Original Article
Dedifferentiated Liposarcoma and Pleomorphic Liposarcoma A Comparative Study of Cytomorphology and MDM2/CDK4 Expression on Fine-Needle Aspiration n Marin ~ o-Enrıquez, MD; Jason L. Hornick, MD, PhD; Paola Dal Cin, PhD; Adria Edmund S. Cibas, MD; and Xiaohua Qian, MD, PhD
BACKGROUND: Dedifferentiated liposarcoma (DDLPS) and pleomorphic liposarcoma (PLPS) are distinct high-grade liposarcomas. DDLPS is a nonlipogenic sarcoma characterized by amplification of MDM2 and CDK4. PLPS is a high-grade sarcoma containing lipoblasts, characterized by a complex karyotype and a more aggressive clinical course. Rarely, DDLPS shows lipogenic differentiation, mimicking PLPS. The cytomorphologic features of DDLPS and PLPS and the utility of ancillary studies have not been systemically analyzed. METHODS: Cytologic preparations of 25 DDLPS and 13 PLPS, all histologically confirmed, were retrospectively reviewed along with clinical and cytogenetic data. Sample cellularity, vascular architecture, background material, predominant cell morphology, quality of the cytoplasm, and nuclear pleomorphism were compared for both tumor types. Immunohistochemistry for MDM2 and CDK4 was performed on cell blocks and/or core needle biopsies. RESULTS: Fine-needle aspirate smears from both DDLPS and PLPS were variably cellular, composed of cellular clusters and noncohesive cells. Abundant myxoid stroma was present in 25% of DDLPS and PLPS cases, whereas branching curvilinear vessels were more common in DDLPS than in PLPS (7 of 25 versus 2 of 13). Tumors were composed of predominantly spindled (18 of 25 DDLPS versus 3 of 13 PLPS) or epithelioid cells (7 of 25 DDLPS versus 6 of 13 PLPS). Pleomorphic cells were predominant in 3 PLPS, and were frequent in both (13 of 25 DDLPS versus 10 of 13 PLPS). The cytoplasm was mostly fibrillary and often vacuolated in both entities. Other features included necrosis, mitoses, and a prominent inflammatory infiltrate. The main cytologic differences were the presence of marked pleomorphism, abundant lipoblasts, and cells with microvacuolated cytoplasm in most PLPS. A total of 24 (96%) and 20 (80%) cases of DDLPS expressed MDM2 and CDK4, respectively, whereas none of the PLPS expressed both markers. Six DDLPS tested showed ring or giant marker chromosomes and/or MDM2 amplification by fluorescence in situ hybridization; 2 PLPS had complex karyotypes. CONCLUSIONS: DDLPS and PLPS exhibit variable and occasionally overlapping cytologic features. The presence of lipoblasts, cells with microvacuolated cytoplasm, and marked pleomorphism are more suggestive of PLPS, but these characteristics can be present in DDLPS. Coexpression of MDM2 and CDK4 distinguishes DDLPS from C 2013 American Cancer Society. PLPS. Cancer (Cancer Cytopathol) 2014;122:128-37. V
KEY WORDS: sarcoma; fine-needle aspirate; dedifferentiated liposarcoma; pleomorphic liposarcoma; MDM2; CDK4; lipoblast; cytology; adipocytic; soft tissue.
Corresponding author: Xiaohua Qian, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115; Fax: (617) 582-6015; [email protected] Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts Presented in part at the 102nd Annual Meeting of the United States and Canadian Academy of Pathology (USCAP), Baltimore, MD; March 2013. The authors thank Mei Zheng for outstanding technical support and Nick Hoover for his early work on the project. Received: August 9, 2013; Revised: September 14, 2013; Accepted: September 16, 2013 Published online November 12, 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21362, wileyonlinelibrary.com
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~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
INTRODUCTION Liposarcomas are a heterogeneous group of malignant adipocytic neoplasms that consist of 3 distinct clinicopathological entities: well-differentiated/dedifferentiated liposarcoma, myxoid liposarcoma, and pleomorphic liposarcoma (PLPS). Collectively, liposarcomas account for approximately 20% of all adult sarcomas.1 Dedifferentiated liposarcoma (DDLPS) results from the progression of a well-differentiated liposarcoma (WDLPS) to a nonlipogenic sarcoma of variable histologic grades and morphologic patterns that acquires metastatic potential.2,3 Dedifferentiated areas in DDLPS have the appearance of a high-grade sarcoma in approximately 90% of cases, and show a variety of growth patterns including, most frequently, nondistinct spindle cell and pleomorphic patterns, and less commonly, inflammatory, giant cell, round cell, or meningothelial-like patterns. The remaining 10% of DDLPS are morphologically low grade, characterized by uniform spindle cells with mild nuclear atypia, often organized in a fascicular pattern and exhibiting lower cellularity than the usual high-grade areas.3 DDLPS exhibits heterologous differentiation in approximately 5% to 10% of cases, which does not affect the clinical outcome.4 Adipocytic differentiation within the high-grade component of DDLPS, traditionally excluded by definition, has been recently recognized and designated “homologous” lipoblastic differentiation.5,6 PLPS is rare, representing 5% of liposarcomas.7 Morphologically, PLPS is a high-grade pleomorphic sarcoma with varying numbers of multivacuolated lipoblasts.4,7–9 Because of its wide morphologic spectrum, PLPS can be confused with numerous adipocytic and nonadipocytic neoplasms, including DDLPS and other high-grade pleomorphic sarcomas. PLPS pursues a consistently more aggressive clinical course than DDLPS.7–9 Cytogenetic techniques such as karyotyping, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) have unraveled certain aspects of the biology of liposarcomas,10,11 prompting the development of new diagnostic tests that are now available for routine clinical practice.12 Given its high sensitivity and specificity, the combined detection of MDM2 (mouse double minute 2) and CDK4 (cyclin-dependent kinase 4) overexpression by immunohistochemistry has become a particularly useful ancillary technique to support the diagnosis of WDLPS/DDLPS on surgical resecCancer Cytopathology
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tion samples.13–15 The molecular basis for the overexpression of these cell cycle–related proteins is the presence of 12q13-q15 amplicons in supernumerary ring and/or giant markers chromosomes with constant amplification of MDM2 and HMGA2, and frequent amplification of CDK4, that play an essential role in the pathogenesis of WDLPS/DDLPS.16–20 A recent study demonstrated that detection of MDM2 amplification by FISH is more sensitive and specific than MDM2 immunohistochemistry to differentiate WDLPS from various benign lipomatous tumors on core needle biopsy.21 The utility of these ancillary studies in diagnosing high-grade liposarcomas on small-needle biopsy has not yet been systematically evaluated. In contrast to DDLPS, the molecular features of PLPS are still poorly understood. At the cytogenetic level, PLPS is characterized by a complex karyotype with multiple chromosomal gains and losses that do not typically include the 12q13-q15 region.22 The resulting gene expression profiles of PLPS differ significantly from those of WDLPS/DDLPS.23 These features reflect the generally anaplastic nature of pleomorphic sarcomas and place PLPS nosologically closer to other high-grade nonlipogenic sarcomas, such as conventional osteosarcoma, high-grade leiomyosarcoma, or chondrosarcoma, than to DDLPS.9,22 In some situations, the morphology of DDLPS and PLPS is remarkably similar, and inadequate sampling can lead to misdiagnosis. In particular, DDLPS with “homologous” lipoblastic differentiation is indistinguishable from PLPS on morphologic grounds. The clinical and biological differences between these 2 entities facilitates the differential diagnosis: a previous diagnosis of WDLPS or the presence of WDLPS areas contiguous to or admixed with a high-grade sarcoma should prompt the diagnosis of DDLPS; conversely, lipoblastic differentiation in a high-grade pleomorphic sarcoma favors the diagnosis of PLPS, but the status of MDM2 and CDK4 should be determined by immunohistochemistry or FISH, to rule out a rare case of DDLPS with homologous lipoblastic differentiation.5,6 The cytologic descriptions of DDLPS and PLPS in the literature are included in single case reports or heterogeneous series. Whereas most reported DDLPS cases were recognized in light of a concurrent or prior history of WDLPS, in approximately half of the cases of PLPS the authors initially favored other diagnostic possibilities, including poorly differentiated carcinoma and high-grade 129
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sarcoma without specifying the line of differentiation.24–27 In this study, we analyze the cytologic aspirates of 38 histologically confirmed high-grade liposarcomas, including 25 DDLPS and 13 PLPS, correlating the cytomorphology with clinical, histologic, immunohistochemical, and genetic data.
