Diagnosis of soft tissue tumors by fine-needle aspiration with combined cytopathology and ancillary...

Diagnosis of Soft Tissue Tumorsby Fine-Needle Aspiration WithCombined Cytopathology andAncillary TechniquesZoltan Sapi, M.D., Ph.D.,

1* Imre Antal, M.D.,2 Zsuzsa Papai, M.D.,

3

Miklos Szendroi, M.D., D.Sc.,2 Arpad Mayer, M.D., D.Sc.,

4 Klara Jakab, M.D.,5

Laszlo Pajor, M.D., Ph.D.,6 and Miklos Bodo, M.D., D.Sc.

1

The diagnosis of mesenchymal neoplasm by fine-needle aspirationbiopsy (FNAB) has presented a diagnostic challenge. Most reportsclaim an accuracy approaching 95%, but while they distinguishbenign and malignant lesions, the most problematic group, theintermediary malignant group, is omitted. The purpose of thisstudy was to determine whether rapid cytologic diagnosis of soft-tissue tumors could guide surgeons in therapeutic decisions with-out the need for a tissue biopsy. Ninety-four FNA cytologic spec-imens were examined by the National Soft Tissue Consortium ofHungary and compared with the corresponding histology. Ordi-nary lipomas were excluded. Morphologic evaluation was supple-mented by ancillary techniques such as fluorescence in situ hy-bridization (FISH), DNA cytometry, and immunocytochemistry.From a practical clinicopathological point of view, the cases weregrouped in the following categories: 1) tumors with definitivediagnosis: a) high-grade malignant neoplasms (high-grade sarco-mas, metastatic carcinomas, lymphoma), b) tumors with precisehistogenetic origin by cytogenetics, c) benign tumors; 2) tumors ofquestionable nature. In the first group there were 74 tumors: 22high-grade sarcomas, six metastatic carcinomas, one malignantlymphoma, 16 malignant tumors in which the precise histogeneticorigin could be established by cytogenetic studies, and 29 benignsoft-tissue tumors other than lipomas. In the second group there

were 20 tumors comprising benign and malignant soft tissuetumors of low grade, wherein the precise nature of the neoplasmcould not be established with confidence on cytologic study, evenusing ancillary techniques. FNAB of soft-tissue tumors combinedwith ancillary techniques should be considered a viable diagnostictechnique for therapeutic protocols. Although the second group isfairly large, we have reliable, well-characterized categories whichprovide great freedom for preoperative and surgical treatment,thus providing the best chance for healing. Diagn. Cytopathol.2002;26:232–242. © 2002 Wiley-Liss, Inc.

Key Words: soft tissue tumors; fluorescence in situ hybridization;DNA content; immunocytochemistry

Fine-needle aspiration biopsy (FNAB) has been extensivelyused in the diagnosis of soft-tissue tumors (STTs).1–4 Theaccuracy of this approach may benefit from the use ofancillary techniques, such as fluorescence in situ hybridiza-tion (FISH), analysis of genetic abnormalities by polymer-ase chain reaction (PCR), karyotyping, DNA quantitationby flow cytometry, and immunocytochemistry.5–9 DNA cy-tometry and assessment of proliferating activity (Ki-67)have been reported as prognostic factors but were not usedfor diagnostic purposes.10–14 In this article we describe theapplication of a number of ancillary techniques supplement-ing the cytologic diagnosis of STT for purposes of betterpreoperative diagnosis and, hence, optimization of treat-ment.

Materials and MethodsSince 1996, 94 FNAB of STT were examined and treated bythe National Soft Tissue Tumor Team of Hungary. Ordinarylipomas and tumors with inadequate material for ancillarymethods were excluded from this study (47 lipomas and 12other STT). The 94 tumors were either malignant or benignbut causing diagnostic problems by their cytomorphologyand/or clinical appearance. Eight samples were obtainedunder CT-guided FNAB from the retroperitoneum and kid-

1Department of Oncopathology, Semmelweis University of HealthSciences, Budapest, Hungary

2Department of Orthopaedics, Semmelweis University of HealthSciences, Budapest, Hungary

3Department of Chemotherapy, National Institute of Oncology,Budapest, Hungary

4Department of Oncoradiology, Uzsoki Street Hospital, Budapest,Hungary

5Department of Radiology, St. Stephan Hospital, Budapest, Hungary6Department of Pathology, University of Health Sciences, Pecs,

HungaryPresented in part at the XXIII International Congress of the International

Academy of Pathology, Nagoya, Japan, 15–20 October 2000.*Correspondence to: Zoltan Sapi, MD, PhD, Semmelweis University of

Health Sciences, Department of Oncopathology, 1125 Budapest, Dios arok1-3, Hungary. E-mail: [email protected]

Received 14 December 2001; Accepted 19 December 2001Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/dc.10096

232 Diagnostic Cytopathology, Vol 26, No 4 © 2002 WILEY-LISS, INC.

ney (examined tumors proved to be not epithelial). All 94FNAB were performed by one cytopathologist (Z.S.) onoutpatients and from each case 8–10 additional smears(fixed in cold acetone for 10 min and stored at �20°C) werepreserved for the ancillary techniques, but usually (morethan 90% of cases) we performed the different ancillarytechniques on the same day.

