EURURO-6262; No. of Pages 3 EUROPEAN UROLOGY XXX (2015) XXX–XXX
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Brief Correspondence
Carbonic Anhydrase IX as a Diagnostic Urinary Marker for Urothelial Bladder Cancer Michela de Martino a, Ilaria Lucca a, Aurelie Mbeutcha a, Helene G. Wiener b, Andrea Haitel b, Martin Susani b, Shahrokh F. Shariat a,c,d, Tobias Klatte a,* a
Department of Urology, Medical University of Vienna, Vienna General Hospital, Vienna, Austria;
b
Clinical Institute of Pathology, Medical University of
c
Vienna, Vienna General Hospital, Vienna, Austria; Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX, USA; d Department of Urology, Weill Cornell Medical College, New York-Presbyterian Hospital, New York, NY, USA
Article info
Abstract
Article history: Accepted June 14, 2015
Urinary biomarkers are needed to improve the management and reduce the cost of urothelial bladder cancer (UBC); however, none have been recommended yet for clinical practice. This study evaluated carbonic anhydrase IX (CAIX) as a diagnostic urinary biomarker for UBC. CAIX was analyzed by quantitative polymerase chain reaction in urine samples of 196 patients with UBC and 123 controls with hematuria. Paired samples from urine and tumor tissue were evaluated in 16 cases. Data were validated in 155 independent samples. The sensitivity and specificity of CAIX for UBC detection were 86.2% and 95.1%, respectively (area under the curve [AUC]: 90.5%). There was a significant association of CAIX expression between the paired urine and tumor specimens (p = 0.002). CAIX showed a significantly higher predictive accuracy than urinary cytology (90.5% vs 71.7%), specifically in low-grade tumors (90.0% vs 61.8%). CAIX expression decreased with increasing tumor stage and grade. Analyses in an independent validation cohort confirmed the high accuracy of CAIX for diagnosing UBC (AUC: 88.3%). Patient summary: We evaluated carbonic anhydrase IX (CAIX) as a urinary marker for bladder cancer (BCa) using a large series of patients from a single hospital. We found that urinary CAIX has a high sensitivity and specificity for diagnosing BCa. # 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Associate Editor: James Catto Keywords: Urinary marker Cytology CA9 Carbonic anhydrase IX Diagnosis Cystoscopy Recurrence Bladder cancer
* Corresponding author. Department of Urology, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. Tel. +43 1 40400 26020; Fax: +43 1 40400 23320. E-mail address: [email protected] (T. Klatte).
Intense research is being performed on urinary biomarkers with the goal of enhancing the detection of noninvasive early urothelial bladder cancer (UBC), allowing replacement, delay, or complementation of cystoscopy. Numerous biomarkers have shown a higher sensitivity than urinary cytology but lower specificity [1]. At present, none of these biomarkers has been recommended by guidelines for routine clinical care [1,2]. Carbonic anhydrase IX (CAIX) is an enzyme of the carbonic anhydrase family that regulates the pH value as a response to hypoxia [3]. In a previous immunohistochemical
study, normal urothelial tissue samples were all negative for CAIX; >70% of UBCs stained positive. Expression levels were markedly increased in Ta and low-grade tumors [4]. We hypothesized that urinary levels of CAIX were significantly elevated in UBC compared with controls and that CAIX is associated with UBC pathology. A total of 319 urine samples and associated clinical and pathologic information were collected prospectively from UBC patients and controls between 2009 and 2011 (training cohort). The UBC group consisted of patients who were
http://dx.doi.org/10.1016/j.eururo.2015.06.015 0302-2838/# 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: de Martino M, et al. Carbonic Anhydrase IX as a Diagnostic Urinary Marker for Urothelial Bladder Cancer. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.06.015
EURURO-6262; No. of Pages 3 2
EUROPEAN UROLOGY XXX (2015) XXX–XXX
