Clinicopathologic features and molecular spectrum of spindle cell and sclerosing rhabdomyosarcomas in the head and neck region

Abstract

Recently, spindle cell/sclerosing rhabdomyosarcoma (SRMS/ScRMS) has been recognized as a stand-alone entity in the latest edition of WHO Classification of Tumors of Soft Tissue and Bone. As SRMS/ScRMS have a predilection for the head and neck, we evaluated the clinicopathologic and molecular features of 20 cases of SRMS/ScRMS (13 SRMS and 7 ScRMS) arising in the head and neck region. 10 patients were men, and 10 were women, and their ages ranged from 2 months to 57 years. Tumor size ranged from 1.5 to 20 cm. By immunohistochemistry, all tumors showed diffuse desmin expression, and MYOD1 immunostaining was diffuse to multifocally positive: 16 cases showed myogenin positive immunostaining. 2 patients had local recurrences, and 5 patients developed distant metastases. So far, 10 patients have died of the disease. 7 of 13 SRMS and 4 of 7 ScRMS showed PIK3CA mutations, while 8 of 13 SRMS and all 7 ScRMS showed MYOD1 mutations. A novel p.R524K hotspot mutation in 8 of 11 cases showed PIK3CA mutations. SRMS/ScRMS shares similar clinicopathological and molecular features. Diagnostic pitfalls from other spindle or sclerosing sarcomas should be avoided with the use of appropriate immunohistochemical stains and relevant clinical information. Co-occurrence of PIK3CA and MYOD1 mutations are associated with unfavorable clinical outcomes.

Introduction

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and adolescents [1]. Traditionally it has three morphologically and clinically distinct subtypes: embryonal RMS (ERMS), alveolar RMS (ARMS), and pleomorphic rhabdomyosarcoma (PRMS). Spindle cell RMS (SRMS) is a rare morphologic subtype of Rhabdomyosarcoma (RMS), which was initially grouped under ERMS. Sclerosing RMS (ScRMS) usually has clinical and pathological presentations that are similar to SRMS, so, recently, the World Health Organization (WHO) classification of Soft Tissue and Bone merged SRMS and ScRMS into a single pathologic entity.

Some recent studies have discussed the clinicopathological and genetic features of this rare tumor entity. They found that SRMS/ScRMS usually has a predilection for paratesticular and head and neck sites, and that children, were likely to have a more favorable prognosis. But some discrepancies still exist among research groups from different countries. Cavazzana et al. [2] from Italy first described SRMS as a variant of ERMS in children which had a relatively favorable prognosis, but Yasui et al. [3] from Japan suggested a worse prognosis for SRMS/ScRMS compared to the pediatric ERMS. And Owosho et al. [4] from the USA also reported that the 5-year overall survival rate for ERMS patients was significantly higher (82%) compared to SRMS/ScRMS (50%) [SRMS (75%); ScRMS (30%)]. Because of the rarity of SRMS/ScRMS, it is necessary to extend samples and focus on a detailed clinicopathologic and survival analysis of SRMS/ScRMS.

MyoD1, desmin and Myogenin et al. [5] are vital immunohistochemical markers to distinguish RMS from its mimics. MyoD1, as a myogenic transcriptional regulatory protein which is expressed in early skeletal muscle differentiation, is rarely expressed in mature skeletal muscle tissue. The sustained expression of MYOD1 in RMS might be caused by gene mutation or other mechanisms. However, its exact mechanism is still unknown. Previously, Van Antwerp et al. [6] described how the MYOD1 p.L122R mutation occurs in the conserved DNA binding domain, leading to transactivation and MYC-like functions, which result in sustained proliferation and the blocking of the myogenic differentiation process. MYOD1 p.L122R as a mutation hotspot can be detected in 38%-56% of SRMS/ScRMS and has never been detected in other types of RMS [7,8]. But the mutation rate of MYOD1 seems inconsistent with the positive expression rate of the MYOD1 protein, which indicates that there are other factors influencing the expression level of MYOD1 protein.

