Specificity of Brachyury in the Distinction of Chordoma from Clear Cell Renal Cell Carcinoma and Germ Cell Tumors: a Study of 305 Cases

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Specificity of brachyury in the distinction of chordoma from clear cell renal cell carcinoma and germ cell tumors: a study of 305 cases

Ankur R Sangoi1,2, Jason Karamchandani1, Brooke Lane1, John P Higgins1, Robert V Rouse1,3, James D Brooks4 and Jesse K McKenney1,4

1Department of Pathology, Stanford University, Stanford, CA, USA; 2Department of Pathology, El Camino Hospital, Mountain View, CA, USA; 3Department of Pathology, Veterans Affairs Palo Alto Medical Center, Palo Alto, CA, USA and 4Department of Urology, Stanford University, Stanford, CA, USA

Brachyury is recognized as a specific marker for notochord-derived tissues and neoplasms, and has become a defining immunohistochemical feature of chordoma. The main differential diagnostic consideration for chordoma is chondrosarcoma, which is known to lack brachyury expression. However, within the spectrum of genitourinary neoplasia, metastatic germ cell tumors and clear cell renal cell carcinoma may also be close morphological mimics of chordoma, particularly given the increasing prevalence of small tissue samples from image-guided biopsies. Although immunoreactivity for brachyury has been reported in a few germ cell tumors, a thorough characterization of staining by specific subtype has not been performed in a large series. Additionally, brachyury expression in clear cell renal cell carcinoma has not been well studied. In this study, immunohistochemical expression with the brachyury antibody was evaluated in 111 germ cell tumors, 30 non-neoplastic and neoplastic (non-germ cell) testicular tissues, and 184 metastatic clear cell renal cell carcinomas using tissue microarray technology. In addition, immunoreactivity for PAX-8 and SALL-4 was evaluated in 12 chordomas on whole section. No nuclear brachyury expression was identified in any of the 101 germ cell tumors within the tissue microarray (including choriocarcinoma (1), embryonal carcinoma (20), intratubular germ cell neoplasia unclassified (2), seminoma (64), spermatocytic seminoma (1), teratoma (5) and yolk sac tumor (8)), in any of the 30 non-neoplastic and neoplastic (non-germ cell) testicular tissues, or in any of the 10 whole-section seminomas. All 184 metastatic clear cell renal cell carcinomas were also non-reactive for brachyury. All 12 chordomas showed strong nuclear immunoreactivity for brachyury, but no expression of SALL-4. In all, 1 of 12 chordoma cases showed patchy, 1 þ nuclear immunoreactivity for PAX-8. This study confirms the specificity of brachyury for chordoma in the differential diagnostic distinction from the potential genitourinary mimics, germ cell tumors and metastatic clear cell renal cell carcinoma. Modern Pathology (2011) 24, 425–429; doi:10.1038/modpathol.2010.196; published online 19 November 2010

Keywords: brachyury; chordoma; germ cell tumor; metastatic; renal cell carcinoma

Brachyury is recognized as a key transcription factor been thoroughly evaluated in previous studies expressed in notochord-derived tissues and neo- and does not express brachyury.2,5–9 However, with- plasms. Although expression of the novel antigen in the spectrum of genitourinary neoplasia, meta- brachyury has come to be considered a defining static germ cell tumors and clear cell renal cell diagnostic feature of chordoma,1–6 few studies carcinomas may also be close morphological mimics have thoroughly addressed specificity of the anti- of chordoma, particularly on a small biopsy sample. body. Chondrosarcoma, the most recognized differ- Immunoreactivity with the brachyury antibody has ential diagnostic consideration for chordoma, has been reported in a few germ cell tumors, either in cases without reported subtype5 or in seminomas;3 however, a full characterization of staining by Correspondence: Dr AR Sangoi, MD, Department of Pathology, tumor subtype has not been evaluated in a large El Camino Hospital, GC-33, Mountain View, CA, 94040, USA. series. Additionally, brachyury expression in clear E-mail: [email protected] Received 15 July 2010; revised 30 August 2010; accepted 30 cell renal cell carcinoma has not been thoroughly August 2010; published online 19 November 2010 studied. www.modernpathology.org Brachyury in germ cell tumors and renal cell carcinoma 426 AR Sangoi et al

