Original Article | Published:

Discovery of molecular subtypes in leiomyosarcoma through integrative molecular profiling

Oncogene volume 29, pages 845854 (11 February 2010) | Download Citation

This material was presented in part at the 2009 United States and Canadian Academy of Pathology Meeting in Boston, MA, USA.

Abstract

Leiomyosarcoma (LMS) is a soft tissue tumor with a significant degree of morphologic and molecular heterogeneity. We used integrative molecular profiling to discover and characterize molecular subtypes of LMS. Gene expression profiling was performed on 51 LMS samples. Unsupervised clustering showed three reproducible LMS clusters. Array comparative genomic hybridization (aCGH) was performed on 20 LMS samples and showed that the molecular subtypes defined by gene expression showed distinct genomic changes. Tumors from the ‘muscle-enriched’ cluster showed significantly increased copy number changes (P=0.04). A majority of the muscle-enriched cases showed loss at 16q24, which contains Fanconi anemia, complementation group A, known to have an important role in DNA repair, and loss at 1p36, which contains PRDM16, of which loss promotes muscle differentiation. Immunohistochemistry (IHC) was performed on LMS tissue microarrays (n=377) for five markers with high levels of messenger RNA in the muscle-enriched cluster (ACTG2, CASQ2, SLMAP, CFL2 and MYLK) and showed significantly correlated expression of the five proteins (all pairwise P<0.005). Expression of the five markers was associated with improved disease-specific survival in a multivariate Cox regression analysis (P<0.04). In this analysis that combined gene expression profiling, aCGH and IHC, we characterized distinct molecular LMS subtypes, provided insight into their pathogenesis, and identified prognostic biomarkers.

Introduction

Cancer can be broadly divided into three main classes: leukemias/lymphomas (derived from cells of the hematopoetic system), carcinomas (derived from epithelial cells) and sarcomas (derived from mesenchymal tissues, including bone, muscle and cartilage) (Abbas et al., 2005). Within each of these broad categories, tumors have traditionally been further subdivided into specific diagnostic subtypes based primarily on their clinical and histopathological features (Lakhani and Ashworth, 2001; Ackerman and Rosai, 2004). In the past decade, gene expression profiling has been used to discover novel cancer subtypes in a variety of hematological (Alizadeh et al., 2000; Bullinger et al., 2004) and epithelial (Sorlie et al., 2001; Lapointe et al., 2004) malignancies. It is hoped that the further subclassification of cancer based on molecular features will facilitate the identification of prognostic and predictive biomarkers (Sidransky, 2002; Beck et al., 2009b), the development of therapies targeted at oncogenic pathways altered in particular subtypes (Potti et al., 2006), and ultimately, the application of a more personalized form of medicine to improve the lives of cancer patients (Sotiriou and Piccart, 2007).

Soft tissue sarcomas account for approximately 1% of all malignancies diagnosed annually, and there are 100 recognized sarcoma diagnostic subtypes (Weiss and Goldblum, 2008). Sarcomas can be subdivided in two groups, one wherein each tumor type is characterized by a unique simple recurrent genetic abnormality such as a chromosomal translocation and the other in which highly complex genetic abnormalities are present (Helman and Meltzer, 2003). Leiomyosarcomas (LMS) belong to the latter group and are malignant neoplasms of smooth muscle, which most frequently occur in the uterus or retroperitoneum but can occur throughout the body (Fletcher et al., 2002). They account for 24% of soft tissue sarcomas, making LMS the most common soft tissue sarcoma subtype (Toro et al., 2006). Although significant advances have been made in the molecular understanding of sarcoma subytpes with simple recurrent genetic abnormalities (Helman and Meltzer, 2003), the molecular pathogenesis and heterogeneity of LMS are poorly understood. Currently, the diagnosis of LMS is made based on the demonstration of smooth muscle differentiation in a histologically malignant neoplasm (Fletcher et al., 2002). Clinical management typically consists of surgery with adjuvant doxorubicin-based chemotherapy with consideration for the addition of ifosfamide and radiotherapy in selected cases (Borden et al., 2003). Doxorubicin-based chemotherapy has shown a marginal association with improved overall survival (Cochrane Database Syst Rev, 2000) and the addition of ifosfamide has been shown to strengthen the association with improved survival (Pervaiz et al., 2008). The overall prognosis for soft tissue sarcomas is poor, with reported rates of 12-year disease-specific survival (DSS) of 64% (Kattan et al., 2002). There are currently no effective targeted therapies available for LMS that are directed at molecular aberrations in specific LMS subtypes. It has been shown that gene expression signatures may be able to predict metastasis in LMS (Lee et al., 2004). Our laboratory has shown earlier that macrophage infiltration correlates with poor outcome in LMS (Lee et al., 2008; Espinosa et al., 2009). There are currently no molecular biomarkers used in the routine prognostication or determination of treatments in LMS.

