Genomic profiling of pleomorphic rhabdomyosarcoma reveals a genomic signature distinct from that of embryonal rhabdomyosarcoma

Abstract

Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive sarcoma, occurring mostly in the deep soft tissues of middle-aged adults and showing a variable degree of skeletal muscle differentiation. The diagnosis is challenging as pathologic features overlap with embryonal rhabdomyosarcoma (ERMS), malignant Triton tumor and other pleomorphic sarcomas. As recurrent genetic alterations underlying PRMS have not been described to date, ancillary molecular diagnostic testing is not useful in subclassification. Herein, we perform a genomic profiling of a well-characterized cohort of 14 PRMS, compared to a control group of 23 ERMS and other pleomorphic sarcomas (undifferentiated pleomorphic sarcoma and pleomorphic liposarcoma) using clinically validated DNA-targeted NGS panels (MSK-IMPACT). The PRMS cohort included 8 males and 6 females, with a median age of 53 years (range 31–76 years). Despite similar tumor mutation burdens, the genomic landscape of PRMS, with high frequency of TP53 (79%) and RB1 (43%) alterations, stood in stark contrast to ERMS, with 4% and 0%, respectively. CDKN2A deletions were more common in PRMS (43%) compared to ERMS (13%). In contrast, ERMS harbored somatic driver mutations in the RAS pathway and loss of function mutations in BCOR, which were absent in PRMS. Copy number variations in PRMS showed multiple chromosomal arm level changes, most commonly gains of chr17p and chr22q and loss of chr6q. Notably, gain of chr8, commonly seen in ERMS (61%) was conspicuously absent in PRMS. The genomic profiles of other pleomorphic sarcomas were overall analogous to PRMS, showing shared alterations in TP53, RB1 and CDKN2A. Overall survival and progression-free survival of PRMS were significantly worse (p<0.0005) than that of ERMS. Our findings revealed that the molecular landscape of PRMS aligns with other adult pleomorphic sarcomas and is distinct from that of ERMS. Thus, NGS assays may be applied in select challenging cases towards a refined classification. Finally, our data corroborate inclusion of PRMS in the therapeutic bracket of pleomorphic sarcomas given that their clinical outcomes are comparable.

INTRODUCTION:

Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive pleomorphic sarcoma, occurring almost exclusively in adults and displaying variable morphologic evidence of skeletal muscle differentiation. Most PRMS occur in the deep soft tissue of the lower extremities; rare cases have been reported in a wide variety of sites, including the chest, upper extremities, retroperitoneum and head and neck^1–3. Men are more frequently affected than women, with a median age at diagnosis in the 6^th-7^th decade³. The diagnosis of PRMS remains challenging as morphologically these tumors resemble undifferentiated pleomorphic sarcoma (UPS) more closely than rhabdomyosarcoma (RMS), exhibiting only focal rhabdomyoblastic differentiation, especially in limited biopsy samples². However, due to their morphologic and immunohistochemical overlap, these tumors can be mistaken for embryonal rhabdomyosarcoma (ERMS) with anaplasia, and malignant peripheral nerve sheath tumor with rhabdomyoblastic differentiation (malignant Triton tumor, MTT). The pathogenesis of PRMS is poorly understood, with no large, focused comprehensive genomic studies conducted to date. Historically, cytogenetic studies have revealed a complex karyotype with numerical and unbalanced structural changes, but no recurrent structural alterations⁴. In addition, comparative genomic hybridization has identified multiple chromosome arm level losses (10q23, 15q21–22), gains (1p22–23, 7p) and amplifications, a profile resembling that reported in undifferentiated pleomorphic sarcoma (UPS) and osteosarcoma^5,6. Thus, molecular interrogation in the clinical setting has not been found useful as an adjunct diagnostic test in these lesions. The distinction from ERMS is a common challenge, in particular due to the significant differences in therapeutic strategies applied.

In this study, we apply a conclusive genomic profiling of mutations, gene-level and arm-level copy number alterations, to investigate a well-characterized cohort of PRMS along with a control group of ERMS and other pleomorphic sarcomas, in order to enhance our understanding of their pathogenesis.