scheduled for a transurethral resection of the bladder (TURB). The control group was composed of patients with hematuria but with other urologic conditions (Supplementary Table 1). These patients were evaluated with urinalysis, ultrasound studies, as well as urinary cytology, cystoscopy, TURB, and a computed tomography scan of the upper tract, if appropriate. An independent validation cohort of 155 subjects was prospectively accrued in 2014. Total RNA was isolated from 50 ml of midstream-voided urine samples that were collected in a sterile cup before TURB, further instrumentation, or contrast studies. Total RNA was further extracted from 16 randomly selected fresh snapfrozen UBC specimens. Following reverse transcription, CAIX presence and CAIX quantity were analyzed by quantitative polymerase chain reaction using TaqMan Gene Expression Assays (Supplement). CAIX was detected in 169 of the 196 patients with UBC (86.2%) and in only 6 of 123 controls (4.9%; p < 0.001). The sensitivity, specificity, and positive and negative predictive values of CAIX for UBC detection were 86.2%, 95.1%, 96.6%, and 81.2%, respectively (area under the curve [AUC]: 90.5%). Comparably, the AUC for continuously coded CAIX expression levels was 90.8% (Supplementary Fig. 1). Paired samples from urine and tumor tissue were evaluated in 16 cases. Of the 13 CAIX-positive urine samples (81.3%), all expressed CAIX within the tumor. One patient with CAIX in the tumor did not show CAIX in the urine, and two patients had both negative CAIX in the urine and tumor (p = 0.002). A cytologic examination of voided urine samples was performed in 246 of the 319 participants including 184 with UBC and 62 controls. The sensitivity and specificity of urinary cytology were 43.5% and 100%, respectively (AUC: 71.7%). CAIX showed significantly higher predictive
Table 1 – Comparisons of sensitivity, specificity, and area under the curve for cytology and carbonic anhydrase according to grade Overall Sensitivity, % Cytology CAIX Specificity, % Cytology CAIX AUC, % Cytology CAIX p value*
Low grade
High grade
43.5 86.2
23.6 84.5
70.5 88.4
100.0 95.1
100.0 95.1
100.0 95.1
71.7 90.5
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Brief Correspondence
Carbonic Anhydrase IX as a Diagnostic Urinary Marker for Urothelial Bladder Cancer Michela de Martino a, Ilaria Lucca a, Aurelie Mbeutcha a, Helene G. Wiener b, Andrea Haitel b, Martin Susani b, Shahrokh F. Shariat a,c,d, Tobias Klatte a,* a
Department of Urology, Medical University of Vienna, Vienna General Hospital, Vienna, Austria;
b
Clinical Institute of Pathology, Medical University of
c
Vienna, Vienna General Hospital, Vienna, Austria; Department of Urology, University of Texas Southwestern Medical Center, Dallas, TX, USA; d Department of Urology, Weill Cornell Medical College, New York-Presbyterian Hospital, New York, NY, USA
Article info
Abstract
Article history: Accepted June 14, 2015
Urinary biomarkers are needed to improve the management and reduce the cost of urothelial bladder cancer (UBC); however, none have been recommended yet for clinical practice. This study evaluated carbonic anhydrase IX (CAIX) as a diagnostic urinary biomarker for UBC. CAIX was analyzed by quantitative polymerase chain reaction in urine samples of 196 patients with UBC and 123 controls with hematuria. Paired samples from urine and tumor tissue were evaluated in 16 cases. Data were validated in 155 independent samples. The sensitivity and specificity of CAIX for UBC detection were 86.2% and 95.1%, respectively (area under the curve [AUC]: 90.5%). There was a significant association of CAIX expression between the paired urine and tumor specimens (p = 0.002). CAIX showed a significantly higher predictive accuracy than urinary cytology (90.5% vs 71.7%), specifically in low-grade tumors (90.0% vs 61.8%). CAIX expression decreased with increasing tumor stage and grade. Analyses in an independent validation cohort confirmed the high accuracy of CAIX for diagnosing UBC (AUC: 88.3%). Patient summary: We evaluated carbonic anhydrase IX (CAIX) as a urinary marker for bladder cancer (BCa) using a large series of patients from a single hospital. We found that urinary CAIX has a high sensitivity and specificity for diagnosing BCa. # 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Associate Editor: James Catto Keywords: Urinary marker Cytology CA9 Carbonic anhydrase IX Diagnosis Cystoscopy Recurrence Bladder cancer
* Corresponding author. Department of Urology, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. Tel. +43 1 40400 26020; Fax: +43 1 40400 23320. E-mail address: [email protected] (T. Klatte).
