Clinical genomic sequencing of pediatric and adult osteosarcoma reveals distinct molecular subsets with potentially targetable alterations

Abstract

Purpose:

While multimodal chemotherapy has improved outcomes for patients with osteosarcoma (OS), the prognosis for patients who present with metastatic and/or recurrent disease remains poor. In this study, we sought to define how often clinical genomic sequencing of OS samples could identify potentially actionable alterations.

Experimental Design:

We analyzed genomic data from 71 OS samples from 66 pediatric and adult patients sequenced using MSK-IMPACT, a hybridization capture-based large panel NGS assay. Potentially actionable genetic events were categorized according to the OncoKB precision oncology knowledge base, of which Levels 1–3 were considered clinically actionable.

Results:

We found at least one potentially actionable alteration in 14/66 patients (21%), including amplification of CDK4 (n=9, 14%: Level 2B) and/or MDM2 (n=9, 14%: Level 3B), and somatic truncating mutations/deletions in BRCA2 (n=3, 5%: Level 2B) and PTCH1 (n=1, Level 3B). Additionally, we observed mutually exclusive patterns of alterations suggesting distinct biological subsets defined by gains at 4q12 and 6p12–21. Specifically, potentially targetable gene amplifications at 4q12 involving KIT, KDR and PDGFRA were identified in 13 of 66 patients (20%), which showed strong PDGFRA expression by immunohistochemistry. In another largely non-overlapping subset of 14 patients (24%) with gains at 6p12–21, VEGFA amplification was identified.

Conclusions:

We found potentially clinically actionable alterations in approximately 21% of OS patients. Additionally, at least 40% of patients have tumors harboring PDGFRA or VEGFA amplification, representing candidate subsets for clinical evaluation of additional therapeutic options. We propose a new genomically-based algorithm for directing OS patients to clinical trial options.

Introduction

Osteosarcoma, the most common primary malignant bone tumor, accounts for approximately 1% of all cancer cases in the United States^1,2. The incidence of OS shows a bimodal distribution with one peak in childhood/adolescence and the other in adults over 50 years of age¹. The current standard therapies, which include combination chemotherapy and surgical resection, were originally developed in the 1980s and have significantly improved the 5-year disease-free survival of OS patients to approximately 70%^3,4. Furthermore, the response to preoperative combination chemotherapy is highly prognostic in patients with localized disease⁵. However, 20–30% of patients remain refractory to conventional treatment and the survival rate for patients presenting with localized disease has remained essentially unchanged for over 20 years^4,6. Patients with unresectable primary tumors or metastases have poor clinical outcomes^7,8. Older studies have reported on kinases or their ligands including VEGF, IGF1, PDGF, HER2 and MET as potential therapeutic targets in OS based on their overexpression by immunohistochemical analysis⁹.

Next generation sequencing (NGS) technology has made the comprehensive analysis of cancer-related genes more clinically accessible, opening new avenues in treatment modalities for a variety of tumor types^10,11. The implementation of precision medicine for the treatment of rare tumors such as OS has been difficult due to a lack of targetable driver mutations or fusions involving well-established drug targets such as kinases¹². In the present study, we analyzed clinical sequencing data in OS using the MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) panel assay¹¹ to identify the proportion of patients with potential somatic actionable alterations as defined by the OncoKB precision oncology knowledge base¹³.

Materials and methods

Patients and samples:

This project was approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (MSKCC) and was conducted in accordance with the U.S. Common Rule. A total of 92 formalin-fixed paraffin-embedded OS samples from patients treated at MSKCC between 2004 and 2016 were submitted for clinical sequencing using the MSK-IMPACT panel¹¹. In all cases, the diagnosis of OS was confirmed by sarcoma pathologists. The MSK-IMPACT assay generated data for 81 of the 92 OS samples (Supplemental Table 1), with the remaining 11 samples (12%) being insufficient or inadequate for NGS. This percentage is in keeping with our general experience with MSK-IMPACT testing, where approximately 9% of samples overall are found to have insufficient tumor or insufficient DNA extracted to proceed with MSK-IMPACT NGS¹¹. The remaining 80 cases consisted of 71 samples of classic high-grade OS (including 6 samples of post-radiation OS) that were used for the analyses of genomic and clinicopathologic correlates, and a separate group of 9 cases of special OS subtypes (extra-skeletal OS, n=7; dedifferentiated OS, n=2) that were excluded from further analysis in this study (Supplemental Table 1).