MATERIAL AND METHODS:

Case selection and study cohort

After approval from the Institutional Review Board, the files of the Department of Pathology and the http://cbioportal.mskcc.org/ were searched for PRMS cases with available molecular data, between 2013 and 2023. Diagnoses were individually confirmed by review of morphology and supporting immunostains of the biopsies and subsequent resections, when available. Although the overwhelming number of patients were adults, the few cases arising in children were excluded, to ensure its distinction from other RMS types. In addition, the only case of PRMS arising in the gynecologic tract was also excluded. Moreover, cases lacking a pre-therapy biopsy for review, with only the post neoadjuvant chemotherapy resection available showing pleomorphic rhabdomyoblasts, were excluded. Clinical and radiologic data was extracted from the electronic charts. The macroscopic and morphologic features, mitotic count and pre-and post -treatment percentage of necrosis were noted.

A control group of conventional ERMS (n=23) tested on the same targeted sequencing platform was used for comparative analysis. We opted not to include the ERMS botryoides-subtype in our control group due to its distinctive clinical presentation (infants/young children, involving mucosal-lined luminal organs, mostly vaginal), dissimilar pathologic features (grape-like growth, with expansile myxoid stroma and subepithelial ‘cambium layer’ cellular condensation), as well as its distinct genomic landscape, with increased germline and somatic DICER1 alterations⁷, which are very different from PRMS and are rarely included in the differential diagnosis.

Furthermore, a large cohort of other pleomorphic sarcoma cases comprised of UPS (n=273) and pleomorphic liposarcoma (PLPS, n=21) were also included in the comparative genomic evaluation.

Targeted DNA sequencing, mutational profiling and data analysis

Detailed descriptions of MSK-IMPACT workflow and data analysis, a hybridization capture-based targeted DNA NGS assay for solid tumor were described previously ⁸. The samples submitted for MSK-IMPACT were all primary tumor samples, except for case 1 which was a lung metastasis from a laryngeal primary. Data analysis was performed using R version 4.1.0. R package “ComplexHeatmap” version 2.14.0 was used to visualize gene-level mutations and copy number variations⁹.

Survival analysis

The clinical charts were manually reviewed and date of initial presentation, disease progression and survival status were documented. The time to disease progression was defined as the time between initial presentation (first time tumor felt on physical exam or seen on radiology) and first event of tumor recurrence/ metastasis after surgical resection and/or therapy with radiologic evidence of no residual disease. Survival analysis was performed using R packages “survminer” version 0.4.9 and “survival” version 3.2.13 by comparison of hazard ratios using log rank P testing and Kaplan-Meier curves.

RESULTS

Clinico-radiologic findings

Of the 14 patients with PRMS, eight patients were male and six were female with a median age of 53 years (range 31–76 years). All tumors arose in the soft tissue. Case 3 presented with extensive involvement of the tibia and fibula and the surrounding soft tissue; the origin of the tumor could not be determined. The pelvis was the most common site (n=4), followed by soft tissue of the thigh (n=2) and arm (n=2). The tumors ranged from 3.2 to 27.0 cm, with a median size of 8.5 cm. Radiographically, the tumors showed a heterogenous signal intensity on magnetic resonance imaging (MRI) (Supplementary Fig. 1A–1B), with a subset showing central area of non-enhancement corresponding to central necrosis. The tumors were heterogeneously enhancing to intensely FDG-avid on positron emission/computed tomography scans (PET/CT). The clinicopathologic data of PRMS cohort is summarized in Table 1.

Table 1.

Clinical summary and follow-up of Pleomorphic RMS cohort.

