Clinicopathological Evaluation of Rhabdomyosarcoma with Emphasis on Myogenin Expression and FOXO1 Gene Fusion in Various Morphological Subtypes: A Cohort Study

Introduction: Rhabdomyosarcoma (RMS) is a prevalent paediatric soft-tissue sarcoma. Among the four histomorphological subtypes of RMS, distinguishing the dense pattern of Embryonal RMS (ERMS) from the solid pattern of Alveolar RMS (ARMS) solely based on morphology is challenging and necessitates ancillary techniques. Aim: To study the demographics, classify the histomorphological subtypes and reclassify the morphologically overlapping embryonal and ARMS cases into specific subtypes based on the intensity of myogenin expression and the FOXO1 gene fusion status. Materials and Methods: This cohort study was conducted at the Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India, from January 2019 to December 2022. The study included 71 cases of RMS. Clinical data such as age, gender, tumour site, size and clinical stage, along with histomorphological types, were analysed. A panel of immunomarkers was performed based on morphological differentials, which included desmin, myogenin, MyoD1, CD99, synaptophysin, chromogranin, PanCK, NKX2.2, INI1, CD56, S100, CD34 and SMA. Myogenin expression was scored based on the extent of tumour cell positivity. Interphase Fluorescence In Situ Hybridization (FISH) analysis was conducted on all ARMS and morphologically unclassified cases using the CytoTest Break Apart Probe for the FOXO1 gene. One hundred tumour cells were analysed and split signals, with or without amplification signals in at least 15 cells, were considered positive. Results: A male preponderance was noted (43/71, 60.6%), with a higher incidence among children under 14 years (42/71, 59.2%). ERMS was the most common histological subtype (26/71, 36.6%), followed by ARMS (13/71, 18.3%) and Spindle Cell RMS (SCRMS) (7 cases, 9.9%). The head and neck regions were frequently involved (24 cases, 33%). Twenty-three cases of unclassified RMS were reclassified into ARMS (fusion-positive) (43.4%, n=10) and reclassified ERMS (fusion-negative) (34.7%, n=8). Notably, the correlation between myogenin expression and FOXO1 fusion showed that 94.7% of fusion-positive cases exhibited 4+ myogenin expression (p-value <0.001). Overall, ARMS had the worst overall survival (OS) rate (26.1%). The reclassified ERMS and the classic ERMS cases showed almost similar survival rates (62.5% vs 64.2%, respectively) (p-value=0.025). Conclusion: The study highlights that the myogenin immunomarker is useful in differentiating between ERMS and ARMS in resource-constrained settings and emphasises the need for fusion testing in ARMS and unclassified RMS cases for accurate risk assessment and tailored treatment strategies.