MATERIALS AND METHODS Histologically confirmed cases of DDLPS and PLPS diagnosed between 1990 and 2012, with both cytologic and histologic materials available for review, were retrieved from consultation and pathology files of the Department of Pathology of the Brigham and Women’s Hospital, Boston, Massachusetts. There were 25 DDLPS and 13 PLPS with at least one satisfactory fine-needle aspirate (FNA) and corresponding histologic preparations, either core biopsies or surgical resections. These 38 paired samples corresponded to 35 patients: repeat matching cytologic and histologic samples were obtained at 2 different time points for 3 patients (1 DDLPS, 2 PLPS) and were analyzed as independent cases for the purposes of this study. FNAs consisted of cytologic smears (n 5 30) and/or cell block preparations (n 5 32). Air-dried smears were stained with Hemacolor (Harleco, EM Science, Gibbstown, NJ); alcohol-fixed slides were fixed in 95% ethanol and stained with a modified Papanicolaou stain. Cell block pellets were prepared from the centrifuged needlerinse specimens using the plasma–thrombin method whereby the pellet is congealed with several drops each of plasma and thrombin. The resulting clot was fixed in formalin, embedded in paraffin, and processed for histologic sections. For histologic evaluation, 4-lm-thick hematoxylin-and-eosin–stained sections were examined. The following cytomorphologic features were evaluated: cellularity, vascular architecture, cell morphology, quality of the cytoplasm, nuclear pleomorphism, background material, presence of lipoblasts, and multinucleate giant cells. The cell morphology of each sample was classified as epithelioid, spindled, or pleomorphic based on the predominant cell type (> 50%). Clinical information was obtained from the electronic medical record and the pathology reports. Immunohistochemistry was performed on 4-lmthick formalin-fixed paraffin-embedded tissue sections (n 5 32) and/or cell blocks (n 5 25) using pressure cooker heat-induced epitope retrieval (0.01 M citrate buffer, pH 130
6.0) and antibodies against MDM2 (Calbiochem, San Diego, Calif; clone 1F2; dilution 1:50) and CDK4 (Biosource/Invitrogen, Carlsbad, Calif; clone DCS-31; dilution 1:250). The Envision Plus detection system (Dako, Carpinteria, Calif) was used. Appropriate positive and negative controls were used throughout. Concordance between cytologic and histologic preparations was analyzed when both type of materials were available for staining (n 5 19). Karyotypes were retrospectively reviewed when available (n 5 6). FISH analysis was performed on 4 selected DDLPS cases on 4-lm sections from paraffin blocks using a commercially available MDM2 probe (LPS 016-A; Cytocell, Cambridge, UK) following standard protocols in our laboratory.28 The study was approved by the Institutional Review Board at Brigham and Women’s Hospital.
RESULTS Clinical Information
Clinical data are summarized in Figure 1 and Table 1. Seventeen DDLPS patients were male and 8 were female, ranging in age from 43 to 81 years (median, 67 years). Tumors occurred most commonly in the retroperitoneum (12 patients, 48%); other deep locations included intra-
FIGURE 1. Anatomic distribution of 25 dedifferentiated liposarcomas (LPS) and 13 pleomorphic LPS. White squares represent primary tumors, white circles represent tumor recurrences, and black circles represent metastases.
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~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
Table 1. Clinical, Cytologic, and Immunohistochemical Features of 25 Dedifferentiated Liposarcomas (DDLPS) and 13 Pleomorphic Liposarcomas (PLPS).
Case ID
Age
Sex
Site (Side)a
Type of Lesionb
DDLPS-1 DDLPS-2 DDLPS-3 DDLPS-4 DDLPS-5 DDLPS-6 DDLPS-7 DDLPS-8 DDLPS-9 DDLPS-10 DDLPS-11 DDLPS-12 DDLPS-13 DDLPS-14 DDLPS-15 DDLPS-16 DDLPS-17 DDLPS-18 DDLPS-19 DDLPS-20 DDLPS-21 DDLPS-22 DDLPS-23 DDLPS-24 DDLPS-25 PLPS-1 PLPS-2 PLPS-3 PLPS-4 PLPS-5 PLPS-6 PLPS-7 PLPS-8 PLPS-9 PLPS-10 PLPS-11 PLPS-12 PLPS-13
43 74 74 67 70 63 60 66 64 64 62 73 68 51 81 81 59 59 44 67 70 77 68 79 59 41 32 65 65 51 22 80 45 71 59 59 53 53
F M M M M M F M F F F M F M M M M M F M M M F M M M M M M M F M M M M M F F
RP RP RP RP RP Intra-abdominal Liver Chest wall Intra-abdominal RP RP Intra-abdominal Thigh (R) Liver RP RP Thigh (L) Liver Thigh (R) RP Lung RP Lung RP Chest wall Pancreas Liver Calf (R) Mediastinal Thigh (L) Mediastinal Arm (R) Thigh (L) Pancreas Calf (L) Thigh (L) Humerus (L) Knee (R)
Rec Rec Rec Rec Rec Rec Met Met Rec Rec Met Rec Rec Met Rec Rec P Met P Rec Met P Met Rec Rec Met Met P Met Rec P P Met P P Rec P P
Lipoblasts
Vacuolated cells
IHC Cytology MDM2/ CDK4c
IHC Surgical MDM2/ CDK4c
N N N N N N N N N N N N N N N N N N N N N N N Y Y N Y Y Y Y N Y Y Y Y N Y Y
N N N Y N Y N N Y N N N Y N N Y N N N Y Y Y N Y Y N Y Y Y Y Y Y Y Y Y Y Y Y
1/1 NA 1/– 1/– 1/– 1/1 1/1 NA 1/1 NA NA NA 1/1 1/1 1/NA 1/1 –/– 1/1 1/1 NA NA 1/1 NA 1/1 1/1 –/– NA NA NA –/– NA –/– –/– –/– –/weak NA –/– –/–
NA 1/1 1/– NA 1/– 1/1 1/1 1/1 1/1 NA 1/1 1/1 1/1 1/1 1/1 1/1 –/– 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 NA –/– –/– –/– –/– –/– –/– –/– NA NA –/– –/– –/–
a
L, left; R: Right; RP, retroperitoneum. P, primary; Rec, recurrence; Met, metastasis. c IHC, immunohistochemistry; (1), positive; (–), negative; NA, not available. b
abdominal soft tissue (3 patients), liver (3 patients), proximal lower extremity (3 patients), lung (2 patients), and chest wall (2 patients). Three lesions were primary tumors, 15 were recurrences, and 7 were metastases. A total of 17 patients had a previous diagnosis of WDLPS (16 patients) or DDLPS (1 patient). In 22 tumors, the diagnosis of DDLPS was suggested on FNA alone, including 17 cases with a prior history of WDLPS/DDLPS, and 5 initial diagnoses; the 3 remaining cases were diagnosed in conjunction with the concurrent core needle biopsy. Ten PLPS patients were male and 3 were female, ranging in age from 22 to 80 years (median, 56 years). PLPS involved the deep soft tissue of the extremities (8 patients), mediastinum (2 patients), pancreas (2 patients), Cancer Cytopathology
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and liver (1 patient). Six lesions were primary tumors, 2 were recurrences, and 5 were metastases. Eight of these tumors were diagnosed on FNA alone, including 7 cases with a known history of PLPS and 1 initial diagnosis; in 5 cases, a diagnosis of pleomorphic malignancy was rendered on FNA, and further classification was made on the concurrent core needle biopsy. Cytologic Features of DDLPS
FNA smears of DDLPS were variably cellular, composed of both ill-defined cellular clusters and variable amounts of noncohesive cells. The cellular clusters were mainly syncytial-like (Fig. 2a) and, occasionally, loosely cohesive cell aggregates. Abundant myxoid stroma in the 131
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FIGURE 2. Cytomorphologic features of dedifferentiated liposarcomas (DDLPS) (a,c-h: Hemacolor stain; b,i: Papanicolaou stain). (a) Smears were composed of syncytial-like cellular clusters or loosely cohesive aggregates. (b) Six DDLPS showed myxoid stromal fragments and (c) 7 showed prominent curvilinear branching vessels. Highly atypical pleomorphic cells (d) were usually scant but occasionally abundant. The cytomorphology was (d) predominantly spindled in 18 cases and (e) epithelioid, polygonal, or plasmacytoid cells in 7 cases. (f, g, h) Cells with microvacuolated cytoplasm were present in 43% of DDLPS. (i) Variable numbers of lipoblasts with multivacuolated cytoplasm and hyperchromatic scalloped nuclei were observed in 2 DDLPS with “homologous” lipoblastic differentiation.
background was seen in 6 cases (Fig. 2b), and prominent curvilinear branching blood vessels in 7 cases (Fig. 2c). Most tumors showed a combination of different cell morphologies with predominantly spindle cells in 18 cases (Fig. 2d) and epithelioid, polygonal, or plasmacytoid cells in 7 cases (Fig. 2e). Isolated highly atypical pleomorphic cells with bizarre, hyperchromatic nuclei, irregular nuclear contours, and prominent nucleoli were observed in most cases and were particularly numerous in 13 (54%) cases (Fig. 2f). Multinucleated giant cells were present in 4 cases (17%). Cytoplasmic microvacuoles or a “bubbly” cytoplasm was noted in 10 cases (40%), usually in epithelioid or pleomorphic cells (Fig. 2f,g,h). Scattered lipoblasts with single or multiple sharply circumscribed cytoplasmic vacuoles and scalloped hyperchromatic nuclei were pres132
ent in 2 cases of DDLPS (8%) (Fig. 2i). On histologic examination, these 2 cases corresponded to DDLPS with high-grade spindle cell morphology and “homologous” lipoblastic differentiation, showing numerous large multivacuolated lipoblasts with scalloped hyperchromatic nuclei within the high-grade component. Necrosis, mitoses, and a prominent inflammatory infiltrate were present in 6, 3, and 5 cases, respectively. Cytologic Features of PLPS
FNA smears of PLPS were variably cellular, composed of numerous irregular cellular clusters (Fig. 3a,b), and abundant isolated, noncohesive cells. In 3 cases, there was an abundant myxoid stroma. 2 cases showed prominent curvilinear branching blood vessels (Fig. 3c). In 12 cases of Cancer Cytopathology
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~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
FIGURE 3. Cytomorphologic features of pleomorphic liposarcomas (PLPS) (a: Papanicolaou stain; b-j: Hemacolor stain). (a) Cellular, tight clusters of cohesive cells are characteristic, often (b) combined with irregular cellular aggregates and abundant isolated, noncohesive cells. (c) Epithelioid cytomorphology was predominant in half of the cases, and branching thin vessels were uncommon. (d-h) Twelve PLPS showed cells with microvacuolated cytoplasm. (f-h) Variable numbers of lipoblasts (often numerous) with multivacuolated cytoplasm and hyperchromatic scalloped nuclei were identified in 10 PLPS. (i) Highly atypical pleomorphic cells were abundant in 10 cases. (j) Multinucleated giant cells were present in 6 cases.
