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Proteins expressed in osteosarcoma and serum levels as prognostic factors
The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17 www.elsevier.com/locate/ijbcb
Review
Proteins expressed in osteosarcoma and serum levels as prognostic factors Klemens Trieb *, Rainer Kotz Department of Orthopedics, Uni6ersity of Vienna, Wa¨hringergu¨rtel 18 -20, A-1090 Vienna, Austria Received 3 May 2000; accepted 15 September 2000
Abstract Osteosarcoma is the most frequent highly malignant bone-tumor with a peak manifestation during the second and third decade of life. Although survival rate increased up to 60 – 70% within the last 20 years, the problem of non-response to chemotherapy remains. Initial tumor size and response to neoadjuvant chemotherapy are the most accepted prognostic factors used for postoperative stratification of chemotherapy. The identification of patients with a bad response to therapy at the time of diagnosis would facilitate already a preoperative stratification of chemotherapy or a more aggressive regime to increase survival. This review reflects on recently described molecular markers but not on clinical parameters in human osteosarcoma with respect to their prognostic potential. This includes p53, the p-glycoprotein, the multidrug resistance gene, the humen epidermal growth factor receptor and metallothionein expression. Heat shock proteins have recently become important in osteosarcoma because of their prognostic value and their role in drug resistance. A short overview of serological markers is also given. Further results on drug resistance and survival may be provided by ongoing studies, which investigate the role of proteins of the apoptotic and antiapoptotic families in human osteosarcoma. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Osteosarcoma; Prognosis; Multidrug resistance; Heat shock protein; Apoptosis
Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2. Human epidermal growth factor receptor 2 (HER2) . . . . . . . . . . . . . . . . . .
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3. p53 protein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: + 43-1-404004070; fax: + 43-1-404004077. E-mail address: [email protected] (K. Trieb). 1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 6 6 - 2
K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
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4. Multidrug resistance: p-glycoprotein and multidrug resistance-1 (MDR1) gene . . .
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5. Metallothionein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6. Cytogenetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7. Heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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8. Antibodies to heat shock proteins in sera. . . . . . . . . . . . . . . . . . . . . . . . .
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9. Serological markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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10. Inhibitors of apoptotic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Osteosarcoma is the most frequent highly malignant tumor of bone and exhibits a peak in manifestation during the second and third decade of life. As a result of the introduction of neoadjuvant chemotherapy 20 years ago [1 – 6] an improvement in long-term survival rate from 10 – 20% to nearly 60 – 70% could be achieved [7]. Because then osteosarcoma patients receive full neoadjuvant multiagent chemotherapy [8] immediately following diagnosis, i.e. biopsy. Surgical margins are defined histologically according to the criteria of Enneking (wide, marginal and intralesional; [9]). Initial tumor size is a prognostic factor [10] and serves together with response to chemotherapy as a basis for risk-adapted postoperative chemotherapy. Histologic analysis is done on surgically resected tumors with regard to tumor response to preoperative chemotherapy according to different classifications [11 – 13]. Response to preoperative chemotherapy is considered as good when no or less than 10% viable tumor cells could be found whereas response is considered as poor when \10% viable tumor cells are found. However, it is available only after preoperative therapy and, therefore, its practical value is limited to determination of different post-
operative therapeutic regimes [1,5,6,14–18]. Due to the lack of reliable markers predicting response to chemotherapy at the time of diagnosis, preoperative risk-adaption is not possible to this date. The improved survival reveals long-term side effects of chemotherapy, a special problem in young patients. Thus, on the one hand, chemotherapy should be as minimal as possible to avoid late toxic effects such as cardiotoxicity [19], nephrotoxicity, secondary acute leukemia and ototoxicity gaining in importance with improved survival [6]. On the other hand, non-responder must have an intensive chemotherapy to avoid relapse, metastasis and death. Although prognosis increased dramatically, drug resistance is still one of the major problems in the treatment of cancers [20,21]. A lot of clinical prognostic parameters beside the mentioned above are established in the therapy of osteosarcoma. To discuss all of them would be beyond the scope of this review. So far, a marker is needed able to predict response to chemotherapy at the time of diagnosis thereby facilitating modified chemotherapeutic regimens already preoperatively. Therefore, research for new prognostic markers is very important. This review describes different proteins in tumor tissue or serum with regard to their prognostic and/or predictive value.
