A Rapid and Cost-Effective Gene Expression Assay for the Diagnosis of Well-Differentiated and Dedifferentiated Liposarcomas

Histologic examination neither reliably distinguishes benign lipomas from atypical lipomatous tumor/well-differentiated liposarcoma, nor dedifferentiated liposarcoma from other pleomorphic sarcomas, entities with different prognoses and management. Molecular confirmation of pathognomonic 12q13-15 amplifications leading to MDM2 overexpression is a diagnostic gold standard. Currently the most commonly used assay for this purpose is fluorescence in situ hybridization (FISH), but this is labor intensive. This study assessed whether newer NanoString-based technology could allow for more rapid and cost-efficient diagnosis of liposarcomas on standard formalin-fixed tissues through gene expression. Leveraging large-scale transcriptome data from The Cancer Genome Atlas, 20 genes were identified, most from the 12q13-15 amplicon, that distinguish dedifferentiated liposarcoma from other sarcomas and can be measured within a single NanoString assay. Using 21 cases of histologically ambiguous low-grade adipocytic tumors with available MDM2 amplification status, a machine learning–based analytical pipeline was built that assigns a given sample as negative or positive for liposarcoma based on quantitative gene expression. The effectiveness of the assay was validated on an independent set of 100 sarcoma samples (including 40 incident prospective cases), where histologic examination was considered insufficient for clinical diagnosis. The NanoString assay had a 93% technical success rate, and an accuracy of 97.8% versus an MDM2 amplification FISH gold standard. NanoString had a considerably faster turnaround time and was cheaper than FISH. Histologic examination neither reliably distinguishes benign lipomas from atypical lipomatous tumor/well-differentiated liposarcoma, nor dedifferentiated liposarcoma from other pleomorphic sarcomas, entities with different prognoses and management. Molecular confirmation of pathognomonic 12q13-15 amplifications leading to MDM2 overexpression is a diagnostic gold standard. Currently the most commonly used assay for this purpose is fluorescence in situ hybridization (FISH), but this is labor intensive. This study assessed whether newer NanoString-based technology could allow for more rapid and cost-efficient diagnosis of liposarcomas on standard formalin-fixed tissues through gene expression. Leveraging large-scale transcriptome data from The Cancer Genome Atlas, 20 genes were identified, most from the 12q13-15 amplicon, that distinguish dedifferentiated liposarcoma from other sarcomas and can be measured within a single NanoString assay. Using 21 cases of histologically ambiguous low-grade adipocytic tumors with available MDM2 amplification status, a machine learning–based analytical pipeline was built that assigns a given sample as negative or positive for liposarcoma based on quantitative gene expression. The effectiveness of the assay was validated on an independent set of 100 sarcoma samples (including 40 incident prospective cases), where histologic examination was considered insufficient for clinical diagnosis. The NanoString assay had a 93% technical success rate, and an accuracy of 97.8% versus an MDM2 amplification FISH gold standard. NanoString had a considerably faster turnaround time and was cheaper than FISH. Soft tissue tumors are a broad group of neoplasms that can arise in mesenchymal tissues throughout the body.1Barretina J. Taylor B.S. Banerji S. Ramos A.H. Lagos-Quintana M. Decarolis P.L. et al.Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy.Nat Genet. 2010; 42: 715-721Crossref PubMed Scopus (484) Google Scholar Malignant soft tissue tumors—sarcomas—are collectively uncommon relative to other cancer types, and have been classified into over 60 different subtypes.2De Vita A. Mercatali L. Recine F. Pieri F. Riva N. Bongiovanni A. Liverani C. Spadazzi C. Miserocchi G. Amadori D. Ibrahim T. Current classification, treatment options, and new perspectives in the management of adipocytic sarcomas.Onco Targets Ther. 2016; 9: 6233-6246Crossref PubMed Scopus (33) Google Scholar Diagnosis typically requires subspecialty pathology, in the context of detailed imaging and clinical information at designated sarcoma centers. Even then, molecular genetic testing is likely obligatory for accurate diagnosis of several types of sarcoma,3Italiano A. Di Mauro I. Rapp J. Pierron G. Auger N. Alberti L. Chibon F. Escande F. Voegeli A.C. Ghnassia J.P. Keslair F. Laé M. Ranchère-Vince D. Terrier P. Baffert S. Coindre J.M. Pedeutour F. Clinical effect of molecular methods in sarcoma diagnosis (GENSARC): a prospective, multicentre, observational study.Lancet Oncol. 2016; 17: 532-538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar including liposarcomas—the most common histotype of soft tissue sarcomas.4Dei Tos A.P. Liposarcomas: diagnostic pitfalls and new insights.Histopathology. 2014; 64: 38-52Crossref PubMed Scopus (105) Google Scholar Liposarcomas primarily affect adult patients and are concentrated in deep soft tissues,5Robbins S.L. Cotran R.S. Kumar V. Abbas A.K. Aster J.C. Pathologic basis of disease.ed 9. Saunders Elsevier, Philadelphia, PA2015Google Scholar falling into three main categories based on molecular etiology: i) well-differentiated/dedifferentiated liposarcomas (WDLS/DDLS); ii) myxoid liposarcomas, which have characteristic histologic examination and diagnostic translocations; and iii) pleomorphic liposarcomas, many of which have complex karyotypes.6Fletcher C.D.M. The evolving classification of soft tissue tumours - an update based on the new 2013 WHO classification.Histopathology. 