1+	Heterogeneous, 1+	negative	negative
4	GBM	1+	Homogeneous, 2+	FGFR3ex17::TACC3ex11	—
5	GBM	2+	Homogeneous, 2+	FGFR3ex17::TACC3ex11	—
6	GBM	2+	Heterogeneous, 2+	FGFR3ex17::TACC3ex11	—
7	GBM	1+	Homogeneous, 2+	FGFR3ex17::TACC3ex8	—
8	GBM	2+	Homogeneous, 2+	FGFR3ex17::TACC3ex11	—
9	GBM	1+	Heterogeneous, 2+	FGFR3ex17::TACC3ex11	—
10	GBM	1+	Heterogeneous, 1+	negative	N/A
11	GBM	1+	Heterogeneous, 2+	negative	FGFRex17::TACC3ex12
12	GBM	1+	Heterogeneous, 1+	negative	negative
13	GBM	1+	Heterogeneous (very focal), 1+	negative	negative

RT-PCR Assay for Detection of FGFR3::TACC3 Fusions on FF/FFPE

FGFR3::TACC3 RT-PCR was performed on the 13 FGFR3 IHC-positive samples to check for the presence of a fusion. Together, the RT-PCR assay on 13 FFPE samples revealed 7 FGFR3::TACC3 positive samples (Table 1) consisting of the 3 most commonly observed fusion variants: FGFR3ex17::TACC3ex11 (n = 5), FGFR3ex17::TACC3ex8 (n = 1), and FGFR3ex17::TACC3ex10 (n = 1). An example of each is shown in Figure 3. The PCR products including the breakpoints of the 3 fusion variants were validated by Sanger sequencing (Supplementary Figure 2). For 5 cases for which FF material was available, the presence of the fusion was confirmed using RT-PCR on FF tissue.

Additional Molecular Analyses to Explain FGFR3 Overexpression

To investigate the possible alternative explanations of FGFR3 overexpression in the 6 FGFR3 IHC positive but FGFR3::TACC3 fusion-negative samples, FISH was performed to assess for FGFR3 copy-number gain or FGFR3 amplification. All 6 samples showed a normal (n = 2) FGFR3 gene copy number. To check whether the FGFR3 overexpression in these samples could be explained by an activating point mutation in FGFR3, the samples were subjected to NGS using a Cancer Hotspot panel. In one of the 5 samples, an inactivating FGFR3 mutation was detected: Case 13, FGFR3 mutation p.(Asp785Argfs*31), which does not explain FGFR3 expression with IHC. The other genetic alterations that were detected through NGS are reported in Supplementary Table 1. Finally, RNA sequencing revealed 1 additional case with an FGFR3::TACC3 fusion: Case 11 showed variant FGFR3ex17::TACC3ex12, which is not covered by the 5 primer sets we used in the RT-PCR assay for FFPE material.

Sensitivity, Specificity, and Predictive Value of RT-PCR on FFPE

Sensitivity, specificity, and predictive value could only be calculated for the limited number of cases for which both RT-PCR on FFPE and RT-PCR on FF or RNA sequencing were performed (n = 9). This analysis does not include 2 FGFR3 IHC-positive cases for which RNA sequencing was not successful and 2 fusion-positive cases upon RT-PCR on FFPE, for which no FF tissue was available.

Positive predictive value and negative predictive value for RT-PCR test on FFPE were both found to be 100%. Also, the specificity was 100%. As expected (because of the fact that the current RT-PCR method for FFPE does not cover all possible fusion variants), sensitivity was 83.3% (Supplementary Table 3).

Discussion and Conclusions

The occurrence of FGFR aberrations in GBM IDH-wild-type is relatively low: frequencies in the range of ~3% for FGFR rearrangements⁵ to ~8% for all types of FGFR aberrations²⁷ are reported. FGFR3 is most frequently altered in this context, but several types of tests would be needed to detect all possible changes involving FGFR3 (i.e., mutations, amplifications, and translocations). Meanwhile, as FGFR3 accumulates at high levels in FGFR3::TACC3 fusion-positive cases, positive FGFR3 immunostaining can be used for prescreening of cases that require further analysis to sort out the exact nature of the FGFR3 alterations.

In this study, we showed that RT-PCR for the detection of FGFR3::TACC3 fusions can successfully be performed on FFPE material, as well as on FF material. Out of 14 samples with FGFR3 overexpression on IHC, an FGFR3::TACC3 fusion variant was detected using RT-PCR in 8 FFPE samples. For 5 of these, FF was available, and the result was confirmed using FF material. RNA sequencing revealed one more sample with an FGFR3ex17::TACC3ex12 fusion, which was not included in our RT-PCR assay. In 1 sample with FGFR3 overexpression but no FGFR3::TACC3 fusion on RT-PCR and RNAseq, an inactivating FGFR3 mutation was found. It was previously suggested that activating FGFR3 mutations do not occur in diffuse gliomas due to suppression of FGFR3 protein expression by miRNA-99A.¹¹ However, recently, 3 cases were described in which an FGFR3::TACC3 fusion co-existed with an FGFR3 K650T mutation.²⁸ The prognostic and predictive meaning of the FGFR3 mutation and FGFR3 overexpression in this case without FGFR3::TACC3 fusion remains unknown. Currently, this RT-PCR assay for FFPE material tests for 5 common fusion variants. It is still possible that the 2 samples with positive FGFR3 staining on IHC, but no fusion detected on RT-PCR (Case 1 and Case 11) do harbor an FGFR3::TACC3 fusion as RNAseq was unsuccessful in these cases.

