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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Histopathology. 2015 Jan 12;66(5):627–638. doi: 10.1111/his.12466

Progressive Loss Of Myogenic Differentiation In Leiomyosarcoma Has Prognostic Value

Elizabeth G Demicco 1, Genevieve M Boland 2, Kari J Brewer Savannah 2, Kristelle Lusby 2, Eric D Young 2, Davis Ingram 3, Kelsey L Watson 2, Marshall Bailey 2, Xiangqian Guo 6, Jason L Hornick 5, Matt van de Rijn 6, Wei-Lien Wang 2,3, Keila E Torres 2,4, Dina Lev 2,7, Alexander J Lazar 2,3
PMCID: PMC4248015  NIHMSID: NIHMS599739  PMID: 24889065

Abstract

Aims

Well-differentiated leiomyosarcoma show morphologically recognizable smooth muscle differentiation, while poorly differentiated tumors may form a spectrum with a subset of undifferentiated pleomorphic sarcomas. Expression of certain muscle markers has been reported to have prognostic impact. We investigated the correlation between morphologic spectrum and muscle-marker expression profile of leiomyosarcoma and the impact of these factors on patient outcomes.

Methods and Results

Tissue microarrays including 203 non-uterine and 181 uterine leiomyosarcoma with a spectrum of tumor morphologies were evaluated for expression of immunohistochemical markers of muscle differentiation. Poorly differentiated tumors frequently lost one or more conventional smooth muscle markers (smooth muscle actin, desmin, h-caldesmon and smooth muscle myosin (p<0.0001)), as well as more recently described markers SLMAP, MYLK and ACTG2 (p<0.0001). In primary tumors, both desmin and CFL2 expression predicted improved overall survival in multivariate analyses (p=0.0111 and p=0.043, respectively). Muscle-marker enriched tumors (expressing all 4 conventional markers or any 3 of ACTG2, CFL2, CASQ2, MYLK, and SLMAP, had improved overall survival (p<0.05) in univariate analyses.

Conclusions

Morphologically and immunohistochemically, poorly differentiated leiomyosarcoma can masquerade as undifferentiated pleomorphic sarcoma with progressive loss of muscle markers. Expression of muscle markers has prognostic significance in primary leiomyosarcoma independent of tumor morphology.

Keywords: Leiomyosarcoma, differentiation, immunohistochemistry, prognosis, desmin

Introduction

Leiomyosarcomas account for up to 20% of all adult soft tissue sarcomas,1–7 and are characterized by an aggressive clinical course and relative insensitivity to cytotoxic chemotherapy.4, 5 Leiomyosarcomas demonstrate smooth muscle differentiation, and are typically subclassified for diagnostic and therapeutic purposes as uterine and non-uterine (somatic) origin. Uterine leiomyosarcoma is distinguished from the more common myometrial leiomyomas, by a combination of mitotic activity at least 5/10 high power fields in the presence of necrosis and cellular atypia, or mitotic activity >10/10 high power fields in the absence of necrosis or atypia.8–11 In somatic tissues outside of the dermis or gastrointestinal tract, leiomyomas are extremely rare. Thus, smooth muscle tumors with any mitotic activity are generally considered to be leiomyosarcoma, and commonly graded using the Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC) criteria.6

Well-differentiated leiomyosarcomas demonstrate a growth pattern of perpendicularly-intersecting cellular fascicles composed of elongated spindle-shaped tumor cells with abundant, brightly eosinophilic, fibrillary cytoplasm. Nuclei generally have an elongated, blunt-ended oval “cigar-like” shape. Variable numbers of markedly pleomorphic “monster” cells may be seen. In poorly differentiated tumors, classic features may be focal or difficult to appreciate as the proportion of pleomorphic cells increases. Additional morphologic variants include epithelioid and myxoid types, which are more commonly associated with uterine origin. While the majority of cases can be diagnosed on biopsy based on classic histologic features on H&E stained sections, morphologic variants, poorly differentiated tumors, and those with unusual features may be difficult to distinguish from other sarcomas and may require additional immunohistochemical evaluation. In anaplastic variants, or extremely poorly differentiated tumors, myogenic markers may be lost.12

