In situ analysis of FGFR2 mRNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients

Abstract

Background: Fibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.

Methods: We used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).

Results: We successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification (FGFR2:CEN10 <10; ≥2) was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression (dense clusters of the signal visible under a 1× objective). Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0-3 patients.

Conclusion: RNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.

Keywords: Fibroblast growth factor receptors 2; Gene amplification; In situ hybridization; Molecular targeted therapy; Stomach neoplasms.

Fig. 1

Patient samples flow diagram. DISH dual-color in situ hybridization, FGFR fibroblast growth factor receptor

Fig. 2

Fibroblast growth factor receptor (FGFR)2 mRNA analysis. FGFR2 mRNA expression was determined by RNAscope 2.0 following manufacturer’s instructions (Advanced Cell Diagnostics, Hayward, CA, USA) using FGFR2 and peptidyl prolyl isomerase B (PPIB) target probes. FGFR2 expression was scored on a scale from 0 to 4. a Representative images. b Prevalence of FGFR2 mRNA expression in 718 gastric cancer cases. Samples with no PPIB or FGFR2 signal were excluded from the analysis

Fig. 2

Fibroblast growth factor receptor (FGFR)2 mRNA analysis. FGFR2 mRNA expression was determined by RNAscope 2.0 following manufacturer’s instructions (Advanced Cell Diagnostics, Hayward, CA, USA) using FGFR2 and peptidyl prolyl isomerase B (PPIB) target probes. FGFR2 expression was scored on a scale from 0 to 4. a Representative images. b Prevalence of FGFR2 mRNA expression in 718 gastric cancer cases. Samples with no PPIB or FGFR2 signal were excluded from the analysis

Fig. 3

Intratumor heterogeneity of FGFR2 in gastric cancer. The percentage of tumor cells showing FGFR2 staining in either RNAscope (mRNA) or DISH (gene amplification) was determined on tissue slides. Samples with score 3 and 4 RNAscope level were analyzed. Score 5 RNA samples with dense clusters of RNA signal were analyzed separately

Fig. 4

FGFR2 gene amplification analysis. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH) using FGFR2 and CEN10 target probes. High FGFR2 gene amplification was defined as a FGFR2:CEN10 ratio >10; moderate gene amplification was defined as FGFR2:CEN10 <10 but ≥2. There were no samples with high chromosome 10 polysomy >4 per cell. Data were generated for 578 gastric cancer samples. a Prevalence of FGFR2 gene amplification. b Prevalence of FGFR2 mRNA expression by RNAscope in samples with high, moderate, or no FGFR2 gene amplification c FGFR2 RNA scope (left) and DISH data (right) from the same sample. Arrows point to tumor areas with or without FGFR2 mRNA. Inserted square shows a high-magnification image of the DISH staining. Red arrow points to the CEN10 signal in red. FGFR2 signal is black

Fig. 5

Gastric cancer histology versus FGFR2 gene amplification. Histological subtypes were determined following the Japanese classification of gastric carcinoma. Prevalence of histological subtypes in samples with high, moderate, or no FGFR2 gene amplification is plotted

Fig. 6

Patient outcomes according to FGFR2 mRNA level. a Kaplan–Meier plot for recurrence-free survival (RFS) for the FGFR2 RNA levels determined by RNAscope (0–4). b Kaplan–Meier plot for overall survival (OS) for the FGFR2 RNA levels determined by RNAscope (0–4). c Kaplan–Meier plot for RFS in the FGFR2 RNAscope groups score 3 and 4 with heterogeneous (≤30% of tumor cells show expression) and homogenous expression (≥30%). d Kaplan–Meier plot for OS in the FGFR2 RNAscope groups score 3 and 4 with heterogeneous and homogenous expression

Fig. 7

Patient outcomes according to FGFR2 gene amplification. a Kaplan–Meier plot for RFS for the FGFR2 gene amplification levels determined by DISH. b Kaplan–Meier plot for OS for the FGFR2 gene amplification levels determined by DISH