PLPS (92%), there were abundant cells featuring microvacuolated cytoplasm with a “bubbly” apperance, which is best appreciated on air-dried, Hemacolor-stained smears (Fig. 3c-h). Lipoblasts were identified in 10 cases (77%) (Fig. 3c,d,f-h). A total of 10 samples also showed numerous pleomorphic cells with abundant cytoplasm and bizarre nuclei (Fig. 3i). The nonpleomorphic tumor cells were predominantly epithelioid in 6 cases (Fig. 3c) and spindled in 3. Multinucleated giant cells were present in 6 cases (46%) (Fig. 3j). Necrosis was present in 5 cases; mitoses and a prominent inflammatory infiltrate each were seen in 3 cases. The main cytomorphologic features of DDLPS and PLPS are summarized and compared in Figure 4. Cancer Cytopathology
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FIGURE 4. Comparative frequency of major cytomorphologic features present in 25 dedifferentiated liposarcomas (DDLPS) and 13 pleomorphic liposarcoma (PLPS) fine-needle aspirate smears.
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FIGURE 5. Immunohistochemical expression of MDM2 and CDK4 in dedifferentiated liposarcomas (DDLPS), present in 24 (96%) and 20 (80%) cases, respectively.
Immunohistochemistry, Cytogenetics, and FISH
The immunohistochemical findings of DDLPS and PLPS are summarized in Table 1. A total of 24 DDLPS cases expressed MDM2 (96%) and 20 expressed CDK4 (80%) by immunohistochemistry on cell block or tissue sections (Fig. 5). A single case (DDLPS-17) was negative for both markers. Karyotyping detected the presence of ring and/ or giant marker chromosomes in all 4 cases analyzed. FISH showed high-level amplification of the MDM2 locus in 2 of the karyotyped cases, and in 2 additional DDLPS, including case DDLPS-17 which was negative for MDM2 and CDK4 by immunohistochemistry. Both cases of DDLPS with “homologous” lipoblastic differentiation showed MDM2 amplification by FISH. In contrast, all PLPS cases were negative for MDM2 and CDK4 by immunohistochemistry, except for 1 case that was weakly positive for CDK4 (7%). Karyotyping showed a complex karyotype with multiple numeric and structural aberrations in the 2 PLPS cases analyzed. There was 100% agreement between the results of the immunohistochemical stains performed on cytologic and surgical materials in the 19 cases that were evaluated.
DISCUSSION Some aspects of liposarcoma cytopathology have been discussed in previous reports, mostly in the context of benign lipomatous lesions25,26,29,30 or other soft tissue sarcomas.31,32 In particular, 1) the differential diagnosis of 134
well-differentiated liposarcoma versus benign adipocytic tumors,25,26,29,30 including radiologic correlates,33,34 and 2) the morphologic features of myxoid liposarcoma and its high-grade counterpart, known as round cell liposarcoma, have been reasonably well defined.24,25,29,35 Few studies, however, have analyzed the cytomorphology of DDLPS and PLPS, resulting in limited cytologic descriptions available, as recently reviewed by Dodd.36 We focused on characterizing the cytomorphologic features of these 2 biologically distinct groups of highgrade liposarcoma, DDLPS and PLPS, in a series of 25 and 13 cases, respectively. To distinguish them is relevant, given the differences in clinical management and prognosis,4 and the increasing availability of targeted therapies only effective in tumors with specific molecular alterations, such as MDM2 inhibitors for DDLPS.37 Despite notable biological and clinical differences, DDLPS and PLPS may exhibit very similar cytomorphology. Both share features of a high-grade sarcoma, yielding moderateto-highly cellular smears showing cellular and nuclear pleomorphism, frequent necrosis with associated inflammation, and high mitotic counts, including occasional atypical mitoses. Highly atypical cells with bizarre nuclei and multinucleated giant cells, although more commonly seen in PLPS, were also present in DDLPS. Traditionally, the identification of lipoblasts in a smear showing features of high-grade pleomorphic sarcoma was considered sufficient to diagnose PLPS.24,25,29,35 The recent description of “homologous” lipoblastic differentiation in DDLPS, however, has complicated the interpretation of lipoblasts in this context. Therefore, the differential diagnosis between DDLPS and PLPS should always be entertained when encountering a high-grade sarcoma with lipoblasts. That being said, the presence of lipoblasts is far more frequently associated with PLPS than DDLPS (77% versus 8% in this study; Fig. 4). In addition, lipoblasts in PLPS are often numerous and strikingly atypical, with bizarre nuclei and irregular nuclear membranes; some are actually multinucleated giant cells. Although nonspecific, other morphologic clues that may guide the differential diagnosis are the predominant cell morphology, the proportion of highly atypical or bizarre cells, and the degree of pleomorphism. In DDLPS, we have found, there are usually few bizarre cells, scattered among other tumor cells, which are often spindled; in PLPS, bizarre atypical tumor cells are more abundant, admixed with tumor cells that are more often epithelioid. Also, the degree of cellular Cancer Cytopathology
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~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
pleomorphism is (unsurprisingly) greater in PLPS than in DDLPS, seen in both lipoblasts and nonlipogenic tumor cells. In addition, correlation with clinical and radiologic data is essential. In particular, history of a previously documented well-differentiated liposarcoma, suspicious-looking fatty areas on imaging studies, and a retroperitoneal location are highly suggestive of DDLPS; location in the extremities is more common in PLPS (Fig. 1). An interesting finding in this study is the presence of atypical cells with abundant microvacuolated cytoplasm in 40% of DDLPS and 92% of PLPS smears. As shown in Figs. 2f-h and Figs. 3e-h, these cells do not satisfy the morphologic definition of a lipoblast, which requires that hyperchromatic nuclei be indented by cytoplasmic vacuoles (frequently described as “scalloped nuclei”). Furthermore, the microvacuolization of the cytoplasm consists of innumerable minute vacuoles, conferring the appearance of a “bubbly cytoplasm,” which contrasts with the less numerous and usually larger cytoplasmic vacuoles in unequivocal lipoblasts. Cells with similar cytoplasmic vacuoles have been described in hibernoma,24,25 highgrade myxoid liposarcoma35 (formerly designated round cell liposarcoma), and in a case of so-called malignant fibrous histiocytoma.31 These peculiar microvacuolated cells have not been associated previously with DDLPS or PLPS in the cytology literature. In hibernoma, the main criteria proposed to differentiate them from lipoblasts are the round, nonscalloped nuclei and the uniformity of the small-sized cytoplasmic vacuoles.24,25 In high-grade myxoid liposarcoma, these microvacuolated cells constitute a monomorphic population of noncohesive atypical cells (see below).35 In the context of DDLPS and PLPS, we observed a pleomorphic population of microvacuolated atypical cells, with hyperchromatic nuclei of variable sizes and shapes. The fact that these cells are present in all 3 types of high-grade liposarcoma suggests that they may be related to true lipoblasts and raises the possibility of a previously unrecognized subtle manifestation of adipocytic differentiation in cytology specimens. Regardless of their nature, a pleomorphic population of atypical microvacuolated cells is common in DDLPS and very common in PLPS, and hence may be a helpful diagnostic clue to classify a highgrade sarcoma of uncertain differentiation. Immunohistochemistry for MDM2 and CDK4 is very useful for diagnosis: most DDLPS coexpress these 2 markers that reflect their genetic make-up, whereas virtually all PLPS are negative for both.13–15 In the present Cancer Cytopathology
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series, we observed strong and diffuse expression of MDM2 and/or CDK4 in all but one DDLPS cases. The exception (DDLPS-17) was a 19-cm primary DDLPS arising in the thigh of a 59-year-old male, which was repeatedly negative for MDM2 and CDK4 by immunohistochemistry in the cytology specimens and core biopsy. We confirmed the diagnosis of DDLPS by FISH, which demonstrated high-level amplification of the MDM2 locus. In addition, on the subsequent surgical excision the tumor showed small areas of WDLPS. All the PLPS cases were negative for MDM2 by immunohistochemistry, and only one case showed weak CDK4 expression. We observed absolute correlation between the cell block preparations and the core biopsy and/or surgical resection materials. These results underscore the diagnostic value of detecting MDM2 and CDK4 expression for the differential diagnosis of DDLPS versus PLPS on FNA cytology. Discussions regarding the sensitivity and specificity of FNA for the diagnosis of DDLPS and PLPS in the context of a broader differential diagnosis are beyond the scope of this study. We would like to mention, however, that some of the cytomorphologic findings in our series highlight an obvious potential overlap with myxoid liposarcoma: smears from 6 DDLPS and 3 PLPS showed a prominent myxoid background with rich vascularization (Figs. 2c and 3b), often with curvilinear arborizing vessels similar to those observed in myxoid liposarcoma.24,35 A similar phenomenon has been recently documented in surgical specimens.38 A very illustrative case in this regard is DDLPS-25, one of the DDLPS cases with lipoblastic differentiation, which showed large myxoid stromal fragments with prominent branching thin-walled vessels, and relatively small lipoblasts located at the vessel bifurcations (Fig. 2c). In our experience, however, the cytomorphology of myxoid liposarcoma is characteristic and quite different from that of DDLPS or PLPS: in low-grade cases, smears are dominated by abundant fragments of vascularized myxoid stroma and relatively low cellularity, consisting of uniform, bland cells displaying only subtle atypia; high-grade cases show uniform round cell cytomorphology with occasional uni- or bi-vacuolated lipoblasts. Typically, myxoid liposarcoma can be ruled out based on the presence of marked cellular pleomorphism typically seen in PLPS and DDLPS. In problematic cases, immunohistochemical detection of MDM2 and CDK4 and/or molecular/genetic analyses are useful ancillary techniques. 135
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In summary, we have characterized and compared the cytomorphologic features of DDLPS and PLPS in 38 FNA samples with correlation of immunoprofile and cytogenetic data. Atypical cells with abundant microvacuolated cytoplasm and/or lipoblasts are more frequently present in PLPS, but can also be seen in DDLPS; therefore, they do not necessarily discriminate between these 2 distinct high-grade liposarcomas. The presence of marked pleomorphism, combined with highly atypical lipoblasts, favors the diagnosis of PLPS. The detection of MDM2 and CDK4 expression by immunohistochemistry has great value in establishing the diagnosis of DDLPS, which is applicable to FNA cytology. FUNDING SOURCES No specific funding was disclosed.
