MYOD1 Mutations Research Articles

Clinicopathological Analysis of a European Cohort of MYOD1 Mutant Rhabdomyosarcomas in Children and Young Adults.

Patients with PAX3/7-FOXO1 fusion-negative rhabdomyosarcomas (fnRMS) harbouring the rare L122R MYOD1 mutation have significantly poorer prognosis than other fnRMS. We undertook a detailed clinicopathological evaluation of a cohort of patients with MYOD1 mutated fnRMS in order to improve risk stratification and treatment options. Histological, mutational and clinical data from a cohort of patients with MYOD1 mutant RMS treated in Europe were analysed. Thirty-two cases with mutant MYOD1 RMS were identified from patients enrolled in sequential European rhabdomyosarcoma clinical trials from 1992 to 2022 (n = 22) and non-trial cohorts (n = 10). Thirty cases had the recurrent L122R missense mutation, one case harboured a K124E mutation and one case had a truncating mutation (S63X). Increased MyoD1 and reduced MYF4 immunostaining were consistent features of MYOD1L122R-mutated RMS. Applying the risk stratification of the European paediatric Soft tissue sarcoma Study Group (EpSSG) RMS2005 trial, among 20 localised RMS cases that could be assigned a risk category, one was Very High Risk, 13 were High Risk and six were Standard Risk. Eight patients had distant metastases at diagnosis. Of the 25 patients with adequate clinical follow-up data, 15/25 (60%) patients had an event at a median time of 9months (12/15 included failure of local control) and 13/25 (52%) died of disease. This MYOD1 mutant cohort demonstrates increased MYOD and reduced MYF4 immunostaining, high risk of local failure and poor survival in agreement with other studies. Increased treatment intensity and improved local control should be considered for these patients.

Improving Individualized Rhabdomyosarcoma Prognosis Predictions Using Somatic Molecular Biomarkers.

Molecular markers, such as FOXO1 fusion genes and TP53 and MYOD1 mutations, increasingly influence risk-stratified treatment selection for pediatric rhabdomyosarcoma (RMS). This study aims to integrate molecular and clinical data to produce individualized prognosis predictions that can further improve treatment selection. Clinical variables and somatic mutation data for 20 genes from 641 RMS patients in the United Kingdom and the United States were used to develop three Cox proportional hazard models for predicting event-free survival (EFS). The 'Baseline Clinical' (BC) model included treatment location, age, fusion status, and risk group. The 'Gene Enhanced 2' (GE2) model added TP53 and MYOD1 mutations to the BC predictors. The 'Gene Enhanced 6' (GE6) model further included NF1, MET, CDKN2A, and MYCN mutations, selected through LASSO regression. Model performance was assessed using likelihood ratio (LR) tests and optimism-adjusted, bootstrapped validation and calibration metrics. The GE6 model demonstrated superior predictive performance, offering 39% more predictive information than the BC model (LR p<0.001) and 15% more than the GE2 model (LR p<0.001). The GE6 model achieved the highest discrimination with a C-index of 0.7087, a Nagalkerke R2 of 0.205, and appropriate calibration. Mutations in TP53, MYOD1, CDKN2A, MET, and MYCN were associated with higher hazards, while NF1 mutation correlated with lower hazard. Individual prognosis predictions varied between models in ways that may suggest different treatments for the same patient. For example, the 5-year EFS for a 10-year-old patient with high-risk, fusion-negative, NF1-positive disease was 50.0% (95% confidence interval: 39-64%) from BC but 76% (64-90%) from GE6. Incorporating molecular markers into RMS prognosis models improves prognosis predictions. Individualized prognosis predictions may suggest alternative treatment regimens compared to traditional risk-classification schemas. Improved clinical variables and external validation are required prior to implementing these models into clinical practice.

Clinicopathological and treatment response characteristics of updated rhabdomyosarcoma histomolecular subtypes: An Asian population-based study.

New histomolecular subtypes of rhabdomyosarcoma have recently been defined but their corresponding clinical characteristics are not well described. Also, these clinical phenotypes vary greatly by age and ethnicity but have not been profiled in Asian populations. Thus, we sought to determine the landscape of rhabdomyosarcoma subtypes in a national Asian cohort and compare clinical characteristics among age groups and molecular subtypes. We performed a retrospective population-based study of all rhabdomyosarcoma patients in Singapore public hospitals from 2004 to 2014 (n=67), and assigned histomolecular subtypes according to the updated 2020 WHO classification of soft tissue tumors following central pathology review and molecular profiling. Age-specific prevalence followed a tri-modal peak. There were significantly more embryonal and alveolar (p=0.032) and genitourinary (non-bladder/prostate) tumors (p=0.033) among children. Older age was associated with complete resection among spindle cell/sclerosing tumors (p=0.027), with the omission of chemotherapy among embryonal tumors (p=0.001), and with poorer survival among embryonal and alveolar tumors (p=0.026, p=0.022, respectively). Overall survival differed with stage, group, and surgical resection, adjusted for age group (p=0.004, p=0.001, p=0.004, respectively). Spindle-cell/sclerosing tumors showed an indolent phenotype with a significantly lower incidence of nodal metastasis (p=0.002), but two of 15 patients with MYOD1 mutations had a contrastingly aggressive disease. Disease and treatment response profiles of rhabdomyosarcoma subtypes vary significantly between adults and children, especially surgical resectability. In our Asian population, poorer outcomes were observed in adults with embryonal and alveolar tumors, while activating mutations influence the behavior of otherwise favorable spindle cell/sclerosing tumors.

