Virchows Arch DOI 10.1007/s00428-014-1568-8

ORIGINAL ARTICLE

Atypical spindle cell lipoma: a clinicopathologic, immunohistochemical, and molecular study emphasizing its relationship to classical spindle cell lipoma David Creytens & Joost van Gorp & Suvi Savola & Liesbeth Ferdinande & Thomas Mentzel & Louis Libbrecht Received: 20 October 2013 / Revised: 22 January 2014 / Accepted: 6 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract We studied a series of spindle cell lipomas arising in atypical sites and showing unusual morphologic features (which we called atypical spindle cell lipoma) to assess if these lesions have the same chromosomal alterations as classical spindle cell lipoma but different from those found in atypical lipomatous tumor/well-differentiated liposarcoma. We investigated alterations of different genes in the 13q14 region and the amplification status of the MDM2 and CDK4 genes at 12q14-15 by multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) analysis. In the atypical spindle cell lipomas, MLPA revealed deletions in the two nearest flanking genes of RB1 (ITM2B and RCBTB2) and in multiple important exons of RB1. In contrast, in classical spindle cell lipomas, a less complex loss of RB1 exons was found but no deletion of ITM2B and RCBTB2. Moreover, MLPA identified a deletion of the DLEU1 gene, a finding which has not been reported earlier. We propose an immunohistochemical panel for lipomatous tumors which comprises of MDM2, CDK4, p16, Rb, which we have found useful in discriminating between atypical or classical spindle cell lipomas and other adipocytic neoplasms, especially atypical lipomatous tumor/well-differentiated D. Creytens (*) : L. Ferdinande : L. Libbrecht Department of Pathology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium e-mail: [email protected] D. Creytens e-mail: [email protected] J. van Gorp Department of Pathology, Diakonessenhuis Utrecht, Utrecht, The Netherlands S. Savola MRC-Holland, Amsterdam, The Netherlands T. Mentzel Dermatopathologie Bodensee, Friedrichshafen, Germany

liposarcoma. Our findings strengthen the link between atypical spindle cell lipoma and classical spindle cell lipoma, and differentiate them from atypical lipomatous tumor/welldifferentiated liposarcoma. Keywords Atypical spindle cell lipoma . Spindle cell lipoma . Atypical lipomatous tumor/well-differentiated liposarcoma . RB1 . Immunohistochemistry . Multiplex ligation-dependent probe amplification . Fluorescence in situ hybridization

Introduction In 2010, Mentzel et al. reported a consistent deletion of the retinoblastoma gene (RB1) by fluorescence in situ hybridization (FISH) in six cases of an “atypical spindle cell lipomatous tumor” (“well-differentiated spindle cell liposarcoma”), which may represent the atypical/low-grade counterpart of spindle cell lipoma [1]. Spindle cell liposarcoma, first described by Dei Tos et al. [2], has been a confusing and evolving entity. These tumors have been considered variants of welldifferentiated liposarcoma, myxoid liposarcoma, and spindle cell lipoma, suggesting that it is heterogeneous a group of lesions [1–10]. As things stand in 2014, a consensus is yet to be reached on the precise classification of welldifferentiated spindle cell liposarcoma. The WHO 2013 classification currently considers well-differentiated spindle cell liposarcoma as an uncommon variant of atypical lipomatous tumor/well-differentiated liposarcoma [11]. These adipocytic tumors are clinically characterized by locally aggressive growth and a tendency to recur locally, whereas metastasis does not occur and dedifferentiation only very rarely [9]. While the cytogenetic and molecular patterns of atypical lipomatous tumor/well-differentiated liposarcoma are well established, featuring the consistent presence of supernumerary ring or giant chromosomes containing amplified material from chromosome 12q14-15 which includes the MDM2 and

