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Keywords:

  • fluorescence in situ hybridization;
  • formalin-fixed paraffin-embedded tissue sections;
  • genetic abnormalities;
  • soft tissue tumors

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

For the detection of chromosome translocations/chimeric genes and specific genetic abnormalities in soft tissue tumors, we conducted fluorescence in situ hybridization (FISH) analysis on 280 cases of soft tissue and other tumors using formalin-fixed paraffin-embedded tissue sections. The detection rate of the FISH split-signal was 84% (129/154 cases) for the translocation-associated soft tissue tumors, such as Ewing's sarcoma/primitive neuroectodermal tumor, synovial sarcoma, alveolar rhabdomyosarcoma, myxoid liposarcoma, clear cell sarcoma and so forth. Positive split-signals from EWSR1, SS18 and FOXO1A probes were detected in 3% (2/64) of various histological types of carcinoma, lymphoma, melanoma, meningioma and soft tissue tumors. In FISH using the INI1/CEP22 probe, the INI1 deletion signal was detected in 100% (9/9) of epithelioid sarcoma. In well-differentiated and dedifferentiated liposarcomas, detection of MDM2 amplification signals in FISH using the MDM2/CEP12 probe were both as high as 85% (11/13) and 100% (13/13), respectively. In other adipocytic and non-adipocytic tumors requiring differentiation from these types, detection was only 13% (5/39), and CEP12 polysomy was frequently detected. As these results demonstrate the high sensitivity and specificity of FISH, we concluded FISH to be a useful pathological diagnostic adjunct for definite and differential diagnosis of soft tissue tumors.

Soft tissue tumors are diagnosed comprehensively on the basis of clinical, imaging, and pathological findings. However, many tumors share similar morphological features and have indistinct cellular differentiation. Due to this diversity in pathological findings, diagnosis is often extremely difficult. Despite technological advancements in genetic analysis, many genetic abnormalities specific to certain histological types of soft tissue tumor have been reported in recent years. Of the soft tissue tumors, a group of tumors is characterized by chromosomal abnormalities based on translocation that leads to a genetic abnormality known as a chimeric gene. These abnormalities include Ewing's sarcoma/primitive neuroectodermal tumor (PNET), synovial sarcoma, and alveolar rhabdomyosarcoma1,2 (Table 1). Furthermore, MDM2 gene amplification are known to exist in well-differentiated and dedifferentiated liposarcomas,3 and deletion abnormalities in the INI1 gene are known to exist in epithelioid sarcoma and malignant rhabdoid tumors (MRTs).4 These genetic abnormalities can be detected by fluorescence in situ hybridization (FISH) analysis. Interphase FISH can detect coarse chromosomal or genomic changes including gene amplification and deletion but not subtle genomic abnormalities such as point mutation and cryptic deletion or insertion and its specificity against different histological types is high. Therefore, development of diagnostic chimeric genes has greatly contributed to the diagnosis of soft tissue tumor groups with reported detection of chromosome translocation-derived chimeric genes.5–8 However, there have been no reports to date on an exhaustive FISH analysis of each histological type of soft tissue tumor group containing characteristic genetic abnormalities.

Table 1.  Fluorescence in situ hybridization (FISH) analysis on a chimeric gene-forming group of soft tissue tumors Thumbnail image of

We therefore used formalin-fixed paraffin-embedded tissue sections to investigate the effectiveness of FISH as a pathological diagnostic adjunct for detecting characteristic genetic abnormalities in soft tissue tumors.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Case selection

A total of 280 cumulative cases of soft tissue and other tumors that were pathologically diagnosed between 2005 and 2010 at the Sapporo Medical University Hospital were selected for study. FISH analysis was conducted on formalin-fixed paraffin-embedded tissue sections of the samples. The analyzed histological types included 154 cases in the soft tissue tumor group, which had chimeric genes derived from chromosome translocations, such as Ewing's sarcoma/PNET, synovial sarcoma, alveolar rhabdomyosarcoma, myxoid liposarcoma, clear cell sarcoma and so forth, 26 cases of well-differentiated and dedifferentiated liposarcoma in which the MDM2 gene had been amplified, nine cases of epithelioid sarcoma in which the INI1 gene had been deleted, and 91 cases in other tumor groups that required differentiation from these types.

FISH analysis

FISH was conducted using the PathVysion HER-2 DNA Probe Kit (PathVysion kit) by Abbott Molecular (Abbott Park, IL, USA). Sliced tissue sections (4 µm) were placed on silanized slides, which were deparaffinized after baking for 1 h at 60°C. For target gene activation, sections were immersed in 0.2 M HCl for 20 min followed by immersion in pretreatment solution (Abbott Molecular) for 30 min at 80°C. For enzyme treatment, sections were immersed in protease solution (Abbott Molecular) pre-warmed to 37°C for 60 min. Sections were then re-fixed over 10 min in 10% formalin neutral buffer solution at room temperature. Target genes were denatured by immersion in denaturation solution (Abbott Molecular) heated to 72°C for 5 min. Sections were then washed and dehydrated by immersing for 1 min each in 70%, 85%, and 100% ethanol. For hybridization, 10 µL of DNA probe solution were added and sealed by cover glass. DNA probes were denatured at 90°C for 5 min in the hybridizer, followed by hybridization at 37°C for over 48 h. After removing the cover glass, sections were washed in post-hybridization wash buffer (Abbott Molecular) at 72°C for 2 min. To counterstain, 10 µL of 4′,6-diamidino-2-phenylindole (DAPI; Abbott Molecular) was added. Slides were coverslipped for viewing under a fluorescence microscope.

