A preliminary investigation of Beta-hCG expression in patients with osteosarcoma

Transcription

1 ONCOLOGY A preliminary investigation of Beta-hCG expression in patients with osteosarcoma K. Z. Masrouha, R. Khattab, A. Tawil, A. Abdallah, S. Saghieh, R. Haidar, M. Abboud, N. J. Khoury From American University of Beirut Medical Center, Beirut, Lebanon K. Z. Masrouha, MD, Resident S. Saghieh, MD, Associate Professor of Clinical Orthopaedic Surgery R. Haidar, MD, Professor of Clinical Orthopaedic Surgery Medical Center, Division of Orthopaedic Surgery, P.O. Box , Riad El-Solh, Beirut , Lebanon. R. Khattab, MD, Resident A. Tawil, MD, Professor of Clinical Pathology Medical Center, Department of Pathology and Laboratory Medicine, P.O. Box , Riad El-Solh, Beirut , Lebanon. A. Abdallah, MD, Resident M. Abboud, MD, Professor of Pediatric Hematology/ Oncology Medical Center, Department of Pediatrics and Adolescent Medicine, P.O. Box , Riad El-Solh, Beirut , Lebanon. N. J. Khoury, MD, Associate Professor of Clinical Radiology Medical Center, Department of Diagnostic Radiology, P.O. Box , Riad El-Solh, Beirut , Lebanon. Correspondence should be sent to Dr N. J. Khoury; nk01@aub.edu.lb 2012 British Editorial Society of Bone and Joint Surgery doi: / x.94b $2.00 J Bone Joint Surg Br 2012;94-B: Received 31 May 2011; Accepted after revision 17 November 2011 There are eight reported cases in the literature of osteosarcomas secreting β-hcg. Our primary aim was to investigate the rate of β-hcg expression in osteosarcoma and attempt to understand the characteristics of osteosarcomas that secrete β-hcg. We reviewed 37 histopathology slides (14 biopsies and 23 surgical specimens) from 32 patients with osteosarcoma. The slides were retrospectively stained for β-hcg expression. Patient and tumour characteristics, including age, gender, tumour location, subtype, proportion of necrosis, presence of metastases and recurrence were recorded. A total of five of the 32 tumours were found to be positive for β-hcg expression (one strongly and four weakly). This incidence of this expression was found in tumours with poor histological response to neoadjuvant chemotherapy. The use of β-hcg expression as a diagnostic, prognostic or follow-up marker is questionable and needs further investigation with a larger sample size. Human chorionic gonadotropin (hcg) is a glycoprotein which is usually produced by the placental syncytiotrophoblasts in healthy pregnant women from six to eight days after conception. 1 It is composed of two subunits which are non-covalently linked: alpha and beta. The alpha subunit is biochemically similar to pituitary gonadotropins (follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) and thyroid stimulating hormone (TSH), making it non-specific and of little use as a tumour marker. On the other hand, the beta (β) subunit, which is 80% homologous, has strong specificity and is used to diagnose pregnancy and many obstetric and gynaecological disorders including hydatiform moles, choriocarcinoma and ovarian neoplasms. 1 In addition, β-hcg may be produced by nongynaecological organs, including the testes, 2, colon, 1 liver, 1 lungs, 1,3 stomach, 1 bone 1,3-8 and breast. 9 Therefore, increased blood levels might be found in patients with tumours arising from these organs, particularly of the testes, bladder, and pancreas. 4,7 There are only eight patients reported in the literature with a diagnosis of osteosarcoma who were found to have immunohistochemical proof of β-hcg expression. 1,3-8 In six of these there were elevated serum levels of β-hcg. 1,4-8 Reports suggest the potential use of this glycoprotein as both a prognostic and follow-up marker in patients with osteosarcoma. 3,6 However, this has not been extensively studied in these patients. If a correlation is established between β-hcg expression and osteosarcoma, it might serve as a useful tool in the management of these patients. We present the results of immunohistochemical staining for β-hcg expression in histology slides from patients with osteosarcoma to assess the incidence of β-hcg positivity. Patients and Methods After obtaining Institutional Review Board approval we retrospectively reviewed histology slides from patients with osteosarcoma treated at the Children s Cancer Center of Lebanon, between 2002 and Initially, 37 cases of osteosarcoma were retrieved. The studied tumour tissues were originally fixed with formalin for four to six hours for open biopsy and 24 hours for excision biopsy specimens. These were then decalcified using a solution of hydrochloride and acetic acids (Surgipath Decalcifier II; Leica Biosystems, Richmond, Illinois) for a period of three to four hours for open biopsy and 12 to 24 hours for excisional biopsy specimens. The haematoxylin and eosin-stained sectioned slides were reviewed by an attending pathologist (AT) and a pathology resident (RK), taken from all available specimens, whether open biopsy at the time of diagnosis and/or excised tumour. The slides of all tumours were reviewed to select appropriate blocks. For each patient, the slide that demonstrated the greatest proportion of viable VOL. 94-B, No. 3, MARCH

