Breaking Up Isn't Hard to Do: Isolating Cell-free DNA Fragments in Osteosarcoma.

The novel use of blood-based biospecimens from a retrospective cohort of 50 patients with osteosarcoma was recently studied. The potential clinical utility of sorting cell-free DNA by fragment size was defined, with shorter tumor-specific DNA enrichment providing prognostic value and allowing for streamlined molecular profiling of circulating tumor material. See related article by Udomruk et al., p. 2085.

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

PURPOSE: Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS: In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS: In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS (P = .04). The most frequently altered genes in the primary cohort were TP53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort (P = .015) and the validation cohort (P = .012). CONCLUSION: The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Acetophenone protection against cisplatin-induced end-organ damage.

Cisplatin is a widely used and efficacious chemotherapeutic agent for treating solid tumors, yet it causes systemic end-organ damage that is often irreversible and detrimental to quality of life. This includes severe sensorineural hearing loss, hepatotoxicity, and renal injury. Based on the hard-soft acid-base theory, we recently developed two acetophenone-derived, enol-based compounds that directly interfere with the side effects of cisplatin. We investigated organ-specific and generalized toxicity in order to define dose-dependent responses in rodents injected with cisplatin with or without the protective compounds. All metrics that were used as indicators of toxicity showed retention of baseline or control measurements when animals were pre-treated with acetophenones prior to cisplatin administration, while animals injected with no protective compounds showed expected elevations in toxicity measurements or depressions in measurements of organ function. These data support the further investigation of novel acetophenone compounds for the prevention of cisplatin-induced end-organ toxicity.

A phase I trial of metformin in combination with vincristine, irinotecan, and temozolomide in children with relapsed or refractory solid and central nervous system tumors: A report from the national pediatric cancer foundation.

BACKGROUND: Patients with relapsed and refractory solid and central nervous system (CNS) tumors have poor outcomes and need novel therapeutic options. Vincristine, irinotecan, and temozolomide (VIT) is a common chemotherapy regimen in relapsed pediatric tumors with an established toxicity profile. Metformin shows preclinical anti-cancer activity through multiple pathways. METHODS: The objective of this Phase I trial was to establish the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of metformin in combination with VIT in children with relapsed and refractory solid and CNS tumors. A 3 + 3 design was used to test the addition of metformin at five dose levels (666, 999, 1333, 1666, and 2000 mg/m2 /day). Therapy toxicity, pharmacokinetics, and radiologic response to treatment were evaluated. RESULTS: Twenty-six patients (median age 13 years, range 2-18 years) were enrolled with 22 evaluable for toxicity. The most common diagnoses were Ewing sarcoma (n = 8), rhabdomyosarcoma (n = 3) and atypical teratoid/rhabdoid tumor (n = 3). The MTD was exceeded at Dose Level 5 due to two dose-limiting toxicities; both were Grade 3 diarrhea requiring prolonged hospitalization and intravenous fluids. The MTD was not determined due to study closure with less than six patients enrolled at Dose Level 4. Frequently observed toxicities were gastrointestinal (most notably diarrhea) and hematologic. Amongst 16 patients evaluable for best overall response, there was one complete response (Ewing sarcoma), three partial responses (Ewing sarcoma, glioblastoma multiforme, and alveolar rhabdomyosarcoma), and five patients with stable disease. CONCLUSIONS: The MTD of VIT with metformin was not determined due to premature study closure. We recommend an RP2D of Dose Level 4, 1666 mg/m2 /day. Radiographic responses were seen in multiple tumor types. Further evaluation for efficacy could be investigated in a Phase II trial.

Omicron variant of SARS-CoV-2, an epidemiologic assessment of pediatric oncology patients in the Bronx.

BACKGROUND: Children receiving cytotoxic therapy for cancer have increased risk of infection due to drug-induced neutropenia and are therefore treated empirically for bacteremia when febrile or ill-appearing. However, viral infections, which are not frequently life-threatening, are the most common etiology of febrile episodes and there has been increased effort to differentiate patients who may have a higher risk for adverse outcomes. CASE: We performed a retrospective chart review of pediatric oncology patients diagnosed with COVID-19 between December 20, 2021 and February 22, 2022 during the Omicron (B.1.1.529) surge at The Children's Hospital at Montefiore, a tertiary care center in the Bronx. CONCLUSION: We found that no patients in our cohort developed respiratory distress, bacteremia, or serious illness after COVID-19 infection during the Omicron surge. Future studies will aid in understanding the relationship between community-acquired infections and bacteremia, and this knowledge can then be applied to develop optimal infection prevention clinical care guidelines.

Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

Donor-derived vasculature is required to support neocortical cell grafts after stroke.

