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NPV.7 9 Using the test, »17% of PFBC from our vali-dation cohort could safely skip cystoscopy. The remain-ing PFBC should undergo cystoscopy. This is not a major problem, since in normal daily practice PFBC would have undergone a cystoscopy anyway. Using our 8-gene classifier, 6 recurrent PFBC were wrongly diagnosed as not having a recurrence; 4 of them had TaLG, 1 had a TIS and 1 had a T1HG tumor. Of note, cytology also missed these 6 tumors. Further-more, it is known that cystoscopy, our gold standard, misses upto 15% of the papillary and upto 30% of the flat lesions.22
It should be considered that a high proportion of patients who presented non high-risk tumors are being long-term monitored. In our series, 60% of the BC samples analyzed are non high-risk NMIBC and a SN of 94% and NPV of 98% was found in this sub-group of patients. This population may be hugely benefited from the avoidance of continuing surveil-lance procedures which are invasive. Of importance, the 8-gene classifier maintains a high SN and NPV in high-risk NMIBC disease (97% and 99%, respectively) guarantying the detection of potential life-threatening tumors. Lastly, the accuracy of our 8-gene classifier was not affected by number of tumors neither did it correlate with tumor size, further supporting its useful-ness in the FU of BC patients.
We found that the 8 genes composing the classifier regulate the carcinogenesis process via different mechanisms. This is shown by the fact that the genes of the model have no direct interactions and derive from different pathways. This 8-gene classifier
82 Montalbo et al Translational Research
Fig 4. Performance of the 8-gene classifier. (A) ROC curve of 8-gene model in training set. (B) Risk of tumor probability representation according to the 8-gene expression classifier in training and (C) testing sets (cut-off = 0.425).
Abbreviations: AUC, area under the curve; BC, Dorsomorphin (Compound C) cancer; NMIBC, Non muscle-invasive bladder cancer; NR-PFBC, non-recurrent con-trol patients in follow-up for BC. (Color version of figure is available online.)
incorporates 3 genes not previously included in our reported classifiers: LCN2, RPS21, and KIFC3. LCN2 facilitates tumorigenesis by promoting sur-vival, growth, and metastasis23 and it has been reported several cancers.24 Preliminary evidence also suggests that LCN2 may be used as a prognostic uri-nary biomarker of breast cancer.25 According to our results, Arthurs et al26 found that RPS21 presented significantly raised levels of expression in malignant prostate cancer tissue. Finally, KIFC3 plays a role in the formation, maintenance, and remodeling of the bipolar mitotic spindle. It has been not related previ-ously with carcinogenetic processes. Gene ontology analysis confirmed that the genes included in the classifier are highly related to bladder malignancies,
corroborating the suitability of these genes to be use-ful for the detection of BC.
The strengths of this study lie in the fact that we have chosen a 3-stage approach using PFBC, the patients in whom the test should be applied. We have also used a blind approach to identify genes particularly related with non high-risk BC patients. Furthermore, the use of voided urine samples to analyze gene expression allows for the development of a non-invasive BC diagnostic tool with an easy translation into clinical practice. Lastly, the test is based on a multiplexing format directly quantifiying by a hybridization gene expression. However, some limitations should be mentioned. To avoid ineffi-ciency, patients with recurrence were oversampled by also recruiting patients who were scheduled for
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a bladder transurethral resection of a proven bladder tumor. Consequently, some of the patients had pri-mary tumors. Moreover, the number of NR-PFBC was misrepresented in the validation series and we had to make an estimation to calculate the number of cystoscopies that could be skipped. Second, 8.5% of samples had to be excluded due to technical fail-ures. Eventually, the low SP of the test should be improved to prevent unnecessary cystoscopies.