Alterations of p53 and PIK3CA/AKT/mTOR Pathways in Angiosarcomas: A Pattern Distinct from Other Sarcomas with Complex Genomics

Abstract

Background

The p53 and the PIK3CA/AKT/mTOR pathways are frequently altered in sarcoma with complex genomics such as leiomyosarcoma (LMS) or undifferentiated pleomorphic sarcoma (UPS). The scale of genetic abnormalities in these pathways remains unknown in angiosarcoma.

Methods

We investigated the status of critical genes involved in the p53 and the PIK3CA/AKT/mTOR pathways in a series of 62 AS.

Results

The mutation and deletion rates of TP53 were 4% and 0, respectively. p53 overexpression was detected by immunohistochemistry in 49% of cases and was associated with inferior disease-free survival. Although p14 inactivation or HDM2 overexpression are frequent in LMS and UPS and could substitute for TP53 mutation or deletion, such alterations were rare in angiosarcomas. pS6K and/or p-4eBP1 overexpression was observed in 42% of cases suggesting frequent activation of the PIK3CA/AKT/mTOR pathway in angiosarcomas. Activation was not related to intragenic deletion of PTEN, an aberration that is frequent in LMS and UPS, but absent in angiosarcomas.

Conclusion

Angiosarcomas constitute a distinct subgroup among sarcomas with complex genomics. Although TP53 mutation and PTEN deletion are frequent in LMS and UPS, these aberrations are rarely involved in the pathogenesis of AS.

INTRODUCTION

Angiosarcomas (AS) represent a rare (<2%) subgroup of soft tissue sarcomas (STS) characterized by an aggressive clinical behavior and occurring not only in different anatomical locations, but also in distinct clinical settings, such as de novo (primary AS), or after radiation therapy or chronic lymphedema (secondary AS) ¹. Radical surgery and adjuvant radiotherapy when indicated represent the cornerstone of treatment for patients with localized disease. However, despite an adequate loco-regional treatment, half or more of patients will develop metastatic relapse and will die of disease ². The genetic aberrations involved in AS genesis are still poorly understood. Until recently, data were limited to case reports or small case series showing a complex pattern of numerical and structural genetic aberrations ^3-11. We and others have recently identified MYC amplification as a recurrent genetic aberration in secondary AS ^{12, 13}, but no specific recurrent event has been reported in primary AS to date.

Alterations of TP53 or PTEN are among the most frequent genetic events in human cancer and the role of their combined inactivation in tumorigenesis has been well examined in several contexts, including lymphoma, prostate cancer, glioblastoma, and medulloblastoma ^14-20. Interestingly, the p53 and the PIK3CA/AKT/mTOR pathways are frequently altered in sarcoma with complex karyotypes. Mutations and/or deletions of the TP53 gene have been recently shown to occur in approximately 80% of leiomyosarcomas (LMS), 70% of undifferentiated pleomorphic sarcoma (UPS), and 60% of pleomorphic liposarcomas ^21-23. Large deletions on chromosome 10 including the tumor suppressor gene PTEN have also been observed in approximately 40-50% of LMS and 20-25% of UPS ^{24, 25}. Moreover, in vitro and in vivo studies ^{25, 26} suggest the crucial role of the PIK3CA/AKT/mTORpathway in LMS genesis. Due to its rarity, studies regarding the incidence of p53 and PIK3CA/AKT/mTOR pathway aberrations in AS have used heterogeneous methods and are based on single case reports or small case series ^27-35. Therefore, we investigated the status of key genes involved in these pathways in a series of primary and secondary AS by sequencing, array CGH and immunohistochemistry.

PATIENTS AND METHODS

Patient and samples

Sixty-two AS (30 primary and 32 secondary) were included in this study on the basis of availability of tumor material for genetic studies. The clinico-pathologic characteristics are listed in Table 1. Diagnoses were established according to the World Health Organization Classification of Tumors ¹. This study was approved by the institutional review board (IRB-protocol 02-060).

