Identification of an Actionable Mutation of KIT in a Case of Extraskeletal Myxoid Chondrosarcoma

Extraskeletal myxoid chondrosarcoma (EMC) is an extremely rare soft tissue sarcoma, marked by a translocation involving the NR4A3 gene. EMC is usually indolent and moderately sensitive to anthracycline-based chemotherapy. Recently, we reported on the therapeutic activity of sunitinib in a series of EMC cases, however the molecular target of sunitinib in EMC is unknown. Moreover, there is still the need to identify alternative therapeutic strategies. To better characterize this disease, we performed whole transcriptome sequencing in five EMC cases. Peculiarly, in one sample, an in-frame deletion (c.1735_1737delGAT p.D579del) was identified in exon 11 of KIT. The deletion was somatic and heterozygous and was validated both at DNA and mRNA level. This sample showed a marked high expression of KIT at the mRNA level and a mild phosphorylation of the receptor. Sanger sequencing of KIT in additional 15 Formalin Fixed Paraffin Embedded (FFPE) EMC did not show any other mutated cases. In conclusion, exon 11 KIT mutation was detected only in one out of 20 EMC cases analyzed, indicating that KIT alteration is not a recurrent event in these tumors and cannot explain the EMC sensitivity to sunitinib, although it is an actionable mutation in the individual case in which it has been identified.

Despite the high risk of metastases (about 40%), EMC are usually indolent with 10-year survival ranging between 65% and 85% [8,9].Generally, these sarcomas are moderately sensitive to anthracycline-based chemotherapy [10], and therefore there is an urgent need to identify alternative therapeutic strategies [8][9][10].Recently, we reported the therapeutic activity of sunitinib in a cohort of 10 EMC patients with 6 partial responses, 2 stable disease, and 2 showed progression.In that series, the two progressive patients upon treatment were those carrying a TAF15-NR4A3 fusion, while all the others were EWSR1-positive [11,12].EMC tumor specimens showed RET proto-oncogene expression and activation, while no other predictive biological markers of response were identified [12,13].To further investigate the molecular alterations present in EWSR1-positive EMC cases, we performed whole transcriptome sequencing (WTS) in a small series of EMC, identifying a case carrying an activating KIT mutation and the recurrence of this event was evaluated in a larger series.

Results and Discussion
WTS was performed on five cases of EMC positive for EWSR1-NR4A3 fusion.Presence of the chimeric mRNA was confirmed and no additional fusion event was identified.In concordance with previous findings, as reported in the COSMIC database (available online: cancer.sanger.ac.uk/cosmic), exon12/exon3 EWSR1-NR4A3 fusion was the most frequent breakpoint in our cohort.In particular, the exon12/exon3 junction was detected in three out of five cases (#2, #3, #5) while exon13/exon3 and exon7/exon2 were detected respectively in samples #4 and #1 (Figure 1).All fusions retained the coding frame for creating a chimeric protein, with the exception of sample #1, in which EWSR1 fuses with exon2 of NR4A3, an exon upstream of the start codon.
Despite the high risk of metastases (about 40%), EMC are usually indolent with 10-year survival ranging between 65% and 85% [8,9].Generally, these sarcomas are moderately sensitive to anthracycline-based chemotherapy [10], and therefore there is an urgent need to identify alternative therapeutic strategies [8][9][10].Recently, we reported the therapeutic activity of sunitinib in a cohort of 10 EMC patients with 6 partial responses, 2 stable disease, and 2 showed progression.In that series, the two progressive patients upon treatment were those carrying a TAF15-NR4A3 fusion, while all the others were EWSR1-positive [11,12].EMC tumor specimens showed RET proto-oncogene expression and activation, while no other predictive biological markers of response were identified [12,13].To further investigate the molecular alterations present in EWSR1-positive EMC cases, we performed whole transcriptome sequencing (WTS) in a small series of EMC, identifying a case carrying an activating KIT mutation and the recurrence of this event was evaluated in a larger series.

