IDH mutations in G2-3 conventional central bone chondrosarcoma: a mono institutional experience

Background Heterozygous isocitrate dehydrogenase (IDH) mutations occur in about half of conventional central bone chondrosarcomas (CCBC). Aim of this study was to assess the frequency and prognostic impact of IDH mutations in high grade CCBC patients. Methods 64 patients with G2 and G3 CCBC were included. DNA extraction, PCR amplification of IDH1/2 exon 4s, and sequencing analysis with Sanger were performed. Results IDH mutations were detected in 24/54 patients (44%): IDH1 in 18, IDH2 in 4, and both IDH1/2 in 2 patients. The frequency of mutations was 37% in G2 vs. 69% in G3 (p = 0.039), and 100% in three Ollier disease associated chondrosarcoma. 5-year overall survival (OS) at 124 months (range 1-166) was 51%, with no significant difference based on the IDH mutational status: 61% in IDHmut vs. 44% in IDH wild type (IDHwt). The 5-year relapse free survival (RFS) was 33% (95% CI:10–57) for IDHmut vs. 57% (95%CI: 30–77) for IDHwt. Progression free survival (PFS) was 25% (95%CI:1–65) IDHmut vs. 16% (95%CI: 0.7–52) IDHwt. 55% (5/9) of IDHmut G2 became higher grade at the recurrence, as compared with 25% (3/12) of G2 IDHwt. Conclusions This study shows a higher frequency of IDH mutations in G3 CCBC as compared with G2. No significant differences in OS, RFS, and PFS by mutational status were detected. After relapse, a higher rate of G3 for IDH mutated CCBC was observed.


Background
Cartilaginous bone sarcomas are the second most frequent bone tumors in adults with roughly 3 new cases per 10 6 population per year, while in children and young adults they represent only 3% of bone malignancies [1].They include different histologic subtypes, all characterized by the production of a hyaline cartilaginous matrix, and conventional chondrosarcoma is the most common (85-90%) [2].Conventional central bone chondrosarcoma (CBCC) arises from the inner bone, while peripheral chondrosarcoma from the surface.Both can rise as primary, de novo, lesions or from the transformation of pre-existing benign lesions, through mutations in different genes: central chondrosarcoma from enchondroma (IDH mutations), peripheral from osteochondroma (EXT1, EXT2 mutations) [3].Dedifferentiated chondrosarcoma arises from conventional chondrosarcoma and behaves more aggressively; in the last WHO classification [2] it is considered a separate high-grade chondrosarcoma subtype, defined by a bimorphic histological appearance of a conventional chondrosarcoma component and a high-grade non-cartilaginous sarcoma (more often undifferentiated pleomorphic sarcoma or osteosarcoma, rarely it demonstrates features of angiosarcoma, leiomyosarcoma, rhabdomyosarcoma, or shows epithelial differentiation) [2].The cartilaginous and sarcomatous components are juxtaposed, generally with an abrupt transition between them, and IDH mutations have been described in both components.In the largest series reported in literature, the percentage of dedifferentiation occurring in conventional chondrosarcomas is around 15% [2,[4][5][6].
Biological mechanisms by which cancerogenesis is induced by mutant IDH are to be fully elucidated.Mutant isoforms 1 and 2 of IDH form with the wild-type protein a heterodimeric enzyme that gains a neomorphic activity, which consists in the NADPH dependent reduction of α-ketoglutarate to D-2-hydroxyglutarate (D-2-HG).D-2-HG behaves as an oncometabolite, competitively inhibiting α-Kg-dependent dioxygenases, inducing DNA methylation rewiring, HIF1α a stabilization independently of oxygen levels, and alteration of the cellular redox balance [23][24][25][26].
IDH prognostic role is controversial in different reported series of chondrosarcoma patients [27][28][29][30][31][32][33] However, a limitation of most of these studies is the inclusion of patients with different histological types and different histological grade [34].In addition, some of these studies are multicentric [34].The aim of our study is to assess the frequency of IDH mutations, their relationship with clinical characteristics, and their prognostic role in patients with high grade CBCC treated in the same institution.

