Early diagnosis for UTUC has important clinical implications for improving patient outcomes . However, accurate diagnosis is still challenging owing to the difficult accessibility of the upper tract, causing clinical decision-making intractable, especially when radiologic imaging or biopsies are inconclusive or not diagnostic. We have previously identified a urine-based non-invasive UTC assay which detects bladder UC with high sensitivity and specificity . Accordingly, this study aimed to assess the value of the UTC assay as a diagnostic tool for UTUC. Our results show that UTC assay identified UTUC with 85.0% sensitivity in void urine samples, maintaining satisfactory specificity. Furthermore, UTC assay showed excellent performance in subgroups of stage and grade. Remarkably, UTC assay increased the sensitivity rates as compared to FISH across non-muscle-invasive and LG UTUC.
Previously reported data agreed that conventional urine cytology has little value in the diagnosis of UTUC . Although the collection of urine specimens by ureteral catheterization can improve the sensitivity to some degree, the sensitivity of selective upper tract urinary cytology is 53.1%, specifically, 45.6% for LG tumors and 69.9% for HG tumors , still not good enough, and catheterization may increase false-positive rate. Voided urine collection is convenient and comfortable, particularly for patients under long surveillance. Therefore, we selected voided urine as the research specimen for UTC assay.
FISH was first reported to be feasible and effective for the diagnosis of UTUC using voided urine specimens by Marín‑Aguilera et al. with overall sensitivity of 76.7% and specificity 94.7% . A few years later, Mian et al. reported a FISH analysis with 100% sensitivity using washing samples of the renal pelvis or ureter in a small UC cohort . Three FISH studies reported sensitivities of 78.9%, 84.0% and 85.7% respectively on voided urine specimens [14,15,16]. Sensitivity of FISH in the present study was somewhat lower (73.3%), the discrepancy might be attributed to the different interpretation standard for positive FISH test (e.g., cut-off value and quality control), difference in patient selection, and more importantly the “voided urine” sample collection method. Indeed, there were even more disappointing sensitivities of voided FISH in previous literature, and some were as low as 52% and 54%, questioning its usefulness for UTUC diagnosis and surveillance [17–18]. It is of note that FISH is much less sensitive in detecting LG UTUC. Our results are consistent with the previous findings that FISH has a low sensitivity in non-invasive and LG tumors [12, 17–18].
One possible reason for the poor FISH results in LG and non-muscle-invasive UTUC is these relatively low-burden tumors are less prone to shedding tumor cells into the collecting system . Correspondingly, our final analysis found that one FISH negative LG case did show genetic abnormalities (p16-, + 17, +7, + 3), but only in one cell, not reaching positive threshold. Likewise, for two of the three both negative (UTC assay and FISH) cases, surgical records indicated twisted and narrowed ureters resulting in procedures failure during preoperative ureteroscopy. Urinary obstruction caused by tumor or inflammation might prevent the dissemination of exfoliated tumor cells to urine . We also speculated that reduced urine flushing due to reduced glomerular filtration rate of the ipsilateral kidney or decreased ureteral motility may further retard cell shedding. Optimization of cell collection either by washing urine or from voided urine is crucial . It was reported that the result of immunocytology was dependent on the technique of slide preparation . One of the advantages of UTC assay is the urine exfoliated cell enrichment nanotechnology. Among the UTC assay treated 60 UTUC voided urine specimens, the median enriched and high-throughput analyzed cell number was 8,384, the most one being 525,468 cells. However, during FISH judgment process, only about 100 tested cells in one analysis sample are observed in the view of microscope in routine process of our pathology department. Thus, UTC assay improves sample collecting from another aspect, that is, greatly increase the number of tested cells. By making full use of the exfoliated cells from upper tract, UTC assay can greatly increase the probability of capturing cancer cells. Meanwhile, after the efficient, strong, and rapid attachment of tumor cells in urine, the nanostructured substrates could retain morphology of the binding cells for further judgement. Another possible explanation is that LG tumors in these patients were generally diploid or near-diploid tumors with relatively few chromosomal abnormalities . Molecular genetic studies suggest that LG pTa tumors have relatively few chromosomal alterations other than deletions of part or all of chromosome 9 .
The Immunocyt test which targets the monoclonal antibodies M344, LDQl0 and 19A211 was reported to be sensitive for all grades and stages bladder UC and high sensitivity to LG UTUC [24, 25]. Messing et al. confirmed the performance of Immunocyt for detecting LG, superficial, small tumors . Intrinsically, the UTC assay is an immunocytology method applying immunoreaction against tumor associated antigen CK20 based on cell enrichment nanotechnology and high-throughput analysis. CK20 expression in exfoliated urine cells has been extensively studied either with immunocytochemistry or RT-PCR [21, 27,28,29,30,31]. Bhatia et al. reported that CK20 is an important biomarker that can be used to identify UC in urine cytology smears, especially useful in those cases where the atypical cells are fewer in number or obscured by the inflammatory cells . Buchumensky et al. reported a CK20 RT-PCR-based study of voided urine samples and observed a sensitivity of up to 91% . Importantly, CK20 showed high diagnostic efficiency for LG tumors [28, 32], which may be another important interpretation accounting for high sensitivity of UTC assay in LG UTUC.
Apart from standard treatment of nephroureterectomy with a bladder cuff for UTUC, endoscopic treatment of biopsy-proven LG UTUC in selected patients such as those with solitary kidney, renal insufficiency, bilateral upper tract cancer or clinically low-risk cancer is reasonable . The patients underwent endoscopic ablation and surveillance are the primary candidates to potentially benefit from a urine marker that could predict therapeutic effect and recurrence. The diagnostic dilemmas created by LG urothelial neoplasms of the upper tract are difficult to resolve by conventional cytology, nor FISH [5, 17–18, 33]. J. E. Freund el al evaluated FISH in 1 mL of passively collected selective urine as a triage test for kidney sparing surgery of UTUC, reporting high sensitivity of 90% and a specificity of 80%, however the sample size of the study is small . In contrast, FISH showed disappointing sensitivity for LG UTUC in our study, indicating that the usefulness of voided FISH in this patient population is unsuitable, which is similar to the study of James et al. . The encouraging results of UTC assay in the present study suggest that it is an alternative diagnostic option in selected patients, especially in the surveillance of patients treated endoscopically for LG UTUC.
UTC assay has several additional clinical advantages. The accuracy of the cytology and immunocytology is dependent on cytopathologists’ experience and skills. For FISH, the counting and interpretation process is troublesome. In this regard, UTC assay can reduce interobserver difference by using fully automated high-resolution cell imaging system, which allow maximum analysis of fluorescent staining signal and high-throughput analysis of cell structure. The examination time of UTC assay is shorter as the immunostaining technique is simpler and easier compared with FISH. Last but not least, the cost of UTC assay is lower, which is nearly half of that of FISH.
The limitation of the current study was the retrospective design and relatively small number of patients from a single institution. Our preliminary results warrant further larger and multi-center studies, particularly, for LG UTUC. Additional well-designed prospective studies on UTC assay are needed for validation.