GRIN3A: A biomarker associated with a cribriform pattern and poor prognosis in prostate cancer

Abstract

Prostate cancer with a cribriform pattern, including invasive cribriform carcinoma (ICC) and/or intraductal carcinoma (IDC) is associated with a poor prognosis, and the underlying mechanisms are unclear. Therefore, we aimed to identify biomarkers for this feature. Using a radical prostatectomy cohort, we performed within-patient differential expression analyses with RNA sequencing data to compare samples with a cribriform pattern to those with non-cribriform Gleason pattern 4 (NcGP4; n=13). ACSM1, GRIN3A, PCDHB2, and REG4 were identified as differentially expressed, and validation was performed using real-time reverse transcription polymerase chain reaction (n=99; 321 RNA samples) and RNA in situ hybridization on tissue microarrays (n=479; 2047 tissue cores). GRIN3A was significantly higher expressed in cribriform pattern vs. NcGP4, when assessed within the same patient (n=27; p=0.005) and between different patients (n=83; p=0.001). Tissue cores with IDC more often expressed GRIN3A compared to ICC, NcGP4, and benign tissue (52 % vs. ≤ 32 %). When IDC and NcGP4 was compared within the same patient (173 pairs of tissue cores; 54 patients), 38 (22 %) of the tissue microarray core pairs had GRIN3A expression in only IDC, 33 (19 %) had expression in both IDC and NcGP4, 14 (8 %) in only NcGP4 and 88 (51 %) were negative in both entities (p=0.001). GRIN3A was as well associated with biochemical recurrence (log-rank, p=0.002). In conclusion, ectopic GRIN3A expression is an RNA-based biomarker for the presence of cribriform prostate cancer, particularly for IDC.

Keywords: Biomarker; Intraductal carcinoma; Invasive cribriform carcinoma; Prostate cancer; RNA in situ hybridization; RNA sequencing.

Fig. 1

Patient inclusion in the different analyses. Schematic illustration showing overlap of patient inclusion for the different analyses. Abbreviations: RNA-seq, RNA sequencing; RT-PCR, reverse transcription polymerase chain reaction.

Fig. 2

Assessment of GRIN3A expression levels. (A) Plot showing the correlation between GRIN3A expression levels as determined by RNA sequencing and real-time RT-PCR. Each dot represents one sample from a patient that was included in the differential gene expression analysis with a paired sampling analysis of cribriform pattern versus non-cribriform Gleason pattern 4. (B) Paired analysis (intrapatient pairwise comparison) comparing log₂ fold changes for GRIN3A expression in cribriform samples to non-cribriform Gleason pattern 4 samples. Samples from patients in an independent subcohort were included (n = 27). Each dot represents one sample, and gray lines between samples indicate the sample pair being compared. Box plots show median relative log₂ expression, first and third quartile, and maximum and minimum log₂ values. (C) Unpaired analysis (interpatient pairwise comparison) comparing log₂ fold changes for GRIN3A expression in cribriform samples to non-cribriform Gleason pattern 4 samples (n = 83). Abbreviations: GP3, Gleason pattern 3; NcGP4, non-cribriform Gleason pattern 4; RT-PCR, reverse transcription polymerase chain reaction; * statistical significance.

Fig. 3

Assessment of ACSM1, GRIN3A, PCDHB2, and REG4 expression by RNA in situ hybridization. (A) Example images of “positive” staining for the four candidate genes (ACSM1, GRIN3A, PCDHB2, and REG4), at 10x magnification for ACSM1,GRIN3A and REG4, and 20x magnification for PCDHB2. (B) Stacked bar charts showing the percentage of samples with either “positive” or “negative” expression of the candidate genes. Classification of the tissue cores by morphological feature is shown on the x-axis, and colored according to whether they are from radical prostatectomy specimens with (purple) or without (green) a cribriform pattern. (C) Example images (10x magnification) of a selection of the different morphological features and their GRIN3A expression in descending order of frequency. Abbreviations: ICC, invasive cribriform carcinoma; IDC, intraductal carcinoma; NcGP4, non-cribriform Gleason pattern 4; RP, radical prostatectomy.

Fig. 4

Biochemical recurrence-free and clinical-recurrence-free survival in patients treated with radical prostatectomy stratified by GRIN3A expression status in malignant samples. Kaplan-Meier curves for (A) biochemical recurrence-free survival and (B) clinical recurrence-free survival, stratified by GRIN3A expression status in malignant samples. A patient was classified as GRIN3A-positive (+) if at least one malignant tissue core from that patient was scored as “positive,” as determined by RNA in situ hybridization. Abbreviations: BCR, biochemical recurrence; CR, clinical recurrence.