Background
The development of high‐throughput drug screening (HTS) using primary cultures provides a promising, clinically translatable approach to tailoring treatment strategies for patients with ...cancer. However, this has been challenging for solid tumors because of often limited amounts of tissue available. In most cases, in vitro expansion is required before HTS, which may lead to overgrowth and contamination by non‐neoplastic cells.
Methods
In this study, hematoxylin and eosin staining and immunohistochemical staining were performed on 129 cytopathology cases from 95 patients. These cytopathology cases comprised cell block preparations derived from primary tumor specimens or patient‐derived xenografts as part of a pediatric precision oncology trial. Cytopathology cases were compared with the morphology and immunohistochemical staining profile of the original tumor. Cases were reported as tumor cells present, equivocal, or tumor cells absent. The HTS results from cytopathologically validated cultures were incorporated into a multidisciplinary tumor board report issued to the treating clinician to guide clinical decision making.
Results
On cytopathologic examination, tumor cells were present in 77 of 129 cases (60%) and were absent in 38 of 129 cases (29%), whereas 14 of 129 cases (11%) were equivocal. Cultures that contained tumor cells resembled the tumors from which they were derived.
Conclusions
Cytopathologic examination of tumor cell block preparations is feasible and provides detailed morphologic characterization. Cytopathologic examination is essential for ensuring that samples submitted for HTS contain representative tumor cells and that in vitro drug sensitivity data are clinically translatable.
Routine cytopathology is able to establish the presence or absence of tumor cells in primary cultures derived from patients with pediatric cancer. Cytopathologic examination ensures the clinical relevance of in vitro drug sensitivity profiling in a pediatric precision oncology platform.
Iron-refractory iron-deficiency anemia (IRIDA) is an autosomal recessive disorder caused by mutations in TMPRSS6. Patients have hypochromic microcytic anemia refractory to oral iron and are only ...partially responsive to parenteral iron administration. We report a French-Canadian kindred in which 2 siblings presented in early childhood with severe microcytic anemia, hypoferremia, and hyperferritinemia. Both children have been successfully treated solely with low-dose oral iron since diagnosis. Clinical and biological presentation did not fit any previously described genetic iron-deficiency anemia. Whole exome sequencing identified in both patients compound heterozygous mutations of TMPRSS6 leading to p.G442R and p.E522K, 2 mutations previously reported to cause classic IRIDA, and no additional mutations in known iron-regulatory genes. Thus, the phenotype associated with the unique combination of mutations uncovered in both patients expands the spectrum of disease associated with TMPRSS6 mutations to include iron deficiency anemia that is accompanied by hyperferritinemia at initial presentation and is responsive to continued oral iron therapy. Our results have implications for genetic testing in early childhood iron deficiency anemia. Importantly, they emphasize that whole exome sequencing can be used as a diagnostic tool and greatly facilitate the elucidation of the genetic basis of unusual clinical presentations, including hypomorphic mutations or compound heterozygosity leading to different phenotypes in known Mendelian diseases.
The mitogen-activated protein kinase (MAPK) pathway signaling pathway is one of the most commonly mutated pathways in human cancers. In particular, BRAF alterations result in constitutive activation ...of the rapidly accelerating fibrosarcoma-extracellular signal-regulated kinase-MAPK significant pathway, leading to cellular proliferation, survival, and dedifferentiation. The role of BRAF mutations in oncogenesis and tumorigenesis has spurred the development of targeted agents, which have been successful in treating many adult cancers. Despite advances in other cancer types, the morbidity and survival outcomes of patients with glioma have remained relatively stagnant. Recently, there has been recognition that MAPK dysregulation is almost universally present in paediatric and adult gliomas. These findings, accompanying broad molecular characterization of gliomas, has aided prognostication and offered opportunities for clinical trials testing targeted agents. The use of targeted therapies in this disease represents a paradigm shift, although the biochemical complexities has resulted in unexpected challenges in the development of effective BRAF inhibitors. Despite these challenges, there are promising data to support the use of BRAF inhibitors alone and in combination with MEK inhibitors for patients with both low-grade and high-grade glioma across age groups. Safety and efficacy data demonstrate that many of the toxicities of these targeted agents are tolerable while offering objective responses. Newer clinical trials will examine the use of these therapies in the upfront setting. Appropriate duration of therapy and durability of response remains unclear in the glioma patient cohort. Longitudinal efficacy and toxicity data are needed. Furthermore, access to these medications remains challenging outside of clinical trials in Australia and New Zealand. Compassionate access is limited, and advocacy for mechanism of action-based drug approval is ongoing.