Study design
The aim of the study was to describe the frequency and type of IDH mutations in G2 and G3 CBCC and identify correlations with clinical characteristics and outcome.Inclusion criteria: surgery of primary tumor at our institution from 2002 to 2012, confirmed diagnosis of G2 or G3 conventional chondrosarcoma, availability of both paraffin-embedded and fresh frozen tissue samples.A comprehensive written informed consent was signed for the surgical procedure and related diagnostic procedures in accordance with the standard institutional procedure.After Ethical Institutional Committee approval, clinical data on the diagnosis, treatments, and follow-up (according to institutional guidelines) were collected retrospectively from the patient charts.Surgical margins were defined according to the Enneking score system [33].
Molecular analyses were performed on formalin-fixed and paraffin-embedded tissues (FFPET) and/or frozen tumor samples of patients included in the study.All information regarding the human material was managed using anonymous numerical codes, clinical data were not used, and samples were handled in compliance with the Helsinki declaration.

DNA extraction
Total DNA extraction from FFPET samples was performed as previously described [33].A representative tumor area selected on hematoxylin-eosin by the pathologist, which has more than 80% tumor cells, was selected on paraffin block using Multi-Purpose Sampling Tool (Harris UNI-CORE, TedPella Inc, USA) which allows to take tissue cores from the sample, purifying DNA from two cores for each sample.With some modifications, the same QIAamp FFPE Tissue kit (Qiagen) was used for DNA purification of frozen samples.In brief, a fragment of less than 10 mg of tumor tissue was selected using scalpel blades.For each sample 180 µl of lysis buffer and 20 µl of Proteinase K Solution were added.After mechanical stirring, samples were incubated in a water bath overnight at 56 °C.Hematoxylin-eosin cryostat sections were performed to evaluate if the area of tissue selected was representative of the tumor.Quantitative and qualitative analysis was carried out on DNA obtained from both FFPET and frozen tissue by measuring the absorbance (A) in a spectrophotometer to determine the concentration and purity.DNA was considered suitable for molecular analysis only if the A260/A280 ratio was greater than 1.8.

Sequencing of polymerase chain reaction products
The PCR conditions have been previously described [33].The Sanger sequencing was performed by Bio-Research Fab (www.biofabresearch.it,Rome, Italy).Mutation analysis was conducted with Basic Local Alignment Search Tool (BLAST), in the NCBI database "National Center of Biotechnology Information Database" (http://www.ncbi.nlm.nih.gov/BLAST).Electropherograms were exported to fasta format and were aligned to the NCBI BLAST sequences.

Statistical analysis
Continuous variables were summarized as mean and range; discrete and categorical variables were summarized using frequencies and percentages.Patient characteristics at diagnosis (age, site, grade, margins according to Enneking [35], and stage), were compared between IDH mutation using Chi-Square test.OS was defined as the time from the date of surgery to the date of death.Patients who did not experience the outcome of interest were censored at the time of last follow-up.RFS was calculated in localized patients and was defined as the time from date of surgery to the date of first relapse.PFS was calculated in metastatic patients and was defined as the time from date of surgery to the date of progression.Patients who died for surgery complications were excluded from survival analysis.Kaplan-Meier methods were used to estimate OS, RFS and PFS and the curves were compared using log-rank test.A value of p < 0.05 was considered statistically significant.All p values were 2-sided.Data were analyzed using the SAS 9.4 software (SAS Institute).