Commonly used markers of muscle differentiation in clinical practice include desmin, seen in all types of muscle, smooth muscle actin (SMA), most commonly present in smooth muscle or myofibroblasts, h-caldesmon and smooth muscle myosin (SMMS), both of which are relatively specific for smooth muscle. More recently, expression profiling studies identified an additional set of muscle markers, including smooth muscle gamma actin (ACTG2, typically expressed in enteric smooth muscle), calsequestrin 2 (CASQ2, cardiac and skeletal muscle), human muscle cofilin 2 (CFL2, skeletal muscle), myosin light chain kinase (MYLK, smooth muscle) and sarcolemmal membrane associated protein (SLMAP, all muscle types) as being associated with improved outcomes when 3 or more were co-expressed – so called “muscle-enriched” leiomyosarcomas.13

Few studies have investigated the utility of muscle marker expression as a more objective measure of differentiation status compared to histologic appearance on H&E sections. We therefore queried how well muscle marker expression correlated with histologic assessment of tumor differentiation, and if muscle marker expression could independently predict tumor behavior and survival outcomes in primary leiomyosarcoma.

Materials and Methods

Patents and tumor tissues

Acquisition of tissue specimens and clinical information and subsequent analyses were approved by the Institutional Review Board (IRB) of The University of Texas M. D. Anderson Cancer Center (UTMDACC).

Tissue microarray construction

All available formalin-fixed, paraffin-embedded leiomyosarcoma specimens collected between 1993 and 2010 were retrieved from UTMDACC pathology archives. Hematoxylin and eosin (H&E)-stained sections were reviewed to confirm the diagnosis, define areas of viable tumor, and select one or more areas (if there was morphologic variability) for inclusion in tissue microarrays. Extremely poorly differentiated tumors or those that had heterologous elements were only included in the study if they had a prior documented history leiomyosarcoma with at least focal typical immunohistochemical and/or morphologic features.

An automated tissue microarray apparatus (ATA-27, Beecher Instruments, Sun Prairie, WI) was used to obtain and format paired 1.2 mm punch samples from each case into recipient blocks.14 H&E staining of 4 μm tissue microarray sections was used to verify all samples. Individual cores on the tissue microarray were screened by an experienced soft tissue pathologist and classified by differentiation score as 1) well, 2) moderately or 3) poorly differentiated (Figure 1).6 Tumors with myxoid or epithelioid morphology were considered to be moderately and poorly differentiated, respectively. Only the most poorly differentiated pair of cores from each case was considered in data analysis.

Figure 1. Morphologic Variation in Leiomyosarcoma.

Figure 1

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A) Well differentiated tumor demonstrating intersecting fascicles of slightly atypical eosinophilic spindle cells. B) Moderately differentiated tumor with nuclear variability and increased disorganization of fascicles. C) Moderately differentiated myxoid tumor with bland spindle cells in a storiform to fascicular arrangement. D) Moderately differentiated tumor with scattered “monster cells.” E) Poorly differentiated tumor with loss of fascicular architecture, and increased rounded to epithelioid cells. F) Poorly differentiated tumor with epithelioid features. G) Poorly differentiated tumor showing therapy effect and marked nuclear pleomorphism. H) Poorly differentiated tumor with pleomorphism, loss of architecture, and numerous mitoses. I) Poorly differentiated “undifferentiated pleomorphic sarcoma-like” tumor showing storiform growth, marked pleomorphism and scattered inflammatory infiltrate. (All panels at 200X).

Clinicopathologic Data Collection

Patient and tumor variables, including age, gender, tumor site, size, and disease status were recorded. Sites of primary tumors were categorized thusly: uterine; vascular (apparently arising from or extensively invading a large caliber vein); retroperitoneal/pelvic (not clearly arising in association with a specific intra-abdominal organ or vascular structure); extremity (leg or arm, including dermal tumors); trunk (chest wall, superficial abdominal wall, back, paraspinal); uterine; or other miscellaneous sites (including viscera, bone and head and neck).

Outcome data including survival and disease recurrence were tabulated for primary tumors only. Complete FNCLCC grading criteria (mitotic figures / 10 high power fields, percent necrosis) were not tabulated because these were not equivalently applicable in primary uterine leiomyosarcoma.

