Few studies have explored the ability of urinary biomarkers in predicting the progression of high risk NMIBC to a muscle-invasive state.

The evaluation of the Urovision® Test before BCG treatment failed to differentiate patients who later progressed from those who did not. [22] Kamat et al. found a notable disease progression variance in High Risk NMIBC patients with a positive Urovision® Test (19.8% vs. 4.4%), lacking reliability for clinical use [23]. Piaton et al. in 2014 highlighted using p16INK4a and Ki-67, showing 100% progression likelihood in pTa/pT1 HG BC patients with both markers. Despite this, the test’s sensitivity and specificity did not exceed traditional cytology, making it unsuitable as a clinical substitute [4].

As far as we know, the CUBN/MPO ratio has never been previously studied as a promising biomarker or prognostic factor for urinary cancer or any other type of cancer. However, cubilin (CUBN) has been identified and validated as a marker capable of classifying renal cell carcinoma (RCC) patients into low- and high-risk groups. Loss of CUBN expression has been significantly and independently correlated with less favourable patient outcomes concerning overall survival. Conversely, stratification of patients according to CUBN positivity showed a significant benefit for patients with CUBN-positive tumours in overall survival and RCC-specific survival [16].

Although these results apply to RCC, they point in the same direction as our findings, suggesting that the loss of expression of this protein is associated with a poorer outcome. According to our results, patients with upregulated cubilin did not show disease progression, apart from only one patient who had recurrence. On the contrary, of the patients who exhibited downregulated cubilin (eight patients), seven had recurrence and five had disease progression.

On the other hand, myeloperoxidase (MPO) can support a hypermutagenic environment through the action of MPO-derived oxidants that are able to oxidize and modify DNA. Furthermore, MPO has been involved in pathways of apoptosis, cell migration, tumour growth and adaptive immunity in cancer [12]. Also, it is already known that MPO interferes in the activation of procarcinogens, included in tobacco smoke, such as polycyclic aromatic carcinogens, aromatics amines, heterocyclic amines and the endogenous formation of carcinogenic free radicals [24]. The variant genotypes of MPO may be related to low enzyme activity and polymorphisms in MPO that was related to a decreased risk of bladder cancer developing in Tunisian population [25].

This finding emphasizes the potential importance of MPO in BC carcinogenesis.

Although the protein has not been validated as a severity biomarker in bladder cancer, Valadez-Cosmes et al. showed that MPO expression was associated with lower survival in non-small cell lung cancer patients [26]. Indeed, in our study, the results indicate that patients with downregulated MPO in urine tend to have a better outcome during follow-up, with lower rates of recurrence and disease progression and patients with upregulated MPO tend to have shorter overall survival.

The value in ratio use for both proteins as a biomarker allowed to add discriminatory value in patient selection, mostly in those who had one of the proteins excessively down- or upregulated. Here, we demonstrated that the mean ratio value of CUBN/MPO was significantly lower if patients had experienced bladder tumour recurrence or progression. This result suggests that the lower the CUBN/MPO ratio, the worse will be the prognosis.

In our study, the results showed that the risk of having recurrence and progression was respectively and approximately 13 and 8 times higher in patients with low ratio of CUBN/MPO compared to patients with high ratio of CUBN/MPO. In the univariable Cox regression analysis, it was not possible to demonstrate a statistically significant risk association for recurrence-free survival or progression-free survival for other clinically validated variables known as prognostic factors, such as tumour size and the number of tumours. These results were likely due to the small size of the patient cohort studied, which precluded performing a multivariable analysis. Therefore, this work must be viewed as an exploratory and preliminary study that will need validation in a larger patient population.

An important limitation of this study was the lack of a representative sample of female patients. Only one woman was included in the study. This could have been the reason why the female gender showed a discrepancy in the risk of recurrence-free survival (HR 0.186 (0.022–1.591)) compared to that of progression-free survival 21.677 (0.00–436,032,183.4). A more representative number of both genders should be analysed in further studies, also to allow evaluate possible sex-dependent results. Importantly, age may also play a factor, in that the low CUBN/MPO ratio group was older compared to the high ratio group.

Another limitation of the study, as previously mentioned, was that not all patients underwent a complete initial TURB. Nonetheless, from the interpretation of the survival analysis, it appears that the risk of recurrence was lower in patients who had a complete TURB compared to the group of patients in whom this did not occur. However, it was not statistically significant (p = 0.370).