FISH and Dual-Color FISHProbes used in the analysis included biotin-labeled alphasatellite centromeric DNA probes for chromosome 2(D2Z1), chromosome 8 (D8Z1), chromosome 11 (D11Z1),chromosome 12 (D12Z1), chromosome 18 (D18Z1), anddigoxigenin-labeled painting DNA probes for total chromo-some X (COTASOME X), chromosome 16 (COTASOME16), and chromosome 22 (COTASOME 22). All probeswere from Oncor (Gaithersburg, MD, USA). To documentnumerical chromosome alterations, only alpha satellite cen-tromeric probes were used. To document translocations, amixture of one digoxigenin-labeled painting probe (paintingthe whole chromosome) and one biotin-labeled centromericprobe (i.e., X and 18 for synovial sarcoma; 12 and 16 formyxoid liposarcoma, etc.) were used simultaneously as adual-color FISH. The nuclei and the probes were denaturedsimultaneously for 3 min at 80°C and hybridized overnightat 37°C. For visualization, hybridized probes were furtherconjugated with FITC-labeled avidin and rhodamine-la-beled anti-digoxigenin. For identification of topographicrelationships between biotin-labeled and digoxigenin-la-beled DNA fragments, nuclei were stained with DAPI/antifade (Oncor). The analysis of fluorescent signals wasperformed by the method described by Yang et al.,15 brieflyas follows: hybridized nuclei were viewed under a magni-fication of �1,000 by a fluorescence microscope equippedwith a triple-bandpass filter (Olympus) and photographed.The topographical relationship between FITC-labeled alphacentromeric (in yellow-green) and rhodamine-labeled chro-mosomal painting signals (in red) in each nucleus wasjudged to be either juxtaposed, if an intimate topographicalarrangement was observed, or separate, if their arrangementwas not related topographically. A total of 50–100 nucleifrom each case were counted for each pair of probes.

DNA Quantitation by Image CytometryFNAB smears were fixed for 5 min in Carnoy’s solution(60v.% pure alcohol, 30v.% chloroform, 10v.% concen-trated acetic acid) and postfixed in buffered formalin for 30min. Acid hydrolysis with 5N HCL was performed at 27°Cfor 1 h, before Feulgen staining with Schiff’s reagent(Merck, KGaA, Darmstadt, Germany) for 1 h. The measur-ing principle was based on the digital processing of a TVimage of microscopic samples using mathematical morphol-ogy. The integrated optical density of Feulgen-stained in-ternal reference cells (lymphocytes) with DNA content of

2c was correlated with the tumor cells to be measured; thus,the relative DNA content was determined (DI). At least 20lymphocytes and 100 tumor cells were measured to deter-mine the diploid value (lymphocytes) and the stem line oftumor cells. A stem line is considered DNA aneuploid if itsvalue, registered in the Kolmogoroff-Smirnow test, is sig-nificantly (P � 0.001) different from the diploid referencecells, and the DNA index is not between 0.90–1.10. Themeasurement was accepted if its CV (coefficient of varia-tion) was �5%. The so-called 2c deviation index (2c – DI)and 5c exceeding rate were determined also, but we did notconsider these values because measuring 100 tumor cells issufficient only for determination of ploidy.

ImmunocytochemistryMore than 90% of the immunocytochemical stains wereperformed on the same day as the FNAB was taken, to avoidfalse negativity. Smears were fixed in cold acetone for 10min. Immunocytochemistry was performed with VentanaNexis (fully automated staining procedure) to have standardcircumstances. In each case, Ki-67 (MIB-1) (NovocastraLaboratories, Newcastle Upon Tyne, UK) was performed toassess proliferating activity and to determine PI (prolifera-tion index; percentage of 100 cells). The following antibod-ies were used as necessary: vimentin, cytokeratin (PAN),LCA, desmin, �-smooth muscle actin, S-100 (monoclonal),HMB-45, Melan-A, NK1-C3, (Novocastra Laboratories)and CD-117 (C-kit), CD-99, CD-68, CD-34, P-53 (DAKOA/S, Glostrud, Denmark).

Grading (Only on Tissue Sections)In our study we used the French Federation of CancerCenters grading system,16 which proposes three parameters(differentiation, mitosis count, and tumor necrosis) andcombines them, using a scoring system: Differentiation:Score 1: sarcomas closely resembling normal adult tissue(e.g., well-differentiated liposarcoma); Score 2: other sar-comas of certain histologic type (e.g., myxoid liposarcoma);Score 3: embryonal sarcomas, synovial sarcomas, and sar-comas of doubtful tumor type. Mitosis count: the mitosiscount is made in most mitotic areas in 10 high-power fields;Score 1: 0–9 mitosis; Score 2: 10–19 mitosis; Score 3: morethan 19 mitosis. Tumor necrosis: Score 0: no necrosis;Score 1: less than 50% necrosis; Score 2: more than 50%necrosis. The total sum of the above scores gives the grade:combined score 2–3 � Grade 1; combined score 4–5 �Grade 2; combined score 6–8 � Grade 3.

