Current challenges and practical aspects of molecular pathology for bone and soft tissue tumors

Vol.:(0123456789) Virchows Archiv (2024) 484:353–367 https://doi.org/10.1007/s00428-024-03736-5 REVIEW ANDPERSPECTIVES Current challenges andpractical aspects ofmolecular pathology forbone andsoft tissue tumors EnriquedeÁlava1,2,3 Received: 23 November 2023 / Revised: 2 January 2024 / Accepted: 4 January 2024 / Published online: 16 January 2024 © The Author(s) 2024 Abstract This review shows the extraordinary change molecular pathology has induced in the classification, diagnosis, and clinical practice of molecular pathologists dealing with sarcomas. We have primarily focused on the practical aspects of molecular studies and the current and mid-term challenges for our subspecialty, ending with ten tips for the next generation of sarcoma molecular pathologists. Keywords Molecular pathology· Sarcomas· Biomarkers· Diagnostics· Genomics Introduction Brief description ofmolecular pathology andits significance inoncology Mesenchymal neoplasms constitute a large group of tumor entities characterized by their taxonomic complexity and complex management. The publication of the 5th edition of the WHO classification of bone and soft tissue tumors in 2020 [1] and subsequent articles between 2020 and 2023 reflects the enormous progress in our knowledge of these tumors [2, 3]. The updating of taxonomic classifications, the redefinition of diagnostic criteria, the invention of new molecular diagnostic techniques, the development of prognostic indices, and the design of therapeutic targets correspond to the most critical innovations in translational research in the molecular pathology of mesenchymal neoplasms and, by extension, in the fight against cancer. Therefore, integrating molecular pathology in managing these tumors is a critical tool that links the development of scientific knowledge with diagnostic and therapeutic improvements in managing patients with mesenchymal neoplasms. Contextualization ofsoft tissue andbone tumors inmolecular pathology Molecular pathology has revolutionized our understanding of tumor biology to the point of exponentially increasing our knowledge of the natural history of neoplasms. Although each type of mesenchymal neoplasm usually presents several molecular alterations, some of them carry a greater weight in tumor biology. Basically, we can differentiate three groups of mesenchymal neoplasms based on their molecular pathology [3] (Table1). The first group refers to the group of tumors that do not present a specific molecular alteration of clinical interest; i.e., there is no alteration sufficiently relevant for it to be considered of diagnostic and/or therapeutic importance (this is the case, for example, of undifferentiated pleomorphic sarcoma). In the second group, we would find neoplasms with recurrent molecular alterations, among which we can highlight gene fusions, point mutations, deletions, and amplifications (i.e., Ewing sarcoma). Finally, we would have a group of neoplasms with complex karyotypes. These can occur de novo or, more frequently, due to the degeneration of previously existing neoplasms with more favorable characteristics (i.e., malignant peripheral nerve sheath tumor). The presence of a complex karyotype is believed to originate in the loss of tumor suppressor genes * Enrique de Álava ealav[email protected] 1 Institute ofBiomedicine ofSevilla, IBiS/Virgen del Rocio University Hospital/CSIC/University ofSevilla/CIBERONC, 41013Seville, Spain 2 Department ofNormal andPathological Cytology andHistology, School ofMedicine, University ofSeville, 41009Seville, Spain 3 Department ofPathology, Virgen del Rocío University Hospital, Manuel Siurot S/N, 41013Seville, Spain 354 Virchows Archiv (2024) 484:353–367 of enormous importance, such as RB1, NF1, and P53, and in the phenomena of chromoanagenesis [4]. The mortality rate for high-grade metastatic sarcomas remains very high [5]. Sarcomas are highly heterogeneous morphologically, genetically, and in their behavior, so in addition to chemotherapy, which has a limited role in disease control, new strategies are needed for their treatment. In this sense, applying precision medicine strategies, which must start from a more