Global Journal of Science Frontier Research, G: Bio-Tech & Genetics, Volume 22 Issue 2

of the translocation, as known through synovial sarcoma, SMART-seq could easily identify the variants, while having the added benefit of transcriptomic data for each cell linked to any one variant. Importantly, regardless of grade, MLPS has potential to metastasize. A key question to explore using SCS would be to identify if there exists a cell state or subclone within the lesion that has a higher propensity for metastasis. Feasibility of using SCS with fatty tissues, such as liposarcoma, is an important issue to resolve. Two major concerns with adipocytes are their large and fragile nature, which has proven to be a problem with SCS technologies. An alternative strategy to certain SCS methods, like scRNA-seq, which typically uses whole cells, is to use the nucleus – termed single-nucleus RNA-seq (snRNA-seq) 154 . SnRNA-seq has previously been leveraged for various mouse and human adipose tissue 155-157 . Recently, an atlas of white adipose tissue demonstrated that only snRNA-seq was capable of sequencing and detecting adipocytes, which were not present in the scRNA-seq data from the same tissue 158 . Interestingly, while many of the other cells within the microenvironment were also present in snRNA-seq, there were also a lower abundance of endothelial and immune cells. Overall, this suggests that sequencing liposarcoma, where cases with WDLPS tend to be fattier, may require nucleus rather than whole cell. In that regard, techniques using scDNA-seq or scATAC-seq should not be affected since the nucleus is typicallly the default input. b) Cell-Free tumor DNA in Liposarcoma Detection of possible recurrence or metastasis in patients with liposarcoma that have undergone complete resection can be difficult and costly. Because there are no diagnostic biomarkers associated with possible recurrence or metastasis, clinical examinations with frequent imaging throughout the body is the only alternative. Recently, cell-free tumor DNA (ctDNA) has emerged as a novel method to interrogate cancer biology and etiology in a feasible manner by profiling tumor-derived materials, such as blood, cerebral spinal fluid, and urine 159 . CtDNA often contain genetic material that had been shed from tumor cells, where such materials should reflect the tumor genome in some capacity. At a molecular level, somatic mutations, copy number alterations, methylation, and point mutations can be detected in ctDNA by sequencing methods. In that regard, ctDNA is a useful diagnostic tool that could detect early diagnosis and predict tumor burden and activity and overcome the hurdles of traditional diagnostic methods such as imaging and traditional biopsies. Given that MLPS has a well-defined molecular diagnostic feature, the FUS-DDIT3 or the alternative EWSR1-DDIT3 translocation, ctDNA has recently been used to monitor disease activity of patients with MLPS 160 . Quantification of ctDNA of the t(12;16) breakpoint for multiple patients using digital droplet PCR demonstrated a correlation of ctDNA concentration with tumor volume and stage. Upon resection there was an observed drop-off of t(12;16) ctDNA, while recurrence or metastases was associated with an increase of t(12;16) ctDNA concentration. Interestingly, unlike MLPS where the translocation was detected by ctDNA, genotyping WDLPS and DDLPS was more difficult. While these tumors harbor amplifications resulting in multiple copies of MDM2, CDK4, and HMGA2 , the method for detection by digital droplet PCR in a recent study was not nearly as sensitive 160 . CtDNA derived from the peripheral blood from five WDLPS/DDLPS patients were collected and primers for MDM2 and a control gene, EIF2C1 , were used to genotype. The MDM2/EIF2C1 ratio was 1.21 (range of 1.14-1.38), whereas health patients had a ratio of 1.09 (range of 0.69–1.41), which had no statistical significance, suggesting that PCR may not have enough specificity and sensitivity to detect the CNAs. On the other hand, a separate study used shallow whole- genome sequencing, which is well-established for genotyping with low-coverage, to detect MDM2 in ctDNA from the plasma of WDLPS and DDLPS patients 161 . Interestingly, only two out of three DDLPS patients had readily detectable MDM2 amplification. This seemed to correlate with tumor size, where the undetected patient had a tumor size of 14 cm v. 19 and 25 cm. Moreover, no WDLPS patients had detectable MDM2 amplification in ctDNA, perhaps due to the lower cellularity content of these tumors as opposed to DDLPS. In addition, a longitudinal study showed that MDM2 levels decreased after tumor resection. Overall, these data suggests that MDM2 amplification could be detected for DDLPS patients by shallow whole-genome sequencing from the plasma. While PLPS is an aggressive sarcoma with high recurrences, it does not have a unifying genetic alteration that could be easily detected for disease monitoring. Over 50% of patients diagnosed with PLPS will eventually have metastatic disease 22 . A study evaluating PLPS for biomarkers failed to identify prognostic biomarker for patients whose follow-up information was available (n=22) 12 . Despite the lack of distinctive genetic alterations, patient-specific gene variants found within the ctDNA could be a possible avenue for detecting residual disease or possible recurrence. One strategy would be to perform deep NGS sequencing on tumor tissue from surgical resection to discover patient specific alterations. Paired analysis of patient plasma from ctDNA using a targeted approach, like molecular tag-based sequencing, may reveal concordant mutations with the tumor tissue that could be used for disease monitoring during follow-up. In a recent study that monitored patient-specific ctDNA 1 Year 2022 25 © 2022 Global Journals Global Journal of Science Frontier Research Volume XXII Issue ersion I VII ( G ) The Genomics of Liposarcoma: A Review and Commentary