Purpose: Osteosarcoma (OS) and skeletal Ewing’s sarcoma (EWS), the most common pediatric primary bone tumors, are aggressive malignancies with a tendency for early pulmonary metastasis. Advances in therapy have increased the overall 5-year survival to approximately 70%; however, patients who relapse often fail to respond to salvage therapy. Thus, more effective adjuvant chemotherapy is needed for these patients. Several reports have claimed expression of the HER2/neu (c-erbB-2) gene in a high percentage of OSs and that its expression is a poor prognostic factor and have advocated monoclonal antibody therapy in those cases.

Experimental Design and Results: To validate the expression of HER2/neu in these tumors, archival cases of OS (n = 66) and EWS (n = 11) from 44 patients were assessed for HER2/neu gene expression by immunohistochemistry (DAKO rabbit polyclonal antibody A0485). Thirty-four cases (44%) demonstrated granular cytoplasmic staining, but none showed the distinct membranous staining characteristic of expression exhibited by breast carcinomas. To validate these immunohistochemical results, reverse transcription-PCR using RNA derived from archival material (n = 48) and several different primer pairs also failed to demonstrate the presence of amplifiable HER2/neu mRNA, although a known HER2/neu-positive breast carcinoma showed amplifiable mRNA.

Conclusions: Thus, in contrast to previous reports, our results demonstrate an absence of HER2/neu expression in OSs and EWSs. Our results show that HER2/neu is not expressed by these sarcomas, and we conclude that HER2/neu is therefore not an important prognostic factor and that anti-HER2/neu monoclonal antibody therapy is not appropriate for these patients.

OS3 and EWS, the most common bone tumors with a peak incidence between 10 and 30 years of age in 75% of cases, are highly aggressive mesenchymal tumors that produce osteoid and bone (1). OS occurs most commonly in the metaphysis of the long bones, the distal and proximal femur and the proximal humerus. The natural history of OS is characterized by a rapidly progressive course with early metastases primarily to lungs, which generally occurs within 1–2 years even after amputation and portends a poor prognosis.

EWS, unlike OS, is characterized by the presence of small round uniform cells with no evidence of matrix formation (1). Despite the aggressive nature of these neoplasms, early adjuvant treatment with pre- and postsurgical chemotherapy coupled with limb-sparing surgery has achieved a 5-year disease-free survival of approximately 60% in patients without pulmonary metastasis at presentation (2). Although this represents a significant advance in the management of these patients, those who relapse often fail to respond to salvage therapy. Attempts to intensify therapy based on less than optimal histological response to preoperative chemotherapy have not improved outcome. Thus, there is a need for alternate adjuvant chemotherapy for these patients (3).

The HER2/neu proto-oncogene (also called c-erbB-2), located at 17q21, encodes a Mr 185,000 transmembrane glycoprotein (p185HER2/neu) with tyrosine kinase activity (4). Originally isolated from ethylnitrosourea-induced rodent neuroblastoma (5), HER2/neu shares extensive sequence homology with the epidermal growth factor receptor gene and other members of the epidermal growth factor receptor family, which have been extensively reviewed and function as receptor tyrosine kinase (6). Since its identification in 1981, HER2/neu has been shown to be overexpressed in a variety of primary human carcinomas, including breast (7), ovarian and endometrial (8), and lung carcinomas (9). The oncogenic potential of HER2/neu in human neoplasms has been shown to be due to gene amplification and overexpression of the gene product and is not associated with a somatic mutation. The overexpression of HER2/neu has been recognized as an important prognostic factor, and therapies using recombinant humanized anti-HER2/neu antibodies have been approved as therapy for patients with BC with HER2/neu overexpression (10).

Overexpression of proto-oncogenes (c-myc, c-myb, c-met, c-sis, c-raf, and c-gli) in OS and EWS has been reported (11, 12, 13, 14, 15), whereas mutations in tumor suppressor genes RB and p53 also have been observed (16). Recently, several reports (17, 18, 19, 20) have demonstrated a high incidence (approximately 40%) of HER2/neu expression in OS and have postulated a role for Herceptin (recombinant humanized recombinant antibodies) for salvage therapy in primary bone tumors. Akatsuka et al.(20) have suggested that loss of HER2/neu expression is associated with pulmonary metastasis. However, an earlier study looking at a large cohort of pediatric sarcomas showed that HER2/neu overexpression was a rare event in these mesenchymal tumors (21).

To further investigate the possibility of HER2/neu overexpression in OS and EWS, we examined its expression in 33 patients with OS and 11 patients with EWS by immunohistochemistry and RT-PCR.