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CDK4 genes, the data concerning genomic alterations of welldifferentiated spindle cell liposarcoma are scarce [1, 10]. RB1, located on the long arm of chromosome 13 (13q14), is one of the best characterized tumor suppressor genes and its inactivation has been noted in a variety of human tumors [12–14]. Cytogenetic analysis and FISH studies on spindle cell/pleomorphic lipomas, cellular angiofibromas and mammary-type myofibroblastomas have demonstrated consistent deletions of the long arm of chromosome 13, including the RB1 gene, suggesting that these entities form a family of histologically and cytogenetically related neoplasms [15–21]. Recently, Chen et al. reported that these 13q deletions are associated with loss of Rb protein expression in spindle cell lipomas, pleomorphic lipomas, cellular angiofibromas, and mammary-type myofibroblastomas. This illustrates the correlation between RB1 deletion and loss of immunohistochemical Rb expression and the potential diagnostic utility of Rb staining as this is maintained in histologic mimics [22]. Thway et al. and more recently Gonzalez et al. described p16 as a sensitive and specific marker for distinguishing atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma from its adipocytic mimics [23, 24]. The p16 INK4a (CDKN2A) locus on the short arm of chromosome 9 (9p21.3) is one of the most frequently altered sequences in cancer and p16 is an important tumor suppressor gene [25]. In sarcomas, methylation of the p16 gene appears to be a dominant silencing mechanism [26–28]. The precise role of the p16 gene in the biology of liposarcoma is still unknown. The aim of the present study was to investigate spindle cell lipomas arising in atypical sites and showing unusual morphologic features (for which we propose the term atypical spindle cell lipoma to contrast them from the classical type), to assess if these lesions share the same chromosomal alteration and if these are different from those of atypical lipomatous tumor/well-differentiated liposarcoma, especially the sclerosing and lipoma-like subtypes. To this end, we studied alterations of the 13q14 region, including RB1, deleted in lymphocytic leukemia (DLEU1), integral membrane protein 2B (ITM2B) and regulator of chromosome condensation and BTB domain containing protein 2 (RCBTB2) genes, as well as the amplification status of the MDM2 and CDK4 genes, by multiplex ligation-dependent probe amplification (MLPA) and FISH analysis. We also investigated the value of a panel of immunohistochemical markers, including Rb, p16, MDM2 and CDK4, in the diagnosis of these neoplasms.

well-differentiated liposarcoma (including 6 sclerosing and 4 lipoma-like subtypes) were included in this study. Six cases of atypical spindle cell lipoma were previously published by Mentzel et al. as atypical spindle cell lipomatous tumors (well-differentiated spindle cell liposarcoma) [1]. The other cases were retrieved from the files of the pathology department of the Ghent University Hospital, the Soft Tissue Tumor Registry Utrecht, and the referral files of one of the authors (DC). Follow-up information was obtained from clinicians and pathologists. Clinical data and diagnostic criteria for the different entities are summarized in Tables 1 and 2. Immunohistochemistry For each case, 4-μm-thick sections from a representative block of formalin-fixed, paraffin-embedded tumor tissue were used for immunohistochemical analysis. Immunohistochemistry was performed using an immunostainer (Benchmark XT, Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturer’s instructions. The sections were immunostained with primary monoclonal antibodies against CD34 (1:100; QBEnd10; Dako, Glostrup, Denmark), p16 (ready-touse; E6H4; CINtec MTM laboratories-Roche, Tucson, AZ, USA), Rb (1:50; G3-245; BD Pharmingen, San Diego, CA, USA), CDK4 (1:400; DC9-31; Biosource, Carlsbad, CA, USA), and MDM2 (1:10; IF2; Invitrogen, Carlsbad, CA, USA). Heat-induced epitope retrieval was performed using Cell Conditioning 1 (Ventana Medical Systems) for CD34, p16, Rb, CDK4, and MDM2. Visualization was achieved with ultraView Universal DAB Detection kit (Ventana Medical Systems, Tucson, AZ, USA). Appropriate positive and negative controls were used throughout the study. Rb nuclear immunoreactivity was classified as deficient [negative (80 % of tumor cells with nuclear staining)] [22]. Expression of Rb in nuclei of lymphocytes and endothelial cells served as internal positive control. Criteria for evaluating p16 were as described by He et al. [29]. p16 staining was classified into four categories: negative (no nuclear staining), 1+ (less than 20 % of tumor cells with nuclear staining), 2+ (20-80 % of tumor cells with nuclear staining) and 3+ (> 80 % of tumor cells with nuclear staining). Following Binh et al., we considered a tumor as MDM2 or CDK4 positive when at least one nucleus was stained per high power field [30].