Table 1.

Clinical and pathologic characteristics of angiosarcoma patients

Case ID	Age	Gender	Previous radiation therapy	Chronic Lymphedema	Primary location
AS3δ	58	M	No	No	Femur
AS4	63	F	Yes	No	Breast
AS5	82	F	No	No	Thigh
AS6	49	F	No	No	Humerus
AS9δ	70	M	No	No	Thigh
AS10δ	70	F	Yes	No	Breast
AS11δ	50	M	No	No	Spleen
AS12	66	M	No	No	Kidney
AS13	67	F	No	Yes	Forearm
AS15δ	61	F	Yes	No	Breast
AS14	40	F	No	No	Breast
AS16	34	M	No	No	Pelvis
AS17	74	F	Yes	No	Breast
AS19	62	F	Yes	No	Breast
AS20δ/AS21	56	F	Yes	No	Breast
AS22	67	F	No	No	Breast
AS25	70	F	Yes	No	Chest wall
AS26	34	F	No	No	Breast
AS27	37	F	No	No	Breast
AS28	54	M	No	No	Pelvis
AS29δ	75	F	Yes	No	Breast
AS30δ	38	F	Yes	No	Head and Neck
AS31	79	M	No	No	Scalp
AS32δ	76	F	Yes	No	Breast
AS33	69	M	No	No	Thigh
AS34	43	F	No	No	Breast
AS36	77	F	No	No	Breast
AS37	71	F	No	No	Mediastinum
AS38δ	74	F	No	Yes	Forearm
AS39δ	84	M	No	Yes	Arm
AS44	63	F	Yes	No	Breast
AS45	66	F	Yes	No	Breast
AS47	65	F	Yes	No	Breast
AS48	74	F	Yes	No	Breast
AS49	43	F	Yes	No	Chest wall
AS56	64	F	No	No	Breast
AS57	66	F	Yes	No	Thigh
AS58	74	M	No	No	Scalp
AS59	79	M	No	No	Nostril
AS61	83	F	Yes	No	Breast
AS62	52	F	No	No	Liver
AS66	77	M	No	No	Femur
AS68δ	80	F	Yes	No	Breast
AS69	60	F	Yes	No	Breast
AS70δ	38	F	No	No	Breast
AS71	41	F	Yes	No	Spleen
AS72	66	F	No	Yes	Breast
AS73δ	83	F	Yes	No	Breast
AS75	80	M	No	No	Head and Neck
AS76δ	76	M	Yes	No	Bladder
AS77	73	M	No	No	Retroperitoneum
AS79	48	F	No	No	Spine
AS83	65	M	Yes	No	Breast
AS85	76	F	Yes	No	Breast
AS100	60	M	No	No	Chest wall
AS101	62	F	Yes	No	Breast
AS109	63	M	No	No	Spleen
AS117	38	F	Yes	No	Head and Neck
AS123δ	76	F	Yes	No	Breast
AS124δ	57	M	No	No	Head and Neck
AS125δ	73	F	No	No	Breast

^δtumors studied by aCGH.

Array Comparative Genomic Hybridization (CGH)

Genomic DNA was isolated from frozen tumor tissue by phenol/chloroform extraction and quality was confirmed by spectrophotometry and electrophoresis. Array CGH analysis was performed as described previously (Thomas et al., 2011). Briefly, tumor and reference DNA samples were labeled with Cyanine-3-dUTP and Cyanine-5-dUTP, respectively, by random priming (Agilent Enzymatic Labeling Kit, Agilent Technologies, Santa Clara, CA). The reference sample comprised a pool of DNA from multiple clinically healthy donors (Promega, Madison, WI) of the same gender as the AS patient. The labeled probes were combined and hybridized to a ~180,000-feature, genome-wide oligonucleotide CGH array (design 052252, Agilent Technologies). Arrays were scanned at 3μm resolution using an Agilent G2565CA scanner. Image data were processed with Feature Extraction version 10.10 and Genomic Workbench version 6.5 (Agilent Technologies). Data were filtered to exclude probes exhibiting non-uniform hybridization or signal saturation and were normalized using the centralization algorithm with a threshold of six. The ADM2 algorithm was used to define DNA copy number aberrations using a ‘three probes minimum’ filter and a threshold of six with a fuzzy zero correction, with genomic gain and loss defined as log₂ test:reference DNA signal intensity >0.2 and <−0.2, respectively. A genomic copy number amplification was defined as a region within which the log₂ ratio of tumor DNA to reference DNA exceeded 2.0.