In this study, we performed global gene expression analysis of 51 LMS samples to identify three distinct LMS subtypes. We then performed array comparative genomic hybridization (aCGH) to characterize the genomic changes seen in two of the LMS subtypes. On the basis of our gene expression findings, we identified biomarkers for the most distinct LMS subtype and evaluated their pattern of expression and association with clinicopathologic variables by performing immunohistochemistry (IHC) on tissue microarrays (TMAs) containing 377 LMS samples. In this integrative analysis, we characterized distinct molecular LMS subtypes, provided insight into their pathogenesis, and identified prognostic biomarkers.

Results

Gene expression profiling

Unsupervised hierarchical clustering was performed on median centered gene expression data using the 3038 gene spots that had a s.d. of at least 1 across the 51 LMS samples and passed quality-based filtering criteria (described in Supporting Information Materials and Methods). This analysis showed three predominant clusters of LMS samples (Figure 1a). Cluster 1 contained 13 samples derived from 11 patients, cluster 2 contained 12 samples derived from 12 patients and cluster 3 contained 26 samples derived from 23 patients (Supplementary Information Table 1). For the five patients with paired primary, recurrent, and/or metastatic samples in the analysis, the matched pairs clustered into the same group (Figure 1a), suggesting that the molecular subtype is preserved during metastasis, as has been observed in other malignancies (Bernards and Weinberg, 2002; Ramaswamy et al., 2003; Weigelt and van’t Veer 2004; Weigelt et al., 2005). To assess the similarity of each LMS sample to its cluster's centroid, we defined a centroid for the three predominant LMS clusters and assessed the correlation of each LMS sample with each LMS group centroid. In all, 48 out of 49 samples showed the highest correlation with the sample's LMS group centroid, which provides further support of the clusters’ robustness (Supplementary Figure S1). Neither of the two ‘outlier’ cases from unsupervised hierarchical cluster analysis showed strong correlation with any of the LMS group centroids (correlation <0.35).

Figure 1
Figure 1

Unsupervised clustering of 51 leiomyosarcoma (LMS) samples reveals three reproducible molecular subtypes.(a) Unsupervised hierarchical clustering was performed on 51 LMS samples with 3038 genes that showed at least one s.d. across the samples. The 20 samples that were also profiled for DNA copy number changes with array comparative genomic hybridization (aCGH) are indicated by an asterisk. The five paired primary-metastasis samples are indicated by a paired symbol (#, $, &, !, ). On the sample dendrogram, the group I cases are highlighted in red, group II blue and group III green. The two cases that did not cluster into a group are indicated in black. Within the heatmap, yellow indicates relatively increased expression, black indicates median expression and blue indicates relatively decreased expression. (b) Principal component analysis of the 51 LMS samples with 3038 genes. Each sample is represented in the figure by a colored box. The color indicates the clustering designation made by hierarchical clustering: red=group I, blue=group II and green=group III. A majority of the variance between the three groups are captured in the first two principal components. (c) Sparse principal component analysis. The 51 LMS samples were plotted against the sparse principal component analysis (PCA) coordinate 1 (containing 45 genes) and sparse PCA coordinate 2 (containing 40 genes). Each sample is represented by a colored circle, and the color indicates the clustering designation made by hierarchical clustering: red=group I, blue=group II and green=group III. A majority of the variance between the three LMS molecular subtypes are explained by these two sparse principal components.