Materials and methods Fluorescence in situ hybridization Selection of cases Ten cases of atypical spindle cell lipoma, 7 cases of classical spindle cell lipoma and 10 cases of atypical lipomatous tumor/

FISH analysis for the detection of RB1, located on 13q14.2, was performed with a Vysis LSI13 (RB1) 13q14 direct spectrum orange-labeled probe (Abbott, Bergisch Gladbach,

Virchows Arch Table 1 Clinical data of atypical spindle cell lipomas, spindle cell lipomas and well-differentiated liposarcomas

Age (years) Atypical spindle cell lipoma Case 1 75 Case 2 59 Case 3 85 Case 4 70 Case 5 60 Case 6 62 Case 7 52 Case 8 72 Case 9 69 Case 10 63 Spindle cell lipoma Case 11 41 Case 12 51 Case 13 45 Case 14 39 Case 15 53 Case 16 41 Case 17 46 Well-differentiated liposarcoma Case 18 65 Case 19 74 Case 20 70

F female, M male, NA data non available, NSR no sign of recurrence, R local recurrence

Case 21 Case 22 Case 23 Case 24 Case 25 Case 26 Case 27

49 62 55 52 61 67 50

Sex

Site

Size (cm)

Follow-up

M M F M

Right shoulder Paratesticular Lower leg Chest wall

1,5 10,0 2,8 10,0

NSR at 12 months NSR at 24 months NSR at 24 months NA

F M F M M F

Back of hand Left thigh Lower leg Shoulder Shoulder Lower leg

3,5 9,1 6,0 2,5 2,0 5,5

R at 6 months; NSR 9 months later NA NSR at 15 months NSR at 18 months NSR at 32 months NSR at 24 months

F M F M M M M

Neck Neck Shoulder Shoulder Neck Neck Neck

4,0 4,0 2,5 2,0 3,5 2,0 3,0

NSR at 48 NSR at 42 NSR at 36 NSR at 28 NA NA NSR at 24

F F F

Lower leg Lower leg Shoulder

6,0 4,5 7,0

R at 26 months NSR at 21 months NA

M F M M F M M

Thigh Thigh Thigh Inguinal Lower leg Lower leg Lower leg

7,0 5,0 5,0 6,0 5,0 8,0 5,5

NSR at 29 months NSR at 22 months NSR at 42 months R at 18 months NA NSR at 20 months NSR at 28 months

Germany) as previously described [1]. A case was interpreted as deleted if only 1 fusion signal was detected in more than 22 % of the nuclei evaluated [28]. In addition, dual-color FISH using MDM2 and CDK4 specific probes with a centromere-specific probe for chromosome 12 was performed, as previously described [1]. Cases with a MDM2/CEP12 ratio>2 and CDK4/CEP12 ratio>2 were considered amplified for the MDM2 gene and CDK4 gene respectively [31]. RB1 deletion by multiplex ligation-dependent probe amplification MLPA analysis on approximately 100 ng of genomic DNA from 18 cases was performed as described by Schouten et al. [32–34]. The RB1 probemix (SALSA MLPA P047 RB1 probemix) contains 40 different probes with amplification products

months months months months

months

between 130 and 481 nucleotides, including probes for 25 of the 27 RB1 exons and including 2 probes for exon 27 but not for exon 15 in view of its close proximity to other exons. Two probes are present for ITM2B (exon 3) and RCBTB2 (exon 6) genes at close distance of RB1 (48 kb upstream; 72 kb downstream respectively) as well as a probe for the DLEU1 (exon 2) gene located at a distance of 1.6 Mb from RB1. This DLEU1 probe is included in the probemix to facilitate determination of the extent of a RB1 deletion, especially in chronic lymphocytic leukemia (CLL), which is one of the established clinical applications of this probe mix. In addition, 11 reference probes for other human genes located on different chromosomes are included (DYSF-2p13.2, IL1RN-2q13, ACAP23q29, OCIAD1-4p11, IL4-5q31.1, PCSK5-9q21.13, ZNF2510p11.1, ALX4-11p11.2, RPGRIP1-14q11.2, RUNX121q22.12 and PPIL2-22q11.21). Genomic human DNA (Promega, Fitchburg, WI, USA) and DNA extracted from normal fatty tissue were used as normal reference. Data

