Dermatofibrosarcoma protuberans (DFSP) is a spindle-cell

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1 ORIGINAL ARTICLE Apo D in Soft Tissue Tumors A Novel Marker for Dermatofibrosarcoma Protuberans Robert B. West, MD, PhD,* Jeff Harvell, MD,* Sabine C. Linn, MD, PhD,* Chih Long Lui, Wijan Prapong, Tina Hernandez-Boussard, PhD, Kelli Montgomery,* Torsten O. Nielsen, MD, PhD, Brian P. Rubin, MD, PhD, Rajiv Patel, MD, John R. Goldblum, MD, Patrick O. Brown, MD, PhD, and Matt van de Rijn, MD, PhD* Abstract: Using gene microarray expression profiling, we previously found that apolipoprotein D (Apo D) was highly expressed in dermatofibrosarcoma protuberans (DFSP). In this study, we confirm that Apo D is highly and relatively specifically expressed in DFSP using immunohistochemistry. A tissue microarray containing 421 soft tissue tumors was constructed and stained with antibodies against Apo D and CD34. Cytoplasmic immunostaining for Apo D was found in 9 of 10 typical DFSPs. In addition, 3 of 3 Bednar tumors and 2 of 3 giant cell fibroblastomas stained in conventional sections. In contrast, Apo D was immunoreactive in only a very small subset of a diverse collection of other soft tissue tumors, including Malignant Fibrous Histiocytoma (MFH), glomus tumor, neurofibroma, and malignant peripheral nerve sheath tumors. Immunostains for Apo D were negative in conventional sections of 16 fibrous histiocytomas, and an additional 12 variants of fibrous histiocytoma. Digital images of all immunohistochemical and hematoxylin and eosin tissue microarray stains are available at the accompanying website ( We conclude that Apo D is strongly expressed in DFSPs and neural lesions and may be useful in differentiating DFSP from fibrous histiocytoma. Key Words: dermatofibrosarcoma protuberans, soft tissue tumors, gene microarray expression, apolipoprotein D (Am J Surg Pathol 2004;28: ) From the Departments of *Pathology and Biochemistry and Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, CA; Department of Pathology and Genetic Pathology Evaluation Centre, Vancouver General Hospital, Vancouver, British Columbia, Canada; Department of Anatomical Pathology, University of Washington Medical Center, Seattle, WA; and Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, OH. Supported by NIH grants CA85129 and CA84967 and the Howard Hughes Medical Institute. P.O.B. is an Associate Investigator of the Howard Hughes Medical Institute. S.C.L. was a recipient of a Dutch Cancer Society Postdoctoral Research Fellowship. Reprints: Matt van de Rijn, Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA ( mrijn@ stanford.edu). Copyright 2004 by Lippincott Williams & Wilkins Dermatofibrosarcoma protuberans (DFSP) is a spindle-cell tumor of uncertain histogenesis. The clinical behavior of this lesion is characterized by multiple, often aggressive, local recurrences. A translocation of chromosomes 17 and 22, resulting in fusion of the collagen type I alpha I (COL1A1) and platelet-derived growth factor beta (PDGF- ) genes is thought to be the transforming event for DFSPs. 16,19 Because of this translocation, the high activity of the promoter of COL1A1 leads to the high expression of PDGF-. 4,10,18 The main lesion in the differential diagnosis is fibrous histiocytoma (FH), a benign spindle cell proliferation. These uncommon tumors can have similar and overlapping morphologic findings, but the histologic distinction between the two entities is clinically relevant. The most commonly used histochemical marker to aid in this distinction is CD34, which reacts with DFSP but not with FH. In normal tissue, CD34 reacts with a number of different cell types, including endothelial cells and interstitial dendritic cells found within the gastrointestinal tract and in the skin. 20,21 However, CD34 does not react with all DFSPs and, more importantly, CD34 is also expressed in a number of other soft tissue tumors, such as gastrointestinal stromal tumor, solitary fibrous tumor, and a variety of vascular and neural lesions. 23 Recently, several reports have described molecular characterization of soft tissue tumors using gene arrays. 