To visualize the variability within LMS by an additional unsupervised technique, we performed principal component analysis, which showed that the molecular subtypes observed in hierarchical clustering were largely captured in the first two principal components (Figure 1b). To identify a sparse set of genes composing the first two principal components, we performed sparse principal component analysis (Zou et al., 2006) and identified 45 genes in component 1 and 40 genes in component 2. Plotting the LMS samples along these two sparse principal components largely recreates the clustering structure observed in the hierarchical clustering analysis (Figure 1c), and the classifications into three subtypes made using a simple nearest neighbor classifier based on these two principal components is concordant with the classifications made by unsupervised hierarchical clustering for 48 out of 49 LMS samples (Figure 1c).

Significance analysis of microarrays (Tusher et al., 2001) was performed to identify sets of genes highly differentially expressed between the three LMS subtypes. The set of genes most highly and differentially expressed in LMS group I was significantly enriched for functional annotation terms relating to muscle contraction and the actin cytoskeleton (including CALD1, SLMAP, DMD, ACTG2, CASQ2,CFL2, MYLK and LPP) (Supplementary Information Table 1). As this gene set is highly enriched for genes encoding proteins involved in muscle differentiation and function, we refer to this LMS group as the ‘group I/muscle-enriched’ LMS molecular subtype. This gene set also showed significant enrichment for phosphoproteins, protein kinases and kinase-binding proteins (Supplementary Information Table 1). The set of genes most highly and differentially expressed in LMS group II was significantly enriched for functional annotation terms relating to protein metabolism, regulation of cell proliferation and organ development (Supplementary Information Table 1). The set of genes most highly and differentially expressed in LMS group III was significantly enriched for annotation terms relating to organ and system development, metal binding, extracellular proteins, proteins involved in the wound response and ribosomal proteins involved in protein synthesis (Supplementary Information Table 1). Of the three LMS subtypes, the group III gene set contained the maximum number of genes (23) and the highest proportion of genes from the CSF1 response gene expression signature (Beck et al., 2009a). In a previous study, the CSF1 response gene expression signature was shown to be present in a subset of LMS, and the expression of four CSF1 response signature-associated proteins was associated with poor prognosis in LMS (Espinosa et al., 2009).

To compare the pattern of gene expression seen in these LMS subtypes with other sarcomas, we performed unsupervised clustering of a large and diverse set of soft tissue tumors (STTs, n=291, spanning 25 diagnostic subtypes) analysed in our laboratory with the 51 LMS samples using the same gene list that was used above to cluster only the LMS samples. In this analysis, samples from the group I/muscle-enriched cluster continue to cluster together, while cases from the other LMS clusters were interspersed in the dendrogram with other STT types (Supplementary Figure S2a). We performed sparse principal component analysis on the same set of LMS and STTs and similarly found that the group I/muscle-enriched LMS samples formed the most distinct cluster of LMS cases, whereas the other subtypes of LMS were intermixed with other STTs (Supplementary Figure S2b). These results suggest that of the three LMS subtypes, group I/muscle-enriched shows the most distinct and specific gene expression profile.

To evaluate the reproducibility of the LMS molecular subtypes in an independent data set, we searched the gene expression omnibus for publically available sarcoma gene expression data sets and identified one data set containing >15 LMS samples (Baird et al., 2005 (GSE2553) contains 17 LMS samples). We used the clusterRepro algorithm (Kapp and Tibshirani, 2007) by training it on our LMS data set and testing it on the Baird set of 17 LMS samples. In this analysis, 12 of the 17 Baird LMS samples were classified as group I/muscle-enriched LMS, and the group I LMS achieved statistically significant cluster reproducibility (IGP=1, P=0.03). Only 1 of the 17 cases was classified as group II and 4 of the 17 cases were classified as group III. Neither LMS group II nor III achieved statistically significant reproducibility on this data set (IGP<0.5, P>0.5). These findings suggest that the group I/muscle-enriched LMS subtype is significantly reproducible in the Baird data set, and most LMS cases from this data set are best characterized as group I/muscle-enriched LMS (Supplementary Figure S3). Although the Baird data set provides no definite support for group II or III LMS, this may be partially explained by the data set's relatively small number of LMS samples and the relatively low level of variability observed in the LMS samples in this data set (Baird et al., 2005).

