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Current intensive treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has substantial side effects, highlighting a need for novel biomarkers to improve risk stratification. Canonical biomarkers, such as genetics and immunophenotype, are largely not used in pediatric T-ALL stratification. This study aimed to validate the prognostic relevance of DNA methylation CpG island methylator phenotype (CIMP) risk stratification in 2 pediatric T-ALL patient cohorts: the Nordic Society of Paediatric Haematology (NOPHO) ALL2008 T-ALL study cohort (n = 192) and the Dutch Childhood Oncology Group (DCOG) ALL-10/ALL-11 validation cohorts (n = 156). Both cohorts revealed that combining CIMP classification at diagnosis with measurable residual disease (MRD) at treatment day 29 (D29) or 33 (D33) significantly improved outcome prediction. The poor prognosis subgroup, characterized by CIMP low/D29 or D33 MRD ≥ 0.1%, had a cumulative incidence of relapse (pCIR5yr) of 29% and 23% and overall survival (pOS5yr) of 59.7% and 65.4%, in NOPHO and DCOG, respectively. Conversely, a good prognosis subgroup was also identified representing CIMP high/D29 or D33 MRD < 0.1% with pCIR5yr of 0% and 3.4% and pOS5yr of 98.2% and 94.8%, in NOPHO and DCOG, respectively. For NOPHO, MRD was also evaluated on D15, and the relapse prediction accuracy of CIMP/D29 MRD (0.79) and CIMP/D15 MRD (0.75) classification was comparable, indicating potential for earlier stratification. The evaluation of the biology behind the CIMP subgroups revealed associations with transcriptome profiles, genomic aberrations, and mitotic history, suggesting distinct routes for leukemia development. In conclusion, integrating MRD assessment with the novel CIMP biomarker has the potential to improve risk stratification in pediatric T-ALL and guide future therapeutic decisions.

STUDY QUESTION: Which spermatogonial differentiation states are present in prepubertal testes under normal conditions and following chemotherapy-induced depletion of spermatogonia in paediatric patients with cancer?

SUMMARY ANSWER: Single-cell transcriptomic analysis reveals that only undifferentiated spermatogonia are present in prepubertal boys, while differentiated states emerge during puberty, with reduced protein expression of advanced spermatogonial markers observed in younger patients, those treated with alkylating agents, or those with a diminished spermatogonial pool. WHAT IS

KNOWN ALREADY: Paediatric oncology treatments often involve gonadotoxic therapies that can impair spermatogonial stem cells, increasing the risk of subfertility. While five distinct spermatogonial subpopulations have been identified in adult testes via single-cell RNA sequencing, their presence in prepubertal testes of childhood cancer patients remains to be confirmed through marker protein expression.

STUDY DESIGN, SIZE, DURATION: Gene expression profiles of spermatogonial subpopulations were investigated using single-cell RNA sequencing data from six testicular samples of healthy boys aged 0-17 years. Protein expression patterns were examined via immunofluorescence staining in 14 biobank control samples (median age: 4.9 years; range: 0.6-13.1 years) and in 31 prepubertal testicular tissue samples of paediatric patients with cancer (median age: 6.8 years; range: 0.7-13.1 years).

PARTICIPANTS/MATERIALS, SETTING, METHODS: Gene expression profiles of UTF1 (states 0-1), ID4 (states 0-1), PIWIL4 (states 0-1), FGFR3 (states 0-2), and KIT (state 4), were analysed in testicular cells of paediatric origin obtained from our previously published open-access data source (GSE134144 and GSE120508). The protein expression of these spermatogonial subpopulation markers was evaluated by counting immunofluorescence-positive cells per analysed area. Marker expression was correlated with prior chemotherapy exposure and spermatogonia numbers. Exposure to alkylating agents was quantified as the cumulative cyclophosphamide equivalent dose (CED), and anthracycline exposure as the cumulative doxorubicin isoequivalent dose equivalents (DIE). A depleted spermatogonia pool was defined as having S/T Z-scores lower than -7 SD.