Case	Age (y)/Sex	Primary site	Size (cm)	Initial resection margin status	Treatment Modalities	Metastasis/Recurrence	Outcome	Follow-up period (months)
1	51/ M	Larynx-ST	3.5	Not known	- Neoadjuvant RT - Surgical resection - Adjuvant CT (AIM)	Lung	DOD	39
2	49/ F	Pelvis- ST	6.5	Negative	- Neoadjuvant CT (AIM) and RT - Surgical resection	-	NED	95
3	67/ M	Ankle- ST extending to bone	17.0	Negative	- Neoadjuvant CT (VAC/IE) - Surgical resection - Adjuvant CT (docetaxel, gemcitabine)	Lung	DOD	14
4	46/ M	Retropharyngeal-ST	14.1	Not resected	- CT (doxorubicin, olaratumab) and RT	Lung	DOD	22
5	65/ M	Pelvis- ST	12.5	Not resected	- CT (doxorubicin, docetaxel, gemcitabine) and IT (pembrolizumab)	Lung (at presentation)	DOD	19
6	31/ F	Knee- ST	3.2	Negative	- Surgical resection - Adjuvant CT (VAC, mesna)	-	NED	39
7	63/ F	Pelvis- ST	8.0	Negative	- Surgical resection - Adjuvant CT (ifosfamide, mesna)	Spine	DOD	9
8	65/ M	Arm- ST	13	Negative	- Surgical resection - Adjuvant CT (AIM)	Arm (recurrence)	DOD	5
9	76/ M	Thigh- ST	3.4	Negative	- Surgical resection - Adjuvant CT (AIM)	Thigh (recurrence)	AWD	11
10	53/ F	Arm- ST	4.4	Negative	- Neoadjuvant CT (AIM) and RT - Surgical resection	-	NED	7
11	48/ M	Flank- ST	9.0	Positive	- Surgical resection - Adjuvant CT (VDC/IE, mesna)	Lung (at presentation)	DOD	4
12	48/ F	Mesentery- ST	14.4	Negative	- Neoadjuvant CT (AIM) - Surgical resection - Adjuvant CT (gemcitabine, vinorelbine)	Abdomen (recurrence) Lung	DOD	20
13	61/ M	Pelvis- ST	27	Positive	- Neoadjuvant CT (AIM) - Surgical resection	Pelvis (recurrence) Lung	DOD	16
14	53/ F	Thigh- ST	5.2	Negative	- Surgical resection - Adjuvant CT (VDC) and RT	-	NED	5

ST, soft tissue; CT, chemotherapy; RT, radiotherapy; IT, immunotherapy; AWD, alive with disease; DOD, died of disease; NED, no evidence of disease; AIM, doxorubicin, ifosfamide, mesna; VAC, vincristine, actinomycin-D, cyclophosphamide; IE, ifosfamide, etoposide; VDC, vincristine, doxorubicin, cyclophosphamide

The clinicopathologic data of the ERMS control cohort is summarized in Supplementary Table 1. There were 13 males and 10 females, with a median age of 7 years (range=0.8–48 years). The genital tract was the most common site involved (n=10), followed by the pelvis/abdomen (n=7) and head and neck (n=6). The tumors ranged from 1.8 to 15.3 cm, with a median size of 3.8 cm.

Histopathologic features

Macroscopically, the tumors were well-circumscribed, surrounded by a pseudocapsule with a fleshy, tan-white to yellow cut surface and variable areas of hemorrhage and necrosis (Supplementary Fig. 1C–1D). Microscopically, the tumors were composed of loosely arranged cells in a haphazard pattern and showing undifferentiated spindle to epithelioid cells with hyperchromatic, pleomorphic nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (Fig. 1A–1B). Scattered to rare tadpole-shaped cells with densely eosinophilic cytoplasm were present in 13 cases (93%). Cross striations were not seen. One case (case 7) showed a predominant rhabdoid morphology, characterized by sheets of variably sized cells focally arranged in a pseudoalveolar pattern and exhibiting eccentric vesicular nuclei, prominent nucleoli and eosinophilic cytoplasm (Fig. 1C–1D). In one case (case 6), the cells were arranged in a fascicular pattern, resembling pleomorphic leiomyosarcoma (Fig. 2A). Two cases (8 and 12) displayed prominent myxoid stroma, resembling myxofibrosarcoma (MFS), one of which (case 8), was composed exclusively of epithelioid cells with abundant eosinophilic cytoplasm, akin to epithelioid MFS (Fig. 2B–2D). Brisk mitotic activity (14 to 54/10 high-power fields) and tumor necrosis were appreciated (5–40%).

Fig 1A-1D: Histopathologic features of pleomorphic rhabdomyosarcoma (PRMS).

A-B, PRMS characterized by undifferentiated spindle and epithelioid cells with pleomorphic nuclei and abundant eosinophilic cytoplasm, arranged in a loose haphazard pattern. Scattered tadpole to rhabdoid pleomorphic rhabdomyoblasts with dense eosinophilic cytoplasm (A, case 3, 200x; B, case 4, 200x). C-D, PRMS showing a predominant proliferation of variably sized pleomorphic rhabdomyoblasts arranged in sheets and focally in alveolar-like pattern. In addition, multinucleated forms are seen. (case 7, 100x).

