Recent advances in soft tissue sarcoma and its applications

Recent advances in soft tissue sarcoma

1. RECENT ADVANCES IN ANCILLARY TECHNIQUES IN SOFT TISSUE               SARCOMA                                                  - D.VINOTH KUMAR ( Ist year PG)

2. OVERVIEW A n c illary techniques used in soft tissue tumours . New IHC markers Molecular techniques AI

3. A n c illary techniques used in soft tissue tumours   Reverse Transcription Polymerase Chain Reaction (RT-PCR )  Immunohistochemistry (IHC) Fluorescence In Situ Hybridization (FISH) Next-Generation Sequencing (NGS)

4. Existing IHC markers Categories Markers Adipocytic tumours MDM2,CDK4 Fibroblastic and myofibroblastic tumours SMA, CD34, ERG, STAT6, ALK, MUC4. So-called fibrohistiocytic tumours SMA Vascular tumours CD34, CD31, ERG , FLI1, EMA , D2-40, VEGFR3, HHV8. Pericytic (perivascular) tumours SMA

5. Existing IHC markers Categories Markers S mooth muscle tumours SMA, D esmin and h-caldesmon Skeletal muscle tumours M yogenin (MYF4) and MYOD1 Gastrointestinal stromal tumour KIT(CD117) and DOG1 Peripheral nerve sheath tumours S100, SOX10, GFAP, Inhibin, Calretinin, HMB45, Melan-A and EMA Tumours of uncertain differentiation HMGA2 , Calponin, CD56, ERG, FGFR1, TLE1 , SATB2, SALL4, Glypican-3 (GPC3)

6. Newer IHC markers

7. ADIPOCYTIC TUMOUR

8. DDIT3 : DNA Damage Inducible Transcript 3 Myxoid liposarcoma. Diffuse nuclear positivity . S ensitivity: 85% and S pecificity: 89%. Other: Ewings sarcoma.

9. FIBROBLASTIC AND MYOFIBROBLASTIC TUMOUR

10. pan-TRK: pan - Tyrosine Receptor Kinase Infantile fibrosarcoma . Nuclear expression in a tumour with ETV6-NTRK3 fusion. S ensitivity: 92% and S pecificity: 95%. Other: Paediatric spindle cell tumour.

11. Vascular tumors

12. FOSB Pseudomyogenic hemangioendothelioma (PHE) . D iffuse nuclear positivity . S ensitivity: 96% and Specficity: 100% . Other: EHE(Epithelioid haemangioendothelioma).

13. Skeletal muscle tumors

14. CAMTA1 : Calmodulin-binding transcription activator 1 Epithelioid hemangioendothelioma [WWTR1::CAMTA1 (85–90%) ] Strong nuclear. S ensitivity: 91.2% and Specificity: 100%. Other: Gliomas

15. GIST

16. SDHB : Succinate Dehydrogenase B SDHB-mutant GIST(d) . L oss of cytoplasmic expression of SDHB (e) . S ensitivity: 100% and Specificity: 100%. Other: Paragangliomas.

17. Peripheral nerve sheath tumours

18. PRAME: PReferentially expressed Antigen in MElanoma . P rimary cutaneous melanoma . Diffuse nuclear staining . Sensitivity: 67-91.7% and Specificity: 93.5-100%. Other: Leukaemias and Lymphomas.

19. PRKAR1A: Protein Kinase cAMP dependent type I Regulatory subunit Alpha Melanotic malignant nerve sheath tumour. Complete loss of cytoplasmic expression. Sensitivity: 60-80% and Specificity: 70-90%. Other: Carcinoid tumours.

20. H3K27me3 : Trimethylation of histone H3 at lysine 27. M alignant peripheral nerve sheath tumour. Loss of nuclear staining pattern. S ensitivity: 44% and Specificity: 92% . Other: DIPG (Diffuse Intrinsic Pontine Gliomas).

21. Tumours of uncertain differentiation

22. SS18-SSX Synovial sarcoma . Diffuse n uclear staining . S ensitivity: 90 -95% and Specificity: 85%-95%.

23. WT1 (C-terminus) : Wilms Tumour protein 1 D esmoplastic small round cell tumour. Nuclear staining . Sensitivity: 90% and Specificity: 85%. Other: Wilms tumour.

24. FGFR1: Fibroblast Growth Factor Receptor 1 Phosphaturic mesenchymal tumour. Cytoplasmic staining pattern. Sensitivity: 80% and specificity: 90%. Other: SCC of Head and neck region.

25. Undifferentiated Small Round Cell Sarcomas

26. DUX4 CIC-rearranged sarcoma[CIC::DUX4 (95%)] . Nuclear staining . S ensitivity: 90% and Specificity: 85% . Other: Ewing sarcoma, Desmoplastic small round cell tumour.