Epithelioid and spindle rhabdomyosarcoma with TFCP2 rearrangement in abdominal wall: a distinctive entity with poor prognosis

BackgroundEpithelioid and spindle rhabdomyosarcoma (ES-RMS) with TFCP2 rearrangement is a recently discovered rare variant of rhabdomyosarcoma composed of epithelioid and spindle cells, because it shows extraordinarily adverse prognosis and is easily misdiagnosed as other epithelioid or spindle cell tumors.MethodsA rare case of ES-RMS with TFCP2 rearrangement was presented and English literatures in Pubmed online up to 01 July 2022 were gathered by two authors for a systematic review according to the inclusion and exclusion criteria.Case presentation/resultsWe report a case of ES-RMS in an early 30s-years-old female, the neoplastic cells are remarkably immunoreactive with CK(AE1/AE3), and partially with ALK protein. Unexpectedly, the tumor shows TFCP2 rearrangement with coexistence of increased copy numbers of EWSR1 and ROS1 gene and MET gene mutation. Besides, Next-generation sequencing for genetic mutational profiling revealed frequent MET exon14 mutations in chromosome 7, most of which are C > T nonsynonymous SNV, and exon42 of ROS1 in chromosome 6 showed frequent G > T mutation up to 57.54%. In addition, neither MyoD1 mutation nor gene fusions were detected. Moreover, the patient shows high tumor mutational burden (TMB) up to 14.11 counts/Mb. Finally, as many cases of ES-RMS including our case had local progression or metastasis, we find, similar to epithelioid rhabdomyosarcoma (median survival time is 10 month), ES-RMS shows a more aggressive behavior and adverse prognosis (median survival time is 17 month) than spindle cell/sclerosing rhabdomyosarcoma (median survival time is 65 month) according previous studies.ConclusionsES-RMS with TFCP2 rearrangement is a rare malignant tumor and easily confused with other epithelioid or spindle cell tumors, it may harbor additional gene alteration in addition to TFCP2 rearrangement, such as MET mutation, increased copy numbers of EWSR1 and ROS1 gene, high TMB. Most importantly, it may show very poor outcome with extensive metastasis.

Abstract 466: Predicting survival of rhabdomyosarcoma patients based on deep learning of H&amp;E images

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue tumor in children and young adults, accounting for 350-400 new cases annually in the US. Diagnosis of RMS is defined by the expression of genes related to skeletal muscle differentiation and can be further subclassified based on histological patterns (embryonal, ERMS; alveolar, ARMS; spindle/sclerosing, SSRMS). Genetic studies have found that the presence of a PAX fusion gene (FP-RMS), which is present in many ARMS tumors, correlates with poor outcome. Subsequent studies have identified additional genetic alterations (ex. TP53 or MYOD1 mutations) which also display distinct histological features and are associated with poor outcome. As a result, there is a growing need to identify these mutations to improve risk stratification. The goal of this study is to develop and test deep learning algorithms from diagnostic H&amp;E images of RMS tumors which can aid in the diagnosis, mutation prediction and risk stratification for RMS patients. Methods: De-identified RMS patient samples were collected from tissue banking studies from Children’s Oncology Group (n=275), University Hospital Zurich (n=250) and Memorial Sloan Kettering (n=10). H&amp;E stains on whole slides or TMAs were digitally scanned and used for analysis. Clinical information including clinical risk group, event-free survival, and genomic findings were used as available for training and testing. Convolutional neural networks (CNN) using EfficientNet were trained using K-fold cross validation to classify tumor histology, mutation probability, and risk stratification and tested against randomly selected samples or independent datasets when available. Results: The developed AI algorithm was able to classify tissue as ARMS (FP-RMS), ERMS (FN-RMS), stroma and necrosis with an average weighted intersect-over-union of 0.74 when compared to an expert pathologist annotation. A second deep learning algorithm developed specifically for distinguishing FP-RMS from FN-RMS displayed excellent sensitivity (FP-RMS=0.88) and specificity (FP-RMS=0.86) when tested against an independent RMS TMA dataset. Algorithms were also trained to predict mutations in MYOD1, RAS pathway genes, and TP53 and displayed good performance with ROC values of 0.96, 0.68, and 0.64, respectively. Lastly, we developed an algorithm to provide a Cox proportional hazard prediction based on H&amp;E images. The resulting algorithm was capable of predicting EFS with similar accuracy as current clinical risk group assessment with improved ability to distinguish intermediate and high risk patients. Conclusions: Deep learning with convolutional neural networks provides pathologist-independent classification of RMS patients from simple H&amp;E images. These AI algorithms can provide probabilities of prognostically relevant genetic alterations and survival which will ultimately contribute to better risk stratification of RMS patients. Citation Format: David Milewski, Hyun Jung, G. Thomas Brown, Yanling Liu, Jack Collins, Marc Ladanyi, Erin Rudzinski, Javed Khan. Predicting survival of rhabdomyosarcoma patients based on deep learning of H&amp;E images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 466.

