Virchows Archiv

, Volume 446, Issue 2, pp 157–163

Aggressive angiomyxoma: a clinicopathological and immunohistochemical study of 11 cases with long-term follow-up

Authors

  • J. F. Graadt van Roggen
    • Department of PathologyLeiden University Medical Centre
    • Department of Pathology
  • J. A. M. van Unnik
    • Comprehensive Cancer Centre of the Middle Netherlands
  • I. H. Briaire-de Bruijn
    • Department of PathologyLeiden University Medical Centre
    • Department of PathologyLeiden University Medical Centre
Original Article

DOI: 10.1007/s00428-004-1135-9

Cite this article as:
van Roggen, J.F.G., van Unnik, J.A.M., Briaire-de Bruijn, I.H. et al. Virchows Arch (2005) 446: 157. doi:10.1007/s00428-004-1135-9

Abstract

Aims

To report the clinicopathological and immunohistochemical features and longer term biological behaviour of aggressive angiomyxoma, an uncommon mesenchymal neoplasm occurring predominantly in the pelvi-perineal region of adults. Using immunohistochemistry, possible overexpression of CDK4 and MDM2 was analysed, which might point to (cyto)genetic alteration(s) in chromosome region 12q13–15, an area reported to be altered in this tumour entity.

Methods and results

Cases (n=11) of aggressive angiomyxoma were retrieved from the consultation files of the Comprehensive Cancer Centre of the Middle Netherlands (IKMN) panel for soft tissue tumours. Clinical and follow-up information were obtained, and immunohistochemical analysis was performed using antibodies directed against vimentin, cytokeratin AE1/AE3, desmin, α-smooth-muscle actin, CD34, S-100 protein, oestrogen receptors, CDK4 and MDM2. Five patients were female (age range 24–47 years; median 39 years), and six patients were male (age range 36–69 years; median 44.5 years). Of 11 cases, 10 arose in the pelvi-perineal area and 1 arose in the abdominal cavity in close relation to the bladder. Morphology was consistent with previous reports of this entity. Immunohistochemically, 8 of 11 cases were desmin positive (5 of 5 positive in females; 3 of 6 positive in males), 6 of 11 cases were positive for α-smooth-muscle actin, 5 of 11 cases were CD34 positive, 11 of 11 cases, irrespective of gender, were positive for oestrogen receptors and 3 of 11 cases were positive for cytokeratin AE1/AE3. Strong, diffuse nuclear positivity for CDK4 expression was present in all 6 cases tested, while only 1 of 11 cases tested for MDM2 showed weak focal positivity. Clinical follow-up in all cases (range 1–216 months; median 72 months) showed one local recurrence (9%) after 36 months. No metastases or tumour-related deaths were noted.

Conclusions

The sex distribution of cases reported in this study was roughly equal, in contrast to previous reports emphasising the predominance of this tumour in females. Our study confirms the local aggressive nature of aggressive angiomyxoma, although our local recurrence rate is lower than previous reports (9% versus 36–72%); no metastases and/or disease-related patient deaths were documented. All cases arising in females were positive for desmin, while three of the six cases arising in males were negative for desmin, supporting previous findings and indicating that the lesion may be somewhat different in males. The strong diffuse positivity for CDK4 in all six cases tested goes some way in implicating CDK4, either directly or indirectly, in tumourigenesis. The negative immunostaining for MDM2 would argue against functional amplification of this gene.

Keywords

Soft tissue tumourSarcomaAggressive angiomyxomaMyxomaCytogenetics

Introduction

Aggressive angiomyxoma is an uncommon, variably myxoid, mesenchymal neoplasm, occurring predominantly in the pelvi-perineal region of adults, with a reported female to male ratio of approximately 6:1 [1, 4, 7, 11, 17, 18]. The lesion appears to behave in a locally aggressive manner, with tumour recurrence often occurring years after the primary excision [1, 4, 7, 11, 17, 18]. Reported rates of local recurrence in larger series and follow-up reports vary from 36% to 72% [1, 4, 7, 11, 17, 18]. While aggressive angiomyxoma is generally regarded as a benign lesion, two purported cases with metastases and associated tumour-related death have been documented to date [3, 16].