Frequency and type of mutations
DNA extraction was performed on tumor samples derived from the first surgical procedure in 64 patients included in the protocol.
Molecular analysis was not evaluable in 10 patients, for scarcity of extracted DNA, due to aged slides and decalcification procedures.In 54/64 (84%) cases the quality of tissue was adequate: IDH mutations were detected in 24 patients (44%): in 18 IDH1mut only (34%), in 4 IDH2mut only (7%), and in 2 patients both IDH1 and 2mut (3%).IDH1 was mutated on residue 132 of arginine while IDH2 on residue 172, in the substrate-binding site.Details on mutation types are described in Table 1 and Fig. 1.
A comparison of clinical characteristics between IDHmut and IDHwt cohorts is illustrated in Table 2. On histological examination, no different morphological features have been reported for mutant versus wild-type tumors.
As expected, syndromic conditions were confirmed to be a substrate for IDHmut conventional chondrosarcoma.The three chondrosarcomas of patients with Ollier syndrome (100%) harbored a mutation of IDH1, and all 3 cases shared the same mutation: R132C(TGT); none of the patients presented IDH2 mutation.One patient with Maffucci's syndrome had a metacarpal chondrosarcoma IDH1wt and IDH2 not evaluable.
In Table 3 the comparison between G2 and G3 groups is reported.

Evolution/dedifferentiation
We observed a different grade progression over time (at relapse) in the 2 groups (IDH1/2mut or wt): IDHmut chondrosarcoma had a higher rate of grade progression at relapse, as compared to IDHwt.In particular 25 patients (21 cases presenting with G2 at diagnosis and 4 presenting with G3) had a new histologic evaluation at recurrence/progression: 5/9 (55%) of G2 IDH1/2mut tumors became higher grade at recurrence (1 became G3 and 4 dedifferentiated); while only 3/12 (25%) of G2 IDH1/2wt acquired a higher grade in the recurrence (3 became dedifferentiated).Two IDH1wt patients developed second malignancies: 1 desmoid fibromatosis (and had had a previous prostatic adenocarcinoma) and 1 non-Hodgkin lymphoma.
Two patients with IDHmut developed a second malignancy: 1 prostatic adenocarcinoma and 1 patient with Maffucci Syndrome developed second chondrosarcoma at a different site.Three IDHmut patients with Ollier disease, had evidence of pre-existing enchondroma.Moreover, one IDH1/2 patient had evidence of enchondroma, G2 chondrosarcoma, and dedifferentiated chondrosarcoma recapitulating in his history all the phases of cancerogenesis.Treatment at recurrence/progression consisted of surgery (19 patients) chemotherapy (9 patients), and radiotherapy (8 patients).

Treatment
Number and duration of chemotherapy lines are summarized in Fig. 3.

Outcome
Survival analysis was performed on 50 patients: 4 patients died of surgical complications and were excluded from the outcome analysis.After a median follow-up of 124 months (range 1-166) relapse occurred in 21/40 localized patients (10 IDHmut): 11 local recurrences, 5 lung metastases, 1 lymph node metastases, and 4 both local relapse and metastasis.Disease progression was reported for all metastatic patients.
The 5-years OS in IDHmut vs. IDHwt was not significantly different in the general population, nor in the split group of G2 and G3, as reported in Fig. 4a, 4b, 4c.No differences between IDHmut and IDHwt in the 5-years RFS and PFS for localized and metastatic patients respectively has been detected, as reported in Fig. 5a and 5b).