Information on patient age, tumor grade, tumor histological subtype, primary tumor site, tumor status (primary, recurrence, metastasis) and patient treatment were available for the LMS cases in the gene expression analysis (Supplementary Information Table 1). There was no significant difference in patient age or tumor grade between the three molecular subtypes (P>0.15). Group I LMS tended to be conventional LMS histological subtype (8/11, 73%), group II LMS were relatively evenly distributed between conventional (5/12, 42%) and pleomorphic/mixed (7/12, 58%) and group III LMS tended to be pleomorphic/mixed histological subtype (16/21, 76%) (P=0.03). Group I LMS (10/11, 91% extra-uterine) and group II LMS (9/12, 75% extra-uterine) tended to have an extra-uterine primary tumor site, while group III LMS was relatively evenly distributed between uterine and extra-uterine primary sites (9/19, 47% extra-uterine) (P=0.04). In all, 9 out of 13 group I samples and 10 out of 12 group II samples analysed by gene expression profiling were from the primary tumor site, while only 6 out of 23 group III tumors were from the primary site (P=0.002) and the remainder came from a recurrence (7/23) or a metastasis (10/23). (Although there are a total of 24 group III samples, the site for one of the samples (STT4401) was not known to be primary or metastasis/recurrence, and this sample was excluded from this analysis). Information on patient treatment with radiotherapy and/or chemotherapy was available for 45 of the group I, II and III samples. There was no significant difference in proportion of treatment with radiotherapy and/or chemotherapy in the molecular subtypes: 11 out of 13 group I samples, 11 out of 12 group II samples and 14 out of 20 group III samples were resected from patients with no history of previous treatment with radiotherapy or chemotherapy (P=0.3). Three patients contributed tumor samples removed pre- and post-treatment with chemotherapy and/or radiotherapy, and in all three cases the pre- and post-treatment samples clustered into the same LMS molecular subtype.

Array comparative genomic hybridization

Array comparative genomic hybridization was performed to characterize genomic changes in 20 LMS samples and 4 normal smooth muscle samples: 12 of the LMS samples were from group I/muscle-enriched (derived from 10 patients), 7 from group III (derived from six patients) and 1 from group II. To identify regions of genomic gain and loss, the fused lasso technique was performed (Tibshirani and Wang, 2008) with an FDR threshold of 10% for calling regions of gain/loss (Supplementary Information Table 1).

The group I/muscle-enriched and group III LMS samples showed distinct patterns of genomic gain and loss. The group I samples showed significantly increased genomic gains and losses compared with the group III samples (mean proportion of genome involved by gain/loss=17% in group I samples vs 2% in group III samples, P=0.04) (Figure 2). Analyzing the seven group III samples together showed no statistically significant shared regions of gain or loss (all consensus FDR >5%). In contrast, analyzing the 12 group I/muscle-enriched samples together showed 691 spots that each had a consensus FDR <5% (Supplementary Information Table 1). Taken together, these findings suggest distinct pathways of oncogenesis in the two LMS subtypes, with decreased genomic stability in group I/muscle-enriched LMS.

Figure 2
Figure 2

Array comparative genomic hybridization (aCGH) of 20 leiomyosarcoma (LMS) samples. The 20 samples are arranged along the y axis and ordered according to amount of DNA copy number changes. Chromosomal locations are indicated along the x axis. Copy number changes were called using the cghFLasso algorithm with an overall false discovery rate of 0.10. Regions of genomic gain are indicated in red and loss in green. The proportion of genome showing gain or loss is indicated to the left of each row. The gene expression defined molecular subtype is indicated on the color bar on the left: red=group I, blue=group II and green=group III. The group I cases show significantly increased regions of genomic gain/loss compared with the group III cases (P=0.04).

