Driver mutations in central conventional and dedifferentiated chondrosarcomas
Profiling a total of 350 CS cases for IDH1 and IDH2, using ddPCR (n = 282) and WGS (n = 68), we verified previous findings that IDH2 are less frequent than IDH1 mutations (IDH1: 51%, IDH2: 14%, IDHwt: 35%) [16, 26, 41]. In cases with grading information (n = 343), we found that IDH1 mutations were equally frequent across grades (ACT/G1: 51%, G2/3: 53%, DD CS: 55%; p = 0.9) and that IDH2 mutations were more frequent in higher grade disease (ACT/G1: 7%, G2/3: 14%, DD: 25%; p = 0.005). IDHwt was negatively associated with increasing grade (%IDHwt; ACT/G1: 41%, G2/3: 32%, DD CS: 19%; p = 0.01). These data imply that the progression to DD CS is more common in tumours with IDH2 mutations and least common in IDHwt tumours.
Canonical mutations and structural changes near the TERT promoter have been reported in approximately 20% of CS and found to correlate with high grade disease [24, 26, 42]. We found a similar frequency in our cohort (23%, 74 with C228T, one with C250T, four with structural changes near the TERT promoter (Additional file 3: Supplementary Fig. 1). TERT mutations were rare in well differentiated tumours and increased in frequency across grades (ACT/G1: 3%, G2/3: 22%, DD: 56%; p = 2e-14, Fig. 1A). We found that the TERT promoter was hypermethylated in 19/57 (33%) of cases analysed on methylation arrays, excluding DD CS cases (Fig. 1B). Seven of these cases, all high grade, also harboured TERT C228T promoter mutations. There was no significant difference between the number of cases with both TERT promoter mutations and hypermethylation across IDH1 and IDH2 tumours (Fig. 1B). Rare alterations involving ATRX have been reported previously [26], but in the 100KGP cohort (n = 68), we found no such alterations in this cohort. These data confirm that TERT mutations, and possibly methylation, have distinct roles in CS progression.
We next examined mutations in other key driver genes reported in CS, utilising the 100KGP cohort (Fig. 1A). Our findings were largely similar to those previously reported [13, 43]. Monosomies of 17p and pathogenic SNVs/indels in TP53 were found in 22% of cases in line with previous reports [12]. TP53 mutations were absent from all but one well-differentiated tumour. COL2A1 mutations were common and marginally anti-correlated with increasing grade (ACT/G1: 100%, G2/3: 54%, DD CS: 44%; p = 0.03). As previously reported [17, 19, 21], pathogenic SNVs/indels and/or bi-allelic deletions of CDKN2A and CDKN2B were common in CS and enriched in G2/3 and DD CS, though not significantly in this dataset. Hypermethylation of these genes was not detected. CDK4 and CDK6 gains were found in 12 cases, and a single case had a pathogenic SNV in CDK6. These frequencies are similar to previous reports [12]. MYC amplifications were found in five high-grade tumours establishing its status as a driver of CS [44, 45]. MDM2 alterations were identified in three high grade IDHwt cases, two of which were amplifications (one 8 copies, one 31 copies, confirmed using fluorescence in situ hybridisation), and one was a structural alteration involving intron 7 of MDM2 and an intragenic region on chr4q28.3. The latter did not result in amplification of MDM2 but removal of the zinc finger binding domains, which has been suggested to have an oncogenic effect [46]. All three mutations were mutually exclusive of TP53 mutations. These data support the premise that MDM2 is a potential driver gene in CS [26], though any biological effects require further exploration. Homozygous deletions of PTEN were present in three high grade cases. PTEN promoter hypermethylation was found in 13/57 cases, all high grade. Analysis utilising dNdS [47] returned no previously unknown drivers, implying that all prominent somatically mutated genes driving CS have likely been identified (Additional file 2: Supplementary Methods, Additional file 3: Supplementary Fig. 2).
IDH1, IDH2, and TERT define key genetic subgroups
Analysis of all mutation calls (n = 350) revealed that the frequency of TERT mutations was different across IDH1, IDH2, and IDHwt cases (Fig. 1C). IDH2 mutations were strongly associated with TERT mutations (IDHwt: 5%, IDH1: 24%, IDH2: 58%, p = 6e−13; IDH1 vs IDH2: p = 1e−5). This association was observed in G2/3 (IDH1 vs IDH2: p = 7e−6) but not in DD CS (IDH1 vs IDH2: p > 0.99), implying that although TERT is associated with high-grade tumours, this is not equal in the context of IDH mutation status.
Hypermethylation across IDH1- and IDH2-mutated tumours
CpG island DNA hypermethylation has been reported to distinguish between cartilaginous IDH and IDHwt tumours [14, 15]. However, utilising the larger numbers available in this study, we found 3468 differentially methylated probes (DMPs) across IDH1 and IDH2 tumours, excluding DD CS (n = 31, p = 0.002, Additional file 3: Supplementary Fig. 2, Supplementary Tables 4-6). The overall methylation level across all probes also revealed significant differences between IDH1 and IDH2 tumours (p = 0.002) indicating that the former are globally hypermethylated compared to IDH2 and IDHwt tumours.