Patients.

FFPE blocks originally derived from primary bone tumors were obtained from the files of the Department of Pathology, University of Michigan Medical Center (Ann Arbor, MI). Pertinent history was extracted from the patients’ medical records. Pre- and posttreatment samples, as well as pulmonary metastatic lesions, were included where available. Institutional review board approval was obtained.

Immunohistochemical Staining for HER2/neu.

Deparaffinized sections of tumors were microwave-pretreated in citric acid buffer to retrieve antigenicity. Sections were incubated with blocking solution for 60 min at room temperature before being exposed to the primary antibody (rabbit polyclonal antibody, catalogue number A0485; DAKO, Carpinteria, CA; 1:400) for 30 min at room temperature. The immunocomplex was visualized by the immunoglobulin enzyme bridge technique using Vector ABC-peroxidase kit (Vector Laboratories, Burlingame, CA). The enzyme substrate 3,3′-diaminobenzidine tetrachloride was used, resulting in a brown reactant. Sections were weakly counterstained with 0.1% hematoxylin. Concurrent sections were stained with antibodies to vimentin to assess antigen preservation. Appropriate negative (no primary antibody) and positive (a known HER2/neu-overexpressing BC) controls were stained in parallel with each set of sarcomas studied. Several BC specimens that had metastasized to bone were also assessed for HER2/neu immunoreactivity after decalcification. These assays demonstrated that decalcification was not a deterrent to staining. The immunohistochemical stains were scored by a five-tier (negative, cytoplasmic, low-positive, medium-positive, and high-positive) modification of the normal grading scheme described previously by Wang (22). Low-, medium-, and high-positivity are characterized by complete membranous staining.

Expression of HER2/neu by RT-PCR.

Total RNA was isolated from FFPE tissue using the Ambion Paraffin Block RNA isolation kit (Ambion, Austin, TX) in accordance with the manufacturer’s protocols. cDNA was synthesized from 1 μg of RNA using a first-strand synthesis kit for RT-PCR (Retroscript; Ambion) with random decamer primers. RT-PCR amplification used the Bieche et al.(23) method to analyze HER2/neu expression. PCR primers were designed using Primer Express software (Applied Biosystems, Foster City, CA), and the sequences are given in Table 1. To amplify cDNAs, 2-μl aliquots of the reverse-transcribed cDNA were subjected to PCR in 50 μl of PCR buffer [10 mm Tris-HCl buffer (pH 8.3), 50 mm KCl, and 2.5 mm MgCl2] containing 0.8 mm deoxynucleotide triphosphates, 0.5 mm of each set of primers, and 2.5 units of platinum Taq DNA polymerase or native Taq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). PCR conditions for GAPDH were 35 cycles of denaturation at 94°C for 45 s, annealing at 57°C for 45 s, and chain extension at 72°C for 90 s. For HER2/neu and TBP-1, cDNA was amplified using a modified touchdown program (24) with annealing temperatures ranging from 72°C down to 64°C. Specifically, there were 5 cycles of each annealing temperature for a total of 25 cycles, with a final 15 cycles of amplification with an annealing temperature of 62°C. PCR products were resolved by PAGE using 8% gels, stained with SYBR Green I nucleic acid gel stain (FMC, Rockland, ME), and photographed under UV conditions. A known HER2/neu-overexpressing BC also was extracted under identical conditions and run in parallel as a positive control. Specimens were scored for the presence of the appropriately sized band. TBP-1, a component of the DNA-binding protein complex transcription factor IID, is expressed at a similar abundance to HER2/neu in BCs without HER2/neu amplification (23, 25).

Clinicopathological Data.

The OS cases (n = 33) had an age range of 6–75 years, with a mean age of 21.7 years and a male:female ratio of 16:17. There were 25 primary biopsies, 29 resections after chemotherapy [3 with Huvos grade IV chemotherapy response (100% necrosis) that were not assessed for HER2/neu immunohistochemistry], 9 pulmonary metastatic lesions, and 6 other lesions. There were 17 OSs from the femur, 10 OSs from the tibia, 2 OSs each from the maxilla and pelvis, and 1 OS each from the humerus and rib. The EWS (n = 11) cases had an age range of 16 months to 18 years, with a mean age of 14.4 years and a male:female ratio of 5:6. There were eight primary and three metastatic lesions.

Expression of the Erb-B2 Protein in OS and EWS.

