Influencing factors on the NMP-22 urine assay: an experimental model

Abstract

Background: The commercial NMP-22 urine assays for bladder cancer (BCa) detect nuclear mitotic apparatus protein 1 (NUMA1) using monoclonal antibodies. It remains unclear whether these assays are monitoring a tumor antigen or some other phenomenon associated with the disease state. In this study, we investigated the influence of urinary cellular and protein concentration, and hematuria on the performance of the NMP-22 tests in an experimental model.

Methods: Pooled urine from healthy subjects were spiked with varying concentrations of benign (UROtsa) cells, cancer cells (RT4, T24, KU-7 and UM-UC-14), whole blood or serum, prior to analysis with both NMP22® Bladder Cancer ELISA test and the NMP22® BladderChek® point-of-care test.

Results: Urines from control subjects were negative for NMP-22. The addition of whole blood at 50ul/10 ml, but not serum, resulted in a false-positive result. Furthermore, the addition of a high concentration of benign urothelial cells (10(6)) or the cell lysate from these cells (306 μg protein) resulted in a false-positive result. High concentrations of pooled-cancer cells (10(6)) or cell lysate (30.6 μg and above) resulted in a positive NMP-22 assay. Concordance between the NMP-22 ELISA assay and the NMP-22 point of care assay was >90%.

Conclusions: Rather than detecting a specific tumor antigen, urinary NMP-22 assays may be measuring the cellularity or amount of cell turnover that may be introduced into the urine by a variety of conditions, including surface shedding from bladder tumors. The absence of significant urinary cellularity in some cases due to lesion characteristics or the timing of sampling may result in false-negative NMP-2 assays.

Figure 1

Schematic of experimental model. Low concentration (1×10⁴), medium concentration (1× 10⁵) and high concentrations (1× 10⁶) of UROtsa benign human bladder cells, or a mixture of human bladder cancer lines, RT4, T24, UC-UM-14 and KU-7 were added to 10 ml of pooled urine from three healthy controls. The cellular lysate associated with low protein concentration (3.06 μg), medium concentration (30.6 μg) and high concentrations (306 μg) of UROtsa benign human bladder cells or a mixture of human bladder cancer lines, RT4, T24, UC-UM-14 and KU-7 were added to 10 ml of pooled urine from three healthy controls. In addition, whole blood (1, 5, 20 and 50 μl) and serum (3.06, 30.6 and 306 μg protein) were added to 10 ml of pooled urine from healthy controls.

Figure 2

NMP-22 ELISA assay of human bladder cell lines. The human bladder cancer cell lines, RT4, T24 and UM-UC-14 had elevated NMP-22 levels compared to the benign UROtsa human cell line. Levels of NMP-22 in the KU-7 human bladder cancer cell line were not elevated compared to UROtsa. Significance (p < 0.05) was assessed by Student t test. n.s., not significant.

Figure 3

NMP-22 ELISA assay of the experimental model. Positive NMP assays were elicited by high concentrations of UROtsa cells, pooled cancer cells, cell lysate from UROtsa, and medium and high concentrations of cell lysate from pooled cancer cells. Mean levels are depicted by long horizontal lines. Error bars indicate standard deviations.

Figure 4

Immunostaining of NMP-22 (NUMA1) in normal urothelia and bladder cancer tissue. Images originate from the Human Protein Atlas web portal. Top panels show magnified insets of tissue microarray images directly below. A, normal urinary bladder tissue from a male of 37 years. B, normal tissue from a male of 51 years. C, bladder carcinoma tissue from a male of 51 years. D, bladder carcinoma tissue from a male of 78 years. The normal urothelial layer, and carcinoma tissues display strongly positive nuclear staining and less intense cytoplasmic staining.