In addition to the above three markers, 15-18% [8,9] of SRMS/ScRMS also showed phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations, including PIK3CA p.E542V, p.E542K, p.E545K, p.H1047R and p.G1049R, along with MYOD1 mutations, which may activate the PI3K/AKT signaling pathway and enhance proliferation. Remarkably, in previous reports, all the cases which harbored MYOD1 and PIK3CA coexisting mutations showed sclerosing morphology. However, PIK3CA mutations can also been detected in ERMS and ARMS [10,11]. Therefore, the exact role of PIK3CA mutations in SRMS/ScRMS still needs further research.

To sum up, in order to know more about this rare type of RMS, we present a clinicopathological retrospective study of SRMS/ScRMS in our institution during the period 1995-2016 to detect the mutation of MYOD1 and PIK3CA and their correlation with the prognosis.

Materials and methods

Patient selection

We retrieved 166 cases diagnosed with primary head and neck RMS in the Department of Pathology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, diagnosed between 1995 and 2016. The clinicopathological and follow up details were collected through the electronic medical records and telephonically. In each case, the diagnosis was confirmed by reviewing the hematoxylin-eosin (H&E) slides and the immunohistochemical stains. All the cases included in the study were reviewed by two independent pathologists, with the classification based on current WHO criteria as SRMS/ScRMS, which is characterized by a prominent fascicular spindle cell morphology and hyalinized stroma, and primitive-appearing tumor cells set in cords and nests. In several cases, the tumor displayed focal areas of sclerosing RMS and vice versa. The clinicopathologic feature data included: age at diagnosis, gender, primary site, tumor size, therapy method (surgical resection, chemotherapy, and/or radiation therapy) and pre-therapy stage, which was based on the Intergroup Rhabdomyosarcoma Study Group (IRSG) pretreatment staging system. The study procedures were approved by our hospital’s ethical committee.

Immunohistochemistry of MYOD1, desmin and myogenin

After heating antigen retrieval (water bath/Tris-EDTA) on FFPE tissue sections, immunohistochemical studies were performed by the Envision^TM method (Dako, Glostrup, Denmark) according to the manufacturer’s protocol. Monoclonal antibodies including myogenin (Dako, cl: F5D; 1:300), MYOD1 (Dako, cl: 5.8A; 1:40) and desmin (Dako, cl: D33; 1:200) were adopted. PBS was used to replace primary antibodies as a blank control. The number of immunoreactive cells in each section was semi quantitatively evaluated as follows: ++++, >75% positive cells; +++, 75-50% positive cells; ++, 50%-25% positive cells; +, <25% positive cells; and -, no positively stained cells.

PCR and sanger sequencing

Genomic DNA was isolated from FFPE samples using a TIANamp FFPE DNA Kit (TIANGEN, Beijing, China), following the manufacturer’s instructions. An ultraviolet spectral photometer (NanoDrop 2000c, Thermo Scientific) was used to determine the concentration of DNA. The primer sequences for PCR amplifications of PIK3CA and MYOD1 were based on the study of Agaram et al. [8]. Targeted PCR was performed for the known MYOD1 exon 1 (122) hot spot mutation, PIK3CA exon 10 (542/545), and the exon 21 mutation (1047) by 2 μl primers in a 25 μl reaction volume on a CFX 96 Real-Time PCR detection system (Bio-Rad, USA). PCR conditions were used: initial hotstart denaturation step of 1 min at 96°C, followed by 30 cycles of 10 s at 96°C, 5s at 50°C, and 2 min at 60°C. Using the QIAquick PCR purification kit (Qiagen, Hilden, Germany), purified PCR products were sequenced on the ABI3500DX DNA sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The data were further analyzed by using Chromas software v 2.6.4 (http://www.technelysium.com.au/chromas.html).

Statistical analysis

The data were analyzed using SPSS 20.0.0 (IBM Corp, NY, USA). The associations between the clinicopathologic features of SRMS/ScRMS with PIK3CA and the MYOD1mutation were calculated using Fisher’s exact test. The relationship between the MYOD1 mutation with its expression was performed using the Mann-Whitney U-test. Survival analyses were based on the Kaplan-Meier method and calculated using the log-rank test (calculated as the time of diagnosis). The threshold for statistical significance was set at P≤0.05.