Sample collection and sequencing:

Among the 71 high-grade OS samples (from 66 patients), 54 samples (from 49 patients) underwent clinical sequencing in a prospective manner while 17 samples (from 17 patients) were selected and sequenced retrospectively. To confirm and select the tumor and corresponding normal tissue for the retrospective group, slides from all the tissue blocks were reviewed by a sarcoma pathologist (MH). In the prospective group, matched blood was used as the germline sample after obtaining patient consent. Tumor and germline DNA were sequenced using MSK-IMPACT, an US Food and Drug Administration (FDA)-cleared, hybridization capture-based NGS assay capable of detecting all somatic protein-coding mutations, copy number alterations (CNAs), and select promoter mutations and structural rearrangements in a panel consisting of 341 cancer-related genes (Version 1) later expanded to 410 (Version 2) and then 468 genes (Version 3)¹¹. Of the genes discussed in this study, only VEGFA was not present in all 3 versions (versions 2 and 3 only). The sequence read alignment processing, non-synonymous mutations and rearrangements were determined as previously described¹¹.

Copy number aberrations were identified using an in-house developed algorithm by comparing sequence coverage of targeted regions in a tumor sample relative to a standard diploid normal sample¹¹, as extensively validated for ERBB2 (HER2) amplification¹⁴. Specifically, coverage values were normalized for the overall coverage of the sample, square root transformed, and adjusted for the GC content of each target region using Loess normalization¹⁴. The following criteria were used to determine significance of whole-gene gain or loss events: fold change >2.0 (gain) or <−2.0 (loss), P < 0.05 (false discovery rate–corrected for multiple testing).

Somatic structural rearrangements including putative gene fusions were identified by Delly (v0.6.1)¹⁵ based on supporting read pairs and split reads¹⁶. Candidate rearrangements were flagged for manual review if the tumor harbored ≥3 discordant reads with a mapping quality of ≥5 and the matched normal sample harbored ≤3 discordant reads (sites of known recurrent rearrangements) or if the tumor harbored ≥5 discordant reads with mapping quality of ≥20 and the matched normal sample harbored ≤1 discordant read (novel rearrangement sites). All candidate somatic structural rearrangements were annotated using in-house tools and manually reviewed using the Integrative Genomics Viewer¹⁷.

The somatic genomic alterations in the sequenced OS samples were then analyzed using cBioPortal for Cancer Genomics tools^18,19. Germline alterations in cancer susceptibility genes were not evaluated in this study as consent issues did not allow germline variant calling across this entire set of OS patients. A systematic analysis of germline cancer susceptibility across pediatric solid cancers (including OS) in the MSK-IMPACT dataset is in progress and will be published separately.

Identification of potentially actionable alterations by OncoKB:

Potentially actionable genetic events were categorized into one of four levels using MSK-Precision Oncology Knowledge base (OncoKB) (www.OncoKB.org)¹³. The level of evidence on a specific molecular alteration is based on FDA labeling, National Comprehensive Cancer Network (NCCN) guidelines, disease-focused expert group recommendations and scientific literature¹³. Tumors with two or more Level 1–4 oncogenic drivers were grouped with the highest level actionable driver alteration per the following OncoKB criteria. Individual mutational events are annotated by the level of evidence that supports the use of a certain drug in an indication that harbors that mutation. The levels of evidence are tiered as follows-

OncoKB Level 1: FDA–recognized biomarkers that are predictive of response to an FDA-approved drug in a specific indication.

OncoKB Level 2A: Standard care biomarkers that are predictive of response to an FDA-approved drug in a specific indication.

OncoKB Level 2B: FDA-approved biomarkers predictive of response to an FDA approved drug detected in an off-label indication.

OncoKB Level 3A: FDA- or non–FDA-recognized biomarkers that are predictive of response to novel targeted agents that have shown promising results in clinical trials in a specific indication.

OncoKB Level 3B: FDA- or non–FDA-recognized biomarkers that are predictive of response to novel targeted agents that have shown promising results in clinical trials for another indication.