K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
2. Human epidermal growth factor receptor 2 (HER2) The HERs is encoded by the c-erbB-2 protooncogene and represents a protein structurally homologous to the epidermal growth factor receptor. Overexpression of the HER2 by transfection with the c-erbB-2 proto-oncogene in mouse fibroblasts results in malignant transformation and tumorgenic capacity [22]. A correlation between HER2/erbB-2 overexpression and decreased outcome has first been described for breast cancer. In human osteosarcoma c-erbB-2 is expressed in 40–45% and correlates with poor prognosis and early pulmonary metastases [23,24]. Additionally the expression correlated with a poor response to preoperative chemotherapy [23]. Overexpression of HER2 was not associated with amplification or gene rearrangement [23].
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been investigated by several studies, their prognostic potential remains controversial. Studies on p-glycoprotein expression, one of them with a large number of patients, showed a decreased outcome of patients with p-glycoprotein positive osteosarcomas [28–31], although no correlation between p-glycoprotein expression and response to preoperative chemotherapy was found. A recent study suggests, that only doxorubicin is related to p-glycoprotein mediated drug resistance [32]. The difference in success rates between multidrug chemotherapy and single-agent doxorubicin is greater for p-glycoprotein positive tumors than with p-glycoprotein negative specimens. Other studies could not find any correlation between p-glycoprotein expression and clinical outcome in patients with osteosarcoma [24,33].
5. Metallothionein expression 3. p53 protein expression Expression of the p53 protein and its mutations has been reported to be associated with poor prognosis and response to chemotherapy in many human malignancies. This could not be confirmed for human osteosarcoma. Several studies did not find a correlation between p53 expression (between 15 and 25% positive) and the clinical outcome and response to chemotherapy, suggesting that p53 fails as a marker for osteosarcoma [24–27].
Metallothioneins are intracellular cytoplasmatic proteins containing high amounts of thiol groups enabling them to bind heavy metals. They play a physiological role in the elemination of cytotoxic heavy metals and in the metabolism of trace metals. They are able to bind chemotherapeutics, for instance cisplatin. So far, expression of metallothionein in osteosarcoma did not show any correlation with the outcome [33,34].
6. Cytogenetic studies 4. Multidrug resistance: p-glycoprotein and multidrug resistance-1 (MDR1) gene The main problem in the treatment of osteosarcoma is nonresponsiveness to chemotherapy, i.e. drug resistance, which is related to many factors. One of the best described models is the plasma membrane protein p-glycoprotein, the product of the MDR1 gene, responsible for the efflux of many cytotoxic molecules from the cell including chemotherapeutic agents. Although expression of p-glycoprotein and MDR1 has
Genetic aberrations have been detected in osteosarcoma. The chromosomal changes in osteosarcoma are very complex and numerous. For instance an increased amplification was reported for 8q and 17p12, whereas a loss of 13q14 and 13q21 could be detected [35–38]. In these studies none of the chromosomal aberrations was associated with clinical risk factors or response to chemotherapy. To confirm their value as prognostic factors, further studies with a larger number of patients are needed.
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K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
7. Heat shock proteins Heat shock proteins are physiologically essential, highly conserved proteins executing a variety of vital intracellular chaperoning functions includung protein processing (Fig. 1). In case of harmful events they are upregulated to protect cells from death by protecting DNA and proteins from degradation. This phenomen was seen first in drosophila after elevation of temperature [39]. The mechanism of increased synthesis of hsps is uniform and independent from the stimuli. The hsps consist of constitutive and inducible forms and they are subdivided into families according to their molecular weight. For instance, hsp60 is a mitrochondrial hsp, hsp73 is the cytoplasmic/nuclear constitutive member of the 70-kDa family and hsp72 the stress-inducible nuclear hsp70 member [40]. Beside these functions, hsps have been shown to be strongly involved in immunologically mediated reactions, they can act as antigen and stimulate immune competent cells. In autoimmunity they can stimulate the immune system via ‘molecular mimikry’ (Fig. 2; [41]). In murine models hsps elicit cancer immunity [42] and additionally, T lymphocytes specific for hsps with cytotoxic potential were described and isolated from human osteosarcomas [43,44]. Mice develop T-cell lymphomas when they express the human hsp 70 gene [45], whereas transfection of tumors with hsp 65 in vivo resulted in the rejec-
Fig. 2.
tion of the tumor by development of an immune response, and tumor cells transfected with hsps in vitro lost in tumorgenicity as compared to untransfected cells [46]. Overexpression of hsp70 and hsp27 by gene transfection results in protection of many apoptotic stimuli as heat shock, anticancer drugs, radiation or tumor necrosis factors [47]. In sarcoma cell lines, hsp72 has been shown to be selectively expressed on their cell surface thereby overcoming protection and acting as the target for natural killer cells [48]. Hsps are frequently overexpressed in different human cancers and correlation of expression with prognosis and resistance or response to chemotherapy is not uniform in all tumors. It has been reported that hsp27 overexpression in human osteosarcomas is related with poor prognosis [34]. The authors group has shown that hsp72 is de-novo expressed in human osteosarcoma [49]. This hsp72 de-novo expression correlates with a good response to neoadjuvant chemotherapy.