Immunohistochemical analysis of 77 OS and EWS samples representing primary and metastatic lesions using the anti-HER2/neu polyclonal antibody showed no characteristic membranous staining normally associated with membrane-bound tyrosine kinases (Fig. 1,D), although 31 OS (47%) specimens and 3 (27%) EWS specimens demonstrated a diffuse granular cytoplasmic staining (Fig. 1, A and B), particularly in tumor giant cells (higher magnification insets in Fig. 1, A and B). Higher magnification of these cells demonstrated that the staining was cytoplasmic in origin, with no evidence of membrane staining. Furthermore, the cytoplasmic staining on higher magnification appeared to be granular. There was excellent concordance between multiple samples that expressed cytoplasmic staining in the primary biopsy, the posttreatment specimen, and, where available, the metastatic lesion. All positive and negative immunohistochemical controls were appropriate.

Expression of HER2/neu by RT-PCR.

To extend the negative membrane immunohistochemical results and to investigate any possible significance of the cytoplasmic immunoreactivity, RT-PCR for HER2/neu, GAPDH, and TBP-1 mRNA was performed using RNA from archival material. None of the specimens (36 OSs and 12 EWSs) demonstrated amplifiable HER2/neu mRNA (Figs. 2 and 3), although all specimens demonstrated amplifiable GAPDH and TBP-1 cDNA. Parallel RT-PCR of a HER2/neu-immunoreactive BC demonstrated a single intense band of the expected size in all PCR reactions.

In this report, we analyzed 33 cases of OS and 11 cases of EWS using routine immunohistochemical methods. Seventy-four separate pre- and posttreatment and metastatic tissue samples were analyzed and showed a uniform absence of membranous HER2/neu immunoreactivity. These results were extended by RT-PCR of a subset (19 OSs and 12 EWSs) of these cases, which showed a uniform absence of amplified HER2/neu mRNA with several different primers, although all of the specimens studied demonstrated GAPDH and TBP-1 mRNA. Thus, we are unable to detect any HER2/neu gene expression at the mRNA or protein level using these methods and conclude that HER2/neu is either not expressed or expressed at low levels in these tumors.

These results do not support the results of others (17, 18) in which a high percentage (up to 42%) of HER2/neu-positive cases was observed using immunological methods. HER2/neu expression in OS was first described by Onda et al.(18) using an immunoblotting technique on frozen tissue and immunohistochemistry on archival FFPE tissue. They demonstrated that 42% of their cases had overexpression of HER2/neu. Furthermore, analysis of HER2/neu gene expression by Southern blot hybridization in their cohort of patients failed to show evidence of gene amplification. Gorlick et al.(17) also demonstrated that 42% of their cases had HER2/neu expression by immunohistochemistry on archival FFPE tissue. The reasons for this discrepancy are unknown, but several possibilities exist. The high percentage of positive cases in these studies raises the possibility that the results are incorrect. Up to 43% of our specimens demonstrated granular cytoplasmic staining. This raises the possibility of overinterpretation in the previous studies of Gorlick et al.(17) and Onda et al.(18). Even in BCs, HER2/neu expression by immunohistochemistry is only seen in approximately 30% of cases (26). Differences in antibodies used may account for some of the discrepancies. Furthermore, unusual immunohistochemical staining patterns may lead to erroneous interpretation. Immunohistochemistry is a routine laboratory technique that offers several advantages over other tests measuring protein expression (27). Although it is a routine method, several disadvantages should be noted. The results of immunohistochemistry may be influenced by both the length and method of fixation, as well as by the degree of antigen retrieval (28). Secondly, the results are dependent on the antibody used. Press et al.(29) evaluated a panel of 28 antibodies (7 polyclonal and 21 monoclonal antibodies) on 187 FFPE BC cases previously shown to be HER2/neu positive and demonstrated that the ability to detect HER2/neu-positive cells varied from 6% to 82%. Gancberg et al.(30) has also demonstrated similar specificity and sensitivity results. Caution should also be exercised in interpreting immunohistochemical data because of the well-studied phenomenon of interobserver inconsistency, even with standardized reference material (31). In BCs, HER2/neu oncogene amplification has typically been associated with HER2/neu overexpression, as evidenced by membrane staining (29, 32). The importance of restricted cytoplasmic staining has been questioned (33). It should be noted that a study using the HER2/neu antibody 20N (a rabbit polyclonal antibody raised against a synthetic peptide antigen) has shown that cytoplasmic staining was correlated with HER2/neu amplification (33). However, a second antibody reported in the same study (33) recognizing a membrane-bound component of HER2/neu was thought to be superior in predicting prognosis in BC. Cytoplasmic staining may represent an alteration in the processing or stability of HER2/neu mRNA and p185HER2/neu or both, or it may represent gene amplification (34); conversely, it may represent, as several authors have suggested, a similar epitope on a mitochondrial protein (35, 36). HER2/neu also shares a common epitope with MDR-1 (37), a protein responsible for chemoresistance to a wide range of therapeutic agents and that has been shown to be overexpressed in OSs (38). The epitope in question maps to the NH2 terminus of the HER2/neu protein (37). It is not suggested that the monoclonal antibody used in the previous studies on OSs (5B5; PharMingen, San Diego, CA) is recognizing a foreign epitope but rather emphasizes the care and caution needed when interpreting data using a monoclonal antibody for immunohistochemistry. Interpretation of antibody 5B5 staining has long been recognized as problematic, with granular cytoplasmic staining in many BCs in addition to the membrane staining (39). It should be noted that Gorlick et al.(17) also studied the expression of MDR in their cohort of OSs and did not find a correlation between their HER2/neu and MDR staining. However, the antibody used in the present study is a polyclonal antibody and should not have the problems associated with the small epitopes associated with monoclonal antibodies. Furthermore, the absence of amplifiable mRNA for three different sets of primers for HER2/neu suggests that a truncated protein may not be responsible for the cytoplasmic staining seen in 45% of our cases.