The histogenesis of this tumour remains enigmatic. Recent cytogenetic and molecular studies of aggressive angiomyxoma have identified a variety of genetic alterations, including chromosomal translocations, involving the long arm of chromosome 12, particularly in the chromosomal region 12q13–15 [2, 10, 12, 13, 14]. This genetic region appears to contain a number of genes implicated in the tumourigenesis of a number of mesenchymal tumours, including aggressive angiomyxoma [9]. HMGIC, a gene encoding a member of the high-mobility group of proteins, appears to be involved, possibly by amplification or translocation in most cases, although the molecular mechanisms by which the genetic heterogeneity in tumourigenesis might be explained remain elusive.

Using detailed long-term clinical follow-up information, we have described the clinicopathological features of 11 cases of aggressive angiomyxoma, particularly with respect to their biological behaviour. Additionally, using immunohistochemistry to assay protein expression—and thereby determining possible alteration in gene expression—of MDM2 and CDK4, genes flanking the chromosomal region 12q13–15 and spanning the HMGIC locus, we have attempted to ascertain whether possible genetic alterations (e.g. gene amplification) might be present in this region.

Materials and methods

Cases (n=11) of aggressive angiomyxoma were retrieved from the consultation files of the Comprehensive Cancer Centre of the Middle Netherlands (IKMN) panel for soft tissue tumours, under the auspices of one of the authors (J.A.M.vU). Additional clinical and follow-up information was obtained from the original submitting pathologist following consultation with the treating physician (see Acknowledgements).

Representative formalin-fixed, paraffin-embedded material and/or unstained slides were available for routine haematoxylin and eosin (H&E) and alcian blue staining and immunohistochemical analysis in all cases. No frozen material or cell culture material was available for ancillary (cyto)genetic study.

H&E sections 4-μm thick were collectively re-examined in each case by the three pathologists involved in the paper (J.F.G.vR, J.A.M.vU, P.C.W.H). Staining for alcian blue was performed at pH 2.5. All immunohistochemical stains were performed using the avidin-biotin-peroxidase complex method. The antibodies, their sources, antigen retrieval method and dilutions used are documented in Table 1. All cases (n=11) were stained for cytokeratin AE1/AE3, desmin, α-smooth-muscle actin, S-100 protein, CD34, MDM2, vimentin and oestrogen receptor expression. Of 11 cases, 6 were screened for CDK4 expression. Appropriate positive and negative controls were used throughout.
Table 1

Antibodies used for immunohistochemical analysis

Antibody

Clone

Dilution

Antigen retrieval

Source

Vimentin

1:2000

None

Eurodiagnostika

Cytokeratin AE1/AE3

1:4000

Citrate pretreatment

Chemicon International

Desmin

33

1:400

None

Sanbio

CD34

Qbend10

1:800

None

Neomarkers AL

Smooth-muscle actin

ASM-1

1:2000

None

Progen Immuno Diagnostika

S-100 protein

1:8000

None

Dako

MDM 2

1:400

Citrate pretreatment

Neomarkers AL

CDK4

1:10

Ethylenediaminetetraacetate pretreatment

Transduction lab AL

Oestrogen receptor

1D5

1:200

Citrate pretreatment

Dako

In scoring the immunohistochemical stains, lesional positivity of more or less than 25% of the cells stained per mm2 was empirically used to distinguish between weak and stronger expression per case. The mitotic rate was expressed as the number of mitoses in 10 consecutive high-power fields (HPF; 1 HPF=0.159 mm2 on the microscope used), counted in the most cellular areas.

Results

Clinical findings

The clinical features of the lesions are summarised in Table 2. The study group comprised 6 males (age range 36–69 years; median 44.5 years) and 5 females (age range 24–47 years; median 39 years); age range for the whole group was 24–69 years (median 39 years). All cases, except one (AM 14), were located in the soft tissues of pelvi-perineal area. Case AM 14 presented as a mass lesion within the pelvic cavity, in intimate association with the bladder. Specific details concerning clinical presentation were not available. Primary surgical excision was performed in all 11 cases. Due to the anatomic location, wide excision was only achieved in 2 cases (AM 03, AM 13). Surgical excision in the remaining 9 cases was intralesional (AM 04–08, AM 10–12, AM 14). It was not possible to quantitatively ascertain the residual tumour bulk following primary surgery in those cases with intralesional excision.
Table 2

Clinical details of 11 cases of aggressive angiomyxoma. NED no evidence of disease, LR local recurrence