Immunohistochemical analysis

Immunohistochemical staining was performed on 4-μm-thick tissue microarray sections using an automated stainer (Dako, Carpinteria, CA) following the manufacturer’s instructions, and commercially available antibodies (Table 1). Positive and negative controls were run in parallel. Sections were counterstained with hematoxylin. SMA, desmin, SMMS, and h-caldesmon were scored as absent (0), focal (<10%) or diffuse (≥10%). ACTG2, CASQ2, CFL2, MYLK and SLMAP were scored as completely absent (0), or by intensity if any staining present as weak (1) or strong (2). For the purposes of outcome analysis, only diffuse/strong staining was considered positive. Muscle enriched tumors were defined as those expressing 3 or more of: ACTG2, CASQ2, CFL2, MYLK and SLMAP.

Table 1.

Antibodies used for immunohistochemistry

Marker Catalog # Company dilution
Smooth muscle actin A2547 Sigma, St Louis, MO 1:80,000
Desmin M0760 Sigma 1:200
Caldesmon M3557 Dako, Carpenteria, CA 1:50
Smooth muscle myosin IR066 Dako 1:200
Smooth muscle gamma actin (AC TG2) H00000072-A01 Novus Biologicals, Littleton CO 1:2,000
Calsequestrin 2 (CASQ2) GTX90833 GeneTex Inc, Irvine, CA 1:100
Human muscle cofilin 2 (CFL2) GTX92818 GeneTex Inc 1:100
Myosin light chain kinase (MYLK) GTX91044 GeneTex Inc 1:25
Sarcolemmal associated protein (SLMAP) GTX94163 GeneTex Inc 1:2,500
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Statistical methods

Associations between histopathological features and immunophenotype were examined using Fisher’s exact test or χ2 test, with an alpha of p≤0.05 (adjusted to p≤0.025 for multiple comparisons) considered significant. For primary tumors only, the method of Kaplan and Meier was used to assess outcomes. Univariable and multivariable Cox proportional hazards regression models were used to estimate the association between histopathologic features or biomarker expression with overall survival (OS), disease-specific survival (DSS) and time to first metastasis, with alpha of 0.05 considered as significant.

Results

Leiomyosarcoma characteristics

Tumors included 384 leiomyosarcoma from 257 patients, including 198 women and 59 men. Primary sites of origin included the uterus (41%), retroperitoneum/pelvis (19%), large vessels (16%), extremities (11%), trunk (3%), and miscellaneous sites (9%). Vascular leiomyosarcoma predominately arose in the inferior vena cava and renal vessels, with a few cases originating from other deep veins. Tumors in the head and neck arose in the nasal cavity, sinuses and mandible, while organs giving rise to leiomyosarcoma included lung/bronchus, bowel, bladder, liver and penis. Extremity tumors included 4 leiomyosarcomas of dermal origin. There were 98 primary leiomyosarcomas, 93 local recurrences (including both those recurring within a prior surgical site and intra-abdominal spread) and 192 distant metastases. The median size of primary leiomyosarcomas was 8 cm (range 0.7 to 30 cm).

When tumors were classified by their microarray core with the least-differentiated histology, there were 130 (34%) well-differentiated leiomyosarcomas, 170 (45%) moderately differentiated and 82 (21%) poorly differentiated. Loss of differentiation correlated with disease progression (p<0.0001), with primary tumors being more frequently well differentiated (53%) than either recurrent (30%, p=0.0013) or metastatic tumors (26%, p<0.0001) (Figure 2). There was no significant further loss of differentiation between recurrent and metastatic tumors.

Figure 2. Histologic Features of Leiomyosarcoma by Site and Stage.

Figure 2

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Tumor status correlates with differentiation. Primary tumors are most frequently well-differentiated (53%) compared to recurrence or metastases (30%, p=0.0013 and 26%, p<0.0001 respectively). Poorly differentiated tumors are most commonly metastases (29%) compared to 19% recurrences (p=0.087), and 9% of primaries (p=0.0001).

Muscle marker expression

Smooth muscle markers SMA and ACTG2 were the most frequently expressed (91% and 90% of cases, respectively), while striated muscle marker CASQ2 was least prevalent (18% of cases) (Table 2). In sum, 235/377 (62%) tumors expressed all 4 conventional muscle markers, and 292 (77%) expressed 3 or more, while 56 (15%) expressed only one or no markers. Similarly, muscle-enriched phenotype was seen in 298/357 cases (83%).