Our results are supported by recent studies by others (40, 41, 42, 43). In an earlier immunohistochemical survey of sarcomas and small round cell tumors of childhood (21), no detectable immunoreactivity for HER2/neu was observed in 10 OSs and 14 EWSs. The study by Zhou et al.(43) suggests that the presence of cytoplasmic staining in pretreatment OS biopsies was associated with later pulmonary metastasis, although no membranous staining was observed as in our study. Maitra et al.(42) demonstrated no evidence of either cytoplasmic staining or the presence of HER2/neu gene amplification by fluorescence in situ hybridization.

Analysis of mRNA extracted from archival FFPE tissue is problematic; however, several recent reviews have demonstrated that this is a feasible method for the analysis of the histopathology archive (44, 45, 46, 47). Formalin fixation of the cell results in the fragmentation of mRNA into fragments of approximately 200–250 bases (48), chemical modification of all 4 bases of RNA, and the additional dimerization of adenine residues by methylene cross-linking (49). Taking these factors into consideration when designing primers and amplicons allows the researcher to look for gene expression in FFPE tissue.

Identification of prognostic factors and alternate therapeutic strategies are essential for the treatment of patients with osteogenic sarcoma and EWS. Although the use of antireceptor therapy has become the standard of care in patients with chemotherapy-refractory BC, it should be pointed out that the use of recombinant humanized anti-p185HER2/neu monoclonal antibody is not without considerable risk to these patients. Several recent studies have examined the safety of this drug and revealed that cardiac dysfunction sometimes leading to fatal arrhythmias is a major risk factor for these patients (50, 51, 52).

In summation, our results, although they represent a small sample population, demonstrate no evidence of HER2/neu expression in OS and skeletal EWS. This small patient population may constitute a Type II error; however, when our results are taken into consideration with the previously mentioned studies (40, 41, 42, 43) using three different antibodies (OS, n = 136 patients; EWS, n = 63 patients), they suggest that HER2/neu expression does not represent an important molecular event in the oncogenesis of either OS or EWS. Furthermore, because of the absence of significant expression of the HER2/neu protein, the use of humanized recombinant antibodies to HER2/neu (Herceptin) may not be useful in these patients.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was presented in part at the 6th Connective Tissue Oncology Society Meeting, Amsterdam, November 2000. D. G. T. is a Walther Cancer Institute Research Scholar at the University of Michigan and acknowledges their kind support.

3

The abbreviations used are: OS, osteosarcoma; EWS, Ewing’s sarcoma; RT-PCR, reverse transcription-PCR; BC, breast carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FFPE, formalin-fixed, paraffin-embedded; TBP-1, TATA box-binding protein.

Fig. 1.

Immunohistochemical staining of HER2/neu in OS and EWS. A, OS, patient 7, 6-year-old male, pretherapy specimen showing focal areas of chondrosarcoma differentiation (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200). Inset, higher magnification of tumor giant cell with cytoplasmic staining (×400). B, OS, patient 8, 13-year-old male, pretherapy specimen (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200). Inset, higher magnification of tumor giant cell with cytoplasmic staining (×400). C, EWS, patient 1, 16-year-old female with recurrent EWS (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200). D, positive control (BC), run in parallel to the bony tumors, demonstrating typical pattern of positivity restricted to the cytoplasmic membrane (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200).

Fig. 1.

