Molecular Diagnostics in Sarcoma Pathology

Although malignant tumors of bone and soft tissue (i.e., sarcomas) are rare (approximately 1% of newly diagnosed cancers), they represent an outsized diagnostic challenge.^1,2 This difficulty stems in part from the impressive diversity of these neoplasms which are comprised of at least 80 distinct types.³ Notably, a significant number of sarcomas harbor a characteristic gene fusion (e.g., SS18 fusions in synovial sarcoma, EWSR1-FL1 and related fusions in Ewing sarcoma, FOXO1 fusions in alveolar rhabdomyosarcoma, etc.).

Increasingly, pathologists employ molecular testing as an aid in sarcoma diagnosis. The intent of this article is to summarize the current state of routine molecular testing in sarcomas. The future direction of this testing is also briefly discussed.

Currently, the mainstay methods for sarcoma diagnosis are morphology and immunohistochemistry (IHC). Indeed, the most recent WHO guidelines for Bone and Soft Tissue tumors emphasize the central diagnostic importance of morphology. In addition to morphology, in many instances, relevant IHC markers are also considered essential for diagnosis of sarcomas.^2,4 Morphology is the foundation of anatomic pathology and the evaluation of protein expression by IHC is familiar to all anatomic pathologists while also being convenient and generally lower cost than most molecular methods. In practice, morphologic evaluation of sarcomas often generates a differential diagnosis for which further evaluation by ancillary methods is required. IHC is the most common accessory method for diagnosis, but also carries significant limitations, including imperfect specificity. Antigens used to help define a pattern of differentiation are often expressed across multiple tumor subtypes within a given category that nonetheless require subtype-specific therapies. For that matter, IHC markers used to help specific subtypes can also have significant false negative rates for molecular alterations such as gene fusions.⁵ To address these limitations, molecular testing (i.e., testing DNA and/or RNA) has become increasingly common in diagnostic testing pathways for sarcomas and is often required for clinical trial protocols.

Molecular diagnostic methods used for sarcomas can be grouped into several categories including targeted gene-level assays (fluorescence in situ hybridization, polymerase chain reaction), copy number arrays, and newer techniques (multiplex fluorescent color-coded probes, sequencing-based assays).³ For targeted gene-level assays, fluorescence in situ hybridization (FISH) is widely used and effectively identifies translocations and amplifications, however, in the case of common breakapart probes (e.g., EWSR1 breakapart FISH) the identity of the fusion partner is not elucidated. Additionally, as FISH usually targets one gene, it generally requires the pathologist to recognize the suspected tumor subtype in the differential diagnosis, uses significant amounts of often limited tissue, and may show false negative results due to small insertions or inversions. In polymerase chain reaction (PCR) methods, primers are designed against specific sarcoma fusion breakpoints. While this method robustly captures known fusion breakpoints, it risks missing alternative fusion partners that are not in the pre-specified fusion breakpoints. Comparative genomic hybridization (CGH) or single nucleotide (SNP) arrays reliably identify copy number alterations of a broad range of sizes. A significant weakness of arrays is a limited ability to identify balanced rearrangements and small coding sequence mutations. A newer method, multiplex fluorescent color-coded probes uses DNA probes with fluorescent molecular barcodes to directly bind target RNA. An advantage of this method is the ability to multiplex hundreds of targets. A limitation of this method is, like PCR, probes are designed against specific fusion breakpoints and cannot identify novel fusion partners. Finally, next-generation sequencing approaches offer the ability to capture hundreds to thousands of fusions and to identify novel fusions and/or fusion partners depending on the method employed. The major disadvantages include greater cost and complexity than other commonly used techniques, as well as, potentially, increased turnaround time.

Several studies have examined the utility of molecular methods in the diagnosis of sarcomas. In the GENSARC study, tumor samples from 384 patients with a diagnosis of sarcoma were profiled with FISH; a subset of specimens also received additional testing with copy number array and/or PCR.⁶ The authors reported that, in 13% of cases, the pathologic diagnosis was refined or changed following integration of the data from molecular testing. The authors conclude that, for all sarcomas with recurrent genetic aberrations (e.g., synovial sarcoma, alveolar rhabdomyosarcoma), molecular testing should be performed.⁶ In another study, whole genome sequencing (WGS) was performed on tumor samples from 83 patients with a sarcoma diagnosis. Significant findings included a revision in diagnosis in 14% of patients and a change in treatment plan in 10% of patients following integration of molecular results. It should be noted that, as a tool for identifying cancer mutations, WGS is currently used mostly in research settings.⁷ Future studies examining relevant, targeted NGS panels for sarcomas should further clarify the diagnostic utility of sequencing-based methods in this tumor type.