Materials and methods Results

In all, 111 germ cell tumors, 184 metastatic clear cell No nuclear brachyury expression was identified renal cell carcinomas and 12 chordomas were in any of the 111 germ cell tumors, including 101 retrieved from the pathology archives of Stanford tissue microarray cases and 10 whole sections of University Medical Center and/or Veterans Affairs seminomas (Figure 1) (Table 1). In the 10 whole Palo Alto Medical Center. Standard whole sections sections of seminoma, brachyury expression was from each were reviewed and diagnoses for all cases also not identified in surrounding non-neoplastic were confirmed on routine hematoxylin and eosin- testis. Similarly, brachyury expression was not stained sections. A tissue microarray consisting of identified in any of the 30 non-germ cell tumor 101 germ cell tumors (including choriocarcinoma (1), testicular tissues present on the germ cell tumor embryonal carcinoma (20), intratubular germ cell microarray (including adenomatoid tumor (1), atro- neoplasia unclassified (2), seminoma (64), spermato- phy (7), cryptorchidism (1), fibrous pseudotumor cytic seminoma (1), teratoma (5) and yolk sac tumor (1), hypospermatogenesis (1), inflamed testis (2), (8)) using 1.2 mm diameter tissue cores was prepared Leydig cell hyperplasia (1), Leydig cell tumor (1), (Stanford tissue microarray 136) and evaluated as liposarcoma (3), normal epididymis (2), normal described in detailed elsewhere.10 Similarly, a tissue testis (9) and serous papillary carcinoma (1)). All microarray of 184 metastatic clear cell renal cell 184 metastatic clear cell renal cell carcinomas from carcinomas from 26 unique sites using 0.7 mm 26 unique sites were non-reactive for brachyury diameter tissue cores was prepared (in duplicate) (Figure 1) (Table 2). In all, 10 non-germ cell tumor and evaluated as above (Stanford tissue microarray malignancies and 5 non-neoplastic tissues present 238). Only metastatic clear cell renal cell carcinoma on the metastatic clear cell renal cell carcinoma cases with a documented prior primary renal cell array were also negative for brachyury (including carcinoma history (by chart review or in-house colorectal adenocarcinoma (1), hepatocellular carci- pathology database review) were included in the noma (1), lung adenocarcinoma (1), mammary study. Additionally, although some cases had syn- ductal carcinoma (1), melanoma (1), normal lymph chronous metastasis in other organs/sites (eg, lung node (1), normal placenta (4), ovarian clear cell plus multiple lymph node metastasis), only one carcinoma (1), prostatic adenocarcinoma (2), urinary representative block per patient was selected to bladder carcinoma (1) and uterine cervical adeno- eliminate data duplication. Immunohistochemical carcinoma (1)). All 12 chordomas showed nuclear staining using anti-brachyury antibody (rabbit poly- reactivity for brachyury, but no expression with clonal (1:50); Santa Cruz Biotechnology, Santa Cruz, anti-SALL-4. In all, 1 of 12 chordomas showed focal CA, USA; Ventana Benchmark XT (Tuscon, AZ, USA) 1 þ nuclear PAX-8 immunoreactivity. autostainer with EDTA retrieval) was performed using standard avidin-biotin technique on 4-mm Discussion thick formalin-fixed, paraffin-embedded tissue microarray sections mounted on charged slides baked at mRNA expression of brachyury, a T-box mesodermal 601C for 1 h. To address the potential staining transcription factor critical in the development of heterogeneity bias of a tissue microarray study, 10 notochord-derived tissues and neoplasms, has been whole sections of seminomas with associated intra- demonstrated in various human carcinomas;11 how- tubular germ cell neoplasia unclassified were also ever, few studies have comprehensively explored stained for brachyury, with the diagnoses confirmed immunohistochemical expression of brachyury in by immunostaining for OCT3/4 (anti-OCT3/4; goat other tissues to address the diagnostic specificity for polyclonal (1:400); Santa Cruz Biotechnology; Leica chordoma in non-notochord derived tumors with Bond Max (Bannockburn, IL, USA) autostainer with clear cytoplasm that may be close morphological citrate retrieval). Whole sections of 12 classic chor- mimics.5–7 Although the morphological overlap domas were used as positive control tissue, and only between chordoma and clear cell renal cell carcino- nuclear reactivity was scored as positive. Expression ma is well known, we have also encountered cases of PAX-8 (anti-PAX8, rabbit polyclonal antibody in which the distinction from central nervous (1:20); Proteintech, Chicago, IL, USA; Dako (Carpin- system germ cell tumors such as seminoma and teria, CA, USA) autostainer with citrate retrieval) and yolk sac tumor was considered, especially in the SALL-4 (anti-SALL4, clone 6E3 (1:200); Abnova, evaluation of poorly differentiated (anaplastic) Walnut, CA, USA; Ventana Benchmark XT autostai- chordomas. Within the spectrum of genitourinary ner with EDTA retrieval) was also evaluated in the 12 neoplasia, rare brachyury immunoreactivity has control chordomas cases. In all, 30 non-neoplastic been reported in testicular germ cell tumors (2/14 and non-germ cell neoplastic testicular tissues pre- cases; most unspecified as to subtype), including sent on the germ cell tumor tissue microarray were focally within non-neoplastic testis.5,7 A study used as negative controls. In all, 10 non-germ cell presented in abstract form has also reported focal tumor malignancies and 5 non-neoplastic tissues brachyury staining in four out of six seminomas,3 were used as negative controls for the metastatic whereas a very recent study reported no staining in clear cell renal cell carcinoma tissue microarray. six additional testicular germ cell tumors (four

