Spindle Cell Variant of uterine embryonal Rhabdomyosarcoma: therapeutic and diagnostic challenges in low resource setting

Rhabdomyosarcoma (RMS), a rare malignant tumor, frequently affects pediatric patients, with 35%-40% occurring in the head and neck. This study analyzes the clinicopathologic profile of pediatric head and neck rhabdomyosarcomas from Brazil, Guatemala, Mexico, and South Africa. We reviewed 44 cases from 10 Oral and Maxillofacial Pathology services, conducting immunohistochemical analyses of desmin, myogenin, Myo-D1, and Ki67, with quantification via QuPath software. Cases with ≥ 50% myogenin expression were tested for fusion status using AP2β, NOS-1, and HMGA2. Statistical analyses included the Kruskal-Wallis test for age and marker expression comparisons, Fisher's exact test for categorical variables, Spearman's rank correlation for marker relationships, and multinomial logistic regression to assess fusion status likelihood. Cases were predominantly from Brazil (40.9%), followed by South Africa (27.3%), Guatemala (22.7%), and Mexico (9.1%). Two-thirds of patients were diagnosed in their first decade with no gender predilection. Nonparameningeal sites (45.5%) were more affected than parameningeal (40.9%) and orbital sites. Microscopically, embryonal RMS (77.3%) was most common, followed by alveolar (18.2%) and spindle cell (2.3%) tumors. Immunohistochemistry revealed positivity for myogenic markers, with significant differences in myogenin expression between embryonal and alveolar RMS variants (p < 0.05). Fusion status prediction identified two potential fusion-positive alveolar RMS cases, while all embryonal RMS and one alveolar RMS case appeared fusion-negative. Significant correlation with positive fusion status was found only between AP2β and NOS1 (p < 0.05). Although there are slight clinical-demographic variations among pediatric head and neck rhabdomyosarcomas in these regions, identifying fusion status through immunohistochemistry remains a diagnostic challenge.

The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrificed after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunohistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunohistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a complete eradication of the tumors. Chemotherapy seemed to upregulate the expression of resistance-associated genes in vitro and in vivo. The mechanism of multidrug resistance depends on the tumor subtype. Therefore, further investigations will be required to evaluate multidrug resistance in patients and to investigate new modalities for a reversal of multidrug resistance.

Embryonal rhabdomyosarcoma (ERMS) of the uterus has recently been shown to frequently harbor DICER1 mutations. Interestingly, only rare cases of extrauterine DICER1-associated ERMS, mostly located in the genitourinary tract, have been reported to date. Our goal was to study clinicopathologic and molecular profiles of DICER1-mutant (DICER1-mut) and DICER1-wild type (DICER1-wt) ERMS in a cohort of genitourinary tumors. We collected a cohort of 17 ERMS including nine uterine (four uterine corpus and five cervix), one vaginal, and seven urinary tract tumors. DNA sequencing revealed mutations of DICER1 in 9/9 uterine ERMS. All other ERMS of our cohort were DICER1-wt. The median age at diagnosis of patients with DICER1-mut and DICER1-wt ERMS was 36 years and 5 years, respectively. Limited follow-up data (available for 15/17 patients) suggested that DICER1-mut ERMS might show a less aggressive clinical course than DICER1-wt ERMS. Histological features only observed in DICER1-mut ERMS were cartilaginous nodules (6/9 DICER1-mut ERMS), in one case accompanied by foci of ossification. Recurrent mutations identified in both DICER1-mut and DICER1-wt ERMS affected KRAS, NRAS, and TP53. Copy number analysis revealed similar structural variations with frequent gains on chromosomes 2, 3, and 8, independent of DICER1 mutation status. Unsupervised hierarchical clustering of array-based whole-genome DNA methylation data of our study cohort together with an extended methylation data set including different RMS subtypes from genitourinary and extra-genitourinary locations (n = 102), revealed a distinct cluster for DICER1-mut ERMS. Such tumors clearly segregated from the clusters of DICER1-wt ERMS, alveolar RMS, and MYOD1-mutant spindle cell and sclerosing RMS. Only one tumor, previously diagnosed as ERMS arising in the maxilla of a 6-year-old boy clustered with DICER1-mut ERMS of the uterus. Subsequent sequencing analysis identified two DICER1 mutations in the latter case. Our results suggest that DICER1-mut ERMS might qualify as a distinct subtype in future classifications of RMS.

