There are approximately 50,000 new cases of urothelial carcinoma with 10,000 deaths each year in the United States. Cystoscopy and cytology have been the primary modalities used to detect and monitor urothelial carcinomas.[2, 3] It has been well demonstrated that cytology has a high specificity but low sensitivity for bladder cancer detection, particularly if the urothelial neoplasia is of a lower grade. Several potential adjunct markers have been used to improve the sensitivity and specificity of the urinary tract cytology diagnoses.[4-9] Here, we have explored usage of ProEx C as an aid in improving the sensitivity of urine cytology cancer diagnoses. ProEx C is a novel antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein 2 (MCM2).[10-12] Both proteins are overexpressed in the cell nucleus during aberrant S-phase induction of neoplastic and human papillomavirus–infected cells.[13-15] Several studies have demonstrated ProEx C as an adjunct marker for assessing dysplasia in gynecologic specimens, especially in cervical biopsies and smears.[15-23] In this study, we have attempted to use ProEx C on urine cytology specimens where a significant number of them are diagnosed as “atypical,” a designation that triggers unwarranted additional procedures that may result in patient discomfort and/or increase in cost. This study was designed to determine the utility of ProEx C in urine cytology samples for improving the precision of detection of urothelial carcinomas.
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- MATERIALS AND METHODS
- FUNDING SOURCES
- CONFLICT OF INTEREST DISCLOSURES
Since the introduction of urine cytology 6 decades ago as a means of detecting urothelial neoplasms, it has remained a cancer screening test despite its well-known limitations.[26-31] When a definitive benign or malignant diagnosis is made by cytology on urinary samples, it is highly reliable. It is, however, limited when a neoplastic lesion, particularly a low-grade tumor, becomes indistinguishable from a reactive process. Therefore, whereas urine cytology has a critical role in the evaluation of urothelial cells, the cytological diagnosis of urothelial carcinoma can be very challenging. In general, urine cytology has been used as a screening tool to detect urothelial cancers in high-risk patients, as an initial test for individuals with hematuria, and as a follow-up test in patients with a previous diagnosis of urothelial cancer to rule out the tumor recurrence. When a diagnosis of “atypical” cytology is made on urinary specimens, invasive procedures may be triggered that result in patient's discomfort, anxiety, and/or increase in cost. To remedy this situation, several markers have been introduced to improve the accuracy of urinary cytology. Cytokeratin-20 has been evaluated as an adjunct marker in cases of atypical cytology. A malignant process is favored when this marker is positive.[34-36] Cheng et al has proposed that prostate stem cell antigen may be another useful adjunct marker to supplement urine cytology. Multiple studies have evaluated the usefulness of NMP-22 (nuclear matrix protein 22).[38-41] Other ancillary tests such as ImmunoCyt[42, 43] and UroVysion[4-6] have also been used; however, they are expensive and require special expertise and certification for the test interpretation. Yet, none of these markers has been accepted as a simple and universal marker of the neoplastic cells in urine cytology. In addition, Burger et al have used MCM2 antibody in surgical specimens to evaluate the risk of recurrence in bladder cancers. They have shown that MCM2 predicts recurrence of the neoplasia in stage Ta/T1 more accurately than cytokeratin-20, ki-67, and histologic grade.
In this study, we have introduced immunocytochemical staining of urothelial cells with ProEx C. This marker, heretofore, has been mainly used as a molecular marker for differentiating neoplastic/dysplastic cells in the gynecological specimens. We demonstrate that this antibody cocktail could also serve as an adjunct marker in urine cytology. As shown in Tables 1 and 4, ProEx C staining results highly correlate with the cytology and histopathology findings where definitive diagnoses have been made in group I. It appears that when a definite diagnosis is made by cytology, there is no need for performing the ProEx C stain on the specimen. The value of the stain becomes apparent in a significant number of the urinary cytology cases (group II, ∼60% of the cases in this study) when a diagnosis of “atypia” is made (Table 2). It appears that a large number of the cases, 27 of 35 (cases #26-#35 and #44-#60, Table 2), have benefited from staining with ProEx C by stratifying the cytology diagnoses. The remaining 8 cases in this group have resulted in the false reactions. In a relative sense, the number of false negative reactions (case #13, Table 1 and cases #37-#43, Table 2) is higher than the false positive reactions in this study (case #36, Table 2), yielding a high specificity and PPV but a lower sensitivity and NPV (Table 4). We presume that the false negative (if they are indeed false negative) reactions are mostly technical in nature due to the variability inherent in the manual methodologies. The basis of our presumption is the general stainability of the normal small parabasal cells with ProEx C. The parabasal cells in 4 of the cases (cases # 39-#42, Table 2) did not exhibit ProEx C staining, which we attribute to isolated technical malfunctions. Because the respective large atypical cells also did not stain with ProEx C, we assume that these 4 cases are outliers resulting from the same technical variability. Henceforth, on the basis of this assumption of technical malfunctions, we removed the 4 cases from Table 4 and retabulated in Table 6, resulting in significant improvements in the assay performance. To aid in circumventing a misinterpretation of a negative stain arising from such technical malfunctions in the future, one may also use a histologic section of normal urothelium with an intact parabasal cell layer as positive control on all cases simultaneously stained with this marker, particularly when using a well-controlled automated platform.
Table 6. Presumptive Test Performance of ProEx C Reactivity in the Combined and Individual Groups After Eliminating 4 of the False Negative Reactions in Group II on the Assumption of Being Outliers
| ||All Groups||Group I||Group II|
|False positive (n)||1||0||1|
|False negative (n)||4||1||3|
In addition, in this study, 8 cases (1 patient in group I and 7 patients in group II) had a low-grade urothelial neoplasia on histopathology follow-up. Four of these patients were classified as false negative for ProEx C; 1 patient in group I (case #13, Table 1) and 3 patients in group II (cases #40, #41, and #43, Table 2). Although an argument can be made for a lower sensitivity of ProEx C in low-grade urothelial cancers, the low number of the low-grade papillary carcinomas in our series does not allow for such a definitive conclusion. Future large-scale studies are required to address this point.
We are confident that in a larger study with longer follow-up periods, all assay performance parameters would be 95% or better provided the staining with ProEx C is performed on a tightly controlled automated platform. The “false positive” reaction (case #36, Table 2) may have been an early indication of neoplastic development even though the immediate histopathology follow-up has been negative. This assumption may be clarified during a longer follow-up period.
In conclusion, ProEx C seems to be a promising and simple adjunct tool in urine cytology and further helps by avoiding expensive and time-consuming tests such as immunofluorescent and/or fluorescence in situ hybridization assays. This stain is particularly useful when the urine samples have scant cellularity with only a few atypical cells present, creating difficulty in cytologic interpretation.