Congenital spindle cell rhabdomyosarcoma: An international cooperative analysis

BackgroundSpindle cell rhabdomyosarcoma (RMS) is a rare variant of RMS accounting for up to 10% of cases in infants. In older children and adults, spindle cell RMS is associated with MYOD1 mutations and a poor prognosis. In infants, it is associated with recurring fusions involving NCOA2 and VGLL2. Reports in the literature suggest a favorable prognosis for this subset, however, little is known about treatment and outcome data of infants with spindle cell RMS. MethodsCharacteristics, treatment, and outcome of an international cohort of 40 patients aged ≤ 12 months with spindle cell RMS treated from 1997 to 2018 were evaluated. ResultsLocalized disease (LD) was diagnosed in 39 patients. The median age at diagnosis was 2.5 months (range 0–12 months). Expert pathologic review confirmed the diagnosis of spindle cell RMS in all patients. Among 26 tumors that had molecular evaluation, 13 had rearrangements of NCOA and/or VGLL. Multimodal treatment of infants with LD included conventional (age adjusted) chemotherapy (n = 37), resection (n = 31) and radiotherapy (RT) (n = 5, brachytherapy in 3). Complete remission was achieved in 37/39 patients. Progressive disease occurred in two infants, relapsed disease in three. Microscopically complete surgical resection was associated with five-year event-free survival (EFS) and overall survival (OS) of 100%. Two patients with tumors ≤ 5 cm were treated with microscopically complete resection only and were alive 1 and 4.2 years after diagnosis. The 5-year EFS and OS for infants with LD were 86% (±11; CI 95%) and 91% (±9; CI 95%), respectively. One patient had metastatic disease (NCOA fusion positive) with primary tumor in head and neck and brain metastases. This patient died despite chemotherapy and delayed resection of the primary tumor due to respiratory failure secondary to cytomegalovirus infection 1.2 years after diagnosis. ConclusionInfants with spindle cell RMS have an excellent prognosis. Multimodal treatment including microscopically complete resection of the tumor is strongly recommended.

Genomic analysis and preclinical xenograft model development identify potential therapeutic targets for MYOD1-mutant soft-tissue sarcoma of childhood.

The myogenic differentiation 1 gene (MYOD1) p.L122R somatic mutation was first discovered in a subset of clinically aggressive embryonal rhabdomyosarcomas and has since been described in both pediatric and adult spindle cell/sclerosing rhabdomyosarcomas. Relatively little is known about the clinical, molecular, and histopathological features of these tumors in children. In order to further characterize the genomic and clinical features of pediatric MYOD1-mutant sarcomas, we evaluated a cohort of soft-tissue sarcoma patients treated at Texas Children's Hospital. Tumor DNA was subjected to next-generation panel sequencing and/or Sanger sequencing of the MYOD1 hotspot mutation. The MYOD1 p.L122R mutation was identified in six tumors, with a variant allele fraction greater than 0.8 in three cases, suggestive of loss of heterozygosity. One sclerosing rhabdomyosarcoma lacking the MYOD1 hotspot mutation was observed to have a MYOD1 copy number gain, also with evidence of loss of heterozygosity. Cancer gene panel sequencing revealed potentially targetable alterations in six of seven (86%) patients with MYOD1 alterations, including four patients with an alteration in the PI3K-AKT pathway: two hotspot PIK3CA mutations and deletions in PTEN and TSC2. On histopathologic review, MYOD1-altered tumors exhibited spindle and/or round cells and varying degrees of hyaline sclerosis. At last follow-up, six patients had died of disease and the seventh progressed early and was subsequently lost to follow-up. Both pre- and post-therapy patient-derived xenograft models were generated from one patient's tumor. These models were confirmed to harbor the MYOD1 and PIK3CA mutations seen in the primary tumor and were shown to be sensitive to PI3K/mTOR inhibition in vitro and in vivo. In conclusion, this study adds to recent reports describing the clinicopathologic and genomic features of MYOD1-altered soft-tissue sarcomas in children, including dismal prognosis and potential molecular targets for therapy. The novel preclinical models developed will facilitate further biological and preclinical study of this rare and aggressive tumor. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Establishment and characterization of NCC-ssRMS2-C1: a novel patient-derived cell line of spindle cell/sclerosing rhabdomyosarcoma.