Results

Clinicopathological features of SRMS and ScRMS

Twenty of 166 cases (13 SRMS and 7 ScRMS) were reclassified as SRMS/ScRMS. The clinicopathologic features and follow-up are summarized in Table 1. The cohort included 10 children (4 males and 6 females) and 10 adults (6 males and 4 females). Their ages ranged from 2 months to 57 years. The mean age was 20.3 years, and the median age was 16.5 years. The most common anatomic location of the neoplasms in our study was the infratemporal fossa (6), with rarer locations including the pterygomandibular space (1), the parotid region (1), the nasolabial area (1), the neck (1), and the lip (1). The tumor sizes ranged from 1.5 to 20 cm the mean size was 5.4 cm, and the median size was 3 cm. According to the IRSG pretreatment classification, 4 of the 20 cases were stage IV, and 16 of the 20 cases were stage I. All the patients underwent surgical resections, and only 5 patients chose chemotherapy and radiotherapy after surgery as adjuvant treatments.

Table 1.

Clinicopathologic features and follow-up

Case No.	Age/sex	Diagnosis	Primary site	Size (cm)	Therapy	Stagea	Follow-Up
1	18 y/male	ScRMS	Masseter region	1.5	Surgery	1	NED (205 mos)
2	22 y/female	ScRMS	Infratemporal fossa	4	Surgery	4	DM (7 mos); DOD (16 mos)
3	20 y/female	SRMS	Soft tissue mandible	20	Surgery	1	DM (10 mos); DOD (18 mos)
4	22 y/female	SRMS	Pterygomandibular space	4	Surgery, CT & RT	4	DM; DOD (4 mos)
5	14 y/female	SRMS	Parotid region	20	Surgery	1
6	34 y/female	ScRMS	Infratemporal fossa	6	Surgery	1	DOD (24 mos)
7	15 y/female	ScRMS	Soft tissue maxilla	2.5	Surgery, CT & RT	4	LR, DM (14 mos); DOD (17 mos)
8	57 y/male	SRMS	Tongue	3.5	Surgery, CT & RT	1	NED (11 mos)
9	36 y/male	SRMS	Soft tissue maxilla	2	Surgery	1	NED (54 mos)
10	11 y/male	ScRMS	Neck	10	Surgery	1	DOD (80 mos)
11	4 y/male	SRMS	Lip	1.5	Surgery	1	NED (149 mos)
12	12 y/female	SRMS	Infratemporal fossa	2.5	Surgery	1	DOD (58 mos)
13	2 mos/male	SRMS	Tongue	2.5	Surgery	1	NED (82 mos)
14	7 y/female	SRMS	Infratemporal fossa	6.5	Surgery	1	DOD (63 mos)
15	29 y/male	SRMS	Infratemporal fossa	9	Surgery	1	DOD (46 mos)
16	47 y/male	ScRMS	Masseter region	4.5	Surgery, CT & RT	1	NED (41 mos)
17	13 y/female	ScRMS	Soft tissue mandible	2	Surgery, CT & RT	1	LR (24 mos); NED (39 mos)
18	8 y/female	SRMS	Nasolabial	2.5	Surgery	1	NED (259 mos)
19	33 y/male	SRMS	Soft tissue maxilla	2	Surgery	4	DM (33 mos); DOD (52 mos)
20	4 y/male	SRMS	Infratemporal fossa	1.5	Surgery	1

CT-chemotherapy, RT-radiotherapy, LR-local recurrence after surgery, DM-distant metastasis after surgery, DOD-died of disease, NED-no evidence of disease.

^aIRSG (Intergroup Rhabdomyosarcoma Study Group) indicates pretreatment staging system.

Completed clinical follow-up information was available on 18 patients (Table 1), ranging from 4-259 months (average = 67.67 months, median = 49 months). 5 patients (33%, 6/18) developed recurrences at 4-33 months after surgery (2 SRMS and 3 ScRMS, Table 1). The regions of distant metastasis included bone and brain. Ten patients (53%, 10/19) died of disease at 4-80 months after surgery, including 4 adolescents and 5 adults. A Kaplan-Meier survival analysis showed that there was no statistical difference among the different age groups in overall survival (OS) (P>0.05, Figure 4). But the OS of patients in whom coexisting PIK3CA and MYOD1 mutations were detected was significantly worse than those merely harboring the MYOD1 mutation (P<0.05, Figure 4), which suggested that SRMS/ScRMS coexisting with the above mutations were associated with an aggressive clinical behavior and a poor outcome.