Fig 2A-2D: Histopathologic features of pleomorphic rhabdomyosarcoma (PRMS).

A, PRMS composed of spindle cells arranged in fascicular pattern, with scattered hyperchromatic cells, resembling high-grade leiomyosarcoma. No pleomorphic rhabdomyoblasts were seen in this case (case 6, 100x). B, PRMS composed of epithelioid cells with vesicular chromatin and abundant eosinophilic cytoplasm embedded in a myxoid stroma, reminiscent of high-grade epithelioid myxofibrosarcoma. Scattered pleomorphic rhabdomyoblasts present (case 8, 200x). C-D, PRMS showing predominantly high-grade undifferentiated pleomorphic sarcoma-like morphology (C) with focal myxofibrosarcoma-like areas (D) (case 12; C, 200x, D, 100x).

All cases diffusely expressed desmin (Fig. 3A–3B) with limited expression of myogenin (Fig. 3C) and/ or MyoD1 (Fig. 3D). SMA was expressed in 6 of 9 (67%) tested cases and S100 was negative in all tested cases (0/11). Cytokeratin was focally expressed in one of nine tested cases (11%). Immunohistochemical findings are summarized in Supplementary Table 2.

Fig 3A-3D: Immunohistochemical features of pleomorphic rhabdomyosarcoma (PRMS).

PRMS (A) showing diffuse and strong expression of desmin (B) and focal expression of myogenin (C) and MyoD1 (D) (case 9, A-D, 100x).

The genomic landscape of PRMS is distinct from ERMS and is analogous to other pleomorphic sarcomas

The most common genetic alteration observed in PRMS was TP53, present in 11 of 14 (79%) cases (3 deletion, 3 missense, 2 structural variant, 1 nonsense, 1 splicing mutation and 1 with both deletion and missense mutation). Alterations in RB1 gene (4 deletion, 1 structural variant and 1 spicing mutation) and CDKN2A gene deletions were the second most common alterations in PRMS accounting for 43% (6 of 14 cases each). Less frequent alterations included mutations in ARID1B and NOTCH3 genes (14% each). Multiple arm level gains and losses were identified, the most common chromosome arm level copy number gains were in chr17p (29%) and chr22q (21%), while losses in chr6q (21%) was the most common chromosome arm level loss. Three patients had diagnostic molecular testing for germline mutations. Among these (case 4), a 46-year-old male with a retropharyngeal tumor, was found to have a pathogenic heterozygous germline TP53 mutation (exon 5 p.R175H (c.524G>A)).

In stark contrast, ERMS harbored alterations in the RAS pathway including somatic driver mutations in NRAS, HRAS, BRAF, NF1 and FGFR4 (12 cases, 52%). Among those, NF1 (5 cases, 22%) and FGFR4 (3 cases, 13%) were the most commonly altered genes. All cases with FGFR4 alterations harbored hot spot mutations in exon 3 p.V550L (c.1648G>C). One case with FGFR4 hotspot mutation had an additional missense (non-hotspot) mutation in FGFR4 (exon12 p.G528C (c.1582G>T)). Two cases (9%) harbored hotspot mutations in NRAS (exon3 p.Q61K (c.181C>A)), one case (4%) revealed a hotspot mutation in HRAS (exon2 p.G12V (c.35G>T)) and one case showed a hotspot mutation in BRAF (exon15 p.V600E (c.1799T>A)). Moreover, loss of function mutations in BCOR (2 frameshift insertion, 2 frameshift deletion and 1 nonsense mutation) were noted in 5 cases (22%). CDKN2A deletions were less frequent among the ERMS group, accounting for 13% (3 cases). In contrast to PRMS, ERMS showed a high frequency gain of chr8 (61%), which were not present in PRMS (0%). Less frequent chromosome arm-level copy number alterations in ERMS include gain in chr20 (22%) and chr22q (13%). The tumor mutation burden was not significantly different between the two groups. Recurrent genetic mutations and gene-level and arm-level copy number alterations in PRMS and ERMS are summarized in Fig. 4.

