Abstract Background Aberrant activation of mesenchymal epithelial transition receptor (MET) contributes to tumorigenesis in pediatric CNS tumors making MET inhibition a potential therapeutic target in this patient population. Methods We conducted a first-in-children multicenter phase 1 trial of the MET inhibitor savolitinib in children with recurrent or refractory MB, HGG and DIPG. The main objectives were to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D) of once-daily oral savolitinib and to characterize its toxicity and pharmacokinetics. Following MTD/RP2D determination, an efficacy expansion cohort limited to participants with MET-altered CNS tumors (including MET mutations, fusions, MET/HGF amplification) was activated. Results 41 participants were enrolled (median age: 12.5 years, range 5.3-21.5); 1 was deemed ineligible. The MTD/RP2D was 350 mg/m2 (dose level 3). Two dose-limiting toxicities (grade 3 fatigue, grade 3 ALT elevation) occurred. The most common grade 3/4 adverse events at least possibly related to savolitinib were neutropenia and lymphopenia (each 3/39, 7.7%), and leukopenia (2/39, 5.1%). In the phase 1 cohort, 1 objective response was observed in a participant with recurrent HGG on dose level 1 (150 mg/m2); they completed 39 treatment courses. Three participants with progressive DIPG remained on treatment for a median of 9 courses (range 4-24); One other participant with recurrent HGG remained on treatment for 7 courses.The median (range) apparent oral savolitinib clearance was 30.4 L/h/m2 (3.8–220.1) and half-life 3.2 hr (1.63–22.2). Among 7 participants with tumors harboring MET aberrations, 2 demonstrated sustained stable disease for 6 courses, while none achieved an objective response. Correlative analyses are underway. Conclusions Savolitinib was well-tolerated in children with recurrent CNS tumors with an MTD/RP2D of 350 mg/m2. Although preliminary antitumor activity was observed in the phase 1 cohort, no objective responses were seen in biomarker-selected participants.

Abstract Background Phosphoproteomics studies have identified a critical role for the protein kinase CK2 in SHH MB growth by affecting the terminal-most components of the pathway. CX-4945 is an oral selective inhibitor of CK2 with promising preclinical data in medulloblastoma models. Methods PBTC conducted the first multicenter prospective clinical trial of CX-4945 in children and adults with recurrent SHH MB. There were 3 components: phase 1 for skeletally immature patients; phase 2 for skeletally mature patients/adults; and a surgical arm for patients undergoing tumor resection. Stratification by skeletal maturity addressed potential growth plate toxicities with SHH pathway inhibition. SHH subgroup was confirmed by methylation profiling. The phase 1 component followed the rolling-6 design, phase 2 used a Simon two-stage minimax design to assess objective response rate. Results Six subjects (median age: 10.8 years, range 8.3-13.9) were enrolled to the phase 1 component. No dose-limiting toxicities occurred at dose level 2 (800 mg/m2 twice a day given continuously); growth plate toxicities were not observed. Twelve participants (of 16 planned for interim efficacy analysis, median age 24.7 years [range 13.3-42]) enrolled in the phase 2 arm, with no objective responses observed. Three of the planned 6 surgical arm subjects had tumors analyzed for CX-4945 brain tissue concentrations, and 2/3 had measurable concentrations (54 and ∼131 ng/ml). Grade 3/4 adverse related events were limited to hypokalemia and nausea experienced by one subject. Two participants achieved sustained stable disease and remained on treatment for 6 and 9 cycles. Trial enrollment was terminated early due to discontinuation of consortium funding. Conclusions CX-4945 was well tolerated in patients with recurrent SHH MB at doses reaching 800 mg/m2 twice daily, a final maximum tolerated dose was not determined. Early study closure restricts definitive conclusions; however the lack of an efficacy signal suggests limited activity in this patient population.

