Evaluation of Urine Aquaporin-1 and Perilipin-2 Concentrations as Biomarkers to Screen for Renal Cell Carcinoma: A Prospective Cohort Study

Abstract

Importance: Historically, early detection of small asymptomatic kidney tumors presages better patient outcome. Screening for asymptomatic renal tumors by abdominal imaging is not cost-effective and cannot reliably distinguish benign from malignant tumors.

Objective: This investigation evaluated the clinical utility, sensitivity, and specificity of urine aquaporin-1 (AQP1) and perilipin-2 (PLIN2) concentrations as unique, noninvasive biomarkers to diagnose malignant clear cell or papillary renal cell carcinoma (RCC) in a screening paradigm.

Design, setting, and participants: From February through December 2012, urine samples were obtained from 720 patients undergoing routine abdominal computed tomography (CT) (screening population), 80 healthy controls, and 19 patients with pathologically confirmed RCC.

Main outcomes and measures: Urine AQP1 and PLIN2 concentrations were measured by sensitive and specific enzyme-linked immunosorbent assay and Western blot procedures, respectively, in all groups. In the otherwise asymptomatic screening population, the absence or presence of a renal mass and RCC were verified by abdominal CT and by postnephrectomy pathologic diagnosis, respectively.

Results: Urine AQP1 and PLIN2 concentrations were significantly higher (all P < .001) in the 19 patients with known RCC (AQP1 median [95% CI], 225.0 [121.0-450.0] ng/mg urine creatinine; and PLIN2 median [95% CI], 37.8 [22.8-83.7] absorbance units/mg creatinine) than in the 80 healthy controls (AQP1 median [95% CI], 1.1 [0.9-1.3] ng/mg urine creatinine; and PLIN2 median [95% CI], 3.1 [2.4-3.7] absorbance units/mg creatinine) and the 720 patient screening population (AQP1 median [95% CI], 0.5 [0.0-1.0] ng/mg urine creatinine; and PLIN2 median [95% CI], 0 [0-0] absorbance units/mg creatinine). The area under the receiver operating characteristic curve for urine AQP1 and PLIN2 concentrations individually or in combination was 0.990 or greater, with 95% or greater sensitivity and 91% or greater specificity compared with controls or the screening population. Of the 720 screened patients, 3 had biomarker concentrations suggestive of RCC and were found to have an imaged renal mass by CT. Two of the patients had pathologically confirmed RCC in further evaluation.

Conclusions and relevance: These results demonstrate the clinical utility, specificity, and sensitivity of urine AQP1 and PLIN2 to diagnose RCC. These tumor-specific proteins have high clinical validity and substantial potential as specific diagnostic and screening biomarkers for clear cell or papillary RCC and in the differential diagnosis of imaged renal masses.

Figures

Figure 1

Dot and box representation of urine AQP1 (A) and PLIN2 (B) concentrations. Depicted are the median, first, second, third and fourth quartiles. Values of individuals within the first through fourth quartile are represented by (O), values of individuals over 1.5 but under 3-times the interquartile range are represented by (+), and individuals exceeding 3-times the interquartile range are represented by (X). Median urine AQP1 concentration in the 19 patients enrolled a priori with confirmed kidney cancer (clear cell or papillary subtypes) is greater (P<0.001) than that of the 80 healthy controls, the 334 patients with no cancer history, and 386 patients with a history of non-kidney cancer. Median urine AQP1 concentration in the healthy controls is less than that of the cancer and no cancer groups (each P<0.001). The urine AQP1concentations in the cancer and no cancer groups are not significantly different (P=0.444). Median urine PLIN2 concentration in the 19 patients enrolled a priori with confirmed kidney cancer is greater (P<0.001) than that of the 80 healthy controls, the 334 patients with no cancer history), and 386 patients with a history of non-kidney cancer. Median urine PLIN2 concentration in the healthy controls is less than that of the no cancer group (P=0.032) but not the group with a history of cancer (P<0.085). Urine PLIN2 concentration in the no cancer and cancer groups is not different (P=0.447). Significance was determined by the Kruskal-Wallis test with least squares difference correction.