Immunohistochemical staining of HER2/neu in OS and EWS. A, OS, patient 7, 6-year-old male, pretherapy specimen showing focal areas of chondrosarcoma differentiation (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200). Inset, higher magnification of tumor giant cell with cytoplasmic staining (×400). B, OS, patient 8, 13-year-old male, pretherapy specimen (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200). Inset, higher magnification of tumor giant cell with cytoplasmic staining (×400). C, EWS, patient 1, 16-year-old female with recurrent EWS (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200). D, positive control (BC), run in parallel to the bony tumors, demonstrating typical pattern of positivity restricted to the cytoplasmic membrane (anti-HER2/neu avidin-biotin-peroxidase complex/hematoxylin; original magnification, ×200).

Close modal
Fig. 2.

RT-PCR of OS for HER2/neu mRNA. Total RNA was isolated from FFPE tissue and subjected to RT-PCR as described in “Materials and Methods.” All specimens demonstrated amplification of GAPDH and TBP-1. None of the OSs demonstrated expression of mRNA for HER2/neu using three different primer sets. A HER2/neu-overexpressing BC also was extracted under identical conditions and run in parallel as a positive control.

Fig. 2.

RT-PCR of OS for HER2/neu mRNA. Total RNA was isolated from FFPE tissue and subjected to RT-PCR as described in “Materials and Methods.” All specimens demonstrated amplification of GAPDH and TBP-1. None of the OSs demonstrated expression of mRNA for HER2/neu using three different primer sets. A HER2/neu-overexpressing BC also was extracted under identical conditions and run in parallel as a positive control.

Close modal
Fig. 3.

RT-PCR of EWS for HER2/neu mRNA. Total RNA was isolated from FFPE tissue and subjected to RT-PCR as described in “Materials and Methods.” All specimens demonstrated amplification of GAPDH. None of the EWSs demonstrated expression of mRNA for HER2/neu. A HER2/neu-overexpressing BC also was extracted under identical conditions and run in parallel as a positive control.

Fig. 3.

RT-PCR of EWS for HER2/neu mRNA. Total RNA was isolated from FFPE tissue and subjected to RT-PCR as described in “Materials and Methods.” All specimens demonstrated amplification of GAPDH. None of the EWSs demonstrated expression of mRNA for HER2/neu. A HER2/neu-overexpressing BC also was extracted under identical conditions and run in parallel as a positive control.

Close modal
Table 1

Oligonucleotide primer sequences used

GeneOligonucleotideLocationSequencePCR product size (bp)
HER2/neu Upper primer 207U 5′-AGCCGCGAGCACCCAAGT  
(5′ end) Lower primer 353L 5′-TTGGTGGGCAGGTAGGTGAGTT 147 
HER2/neu Upper primer 2612U 5′-ACCTGCTGAACTGGTGTATGCA  
(middle) Lower primer 2735L 5′-GTAATTTTGACATGGTTGGGACTCTT 123 
HER2/neu Upper primer 4282U 5′-GAACAAAAGCGACCCATTCAG  
(3′ end) Lower primer 4404L 5′-AAGTAAAAACTAAACAGAAAAGCACTCTGT 122 
TBP-1 Upper primer 706U 5′-CACGAACCACGGCACTGATT  
 Lower primer 353L 5′-TTTTCTTGCTGCCAGTCTGGAC 89 
GAPDH Upper primer  5′-CCATGGAGAAGGCTGGGG  
 Lower primer  5′-CAAAGTTGTCATGGATGACC 208 
GeneOligonucleotideLocationSequencePCR product size (bp)
HER2/neu Upper primer 207U 5′-AGCCGCGAGCACCCAAGT  
(5′ end) Lower primer 353L 5′-TTGGTGGGCAGGTAGGTGAGTT 147 
HER2/neu Upper primer 2612U 5′-ACCTGCTGAACTGGTGTATGCA  
(middle) Lower primer 2735L 5′-GTAATTTTGACATGGTTGGGACTCTT 123 
HER2/neu Upper primer 4282U 5′-GAACAAAAGCGACCCATTCAG  
(3′ end) Lower primer 4404L 5′-AAGTAAAAACTAAACAGAAAAGCACTCTGT 122 
TBP-1 Upper primer 706U 5′-CACGAACCACGGCACTGATT  
 Lower primer 353L 5′-TTTTCTTGCTGCCAGTCTGGAC 89 
GAPDH Upper primer  5′-CCATGGAGAAGGCTGGGG  
 Lower primer  5′-CAAAGTTGTCATGGATGACC 208 

We thank the staff and patients of the Connective Tissue Oncology Program of the University of Michigan Comprehensive Cancer Center, without whom none of this work would be possible.

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