Interestingly, loss of a 291-kb region on 16q24 was seen in 7 of the 12 group I/muscle-enriched samples (all of which showed at least 7% genomic changes) and none of the group II (0/1) or group III (0/7) samples. This genomic region contains several cancer-associated genes, including the Fanconi anemia, complementation group A (FANCA) gene, which is a core Fanconi anemia protein that functions as a signal transducer and DNA-processing molecule in a DNA-damage repair network (Wang, 2007). In acute myeloid leukemia, it has been shown that acquired FANCA dysfunction promotes cytogenetic instability and clonal progression (Lensch et al., 2003). The loss of FANCA may be an important event that is specific for the molecular pathogenesis of the group I/muscle-enriched LMS subtype and suggests an etiology for the increased genomic complexity observed in this LMS subtype. In addition, this region contains CBFA2T3, which is known to be involved in a translocation with RUNX1 (AML1) in a subset of therapy related acute myeloid leukemia (Ottone et al., 2009) and was identified as a putative breast tumor suppressor gene (Kochetkova et al., 2002). The shared deleted region on 16q24 spans a total of 36 genes and includes CDK10, TCF25, FOXF1 and IRF8 in addition to FANCA, CBFA2T3 and others. The full list of genes can be found in the Supplemental Workbook.

The most commonly shared region of gain or loss in the group I/muscle-enriched cases was a 2.5-Mb region on 1p36.32, which spans PRDM16, TNFRSF14, C1orf93 and MMEL1. This region was lost in 8 of the 12 group I/muscle-enriched samples (consensus FDR=0.01). This change was specific to this LMS subtype and there was no loss at 1p36.32 observed in the one group II sample or in the seven group III samples. The PRDM16 gene has recently been shown to control a brown fat/skeletal muscle switch. Loss of PRDM16 from brown fat precursors promotes skeletal muscle differentiation and leads to elevated expression of muscle specific genes (Seale et al., 2008). Group I LMS showed high expression of a diverse set of muscle-associated genes, including genes expressed in smooth, cardiac and skeletal muscle (including ACTG2, MYLK and PDLIM5), genes expressed primarily in cardiac and skeletal muscle (including CFL2, SLMAP), and genes expressed primarily in cardiac muscle (CASQ2). The loss of PRDM16 gene in most group I cases suggests a potential etiology of the ‘muscle-enriched’ pattern of gene expression observed in group I tumors, which includes both genes expressed in smooth and skeletal/cardiac muscle, suggesting either that PRDM16 may have a role in expression of genes involved in skeletal, smooth and cardiac muscle or a separate transcriptional regulatory factor may account for the increased expression of smooth muscle and cardiac muscle associated genes in group I/muscle-enriched LMS. MYOCD amplification has recently been shown to have an important role in LMS pathogenesis (Perot et al., 2009). In our study, a region on 17p11 containing MYOCD (as well as MAP2K4) was amplified in three group I/muscle-enriched tumors and the one group II sample.

Other cancer-associated genes that showed copy number gains in at least six of the group I samples included: TCF12 (15q21), ABL2 (1q24) and the MET oncogene (7q31). MET overexpression has been reported earlier in a variety of sarcomas, including alveolar soft part sarcoma (Jun et al., 2009), osteosarcoma, chondrosarcomas and LMS (Rong et al., 1993). Copy number gain suggests a possible mechanism of MET overexpression in LMS. Additional cancer-associated genes that were lost in at least six group I samples include the alveolar soft part sarcoma chromosome region, candidate 1 (ASPSCR; 17q25), BCL3 (19q13), ERCC2 (19q13), FSTL3 (19p13), RB1 (13q14), STK11 (19p13) and TCF3 (19p13) (An expanded table of cancer-associated genes with the copy number changes observed in our study is provided in the Supplemental Workbook).

There were no recurrent genomic changes seen in >3 of the 7 group III samples, and all of the changes seen in multiple group III samples were also observed in multiple group I/muscle-enriched samples. The only change involving a known gene that was shared in at least 3 of the 7 group III cases was gain at 7q31.2, which includes the CAV-1 gene. This region was also gained in 7 of the 12 group I/muscle-enriched cases and the one group II case. Caveolin-1 is known to be expressed on smooth muscle and has been shown to activate the Akt pathway in an in vitro prostate cancer model (Li et al., 2003). The Akt pathway has been shown to have an important role in LMS (Hernando et al., 2007).