CONFLICT OF INTEREST DISCLOSURE Dr. Mari~ no-Enrıquez is supported by a Career Development Award from the Sarcoma Alliance for Research through Collaboration (SARC). All other authors made no disclosure.
REFERENCES Fletcher CDM. Diagnostic Histopathology of Tumors. 4th ed. Philadelphia, PA: Saunders, Elsevier; 2013. 2. McCormick D, Mentzel T, Beham A, Fletcher CD. Dedifferentiated liposarcoma. Clinicopathologic analysis of 32 cases suggesting a better prognostic subgroup among pleomorphic sarcomas. Am J Surg Pathol. 1994;18:1213-1223. 3. Henricks WH, Chu YC, Goldblum JR, Weiss SW. Dedifferentiated liposarcoma: a clinicopathological analysis of 155 cases with a proposal for an expanded definition of dedifferentiation. Am J Surg Pathol. 1997;21:271-281. 4. Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F. WHO Classification of Tumours of Soft Tissue and Bone. Lyon, France: IARC; 2013. 5. Marino-Enriquez A, Fletcher CD, Dal Cin P, Hornick JL. 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;34:1122-1131. 6. Boland JM, Weiss SW, Oliveira AM, Erickson-Johnson ML, Folpe AL. Liposarcomas with mixed well-differentiated and pleomorphic features: a clinicopathologic study of 12 cases. Am J Surg Pathol. 2010;34:837-843. 7. Hornick JL, Bosenberg MW, Mentzel T, McMenamin ME, Oliveira AM, Fletcher CD. Pleomorphic liposarcoma: clinicopathologic analysis of 57 cases. Am J Surg Pathol. 2004;28:1257-1267. 8. Gebhard S, Coindre JM, Michels JJ, et al. Pleomorphic liposarcoma: clinicopathologic, immunohistochemical, and follow-up analysis of 63 cases: a study from the French Federation of Cancer Centers Sarcoma Group. Am J Surg Pathol. 2002;26:601-616. 9. Downes KA, Goldblum JR, Montgomery EA, Fisher C. Pleomorphic liposarcoma: a clinicopathologic analysis of 19 cases. Mod Pathol. 2001;14:179-184. 10. Coindre JM, Pedeutour F, Aurias A. Well-differentiated and dedifferentiated liposarcomas. Virchows Arch. 2010;456:167-179. 1.
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11. Mertens F, Fletcher CD, Dal Cin P, et al. Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: a report of the CHAMP study group. CHromosomes And MorPhology. Genes Chromosomes Cancer. 1998;22:16-25. 12. Dal Cin P, Qian X, Cibas ES. The marriage of cytology and cytogenetics. Cancer Cytopathol. 2013;121:279-290. 13. Aleixo PB, Hartmann AA, Menezes IC, Meurer RT, Oliveira AM. Can MDM2 and CDK4 make the diagnosis of well differentiated/ dedifferentiated liposarcoma? An immunohistochemical study on 129 soft tissue tumours. J Clin Pathol. 2009;62:1127-1135. 14. Binh MB, Sastre-Garau X, Guillou L, et al. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing welldifferentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol. 2005;29:1340-1347. 15. Dei Tos AP, Doglioni C, Piccinin S, et al. Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours. J Pathol. 2000;190:531-536. 16. Italiano A, Bianchini L, Keslair F, et al. HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas whereas CDK4 belongs to a distinct inconsistent amplicon. Int J Cancer. 2008;122:2233-2241. 17. Italiano A, Bianchini L, Gjernes E, et al. Clinical and biological significance of CDK4 amplification in well-differentiated and dedifferentiated liposarcomas. Clin Cancer Res. 2009;15:5696-5703. 18. Nilbert M, Rydholm A, Mitelman F, Meltzer PS, Mandahl N. Characterization of the 12q13-15 amplicon in soft tissue tumors. Cancer Genet Cytogenet. 1995;83:32-36. 19. Persson F, Olofsson A, Sj€ ogren H, et al. Characterization of the 12q amplicons by high-resolution, oligonucleotide array CGH and expression analyses of a novel liposarcoma cell line. Cancer Lett. 2008;260:37-47. 20. Suster S, Morrison C. Sclerosing poorly differentiated liposarcoma: clinicopathological, immunohistochemical and molecular analysis of a distinct morphological subtype of lipomatous tumour of soft tissue. Histopathology. 2008;52:283-293. 21. Weaver J, Rao P, Goldblum JR, et al. Can MDM2 analytical tests performed on core needle biopsy be relied upon to diagnose welldifferentiated liposarcoma? Mod Pathol. 2010;23:1301-1306. 22. Rieker RJ, Joos S, Bartsch C, et al. Distinct chromosomal imbalances in pleomorphic and in high-grade dedifferentiated liposarcomas. Int J Cancer. 2002;99:68-73. 23. Fritz B, Schubert F, Wrobel G, et al. Microarray-based copy number and expression profiling in dedifferentiated and pleomorphic liposarcoma. Cancer Res. 2002;62:2993-2998. 24. Klijanienko J, Caillaud JM, Lagace R. Fine-needle aspiration in liposarcoma: cytohistologic correlative study including welldifferentiated, myxoid, and pleomorphic variants. Diagn Cytopathol. 2004;30:307-312. 25. Walaas L, Kindblom LG. Lipomatous tumors: a correlative cytologic and histologic study of 27 tumors examined by fine needle aspiration cytology. Hum Pathol. 1985;16:6-18. 26. Akerman M, Rydholm A. Aspiration cytology of lipomatous tumors: a 10-year experience at an orthopedic oncology center. Diagn Cytopathol. 1987;3:295-302. 27. Al-Maghraby HQ, Khalbuss WE, Rao UN, Cieply K, Dacic S, Monaco SE. Fine needle aspiration biopsy diagnosis of dedifferentiated liposarcoma: cytomorphology and MDM2 amplification by FISH. Cytojournal. 2010;7:5. 28. Liegl B, Kepten I, Le C, et al. Heterogeneity of kinase inhibitor resistance mechanisms in GIST. J Pathol. 2008;216:64-74. 29. Nemanqani D, Mourad WA. Cytomorphologic features of fineneedle aspiration of liposarcoma. Diagn Cytopathol. 1999;20:6769.
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30. Kapila K, Ghosal N, Gill SS, Verma K. Cytomorphology of lipomatous tumors of soft tissue. Acta Cytol. 2003;47:555-562. 31. Gonzalez-Campora R, Mu~ noz-Arias G, Otal-Salaverri C, et al. Fine needle aspiration cytology of primary soft tissue tumors. Morphologic analysis of the most frequent types. Acta Cytol. 1992;36: 905-917. 32. Nagira K, Yamamoto T, Akisue T, et al. Reliability of fine-needle aspiration biopsy in the initial diagnosis of soft-tissue lesions. Diagn Cytopathol 2002;27:354-361. 33. Einarsdottir H, Skoog L, S€oderlund V, Bauer HC. Accuracy of cytology for diagnosis of lipomatous tumors: comparison with magnetic resonance and computed tomography findings in 175 cases. Acta Radiol. 2004;45:840-846.
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34. Nikolaidis P, Silverman SG, Cibas ES, et al. Liposarcoma subtypes: identification with computed tomography and ultrasound-guided percutaneous needle biopsy. Eur Radiol. 2005;15:383-389. 35. Vicandi B, Jimenez-Heffernan J, Lopez-Ferrer P, GonzalezPeramato P, Viguer JM. Cytologic features of round cell liposarcoma: a report on five patients. Cancer. 2003;99:28-32. 36. Dodd LG. Update on liposarcoma: a review for cytopathologists. Diagn Cytopathol. 2012;40:1122-1131. 37. M€ uller CR, Paulsen EB, Noordhuis P, Pedeutour F, Saeter G, Myklebost O. Potential for treatment of liposarcomas with the MDM2 antagonist nutlin-3A. Int J Cancer. 2007;121:199-205. 38. Sioletic S, Dal Cin P, Fletcher CD, Hornick JL. Well-differentiated and dedifferentiated liposarcomas with prominent myxoid stroma: analysis of 56 cases. Histopathology 2013;62:287-293.
