Outcomes for Australian children with relapsed/refractory acute lymphoblastic leukaemia treated with blinatumomab.

We report on the Australian experience of blinatumomab for treatment of 24 children with relapsed/refractory precursor B-cell acute lymphoblastic leukaemia (B-ALL) and high-risk genetics, resulting in a minimal residual disease (MRD) response rate of 58%, 2-year progression-free survival (PFS) of 39% and 2-year overall survival of 63%. In total, 83% (n = 20/24) proceeded to haematopoietic stem cell transplant, directly after blinatumomab (n = 12) or following additional salvage therapy (n = 8). Four patients successfully received CD19-directed chimeric antigen receptor T-cell therapy despite prior blinatumomab exposure. Inferior 2-year PFS was associated with MRD positivity (20%, n = 15) and in KMT2A-rearranged infants (15%, n = 9). Our findings highlight that not all children with relapsed/refractory B-ALL respond to blinatumomab and factors such as blast genotype may affect prognosis.

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.

BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

Buccal swabs and treated cards: methodological considerations for molecular epidemiologic studies examining pediatric populations.

Self-collection of buccal cells provides a noninvasive method for obtaining biologic samples for genetic analyses in pediatric studies. Nevertheless, low yields, microbial contamination, and degradation of buccal samples present challenges for epidemiologic studies incorporating genetic investigations. The aims of this study were to compare the quality and yield of DNA extracted from buccal specimens with BuccalAmp swabs (Epicenter BioTechnologies, Madison, Wisconsin) or FTA cards (Whatman, Inc., Clifton, New Jersey) and to investigate the use of whole-genome amplification (WGA) for increasing DNA yields for single nucleotide polymorphism analyses. Buccal specimens were collected from 55 children with acute lymphoblastic leukemia and 52 control children without acute lymphoblastic leukemia in New South Wales, Australia, in 2003-2004. Real-time polymerase chain reaction was used to evaluate polymorphisms in the genes encoding the cytochrome p450 enzyme CYP3A4 (CYP3A4 A392G, also known as CYP3A4*1B) and the steroid xenobiotic receptor (SXR C25385T). Results showed that DNA could be isolated from buccal specimens collected by use of both methods and that yields could be substantially improved with WGA without introducing genotyping error. However, DNA quality was poorer in samples collected by BuccalAmp swabs, and the presence of polymerase chain reaction inhibitors in these samples reduced the sensitivity of quantitative real-time PCR analysis. These findings show that different methods for collecting buccal samples impact on the downstream success of genetic investigations and influence DNA quality after WGA.

Relapse in children with acute lymphoblastic leukemia involving selection of a preexisting drug-resistant subclone.

Relapse following remission induction chemotherapy remains a barrier to survival in approximately 20% of children suffering from acute lymphoblastic leukemia (ALL). To investigate the mechanism of relapse, 27 matched diagnosis and relapse ALL samples were analyzed for clonal populations using polymerase chain reaction (PCR)-based detection of multiple antigen receptor gene rearrangements. These clonal markers revealed the emergence of apparently new populations at relapse in 13 patients. More sensitive clone-specific PCR revealed that, in 8 cases, these "relapse clones" were present at diagnosis and a significant relationship existed between presence of the relapse clone at diagnosis and time to first relapse (P < .007). Furthermore, in cases where the relapse clone could be quantified, time to first relapse was dependent on the amount of the relapse clone at diagnosis (r = -0.84; P = .018). This observation, together with demonstrated differential chemosensitivity between subclones at diagnosis, argues against therapy-induced acquired resistance as the mechanism of relapse in the informative patients. Instead these data indicate that relapse in ALL patients may commonly involve selection of a minor intrinsically resistant subclone that is undetectable by routine PCR-based methods. Relapse prediction may be improved with strategies to detect minor potentially resistant subclones early during treatment, hence allowing intensification of therapy.

Activity of postoperative carboplatin, etoposide, and high-dose methotrexate in pediatric CNS embryonal tumors: results of a phase II study in newly diagnosed children.

BACKGROUND: Chemotherapy is used as an alternative to irradiation or to minimize the irradiation exposure among infants with medulloblastoma or other CNS embryonal tumors. Adjuvant chemotherapy is commonly used in older children with high-risk medulloblastoma to improve survival or to allow a reduction in the craniospinal irradiation dose in standard-risk patients. However, optimal multimodality therapy, including the precise role of chemotherapy, has not been defined for these groups of patients. The objective of the present study is to assess the efficacy and toxicity of four postoperative courses of carboplatin, etoposide, and high-dose methotrexate in newly diagnosed children with medulloblastoma or other CNS embryonal tumors. PROCEDURE: Twenty-eight children, aged from 0.3 to 15.9 years (median, 6.2 years) with post-operative measurable residual CNS embryonal tumors were enrolled, comprising medulloblastoma (n = 19), supratentorial PNET (n = 7), and pineoblastoma (n = 2). Post-operative chemotherapy comprised carboplatin 350 mg/m(2) and etoposide 100 mg/m(2) on Days 1 & 2, and methotrexate 8 g/m(2) on Day 3, repeated at 21-28-day intervals for a total of four courses. Therapy following completion of the initial Phase II study was influenced by patient age and investigator preference. RESULTS: The combined complete response rate (CR, 7/19) and partial response rate (PR, 7/19) was 74% in patients with medulloblastoma, 89% for patients with PNET/pineoblastoma (CR, 2/9 and PR, 6/9), and for all patients it was 79%. Patients aged < 3 years at diagnosis had a combined PR and CR rate of 71% compared to 81% in patients aged > 3 years. Treatment was well tolerated although myelosuppression and thrombocytopenia were common. CONCLUSIONS: The combination of carboplatin, etoposide, and high-dose methotrexate is highly active in pediatric patients with CNS embryonal tumors.