DNA probes

For detecting chimeric genes derived from chromosome translocations, the following commercially available dual-color split-signal probes were used: EWSR1 [22q12], FUS [16p11], SS18/SYT [18q11.2], FOXO1A/FKHR [13q14], and DDIT3/CHOP [12q13] (Abbott Molecular) (Table 1, Fig. 1). Similarly, the following in-house probes were used: NR4A3 [9q31.1], PDGFB [22q13.1], and TFE3 [Xp11.23] (Table 1, Fig. 1). For detecting INI1 gene deletion and MDM2 gene amplification, the following in-house dual-color probes were used: INI1 [22q11.23]/CEP22, and MDM2 [12q15]/CEP12 (Fig. 1).

image

Figure 1. Probe map of in-house probes. NR4A3, PDGFB, and TFE3 probes are dual-color split-signal probes that detect chimeric genes formed by chromosome translocation. The MDM2/CEP12 probe detects MDM2 gene amplification, and the INI1/CEP22 probe detects INI1 gene deletion. These probes consist of red probes labeled by SpectrumOrange and green probes labeled by SpectrumGreen. The size and target gene (yellow) of each probe is shown.

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In-house DNA probes were prepared by using an Escherichia coli strain (BAC clone) obtained from the Children's Hospital Oakland Research Institute (Oakland, CA, USA) and labeled with the following two fluorescent dyes: SpectrumOrange (red) and SpectrumGreen (green) (Abbott Molecular) using the Nick Translation Kit (Roche Applied Science, Indianapolis, IN, USA). The in-house probe specificity was confirmed by chromosomal mapping on metaphase spreads.

Positive determination of FISH results

Hybridization signals were counted in at least 50 morphologically intact, non-overlapping tumor cell nuclei to avoid misinterpretation in signal counts or false positivity due to nuclear truncation. In accordance with our previous reports,5–8 we defined the presence of split-signals as reflecting gene rearrangement after chromosome translocation if more than 10% of cells, other than those that presented fused signals, showed signals separated by more than two- to three-fold the signal diameter. In alveolar rhabdomyosarcoma, cells presenting excessive green signals were determined to have increased copy numbers of the FOXO1A gene centromeric region, as reported previously.6 Furthermore, cells presenting more than three pairs of red (FOXO1A telomeric region) and green signals were determined to have polysomy. In dermatofibrosarcoma protuberans (DFSP), cells presenting excessive green signals were determined as having increased copy numbers of the PDGFB gene centromeric region. The green/red signal ratio was calculated from the total number of green and red (PDGFB telomeric region) signals in 50 tumor cells. In alveolar soft part sarcoma (ASPS), the number of X chromosomes in which TFE3 is located differs by sex; therefore, red signals indicating the TFE3 telomeric region and green signals indicating the centromeric region are detected as two fused signals in normal females and as one fused signal in normal males. Although in this study, all cases in which a red signal was detected with fused signals or split-signals (for a red : green signal ratio of 2:1) were defined as chromosome translocation regardless of sex. Furthermore, the ratio of signal patterns detected, excluding normal signals, was calculated.

When performing FISH with the INI1/CEP22 probe, the normal signal was detected as two red signals (indicating the INI1 region) and two green signals (indicating the centromeric region of chromosome 22). Other than this normal signal, some tumor cells presented one red signal against one or two green signals (heterozygous deletion), while others presented no red signal against one or two green signals (homozygous deletion). Therefore, we determined that the cells that exhibited a loss of red signal had INI1 gene deletion. The detection rate and ratio of deletion signal pattern of the INI1 deletion signal were calculated in 50 tumor cells.

When performing FISH with the MDM2/CEP12 probe, the normal signal was detected as two red signals (indicating the MDM2 region) and two green signals (indicating the centromeric region on chromosome 12). Of the tumor cells presenting an excessive red signal against two green signals, those with greater than twice the amount of red-to-green signals were determined to have MDM2 gene amplification. From 20 MDM2 gene-amplified cells, the MDM2/CEP12 ratio was calculated based on the total number of red signals and total number of green signals. We further determined that tumor cells with greater than three green signals had CEP12 polysomy.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Detection of chromosome translocation/chimeric genes

In the soft tissue tumor group in which chimeric genes were formed by chromosomal translocation, FISH analysis using various dual-color split-signal probes revealed the occurrence of split-signals in 84% (129/154) of cases (Table 1).

The EWSR1 split-signal detection rate for Ewing's sarcoma/PNET was 97% (29/30 cases). The rate was 100% in clear cell sarcoma of soft parts (3/3 cases) and in desmoplastic small round cell tumor (2/2 cases). In extraskeletal myxoid chondrosarcoma, the EWSR1 split-signal was detected in 76% (16/21) of cases and the NR4A3 split-signal was detected in 71% (15/21) of cases. The EWSR1 split-signal detection rate in angiomatoid fibrous histiocytoma was 90% (9/10 cases), and the FUS split-signal was detected in a single case in which EWSR1 gene rearrangement was unconfirmed. Therefore, EWSR1 or FUS gene rearrangements were found in 100% (10/10) of cases in this histological type. The SS18 split-signal detection rate was 97% (30/31 cases) in synovial sarcoma (Fig. 2). The DDIT3 split-signal detection rate was 100% (12/12 cases) in myxoid liposarcoma. In low grade fibromyxoid sarcoma, the FUS split-signal was detected in 83% (5/6) of cases.

image

Figure 2. Synovial sarcoma. (a) Monophasic type presenting a fascicular pattern of short uniform spindle-shaped cells along with epithelioid cells. (b) In fluorescence in situ hybridization (FISH) analysis using SS18 probe, a split-signal (white arrow) consisting of a green signal indicating the SS18 gene centromeric region and red signal indicating telomeric region is observed.

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The FOXO1A split-signal detection rate was 94% (16/17 cases) in alveolar rhabdomyosarcoma. Furthermore, increased copy numbers of FOXO1A gene centromeric regions were found in 65% (11/17) of cases. Polysomy was found in these 65% (11/17) of cases.