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Dedifferentiated Liposarcoma and Pleomorphic Liposarcoma A Comparative Study of Cytomorphology and MDM2/CDK4 Expression on Fine-Needle Aspiration n Marin ~ o-Enrıquez, MD; Jason L. Hornick, MD, PhD; Paola Dal Cin, PhD; Adria Edmund S. Cibas, MD; and Xiaohua Qian, MD, PhD
BACKGROUND: Dedifferentiated liposarcoma (DDLPS) and pleomorphic liposarcoma (PLPS) are distinct high-grade liposarcomas. DDLPS is a nonlipogenic sarcoma characterized by amplification of MDM2 and CDK4. PLPS is a high-grade sarcoma containing lipoblasts, characterized by a complex karyotype and a more aggressive clinical course. Rarely, DDLPS shows lipogenic differentiation, mimicking PLPS. The cytomorphologic features of DDLPS and PLPS and the utility of ancillary studies have not been systemically analyzed. METHODS: Cytologic preparations of 25 DDLPS and 13 PLPS, all histologically confirmed, were retrospectively reviewed along with clinical and cytogenetic data. Sample cellularity, vascular architecture, background material, predominant cell morphology, quality of the cytoplasm, and nuclear pleomorphism were compared for both tumor types. Immunohistochemistry for MDM2 and CDK4 was performed on cell blocks and/or core needle biopsies. RESULTS: Fine-needle aspirate smears from both DDLPS and PLPS were variably cellular, composed of cellular clusters and noncohesive cells. Abundant myxoid stroma was present in 25% of DDLPS and PLPS cases, whereas branching curvilinear vessels were more common in DDLPS than in PLPS (7 of 25 versus 2 of 13). Tumors were composed of predominantly spindled (18 of 25 DDLPS versus 3 of 13 PLPS) or epithelioid cells (7 of 25 DDLPS versus 6 of 13 PLPS). Pleomorphic cells were predominant in 3 PLPS, and were frequent in both (13 of 25 DDLPS versus 10 of 13 PLPS). The cytoplasm was mostly fibrillary and often vacuolated in both entities. Other features included necrosis, mitoses, and a prominent inflammatory infiltrate. The main cytologic differences were the presence of marked pleomorphism, abundant lipoblasts, and cells with microvacuolated cytoplasm in most PLPS. A total of 24 (96%) and 20 (80%) cases of DDLPS expressed MDM2 and CDK4, respectively, whereas none of the PLPS expressed both markers. Six DDLPS tested showed ring or giant marker chromosomes and/or MDM2 amplification by fluorescence in situ hybridization; 2 PLPS had complex karyotypes. CONCLUSIONS: DDLPS and PLPS exhibit variable and occasionally overlapping cytologic features. The presence of lipoblasts, cells with microvacuolated cytoplasm, and marked pleomorphism are more suggestive of PLPS, but these characteristics can be present in DDLPS. Coexpression of MDM2 and CDK4 distinguishes DDLPS from C 2013 American Cancer Society. PLPS. Cancer (Cancer Cytopathol) 2014;122:128-37. V
KEY WORDS: sarcoma; fine-needle aspirate; dedifferentiated liposarcoma; pleomorphic liposarcoma; MDM2; CDK4; lipoblast; cytology; adipocytic; soft tissue.
Corresponding author: Xiaohua Qian, MD, PhD, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115; Fax: (617) 582-6015; [email protected] Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts Presented in part at the 102nd Annual Meeting of the United States and Canadian Academy of Pathology (USCAP), Baltimore, MD; March 2013. The authors thank Mei Zheng for outstanding technical support and Nick Hoover for his early work on the project. Received: August 9, 2013; Revised: September 14, 2013; Accepted: September 16, 2013 Published online November 12, 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21362, wileyonlinelibrary.com
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~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
INTRODUCTION Liposarcomas are a heterogeneous group of malignant adipocytic neoplasms that consist of 3 distinct clinicopathological entities: well-differentiated/dedifferentiated liposarcoma, myxoid liposarcoma, and pleomorphic liposarcoma (PLPS). Collectively, liposarcomas account for approximately 20% of all adult sarcomas.1 Dedifferentiated liposarcoma (DDLPS) results from the progression of a well-differentiated liposarcoma (WDLPS) to a nonlipogenic sarcoma of variable histologic grades and morphologic patterns that acquires metastatic potential.2,3 Dedifferentiated areas in DDLPS have the appearance of a high-grade sarcoma in approximately 90% of cases, and show a variety of growth patterns including, most frequently, nondistinct spindle cell and pleomorphic patterns, and less commonly, inflammatory, giant cell, round cell, or meningothelial-like patterns. The remaining 10% of DDLPS are morphologically low grade, characterized by uniform spindle cells with mild nuclear atypia, often organized in a fascicular pattern and exhibiting lower cellularity than the usual high-grade areas.3 DDLPS exhibits heterologous differentiation in approximately 5% to 10% of cases, which does not affect the clinical outcome.4 Adipocytic differentiation within the high-grade component of DDLPS, traditionally excluded by definition, has been recently recognized and designated “homologous” lipoblastic differentiation.5,6 PLPS is rare, representing 5% of liposarcomas.7 Morphologically, PLPS is a high-grade pleomorphic sarcoma with varying numbers of multivacuolated lipoblasts.4,7–9 Because of its wide morphologic spectrum, PLPS can be confused with numerous adipocytic and nonadipocytic neoplasms, including DDLPS and other high-grade pleomorphic sarcomas. PLPS pursues a consistently more aggressive clinical course than DDLPS.7–9 Cytogenetic techniques such as karyotyping, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) have unraveled certain aspects of the biology of liposarcomas,10,11 prompting the development of new diagnostic tests that are now available for routine clinical practice.12 Given its high sensitivity and specificity, the combined detection of MDM2 (mouse double minute 2) and CDK4 (cyclin-dependent kinase 4) overexpression by immunohistochemistry has become a particularly useful ancillary technique to support the diagnosis of WDLPS/DDLPS on surgical resecCancer Cytopathology
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tion samples.13–15 The molecular basis for the overexpression of these cell cycle–related proteins is the presence of 12q13-q15 amplicons in supernumerary ring and/or giant markers chromosomes with constant amplification of MDM2 and HMGA2, and frequent amplification of CDK4, that play an essential role in the pathogenesis of WDLPS/DDLPS.16–20 A recent study demonstrated that detection of MDM2 amplification by FISH is more sensitive and specific than MDM2 immunohistochemistry to differentiate WDLPS from various benign lipomatous tumors on core needle biopsy.21 The utility of these ancillary studies in diagnosing high-grade liposarcomas on small-needle biopsy has not yet been systematically evaluated. In contrast to DDLPS, the molecular features of PLPS are still poorly understood. At the cytogenetic level, PLPS is characterized by a complex karyotype with multiple chromosomal gains and losses that do not typically include the 12q13-q15 region.22 The resulting gene expression profiles of PLPS differ significantly from those of WDLPS/DDLPS.23 These features reflect the generally anaplastic nature of pleomorphic sarcomas and place PLPS nosologically closer to other high-grade nonlipogenic sarcomas, such as conventional osteosarcoma, high-grade leiomyosarcoma, or chondrosarcoma, than to DDLPS.9,22 In some situations, the morphology of DDLPS and PLPS is remarkably similar, and inadequate sampling can lead to misdiagnosis. In particular, DDLPS with “homologous” lipoblastic differentiation is indistinguishable from PLPS on morphologic grounds. The clinical and biological differences between these 2 entities facilitates the differential diagnosis: a previous diagnosis of WDLPS or the presence of WDLPS areas contiguous to or admixed with a high-grade sarcoma should prompt the diagnosis of DDLPS; conversely, lipoblastic differentiation in a high-grade pleomorphic sarcoma favors the diagnosis of PLPS, but the status of MDM2 and CDK4 should be determined by immunohistochemistry or FISH, to rule out a rare case of DDLPS with homologous lipoblastic differentiation.5,6 The cytologic descriptions of DDLPS and PLPS in the literature are included in single case reports or heterogeneous series. Whereas most reported DDLPS cases were recognized in light of a concurrent or prior history of WDLPS, in approximately half of the cases of PLPS the authors initially favored other diagnostic possibilities, including poorly differentiated carcinoma and high-grade 129
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sarcoma without specifying the line of differentiation.24–27 In this study, we analyze the cytologic aspirates of 38 histologically confirmed high-grade liposarcomas, including 25 DDLPS and 13 PLPS, correlating the cytomorphology with clinical, histologic, immunohistochemical, and genetic data.