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.

Loss of H3K27me3 occurs in a large subset of embryonal rhabdomyosarcomas: Immunohistochemical and molecular analysis of 25 cases

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Pathologic quiz case: Ovarian mass in a 2-year-old girl presenting with pleural effusions.

Uterine sarcomas.

Rhabdomyosarcoma is the fifth most common type of soft tissue solid tumor in children and the most common in the last two decades. Rhabdomyosarcoma of the urogenital organ is a rare mesenchymal tumor, covering 22% of all Rhabdomyosarcoma cases. The two most common histologic types are alveolar and embryonal, whereas botryoid and spindle cells are rarely found. We reported a case of embryonal Rhabdomyosarcoma of the prostate. In this study, we improved the understanding of Embryonal Rhabdomyosarcoma of the prostate on 23 years old male who had a history of swelling in the perineal area and a history of falling from a height in the groin area 5 months before. The patient complains of the difficulty of urinating and hematuria one month after falling. We underwent drainage of the swelling area, found pus 100cc and took a sample for pathological examination. The result was embryonal rhabdomyosarcoma. Prostate volume was 122cc, PSA 5,32 and PSAD 0,04. The CT scan result was solid mass size 15x8x18 cm at perineum enhance to the pelvic cavity, push the bladder to the superior, rectum to posterior, and urethra posterior to the left side. We diagnosed this patient as Embryonal Rhabdomyosarcoma prostate T2bG1N0M0 (stage 3) group 3 and intermediate-risk group. The patient underwent VAC chemotherapy based on D.9803 (IRS V) protocol and planned radiotherapy, but it stopped at halfway because of profuse bleeding. Embryonal Rhabdomyosarcoma is an aggressive tumor, especially in adults. However, there was limited evidence and guideline to diagnose and manage the disease.

DICER1-associated sarcomas: towards a unified nomenclature.

BackgroundEmbryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.MethodsEmbryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.ResultsHere we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.ConclusionsThese results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

We present the largest series of an unclassified subtype of renal cell carcinoma, which seems to be a distinct morphological entity and which is sometimes designated as spindle and cuboidal renal cell carcinoma. Eleven cases of spindle and cuboidal renal cell carcinoma were found among 7000 primary renal cell tumours in Pilsen's routine and consultation files. The patients were five men and six women. They ranged in age from 22 to 65 years (mean 56.8). Microscopically, the tumours were composed of two main populations of cells. First, the preponderant type of cells was formed by flattened, spindle cells with sparse cytoplasm. The second cell type was a small cuboidal cell with clear to light eosinophilic cytoplasm. Spindle-shaped cells were arranged in a fascicular pattern often reminiscent of low-grade smooth muscle tumours. Solid areas of spindle cells were also present. Small cuboidal cells formed sparse tubular structures lined by a row of single cells. In addition to all previous published cases of spindle and cuboidal renal cell carcinoma we observed an association of nephrolithiasis in our cases. It was seen in 3/11 of our patients. A previously unreported feature is the occurrence of a conventional renal cell carcinoma component in one of our cases. Seven of our patients are currently well without signs of recurrence or metastasis, one had metastasis in a regional lymph node at the time of nephrectomy, one died of an unrelated condition, and two were lost to follow-up. We present 11 cases of spindle and cuboidal renal cell carcinoma, which is believed to be a distinctive morphological entity. Our cases were histologically, immunohistochemically and ultrastructurally similar to the previously reported cases of spindle and cuboidal renal cell carcinoma. In contrast to all previously reported cases of spindle and cuboidal renal cell carcinoma, we observed an association with nephrolithiasis in three of our cases; moreover, one of our tumours had a conventional renal cell carcinoma component and another revealed a metastatic focus in a regional lymph node. None of our patients died of the disease. This study confirms that spindle and cuboidal renal cell carcinoma has a low malignant potential.