In DFSP (6/13 cases contained areas of fibrosarcoma (DFSP-FS)), the PDGFB split-signal detection rate was 100% (7/7 cases) (Fig. 3). The split-signal detection rate was 100% (7/7 cases) in conventional DFSP, 80% (4/5 cases) in DFSP areas of DFSP-FS, and 83% (5/6 cases) in fibrosarcoma areas of DFSP-FS (Table 2). Furthermore, increased copy numbers of the PDGFB gene centromeric region were found in 85% (11/13) of cases. The median green/red signal ratio was 2.0 (1.0–2.5) in conventional DFSP, 2.4 (1.0–3.3) in DFSP areas of DFSP-FS, and 1.6 (1.0–3.7) in fibrosarcoma areas of DFSP-FS (Table 2).

image

Figure 3. Dermatofibrosarcoma protuberans (DFSP) with fibrosarcoma area (DFSP-FS). (a) DFSP area in which uniform short spindle-shaped cells form a storiform and cartwheel pattern. (b) Fibrosarcoma area in which spindle-shaped cells form a herringbone pattern, which intersects in a fascicular pattern. (c) In fluorescence in situ hybridization (FISH) analysis using the PDGFB probe on the fibrosarcoma area of DFSP, a split-signal (white arrow) consisting of green signal indicating PDGFB gene centromeric region and red signal indicating telomeric region is observed, together with excess green signal (green arrow). The increased copy number of the centromeric region of the PDGFB gene is detected from two red signals against seven green signals (green/red signal ratio of 3.5).

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Table 2.  Fluorescence in situ hybridization (FISH) analysis using PDGFB probe on conventional dermatofibrosarcoma protuberans (DFSP) and DFSP with fibrosarcoma area (DFSP-FS)
Histologic type (no. cases)Green/red signal ratio range (median)Split-signal detection rate (%)
Conventional DFSP (n= 7)1.0–2.5 (2.0)7/7 (100)
DFSP-FS (n= 6)1.0–3.7 (1.6)6/6 (100)
 DFSP area (n= 5)1.0–3.3 (2.4)4/5 (80)
 Fibrosarcoma area (n= 6)1.0–3.7 (1.6)5/6 (83)
Total (n= 13)1.0–3.7 (1.8)13/13 (100)

In ASPS, the TFE3 split-signal detection rate was 100% (9/9 cases) (Fig. 4). In addition to signal patterns with a red : green signal ratio of 2:2 or 1:1, many variations were observed (Table 3). In a female patient (case 7), unbalanced chromosome reciprocal translocation was demonstrated by one fusion and one deleted green signal (Fig. 4c). In two male patients (cases 4 and 8), who should present one red and one green signal, an unbalanced chromosome reciprocal translocation of the same signal pattern to that in the female patient was observed in addition to a unique pattern of one split-signal and one deleted green signal, indicating balanced chromosome reciprocal translocation (Fig. 4b). In male patient cases, split-signals were even detected in cells having two red and two green signals.

image

Figure 4. Alveolar soft part sarcoma (ASPS). (a) Large round to polygonal cells forming an alveolar pattern. (b) In fluorescence in situ hybridization (FISH) analysis using the TFE3 probe (case 8, male), a split-signal (white arrow) consisting of green signal indicating TFE3 gene centromeric region and red signal indicating telomeric region is observed. Furthermore, one green signal is missing due to partial deletion of the translocated X chromosome. (c) In a different tumor (case 7, female), green signal is missing and one red signal (red arrow) is observed, in addition to a fused signal (white arrow) made of one red and one green signal, due to unbalanced chromosome reciprocal translocation.

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Table 3.  Fluorescence in situ hybridization (FISH) signal pattern using TFE3 probe and split-signal detection rate (%) in alveolar soft part sarcoma
CaseSexAge (years)Split-signal cell number/counted cell number by red : green signal patternsSplit-signal detection rate (%)
1:01:12:02:12:23:13:23:34:24:3
1Female233/193/315/2821/50 (42)
2Female23/204/49/2616/50 (32)
3Female341/218/88/2117/50 (34)
4Male404/1812/1212/2328/53 (53)
5Female280/163/311/300/114/50 (28)
6Female30/23/312/4515/50 (30)
7Female300/10/60/416/161/160/11/20/10/10/218/50 (36)
8Male280/20/50/334/341/635/50 (70)
9Female250/20/20/217/173/230/420/50 (40)

Valuation of EWSR1, SS18, and FOXO1A probe specificities

The split-signal detection rate of EWSR1 and SS18 was 11% (2/18 cases) and 0% (0/23 cases), respectively, among carcinomas, malignant lymphoma, malignant melanoma, meningiomas, and other soft tissue tumors requiring differential diagnosis from Ewing's sarcoma/PNET and synovial sarcoma. Although, the two cases positive for EWSR1 split-signals were myoepithelial carcinoma and malignant lymphoma (lymphoblastic lymphoma) (Table 4). The FOXO1A split-signal detection rate in embryonal rhabdomyosarcoma was 0% (0/23 cases) (Table 4).

Table 4.  Specificity valuation of fluorescence in situ hybridization (FISH) analysis using EWSR1, SS18, and FOXO1A probes
Histological type (no. cases)Split-signal detection rate (%)
EWSR1SS18FOXO1A
  1. MFH, malignant fibrous histiocytoma; PNET, primitive neuroectodermal tumor.

Carcinoma (n= 9)   
 Carcinosarcoma (n= 2)0/1(0)0/2(0)
 Small cell carcinoma(n= 1)0/1(0)
 Merkel cell carcinoma(n= 2)0/1(0)0/1(0)
 Myoepithelial carcinoma (n= 1)1/1(100)0/1(0)
 Metastatic carcinoma(n= 1)0/1(0)
 Spindle cell carcinoma (n= 1)0/1(0)
 Thymic carcinoma(n= 1)0/1(0)
Malignant lymphoma(n= 1)1/1(100)
Malignant melanoma(n= 1)0/1(0)
Meningioma(n= 2)0/2(0)
Soft tissue tumor(n= 47)   
 Embryonal rhabdomyosarcoma(n= 25)0/2(0)0/1(0)0/23(0)
 Ewing's sarcoma/PNET (n= 4)0/4(0)
 Synovial sarcoma(n= 4)0/4(0)
 Malignant peripheral nerve sheath tumor(n= 3)0/1(0)0/3(0)
 Solitary fibrous tumor(n= 2)0/2(0)
 Chondroma(n= 1)0/1(0)
 Chondrosarcoma(n= 1)0/1(0)
 Neuroblastoma(n= 1)0/1(0)
 Undifferentiated sarcoma(n= 1)0/1(0)
 Fibrosarcoma(n= 1)0/1(0)
 Pleomorphic MFH(n= 1)0/1(0)
 Myxofibrosarcoma(n= 1)0/1(0)
 Mesenchymal chondrosarcoma(n= 1)0/1(0)
 Epithelioid sarcoma(n= 1)0/1(0)
 2/18(11)0/23(0)0/23(0)

Detection of INI1 deletion in epithelioid sarcoma

Fluorescence in situ hybridization analysis of nine cases of epithelioid sarcoma (five proximal-type, four distal-type) using the INI1/CEP22 probe revealed an INI1 gene deletion signal of 100% (9/9 cases) (Fig. 5). The deletion signal detection rate and deletion signal pattern ratios are shown in Table 5.

