Subclonal somatic copy number alterations emerge and dominate in recurrent osteosarcoma

Abstract

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma.

Keywords: clonal evolution; intratumor heterogeneity; osteosarcoma; somatic copy number alterations; whole-genome sequencing.

Figure 1.

Characteristics of the patients and samples included in the analysis cohort. A, Oncoprint of sample and patient level details for each patient. Samples from the same patient are connected by dots and lines on bottom of figure and are in chronological order of time obtained (earliest on left). Sample Name Key: Bx = biopsy, Rx = Resection, Sample Ending in 0 = metastatic site present at diagnosis, sample ending in a number >0 indicates number of relapses. L/R = laterality, d = distal, p=proximal, f=femur, t=tibia, cw=chest wall, H=heart, ul=upper lobe, ll=lower lobe, di=diaphragm, rp=retroperitoneal, l=lobe. B, Summary of the number of samples per timepoint for each patient, darker shades of blue represent higher number of samples at a respective time point. C, Age and sex assigned at birth for each patient, patient is on the x-axis, age is on the y-axis, and sex assigned at birth is plotted on the chart as blue for male and pink for female. D, Percent necrosis at time of primary resection for each patient (Note OSCE2/OSCE10 both have 45% necrosis). E, Patients are listed on the y-axis and are ordered from longest disease course at the top to shortest at the bottom of the figure. The light blue bars represent length of disease course in months. Events are marked as depicted in the legend with different shapes and colors and plotted along the disease course bar at the time in months that the event occurs.

Figure 2.

Subclonal copy number clones emerge at relapse. A, B, C, D, E, and F, For each patient there is a panel of three figures. The figure on the left is an oncoprint featuring clone specific copy number alterations in recurrently altered genes of interest in osteosarcoma. The top figure is a plot of allele specific copy number alterations for each clone with significant events for each clone circled and highlighted (note y-axis scales are unique for each patient). Clone 1 is the magenta clone, clone 2 is the teal clone, and clone 3 in OSCE10 is the gray clone. The major allele is plotted above 0 and the minor allele is plotted below 0. The bottom figure in each panel is a TimeScape plot of the prevalence of each clone at different timepoints throughout a patient’s disease course. G, Combined genome-wide copy number alterations across all patients in the cohort with recurrently altered genes highlighted.

Figure 3.

Chromosomal duplication timing analysis reconstructs evolutionary past of genomic instability events. A, B, C, Ridgeline plots of the density of duplication events over molecular time for each sample for the selected patients. Notably the highest peak in duplications occurs before diagnosis. D, Chromosomal duplication timing of 5 primary site samples. Whole genome duplication events as called by HATCHet appear to be a late event in our cohort. E, Plot of tumor size (by volume) verse ploidy called by HATCHet. Median size (y-axis) and ploidy (x-axis) values are plotted with dark black lines, with the shaded gray areas representing the range between the lower and upper quartile for each metric. F, Plot of duplication and rearrangement events in molecular time. Left side of figure is plot of duplication events in blue and LOH events in orange for select primary site samples. The median duplication time is highlighted for each sample. The plot on the right side of the figure are complex amplicon events in teal and complex rearrangement events in yellow. Y-axis for both plots is molecular time. The samples are in the same order for each plot. Each plotted event represents an affected chromosomal arm.

Figure 4.

SNV based phylogenies highlighting temporal evolution with clonal mutational signature composition. A, B, C, and D, Upper figure in each panel is a TimeScape plot of the inferred evolutionary phylogeny, highlighting clonal proportions over time. The prevalence of different clones is shown over time on the vertical axis, with the different clones represented by different colors. The horizontal axis represents the timepoints, which are represented by gray lines. The evolutionary relationships between the clones are shown on the phylogenetic tree and in the TimeScape layout. The bottom right of each panel is a stacked bar plot of the total number of mutations assigned to each clone. Colors represent total number of mutations attributed to each mutational signature with color legend at bottom of figure. Patient level metastatic seeding patterns are denoted by the brackets at the top of the page.

Figure 5.

SNV based phylogenies highlighting spatial evolution and descriptions of metastatic seeding patterns at the sample and patient level. A, B, C, and D, Spatially and in some cases temporally distinct samples are indicated on the anatomic sites from where the sample originated. The colors represent different clones, and the phylogenetic trees show the evolutionary relationships between these clones. The prevalence of each clone at a particular site is proportional to the colored area of the cellular aggregate representation. E, Sample and patient level dissemination patterns are characterized in these charts. Monoclonal dissemination: single subclone within the primary tumor seeds one or more metastatic lesions, polyclonal dissemination: multiple distinct subclones from the primary tumor seed one or more metastatic lesions, monophyletic origin: all metastatic clones are derived from a recent common ancestor, polyphyletic origin: metastasizing clones are more similar to other subclones within the primary tumor than they are to each other. These descriptions can be considered at the sample level, focused on the clonal make up of a single metastatic site compared to the primary tumor, or taken as a whole, evaluating all spatially or temporally separated samples and how they relate back to the primary tumor. F, Multi-region sequencing was performed on a primary resection sample from OSCE10. Regions D, E, M, O, P were sequenced from the specimen grid depicted. A table of Jaccard similarity indexes based on shared SNVs for these samples is shown in the upper left inset.

Figure 6.

Mutational signature patterns across the cohort. A, Stacked line chart of mutational signature contribution by total number of mutations attributed to each signature. Colors represent the different signatures. B, Stacked bar chart of the relative contribution of HRD-related SBS3 (green) & cisplatin (blue) signature in metastatic and relapse samples. C, Stacked bar chart of the relative contribution of HRD (green), clock-like (pink), reactive oxygen species damage (red), late replication errors (beige), somatic hypermutation (orange), and APOBEC (teal), in primary site samples. D, Stacked bar chart of the probability that driver gene SNVs were attributed to a mutational signature. Pretreatment samples from patients with driver SNVs were included and the primary resection sample from OSCE9 since no pretreatment sample was available. Colors represent the different signatures.