Longitudinal heterogeneity in glioblastoma: moving targets in recurrent versus primary tumors

Abstract

Background: Molecularly targeted therapies using receptor inhibitors, small molecules or monoclonal antibodies are routinely applied in oncology. Verification of target expression should be mandatory prior to initiation of therapy, yet, determining the expression status is most challenging in recurrent glioblastoma (GBM) where most patients are not eligible for second-line surgery. Because very little is known on the consistency of expression along the clinical course we here explored common drug targets in paired primary vs. recurrent GBM tissue samples.

Methods: Paired surgical tissue samples were derived from a homogeneously treated cohort of 34 GBM patients. All patients received radiotherapy and temozolomide chemotherapy. Verification of common drug targets included immunohistological analysis of PDGFR-β, FGFR-2, FGFR-3, and mTOR-pathway component (phospho-mTOR^Ser2448) as well as molecular, MLPA-based analysis of specific copy number aberrations at the gene loci of ALK, PDGFRA, VEGFR2/KDR, EGFR, MET, and FGFR1.

Results: Paired tumor tissue exhibited significant changes of expression in 9 of the 10 investigated druggable targets (90%). Only one target (FGFR1) was found "unchanged", since dissimilar expression was observed in only one of the 34 paired tumor tissue samples. All other targets were variably expressed with an 18-56% discordance rate between primary and recurrent tissue.

Conclusions: The high incidence of dissimilar target expression status in clinical samples from primary vs. recurrent GBM suggests clinically relevant heterogeneity along the course of disease. Molecular target expression, as determined at primary diagnosis, may not necessarily present rational treatment clues for the clinical care of recurrent GBM. Further studies need to analyze the therapeutic impact of longitudinal heterogeneity in GBM.

Keywords: EGFR; Glioblastoma; Heterogeneity; MLPA; Targeted therapy.

Fig. 1

Target investigation by immunohistochemistry. a Typical examples of strongly immunoreactive targets (scale bar 50 µm). b Shifting target expression as revealed by quantitative scoring (left panel), illustrated by changes per target (right panel). The thickness of lines indicates the number of patients. If the status changed between primary and recurrent tumor tissue, the percentage of affected patients was indicated in circles in the graph (numbers rounded). For PDGFR-β, FGFR-2, and FGFR-3 the numbered boxes represent the portion of positively labeled tumor cells: box 0 = negative; box 1 ≤ 10%; box 2 = 10–90%; box 3 ≥ 90%. Due to spatial/intratumoral inhomogeneous staining results of phosphor-mTOR^Ser2448: box 0 = negative; box 1 = smaller groups, but < 50% of tumor cells; box 2 = major groups, > 50% of tumor cells; box 3 = nearly all tumor cells positive

Fig. 2

Target investigation by Multiplex ligation-dependent probe amplification (MLPA). a Overview of cases and the respective copy number aberrations status of the selected targets. b Graphs illustrate correlation of target status in the investigated cohort of paired tissue samples. The thickness of lines visualizes the number of affected patients. A status change between primary and recurrent tumor tissue is indicated by encircled numbers, reflecting the percentage of affected patients (numbers rounded). The boxes indicate the portion of copy number aberrations: No CNA, low gain, high gain, and focal amplification