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma (RMS) that has fascicular spindle cell and/or sclerosing morphology. SsRMS has a diverse molecular background and is categorized into three groups: congenital/infantile ssRMS with a gene fusion involving the NCOA2 and VGLL2, ssRMS with the MYOD1 mutation, and ssRMS with no recurrent identifiable genetic alterations. Because ssRMS is a newly defined disease concept of RMS, the optimal treatment methods have not been determined. This results in unfavorable prognosis and consequently signals the urgent need for continuous research. Patient-derived cell lines are essential tools in basic and translational research. However, only two ssRMS cell lines with the MYOD1 mutation have been reported to date. Thus, we established a novel ssRMS cell line named NCC-ssRMS2-C1 using a surgically resected tumor tissue from an adult ssRMS patient. NCC-ssRMS2-C1 cells retained the copy number alterations corresponding to the original tumor and are categorized into the group with no recurrent identifiable genetic alterations. NCC-ssRMS2-C1 cells demonstrated constant proliferation, spheroid formation, and capability for invasion in vitro, reflecting the malignant features of the original tumor tissue. In a drug screening test, ssRMS demonstrated remarkable sensitivity to romidepsin, trabectedin, actinomycin D, and bortezomib. Hence, we conclude that the NCC-ssRMS2-C1 cell line is the first ssRMS cell line which belongs to the group with no recurrent identifiable genetic alterations, and it will be a useful resource in both basic and translational studies for ssRMS.

MYOD1 as a prognostic indicator in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis. To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (<18years of age) and adult (age range ≥18 to 35years) ERMS and SRMS cases for presence or absence of MYOD1 immunoreactivity in correlation with clinical outcome and MYOD1 L122R mutations. Lack of MYOD1 immunoreactivity, identified in 23.8% of nonalveolar RMS (non-ARMS) cases, was more prevalent in SRMS (44%) than ERMS (17.2%) and was significantly associated with low overall survival and unfavorable tumor sites (p<.05). Lack of MYOD1 immunoreactivity was not associated with MYOD1 L122R mutations, which were identified in 3/37 (8%) cases including only two of 31 (6.5%) pediatric cases, one of 11 or 9% pediatric SRMS, and one case of infant ERMS. These studies highlight the prognostic role of MYOD1 in non-ARMS. Lack of MYOD1 immunoreactivity is associated with poor prognosis in ERMS and SRMS. MYOD1 gene mutations are generally infrequent in pediatric RMS. Although mutations are predominant in SRMS, they may exceptionally occur in infantile ERMS.

Novel Gene Fusions in Rhabdomyosarcoma

Abstract Introduction/Objective Rhabdomyosarcoma (RMS) encompasses a heterogeneous group of tumors with striated muscle morphology and/or immunophenotype. Molecular analysis of rhabdomyosarcoma has identified a number of aberrations useful for classification. Initially, fusion associated RMS was restricted to tumors with FOXO1 rearrangements, confirming the diagnosis of alveolar RMS, and identifying patients with poor-outcome. Spindle cell RMS, have been described with VGLL2 fusions, EWSR1/FUS-TFCP2 rearrangements, and myoD1 mutations. In addition, NCOA2-MEIS1 fusion gene was recently described in two primary intraosseous RMSs sharing primitive histology. Herein we report two cases of spindle cell RMS, harboring different novel fusion genes, one presenting EP300-VGLL3, and the second with NCOA2-MEIS1 and CAV1-MET. Methods Two cases of RMS were retrieved from the pathology files at the University of Miami Hospital (UMH) and Jackson Memorial Hospital (JMH). Morphology and immunohistochemistry were reviewed. A commercial genomic profiling test for both cases was also analyzed. The study was approved by the Institutional Review Board at UMH. Results Case #1 is a 36-year-old man presented with a 1.5 cm submucosal mass in the ventral left tongue. The biopsy revealed a malignant spindle cell neoplasm and immunohistochemistry showed tumor cells positive for desmin, SMA, myogenin and MYOD-1. A partial glossectomy was performed, achieving negative resection margins. Next- generation sequencing (NGS) identified an EP300-VGLL3 fusion. The patient is currently being followed and has been disease-free for 6 months. Case #2 is a 19-years-old male with no medical history admitted with lower extremities weakness and a large expansile pelvic bone lesion and multiple spinal metastases. Biopsy from the pelvic lesion revealed malignant primitive round cells and short spindle cells arranged in fascicles which were positive for desmin, MYOD-1, TLE-1 and CD99 and focally positive for myogenin and WT-1. NGS identified two fusion genes, NCOA2- MEIS1 and CAV1-MET. Conclusion Different types of rhabdomyosarcoma are associated with a variety of different gene fusions and some may be of prognostic significance. Our two cases are the first reported with EP300-VGLL3 and CAV1-MET fusion genes. CAV1-MET oncogenic potential has been described only in lung cancer. The prognostic significance of rhabdomyosarcoma with novel gene fusions requires longer follow-up and the identification of additional cases.