ResultsFrom a practical clinicopathological point of view, the caseswere grouped in the following categories:

1. Tumors with definitive diagnosis: a) high-grade ma-lignant neoplasms, high-grade sarcomas, metastatic

CYTOPATHOLOGY OF SOFT TISSUE TUMORS

Diagnostic Cytopathology, Vol 26, No 4 233

carcinomas, lymphoma; b) tumors with precise histo-genetic origin by cytogenetics; c) benign tumors.

2. Tumors of a questionable nature.

In the first group there were 74 cases (78.7%) and nofalse-positive or -negative cases. The following cut-levelswere made, using DNA cytometry and determining PI.

1a. High-Grade SarcomasPI 15% or more, clearly aneuploid DI (usually with twosubpopulations), and characteristic anaplastic or pleomor-phic cytomorphology. In this group there were 22 sarcomas(Table I). All 22 sarcomas proved to be Grade III tumors byhistology, except one myxofibrosarcoma and two leiomyo-sarcomas. They were Grade II sarcomas. Of course, in thisgroup we did not want to give the histogenetic origin (infact, in many cases we had a strong suspicion of some kindof sarcoma) because the only important thing was the highlymalignant nature of these tumors. It is interesting that wehad one synovial sarcoma in this group, because there wasno specific t(X;18). The cytogenetics are very complex inthis subgroup (including several numerical chromosomalalterations and no specific translocations); therefore, it is notimportant.

We had 18 cases with two subpopulations; the average DIwas 1.36 and 1.72, respectively. The remaining four caseshad an average DI of 1.41. Concerning PI, which is a veryimportant factor in this group, the average PI was 36.5% (22cases), the lowest was 18% (leiomyosarcoma) (Figs. 1, 2,C-1).

Carcinomas and LymphomaCarcinomas and lymphoma were ruled out as STTs by ICC(immunocytochemistry) (cytokeratin positivity and vimen-tin negativity in carcinomas and LCA positivity in lym-phoma) and by finding the primary tumors (in the cases ofcarcinomas) later. Nevertheless, these carcinomas and lym-phoma appeared as primary large STTs (Table II).

1b. Tumors With Precise Histogenetic Origin byCytogeneticsThe cut levels in this subgroup are unimportant and notcharacteristic for DI and PI (PI range 6 –34%, 10 diploid

Table I. Clinical, Pathologic, Cytogenetic Findings in Cases Studied, High-Grade Sarcomas

Cyt. diagnosis Age, sex Anatomic loc. DI PI FISH Immunocyt. Histology

1. HGS 29 M r. thigh 1.40; 1.65 28% ND Vim: �; Ker:� MFH grade III2. HGS 61 F l. thigh 1.39; 1.61 23% no t(12; 16) Vim: �; Ker:� Myxofibrosc. grade II.3. HGS 75 F r. thigh 1.36; 1.70 35% ND Vim: �; Ker:� Myxofibrosc. grade III.4. HGS 60 M l. thigh 1.37; 1.66 31% ND Vim: �; Ker:� MFH grade III5. HGS 57 M r. leg 1.41; 1.82 41% ND Vim: �; Ker:� Myxofibrosc. grade III.6. HGS 39 F r. upper arm 1.38; 1.70 30% ND Vim: �; Ker:� Myxofibrosc. grade III.7. HGS 41 M pelvis 1.35; 1.65 38% ND Vim: �; Ker:� MFH grade III8. HGS 58 M r. thigh 1.49; 1.89 38% ND Vim: �; Ker:� Myxofibrosc. grade III.9. HGS 64 M l. upper arm 1.34; 1.74 40% no t(12;16) Vim: �; Ker:� Myxofibrosc. grade III.10. HGS 55 F r. forearm 1.41; 1.73 37% ND Vim: �; Ker:� MFH grade III11. HGS 30 M l. leg 1.34 27% ND Vim: �; Ker:� Alveolar soft part

sarcoma12. HGS 18 M r. forearm 1.39 43% ND Vim: �, Des: � Rhabdomyosc.13. HGS 21 F retroperit. 1.43; 1.81 39% ND Des: � Rhabdomyosc.14. HGS 22 M r. ankle 1.28; 1.72 20% ND �-smooth-actin � Leiomyosc. grade: II.15. HGS 54 M l. thigh 1.28; 1.64 18% ND �-smooth-actin � Leiomyosc. grade: II.16. HGS 48 F pelvis 1.37; 1.62 40% ND �-smooth-actin � Leiomyosc. grade: III.17. HGS 21 F l. thigh-pelvis 1.51 37% ND Vim: �, Ker: � Epithelioid sc.18. HGS 24 M r. popliteal space 1.24 31% no t(X; 18) Vim: �, Ker: � EMA � Synovial sc.19. HGS 18 F l. leg 1.35; 1.83 41% ND S-100: � MPNST20. HGS 58 M retroperit. 1.39; 1.90 39% ND Vim: �, Ker:� Liposarcoma grade III:21. HGS 71 M retroperit. 1.35; 1.84 33% ND Vim: �, Ker:� Liposarcoma grade III:22. HGS 63 M l. leg 1.28; 1.70 22% ND Vim: �; Ker:� Myxofibrosc. grade III.