Table 2.

Muscle marker expression by differentiation status.

Total (n=384) Well differentiated (n=131) Moderately differentiated (n=170) Moderately vs. well- differentiated (p value) Poorly differentiated (n=82) Poorly vs. well- differentiated (p value) Poorly vs. moderately differentiated (p value)
SMA 91% 99% 93% 0.0082 73% <0.0001 <0.0001
Desmin 74% 89% 75% 0.0045 50% <0.0001 <0.0001
SMMS 76% 91% 79% 0.0065 47% <0.0001 <0.0001
Caldesmon 78% 94% 78% 0.00013 51% <0.0001 <0.0001
SLMAP 83% 93% 84% 0.028 63% <0.0001 0.003
CASQ2 18% 23% 17% 0.24 12% 0.044 0.34
ACTG2 90% 99% 94% 0.047 66% <0.0001 <0.0001
CFL2 69% 77% 64% 0.0196 64% 0.0537 1
MYLK 82% 95% 77% <0.0001 71% <0.0001 0.42
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Expression of muscle markers correlated with morphologic differentiation (Figure 3). Expression of all four conventional muscle markers decreased from 82% (106/129) in well to 60% (100/167) in moderate to 36% (24/81) in poorly differentiated tumors (p<0.0001). The most dramatic loss of expression was seen in SMMS, which was expressed in 118/130 (91%) well differentiated tumors, 133/169 (79%) moderately differentiated tumors and only 38/81 (47%) poorly differentiated, while SMA was retained at the highest levels, being present in 129/130 (99%) of well-differentiated, 158/170 (93%) moderately differentiated and 60/82 (73%) poorly differentiated tumors. Statistically significant loss of expression of SLMAP, ACTG2, MYLK, and COFL2 was also seen as tumors became more poorly differentiated.

Figure 3. Expression of Markers of Smooth Muscle Differentiation in Leiomyosarcoma.

Figure 3

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A) Diffuse expression of smooth muscle actin, desmin, smooth muscle myosin, and caldesmon are reduced in less-well-differentiated tumors. B) Total number of conventional muscle markers with diffuse expression is reduced in more poorly differentiated tumors, p<0.0001. C) Expression of SLMAP, CASQ2, ACTG2, MYLK and CFL2 is less frequent in poorly differentiated tumors. D) Muscle-enriched phenotype (3 or more of SLMAP, CASQ2, ACTG2, MYLK, CFL2) is less frequent in more poorly differentiated tumors (p=0.0089).

Prognostic utility of differentiation markers in primary leiomyosarcoma

Clinical follow-up was available for 97 primary tumors, and ranged from 2.6-258 months (median 50). Five- and 10-year OS were 54% and 33%, respectively, while 5-and 10-year DSS were 60 % and 41% (Figure 4a). Four patients had distant metastases at the time of diagnosis, while 52/97 patients developed metastases during the course of follow-up. Of these 52 patients, median time to development of first metastasis after primary resection was 15 months (range 8–167 months).

Figure 4. Survival Outcomes in Primary Leiomyosarcoma.

Figure 4

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A) Overall survival, disease-specific survival and time to development of first metastasis for entire cohort. B) Patients with well-differentiated leiomyosarcoma have improved overall survival than those with poorly differentiated tumors. C) Expression of desmin correlates with improved overall survival. D) Moderately differentiated tumors expressing desmin have identical survival to well differentiated tumors, while moderately differentiated tumors without desmin behave like poorly differentiated tumors.

There was no difference in outcome by sex or site (uterine vs. somatic). Primary tumor size >5 cm was associated with poor OS and DSS but not time to first metastasis (Tables 3–5). Histopathological differentiation status alone was a modest predictor of clinical outcomes (Figure 4b). Poor differentiation predicted a worse OS compared to well-differentiated tumors (HR, 3.281 [1.311–8.212], p=0.0112), and trended to shorter time to first metastasis (p=0.0587), but was not predictive of DSS (p=0.0897). There was no significant difference in outcome between well- and moderately-differentiated tumors.

Table 3.