Spindle cell/sclerosing rhabdomyosarcoma: a clinicopathological study of 20 cases

Objective: To study the clinicopathological features and immunophenotype of spindle cell/sclerosing rhabdomyosarcoma (SRMS) in adults and children, as well as its correlation with the expression and gene-mutations of MYOD1. Methods: Twenty cases of SRMS were collected at Xijing Hospital, Fourth Military Medical University from 2009 to 2019. These cases were evaluated for clinical, pathological, and immunohistochemical features. MYOD1 gene sequencing was performed on 12 cases with available tissue and sufficient DNA quantity using Sanger sequencing. Results: The 20 patients included 12 children and 8 adults, 11 males and 9 females, with an age range of 8 months to 85 years (mean 22 years). Most of them presented with a painless, progressively enlarged solid mass. The tumors occurred in head and neck (7 cases), abdominal and pelvic cavity (7 cases, including 4 in abdominal cavity, 2 in pelvic cavity, 1 in abdominal and left thoracic cavity), upper limb (5 cases, including 2 in left shoulder, 1 in right armpit, 1 in right humerus, and 1 in left forearm), and the back (1 case). The diameter of these tumors ranged from 2.5 to 20 cm, with a mean of 6.2 cm. Histologically, all of the tumors were mainly composed of spindle cells arranged in fascicles, and in 7 cases, at least in part, arranged in herringbone pattern, resembling adult fibrosarcoma. Foci reminiscent of interstitial sclerosing were presented in 4 cases, pseudovascular structures in 2 cases, loosely myxoid stroma in 4 cases, and varying degree of necrosis in 9 cases. A various number of spindled or polygonal rhabdomyoblasts were observed between spindle cells in 3 cases.Among them,16 cases showed spindle cell morphology, 2 cases showed scleroisng morphology, and 2 cases showed a hybrid phenotype of spindle, sclerosing and primitive undifferentiated areas. Immunohistochemically, the tumor cells were positive for desmin, Myogenin and/or MyoD1, but negative for CKpan, ALK1, CD34, EMA, HMB45, SMA, H-cald and S-100. Four cases (4/12) harbored a homozygous or heterozygous MYOD1 (p.L122R) mutation. MYOD1-mutant SRMS usually had diffuse and strong nuclear MyoD1 positivity. Follow-up was available in 12 cases, ranged from 1 to 51 months. At the end of follow-up period, 3 patients died of the disease, 3 patients developed local recurrences, 2 patients survived with disease. Conclusions: SRMS is a rare type of rhabdomyosarcoma, and more commonly occurs in the head and neck of children than adults. MYOD1-mutant SRMS usually had diffuse and strong nuclear MyoD1 positivity, frequently associated with a more aggressive behavior.

Soft Tissue Special Issue: Skeletal Muscle Tumors: A Clinicopathological Review.

Skeletal muscle tumors are classified into rhabdomyoma and embryonal, alveolar, spindle cell/sclerosing and pleomorphic rhabdomyosarcoma according to WHO classifications of tumors. These tumors arise mostly in the head and neck and, in childhood, represent the largest subset of soft tissue tumors. Although these skeletal muscle tumors show common immunoexpression of two myogenic regulatory factors, MyoD1 and myogenin, their molecular biological backgrounds are quite different. Therefore, treatment regimens vary a great deal depending on the histological subtype. Histopathologically, rhabdomyoma is characterized by well-demarcated lesions with no invasion of the surrounding tissue. Embryonal rhabdomyosarcoma is composed of primitive mesenchymal cells in various stages of myogenesis and shows heterogeneous nuclear staining for myogenin. Alveolar rhabdomyosarcoma, on the other hand, shows a proliferation of uniform primitive round cells arranged in alveolar patterns. The tumor cells at the periphery of alveolar structures adhere in a single layer to the fibrous septa. Diffuse and strong nuclear immunoexpression for myogenin is observed. In genetic backgrounds, almost all alveolar rhabdomyosarcomas contain a characteristic fusion gene such as PAX3/7-FOXO1. Spindle cell/sclerosing rhabdomyosarcoma is characterized by fascicularly arranged spindle-shaped cells or dense hyalinized collagenous matrix. NCOR2- or VGLL2-related gene fusions or MYOD1 (p.L122R) mutation is commonly recognized. Epithelioid rhabdomyosarcoma is a rare variant of rhabdomyosarcoma that shows a proliferation of epithelioid tumor cells having large vesicular nuclei, prominent nucleoli, and amphophilic to eosinophilic cytoplasm arranged in sheets. As these characteristic histological and molecular features are present in each subtype, it is possible to diagnose skeletal muscle tumors accurately.

