Implementation of next generation sequencing into pediatric hematology-oncology practice: moving beyond actionable alterations

Abstract

Background: Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated.

Methods: The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient's electronic medical record.

Results: NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients.

Conclusions: Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases.

Keywords: Pediatric oncology; Precision medicine; RNA sequencing; Whole exome sequencing.

Fig. 1

PIPseq overview. An overview of the PIPseq patients sequenced is presented on the left and a pie chart showing the distribution of diagnostic categories on the right

Fig. 2

Somatic mutation load by diagnostic category. Box plots comparing overall somatic mutation rates across solid tumors and hematologic conditions detected by NGS. The top and bottom ends of the boxes represent the 25th and 75th percentile values, respectively, and the segment in the middle is the median. The top and bottom extremes of the bars extend to the minimum and maximum values. The box plot depicts the total mutation load excluding four outliers (one solid tumor and three hematologic). See Additional file 4: Figure S1 for inclusive dataset with outliers. The total mutational load (prior to filtering or orthogonal validation) for solid tumors was 4972 variants (mean, 84.3; SD, 43.9; median, 85; range, 15–214) and for hematologic conditions was 1478 variants (mean, 56.85; SD, 34.9; median, 47; range, 14–149)

Fig. 3

Summary of informative results from the PIPseq program. A matrix representation of findings with biological significance from the sequencing results are presented. Data are derived from all 101 patients that underwent WES of tumor-normal sample pairs, exome sequencing of germline DNA, transcriptome analysis of tumor, CNV of tumor, and targeted panel sequencing of tumor only. Deleterious mutations were loss of function mutations and activating mutations refers to recurrent, previously reported activating mutations in oncogenes or variants with published in vitro evidence as being activating

Fig. 4

Clinically impactful results. The PIPseq experience yielded clinically impactful results in 67/101 cases. The Venn diagrams depict the complexity of overlapping findings within patients. That is, a patient may have a single finding fitting more than one category, whereas another patient may have a finding fitting one category and another finding fitting a different category. For example, results categorized as Targetable/ Diagnostic (n = 6) are as follows: BCR-ABL1; IDH1; PIK3CA; EML4-NTRK3; [STAT5B, KRAS, JAK1/ STAT5B, i7q]; and [TMEM106B-BRAF/ gain chr 7, LOH 9p], with non-bracketed results representing a single finding fitting two categories and results within brackets representing those that were Targetable/ Diagnostic, respectively. Similarly, results categorized as Targetable/ Prognostic (n = 7) are as follows: FOXP1-ABL1; [TET2/ CEBPA]; [H3F3A, FGFR1/ H3F3A]; [NRAS/ MYCN amp, del 1p and 11q, gain 17q]; [c-KIT, TET2, FLT3, NRAS/ CBFB-MYH11]; [KRAS/ No LOH 1p11q]; and [Gain 12q.14.1 involving CDK2/ H3F3A]. Individual patient results are provided in Tables 2, 3, and 4

Fig. 5

Clinical impact of WES and RNA-seq by sequencing technology. Sixty patients had full tumor/normal WES (including CNV) and RNA-seq (cWES) performed. A total of 72 clinically impactful results were found in 45/60 cases (75%). A pie chart of the overall clinical impact of cWES is presented on the left with a pie chart and table showing the number of impactful findings by sequencing technology on the right. For six patients, CNV and overexpression together yielded prognostic information in four patients with neuroblastoma and two patients with medulloblastoma