Figure 2

Correlation between tumor size and urine biomarkers in the 19 patients enrolled a priori with kidney cancer (clear cell or papillary subtypes). (A) AQP1 concentrations (Spearman correlation coefficient 0.78, P<0.001) or (B) PLIN2 concentrations (Spearman coefficient 0.72, P<0.001).

Figure 3

Dot and box representation of urine AQP1 (A) and PLIN2 (B) concentrations (ng or absorbance units per mg urine creatinine, respectively). Depicted are the median, first, second, third and fourth quartiles. Values of individuals within the first through fourth quartile are represented by (O), values of individuals over 1.5 but under 3-times the interquartile range are represented by (+), and individuals exceeding 3-times the interquartile range are represented by (X). The median urine AQP1 concentration for the 19 patients enrolled a priori with confirmed kidney cancer (clear cell or papillary subtypes) is significantly different (P<0.0001) from that of the 386 patients with a history of cancer in various non-kidney tissue/organs. These include lung (n=89), prostate (n=12), colorectal (n=25), gastrointestinal (n=11), uterine (n=16), ovarian (n=25), pancreatic (n=13), lymphoma (n=38), breast (n=44), and various other organs/tissues (n=95). Significant differences, or lack thereof in urine AQP1 and PLIN2 concentrations between all these different tissues/organs are summarized in eTable 1.

Figure 4

ROC analysis of urine AQP1 and PLIN2. AQP1 (A) and PLIN2 (B) concentrations in 22 patients with an imaged renal mass (19 enrolled a priori with a known renal mass and 3 incidentally discovered during CT screening - see Figure 5) compared with the 80 healthy controls. AQP1 (C) and PLIN2 (D) concentrations in the 22 patients with an imaged renal mass compared with the 717 patients without an imaged renal mass. AQP1 and PLIN2 concentrations which were added (E) compared with the 717 patients without an imaged renal mass

Figure 4

ROC analysis of urine AQP1 and PLIN2. AQP1 (A) and PLIN2 (B) concentrations in 22 patients with an imaged renal mass (19 enrolled a priori with a known renal mass and 3 incidentally discovered during CT screening - see Figure 5) compared with the 80 healthy controls. AQP1 (C) and PLIN2 (D) concentrations in the 22 patients with an imaged renal mass compared with the 717 patients without an imaged renal mass. AQP1 and PLIN2 concentrations which were added (E) compared with the 717 patients without an imaged renal mass

Figure 4

ROC analysis of urine AQP1 and PLIN2. AQP1 (A) and PLIN2 (B) concentrations in 22 patients with an imaged renal mass (19 enrolled a priori with a known renal mass and 3 incidentally discovered during CT screening - see Figure 5) compared with the 80 healthy controls. AQP1 (C) and PLIN2 (D) concentrations in the 22 patients with an imaged renal mass compared with the 717 patients without an imaged renal mass. AQP1 and PLIN2 concentrations which were added (E) compared with the 717 patients without an imaged renal mass

Figure 5

Urine AQP1 (A) and PLIN2 (B) concentration in individual patients enrolled between February and December 2012. The biomarker concentration (ng/mg creatinine for AQP1 and absorbance units/mg creatinine for PLIN2) is shown for each of the 720 patients screened who also underwent abdominal CT (open circles), and the 19 patients with confirmed kidney cancer (clear cell or papillary subtypes) (filled circles). Three of the 720 screened patients (shaded circles) were subsequently found to have an imaged renal mass based on their CT. The dotted lines represents the cutoff values (52 ng/mg urine creatinine for AQP1 and 9.8 absorbance/mg urine creatinine for PLIN2) derived from the receiver operating characteristic analysis shown in Figure 4.