Cytogenetic and Molecular Study of an Adult Sclerosing Rhabdomyosarcoma of the Extremity: MYOD1-mutation and Clonal Evolution

Spindle cell/sclerosing rhabdomyosarcoma is a genomically heterogeneous, uncommon subtype of rhabdomyosarcoma, particularly rare in adults. Its MYOD1-mutant variant is aggressive irrespective of age. Cytogenetic data on spindle cell/sclerosing rhabdomyosarcoma are sparse and disparate. Cytogenetic and molecular analyses were performed on an adult sclerosing rhabdomyosarcoma. The karyotype of the sclerosing rhabdomyosarcoma displayed clonal evolution corresponding to two hyperdiploid clones: 48,XY,+i(19)(p10),+22/48,idem,der(9)t(2;9)(q21~22;p21). The changes were gain of chromosome 19 with the overrepresentation of 19p arm, gain of chromosome 22, gain of the 2q arm, and loss of 9p21. Mutation analysis revealed a homozygous c.T365G (p.L122R) mutation of the MYOD1 gene, but none of PIK3CA. To our knowledge, this is the first adult MYOD1-mutant sclerosing rhabdomyosarcoma studied cytogenetically. The only other reported sclerosing rhabdomyosarcoma with MYOD1 mutation and abnormal karyotype was pediatric. Since these tumors are highly aggressive, further studies unravelling their cytogenetic and molecular characteristics are warranted.

The current landscape of rhabdomyosarcomas: an update.

Rhabdomyosarcomas are malignancies associated with a rhabdomyoblastic phenotype which can be demonstrated morphologically or by immunohistochemistry for MYOD1 and myogenin. Rhabdomyosarcomas are currently subdivided into 4 types in the 2013 WHO classification of tumors of soft tissue and bone, including embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, spindle cell/sclerosing rhabdomyosarcoma, and pleomorphic rhabdomyosarcoma. Recent studies have significantly impacted this classification with the emergence of three distinct new subtypes of rhabdomyosarcomas, namely rhabdomyosarcoma with MYOD1 mutations, rhabdomyosarcoma with TFCP2 fusions, and rhabdomyosarcoma with VGLL2/NCOA2 fusions. Although all these tumors share the terminology "rhabdomyosarcoma," their morphology, clinical behavior, and underlying molecular alterations are dramatically different. Finally, the presence of a rhabdomyoblastic phenotype within a tumor is by no means a diagnostic of a rhabdomyosarcoma, as this may be seen in many other mesenchymal malignancies, such as mesenchymal chondrosarcomas, malignant peripheral nerve sheaths tumors, and biphenotypic sinonasal sarcomas. In this review, we present the main clinical, morphological, and molecular features of these tumors and discuss the evolution of the current classification.

Beyond "Triton": Malignant Peripheral Nerve Sheath Tumors With Complete Heterologous Rhabdomyoblastic Differentiation Mimicking Spindle Cell Rhabdomyosarcoma.