Partial haploidisation followed by genome doubling is common in IDHwt tumours
We compared the mutational profiles across each IDH group (complete summary of 100KGP data shown in Fig. 1D) and found that the frequency of common drivers, excluding TERT, was similar across IDH1 and IDH2 and IDHwt tumours. We did not find that mutations in CDKN2A/B and TP53 were enriched in IDH1/2 cases, as previously reported [48] but contrasting another study [26]. The total number of SVs was not statistically different across the IDH groups nor was the number of SVs that fell into gene regions. We did not find any common structural variants affecting the same gene more than 25% of cases, although none of these were cancer-related genes (Additional file 2: Supplementary Methods).
The genetic alterations initiating development of IDHwt CS remains unknown, but previous reports of near-haploid (HP) and hyperhaploidy in CS and in other sarcoma subtypes including undifferentiated sarcomas and malignant peripheral nerve sheath tumours prompted us to investigate this [21, 22, 49, 50]. We found 23 tumours with GD and seven with HP in the 100KGP cohort (n=68, Additional file 3: Supplementary Fig. 4, Additional file 2: Supplementary Methods). Most GD events (16/23, 69%) occurred in the absence of HP, whereas HP always occurred with GD (Fig. 1D). GD was highly enriched in IDHwt tumours (GD%, IDH1: 24%, IDH2: 9%, IDHwt: 63%, IDHwt vs IDH1 p = 0.0005, Fig. 1E), and HP was exclusive to this group (HP%, IDH1: 0%, IDH2: 0%, IDHwt: 37%, IDHwt vs IDH1 p = 8e-5, Fig. 1E). Timing analysis demonstrated that GD events tended to occur at a similar relative time in IDHwt and IDH1 cases implying that it could be an intermediate or late event in evolutionary timelines of both tumour groups (Fig. 1E, Additional file 2: Supplementary Methods). The six cases of IDHwt tumours without HP/GD events, harboured mutations in TP53 and CDKN2A, although alterations in these genes were not mutually exclusive with the absence of GD and HP (TP53: 3/6, 50%, CDKN2A/B: 5/6, 83%, Fig. 1D). One of these cases was ACT/G1, pointing to a possible initiating role of TP53 and CDKN2A in some IDHwt tumours.
Mutational signatures across IDH1, IDH2, and IDHwt groups
Analysis of mutational signatures in the 100KGP cohort (n = 52, Fig. 1F, Additional file 3: Supplementary Fig. 5) revealed nine active signals, with SBS1, SBS5, and SBS8 being ubiquitous and most prominent across IDH1, IDH2, and IDHwt tumours. Five signatures (SBS2, SBS12, SBS13, and SBS17a/b) were principally exclusive to IDHwt tumours. SBS2 and SBS13 have been associated with APOBEC and were simultaneously active in five IDHwt cases (18%). We did not observe any difference in SNV burden in tumours with active SBS2 and SBS13. SBS12 was found in one IDH1 case and three IDHwt cases. SBS17a/b, signatures with unknown aetiology, were found only in IDHwt cases. SBS40, also of unknown aetiology, was found in 28% of IDH1 cases, 25% of IDH2 cases, and 81% of IDHwt. These data demonstrate that IDH1 and IDH2 tumours are comparable in terms of mutational signatures, whereas IDHwt tumours exhibit more heterogeneous mutational processes.
The genetic distinction between central conventional and dedifferentiated chondrosarcoma
We next analysed the DD CS for specific alterations that may explain their histological phenotype and their poor clinical outcomes. We confirmed that metastatic disease was most common in DD CS (60%, compared to 27% in G2/3 and <1% in ACT/G1 (G2/3 vs DD: p = 1e−5). Analysing the 100KGP data (DD: n = 16, G2/3: n = 41), the frequency of identified known drivers in DD CS and G2/3 revealed no difference except for IDH2 and TERT, which were enriched in DD (IDH2: p = 0.05, TERT: p = 3e−6). However, we found differences in total driver burden (p = 2e−8), SNV burden (p = 0.009), number of chromosome segments (p = 0.01), and SV burden (p = 0.01) (Additional file 3: Supplementary Fig. 6). We next explored whether the increased segment counts were attributable to chromothripsis. Using a previously published method [51], we found only one instance of chromothripsis (WGS_21) which overlapped with the SV identified at the TERT loci (Fig. 1D, Additional file 3: Supplementary Fig. 1). Examining the number more broadly, the average number of chromosomes with high breakage was higher in DD CS compared to G2/3 (median, G2/3: 0, DD CS: 2.5, p = 0.03, see Additional file 2: Supplementary Methods). There were no specific chromosome arms enriched amongst those with high fragmentation, although three cases (19%) had fragmentation across chromosome 12q, which has also been reported in dedifferentiated liposarcoma [52]. Previous studies have reported that aberrations of chromosome 5q and trisomy of chromosome 19 distinguish G2/3 from DD CS [53]. Twenty-five percent DD CS harboured 19p/q gains which is less than the 50% previously reported [53]. Examining losses and gains across all chromosome arms revealed no events unique to DD CS although losses at 15q were more common in this subtype (15q loss, G2/3, 10%, DD CS: 38%, p = 0.05). Together, these analyses suggest that the primary genetic difference between G2/3 and DD CS is the number of accrued SNVs and the degree of chromosomal instability.