OncoKB Level 4: Non–FDA recognized biomarkers that are predictive of response to novel targeted agents on the basis of compelling biologic data.

Results

Clinicopathological characteristics

The clinical characteristics of the 67 patients with high grade OS are summarized in Table 1 while clinical, pathologic and predominant molecular characteristics of all OS cases with DNA sequencing belonging to multiple cohorts are shown in Supplemental Tables 1 and 7. The cutoff age of disease presentation for pediatric OS was defined as up to 18 years. The median age at diagnosis was 14 for the pediatric group (n=33, age range 8–18) and 32 for the adult group (n=34, age range 19–80). 38 (56.7%) of the patients were male and 29 (43.3%) were female. The primary sites included extremities (n=53, 79.1%), trunk (n=9, 13.4%) and other (n=5, 7.5%). The histological subtypes for high grade OS and all sequenced cohorts are shown in Supplemental Table 1. Thirty-five samples were collected from the primary site, 5 from local recurrences, and 32 from metastatic lesions. Upon NGS, one sample (#40) failed QC metrics for tumor content (flat copy number profile + no non-synonymous somatic variants + no silent somatic variants) and therefore the subsequent MSK-IMPACT data analyses were performed on the remaining 71 OS samples from 66 patients.

Table 1:

Clinicopathologic characteristics of 72 osteosarcoma samples (67 patients)

Features	No. of cases (%)	Total
Age (in years)		67
Range	8–80
Median	19
Gender		67
Male	38 (56.7%)
Female	29 (43.3%)
Primary Site		67
Extremity	53 (79.1%)
Trunk	9 (13.4%)
Other	5 (7.5%)
Type		72
High-grade osteosarcoma	66 (91.7%)
Post-radiation osteosarcoma	6 (8.3%)
Histological subtype		72
Osteoblastic	32 (44.5%)
High grade-NOS	13 (18.2%)
Telangiectatic	8 (11.2%)
Chondroblastic	7 (9.7%)
Fibroblastic	6 (8.3%)
Pleomorphic	2 (2.7%)
Giant cell rich	2 (2.7%)
Spindle	2 (2.7%)
Sample type		72
Primary	35 (48.7%)
Local recurrence	5 (6.9%)
Metastasis	32 (44.4%)

Somatic mutations

Somatic alterations detected by MSK-IMPACT in the 71 high grade OS samples from 66 patients are shown in Figure 1A and listed in Supplemental Tables 2 and 3. Among the common mutations, TP53 mutations were identified in 22 samples (31%) (Figure 1A and Supplemental Table 2). As MSK-IMPACT is not designed to pick up TP53 intron 1 rearrangements, recently reported in OS²⁰, the prevalence of TP53 mutations may even higher. We also identified alterations in ATRX (9 mutations in 7 samples, 10%), RB1 (7 mutations in 7 samples, 10%), and SETD2 (5 mutations in 5 samples, 7%) (Supplemental Table 2). Approximately 13% of samples (9/71) did not show alterations in any of the genes in Figure 1A but did show other somatic mutations and/or CNAs. Tumor adequacy was not deemed to be an issue in these cases since they showed similar tumor mutational burdens as the cases with the more common alterations (range 0.9–16.7 mutations/Mb). The mutations seen in these 9 cases are listed in Supplemental Table 8.

Figure 1:

(A) Oncoprint of commonly occurring and potential targetable somatic alterations and tumor mutational burden (TMB) in 71 osteosarcoma samples. As VEGFA was not present on the first version of MSK-IMPACT, some samples are missing data for VEGFA. TMB estimation was not possible in samples that showed no somatic mutations in the MSK-IMPACT panel.

(B) Copy number plot of an osteosarcoma case (Sample 4) showing 4q12 gene amplification

(C) Copy number plot of an osteosarcoma case (Sample 7) showing 6p12–21 and 12q14 gene amplification

CNAs

With respect to CNAs (Figure 1A and Supplemental Table 3), amplifications at 6p12–21 harboring VEGFA (n=17 / 64 samples [27%]), often also including CCND3, were the most frequent CNAs. Deletions at 9p21 involving CDKN2A (n=16 [22%]) and CDKN2B (n=16 [22%]) were the second most frequent CNAs (Table 2). Amplifications at 12q14 harboring MDM2 (n=11 [15%]) and CDK4 (n=9 [13%]) were frequent (Figures 1 and 2, Table 2 and Supplemental Table 4). As expected, MDM2 and CDK4 amplification were mutually exclusive with TP53 and CDKN2A alterations, respectively, (Supplemental Figure 1, Supplemental Tables 5 and 6), consistent with previous data in OS^21,22. Furthermore, CDK4 and CDKN2A alterations were mutually exclusive with RB1 alterations, such that, in aggregate, this pathway was altered in about half of OS samples. Likewise, the TP53/MDM2 pathway is altered in at least half of cases.