Because FF material is not always available in the diagnostic setting, it is helpful that RT-PCR can also be performed on FFPE material, using the same RT-PCR protocol and reagents, but different primers: 1 primer pair detecting large amplicons in the range of 1000 bp for FF, and 5 primer pairs detecting amplicons in the range between 100 and 200 bp for FFPE. The presence of nonspecific amplicons and the possibility of false negative results as a result of RNA degradation, warrant caution when performing RT-PCR on FFPE material. In our experience, RT-PCR was more challenging on older archival FFPE material, but will likely be easier on FFPE material during the initial diagnostic process as the tissue block is freshly prepared, making RNA fragmentation less severe. In an FFPE assay, it is essential to include controls to check for RNA fragmentation. It would be ideal to have a primer set covering all 15 previously described fusion variants of FGFR3::TACC3¹⁸ for FPPE, so that in the absence of FF material, a complete diagnostic test for FGFR3::TACC3 fusion can still be performed. The fact that in this study we only had 5 primer sets, allowing for the detection of 5 FGFR3:TACC3 fusion variants, is a limitation of this study. By adding 2 additional sets of primers (FGFR3ex16-TACC3ex3 and FGFR3ex18::TACC3ex11) to the currently described assay, for which only 2 new primers would have to be designed, 77% of fusion variants could be detected. If available, FF is still preferred over FFPE because of the above-mentioned challenges when using FFPE and because of the high input required for successful RT-PCR when FFPE tissue is used.

It has been suggested to use a combined immune-reactivity score (IRS, staining intensity [range 0–3] × staining quantification (range 1–4 based on percentage of tumor cells that stain, at 25% intervals), with a cutoff value for overexpression set at an IRS of 7 or more.²⁹ In our experience, it is worth testing for FGFR3 fusions in all cases with some staining upon FGFR3 IHC. Several of our cases with heterogeneous, low staining intensity did harbor an FGFR3::TACC3 fusion, while these cases might not have reached the cutoff of IRS score 7 for FGFR3 expression. In this study, all cases with 2+ staining on IHC were confirmed to have an FGFR3::TACC3 fusion, suggesting that additional testing for cases with clear expression of FGFR3 on IHC might not be necessary. However, since the numbers in this study are low, and positive staining for FGFR3 is rare, it might be worth testing all samples that show some positive staining.

In line with previously reported incidence, we found 9 samples with FGFR3::TACC3 fusions in a cohort of 353 cases (2.5%; 2.9% of 308 cases with interpretable FGFR3 IHC), 8 of which we were able to detect using RT-PCR. The small sample size is another limitation of this study. For implementation of this technique in clinical practice, it would be desired to further validate the technique on a larger number of samples, for example, by temporarily running the RT-PCR assay alongside another detection method. Higher numbers of FGFR3::TACC3 fusion-positive samples could be achieved if several labs combine their data. For the implementation of RT-PCR assay in routine clinical practice, we would advise to screen for FGFR3 overexpression using IHC, and to test samples with any overexpression in tumor cells. For the time being, testing for this aberration would be most clinically relevant for patients who have a chance to enter current or future clinical trials.

In conclusion, in the absence of FF material, RT-PCR to test for the presence of an FGFR3::TACC3 fusion can still be performed on FFPE material, using the same protocol with different primer sets and a higher cDNA input. In this way, eligibility for treatment with an FGFR3 inhibitor can still be assessed, also when only a routinely processed tumor tissue sample is available.

Supplementary material

Supplementary material is available online at Neuro-Oncology (https://academic.oup.com/neuro-oncology).

Figure 3.

(A) RT-PCR results of FFPE material of 3 glioma samples, showing the 3 most frequently observed fusion variants as detected in Case 4, Case 10, and Case 2, respectively. Per control lane, the length (bp) that the product is expected to have is presented in the signal. Nonspecific products below 100 bp are considered to be primer-dimers. 1-kb DNA size ladder. (B) RT-PCR results of 5 fresh frozen GBM samples (Cases 2, 4, 5, 6, and 8) revealing FGFR3::TACC3 fusions. FGFR3ex17TACC3ex11 means that exon 17 of FGFR3 gene is fused to exon 11 of TACC3 gene. Positive control: GBM-1123 cell line (Singh et al.⁵); negative control: sample with an EWS-FLI1 translocation. 1-kb DNA size ladder.

Funding

No funding was acquired for this research.

Conflict of interest statement The authors declare no conflict of interest.

Author contributions

J.v.K. and A.G.M.H. (acknowledgements) set up the RT-PCR for FFPE samples under supervision of W.E.J.d.L. P.A.R. selected and gathered the samples for the creation of the tissue micro-array (TMA). W.v.H. scored FGFR3 IHC stainings on TMA and whole slide. J.v.K., A. G.M.H., and L.P.P.-A. performed the laboratory testing. L.P.P.-A. wrote the manuscript with input from all authors and created visual representations of the outcomes. All authors read and approved the manuscript.