Overall survival, univariate and multivariate

Variable Level N # all cause deaths Hazard Ratio 95% CI Lower 95% CI upper P- Value
Univariate analyses
Actin, smooth muscle >10% pos 96 51 1.2191 0.1673 8.8841 0.845
Caldesmon >10% pos 97 52 0.4154 0.2011 0.8583 0.0177
Desmin >10% pos 97 52 0.3058 0.1617 0.5783 0.0003
Myosin, smooth muscle >10% pos 95 51 0.7951 0.3867 1.635 0.5331
Standard muscle markers all 4 markers expressed 94 50 0.4721 0.2576 0.865 0.0151
ACTG2 strong, diffuse 96 51 1.8255 0.2507 13.2914 0.5524
CASQ2 strong, diffuse 96 51 0.8104 0.4433 1.4816 0.4946
CFL2 strong, diffuse 91 48 0.4191 0.2288 0.7676 0.0049
MYLK strong, diffuse 95 50 0.5227 0.2046 1.3356 0.1753
SLMAP strong, diffuse 97 52 0.6123 0.2981 1.2576 0.1816
Muscle enriched phenotype any 3/5 94 49 0.4177 0.1763 0.9897 0.0473
differentiation score moderate
poor 		97 52 1.4259
3.2807 		0.7843
1.3107 		2.5923
8.2119 		0.2447
0.0112
gender (M/F) M 97 52 1.068 0.6086 1.8742 0.8186
site (U/S) Uterine vs all other 97 52 1.2194 0.5489 2.709 0.6262
Size >5 cm 97 52 2.7263 1.1624 6.3944 0.0211
Multivariate analyses
 Differentiation Moderate poor 91 48 0.9084
3.5215		 0.4373
1.2365		 1.8869
10.0286		 0.7967
0.0184
  Desmin >10% pos 91 48 0.3077 0.1216 0.7788 0.0129
   CFL2 strong, diffuse 91 48 0.4943 0.2615 0.9343 0.0301
   Size >5 cm 91 48 4.5457 1.7251 11.9782 0.0022
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Table 5.

Time to first metastasis, univariate analyses

Variable Level N # with metastases Hazard Ratio 95% CI Lower 95% CI upper P- Value
Univariate analyses
Actin, smooth muscle >10% pos 92 51 0.9286 0.1276 6.7582 0.9417
Caldesmon >10% pos 93 52 0.6418 0.288 1.4304 0.2781
Desmin >10% pos 93 52 0.4643 0.2407 0.8957 0.0221
Myosin, smooth muscle >10% pos 91 50 0.8576 0.4156 1.7696 0.6777
Standard muscle markers all 4 markers expressed 90 49 0.5986 0.3255 1.101 0.0988
ACTG2 strong, diffuse 92 51 1.789 0.2462 13.0021 0.5654
CASQ2 strong, diffuse 92 51 0.6913 0.367 1.302 0.253
CFL2 strong, diffuse 87 46 0.559 0.2876 1.0868 0.0864
MYLK strong, diffuse 91 50 0.9674 0.3463 2.7026 0.9496
SLMAP strong, diffuse 93 52 0.8091 0.3794 1.7254 0.5835
Type-1 leiomyosarcoma markers muscle enriched (any 3/5) 90 49 0.8023 0.2872 2.2412 0.6743
differentiation score moderate
poor		 93 52 1.104
2.4072		 0.6043
0.9684		 2.0171
5.9836		 0.7476
0.0587
gender (M/F) M 93 52 1.324 0.766 2.2886 0.3148
site (U/S)
Size		 Uterine vs all other
>5 cm		 93
93		 52
52		 0.5359
1.7609		 0.1927
0.881		 1.4903
3.5196		 0.2319
0.1093
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Muscle enriched primary leiomyosarcoma predicted improved OS in univariate analysis (HR 0.4177 [0.1763–0.9897], p=0.0473), as did expression of all 4 conventional smooth muscle (HR 0.4887 [0.2711–0.8809], p=0.0172). As individual markers, CFL2, desmin, and h-caldesmon all associated with improved survival (p=0.0049, p=0.0003, and p-0.0177, respectively) (Table 3). The small sample size limited multivariate analyses of OS, and only the 2 known risk factors (tumor size, differentiation score) and 2 muscle markers (desmin and CFL2) were included in the model. All 4 retained independent prognostic significance.

