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© 1999 American Society for Clinical Oncology Expression of HER2/erbB-2 Correlates With Survival in OsteosarcomaFrom the Departments of Pediatrics, Pathology, Epidemiology and Biostatistics, Surgery, Orthopaedic Service, and Pathology, Memorial Sloan-Kettering Cancer Center; New York Presbyterian Hospital; and Cornell University Medical College, New York, NY; and Department of Pediatrics, Yale University Medical Center, New Haven, CT. Address reprint requests to Paul A. Meyers, MD, Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, Box 471, New York, NY 10021; email meyersp{at}mskcc.org
PURPOSE: In osteosarcoma, prognostic factors at diagnosis other than clinical stage have not been clearly identified. The aim of this study was to determine whether human epidermal growth factor receptor 2 (HER2)/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or event-free survival. PATIENTS AND METHODS: We performed a retrospective immunohistochemical study on material obtained from patients treated on the Memorial Sloan-Kettering Cancer Center T12 protocol between 1986 and 1993. Paraffin-embedded tissue was identified from 53 patients (73% of patients enrolled onto protocol) and stained for HER2/erbB-2, p53, and p-glycoprotein expression using standard monoclonal antibodies and methods. RESULTS: At the time of initial biopsy, 20 (42.6%) of 47 samples demonstrated high levels of HER2/erbB-2 expression. Higher frequencies of expression were observed in samples from patients with metastatic disease at presentation and at the time of relapse. Expression of HER2/erbB-2 correlated with a significantly worse histologic response (P = .03). In patients presenting with nonmetastatic disease, expression of HER2/erbB-2 at the time of initial biopsy was associated with a significantly decreased event-free survival (47% v 79% at 5 years, P = .05). p53 and p-glycoprotein expression did not correlate with histologic response or patient event-free survival. CONCLUSION: The correlation of HER2/erbB-2 expression with histologic response to preoperative chemotherapy and event-free survival in this study suggests that HER2/erbB-2 should be evaluated prospectively as a prognostic indicator. The correlation also suggests that clinical trials of antibodies that target this receptor, such as recombinant humanized anti-HER2 monoclonal antibody (Herceptin; Genentech, San Francisco, CA), should be considered for the treatment of osteosarcoma.
THE IDENTIFICATION OF effective chemotherapy for patients with osteosarcoma (OS) has led to significant improvement in patient outcome over the last several decades.1 Current protocols for the treatment of OS, which use a combination of surgery and chemotherapy, achieve a 5-year disease-free survival of approximately 70% in patients who do not have metastatic disease at presentation.2,3 Although this represents a high proportion, patients who relapse rarely respond to salvage treatment.1 One of the most important predictors of outcome in OS is the histologic response of tumors to preoperative chemotherapy.2-6 Attempts at intensifying therapy based on less than optimal histologic response have not improved outcome.4,7 The use of more intensive preoperative chemotherapy in subsequent clinical trials has increased the percentage of patients with good histologic response but has not changed the event-free survival (EFS).4 Outcome in OS, therefore, has not improved significantly over the last decade. A need exists for the capability to stratify patients at diagnosis into high- and low-risk subgroups. In many malignancies, such as acute lymphoblastic leukemia and neuroblastoma, biologic and clinical features at diagnosis provide a determination of patient risk for disease recurrence. This allows stratification of therapy, which may improve the ability to cure high-risk patients and minimize toxicity to the low-risk patients. At the present time, the ability to predict prognosis at diagnosis in OS is limited. The most important predictor of outcome at diagnosis is the presence or absence of metastases.1 The 15% to 20% of patients who present at diagnosis with metastatic disease have an extremely poor prognosis, with approximately 10% achieving a long-term EFS, although one recent study suggests this group of patients may have a higher survival rate.8-10 The metastases predictor is of no use in the 80% to 85% of patients who present with local disease only. In these patients, a limited number of determinants that predict outcome have been identified. The determinants are related to the size of the tumor (either by direct measurement or reflected in serum chemistries, such as lactate dehydrogenase or alkaline phosphatase) or the site of presentation (most likely related to the resectability of the tumor).11,12 Unfortuantely, these determinants are of insufficient predictive value to allow stratification of therapy. Some of the biologic factors evaluated thus far as potential prognostic factors in OS are the expression of p-glycoprotein and erbB-2.13,14 The expression of p-glycoprotein, the protein product of the multidrug resistance-1 (MDR1, PGY1) gene, results in the efflux of numerous natural compounds, including doxorubicin, which is a major component of OS therapy.13 In a previous study of 92 OS tumor samples, 30% were found to have expression of