Differential diagnosis of lipoma and atypical lipomatous tumor/well‐differentiated liposarcoma by cytological analysis

1 INTRODUCTION

Adipocytic tumors are the most common soft tissue tumors and mostly comprise lipomas and atypical lipomatous tumor/well-differentiated liposarcomas (ALT/WDL). ALT and WDL primarily differ on the anatomical location and resectability of the tumor. Tumors that occur in the limbs or trunk and can be resected are called ALT, while those that occur in the retroperitoneum or mediastinum and must be resected at the margins are called WDLs. The fifth edition of the WHO classification classifies ALT and WDL into the same category.1

ALT/WDL can become highly malignant by dedifferentiation or recurrence, thereby making it important to differentiate them from lipomas, which are benign tumors, for appropriate treatment and determination of the follow-up period after tumor resection.2 The genomic abnormalities described in dedifferentiated liposarcoma support the fact that this tumor corresponds to a malignant adipocytic tumor showing progression from ALT/WDL to non-lipogenic sarcoma of variable aspect and grade.3 The mouse double minute 2 homolog (MDM2) protein suppresses TP53, a tumor suppressor gene. Fluorescence in situ hybridization (FISH) analysis of formalin-fixed paraffin-embedded (FFPE) tissues has revealed MDM2 amplification in ALT/WDL and is currently used to differentiate these tumors.4-6 With the development of new molecular tests showing high diagnostic specificity, fine-needle aspiration cytology (FNAC) has gained acceptance for the preoperative assessment of soft tissue tumors.7 FNAC represents a versatile, low-cost, well-tolerated diagnostic strategy with advantages over histological biopsies.8 However, a detailed comparison of the cytological findings of lipoma and ALT/WDL with MDM2 amplification has not yet been reported. In this study, we compare the cytological features of lipoma and ALT/WDL and use cytological findings for differential diagnosis. We also performed FISH to examine MDM2 amplification in cytological specimens and evaluate the usefulness of cytology for the differential diagnosis of adipocytic tumors.

2 MATERIALS AND METHODS

We reviewed the clinical and histological data of 20 patients with lipoma and ALT/WDL who had undergone resection at Kanagawa Cancer Center between 2018 and 2020. One ALT/WDL case showed partially dedifferentiated areas. We evaluated age, sex, and maximum tumor diameter as clinical parameters. Tissue samples (2–3 mm in size) were randomly collected from the center of the surgical specimens, mimicking FNAC, and subjected to Pap-staining. If the case showed a dedifferentiated area, the sample was collected from the ALT/WDL area. Six cytotechnologists (CTs) evaluated the cell morphology, number of lipoblasts, size of adipocytes, nuclear pleomorphism, intranuclear vacuoles, multinucleated cells, nuclear enlargement, unequal size of nuclei, irregular nuclear borders, hyperchromasia, prominent nucleoli, large atypical cells, and background necrosis as a cytology routine observation. (Figure 1A–G). Large atypical cells were defined as cells with hyperchromasia and irregular nuclear enlargement (Figure 1H–l).

image

Cytological findings from Papanicolaou-stained tissue sample. (A) The absence of nuclear pleomorphism at low magnification. (B) Prominent nuclear pleomorphism observed at low magnification. (C) Cells with intranuclear vacuoles observed at high magnification. (D) Multinucleated cells observed at high magnification. (E) Cells with nuclear enlargement and unequal nuclear size observed at low magnification. (F) Cells with nuclear enlargement and prominent nucleoli observed at high magnification. (G) A cell with hyperchromasia observed at high magnification. (H–L) Large atypical cells are defined as cells with hyperchromasia and enlarged irregular nucleus observed at high magnification. (Scale size: 50 μm)

The morphology of each cell was evaluated on a scale of 1 to 4 (1: almost none, 2: a little, 3: common, 4: prominent), and background necrosis was assessed on a scale of 0 to 1 (0: absent, 1: present). Furthermore, each CT estimated the histological type of the lipoma or ALT/WDL based on cytological findings. More than 200 cells were examined using a WinROOF2018 image analyzer (MITANI Corporation, Tokyo, Japan), and the short nuclear diameter was measured for samples subjected to Pap staining.