Fig 4: Recurrent mutations, gene-level and arm level copy number alterations across pleomorphic rhabdomyosarcoma (PRMS) and embryonal rhabdomyosarcoma (ERMS).

Oncoplot comparing mutations, gene-level and arm-level copy number alterations among PRMS and ERMS.

The genomic profiling of UPS and PLPS revealed overall similar genomic signature showing common alterations in TP53, followed by RB1 and ATRX. Except for a lower frequency of ATRX alterations (7%) and relatively higher frequency of CDKN2A deletion (43%) observed among PRMS, the genomic signature of PRMS is indistinguishable from that of other pleomorphic sarcomas. The tumor mutation burden was not significantly different between the different pleomorphic sarcomas. Recurrent genetic alterations in UPS and PLPS in comparison to PRMS are summarized in Fig. 5.

Fig 5: Recurrent gene-level mutations and copy number alterations across different types of pleomorphic sarcoma.

Oncoplot showing mutations and copy number alterations in undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and pleomorphic liposarcoma.

Outcome and survival

Two patients with PRMS presented with metastatic disease at presentation, while the remaining patients presented with localized disease. All except one PRMS patients were managed by adult sarcoma medical oncologists, while all ERMS patients were treated by pediatric sarcoma medical oncologists including three adult patients. Six patients (43%) with PRMS (age range, 48–67) received neoadjuvant chemo- and/or radiotherapy. The chemotherapy regimens included AIM (doxorubicin, ifosfamide, mesna), VAC (vincristine, actinomycin-D, cyclophosphamide) and IE (ifosfamide, etoposide). All except two patients (86%) underwent surgical resection. Histologic treatment response, seen as fibrosis and necrosis, varied between the cases and ranged from 5 to > 95% with a median of 20%. Nine patients (65%) received adjuvant treatment, chemotherapy in eight patients (57%) and chemoradiation therapy in one patient (7%). Adjuvant chemotherapy regimens included AIM, VAC, VDC (vincristine, doxorubicin, cyclophosphamide), IE, docetaxel, gemcitabine, vinorelbine. Ten patients (72%) showed disease progression: two with local recurrence, six with distant metastasis, and two with local recurrence and distant metastasis. The lung was the most common site of metastasis (88%). Nine patients (65%) died of disease, one patient (7%) was alive with disease and four patients (28%) had no evidence of disease. Median follow-up was 15 months (4–95 months).

In the ERMS group, 8 patients (34%) had locoregional or distant metastatic disease at presentation. Four patients had locoregional lymph node metastasis, two patients had lung metastasis, one patient had intra-abdominal metastasis and one patient had osseous metastasis. Using the Children Oncology Group (COG) RMS risk stratification¹⁰, 13 patients with ERMS were classified as low risk, 6 as intermediate risk and 4 as high risk. All patients were treated with standard RMS chemotherapy regimens (VAC, VDC), in addition to other agents in a subset of patients (mesna, irinotecan, temozolomide, etoposide). Seven patients (30%) showed evidence of disease progression after remission, with median time to progression of 21 months. Seven patients (30%) died of disease, while the remaining 16 had no evidence of disease with median follow-up of 37 months (7–122 months). The median time to death was 36 months (7–55 months).

Survival comparative studies showed that the overall survival and progression-free survival in PRMS were significantly worse compared to ERMS (p<0.0005) (Fig. 6).

Fig 6A-6B: Overall and progression-free survival among pleomorphic rhabdomyosarcoma (PRMS) and embryonal rhabdomyosarcoma (ERMS).

Kaplan-Meier survival curves of PRMS and ERMS: A, overall survival and B, progression free survival. p-values based on log-rank test.

