American Journal ofPathology, Vol. 13 7, No. 2, August 1990 Copyright © American Association ofPathologists

Chordomas with Malignant Spindle Cell Components A DNA Flow Cytometric and Immunohistochemical Study with Histogenetic Implications

Ralph H. Hruban, Frank Traganos, Victor E. Reuter, and Andrew G. Huvos

epithelial and mesenchymal pathways. (Am J Pathol 1990; 13 7:435-44 7)

From the Department ofPathology, Memorial SloanKettering Cancer Center, New York, New York

The authors studied four chordomas with malignant spindle cell components (SCs) and 12 conventional chordomas (CCs) by DNA flow cytometry using paraffin-embedded tissue. In addition, immunohistochemical stains for a variety of epithelial and mesenchymal markers were performed. Thefour SCs contained areas histologically identical to conventional chordomas, as well as a highgrade malignant spindle cell component. All four (100%) SCs had an aneuploid-multiploid DNA content. Of interest, the conventional chordoma areas in these tumors had DNA contents different from those containing the high-grade malignant spindle cells. In contrast, only three (27%) of the 11 conventional chordomas with analyzable histograms had an aneuploid-multiploid DNA content. Immunohistochemical studies performed on the four SCs showed the high-grade malignant spindle cells to stain strongly for vimentin and weakly for cytokeratin, S-100 protein, and epithelial membrane antigen (EMA), whereas the areas ofconventional chordoma in these same neoplasms stained moderately for vimentin and S- 100 protein, and stronglyfor cytokeratin and EMA. In two cases, the staining for EMA and cytokeratin highlighted a gradual transition between the areas of conventional chordoma and the spindle cell areas. The immunohistochemical staining pattern of the 12 conventional chordomas was similar to that seen in the conventional chordoma components ofthefour chordomas with malignant spindle cell components. These results suggest that: 1) aneuploidy is more common in SCs than in CCs, and 2) some SCs are multipotential neoplasms in which the neoplastic cells are capable of differentiation along both

Chordomas are rare neoplasms believed to arise from ectopic rests of notochordal tissue.1-3 They can be divided into three distinct, but often overlapping, groups: 1) classic or conventional chordomas, 2) chondroid chordomas, and 3) chordomas with a malignant spindle cell component.4-7 Conventional chordomas are lobated neoplasms composed of polyhedral cells with abundant eosinophilic cytoplasm.4'5 The cells are characteristically arranged in cords, are embedded in an abundant mucinous matrix, and are admixed with physaliferous cells. Conventional chordomas, although they can metastasize, are generally slowly growing neoplasms that recur locally after excision.4'5 Chondroid chordomas are neoplasms characterized by the presence of a significant cartilaginous component intermingled with areas more typical of conventional chordoma.6 These are usually less aggressive than classic chordomas,6 and although they were originally believed to be limited to the spheno-occipital region, they also have been reported in the lumbosacral area. Chordomas with high-grade malignant spindle cell components are often referred to as 'dedifferentiated' chordomas. They are neoplasms characterized by the presence of a significant atypical spindle cell component intermixed with, or associated with, areas of conventional chordoma.7 10 The spindle cell component in these neoplasms is remarkable for high cellularity, marked nuclear pleomorphism, and a high mitotic rate. These neoplasms behave in an aggressive manner and often metastasize.7'10 Several theories have been proposed to explain the association of chordomas and high-grade spindle cell Accepted for publication March 28, 1990. Address reprint requests to Dr. Ralph H. Hruban, Department of Pathology, The Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21205.

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tumors, including: 1) a collision of two unrelated tumors, 2) a postradiation sarcoma arising adjacent to an irradiated chordoma, 3) a neoplasm in which differing rates of growth causes two distinct morphologic patterns, and 4) a sarcoma arising from the chordoma itself.11-15 This study used DNA flow cytometry and immunohistochemical stains for a variety of mesenchymal and epithelial markers to define chordomas with malignant spindle cell components.