The FFPE blocks of surgical samples were prepared for histological examination. The resected specimens were fixed in 10% neutral-buffered formalin. At least one block per centimeter of the largest diameter of the tumor was prepared for histological evaluation. If the maximum diameter was 10 cm, more than 10 FFPE blocks were prepared for histological evaluation. The two pathologists evaluated the nuclear atypia of adipocytes and atypical stromal cells and made a histological diagnosis. Immunostaining for MDM2 and CDK4 and FISH examination for MDM2 were performed in all cases using FFPE specimens. In addition, in four cases, FISH for MDM2 was performed using Pap-stained specimens.

The histological type determined via at least four of the six CTs was used as the cytological diagnosis result for analysis. We used the Mann–Whitney U test to analyze the association between clinical findings, histological diagnosis, cytological diagnosis, and length of the short diameter of the cell nuclei and the presence of MDM2 amplification. We analyzed the association between each cytological finding and each diagnosis based on Pap-stained specimens using Spearman's correlation and then examined the mean values. We also analyzed the association between the total score of the cytological findings of the six CTs and the presence of MDM2 amplification using Spearman's correlation. Statistical analysis was performed using SPSS version 26 software (SPSS Inc., Chicago, IL). Statistical significance was set at p < .05.

2.1 Immunohistochemistry

Deparaffinized tumor sections were stained for CDK4 (Clone DCS-31, Thermo Fisher Scientific, Waltham, MA) and MDM2 (Clone IF2, Thermo Fisher Scientific) using the heat-induced epitope retrieval method. Appropriate positive and negative controls were used for all analyses. Immunostaining was evaluated based on the intensity and proportion of the tumor cells in each specimen. The intensity of staining was defined by applying Allred scoring9 as follows: 3+, strong; 2+, moderate; 1+, weak; −, no staining. The proportion of staining was measured for each specimen and classified by applying Allred scoring as follows: 5, > 66%; 4, 66%–33%; 3, 33%–10%; 2, 10%–1%; 1, < 1%; 0, 0%. Cases were defined as MDM2-positive if the Allred score of the marker (defined as the combined value of the intensity score and proportion score) was more than 1. Moreover, cases were considered CDK4-positive if the Allred score of the marker was more than 5.

2.2 FISH analysis

FISH for MDM2 was performed in all 20 cases using FFPE tissues with the Vysis® LSI® MDM2 SpectrumOrange Probe (CEP® 12 [D12Z3], Abbott Molecular, Des Plaines, IL) according to the manufacturer's protocol. The probe cocktail decorates the human chromosomal region harboring MDM2 with an orange signal and the centromeric region of chromosome 12 with a green signal. The signals were scored by counting a minimum of 20 non-overlapping nuclei per case, and the average of MDM2 and centromere 12 signals was calculated. An MDM2/chromosome 12 signal ratio of >2.0 was considered to represent MDM2 amplification (amplification-positive).

FISH for MDM2 was performed using Pap-stained specimens to evaluate the cytological findings in four cases. Using FISH in FFPE samples, two cases showed MDM2 amplification, whereas two did not. After xylene was removed using 100% ethanol, the samples were destained with ethanol hydrochloride for 2–3 h. Then, the samples were washed with 100% ethanol and incubated overnight at room temperature (20–30°C). The specimens were immersed in 0.2% hydrochloride for 20 min, followed by immersion in distilled water for 1 min, a wash buffer for 5 min, and finally a protease solution (Abbott Molecular; pre-warmed to 37 ± 1°C) for 10 min. The samples were again immersed in wash buffer for 5 min, and the procedure was repeated. Next, the samples were immersed in 10% neutral-buffered formalin for 10 min, followed by immersion in wash buffer for 5 min, and the procedure was repeated. Finally, the Vysis® LSI® MDM2 SpectrumOrange Probe was added to the denatured DNA, and hybridization was carried out at 73°C for 3 min, followed by overnight incubation at 37°C. The cells were washed to eliminate nonspecific signals by immersing them in hybridization wash buffer (2X SSC/0.3% NP-40; Abbott Molecular) preheated to 72 ± 1°C for 2 min. The specimens were immersed in wash buffer and DAPI was added, followed by observation with a fluorescence microscope Ti-E equipped with a triple bandpass filter set, DAPI/Green/Orange v2 (Nikon Corporation, Tokyo, Japan).