Intense research is being performed on urinary biomarkers with the goal of enhancing the detection of noninvasive early urothelial bladder cancer (UBC), allowing replacement, delay, or complementation of cystoscopy. Numerous biomarkers have shown a higher sensitivity than urinary cytology but lower specificity [1]. At present, none of these biomarkers has been recommended by guidelines for routine clinical care [1,2]. Carbonic anhydrase IX (CAIX) is an enzyme of the carbonic anhydrase family that regulates the pH value as a response to hypoxia [3]. In a previous immunohistochemical
study, normal urothelial tissue samples were all negative for CAIX; >70% of UBCs stained positive. Expression levels were markedly increased in Ta and low-grade tumors [4]. We hypothesized that urinary levels of CAIX were significantly elevated in UBC compared with controls and that CAIX is associated with UBC pathology. A total of 319 urine samples and associated clinical and pathologic information were collected prospectively from UBC patients and controls between 2009 and 2011 (training cohort). The UBC group consisted of patients who were
http://dx.doi.org/10.1016/j.eururo.2015.06.015 0302-2838/# 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: de Martino M, et al. Carbonic Anhydrase IX as a Diagnostic Urinary Marker for Urothelial Bladder Cancer. Eur Urol (2015), http://dx.doi.org/10.1016/j.eururo.2015.06.015
EURURO-6262; No. of Pages 3 2
EUROPEAN UROLOGY XXX (2015) XXX–XXX
scheduled for a transurethral resection of the bladder (TURB). The control group was composed of patients with hematuria but with other urologic conditions (Supplementary Table 1). These patients were evaluated with urinalysis, ultrasound studies, as well as urinary cytology, cystoscopy, TURB, and a computed tomography scan of the upper tract, if appropriate. An independent validation cohort of 155 subjects was prospectively accrued in 2014. Total RNA was isolated from 50 ml of midstream-voided urine samples that were collected in a sterile cup before TURB, further instrumentation, or contrast studies. Total RNA was further extracted from 16 randomly selected fresh snapfrozen UBC specimens. Following reverse transcription, CAIX presence and CAIX quantity were analyzed by quantitative polymerase chain reaction using TaqMan Gene Expression Assays (Supplement). CAIX was detected in 169 of the 196 patients with UBC (86.2%) and in only 6 of 123 controls (4.9%; p < 0.001). The sensitivity, specificity, and positive and negative predictive values of CAIX for UBC detection were 86.2%, 95.1%, 96.6%, and 81.2%, respectively (area under the curve [AUC]: 90.5%). Comparably, the AUC for continuously coded CAIX expression levels was 90.8% (Supplementary Fig. 1). Paired samples from urine and tumor tissue were evaluated in 16 cases. Of the 13 CAIX-positive urine samples (81.3%), all expressed CAIX within the tumor. One patient with CAIX in the tumor did not show CAIX in the urine, and two patients had both negative CAIX in the urine and tumor (p = 0.002). A cytologic examination of voided urine samples was performed in 246 of the 319 participants including 184 with UBC and 62 controls. The sensitivity and specificity of urinary cytology were 43.5% and 100%, respectively (AUC: 71.7%). CAIX showed significantly higher predictive
Table 1 – Comparisons of sensitivity, specificity, and area under the curve for cytology and carbonic anhydrase according to grade Overall Sensitivity, % Cytology CAIX Specificity, % Cytology CAIX AUC, % Cytology CAIX p value*
Low grade
High grade
43.5 86.2
23.6 84.5
70.5 88.4
100.0 95.1
100.0 95.1
100.0 95.1
71.7 90.5