Results and Discussion
WTS was performed on five cases of EMC positive for EWSR1-NR4A3 fusion.Presence of the chimeric mRNA was confirmed and no additional fusion event was identified.In concordance with previous findings, as reported in the COSMIC database (available online: cancer.sanger.ac.uk/cosmic), exon12/exon3 EWSR1-NR4A3 fusion was the most frequent breakpoint in our cohort.In particular, the exon12/exon3 junction was detected in three out of five cases (#2, #3, #5) while exon13/exon3 and exon7/exon2 were detected respectively in samples #4 and #1 (Figure 1).All fusions retained the coding frame for creating a chimeric protein, with the exception of sample #1, in which EWSR1 fuses with exon2 of NR4A3, an exon upstream of the start codon.Pathogenic single nucleotide variants (SNV) and insertions and deletions (INDEL) were searched starting from WTS data.Interestingly, in one case an in-frame deletion (c.1735_1737delGAT p.D579del) was detected in the juxtamembrane domain (exon 11) of the KIT receptor (Figure 2A).The mutation was somatic and heterozygous, and was validated both at the DNA and mRNA level through Sanger sequencing (Figure 2B).This EMC case showed epithelioid morphology with cells arranged in cords and strands (Figure 2C), with diffuse cytoplasmic and membranous immunohistochemical positivity for KIT (Figure 2D).We evaluated the KIT expression using WTS data, finding that sample #1 had an extremely high mRNA level, about 9.8-fold greater than in the other EMC cases (Figure 2E).At the protein level, KIT was found to be expressed in all three EMC samples tested, while a mild phosphorylation of KIT was detected only in the KIT mutated case (Figure 2F).
Pathogenic single nucleotide variants (SNV) and insertions and deletions (INDEL) were searched starting from WTS data.Interestingly, in one case an in-frame deletion (c.1735_1737delGAT p.D579del) was detected in the juxtamembrane domain (exon 11) of the KIT receptor (Figure 2A).The mutation was somatic and heterozygous, and was validated both at the DNA and mRNA level through Sanger sequencing (Figure 2B).This EMC case showed epithelioid morphology with cells arranged in cords and strands (Figure 2C), with diffuse cytoplasmic and membranous immunohistochemical positivity for KIT (Figure 2D).We evaluated the KIT expression using WTS data, finding that sample #1 had an extremely high mRNA level, about 9.8-fold greater than in the other EMC cases (Figure 2E).At the protein level, KIT was found to be expressed in all three EMC samples tested, while a mild phosphorylation of KIT was detected only in the KIT mutated case (Figure 2F).KIT encodes a receptor tyrosine kinase, which is often mutated in gastrointestinal stromal tumors (GIST) and represents the target and rationale of tyrosine kinase inhibitors (TKI) treatment in this tumor.While 85% of GISTs are characterized by alteration of KIT, more rarely (5-10% of cases) they can harbor activating mutations of platelet derived growth factor receptor alpha (PDGFRA) [14,15].KIT encodes a receptor tyrosine kinase, which is often mutated in gastrointestinal stromal tumors (GIST) and represents the target and rationale of tyrosine kinase inhibitors (TKI) treatment in this tumor.While 85% of GISTs are characterized by alteration of KIT, more rarely (5-10% of cases) they can harbor activating mutations of platelet derived growth factor receptor alpha (PDGFRA) [14,15].
With the aim to evaluate the frequency of the involvement of KIT or PDGFRA alterations in EMC, an additional cohort of 15 EMC were collected.Through Sanger sequencing, we analyzed all the known hotspot exons of KIT and PDGFRA throughout the entire cohort, however we failed to detect any other mutated case in addition to sample #1.These data supported the hypothesis that alterations of KIT/PDGFRA are extremely rare in EMC.
In GIST, the location of the KIT mutation is clinically relevant as it influences the response to imatinib.Mutations of KIT exon 11 are the most frequent GIST (about 70%), and generally they are the most sensitive to imatinib [15].The p.D579del, described in this EMC case, is a known gain-of-function mutation of KIT through disruption of the autoinhibitory function of the juxtamembrane domain [16], and it was already reported in several cases of GIST and of melanoma (COSM1294).Moreover, it was also described in a metastatic case of sinonasal carcinoma and in a heavily pretreated thymic carcinoma.Noticeably, also in these two types of tumors, authors observed a relevant disease stabilization under imatinib [17,18].The EMC patient with a KIT exon 11 mutation described here never received imatinib.Thus, the therapeutic role of this agent in this case is still to be defined.Interestingly the same patient had been treated with sunitinib with a prolonged response.Unfortunately, the KIT exon 11 mutation was found only in one case out of 20, and therefore cannot explain the other high frequency of responses to sunitinib that have been observed, although it cannot be ruled out that it may have favored the response in this single case.

Samples
Five EWSR1-NR4A3 positive EMCs were collected for WTS analysis.As validation analysis, we evaluated the frequency of a KIT alteration in EMC in an additional retrospective cohort of 15 FFPE cases.The study was approved by the local ethics committee, and informed written consent was obtained in all cases, in accordance with national legislation and the Helsinki Declaration (27 May 2014, Prot.INT 54/13).Sample characteristics are shown in Supplementary Materials Table S1.