DISCUSSION

In this study, we performed a genomic profiling of a well characterized cohort of PRMS in comparison to a control group of ERMS and a large cohort of pleomorphic sarcomas including UPS and PLPS. The epidemiologic characteristics of our PRMS patients were in keeping to other published series showing a median age of 53 years (31–76 years) and a slight male predominance (M: F = 1.8:1)³. Although PRMS is the most common RMS subtype in adults, ERMS accounts for up to 21% of adult RMS, which was reflected in our control group (13%) (Supplementary Fig. 2A–2B)¹¹. Similar to prior reports, we demonstrated that PRMS follows a highly aggressive clinical course showing an overall poor prognosis. In the largest series with available follow-up data to date, the median survival for patients with localized disease and metastatic disease were 12.8 and 7.1 months, respectively, while the median relapse-free survival was 7.3 months³. This was concordant with our findings showing a median time to progression of 7 months and median time to death of 16 months. However, this clinical outcome was significantly worse compared to our control ERMS group, which showed a median time to progression and death of 21 and 36 months, respectively. In our cohort, all except two PRMS patients were treated with standard clinical management for advanced soft tissue sarcoma in adults, consisting primarily of surgical resection with neoadjuvant or adjuvant chemotherapy and/or radiation^12,13. The remaining 2 patients were treated with chemotherapy and/or immunotherapy, as the tumors were deemed unresectable even after treatment. Ten patients (71%) were treated with the recommended chemotherapy regimen by the National Comprehensive Cancer Network (NCCN) Guidelines¹³ for soft tissue sarcoma with non-specific histologies (AIM, doxorubicin…), while four patients were treated with other regimens. One of those (case 3) was treated at an outside facility and was transferred to our institution for surgical resection of the tumor. A second patient (case 6), the youngest in our cohort, was managed by a pediatric oncologist and treated according to ERMS protocol. Despite having a small subset of patients treated with non-standard chemotherapy regimens, the survival was poor regardless of the regimen used. Of note, in our institution PRMS patients are managed with a similar approach as for other pleomorphic sarcomas, following the guidelines outlined by the NCCN, which is reflected by the practice observed in the majority of cases in this cohort. On the other hand, all ERMS patients were treated with standard rhabdomyosarcoma chemotherapy regimens (VAC, VDC), according to COG protocols, including the three adult patients. Given the significant difference in clinical management and prognosis, the diagnostic distinction between the two groups is crucial for appropriate clinical care.

Histologically, the tumors showed heterogenous morphology and a variable proportion of large pleomorphic tumor cells with deeply eosinophilic cytoplasm (so-called pleomorphic rhabdomyoblasts). The majority (n=10) resembled UPS, showing haphazardly-oriented large epithelioid to spindle cells with abundant eosinophilic cytoplasm. In addition, two cases were reminiscent of myxofibrosarcoma and one case resembled pleomorphic leiomyosarcoma. This is not surprising, as the concept of PRMS has changed considerably since its introduction by Stout in 1946, where the diagnosis was based solely on morphologic appearance and identification of strap cells, which likely encompassed other sarcomas including UPS, pleomorphic leiomyosarcoma and MTT^14,15. In recent decades, an immunohistochemical approach was adopted as the gold standard in diagnosing PRMS, with retrospective studies showing that tumors initially classified as PRMS were reclassified as UPS, leiomyosarcoma and liposarcoma^16–20. Moreover, the MFS-like morphologic variant seen in 2 of our cases appears to be a rare event, with only one case to our knowledge, previously reported²¹. Additionally, in one case the pleomorphic cells showed a distinctive rhabdoid morphology with focal alveolar-like pattern. Anaplasia in ERMS is defined as large, lobated and hyperchromic nuclei, at least 3-times the size of neighboring nuclei (Supplementary Fig. 2C–2D)²². In general, the presence of cross-striation and the presence of more conventional ERMS areas, characterized by primitive round to ovoid cells with alternating cellular and myxoid stroma, favors a diagnosis of ERMS. This distinction is of greater significance in adults, as PRMS is regarded as a controversial entity in the pediatric population. In early literature, up to half of PRMS presenting in childhood were found to exhibit ERMS areas^23–25; an observation that led to a conclusion by the Intergroup Rhabdomyosarcoma Study I/II that most childhood pleomorphic/anaplastic RMS likely represent the embryonal type with anaplastic rhabdomyoblasts rather than constituting a distinct clinicopathologic subtype^24,26. Moreover, comprehensive molecular studies have not been specifically undertaken towards investigating this issue.