3 RESULTS

The participants included 13 males and 7 females, with a mean age of 56 years. The most commonly affected site was the thigh (n = 10), and the mean maximum tumor diameter was 133 mm. Seven cases were histologically diagnosed as lipomas and 13 as ALT/WDLs. The mean nucleus short diameter of tumor cells measured from the Pap-stained samples was 4.02 μm, with a median of 3.75 μm. The short diameter of the nucleus in most tumor cells observed in Pap-stained samples was <5 μm, and a few tumor cells had nuclei with a short diameter of more than 10 μm. Immunostaining evaluation using FFPE tissues revealed nine MDM2-positive cases and 11 CDK4-positive cases. In 12 cases, MDM2 amplification was observed by FISH on FFPE tissues (Table 1). Histologically, no necrotic findings were observed.

TABLE 1. Clinicopathological findings of patients with lipomas and atypical lipomatous tumor/well-differentiated liposarcomas (min, max) Male/female, n 13/7 Age, yearsa 56 ± 15 (19, 90) Tumor size, mma 133 ± 73 (50, 350) Location, n Thigh 10 Neck 3 Head 1 Buttocks 1 Foot 1 Inguinal region 1 Lower leg 1 Upper arm 1 Retroperitoneum 1 Histological diagnosis Lipoma 7 ALT/WDL 13 Nucleus short diameter, μma Average 4.02 ± 0.53 (3.26, 5.03) (Papanicolaou staining) Maximum diameter 10.07 ± 2.62 (6.21, 14.82) Proportion of nucleus diameter, %a ≥5 μm 20.4 ± 10.8 (3.4, 38.2) (Papanicolaou staining) ≥6 μm 9.6 ± 7.7 (0.5, 24.8) ≥7 μm 4.9 ± 5.0 (0, 18.3) ≥8 μm 2.3 ± 2.8 (0, 9.2) ≥9 μm 1.1 ± 1.5 (0, 4.2) ≥10 μm 0.6 ± 0.9 (0, 2.9) MDM2 FISH (20 cells)b MDM2 signals total 168.5 (33–230) (28, 449) MDM2/CEP®12 ratio 4.95 (0.98–6.70) (0.8, 13.6) Amplification +/− 12/8 Abbreviations: ALT/WDL, atypical lipomatous tumor/well-differentiated liposarcoma; FISH, fluorescence in situ hybridization.

In nine cases, at least four out of six CTs predicted ALT/WDL based on the cytological characteristics of Pap-stained tissue samples. Moreover, MDM2 amplification was observed using FISH in each of the nine cases. The nucleus short diameter was significantly longer (p < .001), with a larger standard deviation, in cases with MDM2 amplification than in those without MDM2 amplification, indicating greater variation in nuclear size. Cells in which the short diameter of the nucleus was greater than 9 μm were not observed in cases in which MDM2 was not amplified (Table 2).

TABLE 2. Association between clinical findings, immunostaining results, and the length of the short diameter of cell nuclei in cases with and without MDM2 amplification detected using FISH MDM2 amplification (+) MDM2 amplification (−) p-value 12 cases 8 cases MDM2 signal (20 cells)a 247.7 ± 89.6 31.9 ± 2.1 CEP signal (20 cells)a 36.0 ± 8.3 33.6 ± 2.9 MDM2 / CEP12 ratioa 7.24 ± 3.02 0.95 ± 0.1 Percentage of cells with MDM2/CEP12 ratio > 2.0a 80.8 ± 10.4 0 ± 0 Male/Female, n 8 / 4 5 / 3 .910 Age, yearsa 58 ± 15 51 ± 15 .427 Tumor size, mma 156 ± 80 97 ± 44 .082 Histologic specimen Histological diagnosis ALT/WDL, cases 12 1 < .001 Immunohistochemistry MDM2 9 0 .004 Positive casesb CDK4 11 0 < .001 Cytologic specimen Cytological diagnosis ALT/WDL, casesc 9 0 .004 Nucleus short diameter, μma Average 4.32 ± 0.44 3.57 ± 0.29 < .001 SD 1.61 ± 0.30 1.11 ± 0.15 Maximum diameter 11.6 ± 2.2 7.76 ± 0.87 Proportion of nucleus diameter, %a ≧ 5 μm 26.4 ± 9.2 11.3 ± 5.4 .001 ≧ 6 μm 14.0 ± 7.2 3.4 ± 2.3 < .001 ≧ 7 μm 7.4 ± 5.0 1.1 ± 1.0 < .001 ≧ 8 μm 3.6 ± 3.0 0.3 ± 0.3 .001 ≧ 9 μm 1.9 ± 1.5 0 ± 0 .004 ≧ 10 μm 1.0 ± 0.9 0 ± 0 .012 Abbreviations: ALT/WDL, atypical lipomatous tumor/well-differentiated liposarcoma; FISH, fluorescence in situ hybridization.

