PDGFRA, KIT, and KDR Gene Amplification in Glioblastoma: Heterogeneity and Clinical Significance

NeuroMolecular Medicine https://doi.org/10.1007/s12017-023-08749-y RESEARCH PDGFRA, KIT, and KDR Gene Amplification in Glioblastoma: Heterogeneity and Clinical Significance Bianca Soares Carlotto1 · Patricia Trevisan1,2 · Valentina Oliveira Provenzi3 · Fabiano Pasqualotto Soares4 Rafael Fabiano Machado Rosa1,5,6 · Marileila Varella‑Garcia7 · Paulo Ricardo Gazzola Zen1,5,6 · Received: 3 February 2023 / Accepted: 30 July 2023 This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023 Abstract Glioblastoma (GBM) is the most frequent tumor of the central nervous system, and its heterogeneity is a challenge in treatment. This study examined tumoral heterogeneity involving PDGFRA, KIT, and KDR gene amplification (GA) in 4q12 and its association with clinical parameters. Specimens from 22 GBM cases with GA for the 4q12 amplicon detected by FISH were investigated for homogeneous or heterogeneous coamplification patterns, diffuse or focal distribution of cells harboring GA throughout tumor sections, and pattern of clustering of fluorescence signals. Sixteen cases had homogenously amplification for all three genes (45.5%), for PDGFRA and KDR (22.7%), or only for PDGFRA (4.6%); six cases had heterogeneous GA patterns, with subpopulations including GA for all three genes and for two genes - PDGFRA and KDR (13.6%), or GA for all three and for only one gene - PDGFRA (9.1%) or KIT (4.6%). In 6 tumors (27.3%), GA was observed in focal tumor areas, while in the remaining 16 tumors (72.7%) it was diffusely distributed throughout the pathological specimen. Amplification was universally expressed as double minutes and homogenously stained regions. Coamplification of all three genes PDGFRA, KIT, and KDR, age ≥ 60 years, and total tumor resection were statistically associated with poor prognosis. FISH proved effective for detailed interpretation of molecular heterogeneity. The study uncovered an even more diverse range of amplification patterns involving the 4q12 oncogenes in GBM than previously described, thus highlighting a complex tumoral heterogeneity to be considered when devising more effective therapies. Keywords Glioblastoma · PDGFRA · KIT · KDR · Genetic heterogeneity · FISH 2 Bianca Soares Carlotto Colorado Genetics Laboratory, Department of Pathology, School of Medicine, University of Colorado, Aurora, CO, USA 3 Patricia Trevisan Pathology Section, Grupo Hospitalar Conceição (GHC), Porto Alegre, RS, Brazil 4 Valentina Oliveira Provenzi Regenerar - Centro de Medicina da Dor, Porto Alegre, RS, Brazil 5 Department of Internal Medicine, Clinical Genetics, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil 6 Irmandade da Santa Casa de Misericórdia de Porto Alegre (ISCMPA), Porto Alegre, RS, Brazil 7 Department of Medicine, Medical Oncology Division, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA * Marileila Varella‑Garcia Fabiano Pasqualotto Soares Rafael Fabiano Machado Rosa Paulo Ricardo Gazzola Zen 1 Graduate Program in Pathology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil 13 Vol.:(0123456789) NeuroMolecular Medicine Introduction Glioblastoma (GBM) is the most common primary malignant lesion in the brain and other central nervous system (CNS) organs (14.3% of all tumors and 49.1% of malignant tumors); it represents most of gliomas (58.4%) and associates with median survival of 8 months (Ostrom et al., 2021). GBM incidence is higher in older and in male adults; female sex and older age (40+ years) were associated with poorer survival (Ostrom et al., 2020, 2021). The average annual age-adjusted incidence rate of malignant brain and other CNS tumors was 7.06 per 100,000 between 2014 and 2018 in the US (Ostrom et al., 2021). In Southern Brazil (state of Rio Grande do Sul), incidence of CNS tumors in 2020 was estimated at 9.05 and 7.58 per 100,000, respectively in male and female adults (Instituto Nacional de Câncer José Alencar Gomes da Silva, 2019). GBMs are highly heterogeneous tumors exhibiting regional and cellular genotypic and phenotypic variations (Park et al., 1995). Tumor heterogeneity is one of the significant barriers to the development of effective therapeutic approaches in solid tumors (Hanahan & Weinberg, 2011) and this high-grade glioma is well known by therapeutic resistance and aggressiveness (Cantanhede & de Oliveira, 2017). The GBM heterogeneity not only manifests as a genetic and phenotypic variation in different individuals (intertumoral heterogeneity), but also as a simultaneous display of subclonal diversity (intermingled or spatially separated) within a tumor (intratumoral heterogeneity - IH) (Burrell et al., 2013). Heterogeneity is expressed as different gene, molecular and cellular features leading to lesions with distinct genetic, molecular, and morphological profiles, transcription and expression patterns, configurations of vascular proliferation, metabolism, micro-environment, and metastatic potential; all factors playing a key role in tumor progression and therapeutic resistance (Belsuzarri et al., 2018; Hanahan & Weinberg, 2011; Little et al., 2012; Snuderl et al., 2011). The striking IH originates from the combination of regional genetic variation and cellular hierarchy, frequently controlled by distinct groups of cancer stem cells (Schonberg et al., 2014). Genomic and in situ fluorescent (FISH) and chromogenic hybridization (CISH) studies in GBM have detected a profound IH in the amplification patterns of receptor tyrosine kinases (RTK) and drug target genes, such as EGFR, MET, and PDGFRA (Burford et al., 2013; Little et al., 2012; Snuderl et al., 2011; Szerlip et al., 2012). PDGFRA maps at 4q12 and is contiguous to KIT and KDR, two other RTK and drug target genes (Burford et al., 2013). PDGFRA, KIT, and KDR apparently developed from a common ancestral gene and frequently coamplify in GBM (Joensuu et al., 2005). 13 We used FISH to examine rearrangements in ROS1 and NTRK1, amplification of PDGFRA, KIT, and KDR, and deletion of RB1, and to verify their potential clinical significance in a Brazilian cohort of adult GBM patients (Trevisan et al., 2019). RB1 was deleted in 16% of cases and PDGFRA was amplified in 20%, often coamplified with KDR (> 90%) and KIT (> 60%). Complications after surgery, older age and right-sided tumors were independent variables associated with patient survival. Subsequently, we re-investigated in detail the patterns of gene amplification (GA) of PDGFRA, KIT, and KDR in those GBM specimens carrying GA, and its association with RB1 deletion and other clinical variables. These data are presented here. Materials and Methods Datasets and FISH From 113 cases classified as GBM NOS (Trevisan et al., 2019), according to OMS 2016 guidelines (Ohgaki et al., 2016), a retrospective cohort of 22 cases with GA in (...truncated)