Plasma Glycosaminoglycans as Diagnostic and Prognostic Biomarkers in Surgically Treated Renal Cell Carcinoma

Abstract

Background: Plasma glycosaminoglycan (GAG) measurements, when aggregated into diagnostic scores, accurately distinguish metastatic clear-cell renal cell carcinoma (RCC) from healthy samples and correlate with prognosis. However, it is unknown if GAG scores can detect RCC in earlier stages or if they correlate with prognosis after surgery.

Objective: To explore the sensitivity and specificity of plasma GAGs for detection of early-stage RCC and prediction of recurrence and death after RCC surgery.

Design, setting, and participants: This was a retrospective case-control study consisting of a consecutive series of 175 RCC patients surgically treated between May 2011 and February 2014 and 19 healthy controls.

Outcome measurements and statistical analysis: Plasma GAGs in preoperative and postoperative RCC and healthy samples were measured using capillary electrophoresis with laser-induced fluorescence in a single blinded laboratory. A discovery set was first analyzed to update the historical GAG score. The sensitivity of the new GAG score for RCC detection versus healthy subjects was validated using the remaining samples. The correlation of the new GAG score to histopathologic variables, overall survival, and recurrence-free survival was evaluated using nonparametric and log-rank tests and multivariable Cox regression analyses.

Results and limitations: The RCC cohort included 94 stage I, 58 stage II-III, and 22 stage IV cases. In the first discovery set (n=67), the new GAG score distinguished RCC from healthy samples with an area under the receiver operating characteristic curve (AUC) of 0.999. In the validation set (n=108), the GAG score achieved an AUC of 0.991, with 93.5% sensitivity. GAG scores were elevated in RCC compared to healthy samples, irrespective of and uncorrelated to stage, grade, histology, age, or gender. The total chondroitin sulfate concentration was an independent prognostic factor for both overall and recurrence-free survival (hazard ratios 1.51 and 1.25) with high concordance when combined with variables available at pathologic diagnosis (C-index 0.926 and 0.849) or preoperatively (C-index 0.846 and 0.736). Limitations of the study include its retrospective nature and moderate variability in GAG laboratory measurements.

Conclusions: Plasma GAGs are highly sensitive diagnostic and prognostic biomarkers in surgically treated RCC independent of stage, grade, or histology. Prospective validation studies on GAG scores for early detection, prediction, and surveillance for RCC recurrence are thus warranted.

Patient summary: In this study, we examined if a new molecular blood test can detect renal cell carcinoma in the early stages and predict if the cancer might relapse after surgery. The trial is registered on ClinicalTrial.gov as NCT03471897.

Keywords: Diagnostic biomarkers; Liquid biopsy; Prognostic biomarkers; Renal cell carcinoma.

Fig. 1 –

Clustering analysis of plasma chondroitin sulfate (CS) and heparan sulfate (HS) profiles for 175 preoperative renal cell carcinoma (RCC) samples and 19 samples from healthy subjects. (A) Principal component analysis (PCA) based on quantification of the CS and HS profile in plasma samples from RCC patients and healthy subjects. Each point represents an individual sample. The percentage indicates the proportion of variance explained along the axis of each principal component (PC). The ellipses delimit the area in which samples belonging to a certain group are expected to be located at a 95% confidence level assuming a multivariate t distribution. (B) Unsupervised hierarchical clustering based on between-sample correlation in the CS and HS profiles. Each row represents an individual CS or HS property. Each column represents a sample. The annotation above provides information on the principal diagnosis, the Fuhrman nuclear grade (if applicable and available) and the TNM stage. For each row, values for the corresponding CS or HS property were normalized as z scores.

Fig. 2 –

Selected glycosaminoglycan properties in plasma samples in the discovery set, comprising 38% of the current cohort (RCC 67, healthy 19) and two historical cohorts from Sweden (RCC 26, healthy 20) and Italy (RCC 23, healthy 5) [14]; see also Table 3. RCC = renal cell carcinoma; CS = chondroitin sulfate; HS = heparan sulfate; tot = total.

Fig. 3 –

(A) Boxplot of the new plasma glycosaminoglycan (GAG) score for 19 healthy samples versus 67 preoperative renal cell carcinoma (RCC) samples comprising the discovery set versus 108 preoperative RCC samples comprising the validation set. The horizontal line indicates the cutoff score corresponding to maximum accuracy for the discovery set. (B) Corresponding receiver operating characteristic curve for classification for the discovery and validation sets. Note that 12 samples with scores greater than 2 in the validation set were omitted from display to prevent shrinkage of the plot.

Fig. 4 –

Kaplan-Meier curves for overall survival for low versus high risk according to (A) the preoperative total chondroitin sulfate (CS_tot) value alone and (B) the preoperative CS_tot value combined with tumor size >5 cm among 175 patients with renal cell carcinoma.

Fig. 5 –

Kaplan-Meier curves for recurrence-free survival for low versus high risk according to (A) the preoperative total chondroitin sulfate (CS_tot) value alone and (B) the preoperative CS_tot combined with tumor size >5 cm among 152 patients with nonmetastatic renal cell carcinoma.