Accurate assessment of renal function is essential in treating pediatric patients dosed with nephrotoxic chemotherapy. The validity of the bedside Schwartz, 5-covariate St. Jude (5SJ), CKiD-CysC-U25, combined Cr-CysC-based CysPed, and the serum creatinine-BUN-cystatin C-based CKiD (CKiD Cr-CysC) equations were evaluated in pediatric hematology and oncology patients. A retrospective analysis was conducted comparing estimated glomerular filtration rate (eGFR) to measured GFR (mGFR) obtained from technetium- 99 m diethylenetriaminepentaacetic acid (99 mTc-DTPA) clearance between January 2016 and May 2022. The influence of corticosteroid use and inflammation in our patient population was evaluated for effect on serum cystatin C (CysC) concentrations and mGFR. All equations agreed within 2 SD of the mean difference with mGFR, but the 5SJ equation had the smallest bias followed closely by the CysPed equation. Overall accuracy (P30) was assessed, and the 5SJ, CKiD Cr-CysC, CysPed, and CKiD-Cys-U25 exhibited comparable performance. In our patient population, we did not observe an effect of corticosteroids (cumulative dosage of > 0.5 mg/kg within the past 14 days) or the presence of inflammation (CRP > 1.2 mg/L) on cystatin C concentrations or mGFR. In our pediatric hematology and oncology patient population, no one estimating equation demonstrated superior accuracy and bias overall and in all subgroups. Neither corticosteroid use nor elevated CRP influenced serum CysC concentrations or eGFR.

Effective therapies for recurrent brain tumors remain scarce, leading to persistently high mortality rates following relapse. With cell cycle dysregulation playing a pivotal role in disease pathology, preclinical studies investigating CDK4/6 inhibitors (CDK4/6i) have elicited survival benefit across multiple brain tumor models. These studies simultaneously cautioned against prolonged single-agent treatment due to concerns for tumor progression. Combination therapy featuring the highly selective, brain-penetrant CDK4/6i, ribociclib, was evaluated in 68 relapsed/refractory brain tumor patients enrolled on SJDAWN (NCT03434262). Patients were molecularly stratified into 3 treatment groups: n=33 in A (Group 3/4 medulloblastoma, ependymoma); n=28 in B (high-grade glioma, other); and n=7 in C (skeletally mature SHH-activated, TP53 wildtype medulloblastoma). Safety, tolerability, and recommended phase 2 dosing for ribociclib combinations with gemcitabine (A), trametinib (B), and sonidegib (C) were previously reported. 12-month progression-free survival (PFS) was 18% (95% CI: 7-33%) in A, 4% (95% CI: 0-15%) in B, and 43% (95% CI: 10-73%) in C. 24-month PFS declined to 12% (95% CI: 4%-26%) in A and 0% in B and C. To investigate whether resistance contributed to disease progression, we analyzed n=143 cell-free DNA (cfDNA) methylomes derived from serial cerebrospinal fluid of 34 of the 68 patients. From trace amounts of cfDNA (<500 picograms), we were able to observe emerging resistance mechanisms marked by focal amplifications in positive cell cycle regulators (i.e., CDK6, CCND2, and MYCN), coinciding with a shift in cell cycle based on computational inference. These findings notably foreshadowed radiographic progression by 6 to 13 months. For patients who achieved PFS >18 months, cfDNA analyses predicted stable disease until tumor evolution. Further analyses resolving tumor microenvironment dynamics from cfDNA are underway. Collectively, ribociclib combination therapy exhibited clinical benefit in select patients before resistance emerged. cfDNA assessment enabled early detection of resistance, unveiling a window for therapeutic adjustment.