Virchows Arch Table 2 Clinical and pathological diagnostic criteria for spindle cell lipoma, atypical spindle cell lipoma and sclerosing type of well-differentiated liposarcoma Diagnosis

Spindle cell lipoma

Atypical spindle cell lipoma

Age Localization

Fifth to sixth decade –Subcutis posterior neck, back and shoulder region –Rare: trunk, extremities, orbit –Extremely rare if ever in deep soft tissue –Well circumscribed and encapsulated

Sixth to ninth decade Fifth to eighth decade –Subcutis thigh, lower extremities, –Retroperitoneum, inguinal region shoulder, chest wall, paratesticular region –Less common: deep soft tissue –Less common: deep soft tissue lower extremity, thigh

Tumor border Morphological features

–“Ropey-like” hyaline collagen bundles –Variable cellularity –Bland bipolar spindle cells –No significant variation in size and shape of lipogenic cells, rare lipoblasts

Immunohistochemistry MDM2−, CDK4−, p16-, Rb loss (most common profile) Cytogenetic features –Deletion 13q (including RB1) –No 12q13-15 alterations (MDM2, CDK4)

–Less well circumscribed and not completely encapsulated –Collagenous, but not “ropey-like” hyaline stroma –Variable cellularity –Cytonuclear atypical, hyperchromatic spindle cells –Significant variation in size and shape of lipogenic cells, lipoblasts relatively common MDM2−, CDK4−, p16-, Rb loss

Well-differentiated liposarcoma (sclerosing type)

–Less well circumscribed and not completely encapsulated –Dense, collagenous but not “ropey-like” stroma –Low cellularity –Mildly pleomorphic, hyperchromatic spindle and multipolar cells –Rare lipoblasts

MDM2+, CDK4+, p16+, Rb+ (“intact”) –Deletion 13q (including RB1) –Amplification 12q13-15 –No 12q13-15 alterations (MDM2, CDK4) (MDM2, CDK4) –No deletion 13q (RB1)

analysis was performed with Coffalyser NET software [35]. The peak areas achieved using RB1 specific probes in each patient sample were first normalized by the average of peak areas achieved by control probes specific for locations different from chromosome 13. A final ratio was then calculated by dividing the value from the patient samples by matching the value from the pool of normal reference samples. If this ratio was ≤0.70, the assay was scored as having reduced copy number (deletion). If the ratio was ≥1.30, the assay was scored as having increased copy number [36]. For RB1, a RB1 median value across all RB1 probes was calculated for each sample: a median RB1 ratio of ≤0.70, 0.70–0.80, 1.20–1.30, and ≥1.30 was respectively scored as loss, low level loss, low level gain and gain of RB1.

Results The clinical, immunohistochemical and molecular data are summarized in Tables 1 and 3. All atypical spindle cell lipomas were (re-)excised with tumor-free margins and no adjuvant treatment was given. A local recurrence was seen at 6 months in case 5 and was excised completely. No metastasis was noted, and all patients are alive and free of tumor (followup ranged from 12 to 32 months for the eight cases of which information was available). The seven spindle cell lipomas included in this study showed considerable clinicopathological differences. In comparison with atypical spindle cell lipomas, classical spindle cell lipomas were characterized by a narrower anatomic distribution (neck and shoulder location), a