Modern Pathology (2011) 24, 425–429 Brachyury in germ cell tumors and renal cell carcinoma AR Sangoi et al 427

Figure 1 (a) Hematoxylin and eosin (H&E), nested and cord-like growth typical of chordoma with intracytoplasmic vacuolization; (b) strong nuclear immunoreactivity for brachyury was seen in all 12 chordoma cases (highlighted in high power inset); (c) H&E, clear cell renal cell carcinoma showing the characteristic nested pattern with surrounding vascular septa; (d) no immunoreactivity for brachyury was seen in renal cell carcinoma (n ¼ 184); (e) H&E, classic seminoma with clear cytoplasm; (f) no immunoreactivity for brachyury was seen in any of the germ cell tumors (n ¼ 111). seminomas and two mixed germ cell tumors).7 hensive series of cases examined for brachyury The current study was undertaken because of the expression. potential morphological overlap of chordoma with In this study, brachyury expression was not these genitourinary tumors, the emerging recogni- identified in any of the 111 germ cell tumors or 30 tion of true primary peripheral chordomas in non-germ cell tumor testicular tissues investigated, unusual sites,5,8,12,13 and the lack of a large compre- including a lack of staining in the surrounding

Modern Pathology (2011) 24, 425–429 Brachyury in germ cell tumors and renal cell carcinoma 428 AR Sangoi et al

Table 1 Immunostaining results with anti-brachyury antibody in Table 2 Immunostaining results with anti-brachyury antibody in germ cell tumors metastatic CC-RCCa