precise diagnosis, is of extraordinary interest in such a heterogeneous group of tumors. Soft tissue tumors: practical molecular aspects The pivotal role ofdiagnostic biomarkers inmolecular pathology ofsoft tissue tumors Soft tissue tumors represent a diverse group of neoplasms with complex molecular underpinnings. Advances in molecular pathology have revolutionized our understanding and classification of these tumors, primarily due to the discovery and understanding of specific diagnostic biomarkers (see Table2). • Lipogenic neoplasms [6]: Atypical pleomorphic and spindle-cell lipomatous tumors, previously variants of well-differentiated liposarcomas, are now distinctively identified by the absence of MDM2 and CDK4 gene amplification and, notably, the deletion of 13q14 and loss of the RB1 gene in many cases. • Fibroblastic and myofibroblastic neoplasms: The presence of the NCOA2 gene rearrangement, leading to AHRR::NCOA2 fusion in most angiofibroma of soft tissues [7], pinpoints its molecular pathology. Another critical discovery is the EWSR1::SMAD3 fusion in EWSR1-positive fibroblastic tumors [8], providing clear diagnostic criteria. • Fibrohistiocytic neoplasms: Molecular insights have led to a shift in classification. For instance, the once-termed malignant fibrous histiocytomas have been divided into multiple distinct entities. • Vascular neoplasms: The identification of GNAQ or GNA14 gene mutations in anastomosing hemangioma and the discovery of two main fusion types in epithelioid hemangioendothelioma, WWTR1::CAMTA1 and YAP1::TFE3 [9], have significantly advanced our diagnostic accuracy. • Smooth muscle neoplasms: The expression of the viral EBER RNA in Epstein-Barr virus–positive smooth muscle tumors [10], leading to MYC overexpression, underlines its diagnostic significance. Additionally, in most cases, inflammatory leiomyosarcomas showcase a near-haploid karyotype, further refining our diagnostic approach. • Striated muscle neoplasms: Newly identified fusions like TFCP2 with FUS or EWSR1 and MEIS1::NCOA2 in rhabdomyosarcomas [11] have shifted our understanding of their origins and aggressiveness. • Osteochondrogenic neoplasms: For instance, soft tissue chondromas are now known to harbor FN1::FGFR gene fusions in up to 50% of cases [12]. • Neoplasms of the nerve sheath: The malignant melanotic tumor of the nerve sheath is linked to Carney’s complex in a significant proportion of cases, underpinned by the loss of the tumor suppressor gene PRKAR1A [13]. • Other soft tissue neoplasms: The discovery of NTRK gene rearrangements (reviewed in 6) has been groundbreaking due to their therapeutic implications. The presence of these rearrangements is a pivotal diagnostic criterion for certain tumors (Fig.1). • Undifferentiated round cell sarcomas [14]: The emergence of advanced high-throughput methodologies has significantly reshaped our understanding and categorization of small round cell sarcomas (SRCSs). This evolution, fueled by the integration of extensive genetic, epigenetic, and transcriptomic insights along with progressive clinicopathological data and experimental frameworks, culminated in the establishment of a novel chapter dedicated to “undifferentiated SRCSs of Table 1 Three main molecular types of bone and soft tissue sarcomas Type of mesenchymal neoplasm Molecular alterations Examples Tumors without specific molecular alterations None Undifferentiated pleomorphic sarcoma Neoplasms with recurrent molecular changes Gene fusions, mutations, deletions, amplifications Ewing sarcoma Neoplasms with complex karyotypes Loss of tumor suppressor genes (e.g., RB, NF1, P53) Malignant peripheral nerve sheath tumor 355Virchows Archiv (2024) 484:353–367 bone and soft tissue” in the 2020 WHO classification for soft tissue and bone tumors. As these technologies evolve, they are expected to uncover even more uncommon SRCS variants. • Predominantly, the most common fusion-driven entities that resemble Ewing sarcoma in morphology include round cell sarcomas characterized by the fusion of EWSR1 or FUS with non-ETS family genes (notably EWSR1::NFATC2, FUS::NFATC2, and EWSR1::PATZ1), sarcomas with CIC rearrangements (primarily CIC::DUX4), and sarcomas exhibiting BCOR genetic changes (chiefly