Neural precursor cells (NPCs) transplanted into the adult neocortex generate neurons that synaptically integrate with host neurons, supporting the possibility of achieving functional tissue repair. However, poor survival and functional neuronal recovery of transplanted NPCs greatly limits engraftment. Here, we test the hypothesis that combining blood vessel-forming vascular cells with neuronal precursors improves engraftment. By transplanting mixed embryonic neocortical cells into adult mice with neocortical strokes, we show that transplant-derived neurons synapse with appropriate targets while donor vascular cells form vessels that fuse with the host vasculature to perfuse blood within the graft. Although all grafts became vascularized, larger grafts had greater contributions of donor-derived vessels that increased as a function of their distance from the host-graft border. Moreover, excluding vascular cells from the donor cell population strictly limited graft size. Thus, inclusion of vessel-forming vascular cells with NPCs is required for more efficient engraftment and ultimately for tissue repair.

Therapeutic Targeting of Exportin-1 in Childhood Cancer.

Overexpression of Exportin-1 (XPO1), a key regulator of nuclear-to-cytoplasmic transport, is associated with inferior patient outcomes across a range of adult malignancies. Targeting XPO1 with selinexor has demonstrated promising results in clinical trials, leading to FDA approval of its use for multiple relapsed/refractory cancers. However, XPO1 biology and selinexor sensitivity in childhood cancer is only recently being explored. In this review, we will focus on the differential biology of childhood and adult cancers as it relates to XPO1 and key cargo proteins. We will further explore the current state of pre-clinical and clinical development of XPO1 inhibitors in childhood cancers. Finally, we will outline potentially promising future therapeutic strategies for, as well as potential challenges to, integrating XPO1 inhibition to improve outcomes for children with cancer.

Germline Sequencing Improves Tumor-Only Sequencing Interpretation in a Precision Genomic Study of Patients With Pediatric Solid Tumor.

PURPOSE: Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS: Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS: One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION: A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.

XPO1 inhibition with selinexor synergizes with proteasome inhibition in neuroblastoma by targeting nuclear export of IkB.

Across many cancer types in adults, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Using proteomic profiling of primary tumor material from patients with high-risk neuroblastoma, as well as gene expression profiling from independent cohorts, we have demonstrated that XPO1 overexpression correlates with poor patient prognosis. Neuroblastoma cell lines are also sensitive to selinexor in the low nanomolar range. Based on these findings and knowledge that bortezomib, a proteasome inhibitor, blocks degradation of XPO1 cargo proteins, we hypothesized that combination treatment with selinexor and bortezomib would synergistically inhibit neuroblastoma cellular proliferation. We observed that selinexor promoted nuclear retention of IkB and that bortezomib augmented the ability of selinexor to induce cell-cycle arrest and cell death by apoptosis. This synergy was abrogated through siRNA knockdown of IkB. The synergistic effect of combining selinexor and bortezomib in vitro provides rationale for further investigation of this combination treatment for patients with high-risk neuroblastoma.

Preclinical Metrics Correlate With Drug Activity in Phase II Trials of Targeted Therapies for Non-Small Cell Lung Cancer.

Novel oncology drugs often fail to progress from preclinical experiments to FDA approval. Therefore, determining which preclinical or clinical factors associate with drug activity could accelerate development of effective therapies. We investigated whether preclinical metrics and patient characteristics are associated with objective response rate (ORR) in phase II clinical trials of targeted therapies for non-small cell lung cancer (NSCLC). We developed a reproducible process to select a single phase II trial and supporting preclinical publication for a given drug-indication pair, which we defined as the pairing of a small molecule inhibitor (e.g., crizotinib) with the specific patient population for which it was designed to work (e.g., patients with an ALK aberration). We demonstrated that robust drug activity in mice, as measured by change in tumor size, is independently associated with improved ORR in phase II clinical trials. The number of mice utilized in experiments, the number of publications referencing the drug for NSCLC before the phase II clinical trial, and whether the drug was approved for a cancer other than NSCLC also significantly correlated with ORR. Among clinical characteristics, sex, race, histology, and smoking history were significantly associated with ORR. Further research into metrics that correlate with drug activity has the potential to optimize selection of novel therapies for clinical trials and enrich the drug development pipeline, particularly for patients with targetable genetic aberrations and rare cancers.