Spindle cell rhabdomyosarcoma (RMS) is an aggressive sarcoma type with a predilection for the head and neck and frequent transactivating MYOD1 mutations. Malignant peripheral nerve sheath tumors (MPNST) show heterologous (most often rhabdomyoblastic) differentiation in 10% of cases; such tumors have been referred to as malignant "Triton" tumors. MPNST frequently harbors inactivating mutations in SUZ12 or EED, resulting in PRC2 dysfunction and loss of histone H3 lysine 27 trimethylation (H3K27me3), most often seen in sporadic and radiation-associated, high-grade tumors; immunohistochemistry (IHC) for H3K27me3 is a useful diagnostic marker. We recently encountered a tumor showing H3K27me3 loss but with otherwise typical features of spindle cell RMS. The purpose of this study was to evaluate H3K27me3 in spindle cell RMS and further investigate putative spindle cell RMS with loss of H3K27me3. IHC for H3K27me3 was performed on 50 tumors diagnosed as spindle cell RMS. Targeted sequencing of all exonic and selected intronic regions of ~450 genes was performed on the tumors with H3K27me3 loss using hybrid capture with a custom probe set and massively parallel (next-generation) sequencing (NGS). Of the 50 patients, 32 were male and 18 were female with a median age of 33 years (range, 6 wk to 77 y). Tumors most often involved head and neck (N=23), extremities/limb girdles (N=11), and trunk wall (N=5). Three cases (6%) showed loss of H3K27me3; based on all available evidence, we believe at least 2 of these cases in fact represent MPNST with complete heterologous rhabdomyoblastic differentiation: a deep-seated groin mass in a 76-year-old female and a paratesticular mass in a 22-year-old male (neither of whom had a history or signs of type 1 neurofibromatosis). The tumors showed similar histologic appearances: fascicular architecture, marked nuclear atypia, eosinophilic cytoplasm, and a high mitotic rate; rhabdomyoblasts were not apparent. One tumor showed focal areas with scant myxoid stroma and alternating hypocellularity and hypercellularity. By IHC, the tumors showed diffuse staining for desmin, myogenin, and MyoD1, whereas S100 protein and SOX10 were negative. NGS on 2 tumors revealed (1) 2-copy deletion of NF1, CDKN2A, and SUZ12 and a TP53 mutation with arm-level loss of 17p; and (2) 2-copy deletion of CDKN2A and an NF1 mutation with loss of 17q11, findings characteristic of MPNST. NGS on the third tumor showed no distinctive alterations. MPNST may occasionally show complete heterologous rhabdomyoblastic differentiation without histologic evidence of residual conventional MPNST, closely mimicking spindle cell RMS. IHC for H3K27me3 reliably distinguishes MPNST from spindle cell RMS.

CONGENITAL-INFANTILE SPINDLE CELL AND SCLEROSING RHABDOMYOSARCOMAS: UNIQUE VARIANTS DEfiNED BY MOLECULAR FEATURES

Objectives Congenital-infantile spindle cell (SpRMS) and sclerosing (ScRMS) rhabdomyosarcomas with tumor-defining molecular features in the head and neck region will be described. These tumors may be confused with more commonly occurring spindle cell tumors (myofibroma, infantile fibrosarcoma) in infants. NCOA2 and VGLL2 rearrangements, and MyoD1 mutations are characteristically identified in SpRMS and ScRMS. NCOA2 and VGLL2 rearrangements are more common in SpRMS, while MyoD1 mutations are more common with ScRMS. NCOA2 or VGLL2 RMS tend to have favorable outcomes, but MyoD1 mutation RMS may have aggressive disease with dismal outcome. Findings 5 neonates and infants were diagnosed with head and neck SpRMS (n=3, 2 males, 1 female, ages 2 weeks to 6 months, 2 maxillary sinus, 1 neck) and ScRMS (n=2, 2 males, ages 5 weeks and 8 months, 1 perinasal, 1 mandible). SpRMS were characterized by malignant spindle cells that were compactly apposed, and closely resembled infantile fibrosarcoma. ScRMS were composed of small round cells in a prominent sclerotic matrix. Both SpRMS and ScRMS lacked rhabdomyoblastic differentiation on HE while NCOA2 RMS responded favorably to oncologic management. Conclusions SpRMS and ScRMS occurring in neonates and infants require molecular characterization for diagnosis, initiating appropriate oncologic and surgical management, and predicting outcome. SpRMS may mimic infantile fibrosarcoma closely, and it is recommended that spindle cell tumors in neonates and infants be assessed for myogenic differentiation.

MYOD1-mutant spindle cell and sclerosing rhabdomyosarcoma: an aggressive subtype irrespective of age. A reappraisal for molecular classification and risk stratification

Sclerosing and spindle cell rhabdomyosarcoma is a rare histologic subtype, designated in the latest WHO classification as a stand-alone pathologic entity. Three genomic groups have been defined: an infantile subset of spindle cell rhabdomyosarcoma harboring VGLL2-related gene fusions, a MYOD1-mutant subset commonly associated with sclerosing morphology, and a subset lacking recurrent genetic abnormalities. In this study, we focus on MYOD1-mutant rhabdomyosarcoma to further define their clinicopathologic characteristics and behavior in a larger patient cohort. We investigated 30 cases of MYOD1-mutant rhabdomyosarcoma (12 previously reported and 18 newly diagnosed) with an age range of 2–94 years, including 15 children. All cases showed morphology within the spectrum of spindle cell/sclerosing rhabdomyosarcoma (8 cases showing pure sclerosing morphology, 8 cases showing pure spindle cell morphology and 14 cases showing a hybrid phenotype of spindle, sclerosing and primitive undifferentiated areas). All tumors harbored either homozygous or heterozygous MYOD1 (p.L122R) exon 1 mutations. In 10 (33%) cases, a co-existent PIK3CA mutation was identified. Hot-spot mutations in NRAS and HRAS were each identified in a single case, respectively. Follow-up was available on 22 (73%) patients with a median duration of 28 months. Local recurrence was seen in 12 (55%) and distant recurrence in 12 (55%) cases, despite multimodality chemoradiation therapy. At last follow-up, 15 (68%) patients died of the disease, one patient was alive with disease and five had no evidence of disease. The prognosis was equally poor in pediatric and adult patients. In conclusion, MYOD1 mutation defines an aggressive rhabdomyosarcoma subset, with poor outcome and response to therapy, irrespective of age. Given that this distinct molecular subtype is characterized by an aggressive biologic behavior compared to other genetic subtypes of spindle and sclerosing rhabdomyosarcoma, the MYOD1 genotype should be used as a molecular marker in both subclassification and prognostication of rhabdomyosarcoma.