significantly younger age (mean 45 years) and a smaller size (ranging from 2.0 to 4.0 cm; mean 3,0 cm). Histologically, most atypical spindle cell lipomas (Figs. 1a, b, c; 2a, b, c, d; and 3a, b, c, d, e) showed a nodular or multinodular growth pattern but none were completely encapsulated, in contrast to classical spindle cell lipomas, which were always completely encapsulated. In four cases, an infiltrative growth pattern was seen. In two deep-seated tumors infiltration of skeletal muscle was observed (Fig. 2d). All cases were composed of atypical lipogenic tumor cells, which varied in size and shape with scattered and hyperchromatic nuclei, and vacuolated lipoblasts, showing a mono- and/or multivacuolated cytoplasm and scalloped, hyperchromatic nuclei (Figs. 1c and 3c, d, e). Numerous spindle-shaped tumor cells, featuring an ill-defined, pale eosinophilic cytoplasm with hyperchromatic nuclei, were irregularly admixed with the lipogenic tumor cells (Fig. 1b, c). The spindle-shaped tumor cells were set in a collagenous stroma, but obvious ropey-like collagen fibers, typical of classical spindle cell lipoma, were not seen. Mitotic activity was very low in all studied cases and tumor necrosis was absent. Myxoid changes were seen in four cases and were associated with scattered mast cells (Fig. 3a, b, c, d, e). Three cases showed a more fascicular growth pattern. Immunohistochemically, spindle-shaped tumor cells in all atypical and classical spindle cell lipomas stained positive for CD34. Nuclear Rb expression was deficient in all atypical and classical spindle cell lipomas (Fig. 3f). No nuclear p16 expression was seen in the adipocytes or the spindle-shaped tumor cells (Fig. 3g) of atypical or classical spindle cell lipomas. Only in two atypical spindle cell lipomas a few

− − − −

+ (few cells) −



+ (few cells) −



















Case 3

Case 4

Case 5

Case 6

Case 7

Case 8

Case 9

+

+

Case 19 +

Case 20 +

Case 15 − − Case 16 − No data available Case 17 − No data available Well-differentiated liposarcoma Case 18 + +

− −

Case 13 − Case 14 −

Intact

Intact

AMP

AMP

AMP

Intact

Deficient NAMP Deficient NAMP

Deficient NAMP

Deficient NAMP

Deficient NAMP

Deficient NAMP

Deficient NAMP

Deficient NAMP

Deficient NAMP

Deficient NAMP

3+

3+

NAMP

AMP

AMP

AMP

NAMP NAMP NAMP

NAMP NAMP

NAMP

NAMP

NAMP

NAMP

NAMP

NAMP

NAMP

NAMP

Deficient No data available NAMP

Deficient NAMP

Deficient NAMP Deficient NAMP Deficient NAMP

2+

NAMP

CDK4 FISH

Deficient No data available NAMP

Deficient NAMP

MDM2 FISH

− − −

− −

















Case 12 −

Case 10 − Spindle cell lipoma Case 11 −







Case 2





P 16 IHC Rb IHC

Atypical spindle cell lipoma Case 1 − −

MDM2 IHC CDK4 IHC

RB1 MLPA

No deletion

No deletion

No deletion

Quality DNA insufficient Quality DNA insufficient Deletion in 24/45 nuclei

Deletion in 19/40 nuclei Deletion in 16/30 nuclei

Deletion in 24/45 nuclei

Deletion in 22/35 nuclei

Deletion in 20/35 nuclei

Deletion in 27/55 nuclei

Deletion in 30/55 nuclei

Deletion in 32/50 nuclei

Deletion in 34/49 nuclei

RB1 median ratio 1.16. No loss of RB1 exons. No loss DLEU1, ITM2B and RCBTB2 RB1 median ratio 0.97. No loss of RB1 exons. No loss DLEU1, ITM2B and RCBTB2 RB1 median ratio 1.16. No loss of RB1 exons. No loss DLEU1, ITM2B and RCBTB2

RB1 median ratio 0.76. Loss exons 7–10,20,21,25 Loss of DLEU1 (low level ratio 0.77) RB1 median ratio 0.77. Loss exons 7–9,12,13,24,25 Loss of DLEU1 (low level ratio 0.73) RB1 median ratio 0.84. Loss exons 7,10. RB1 median ratio 0.85. Loss exons 6,7,8,10,21,25 (low level ratios). Loss of DLEU1 (low level ratio 0.72) Quality DNA insufficient Quality DNA insufficient Quantity DNA insufficient