1,8,12,15 These techniques determine mrna expression on a genomewide basis and can discern patterns of gene expression that are specific for certain histologic entities. Antisera or monoclonal antibodies reacting with the gene products of interest can then be used in immunohistochemical studies to search for practical markers in surgical pathology practice. Tissue microarrays (TMAs) allow large numbers of tissues to be stained with a novel antibody on a single glass slide. 9 This method can evaluate antibodies with potential for diagnostic 5,13 or prognostic 22 use. Recently, DNA microarrays were used to profile gene expression in DFSP. 10 In analysis of a series of 36 soft tissue tumors, several genes were highly expressed in 9 cases of DFSP when compared with other spindle cell neoplasms. One Am J Surg Pathol Volume 28, Number 8, August

2 West et al Am J Surg Pathol Volume 28, Number 8, August 2004 of these genes was Apo D, a 33-kDa glycoprotein component of high-density lipoprotein in human plasma. To corroborate and extend the DNA microarray results, a TMA containing 421 soft tissue tumors was constructed and stained with antibodies against Apo D and CD34. Here we report that Apo D is consistently expressed in DFSP and peripheral nerve sheath tumors but is not detectably expressed in most other soft tissue tumors. MATERIALS AND METHODS TMA Construction A TMA of soft tissue tumors was constructed using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD) following previously described techniques. 9,13 A set of 2 TMAs was constructed that represented over 50 different soft tissue tumor entities by a total of 460,600-µm cores in duplicate (a total of 920 cores). These cores represent material from a total of 421 patients with several patients having more than one specimen appearing on the arrays. The 920 cores were arranged in two TMAs, TA34 and TA35. Duplicate 600-µm cores were taken from soft tissue tumor samples archived at the Stanford University Medical Center Department of Pathology between 1995 and The cores were taken from areas in the paraffin block that were representative of the diagnostic tissue. In several instances, multiple resection specimens from the same patient were included. For example, 15 specimens of malignant peripheral nerve sheath tumors (MPNSTs) were derived from 5 patients. Throughout the manuscript, the numbers refer to the patients and not the surgical resection specimens. Over 50 different soft tissue tumor diagnostic entities were represented on the array (Table 1). The total number of scorable cores for any stain can be less than the total number of cores in the array as some cores fall off in each slice of the array. A single section of a synovial sarcoma tissue microarray 14 containing 46 synovial sarcomas was also stained for Apo D. Immunohistochemistry Primary antibodies toward Apo D (clone 36C6, 1:40 dilution, Novocastra, Newcastle, UK) and CD34 (1:20 dilution, BD Biosciences) were used. Briefly, serial sections of 4 µm were cut from the TMA blocks, deparaffinized in xylene, and hydrated in a graded series of alcohol. The slides were pretreated with citrate buffer and a microwave step. Staining was then performed using the DAKO EnVision+ System, Peroxidase (DAB), (DAKO, Cambridgeshire, UK). Digital images of all cores stained by hematoxylin and eosin or immunohistochemistry were collected using the BLISS system from Bacuslabs (Lombard IL, com). All digital images of the TMA cores are available at the accompanying website: edu/tma_portal/apod/. Scoring of Immunohistochemistry Results were interpreted as follows: staining was interpreted as negative when no more than 20% of tumor cells showed light staining. A score of weak positive was given for light brown staining in greater than 20% of tumor cells, or dark brown staining in up to 50% of tumor cells. A score of strong positive was given for staining in greater than 50% of tumor cells. Non-tumor cells or cells of unknown origin were not scored. Cores in which no diagnostic material was present were omitted from further