Real-Time Quantitative Reverse Transcription-PCR

One μg of total RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) at 25°C for 10 min, 37°C for 120 min, 85°C for five min and hold at 4°C. Twenty ng/μl of resultant cDNA was used in a quantitative-PCR using an 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) and pre-designed TaqMan ABI Gene expression Assays (Hs_00234753_m1 for HDM2; Hs_99999189_m1 for P14/CDKN2A). Amplification was carried out at 95°C for 10 min, and 40 cycles (95°C for 15 sec, 60°C for one min). To calculate the efficiency of the PCR and to assess the sensitivity of each assay, we also performed a five point standard curve (80, 26.67, 8.88, 2.96, and 0.98 ng/ul). Triplicates CT values were averaged and amounts of target were interpolated from the standard curves and normalized to GAPDH (reference gene).

Mutation Screening

The mutational status of TP53 (exon 2 to exon 11), PIK3CA (exons 3, 10, 20), BRAF (exon 15) and NRAS (exons 2 and 3) was assessed by direct sequencing of genomic DNA. Protocols and primers are available on request. Sequence analysis was performed with Applied Biosystems Sequence ScannerTM v1.0.

Enhanced Mismatch Mutation Detection Analysis (EMMA) and Copy Number Analysis by Quantitative Multiplex PCR (QMP)

A screening of genomic variants of PTEN was performed by using Enhanced Mismatch Mutation Detection Analysis (EMMA), an electrophoretic heteroduplex analysis based on the use of a matrix increasing the electrophoretic mobility between homoduplex and heteroduplex ³⁶. Primers were designed to include flanking intronic sequences (at least 50 base-pairs from splices sites) and to avoid known polymorphism and prevent mis-priming (primer sequences are available upon request). PTEN coding sequences were divided into nine amplicons, analyzed in three multiplex PCRs. EMMA was performed as previously described using EMMA polymer and according to the manufacturer’s instructions ³⁷. Amplicons showing abnormal profiles were sequenced and analyzed on the ABI PRISM 3130XL Genetic Analyser using the Collection and Sequence Analysis software package (Applied Biosystems, Courtaboeuf, France). Nucleotide position was indicated on the basis of the coding sequence NM_000314.4 and according to the recommended guidelines (available at http://www.emqn.org/emqn.php and www.hgvs.org/mutnomen).

Quantitative multiplex PCR (QMP) was carried out with 200 ng of tumor DNA sample, measured on a NanoDrop (ND-1000, Labtech International, France). All primers were designed in order to include flanking intronic sequences and prevent the detection of the processed pseudogene (PTEN1) that presents 98.6% homology to the cDNA of PTEN (the sequences of primers are available upon request). Amplicon sizes ranged from 111-347 bp. Exons of the PTEN gene were analyzed by two assays consisting of four and five PTEN amplicons, one amplicon of two genes proximal to PTEN (PAPSS2 and KILLIN) and amplicons of the MSH2 and BRCA1 genes used as internal control for copy number analysis. Multiplex PCRs were performed with the Qiagen Multiplex PCR kit, according to the manufacturer’s recommendations (Qiagen, Courtaboeuf, France), and 1 μl of 10X primer mix (final concentration from one to 10 μmol/L of each primer), one of them carrying a 6-FAM label. Eight per cent DMSO was added for the assay including PTEN exon 1 and KILLIN amplicons. Amplification was performed as recommended by the manufacturer (Qiagen) and 24 cycles were performed for quantitative PCR. DNA fragments generated by QMP were separated by capillary electrophoresis on the ABI 3130XL Genetic Analyser (Applied Biosystems). Data were analyzed with the GeneMapper Software V4.0 and copy number variation was evaluated for each amplicon by calculating the relative peak height for each sample with respect to the mean value of the relative peak height observed in all samples.