Whole Transcriptome Sequencing
For WTS analysis, total RNA was extracted from tumor specimens with RNeasy Mini Kit (Qiagen, Milan, Italy), then cDNA libraries were synthesized from 250 ng of total RNA with TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer's recommendations.Briefly, poly(A)-RNA molecules were purified using oligo-dT magnetic beads, then mRNA was fragmented and randomly primed for reverse transcription, followed by second-strand synthesis to create double-stranded cDNA fragments.The generated cDNA fragments went through a terminal-end repair process and ligation using paired-end sequencing adapters, then amplified to create the final cDNA library.WTS libraries were quality-checked and sized with Agilent DNA 7500 chips on the Bioanalyzer 2100 (Agilent Technologies, Milan, Italy), then quantified using a fluorometric assay (QuantITPicogreen assay, Thermo Fisher, Monza, Italy).Then, 12 pM paired-end libraries were amplified and ligated to the flowcell by bridge PCR, and sequenced at 2 × 80 bp read length for WTS, using Illumina Sequencing by synthesis (SBS) technology.

Sanger Sequencing
KIT and PDGFRA hotspot exons were sequenced through Sanger method in these cases and in an additional cohort of 15 EMC FFPE samples.DNA was extracted with a QiAmp DNA Mini Kit (Qiagen, Milan, Italy) from fresh frozen tissue and with QiAmp DNA Micro Kit (Qiagen)) for FFPE.Respectively, 10 and 20 ng of DNA was used for PCR using primers specific for exons 8, 9, 11, 13, 14, and 17 of KIT, and for exons 12, 14, and 18 of PDGFRA (Supplementary Materials Table S2).Amplicons were then purified and sequenced on a ABI3730 Genetic Analyzer (Applied Biosystems, Monza, Italy).

Immunohistochemistry
KIT protein expression was assessed by immunohistochemistry on FFPE 3 µm slides using c-kit (Ab CD117, A4502, Agilent Technologies Dako, Milan, Italy) with an automated immunostainer (BenchMark Ultra, Ventana Medical Systems Inc., Tucson, AZ, USA) according to manufacturer's instructions.

Conclusions
In conclusion, this is the first report of the presence of KIT exon 11 mutations in EMC.This event was detected in one out of 20 EMC cases analyzed, indicating that a KIT alteration is not a recurrent event in EMC, and cannot explain the EMC sensitivity to sunitinib, although it is an actionable mutation in the individual case in which it has been identified.Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7/1855/s1.

Figure 2 .
Figure 2. Identification of a KIT exon 11 deletion in one case of EMC.(A) WTS reads supporting the deletion.Blue and pink bars represented the reads (respectively sense and antisense to the reference).Black lines that interrupt the alignment of the reads represent the deletion of three bases, corresponding to p.D579del.(B) Sanger sequencing validation of the mutation at mRNA and DNA level (upper and lower panel).In tumor specimens, the overlapping of two signals, starting after the TAT codon, demonstrated the presence of the heterozygous deletion corresponding to p.D579del.(C) Hematoxylin and eosin stain, and (D) immunohistochemistry evaluation of KIT in Sample #1.Scale-bars indicate 50 μm (E) Expression level of KIT mRNA was evaluated through counts per million (CPM) analysis from WTS data.Each red circle correspond to one of the five EMC case analyzed: sample #1 is indicated.(F) Detection of KIT protein expression and phosphorylation through western blot on three EMC cases.Relative quantification of band intensity is shown under each blot.Actin was used as the loading control.

Figure 2 .
Figure 2. Identification of a KIT exon 11 deletion in one case of EMC.(A) WTS reads supporting the deletion.Blue and pink bars represented the reads (respectively sense and antisense to the reference).Black lines that interrupt the alignment of the reads represent the deletion of three bases, corresponding to p.D579del.(B) Sanger sequencing validation of the mutation at mRNA and DNA level (upper and lower panel).In tumor specimens, the overlapping of two signals, starting after the TAT codon, demonstrated the presence of the heterozygous deletion corresponding to p.D579del.(C) Hematoxylin and eosin stain, and (D) immunohistochemistry evaluation of KIT in Sample #1.Scale-bars indicate 50 µm (E) Expression level of KIT mRNA was evaluated through counts per million (CPM) analysis from WTS data.Each red circle correspond to one of the five EMC case analyzed: sample #1 is indicated.(F) Detection of KIT protein expression and phosphorylation through western blot on three EMC cases.Relative quantification of band intensity is shown under each blot.Actin was used as the loading control.