analysis. The cores were reviewed independently by two pathologists (R.B.W. and M.vdR.), and disagreements were reviewed together to achieve a consensus score. In rare cases, the number of tumor cells in the core was too low to score with confidence; examples of these included some lipomatous tumors. MFHs were likewise difficult to score because in some instances only the macrophage-like cells within the tumor were reactive while the pleomorphic cells were not. In these cases, only the clearly malignant and pleomorphic tumor cells were scored. Scoring of the arrays was analyzed using the Deconvoluter software as previously described, 11 with a tumor receiving the highest score for either of the two cores. In some instances, the data were further analyzed using the Compressor software. This software, currently in development, allows for the compression of multiple datapoints for a case (eg, when duplicate cores are used) into a single value (Liu et al, manuscript in preparation). RESULTS Soft tissue tumors analyzed for gene expression using cdna gene microarrays show a striking variation in expression levels for a large number of genes in several studies. 1,8,12,15 In a separate study, DFSP showed relatively high levels of Apo D mrna, while gastrointestinal stromal tumors, leiomyosarcomas, and synovial sarcomas showed relatively low levels of expression. 10 The gene expression pattern for Apo D in these tumors was measured using two separate cdna clones, AA and H15842, on each microarray, giving the results shown in Figure 1A; commercial antiserum was available to confirm and extend the apparent specificity on archived tissue. The expression of Apo D protein in soft tissue tumors was evaluated in the present study using two TMAs containing tissues from 421 patients. Major diagnostic categories (including leiomyosarcoma, malignant FH, and lipomatous tumors) were represented by more than 30 different tumor samples each (Table 1). The soft tumor TMA was stained with antibodies against Apo D (Fig. 2A) and CD34. Apo D was strongly immunoreactive in only a small subset of soft tissue tumors. Apo D antibody gave diffuse cytoplasmic staining of the tumor cells in DFSP (9 of 10 cases, 90%, Fig. 2B and Fig. 3A). Significant nuclear staining was not noted. There was no strong staining in the leiomyosarcomas or gastrointestinal stromal tumors. A single case of syno Lippincott Williams & Wilkins

3 TABLE 1. Tumors Represented on Soft Tissue Tumor TMA Diagnosis No. of Cases* Alveolar soft part sarcoma 2 Angiomyxoma 1 Angiosarcoma 14 Atypical lipomatous tumor 40 Carcinosarcoma 7 Chondrosarcoma 5 Clear cell sarcoma 3 Desmoplastic small round cell tumor 4 DFSP 10 Elastofibroma 1 Embryonal sarcoma 1 Endometrial stromal sarcoma 9 Eosinophilic granuloma 1 Epithelioid sarcoma 2 Ewing s sarcoma/pnet 10 Fasciitis 7 Fibroma/fibroblastoma 3 Fibromatosis 18 Fibromyxoid sarcoma 1 Fibrosarcoma 4 Fibroxanthoma 4 GIST 36 Glomangioma/glomus tumor 9 Granular cell tumor 12 Hemangioendothelioma 10 Hemangioma 2 Hemangiopericytoma 1 Hibernoma 1 Inflammatory pseudotumor 3 Kaposi s sarcoma 3 Leiomyoma 8 Leiomyosarcoma 37 Lipoblastomatosis 3 Lymphoangioma 3 Masson s lesion 1 MFH 53 MPNST 5 Myofibrosarcoma 1 Myxofibrosarcoma 2 Myxoma 8 Neuroblastoma 1 Osteosarcoma 14 Paraganglioma 1 Peripheral nerve sheath tumor, benign 5 Rhabdomyoma 1 Rhabdomyosarcoma 13 Rosai-Dorfmann disease 1 Sarcoma, NOS 10 SFT 11 Synovial sarcoma 10 Tenosynovial giant cell tumor 9 *Numbers indicate number of patients presented, not number of specimens, as some specimens are from the same patient. vial sarcoma (STT865) had high Apo D expression by cdna microarray. 