BCOR::CCNB3). The consequences of these fusions on intracellular signaling pathways emphasize the shift in our understanding of tumor biology based on molecular findings [14]. After the 5th edition of the WHO classification (2020–2023), the realm of soft tissue tumor molecular pathology witnessed further revelations: • Fibrogenic neoplasms: Sarcomas with KMT2A gene rearrangements and fibroblastic tumors with PRRX1::NCOA1 fusion have enriched the spectrum of soft tissue tumors [15]. • Fibrohistiocytic neoplasms: The distinction of soft tissue giant cell tumors from their bony counterparts based on the absence of H3-3/H3F3 gene mutations is another testament to the precision offered by molecular biomarkers [16]. • Striated muscle neoplasms: The inflammatory rhabdomyoblastic tumor, a newly recognized entity, embodies the influence of molecular pathology in refining our understanding of soft tissue tumor classification [17]. • Other soft tissue neoplasms: The identification of EWSR1::SSX2 fusion in a subset of undifferentiated soft tissue sarcomas [18], NUTM1 gene rearrangements in colorectal sarcomas, and FN1 gene rearrangements in chondroid neoplasms underscores the relentless evolution of soft tissue tumor classification. Glioma-associated oncogene 1 (GLI1), a transcription factor activated by the Sonic hedgehog pathway, plays a role in the development of various tumors, including gliomas, alveolar rhabdomyosarcomas, and osteosarcomas. GLI1 amplifications and gene fusions are also found in diverse mesenchymal tumors like pericytoma with t(7;12), gastroblastoma, plexiform fibromyxoma, and a new category of GLI1- altered mesenchymal neoplasms. This group includes “nested glomoid neoplasm,” a new tumor type with Table 2 Soft tissue neoplasms and recently described genetic alterations Neoplasm group Tumor subtype Genetic alterations Lipogenic neoplasms Atypical pleomorphic and spindle-cell lipomatous tumors Absence of MDM2 and CDK4 gene amplification, deletion of 13q14 and loss of RB1 gene in many cases Fibrogenic and myofibrogenic neoplasms Most angiofibroma of soft tissues, EWSR1-posi- tive fibroblastic tumors NCOA2 gene rearrangement, leading to AHRR::NCOA2 fusion; EWSR1::SMAD3 fusion; sarcomas with KMT2A gene rearrangements; PRRX1::NCOA1 fusion Fibrohistiocytic neoplasms Soft tissue giant cell tumors, malignant fibrous histiocytomas Distinction based on absence of H3-3/H3F3 gene mutations Vascular neoplasms Anastomosing hemangioma, epithelioid hemangioendothelioma GNAQ or GNA14 gene mutations; WWTR1::CAMTA1 and YAP1::TFE3 fusions Smooth muscle neoplasms Epstein-Barr virus-positive smooth muscle tumors, inflammatory leiomyosarcomas Viral EBER RNA expression in Epstein-Barr virus-positive tumors; near-haploid karyotype in inflammatory leiomyosarcomas Striated muscle neoplasms Rhabdomyosarcomas, inflammatory rhabdomyoblastic tumor TFCP2 with FUS or EWSR1 and MEIS1::NCOA2 fusions in rhabdomyosarcomas Osteochondrogenic neoplasms Soft tissue chondromas FN1::FGFR gene fusions in up to 50% of cases Neoplasms of the nerve sheath Malignant melanotic tumor of the nerve sheath Linked to Carney’s complex, loss of PRKAR1A gene Other soft tissue neoplasms NTRK sarcoma NTRK gene rearrangements with therapeutic implications GLI-1 altered mesenchymal neoplasms GLI-1 rearrangements or amplifications Undifferentiated round cell sarcomas Ewing sarcoma FET::ETS fusions EWSR1::non-ETS sarcomas EWSR1::NFATC2, EWSR1::PATZ1 CIC-rearranged sarcomas CIC::DUX4 Sarcomas with BCOR alterations BCOR::CCNB3 356 Virchows Archiv (2024) 484:353–367 unique architecture, and a range of low to high-grade neoplasms, some resembling myoepithelial carcinoma. Pericytomas with t(7;12) and nested glomoid neoplasms have distinct morphologies and immunohistochemical profiles, expressing markers like S100, SMA, CDK4, and MDM2. GLI1 immunohistochemistry can aid in diagnosing these rare tumors, potentially eliminating the need for molecular testing [19]. Integrating diagnostic biomarkers into the classification of soft tissue tumors has transformed the landscape of tumor diagnosis, prognosis, and treatment. It underscores the importance of a multidisciplinary approach, combining histopathology with molecular pathology, for the optimal management of