Impact of Long-Term Tyrosine Kinase Inhibitor Exposure on Spermatogenesis in Juvenile Rats
Authors: Girke Vera , Tauer Josephine Tabea , Glauche Ingmar , Hofbauer Lorenz , Suttorp Meinolf 
Published: October 2019

Abstract
Background: Pediatric patients with chronic myeloid leukemia (CML) are exposed to off-target side effects from long-term treatment with tyrosine kinase inhibitors (TKIs) which have been observed with increasing watchfulness in the last years (Hijiya N, et al. BLOOD 127:392, 2016). TKIs inhibit c-kit and platelet-derived growth factor receptors (PDGF-R alpha/beta) which are known to regulate spermatogenesis (Zhang M, et al. SCI REP 4:5936, 2014). The influence of TKIs on spermatogenesis in pediatric patients with CML is not fully understood yet (Samis J, et al. PEDIATR BLOOD CANCER 63:1332, 2016). Therefore, we studied testicular tissue in juvenile rats following exposure to TKIs imatinib (IMA) and dasatinib (DASA) in a time and dose-dependent manner.
Methods: Using an established model (Tauer JT, et al. PLOS ONE 10:e0131192, 2015) of juvenile still growing Wistar rats, animals (age: 4 weeks [w]) were exposed to IMA or DASA at different dosages for 10 w (low dose [LD], high dose [HD], intermittently high dose [ID]; total number of rats: 20 to 32 animals, 5 - 8 rats per cohort). At defined developmental stages, that is at prepubertal age (6 w), pubertal age (8 w), and postpubertal age (14 w), testis weight as well as cellularity (spermatogonia, spermatocytes, spermatids, Ki-67 positive cells) were evaluated histopathologically in seminiferous tubule microscopic cross sections after continuous IMA treatment. Expression of genes involved in spermatogenesis comprising SCF and PDGF-alpha/beta as well as their corresponding receptors c-kit and PDGFR-alpha/beta (Nurmio M, et al. REPRODUC TOXICOL 25:442, 2008) was studied after continuous DASA exposure.
Results: Testis weight remained unchanged compared to non-exposed controls by exposure to any TKI. However, spermatogenic cell counts decreased significantly by 10% after IMA HD-exposure. In spermatogenesis cell cycle, the stage of the dominant cell proportion (stage VII according to Perey B, et al. AM J ANAT 108:47, 1961) was shifted to more immature stages (stage II/III) as well. LD- and ID-exposure with IMA attenuated these findings. Cell proliferation as investigated by Ki-67+ expression was significantly lowered by 10% - 20% at all applied IMA doses. Long-term DASA treatment at LD, HD and ID resulted in significantly reduced gene expression of SCF, c-kit and PDGF-R alpha/beta. Gene expression of PDGF-alpha was significantly decreased in HD and ID but not LD, whereas PDGF-beta showed no significant reduction postpubertally.
Conclusion: Long-term TKI toxicity in still growing organisms can easily be modelled in juvenile rats and emulates well the so-far clinical experience with regard to osseous side effects of TKIs (Millot F, et al. EUR J CANCER, 50:3206, 2014). Assessment of gonadal toxicity by isolated determination of testis weight represents a rather unspecific approach and will neglect subtle histopathological changes. With regard to spermatogenesis long-term TKI exposure resulted in reduced progenitor cell proliferation and downregulation of involved genes in a cumulative dose-dependent fashion. Thus, at least in juvenile, still growing Wistar rats a long-lasting negative effect of long-term TKI exposure on spermatogenesis has to be taken into account. Improved preclinical testing in well-established leukemia models should help to prioritize TKI agents in the clinical studies pipeline for pediatric patients with CML. Long-term follow-up of pediatric and adolescent patients who are given new targeted agents is mandatory and will prospectively explore potential late effects, and hopefully also provide corrective or preventive measures.

Disclosures
Glauche: Bristol Meyer Squib: Research Funding. Suttorp:Novartis, Bristol Meyer Squib, Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.