10 This same tumor was also immunoreactive for Apo D when evaluated on a separate synovial sarcoma TMA where it was the only 1 of 46 synovial sarcoma cases tested 14. In addition, all 10 synovial sarcomas studied on the current TMA were immunonegative. All DFSPs were CD34 immunoreactive, consistent with previously published data 25,27 (accompanying website: edu/tma_portal/apod/). Full section staining of rare subtypes of DFSP was performed. Three of three Bednar tumors (pigmented DFSPs) and two of three giant cell fibroblastomas were immunoreactive with Apo D. Three of three plaque stage DFSPs were immunoreactive. One of one myxoid DFSP was immunoreactive for Apo D. Three of six DFSPs with fibrosarcomatous transformation demonstrated staining in the fibrosarcomatous elements (data not shown). Fifteen specimens of MPNST, obtained from different resection specimens from 5 patients, were represented on the TMA (Fig. 2B). Material from 2 of 5 patients reacted strongly for Apo D. This, taken together with the observed reactivity for one benign nerve sheath tumor on the TMA, prompted us to examine conventional sections of neurofibroma for Apo D reactivity; 6 of 8 showed strong staining (Fig. 3B; Table 2). Other tumors that had strong Apo D immunoreactivity on the TMAs included alveolar soft part sarcoma (100%, 2 of 2 cases), glomus tumor (60%, 3 of 5 cases), granular cell tumor (37%, 4 of 11 cases), endometrial stromal sarcoma (22%, 2 of 9 cases), angiosarcoma (21%, 3 of 14 cases), benign peripheral nerve sheath tumors (20%, 1 of 5 cases), and atypical lipomatous tumors (6%, 2 of 34 cases). Normal histiocyte-like cells in a variety of lesions showed strong staining. This usually did not present a scoring problem as they were easily distinguished from tumor cells. Only in cases of MFH did this raise an issue. In these lesions, only clearly pleomorphic cells were scored. The reactivities of Apo D antibodies for a variety of soft tissue tumors in this soft TMA are listed in Table 3. Weak immunohistochemical staining was observed in the solitary fibrous tumor group. None of the 11 tumors had strong immunoreactivity for Apo D, and only four tumors (36%) had faint reactivity. These tumors were capable of good antibody reactivity, as strong CD34 staining was demonstrated in the majority of SFT samples (data shown at accompanying website). Given the strong Apo D immunoreactivity in DFSPs, we evaluated whether FHs (dermatofibromas), a major differential diagnostic category, were also immunoreactive for Apo D. A single case of FH (STT 169) was examined on gene microarray and showed relatively high Apo D mrna levels (Fig. 1). The same FH run on the gene arrays was however negative by immunohistochemistry 10 (see accompanying website), demonstrating a discrepancy between DNA microarray and immunohistochemistry findings. In conventional sections from 16 other cases of FH, Apo D failed to react with the spindle com Lippincott Williams & Wilkins 1065

4 West et al Am J Surg Pathol Volume 28, Number 8, August 2004 FIGURE 1. The levels of of Apo D mrna expression for various soft tissue tumors as determined by gene microarrays and measured by two separate cdna fragments for Apo D. Each row represents the relative level of expression, centered at the geometric mean of its expression level across 40 samples. Each column shows the expression levels for a single sample. The red or green colors indicate high or low expression, respectively, relative to the mean. The intensity of the colors indicates the magnitude of deviation from the mean, with bright red indicating the highest levels and bright green the lowest levels. Gray denotes missing data. Gene array data are taken from dataset previously published. 10 FIGURE 2. A, Immunohistochemistry for Apo D on the entire soft tissue tumor tissue microarray (see accompanying website for complete imageset, including hematoxylin and eosin, Apo D, and CD34 stains [ DFSPs are boxed in red, MPNSTs are boxed in blue, and SFTs are boxed in green. B, Isolated images of the tumor clusters for MPNST, DFSP, and SFT as stained for Apo D. Multiple biopsies were represented from several MPNST patients; only specimens from 2 of 5 MPNST patients reacted Lippincott Williams & Wilkins