10007 Background: Talazoparib (TAL), a potent PARP inhibitor, demonstrated preclinical activity in Ewing sarcoma when combined with irinotecan (IRN) and temozolomide (TMZ). A phase I study (BMNIRN, NCT02392793) showed clinical benefit of TAL + IRN + TMZ; but dose escalation was limited due to hematologic and gastrointestinal toxicity. We therefore evaluated, for the first time in children, nanoliposomal irinotecan (nal-IRI) in combination with either TAL or TMZ. Methods: Patients (pts) aged 1-30 with recurrent or refractory solid tumors were eligible to receive nal-IRI + TAL (Arm A) or nal-IRI + TMZ (Arm B) using a Bayesian keyboard design. Nal-IRI dosage was escalated with fixed TAL and TMZ doses. Each cycle was 21 days. Maximum tolerated dosage(s) (MTD) and recommended phase 2 dosage(s) (RP2D) were determined. Nal-IRI and TAL plasma pharmacokinetics (PK) were evaluated. Toxicities were assessed using CTCAE v.5 and responses were evaluated by RECIST 1.1. UGT1A1 polymorphisms and serial circulating tumor DNA (ctDNA) samples were evaluated. Results: Forty-six pts enrolled at 5 sites. The first 9 (5 Arm A; 4 Arm B) received nal-IRI days 1, 8. Four pts had dose limiting toxicities (DLTs), so day 8 nal-IRI was removed (Amend 1). After Amend 1, 37 pts (23 male; median age 14 years, range 1-23) enrolled (19 Arm A; 18 Arm B). The most common diagnosis was Ewing sarcoma (n = 16, 35%). Table 1 summarizes DLTs in Cycle 1 and response. The most common serious adverse events included febrile neutropenia (12 Arm A; 2 Arm B), colitis (4 Arm A; 2 Arm B), vomiting (4 Arm A), and diarrhea (3 Arm A). Confirmed responses were seen in five Arm A (1 CR, 4 PR) and five Arm B (1 CR, 4 PR) pts with Ewing sarcoma, CIC-DUX4 sarcoma, synovial sarcoma, and rhabdomyosarcoma. Seven pts (5 Arm A; 2 Arm B) had stable disease. Results of PK, UGT1A1 and ctDNA will be presented. Conclusions: The MTDs were nal-IRI 160mg/m 2 plus TAL (Arm A) and nal-IRI 200mg/m 2 plus TMZ (Arm B). The RP2Ds are pending FDA review. These regimens are feasible with evidence of anti-tumor activity and warrant further investigation. Clinical trial information: NCT04901702 . Dose level Nal-IRImg/m 2 IV Arm ANal-IRI + TAL(po, 600mcg/m 2 /dose)Days (D) # of pts Arm BNal-IRI + TMZ(po, 100mg/m 2 )Days (D) # of pts DLT Cycle 1 (# of pts) Confirmed Response(CR, PR, SD, PD) 1 120 D 1: nal-IRI + TAL divided BIDD 2-6: TAL daily 7 D 1: nal-IRI + TMZD 2-5: TMZ daily 3 Arm A: 1 pt - neutropenia (1) Arm A: CR (1), SD (1), PD (2)Arm B: PR (1), SD (1), PD (1) 2 160 D 1: nal-IRI + TAL divided BIDD 2-6: TAL daily 10 D 1: nal-IRI + TMZD 2-5: TMZ daily 3 Arm A: 3 pts - anemia (1), thrombocytopenia (1), sepsis (1), colitis (1) Arm A: PR (3), SD (4), PD (3)Arm B: PR (1), PD (1) 3 200 D 1: nal-IRI + TAL divided BIDD 2-6: TAL daily 2 D 1: nal-IRI + TMZD 2-5: TMZ daily 12 Arm A: 2 pts – abd pain (1), thrombocytopenia (1), neutropenia (1), diarrhea (1)Arm B: 3 pts – nausea (1), neutropenia (1), sepsis (1), thrombocytopenia (1) Arm A: PR (1)Arm B: CR (1), PR (2), SD (1), PD (6)

Background PBTC-029B was a phase 2 trial evaluating efficacy of selumetinib in children with recurrent/progressive low-grade glioma. We report results of strata 2, 5, and 6 with updated survivals for strata 1, 3, and 4.Methods Stratum 2 included recurrent/progressive pilocytic astrocytoma (PA) not associated with neurofibromatosis type-1 (NF1) that screened negative for the BRAF-KIAA1549 fusion and BRAFV600E mutation. Stratum 5 enrolled non-PA that screened positive for one of the BRAF aberrations. Stratum 6 enrolled children who consented to tissue screening, but there was an assay failure. For long-term survivals, stratum 1 included non-NF1 PA positive for one of the BRAF aberrations; stratum 3 included NF1-associated pLGG; and stratum 4 included non-NF1 optic pathway/hypothalamic tumors.Results Stratum 2: among 14 evaluable patients, there was 1 partial response (PR), 7 stable disease (SD), and 6 progressive disease (PD); the overall response rate (ORR) was 7.1%. Two-year progression-free survival (PFS)/overall survival (OS) were 57.1%/100%, respectively. Stratum 5: among 23 evaluable patients, there was 1 complete response (CR), 4 PR, 12 SD, and 6 PD; ORR was 21.7%. Two-year PFS/OS were 74.8%/100%, respectively. Stratum 6: among 26 evaluable patients, there were 7 PR, 14 SD, and 5 PD; ORR was 26.9%. Two-year PFS/OS were 72.0%/100%, respectively. The median follow-up for patients on strata 1, 3, and 4 without events are 60.4, 60.4, and 58.1 months, and 5-year PFS/OS were 30.8%/88.9%, 54.2%/100%, and 51.0%/100%, respectively.Conclusions Selumetinib provided stability and responses across many pLGG subgroups, and some patients achieved prolonged disease control without additional therapy.