Germ cell tumor Positive cases Metastatic CC-RCC site Positive cases

Choriocarcinoma 0/1 Bone 0/39 Embryonal carcinoma 0/19 Lung 0/38 Embryonal carcinoma (pure) 0/1 Lymph node 0/30 ITGCNUa 0/2 Soft tissue 0/17 Seminoma 0/52 Brain 0/10 Seminoma (pure) 0/12 Vessel 0/7 Seminoma+ITGCNUb 0/10 Skin 0/6 Spermatocytic seminoma 0/1 Adrenal 0/5 Teratoma (immature) 0/2 Parotid 0/5 Teratoma (mature) 0/3 Pleura 0/4 Yolk sac tumor 0/8 Nose/nasal 0/3 Total 0/111 Bronchus 0/2 Chest wall 0/2 aIntratubular germ cell neoplasia unclassified. Diaphragm 0/2 b Pancreas 0/2 Whole-section analysis. Breast 0/1 Heart 0/1 Small bowel 0/1 non-neoplastic testis of 10 whole-section semino- Liver 0/1 mas. Although this finding contrasts with other Omentum 0/1 reports of brachyury immunoreactivity in germ cell Pituitary 0/1 tumors (both seminoma3 and unspecified subtypes5) Retroperitoneum 0/1 5,7 Spleen 0/1 and non-neoplastic testis (spermatogonia), it Testicle 0/1 should be noted that both studies describe and/or Thyroid 0/1 depict immunoreactivity that is very focal in nature. Tonsil 0/1 Given the reports of high brachyury mRNA expres- Total 0/184

sion in testicular carcinoma (type unspecified) with a low level expression in normal testis,11 theoretically, Clear cell renal cell carcinoma. it would not be totally unexpected to find immunoreactivity for brachyury in paraffin sections. The different results in these studies may potentially be The main goal of this study was to confirm the secondary to differences in staining methodology; specificity of brachyury for chordoma in the differ- however, our chordoma controls showed strong and ential diagnostic distinction from these metastatic diffuse nuclear reactivity with brachyury and we genitourinary mimics. Other immunohistochemical utilized both the same antibody and the same stains would obviously be useful adjuncts in this dilution as reported previously. In order to address differential diagnostic setting. The widely available potential sampling bias in our study due to the renal cell carcinoma marker PAX-818,19 and the novel evaluation of small core tissue samples, we also germ cell marker SALL-420–22 would be useful in an evaluated brachyury staining in whole-section semi- immunohistochemical panel when renal cell carcinomas. Despite the fact that seminoma is the noma or a germ cell tumor are considered, respec- subtype most frequently reported to show brachyury tively. Although no chordomas expressed SALL-4 in staining, we again found no immunoreactivity in this study, 1 of 12 cases had patchy weak nuclear these additional cases. immunoreactivity for PAX-8. This finding under- Similarly, immunohistochemical expression of bra- scores the importance of using a panel of antibodies chyury was not identified in any of the 184 metastatic in this differential diagnostic setting. clear cell renal cell carcinomas. Brachyury immuno- In conclusion, unlike previous results, this study reactivity in renal cell carcinoma has, to the authors’ confirms the specificity of the brachyury antibody knowledge, previously been studied in only one for chordoma in the differential diagnostic distinc- series with six cases that were all negative.6 Particu- tion from the genitourinary mimics clear cell renal larly, given the rising incidence of metastatic renal cell carcinoma and germ cell tumors. cell carcinoma,14,15 the increasing use of targeted therapies, and the propensity for metastasis to virtually any body site,16,17 distinction of metastatic Acknowledgement clear cell renal cell carcinoma from chordoma is critical. Moreover, the high incidence of bony We acknowledge Shirley Kwok and Kelli Montgomery metastasis in this study (Table 2), an incidence for excellence in tissue microarray construction. supported by a previous study,16 makes metastatic clear cell renal cell carcinoma a particularly plausible Disclosure/conflict of interest morphological mimic of chordoma (both axial and extra-axial), particularly on a small biopsy sample. The authors declare no conflict of interest.

Modern Pathology (2011) 24, 425–429 Brachyury in germ cell tumors and renal cell carcinoma AR Sangoi et al 429

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