Immunohistochemistry

Immunohistochemistry (IHC) for p53 (1:800, DO7, Dako, Carpinteria, CA), HDM2 (1:50, clone# 1F2,Calbiochem), p14 (1:1000, clone 4C6/4, Cell Signaling, Danvers, MA), p-S6K (1:50, clone Ser235/236, Cell Signaling), p-4eBP1 (1:500, clone Thr37/46, Cell Signaling), and p44/p-ERK (1:50; clone Thr202/Tyr204, Cell Signaling) was performed according to manufacturer’s recommendations. The immunoreactivity scoring was counted as the percentage of nuclear staining per 10 high-power fields, in several areas, regardless of staining intensity. A 20% cutoff value for detection of positive nuclear reactivities was selected for all antibodies. ^{38, 39}

Statistical analysis

Descriptive statistics were used to show the distribution of variables in the population. Differences between groups were evaluated by χ² test or Fisher’s exact test for categorical variables and t test for continuous variables. The statistical analysis of baseline demographics and clinical outcome is based on all data available up to the cut-off date, 31 August 2011. Disease-free survival was defined as the length of time after diagnosis during which patients had no evidence of disease and was estimated with the use of the Kaplan–Meier method.

RESULTS

TP53 gene alterations are very rare in angiosarcomas

TP53 deletions have been reported to be frequent events in sarcomas with complex karyotypes, especially LMS and UPS. To assess the frequency of this aberration in AS, we analyzed 18 tumors (6 primary and 12 secondary) by array CGH, none of which exhibited copy number loss of the TP53 gene. We then analyzed the mutational status of TP53 by sequencing exons 2-11 on genomic DNA in 52 tumors. We found a TP53 mutation in only two cases (4%) (A138S exon 5, R238Q exon 7), which were not present in the corresponding germline DNA from the same patient. Both cases with TP53 mutation occurred in patients with a secondary breast AS developed in a previously irradiated field (AS10, AS17). Both tumors carried MYC amplification and one of them a KDR A1065T exon 24 mutation (AS17), as previously reported 40. Moreover, we observed a R72P polymorphism in TP53 exon 4 in 30 cases (58%), including 19 secondary and 11 primary AS. P53 protein expression was then assessed by IHC in 46 cases. Twenty-three tumors (50%) were negative for p53 expression and 23 (50%) were positive (including the two tumors with TP53 mutation). There was no significant difference of p53 IHC status between primary and secondary AS. Moreover, there was no correlation between the presence of the R72P polymorphism and protein expression.

p53 protein expression correlates with disease-free survival in angiosarcoma

Since p53 protein status assessed by IHC was previously associated with outcome in several tumor types, including sarcomas with complex genomics, we decided to analyze its correlation with clinical behavior in AS. We found that p53 positivity (defined as >20% nuclear positivity) was significantly associated with worse disease-free survival: 3.4 months (95% CI: 0.8-6) versus 14.9 months (95% CI: 2.5-27.4), p= 0.002 (Figure 1).

Figure 1.