2 420 K. Z. MASROUHA, R. KHATTAB, A. TAWIL, A. ABDALLAH, S. SAGHIEH, R. HAIDAR, M. ABBOUD, N. J. KHOURY Table I. Patient and biopsy details Patient/ Gender/ Age (yrs) Location Subtype Specimen Beta-hCG +/- Tumour necrosis (%) * Metastasis/ recurrence 1 / F / 17 Left tibia Telangiectatic Excised tumour Strong positive 25 Lung 2 / M / 21 Left femur Parosteal Excised tumour N/A - 3 / M / 11 Right tibia Osteoblastic/Chondroblastic Open biopsy 0 Lung 4 / M / 16 Right tibia Osteoblastic Excised tumour 35 Recurrence 5 / M / 13 T6 vertebra Osteoblastic/Fibroblastic Open biopsy 0-6 / M / 11 Left femur Osteoblastic Excised tumour 50-7 / F / 8 Right tibia Telangiectatic Open biopsy N/A - 8 / M / 17 Left humerus Osteoblastic Excised tumour N/A Lung 9 / M / 14 Left ilium Osteoblastic Excised tumour Weak positive 50 Lung 10 / F / 15 Right femur Osteoblastic/Fibroblastic Open biopsy N/A - 11 / M / 12 Left femur Osteoblastic Excised tumour 80 Lung 12 / M / 10 Right femur Osteoblastic Open biopsy N/A Lung and bone 13 / M / 9 Left tibia Osteoblastic Excised tumour / F / 11 Left femur Osteoblastic Excised tumour / M / 14 Left femur Osteoblastic Excised tumour / M / 15 Right femur Chondroblastic Excised tumour 5 Lung 17 / F / 19 Left femur Osteoblastic/Chondroblastic Core biopsy N/A - 18 / F / 18 Right femur Fibroblastic Excised tumour 80 Lung 19 / F / 16 Left femur Osteoblastic Excised tumour < 5 Lymph node and recurrence 20 / F / 18 Left femur Osteoblastic Open biopsy N/A - Excised tumour / F / 6 Right femur Osteoblastic Open biopsy N/A Lung 22 / F / 17 Right tibia Osteoblastic Open biopsy Weak positive N/A - Excised tumour / M / 12 Right femur Osteoblastic/Chondroblastic Excised tumour / M / 20 Right femur Parosteal Open biopsy N/A Recurrence Excised tumour N/A 25 / M / 14 Right tibia Fibroblastic Core biopsy N/A - 26 / M / 17 Left femur Osteoblastic Excised tumour / F / 11 Left femur Osteoblastic Open biopsy N/A - 28 / F / 24 Left femur Parosteal Excised tumour 0-29 / F / 9 Left femur Chondroblastic Excised tumour 35 Lung 30 / F / 17 Left femur Osteoblastic Excised tumour 30 Lung 31 / M / 17 Right tibia Osteoblastic Open biopsy N/A Lung and bone Excised tumour Weak positive / M / 5 Left femur Telangiectatic Core biopsy Weak positive N/A Lung Excised tumour Weak positive 50 * N/A, not applicable tumour cells was chosen for staining. A total of five cases were excluded, four of which had complete necrosis of the tumour and no viable cells to stain, and one case that was an initial biopsy from another institution for which the specimen block was not available. Beta-hCG immunostaining was performed on the blocks of the remaining 32 cases. Data for each patient, including age, gender, location of tumour, subtype, source of specimen (biopsy and/or excised tumour), proportion of the specimen which was necrotic, and the presence or development of metastases or recurrence was recorded. Of the 32 patients included in the study, 18 were male and 14 were female, with a mean age of 14.2 years (5 to 24). The mouse anti-β-hcg antibody clone: ZSH17 (Zymed, San Francisco, California) was used on formalin-fixed, paraffin-embedded tissue sections. No antigen retrieval was required. Appropriate positive control tissue (normal placental tissue) was used. Negative controls consisted of additional tissue sections of tumour tissue, stained similarly but without the anti-β-hcg antibody. As there is no established grading system for staining patterns in osteosarcoma, the interpretation of staining was based on both the strength and intensity of staining compared with the positive control (weak versus strong intensity). Results The patient population and results are summarised in Table I. There were 23 slides from excised tumour specimens, after neoadjuvant chemotherapy, and 14 slides from biopsy specimens taken at the time of diagnosis. A total of five patients had slides from specimens of both the biopsy and excised tumours. In 18 patients in whom we only analysed the excised tumour, the diagnostic biopsy specimens THE JOURNAL OF BONE AND JOINT SURGERY

