Subclinical diagnosis of cisplatin-induced ototoxicity with biomarkers

A successful biomarker for cisplatin-induced ototoxicity is capable of detecting, diagnosing and monitoring disease progression18. In this study, prestin was significantly elevated in the serum of R1 mice before the onset of significant ABR threshold changes and OHC loss, demonstrating its ability to detect and diagnose subclinical cisplatin-induced ototoxicity. . These findings are consistent with previous reports in the literature showing an elevation of serum prestin before changes in hearing occur.12.15.

In our study, prestin was also found to correlate with the severity of ABR threshold changes. Prestin levels increased significantly in serum as the ABR threshold rose with each cycle of CDDP (FIG. 4). This finding demonstrates that prestin has the potential to monitor disease progression in acute phases of inner ear stress. In 2016, Parham and Dyhrfjeld-Johnsen also showed similar findings with a positive linear relationship between serum prestin concentration and ABR threshold changes.eleven. Another study showed that serum prestin levels increased in response to high-dose versus low-dose cisplatin injection. However, in this study, the low dose of CDDP may not have reached a threshold that significantly challenged the inner ear in their animal model.19.

By measuring the concentration of CDDP in the perilymph, we found that after each cycle of systemic CDDP, there was a gradual increase in CDDP in the perilymph of the mice. Mice with higher levels of CDDP in their perilymph had increased ABR threshold changes and higher levels of prestin in their serum (Fig. 3). We believe that elevated levels of CDDP in the inner ear cause additional ototoxic damage and inner ear stress leading to increased gene expression and serum prestin release. However, the etiology of OHC prestin release is beyond the scope of this study. In 2017, Breglio et al. also found that CDDP increases in a stepwise manner in the cochlea of ​​mice after each systemic dose of CDDP using a similar clinically relevant CDDP mouse model.twenty. However, in their study, they measured CDDP throughout the cochlea and found that CDDP was retained indefinitely within the stria vascularis, organ of Corti, and spiral ganglion with no significant change after 60 days of rest.twenty. In our study, we found that within 24 days of CDDP exposure, CDDP levels from R4 mice returned to non-significant levels in the perilymph. Although R4 levels were still slightly elevated and almost significant compared to control, we believe that the drop in CDDP concentration is likely due to differences in permeability between inner ear endolymph and perilymph, and is likely that CDDP is still sequestered in other locations in the cochlea. knittingtwenty-one. This would also explain why the R4 group had hearing threshold changes and morphological changes that continued to worsen compared to R3, suggesting that CDDP accumulation led to further damage and loss of outer hair cells over time. However, the possible efflux or redistribution of CDDP within the inner ear during this resting period may also play a role in the decreased expression of prestin. Our results show that as CDDP levels fell in perilymph, serum prestin concentration returned to baseline.

Taken together from our current study and others, the data suggest that following systemic administration, CDDP may enter the perilymphatic fluid before being sequestered by other inner ear structures causing ototoxicity. The mechanism of CDDP entry into the inner ear may provide an important approach to blocking inner ear ototoxicity through perilymphatic dialysis.

Our study is the first to capture the relationship of prestin to sequential ototoxic CDDP exposures, mimicking human dosage protocols. Previous studies used a single high-dose bolus of CDDP to cause hearing damage without further exposure after prestin returned to baseline11,12,15. Our findings suggest that acutely elevated serum prestin is likely due to increased prestin expression in response to cisplatin-induced cellular stress rather than outer hair cell apoptosis alone. This is supported by the negligible changes in outer hair cell counts in samples with elevated serum prestin levels (Fig. 5). Although we did not observe obvious changes in prestin expression in our histological analysis, we did not quantify fluorescent intensity due to the limitations of this method, and therefore small changes in prestin expression would not be detected. More importantly, outer hair cell apoptosis alone would likely cause a single spike in prestin with a return to baseline levels after the cells have shed their contents. Previous literature also supports this concept by showing that in response to noise-induced hearing loss, prestin increased between 32% and 58% before returning to baseline 4 weeks after noise with hair cells. intact external.22.23. In another study, protein and mRNA expression of prestin increased threefold in response to two weeks of exposure to salicylate (a known ototoxic agent) and returned to baseline 4 weeks after cessation without permanent hearing loss.24.