2014; 64: 2-11Crossref PubMed Scopus (259) Google Scholar,7WHO Classification of Tumours Editorial BoardSoft tissue and bone tumours.ed 5. WHO classification of tumours series. International Agency for Research on Cancer, Lyon, France2020Google Scholar This project was focused on WDLS/DDLS, which is not only the most prevalent liposarcoma but also the type that most commonly causes diagnostic difficulties. WDLS/DDLS are karyotypically characterized by the presence of a supernumerary ring and/or giant rod chromosomes containing amplifications of chromosome segment 12q13-15.8Micci F. Teixeira M.R. Bjerkehagen B. Heim S. Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH.Cytogenet Genome Res. 2002; 97: 13-19Crossref PubMed Scopus (33) Google Scholar,9Italiano A. Bianchini L. Keslair F. Bonnafous S. Cardot-Leccia N. Coindre J.M. Dumollard J.M. Hofman P. Leroux A. Mainguené C. Peyrottes I. Ranchere-Vince D. Terrier P. Tran A. Gual P. Pedeutour F. HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas whereas CDK4 belongs to a distinct inconsistent amplicon.Int J Cancer. 2008; 122: 2233-2241Crossref PubMed Scopus (149) Google Scholar This locus encodes several oncogenes, including the cell-cycle regulator CDK4, as well as MDM2, the main negative regulator of the key tumor suppressor, p53.10Yap D.B. Hsieh J.K. Chan F.S. Lu X. MDM2: a bridge over the two tumour suppressors, p53 and Rb.Oncogene. 1999; 18: 7681-7689Crossref PubMed Scopus (79) Google Scholar,11Okoro D.R. Arva N. Gao C. Polotskaia A. Puente C. Rosso M. Bargonetti J. Endogenous human MDM2-C is highly expressed in human cancers and functions as a p53-independent growth activator.PLoS One. 2013; 8: e77643Crossref PubMed Scopus (24) Google Scholar The identification of WDLSs, also termed atypical lipomatous tumor, is difficult as they are often histologically similar to lipomas.12O’Donnell P.W. Griffin A.M. Eward W.C. Sternheim A. White L.M. Wunder J.S. Ferguson P.C. Can experienced observers differentiate between lipoma and well-differentiated liposarcoma using only MRI?.Sarcoma. 2013; 2013: 982784Crossref PubMed Scopus (21) Google Scholar Lipomas are common and completely benign, whereas WDLSs have a potential to recur and a risk for progressing to DDLS, an aggressive malignancy that itself can be histologically difficult to distinguish from other high-grade sarcomas. Misdiagnosis can result in delayed or inadequate treatment of liposarcomas, inappropriately long-term postoperative follow-up or overtreatment of lipomas, with serious implications for patient morbidity. Based on the most recent World Health Organization Blue Book recommendations, retroperitoneal fatty tumors and deep-seated fatty tumors of >10 cm require molecular testing.7WHO Classification of Tumours Editorial BoardSoft tissue and bone tumours.ed 5. WHO classification of tumours series. International Agency for Research on Cancer, Lyon, France2020Google Scholar Fluorescence in situ hybridization (FISH) and immunohistochemical (IHC) analysis for MDM2 are the main tools, each with its own limitations, that are currently used as front-line molecular tests to aid in the clinical diagnosis of liposarcomas. FISH for the diagnosis of WDLS/DDLS relies on the detection of MDM2 amplifications.13Tanas M.R. Goldblum J.R. Fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms: a review.Adv Anat Pathol. 2009; 16: 383-391Crossref PubMed Scopus (56) Google Scholar In FISH, fluorescence DNA probes hybridize to specific chromosomal targets in cell nuclei, and specialized cytotechnologists count the resulting hybridization signals under fluorescence microscopy to directly determine the amplification status of the target gene relative to different-colored probes hybridizing to its chromosomal centromere.8Micci F. Teixeira M.R. Bjerkehagen B. Heim S. Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH.Cytogenet Genome Res. 2002; 97: 13-19Crossref PubMed Scopus (33) Google Scholar FISH is a tool valuable for diagnosing WDLS/DDLS, but is relatively expensive, at approximately US $380 per assessed locus; has turnaround constrained by the availability of microscopes and cytotechnologists; requires relatively tedious manual counting time; and measures only one target region per assay. IHC is similarly limited, as it also measures only the protein product of one gene per assay. Liposarcomas are substrates difficult for IHC applications because scant amounts of tissue survive processing and lack specific markers.14Yang L. Wang K. Hong L. Wang X. The of in diagnosing a and 97: PubMed Scopus Google Scholar MDM2 can be its is and difficult to in lipomas and S. A. K. M. of from One. PubMed Scopus Google Scholar for the of from other pleomorphic sarcomas. as by et A. T. M. N. T. and of of CDK4, and MDM2 and in situ hybridization of MDM2 for the diagnosis of cases of dedifferentiated PubMed Scopus Google Scholar the of IHC analysis of MDM2 is only which can result in the MDM2 protein is expressed to in most a quantitative in and protein to 12q13-15 and is assessed with the methods that are quantitative over a broad in the context of molecular with amplification and liposarcoma This an assay for WDLS/DDLS that NanoString technology for as a for chromosomal a at the for amplifications in J. S. S. J. J. J. gene to Full Text Full Text PDF PubMed Scopus Google Scholar The NanoString is a hybridization assay for of or amplification B. T. N. S. T. M. P. T. L. of gene with 2008; PubMed Scopus Google Scholar This hybridization basis the for the relatively in standard clinical in a rapid and S. A. K. M. of from One. 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HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas whereas CDK4 belongs to a distinct inconsistent amplicon.Int J Cancer. 2008; 122: 2233-2241Crossref PubMed Scopus (149) Google L. S. P. Wang C. in and molecular alterations and clinical Cancer. PubMed Scopus Google M. P. J. A.M. S. 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