Purpose
We undertook a multi-dimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome guided precision therapy.
Experimental Design
Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with Food and Drug Administration approved drugs.
Results
Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7-months-old to 25-years-old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation.
Conclusions
We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors.
The blood-brain barrier (BBB) limits entry of most chemotherapeutic agents into the CNS, resulting in inadequate exposure within CNS tumor tissue. Intranasal administration is a proposed means of delivery that can bypass the BBB, potentially resulting in more effective chemotherapeutic exposure at the tumor site. The objective of this study was to evaluate the feasibility and pharmacokinetics (plasma and CSF) of intranasal delivery using select chemotherapeutic agents in a non-human primate (NHP) model. Three chemotherapeutic agents with known differences in CNS penetration were selected for intranasal administration in a NHP model to determine proof of principle of CNS delivery, assess tolerability and feasibility, and to evaluate whether certain drug characteristics were associated with increased CNS exposure. Intravenous (IV) temozolomide (TMZ), oral (PO) valproic acid, and PO perifosine were administered to adult male rhesus macaques. The animals received a single dose of each agent systemically and intranasally in separate experiments, with each animal acting as his own control. The dose of the agents administered systemically was the human equivalent of a clinically appropriate dose, while the intranasal dose was the maximum achievable dose based on the volume limitation of 1 mL. Multiple serial paired plasma and CSF samples were collected and quantified using a validated uHPLC/tandem mass spectrometry assay after each drug administration. Pharmacokinetic parameters were estimated using non-compartmental analysis. CSF penetration was calculated from the ratio of areas under the concentration-time curves for CSF and plasma (AUC). Intranasal administration was feasible and tolerable for all agents with no significant toxicities observed. For TMZ, the degrees of CSF drug penetration after intranasal and IV administration were 36 (32-57) and 22 (20-41)%, respectively. Although maximum TMZ drug concentration in the CSF (C) was lower after intranasal delivery compared to IV administration due to the lower dose administered, clinically significant exposure was achieved in the CSF after intranasal administration with the lower doses. This was associated with lower systemic exposure, suggesting increased efficiency and potentially lower toxicities of TMZ after intranasal delivery. For valproic acid and perifosine, CSF penetration after intranasal delivery was similar to systemic administration. Although this study demonstrates feasibility and safety of intranasal drug administration, further agent-specific studies are necessary to optimize agent selection and dosing to achieve clinically-relevant CSF exposures.
<p>Tab 1: Patient Demographics at Enrollment. Tab 2: Details of Multiple Samples from Individual Patients , Tab 3: Sample Data of Patients and Sequencing Data of Each Patient Sample, Tab 4: Sample Depth and Coverage, Tab 5: Somatic SNVs in Tumor Samples, Tab 6: Somatic Indels in Tumor Samples, Tab 7: Germline SNVs and Indels in Normal Samples, and Tab 8: Fusion Genes in Defuse, and Tab 9: Fusion Genes in TopHat</p>
<p>Imaging from two clinical vignettes highlighting the importance of sequencing at diagnosis and relapse. A. An epithelioid inflammatory myofibroblastic sarcoma (Patient NCI0244) positive for a RANBP2-ALK fusion leading to activation of ALK was treated with targeted monotherapy. PET scans are shown in the top row. At initial diagnosis, abdominal tumors were located in the left upper quadrant with extensive FDG-avid peritoneal lesions. Clinical response after 8 months of targeted therapy with crizotinib was noted. First relapse with development of a recurrent nodule noted in between the right kidney and duodenum with pulmonary and hepatic metastases was observed after 14 months of crizotinib therapy. A secondary clinical response after treatment with ceritinib was obtained after 1 month of therapy, but was not maintained. The primary tumor sample derived cell line, labeled Tumor.1, did not contain an ALK I1171T mutation. Sanger validation confirmed the presence of an ALK p.I1171T mutation after targeted therapy with crizotinib and ceritinib. The patient's tumor samples all contained a RANBP2-ALK fusion, which was not present in the germline. B. A GNAQ Q209L mutation-positive melanoma (Patient NCI0155), treated with trametinib. The melanoma originated from the left frontotemporal scalp and left orbit, and was metastatic to regional lymph nodes, dura mater, liver, bone and lung as shown on CT (first column, first row) with PET avidity in metastases (first column, second row). WES and WTS of her tumor revealed a GNAQ Q209L mutation. The patient was treated with trametinib and had an initial mixed response, with loss of PET avidity of several liver metastases (second column, second row). However, progressive disease of the primary tumor was noted 5 months after initiating therapy, and PET/CT scans showed increased size of the primary tumor in the scalp (third column, first row)</p>
<p>Using transcriptome data for expression profiling of tumors. Heatmap of transcriptome data allowing for expression profiling and clustering of diagnoses based on the 2000 most highly differentially expressed genes in comparison to the other cohort samples to validate tumor diagnosis. The diagnostic abbreviations are the same as in Figure 1 with the addition of EWLS for Ewing-like sarcoma.</p>
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