Original articles
DD23 Biomarker: A prospective clinical assessment in routine urinary cytology specimens from patients being monitored for TCC

https://doi.org/10.1016/S1078-1439(02)00188-6Get rights and content

Abstract

Background: A prospective clinical study was conducted to assess the ability of the DD23 murine monoclonal antibody to enhance detection of bladder cancer in routine alcohol fixed urine cytology samples. Methods: Prospectively, 308 bladder cytology specimens were obtained from patients with a history of bladder cancer with a mean age of 71.4±11.9 (27% female, 73% male). Data included 121 biopsy-confirmed results and 187 cystoscopy results to assess presence or absence of cancer. Thirty-five normal cytology specimens were obtained from asymptomatic men and women between 55–85 years of age. Separate slides from the alcohol fixed cytology specimens were stained using the Papanicolaou (Pap) and Feulgen staining procedures. The DD23 assay was performed using an avidin-biotin alkaline phosphatase immunocytochemical procedure, with a single urothelial cell exhibiting intense immunostaining sufficient to make a positive call. RESULTS: Pap-Feulgen cytopathology for the 308 cases yielded an overall sensitivity of 65.5% and a specificity of 85.1%, and the DD23 biomarker alone yielded a sensitivity of 80.5% and a specificity of 59.7%. Analysis of the voided urines only (n=164) yielded sensitivities of 61.0% and 73.2% and specificities of 86.2% and 67.5% for cytopathology and DD23 alone, respectively. Results in 49 bladder wash urine cytology cases produced a sensitivity of 70.2% and 100% and specificities of 92.3% and 61.5% for cytopathology and DD23 alone, respectively. In 133 patients that underwent biopsy or had positive cystoscopy results, cytopathology yielded a sensitivity of 65.5% and a specificity of 69.6% while DD23 yielded a sensitivity of 80.5% and a specificity of 58.7%. In 25 biopsy-confirmed low-grade cancers, DD23 improved cancer detection from 32% to 72% when compared to cytopathology. The DD23 biomarker had a specificity of 85.7% in 35 age-matched normal asymptomatic control specimens. Conclusions: The DD23 biomarker is an adjuvant test that provides improved detection of bladder cancer in cytology specimens and enhances the sensitivity of the cytopathology diagnosis, especially in low-grade cancers.

Introduction

A prospective clinical validation study was performed to evaluate the diagnostic performance of the DD23 monoclonal antibody as a biomarker for urinary cytology to detect bladder cancer in patients monitored for recurrence. The assay was an immunocytochemistry chromogen-based cytology stain, applied to routine clinical samples fixed in buffered alcohol. The assay proved to be an excellent adjuvant test for bladder cytology, enhancing the detection of all grades of bladder cancer.

Bladder cancer is the sixth most common cancer in the United States and in 2001 it is estimated that there will be 54,000 new cases reported with more than 12,400 deaths [1]. Approximately 75% of bladder cancers present as superficial disease (Ta, Tis (CIS), or T1 lesions), while the remaining 25% are muscle invasive (T2-T4) 2, 3. The sex ratio for incidence of the disease has remained constant at about 3.0 males per female case reported, with peak prevalence at an age of 60-70 years old 1, 3. Bladder cancer occurs nearly twice as often in Caucasians compared to African Americans [2].

The main risks associated with superficial bladder tumors are recurrence (70–80%) and progression (5–30%). The risk of recurrence and/or progression is driven by the pathology and biology of the primary tumor (e.g. grade, stage, number and size of primary) 3, 4, 5. Hence, bladder cancer patients require diligent monitoring utilizing direct bladder visualization (cystoscopy) and urinary cytopathology 2, 4, 5, 6. This approach permits early detection and characterization of significant changes in the tumor relevant to patient management [6].