patients with these neoplasms. Bone tumors: practical molecular aspects Mesenchymal bone neoplasms are a group of tumors originating from the bone’s mesenchymal tissue. These neoplasms vary in their aggressiveness, clinical presentation, histology, and genetics. Below is a comprehensive review of the different categories of these neoplasms and their associated molecular characteristics (see also Table3): 1. Chondrogenic neoplasms Fig. 1 A 42-year-old man with an NTRK sarcoma. A Imaging shows a rapidly growing mass in the pectoral region. B It corresponds to a spindle/epithelioid cell sarcoma (H&E × 400). C Tumor cells show pan-NTRK expression (× 100). D Targeted sequencing shows TPM3- NTRK1 fusions Table 3 Bone neoplasms and recently described genetic alterations Neoplasm group Tumor subtype Genetic alterations Chondrogenic neoplasms Chondromyxoid fibroma Linked to rearrangements of the GRM1 gene Synovial chondromatosis Associated with FN1::ACVR2A and ACVR2A::FN1 fusions Osteogenic neoplasms Osteoid osteoma FOS rearrangement in most cases Osteoblastoma Shows rearrangement of FOS and less frequently FOSB Giant cell–rich neoplasms Non-ossifying fibroma Mutations in KRAS and FGFR1 Notochordal neoplasms Poorly differentiated chordoma Homozygous SMARCB1/INI1 gene deletion Other bone neoplasms Adamantinoma Displays numerical and structural chromosomopathies 357Virchows Archiv (2024) 484:353–367 Chondromyxoid fibroma: This neoplasm is linked to rearrangements of the GRM1 gene. Overexpression ofGRM1 indicates the diagnosis of chondromyxoid fibromas [20]. However, a small percentage does not show this overexpression, suggesting the possibility of other genetic alterations. Synovial chondromatosis: associated with the FN1::ACVR2A and ACVR2A::FN1 fusions [20]. These fusions are present in most benign synovial chondromatosis and some malignant cases. 2. Osteogenic neoplasms Osteoid osteoma: Characterized by the presence of FOS rearrangement in most cases [21]. A common neoplastic spectrum with osteoblastoma and epithelioid hemangioma is postulated. The FOS family plays a crucial role in cellular transcription. Although the diagnosis of almost all osteoid osteomas does not require a demonstration of FOS rearrangements, their detection can be useful in selected cases in which a clear radiology-pathology correlation is missing. Osteoblastoma: Similar to osteoid osteoma, it shows rearrangement of FOS. Also observed, albeit less frequently is the rearrangement of FOSB [21]. 3. Giant cell–rich neoplasms Non-ossifying fibroma: A subset of these tumors originates due to neurofibromatosis type 1 and Jaffe- Campanacci syndrome. They are associated with mutations in the NF1 and KRAS genes. Additionally, they are characterized by mutations in KRAS andFGFR1 [22]. 4. Notochordal neoplasms Poorly differentiated chordoma: This neoplasm shows a homozygous SMARCB1/INI1 gene deletion. A small fraction of cases show gene loss without detectable mutation [23]. Furthermore, some cases have a codeletion of the EWSR1 gene. 5. Other bone neoplasms Adamantinoma: This neoplasm displays both numerical and structural chromosomopathies. They are associated with trisomies and chromosomal translocations. The dedifferentiation process in these neoplasms is linked to the loss of P53 and the acquisition of a complex karyotype. Identifying and understanding these neoplasms’ genetic and molecular alterations provide a deeper insight into their biology and potential therapeutic interventions. Molecular diagnostic techniques inclinical practice ofbone andsoft tissue pathology Molecular diagnostics have markedly enhanced our ability to classify and diagnose neoplastic entities, particularly with the refinement of tumor taxonomy. This progress is exemplified by the GENSARC study [24], which indicated that molecular investigations altered initial diagnostic conclusions in about 14% of sarcoma cases made by specialized pathologists. Let us delve deeper into the panorama of molecular diagnostic techniques, both traditional and avant-garde. Traditional molecular techniques (reviewed in[25]) • Karyotyping: A foundational technique, karyotyping was paramount in identifying the earliest chromosomal deviations in sarcomas. Nevertheless, its