Selinexor (KPT-330), a first-in-class, CNS-penetrant oral inhibitor of Exportin-1, disrupts the nuclear export of tumor suppressor proteins, promoting their accumulation and inducing cancer cell death. In this study, a reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to quantify selinexor concentrations in human plasma. A standard solid-phase extraction method using an Oasis HLB μElution plate was utilized to isolate selinexor and its internal standard, selinexor-d3, from human plasma. The chromatographic separation was executed on a reversed-phase analytical column with a binary gradient of water and acetonitrile, both containing 0.1 % formic acid, at a flow rate of 0.5 mL/min. Mass spectrometry detection was performed in positive ion mode by tracking the mass transitions of 444.0 > 334.0 for selinexor and 447.0 > 333.9 for selinexor-d3. The developed LC-MS/MS assay for selinexor was rigorously validated over a wide range of clinically relevant concentrations (1-1000 ng/mL, r2 ≥ 0.99) in accordance with FDA bioanalytical method validation guidelines. The method exhibited inter-day accuracy, expressed as relative error (R.E.), ranging from 2.28 % to 4.38 %, with precision values not exceeding 5.92 %. Intra-day accuracy showed R.E. values between 0.24 % and 7.30 %, accompanied by precision values ≤4.81 %. Additionally, the method demonstrated high extraction recovery, ranging from 82.80 % to 87.87 %, and a negligible matrix effect. The pH adjustments applied to the plasma prior to storage and processing maintained the stability of selinexor under several experimental conditions, including multiple freeze-thaw cycles and long-term storage at -80 °C. As proof of principle, the LC-MS/MS assay was successfully applied to a phase I clinical pharmacokinetic study of selinexor in pediatric patients with non-rhabdomyosarcoma soft tissue sarcomas, yielding reliable and reproducible measurements of selinexor concentrations in plasma.

Abstract BACKGROUND Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG), are a leading cause of central nervous system tumor-related morbidity and mortality in children. Neuronal activity promotes growth of HGGs; one key mechanism is neuronal activity-regulated shedding of neuroligin-3 into the glioma microenvironment, mediated by the protease (A Disintegrin and Metalloprotease) ADAM10. ADAM10 inhibition slows tumor growth in preclinical pHGG models. Here, we report results of the clinical trial PBTC-056 (NCT04295759) evaluating safety and tolerability of INCB007839, an inhibitor of the ADAM 10 and 17 proteases. METHODS Patients aged 3-21 years old with recurrent/progressive pHGGs, including DIPG, were eligible. Additional eligibility criteria included measurable disease and failure of at least 1 standard treatment. One dose level (120mg/m2/dose twice daily [BID]) was tested, and the trial was subsequently amended to require prophylactic anticoagulation with enoxaparin due to an unanticipated toxicity. The primary objective was to assess the safety and tolerability of INCB007839 for children with pHGGs. RESULTS 12 of 13 eligible subjects were evaluable. Median age was 13.5 years (4.9-20.7 years). Diagnoses included: DIPG (54%), glioblastoma multiforme (31%), anaplastic astrocytoma (8%) and CNS primary tumor NOS (8%). All patients were treated at DL1 and remained on study for 1-4 courses. The most common toxicities were lymphopenia, elevated transaminases, and fatigue. There were 3 dose-limiting toxicities: Grade 5 cerebral venous thrombosis (n=1), Grade 3 alanine aminotransferase increase (n=1) and Grade 2 thrombocytopenia (n=1). Ten patients progressed/relapsed during active treatment, 1 patient died on treatment, and 2 patients withdrew (1 prior to starting therapy). CONCLUSIONS INCB007839 was generally well tolerated with a recommended phase 2 dose of 120mg/m2/dose BID and concurrent prophylactic anti-coagulation. These data, combined with foundational preclinical studies targeting neuron-cancer interactions, open the door to possible cancer neuroscience strategies, including combination therapy, for pediatric high-grade gliomas.