Table 2:

Frequent copy number alterations (CNAs) in 71 osteosarcomas

Gene	Cytoband	CNA	No. of CNAs	Fieq
JUN	1p32-p31	AMP	4	6%
MCL1	1q21	AMP	6	8%
TMEM127	2q11.2	AMP	4	6%
KDR*	4q11-q12	AMP	11	15%
PDGFRA*	4q12	AMP	13	18%
KIT*	4q12	AMP	11	15%
FAT1	4q35	DEL	6	8%
TERT	5p15.33	AMP	4	6%
VEGFA*	6p12	AMP	17	24%
CCND3*	6p21	AMP	13	18%
PIM1	6p21.2	AMP	6	8%
CARD11	7p22	AMP	4	6%
RAD21*	8q24	AMP	5	7%
MYC*	8q24.21	AMP	6	8%
CDKN2A*	9p21	DEL	16	22%
CDKN2B*	9p21	DEL	16	22%
CCND1*	11q13	AMP	4	6%
FGF3*	11q13	AMP	4	6%
FGF19*	11q13.1	AMP	4	6%
FGF4*	11q13.3	AMP	4	6%
GLI1	12q13.2-q13.3	AMP	4	6%
CDK4*	12q14	AMP	9	13%
MDM2*	12q14.3-q15	AMP	11	15%
RB1	13q14.2	DEL	7	10%
NCOR1*	17p11.2	AMP	8	11%
FLCN*	17p11.2	AMP	7	10%
MAP2K4*	17p12	AMP	4	6%
TP53	17p13.1	DEL	7	10%
ALOX12B*	17p13.1	AMP	4	6%
AURKB*	17p13.1	AMP	4	6%
CCNE1	19q12	AMP	6	8%
DNMT1*	19p13.2	AMP	4	6%
KEAP1*	19p13.2	AMP	4	6%
INSR*	19p13.3-p13.2	AMP	4	6%

^*Significant co-occurrent CNAs

Figure 2:

PDGFRA immunohistochemical staining in cases identified with 4q12 amplification

(A) H&E and PDGFRA immunohistochemistry in a case of telangiectatic osteosarcoma (Sample 57) showing strong PDGFRA expression

(B) Copy number plot of (A) showing 4q12 amplification

(C) H&E and PDGFRA immunohistochemistry in a case of osteoblastic osteosarcoma (Sample 17) showing strong PDGFRA expression

(D) H&E and PDGFRA immunohistochemistry in a case of pleomorphic osteosarcoma (Sample 55) showing strong PDGFRA expression

Notably, we also identified a subset of tumors with 4q11–12 amplification, including KIT (n=11 [15%]), KDR (n=11 [15%]) and PDGFRA (n=13 [18%]). Consistent with their chromosomal proximity, amplifications of PDGFRA and KDR frequently co-occurred with KIT amplification (p<0.001) (Figures 1A and 1B, Table 2 and Supplemental Table 4). Tumors with 4q11–12 amplification were mutually exclusive from those with 6p12–21 amplification with the exception of a single 4q12-amplified case that also showed borderline 6p12 gain (Figure 1A). In addition, cases with 4q12 gene amplification were not only mutually exclusive with 6p12–21 amplification, but also with 12q14 gene amplification involving MDM2 (Supplemental Tables 5 and 6). Perhaps not unexpectedly, given that cases with 4q12 gain were mutually exclusive with MDM2 amplification, they appeared enriched for TP53 alterations. Additionally, four cases with 11q13 gene amplification involving CCND1 and the FGF cluster were non-overlapping with CCND3 gains at 6p12 and PDGFRA/KIT/KDR gains at 4q12 (Supplemental Table 6). Other less common regions of recurrent amplification are shown in Figure 1A and Supplemental Table 3).