MAIN RESULTS AND THE ROLE OF CHANCE: Transcriptomic analysis confirmed that germ cells in the prepubertal testis consist solely of undifferentiated spermatogonia. The expression of KIT protein, defining differentiated spermatogonia, was positively correlated with age (P < 0.001). A reduction in the number of spermatogonia expressing ID4 protein was associated with higher CED (P = 0.001), and spermatogonia expressing KIT protein with higher CED and DIE exposure (P = 0.005, and P = 0.035, respectively). A depleted spermatogonia pool (S/T Z-score <-7 SD) correlated with fewer spermatogonia expressing ID4 (P = 0.033), FGFR3 (P = 0.050), and KIT (P = 0.051) proteins. These results indicate that distinct protein expression patterns were observed following chemotherapy-induced reduction of the spermatogonial pool, with reduced expression of ID4, FGFR3, and KIT proteins. Numbers of spermatogonia positive for markers indicating more naïve, undifferentiated states, such as UTF1 and PIWIL4, did not correlate with spermatogonial pool reduction.

LIMITATIONS, REASONS FOR CAUTION: The study population was heterogeneous in terms of age and treatment exposure. Moreover, the impact of specific cancer treatments could not be individually assessed. Limited tissue availability reduced the statistical power of the study, and repeated double or triple immunofluorescence staining could not be performed. As a result, the correlations between the expression of different spermatogonial markers can only be considered indicative trends. Child testicular control tissue samples were considered normal for inclusion if no testicular pathology was reported. However, detailed information on prior medical treatments or testicular volumes for the patients in this biobank was unavailable.

WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggest that alkylating agents have dose-dependent effects on all spermatogonial subpopulations. However, spermatogonial subtypes expressing the protein markers UTF1 and PIWIL4 were more resistant to chemotherapy-induced depletion of the spermatogonial pool, potentially representing true reserve stem cells. The identification of reserve stem cells could provide a valuable method for evaluating the fertility potential of testicular tissue collected for fertility preservation in prepubertal and peripubertal boys.

Age and sex have historically been associated with differences in the survival of patients with acute lymphoblastic leukemia (ALL). The NOPHO ALL2008 trial included patients aged 1-45 years with BCR::ABL1-negative B-precursor and T-cell ALL, but neither sex nor age was integrated into risk group allocation. Among 1,771 trial patients stratified into protocol- appropriate risk groups, we estimated the impact of age and sex on survival (even after relapse) and toxicities prospectively registered at 3-month intervals. In multivariate Cox regression analysis adjusted by sex, age group, and risk group, age but not sex was an independent risk factor for reduced 5-year event-free survival (EFS): hazard ratio=1.57 (95% confidence interval: 1.15-2.14) for patients 10-17.9 years, and 2.70 (95% confidence interval: 2.03-3.58) for patients 18-45 years, compared to patients <10 years old at diagnosis. The overall 5-year pEFS was 0.83. For standard-risk patients (B-lineage, white cell count <100x109/L, no risk genetics, minimal residual disease day 29 <0.1%), an inferior 5-year EFS was observed among patients 18-45 years (pEFS 0.78, P<0.001) and 10-17.9 years (pEFS 0.86, P=0.002) compared to patients <10 years at diagnosis (pEFS 0.93). For the intermediate-risk and high-risk groups, EFS was worse for patients 18-45 years compared to patients <10 years: pEFS 0.69 versus 0.89 (P<0.001) and pEFS 0.55 versus 0.71 (P=0.005), respectively. Osteonecrosis and veno-occlusive disease were associated with female sex in the standard-risk group, and age ≥10 years was associated with osteonecrosis, thrombosis, and pancreatitis in sex- and treatment-group-adjusted analyses. In conclusion, this study indicates that risk-grouping and/or treatment-intensity criteria should differ across age groups and that age-adapted strategies to mitigate toxicities are needed.