Immunohistochemical (IHC) results for p53 (A,B,C), p14 (D), HDM2 (E) and prognostic impact of p53 expression (F). (A,B) Diffuse and strong nuclear reactivity for p53 in a secondary AS associated with a TP53 mutation and KDR mutation (A: AS17, 200x) and a primary AS without TP53 mutation (B: AS59, 200x). (C) Absence of expression of p53 in a case of secondary AS (AS19, 200x). (D) Diffuse nuclear reactivity for p14 in a secondary AS (AS20, 200x). (E) Absence of expression of HDM2 in a case of secondary AS (AS39, 200x). (F) Kaplan Meier curve for disease-free survival according to p53 IHC status (blue line: negative; green line: positive).

p14 inactivation or HDM2 overexpression does not substitute for TP53 inactivation in angiosarcoma

Since p14 is implicated in p53 degradation by proteasome through its interaction with HDM2, we analyzed the p14 and MDM2 copy number alterations by array CGH and their expression level (real-time PCR, IHC) in order to identify the potential loss of p14 expression or HDM2 overexpression, which could compensate for a lack of TP53 inactivation. However, no deletion of the CDKN2A locus encoding for P14 or MDM2 amplification was observed by array CGH. Real-time RT-PCR was performed for 19 tumors (10 secondary AS and nine primary AS) and did not reveal any significant difference in P14 or MDM2 expression between primary and secondary AS or between AS and other vascular tumors (data not shown). Immunohistochemistry was performed for 43 tumors. We observed P14 and HDM2 expression in 36 tumors out of 40 (89%) and 6 tumors out of 40 (15%) with interpretable results, respectively. We found a concordance between the presence/absence of p14 overexpression and absence/presence of HDM2 expression in 81% of cases.

No PTEN intragenic mutations or deletions are detected in angiosarcoma

PTEN deletion is a frequent event in sarcomas with complex karyotypes. In order to assess the incidence of this genomic aberration in AS, we analyzed a series of 26 tumors using QMP to detect gross genomic rearrangements and by array CGH to detect smaller deletions. Six cases revealed a duplication of all the exons of PTEN, whereas eight cases revealed deletion of all the exons. No case with intragenic rearrangement or deletion was identified. These results indicate the presence of large genomic imbalance involving chromosome 10 but do not favor PTEN as a target gene. We then used EMMA in order to detect point mutations that may inactivate the function of PTEN and substitute the absence of specific deletion. We did not find PTEN mutations in this series of AS. We analyzed the expression of PTEN gene using published expression data from a series of 21 AS and 3 other vascular tumors previously studied on the U133A Affymetrix platform⁴⁰. Although the expression of PTEN was relatively low, it was uniform across the samples and similar between AS and other vascular tumors.

The PIK3CA/AKT/mTOR pathway is activated in a subset of angiosarcoma

Previous studies suggested a crucial role of the PIK3CA/AKT/mTOR pathway in the malignant transformation of endothelial cells. Although, PTEN did not appear to be specifically altered in AS we hypothesized that the PIK3CA/AKT/mTOR pathway was activated through alternative mechanisms in at least a subset of AS. We therefore performed further immunohistochemical analyses to evaluate the expression of p-S6K and p-4eBP1 (4E-binding protein 1), two downstream targets of mTORC1. We also evaluated the activation of the ERK pathway due to its multiple interactions with the PIK3CA/AKT/mTOR pathway and its potential involvement in the phosphorylation of S6 ribosomal protein. We found that 17 tumors out of 40 AS (42%) were positive for pS6K and/or p-4eBP1. We observed also that 12 tumors of 39 (31%) were positive for p44/p-ERK. Eleven of the 17 tumors positive for p-S6K and/or p-4eBP1 were also positive for p44/p-ERK (Pearson correlation coefficient 0.6; P=0.02). Then we investigated the incidence of point mutations in PIK3CA by screening this gene for 13 common hot spot mutations in exons 3, 10 and 20 in 33 cases of AS (22 secondary and 11 primary). We found a recurrent truncating frame shift mutation in exon 10 of the PI3KCA gene (S553fs) in 13 cases (39%) including five primary and eight secondary AS. This mutation was then confirmed by PCR using mutant-specific allele exon 10 primers (Fwd: 5′-CAGTTACTATTCTGTGACTGGTGT-3′; Rev: 5′-AGATTTTCTATGGACCACAGG-3′, Fig. S1). In order to explore further the biological significance of this unusual mutation, we sequenced the cDNA of three mutated cases with available RNA. However, no mutation was identified suggesting that the mutant PIK3CA transcript undergoes a nonsense-mediated decay and therefore is not detectable at the cDNA level (Fig. S1). Moreover, there was no correlation between the PI3KCA mutational status and the p-S6K and/or p-4eBP1 immunohistochemistry results.