DNA methylation array profiling for classifying pediatric central nervous system (CNS) tumors is a valuable adjunct to histopathology. However, unbiased prospective and inter-laboratory validation ...studies have been lacking. The AIM BRAIN (AB) diagnostic trial involving 11 pediatric cancer centers in Australia and New Zealand was designed to test the feasibility of routine clinical testing and ran in parallel with the Molecular Neuropathology 2.0 (MNP2.0) study at Deutsches Krebsforschungszentrum (DKFZ) (German Cancer Centre Heidelberg, Germany). CNS tumors from 269 pediatric patients were prospectively tested on Illumina EPIC arrays including 104 cases co-enrolled on MNP2.0. Using MNP classifier versions 11b4 and 12.5, we report classifications with a probability score ≥ 0.90 in 176/265 (66.4%) and 213/269 (79.2%) cases, respectively. Significant diagnostic information was obtained in 130/176 (74%) (11b4) and 12/174 (7%) classifications were discordant with histopathology. Cases prospectively co-enrolled on MNP2.0 gave concordant classifications (99%) and score thresholds (93%), demonstrating excellent test reproducibility and sensitivity. Overall, DNA methylation profiling is a robust single work-flow technique with an acceptable diagnostic yield that is considerably enhanced by the extensive subgroup and copy number profile information generated by the platform. The platform has excellent test reproducibility and sensitivity and contributes significantly to CNS tumor diagnosis.
Children with low‐grade gliomas have excellent long‐term survival outcomes. The development of therapies targeted to the driver mutations along the Mitogen Activated Protein (MAP) kinase signalling ...pathway are providing long‐term stability for many patients with these tumours. Given the frequency of these tumours residing within or near the suprasellar region, our patients commonly suffer from hormone deficiencies. In Australia, the Pharmaceutical Benefits Scheme currently restricts growth hormone therapy to patients who are not being actively treated for cancer, including those receiving targeted therapies. This viewpoint hopes to facilitate an important discussion amongst our colleagues as to whether this should be changed to allow growth hormone to become available to children on chronic tumour suppressive therapy.
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.
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.
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.
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.
Abstract
Background
ZFTA-RELA (formerly known as c11orf-RELA) fused supratentorial ependymoma (ZFTAfus ST-EPN) has been recognized as a novel entity in the 2016 WHO classification of CNS tumors and ...further defined in the recent 2021 edition. ZFTAfus ST-EPN was reported to portend poorer prognosis when compared to its counterpart, YAP1 ST-EPN in some previously published series. The aim of this study was to determine the treatment outcome of molecularly confirmed and conventionally treated ZFTAfus ST-EPN patients treated in multiple institutions.
Methods
We conducted a retrospective analysis of all pediatric patients with molecularly confirmed ZFTAfus ST-EPN patients treated in multiple institutions in 5 different countries (Australia, Canada, Germany, Switzerland, and Czechia). Survival outcomes were analyzed and correlated with clinical characteristics and treatment approaches.
Results
A total of 108 patients were collated from multiple institutions in 5 different countries across three continents. We found across the entire cohort that the 5- and 10-year PFS were 65% and 63%, respectively. The 5- and 10-year OS of this cohort of patients were 87% and 73%. The rates of gross total resection (GTR) were high with 84 out of 108 (77.8%) patients achieving GTR. The vast majority of patients also received post-operative radiotherapy, 98 out of 108 (90.7%). Chemotherapy did not appear to provide any survival benefit in our patient cohort.
Conclusion
This is the largest study to date of contemporaneously treated molecularly confirmed ZFTAfus ST-EPN patients which identified markedly improved survival outcomes compared to previously published series. This study also re-emphasizes the importance of maximal surgical resection in achieving optimal outcomes in pediatric patients with supratentorial ependymoma.
The identification of rearrangements driving expression of neurotrophic receptor tyrosine kinase (
) family kinases in tumors has become critically important because of the availability of effective, ...specific inhibitor drugs. Whole-genome sequencing (WGS) combined with RNA sequencing (RNA-seq) can identify novel and recurrent expressed fusions. Here we describe three
fusions identified in two pediatric central nervous system cancers and an extracranial solid tumor using WGS and RNA-seq. These fusions arose either through a simple balanced rearrangement or in the context of a complex chromoplexy event. We cloned the
fusion directly from a patient sample and showed that enforced expression of this fusion is sufficient to promote cytokine-independent survival and proliferation. Cells transformed by
expression are sensitive to a TRK inhibitor drug. We report here that
fusions can arise in a range of pediatric cancers. Although WGS and RNA-seq are not required to detect
fusions, these techniques may be of benefit when
fusions are not suspected on clinical grounds or not identified by other methods.
The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) ...and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.
Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To ...explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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