p-glycoprotein by immunohistochemistry. Elevated p-glycoprotein was associated with a decreased probability of EFS (P = .002). This study failed to show a correlation between p-glycoprotein expression and histologic response to preoperative chemotherapy or histologic response and outcome.13 The c-erbB-2 proto-oncogene, which encodes the human epidermal growth factor receptor 2 (HER2), encodes a protein structurally homologous to the epidermal growth factor receptor, although its actual ligand has not yet been identified.14 Overexpression of human c-erbB-2 induces malignant transformation of rodent fibroblasts. In a study of 26 OS tumor samples, 42% expressed erbB-2.14 Protein expression was associated with the presence of pulmonary metastases and decreased survival as well as a poor histologic response to preoperative chemotherapy.14 Alterations in p53 are known to occur in approximately 20% to 25% of OS.14-17 In many malignancies, such as breast, bladder, and colorectal cancer, abnormalities in p53 predict prognosis as well as chemotherapy responsiveness.18-20 This relationship has not been demonstrated in OS.14,17,21 This study was designed to identify prognostic factors assessable at diagnosis in OS by determining whether HER2/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or EFS. Archival paraffin-embedded tissue was identified from a cohort of patients treated uniformly at a single institution. The archival material was stained immunohistochemically for p-glycoprotein, HER2/erbB-2, and p53. Protein expression was related to histologic necrosis in tumors after preoperative chemotherapy and to EFS.
Patients All patients who presented to Memorial Sloan-Kettering Cancer Center with newly diagnosed, previously untreated, high-grade, fully malignant OS from 1986 to 1993 were offered the opportunity to participate in the T12 protocol.4 The pathology department reviewed all material. Written informed consent was obtained from all patients or their guardians before participation in the protocol. The Institutional Review Board of Memorial Sloan-Kettering Cancer Center reviewed the study. Patients were randomly assigned to one of two treatment regimens at the time of study entry. The results of this clinical trial have been published previously.4 Briefly, the standard arm was modeled after the Memorial Sloan-Kettering Cancer Center T10 protocol.2,6 The experimental arm of the study was designed to administer more chemotherapy before definitive surgery. Both regimens included chemotherapy with high-dose methotrexate, cisplatin, doxorubicin, bleomycin, cyclophosphamide, and dactinomycin. Patients with pulmonary metastasis underwent thoracotomy after surgery on the primary tumor. No significant difference was observed in patient EFS based on regimen assignment (78% v 73%); this allowed the entire group to be investigated as a single cohort.4 Histologic necrosis after preoperative chemotherapy was determined by a single pathologist using the Huvos grading system, as previously described.6,22 Briefly, grade 1 indicates no evidence of necrosis, grade 2 indicates areas of necrotic material with other areas of histologically viable tumor, grade 3 indicates only scattered foci of viable tumor seen, and grade 4 indicates no viable tumor seen in extensive sampling.6,22 Grades 3 and 4 are associated with a superior probability of EFS as compared with grades 1 and 2.2,6
Immunohistochemistry After rinses with phosphate-buffered saline for 30 minutes, slides were incubated with peroxidase-conjugated streptavidin (Dako) at a 1/500 dilution in 1% bovine serum albumin/phosphate-buffered saline for 45 minutes, then rinsed again in phosphate-buffered saline for 30 minutes. Color was developed by incubating slides in 0.06% diaminobenzidine in phosphate-buffered saline for 15 minutes. Slides were then rinsed with tap water, counterstained with Harris modified hematoxylin (Fisher, Pittsburgh, PA), rinsed for 2 seconds in 1% acid alcohol, rinsed for 15 seconds in ammonia water, dehydrated, and coverslipped. Positive controls, constituted by normal adrenal gland for p-glycoprotein and cases showing more than an 80% positive stain for p53 and HER2/erbB-2, as well as negative controls, in which the primary antibody was omitted, were included with each run. For HER2/erbB-2, expression was confirmed using the Herceptest kit (Dako) according to manufacturer's instructions. Each case was scored by a pathologist blinded to patient identity. The cases were scored as 0 (no staining), 1+ (1% to 25% of the cells staining positive), 2+ (26% to 50% of the cells staining positive), 3+ (51% to 75% of the cells staining positive), and 4+ (76% to 100% of the cells staining positive). For p-glycoprotein and HER2/erbB-2, the staining localized to the membrane. For p53, the staining was nuclear or cytoplasmic. For p-glycoprotein and p53, any staining was considered positive. For HER2/erbB-2, 2+ or greater staining was considered positive. A 2+ or greater staining with the 5B5 antibody was correlated with at least 1+ staining using the controls and criteria provided in the Herceptest kit. This staining intensity was the minimum level required for a breast cancer patient to be eligible for enrollment onto the clinical trials of recombinant humanized anti-HER2 monoclonal antibody (rhuMAB HER2, trastuzumab [Herceptin; Genentech, San Francisco, CA]).