resolution, limited to around five megabases, impedes the recognition of specific gene mutations, relegating it to a more ancillary role in contemporary diagnostics. • Comparative genomic hybridization (CGH): This technique can spotlight DNA amplifications and deletions. However, its limited prowess in pinpointing point mutations and gene fusions has confined its use to specific cases and research contexts. • Fluorescence insitu hybridization (FISH): Widely employed for detecting translocations and amplifications, FISH, in tandem with PCR, stands as a cornerstone in the current diagnostic landscape (Fig.2). Yet, its blind spots include an inability to discern discrete gene mutations and some proximate gene fusions [14] (Fig.3). • Polymerase chain reaction (PCR): A linchpin in many diagnostic labs, PCR has served as the molecular technique of choice for routine diagnostic procedures. However, it requires a prior understanding of the mutation under scrutiny and has constraints in detecting alternate partner genes. Modern molecular techniques • Next-generation sequencing (NGS): A quantum leap in genetic analysis, NGS allows for the parallel sequencing of vast DNA fragments. Its precision is further honed with targeted versions such as “gene panels by hybridization and capture” and “massive amplicon sequencing,” both of which can accurately identify alternate fusion partner genes (Fig.4) [26]. 358 Virchows Archiv (2024) 484:353–367 • NanoString: A paradigm shift, this method quantifies RNA directly, bypassing the need for retrotranscription or prior amplification, using DNA probes adorned with fluorescent barcodes [27]. Especially adept at analyzing subpar RNA samples, it parallels mass sequencing in identifying a diverse array of partner gene fusions. • Non-targeted massive sequencing: Offering an expansive purview, these techniques can decode any genetic rearrangement without prior knowledge. This category encompasses the holistic RNA-Seq, WES, and WGS techniques. The “nanopore” method is a groundbreaking offshoot, which deduces mutations by discerning ionic current shifts as DNA fragments traverse a nanoporous protein membrane. • Methylome studies: These studies probe methylation patterns beyond mere genetic material, offering a more resilient and granular analysis [28]. Their binary approach, focusing on methylation or its absence, furnishes a unique lens for diagnostics. Recommendation formolecular testing inbone andsoft tissue sarcomas • Consider any molecular result in the right clinical and pathological context. Although some gene fusions are very specific to a particular tumor type (e.g. EWSR1::FLI1 in Ewing sarcoma), other gene fusions are much more non-specific, such as ETV6::NTRK3, which can be seen not only in sarcomas but also in leukemias or carcinomas. Molecular findings should, therefore, never be evaluated in isolation, but always in the appropriate clinical and morphological context. • Perform a foundational assessment with traditional techniques. Begin with FISH, which is widely used for detecting translocations and amplifications, especially when specific genetic alterations or translocations are suspected. Use PCR where a particular mutation is suspected, given its widespread use and the requirement of prior knowledge of the mutation. • Make a judicious use of advanced analysis with modern techniques. Next-generation sequencing (NGS) should be considered, primarily with a targeted approach. These allow for a comprehensive genetic analysis, are adept at identifying alternative fusion partner genes, and could be Fig. 2 A 70-year-old male with mass in the femur and soft tissues and lesions in the lung and spleen. A Epithelioid cells with prominent nucleoli (H&E × 100). B CD31 expression (× 100). C Cytoplasmic lumina with red blood cells (H&E × 100). D ERG expression (× 100). E FISH analysis shows WWTR1 rearrangements, confirming the diagnosis of epithelioid hemangioendothelioma 359Virchows Archiv (2024) 484:353–367 invaluable when a broader genetic landscape needs examination. In cases with low-quality RNA samples or when a more expansive genetic view is needed, NanoString is recommended due to its ability to quantify RNA and identify diverse partner gene fusions directly. For sarcomas