MATERIALS AND METHODS Histologically confirmed cases of DDLPS and PLPS diagnosed between 1990 and 2012, with both cytologic and histologic materials available for review, were retrieved from consultation and pathology files of the Department of Pathology of the Brigham and Women’s Hospital, Boston, Massachusetts. There were 25 DDLPS and 13 PLPS with at least one satisfactory fine-needle aspirate (FNA) and corresponding histologic preparations, either core biopsies or surgical resections. These 38 paired samples corresponded to 35 patients: repeat matching cytologic and histologic samples were obtained at 2 different time points for 3 patients (1 DDLPS, 2 PLPS) and were analyzed as independent cases for the purposes of this study. FNAs consisted of cytologic smears (n 5 30) and/or cell block preparations (n 5 32). Air-dried smears were stained with Hemacolor (Harleco, EM Science, Gibbstown, NJ); alcohol-fixed slides were fixed in 95% ethanol and stained with a modified Papanicolaou stain. Cell block pellets were prepared from the centrifuged needlerinse specimens using the plasma–thrombin method whereby the pellet is congealed with several drops each of plasma and thrombin. The resulting clot was fixed in formalin, embedded in paraffin, and processed for histologic sections. For histologic evaluation, 4-lm-thick hematoxylin-and-eosin–stained sections were examined. The following cytomorphologic features were evaluated: cellularity, vascular architecture, cell morphology, quality of the cytoplasm, nuclear pleomorphism, background material, presence of lipoblasts, and multinucleate giant cells. The cell morphology of each sample was classified as epithelioid, spindled, or pleomorphic based on the predominant cell type (> 50%). Clinical information was obtained from the electronic medical record and the pathology reports. Immunohistochemistry was performed on 4-lmthick formalin-fixed paraffin-embedded tissue sections (n 5 32) and/or cell blocks (n 5 25) using pressure cooker heat-induced epitope retrieval (0.01 M citrate buffer, pH 130
6.0) and antibodies against MDM2 (Calbiochem, San Diego, Calif; clone 1F2; dilution 1:50) and CDK4 (Biosource/Invitrogen, Carlsbad, Calif; clone DCS-31; dilution 1:250). The Envision Plus detection system (Dako, Carpinteria, Calif) was used. Appropriate positive and negative controls were used throughout. Concordance between cytologic and histologic preparations was analyzed when both type of materials were available for staining (n 5 19). Karyotypes were retrospectively reviewed when available (n 5 6). FISH analysis was performed on 4 selected DDLPS cases on 4-lm sections from paraffin blocks using a commercially available MDM2 probe (LPS 016-A; Cytocell, Cambridge, UK) following standard protocols in our laboratory.28 The study was approved by the Institutional Review Board at Brigham and Women’s Hospital.
RESULTS Clinical Information
Clinical data are summarized in Figure 1 and Table 1. Seventeen DDLPS patients were male and 8 were female, ranging in age from 43 to 81 years (median, 67 years). Tumors occurred most commonly in the retroperitoneum (12 patients, 48%); other deep locations included intra-
FIGURE 1. Anatomic distribution of 25 dedifferentiated liposarcomas (LPS) and 13 pleomorphic LPS. White squares represent primary tumors, white circles represent tumor recurrences, and black circles represent metastases.
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Table 1. Clinical, Cytologic, and Immunohistochemical Features of 25 Dedifferentiated Liposarcomas (DDLPS) and 13 Pleomorphic Liposarcomas (PLPS).
Case ID
Age
Sex
Site (Side)a
Type of Lesionb
DDLPS-1 DDLPS-2 DDLPS-3 DDLPS-4 DDLPS-5 DDLPS-6 DDLPS-7 DDLPS-8 DDLPS-9 DDLPS-10 DDLPS-11 DDLPS-12 DDLPS-13 DDLPS-14 DDLPS-15 DDLPS-16 DDLPS-17 DDLPS-18 DDLPS-19 DDLPS-20 DDLPS-21 DDLPS-22 DDLPS-23 DDLPS-24 DDLPS-25 PLPS-1 PLPS-2 PLPS-3 PLPS-4 PLPS-5 PLPS-6 PLPS-7 PLPS-8 PLPS-9 PLPS-10 PLPS-11 PLPS-12 PLPS-13
43 74 74 67 70 63 60 66 64 64 62 73 68 51 81 81 59 59 44 67 70 77 68 79 59 41 32 65 65 51 22 80 45 71 59 59 53 53
F M M M M M F M F F F M F M M M M M F M M M F M M M M M M M F M M M M M F F
RP RP RP RP RP Intra-abdominal Liver Chest wall Intra-abdominal RP RP Intra-abdominal Thigh (R) Liver RP RP Thigh (L) Liver Thigh (R) RP Lung RP Lung RP Chest wall Pancreas Liver Calf (R) Mediastinal Thigh (L) Mediastinal Arm (R) Thigh (L) Pancreas Calf (L) Thigh (L) Humerus (L) Knee (R)
Rec Rec Rec Rec Rec Rec Met Met Rec Rec Met Rec Rec Met Rec Rec P Met P Rec Met P Met Rec Rec Met Met P Met Rec P P Met P P Rec P P
Lipoblasts
Vacuolated cells
IHC Cytology MDM2/ CDK4c
IHC Surgical MDM2/ CDK4c
N N N N N N N N N N N N N N N N N N N N N N N Y Y N Y Y Y Y N Y Y Y Y N Y Y
N N N Y N Y N N Y N N N Y N N Y N N N Y Y Y N Y Y N Y Y Y Y Y Y Y Y Y Y Y Y
1/1 NA 1/– 1/– 1/– 1/1 1/1 NA 1/1 NA NA NA 1/1 1/1 1/NA 1/1 –/– 1/1 1/1 NA NA 1/1 NA 1/1 1/1 –/– NA NA NA –/– NA –/– –/– –/– –/weak NA –/– –/–
NA 1/1 1/– NA 1/– 1/1 1/1 1/1 1/1 NA 1/1 1/1 1/1 1/1 1/1 1/1 –/– 1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1 NA –/– –/– –/– –/– –/– –/– –/– NA NA –/– –/– –/–
a
L, left; R: Right; RP, retroperitoneum. P, primary; Rec, recurrence; Met, metastasis. c IHC, immunohistochemistry; (1), positive; (–), negative; NA, not available. b
abdominal soft tissue (3 patients), liver (3 patients), proximal lower extremity (3 patients), lung (2 patients), and chest wall (2 patients). Three lesions were primary tumors, 15 were recurrences, and 7 were metastases. A total of 17 patients had a previous diagnosis of WDLPS (16 patients) or DDLPS (1 patient). In 22 tumors, the diagnosis of DDLPS was suggested on FNA alone, including 17 cases with a prior history of WDLPS/DDLPS, and 5 initial diagnoses; the 3 remaining cases were diagnosed in conjunction with the concurrent core needle biopsy. Ten PLPS patients were male and 3 were female, ranging in age from 22 to 80 years (median, 56 years). PLPS involved the deep soft tissue of the extremities (8 patients), mediastinum (2 patients), pancreas (2 patients), Cancer Cytopathology
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and liver (1 patient). Six lesions were primary tumors, 2 were recurrences, and 5 were metastases. Eight of these tumors were diagnosed on FNA alone, including 7 cases with a known history of PLPS and 1 initial diagnosis; in 5 cases, a diagnosis of pleomorphic malignancy was rendered on FNA, and further classification was made on the concurrent core needle biopsy. Cytologic Features of DDLPS
FNA smears of DDLPS were variably cellular, composed of both ill-defined cellular clusters and variable amounts of noncohesive cells. The cellular clusters were mainly syncytial-like (Fig. 2a) and, occasionally, loosely cohesive cell aggregates. Abundant myxoid stroma in the 131
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FIGURE 2. Cytomorphologic features of dedifferentiated liposarcomas (DDLPS) (a,c-h: Hemacolor stain; b,i: Papanicolaou stain). (a) Smears were composed of syncytial-like cellular clusters or loosely cohesive aggregates. (b) Six DDLPS showed myxoid stromal fragments and (c) 7 showed prominent curvilinear branching vessels. Highly atypical pleomorphic cells (d) were usually scant but occasionally abundant. The cytomorphology was (d) predominantly spindled in 18 cases and (e) epithelioid, polygonal, or plasmacytoid cells in 7 cases. (f, g, h) Cells with microvacuolated cytoplasm were present in 43% of DDLPS. (i) Variable numbers of lipoblasts with multivacuolated cytoplasm and hyperchromatic scalloped nuclei were observed in 2 DDLPS with “homologous” lipoblastic differentiation.
background was seen in 6 cases (Fig. 2b), and prominent curvilinear branching blood vessels in 7 cases (Fig. 2c). Most tumors showed a combination of different cell morphologies with predominantly spindle cells in 18 cases (Fig. 2d) and epithelioid, polygonal, or plasmacytoid cells in 7 cases (Fig. 2e). Isolated highly atypical pleomorphic cells with bizarre, hyperchromatic nuclei, irregular nuclear contours, and prominent nucleoli were observed in most cases and were particularly numerous in 13 (54%) cases (Fig. 2f). Multinucleated giant cells were present in 4 cases (17%). Cytoplasmic microvacuoles or a “bubbly” cytoplasm was noted in 10 cases (40%), usually in epithelioid or pleomorphic cells (Fig. 2f,g,h). Scattered lipoblasts with single or multiple sharply circumscribed cytoplasmic vacuoles and scalloped hyperchromatic nuclei were pres132
ent in 2 cases of DDLPS (8%) (Fig. 2i). On histologic examination, these 2 cases corresponded to DDLPS with high-grade spindle cell morphology and “homologous” lipoblastic differentiation, showing numerous large multivacuolated lipoblasts with scalloped hyperchromatic nuclei within the high-grade component. Necrosis, mitoses, and a prominent inflammatory infiltrate were present in 6, 3, and 5 cases, respectively. Cytologic Features of PLPS
FNA smears of PLPS were variably cellular, composed of numerous irregular cellular clusters (Fig. 3a,b), and abundant isolated, noncohesive cells. In 3 cases, there was an abundant myxoid stroma. 2 cases showed prominent curvilinear branching blood vessels (Fig. 3c). In 12 cases of Cancer Cytopathology
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FIGURE 3. Cytomorphologic features of pleomorphic liposarcomas (PLPS) (a: Papanicolaou stain; b-j: Hemacolor stain). (a) Cellular, tight clusters of cohesive cells are characteristic, often (b) combined with irregular cellular aggregates and abundant isolated, noncohesive cells. (c) Epithelioid cytomorphology was predominant in half of the cases, and branching thin vessels were uncommon. (d-h) Twelve PLPS showed cells with microvacuolated cytoplasm. (f-h) Variable numbers of lipoblasts (often numerous) with multivacuolated cytoplasm and hyperchromatic scalloped nuclei were identified in 10 PLPS. (i) Highly atypical pleomorphic cells were abundant in 10 cases. (j) Multinucleated giant cells were present in 6 cases.