5 Am J Surg Pathol Volume 28, Number 8, August 2004 Apo D in Soft Tissue Tumors FIGURE 3. A, Typical immunostaining of DFSP with Apo D. B, Neurofibroma staining for Apo D. C, Conventional section of a fibrous histiocytoma showing lack of staining for Apo D. D, Atypical cells in an atypical fibrous histiocytoma are immunoreactive for Apo D. ponent of these lesions (Fig. 3C; stanford.edu/tma_portal/apod/). An additional 10 cases of variants of FH were negative for Apo D (Table 2). In 2 cases of atypical FH (also known as dermatofibroma with monster cells ), the atypical cells were immunoreactive with the Apo D antibody (Fig. 3D; edu/tma_portal/apod/). All TMA immunostains used for the study are available for review on the accompanying website. TABLE 2. Results From Immunostaining for Apo D on Conventional Sections No. NF 6/8 Usual type FH 0/16 Angiomatoid FH 0/3 Epithelioid FH 0/2 Plexiform FH 0/2 Atypical FH 0/4* Aneurysmal FH 0/1 Bednar tumor 3/3 Giant cell fibroblastoma 2/3 Plaque stage DFSP 3/3 Myxoid DFSP 1/1 DFSP with fibrosarcomatous transformation 3/6 *In the atypical FH, only scattered atypical cells stained. DISCUSSION The diagnosis of soft tissue tumors is complicated by the large number of recognized diagnostic entities (>100), the rarity of these neoplasms, and the subtle distinguishing morphologic features of tumors with distinctly different clinical behavior. 26 The challenge is exacerbated by the dearth of immunohistochemical markers to aid pathologists in difficult cases. Moreover, the available markers are often reactive with a number of entities. As a first step toward tackling these issues, we have analyzed global gene expression patterns in a number of soft tissue tumors with DNA microarrays. 10,15 These studies revealed that Apo D mrna was abundant in DFSP and, to a lesser extent, in SFT (West et al, manuscript in preparation). We now show that Apo D may be a useful marker in the diagnosis of DFSP as a complement to CD34, the marker most often used to identify DFSP. Using immunohistochemistry to analyze Apo D expression in TMAs of soft tissue tumors, we found strong staining for Apo D in a majority of DFSP (9 of 10 usual DFSPs and 12 of 16 variant DFSPs), and in a small subset of other tumors including benign and MPNSTs, malignant FHs, glomus tumor, granular cell tumor, tenosynovial giant cell tumor, and atypical lipomatous tumors; 28 cases of FH were immunonegative on conventional slides except for the atypical cells in 2 cases of atypical FH. In dermatopathology, the distinction between FH (dermatofibroma) and DFSP is a frequent challenge, especially in superficial biopsies that do not adequately sample the subcutaneous fat. The distinction is of clinical relevance as DFSP requires a wide excision with negative margins, whereas FH can be locally excised. Immunohistochemisty with CD34 has proven very useful in this differential diagnostic scenario, but it is only approximately 80% to 95% sensitive for DFSP. 25,27 In addition, CD34 has been reported to react with a wide variety of soft tissue neoplasms, including focal reactivity in FHs. 3 Likewise, factor XIIIa is not specific or sensitive for FHs. In the current study, CD34 and Apo D stained the majority of DFSPs (including Bednar tumors and giant cell fibroblastomas), and Apo D stained none of the FHs. Two cases of atypical FH (also known as dermatofibroma with monster cells ) showed focal reactivity with Apo D, but only within the atypical (monster) cells while the bland spindled cells were Apo D immunonegative. These monster cells rarely occur in DFSP and are unlikely to lead to confusion in the distinction between DFSP and FH. An additional 10 cases of variants of FH also failed to stain with antibodies for Apo D. Thus, like CD34 and factor XIIIa, Apo D appears to represent a sensitive and specific marker for distinguishing between DFSP and FH. In this study, all the DFSPs examined were CD34 immunoreactive. A larger number of cases with some CD34 immunonegative specimens will need to be examined to evaluate the staining differences between CD34 and Apo D Lippincott Williams & Wilkins 1067