Statistical Methods
Seventy-three patients were entered onto the Memorial Sloan-Kettering Cancer Center T12 protocol.4 Archival pathologic material could be identified from 53 patients (73% of patients enrolled onto the protocol). Initial biopsy material could be identified from 47 patients. The initial characteristics of the patients with identified pathologic material were not markedly different from those of the patients with no available archival material in terms of age, sex, presence of clinically detectable metastasis, and baseline alkaline phosphatase or lactate dehydrogenase (Table 1). If we could not identify biopsy material, we attempted to identify material from definitive surgery or recurrence. Patients with grade 4 histologic necrosis had no usable tissue at definitive surgery and a low probability of recurrence. For this reason, patients with grade 4 necrosis were relatively overrepresented in the nonassessable group. When we limited the analysis to patients for whom biopsy material was available, there was no difference between assessable and nonassessable patients. This group was, therefore, considered representative of the entire cohort of patients treated on the T12 protocol.
The patient clinical characteristics and the results of the immunohistochemical staining are listed in Table 2. Representative data are shown in Fig 1. In 12 (22.6%) of the 53 patients, p-glycoprotein expression was detected. In eight (15.1%) of the 53 patients, p53 expression was detected. In 24 (45.3%) of the 53 patients, HER2/erbB-2 protein overexpression was detected. At the time of initial biopsy, 20 (42.6%) of 47 samples demonstrated high levels of HER2/erbB-2 expression. Three of six patients who presented with lung metastases had HER2/erbB-2 expression. Ten (76.9%) of 13 resection samples obtained at the time of recurrence demonstrated HER2/erbB-2 overexpression.
Thirty-three of the biopsy samples that had been stained with the 5B5 antibody were stained using the Herceptest kit. The samples were selected so that approximately equal numbers had been graded from 0 through 4+ in prior staining. Of the 33 samples, 29 had an equivalent grading between the two antibodies. The remaining four biopsy samples had greater staining with the 5B5 antibody compared with Herceptest kit staining. No significant correlation was observed between p-glycoprotein or p53 expression and histologic necrosis after preoperative chemotherapy or EFS (Table 3). Overexpression of HER2/erbB-2 correlated significantly with inferior histologic necrosis after preoperative chemotherapy (P = .02, Table 3). High levels of HER2/erbB-2 expression at diagnosis were associated with a significantly worse EFS (78% v 40% at 5 years, P = .01). Excluding the patients who presented with metastatic disease, this difference in EFS remained significant (79% v 47% at 5 years, P = .05; Fig 2).