of uncertain classification or when a holistic view of the genetic material is required, nontargeted massive sequencing techniques like RNA-Seq, WES, and WGS can be employed. The “nanopore” method [29], while more avant-garde, can offer a unique perspective by deducing mutations from ionic current shifts. • Have supplemental analysis available: Methylome studies can be considered an auxiliary diagnostic tool, especially when the genetic material is compromised, providing a robust and detailed analysis based on methylation patterns. • Always have a holistic or integrative consideration: Despite the advancements in molecular diagnostics, the foundation of sarcoma diagnosis remains rooted in histopathological findings. Molecular pathology should be used as a complementary tool, enhancing the specificity and accuracy of the diagnosis (Fig.5). • Be aware of cost and infrastructure: While modern techniques might seem resource-intensive, their potential efficiency, especially in complex sarcoma cases, could render them more cost-effective in the long run. When selecting a testing strategy, balancing the cost, available infrastructure, and diagnostic precision are essential. • Immunohistochemistry can constitute an excellent surrogate of molecular genetics. Over the last decade, advancements in molecular genetics have revolutionized diagnostic approaches, leading to the development of novel, cost-effective, and rapid diagnostic tests using immunohistochemical stains. These new immunohistochemical markers are broadly classified into three categories: proteins indicative of genetic alterations such as PDGFRA, SMARCB1 [INI1], H3K27me3, SMARCA4 [BRG1], β-catenin, MDM2, MYC, RB1, CDK4, and SDHB; protein products resulting from gene fusions including STAT6, TFE3, ALK, FOSB, BCOR, DDIT3, SS18::SSX, CAMTA1, CCNB3, and pan-TRK; and diagnostic markers identified by gene expression profiling, such as MUC4, DOG1, NKX2-2, Fig. 3 A 75-year-old man with a high-grade extraskeletal myxoid chondrosarcoma. A A 6-cm intramuscular mass in the abdominal wall. B Epithelioid high-grade neoplasm (H&E × 400). C FISH analysis showing NR4A3 rearrangements. D EWSR1::NR4A3 fusions were detected by targeted sequencing 360 Virchows Archiv (2024) 484:353–367 TLE1, SATB2, and ETV4. These advancements have significantly enhanced the speed and precision of diagnostics, particularly in the realm of sarcoma identification and classification [2, 3]. In summary, a layered approach, integrating traditional and modern techniques, can provide a comprehensive and accurate molecular diagnosis for bone and soft tissue sarcomas. Reporting The results of ancillary tests (e.g., immunohistochemistry (IHC) or molecular evaluations) should be included in the report where relevant. This is the case, for example, for the detection of translocations in round cell sarcomas, isocitrate dehydrogenase (IDH1 and IDH2) mutations in conventional chondrosarcoma, and MDM2 amplification in low-grade intramedullary and parosteal osteosarcoma. Fig. 4 A 19-year-old woman with a vertebral mass. A Small cell sarcoma with round to oval cells. B Patchy CD99 staining (× 100). C Intense and diffuse expression of NKX2.2 (× 100). D FISH analysis with an EWSR1 breakapart probe did not show EWSR1 gene rearrangements. E Targeted sequencing, however, showed FUS::ERG fusions, confirming Ewing sarcoma diagnosis and showing a common pitfall of EWSR1 breakapart FISH probes in ERG-rearranged Ewing sarcomas Fig. 5 Diagnostic algoritm of small round cell sarcomas in our instituton integrates immunohistochemistry, FISH, and NGS assays to achieve a precise diagnosis 361Virchows Archiv (2024) 484:353–367 The International Collaboration for Cancer Reporting [30] provides guidelines for standardized pathology reporting of soft tissue sarcomas [31]. It reminds us that molecular test results should be integrated into biopsy or resection reports of osteoarticular tumor pathology. Current challenges faced bymolecular pathologists inbone andsoft tissue Pathology(BSTPath) andsignificant concerns Managing discrepancies In cases where sarcoma diagnosis reveals discrepancies between results from different molecular techniques (like FISH vs NGS) or between molecular and histological/ immunohistochemical findings, a multifaceted and cautious approach is recommended: 1. Multidisciplinary review: Engage a multidisciplinary team including pathologists, molecular biologists, radiologists, and oncologists. This team can provide diverse perspectives and expertise, comprehensively analyzing all findings. 