Patient

Sex

Age, years

Site

Size, cm

Primary excision

Duration follow-up, months

Follow-up

AM 03

Male

57

Scrotum

5

Wide excision

72

NED

AM 04

Male

36

Left inguinal area

9.5

Intralesional

72

NED

AM 05

Male

44

Right epididymis

8

Intralesional

12

NED

AM 06

Female

39

Vulva

17

Intralesional

84

NED

AM 07

Female

31

Anterior abdominal wall/genital area

7

Intralesional

50

NED

AM 08

Female

39

Ligamentum latum

20

Intralesional

216

NED

AM 10

Male

38

Perineum

5

Intralesional

144

NED

AM 11

Female

24

Left vulva

5

Intralesional

48

LR (36 months), wide excision, NED 12 months later

AM 12

Female

47

Vagina

10

Intralesional

144

NED

AM 13

Male

69

Testis

6

Wide excision

1

Died, unrelated causes

AM 14

Male

45

Anterior to bladder: cavum retzii

15

Intralesional

72

NED

Follow-up information was available in all 11 cases (Table 2), with a median interval of 72 months (range 1–216 months). One of the two patients who underwent wide local excision had no evidence of disease 72 months after the initial surgery (AM 03); the other patient (AM 13) died 1 month after the surgical excision of causes unrelated to the tumour or the surgical procedure. The follow-up interval available for seven patients (AM 04, AM 06–08, AM 10, AM 12, AM 14) with primary intralesional tumour resection and who were free of disease at the last medical visit ranged from 50 months to 216 months (median 84 months). One patient with intralesional excision of the primary tumour (AM 11) developed a local recurrence 36 months after the initial surgery. The recurrence was excised with free margins, and the patient was disease free 12 months later (last medical visit). None of the patients developed metastases. There were no tumour-related deaths.

Pathological findings

Gross features

Tumours were unencapsulated and generally poorly circumscribed, varying in diametre from 5 cm to 20 cm (median size 8 cm) (Table 2); cut surfaces were variably mucoid.

Microscopic features

On microscopic examination, the tumours were generally composed of a uniform population of small spindle-shaped cells with ovaliform nuclei and poorly demarcated cytoplasm, set in a variably myxoid and collagenous stroma (Fig. 1A–F). Nuclear atypia was not present, and no mitoses were seen. Lesional cellularity was low to moderate; focally, more cellular foci were occasionally present. A single case had a more reticular architecture. The vascular architecture was variable, consisting of capillaries and larger vessels dispersed throughout the lesion focally, with perivascular hyalinisation. All tumours were poorly circumscribed and exhibited infiltration in the adjacent soft tissue. In the majority of cases, delicate bundles of smooth muscle could be identified. The smooth-muscle bundles were often near vascular structures but did not appear to arise from these vessels. Those lesions stained with alcian blue (pH 2.5) demonstrated variable stromal positivity.
Fig. 1A–F

The typical histological features of aggressive angiomyxoma. Lesions were composed of spindle-shaped cells with little or no nuclear polymorphism and variably elongated cytoplasm, set in a muco-myxoid stroma. Short bundles of spindle-shaped cells were occasionally present, often near, but not in continuity with vessels. Vascularity was variably composed of delicate to more hyalinised vessels (haematoxylin and eosin, A and B magnification ×100; CF magnification ×200)

Immunohistochemical features

The immunohistochemical data are summarised in Table 3. All 6 cases tested exhibited variably strong diffuse positivity for CDK4 (Fig. 2A), while only a single case out of 11 tested demonstrated focal weak expression for MDM2. Desmin was positive in the majority of cases (8 of 11). In 2 cases, 10–20% of lesional cells were positive, while, in the remaining cases, lesional positivity was more diffuse (Fig. 2B). Desmin was not expressed in 3 cases. All tumours demonstrated positivity for oestrogen receptors (Fig. 2C). Variable positivity was seen with the other markers used (Table 3); S-100 protein expression was absent in all cases.
Table 3

Immunohistochemical data in 11 cases of aggressive angiomyxoma

Antigen/antibody

Number positive/number tested

Number positive/number tested

Total number cells positive/number tested

<25% cells positive/mm2

>25% cells positive/mm2

Desmin

1/11

7/11

8/11

Smooth-muscle actin

3/11

3/11

6/11

CD34

2/11

3/11

5/11

Cytokeratin AE1/AE3

2/11

1/11

3/11

Vimentin

3/11

7/11

10/11

S-100 protein

0/11

0/11

0/11

Oestrogen receptor

2/11

9/11

11/11

MDM2

0/11

1/11

1/11

CDK4

0/6

6/6

6/6

Fig. 2A–C

Typical nuclear CDK4 (A), cytoplasmic desmin (B) and nuclear oestrogen (C) immunopositivity (magnification ×200)

Discussion

Myxoid tumours of soft tissue encompass a heterogeneous group of lesions characterised by a variable abundance of extracellular mucoid (myxoid) matrix. The predominant extracellular mucosubstances in soft tissue tumours are hyaluronic acid, keratan sulphate and chondroitin sulphate. An increase in the extracellular matrix, relative to the cellular component, accounts for the mucoid/myxoid lesional character and is clearly demonstrable using alcian blue staining. This group of tumours demonstrate significant variability in their biological behaviour and include benign lesions, tumours with a tendency to recur but not metastasise and frankly malignant neoplasms [6].