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Figure 5. Epithelioid sarcoma (distal-type). (a) Granulomatous pattern in which epithelioid cells with abundant cytoplasm surrounds the necrotic focus. (b) In fluorescence in situ hybridization (FISH) analysis using the INI1/CEP22 probe, homozygous deletion demonstrated by two missing red signals indicating INI1 against two green signals (green arrow) showing the centromeric region is detected. (c) In a different case, heterozygous deletion demonstrated by the lack of one green (green arrow) and one red (red arrow) signal becomes detected.

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Table 5.  Fluorescence in situ hybridization (FISH) signal pattern using INI1/CEP22 probe and deletion signal detection rate (%) in epithelioid sarcoma
Histologic typeCaseProportion of INI1(red) : CEP22(green) signal patternsDeletion signal detection rate (%)
Heterozygous deletion rate (%)Homozygous deletion rate (%)Normal rate (%)
1:21:10:10:22:2
Proximal-type18261884060
212268104456
3423650892
413211454753
5121819213070
Mean (cases 1–5)9.818.61918.833.866.2
Distal-type6262816121882
74622521684
80028720100
962620202872
Mean (cases 6–9)91521.53915.584.5
Mean (cases 1–9)9.41720.127.825.774.3

The deletion signal detection rate in proximal and distal-types was 66.2% and 84.5%, respectively, and 74.3% in all cases. Apart from the normal signal in which two INI1 (red) and two CEP22 (green) were detected (when INI1 : CEP22 = 2:2), the INI1 heterozygous deletion signal (when INI1 : CEP22 = 1:2 or 1:1) was detected in 28.4% of proximal-type and 24% of distal-type epithelioid sarcoma cases. Similarly, the homozygous deletion signal (when INI1 : CEP22 = 0:1 or 0:2) was detected in 37.8% of proximal-type and 60.5% of distal-type cases. Heterozygous and homozygous deletion was found in 26.4% and 47.9% of all cases, respectively.

Detection of MDM2 gene amplification in well-differentiated and dedifferentiated liposarcoma

Fluorescence in situ hybridization analysis was conducted with the MDM2/CEP12 probe on well-differentiated liposarcoma, dedifferentiated liposarcoma, and other histological types of soft tissue tumors that require differential diagnosis from the other two (Table 6).

Table 6.  Fluorescence in situ hybridization (FISH) analysis using MDM2/CEP12 probe in well-differentiated liposarcoma, dedifferentiated liposarcoma and soft tissue tumors requiring differential diagnosis from these
Histological type (no. cases)Amplification signal detection rate (%)MDM2/CEP12 ratio range (mean)CEP12 polysomy detection rate (%)
  1. MFH, malignant fibrous histiocytoma.

Liposarcoma (n= 41)   
 Well-differentiated (n= 13)11/13(85)1.08–21.14(11.47)2/13(15)
 Dedifferentiated(n= 13)13/13(100)10.69–25.15(18.59)0/13(0)
 Myxoid(n= 9)0/9(0)0.93–1.13(1.04)0/9(0)
 Pleomorphic(n= 3)1/3(33)1.19–4.08(2.2)3/3(100)
 Spindle cell(n= 3)0/3(0)1.05–1.13(1.09)0/3(0)
Lipoma(n= 6)0/6(0)1–1.75(1.13)1/6(17)
Spindle cell lipoma(n= 3)0/3(0)1–1.05(1.03)1/3(33)
Myxofibrosarcoma(n= 12)4/12(33)0.54–16.28(4.28)9/12(75)
Pleomorphic MFH(n= 3)0/3(0)0.51–0.71(0.63)3/3(100)
Total(n= 65)

In well-differentiated liposarcoma, the MDM2 gene amplification signal was detected in 85% (11/13) of cases (Fig. 6). The ratio of MDM2/CEP12 was 11.47 (1.08–21.14), and CEP12 polysomy was found in 15% (2/13) of cases. The MDM2 gene amplification signal was detected in 100% (13/13) of dedifferentiated liposarcoma cases (Fig. 7), where the MDM2/CEP12 ratio was 18.59 (10.69–25.15). In other histological types of liposarcoma (myxoid and spindle cell), no amplification signals or CEP12 polysomy were detected. The amplification signal was detected in 33% (1/3) of pleomorphic liposarcoma cases. The MDM2/CEP12 ratio was 2.2 (1.19–4.08), and CEP12 polysomy was found in 100% (3/3) of cases.

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Figure 6. Well-differentiated liposarcoma. (a) Mature adipocytes of varying sizes and multivacuolated lipoblasts proliferate along with atypical stromal cells. (b) In fluorescence in situ hybridization (FISH) analysis using the MDM2/CEP12 probe, MDM2 gene amplification is detected from 30 red signals (showing the MDM2 gene) against two green signals (showing the centromeric region, green arrow), from a MDM2/CEP12 ratio of 15.

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image

Figure 7. Dedifferentiated liposarcoma. (a) Transition from well-differentiated liposarcoma area (top left) to fibrosarcoma area with no lipogenesis (bottom right) is shown. (b) In fluorescence in situ hybridization (FISH) analysis using the MDM2/CEP12 probe, MDM2 gene amplification is detected from 60 red signals (showing the MDM2 gene) against two green signals (showing centromeric region, green arrow), from a MDM2/CEP12 ratio of 30.