Acute myeloid leukemia (AML) with t(7;12)(q36;p13) is a recurrent translocation in AML in infants or very young children and was recently included in the World Health Organization (WHO) Classification of Hematolymphoid Tumors. AML with t(7;12) is reported to involve MNX1 and ETV6 signaling; however, the mechanism of leukemogenesis is not well understood, and the presence of MNX1::ETV6 fusion transcripts has only been confirmed in approximately 50% of cases. In contrast, high expression of MNX1 has been seen in all investigated cases. In this study, we investigated the clinical as well as biological characteristics of 12 pediatric AML with t(7;12) and performed whole transcriptome (WTS) and whole genome sequencing (WGS) on six of these. There was no significant difference in event-free survival or overall survival of these t(7;12) AML patients compared with other AML in the same age group. Interestingly, WTS identified several fusion transcripts involving ETV6 but not together with MNX1. WGS identified the genomic breakpoints and revealed that a common fusion partner on chromosome 7 was NOM1. Principal component analysis (PCA) of the WTS data showed that all t(7;12) AML cases cluster together, separate from all other pediatric AML subtypes; all cases had high expression of MNX1, MNX1-AS1, and MNX1-AS2. Hence, t(7;12) AML, despite expressing different fusion transcripts and with varying translocation breakpoints, constitutes a phenotypically homogenous subgroup. This underlines that the leukemia-driving event most likely is ectopic expression of MNX1 and that this therefore should be the defining Classifying criteria of this type of AML.

This study examines spermatogonial numbers in testicular samples from 43 prepubertal patients undergoing haematopoietic stem cell transplantation (HSCT). High-dose chemotherapy and/or radiation during HSCT can impact spermatogenesis requiring fertility preservation. Results show that 49% of patients have decreased and 19% severely depleted spermatogonial pool prior to HSCT. Patients with Fanconi anaemia exhibit significantly reduced spermatogonial numbers. Patients with immunodeficiency or aplastic anaemia generally present within the normal range, while results in patients with myelodysplastic syndrome or myeloproliferative neoplasm vary. The study emphasizes the importance of assessing spermatogonial numbers in patients with severe haematological diseases for informed fertility preservation decisions.

Background: Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors.

Methods: gWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients.

Findings: The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35).

Interpretation: Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients.

Funding: The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.

Hyperleukocytosis in pediatric acute myeloid leukemia (AML) is associated with severe complications and an inferior outcome. We report results on patients with hyperleukocytosis included in the NOPHO-DBH AML 2012 study. We recommended immediate initiation of full-dose chemotherapy (etoposide monotherapy for 5 days as part of the first course), avoiding leukapheresis and prephase chemotherapy. Of 714 patients included in the NOPHO-DBH AML 2012 study, 122 (17.1%) had hyperleukocytosis, and 111 were treated according to the recommendations with etoposide upfront without preceding leukapheresis or prephase chemotherapy. The first dose was applied the same day as the AML diagnosis or the day after in 94%. Etoposide was administered via peripheral veins in 37% of patients without major complications. After initiation of etoposide the white blood cell counts on days 2-5 were 69%, 36%, 17% and 8%, respectively, of the pre-treatment level. On day 3, 81% of patients had a white blood cell count <100 x109/L. Five-year event-free and overall survival rates for all patients with hyperleukocytosis were 52.9% (95% confidence interval [95% CI]: 44.4-63.0) and 74.1% (95% CI: 66.4-82.6), compared to 64.9% (95% CI: 60.9-69.1) and 78.9% (95% CI: 75.4-82.4) for patients without hyperleukocytosis (P<0.001 for event-free survival, P=0.1 overall survival). Six-week early mortality was 4.1% for all patients with hyperleukocytosis (2.7% for the 111 patients treated with etoposide upfront). We conclude that management of hyperleukocytosis in pediatric AML with immediate etoposide monotherapy without leukapheresis or prephase chemotherapy is feasible, safe and effective. The reduction in white blood cell count during the first days is comparable to the reported results of leukapheresis, and outcomes seem at least equivalent to therapies including leukapheresis. Based on our results, we advocate abandoning leukapheresis for hyperleukocytosis in pediatric AML. Instead, it is crucial to start induction chemotherapy as early as possible.

PURPOSE Measurable residual disease (MRD) by using flow cytometry after induction therapy is strongly prognostic in pediatric AML, and hematopoietic stem-cell transplant (hSCT) may counteract a poor response. We designed a phase III study with intensified response-guided induction and MRD-based risk stratification and treated poor induction response with hSCT. The efficacy of liposomal daunorubicin (DNX) in induction was compared with mitoxantrone.