Potentially actionable alterations annotated by OncoKB

Among the 66 patients with MSK-IMPACT data, 14 (21%) had at least one potentially actionable alteration (Level 2 or 3) as defined by the OncoKB classification (www.OncoKB.org)¹³ (Table 3). Overall, 32 of 66 cases (48%) were annotated as Level 2–4 by OncoKB. None of the alterations were Level 1, reflecting the lack of biomarker-driven FDA approvals in this disease.

Table 3:

Potentially actionable alterations identified by OncoKB in 66 osteosarcoma patients

Gene Name	Mut/CNA	Annotated cases	OncoKB Levels	% of cases
CDK4	Amplification	9 cases	Level2B	14.0%
BRCA2	Deletion/Truncating Mutation	3 cases	Level2B	4.5%
MDM2	Amplification	9 cases	Level 3B	14.0%
PTCH1	Fusion	1 case	Level 3B	1.5%
CDKN2A	Deletion/mutation	18 cases	Level 4	27.0%
PTEN	Deletion/Truncating mutation	2 cases	Level 4	3.0%
NF1	Deletion	1 case	Level 4	1.5%

OncoKB Level 2: Nine patients (14%) with CDK4 amplification were classified as Level 2B potentially actionable somatic alterations by OncoKB. CDK4, an intracellular kinase, is altered by amplification in a diverse range of cancers, including liposarcoma, and CDK4 inhibitors, including abemaciclib () and palbociclib^23,24 are treatment options for patients with well-differentiated and dedifferentiated liposarcomas in the NCCN compendium. A somatic BRCA2 truncating mutation and 2 cases with BRCA2 deletions were annotated as a level 2B alterations. BRCA2 is a tumor suppressor gene involved in DNA damage repair by homologous recombination^25,26. PARP inhibitors olaparib²⁵ and rucaparib²⁶ are currently approved by the FDA for use in the treatment of BRCA2-mutant ovarian cancer. Interestingly, a recent analysis identified a genomic signature of homologous recombination deficiency in approximately 27% of OS samples²⁷.

OncoKB Level 3: MDM2 amplifications, detected in 9 patients (14%), are classified as a Level 3B alteration. MDM2, a ubiquitin ligase that negative regulates p53, is amplified in a diverse range of cancers, including well-differentiated and dedifferentiated liposarcomas^28,29. There are promising clinical data supporting the use of MDM2-inhibitors such as RG7112²⁸ and DS-3032b²⁹ in patients with MDM2-amplified liposarcoma. A GULP1-PTCH1 fusion, likely inactivating, was detected in one case and was classified as a Level 3B potentially actionable alteration by OncoKB. PTCH1, a tumor suppressor gene and inhibitor of the hedgehog pathway, is recurrently mutated in basal cell carcinoma^30,31. Currently, there are promising clinical data to support the use of hedgehog pathway inhibitors such as sonidegib³⁰ and vismodegib³¹ in patients with basal cell carcinoma harboring truncating PTCH1 mutations.

OncoKB Level 4: PTEN deletion and truncating mutation, were identified in 2 of 66 patients (3%). PTEN, a tumor suppressor gene and phosphatase, is one of the most frequently altered genes in cancer. Although there are no FDA-approved or NCCN-compendium listed treatments specifically for patients with PTEN-deleted bone cancer, functional studies and clinical trials using ARQ 751, AZD5363+olaparib, AZD8186, GSK2636771 and palbociclib + gedatolisib are in progress for various malignancies^32–41. CDKN2A alterations were identified in 18 cases (27%) and an NF1 deletion was identified in a single case.

4q12 amplification and overexpression of PDGFRA & KDR

A previously underappreciated prevalence of 4q12 amplification, including KIT, KDR and PDGFRA, was noted in this series, being identified in 13 of 66 patients (20%) (Figures 1A, 1B and 2, Tables 2 and 4). Of the 13 patients with 4q12 amplifications, immunohistochemistry (IHC) was performed for PDGFRA [Clone: 1C10; Novus (NBP2–46357); 1:600, (1.7ug/ml)] on 9 patients with available material: tumors from 8 out of 9 patients showed strong cytoplasmic expression (2+ to 3+ intensity) (Figure 2) while one showed weak expression (1+). IHC was also performed for KDR (VEGF Receptor 2) [Clone: 55B11; Cell Signaling Technology (2479); 1:250, (0.1 ug/mL)] on 5 patients with available material and 2 of these showed focal cytoplasmic expression (Supplementary Figure 1). IHC for KIT [Clone: YR145; Cellmarque (117R); 1:300 (0.1ug/ml)] was negative in this subset of cases.