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No amplification signals were detected in lipoma or spindle cell lipoma, although CEP12 polysomy was detected in 17% (1/6) of lipoma and 33% (1/3) of spindle cell lipoma cases. The amplification signal was detected in 33% (4/12) of myxofibrosarcoma cases. The MDM2/CEP12 ratio was 4.28 (0.54–16.28), and CEP12 polysomy was detected in 75% (9/12) of cases. No amplification signal was undetected in pleomorphic malignant fibrous histiocytoma (MFH), although 100% (3/3) of cases showed CEP12 polysomy (Fig. 8).

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Figure 8. Pleomorphic malignant fibrous histiocytoma (MFH). (a) Spindle-shaped, oval and large polygonal cells are arranged in a storiform pattern. (b) In fluorescence in situ hybridization (FISH) analysis using the MDM2/CEP12 probe, CEP12 polysomy is detected from five amplified green signals (showing centromeric region, green arrow) observed against two red signals (showing MDM2 gene, red arrow).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Having conducted FISH analysis on a soft tissue tumor group with chimeric genes derived from chromosome translocations, as well as on well-differentiated and dedifferentiated liposarcoma in which the MDM2 gene is amplified, and on epithelioid sarcoma in which the INI1 gene is deleted, we detected a high prevalence (83% to 100%) of genetic abnormalities in tumors other than extraskeletal myxoid chondrosarcoma.

In particular, among the soft tissue tumor group with chimeric genes derived from chromosome translocations, split-signals of specific genes were detected at high rates in Ewing's sarcoma/PNET, clear cell sarcoma of soft parts, desmoplastic small round cell tumor, angiomatoid fibrous histiocytoma, synovial sarcoma, myxoid liposarcoma, and low grade fibromyxoid sarcoma, which is in accordance with previous reports on FISH analysis.5,7,9–15

Among these tumor groups, FISH analysis has revealed a high prevalence of EWSR1 gene rearrangement in extraskeletal myxoid chondrosarcoma.16 Conducting FISH on a mixture of new cases as well as on those previously reported8 revealed rearrangement of the EWSR1 gene in 76% (16/21) of cases and rearrangement of the NR4A3 gene in 71% (15/21) cases. Given that the size of the NR4A3 probe is smaller than that of the EWSR1 probe, the signal intensity arising from the NR4A3 probe was slightly less than that of the EWSR1 probe. For this reason, in this study, the split-signal detection rate of NR4A3 was slightly lower than that of EWSR1. In addition, the split-signal detection rate in extraskeletal myxoid chondrosarcoma was found to be slightly lower than that in other histological types. The presence of a novel chimeric gene and weakly established diagnostic criteria for this tumor were estimated reasons for this.

Similarly for alveolar rhabdomyosarcoma, FISH analysis with the FOXO1A probe on a mixture of new cases as well as on those previously reported6 revealed a FOXO1A split-signal in 94% (16/17) of cases and increased FOXO1A centromeric region copy numbers in 65% (11/17) of cases. Previous studies have reported the same genome copy number increase in alveolar rhabdomyosarcoma.17 Protein coded by the PAX3-/PAX7-FOXO1A gene generated by chromosome translocation is a strong transcription factor, and tumor cell proliferation has been shown to be enhanced upon the expression of the PAX3-FOXO1A gene.18 This suggests that PAX3-/PAX7-FOXO1A expression and the increase in copy number mediate the mechanisms of tumor formation and progression.

In accordance with previous reports,19,20 the PDGFB split-signal was detected by FISH with a high prevalence (100%, 13/13 cases) in DFSP. Furthermore, an increased PDGFB gene centromeric region copy number (median green/red signal ratio of 1.8) was detected in 85% (11/13) of cases. Various reports have been made on excess signals being generated due to an increase in chromosome translocation-induced COL1A1-PDGFB gene copy number,19,20 and our results matched with these findings. Abbott et al. showed that by using a dual-color split-signal probe in DFSP-FS, the median COL1A1-PDGFB gene copy number in the fibrosarcoma region (2.8) was higher when compared with the DFSP region (1.7).19 On the other hand, differences in median PDGFB gene centromeric region copy numbers were not observed in this study, as values were 2.0 in normal DFSP, 2.4 in the DFSP part of DFSP-FS, and 1.6 in the fibrosarcoma part of DFSP-FS. These results are in accordance with FISH studies using dual-color, dual-fusion probes.21,22 The present findings suggest that a tumor-forming mechanism, other than COL1A1-PDGFB gene increase, mediates fibrosarcomatous transformation of DFSP.

The TFE3 split-signal was detected in 100% (9/9) of ASPS cases, which was a high detection rate similar to a past report.23 As to immunohistochemical status of TFE3 expression, 95% (18/19) of ASPS demonstrated strong (3+) nuclear staining, and one (5%) demonstrated moderate (2+) nuclear staining.24 In the present study, we determined that cases with one fused and one missing green signal were those of unbalanced chromosome reciprocal translocation, where a fused signal is green (indicating the TFE3 gene centromeric region) and red (indicating the telomeric region). Unbalanced chromosome reciprocal translocation has been known as a characteristic of ASPS.25 As it is difficult to determine such signal patterns as split-signals, care is needed in the analysis of ASPS FISH signals. In addition, previous studies have shown that excess signals have been detected as a result of multiplication of the X chromosome short arm telomeric region, which is fused to ASPSCR1 and located on chromosome 17 due to chromosome interchange.23 We observed many different types of signal patterns in our FISH analysis. Therefore in addition to balanced and unbalanced chromosome reciprocal translocation, partial multiplication or deletion of the X chromosome or chromosome number abnormalities might occur in ASPS.