HGS: high-grade sarcoma; MFH: malignant fibrous histiocytoma; MPNST: malignant periferial nerve sheath tumor.

Fig. 1. Highly polymorphic-pleiomorphic tumor cells in a myxoid back-ground (myxofibrosarcoma Grade III). Inset: high proliferating activitywith Ki-67 (PI: 30%) (Case 7).

SAPI ET AL.

234 Diagnostic Cytopathology, Vol 26, No 4

and 6 aneuploid tumors); however, we were able to provethe specific translocations. The characteristic ICC andcytomorphology allows precise diagnosis (diagnosis ofhistological value). Table III summarizes the results ofspecific translocations and ICC findings with DI and PI.Note that all the tumors would fall into the high-gradesarcomas subgroup considering the PI, except myxoidliposarcoma, which is in fact a low-grade sarcoma. How-

ever, myxoid liposarcoma can be characterized by itsmonomorphic but cellular cytomorphology with the char-acteristic plexiform capillarity and by the specific t(12;16) (Fig. C-2). In this subgroup there were 16 cases. Alldiagnoses in this subgroup were confirmed by histology.The average DI was 1.24 and the average PI was 24.5%.Some representative examples are seen in Figures 3, C-3,C-4, and C-5.

Fig. 2. Characteristic DNA histogram of pleomorphic sarcoma with two subpopulation and with the corresponding G2 phase (DI1: 1.49; DI2: 1.89) (Case 9).

Table II. Clinical, Pathologic, Cytogenetic Findings in Cases Studied; Metastatic Carcinomas and Lymphoma


23. Metastatic cc. 65 F sacral field ND ND ND Ker.: �, EMA: � Metastatic cc.24. Metastatic cc. 72 F l. thigh ND ND ND Ker.: �, EMA: � Metastatic cc.25. Metastatic cc. 67 M retroperit. ND ND ND Ker.: �, EMA: � Metastatic cc.26. Metastatic cc. 58 F supraclavicular field ND ND ND Ker.: �, EMA: � Metastatic cc.27. Metastatic cc. 61 F r. thigh ND ND ND Ker.: �, EMA: � Metastatic cc.28. Metastatic cc. 60 M thorax ND ND ND Ker.: �, EMA: � Metastatic cc.29. Malignant lymphoma 67 F r. thigh ND ND ND LCA: �, Ker: � High grade

MALTlymphoma

Table III. Clinical, Pathologic, Cytogenetic Findings in Cases Studied; Malignant STT With Precise Histogenetic Origin by Cytogenetics


30. Myxoid liposc. 36 M r. thigh 1.04 10% t(12; 16) VIM: � Myxoid liposc. grade I.31. Myxoid liposc. 34 M paravertebral field 1.02 6% t(12; 16) VIM: � Myxoid liposc. grade I.32. Myxoid liposc. 38 M l. popliteal space 1.02 9% t(12; 16) VIM: � Myxoid liposc. grade I.33. Myxoid liposc. 54 M r. thigh 1.10 7% t(12; 16) VIM: � Myxoid liposc. grade I.34. Myxoid liposc. 72 F r. forearm 1.02 9% t(12; 22) VIM: � Myxoid liposc. grade I.35. Synovial sc. 20 M r. shoulder 1.06 29% t(X; 18) Vim: �, Ker: �, EMA: � Synovial sc. grade III.36. Synovial sc. 23 M l. sole 1.24 36% t(X; 18) Vim: �, Ker: �, EMA: � Synovial sc. grade III.37. Synovial sc. 52 M r. thigh 1.04 28% t(X; 18) Vim: �, Ker: �, EMA: � Synovial sc. grade III.38. Synovial sc. 18 F l. heel 0.98 16% t(X; 18) Vim: �, Ker: �, EMA: � Synovial sc. grade III.39. Synovial sc. 40 M l. leg 1.08 31% t(X; 18) Vim: �, Ker: �, EMA: � Synovial sc. grade III.40. Synovial sc. 21 F l. inguinal field 1.12 28% t(X; 18) Vim: �, Ker: �, EMA: � Synovial sc. grade III.41. Clear cell sc. 42 M l. forearm 1.32 30% t(12; 22) CD-117 �, Melan A �, HMB-45 � Clear cell sc. grade III.42. Clear cell sc. 47 M l. foot 1.28 26% t(12; 22) CD-117 �, Melan A �, HMB-45 � Clear cell sc. grade III.43. Ewing/PNET 25 M r. shoulder 1.08 34% t(11; 22) Vim: �, CD-99 �; Ker: � Ewing/PNET44. Ewing/PNET 19 F l. thigh 1.14 30% t(11; 22) Vim: �, CD-99: �, Ker: � Ewing/PNET45. DSRCT 22 M l. thorax 1.09 32% t(11; 22) Vim: �, Ker: �, Desm: � DSRCT of pleura

DSRCT: desmoplastic small round cell tumor.