6 West et al Am J Surg Pathol Volume 28, Number 8, August 2004 TABLE 3. Apo D Immunoreactivity in the Soft Tissue Tumor Tissue Microarray Tumor Total Cases Weak + Strong + Alveolar soft part sarcoma Angiosarcoma Atypical lipomatous tumor Carcinosarcoma DFSP Endometrial stromal sarcoma Eosinophilic granuloma Ewing s sarcoma/pnet Fibromatosis Fibroxanthoma Glomus tumor Granular cell tumor Hemangioendothelioma Inflammatory pseudotumor Leiomyosarcoma Lymphoangioma MFH Osteosarcoma Paraganglioma Peripheral nerve sheath tumor, benign Peripheral nerve sheath tumor, malignant Rhabdomyoma Rhabdomyosarcoma SFT Synovial sarcoma* Tenosynovial giant cell tumor Note: All scorable tumors with any weak or strong immunoreactivity for Apo D are listed. *In addition, a separate synovial sarcoma tissue microarray with 46 cases was stained; only 1 case reacted with Apo D. Interestingly, while cdna microarray results suggested Apo D immunoreactivity in solitary fibrous tumors (West et al, manuscript in preparation), these tumors were largely immunonegative for Apo D. This may at least partially be explained by the lower levels of Apo D mrna found in SFT, compared with DFSP. A single FH analyzed using DNA microarray showed levels of Apo D mrna comparable to the DFSPs. This tumor was nevertheless negative for Apo D by immunohistochemistry in the presence of adequate controls. The reason for this discrepancy between mrna and protein data is unclear. The remainder of the immunohistochemical findings correlated very well with the mrna expression levels measured with DNA microarrays: many DFSPs were strongly immunoreactive while gastrointestinal stromal tumors and leiomyosarcomas were never strongly immunoreactive. Immunoreactivity for synovial sarcomas was a very rare event with only 1 of 56 cases reacting. Apo D is a glycoprotein of 33 kda that acts as a component of the high density lipoprotein structure. It is not involved in the t(17;22) translocation, as it is located at 3q26.2-qter. Apo D mrna has been detected in a number of tissues, including liver, intestine, pancreas, kidney, placenta, adrenal, spleen, and fetal brain. 21 Protein sequence analysis reveals little similarity to other lipoprotein sequences but indicates a small degree of homology with plasma retinol-binding protein. Apo D protein expression has been found in a number of other organ systems. 7 In the nervous system, Apo D is expressed in lowgrade primary tumors, especially in pilocytic astrocytomas. 6 Breast, ovarian, and endometrial carcinomas have also been Lippincott Williams & Wilkins

7 Am J Surg Pathol Volume 28, Number 8, August 2004 Apo D in Soft Tissue Tumors examined for Apo D staining. 24 One paper suggests that the absence of Apo D staining may be a poor prognostic indicator in breast cancer. 2 Recent studies have indicated that DFSP could be sensitive to small molecule kinase inhibitors. 17 The identification of specific drug targets heightens the need for more accurate pathologic diagnoses. A panel of immunohistochemical markers and DNA microarray data will likely be useful to develop more precise molecular diagnostic criteria. The data presented here highlight the means by which partially characterized proteins, such as Apo D, can be exploited for diagnostic purposes. Moreover, in the past, individual tumor types were reported (with low numbers) for immunoreactivity with new markers (eg, CD34 in GIST and SFT 20,21 ). These studies were often followed by papers describing immunoreactivity on other lesions, demonstrating that the specificity of the immunostain was less than originally appreciated. Immunostaining of a large and diverse panel of tumor and normal tissues, under controlled conditions using TMAs, provides an efficient and powerful approach to define the specificity and sensitivity of a candidate marker with respect to a clinically relevant range of diagnostic possibilities. REFERENCES 1. Allander SV, Nupponen NN, Ringner M, et al. Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res. 2001;61: Diez-Itza I, Vizoso F, Merino AM, et al. Expression and prognostic significance of apolipoprotein D in breast cancer. Am J Pathol. 1994;144: Goldblum J, Tuthill R. CD34 and factor-xiiia immunoreactivity in dermatofibrosarcoma protuberans and dermatofibroma. Am J Dermatopathol. 1997;19: Greco A, Fusetti L, Villa R, et al. 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