To determine whether the gene expression defined molecular subtypes could be accurately predicted based solely on aCGH changes, we used three classification techniques: prediction analysis of microarrays, prediction analysis of microarrays-FusedLasso (PAM-FL) and K nearest neighbor. The aCGH-based K nearest neighbor classifier obtained a cross validation error rate of 4 out of 19 (21%, corresponding to a permutation-based P-value of 0.05), the prediction analysis of microarrays classifier obtained a cross validation error rate of 2 out of 19 (11%) and the PAM-FL obtained a cross validation error rate of 1 out of 19 (5%). These findings show that the gene expression defined groups I and III can be predicted with significant accuracy by aCGH changes alone. The PAM-FL centroid, which summarizes genomic changes in group I relative to group III is presented as Supplementary Figure S4.

Tissue microarray analysis

On the basis of the findings from gene expression profiling (which showed that group I/muscle-enriched LMS represented the most distinct molecular subtype) and aCGH (which showed that group I/muscle-enriched LMS had the most recurrent regions of genomic gain and loss), we chose to focus our TMA analysis on evaluating the protein expression of genes with high levels of messenger RNA expression in the group I/muscle-enriched LMS molecular subtype.

The protein expression of five markers highly and differentially expressed in group I/muscle-enriched LMS was examined on LMS TMAs. For the 377 LMS cases represented on the TMAs, there were evaluable results for all five stains for 275 cases. For the purposes of clinicopathologic analysis, we determined the sum total of positive staining markers for each case. Clinicopathologic data (including FNLCC histologic grade, mitotic count, necrosis and presence of CSF1-response protein expression signature (Beck et al., 2009a; Espinosa et al., 2009)), and DSS were available for 124 of the 275 cases. Information on anatomic site (gynecological vs non- gynecological) was available for 273 samples.

The stains showed significant correlation with each other (all pairwise Spearman's rho P<0.005) with a minimum correlation of 0.170 between ACTG2 and CASQ2 and a maximum correlation of 0.658 between ACTG2 and SLMAP (Figure 3). In all, 19% (51/275) of the cases showed coordinate expression of all five evaluable markers, similar to the 25% of cases present in the muscle-enriched cluster by gene expression arrays.

Figure 3
Figure 3

Protein expression of group I markers on leiomyosarcoma (LMS) tissue microarray (TMA). We performed immunohistochemistry (IHC) for five markers that showed high levels of expression in group I LMS in the gene expression analysis (CASQ2, MYLK, CFL2, SLMAP and ACTG2). The LMS TMAs contained a total of 377 samples that were scored as strong positive (bright red in the heatmap), weak positive (dull red) or negative (green). The antibodies are listed along the y axis and the 377 samples along the x axis. Missing data are indicated by white in the heatmap. Pictures of an LMS sample showing strong expression of all five stains is shown to the left of the heatmap (magnification=200 × ). The five stains showed significantly correlated expression (all pairwise Spearman's rho P<0.005, with a minimum correlation of 0.17 between ACTG2 and CASQ2 and a maximum correlation of 0.66 between ACTG2 and SLMAP).

The number of positive markers showed no association with site (mean positive markers=3.1 in both gynecological and non-gynecological LMS), no significant association with grade (mean positive markers=3.1 in grade 1, 3.3 in grade 2 and 2.9 in grade 3; P=0.56), no significant association with mitotic figure count (P=0.335), and there was a trend for a negative association with the presence of necrosis (P=0.07). In a multivariate model incorporating the CSF1-response protein expression signature, the number of positive group I/muscle-enriched markers, grade, tumor site (uterine vs extra-uterine), necrosis and mitotic figures, the CSF1 response signature (summarizing the expression of four CSF1 response signature-associated proteins) and the number of positive group I/muscle-enriched markers were the only two significant predictors of survival, with the CSF1-response protein expression signature showing an association with poor outcome whereas expression of the group I/muscle-enriched markers was associated with a more favorable outcome (Table 1).

Table 1: Multivariate Cox regression analysis of disease-specific survival from LMS tissue microarray (n=124)

The findings from our TMA analysis show that the group I/muscle-enriched markers show correlated protein expression, and the expression of group I/muscle-enriched markers is associated with improved DSS independent of grade, mitotic figures, necrosis, site and the CSF1 response signature.