1c. Benign TumorsIn these tumors, PI was 5% or less, clearly diploid DI (rarelyeuploid poliploidization, especially in Schwannomas), andbland cytomorphology with moderate cellularity. We had29 cases in this group (Table IV). The average DI was 1.04(21 cases) and the average PI was 1.5%, the highest was 4%(nodular fasciitis and benign giant cell tumor of tendonsheaths, respectively). Ordinary lipomas were excludedfrom this group because they usually do not cause diagnos-tic problems either clinically or morphologically. On theother hand, the lipoma variants and lipomas in unusual sitesdo cause problems both clinically and morphologically (i.e.,spindle cell lipomas, intramuscular lipomas, etc.). Repre-sentative examples from this group are shown in FiguresC-6 and 4. While we did not insist on precise cytogeneticorigin in the cytological diagnosis in this subgroup, in manyinstances it was possible because of the characteristic age,localization, and cytomorphology (i.e., fibromatosis colli,benign giant cell tumor of tendon sheaths, etc.). All thetumors in this group proved to be benign by histology.

Cytogenetics is not essential in this subgroup, but may bevery helpful, first to prove the lesion (i.e., trisomies 2 innodular fasciitis, trisomies 8, 18, 20 in fibromatosis, etc.)and, second, to rule out malignant lesions (having no spe-cific translocation), as happened in one nodular fasciitis(case 48).

2. Tumors of Questionable NatureIn the second group there were 20 cases (21.3%) comprisingbenign and malignant STTs of low grade. PI was between5–15% and as to DI, there were 11 diploid and 9 aneuploidcases. Table V summarizes the results. Both benign andlow-grade (Grade I) malignant tumors belonged in thisgroup almost in equal numbers, 11 and 9, respectively. In 14cases we suspected that they were either benign or malig-nant, but we could not prove it with confidence; therefore,we put them into this group. In the remaining seven caseswe simple did not know whether they were benign, inter-mediate malignant, or malignant. All the tumors in thisgroup displayed severe cellularity with slight to moderateatypia. This group clearly shows that, even using ancillarytechniques, some kind of pseudosarcomatous benign STTsand low-grade soft-tissue sarcomas cannot be separated and,therefore, they should be treated preoperatively in a uniformway.

The average DI was 1.28 and the average PI was 9.0%.The lowest PI was 6 (extraabdominal fibromatosis) and thehighest was 14 (malignant hemangiopericytoma). The onlymyxoid liposarcoma was put in this group because, despitethe characteristic cytomorphology, we could not prove thecharacteristic t(12; 16) nor the t(12;22) (Figs. 5, 6a,b, 7).

The recommended therapy for the different groups are asfollows:

Fig. 4. DNA histogram of the tumor shown in Fig. C-6, which is clearlydiploid (DI: 1.04; Case 59).

Fig. 5. Characteristic cytomorphology of myxoid liposarcoma, but wecould not prove the specific t(12;16) of t(12;22), so we put this tumor in thesecond group. Inset: there is no translocation of chromosomes 12 to 16because of the two separated green and red signals (there are no juxtaposedsignals) (arrow: centromeric probe of chromosome 12, arrowhead: paintingprobe of chromosome 16; Case 93).

Fig. 3. The synovial sarcomas proved to be mainly diploid but this tumorwas peridiploid (DI: 1.24), indicating some numerical chromosomal alter-ation. Inset: trisomy of chromosome 8 (FISH) (Case 36).

SAPI ET AL.


1) Group 1, subgroup: “malignant STT with precise his-togenetic origin by cytogenetics”; the precise diagno-sis provides the possibility for any kind of preopera-tive treatment (e.g., chemotherapy or radiotherapy)and/or any kind of surgery!

2) Group 1, subgroup: “high grade sarcomas”; in thisgroup the diagnosis provides the possibility for almostany kind of preoperative treatment (except gene andhistogenetic-specific therapy) and/or any kind of sur-gery!

3) Group 1, subgroup: “benign soft-tissue tumors”; sim-ple excision, even shelling out the tumor, if necessary(i.e., on face, hand, etc.).

4) Group 2; wide excision and wait for the final histol-ogy! No excisional biopsy, no preoperative therapy,

no ablation! If excision is not possible, because of thespecial anatomic localization, intraoperative frozensection is recommended.

