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Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy). Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC50 in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.