Age at diagnosis as a clinical factor in chondrosarcoma
Previous studies of CS have treated IDH1 and IDH2 tumours as one group [14, 54]. Our results, leveraging hundreds of cases, provide evidence that IDH1 and IDH2 mutations lead to distinct downstream genetic events, with differences in the frequency of TERT mutations, GD/HP, methylation profiles, and the number and types of mutational signatures.
We examined the effect of the presence or absence of IDH1 and IDH2 mutations on the clinical behaviour of CS (n = 339, Fig. 2). We showed that patient age at diagnosis increased across grades in these groups and that the median age was highest in those with IDH2 tumours (IDH1: 55 year, IDH2: 67 year, IDHwt: 47 year, IDH1 vs IDH2: p = 0.003, IDH1 vs IDHwt: p = 0.0006, Fig. 2A). The age at diagnosis for each IDH group was similar for ACT/G1 and DD CS, and the difference in age of the G2/3 tumours explained the overall difference in ages (median age G2/3, IDH1: 60 year, IDH2: 71 year, IDHwt: 44 year, IDH1vs IDH2: p = 0.04, IDH1 vs IDHwt: p = 2e−6, Fig. 2B). We considered whether these differences in chronological age at diagnosis were reflected in the mutational signatures active in each group. The total SNV burden correlated with age at diagnosis, as did SBS5, previously been reported as clock-like [55], SBS8, but not SBS1. In G2/3 tumours, the activity of SBS5 was similar in IDH1 and IDH2, but lower in IDHwt (IDH1 vs IDH2 p = 0.4, IDH1 vs IDHwt: p = 0.03, Fig. 2C). By contrast, SBS5 activity was similar in all DD cases (IDH1 vs IDH2 p = 0.7, IDH1 vs IDHwt: p = 0.8, Fig. 2C). These same results were recapitulated when using SBS8 and total SNV burden (Additional file 3: Supplementary Fig. 7). Together, these data imply further differences in the rate of evolution from G2/3 to DD CS across IDH1, IDH2, and IDHwt tumours.
Divergent outcomes in IDH1, IDH2 and IDHwt tumours
Using all available clinical information (n = 342), we found that IDH2 tumours tended to be larger at time of presentation (IDH1 vs IDH2: p = 0.001, IDH1 vs IDHwt: p = 0.4, Fig. 3A), supporting the premise that these tumours evolve over longer time periods, and present in older people. Development in specific anatomical locations was not significantly different (Fig. 3A).
Using all cases with available follow-up data (n = 328), a Cox proportional hazard model demonstrated that ACT/G1 tumours nearly always had a good outcome with no metastatic events being recorded and only one of 98 patients, with a pelvic tumour, succumbing to disease. No patients with tumours in the small bones of the hands and feet died of disease (Fig. 3B). We found that DD CS had a higher frequency of metastatic disease compared with G2/3 disease (G2/3 vs DD CS, p = 9e−7). There were no significant differences in the frequency of metastatic or recurrent disease across IDH1, IDH2, and IDHwt DD CS tumours. However, metastases or recurrent disease appeared to occur less frequently in patients with IDH2 G2/3 tumours compared to IDH1 and IDHwt tumours (% metastases/recurrence, IDH1: 37%, IDH2: 13%, IDHwt: 23%, IDH1 vs IDH2: p = 0.04, Fig. 3B). We also found that the time interval between diagnosis and detection of metastatic disease six months following presentation of the primary tumour was shorter in IDH2 tumours compared to IDH1 (p = 0.04, Fig. 3B). Finally, we found no differences in outcome related to the different IDH1 mutation contexts (R132C/G/H/L/S) but noted that R132S/L variants found in only a minority number of cases, making statistical analysis difficult (Additional file 3: Supplementary Table 3, Supplementary Fig. 8).
Canonical TERT promoter mutations (g.1295113) had an independent hazard ratio (HR) that was equal to that of grade (TERT: HR = 2.2, p = 0.003, tumour grade: HR = 2.2, p = 2e−13, Additional file 3: Supplementary Fig. 8), pointing to the benefit of TERT as a biomarker. We also found that overall outcomes were worse in patients whose tumours had TERT hypermethylation (n = 68, HR = 3.4, p = 0.01). Restricting our analyses to high-grade tumours and excluding tumours in the hands and feet, we found that patients whose tumour harboured both IDH1 and TERT mutations had significantly worse outcomes than those with an IDH1 mutation alone. TERT mutations had no effect on outcome in patients with IDH2 tumours, even though these mutations are found more frequently in combination with IDH2 mutations (Fig. 3C, Additional file 3: Supplementary Methods). This suggests that TERT mutations are context specific and only relevant to outcome predictions in IDH1 tumours.
Given these findings, identification of tumours with IDH1 and TERT mutations has clinical value.