As FUS-DDIT3 gene formation from chromosome translocation t(12;16)(q13;p11) or EWSR1-DDIT3 gene formation from chromosome translocation t(12;22)(q13;q12) are known in myxoid liposarcoma,1FUS/EWSR1-DDIT3 fusion can also be assessed using FUS and EWSR1 probes. In a previous report, the DDIT3, FUS and EWSR1 split-signal was detected in 100% (18/18), 94.4% (17/18) and 0% (0/18) of myxoid liposarcoma cases, respectively.15

In this study, we conducted FISH analysis with EWSR1 and SS18 probes on various histological types of tumors requiring differential diagnosis from Ewing's sarcoma/PNET and synovial sarcoma. Similarly, the FOXO1A probe was used to conduct FISH analysis on embryonal rhabdomyosarcoma, which is often difficult to distinguish from alveolar rhabdomyosarcoma. As reported previously,6 the split-signal of the FOXO1A gene was undetected in embryonal rhabdomyosarcoma. EWSR1 and SS18 gene rearrangements were undetected in not only soft tissue tumors but also carcinoma, malignant lymphoma, malignant melanoma, and meningioma; thus, high specificity was shown. Among these, the EWSR1 split-signal was detected in both myoepithelial carcinoma and malignant lymphoma (lymphoblastic lymphoma) as previously reported.26,27 Therefore, care is required for differentiating these histological types.

Epithelioid sarcoma is classified into the typical distal-type that arises in the limbs of young adults and the proximal-type that arises mainly in the groin and pelvis of middle-aged adults. In comparison to the distal-type, the proximal-type has been reported to be more malignant, leading to a poorer outcome.28 On the other hand, a recent report showed no major difference in the degree of malignancy between these two types.29 Modena et al. demonstrated by FISH analysis of proximal-type (six cases) and distal-type epithelioid sarcomas (five cases) that INI1 gene deletion is found only in proximal-type epithelioid sarcoma (five cases).4 Immunohistochemistry has been used to show frequent loss of the INI1 protein expression in MRTs, and proximal-type and distal-type epithelioid sarcomas.29,30 In our FISH analysis, INI1 gene deletion was observed in 100% of proximal-type (five cases) and distal-type (four cases) epithelioid sarcomas, which supports the INI1 protein loss result, 95% (61/64) of proximal-type and 91% (58/64) of distal-type, obtained by immunohistochemistry.30 On the basis of FISH conducted by Modena et al., the signal pattern of INI1 deletion was mostly homozygous deletion.4 In our study, heterozygous deletion (when INI1 : CEP22 ratio = 1:2 or 1:1) was detected in 28.4% of proximal-type and 24% of distal-type cases, showing no major differences. On the other hand, homozygous deletion (when INI1 : CEP22 ratio = 0:1 or 0:2) in distal-type was 60.5%, which was higher in comparison to the 37.8% in the proximal-type epithelioid sarcomas. We will need to increase the number of cases to further analyze these genetic abnormalities such as INI1 gene deletions, which are characteristic of proximal-type and distal-type epithelioid sarcomas.

Supernumerary ring or giant rod marker chromosomes are observed in well-differentiated and dedifferentiated liposarcoma, and these characteristic chromosomes consist of amplified sequences of the 12q13-15 region containing MDM2, CDK4, HMGA2, and SAS genes.3,31 Weaver et al. detected MDM2 gene amplification in 100% of well-differentiated liposarcoma (13 cases) and dedifferentiated liposarcoma (14 cases) by FISH.32 On the other hand, the MDM2 gene amplification frequency shown by Sirvent et al. was 94% (30/32 cases) in well-differentiated liposarcoma and 100% (8/8 cases) in dedifferentiated liposarcoma.33 In our FISH analysis, MDM2 gene amplification was detected in 85% (11/13) of well-differentiated liposarcoma cases and 100% (13/13) of dedifferentiated liposarcoma cases. In agreement with the reports of Sirvent et al.,33 our results showed a lower detection rate of MDM2 gene amplification in well-differentiated liposarcoma when compared with dedifferentiated liposarcoma. Furthermore, in agreement with the reports of Weaver et al.,32 the mean MDM2/CEP12 ratio was lower in well-differentiated liposarcoma (11.47) compared with dedifferentiated liposarcoma (18.59). Weaver et al. did not detect CEP12 polysomy in well-differentiated liposarcoma,32 although we found CEP12 polysomy in 15% (2/13) of well-differentiated liposarcoma cases. It has been known clinically that dedifferentiated liposarcoma has a poorer outcome in comparison to well-differentiated liposarcoma.34 The correlation between the MDM2 gene status and the biological behavior of well-differentiated and dedifferentiated liposarcomas needs to be examined in further studies.

Immunohistochemically, MDM2 was expressed in 100% (44/44) of well-differentiated and 95.1% (58/61) of dedifferentiated liposarcoma cases.35 We have experienced several cases of well-differentiated liposarcoma with negative MDM2 immunostaining but MDM2 gene amplification. In these situations, the use of FISH for the detection of MDM2 amplification is frequently conclusive.

In terms of histopathology, there are many cases where differentiation of well-differentiated liposarcoma from lipoma, spindle cell lipoma, and other liposarcomas (myxoid, pleomorphic, and spindle cell) becomes problematic. In lipoma, chromosome translocation-induced chimeric genes are formed from the HMGA2 gene that is present within the 12q13-15 region. Known partner genes include LPP (3q27-28), NFIB (9p22), CXCR7 (2q37), EBF1 (5q33), and LFHP (13q12).31 In spindle cell lipoma, which is a subtype of lipoma, deletion of 16q, partial deletion of 13q, and monosomy of chromosome 13 have been detected.31 There are no known characteristic chromosome structures or copy number abnormalities in pleomorphic liposarcoma, where chromosome and genetic abnormalities in this type are highly variant and complex.31,36 Spindle cell liposarcoma is classified as a subtype of well-differentiated liposarcoma, although because of the lack of MDM2 and CDK4 gene amplification, the possibility of being a subtype of a different histological type has been suggested.37 In our FISH analysis, MDM2 gene amplification was detected in 33% (1/3) of pleomorphic liposarcoma cases, although the mean MDM2/CEP12 ratio (2.2) was lower in comparison to well-differentiated (11.47) and dedifferentiated (18.59) liposarcomas. MDM2 gene amplification was undetected (0/21 cases) in myxoid liposarcoma (nine cases), spindle cell liposarcoma (three cases), lipoma (six cases), and spindle cell lipoma (three cases). In addition, CEP12 polysomy was detected in a 100% (3/3) of pleomorphic liposarcoma cases, though it was as low as 5% (2/38 cases) in other liposarcomas (well-differentiated, dedifferentiated, myxoid, and spindle cell). CEP12 polysomy has not been reported previously in lipoma, though we detected this in 17% (1/6) of lipoma cases and 33% (1/3) of spindle cell lipoma cases.