The six CTs identified the samples as lipoma or ALT/WDL based on cytological findings, with a concordance rate of 88.3%. The mean concordance rate of identifying samples as lipoma or ALT/WDL based on cytological and histopathological findings was 76.7% (65% minimum, 90% maximum), whereas that based on cytological findings and MDM2 amplification by FISH was 78.3% (70% minimum, 85% maximum). Cytological findings with a relatively high concordance rate among CTs included lipoblasts, large atypical cells, multinucleated cells, and pleomorphism. The cytological findings that correlated significantly with MDM2 amplification by FISH included pleomorphism, unequal size of adipocytes, irregular nuclear borders, hyperchromasia, unequal size of nuclei, nuclear enlargement, prominent nucleoli, large atypical cells, and multinucleated cells. The relationship between cytological findings and ALT/WDL identification via each CT was relatively strong for pleomorphism, nuclear enlargement, and unequal nuclear size (Table 3). In the group with MDM2 amplification, the total scores of large atypical cells, multinucleated cells, and pleomorphism were higher than those in the group without MDM2 amplification. The mean total score of the above three findings was less than five in the group without MDM2 amplification, although some cases had total scores of <5 in the group with MDM2 amplification. A single CT rescreened a 1 cm2 area of the Pap-stained specimen in each case and counted the number of large atypical cells. Sixty-seven percent (8/12 cases) Of the cases with MDM2 amplification had large atypical cells. No large atypical cells were found in the lipoma cases (Table 4).

TABLE 3. Mean concordance rate between cytological findings among cytotechnologists (CTs) Evaluation concordance rate, %a Cytological diagnosisa MDM2/CEP12 ratio r. p-value r. p-value Lipoblasts 94.9 .204 .432 .312 .181 Large atypical cells 73.4 .680 .022 .601 .005 Multinucleated cells 66.7 .641 .035 .559 .005 Pleomorphism 65.1 .812 < .001 .781 < .001 Unequal size of adipocytes 60.0 .619 .056 .711 < .001 Irregular nuclear borders 60.0 .712 .051 .694 .001 Prominent nucleoli 57.6 .593 .012 .608 .004 Unequal size of nuclear 56.6 .764 .001 .668 .001 Nuclear enlargement 55.1 .780 .001 .640 .002 Hyperchromasia 54.1 .688 .170 .693 .001 Intranuclear vacuoles 52.4 .152 .381 −.116 .625 Cytological diagnosis 88.3 .693 .001 Note: Means determined via the six CTs were used to evaluate the association between cytological findings and each individual's histological type estimated by Papanicolaou-stained specimens. The association between the total score of cytological findings of the six CTs and the presence of MDM2 amplification is shown. TABLE 4. Results of MDM2 amplification with FISH using FFPE specimens and histological diagnosis and cytological impression Case FISH Histological diagnosis Cytological impressiona Cytological morphology Score average (1–4)b Large atypical cell re-examinationc Number of MDM2 MDM2/CEP12 Large atypical cells Multinucleated cells Pleomorphism Total score 1 28 0.8 Lipoma Lipoma 1.3 1.7 1.7 4.7 0 2 30 0.9 Lipoma Lipoma 1.2 1.3 1.7 4.2 0 3 31 1.1 Lipoma Lipoma 1.2 1.2 1.2 3.6 0 4 32 0.9 Lipoma Lipoma 1 1.3 1.2 3.5 0 5 32 1.1 Lipoma Lipoma 1 1 1 3 0 6 33 0.9 Lipoma Lipoma 1 1.3 1 3.3 0 7 34 1 Liposarcoma Lipoma 1.3 1.3 1.8 4.4 0 8 35 0.9 Lipoma Lipoma 1.3 1.2 1.5 4 0 9 144 5.3 Liposarcoma Liposarcoma 2.2 2.5 2.8 7.5 4 10 156 2.6 Liposarcoma Liposarcoma 1.8 2.5 2.7 7 1 11 181 4.8 Liposarcoma Lipoma 1 1 1.3 3.3

留言 (0)

沒有登入
gif