8. Antibodies to heat shock proteins in sera
Fig. 1.
Autoantibodies against heat shock protein 90 in breast cancer have been reported to correlate with survival, the presence of involved lymph nodes and metastatic occurrence [50,51]. Recent results
K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
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Table 1 Association of proteins and antibodies with prognosis and response to neoadjuvant chemotherapy in osteosarcomaa Marker
Abb expr
Prognosis
Response
Reference
HER-2/cerbB-2 p53 p-Glycoprotein Metallothinein Chromosomal hsp 27 hsp72 Anti-hsp90 antibody Anti-hsp60 antibody
+ + +
+ − + − − +
+
[22–24] [25–27] [28–31] [33,34] [35–38] [34] [49] [52] [53]
a
+ + + +
− − − + + −
+, correlation; −, no correlation; abb exp, abberant expression.
suggest that the humoral immune response to hsp90 might be of predictive value in human osteosarcoma, because the presence of anti-hsp90 antibodies correlates with a good response to neoadjuvant chemotherapy and their absence correlates with the occurrence of metastases [52] (Table 1). The mechanism of the protective effect of hsp90 is not yet clarified, it might be accompanied by an immune response to the associated hsp or by a direct protective effect of anti-hsp90 antibodies. Antibody titers against hsp60 have been reported to be increased in patients with osteosarcoma, but there was no correlation with clinical parameters [53].
10. Inhibitors of apoptotic proteins The family of antiapoptotic proteins is growing, ranging from bcl-2, discovered first to newly detected ones like survivin. Apoptosis is a programmed cell death ending — in contrast to necrosis—in typical cell fragmentation. Proteins inducing (like APO-1 or bax) or inhibiting apoptosis have been discovered in the recent years and regulation of apoptosis is important in cancer cell growth. Their expression and prognostic value has been evaluated in many tumors, but so far, although promising canditates, they are not investigated in human osteosarcoma. Preliminary data suggest a poor prognosis for survivin negative high-grade osteosarcomas (Trieb et al., unpublished observations).
9. Serological markers Reports on the prognostic value of the alkaline phosphatase (AP) and lactate dehydrogenase (LDH) serum levels are controversial. Some groups describe a correlation of pretreatment levels of the two enzymes within the normal range with a better outcome [54], whereas other could find such a correlation only for LDH, but not for AP [55]. Such a correlation has been reported only for patients without metastases, but not for those with metastases [56]. In the COSS-80 and COSS-86 studies a correlation of a high AP serum level with a worse outcome was reported. Other serum factors like erythrocyte sedimentation rate were found to be of no prognostic value.