Discussion

The diagnosis of mesenchymal neoplasm by FNAB haspresented a diagnostic challenge. Most reports claim anaccuracy approaching 95%,4,17 but they distinguish, in fact,benign and malignant lesions, and the most problematicgroup, the intermediary malignant group, is omitted. Be-cause the remaining 5% is uncertain, the surgical approachis usually uniform. Regardless of the cytological diagnosissurgeons usually take an excisional biopsy for histologyand, what is sometimes more important, they do not dare

Table IV. Clinical, Pathologic, Cytogenetic Findings in Cases Studied; Benign Soft Tissue Tumors


46. BSTT 31 F r. shoulder 1.08 2% ND Vim: � Nodular fasciitis47. BSTT 48 F l. forearm 1.03 4% ND Vim: � Nodular fasciitis48. Nodular fasciitis 42 M l. thigh 1.05 3% no t(X; 18) Vim: � Nodular fasciitis49. BSTT 28 F r. forearm 1.03 1% ND ND Dermatofibroma50. Juvenile

xanthogranuloma8 F r. thigh 1.07 3% ND CD-68: �, Vim:

�Juvenile

xanthogranuloma51. BSTT 52 M neck 1.04 2% ND ND Dermatofibroma52. BSTT 47 M thorax 1.01 2% ND ND Dermatofibroma53. Fibromatosis

colli3 weeks F neck 1.04 1% ND ND Fibromatosis

colli54. Fibromatosis

colli2 weeks F neck 1.03 1% ND ND Fibromatosis

colli55. Fibromatosis

colli2 weeksM

neck 1.01 1% ND ND Fibromatosiscolli

56. BGTTS 45 F l. hand 1.02 �1% ND ND BGTTS57. BGTTS 48 F l. hand 1.02 1% ND ND BGTTS58. BGTTS 31 M l. wrist 1.06 �1% ND ND BGTTS59. BGTTS 37 F r. foot 1.04 4% ND ND BGTTS60. BGTTS 62 F l. hand 1.05 1% ND ND BGTTS61. BSTT 38 M neck 1.06 1% ND ND Benign

Schwannoma62. Benign

Schwannoma49 F l. upper arm 1.06 1% ND S-100: � Benign

Schwannoma63. Benign

Schwannoma70 F l. leg 1.05 �1% ND S-100: � Benign

Schwannoma64. Elastofibroma 44 F r. shoulder-blade ND �1% ND ND Elastofibroma65. BSTT 38 F r. shoulder-blade ND �1% ND ND Elastofibroma66. Angiolipoma 42 M l. kidney ND �1% ND ND Angiolipoma67. Angiolipoma 29 F l. kidney ND �1% ND �-smooth-actin: � Angiolipoma68. Angiolipoma 51 F r. kidney ND �1% ND ND Angiolipoma69. Granular cell tu. 41 F abdomenal wall 1.03 3% ND S-100: � Granular cell tu.70. Granular cell tu. 62 F l. forearm 1.05 1% ND S-100: � Granular cell tu.71. BSTT 57 F l. bottom ND 1% ND ND Ancient

haematoma72. Intramusc.

lipoma47 F l. shoulder ND �1% ND ND Intramusc.

lipoma73. Intramusc.

lipoma42 M r. thigh ND �1% ND ND Intramusc.

lipoma74. BSTT 35 F r. forearm 1.03 1% ND �-smooth-actin: � Glomangioma

BSTT: benign soft tissue tumor; BGTTS: benign giant cell tumor of tendon sheath; DGTTS: diffuse giant cell tumor of tendon sheath.



Fig. C-1. CT-guided FNAB from retroperitoneum (inset: upper right) with highly polymorphic tumor cells having fairly wide eosinophilic cytoplasma.Inset (lower left): Strong desmin positivity, but we could not perform the MyoD-1 reaction, so we put this tumor into the group “high-grade sarcomas” (Case13).Fig. C-2. Typical picture of myxoid liposarcoma with a myxoid background and with monotoneus tumor cells. Inset (lower right): Typical plexiformcapillary structure. Inset (upper left): Dual-color FISH of t(12;16); note the juxtaposed green and red signals and the two separated red and one green signals(arrow: centromeric probe of chromosome 12, arrowhead: painting probe of chromosome 16; Case 30).Fig. C-3. Fairly uniform, slightly elongated, but atypical tumor cells. Inset (upper left): Strong vimentin positivity. Inset (lower left): Focal cytokeratinpositivity. Inset (lower right): dual-color FISH of t(X;18) characteristic of synovial sarcoma. Besides of the juxtaposed signals there is only one red andone green signal because the patient was male (arrow: centromeric probe of chromosome 18, arrowhead: painting probe of chromosome X; Case 35).Fig. C-4. Typical picture of small round cell tumor. This tumor showed t(11;22), vimentin, and cytokeratin, and desmin positivity, characteristic of desmoplasticsmall round cell tumor. Inset (upper left): Strong CD-99 positivity. Inset (lower right): High proliferating activity with Ki-67 (PI: 32%) (Case 45).Fig. C-5. Cellular pleomorphic tumor cells with brown pigment in the cytoplasm; this would be characteristic of malignant melanoma. However, theosteoclast-type giant cell is unusual in malignant melanoma, but characteristic for clear cell sarcoma of the soft part. (This tumor was a deep-seated largesoft tissue tumor and there was no skin melanoma.) Inset (upper left): Dual-color FISH of t(12;22) characteristic of clear cell sarcoma of soft parts (arrow:centromeric probe of chromosome 12, arrowhead: painting probe of chromosome 22). Inset (lower left): HMB-45 positivity of tumor cells (Case 41).Fig. C-6. Left: Tumor cells with large, finely granulated eosinophilic cytoplasma (granular cell tumor; Case 69); right: monomorph tumor cells withosteoclast-type giant cells (Case 59).


