Main

Myxofibrosarcoma, formerly also known as the myxoid variant of malignant fibrous histiocytoma, is one of the most frequent sarcomas in elderly patients with the majority arising in the extremities, clinically most often presenting as a slowly increasing mass.1, 2, 3 Histologically, myxofibrosarcomas show a wide range from hypocellular grade I tumours with oval to spindle form cells in a myxoid stroma, to hypercellular grade III lesions of solid sheets with pleomorphic nuclei.4 According to the French Fédération Nationale des Centres de Lutte Contre le Cancer (FNLCC) grading system, three histological grades are recognised, that is, grade I, II and III.5 Metastatic potential is closely related to tumour grade, with a preference for lung, bone and lymph nodes. Importantly, increasing grade is seen in subsequent recurrences.4 Cytogenetic data on myxofibrosarcoma are scarce with 30 isolated cases described in the literature. Most often, karyotypes are very complex, including ring chromosomes, deletions, translocations and double minutes. Here, we present the clinicopathologic data and karyotypes of 32 cases of myxofibrosarcoma and discuss our findings in the light of tumour grade and clinical behaviour.

Materials and methods

Patient Data

Thirty-two cases of myxofibrosarcoma were identified on which cytogenetic data were available and collected retrospectively from the files of the Pathology Departments of the University Hospitals of Leuven and Leiden University Medical Center. Patient and tumour characteristics were described in Table 1. In each case, 4 μm-thick sections of formalin-fixed, paraffin-embedded material were stained with haematoxylin and eosin (H&E). Slides were revised and classified according to the 2002 World Health Organization (WHO) criteria and histological grading was performed according to the FNLCC.2, 5

Table 1 Patient and tumour characteristics
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Cytogenetic Data

Chromosomes were obtained using short-term cultures and harvesting procedures as described previously.6 Briefly, nonfixed tissue fragments were disaggregated mechanically and enzymatically and then cultured on chamber slides in DMEM-F12 medium (Gibco, Invitrogen, Breda, The Netherlands), supplemented with 10% fetal bovine serum and 0,5% penicillin–streptomycin (Irvine Scientific, Santa Anna, CA, USA) for 4–9 days. For cases 1–17 and 19–29 the protocol was as follows. At 17 h before harvest, cells were exposed to Colcemid (0.005 μg/ml; Gibco, Invitrogen, Breda, The Netherlands). After hypotonic treatment (0.8% sodium citrate for 25 min), the preparations were fixed three times with methanol–glacial acetic acid (3:1). Cytogenetic analysis was performed on GTG-banded chromosomes and the karyotypes were classified according to the International System for Human Cytogenetic Nomenclature.7

Multicolor Fluorescence In Situ Hybridisation

For cases 18, 30–32, cells were harvested, after which a 48-color fluorescence in situ hybridisation (FISH) staining was carried out, staining every chromosome-arm in a different colour combination, after which digital imaging and analysis was performed as previously described.8, 9, 10 Hybridisations with individual libraries labeled with single fluorochromes were used to confirm the detected rearrangements. Breakpoints were assigned by using inverted DAPI counterstained images of the chromosomes.

Results

Patient Data

The study of myxofibrosarcoma included eight grade I, eight grade II and 16 grade III cases. The morphological spectrum ranging from low- to high-grade morphology is depicted in Figure 1. Patient and tumour characteristics are depicted in Table 1, showing equal sex distribution between males and females with an average age of occurrence of 66 years. In all, 22 cases were primary lesions; nine of these lesions recurred locally and one was a lymph node metastasis. Of the six cases recurring as grade III lesions, the primary lesions were either grade III (four cases), grade II (one case) or grade I (one case). All three grade II recurrences were all primarily grade I. The metastasis was a grade III lesion. Except for this lymph node metastasis and one lesion occurring in the thoracic muscles (case 13), all lesions occurred at the extremities (upper ones: 12; lower: 19). In all, 19 were deeply/intramuscular seated (case 13 included), 11 superficially with one not specified regarding the depth of the lesion (case 20).

Figure 1
figure 1

Light Micrographs displaying the morphological Spectrum of Myxofibrosarcoma. (a–c) Grade I myxofibrosarcoma showing a hypocellular lesion with typical elongated curvilinear blood vessels in a myxoid background with (pseudo)lipoblasts and slight nuclear atypia. (a) HE × 10; (b, c): HE × 40. (d) Grade II myxofibrosarcoma showing a more cellular lesions with increased nucleair atypia. HE × 20. (e, f) Grade III myxofibrosarcoma consisting out of solid sheets of spindled, atypical cells and multinucleated giant cells with abundant eosinophillic cytoplasm. In the same tumour, there is often a remarkable but characteristic sharp transition between hypo-and hypercellular areas. (e) HE × 20; (f) HE × 40.