2. Re-evaluate clinical and radiological data: Reassess the patient’s clinical history and radiological data. Sometimes, additional clinical context or imaging studies can provide insights that help reconcile conflicting results. 3. Repeat or confirm tests: If feasible, repeat the tests that show discrepancies. For instance, if there is a mismatch between FISH and NGS results, consider repeating these tests or employing additional methodologies for confirmation. 4. Integrate histological and molecular data: The histological context can sometimes provide essential insights that guide the interpretation of molecular results. Ensure that molecular findings are correlated with histological and immunohistochemical features. 5. Consider technical limitations: Understand the limitations of each technique. For instance, FISH is highly specific but may miss broader genomic alterations that NGS can detect. Conversely, NGS is comprehensive but may miss focal alterations detectable by FISH. 6. Consultation with external experts: In particularly challenging cases, seeking a second opinion from external experts or reference laboratories can be invaluable. 7. Patient monitoring and follow-up: In cases of unresolved discrepancies, close monitoring of the patient with frequent follow-ups may be necessary. This approach can help detect any progression or changes that might clarify the diagnosis. 8. Document and report findings: Careful documentation of all findings and the decision-making process is crucial. This can be valuable for future reference, especially if the patient’s clinical situation evolves. 9. Continued research and learning: Stay updated with the latest research and advancements in sarcoma diagnostics. New discoveries and technologies might provide solutions to current diagnostic challenges. Setup ofthepre‑analytical phase insarcomas Sarcomas, especially those arising from bone tissues, present unique challenges during the biopsy processing phase. Bone sarcomas, in particular, often require decalcification processes to prepare the tissue for histological examination. However, decalcification can adversely affect the quality of nucleic acids, complicating subsequent molecular analysis [32]. This makes the choice of decalcification agent and duration of the process pivotal. In addition to this, the intrinsic nature of sarcomas being deep-seated tumors further complicates biopsy collection. Proper handling becomes paramount, given the diverse subtypes of sarcomas, each with distinct molecular profiles. Preserving RNA integrity in these samples is essential, especially when gene fusion detection, a hallmark of many sarcoma subtypes, is anticipated. As such, the pre-analytical phase requires careful orchestration of multiple steps, ensuring the best possible preservation of molecular details. Ensuring access tothetests ofchoice forsarcomas With over 50 diverse subtypes, sarcomas present a tapestry of unique genetic alterations. While choosing the proper test is vital (see the “Recommendation for molecular testing in bone and soft tissue sarcomas” section), ensuring that these advanced diagnostic tools are equitably available to the general population becomes equally crucial. Whether it is the specificity of FISH for detecting specific translocations or the comprehensive capability of NGS to survey the broader genomic landscape, the real challenge lies in having access to these tests. Healthcare systems and policies must prioritize the widespread availability of these sophisticated diagnostics. This equitable distribution ensures that every patient, regardless of socio-economic status or geographical location, has a fighting chance at accurate diagnosis and targeted therapy. Moreover, a keen understanding of sarcoma histopathology and its potential molecular underpinnings underlines the importance of continuous training and updates for pathologists and technicians involved in sarcoma diagnostics.