Table 4:

Frequent genomic copy number alterations based on sample type in 71 osteosarcoma samples

Locus	Number of samples	Pre-treatment biopsy samples	Post-treatment resection samples	Post-treatment metastatic/recurrent samples
Total	72 samples	24 samples	11 samples	37 samples
6p12–21 gain	17 samples	2 samples	1 sample	14/34 samples
	23.60%	8.30%	9.10%	41.2%*
9p21 loss	16 samples	4 samples	6 samples	6 samples
	22.20%	16.70%	54.50%	16.20%
4q12 gain	13 samples	5 samples	2 samples	6 samples
	18.10%	20.90%	18.20%	16.20%
12q14 gain	14 samples	4 samples	0 samples	10 samples
	19.40%	16.70%	0%	27%
RB1 alterations	14 samples	4 samples	3 samples	7 samples
	19.40%	16.70%	27.30%	18.90%
TP53 alterations	27 samples	8 samples	5 samples	14 samples
	37.50%	33.30%	45.50%	37.90%

^*statistically significant difference between post-treatment metastatic/recurrent samples and primary samples (pretreatment biopsies and post-treatment resections), p<0.01 (Chi-square test)

These findings may provide a rationale for closer evaluation of multi-kinase inhibitors targeting these kinases. For example, pazopanib and regorafenib both target VEGFR, PDGFR, and KIT^42–44. Interestingly, both agents have been recently shown to produce objective responses in a subset of patients with OS. Furthermore, olaratumab, a monoclonal antibody to PDGFRA⁴⁵, could be evaluated in patients in this 4q12-amplified subset of OS.

6p12 amplification involving VEGFA

VEGFA at 6p12 was amplified in 14 of 59 patients (24%), pointing to angiogenesis pathways as potential targets in this subset of OS patients (Figures 1A and 1C). Several anti-angiogenic agents have shown in vitro and in vivo antitumor activity in OS in association with amplification of VEGF^46–51. Clinical studies have reported activity of anti-angiogenic therapies such as antibodies and small molecule inhibitors which target the VEGF-VEGFR axis in some OS patients^52–54, a subset that we now speculate may represent VEGFA/6p12-amplified cases. Sorafenib has also been shown to produce long-lasting partial responses in a small subset of OS⁵⁵ and, intriguingly, it has also been shown to be effective in VEGFA-amplified hepatocellular carcinoma⁵⁶.

Comparison of alterations between pediatric and adult OS

No significant differences were found between pediatric and adult OS groups in the frequency of potentially actionable alterations, commonly altered genes or distinct molecular subsets. Furthermore, we did not identify any molecular alterations that were unique to pediatric or adult OS cases. However, we did find differences in overall Tumor Mutational Burden (TMB) (see below).

Clinical outcome correlates of genomic alterations

The samples obtained from primary site included samples from pre-treatment biopsies (24 samples) as well as post-treatment resections (11 samples) (Table 4). The frequency of the most common CNAs was then calculated for each of the specimen types. Amplification of 6p12–21 including VEGFA was identified in 14/34 metastatic/recurrent samples (41.2%) as compared to 3/31 primary samples (9.7%; Figure 1A and Table 4). This difference was found to be statistically significant (p<0.01, Chi-square test). Overall, the 37 metastatic/recurrent samples in the cohort were enriched for amplification of 12q14 including MDM2 (10 samples, 27%) but the differences did not reach statistical significance (Figure 1A and Table 4). When cases were divided into two prognostic groups based on the development of recurrence and/or metastasis within 5 years of diagnosis, cases with 6p12–21 gain showed a trend towards faster disease progression (recurrence and/or metastasis within 5 years) when compared to the rest of the cohort (32.1% vs 12.8%, p=0.05, Chi-Square test). No differences were observed in OS or DFS between groups with different genomic alterations (data not shown).