Abstract BACKGROUND SJ-ELIOT (NCT04023669) was a phase 1 trial that explored the combination of the checkpoint kinase inhibitor, prexasertib with the DNA-damaging agents, cyclophosphamide and gemcitabine, in recurrent or refractory medulloblastoma. METHODS Participants ≥ 1 year and < 25 years were stratified to one of two treatment strata: stratum A prexasertib and cyclophosphamide and stratum B prexasertib and gemcitabine. Patients with Group 3/4 medulloblastoma were assigned to either stratum A or B, whereas patients with SHH medulloblastoma were assigned to stratum A. A Rolling-6 design was used. RESULTS Fifteen patients were enrolled on stratum A and six patients on stratum B. The study was closed early due to drug supply issues. In stratum A, three patients were escalated to dose level (DL) 3 and none had dose limiting toxicity (DLT). For stratum B, of the first three patients enrolled on DL1, two had DLTs (grade 3 hypotension) related to drug reactions. Consequently, stratum B was amended to add hydrocortisone prophylaxis. A further three patients were enrolled. One patient had a grade 3 DLT (ALT increase). The remaining two patients electively stopped therapy within the first two courses. One patient on stratum A, with SHH TP53 mutant, MYCN amplified medulloblastoma, achieved a complete sustained response, received 12 cycles, and electively stopped treatment after 408 days. They progressed nine months later. The remaining 14 patients on stratum A progressed. There were no objective responses on stratum B. Median PFS were 1.9 and 2.1 months for stratum A and B respectively. CONCLUSIONS The MTD/RP2D was not established for either arm. In stratum A, DL3 was tolerated, whereas DL1 for Stratum B was not tolerated. Despite robust preclinical data, no efficacy signal was observed. Intriguingly, one patient with a highly aggressive tumor (SHH TP53 mutant, MYCN amplified) appeared to derive a sustained benefit from the combination of prexasertib and cyclophosphamide.

Abstract BACKGROUND PBTC-029B was a phase 2 trial evaluating efficacy of selumetinib (up to 26 courses) in children with recurrent/progressive low-grade glioma. We report new results on strata 2, 5, and 6 as well as update long-term survival outcomes on strata 1, 3, and 4. METHODS Stratum 2 enrolled children with pilocytic astrocytoma (PA) whose tumor screened negative for the BRAF-KIAA1549 fusion and the BRAFV600E mutation. Stratum 5 enrolled children with non-PA whose tumor screened positive for one of these two BRAF aberrations. Stratum 6 enrolled children who consented to tissue screening, but there was an assay failure. Responses were based on T2/FLAIR. RESULTS Stratum 2: among 14 patients, there was 1 partial response (PR), 7 stable disease (SD) and 6 progressive disease (PD) with overall response rate (ORR) of 7.1%. Two-year progression-free survival (PFS)/overall survival (OS) were 57.1/100%, respectively. Stratum 5: among 23 patients, there was 1 complete response, 4 PR, 12 SD and 6 PD with ORR of 21.7%. Two-year PFS/OS were 74.8%/100%, respectively. Stratum 6: among 26 patients, there were 7 PR, 14 SD and 5 PD with ORR of 26.9%. Two-year PFS/OS were 72.0%/100%, respectively. Long-term outcomes have now been evaluated in strata 1, 3, and 4. Stratum 1 enrolled patients with PA whose tumor screened positive for one of the two BRAF aberrations; stratum 3 enrolled patients with neurofibromatosis type-1 (NF1) associated pLGG; and stratum 4 enrolled non-NF1 optic pathway/hypothalamic tumors. Among strata 1, 3, and 4, the current median follow-up for patients without events are 60.4, 60.4, and 58.1 months, respectively. Five-year PFS/OS are 30.8%/88.9%, 54.2%/100%, and 51.0%/100%, respectively. CONCLUSIONS These data demonstrate that selumetinib provides tumor stability and responses among diverse pLGG tumors as well as show that many patients will achieve long-term disease control even several years after stopping treatment.