3 A PRELIMINARY INVESTIGATION OF BETA-HCG EXPRESSION IN PATIENTS WITH OSTEOSARCOMA 421 Fig. 1a Fig. 1b Figure 1a MR STIR sequence sagittal image of the leg of a 17-year-old female (patient 1) showing a large lobulated tumour arising from the distal metadiaphysis of the tibia causing bone destruction and invading the ankle joint. A large soft-tissue component is fungating anteriorly through the skin. The tumour has a heterogeneous signal with areas of both high and low signal intensity. There is associated subcutaneous oedema of dorsum of the foot (arrow). Figure 1b coronal CT image of the lungs showing bilateral diffuse metastases. were unavailable because the patients underwent primary diagnosis at other institutions and were referred to us for further management. In nine patients for whom we only analysed the diagnostic biopsy material, the excisional specimens were not available because the patients were either transferred to the care of other institutions (n = 3), lost to follow-up (n = 3) or resection was not indicated (n = 3). Of the 32 patients, 29 had high-grade tumours histologically, consisting of a conventional type in 26 patients (osteoblastic in 22, chrondroblastic in two and fibroblastic in two) in addition to three patients with a telangiectatic type. The remaining three patients had a low-grade parosteal osteosarcoma. A total of five patients had slides which stained positively for β-hcg expression (Table I). One was strongly positive (patient 1) while four cases had weakly positive staining. Two of these (patients 22 and 31) had one positive specimen and one a negative specimen. Patient 22 had a positive biopsy with a negative excised specimen while patient 31 had a negative biopsy with a positive excised specimen. Four of these patients were adolescents, three of whom were 17 years old and one was 14 years old, while the fifth was five years of age. Clinically aggressive tumour was determined based on the initial presence or development of metastasis and/or tumour recurrence during the follow-up period. Of the 27 patients who had negative staining, 12 had aggressive tumours. Among the five cases that stained positively, four had aggressive tumours. The proportion of tumour necrosis in the five patients with positive staining ranged from 25% to 70%, while in those with negative staining the proportion of necrosis ranged from 0% to 95%. Two patients with a high degree of necrosis ( 5% viable cells) did not show β-hcg staining. The number of patients with positive staining was too small for reliable statistical comparison, but there was a tendency towards increased aggressiveness and reduced tumour necrosis among those that stained positive, since all the patient s specimens which stained positively had a poor histological response with more than 10% viable tumour cells after chemotherapy. The single slide which was strongly positive was from a 17-year-old female patient (patient 1) who was transferred to our institution after undergoing neoadjuvant chemotherapy for osteosarcoma of the tibia that was fungating through the skin. MR imaging revealed a large distal tibial tumour invading the soft tissues and ankle joint (Fig. 1a). There were bilateral pulmonary metastases (Fig. 1b). Following below-knee amputation, histological analysis showed telangiectatic osteosarcoma and 25% tumour necrosis (Fig. 2a). Immunohistochemical staining for β-hcg expression from tumour specimens was strongly positive (Fig. 2b). Of the four cases which had weakly positive staining, the first was from a patient with a high-grade tumour of the osteoblastic subtype in the left ilium (patient 9). He presented with lung metastases and local tumour invasion of the hip. He responded poorly to chemotherapy, with 50% tumour necrosis, and subsequently died while undergoing treatment. The second specimen was from a patient with a clinically non-aggressive tumour without metastases at presentation (patient 22). She responded well to chemotherapy, with 70% tumour necrosis, and had no recurrence at her last follow-up. The slide that stained positively was from the VOL. 94-B, No. 3, MARCH 2012