Myxofibrosarcoma and pleomorphic MFH, which require differential diagnosis from the dedifferentiated part of dedifferentiated liposarcoma, both have characteristic genetic abnormalities. There are no specific chromosome structures or copy number abnormalities in these histological types, although they carry abnormalities such as increase, decrease, and high level amplification of various chromosome regions, which leads to highly complex karyotypes.36 In our FISH analysis, MDM2 gene amplification was detected in 33% (4/12) of myxofibrosarcoma cases, although the mean MDM2/CEP12 ratio (4.28) was lower when compared with dedifferentiated liposarcoma (18.59). MDM2 gene amplification was undetected in pleomorphic MFH (0/3 cases). In previous studies, MDM2 gene amplification was undetected in myxofibrosarcoma (one case), although it was found in 40% (4/10) of pleomorphic MFH cases.32 Furthermore, the detection rate of CEP12 polysomy in dedifferentiated liposarcoma was 0% (0/13 cases) in the present study, though this was high in myxofibrosarcoma (75%; 9/12 cases) and pleomorphic MFH (100%; 3/3 cases).

Regarding the pleomorphic liposarcoma and myxofibrosarcoma cases with MDM2 amplification, unusual myxoid MFH-like, mixed or homologously dedifferentiated subtypes of liposarcoma should be included into differential diagnosis.38–40 However, these cases occurred in the extremities, different from the retroperitoneum typical of dedifferentiated liposarcoma, and lacked well-differentiated liposarcoma components morphologically.