Steeper and Rosai first described nine cases of a distinctive, infiltrative, locally aggressive but non-metastasising fibro-myxoid soft tissue tumour arising in the pelvis and perineal area of young female patients, which they termed aggressive angiomyxoma [17]. In their series, local recurrences occurred as much as 14 years after the primary excision; no metastases were noted. Subsequent reports have validated their findings, confirmed the benign but local aggressiveness of this entity and documented the occurrence of aggressive angiomyxoma in males, albeit far less frequently [1, 4, 7, 11, 18]. The reported female to male ratio varies but is in the region of 6:1. Microscopically, the morphological spectrum has been well documented [1, 4, 7, 11, 17, 18]. Immunohistochemically, the vast majority of cases demonstrate positivity for desmin in the myoid bundles and/or stromal cells, while actins and CD34 may be variably positive. Oestrogen receptor expression is typically present [1, 4, 7, 11, 17, 18]. S-100 protein expression is absent.

Our series is interesting in that the sex distribution was roughly equal (six males, five females). This might represent consultation bias in that typical cases in young females are less likely to be submitted for second opinion, while cases occurring in males are more likely to be submitted for validation. Affected males (range 36–69 years, median age 44.5 years) tended to be somewhat older than females (range 24–47 years, median age 39 years), supporting previous observations [18]. Remarkably, despite the fact that the primary resection in the majority of our cases (9 of 11) was intralesional (with positive resection margins), our series was characterised by a very low rate of local recurrence (9%). None of the nine patients with positive margins following primary surgery received adjuvant radiotherapy, which might otherwise have accounted for this low recurrence rate. A single patient in our series (AM 11) developed a local recurrence 36 months after primary intralesional surgery. The remaining ten cases, with a median follow-up of 72 months (range 1–216 months), did not recur locally. This is in contrast to other series in which recurrence rates varied from 36% to 72% [4, 17]. We suspect that the true recurrence rate for aggressive angiomyxoma probably lies somewhere between 9% and the previously reported recurrence rates for a number of reasons. Our cases were generally not routinely screened with radiological imaging studies at medical follow-up visits and relied on clinical symptomatology and physical examination to detect tumour recurrence. Due to the complex anatomic location of many of these lesions, physical examination alone is probably not sensitive enough to detect early recurrences. Additionally, it is clear from the literature that aggressive angiomyxoma is probably a slowly growing neoplasm with low mitotic activity and may, therefore, only present with local recurrence many years after the initial resection. Consequently, our follow-up period may still be too short to exclude the possibility of very late local recurrence.

Immunohistochemically, 8 of our 11 cases demonstrated significant desmin positivity in the myoid bundles and/or stromal cells. Interestingly, all the cases arising in females were positive for desmin, while 3 of the 6 cases arising in males were negative for desmin, supporting previous findings and indicating that the lesion may be somewhat different in males [18]. Cytokeratin positivity was present in 3 cases, a finding that has not been previously highlighted. This is nevertheless not surprising, since cytokeratin AE1/AE3 is a pankeratin, and numerous mesenchymal tumours are well known to express various cytokeratin subsets [8]. The remaining immunohistochemical findings were unremarkable with CD34 positivity present in 5 of 11 cases (45%) and smooth-muscle actin present in 6 of 11 cases (54%). Oestrogen receptor expression was present in all 11 cases, irrespective of gender, supporting previous reports [4].

Consequently, aggressive angiomyxoma is a mesenchymal neoplasm exhibiting infiltrative growth in the adjacent soft tissue and characterised by a potential for local recurrence, generally many years after the primary excision. Two cases with distant metastatic disease and associated patient death have been reported [3, 16]. While the morphological diagnostic accuracy in one case [16] is difficult to verify, the illustrative features provided in the other case [3] could well fit with metastatic aggressive angiomyxoma, underscoring the need for diligent clinical follow-up.