Patients and Methods The files of the Department of Pathology of the Memorial Sloan-Kettering Cancer Center, and the consultation files of one of us (AGH) were searched for chordomas with malignant spindle cell components. Four cases were identified in which paraffin-embedded material was available for flow cytometry and immunohistochemistry. In addition, 12 cases of conventional chordoma were selected for comparison. The histologic material was reviewed by three of us (RHH, VR, AGH). The Fisher's exact test was used in all statistical analyses.

Flow Cytometry Technique At least one formalin-fixed paraffin-embedded block was processed from each neoplasm. All blocks contained foci of normal connective tissue, epithelial tissue, or inflammatory cells, providing an internal diploid control. In one case (case 7) a block from the patient's primary and a block from the patient's recurrence were processed separately. All four chordomas with malignant spindle cell components contained areas histologically identical to conventional chordoma, in addition to the spindle cell component. In these cases, tissue from the areas that histologically resembled conventional chordoma was analyzed separately from tissue from the malignant spindle cell component. Three 50-,u sections were cut from each block. The nuclear suspensions were prepared by the method described by Hedley et al16 and then stained with 2 ug/ml of 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI; Boehringer Mannheim, Indianapolis, IN) on ice for 20 to 60 minutes. The stained nuclear suspensions then were run immediately on an Ortho ICP-22A flow cytometer (Ortho Diagnostics Systems, Westwood, MA). At least 6900 cells were scanned per case. Ploidy, DNA index, and cell cycle distribution were determined using the Asyst software program (Asyst Software Technologies, Rochester, NY). Two of the DNA histograms (case 16 and the conventional chordoma component of case 4) were not interpretable because of cell debris.

The DNA histograms determined by flow cytometry were divided into three groups. Diploid DNA histograms were observed to contain one major 'peak' which, by definition, 17,18 was considered diploid and assigned a DNA index (Dl) of 1.0. Aneuploid tumors consisted of specimens in which a distinct peak could be identified in addition to the diploid peak. This peak had to differ from the diploid peak by at least 10%. Finally, multiploid tumors consisted of specimens in which more than one aneuploid peak could be identified. The DNA index was calculated as the ratio of the peak channel of the abnormal DNA stemline of cells to the peak channel of the diploid peak. The percentage of proliferating cells was calculated as the percentage of cells between the Gi/GO peak and the end of the G2+M peak. The mean coefficient of variation of the G1/GO peak for the 19 interpretable histograms was 8.1.

Immunohistochemical Techniques Immunohistochemical stains for S-100 protein (Dako Co., Carpinteria, CA), vimentin (Dako Co.), cytokeratins (AE-1, Signet Labs, Dedham, MA; CAM 5.2, Becton-Dickinson, Mountain View, CA, and pooled AE-1 and AE-3, Hybritech, San Diego, CA), and epithelial membrane antigen (Dako Co.) were performed on formalin-fixed paraffin-embedded tissue using the avidin-biotin complex (ABC) technique. Before staining for cytokeratin, sections were pretreated with Pronase (Calbiochem, La Jolla, CA). The relative intensity of staining reaction for each stain was graded on a scale from 0 to 3+ (least to greatest) by one of us (RHH).

Brief Case Reports for Four Patients with Spindle Cell Chordomas Case 1 A 62-year-old man was admitted to Memorial Hospital for evaluation of rectal discomfort. A barium enema revealed a mass involving the sacrum. A partial sacrectomy was performed, examination of which revealed a chordoma with a malignant spindle cell component (Figures 1, 2). Tissue from this resection was used in the flow cytometry and immunohistochemistry study. The largest component of the neoplasm was a malignant spindle cell neoplasm morphologically similar to a malignant fibrous histiocytoma by light microscopy. The second, smaller component was a conventional chordoma. The highgrade component extended to the surgical margin of resection, and the patient received postoperative radiation and chemotherapy. Thirty-four months after diagnosis,

Spindle Cell Chordomas

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Chordomas with malignant spindle cell components. A DNA flow cytometric and immunohistochemical study with histogenetic implications.

The authors studied four chordomas with malignant spindle cell components (SCs) and 12 conventional chordomas (CCs) by DNA flow cytometry using paraff...
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