27. ETV4 CIC-rearranged sarcoma . Diffuse Nuclear staining . Sensitivity: 70-80% and Specificity: 80-90%. Other: Prostate cancer.

28. BCOR : BCL 6 interacting Corepressor BCOR CCNB3 –rearranged sarcoma . Nuclear staining . S ensitivity: 80-90% and Specificity: 85-95%. Other: Ovarian tumours.

29. Molecular techniques

30. Current European Society for Medical Oncology (ESMO) guidelines suggest that the morphologic and immunohistochemical analyses should be complemented by molecular pathology: (1) when the specific histologic diagnosis is uncertain , (2) when the clinicopathologic presentation is unusual , or (3) when the genetic information may have prognostic or predictive relevance.

31. Karyotyping Comparative genomic hybridisation FISH: WWTR1 rearrangements, in diagnosing EHE. Synovial Sarcoma: CGH helps identify chromosomal abnormalities, such as the characteristic translocation t(X;18).

32. F luorescence in-situ hybridisation

33. Multiple x FISH: Multiple fluorescent probes to detect several genetic alterations simultaneously within the same tissue sample. Improves diagnostic accuracy Detecting complex genetic alterations within a single assay. Automated FISH Analysis : Incorporates automated image analysis software to assist in the interpretation . Reduces human error , increases consistency, accuracy and reliability of genetic diagnostics. ** Three-Dimensional Fluorescence In Situ Hybridization for the Diagnosis of Soft Tissue Tumors" published in Human Pathology (2022) ** **Automated Fluorescence In Situ Hybridization Analysis in Soft Tissue Sarcomas: Enhancements in Diagnostic Accuracy" published in The American Journal of Surgical Pathology (2021)**

34. 3D FISH : Provides 3D imaging of chromosomal abnormalities . Improves understanding of the structural organization of chromosomal aberrations. Used in research and diagnostic purposes , for understanding tumor heterogeneity and complex chromosomal arrangements . Integration with NGS : P rovide comprehensive genetic profiling. S imultaneous detection of gene fusions, mutations, and other genomic alterations P owerful diagnostic tool that enhances the precision of genetic classification and guides personalized treatment strategies . **Three-Dimensional Fluorescence In Situ Hybridization (3D FISH) for the Study of Chromosomal Abnormalities in Soft Tissue Sarcomas" published in Modern Pathology (2023)** **Integration of NGS and FISH for Comprehensive Analysis of Genetic Alterations in Soft Tissue Sarcomas" published in Journal of Molecular Diagnostics (2023)**

35. Reverse Transcription Polymerase Chain Reaction (RT-PCR ) High-Throughput RT-PCR: Simultaneous analysis of multiple gene expressions in a single sample, improving the efficiency and scope of gene expression profiling in soft tissue tumors. Digital PCR: Higher precision and sensitivity. It partitions the sample into many small reactions and counts the number of positive reactions. Thus useful for detecting low-abundance transcripts or rare mutations.

36. Reverse Transcription Polymerase Chain Reaction (RT-PCR ) Integration with NGS: It enhances the ability to identify novel biomarkers and genetic alterations. Improved Targeting of Fusion Genes . Quantitative RT-PCR (qRT-PCR): Improved accuracy and reproducibility, allowing more precise measurement of gene expression levels, which aids in prognosis and treatment planning.

37. NEXT GENERATION SEQUENCING

38. Sarcoma panel(UNI NGS) comprises of a. Comprehensive mutation coverage. b. Full exon coverage. c. Copy number variation. d. Gene fusion and expression. e. Hot spot gene

39. Comprehensive mutation coverage (across 86 genes): - E xtensive genetic analysis performed to identify mutations in a wide range of genes involved in sarcomas. Full exon coverage (across 44 genes) : - E ntire coding sequence of each gene in the panel is sequenced - M utations present at low frequencies can also be detected and thus crucial for identifying mutations (Highly sensitive)

40. Copy number variation (across 28 genes) : - D etection and analysis of changes in the number of copies of specific genes or genomic regions within the tumour's DNA . - ALK, BRAF, PDGFRA, FGFR 1/2/3/4 . Gene fusion (Fusion and expression across 98 genes) : - Two different genes join to form a new hybrid gene thus resulting in chromosomal rearrangements such as translocations, deletions, or inversions. - Importance in Sarcomas: Many sarcomas are characterized by specific gene fusions, which can drive tumorigenesis.