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Cytogenetics

The cytogenetics findings are listed in Table 2 showing normal to very complex karyotypes. None of them were published in previous publications including these entities by the authors.11 Eight normal karyotypes were found among these 32 myxofibrosarcoma. Clonal, besides nonclonal, aberrations were found in 22 cases. These aberrations were present in all grades. Complex cytogenetic anomalies were not restricted to grade III, but also seen in grade II and grade I cases. There was no difference in affected chromosomes depending on grade. Although some chromosomes were more involved than others (Figure 2), there were no consistent chromosomal abnormalities. In general, grade III lesions showed more complex karyotypes than lower grades. Remarkably, recurrences (indicated by asterisk) showed more complex karyotypes than both their grade II and grade III primary lesions. Furthermore, we found the presence of a ring chromosome, possibly originating from chromosome 3 (case 7).

Table 2 Cytogenetic analysis of the 32 myxofibrosarcomas
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Figure 2
figure 2

Karyotypic aberrations in 32 myxofibrosarcomas. Red=Grade I, Orange=Grade II, Purple=Grade III. Ideogram showing the distribution of the numerical and structural aberrations of 32 myxofibrosarcoma. Aberrations were found in all grades, including all chromosomes. No tumour or grade specific cytogenetic aberration could be withdrawn. Gains are shown on the right side of each chromosome diagram, losses on the left. Arrows indicate inversions.

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Discussion

Myxofibrosarcoma is one of the most frequent sarcomas in elderly patients, mainly located on the extremities.2 Histologically, they compromise a continuous range from hypocellular tumours, consisting of small spindle cells widely spaced among the myxoid matrix, to more cellular variants with pleomorphic nuclei and pseudolipoblasts.1, 2, 3, 4 A consistent morphologic feature is the presence of curvilinear blood vessels. Enzinger and Weiss3 classified the high-grade variant of these tumours as the myxoid variant of malignant fibrous histiocytoma, besides the storiform-pleomorphic, giant cell type and inflammatory subtypes. Angervall and Kindblom,1 almost simultaneously, first used the term myxofibrosarcoma, as they recognised this tumour as a distinct entity. Nowadays, the recognition as a distinct, separate entity is generally accepted and included in the 2002 WHO classification of tumours.2 According to previous studies, grade II and III tumours are true malignant tumours since they have metastastic potential. This is in contrast to grade I lesions, which are only locally aggressive.4 However, since local recurrence goes together with increase in grade, grade I myxofibrosarcoma may finally gain metastatic potential, warranting close surveillance.

The differential diagnoses of myxofibrosarcoma include other myxoid tumours often hard to distinguish from each other, since differences are often very subtle.12 However, the distinction between all these myxoid subtypes is truly important from a clinical point of view, both for required therapy as for follow-up. Therefore, the existence of a specific (cyto) genetic aberration could serve as a welcome diagnostic marker and a helpful tool to distinguish these subtypes. However, cytogenetic data on myxofibrosarcoma are scarce, with only a few isolated cases described in literature showing complex, but nonspecific cytogenetic aberrations (Table 3). These include the presence of ring chromosomes, deletions and translocations. No specific chromosomal rearrangement has been identified so far.

Table 3 Karyotypes of the 30 Myxofibrosarcoma described in literature
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Here, we investigated the clinicopathologic and cytogenetic data of 32 myxofibrosarcoma in order to find tumour and/or grade specific cytogenetic aberrations. Patient and tumour characteristics were in accordance to previous studies (Table 1).1, 2, 3 Although some authors suggested that deep-seated lesions were of higher grade, a large study of grade I myxofibrosarcoma could not confirm this, which is in accordance to our findings.4, 25, 26 Interestingly, the grade I tumours progressing to grade II were located superficially which shows that progression to recurrence is not exclusive for deep-seated lesions. Normal karyotypes were seen in eight cases (five grade III, two grade II and one grade I lesion). Aberrations occurred in all chromosomes; no consistent tumour-specific cytogenetic rearrangement was identified. Those cases with cytogenetic abnormalities were not restricted to a certain grade with grade I, II and III lesions showing a wide range from normal till very complex, polyploid karyotypes (Figures 2, 4). Grade III tumours had, overall, more complex karyotypes than grade I and grade II lesions. This suggests that these complex karyotypic abnormalities occur rather randomly as secondary events. We showed that local recurrences occurred in all grades, with increase in grade upon recurrence, which is in accordance to the literature.2, 27 We hypothesised that in the same lesion, increase in grade goes together with increase in cytogenetic aberrations. Therefore, we compared the karyotypes of primary lesions to these of the corresponding local recurrences. Recurrences showed increase in grade and more complex aberrations than the primary tumours (Figure 3). These complex aberrations suggest additional chromosomal events, corresponding with an increase in histological grade and, subsequently, increase in malignant potential.