PLPS (92%), there were abundant cells featuring microvacuolated cytoplasm with a “bubbly” apperance, which is best appreciated on air-dried, Hemacolor-stained smears (Fig. 3c-h). Lipoblasts were identified in 10 cases (77%) (Fig. 3c,d,f-h). A total of 10 samples also showed numerous pleomorphic cells with abundant cytoplasm and bizarre nuclei (Fig. 3i). The nonpleomorphic tumor cells were predominantly epithelioid in 6 cases (Fig. 3c) and spindled in 3. Multinucleated giant cells were present in 6 cases (46%) (Fig. 3j). Necrosis was present in 5 cases; mitoses and a prominent inflammatory infiltrate each were seen in 3 cases. The main cytomorphologic features of DDLPS and PLPS are summarized and compared in Figure 4. Cancer Cytopathology
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FIGURE 4. Comparative frequency of major cytomorphologic features present in 25 dedifferentiated liposarcomas (DDLPS) and 13 pleomorphic liposarcoma (PLPS) fine-needle aspirate smears.
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FIGURE 5. Immunohistochemical expression of MDM2 and CDK4 in dedifferentiated liposarcomas (DDLPS), present in 24 (96%) and 20 (80%) cases, respectively.
Immunohistochemistry, Cytogenetics, and FISH
The immunohistochemical findings of DDLPS and PLPS are summarized in Table 1. A total of 24 DDLPS cases expressed MDM2 (96%) and 20 expressed CDK4 (80%) by immunohistochemistry on cell block or tissue sections (Fig. 5). A single case (DDLPS-17) was negative for both markers. Karyotyping detected the presence of ring and/ or giant marker chromosomes in all 4 cases analyzed. FISH showed high-level amplification of the MDM2 locus in 2 of the karyotyped cases, and in 2 additional DDLPS, including case DDLPS-17 which was negative for MDM2 and CDK4 by immunohistochemistry. Both cases of DDLPS with “homologous” lipoblastic differentiation showed MDM2 amplification by FISH. In contrast, all PLPS cases were negative for MDM2 and CDK4 by immunohistochemistry, except for 1 case that was weakly positive for CDK4 (7%). Karyotyping showed a complex karyotype with multiple numeric and structural aberrations in the 2 PLPS cases analyzed. There was 100% agreement between the results of the immunohistochemical stains performed on cytologic and surgical materials in the 19 cases that were evaluated.
DISCUSSION Some aspects of liposarcoma cytopathology have been discussed in previous reports, mostly in the context of benign lipomatous lesions25,26,29,30 or other soft tissue sarcomas.31,32 In particular, 1) the differential diagnosis of 134
well-differentiated liposarcoma versus benign adipocytic tumors,25,26,29,30 including radiologic correlates,33,34 and 2) the morphologic features of myxoid liposarcoma and its high-grade counterpart, known as round cell liposarcoma, have been reasonably well defined.24,25,29,35 Few studies, however, have analyzed the cytomorphology of DDLPS and PLPS, resulting in limited cytologic descriptions available, as recently reviewed by Dodd.36 We focused on characterizing the cytomorphologic features of these 2 biologically distinct groups of highgrade liposarcoma, DDLPS and PLPS, in a series of 25 and 13 cases, respectively. To distinguish them is relevant, given the differences in clinical management and prognosis,4 and the increasing availability of targeted therapies only effective in tumors with specific molecular alterations, such as MDM2 inhibitors for DDLPS.37 Despite notable biological and clinical differences, DDLPS and PLPS may exhibit very similar cytomorphology. Both share features of a high-grade sarcoma, yielding moderateto-highly cellular smears showing cellular and nuclear pleomorphism, frequent necrosis with associated inflammation, and high mitotic counts, including occasional atypical mitoses. Highly atypical cells with bizarre nuclei and multinucleated giant cells, although more commonly seen in PLPS, were also present in DDLPS. Traditionally, the identification of lipoblasts in a smear showing features of high-grade pleomorphic sarcoma was considered sufficient to diagnose PLPS.24,25,29,35 The recent description of “homologous” lipoblastic differentiation in DDLPS, however, has complicated the interpretation of lipoblasts in this context. Therefore, the differential diagnosis between DDLPS and PLPS should always be entertained when encountering a high-grade sarcoma with lipoblasts. That being said, the presence of lipoblasts is far more frequently associated with PLPS than DDLPS (77% versus 8% in this study; Fig. 4). In addition, lipoblasts in PLPS are often numerous and strikingly atypical, with bizarre nuclei and irregular nuclear membranes; some are actually multinucleated giant cells. Although nonspecific, other morphologic clues that may guide the differential diagnosis are the predominant cell morphology, the proportion of highly atypical or bizarre cells, and the degree of pleomorphism. In DDLPS, we have found, there are usually few bizarre cells, scattered among other tumor cells, which are often spindled; in PLPS, bizarre atypical tumor cells are more abundant, admixed with tumor cells that are more often epithelioid. Also, the degree of cellular Cancer Cytopathology
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~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
pleomorphism is (unsurprisingly) greater in PLPS than in DDLPS, seen in both lipoblasts and nonlipogenic tumor cells. In addition, correlation with clinical and radiologic data is essential. In particular, history of a previously documented well-differentiated liposarcoma, suspicious-looking fatty areas on imaging studies, and a retroperitoneal location are highly suggestive of DDLPS; location in the extremities is more common in PLPS (Fig. 1). An interesting finding in this study is the presence of atypical cells with abundant microvacuolated cytoplasm in 40% of DDLPS and 92% of PLPS smears. As shown in Figs. 2f-h and Figs. 3e-h, these cells do not satisfy the morphologic definition of a lipoblast, which requires that hyperchromatic nuclei be indented by cytoplasmic vacuoles (frequently described as “scalloped nuclei”). Furthermore, the microvacuolization of the cytoplasm consists of innumerable minute vacuoles, conferring the appearance of a “bubbly cytoplasm,” which contrasts with the less numerous and usually larger cytoplasmic vacuoles in unequivocal lipoblasts. Cells with similar cytoplasmic vacuoles have been described in hibernoma,24,25 highgrade myxoid liposarcoma35 (formerly designated round cell liposarcoma), and in a case of so-called malignant fibrous histiocytoma.31 These peculiar microvacuolated cells have not been associated previously with DDLPS or PLPS in the cytology literature. In hibernoma, the main criteria proposed to differentiate them from lipoblasts are the round, nonscalloped nuclei and the uniformity of the small-sized cytoplasmic vacuoles.24,25 In high-grade myxoid liposarcoma, these microvacuolated cells constitute a monomorphic population of noncohesive atypical cells (see below).35 In the context of DDLPS and PLPS, we observed a pleomorphic population of microvacuolated atypical cells, with hyperchromatic nuclei of variable sizes and shapes. The fact that these cells are present in all 3 types of high-grade liposarcoma suggests that they may be related to true lipoblasts and raises the possibility of a previously unrecognized subtle manifestation of adipocytic differentiation in cytology specimens. Regardless of their nature, a pleomorphic population of atypical microvacuolated cells is common in DDLPS and very common in PLPS, and hence may be a helpful diagnostic clue to classify a highgrade sarcoma of uncertain differentiation. Immunohistochemistry for MDM2 and CDK4 is very useful for diagnosis: most DDLPS coexpress these 2 markers that reflect their genetic make-up, whereas virtually all PLPS are negative for both.13–15 In the present Cancer Cytopathology
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series, we observed strong and diffuse expression of MDM2 and/or CDK4 in all but one DDLPS cases. The exception (DDLPS-17) was a 19-cm primary DDLPS arising in the thigh of a 59-year-old male, which was repeatedly negative for MDM2 and CDK4 by immunohistochemistry in the cytology specimens and core biopsy. We confirmed the diagnosis of DDLPS by FISH, which demonstrated high-level amplification of the MDM2 locus. In addition, on the subsequent surgical excision the tumor showed small areas of WDLPS. All the PLPS cases were negative for MDM2 by immunohistochemistry, and only one case showed weak CDK4 expression. We observed absolute correlation between the cell block preparations and the core biopsy and/or surgical resection materials. These results underscore the diagnostic value of detecting MDM2 and CDK4 expression for the differential diagnosis of DDLPS versus PLPS on FNA cytology. Discussions regarding the sensitivity and specificity of FNA for the diagnosis of DDLPS and PLPS in the context of a broader differential diagnosis are beyond the scope of this study. We would like to mention, however, that some of the cytomorphologic findings in our series highlight an obvious potential overlap with myxoid liposarcoma: smears from 6 DDLPS and 3 PLPS showed a prominent myxoid background with rich vascularization (Figs. 2c and 3b), often with curvilinear arborizing vessels similar to those observed in myxoid liposarcoma.24,35 A similar phenomenon has been recently documented in surgical specimens.38 A very illustrative case in this regard is DDLPS-25, one of the DDLPS cases with lipoblastic differentiation, which showed large myxoid stromal fragments with prominent branching thin-walled vessels, and relatively small lipoblasts located at the vessel bifurcations (Fig. 2c). In our experience, however, the cytomorphology of myxoid liposarcoma is characteristic and quite different from that of DDLPS or PLPS: in low-grade cases, smears are dominated by abundant fragments of vascularized myxoid stroma and relatively low cellularity, consisting of uniform, bland cells displaying only subtle atypia; high-grade cases show uniform round cell cytomorphology with occasional uni- or bi-vacuolated lipoblasts. Typically, myxoid liposarcoma can be ruled out based on the presence of marked cellular pleomorphism typically seen in PLPS and DDLPS. In problematic cases, immunohistochemical detection of MDM2 and CDK4 and/or molecular/genetic analyses are useful ancillary techniques. 135
Original Article
In summary, we have characterized and compared the cytomorphologic features of DDLPS and PLPS in 38 FNA samples with correlation of immunoprofile and cytogenetic data. Atypical cells with abundant microvacuolated cytoplasm and/or lipoblasts are more frequently present in PLPS, but can also be seen in DDLPS; therefore, they do not necessarily discriminate between these 2 distinct high-grade liposarcomas. The presence of marked pleomorphism, combined with highly atypical lipoblasts, favors the diagnosis of PLPS. The detection of MDM2 and CDK4 expression by immunohistochemistry has great value in establishing the diagnosis of DDLPS, which is applicable to FNA cytology. FUNDING SOURCES No specific funding was disclosed.
