The first issue was to establish whether all the tumors were clonally related or independent from each other. Genomic profile analysis showed that the S2008 tumor was the local recurrence of the S2007A tumor and that the other tumors were not clonally related. Second, the presence of six different sarcomas in the same patient prompted us to look for a common tumorigenic alteration that might be constitutional.
The only common alteration between all tumors was the loss of the CDKN2A/2B genes that resulted from chromosome 9 rearrangements in all non-related tumors. This alteration has already been described in sarcomas with complex genetics 6,18. Analysis of the CDKN2A/2B in non-tumor tissues did not reveal any constitutional alteration of these genes in the patient. Even if the CDKN2A/2B deletion was not constitutional, it was very likely involved in the development of these sarcomas, given that there was a total loss of RNA expression in S2016A, a tetraploid tumor which retained two copies of both genes.
Moreover, in the search by RNA-seq for a common alteration, variations in three genes were detected both in tumor and non-tumor tissues: SLC25A39, GLT8D1 and GATAD2A. Analysis in cBioPortal revealed that mutations in these genes have been very rarely reported in sarcomas 19,20. In the 255 sarcomas of the TCGA PanCancer Atlas Studies, alterations were found for SLC25A39, GLT8D1 and GATAD2A in 0.78%, 1.57%, and 2.75% of cases, respectively 21,22. SLC25A39 is located on chromosome 17q21.31 and encodes a protein required for normal heme biosynthesis 23. Little is known about the functions of SLC25A39 and only its role in erythropoiesis and neural functions have been described 23,24. The SLC25A39 missense mutation observed in the patient was heterozygous with both alleles expressed and occurred in one of the mitochondrial carrier domains of the protein 25, potentially modifying the function of the protein. GLT8D1 (glycosyltransferase 8 domain containing 1), located on chromosome 3p21.1, encodes a glycosyltransferase enzyme of unknown function, ubiquitously expressed and localized in the Golgi apparatus 26. Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disorder, has been ascribed to missense mutations in GLT8D1 27. All mutations found in familial ALS cases and in early-onset sporadic ALS arise in GLT8D1 exon 4, which encodes the substrate-binding domain of GLT8D1 and is associated with reduced enzymatic activity. Thus, pathogenic GLT8D1 mutations are thought to be autosomal dominant mutations that are associated with haploinsufficiency and/or a dominant-negative effect 26. The missense GLT8D1 mutation found in the patient was located in exon 10 and was associated with a deletion of the second allele in five tumors and with homozygous expression in all of them, which is consistent with a loss of function of the GLT8D1 gene. The modified amino acid is located in the glycosyltransferase domain of the protein, like ALS mutations, but in a more C-terminal part 27. Consensus is lacking about the deleterious or minor effects of this mutation. GLT8D1 overexpression was also recently reported in melanoma to be associated with worse overall survival and progression-free survival 28. Unfortunately, no material was available from the melanoma in our patient. GATAD2A (GATA Zinc Finger Domain Containing 2A), located on chromosome 19p13.11, codes for the p66α protein, which is a subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex, itself implicated in transcription regulation through chromatin compaction and decompaction 29. At the transcriptional level, the NuRD complex is recruited by tissue-specific oncogenic transcription factors to repress the expression of tumor suppressor genes, while at the post-translational level, it has been shown to deacetylate p53 to inactivate p53-induced apoptosis 30. It has also been detected at replication forks and ensures proper DNA replication, cellular proliferation and protection of genome integrity 31. Moreover, it has been shown that GATAD2A/NuRD can be recruited to sites of DNA damage to promote repair by homologous recombination 32. The mutation found here occurred at an amino acid not located in an identified functional domain 33. Functional prediction algorithms almost all agree that the mutation in GATAD2A is neutral. While the NuRD complex is known to play several important emerging roles in cancer biology 31, the involvement of GATAD2A in cancer is still poorly understood. Nevertheless, a genome-wide meta-analysis of breast, ovarian and prostate cancer identified three cancer susceptibility loci associated with intronic variants of GATAD2A 30.
Then, CDKN2A/2B were lost in all tumors in this patient, associated with three constitutionally mutated genes of great potential interest. The combination of these mutations could confer a predisposition to cancer development. We hypothesize that, because of its role in the NuRD complex, the GATAD2A mutation may destabilize aging cells in certain cellular contexts and could be implicated in the inception of the tumors by modifying their chromatin conformation and gene expression, which in turn triggers genomic instability. This altered complex could change the accessibility of some structural DNA elements prone to recombination, e.g. the MLLT3 gene, in which a breakpoint was detected in three tumors. MLLT3 is one of the most common fusion partner genes of the MLL gene resulting in the t(9;11)(p22;q23) detected in acute myelogenous leukemia (AML) and in acute lymphocytic leukemia (ALL) 34. Strissel et al. identified several common structural DNA elements between MLLT3 and MLL genes, including breakpoint cluster regions bordered by scaffold-associated regions (SARs) and TopoII cleavage sites. They proposed a DNA breakage and repair model in which a non-homologous chromosomal recombination could be mediated by SARs and TopoII with subsequent DNA repair, resulting in translocations between the two genes 34. Thus, a particular chromatin state of this region in our patient may have promoted the rearrangement of MLLT3, leading to the loss of CDKN2A/2B loci. Moreover, by modifying gene expression, the altered NuRD complex may have generated conflict between the replication and transcription processes leading to DNA breaks 35. This mutation might thus confer an increased sensitivity to DNA breaks or could lead to poor repair of double-strand breaks, a process in which the complex is also implicated 32,36. Cells with loss of CDKN2A/2B genes might present altered p53 and RB1 pathways, thus leading them on the path of tumor transformation. The deletion of the GLT8D1 WT allele might also be an early event acquired during the first steps of the oncogenic process because of its occurrence in five tumors and its loss of expression in all tumors studied, suggesting that its functional loss is necessary in the inception of the tumors. In addition, the cell type in which these deregulations occurred might be at the origin of the different types of cancer developed by the patient. For example, if melanocytes were to be affected, this would result in a melanoma 37. Likewise, if different mesenchymal cell types are impacted, it could lead to the development of LMS, UPS or MFS 38,39. This model needs to be confirmed experimentally to prove its involvement in sarcoma’s inception.