To date, the (cyto)genetic characteristics of five cases of aggressive angiomyxoma have been documented (Table 4) [2, 10, 12, 13, 14]. Three of these five cases demonstrate genetic aberrations involving, amongst others, the long arm of chromosome 12 and, in particular, the region 12q13–15, [2, 12, 14]; the fourth case exhibits monosomy of chromosome 12 [10]. 12q13–15 is a region that has been reported to be rearranged in numerous mesenchymal tumour entities, including, in addition to aggressive angiomyxoma, uterine leiomyomas and (atypical) lipomatous tumours [5, 9, 15]. This region contains a number of candidate genes, including HMGI-C, CDK4, MDM2, SAS, GLI and GAD153 [5, 15]. HMGI-C, currently the candidate gene most favoured to be implicated in the pathobiology of aggressive angiomyxoma, encodes a DNA-binding protein belonging to the high mobility group family of proteins and is important for transcriptional regulation [9]. This gene appears to be intimately involved in the histogenesis of at least three cases of aggressive angiomyxoma (Table 4). CDK4 (encoding a cyclin-dependent kinase) and MDM2 (“murine double minute”, encoding a zinc finger protein that contains a TP53 binding site) are two genes delineating the up- and downstream ends of 12q13–15 (Fig. 3) [5]. CDK4 and MDM2 are known to be amplified in a number of mesenchymal neoplasms. Type-D cyclins bind to and activate cyclin-dependent kinases such as CDK4. Consequently, amplification of CDK4 could potentially promote cell-cycle progression. MDM2 is known to inhibit wild-type TP53 activity, potentially promoting further amplification of MDM2 and stimulating uncontrolled cellular proliferation [5]. All six cases in our series (AM 01–06) tested for immunohistochemical expression of CDK4 demonstrated strong diffuse nuclear positivity. This might support amplification (and consequently involvement) of CDK4. All cases in our series did not demonstrate immunohistochemical expression of MDM2, which would appear to make MDM2 amplification in tumourigenesis of aggressive angiomyxoma unlikely. Unfortunately, fresh tumour tissue was not available for molecular genetic analysis, precluding a detailed analysis of this region (12q13–15) with regard to possible genetic alterations that might be present.
Table 4

(Cytogenetic) alterations described in aggressive angiomyxoma

Author

Genetic alteration

Betz et al. [2]

46,XX,t(7;12)(q22–31;q13–14)

Horsman et al. [10]

45–50,XX,-12,-5,+der(5)t(5q15;?),-16,+der(16) t(5q;16q),-15,+der(15)t(15q13:?)(1–4),+/-frag.

Kazmierczak et al. [12]

46,XX,der(5)t(5;12)(q31;p11.2),der(12)t(5;12) (q31;p11)inv(12)(p11.2q15)

Kenny-Moynihan et al. [13]

45,X,-X

Nucci et al. [14]

46,XX,t(8;12)(p12;q15)

Fig. 3

Approximate cytogenetic location of CDK4 and MDM2 in the region 12q11–12q22, relative to HMGIC (not to scale)

In this study, we have documented a morphological and immunohistochemical profile supporting previous reports. Interestingly, in contrast to previous studies, our series was characterised by a relatively low incidence of local recurrence (9%). Nevertheless, aggressive angiomyxoma has a definite potential for local, destructive recurrence, and follow-up of all cases is mandatory. Strong immunohistochemical expression for CDK4 indicates its implication in tumourigenesis.

Acknowledgements

The Comprehensive Cancer Centre of the Middle Netherlands, Utrecht, The Netherlands, under the auspices of one of the authors (J.A.M.vU) for the use of archival material. The following pathologists are thanked for their kind co-operation in providing the initial material and obtaining additional clinical follow-up information: J.R.J. Elbers, Saint Antonius Hospital, Nieuwegein; J.G. van den Tweel, University Medical Centre Utrecht, Utrecht; D.E. Israel, Lievensberg Hospital, Bergen op Zoom (two cases); C. van Heusden, Co-operative Hospitals East Groningen, Winschoten; U.G.J.M. van Haelst, Radboud Hospital/Nijmegen Academic Medical Centre, Nijmegen; M.C.B.J.E. Tutein Nolthenius-Puylaert, Elkerliek Hospital, Helmond; R.J.M. Parren, Rijnstate Hospital, Arnhem; A.N. van Geel and M. Wijngaarden, Daniël den Hoed Kliniek, Rotterdam (four cases). All experiments performed for this paper comply with the laws as stipulated by the appropriate regularity authorities in the Netherlands.

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© Springer-Verlag 2004