This study was designed to identify prognostic factors assessable at diagnosis in OS by determining whether HER2/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or EFS. In this cohort of patients p-glycoprotein and p53 expression did not correlate with either histologic necrosis after preoperative chemotherapy or patient EFS. The expression of HER2/erbB-2 correlated significantly with a poor histologic response to preoperative chemotherapy and decreased EFS. One major study suggested that p-glycoprotein expression is a prognostic factor in OS. A major concern with that study is that p-glycoprotein expression did not correlate with histologic necrosis after chemotherapy.13 A subsequent study by the same investigators ruled out the possibility that p-glycoprotein expression was associated with a more aggressive tumor behavior.24 The investigators concluded that p-glycoprotein expression must relate to chemotherapy responsiveness,24 making the lack of correlation with histologic necrosis after preoperative chemotherapy in the previous study particularly disconcerting. Another concern with the study was the exclusive reliance on immunohistochemistry. Furthermore, a subsequently published letter about the study raised technical concerns.25 Some later studies have also demonstrated an association between p-glycoprotein expression and outcome, whereas others have failed to demonstrate that relationship.26,27 One potential explanation for these discrepant results is unreliability of immunohistochemistry for p-glycoprotein, particularly on paraffin-embedded materials.27 A study that attempted to correlate p-glycoprotein expression, as measured by immunohistochemistry, with MDR1 PGY1–gene mRNA levels in OS failed to demonstrate an association.28 One suggestion was that, in later studies, the measurement of p-glycoprotein should be performed using multiple methods, such as immunohistochemistry, quantitative reverse transcriptase-polymerase chain reaction, and functional methodologies. It must be acknowledged that a flaw in the present study is the infrequency of positive expression of both p-glycoprotein and p53. As a result, there is limited power to conclude an association does not exist. It also must be acknowledged that not all mutations in p53 are detected through immunohistochemistry. Despite these limitations, the frequency of p-glycoprotein (22.6% in this study and 30% in previous studies) and p53 (15% in this study and 20% to 25% in previous studies) expression was not markedly different from that reported in previous studies.13-17 Certainly p-glycoprotein expression cannot be used as a basis of stratification of therapy until these issues are clarified. Likewise, previously performed limited studies of p53 expression in OS have not demonstrated an association with patient outcome or other clinicopathologic features.14,17,21 The association of overexpression of HER2/erbB-2 with an inferior outcome has been clearly demonstrated in numerous studies of breast cancer.29,30 HER2 overexpression may have a direct role in the pathogenesis and poor clinical course of certain human tumors. Transfection of HER2 into mouse fibroblast cells causes transformation, and the resulting cells are tumorigenic in nude mice.31 A previous study noted high levels of HER2/erbB-2 expression in OS and its association with pulmonary metastases and an inferior outcome.14 The present study has confirmed and extended those findings. In the prior study, 42% of OS samples expressed high levels of HER2/erbB-2, which is comparable to the 45% overexpression observed in the present study. We have observed a higher frequency of HER2/erbB-2 expression in samples from patients with pulmonary metastases at presentation and patients who subsequently have a poor histologic necrosis after preoperative chemotherapy. The present study expands on the previous work by demonstrating, in a larger, uniformly treated cohort of patients, that HER2/erbB-2 overexpression is associated with an inferior outcome even among patients presenting with localized disease. This strongly suggests that HER2/erbB-2 should be evaluated prospectively as a potential prognostic factor. We have confirmed the results of the HER2/erbB-2 immunohistochemistry using the Herceptest kit. The kit is used to determine the eligibility of breast cancer patients for rhuMAB HER2 as a therapeutic agent. It is clear that, in addition to identifying prognostic factors, new agents for the treatment of OS are essential because few agents have demonstrated significant activity in the treatment of this disease. Growth factors are an attractive target for therapy as they are known to be involved in tumor development.31 The results of rhuMAB HER2 in the treatment of breast cancer, as a single agent as well as in combination with other chemotherapy, has been encouraging. As a single agent, objective responses were observed in five of 43 assessable patients with metastatic breast cancer, including one complete remission and four partial remissions.32 Higher response rates, with no increase in toxicity, were seen with a combination of rhuMAB HER2 and cisplatin compared with cisplatin alone.33 These results, along with the determination of the high frequency of HER2/erbB-2 expression in OS and its association with a negative prognosis, have led us to develop phase II trials of rhuMAB HER2 in OS. A trial of weekly rhuMAB HER2 in patients with refractory or relapsed OS is currently planned as a multi-institutional phase II trial. A phase II window study of rhuMAB HER2 in combination with chemotherapy for patients with newly diagnosed metastatic OS is also under consideration.
Supported by Mr. and Mrs. Steven Stern, Mr. and Mrs. Aaron Feldman, the Guy M. Stewart Cancer Fund Inc, the National Children's Cancer Foundation, and the New York Marathon Limb Preservation Fund. R.G. is the recipient of an American Society of Clinical Oncology Career Development Award. We thank Karen Allison, Jackie Simpson, Stephanie Vitolano, and Michael Kellick for helping with the care of the patients treated on this protocol. We also thank John Morrison for data management and the immunohistochemistry core facility for their technical assistance.