NRAS and BRAF are not mutated in angiosarcomas

Given the high proportion of AS being positive for p44/ERK IHC in our series and the potential role of RAS dysregulation in abnormal vascular growth control, we hypothesized that a subset of AS are associated with mutation of the BRAF or NRAS genes. We screened BRAF exon 15 and NRAS exons 2 and 3 in 52 cases of AS, however, no mutations were identified at these hot spots.

DISCUSSION

Recent studies investigating the TP53 pathway in large series of sarcomas with complex genomics (LMS, UPS and pleomorphic liposarcomas) have shown that TP53 alterations are highly recurrent in this group of disease with up to 80% of cases exhibiting at least one TP53 alteration (mutation and/or deletion) ^21-23. Our results showing deletion and mutation rates of 0% and 4%, respectively, indicate that AS display a different TP53 alteration profile than the majority of other sarcomas with complex karyotypes. Our findings are in agreement with previous smaller scale reports suggesting that TP53 mutations are uncommon in AS ^{28, 30}.

Interestingly, a P72R polymorphism in exon 4 was detected in 58% of the analyzed cases. Unlike the deleterious tumor-associated P53 mutations, which occur in about 98% of cases in the DNA-binding-domain of the protein, single nucleotide polymorphisms are expected to be phenotypically silent. The R72P polymorphism occurs in the polyproline domain, which lies between the N-terminal transactivating domain and the DNA-binding domain of p53. This polymorphism has been associated with a modulation of the pro-apoptotic potential of p53 and cancer susceptibility ⁴¹. However, the level of evidence is poor due to important methodological issues and several meta-analyses failed to demonstrate a risk between the R72P polymorphism and any cancer ⁴¹. Interestingly, a high incidence of this polymorphism has been recently reported in atypical vascular lesions occurring after radiotherapy ³⁵. In our series, we have observed this SNP in both primary and secondary AS suggesting the absence of any link between this polymorphism and the etiology of AS.

The rarity of deleterious TP53 mutations or deletions in AS does not necessarily imply that the TP53 pathway is intact and functional in these tumors. p14 acts on the TP53 pathway by interacting with HDM2, an E3 ubiquitin ligase targeting p53 for degradation by the proteasome. This results in a stabilization and activation of p53. Loss of p14 expression and/or HDM2 overexpression are frequent in sarcomas and have been shown to substitute for p53 inactivation in tumors without TP53 gene aberrations. Our results indicate that these genetic aberrations are not commonly involved in AS genesis. Despite these results, we observed a high proportion of AS displaying p53 overexpression. Normal p53 protein has an extremely short half-life and it is not detected by IHC. However, following genotoxic stress, the p53 protein level increases rapidly due to stabilization through post-transcriptional mechanisms. In contrast, mutation of the TP53 gene results in nuclear accumulation of the abnormal protein, which has a longer half-life and can be detected by IHC. Based on the assumption that only stable (presumably mutated) protein will be expressed, TP53 IHC is often used as a surrogate to predict the presence of TP53 mutations. In our study, however, we observed a high level of discordance between the IHC and the sequencing results. Although the two cases harboring a TP53 mutation were found to be positive upon immunostaining, 21 cases harboring wild-type TP53 stained also positive. Such discrepancies have already been reported in several studies suggesting that IHC is not a reliable tool for TP53 mutation screening^{42, 43}. One hypothesis is that this overexpression is the result of specific oncogenic stresses leading to stabilization of the wild-type protein. In this case, we would expect to observe an improved outcome as the result of increased tumor apoptosis, which was not the case. A second hypothesis is that the nuclear accumulation of p53 is dependent on mechanisms different from mutation and reflects functional changes. Interestingly, p53 overexpression by IHC has been identified as a strong prognostic factor in many human cancers, including soft-tissue sarcomas ^{42, 43}. Our data indicate that this biomarker has also a prognostic value in AS.