Prestin R4 concentration returned to baseline and ABR threshold changes remained elevated 24 days after completing cycle three of cisplatin. This finding is also consistent with previous studies, which showed that prestin concentrations returned to baseline or below baseline after 14 days of cisplatin exposure.11,12,15. One theory suggests that prestin levels below baseline may be related to fewer surviving outer hair cells, which would be consistent with our histological data for R4.12.

In our human analysis, we saw significantly lower prestin levels in our cases compared to controls. With a median duration between cisplatin exposure and sample collection of 47.25 days, our human cohort is comparable to R4. Both the R4 and human cases are beyond the 14-day window seen in the literature showing prestin returning to baseline, likely due to a decrease in prestin protein expression as it is cleared. ototoxic stressor. The lower levels of prestin in our human case samples compared to control correlate with our animal model and previous literature.11,12,15. This response demonstrates that human prestin responds in a predictable manner compared to the animal model in our study and in the literature. Another recent study showed that human baseline levels of prestin are negatively correlated with noise exposure, which is in line with our current data showing that prior injury to the inner ear leads to lower baseline levels of prestin.25. Of note, our human control prestin concentrations are also within the ranges established by two studies measuring prestin in human serum: a recent publication on the reliability of serum prestin measurements in humans and a study on sensorineural hearing loss. idiopathic13.26.

In our study, we did not measure patients’ prestin levels during their acute CDDP exposure, which would likely lead to elevated, rather than reduced, serum prestin levels. However, a recent study by Jalali et al., 2021 evaluated serum prestin concentrations in 52 patients actively receiving cisplatin-based chemotherapy. Similar to the acute findings in our animal model, they found elevated early prestin levels in the serum of cisplatin-exposed patients, and prestin levels were also elevated in patients with significant hearing damage.27.

There are several limitations in both the animal and human parts of the present study. For our human samples from the ITMAT biobank, there was a low sample size of 8, and some patients were treated with additional chemotherapy agents such as gemcitabine and etoposide. Although these drugs are not known for ototoxicity, we cannot be sure of their effect when given in combination with cisplatin.4.28. Furthermore, the classification of otological symptoms in the table is challenging and subjective from the point of view of timeline and severity. Without quantifiable hearing data for these patients, we are limited in the conclusions we can draw from this part of the study. We also did not have baseline waistband levels for each individual patient, which is another major limitation, as there is a large variability in baseline levels that correlates with environmental factors such as noise exposure.25. Another limitation when comparing the human samples with our animal model is the discrepancy in the dose of cisplatin. The mouse model in this study uses significantly higher doses to achieve consistent ABR threshold changes, whereas humans generally receive much lower doses. The generalizability of our study to humans may be limited by the predominantly male sex, advanced age, and the small size of the sample available for testing; however, at baseline levels there were no significant differences in human serum based on gender or age.25.

Another limitation of our study is the cross-sectional design used to collect serum for testing in our animal model. Ideally, it would be better to measure serum prestin concentration before and after cisplatin exposure in the same organism over time. However, this is not possible in mice due to their small blood volume. This was controlled for using a sample size of six at each time point and comparing the means of each group. Also, because there were no statistically significant changes between the individual groups after each cycle of cisplatin, we were able to combine groups to make stronger comparisons. Unfortunately, the R4 group only had six mice. A larger sample size during this time point would have helped determine the importance of perilymph cisplatin concentration, as it appears elevated, but the data is not significant. It would also have been useful to evaluate CDDP in other inner ear structures to compare with previous literature and expand on the permeability characteristics of the blood labyrinth barrier. Based on the current study, we plan to conduct a multi-institutional human clinical study to investigate several biomarkers, including prestin, that can be used for the preclinical diagnosis of cisplatin-induced ototoxicity.

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