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K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17 [33] S.D. Shnyder, A.J. Hyes, J. Pringle, C.W. Archer, p-Glycoprotein and metallothionein expression and resistance to chemotherapy in osteosarcoma, Br. J. Cancer 78 (1998) 757 – 759. [34] H. Uozaki, H. Horiuchi, T. Ishida, T. Jijima, T. Imamura, R. Machinami, Overexpression of metallothioneins, gluthatione-S-transferase, heat shock protein 27 and lung-related protein in osteosarcoma, Cancer 79 (1997) 2336 – 2344. [35] O. Feugeas, et al., Loss of heterozigosity of the RB gene is a poor prognostic factor in patients with osteosarcoma, J. Clin. Oncol. 14 (1996) 467–472. [36] T.J. Hulsebos, E.H. Bijleveld, N.T. Osam, A. Westerveld, S. Leenstra, P.C. Hogendoorn, J. Bras, Malignant astrocytoma-derived region of common amplification in chromosomal band 17p12 is frequently amplified in high-grade osteosarcomas, Gen. Chrom. Cancer 18 (1997) 279 – 285. [37] M. Tarkkanen, I. Elomaa, C. Blomqvist, A. Kivioja, P. Kellokumpu-Lehtinen, T. Bo¨hling, J. Valle, S. Knuutila, DNA sequence copy number increase at 8q: a potential new prognostic marker in high-grade osteosarcoma, Int. J. Cancer 84 (1999) 114–121. [38] B. Wadayama, J. Toguchida, T. Shimizu, K. Ishizaki, M.S. Sasaki, Y. Kotoura, T. Yamamuro, Mutation spectrum of the retinoblastoma gene in osteosarcomas, Cancer Res. 54 (1994) 3042–3048. [39] F. Ritossa, A new puffing pattern induced by temperature shock and DNP in Drosophila, Experimentia 18 (1962) 571 – 578. [40] S. Lindquist, The heat-shock response, Ann. Rev. Biochem. 55 (1986) 1151–1191. [41] S. Kaufmann, Heat shock proteins and the immune response, Immunol. Today 11 (1990) 129–133. [42] R. Suto, P. Srivastava, A mechanism for specific immunogenicity of heat shock protein-chaperoned peptides, Science 269 (1995) 1585–1588. [43] K. Trieb, R. Windhager, S. Lang, R. Kotz, S. Dirnhofer, Evaluation of HLA-DR expression and T lymphocyte infiltrate in osteosarcoma, Pathol. Res. Pract. 194 (1998) 679 – 684. [44] K. Trieb, S. Lang, R. Kotz, Heat shock protein reactive T lymphocytes in human osteosarcomas, Pediatr. Hematol. Oncol. 17 (2000) 335–364.
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[45] J. Soo, Y. Park, J. Kim, E. Shim, C. Kim, J. Jang, S. Kim, W. Lee, T cell lymphoma in transgenic mice expressiong the human hsp 70 gene, Biochem. Biophys. Res. Commun. 218 (1996) 582 – 587. [46] K.V. Lukacs, A. Nakakes, C.J. Atkins, D.B. Lowrie, M.J. Colston, In vivo gene therapy of malignant tumours with heat shock protein 65 gene, Gene Ther. 4 (1997) 346 – 350. [47] M. Ja¨a¨ttela¨, Escaping cell death: survival proteins in cancer, Exp. Cell Res. 248 (1999) 30 – 43. [48] G. Multhoff, C. Botzler, L. Jennen, J. Schmidt, J. Ellwart, R. Issels, Heat shock protein 72 on tumor cells, J. Immunol. 158 (1997) 4341 – 4350. [49] K. Trieb, T. Lechleitner, S. Lang, R. Windhager, R. Kotz, S. Dirnhofer, Heat shock protein 72 expression in osteosarcomas correlates with good response to neoadjuvant chemotherapy, Hum. Pathol. 10 (1998) 1050 – 1055. [50] S.E. Conroy, D.S. Latchman, Do heat shock proteins have a role in breast cancer?, Br. J. Cancer 74 (1996) 717 – 721. [51] A. Jameel, M. Law, R.C. Coombes, Y.A. Luqmani, Significance of heat shock protein 90 as a prognostic indicator in breast cancer, Int. J. Oncol. 2 (1993) 1075 – 1080. [52] K. Trieb, R. Gerth, G. Holzer, J. Grohs, P. Berger, R. Kotz, Antibodies to heat shock protein 90 in osteosarcoma patients correlate with response to neoadjuvant chemotherapy, Br. J. Cancer 82 (2000) 85 – 87. [53] K. Trieb, R. Gerth, R. Windhager, G. Grohs, G. Holzer, P. Berger, R. Kotz, Serum antibodies to heat shock protein 60 are elevated in patients with osteosarcoma, Immunobiology 201 (1999) 368 – 376. [54] G. Bacci, S. Ferrari, P. Picci, C. Zolezzi, F. Gherlinzoni, D. Iantorno, A. Cazzola, Methotrexate serum concentration and histological response to multiagent primary chemotherapy for osteosarcoma of the limbs, J. Chemother. 8 (1996) 472 – 478. [55] L. Pochgool, T. Subhadharaphandou, M. Dhanachai, P. Hathirat, D. Sangthawan, R. Pirabul, S. Suporn Onsanit, N. Pornpipatpong, Prognostic factors among 130 patients with osteosarcoma, Clin. Orthop. Rel. Res. 345 (1997) 206 – 214. [56] P.A. Meyers, G. Heller, J.H. Healy, A. Huvos, A. Applewhite, M. Sun, M. LaQuaglia, Osteogenic sarcoma with clinically detectable metastasis at initial presentation, J. Clin. Oncol. 11 (1993) 449 – 453.