4 422 K. Z. MASROUHA, R. KHATTAB, A. TAWIL, A. ABDALLAH, S. SAGHIEH, R. HAIDAR, M. ABBOUD, N. J. KHOURY Fig. 2a Fig. 2b Histological images of the tumour shown in Figure 1a, a) showing telangiectatic osteosarcoma with a haemorrhagic cystic space surrounded by a highly pleomorphic viable tumour with abundant osteoclasts and mitotic figures (haematoxylin & eosin; magnification 50), and b) a β-hcg immunohistochemical stain showing strong nuclear positivity in 1% of the tumour cells (stained brown; magnification 100). biopsy sample taken at the time of diagnosis, while the slide from the excised tumour specimen did not stain. The third specimen was from a patient with an initially clinically non-aggressive tumour that presented with no metastases (patient 31). After the third cycle of chemotherapy, repeat MR imaging revealed an increase in the size of the tibial mass with soft-tissue extension and only 35% tumour necrosis. The patient subsequently developed lung and bony metastasis, but was followed-up at another institution. The fourth specimen was from a patient (patient 32) with an aggressive multifocal tumour that did not respond well to chemotherapy, with 50% tumour necrosis, and eventually developed lung metastases. He died while undergoing treatment. Discussion The potential value of serum β-hcg levels as a marker in osteosarcoma has not been adequately assessed as there are only a few reports of β-hcg osteosarcomas. 1-9 The results of our preliminary study suggest that although the incidence of β-hcg expression in osteosarcoma patients may not be high, it raises the question of the possible use of this marker in a subgroup of these patients. Of the eight reported cases in the literature of osteosarcoma with immunohistochemical evidence of β-hcg expression in tumour cells, six had elevated serum levels of β- hcg. 1-9 Only one report described retrospective immunohistochemical analysis of histological slides, similar to the technique we describe but with a smaller sample size. 7 Our patient whose tissue stained strongly positive for β-hcg expression had a highly aggressive fungating tumour that presented with lung metastases. Of the four cases that stained weakly positive, three were from patients with aggressive tumours that presented with or developed metastases and did not respond well to chemotherapy. This aggressive presentation correlates with cases reported in the literature whereby two presented with a large mass and skin necrosis over the tumour, two others presented with relapses one to two years following treatment, and another presented with a high grade osteosarcoma. 5-8 Our preliminary experience suggests that β-hcg expression may be related to tumour aggressiveness, but the incidence of its expression is too low with our small sample size to permit statistical correlation between the characteristics of tumours that do and those that do not express this marker. The apparent association with tumour aggressiveness led some authors to suggest that β-hcg expression may be due to tumour dedifferentiation, but others have found that dedifferentiated cells in their patients did not reveal β-hcg expression. 5,6,10 In the current study, all of our patients with positive β- hcg expression had histologically high-grade tumours of the osteoblastic (three patients) and telangiectatic (two patients) subtypes. In the literature, three cases were of the osteoblastic subtype, two were of the chrondroblastic subtype, and one was of the telangiectatic subtype. 3,5-8 The reports of patients with positive β-hcg expression, including those in our study, is listed in Table II. All our patients with positive β-hcg stain showed more than 10% vital tumour cells after initial chemotherapy denoting rather a poor histological response. The effect of chemotherapy on tumour cells with regards to β-hcg expression is not known. Earlier studies suggested that serum levels of β-hcg decrease following treatment with chemotherapy in both trophoblastic and non-trophoblastic tumours that have proven β-hcg expression. 1,4 With regards to osteosarcoma, few previous THE JOURNAL OF BONE AND JOINT SURGERY

5 A PRELIMINARY INVESTIGATION OF BETA-HCG EXPRESSION IN PATIENTS WITH OSTEOSARCOMA 423 Table II. Osteosarcoma expressing β-hcg in the literature Cases Positive for β-hcg Serum β-hcg Authors analysed (n) expression (n) Gender Tumour type level Goldstein et al N/A * N/A N/A Gailani et al N/A N/A 3.2 ng/ml Kalra et al F Osteoblastic 5000 miu/ml Ordóñez et al F Telangiectatic miu/ml M Chondroblastic N/A Leidinger et al F Chondroblastic 717 miu/ml Demirtas et al F Osteoblastic 51 miu/ml Tuy et al F Osteoblastic 2177 miu/ml Current study 32 5 F Telangiectatic N/A M Osteoblastic N/A F Osteoblastic N/A M Osteoblastic N/A M Telangiectatic N/A Total * N/A, not available reports with β-hcg expression have shown a decrease in serum levels of β-hcg following neoadjuvant chemotherapy and/or post-surgical resection, which would be considered a good response to treatment. 