Discussion

Leiomyosarcoma is an aggressive malignant neoplasm, and its molecular pathogenesis is poorly understood. Treatment options are limited, and there is a major clinical interest in gaining a better understanding of LMS pathogenesis to facilitate the development of targeted therapies.

Several previous studies have performed gene expression profiling on relatively small numbers (n=3–13) of LMS samples (Nielsen et al., 2002; Shmulevich et al., 2002; Ren et al., 2003; Segal et al., 2003; Skubitz and Skubitz, 2003; Quade et al., 2004; Baird et al., 2005; Henderson et al., 2005; Nakayama et al., 2007). Owing to the small number of cases in each study it is difficult to draw conclusions on the heterogeneity within LMS based on these data. Francis et al. (2007) performed gene expression profiling on 177 STTs, including 40 LMS samples. They identified a distinct cluster of 11 LMSs that clustered together, while the remaining 29 LMS samples showed more heterogeneous patterns of gene expression. The distinct cluster of 11 LMS cases from this data set were reported to show high expression levels of a group of muscle-associated genes, many of which were also identified as highly expressed in group I/muscle-enriched LMS in our study (including CALD1, SLMAP, ACTG2, CFL2, MYLK, ACTA2, MBNL1, TPM1, PPP1R12A, DTNA, FZD6, PPP1R12A, CLIC4, CDC42EP3, BARD1, TPM1, RAB27A, MAP1B and EDIL). We find a similar muscle-enriched LMS cluster in our data set and in the Baird data set (Baird et al., 2005). Our findings and those from the literature suggest that multiple molecular subtypes of LMS exist and that the ‘muscle-enriched’ subtype has been reproducibly identified in at least two of the largest data sets.

Several previous reports have looked at comparative genomic hybridization changes in LMS. Meza-Zepeda et al. (2006) performed aCGH on 12 LMS samples and 7 gastrointestinal stromal tumors and observed that LMS showed more genomic losses than gains with the most frequent minimal regions of loss at 10q21.3 and 13q14.2–q14.3, each detected in 9 of the 12 LMS samples in their study. In our study, we identified loss at 10q21.3 in 5 of the 12 group I/muscle-enriched samples and in none of the group II or III samples. We identified loss at 13q14.2 in 6 of the 12 group I/muscle-enriched samples, 0 of the 1 group II samples and 2 of the 7 group III samples. The common region of 13q14.2 that was lost in all eight samples includes the RB1 gene, a well-characterized tumor-suppressor whose loss has been shown to contribute to sarcomagenesis (Landis-Piwowar et al., 2008). Meza-Zepeda et al. (2006) also noted loss at 16q21.2–q22.1 in 6 of the 12 samples and 1p36.32–p36.21 in 4 of the 12 samples, which are both changes we find in our study, specifically in group I/muscle-enriched LMS. Larramendy et al. (2008) evaluated 102 malignant fibrous histiocytomas and 82 LMS cases by conventional comparative genomic hybridization and identified 11 regions with significantly increased losses in LMS compared with malignant fibrous histiocytomas, including 1p36.1pter (10% of LMS vs 1% of malignant fibrous histiocytomas), and 16qter (34% of LMS vs 3% of malignant fibrous histiocytomas), both of which were identified as lost in most group I/muscle-enriched LMS cases in our analysis. It is to be noted that the 1p36 region contains PRDM16 and the 16q24.3 region contains FANCA. To our knowledge, our study is the first to integrate aCGH data with gene expression analysis.