Review
Proteins expressed in osteosarcoma and serum levels as prognostic factors Klemens Trieb *, Rainer Kotz Department of Orthopedics, Uni6ersity of Vienna, Wa¨hringergu¨rtel 18 -20, A-1090 Vienna, Austria Received 3 May 2000; accepted 15 September 2000
Abstract Osteosarcoma is the most frequent highly malignant bone-tumor with a peak manifestation during the second and third decade of life. Although survival rate increased up to 60 – 70% within the last 20 years, the problem of non-response to chemotherapy remains. Initial tumor size and response to neoadjuvant chemotherapy are the most accepted prognostic factors used for postoperative stratification of chemotherapy. The identification of patients with a bad response to therapy at the time of diagnosis would facilitate already a preoperative stratification of chemotherapy or a more aggressive regime to increase survival. This review reflects on recently described molecular markers but not on clinical parameters in human osteosarcoma with respect to their prognostic potential. This includes p53, the p-glycoprotein, the multidrug resistance gene, the humen epidermal growth factor receptor and metallothionein expression. Heat shock proteins have recently become important in osteosarcoma because of their prognostic value and their role in drug resistance. A short overview of serological markers is also given. Further results on drug resistance and survival may be provided by ongoing studies, which investigate the role of proteins of the apoptotic and antiapoptotic families in human osteosarcoma. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Osteosarcoma; Prognosis; Multidrug resistance; Heat shock protein; Apoptosis
Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2. Human epidermal growth factor receptor 2 (HER2) . . . . . . . . . . . . . . . . . .
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3. p53 protein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: + 43-1-404004070; fax: + 43-1-404004077. E-mail address: [email protected] (K. Trieb). 1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 6 6 - 2
K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
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4. Multidrug resistance: p-glycoprotein and multidrug resistance-1 (MDR1) gene . . .
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5. Metallothionein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6. Cytogenetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7. Heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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8. Antibodies to heat shock proteins in sera. . . . . . . . . . . . . . . . . . . . . . . . .
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9. Serological markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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10. Inhibitors of apoptotic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction Osteosarcoma is the most frequent highly malignant tumor of bone and exhibits a peak in manifestation during the second and third decade of life. As a result of the introduction of neoadjuvant chemotherapy 20 years ago [1 – 6] an improvement in long-term survival rate from 10 – 20% to nearly 60 – 70% could be achieved [7]. Because then osteosarcoma patients receive full neoadjuvant multiagent chemotherapy [8] immediately following diagnosis, i.e. biopsy. Surgical margins are defined histologically according to the criteria of Enneking (wide, marginal and intralesional; [9]). Initial tumor size is a prognostic factor [10] and serves together with response to chemotherapy as a basis for risk-adapted postoperative chemotherapy. Histologic analysis is done on surgically resected tumors with regard to tumor response to preoperative chemotherapy according to different classifications [11 – 13]. Response to preoperative chemotherapy is considered as good when no or less than 10% viable tumor cells could be found whereas response is considered as poor when \10% viable tumor cells are found. However, it is available only after preoperative therapy and, therefore, its practical value is limited to determination of different post-
operative therapeutic regimes [1,5,6,14–18]. Due to the lack of reliable markers predicting response to chemotherapy at the time of diagnosis, preoperative risk-adaption is not possible to this date. The improved survival reveals long-term side effects of chemotherapy, a special problem in young patients. Thus, on the one hand, chemotherapy should be as minimal as possible to avoid late toxic effects such as cardiotoxicity [19], nephrotoxicity, secondary acute leukemia and ototoxicity gaining in importance with improved survival [6]. On the other hand, non-responder must have an intensive chemotherapy to avoid relapse, metastasis and death. Although prognosis increased dramatically, drug resistance is still one of the major problems in the treatment of cancers [20,21]. A lot of clinical prognostic parameters beside the mentioned above are established in the therapy of osteosarcoma. To discuss all of them would be beyond the scope of this review. So far, a marker is needed able to predict response to chemotherapy at the time of diagnosis thereby facilitating modified chemotherapeutic regimens already preoperatively. Therefore, research for new prognostic markers is very important. This review describes different proteins in tumor tissue or serum with regard to their prognostic and/or predictive value.