Significant prognostic biomarkers for DSS were similar to those of overall survival (Table 4). Loss of any one of the conventional smooth muscle markers increased risk (HR 2.5031 [1.3078–4.7916]; p=0.0056), while muscle-enriched tumors trended to improved DSS (p=0.0535). Both CFL2 and desmin were associated with decreased risk (p=0.0076 and p<0.0001), while expression of h-caldesmon trended to be protective (p=0.061) in univariate analyses. In multivariate analysis (including size, expression of all 4 conventional muscle markers, CFL2 and desmin expression in the model), only size >5 cm retained prognostic significance.

Table 4.

Disease specific survival, univariate and multivariate

Variable Level N # all cause deaths Hazard Ratio 95% CI Lower 95% CI upper P-Value
Univariate analyses
Actin, smooth muscle >10% pos 96 40 0.9083 0.1239 6.658 0.9246
Caldesmon >10% pos 97 41 0.4042 0.1567 1.0426 0.061
Desmin >10% pos 97 41 0.2398 0.121 0.4753 <0.0001
Myosin, smooth muscle >10% pos 95 40 0.8018 0.3544 1.8145 0.5961
Standard muscle markers all 4 markers expressed 94 39 0.3995 0.2087 0.7646 0.0056
ACTG2 strong, diffuse 96 40 1.457 0.199 10.6664 0.711
CASQ2 strong, diffuse 96 40 0.6548 0.3087 1.3887 0.2696
CFL2 strong, diffuse 91 37 0.3978 0.2022 0.7829 0.0076
MYLK strong, diffuse 95 39 0.503 0.1761 1.4368 0.1994
SLMAP strong, diffuse 97 41 0.5184 0.2391 1.1239 0.0961
Type-1 leiomyosarcoma markers muscle enriched (any 3/5) 94 38 0.3928 0.1521 1.0141 0.0535
differentiation score moderate
poor		 97 41 1.3427
2.5765		 0.6902
0.8634		 2.6119
7.6884		 0.3854
0.0897
gender (M/F) M 97 41 1.2493 0.6692 2.3325 0.4847
site (U/S) Uterine vs all other 97 41 0.8502 0.3025 2.3895 0.7583
Size >5 cm 97 41 2.561 1.0028 6.5405 0.0493
Multivariate analyses
Standard muscle markers all 4 markers expressed 91 37 0.6245 0.139 2.8055 0.5391
Size >5cm 91 37 4.2788 1.4224 12.8719 0.0097
Desmin >10% pos 91 37 0.2378 0.0474 1.1923 0.0707
CFL2 strong, diffuse 91 37 0.5996 0.2769 1.2986 0.1946
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Desmin was the only significant predictor of time to first metastasis in univariate analysis (HR 0.46343 [0.2407–0.8957]; p=0.0221).

Discussion

Although immunohistochemistry is frequently used to support a diagnosis of leiomyosarcoma, few studies have specifically addressed the correlation between morphologic evidence of smooth muscle differentiation and immunohistochemical markers. We examined muscle marker expression in a diverse array of leiomyosarcomas to better understand the patterns of marker loss over the course of disease progression (from well- to poorly differentiated tumors, and from primary to metastatic disease). Of particular interest was whether immunohistochemical markers of muscle differentiation could provide a more objective measure of tumor differentiation than morphology in predicting patient outcomes.

When we reviewed the leiomyosarcoma tissue cored on our tissue microarray, we identified a number of poorly differentiated cores which showed few, if any, classic features of leiomyosarcoma and which could easily have been mistaken for undifferentiated pleomorphic sarcoma if examined out of context. These cores demonstrated a significant loss of markers of smooth muscle differentiation compared to well and moderately differentiated cores. While Carvalho et al., reported no correlation of muscle marker expression (SMA, desmin, caldesmon, calponin, and myosin) with histologic differentiation in a series of 78 cases,15 other studies report similar finding as ours, including retention of diffuse SMA in poorly differentiated tumors, with loss of both desmin and caldesmon (59% vs. 83% and 19% vs. 68%, respectively), compared with well-to-moderately differentiated tumors,16–18 and loss of smooth muscle marker expression in the less-differentiated areas of pleomorphic and “dedifferentiated” variants of leiomyosarcoma.12, 19, 20