To present descriptive statistics for bladder tumours in children, calculated from the Surveillance, Epidemiology and End Results (SEER) database, as bladder malignancies are relatively uncommon in children, causing difficulty in understanding their incidence and survival. The SEER database was interrogated to report the incidence of bladder malignancies in children from birth to 18 years old. Race and sex differences were assessed. The 5-year survival by disease stage at diagnosis and 5-year conditional survival after surviving for 1-3 years is reported for 1973-2003. The incidence and survival rates for bladder embryonal rhabdomyosarcoma were further characterized. We identified 140 cases of bladder cancers in the selected cohort. Papillary urothelial neoplasm of low malignant potential (PUNLMP) and rhabdomyosarcoma comprised 50.7% and 36.4% of the tumours, respectively. The incidence of bladder malignancies significantly increased between 1973 and 2003. Conditional survival calculated for 1 and 2 years after disease diagnosis was 93.6% and 97.5%. Fifty-one cases of embryonal rhabdomyosarcoma were identified. The male to female incidence ratio was approximately 2:1 for these tumours. The 5-year survival rates were 50-80% over the past three decades. We present the most contemporary survey of the SEER database for the incidence and survival for bladder tumours in children. Depending on the patient's age, PUNLMP and rhabdomyosarcoma predominated in different proportions. The 2-3-year conditional survival and overall survival were excellent. We also identified improving survival for embryonal bladder rhabdomyosarcoma with time.

Infant malignant ectomesenchymoma masquerading as inguinal hernia in two patients

Introduction: Pediatric sarcomas are challenging to accurately classify due to their rarity and the wide diversity of subtypes. The process requires highly specialized pathologists as well as molecular and genetic testing that is expensive, takes time, and is not universally available. Deep neural network models (DNNs) trained on histopathology slides can reduce the time and cost to diagnosis and attenuate disparities in care based on geographical location and socioeconomic status. Here, we demonstrate the efficacy of automated image analysis for assigning sarcoma diagnoses across centers by identifying Ewing Sarcoma (ES), distinguishing rhabdomyosarcoma (RMS) vs non-rhabdomyosarcoma soft tissue sarcomas (NRSTS), as well as classifying alveolar, embryonal, and spindle cell RMS subtypes. Methods: Images were collected from Massachusetts General Hospital, Yale School of Medicine, St Jude Children’s, and the Children’s Oncology Group. A total of 691 images were collected across all centers, including 9 different sarcoma subtypes. Images were harmonized using our published STQ pipeline including focus checking, resolution standardization, and stain normalization. Image tiling and feature extraction was performed comparing multiple deep learning backbones (CTransPath, UNI, CONCH). Tile-level features were transposed to whole slide-level representations using our published SAMPLER method in which each feature is represented as the vector of decile values of its distribution across all tiles. Resulting feature sets were fed into logistic regression models for sarcoma classification tasks. We benchmark this approach against transformer based multi-head self-attention models trained on a V100 GPU. Results: We are able to distinguish ES from all other sarcoma types with an AUROC of 0.966. In the task of NRSTS v. alveolar RMS v. embryonal RMS we achieve an AUROC of 0.939. Restricting to RMS subtypes, we distinguish alveolar from embryonal with an AUROC of 0.95. Also, despite uneven sample representation, we obtain an AUROC of 0.88 for alveolar v. embryonal v. spindle type RMS. Finally, we have developed spatially-resolved attention maps, which provide interpretability for the regions of a slide that contain malignant cells. Conclusion: To our knowledge, we have amassed the largest multicenter pediatric sarcoma imaging dataset with broad representation across subtypes, anatomical locations, race, and sex. Our pipeline allows for further integration of images from any center, which may be co-analyzed to attenuate center-specific batch effects. Our classification accuracies are state of the art for multiple tasks fundamental to clinical sarcoma pathology, including novel multiclass distinction among three different RMS subtypes. Importantly, our pipeline and SAMPLER model can be run with minimal computational requirements, allowing for broad accessibility. Citation Format: Adam Thiesen, Sergii Domanskyi, Ali Foroughi pour, Jingyan Zhang, Todd B. Sheridan, Steven B. Neuhauser, Alyssa Stetson, Katelyn Dannheim, Danielle B. Cameron, Shawn Ahn, Hao Wu, Emily R. Christison-Lagay, Carol J. Bult, Jeffrey H. Chuang, Jill C. Rubinstein. Automated classification of pediatric sarcoma using digital histopathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2423.