MYOD1 (L122R) mutations are associated with spindle cell and sclerosing rhabdomyosarcomas with aggressive clinical outcomes

Recurrent mutations in the myogenic transcription factor MYOD1 and PIK3CA were initially described in a subset of embryonal rhabdomyosarcomas. Recently, two independent studies demonstrated presence of MYODI (L122R) mutations as the basis to re-classify a spindle cell rhabdomyosarcoma, along with a sclerosing rhabdomyosarcoma, distinct from an embryonal rhabdomyosarcoma. We analyzed a much larger cohort of 49 primary rhabdomyosarcoma tumor samples of various subtypes, collected over a period of 9 years, for the presence of MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations, along with immunohistochemical analysis of desmin, myogenin, and MYOD1. Although activating PIK3CA mutations were absent across the sample set analyzed, we report 20% MYOD1 (L122R) mutation in rhabdomyosarcomas, found exclusively in 10 of 21 spindle cell and sclerosing rhabdomyosarcomas, occurring mostly in the head and neck region along with extremity sites (64%), than the paratesticular and intra-abdominal sites. Furthermore, while all 10 MYOD1 mutant spindle cell and sclerosing rhabdomyosarcoma samples showed diffuse and strong MYOD1 immunoexpression, 7 of 31 samples of rhabdomyosarcoma with wild-type MYOD1 were negative for MYOD1 expression. Clinically, a striking correlation was found between MYOD1 mutation and the clinical outcomes available for 15 of 21 cases: 5 of 7 patients with spindle cell and sclerosing rhabdomyosarcomas, harboring MYOD1 mutation, were alive-with-disease and 2 of 8 patients with spindle cell and sclerosing rhabdomyosarcomas, with mutant MYOD1, were free-of-disease. Taken together, we present the first report of MYOD1 (L122R) mutation in the largest cohort of 49 rhabdomyosarcomas reported so far, that are associated with a relatively aggressive clinical course. Moreover, consistent with the earlier two studies, this study further reinforces a relationship between spindle cell and the sclerosing rhabdomyosarcoma—now recognized as a single subtype, distinct from an embryonal rhabdomyosarcoma.

Inside the USCAP Journals

Inside the USCAP Journals

A clinicopathologic study of head and neck rhabdomyosarcomas showing FOXO1 fusion-positive alveolar and MYOD1-mutant sclerosing are associated with unfavorable outcome

BackgroundBased on their distinctive histologic and genetic features, the latest WHO classification of soft tissue tumors includes four pathologic variants of rhabdomyosarcoma (RMS): embryonal (ERMS), alveolar (ARMS), spindle cell-sclerosing (SRMS-ScRMS) and pleomorphic RMS. The aim of this study focused on a detailed clinicopathologic and survival analysis of head and neck RMS (HNRMS) using the latest pathologic and molecular criteria reflecting this new subclassification in a large cohort. Patients and methodsPatients managed for HNRMS in our institution (1996–2015) were analyzed. The presence of a FOXO1 fusion was required for the classification of ARMS. MYOD1 mutations in SRMS-ScRMS were tested when material available. Univariate and multivariate analyses were performed to evaluate variables related to overall survival (OS). ResultsNinety-nine HNRMS patients (52 males and 47 females, mean of 16years) were included in the study after pathologic re-review. The most common location was parameningeal (PM) (n=64), followed by non-orbital/non-PM (n=25) and orbital (n=10). There were 53 ERMS, 33 fusion-positive ARMS and 13 SRMS-ScRMS [SRMS (8); ScRMS (5)]. The 5-year OS rate for ERMS patients was significantly higher (82%) compared to ARMS (53%) and SRMS-ScRMS (50%) [SRMS (75%); ScRMS (30%)]. Univariate analysis showed that survival was dependent on histology (P=0.012), tumor size >5cm (P<0.001), regional lymph node involvement (P=0.002), metastasis at initial presentation (P<0.001), stage (P<0.001), and recurrence (P=0.002). Multivariate analysis confirmed histologic subtype to be significant (P=0.043). ConclusionOur findings reinforce that HNRMS is a heterogenous disease with ARMS and SRMS-ScRMS having an equally unfavorable outcome.