1. Meyers PA, Gorlick R: Osteosarcoma. Pediatr Clin North Am 44:973-990, 1997[Medline] 2. Meyers PA, Heller G, Healey J, et al: Chemotherapy for nonmetastatic osteogenic sarcoma: The Memorial Sloan-Kettering experience. J Clin Oncol 10:5-15, 1992[Abstract]
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Provisor AJ, Ettinger LJ, Nachman JB, et al: Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: A report from the Children's Cancer Group. J Clin Oncol 15:76-84, 1997 4. Meyers PA, Gorlick R, Heller G, et al: Intensification of preoperative chemotherapy for osteogenic sarcoma: The results of the Memorial Sloan-Kettering (T12) protocol. J Clin Oncol 16:2452-2458, 1998[Abstract] 5. Rosen G, Marcove RC, Caparros B, et al: Primary osteogenic sarcoma: The rationale for preoperative chemotherapy and delayed surgery. Cancer 43:2163-2177, 1979[Medline] 6. Rosen G, Caparros B, Huvos AG, et al: Preoperative chemotherapy for osteogenic sarcoma: Selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 49:1221-1230, 1982[Medline] 7. Winkler K, Beron G, Delling G, et al: Neoadjuvant chemotherapy of osteosarcoma: Results of a randomized cooperative trial (COSS-82) with salvage chemotherapy based on histological tumor response. J Clin Oncol 6:329-337, 1988[Abstract] 8. Bacci G, Mercuri M, Bricolli A, et al: Osteogenic sarcoma of the extremity with detectable lung metastases at presentation. Cancer 79:245-254, 1997[Medline]
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Toguchida J, Yamaguchi T, Ritchie B, et al: Mutation spectrum of the p53 gene in bone and soft tissue sarcomas. Cancer Res 52:6194-6199, 1992 17. Lonardo F, Ueda T, Huvos AG, et al: P53 and MDM2 alterations in osteosarcoma: Correlation with clinicopathologic features and proliferative rate. Cancer 79:1541-1547, 1997[Medline] 18. Levesque MA, Yu H, Clark GM, et al: Enzyme-linked immunoabsorbent assay-detected p53 protein accumulation: A prognostic factor in a large breast cancer cohort. J Clin Oncol 16:2641-2650, 1998[Abstract] 19. Cordon-Cardo C, Sheinfeld J, Dalbagni G: Genetic studies and molecular markers of bladder cancer. Semin Surg Oncol 13:319-327, 1997[Medline]
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Ahnen DJ, Feigl P, Quan G, et al: Ki-ras mutation and p53 overexpression predict the clinical behavior of colorectal cancer: A Southwest Oncology Group study. Cancer Res 58:1149-1158, 1998 21. Ueda Y, Dockhorn-Dworniczak B, Blasius S, et al: Analysis of mutant p53 protein in osteosarcomas and other malignant and benign lesions of bone. J Cancer Res Clin Oncol 119:172-178, 1993[Medline] 22. Huvos AG, Rosen G, Marcove RC: Primary osteogenic sarcoma: Pathologic aspects in 20 patients after treatment with chemotherapy, en bloc resection and prosthetic bone replacement. Arch Pathol Lab Med 101:14-18, 1977[Medline] 23. Kalbfleisch JD, Prentice RL: The Statistical Analysis of Failure Time Data. New York, NY, Wiley, 1980
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Andrulis IL, Bull SB, Blackstein ME, et al: neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer: Toronto Breast Cancer Study Group. J Clin Oncol 16:1340-1349, 1998 30. Sjogren S, Inganas M, Lindgren A, et al: Prognostic and predictive value of c-erbB-2 overexpression in primary breast cancer, alone and in combination with other prognostic markers. J Clin Oncol 16:462-469, 1998[Abstract]
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Hudziak RM, Schlessinger J, Ulrich A: Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells. Proc Natl Acad Sci USA 84:7159-7163, 1987
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Baselga J, Tripathy D, Mendelsohn J, et al: Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J Clin Oncol 14:737-744, 1996 33. Pegram MD, Lipton A, Hayes DF, et al: Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 16:2659-2671, 1998[Abstract] Submitted March 18, 1999; accepted May 10, 1999. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