Inter-metastatic heterogeneity

Four cases had two or more samples tested (highlighted samples in Supplemental Table 7). All cases with multiple samples were post-treatment metastatic specimens that lacked matched primary tumor data. In three of four cases, the alterations found were concordant across samples, with some alterations identified at sub-threshold levels that did not meet criteria for clinical reporting (Supplemental Table 7). In one patient, where both samples were post-treatment lung metastases resected 1 and 1.5 years after initial presentation, only one of the two samples showed an MDM2 amplification (samples 34 and 35, Supplemental Table 7).

Tumor Mutational Burden (TMB)

The range of TMB scores, based on the ratio of non-synonymous somatic mutations to sequencing territory (adjusted for MSK-IMPACT version), spanned 0.9 to 16.7 mutations/Mb (Figure 1A). The average TMB for patients with an age of diagnosis up to 18 years was lower (1.9 mutations/Mb) than patients aged 19 years or older at disease presentation (2.9 mutations/Mb) (t-test, p<0.05).

Discussion

Knowledge of a tumor’s genetic profile has proved to be useful in diagnosis, prognosis and targeted therapy selection for a variety of common and rare cancers including sarcomas^11,57–61. High-grade OS are genetically unstable tumors with generally complex, chaotic karyotypes⁶². Their genomic instability is highlighted by high levels of somatic structural variations and many CNAs^63–67. Whole-genome sequencing studies have shown recurrent TP53, RB1 and ATRX somatic mutations^64,68–70. TP53, RB1, CDKN2A/B, CDKN2AP14ARF and CDKN2AP16INK4A have been previously shown to be frequently affected by deletions and/or loss of heterozygosity while MDM2 and VEGFA have been the most frequent amplified genes previously reported^64,68–74.

In the present study, the finding of recurrent gene amplifications of CDK4, MDM2, KIT, PDGFRA, KDR and VEGFA raise the possibility of an umbrella protocol using targeted therapeutics in distinct subsets of OS patients (Figure 3). Approximately 20% of tumors in this study harbored a chromosome 4q12 amplification, encompassing the genes encoding the targetable receptor tyrosine kinases PDGFRA, KDR, and KIT. KIT has been previously proposed as a target in OS⁷⁵. IHC anaysis of this cohort confirmed strong expression of PDGFRA, moderate expression of KDR, and only weak expression of KIT, suggesting a rationale for combined PDGFRA/KDR inhibition. Recent reports have described OS patients with clinical responses to single agent multikinase inhibitors with activity against PDGFRA and KDR^42,76,77. Although correlative genomic data for these responders were not reported, these findings are compelling for a formal trial of combined PDGFRA/KDR inhibition in 4q12-amplified OS. If possible, it would be informative to correlate responses in trials of regorafenib^77,78 and pazopanib () for patients with recurrent OS with the genomic amplification profiles of the tumor specimens. In a recent study by Holme et al, 18 OS cell lines were tested for chemosensitivity to 79 small molecule inhibitors and MG-63, an OS cell line with PDGFRA amplification, showed sensitivity to imatinib and sunitinib⁷⁹.

Figure 3.

Recurrent gene amplifications and their potential for an umbrella protocol of targeted therapeutics in distinct subsets of osteosarcoma patients. Percentages are approximate ranges.