Figure 4.

Kaplan-Meier survival analysis. A. Coexisting PIK3CA and MYOD1 mutation was significantly worse than the cases of merely harboring the MYOD1 mutation in the overall survival (OS). B. Shows the OS among the different age groups.

Immunohistochemical features

The immunohistochemical results are shown in Table 2. All of the cases showed MYOD1 and desmin expression in varying degrees. But the MYOD1 immunostaining did not show a statistically significant difference between the pediatric age group (<18 years) and the adult group (≥18 years) (P = 0.529). 16 of 20 cases showed positive myogenin staining, while most of the cases had a focal presentation and were rarely positive, and only one case showed 50%-75% positive cells.

Table 2.

Immunohistochemical and PIK3CA and MYOD1 mutation status of SRMS/ScRMS

Case No.	Immunohistochemical stains			Molecular
				PIK3CA		MYOD1
	MYOD1	Myogenin	Desmin	Exon 10	Exon 21	Exon 1
1	++++	+++	++++	R524K	WT	L122R
2	++++	+	++++	R524K	WT	L122R
3	++++	+	++	R524K	WT	L122R
4	+++	+	++++	R524K	WT	L122R
5	+	+	++	R524K	WT	WT
6	++++	+	++++	R524K	WT	L122R
7	++++	++++	++	R524K	WT	L122R
8	++	-	++++	R524K	WT	WT
9	+++	+	++++	WT	WT	WT
10	++++	-	++++	WT	WT	L122R
11	++++	+	+++	WT	WT	L122R
12	++++	+	++++	WT	WT	L122R
13	++	+	++++	WT	WT	WT
14	++++	+	+	WT	H1047R	121* & L122RΔ
15	++++	-	++++	WT	WT	L122RΔ
16	++++	++	++++	WT	WT	L122R
17	++++	+++	+++	WT	WT	L122R
18	+++	+	++++	WT	WT	L122R
19	++++	-	++	E545K	WT	L122R
20	+	+	++	WT	G1049D	WT

(+) - Positive, (-) - negative, (f) - focal, WT-wild type;

^*nonsense mutation;

^Δhomozygous mutation.

Analysis of PIK3CA and MYOD1 mutations

PIK3CA mutations were found in 11 of 20 cases (55%). Unpredictably, 8 of 11 cases (4 SRMS cases and 4 ScRMS cases), were found that harbored a novelty p.R524K mutation, which had never been reported in SRMS/ScRMS (Table 2; Figures 1 and 2). Only one case showed the p.E545K mutation, which had previously been reported in SRMS/ScRMS [8]. The remaining two other cases showed the H1047R (Figure 3) and G1049D mutations, respectively. Recurrent MYOD1 p.L122R mutations were detected in 15 of the 20 cases (75%). Of these 15 cases, two presented p.L122R homozygous mutations (Table 2), and one case appeared a p.121 nonsense mutation simultaneously (Figure 3). In addition, 8 of the 20 cases harbored both PIK3CA and MYOD1 mutations (40%). But neither age group (P = 0.34), nor gender (P = 0.26) nor histopathologic subtype (P = 0.41) statistically correlated with the mutation status of above two genes (Table 3). The cases which harbored the MYOD1 p.L122R mutations showed diffusely strong positive MYOD1 staining (P<0.001, Table 3).

Figure 1.

ScRMS of a 22 year-old woman (Case 2). (A) ScRMS characterized by stromal hyalinization with tumor cells arranged in nests, focal alveolar or psuedovascular and trabeculae appearance (H & E, 400×). (B) Immunohistochemical stains show diffuse positivity for MYOD1. (C) Cytoplasmic positive for desmin. (D) Focal myogenin positive immunostaining (400×). Sequencing showing a (E) heterozygous PIK3CA p.R524K mutation and (F) MYOD1 p.L122R mutation.