RB1 median ratio 0.53. Loss exons 3,7,9,10,12-14,16-18,20-27 Loss DLEU1 and RCBTB2 RB1 median ratio 0.57. Loss exons 3–7,9,10,12-14,16-18,20-24, 26,27 Loss DLEU1 and RCBTB2 RB1 median ratio 0.52. Loss exons 2–14,16-24,26,27 Loss DLEU1, ITM2B (low level ratio 0.74) and RCBTB2 RB1 median ratio 0.53. Loss exons 2–14,16-27. Loss DLEU1, ITM2B and RCBTB2 Quantity DNA insufficient

RB1 median ratio 0.66. Loss exons 3,4,6-14,16,18-20,22-27 Loss DLEU1, ITM2B and RCBTB2 Deletion in 41/52 nuclei RB1 median ratio 0.52. Loss exons 2–7,9-14,16-18, 20–27 Loss DLEU1 and RCBTB2 Deletion in 19/52 nuclei RB1 median ratio 0.56. Loss exons 2–7,9-14,16-18,20-27 Loss DLEU1, ITM2B (low level ratio 0.75) and RCBTB2 Deletion in 11/46 nuclei RB1 median ratio 0.52. Loss exons 2–14,16-27 Loss DLEU1 and RCBTB2 (low level ratio 0.72) Deletion in 14/36 nuclei RB1 median ratio 0.55. Loss exons 2–14,16-27 Loss DLEU1 (low level ratio 0.78), ITM2B (low level ratio 0.73) and RCBTB2

Deletion in 35/40 nuclei

RB1 FISH

Table 3 Immunohistochemical and molecular findings in atypical spindle cell lipoma, spindle cell lipoma and well-differentiated liposarcoma

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No data available No data available No data available No data available No data available No data available No data available No data available No data available No data available No data available No data available − negative, + positive, AMP amplification, NAMP no amplification

AMP AMP AMP AMP Intact Intact Intact Intact 3+ 2+ 2+ 3+ No data available No data available No data available No data available + + + + Case 24 Case 25 Case 26 Case 27

No data available No data available No data available AMP No data available AMP No data available No data available No data available AMP AMP AMP Intact Intact Intact + 3+ + 2+ No data available 2+ Case 21 + Case 22 + Case 23 +

MDM2 IHC CDK4 IHC

Table 3 (continued)

P 16 IHC Rb IHC

MDM2 FISH

CDK4 FISH

RB1 FISH

RB1 MLPA

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scattered tumor cells showed nuclear MDM2 expression. No nuclear CDK4 expression was seen in atypical or classical spindle cell lipomas. In contrast, the atypical lipomatous tumors/welldifferentiated liposarcomas showed strong expression of p16, MDM2, and CDK4 in more than 20 % of the nuclei of adipocytes and atypical spindle-shaped stromal cells. Nuclear Rb expression was intact in ten cases of atypical lipomatous tumor/well-differentiated liposarcoma, which included six sclerosing variants, five of which occurred at an unusual site such as the deep soft tissues of thigh and lower leg. Microscopically, these cases had dense fibrotic zones alternating with mature fat, and scattered, mildly pleomorphic spindle and multipolar stromal cells with hyperchromatic nuclei. Only some of these cells showed fatty differentiation and lipoblasts were very scarce. By FISH, a monoallelic deletion of the RB1 gene was detected in the ten atypical spindle cell lipomas and in five classical spindle cell lipomas (in two spindle cell lipomas DNA quality was insufficient for reliable FISH analysis), whereas none of these cases showed amplification of MDM2 or CDK4 (Fig. 1d). Moreover, the RB1 deletion was confirmed by the MLPA RB1 probemix in nine of the ten atypical spindle cell lipomas and two classical spindle cell lipomas. In one atypical spindle cell lipoma and three classical spindle cell lipomas DNA quality or quantity was insufficient for reliable MLPA result. The classical spindle cell lipomas showed consistent loss of RB1 exon 7 and less consistent loss of RB1 exons 25, 10, 9, 21, 8, 12, 13, and 24. Deletion of the DLEU1 gene was seen in three of the four classical spindle cell lipomas. No deletion of flanking genes (RCBTB2 and ITM2B) was seen in the tested classical spindle cell lipomas. The MLPA RB1 mean values in the atypical spindle cell lipomas ranged from 0.52 to 0.66. The RB1 alterations in atypical spindle cell lipomas were more complex, harboring intragenic deletions in multiple exons of the RB1 gene with consistent deletions in exons 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, 20, and 22–24 (Figs. 1e, 2e, 3h). Moreover, less consistent deletions in other exons of the RB1 gene were seen (Table 2). All cases showed a deletion of DLEU1 (mean values ranging from 0.54 to 0.78) and RCBTB2 (mean values ranging from 0.55 to 0.72) genes. Five cases of atypical spindle cell lipoma showed a deletion of ITM2B (mean values ranging from 0.33 to 0.75). By FISH, all atypical lipomatous tumors/welldifferentiated liposarcomas showed amplification of MDM2 and CDK4 but no deletions of RB1 were detected in three tested cases, confirmed by MPLA. By MLPA, no deletion of DLEU1, RCBTB2, or ITM2B were detected.