Approximately 24% of patients in our cohort harbored a 6p12 amplification, involving VEGFA and CCND3. Moreover, our study identified this group of tumors as almost entirely mutually exclusive from tumors harboring 4q12 gene amplifications. Similar to PDGFRA and KDR in 4q12 amplified tumors, VEGFA is a candidate driver that is potentially targetable through kinase inhibition. In IHC studies, the expression of VEGF has been detected in 63 to 74% of OS samples and has been associated with pulmonary metastasis, decreased disease-free and overall survival^46,80. Our study shows a significantly higher proportion of metastatic/recurrent samples harboring VEGFA (14/34 samples, 41.2%) as compared to samples procured from primary sites (3/31 samples, 9.7%; p<0.01). Furthermore, VEGF signaling inhibition has been reported to suppress cell growth and enhance apoptosis in OS cell lines^81,82. In another study, 32 of 50 OS showed VEGFA amplification⁴⁶ which was associated with decreased tumor-free survival and increased microvascular density^46,83. Several anti-angiogenic agents have been shown to have antitumor activity against OS in vitro and in vivo^44–47,49. In particular, pazopanib, which targets VEGF, has shown activity in preclinical mouse models with high expression of VEGF⁸⁴. As mentioned above, recent reports of clinical responses to pazopanib in small patient cohorts have been published⁴². Sorafenib, another multi-kinase inhibitor with activity against VEGF, demonstrated significant clinical activity in a very small subset of patients with recurrent OS⁵⁵. In hepatocellular carcinoma, tumors with VEGFA amplifications are distinctly sensitive to sorafenib⁵⁶. In a recent study by Sayles et al, whole genome sequencing performed on tumor specimens from 23 OS patients showed VEGFA amplification in 23%⁸⁵. In the same study, patient-derived tumor xenografts with VEGFA amplification showed significant decrease in tumor volume on treatment with sorafenib⁸⁵. Together, these findings suggest that OS with 6p12 amplifications may be good candidates for VEGF inhibition^42,76.

Among other potentially targetable alterations, we identified MDM2 amplification in 9 of 66 (14%) patients, including 6 cases (9%) with co-amplification of CDK4 and MDM2. Earlier studies using a variety of methods have reported MDM2 amplification in 6.6–14.3% of OS^21,86–87, and recently whole genome sequencing studies identified MDM2 amplification in 3.1–5.1% of OS⁷⁰. In clinical trials, MDM2 inhibitors have shown significant antitumor activity in liposarcoma patients^23,24. Some MDM2 inhibitors also display significant activity in MDM2-amplified OS cell lines (e.g. SJSA) in comparison to non-MDM2-amplified cell lines^88,89. CDK4 overexpression has been reported in about 10 % of OS^22,87,90. However, to the best of our knowledge, there have been no studies examining the association between CDK4 amplification and the activity of CDK4 inhibitors in OS. In well differentiated and dedifferentiated liposarcomas, several clinical trials have shown that treatment with a CDK4 inhibitor was associated with favorable progression-free survival in patients with CDK4 amplification^23,24. Based on these findings, targeting of MDM2 and CDK4 appears to be a potential therapeutic option for the 12q13 amplified subset of OS patients.

Mutually exclusive genetic alterations often point to important alternative oncogenic pathways. There were several notable relationships of this type in our dataset. The 17 samples with VEFGA/CCND3 amplification at 6p12–21 were mutually exclusive with the 13 samples with amplification of PDGFRA, KIT and KDR,) at 4q12, with one exception (Log odds ratio = −1.87) (Supplemental Table 5). In the single case with gains at both loci, the 4q12 amplification was higher while the 6p12 gain was borderline (results not shown). Amplification of 12q14 (MDM2 and CDK4) was found in 20% (14/71) of the samples and was mutually exclusive with 4q12 amplification (Log odds ratio<−10) (Supplemental Table 5). These mutually exclusive and targetable oncogenic pathways may represent distinct biological subsets of OS with important therapeutic implications. It should be noted that the major copy number gains highlighted in Figure 3 could also be detected by methods other than the one used in the present study, such as FISH or array-based copy number profiling, which might be more widely available. In summary, we were able to identify potentially actionable (OncoKb levels 1–3) somatic alterations in approximately 21% of patients with OS (14/66). Additionally, distinct OS subsets defined by amplification of PDGFRA and KDR at 4q12 or VEGFA at 6p12–21 may offer new therapeutic opportunities.

Statement of Translational Relevance.

The prognosis for patients who present with metastatic and/or recurrent OS remains poor, but the potential of routine comprehensive genomic profiling to define additional therapeutic options in this subset of patients remains unclear. Here, we sought to define how often clinical genomic sequencing of OS samples could identify potentially actionable alterations, based on large panel NGS data obtained from 67 OS patients. This identified currently clinically actionable alterations in approximately 21% of patients. In another 40% of patients, we found a mutually exclusive pattern of PDGFRA or VEGFA amplification, representing candidate subsets for future clinical evaluation of additional therapeutic options. These data inform a proposal for genomically-based algorithm that could be used to direct up to 50% of OS patients to targeted therapy options.