METHODS The study planned to randomly assign 300 patients, but the production of DNX ceased in 2017. One hundred ninety-four patients were randomly assigned to mitoxantrone or experimental DNX in induction 1. Ninety-three non–randomly assigned patients served as an observation cohort. Primary end point was fraction of patients with MRD <0.1% on day 22 after induction 1. Patients with MRD ≥15% after induction 1 or ≥0.1% after induction 2 or FLT3-ITD with NPM1 wildtype were stratified to high-risk therapy, including hSCT.

RESULTS Outcome for all 287 children was good with 5-year event-free survival (EFS5y) 66.7% (CI, 61.4 to 72.4) and 5-year overall survival (OS5y) 79.6% (CI, 75.0 to 84.4). Overall, 75% were stratified to standard-risk and 19% to high-risk. There was no difference in the proportion of patients with MRD <0.1% on day 22 after induction 1 (34% mitoxantrone, etoposide, araC [MEC], 30% DNX, P 5 .65), but the proportion increased to 61% for MEC versus 47% for DNX (P 5 .061) at the last evaluation before induction 2. EFS5y was significantly lower, 56.6% (CI, 46.7 to 66.5) versus 71.9% (CI, 63.0 to 80.9), and cumulative incidence of relapse (CIR) was higher, 35.1% (CI, 25.7 to 44.7) versus 18.8% (CI, 11.6 to 27.2) for DNX. The inferior outcome for DNX was only in standard-risk patients with EFS5y 55.3% (CI, 45.1 to 67.7) versus 79.9% (CI, 71.1 to 89.9), CIR 39.5% (CI, 28.4 to 50.3) versus 18.7% (CI, 10.5 to 28.7), and OS5y 76.2% (CI, 67.2 to 86.4) versus 88.6% (CI, 81.4 to 96.3). As-treated analyses, including the observation cohort, supported these results. For all high-risk patients, 85% received hSCT, and EFS5y was 77.7 (CI, 67.3 to 89.7) and OS5y was 83.0 (CI, 73.5 to 93.8).

CONCLUSION The intensification of induction therapy with risk stratification on the basis of response to induction and hSCT for high-risk patients led to improved outcomes. Mitoxantrone had a superior anti-leukemic effect than liposomal daunorubicin.

A comprehensive international consensus on the cytogenetic risk-group stratification of KMT2A-rearranged (KMT2A-r) pediatric acute myeloid leukemia (AML) is lacking. This retrospective (2005-2016) International Berlin-Frankfurt-Münster Study Group study on 1256 children with KMT2A-r AML aims to validate the prognostic value of established recurring KMT2A fusions and additional cytogenetic aberrations (ACAs) and to define additional, recurring KMT2A fusions and ACAs, evaluating their prognostic relevance. Compared with our previous study, 3 additional, recurring KMT2A-r groups were defined: Xq24/KMT2A::SEPT6, 1p32/KMT2A::EPS15, and 17q12/t(11;17)(q23;q12). Across 13 KMT2A-r groups, 5-year event-free survival probabilities varied significantly (21.8%-76.2%; P < .01). ACAs occurred in 46.8% of 1200 patients with complete karyotypes, correlating with inferior overall survival (56.8% vs 67.9%; P < .01). Multivariable analyses confirmed independent associations of 4q21/KMT2A::AFF1, 6q27/KMT2A::AFDN, 10p12/KMT2A::MLLT10, 10p11.2/KMT2A::ABI1, and 19p13.3/KMT2A::MLLT1 with adverse outcomes, but not those of 1q21/KMT2A::MLLT11 and trisomy 19 with favorable and adverse outcomes, respectively. Newly identified ACAs with independent adverse prognoses were monosomy 10, trisomies 1, 6, 16, and X, add(12p), and del(9q). Among patients with 9p22/KMT2A::MLLT3, the independent association of French-American-British-type M5 with favorable outcomes was confirmed, and those of trisomy 6 and measurable residual disease at end of induction with adverse outcomes were identified. We provide evidence to incorporate 5 adverse-risk KMT2A fusions into the cytogenetic risk-group stratification of KMT2A-r pediatric AML, to revise the favorable-risk classification of 1q21/KMT2A::MLLT11 to intermediate risk, and to refine the risk-stratification of 9p22/KMT2A::MLLT3 AML. Future studies should validate the associations between the newly identified ACAs and outcomes and unravel the underlying biological pathogenesis of KMT2A fusions and ACAs.