apply preoperative chemo- or radiotherapy based on cyto-logical diagnosis. Beyond this, it is well known that the firstsurgical treatment is crucial to the fate of the patient.18,19 Tosolve this problem (and to avoid open biopsy20), we tried tohave a much more clinicopathological approach with thehelp of ancillary techniques. In this context, we created twowell-characterized groups with recommended therapy. Ourresults show that in this way more than three-fourths of themesenchymal tumors were put into the first group. Sub-groups of “high-grade sarcomas” and “malignant STT withprecise histogenetic origin by cytogenetics” allow almostany kind of surgery and/or preoperative treatment. Withsubgroup “benign STTs” (about one-fourth of cases), noth-ing else is needed but to remove the tumor. The secondgroup (again, about one-fourth of cases) is fairly large butthe recommendation is clear: wide excision (removal) of thetumor and no excisional biopsy, which is very important tothe fate of the patient18,19 if we consider the low-gradesarcomas in this group.

Among the ancillary techniques, FISH became a revolu-tionary technique because it makes possible the examinationof interphase cells in large number.15,21 One of the most

suitable materials for this purpose is the fresh materialgained by FNAB. If necessary, we are able to get samplesfrom different parts of a large STT to avoid (or just toprove) intratumor heterogeneity.22 With the help of dual-color FISH we are able to recognize the specific transloca-tion, which is, in many cases, very specific for the differenttypes of soft tissue tumors. They include the well-knownsynovial sarcoma t(X;18), Ewing/PNET tumor t(11;22),myxoid/round cell liposarcoma t(12;16), rarely t(12;22),desmoplastic small round cell tumor t(11;22), alveolar rhab-domyosarcoma t(2;13), rarely t(1;13), clear cell sarcomat(12;22), and extraskeletal myxoid chondrosarcomat(9;22).7,9,20,23,34 We had nice examples of this when thecytomorphologies were very similar, suggesting synovialsarcoma in both cases, but the t(X;18) could be detectedonly in one of them (Figs. 7, 8). Some STTs share the sametranslocation (i.e., Ewing/PNET and the desmoplastic smallround cell tumor or, rarely, myxoid liposarcoma and clearcell sarcoma) but we can specify them further by PCR orRT-PCR25 or we can separate them by ICC with confi-dence.4,26 Our results show the power of the FISH techniquebecause we were able to provide a definitive diagnosis of 16

Table V. Clinical, Pathologic, Cytogenetic Findings in Cases Studied; Tumors of Questionable Nature


75. TQN 27 F l. thigh 1.06 8% ND Vim: �, Ker: � Haemangiopericytoma76. TQN 61 M thorax 1.10 6% ND Vim: �, Ker: � Haemangiopericytoma77. TQN 32 F l. wrist 1.12 9% no t(X; 18) Vim: �, Ker: � DGTTS78. TQN 44 F l. popliteal space 1.08 7% no t(X; 18) Vim: �, Ker: � DGTTS79. TQN 57 F l. shoulder 1.08 9% ND Vim: �, Ker: � Extraabdominal

fibromat.80. TQN 81 M thorax 1.10 6% ND Vim: �, Ker: � Extraabdominal

fibromat.81. TQN 39 M r. bottom 1.05 7% ND Vim: �, Ker: � Extraabdominal

fibromat.82. TQN 22 F r. thigh 1.04 8% ND Vim: �, CD-31:

�Intramusc. haem

(Masson)83. TQN 37 F l. leg 1.08 11% no t(X; 18) S-100: � Sarcomatoid

Schwannoma84. TQN 33 F l. popliteal space 1.04 7% no t(12; 16) Vim: �, Ker: � Spindle cell lipoma85. TQN 48 M r. wrist 1.05 9% no t(X; 18) Vim: �, Ker: �,