Skp2 depletion reduces tumor-initiating properties and promotes apoptosis in synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft-tissue cancer with a poor prognosis and a propensity for local recurrence and distant metastasis. In this study, we investigated whether S phase kinase-associated protein (Skp2) plays an oncogenic role in tumor initiation, progression, and metastasis of SS. Our study revealed that Skp2 is frequently overexpressed in SS specimens and SS18-SSX transgenic mouse tumors, as well as correlated with clinical stages. Next, we identified that genetic depletion of Skp2 reduced mesenchymal and stemness markers, and inhibited the invasive and proliferative capacities of SS cell lines. Furthermore, Skp2 depletion markedly suppressed the growth of SS xenografts tumors. Treatment of SS cell lines with the skp2 inhibitor flavokawain A (FKA) reduced Skp2 expression in a dose-dependent manner and resulted in cell cycle arrest and apoptosis. FKA also suppressed the invasion and tumor-initiating properties in SS, similar to the effects of Skp2 knockdown. In addition, a combination of FKA and conventional chemotherapy showed a synergistic therapeutic efficacy. Taken together, our results suggest that Skp2 plays an essential role in the biology of SS by promoting the mesenchymal state and cancer stemness. Given that chemotherapy resistance is often associated with cancer stemness, strategies of combining Skp2 inhibitors with conventional chemotherapy in SS may be desirable.

An 8-Year-Old Boy With Fever, Splenomegaly, and Pancytopenia.

An 8-year-old boy with no significant past medical history presented to his pediatrician with 5 days of fever, diffuse abdominal pain, and pallor. The pediatrician referred the patient to the emergency department (ED), out of concern for possible malignancy. Initial vital signs indicated fever, tachypnea, and tachycardia. Physical examination was significant for marked abdominal distension, hepatosplenomegaly, and abdominal tenderness in the right upper and lower quadrants. Initial laboratory studies were notable for pancytopenia as well as an elevated erythrocyte sedimentation rate and C-reactive protein. Computed tomography (CT) of the abdomen and pelvis showed massive splenomegaly. The only significant history of travel was immigration from Albania 10 months before admission. The patient was admitted to a tertiary care children's hospital and was evaluated by hematology-oncology, infectious disease, genetics, and rheumatology subspecialty teams. Our multidisciplinary panel of experts will discuss the evaluation of pancytopenia with apparent multiorgan involvement and the diagnosis and appropriate management of a rare disease.

DICER1-associated central nervous system sarcoma in children: comprehensive clinicopathologic and genetic analysis of a newly described rare tumor.

The spectrum of neoplasms associated with DICER1 variants continues to expand, with the recent addition of primary "DICER1-associated central nervous system sarcoma" (DCS). DCS is a high-grade malignancy predominantly affecting pediatric patients. Six pediatric DCS were identified through a combination of clinical diagnostic studies, archival inquiry, and interinstitutional collaboration. Clinical, histologic, immunohistologic, and molecular features were examined. Genomic findings in the 6 DCS were compared with those in 14 additional DICER1-associated tumors sequenced with the same assay. The six patients presented at ages 3-15 years with CNS tumors located in the temporal (n = 2), parietal (n = 1), fronto-parietal (n = 1), and frontal (n = 2) lobes. All underwent surgical resection. Histologic examination demonstrated high-grade malignant spindle cell tumors with pleuropulmonary blastoma-like embryonic "organoid" features and focal rhabdomyoblastic differentiation; immature cartilage was seen in one case. Immunohistochemically, there was patchy desmin and myogenin staining, and patchy loss of H3K27me3, and within eosinophilic cytoplasmic globules, alfa-fetoprotein staining. Biallelic DICER1 variants were identified in all cases, with germline variants in two of five patients tested. DCS demonstrated genomic alterations enriched for Ras pathway activation and TP53 inactivation. Tumor mutational burden was significantly higher in the 6 DCS tumors than in 14 other DICER1-associated tumors examined (mean 12.9 vs. 6.8 mutations/Mb, p = 0.035). Postoperative care included radiation (n = 5) and chemotherapy (n = 3); at the last follow-up, three patients were alive without DCS, and three had died of disease. Our analysis expands the clinical, histologic, immunohistological, and molecular spectrum of DCS, identifying distinctive features that can aid in the diagnosis, multidisciplinary evaluation, and treatment of DCS.

Enolate-forming compounds provide protection from platinum neurotoxicity.