Figure 2.

sRMS of a 15 year-old girl (Case 7). (A) Shows the tumor composed of neoplastic spindle cells arranged in elongated fascicles or storiform patterns (H & E, 400×). (B-D) Immunohistochemical stains show positivity for MYOD1, myogenin, desmin (400×). Sequencing showing a (E) heterozygous PIK3CA p.R524K mutation and (F) MYOD1 p. L122R mutation.

Figure 3.

sRMS of a 7 year-old girl (Case 14). (A) Shows the pathologic features of MYOD1-mutant positive sRMS (H & E, 400×). (B-D) Immunohistochemical stains show positivity for MYOD1, myogenin, desmin (400×). Sequencing showing a heterozygous PIK3CA p.H1047R mutation (E). A MYOD1 p.121 nonsense mutation and a p.L122R homozygous mutation (F).

Table 3.

Correlation between the outcome of SRMS/ScRMS with the PIK3CA and MYOD1 mutations; Correlation between the expression of MYOD1 with MYOD1 mutation

Clinicopathological features	Variable	Frequency (n = 20)	Mutation status of PIK3CA and MYOD1				Value	P-valueΔ
			Wild	PIK3CA mutation	MYOD1 mutation	Coexisted PIK3CA and MYOD1 mutation
Age	Pediatric (<18)	10	1	2	5	2		0.34Δ
	Adult (≥18)	10	1	1	2	6
Gender	Male	10	2	2	4	2		0.26Δ
	Female	10	0	1	3	6
Histopathologic subtype	Srms	13	2	3	4	4		0.41Δ
	Scrms	7	0	0	3	4
	Mutation status of MYOD1
	Positive	Wild		Mutant
MYOD1 immunohistochemistry	+	2		0			74	<0.001†
	++	2		0
	+++	1		2
	++++	0		13

^ΔFisher’s Exact Test;

^†Mann-Whitney U test.

Discussion

We observed clinicopathologic features and the mutation spectrum of PIK3CA and MYOD1 in a relatively large cohort of head and neck SRMS/ScRMS. In doing so, we identified a novel mutation hotspot of PIK3CA of SRMS/ScRMS in the head and neck. We also found that the PIK3CA mutation can exist independently without the MYOD1 mutation in SRMS/ScRMS, a finding not previously described. Furthermore, the co-occurrence of the PIK3CA and MYOD1 mutations in SRMS/ScRMS are associated with unfavorable clinical outcomes.

The spindle cell/sclerosing RMS (SRMS/ScRMS) is one of rare morphologic subtypes accounting for 5-10% of the RMS cases [3]. SRMS provides a diagnostic challenge by virtue of its similarity to other spindle cell neoplasms, including leiomyosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, fibrosarcoma, sarcomatoid carcinoma, and spindle cell melanoma. Sometimes it is extremely difficult to diagnose ScRMS by histopathology alone. The differential diagnosis of ScRMS includes ARMS, sclerosing epithelioid fibrosarcoma, metastatic carcinoma, osteosarcoma, and angiosarcoma [15-17]. Therefore, immunohistochemistry plays a vital role in the diagnosis of SRMS/ScRMS.

SRMS/ScRMS tends to show a strong positivity for MYOD, a relatively weak positivity for myogenin, and a diffuse expression of desmin [3,12]. Most of the cases in our study, like many other studies, have shown the typical immunohistochemical profile of SRMS/ScRMS. Interestingly, all of the cases in our cohort were positive for MYOD1. MYOD1 showed a diffused expression in most ScRMS cases in our study, a finding which corresponded to previous observations [13,14]. Recurring MYOD1 p.L122R mutations in SRMS/ScRMS as well as in a subset of ERMS, which displayed frequent spindle cell morphology, have been reported [7,8,15]. This mutation has never been discovered in other types of RMS. And our study, similar to that of Agaram et al. [8], revealed cases of SRMS/ScRMS with heterozygous, as well as homozygous MYOD1 p.L122R mutations. As a myogenic regulatory factor, the mutation of MYOD1 might act as a key factor in skeletal muscle oncogenesis. Early studies showed the MYOD1 protein with the p.L122R mutation could block wild-type MYOD1 function and bind to MYC consensus sequences, then functionally substitute for MYC protein and stimulate proliferation through MYC-independent pathways. And MYC is one of the downstream target molecules of PI3K-AKT signaling [16]. Our present study also shows that the MYOD1 p.L122R mutation is a common event in SRMS/ScRMS, which suggests that MYOD1 p.L122R might be a specific mutation hotspot in SRMS/ScRMS. In addition, we observed cases which harbored MYOD1 p.L122R mutations showing a strong positive MYOD1 staining (P<0.001). A reasonable explanation for this finding is that the MYOD1 p.L122R mutation may enhance the expression of MYOD1. However, wild-type MYOD1 cases also show expression of MYOD1 to a certain degree, which suggests that this mutation is not the initial cause of the MYOD1 protein expression. And our study, like that of Agaram et al. [8], revealed cases of SRMS/ScRMS with heterozygous, as well as homozygous, MYOD1 p.L122R mutations.