Urine sediment cytology analysis has been the longest standing procedure to monitor bladder cancer recurrence and is the standard against which new biomarkers are compared 6, 7. The application of the Papanicolaou (Pap) stain for urine cytology sediment analysis is tedious, associated with interpretive difficulties, and has a low and highly variable sensitivity (20–40%) for the detection of low grade cancers but a high sensitivity and specificity for the detection of high-grade cancers (85–95%) 7, 8, 9, 10, 11.

Cell- and soluble-based biomarkers have been investigated for their ability to enhance or supplement urine cytology examination 8, 9. At the cellular level, DNA ploidy determined by either Flow Cytometry or Image Analysis has been shown to improve the detection of bladder cancer by urine cytology 8, 9, 12, 13, 14. In particular, DNA image analysis provides incremental information when atypical or dysplastic cells are found in the urine [9].

Grossman et al. reported the development of a murine IgG1 monoclonal antibody (MAb) designated DD23 that recognizes a protein dimer of about 185kD 18, 19. Initial clinical studies using immunohistochemical analysis of frozen bladder tissue sections demonstrated strong positive DD23 staining in TCC [19]. Using bladder cytology preparations with a quantitative fluorescence image analysis system in patients with TCC the DD23 MAb yielded a sensitivity of 85% and a specificity of 95% [20].

This manuscript describes the use of the DD23 MAb to detect bladder cancer in routine alcohol fixed cytology clinical samples in conjunction with Papanicolaou and Feulgen staining.

Section snippets

Patient sample

A total of 308 cases were prospectively collected over a sixteen month period. Confirmation for the presence or absence of concurrent tumor was determined using cystoscopy and bladder biopsy results obtained no more than two months prior and six months following the date of specimen collection. The age range for the entire patient sample was 18–94 years of age (mean=71±12).

Among the 308 specimens provided, 164 were voided urines, while the remaining 144 specimens were obtained via

Results

A positive cytology case was defined as a cytopathologic diagnosis of either dysplasia or TCC. Two populations were assessed for specificity of cytopathology vs. the DD23 biomarker. The specificity results in the symptomatic (n=56) and asymptomatic (n=35) controls were 96.4% and 100% for cytopathology, respectively, and for DD23 were 60.7% and 85.7%, respectively.

Table 1 shows the performance of cytopathology alone, DD23 biomarker alone, and the combination of the two in the entire n=308

Discussion

The application of bladder cancer detection biomarkers provides an opportunity to improve the management of individual patients with bladder cancer. To achieve this end, several critical hurdles must be met. First, the performance data of the test must be clinically relevant and diagnostically significant; second, the intended use of the biomarker should be clear (e.g., detection, prognosis, or determination of treatment response); and third, the data should be sufficiently compelling to alter

Conclusions

The DD23 cellular biomarker is a clinically valuable test for enhanced detection of bladder cancer in cytology specimens obtained from patients being monitored for recurrence. It improves the sensitivity for detecting both high grade and low grade cancers compared with cytology alone, and improves the overall negative predictive value of cytology.

Acknowledgements

The authors wish to express their gratitude to Drs. Stanley Hopkins of Boyton Beach, Fl and Mark Ziffer of Delray Beach, Fl for providing patient follow-up information as well as symptomatic control cases. Also, we want to express our sincere appreciation to the UroCor Labs operations cytotechnology staff, especially Anna Debosz, for assisting in the performance of the DD23 reads.

References (21)

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    When used as a quantitative marker to detect BC, it has a sensitivity and specificity of 85% and 95%, respectively [81]. The DD23 assay test was subsequently developed using an avidin–biotin alkaline phosphatase immunocytochemical procedure [82,83], with a single positive cell considered as a positive test result. In 2 studies in the surveillance setting, sensitivity was 70% to 81% and specificity was 60% [82,83].

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    The combination of DD23 and cytology slightly increases the sensitivity (range 78% to 85%). However, the specificity of DD23 is about 60%, and the specificity is lower in barbotage specimens [71,72]. In summary, DD23 is meant to be used with urine cytology to improve the sensitivity of cytology to detect especially low-grade disease.

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