In conclusion, this study demonstrates the high sensitivity and specificity of FISH for detecting chromosomal and genetic abnormalities (the formation of chimeric genes by chromosome translocation and the deletion or amplification of specific genes) specific to soft tissue tumors. FISH is therefore a useful pathological diagnostic adjunct for analyzing formalin-fixed paraffin-embedded tissue sections of soft tissue tumors. Moreover, definite diagnosis could be obtained by identifying FISH signal patterns characteristic to each histological type.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Romeo S, Dei Tos AP. Soft tissue tumors associated with EWSR1 translocation. Virchows Arch 2010; 456: 21934.
  • 2
    Fisher C. Soft tissue sarcomas with non-EWS translocations: Molecular genetic features and pathologic and clinical correlations. Virchows Arch 2010; 456: 15366.
  • 3
    Coindre J, Pedeutour F, Aurias A. Well-differentiated and dedifferentiated liposarcomas. Virchows Arch 2010; 456: 16779.
  • 4
    Modena P, Lualdi E, Facchinetti F et al. SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas. Cancer Res 2005; 65: 40129.
  • 5
    Yamaguchi U, Hasegawa T, Morimoto Y et al. A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor and other small round cell tumors sharing EWS rearrangement using new fluorescence in situ hybridization probes for EWSR1 on formalin fixed, paraffin wax embedded tissue. J Clin Pathol 2005; 58: 10516.
  • 6
    Matsumura T, Yamaguchi T, Seki K et al. Advantage of FISH analysis using FKHR probes for an adjunct to diagnosis of rhabdomyosarcomas. Virchows Arch 2008; 452: 2518.
  • 7
    Matsumura T, Yamaguchi T, Tochigi N et al. Angiomatoid fibrous histiocytoma including cases with pleomorphic features analysed by fluorescence in situ hybridization. J Clin Pathol 2010; 63: 1248.
  • 8
    Noguchi H, Mitsuhashi T, Seki K et al. Fluorescence in situ hybridization analysis of extraskeletal myxoid chondrosarcomas using EWSR1 and NR4A3 probes. Hum Pathol 2010; 41: 33642.
  • 9
    Bridge RS, Rajaram V, Dehner LP et al. Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: A comparison of two FISH strategies and RT-PCR in malignant round cell tumors. Mod Pathol 2006; 19: 18.
  • 10
    Patel RM, Downs-Kelly E, Weiss SW et al. Dual-color, break-apart fluorescence in situ hybiridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma. Mod Pathol 2005; 18: 158590.
  • 11
    Rossi S, Szuhai K, Ijszenga M et al. EWSR1-CREB1 and EWSR1-ATF1fusion genes in angiomatoid fibrous histiocytoma. Clin Cancer Res 2007; 13: 73228.
  • 12
    Terry J, Barry TS, Horsman DE et al. Fluorescence in situ hybridization for the detection of t(X;18)(p11.2;q11.2) in a synovial sarcoma tissue microarray using a break apart-style probe. Diagn Mol Pathol 2005; 14: 7782.
  • 13
    Sun B, Sun Y, Wang J et al. The diagnostic value of SYT-SSX detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) for synovial sarcoma: A review and prospective study of 255 cases. Cancer Sci 2008; 99: 135561.
  • 14
    Tanas MR, Rubin BP, Tubbs RR et al. Utilization of fluorescence in situ hybridization in the diagnosis of 230 mesenchymal neoplasms. An institutional experience. Arch Pathol Lab Med 2010; 134: 1797803.
  • 15
    Downs-Kelly E, Goldblum JR, Patel RM et al. The utility of fluorescence in situ hybrizidation (FISH) in the diagnosis of myxoid soft tissue neoplasms. Am J Surg Pathol 2008; 32: 813.
  • 16
    Wang WL, Mayordomo E, Czerniak BA et al. Fluorescence in situ hybridization is a useful ancillary diagnostic tool for extraskeletal myxoid chondrosarcoma. Mod Pathol 2008; 21: 130310.
  • 17
    Barr FG, Nauta LE, Davis RJ et al. In vivo amplification of the PAX3-FKHR and PAX7-FKHR fusion genes in alveolar rhabdomyosarcoma. Hum Mol Genet 1996; 5: 1521.
  • 18
    Anderson J, Ramsay A, Gould S et al. PAX3-FKHR induces morphological change and enhances cellular proliferation and invasion in rhabdomyosarcoma. Am J Surg Pathol 2001; 159: 108996.
  • 19
    Abbott JJ, Erickson-Johnson M, Wang X et al. Gains of COL1A1-PDGFB genomic copies occur in fibrosarcomatous transformation of dermatofibrosarcoma protuberans. Mod Pathol 2006; 19: 15128.
  • 20
    Patel KU, Szabo SS, Hernandez VS et al. Dermatofibrosarcoma protuberans COL1A1-PDGFB fusion is identified in virtually all dermatofibrosarcoma protuberans cases when investigated by newly developed multiplex reverse transcription polymerase chain reaction and fluorescence in situ hybridization assays. Hum Pathol 2008; 39: 18493.
  • 21
    Stacchiotti S, Pedeutour F, Negri T et al. Dermatofibrosarcoma protuberans-derived fibrosarcoma: Clinical history, biological profile and sensitivity to imatinib. Int J Cancer. 2011; 129: 176172.
  • 22
    Salgado R, Liombart B, Pujol RM et al. Molecular diagnosis of dermatofibrosarcoma protuberans: A comparison between reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization methodologies. Genes Chromosomes Cancer 2011; 50: 51017.
  • 23
    Aulmann S, Longerich T, Schirmacher P et al. Detection of the ASPSCR1-TFE3 gene fusion in paraffin-embedded alveolar soft part sarcomas. Histopathology 2007; 50: 8816.
  • 24
    Argani P, Lal P, Hutchinson B et al. Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions: A sensitive and specific immunohistochemical assay. Am J Surg Pathol 2003; 27: 75061.
  • 25
    Zhong M, Angelo PD, Osborne L et al. Dual-color, break-apart FISH assay on paraffin-embedded tissues as an adjunct to diagnosis of Xp11 translocation renal cell carcinoma and alveolar soft part sarcoma. Am J Surg Pathol 2010; 34: 75766.
  • 26
    Brandal P, Panagopoulos I, Bjerkehagen B et al. t(19;22)(q13;q12) translocation leading to the novel fusion gene EWSR1-ZNF444 in soft tissue myoepithelial carcinoma. Genes Chromosomes Cancer 2009; 48: 10516.
  • 27
    Jakovljevic G, Nakic M, Rogosic S et al. Pre-B-cell acute lymphoblastic leukemia with bulk extramedullary disease and chromosome 22 (EWSR1) rearrangement masquerading as Ewing sarcoma. Pediatr Blood Cancer 2010; 54: 6069.
  • 28
    Guillou L, Wadden C, Coindre JM et al. ‘Proximal-type’ epithelioid sarcoma, a distinctive aggressive neoplasm showing rhabdoid features: Clinicopathologic, immunohistochemical, and ultrastructural study of series. Am J Surg Pathol 1997; 21: 13046.
  • 29
    Kohashi K, Izumi T, Oda Y et al. Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: A useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor. Hum Pathol 2009; 40: 34955.
  • 30
    Hornick JL, Dal Cin P, Fletcher CDM. Loss of INI1 expression is characteristic of both conventional and proximal-type epithelioid sarcoma. Am J Surg Pathol 2009; 33: 54250.
  • 31
    Nishio J. Contributions of cytogenetics and molecular cytogenetics to the diagnosis of adipocytic tumors. J Biomed Biotechnol 2011; 2011: doi:10.1155/2011/524067.
  • 32
    Weaver J, Downs-Kelly E, Goldblum J et al. Fluorescence in situ hybridization for MDM2 gene amplification as a diagnostic tool in lipomatous neoplasms. Mod Pathol 2008; 21: 9439.
  • 33
    Sirvent N, Coindre JM, Maire G et al. Detection of MDM2-CDK4 amplification by fluorescence in situ hybridization in 200 paraffin-embedded tumor samples: Utility in diagnosing adipocytic lesions and comparison with immunohistochemistry and real-time PCR. Am J Pathol 2007; 31: 147689.
  • 34
    Evans HL. Atypical lipomatous tumor, its variants, and its combined forms: A study of 61 cases, with a minimum follow-up of 10 years. Am J Surg Pathol 2007; 31: 114.
  • 35
    Binh MB, Sastre-Garau X, Guillou L et al. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma subtypes: A comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol 2005; 29: 134047.
  • 36
    Guillou L, Aurias A. Soft tissue sarcomas with complex genomic profiles. Virchows Arch 2010; 456: 20117.
  • 37
    Mentzel T, Palmedo G, Kuhnen C. Well-differentiated spindle cell liposarcoma (‘atypical spindle cell lipomatous tumor’) does not belong to the spectrum of atypical lipomatous tumor but has a close relationship to spindle cell lipoma: Clinicopathologic, immunohistochemical, and molecular analysis of six cases. Mod Pathol 2010; 23: 72936.
  • 38
    Hisaoka M, Morimitsu Y, Hashimoto H et al. Retroperitoneal liposarcoma with combined well-differentiated and myxoid malignant fibrous histiocytoma-like myxoid areas. Am J Surg Pathol 1999; 23: 148092.
  • 39
    Boland JM, Weiss SW, Oliveira AM et al. Liposarcomas with mixed well-differentiated and pleomorphic features: A clinicopathlogic study of 12 cases. Am J Surg Pathol 2010; 34: 83743.
  • 40
    Marino-Enriquez A, Fletcher CD, dal Cin P et al. Dedifferentiated liposarcoma with ‘homologous’ lipoblastic (pleomorphic liposarcoma-like) differentiation: Clinicopathologic and molecular analysis of a series suggesting revised diagnostic criteria. Am J Surg Pathol 2010; 34: 112231.