Acute lymphoblastic leukemia (ALL) is the most prevalent pediatric malignancy (1), accounting for a substantial proportion of childhood cancer diagnoses. Despite marked improvements in frontline therapy over recent decades, with 5-year overall survival (OS) rates exceeding 90% in select cohorts, the prognosis for patients who experience relapse remains poor (1, 2). The testes have long been regarded as a sanctuary site due to the blood-testis barrier, and consequently serve as the second most common site for extramedullary relapse in pediatric ALL. Although the application of contemporary intensive chemotherapy such as high-dose methotrexate has reduced the risk of testicular relapse to 2% or lower (3), it continues to pose a serious therapeutic challenge. Reported survival outcomes following isolated testicular relapse (ITR) vary widely, with OS estimates ranging from 53 to 84% (4, 5).

Management strategies following testicular relapse remain heterogeneous across studies. Salvage approaches have traditionally included systemic chemotherapy, orchiectomy, radiation, and hematopoietic stem-cell transplantation (HSCT). More recently, chimeric antigen receptor modified T-cell (CAR-T) therapy has emerged as a promising immunotherapeutic option, with preliminary reports suggesting efficacy in testicular involvement; however, data from large-scale cohorts and studies with extended follow-up remain scarce (6–11). Comparative analyses of these treatment modalities are limited, and the long-term benefits of orchiectomy in the era of advanced systemic therapy remain poorly defined. A clearer understanding of the therapeutic landscape is critical to optimizing outcomes in patients with testicular relapse.

In the present study, clinical features and treatment outcomes were retrospectively evaluated in children with testicular relapse of ALL from 13 medical centers across China. Survival rates were compared among patients receiving different salvage therapies to identify therapeutic strategies offering superior efficacy with reduced toxicity.

In this study, testicular relapse occurred in 1.3% of pediatric ALL patients initially treated under the CCCG-ALL-2015 protocol across 13 medical centers in China, aligning with previous reports that estimate its incidence at 2% or lower (3). The median interval between initial diagnosis and relapse was 37 months, consistent with prior studies indicating that testicular relapse typically arises more than 6 months after completion of frontline therapy (13). Although the occurrence of testicular relapse has become increasingly uncommon with contemporary treatment protocols, its clinical management remains variable and insufficiently characterized across studies (3). Conventional salvage therapies following testicular relapse, including chemotherapy, testicular irradiation, orchiectomy, and HSCT, have historically constituted the mainstay of treatment, while CAR-T therapy has recently emerged as a promising immunotherapeutic approach. Selection of therapeutic modality often depends on whether the testicular relapse is isolated or concurrent with other sites, yet comparative evaluations of efficacy among these treatment strategies remain limited and incompletely defined.

For patients with ITR, salvage regimens combining chemotherapy with orchiectomy or local irradiation have been commonly applied in previous studies (3, 14, 15). The REC80-ITR protocol, which incorporates both systemic chemotherapy and testicular irradiation, achieved 4-year OS and DFS of 67.7% and 41%, respectively (15). Although no conclusive evidence indicates that orchiectomy provides superior outcomes to testicular irradiation, it remains an appropriate option for patients with unilateral testicular involvement or those declining local irradiation (3, 14). HSCT has also been employed for isolated extramedullary relapse, yielding reported 10-year OS and DFS rates of 67% and 65%, respectively (16). However, fertility impairment remains a major concern, as procedures such as orchiectomy and testicular irradiation, as well as exposure to high cumulative doses of alkylating agents exceeding 4 000 mg/m² cyclophosphamide equivalents or total body irradiation prior to HSCT, can adversely affect reproductive function (3, 17, 18). These limitations underscore the urgent need for novel therapeutic strategies that achieve durable remission while preserving fertility in children with testicular relapse.

CAR-T therapy has recently been explored as an innovative strategy for managing testicular relapse (6–8). Yu et al. first applied CAR-T therapy in ITR, demonstrating potent cytotoxic activity against leukemic infiltration in the testes without apparent impairment of normal testicular function (8). Subsequent clinical investigations have reported encouraging outcomes. In our previous study, CAR-T therapy was applied to treat seven B-ALL patients with ITR, yielding a 1-year event−free survival (EFS) of 83.3% (6). Wang et al. reported a 1-year EFS of 95% in a cohort of 20 ITR patients receiving CAR-T therapy (7). These findings suggest that CAR-T therapy represents a promising and potentially less toxic alternative for patients with ITR. In the present study, 18 patients with ITR received CAR-T therapy without subsequent HSCT, achieving 2-year OS and DFS rates of 92.3% and 94.4%, respectively. One patient who underwent HSCT following CAR-T therapy remained event−free at the end of follow-up. Ten patients treated with orchiectomy exhibited 2−year OS and DFS rates of 100%. Survival outcomes did not differ significantly between the orchiectomy and CAR-T cohorts (both P > 0.05).

Treatment strategies for patients with concurrent testicular and BM relapse are similar to those employed for isolated BM relapse (3). In this cohort, approximately half of the patients with CTR underwent allogeneic HSCT, showing a trend toward improved outcomes (Supplementary Figure S1). However, no statistically significant difference in 2-year OS or DFS was observed, potentially due to limited follow-up duration and the favorable prognosis among patients treated with CAR-T therapy. Notably, all five patients with CTR who received CAR-T without subsequent HSCT remained alive, including one who later developed secondary ITR and achieved remission following HSCT. These findings suggest that HSCT may serve as a viable salvage option for patients experiencing post-CAR-T recurrence, although further clinical evidence is needed to substantiate its role in this context.

This study has several limitations, including its retrospective design, modest sample size, and heterogenous cohort, making it difficult to draw definite conclusions. Assessment of sexual function and gonadal hormone levels was not feasible because of the young age of participants, highlighting the need for continued long-term follow-up to evaluate potential reproductive outcomes. Despite these constraints, this investigation represents the largest multicenter retrospective analysis to date examining clinical characteristics and therapeutic outcomes of ALL patients with testicular relapse in China, encompassing the largest patient cohort and longest observation period following CAR-T-based salvage therapy. Epperly et al. and Holland et al. underscored the necessity of systematic evaluations using advanced imaging techniques, such as ¹⁸Fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography/computed tomography (PET/CT), to evaluate the efficacy of CAR-T in extramedullary disease (10, 11). Future studies should incorporate PET/CT to evaluate the efficacy of CAR-T for testicular relapse. At the same time, future work should also pay attention to patients’ mental states, quality of life, and the prognostic impact of B-cell aplasia.

This study shows that testicular relapse occurred in 1.3% of pediatric ALL patients and mainly occurred 3 years after initial diagnosis, which calls for more attention to the relapse in testes during long-term follow-up. For testicular relapse, CAR-T was effective, which might have an advantage in improving life-quality of survivors. CAR-T without subsequent HSCT holds the promise for achieving durable survival in patients with ITR. Prospective studies are needed to confirm these findings.