Abstract BACKGROUND Pediatric CNS tumors have recurring molecular aberrations that modulate the cell cycle. We hypothesized that ribociclib, a brain-penetrant CDK4/6 inhibitor, given in doublet combinations could benefit patients with CNS malignancies. SJDAWN (NCT03434262) was launched to determine the recommended phase 2 dose (RP2D), safety, pharmacokinetics, and early efficacy of doublets. METHODS Patients >1 and <40 years with recurrent/refractory CNS malignancy were stratified into 3 strata. Escalating dose-levels in 28-day cycles were evaluated in each stratum by rolling-6 design with an expansion cohort at the highest tolerated dose-level. RP2D was declared if ≤3 dose-limiting toxicities (DLTs) occurred in 12 patients. Stratum A evaluated ribociclib/gemcitabine in group3/group4 (Grp3/4) MB and ependymoma (EPN). Stratum B evaluated ribociclib/trametinib in malignancies not eligible for stratum A or C. Stratum C evaluated ribociclib/sonidegib in skeletally mature patients with SHH-MB, TP53 wildtype with chr9q loss and/or PTCH1 mutation. Tumor, blood, and CSF samples were analyzed. RESULTS From 2018-2022, 68 patients enrolled: 33 in A (22 Grp3/4-MB, 11 EPN); 28 in B (15 HGG, 13 other); 7 SHH-MB in C. The RP2D was gemcitabine 1250mg/m2 IV day 1, 15 and ribociclib 350mg/m2 PO 1-21 days for A; trametinib 0.025mg/kg PO days 1-14 and ribociclib 280mg/m2 PO days 7-21 for B; and not established for C. DLTs included neutropenia (A), thrombocytopenia (B), mucositis (B), and rash (C). The 6-, 12-, and 24-month progression-free survival (PFS) was 42%(26%-58%), 18%(8%-33%), and 12%(4%-26%) in A, 18%(7%-34%), 4%(1%-15%), and 0% in B, 71%(30%-92%), 43%(13%-73%), and 0% in C. PFS beyond 2-years was observed in 3 Grp3/4-Subgroup3 MBs and 1 EPN. Amplification of CDK6, CCND2, and MYCN in serial CSF samples suggested a mechanism of resistance. CONCLUSIONS Ribociclib doublets are tolerated, and the lengthy survival of some patients suggests a selective benefit.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. For the alveolar subtype (ARMS), the presence of the PAX3::FOXO1 fusion gene and/or metastases are strong predictors of poor outcome. Metastatic PAX3::FOXO1+ ARMS often responds to chemotherapies initially, only to subsequently relapse and become resistant with most patients failing to survive beyond 8 years post-diagnosis. No curative intent phase II or phase III clinical trial has been available for patients in the past 10 years (ARST0921). Thus, metastatic ARMS represents a significantly unmet clinical need. Chemotherapy resistance in ARMS has previously been attributed to PAX3::FOXO1-mediated cell cycle checkpoint adaptation, which is mediated by an HDAC3-SMARCA4-miR-27a-PAX3::FOXO1 circuit that can be disrupted by HDAC3 inhibition. In this study, we investigated the therapeutic efficacy of combining the epigenetic regulator entinostat, a Class I Histone Deacetylase (HDAC1-3) inhibitor, with RMS-specific chemotherapies in patient derived xenograft (PDX) models of RMS. We identified single agent, additive or synergistic relationships between relapse-specific chemotherapies and clinically relevant drug exposures of entinostat in three PAX3::FOXO1+ ARMS mouse models. This preclinical data provides further rationale for clinical investigation of entinostat, already known to be well tolerated in a pediatric phase I clinical trial (ADVL1513).