Current World Health Organization (WHO) and National Comprehensive Cancer Center (NCCN) guidelines acknowledge the utility of molecular testing in sarcoma diagnosis. Notably, the most recent WHO Classification of Tumours of Soft tissue and Bone (2020) contains a considerable number of new tumor entities defined by a genetic alteration.² Examples of new genetically defined tumors include EWSR1-SMAD3 positive fibroblastic tumor and NTRK-rearranged spindle cell neoplasm, as well as several novel rhadomyosarcoma subtypes: congenital spindle cell rhabdomyosarcoma with VGLL2/NCOA2/CITED2 rearrangement, MYOD1-mutant spindle cell/sclerosing rhabdomyosarcoma, and intraosseous spindle cell rhabdomyosarcoma (with TFCP2/NCOA2 rearrangements).² Additionally, the new WHO guidelines now list “essential” and “desirable” diagnostic criteria for tumors. For all tumors with a defining genetic alteration, molecular confirmation of the alteration falls within the “desirable” category.² This may be due in part to the international audience for these guidelines and the reality that molecular testing is not available in all places. As molecular testing continues to become more commonplace around the world, molecular confirmation may transition to “essential” for the diagnosis of many sarcomas.

Current NCCN Soft Tissue Sarcoma guidelines confirm the diagnostic utility of molecular genetic testing and recommend that such testing should be carried out by a pathologist with expertise in sarcoma diagnosis and molecular diagnostic techniques.⁸ Recurrent gene fusions and other molecular alterations for a number of these tumors are highlighted within the guidelines;⁸ a subset of the more common entities are listed in Table 1. Additionally, NCCN Bone Cancer guidelines suggest consideration of comprehensive genomic profiling (CGP) with a validated and/or FDA-approved assay to identify targeted therapy options in metastatic Ewing sarcoma, metastatic chondrosarcoma, recurrent chordoma, and metastatic osteosarcoma.⁹ Molecular evaluation for the t(11;22) translocation in Ewing sarcoma is also recommended.⁹

Table 1. Common fusion genes in select sarcomas/soft tissue tumors.

While uncovering diagnostic alterations is a large focus of molecular testing in sarcomas, revealing therapeutically targetable alterations is another important application. For example, NTRK inhibitors are indicated for certain NTRK-fusion-positive sarcomas. In these tumors, molecular confirmation of subtype-defining fusions defines approved pharmaceutical indications. An additional tumor type with a molecularly-defined targeted therapy is ALK-rearranged inflammatory myofibroblastic tumors where ALK inhibitors are a preferred treatment regimen.⁸

As sequencing costs continue to decrease, NGS-based approaches to identify diagnostic alterations and targeted therapy options in sarcomas are likely to become more common across the world. An additional, an intriguing, modality that has recently shown promise in classifying sarcomas is methylation profiling.¹⁰ Given the rate of innovation in high-throughput molecular methods, molecular testing of sarcomas should gain increasing importance in the years to come.

While morphology and IHC remain the bedrock for sarcoma diagnosis, molecular methods have shown potential for improving and/or refining diagnosis of these rare tumors. Additionally, molecular genetic testing is finding greater acceptance in diagnostic guidelines for soft tissue and bone tumors. Therefore, it is expected that pathologists will increasingly be called for their expertise in the relevant molecular methods, both traditional and newer, for the sarcomas that they encounter in their clinical practice.

1. Anderson WJ, Jo VY. Diagnostic Immunohistochemistry of Soft Tissue and Bone Tumors: An Update on Biomarkers That Correlate with Molecular Alterations. Diagnostics (Basel, Switzerland). 2021;11(4). doi:10.3390/diagnostics11040690
2. Antonescu CR, et al. WHO Classification of Tumours of Soft Tissue and Bone. International Agency for Research on Cancer, 2020.
3. Wang XQ, Goytain A, Dickson BC, Nielsen TO. Advances in sarcoma molecular diagnostics. Genes Chromosomes Cancer. Published online January 21, 2022. doi:10.1002/gcc.23025
4. Kallen ME, Hornick JL. The 2020 WHO Classification: What’s New in Soft Tissue Tumor Pathology? Am J Surg Pathol. 2021;45(1):e1-e23. doi:10.1097/PAS.0000000000001552
5. Gown AM. Diagnostic Immunohistochemistry: What Can Go Wrong and How to Prevent It. Arch Pathol Lab Med. 2016;140(9):893-898. doi:10.5858/arpa.2016-0119-RA
6. Italiano A, Di Mauro I, Rapp J, et al. Clinical effect of molecular methods in sarcoma diagnosis (GENSARC): a prospective, multicentre, observational study. Lancet Oncol. 2016;17(4):532-538. doi:10.1016/S1470-2045(15)00583-5
7. Schipper LJ, Monkhorst K, Samsom KG, et al. Clinical Impact of Prospective Whole Genome Sequencing in Sarcoma Patients. Cancers (Basel). 2022;14(2). doi:10.3390/cancers14020436
8. NCCN Guidelines, Soft Tissue Sarcoma, Version 3.2021
9. NCCN Guidelines, Bone Cancer, Version 2.2022
10. Koelsche C, Schrimpf D, Stichel D, et al. Sarcoma classification by DNA methylation profiling. Nat Commun. 2021;12(1):498. doi:10.1038/s41467-020-20603-4

Matthew Hiemenz, MD, MS, FCAP, is a Senior Pathologist and Associate Medical Director at Foundation Medicine. His research interests include innovative approaches to biomarker-driven clinical trials and the molecular pathology of pediatric solid tumors. He is board-certified in Molecular Genetic Pathology and Anatomic and Clinical Pathology. He serves on the Personalized Health Care Committee of the College of American Pathologists and the Economic Affairs Committee of the Association for Molecular Pathology.