3,5-7 However, those reports did not demonstrate the effect of chemotherapy on immunohistochemical staining. Furthermore, to our knowledge, there are no reports in the literature that discuss the potential change in the behaviour of osteosarcoma cells at the DNA and/or immunohistochemical levels, which might be induced by chemotherapeutic agents. In the current study, four of five patients with positive β- hcg expression had their specimens taken from the excised tumour subsequent to chemotherapy. However, there was one case (patient 22) with a positive slide from a biopsy specimen, while the excised specimen did not stain for β- hcg. Theoretically this may be related to the effect of chemotherapy and hence may raise the possibility that our negatively-stained excised specimens were false negatives. However, necrosis of the tumour cells that did express β- hcg could have contributed to these negative results. In addition, sampling of different sites of the tumour besides the non-uniform staining of cells may play a role in this discrepancy. In fact, a patient (31) who had negative staining of the initial biopsy had positive staining of the excised specimen probably related to sampling a negative site in the tumour. However, we could not obtain blood samples for serum β-hcg, diagnostic biopsy or surgical specimens in all our patients, which would have improved the analysis of the effect of chemotherapy and enabled firm conclusions. We recognise the drawback inherent in all retrospective studies, and the fact that both excised tumour specimens after chemotherapy, as well as biopsy specimens were used. Additionally we were unable to obtain serum β-hcg levels in our patients. Despite these limitations, we ensured that only slides with viable tumour cells were stained and that rigid technical protocols were used. We recommend that a prospective study is undertaken of a larger sample of patients. In conclusion, we found that the incidence of β-hcg expression in tumour cells of osteosarcoma patients to be low by immunohistochemical staining. Although all our positive cases occurred in histologically high-grade tumours with a poor histological response to neoadjuvant chemotherapy, and four of the five patients had clinically aggressive tumours, the relationship between this aggressive behaviour and positive staining remains to be determined from larger studies. The authors would like to thank the Board of Trustees of the Children s Cancer Center of Lebanon Foundation for their continuous support. We also thank the International Outreach Program at the St. Jude Children s Research Hospital, Memphis, Tennessee, and the Lebanese Ministry of Health for their invaluable support. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. References 1. Gailani S, Chu TM, Nussbaum A, Ostrander M, Christoff N. Human chorionic gonadotrophins (hcg) in non-trophoblastic neoplasms: assessment of abnormalities of hcg and CEA in bronchogenic and digestive neoplasms. Cancer 1976;38: Marcillac I, Troalen F, Bidart JM, et al. Free human chorionic gonadotropin beta subunit in gonadal and nongonadal neoplasms. Cancer Res 1992;52: Demirtas E, Krishnamurthy S, Tulandi T. Elevated serum beta-human chorionic gonadotropin in nonpregnant conditions. Obstet Gynecol Surv 2007;62: Goldstein DP, Kosasa TS, Skarim AT. The clinical application of a specific radioimmunoassay for human chorionic gondotropin in trophoblastic and nontrophoblastic tumors. Surg Gynecol Obstet 1974;138: VOL. 94-B, No. 3, MARCH 2012

6 424 K. Z. MASROUHA, R. KHATTAB, A. TAWIL, A. ABDALLAH, S. SAGHIEH, R. HAIDAR, M. ABBOUD, N. J. KHOURY 5. Kalra JK, Mir R, Kahn LB, Wessely Z, Shah AB. Osteogenic sarcoma producing human chorionic gonadotrophin: case report with immunohistochemical studies. Cancer 1984;53: Leidinger B, Bielack S, Koehler G, et al. High level of beta-hcg simulating pregnancy in recurrent osteosarcoma: case report and review of literature. J Cancer Res Clin Oncol 2004;130: Ordóñez NG, Ayala AG, Raymond AK, et al. Ectopic production of the beta-subunit of human chorionic gonadotropin in osteosarcoma. Arch Pathol Lab Med 1989;113: Tuy BE, Obafemi AA, Beebe KS, Patterson FR. Case report: elevated serum beta human chorionic gonadotropin in a woman with osteosarcoma. Clin Orthop 2008;466: Reisenbichler ES, Krontiras H, Hameed O. Beta-human chorionic gonadotropin production associated with phyllodes tumor of the breast: an unusual paraneoplastic phenomenon. Breast J 2009;15: Mack GR, Robey DB, Kurman RJ. Chondrosarcoma secreting chorionic gonadotropin: report of a case. J Bone Joint Surg [Am] 1977;59-A: THE JOURNAL OF BONE AND JOINT SURGERY