EMA: �Panniculitis ossificans

86. TQN 28 F r. thigh 1.32 14% ND Vim: �, Ker: � Mal. HPC. grade I.87. TQN 42 M r. kidney 1.30 13% ND Vim: �,

�-smooth-actin:�

Mal. angiomyolipoma

88. TQN 37 M l. thigh 1.30 7% no t(12; 16) Vim: �, Ker: � Myxofibrosc. grade I.89. TQN 84 F l. thigh 1.18 13% ND Vim: �, CD-68:

�MFH giant cell type.

grade I.90. TQN 72 F r. bottom 1.28 8% no t(12; 16) Vim: �, Ker: � Myxofibrosc. grade I.91. TQN 67 M l. leg 1.30 9% no t(12; 16) Vim: �, Ker: � Myxofibrosc. grade I.92. TQN 28 M l. shoulder-blade 1.20 10% ND �-smooth-actin: � Myofibroblastic sc.

grade I.93. TQN 49 F l. thigh 1.09 6% no t(12;22)

no t(12;16)Vim: � Myxoid liposc. grade

I.94. TQN 61 M r. leg 1.27 11% ND Vim: �,

�-smooth-actin:�

Leiomyosc. grade I.

TQN: tumors of questionable nature; HPC: haemangiopericytoma.



sarcomas. The concept of getting definitive diagnosis byFNAB is nowadays accepted.17,20 As our knowledge aboutthe specific translocations and numerical alterations in-creases, we hope that we will be able to put more tumorsinto this group.7,27–30

Concerning the total DNA content (ploidy), the mainobjection is the false aneuploidy in some benign STTs (i.e.,nodular fasciitis) and, on the other hand, the diploid orperidiploid nature of some sarcomas.31 All these data arebased mainly on flow cytometry. One advantage of imagecytometry is to pick up the smaller second subpopulationthat can easily be lost by flow cytometry.32 It was the“high-grade sarcomas” subgroup where ploidy had the mostimportant role. The two, mainly aneuploid, subpopulationsare very characteristic for pleomorphic sarcomas with highproliferation index, while cytogenetics is not informative inthese cases.33 We are not aware of any kind of STT whichis benign but aneuploid and has a proliferation rate over15% and pleomorphic or anaplastic in cytomorphologicappearance. Of course, if we had any doubt we put thetumor into the second group. Nevertheless, many articles

emphasize the usefulness of the ploidy determinationamong STTs14,32,34 and in fact the total DNA content com-pleted with FISH provides better insight into the geneticalteration of the tumor and provides complementary infor-mation.35

Immunocytochemistry plays a crucial role in the differ-ential diagnosis, especially in subgroup “malignant STTwith precise histogenetic origin by cytogenetics” to specifythe lesions, and in cases of metastatic carcinomas and pri-mary soft tissue lymphomas, if they appear as primary STT.Based on our experience we want to emphasize the use offresh (not stored) material to avoid false negativity. This isespecially true if we want standard circumstances for count-ing the Ki-67 index, which is very important for the diag-nosis in the first group, except in the “malignant STT withprecise histogenetic origin by cytogenetics” subgroup, andalso important in the second group. For preoperative treat-ment, the PI is important in the “high grade sarcoma” and in

Fig. 6. a: Many osteoclast-type giant cells are obvious but the tumor cellsare more variable and slightly atypical. Inset: strong CD-68 positivity in thetumor cells. b: DNA histogram of MFH, giant cell-type, Grade I withperidiploid value (DI: 1.18) (Case 89).

Fig. 7. Top: This tumor was located on the right wrist and was suspiciousof synovial sarcoma because of cellularity and moderate atypia. Inset: wecould not prove the t(X;18); note the two separated green and one redsignals (patient was male) (arrow: centromeric probe of chromosome 18,arrowhead: painting probe of chromosome X). The tumor was put into thesecond group. Bottom: The tumor proved to be panniculitis ossificans(Case 85).

SAPI ET AL.


the “malignant STT with precise histogenetic origin bycytogenetics” subgroups. If we compare our results of PI tothose of other authors, there is a slight difference (perhapsdue to the fresh material) but mainly agreement, especiallyin subgroup “high-grade sarcomas” and in the secondgroup.11,34 We think it does not matter whether a pleomor-phic Grade III tumor is rhabdo-, leio-, lipo-, or myxofibro-sarcoma because almost the same preoperative treatmentcan be given (chemo- and/or radiotherapy), dependingmainly on the localization and size of the tumor and onother important clinical parameters. The only exception is ifwe have histogenetic-specific therapy or effective gene ther-apy, the most promising type of therapy, in the future.17,36

In conclusion, in good agreement with others,1,20,37,38,39

FNAB of STT combined with ancillary techniques shouldbe considered a viable diagnostic technique for therapeuticprotocols. Although the second group is fairly large, wehave reliable, well-characterized categories which providegreat freedom for preoperative and surgical treatment, thusproviding the best chance for healing.

AcknowledgmentsWe thank Prof. Leopold G. Koss (Montefiore Medical Cen-ter, The University Hospital for the Albert Einstein Collegeof Medicine, Bronx, New York, USA), whose help greatlycontributed to this article.

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