Not only were individual muscle markers frequently lost in poorly differentiated tumors in our series, but the overall total number of muscle markers was reduced, with less than half of poorly differentiated tumors expressing 3 or more of SMA, desmin, h-caldesmon and SMMS, compared to over 90% of well differentiated tumors. This is consistent with reports that myosin and caldesmon expression are frequently co-expressed.15 Similarly to Mills et al.,21 we found that poorly differentiated “undifferentiated pleomorphic sarcoma-like” leiomyosarcoma less frequently expressed a muscle-enriched phenotype—that is, positivity for 3 or more of CASQ2, SLMAP, CFL2, MYLK, and ACTG2, with only 62% of poorly differentiated tumors falling into this category, compared to 98% of well-differentiated.

A significant proportion of so-called undifferentiated pleomorphic sarcomas (UPS, formerly termed malignant fibrous histiocytoma) are likely related to leiomyosarcoma and indeed may represent anaplastic (“dedifferentiated”) leiomyosarcoma. In multiple studies using protein expression analyses,22–26 gene expression analysis27–30 and/or comparative genomic hybridization,31 a subset of UPS consistently cluster with leiomyosarcoma, while up to 5% of UPS demonstrate a muscle enriched phenotype.21 These studies suggest that a subset of UPS represent a form of tumor progression from leiomyosarcoma.26, 27, 31, 32 Effectively differentiating between the two classes may have implications for prognostication and therapeutic selection as we more fully understand disease biology.

In our series, desmin and CFL2 were associated with improved OS independent of histologic differentiation or tumor size. Further analysis of desmin expression demonstrated that the impact of this marker in predicting outcome appeared to rest mainly in its ability to segregate moderately differentiated tumors into 2 groups- those that behaved like well-differentiated tumors and those which behaved like poorly differentiated tumors (Figure 4c and d). We also confirmed, in univariate analyses, the reported association of muscle-enriched phenotype with improved outcome. Of note, our study was underpowered for robust analysis of DSS, due to limited available clinical follow-up. However, in general, prognosticators of OS trended to predict DSS, implying that tumor-related deaths may have been more frequent in our cohort than we were able to confirm.

While we found that increased muscle marker expression predicted improved outcomes in leiomyosarcomas, other studies have found that UPS expressing muscle markers behave more aggressively than those without.22, 33–35 Thus, we are left with the seeming paradox that evidence of myoid differentiation is a positive predictor in leiomyosarcomas, and negative one in true UPS. This contradiction merits further study and reinforces the need to carefully evaluate biomarkers only in context with morphology.

In summary, we assessed the diagnostic and prognostic utility of an array of muscle markers in one of the largest cohorts of uterine and non-uterine leiomyosarcoma to date. We demonstrated that morphologically less-well-differentiated leiomyosarcoma are associated with loss of markers of muscle differentiation, thereby reducing the diagnostic value of immunohistochemistry in biopsies of such cases. Moreover, our findings are congruent with the theory that a subset of UPS represent pleomorphic or very poorly differentiated variants of leiomyosarcoma. Lastly, we identified desmin as an independent predictor of survival, which seems to be of most value in moderately differentiated tumors. Taken together our findings help to more clearly delineate patterns of muscle expression in leiomyosarcoma, which may help to improve diagnostic algorithms, and provide evidence for overlooked prognostic significance of everyday immunohistochemical assays.

Acknowledgments

The authors would like to thank Kim Vu for her invaluable assistance in preparation of the figures. Funding for this research was provided in part by the MD Anderson Physician Scientist Program (A.J. Lazar), a NIH/NCI K08CA160443 (K. Torres), NIH CA 112270 (M. van de Rijn), The Sally M. Kingsbury Sarcoma Research Foundation (K. Torres), NIH/NCI 5T32CA009599-21 (K. Lusby) and NIH/NCI S T32 CA009599-22 training grant (G. Boland). EGD, KET, DL, WLW, and AJL designed the study. GMB, KBS, KL, KLW and MB assembled the clinical database. EDY, DI, XG JLH, MvdR performed the immunohistochemistry. EGD analyzed the data and wrote the paper. WLW, AJL, JLH and MvdR provided manuscript critiques. All authors approved the final version.

Footnotes

Disclosure/ Conflicts of interest: The authors have no conflicts of interest to declare.

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