Figure 4
figure 4

COBRA-FISH in a case of a high-grade myxofibrosarcoma. Example of highly complex cytogenetic aberrations in a case of high-grade myxofibrosarcoma (case 32). For karyotypic aberrations see Table 2.

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Figure 3
figure 3

Karyotypic aberrations in nine myxofibrosarcomas comparing primary lesions to their recurrences. Blue: Primary tumour, Red: Recurrence. Ideogram showing the distribution of the numerical and structural aberrations of primary tumours compared to their local recurrences. Recurrences show more complex cytogenetic aberrations than the primary tumours. Gains are shown on the right side of each chromosome diagram, losses on the left. Arrows indicate inversions. In general, there is gain of cytogenetic aberrations with recurrence though no specific pattern is observed for this phenomenon.

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Besides nonclonal and clonal aberrations, we found the presence of a ring chromosome in one case of low-grade myxofibrosarcoma. The presence of ring chromosomes has also have been described in one case of myxofibrosarcoma possibly originating from 20q.17 Ring chromosomes have been described in many tumours, both benign as well as malignant, including dedifferentiated liposarcoma, a differential diagnosis of grade III myxofibrosarcoma.11, 28 However, this is certainly not a consistent feature and its molecular and biological significance has not been revealed yet. Previous reports described several translocations in myxofibrosarcoma. Although all of them were different and nonspecific, some of these involve interesting regions. For example, Clawson et al13 described a case of grade I myxofibrosarcoma with translocation t(2;15)(p23;q21.2). The band 2p23 is a site of ALK gene, expressed in normal tissue and rearrangements have been described in anaplastic large cell lymphomas, and inflammatory myofibroblastic tumours.29, 30 Translocations involving 2p23 have been found in a case of synovial sarcoma as t(2;15)(p23;q26) and also in one case of ‘malignant fibrous histiocytomas not otherwise specified’ as t(2;5)(p23;q13).31, 32 Frequent loss of 9p21 (p16) has been reported in malignant fibrous histiocytomas including also some cases of myxofibrosarcoma.20, 33, 34 We could not confirm these aberrations in our study. Some studies showed add2(q37) in myxofibrosarcoma and in several types of leukaemia.11 Interestingly, the COL6A1 gene is localised to 2q37, coding for collagen VI alpha-3 polypeptide. At least three other extra cellular matrix genes are also located on 2q: 2 collagen genes, COL3A1 and COL5A2 and the fibronectin gene.35 As described above, myxofibrosarcoma contains areas of abundant extracellular matrix although only little is known about its constitution. Ultrastructural studies showed that the majority of cells in myxoid areas of myxofibrosarcoma showed secretory activity with dilated rough endoplasmatic reticulum. Although further study is needed, alterations in genes coding for matrix proteins might lead to increased or impaired matrix production. Interestingly, a myxofibrosarcoma-like appearance appears to be quite frequent in pleomorphic liposarcoma, thereby including it in the differential diagnosis.36 In the light of this differential diagnosis, the cases reported here were carefully characterised, explicitly excluding contaminations with pleomorphic liposarcoma karyotypes in this series of myxofibrosarcoma. Finally, a recent study using comparative genomic hybridisation (CGH) showed very similar chromosome imbalances between myxoid malignant fibrous histiocytoma and pleomorphic liposarcoma, suggesting partially common genetics and biology.37 This study showed gain of 5p and loss of 4q in myxofibrosarcoma of higher grade compared to tumours of lower grade. Whether these and other myxoid soft tissue tumours, in parallel to their morphologic resemblance, also share genetic overlap is a very interesting hypothesis, which, however, still needs to be confirmed.