The previous molecular analysis also showed PIK3CA p.E542V, p.E542K, p.E545K, p.H1047R and p.G1049R as mutation hotspots in SRMS/ScRMS [8,9,15]. Many solid tumors showed that the PIK3CA missense mutations occur in all domains of p110α but are mainly concentrated on two hotspots, p.E542K and p.E545K in the helical domain and p.H1047R in the kinase domain [17]. Earlier studies described the presence of PIK3CA mutations only co-existing with the MYOD1 mutations in ScRMS which occurred in older children or young adults [8]. In the present study, we observed a novel PIK3CA p.R524K heterozygous mutation hotspot in 8 of 11 SRMS/ScRMS cases which occurred in the head and neck region among the Chinese population. In addition, the PIK3CA mutation can exist independently without the MYOD1 mutation in both SRMS and ScRMS. To our knowledge, it is the first study to report the PIK3CA p.R524K mutation in SRMS/ScRMS. A rare missense mutation in the helical domain of PIK3CA, the p.R524K mutation was detected in HER2-positive breast cancer patients [18]. A possible reason for the difference in the mutation location of PIK3CA between our study and others is the ethnicity.

Previous research shows pediatric SRMS/ScRMS cases seem to have a better prognosis than do pediatric ERMS cases, while in adult patients the disease may have a poor prognosis [19,20]. But in our study, the OS among different age groups did not show any statistical significance (P>0.05, Figure 4). From the point of view of the mutation status, some studies showed SRMS/ScRMS which harbored MYOD1 mutations with or without accompanying PIK3CA mutations, were highly associated with an aggressive outcome such as early distant metastases and high mortality, despite multimodality therapy [4,9,21]. And in our study, the OS of patients in whom we detected a coexisting PIK3CA and MYOD1 mutation was significantly worse than the cases in which patients had only the MYOD1 mutation (P<0.05, Figure 4), which suggested that the cooccurrence of the PIK3CA and MYOD1 mutations are associated with an aggressive clinical behavior and unfavorable clinical outcomes. Kohsaka et al [15] had indicated that either MYOD1 p.L122R or PIK3CA p.H1047R increased the in vivo growth of tumor cells, while the combination of PIK3CA H1047R and MYOD1 p.L122R resulted in more rapidly growing tumors, which suggests the coexisting mutations of PIK3CA and MYOD1, to some extent, prompt the progression of SRMS/ScRMS. As above, we suggest that the cooccurrence of PIK3CA and MYOD1 mutations could be seen as an independent prognostic marker in SRMS/ScRMS.

In conclusion, PIK3CA and MYOD1 mutations occur frequently with SRMS/ScRMS in the head and neck. PIK3CA p.R524K is a specific mutation hotspot of SRMS/ScRMS in the head and neck among the Chinese population. Coexisting mutations of PIK3CA and MYOD1 are associated with a poor prognosis of SRMS/ScRMS and suggest that tumor progression may be influenced by activating the PI3K/AKT signaling pathways and by the blocking of myogenic genes’ transcriptional differentiation. Morphologic observation and genetic testing may provide more comprehensive information in choosing the appropriate therapeutic regimen and in screening the targeted therapy of SRMS/ScRMS.

Disclosure of conflict of interest

None.