Background/Objectives: Palbociclib, an oral CDK 4/6 inhibitor, was evaluated in a Pediatric Brain Tumor Consortium (PBTC) phase 1 (NCT02255461; PBTC-042) study to treat children and young adults with recurrent, progressive, or refractory brain tumors. The objectives of this study were to characterize the palbociclib population pharmacokinetics in children enrolled on PBTC-042, to conduct a population pharmacodynamic analysis in this patient population, and to perform a simulation study to assess the role of palbociclib exposure on neutropenia and thrombocytopenia. Methods: The palbociclib population pharmacokinetics and pharmacodynamics were characterized in this patient population (n = 34 patients; 4.9–21.6 years old). Population pharmacokinetics were modeled using a one-compartment model with first-order absorption and elimination. Covariate analysis was performed, evaluating demographics, laboratory values, and concomitant medications. A pharmacodynamic model was used to describe the relation between palbociclib plasma exposure and changes in the ANC and platelet counts. Results: The population estimates for the apparent oral volume, apparent oral clearance, and absorption rate constant were 664.5 L/m2, 36.8 L/h/m2, and 0.48 h−1, respectively. The palbociclib apparent oral clearance was decreased in patients with higher AST values (p = 0.0066). The ANC and platelet pharmacodynamic models estimated that the median (5th–95th percentile) time individuals had grade 3 or greater neutropenia was 4 (0, 21) days. Simulations showed that given 75 mg/m2 palbociclib, 49% of the individuals were expected to have grade 3 or greater neutropenia. Conclusions: Palbociclib pharmacokinetics and pharmacodynamics were adequately characterized in this patient population, no unexpected adverse reactions were noted, and the drug was well tolerated.

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT- 11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11d10 and SN-38-d3, were chromatographically separated on a reversed-phase C 18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10-5000 ng/mL for T-CPT-11, 2.5-250 ng/mL for NE-CPT-11, and 1-500 ng/ mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde (R)) (R)) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

Abstract BACKGROUND Within the CONNECT1702 trial of unesbulin (tubulin-binding agent causing BMI-1 modulation) with radiotherapy in patients with DIPG/HGG, tumor tissue was analyzed at diagnosis and/or autopsy for biology correlatives, enabling comprehensive, longitudinal molecular characterization. METHODS Whole genome sequencing, RNA-sequencing, and Western blot analyses were performed on tumor, normal brain, and peripheral blood mononuclear cells (PBMCs). RESULTS Tumor tissue from diagnosis (n=11) and/or autopsy (n=7) was analyzed for 16 patients (DIPG=9; HGG=7). Western blot analyses demonstrated 1) BMI-1 overexpression in baseline tumor compared to normal brain and 2) primary on-target effect of BMI1-modulation causing M-phase mitotic arrest when comparing pre- and post-treated PBMCs (increased H3S10-phosphorylation). Among 9 DIPG patients, alterations in the following genes were identified: H3-3A (H3.3) (n=5), H3C2 (H3.1) (n=2, both with co-occurring ACVR1 and PIK3CA mutations), TP53 (n=5), ATRX (n=2), and PPM1D (n=2). Tumors from two longer DIPG survivors were H3.1-, ACVR1-mutant (overall survival [OS]=26 months) and MET-amplified (OS=18 months); one patient with H3.3K27M-DIPG had germline MSH6 mutation (Lynch syndrome) (OS=12.6 months). Among 7 HGG patients, alterations in the following genes were identified: H3-3A (n=5), TP53 (n=3), NF1 (n=3), PDGFRA (n=1), EGFR (n=1), and PIK3CA (n=1). A BRAFV600E mutation was observed in the thalamic HGG patient (H3.3K27M) with greatest progression-free survival (>23 months). Early leptomeningeal metastases occurred in one DIPG patient with BCOR alteration and one HGG patient, with MYCN amplification identified in the spinal metastasis, but not intracranial disease. From gene set enrichment analyses, pathways enriched in patients with longer survival included telomerase regulation and ATP synthesis, whereas pathways enriched in patients with shorter survival included interneuron development, neuron semaphorin signaling, and gamma aminobutyric signaling. CONCLUSIONS Comprehensive molecular profiling of diagnostic and post-mortem tumor tissue provides valuable early biological insight, elucidating potential genomic biomarkers predictive of outcome and response to M-phase alteration and BMI-1 modulation in DIPG/HGG.