Discussion
IDH mutations were first identified in 2006 in colon cancer [36], and next in many other tumor types, but only in glioma a clear correlation between genetic alteration and disease phenotype and prognosis has been established [37].
Amary and Pansuriya pioneering works show that IDH mutations in mesenchymal tumors: 1-are prevalent in cartilaginous tumors rather than in other connective tissue sarcomas; 2-are present in 80% of benign enchondromas and in almost half of conventional chondrosarcomas; 3-occur early in the tumorigenesis; 4-both local and distant recurrences of central conventional chondrosarcoma maintain the same IDH mutational status of the primary lesion; 5-are almost always present in enchondromas of Ollier and Maffuci syndromes [21,22,38].
Other authors investigated on the presence and role of IDH mutations in chondrosarcomas, but most of the reported series include benign with high grade, tumors from different sites, and also different chondrosarcoma histotypes.In these series the number of G2 and G3 chondrosarcoma account from 24 to 51. [27][28][29][30][31][32][33][34].In the current study, we decided to focus on G2-3 CCBC, in order to have a homogeneous population, reporting data on 54 patients all treated in one institution.
Table 2 Patient characteristics according to IDH mutation.Median age was comparable in mutated and wild-type groups.The IDH mutation rate was not different in localized (43%) vs. metastatic (50%) patients; while it was significantly higher in G3 (69%) than in G2 (37%) tumors (p=0.0390)Fig. 2 Frequency of mutations by site.Humerus and tibia were the sites with higher mutation rate, followed by femur and pelvi.One IDHmut vertebral CCBC has been found enchondroma, and developed a dedifferentiated chondrosarcoma.Taken together, our data suggest that IDH mutation is one of the events responsible for low to high grade progression, and that IDH mutant cells are more susceptible to changes toward dedifferentiation.It's reported that p16/CDKN2A copy number variation, in IDHmut CBCC, occurs only after the onset of IDH mutation [38], and that the methylation status changes from low grade and high grade IDHmut chondrosarcoma.[40,41].
The impact of IDH mutation on chondrosarcoma prognosis is still unclear.Stage, grade, size, and surgical margins are the main factors to consider for survival prediction in CCBC.In our series grade and stage were confirmed to be prognostic, but not the mutational status.The 5-year OS was 51%, but dropped to 7% in metastatic patients, and to 29% in G3 patients, but did not differ significantly by mutational status.The 5-year RFS was worse in IDHmut localized patients, but not statistically significant.Similarly, Lugowska et al. [27] found a shorter survival in IDH1/2mut patients, but in that series different histotypes other than CBCC have been included, while Zhou et al. [28] reported longer RFS and metastasis-free survival (MFS) in IDHmut CCBC (51 G2-3), but did not detect a significant impact of mutational status on OS.Cleven et al. [29], and Amary et al. [5] could not find differences in DFS and MFS, and survival period respectively.A meta-analysis using individual patient data from 14 studies found a significant negative impact of IDH1/2 mutations on patient survival, but not on RFS and MFS [34].Possible explanations for the discrepancies in defining IDH mutations prognostic role between available studies could in part rely on the rarity of the disease (small sample size), heterogeneity of included population (patients with different chondrosarcoma subtypes and different grade), the employment of different sequencing tools.Moreover, even if IDH mutation is considered one of the most important drivers of chondrosarcoma, other genes seem to be involved in tumor progression (p53, Rb, histone deacetylase, and others) and may impact on patient prognosis.
Target therapies with potential activity in chondrosarcoma include antiangiogenic agents, mTOR inhibitors, hedgehog inhibitors, histone deacetylase inhibitors, immunotherapy, and, more recently, IDH inhibitors.[18,19].IDH inhibitors are approved for the treatment of IDHmut acute myeloid leukemia.Ivosidenib, a firstin-class selective oral IDH1 inhibitor, has shown clinical activity in preclinical studies [42,43] and in a phase 1, open-label, multicenter study with 168 patients with advanced solid tumors.Chondrosarcoma dose-escalation and expansion cohorts included 21 patients (14 with G2-G3 CCBC) with recurrent/progressing disease: 11 (52%) achieved stable disease, and 4 of them for more than 2.5 years [43].Ongoing trials are still recruiting chondrosarcoma patients [19] to understand the more appropriate setting for IDH1/2 inhibitors.

Fig. 4a
Fig. 4aOverall survival according to mutational status in the general population.No significant difference in survival by mutational status (5-years OS IDHmut 61% vs IDHwt 44%, p=0.6854) was detected

Table 1
Distribution of gene mutation types in positive cases (the sum is > 100% since 2 patients had both IDH1 and IDH2 mut)