Prognosis in LMS is currently predicted using a combination of traditional clinicopathologic features (Kattan et al., 2002). There are currently no molecular biomarkers used in prognostication in LMS in clinical practice. Gene expression microarrays have been used to identify signatures to predict metastasis in LMS (Lee et al., 2004). Our group has previously identified macrophage infiltration (Lee et al., 2008) and the CSF1 response signature (Espinosa et al., 2009) as predictors of poor prognosis in LMS. In this study, we have identified protein markers from the group I/muscle-enriched LMS subtype and showed that their expression correlates with improved DSS. These findings suggest that despite showing increased genomic complexity, group I/muscle-enriched LMS may be intrinsically less aggressive and more differentiated than other LMS subtypes. In a multivariate model, incorporating traditional clinicopathologic features (size, grade, necrosis and site) as well as the CSF1 response signature and the group I/muscle-enriched markers, we find that only the CSF1 response signature and the number of positive muscle-enriched markers emerged as significant predictors of survival, with the CSF1 response signature correlating with poor prognosis and the expression of group I/muscle-enriched markers correlating with improved prognosis. These prognostic biomarkers, which can be measured with IHC on paraffin-embedded formalin fixed tissue, may prove useful for the clinical management of LMS. Ultimately, we hope that the characterization of distinct molecular subtypes in LMS will lead not only to the identification of clinically useful prognostic markers, but also to the development of treatments to target-specific molecular aberrations observed in the subtypes.

Materials and methods

The 51 tumor samples were obtained from 46 patients (five patients each contributed two samples). Clinicopathologic features of these tumors are provided in Supplementary Information Table 1. The studies were performed using the approval by the Institutional Review Board at Stanford University Medical Center.

Briefly, gene expression profiling was performed on 51 LMS samples using 44K spotted complementary DNA microarrays. To identify molecular subtypes, unsupervised hierarchical clustering and principal component analysis were performed. To assess the reproducibility of the clusters in an independent data set, the clusterRepro algorithm (Kapp and Tibshirani, 2007) was used with the LMS samples from the GSE2553 data set (Baird et al., 2005) used as the testing data set. For 20 of the LMS samples containing gene expression data, aCGH was performed on 44K Agilent arrays. The fused lasso algorithm was applied to identify regions of copy number gain and loss (Tibshirani and Wang, 2008). To determine whether the gene expression defined molecular subtypes could be accurately predicted based solely on aCGH changes, three classification techniques were used: prediction analysis of microarrays (Tibshirani et al., 2002), PAM-FL (See Supporting Information Materials and Methods for explanation of PAM-FL), and K nearest neighbor. IHC was performed on LMS TMAs using antibodies for ACTG2, CASQ2, SLMAP, CFL2, and MYLK, and the stains were scored by two surgical pathologists (AHB and RBW). Additional information on the methods for gene expression profiling, aCGH, IHC and statistical analysis are provided in the Supporting Information Materials and methods. All IHC images used in this study are accessible from the accompanying website: http://tma.stanford.edu/tma_portal/LMS_IMP. In addition, gene expression and aCGH data have been deposited in the Gene Expression Omnibus (Edgar et al., 2002) with accession number GSE17555.

Conflict of interest

The authors declare no conflict of interest.

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

Grant support came from NIH grant CA112270, the National Leiomyosarcoma Foundation and the Leiomyosarcoma Direct Research Foundation. The authors dedicate this paper to the memory of Suzanne Kurtz, LMS patient and founder of LMSdr. This work was supported by NIH grant CA112270, the National Leiomyosarcoma Foundation and the Leiomyosarcoma Direct Research Foundation.

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Affiliations

  1. Department of Pathology, Stanford University Medical Center, Stanford, CA, USA

    • A H Beck
    • , B Edris
    • , I Espinosa
    • , S Zhu
    • , R Li
    • , K D Montgomery
    • , R B West
    •  & M van de Rijn
  2. Department of Pathology, Genetic Pathology Evaluation Centre, Vancouver General Hospital and British Columbia Cancer Agency, Vancouver, British Columbia, Canada

    • C-H Lee
  3. Department of Statistics, Stanford University Medical Center, Stanford, CA, USA

    • D M Witten
    • , R Tibshirani
    •  & T Hastie
  4. Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA

    • B C Gleason
    •  & C D Fletcher
  5. Department of Biochemistry, Stanford University Medical Center, Stanford, CA, USA

    • R J Marinelli
  6. Department of Surgery, The University of California, San Francisco, CA, USA

    • D M Jablons
  7. Department of Anatomic Pathology, Pathology and Laboratory Medicine Institute, The Cleveland Clinic and the Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH, USA

    • B P Rubin
  8. Department of Pathology and Laboratory Service, Palo Alto Veterans Affairs Health Care System, Palo Alto, CA, USA

    • R B West

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https://doi.org/10.1038/onc.2009.381

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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