CONFLICT OF INTEREST DISCLOSURE Dr. Mari~ no-Enrıquez is supported by a Career Development Award from the Sarcoma Alliance for Research through Collaboration (SARC). All other authors made no disclosure.
REFERENCES Fletcher CDM. Diagnostic Histopathology of Tumors. 4th ed. Philadelphia, PA: Saunders, Elsevier; 2013. 2. McCormick D, Mentzel T, Beham A, Fletcher CD. Dedifferentiated liposarcoma. Clinicopathologic analysis of 32 cases suggesting a better prognostic subgroup among pleomorphic sarcomas. Am J Surg Pathol. 1994;18:1213-1223. 3. Henricks WH, Chu YC, Goldblum JR, Weiss SW. Dedifferentiated liposarcoma: a clinicopathological analysis of 155 cases with a proposal for an expanded definition of dedifferentiation. Am J Surg Pathol. 1997;21:271-281. 4. Fletcher CDM, Bridge JA, Hogendoorn PCW, Mertens F. WHO Classification of Tumours of Soft Tissue and Bone. Lyon, France: IARC; 2013. 5. Marino-Enriquez A, Fletcher CD, Dal Cin P, Hornick JL. 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;34:1122-1131. 6. Boland JM, Weiss SW, Oliveira AM, Erickson-Johnson ML, Folpe AL. Liposarcomas with mixed well-differentiated and pleomorphic features: a clinicopathologic study of 12 cases. Am J Surg Pathol. 2010;34:837-843. 7. Hornick JL, Bosenberg MW, Mentzel T, McMenamin ME, Oliveira AM, Fletcher CD. Pleomorphic liposarcoma: clinicopathologic analysis of 57 cases. Am J Surg Pathol. 2004;28:1257-1267. 8. Gebhard S, Coindre JM, Michels JJ, et al. Pleomorphic liposarcoma: clinicopathologic, immunohistochemical, and follow-up analysis of 63 cases: a study from the French Federation of Cancer Centers Sarcoma Group. Am J Surg Pathol. 2002;26:601-616. 9. Downes KA, Goldblum JR, Montgomery EA, Fisher C. Pleomorphic liposarcoma: a clinicopathologic analysis of 19 cases. Mod Pathol. 2001;14:179-184. 10. Coindre JM, Pedeutour F, Aurias A. Well-differentiated and dedifferentiated liposarcomas. Virchows Arch. 2010;456:167-179. 1.
136
11. Mertens F, Fletcher CD, Dal Cin P, et al. Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features: a report of the CHAMP study group. CHromosomes And MorPhology. Genes Chromosomes Cancer. 1998;22:16-25. 12. Dal Cin P, Qian X, Cibas ES. The marriage of cytology and cytogenetics. Cancer Cytopathol. 2013;121:279-290. 13. Aleixo PB, Hartmann AA, Menezes IC, Meurer RT, Oliveira AM. Can MDM2 and CDK4 make the diagnosis of well differentiated/ dedifferentiated liposarcoma? An immunohistochemical study on 129 soft tissue tumours. J Clin Pathol. 2009;62:1127-1135. 14. Binh MB, Sastre-Garau X, Guillou L, et al. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing welldifferentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol. 2005;29:1340-1347. 15. Dei Tos AP, Doglioni C, Piccinin S, et al. Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours. J Pathol. 2000;190:531-536. 16. Italiano A, Bianchini L, Keslair F, et al. HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas whereas CDK4 belongs to a distinct inconsistent amplicon. Int J Cancer. 2008;122:2233-2241. 17. Italiano A, Bianchini L, Gjernes E, et al. Clinical and biological significance of CDK4 amplification in well-differentiated and dedifferentiated liposarcomas. Clin Cancer Res. 2009;15:5696-5703. 18. Nilbert M, Rydholm A, Mitelman F, Meltzer PS, Mandahl N. Characterization of the 12q13-15 amplicon in soft tissue tumors. Cancer Genet Cytogenet. 1995;83:32-36. 19. Persson F, Olofsson A, Sj€ ogren H, et al. Characterization of the 12q amplicons by high-resolution, oligonucleotide array CGH and expression analyses of a novel liposarcoma cell line. Cancer Lett. 2008;260:37-47. 20. Suster S, Morrison C. Sclerosing poorly differentiated liposarcoma: clinicopathological, immunohistochemical and molecular analysis of a distinct morphological subtype of lipomatous tumour of soft tissue. Histopathology. 2008;52:283-293. 21. Weaver J, Rao P, Goldblum JR, et al. Can MDM2 analytical tests performed on core needle biopsy be relied upon to diagnose welldifferentiated liposarcoma? Mod Pathol. 2010;23:1301-1306. 22. Rieker RJ, Joos S, Bartsch C, et al. Distinct chromosomal imbalances in pleomorphic and in high-grade dedifferentiated liposarcomas. Int J Cancer. 2002;99:68-73. 23. Fritz B, Schubert F, Wrobel G, et al. Microarray-based copy number and expression profiling in dedifferentiated and pleomorphic liposarcoma. Cancer Res. 2002;62:2993-2998. 24. Klijanienko J, Caillaud JM, Lagace R. Fine-needle aspiration in liposarcoma: cytohistologic correlative study including welldifferentiated, myxoid, and pleomorphic variants. Diagn Cytopathol. 2004;30:307-312. 25. Walaas L, Kindblom LG. Lipomatous tumors: a correlative cytologic and histologic study of 27 tumors examined by fine needle aspiration cytology. Hum Pathol. 1985;16:6-18. 26. Akerman M, Rydholm A. Aspiration cytology of lipomatous tumors: a 10-year experience at an orthopedic oncology center. Diagn Cytopathol. 1987;3:295-302. 27. Al-Maghraby HQ, Khalbuss WE, Rao UN, Cieply K, Dacic S, Monaco SE. Fine needle aspiration biopsy diagnosis of dedifferentiated liposarcoma: cytomorphology and MDM2 amplification by FISH. Cytojournal. 2010;7:5. 28. Liegl B, Kepten I, Le C, et al. Heterogeneity of kinase inhibitor resistance mechanisms in GIST. J Pathol. 2008;216:64-74. 29. Nemanqani D, Mourad WA. Cytomorphologic features of fineneedle aspiration of liposarcoma. Diagn Cytopathol. 1999;20:6769.
Cancer Cytopathology
February 2014
~ o-Enrıquez et al FNA Cytomorphology of DDLPS and PLPS/Marin
30. Kapila K, Ghosal N, Gill SS, Verma K. Cytomorphology of lipomatous tumors of soft tissue. Acta Cytol. 2003;47:555-562. 31. Gonzalez-Campora R, Mu~ noz-Arias G, Otal-Salaverri C, et al. Fine needle aspiration cytology of primary soft tissue tumors. Morphologic analysis of the most frequent types. Acta Cytol. 1992;36: 905-917. 32. Nagira K, Yamamoto T, Akisue T, et al. Reliability of fine-needle aspiration biopsy in the initial diagnosis of soft-tissue lesions. Diagn Cytopathol 2002;27:354-361. 33. Einarsdottir H, Skoog L, S€oderlund V, Bauer HC. Accuracy of cytology for diagnosis of lipomatous tumors: comparison with magnetic resonance and computed tomography findings in 175 cases. Acta Radiol. 2004;45:840-846.
Cancer Cytopathology
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34. Nikolaidis P, Silverman SG, Cibas ES, et al. Liposarcoma subtypes: identification with computed tomography and ultrasound-guided percutaneous needle biopsy. Eur Radiol. 2005;15:383-389. 35. Vicandi B, Jimenez-Heffernan J, Lopez-Ferrer P, GonzalezPeramato P, Viguer JM. Cytologic features of round cell liposarcoma: a report on five patients. Cancer. 2003;99:28-32. 36. Dodd LG. Update on liposarcoma: a review for cytopathologists. Diagn Cytopathol. 2012;40:1122-1131. 37. M€ uller CR, Paulsen EB, Noordhuis P, Pedeutour F, Saeter G, Myklebost O. Potential for treatment of liposarcomas with the MDM2 antagonist nutlin-3A. Int J Cancer. 2007;121:199-205. 38. Sioletic S, Dal Cin P, Fletcher CD, Hornick JL. Well-differentiated and dedifferentiated liposarcomas with prominent myxoid stroma: analysis of 56 cases. Histopathology 2013;62:287-293.
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