Cisplatin (CisPt) and other platinum (Pt)-based antineoplastic drugs (e.g., carboplatin, oxaliplatin) are highly effective and widely used in the treatment of solid tumors in both pediatric and adult patients. Although considered to be life-saving as a cancer treatment, Pt-based drugs frequently result in dose-limiting toxicities such as chemotherapy-induced peripheral neuropathies (CIPN). Specifically, irreversible damage to outer hair cells and injury of sensory neurons are linked to profound sensorineural hearing loss (ototoxicity), which complicates tumor management and can lead to a poor clinical prognosis. Given the severity of CIPN, substantial effort has been devoted to the development of neuroprotective compounds, regardless clinical results have been underwhelming. It is noteworthy that Pt is a highly reactive electrophile (electron deficient) that causes toxicity by forming adducts with nucleophilic (electron rich) targets on macromolecules. In this regard, we have discovered a series of carbon-based enol nucleophiles; e.g., N-(4-acetyl-3,5-dihydroxyphenyl)-2-oxocytclopentane-1-carboxamide (Gavinol), that can prevent neurotoxicity by scavenging the platinum ion. The chemistry of enol compounds is well understood and mechanistic research has demonstrated the role of this chemistry in cytoprotection. Our cell-derived data were corroborated by calculations of hard and soft, acids and bases (HSAB) parameters that describe the electronic character of interacting electrophiles and nucleophiles. Together, these observations indicate that the respective mechanisms of Pt neurotoxicity and antitumor activity are separable and can therefore be affected independently.

Survey of Paediatric Oncologists and Pathologists regarding Their Views and Experiences with Variant Translocations in Ewing and Ewing-Like Sarcoma: A Report of the Children's Oncology Group.

Advances in molecular diagnostics have identified subsets of Ewing and Ewing-like sarcomas driven by variant translocations with unique biology. It is likely that patients with these tumours will have different clinical features and therapeutic outcomes. Nevertheless, the management of these patients both locally and within cooperative group trials depends on the local pathological diagnosis. It is not known what molecular diagnostic approaches are employed by local pathologists or if the exact translocation is commonly determined. In addition, it is not known what therapeutic approaches are employed for these patients or what cooperative trials are deemed appropriate for these patients by expert consensus. To answer these questions, we performed an international survey of oncologists and pathologists to better understand the diagnostic approaches used to identify variant translocations and the influence the findings have on therapy and clinical trial eligibility. An online survey was distributed to oncologists and pathologists primarily in North America. A total of 141 surveys were completed, representing a 28% response rate. The majority of respondents considered EWSR1-ETS gene family translocations (range 61-96%) to be Ewing sarcoma and would include them on the primary arm of a Ewing sarcoma clinical trial. There was a lack of consensus on how to classify and stratify BCOR-CCNB3, CIC-DUX4, and EWSR1+ with non-ETS partner fusions. Most respondents were either unsure how their institution tested, or their institution did not perform the test. In cases with atypical Ewing morphology, most respondents favoured additional fusion transcript testing. There is a lack of consensus regarding the classification and stratification of rare molecular subtypes in Ewing sarcoma. It is not clear how these alternative translocations have impacted outcomes for past clinical studies. This suggests a need for molecular confirmation of diagnoses and centralized or minimum standardization of testing for future trial enrolment.

Progress toward liquid biopsies in pediatric solid tumors.

Pediatric solid tumors have long been known to shed tumor cells, DNA, RNA, and proteins into the blood. Recent technological advances have allowed for improved capture and analysis of these typically scant circulating materials. Efforts are ongoing to develop "liquid biopsy" assays as minimally invasive tools to address diagnostic, prognostic, and disease monitoring needs in childhood cancer care. Applying these highly sensitive technologies to serial liquid biopsies is expected to advance understanding of tumor biology, heterogeneity, and evolution over the course of therapy, thus opening new avenues for personalized therapy. In this review, we outline the latest technologies available for liquid biopsies and describe the methods, pitfalls, and benefits of the assays that are being developed for children with extracranial solid tumors. We discuss what has been learned in several of the most common pediatric solid tumors including neuroblastoma, sarcoma, Wilms tumor, and hepatoblastoma and highlight promising future directions for the field.

Physician perspectives on compassionate use in pediatric oncology.

BACKGROUND: Targeted cancer treatments are almost always first studied in adults, even when there is a biologically plausible potential for efficacy in children. Through compassionate use programs, children who are not eligible for a clinical trial and for whom there are no known effective therapies may obtain access to investigational agents, including drugs under development for adults. However, little is known about pediatric oncologists' experiences with applying for and obtaining compassionate use agents. METHODS: This study surveyed 132 pediatric oncologists to assess awareness and utilization of compassionate use programs, to identify barriers to their use, and to evaluate available institutional support and resources. RESULTS: We found that the process of applying for access to drugs in development is poorly understood, which presents a barrier to obtaining investigational drugs. Fifty-seven percent of the pediatric oncologists applied for compassionate use. Providers from larger institutions or with more than 15 years of clinical experience were more likely to complete an application and obtain investigational agents for their patients. CONCLUSION: Identified perceived and actual barriers to compassionate use application submission suggest pediatric oncologists may benefit from educational resources and support to ensure children with cancer equal access to investigational agents and care.