Discussion Recently, Mentzel et al. found by FISH in six cases of what they called atypical spindle cell lipomatous tumor a consistent

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Fig. 1 Atypical spindle cell lipoma (case 9). Numerous spindle-shaped tumor cells are irregularly admixed with lipogenic tumor cells (a). These spindle-shaped tumor cells, featuring an ill-defined, pale eosinophilic cytoplasm with hyperchromatic nuclei, are set in a non ropey-like

collagenous stroma (b, c). Vacuolated lipoblasts are seen. FISH analysis shows deletion of the RB1 gene (d). MLPA shows complex losses in DLEU1, RCBTB2, ITM2B and multiple exons of the RB1 gene (e)

deletion of RB1, a molecular finding characteristic for spindle cell lipomas, but no amplification of MDM2/CDK4. This suggests that these so-called atypical spindle cell lipomatous tumors represent the atypical/low-grade counterpart of spindle

cell lipoma [1]. To date, only one of our cases has recurred and none has metastasized, one could argue that they should not be at this time considered as a variant of a well-differentiated spindle cell liposarcoma. In view of the uncertainty as to their

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Fig. 2 Atypical spindle cell lipoma (case 7). Atypical lipogenic tumor cells with striking variations in size and shape are associated with spindle-shaped tumor cells containing slightly enlarged and

hyperchromatic nuclei (a, b, c). Infiltration of skeletal muscle is seen (d). MLPA shows complex losses in DLEU1, RCBTB2 and multiple exons of the RB1 gene (e)

behavior, their distinct character relative to other lesions previously described as spindle cell liposarcoma and to underscore the molecular link with classical spindle cell lipoma that emerges from our data we have chosen the term atypical

spindle cell lipoma. Further studies of more cases with longer follow-up are necessary to confirm the intermediate, locally aggressive, non-metastasizing biological behavior of this group of atypical spindle cell lipomas.

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Fig. 3 Atypical spindle cell lipoma (case 2). A deep-seated, not encapsulated lipogenic neoplasm with a multinodular growth pattern is seen (a). Numerous atypical spindle-shaped tumor cells with hyperchromatic nuclei are admixed with scattered mono- and multivacuolated lipoblasts.

The stroma shows myxoid changes (b, c, d, e). Endothelial cells show intact nuclear Rb expression, whereas tumor cells are deficient (f). Tumor cells are negative for p16 (g). MLPA shows complex losses in DLEU1, RCBTB2 and multiple exons of the RB1 gene (h)

Our data confirms the chromosomal abnormalities associated with loss of the 13q14 region, including the RB1 gene, in atypical spindle cell lipoma. We found this deletion in all

tested cases by FISH, as monoallelic presence of the RB1 locus, and confirmed this by MLPA. As supportive evidence, deficient immunohistochemical expression of Rb protein was