The genomic profile of PRMS was in sharp contrast to that of ERMS. Pathogenic alterations in genes involved in cell-cycle regulation including TP53, RB1 and CDKN2A were the most common, seen in 43%–79% of PRMS, while these alterations were rare to absent in ERMS (0–13%). These findings are in concordance with the very limited prior genomic data in PRMS. In a study by Beird et al, 71% (12/17) of PRMS cases, studied by whole genome sequencing, harbored TP53 mutations, and 47% (7/17) had RB1 mutations²⁷. Additionally, in the study by Chelsky et al., 70% (7/10) of cases, studied by targeted DNA NGS, showed TP53 mutations²⁸. Akin to prior reports, the spectrum of TP53 alterations included deep deletions, point mutations and truncating mutations. Although only adult patients were included in our cohort, one patient, a 46-year-old male with a retropharyngeal mass, harbored a pathogenic TP53 germline mutation. Notably, this patient had a prior diagnosis of PLPS three years prior, treated with resection and adjuvant radiotherapy, with no subsequent evidence of disease progression. Genomic profiling using the MSK-IMPACT was performed on both lesions, which revealed a distinct landscape of mutations and copy number changes in keeping with two clonally unrelated primaries. Previous studies have shown that individuals with germline TP53 mutations tend to develop so-called “anaplastic/pleomorphic” RMS, at a young age^29–31. Tumors described in these studies occurred in children and adolescents and showed anaplastic histology. As discussed above, these are best considered as ERMS with anaplastic rhabdomyoblasts and reports of PRMS occurring in adult patients with TP53 germline mutations are not described to our knowledge in the literature. For example, in the study of Beird et al, 14 cases of PRMS occurring in adults were tested for germline TP53 and RB1 mutations, yet none were identified as carrying such mutations²⁷.

The frequency of TP53 mutation in ERMS control group was relatively lower than the recently reported 13% by Shern et al.³². This difference could be attributed to the smaller sample size in our control group. Of note, prior and recent literature did report a lower prevalence of TP53 mutation across pediatric RMS (5–9%) and in ERMS subtype (2.6%)^33–35. Shern et al, suggested that the higher frequency of TP53 mutations reported was likely due to the higher sequencing depth used by the targeted assay approach³⁵. Anaplasia was seen in 6 cases (26%) in our ERMS group, one of which showed diffuse anaplasia. This result is concordant with the reported frequency of anaplasia in ERMS of 27%³⁵. Of note, none of our ERMS cases displaying anaplasia harbored TP53 mutations and the single case with TP53 mutation did not reveal histologic evidence of anaplasia. The genomic profile of ERMS cases with anaplasia was similar to those without anaplasia, except for the notable absence of BCOR and NF1 alterations in the former. Shenoy et al reported that only 24% (9/38 cases) of RMS (all subtypes) with anaplasia harbored TP53 mutation, while the majority of tumors with TP53 mutations (69%, 9/13 cases) demonstrated histologic anaplasia³⁵. In addition, TP53 mutations may be rarely seen in patients with no histologic anaplasia (3.7%, 4/108 cases). Thus, histologic anaplasia and its association with underlying TP53 mutations requires further investigation. Although at lower frequencies compared to PRMS, loss of CDKN2A may occur in ERMS which is consistent with our findings^36,37. It has been suggested that concurrent biallelic inactivation of TP53 and CDKN2A is a marker of poor prognosis in PRMS, an observation seen in other malignancies such as melanoma, lymphoma and squamous cell carcinoma^28,38–40. For instance, in the cohort by Chelsky et al, the three PRMS cases (of 10) with the shortest overall survival had concurrent biallelic inactivation of TP53 and CDKN2A²⁸. However, our study did not show a significant difference in survival between cases with coexisting mutations and those without. Nonetheless, this discrepancy is likely related to the limited number of cases in both series, emphasizing the need for larger, multi-institutional cohorts with integrated multiomics data for more accurate prognostication.

Notably, none of the PRMS in the current series harbored alterations known to be drivers in ERMS, including mutations in the RAS pathway (NRAS, KRAS, HRAS, NF1, FGFR4), effectors of PI3K (PTEN, PIK3CA) or genes that control the cell cycle (FBXW7, CTNNB1)⁴¹. Abnormalities in the RAS pathway is one of the leading mechanisms of RMS pathogenesis, as it induces uncontrollable proliferation of cancer cells and prolong their survival^42,43. Age-related associations in RAS-mutated RMS were described by Shern et al., with HRAS mutations being prevalent in infants, KRAS mutations in toddlers, and NRAS mutations in adolescents⁴¹. In addition, loss of function mutations in BCOR were absent in our PRMS cohort, while being one of the most common alterations detected in ERMS control group (22%). The latter frequency is relatively higher than previously reported 7.4% of BCOR alterations in fusion-negative RMS⁴¹. BCOR functions as a transcriptional repressor that has been shown to interact with class I and II histone deacetylases and BCOR somatic mutations have been reported in a number of pediatric tumors including acute myeloid leukemia, retinoblastoma and medulloblastoma^44–47.