41. Gene Expression (Fusion and expression across 98 genes) : - Information from a gene is used to synthesize functional gene products, proteins. - Importance in Sarcomas: Abnormal gene expression such as overexpression or underexpression of tumor suppressor genes can influence tumor growth and response to treatment. BCOR, CAMTA1, STAT6 , ALK. Hot spot gene : - S pecific hotspots regions are frequently mutated in sarcomas that drive s cancer development and progression.

42. NGS , is utilised in the development of targeted therapies. e g., D etecting NTRK fusions enables the use of TRK inhibitors, which have shown high efficacy in treating tumors with these genetic alterations. Tumor Mutation Burden (TMB ): It is a measure of the total n o. of mutations / megabase of tumor DNA . It is used as a biomarker to predict response to immunotherapies. High TMB is associated with better responses to immune checkpoint inhibitors. The application of NGS in measuring TMB helps in selecting suitable candidates for these therapies .

43. Non-targeted massive sequencing This techniques can decode any genetic rearrangement without prior knowledge. It encompasses the holistic RNA-Seq, WES, and WGS techniques.

44. Nanopore The “ nanopore - long read sequencing ” method uses to drive DNA strands or single nucleotides through a very small hole embedded in a membrane. An enzyme motor controls the rate at which a DNA molecule passes through the nanopore. The sequence is determined in real-time based on the extent to which the nucleotides disrupt the current flowing through a nanopore sensor. Advantages: No RNA splicing / amplication.

45. Nanostring technology It is a single molecule counting system. It works by attaching molecular barcodes to target molecules of interest by nucleic acid base pairing. This method quantifies RNA directly, bypassing the need of prior amplification. Adapts to analyse even subpar RNA samples.

46. Methylome studies These studies deals with analysing the methylation patterns that o f fer s a more resilient and granular analysis . Their binary approach, focusing on methylation or its absence, furnishes a unique diagnostic approach to a particular tumour.

47. The emerging field of liquid biopsies includes- a. Cell free DNA(cfDNA) b. Circulating tumour DNA(ctDNA) c. Circulating tumour cells (CTCs) These non invasive tests are primarily derived from blood samples, without need for tissue biopsies. These liquid biopsies aid in early diagnosis, monitoring treatment responses and detecting recurrences. Sensitivity: affected by deep seated nature of many sarcomas because the amount of ctDNA shed into the blood stream may be low. Circulating biomarkers in sarcomas ** Circulating Biomarkers in Soft Tissue Sarcomas: Current Applications and Future Directions" published in Cancer Research (2023) **

48. AI in soft tissue pathology

49. R ecent study from the French Sarcoma Group : Showcased p otential of deep learning (DL) in predicting the progression risk of localized GIST. DL can detect somatic mutations, notably the specif i c PDGFRA exon 18 D842V mutation. This DL method can expedite treatment decisions, particularly for patients with intermediate-risk Miettinen GIST, who typically donot require adjuvant treatment, and high-risk Miettinen GIST, where avapratinib treatment is essential.

50. Enhanced Diagnostic Accuracy. Automated Detection of Tumor Subtypes: AI model that accurately classified various sarcoma subtypes, such as liposarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma, by analyzing whole-slide images. ** Artificial Intelligence in soft tissue tumor pathology: current applications, limitations, and future directions (2023)**

51. Identification of Genetic Mutations and Biomarkers: AI tool developed by PathAI was used to analyze histopathological slides of soft tissue tumors and identify genetic mutations such as MDM2 amplifications in liposarcomas, providing insights into prognosis and potential therapeutic targets. Quantitative Analysis of Tumor Characteristics : A deep learning (DL) model was used to quantify the mitotic rate in histopathological images of soft tissue sarcomas, providing an objective measure that correlates with tumor aggressiveness and patient prognosis. ** Artificial Intelligence in soft tissue tumor pathology: current applications, limitations, and future directions (2023)**

52. Integration with Molecular Data: Recently developed AI platform combines histopathological images with genomic sequencing data to identify mutations in soft tissue sarcomas, guiding targeted therapy decisions. Reduction in Diagnostic Time: An AI-powered digital pathology system developed by Proscia was shown to reduce the time taken to diagnose soft tissue tumors by automatically screening slides and prioritizing cases that require urgent attention. ** Artificial Intelligence in soft tissue tumor pathology: current applications, limitations, and future directions (2023)**

53. References: 1. The Recent advances in molecular diagnosis of soft tissue tumors. 2. Current challenges and practical aspects of molecular pathology. 3. Immunohistochemistry Update in Dermatopathology and Bone and Soft Tissue Pathology. 4. Diagnostic Immunohistochemistry of Soft Tissue and Bone Tumors: An Update on Biomarkers That Correlate with Molecular Alterations.

54. THANK YOU