A Pilot Study Using Next-Generation Sequencing in Advanced Cancers: Feasibility and Challenges

<div><p>Purpose</p><p>New anticancer agents that target a single cell surface receptor, up-regulated or amplified gene product, or mutated gene, have met with some success in treating advanced cancers. However, patients' tumors still eventually progress on these therapies. If it were possible to identify a larger number of targetable vulnerabilities in an individual's tumor, multiple targets could be exploited with the use of specific therapeutic agents, thus possibly giving the patient viable therapeutic alternatives.</p><p>Experimental Design</p><p>In this exploratory study, we used next-generation sequencing technologies (NGS) including whole genome sequencing (WGS), and where feasible, whole transcriptome sequencing (WTS) to identify genomic events and associated expression changes in advanced cancer patients.</p><p>Results</p><p>WGS on paired tumor and normal samples from nine advanced cancer patients and WTS on six of these patients' tumors was completed. One patient's treatment was based on targets and pathways identified by NGS and the patient had a short-lived PET/CT response with a significant reduction in his tumor-related pain. To design treatment plans based on information garnered from NGS, several challenges were encountered: NGS reporting delays, communication of results to out-of-state participants and their treating oncologists, and chain of custody handling for fresh biopsy samples for Clinical Laboratory Improvement Amendments (CLIA) target validation.</p><p>Conclusion</p><p>While the initial effort was a slower process than anticipated due to a variety of issues, we demonstrate the feasibility of using NGS in advanced cancer patients so that treatments for patients with progressing tumors may be improved.</p></div

Sequencing outcomes.

<p>Sequencing outcomes.</p

Circos plot for WGS results for ONB.

<p>This figure depicts the genomic location in the outer ring and chromosomal copy number in the inner ring. The SNVs and indels are marked on the outer ring in their respective genomic locations. In the inner ring, copy gains are shown in red, while copy losses are shown in green. No interchromosomal translocations were observed by assessing counts of anomalous read pairs between specific regions of the genomes, noting that the use of shorter-paired end sequencing may limit our ability to detect these events with this analysis.</p

Validation of mutations in previously collected archival FFPE samples from the ONB patient.

<p>Key: Validated mutations compared to reference codon depicted in bold italics.</p

Patient 9 PET/CT images.

<p>18-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) images depicting (A) axial slice at baseline and (B) axial slice after 30 days on treatment. The yellow arrow is pointing to two hypermetabolic lymph nodes in B and C. In B, the standard uptake value (SUV) of the left lymph node is 9.8 and the lymph node on the right is 7.5, while the SUV of the left lymph node is 3.1 and the lymph node on the right is 3.5 in C.</p

CONSORT schematic on patient enrollment.

<p>*-Prospective enrichment analysis was performed prior to discussing the results with the patient and treating oncologist for the last three patients enrolled.</p

Single nucleotide variations in the seven genes chosen for validation by Sanger sequencing.

<p>Key: *Low confidence, Mutated Codon column-somatic mutation depicted in bold face.</p

Additional file 1 of Molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancers: a Beat Childhood Cancer Research Consortium trial

Additional file 1: Table S1. Cell lines and mice models established. A total of 96 patients enrolled onto the NMTRC009 MGT trial had at least one tumor cell line generated in the laboratory setting. Since many subjects underwent multiple tumor biopsies and/or bone marrow biopsies, subjects may have >1 unique cell line, either from the same tumor obtained from different biopsy dates or from a different disease site (bone marrow). 47 subjects’ tumors underwent successful implantation into a NOD-SCID mouse to generate at least one PDX model. A total of 56 unique PDX models were generated

Indels and SNVs identified through WGS.

a<p>Effects were determined using SIFT/Polyphen-2.</p>b<p>NMD = nonsense mediated decay.</p

Selected^a differentially expressed genes identified using RNAseq^b.

a<p>Selected genes are genes that are reported in COSMIC.</p>b<p>RNAseq was performed on patients 2 and 3.</p