The profiles of arm-level copy number alterations were markedly distinct between the two groups. ERMS exhibited chromosome 8 gains in 61% of cases, while PRMS displayed multiple arm level copy number gains and losses (gains in 17p and chr22q and loss in chr6q). Previous cytogenetic reports revealed highly complex and unbalanced karyotypes in PRMS; nevertheless, no recurrent events were observed^48,49. The most frequently identified abnormalities included losses in regions 3p, 5q, 10q23, 15q21, 3p, 5q32, and 13 and gains in 1p22, 3p12, 7p, 18/18q, 20/20p, 22q^4,5. On the other hand, polysomy 8 is a frequent abnormality seen in ERMS, followed by extra copies of chromosomes 2, 11, 12, 13 and/or 20⁵⁰. Furthermore, loss of heterozygosity (LOH) at numerous loci of the 11p15.5 region, via loss of a chromosome, deletion, or uniparental disomy, is frequently observed in ERMS⁵¹.

Recent genomic efforts, including large integrated datasets (WES, WGS and RNAseq) from TCGA and other groups, have shown that sarcomas with complex genetic alterations are characterized predominantly by copy-number changes, with low mutational loads and only a few, highly recurrent gene mutations (TP53, ATRX, RB1) across sarcoma histotypes^27,52,53. Although PRMS was not included in these large TCGA or comprehensive sequences efforts, likely due to their rare incidence, our current results support its genomic overlap with most other molecularly complex sarcomas, such as UPS and PLS, harboring frequent alterations in tumor suppressor genes TP53, RB1 and CDKN2A. Despite infrequent ATRX alterations in PRMS in our study, other studies from the literature showed a frequency of ATRX alterations in PRMS (20% and 29%) as comparable to that of other pleomorphic sarcomas^27,28.

In summary, using clinically validated genomic profiling, we demonstrate that PRMS are characterized by genomic landscapes distinct from ERMS but highly overlapping with other pleomorphic sarcomas. Moreover, PRMS patients are associated with significantly poor overall, and progression free survival compared to ERMS, supporting the current therapeutic approach of PRMS similar to other pleomorphic sarcomas. Thus, NGS may be applied in selective cases for a refined classification.

Supplementary Material

Supinfo

Supplementary Fig 1A-1D: Radiologic and gross pathologic features of pleomorphic rhabdomyosarcoma. A, T2-weighted axial magnetic resonance imaging (MRI) showing a lobulated well defined mass with intermediate T2 signal and mild surrounding edema extending along the fascia (case 6). B, T2-weighted axial MRI showing a fusiform mass with intermediate to hyperintense T2 signal along the medial aspect of the arm. The mass displaces the long head of the triceps muscle and abuts the medial head (case 10). C, Gross pathology specimen showing a well-circumscribed mass, with tan-white cut surface, surrounded by a pseudocapsule (case 10). D, Gross pathology specimen showing a well demarcated mass with tan-yellow cut surfaces and areas of hemorrhage and necrosis (case 12).

Supplementary Fig 2A-2D: Embryonal rhabdomyosarcoma (ERMS). A-B: ERMS occurring in the pelvis (C, case 6) and testis (D, case 23) of two adult patients. Histologic sections show typical morphology of ERMS composed of spindle and round primitive cells with rare mature rhabdomyoblasts (A, 200x; B, 100x). C-D: ERMS with anaplasia occurring in the pelvis of a pediatric female patient (case 3). Histologic sections show round and spindle cells with scattered cells showing enlarged nuclei and marked pleomorphism and tad-pole cytoplasm, reminiscent to pleomorphic rhabdomyoblasts (C-D, 200x).

DATA AVAILABILITY STATEMENT:

Study dataset is available upon request from the corresponding author.