We have also shown that in contrast with other sarcomas with complex genomics, PTEN is neither deleted nor mutated in AS. Moreover, our expression data showing similar level of PTEN expression across the AS cases and other vascular tumors suggest that epigenetic silencing or transcriptional defect of PTEN is unlikely. All together, our results are in agreement with previous reports showing that constitutional biallelic inactivation of PTEN was associated with vascular malformation, but not with malignant vascular tumors ⁴⁴.

Despite the lack of PTEN alteration in AS, we have identified an activation of the PIK3CA/AKT/mTOR pathway in a significant proportion of cases. Several studies have already demonstrated the crucial role of PIK3CA/AKT/mTOR signaling in endothelial cell homeostasis ⁴⁵. Moreover, embryonal fibroblasts transformed with the PI3KCA gene have been shown to induce AS at the site of injection in chicken ^{46, 47}. Interestingly, we have found a recurrent frameshift mutation affecting the helical domain of PI3KCA in about 40% of cases. The same mutation has been recently reported at a low incidence rate in one case of large B cell lymphoma ⁴⁸ and one case of synovial sarcoma ⁴⁹. Our results showing that this mutation is not detectable at cDNA level strongly suggest that this aberration does not have functional consequence. Therefore, further studies are needed to identify the mechanisms involved in the activation of PIK3CA/AKT/mTOR activation in AS. Indeed, the patterns of regulation of the PIK3CA/AKT/mTOR pathway in cancer cells are very complex and cross-talk with several other pathways. For instance, it is now recognized that PI3KCA and MAPK signals converge upstream and downstream of mTORC1 ⁵⁰. Interestingly, we found a good correlation between the activation of these two pathways in our series with the majority of tumors positive for p-S6K and/or p-4eBP1 being also positive for p44/p-ERK. Overall, our findings may have therapeutic implications, specifically that PI3KCA and/or mTOR antagonists represent a promising approach in the systemic treatment of AS, as already suggested by a recent in vitro study and preliminary clinical data ^{51, 52}. They suggest also that, besides the inhibition of VEGFR receptors, the inhibition of MAPK signaling may be a relevant approach in AS as suggested by preclinical data ⁵³ and may also contribute to the recently described clinical activity of multi-target tyrosine kinase inhibitors such as sorafenib in AS ⁵⁴. Moreover, synergy is suggested with the combination of mTOR and MAPK inhibitors ⁵⁰.

In summary, we have shown here that AS represents a distinct subgroup among sarcomas with complex karyotypes. Although TP53 mutation and PTEN deletion are frequent in LMS and UPS, these aberrations are not involved in the pathogenesis of AS. However, AS are characterized by a complex pattern of alterations of the TP53 and PIK3CA/AKT/mTOR pathways that is not dependent on the MYC amplification status. These results underscore the complex coordinated regulation of the survival and growth-control pathways in AS. Our results represent an important basis for further investigations with the aim of determining how the activities of these pathways are coordinated in AS and for the design of new therapeutic strategies.

Supplementary Material

Supp Fig S1

Figure S1. Detection of truncating frame shift mutation in exon 10 of the PIK3CA gene (S553fs) by DNA PCR and direct sequencing (A), further confirmed by specific mutant allele PCR (B), but not detected by RT-PCR at cDNA level (C).

Figure 2.

Immunohistochemical (IHC) results for p-S6K (A), p-4eBP1 (B) and p44/p-ERK (C). Diffuse and strong reactivity for pS6K (A, 200x), p-4eBP1 (B, 200x) and p44/p-ERK (C, 200x) in a primary angiosarcoma (AS9).