Regarding the analysis for progression-free survival, the p value for the complete TURB variable almost reached the threshold of statistical significance (HR 0.202 (0.040–1.008), p = 0.051), suggesting that the group of patients with low CUBN/MPO may have been impacted in disease progression due to presenting more extensive tumours, as the size of tumour was so large that complete resection was not achieved in the initial surgery.

However, it is necessary to reinforce that two of the patients with incomplete TURB belonged to the low CUBN/MPO ratio group (28.6%) and three patients to the high CUBN/MPO group (13%), which represents a not very disparate percentage between the groups.

Additionally, the fact that several patients did not undergo a second TURB or showed no evidence of muscle in the first TURB sample raises suspicions that some of these patients may have been sub-staged. However, upon analysing Table 1, it is observed that in the group of patients with a low CUBN/MPO ratio, the proportion of patients who underwent a second TURB is similar to the proportion found in the high CUBN/MPO ratio group, 57.1% and 52.2%, respectively. Also, in the univariable Cox regression analysis, the performance of the second TURB did not show statistical significance for recurrence-free survival and progression-free survival, despite maintaining a beneficial trend associated with lower risk.

Contrary to what the study by Baltaci et al. showed, in our study, an increase in the time between the first and second TURB appears to exhibit a trend towards a lower risk of recurrence and progression. This may be because our study included patients with an incomplete first TURB, which in some way influenced the timing of the second TURB [27]. However, the reasons for the extended duration of the second TURB were not evaluated, which is another limitation.

Proteome analysis plays a key role in the understanding of the carcinogenesis involved in bladder carcinoma. The use of urinary proteins as potential diagnostic biomarkers has been investigated for years [5]. However, this is the first time a ratio between a supposed protective protein (cubilin) and a cancer aggressiveness promoter (MPO) measured in the urine of patients has been studied as a prognostic risk factor to predict recurrence and progression to muscle-invasive T1 bladder tumours.

Years ago, it was considered standard to follow NMIBC patients until muscle invasion occurs and then perform radical cystectomy. Nowadays, we know that some high-grade T1 bladder tumours may progress to aggressive muscle-invasive tumours, and this is considered a deleterious prognostic as they are at serious risk of having systemic nonclinical disease at the time of cystectomy. Also, when there is progression to muscle-invasive bladder cancer (MIBC), the delay of cystectomy has been associated with lower survival [2, 3]. However, there is a lack of tumour prognosis biomarkers that can help clinicians in the selection of those patients who will have progression in the future and intervene with more aggressive treatments before the actual development of muscle invasion. This may improve the decision in individualized treatment.

A study investigated Oncuria™, an assay for diagnosing, predicting, and monitoring BCa response to intravesical BCG treatment. This test evaluated intermediate- to high-risk NMIBC patients before BCG treatment. After BCG therapy, BC recurrence was associated with elevated levels of specific proteins (ANG, APOE, A1AT, CA9, MMP9, MMP10, PAI1, SDC1, VEGFA) in the Oncuria™ panel, demonstrating its potential for assessing the effectiveness of intravesical BCG treatment and predicting relapse risk in BC patients [22].

Shariat et al. developed a tool that combines NMP22® with age, gender and cytology to predict recurrences in specific bladder conditions (pTa G3, pT1 and CIS) with high accuracy. Despite its reliability, this model is not widely used in clinical practice, possibly due to uncertainties about its threshold for positivity, adaptability across institutions and a relatively high rate of false positives [28].

Despite the efforts to identify precise biomarkers predicting oncologic results in high-risk NMIBC, conflicting or ambiguous findings hinder the incorporation of these supplementary methods into clinical practice.

Our results suggest that low CUBN/MPO ratio could be a biomarker to select aggressive high-grade T1 bladder cancer patients to be candidates to early cystectomy and improve their oncologic outcome; however, a prospective trial will be necessary. This retrospective study has many limitations including the low number of patients, the inclusion of patients with incomplete first TURB and with no detrusor in specimen and patients that did not undergo a second resection or treatment with BCG. However, despite these limitations, these results must be considered as a preliminary work and further work should be conducted to evaluate and validate the role of CUBN/MPO ratio and to find the biological mechanisms underlying the relationship between CUBN and MPO in high-grade T1 bladder cancer.