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Fig. 3 (continued)

seen in all cases. Moreover, we found in atypical spindle cell lipomas complex losses across the whole coding region of the RB1 gene, notably in exons located in the functionally important pocket domains, composed of the so-called A (exon 12, 13, 14, 15, 16, 17) and B (exon 19, 20, 21, 22) boxes, essential for the interaction of Rb with other proteins notably those of the E2F-family [37]. Mutations affecting the A and B boxes of the pocket domain give rise to Rb proteins with disturbed function and stability [38] which cannot properly bind to E2F. As a result E2F is released, which promotes E2Fmediated transcription and expression of genes required for cell division and proliferation. We also found a deletion of the RCBTB2 and ITM2B genes, which flank RB1, in nine and five atypical spindle cell lipomas, respectively. The ITM2B gene encodes a mitochondrial membrane protein with a Bcl-2 homology 3 (BH3) domain, which is ubiquitously expressed. ITM2B protein may be involved in processing an amyloid precursor protein, is thought to be involved in nerve cell function in the early development of the brain and has been associated with the development of hereditary cerebral amyloid angiopathy [39, 40]. The role of the ITM2B protein in

neoplasia is unclear but it might play a role in a p53independent apoptosis [41–43]. The RCBTB2 gene (also known as CHC1L) encodes a guanine nucleotide exchange factor protein for ras-related GTPase, shows a strong homology with RCC1 and might be a candidate tumor suppressor gene in B-CLL, prostate and bladder cancer [44–47]. We hypothesize that functional loss of these RB1 flanking genes might provide a growth advantage to these atypical spindle cell lipomas by directly regulating cell proliferation and/or cell death. The RB1 deletion in exon 7, consistently seen in all atypical and classical spindle cell lipomas included in this study, might represent an early event in both types of neoplasm. Additional deletions in ITM2B, RCBTB2, and more complex deletions in functionally important exons of the large RB1 gene would then be necessary for the progression to atypical spindle cell lipoma. This implies that some cases of atypical spindle cell lipoma might arise in a pre-existing spindle cell lipoma. This hypothesis of a biological continuum of benign, atypical and malignant lipogenic neoplasms, as proposed by Mentzel et al., should be substantiated in further molecular studies [48], notably as regards the deletions of the

Virchows Arch

ITM2B and RCBTB2 genes. Another molecular feature that links these two entities is the consistent deletion of the DLEU1 gene, located on 13q14 at a distance of 1.6 Mb from RB1, in all atypical spindle cell lipomas and in three of the four classical spindle cell lipomas. DLEU1, of which a deletion has been reported in CLL [51, 52], encodes a long non-coding RNA and is one of the smallest splice variants of B-cell neoplasia-associated gene with multiple splice variants (BCMS), one of the most potent tumor suppressor genes [49, 50]. Its exact role in the development of atypical and classical spindle cell lipomas remains to be elucidated, as well as its potential diagnostic value. Recently, Deyrup et al. [53] reported a group of spindle cell lipomatous tumors, morphologically and genetically different from other forms of liposarcoma, showing a distinctive flat comparative genomic hybridization (CGH) pattern without gains or losses and lacking molecular aberrations characteristic of other lipomatous tumors. Based on our extensive morphological and molecular-genetic analyses which confirm a consistent RB1 deletion, we believe that these so-called “fibrosarcoma-like lipomatous neoplasms” are unrelated to atypical spindle cell lipoma. In summary, we report that RB1 is deleted in atypical spindle cell lipoma, a group of spindle cell lipomas with unusual topography and morphology. This confirms their association with classical spindle cell lipoma and differentiates them from atypical lipomatous tumor/well-differentiated liposarcoma. We report deletions in multiple functionally important exons of RB1 and the two nearest RB1 flanking genes ITM2B and RCBTB2 in these tumors, in contrast to classical spindle cell lipomas that show less complex deletions of functionally important RB1 exons. We also report a deletion of the DLEU1 gene in atypical spindle cell lipoma. Finally, we propose an immunohistochemical panel of MDM2, CDK4, p16 and Rb as a useful aid in the histologic diagnosis of atypical and classical spindle cell lipomas, which will allow to differentiate them from atypical lipomatous tumor/welldifferentiated liposarcoma. Acknowledgments The authors would like to thank Chris Hettinga for technical assistance. Conflict of interest statement The authors declare that they have no conflict of interest.

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Atypical spindle cell lipoma: a clinicopathologic, immunohistochemical, and molecular study emphasizing its relationship to classical spindle cell lipoma.

We studied a series of spindle cell lipomas arising in atypical sites and showing unusual morphologic features (which we called atypical spindle cell ...
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