Kristopher R. Bosse

Copy number variation at 1q21.1 associated with neuroblastoma

Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in human cancer, we performed a genome-wide association study of CNVs in the childhood cancer neuroblastoma, a disease in which single nucleotide polymorphism variations are known to influence susceptibility. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Caucasian controls at ∼550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. Here we describe the identification of a common CNV at chromosome 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets. This CNV was validated by quantitative polymerase chain reaction, fluorescent in situ hybridization and analysis of matched tumour specimens, and was shown to be heritable in an independent set of 713 cancer-free parent–offspring trios. We identified a previously unknown transcript within the CNV that showed high sequence similarity to several neuroblastoma breakpoint family (NBPF) genes and represents a new member of this gene family (NBPF23). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and the expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a previously unknown neuroblastoma breakpoint family gene in early tumorigenesis of this childhood cancer.

Common variations in BARD1 influence susceptibility to high-risk neuroblastoma

We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma, we restricted our analysis here to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 locus (Pallelic = 2.35 × 10−9–2.25 × 10−8). We confirmed each SNP association in a second series of 189 high-risk cases and 1,178 controls (Pallelic = 7.90 × 10−7–2.77 × 10−4). We also tested the two most significant SNPs (rs6435862, rs3768716) in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds ratios of 1.68 each (P = 8.65 × 10−18 and 2.74 × 10−16, respectively). We also found significant association with known BARD1 nonsynonymous SNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.

Integrative genomics identifies LMO1 as a neuroblastoma oncogene

Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC–Neuroblastoma-related (MYCN)–amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

Common Variation at BARD1 Results in the Expression of an Oncogenic Isoform That Influences Neuroblastoma Susceptibility and Oncogenicity

The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma.

Advances in the translational genomics of neuroblastoma: From improving risk stratification and revealing novel biology to identifying actionable genomic alterations

Neuroblastoma is an embryonal malignancy that commonly affects young children and is remarkably heterogenous in its malignant potential. Recently, the genetic basis of neuroblastoma has come into focus and not only has catalyzed a more comprehensive understanding of neuroblastoma tumorigenesis but also has revealed novel oncogenic vulnerabilities that are being therapeutically leveraged. Neuroblastoma is a model pediatric solid tumor in its use of recurrent genomic alterations, such as high‐level MYCN (v‐myc avian myelocytomatosis viral oncogene neuroblastoma‐derived homolog) amplification, for risk stratification. Given the relative paucity of recurrent, activating, somatic point mutations or gene fusions in primary neuroblastoma tumors studied at initial diagnosis, innovative treatment approaches beyond small molecules targeting mutated or dysregulated kinases will be required moving forward to achieve noticeable improvements in overall patient survival. However, the clonally acquired, oncogenic aberrations in relapsed neuroblastomas are currently being defined and may offer an opportunity to improve patient outcomes with molecularly targeted therapy directed toward aberrantly regulated pathways in relapsed disease. This review summarizes the current state of knowledge about neuroblastoma genetics and genomics, highlighting the improved prognostication and potential therapeutic opportunities that have arisen from recent advances in understanding germline predisposition, recurrent segmental chromosomal alterations, somatic point mutations and translocations, and clonal evolution in relapsed neuroblastoma. Cancer 2016;122:20–33.

Suspected posaconazole toxicity in a pediatric oncology patient.

To the Editor: We report a case of posaconazole toxicity in a 13 year old female with an osteosarcoma-like tumor involving the maxillary sinus. The patient underwent an upfront resection of her primary tumor and subsequently received systemic therapy with cisplatin, doxorubicin, and methotrexate. Inability to achieve a complete surgical excision led to radiation of the primary tumor bed. Her clinical course was further complicated by disseminated candidiasis and hemophagocytic lymphohistocytosis, and after completion of all cancer-directed therapy, she also developed a Coniothyrium fuckelii sinus infection. As there are limited data for in vitro sensitivities for Coniothyrium fuckelii, she was initiated on broad antifungal therapy with voriconazole and liposomal amphotericin B, along with sinus debridement. After significant clinical improvement, she was transitioned to posaconazole suspension 400mg BID. After 3 days of inpatient treatment with posaconazole suspension, she was inadvertently prescribed posaconazole delayed release (DR) tablets (400mg BID) for outpatient treatment. Approximately 2 weeks after discharge, she developed fatigue, decreased appetite, and musculoskeletal pain. Serum chemistries were unremarkable except for a potassium of 2.7 mEq/L and oral potassium supplements were prescribed. In the ensuing weeks, she developed worsening nausea, fatigue, bone pain, and decreased enteral intake. In addition to continued hypokalemia (3.1 mEq/L), she developed progressive anemia as her hemoglobin dropped from 9.1 g/dL to 8.3 g/dL. Because of her minimal enteral intake, there was concern for recrudescence of her disseminated candidiasis due to inadequate posaconazole absorption. Admission for further diagnostic evaluation and initiation of intravenous antifungals was considered. However, review of her medications revealed she had been inadvertently started on posaconazole DR tablets upon her discharge from the hospital 2 months prior and thus there was concern her clinical presentation was consistent with posaconazole toxicity. She was instructed to stop taking the drug and a posaconazole level drawn at a clinic visit 3 days after her last dose was 9.5 mcg/mL. The patient’s symptoms resolved over one week’s time. Posaconazole was initially formulated as a suspension in 2006. The suspension is well known to have poor absorption and it is recommended that it be administered with a full meal, liquid nutritional supplement, or acidic carbonated beverage.[1] In November 2013, posaconazole DR tablets were approved at a dose of 300mg BID for 2 doses, then 300mg daily for prophylaxis of fungal infections in patients 13 years of age.[1] Due to improved absorption, the DR tablet formulation achieved an AUC approximately 4 times that of posaconazole suspension dosed at 400mg BID.[1] This improved absorption and different dosing regimen poses a risk for posaconazole toxicity, which was highlighted in a recent Institute of Safe Medication Practices newsletter.[2] To help prevent future errors, the Computerized Physician Order Entry system at our institution was updated to include DR tablets; dosing defaults to 300mg daily with a maximum dose alert of 300mg. The hospital’s formulary also now highlights dosing differences between formulations. Practitioners prescribing posaconazole DR tablets should be aware of specific dosing recommendations for this formulation and the potential for toxicity presenting with symptoms that can include fatigue, nausea, anorexia, hypokalemia, anemia, and musculoskeletal pain.

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes

ABSTRACT CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 high-affinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibody-drug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05–1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics.

Improving Patient Outcomes With Cancer Genomics: Unique Opportunities and Challenges in Pediatric Oncology

Precision medicine, the individualization of health care based on unique patient-specific variables, is not new, especially within oncology. Historically, there has been a “depersonalization” of cancer care by defining histotype-specific standard-of-care treatments for the majority of malignancies, although in contrast oncologists have been adept at individualizing therapy, especially when confronted with disease relapse. Practicing oncologists seek evidence-based approaches to improve patient outcomes, but much of the current personalization of care remains largely empirical. Nevertheless, over the past decade there has been increasing enthusiasm for using genomic data to more precisely diagnose cancer, predict outcomes, and prescribe “targeted” therapies. Although substantial progress has been made, both anticipated and unanticipated barriers exist in integrating sequencing technologies into the care of patients with cancer. Clearly, for multiple reasons, many challenges remain to prove that personalized genomic medicine can substantively improve outcomes for patients with cancer. These challenges are further accentuated with childhood cancers.