K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
2. Human epidermal growth factor receptor 2 (HER2) The HERs is encoded by the c-erbB-2 protooncogene and represents a protein structurally homologous to the epidermal growth factor receptor. Overexpression of the HER2 by transfection with the c-erbB-2 proto-oncogene in mouse fibroblasts results in malignant transformation and tumorgenic capacity [22]. A correlation between HER2/erbB-2 overexpression and decreased outcome has first been described for breast cancer. In human osteosarcoma c-erbB-2 is expressed in 40–45% and correlates with poor prognosis and early pulmonary metastases [23,24]. Additionally the expression correlated with a poor response to preoperative chemotherapy [23]. Overexpression of HER2 was not associated with amplification or gene rearrangement [23].
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been investigated by several studies, their prognostic potential remains controversial. Studies on p-glycoprotein expression, one of them with a large number of patients, showed a decreased outcome of patients with p-glycoprotein positive osteosarcomas [28–31], although no correlation between p-glycoprotein expression and response to preoperative chemotherapy was found. A recent study suggests, that only doxorubicin is related to p-glycoprotein mediated drug resistance [32]. The difference in success rates between multidrug chemotherapy and single-agent doxorubicin is greater for p-glycoprotein positive tumors than with p-glycoprotein negative specimens. Other studies could not find any correlation between p-glycoprotein expression and clinical outcome in patients with osteosarcoma [24,33].
5. Metallothionein expression 3. p53 protein expression Expression of the p53 protein and its mutations has been reported to be associated with poor prognosis and response to chemotherapy in many human malignancies. This could not be confirmed for human osteosarcoma. Several studies did not find a correlation between p53 expression (between 15 and 25% positive) and the clinical outcome and response to chemotherapy, suggesting that p53 fails as a marker for osteosarcoma [24–27].
Metallothioneins are intracellular cytoplasmatic proteins containing high amounts of thiol groups enabling them to bind heavy metals. They play a physiological role in the elemination of cytotoxic heavy metals and in the metabolism of trace metals. They are able to bind chemotherapeutics, for instance cisplatin. So far, expression of metallothionein in osteosarcoma did not show any correlation with the outcome [33,34].
6. Cytogenetic studies 4. Multidrug resistance: p-glycoprotein and multidrug resistance-1 (MDR1) gene The main problem in the treatment of osteosarcoma is nonresponsiveness to chemotherapy, i.e. drug resistance, which is related to many factors. One of the best described models is the plasma membrane protein p-glycoprotein, the product of the MDR1 gene, responsible for the efflux of many cytotoxic molecules from the cell including chemotherapeutic agents. Although expression of p-glycoprotein and MDR1 has
Genetic aberrations have been detected in osteosarcoma. The chromosomal changes in osteosarcoma are very complex and numerous. For instance an increased amplification was reported for 8q and 17p12, whereas a loss of 13q14 and 13q21 could be detected [35–38]. In these studies none of the chromosomal aberrations was associated with clinical risk factors or response to chemotherapy. To confirm their value as prognostic factors, further studies with a larger number of patients are needed.
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K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
7. Heat shock proteins Heat shock proteins are physiologically essential, highly conserved proteins executing a variety of vital intracellular chaperoning functions includung protein processing (Fig. 1). In case of harmful events they are upregulated to protect cells from death by protecting DNA and proteins from degradation. This phenomen was seen first in drosophila after elevation of temperature [39]. The mechanism of increased synthesis of hsps is uniform and independent from the stimuli. The hsps consist of constitutive and inducible forms and they are subdivided into families according to their molecular weight. For instance, hsp60 is a mitrochondrial hsp, hsp73 is the cytoplasmic/nuclear constitutive member of the 70-kDa family and hsp72 the stress-inducible nuclear hsp70 member [40]. Beside these functions, hsps have been shown to be strongly involved in immunologically mediated reactions, they can act as antigen and stimulate immune competent cells. In autoimmunity they can stimulate the immune system via ‘molecular mimikry’ (Fig. 2; [41]). In murine models hsps elicit cancer immunity [42] and additionally, T lymphocytes specific for hsps with cytotoxic potential were described and isolated from human osteosarcomas [43,44]. Mice develop T-cell lymphomas when they express the human hsp 70 gene [45], whereas transfection of tumors with hsp 65 in vivo resulted in the rejec-
Fig. 2.
tion of the tumor by development of an immune response, and tumor cells transfected with hsps in vitro lost in tumorgenicity as compared to untransfected cells [46]. Overexpression of hsp70 and hsp27 by gene transfection results in protection of many apoptotic stimuli as heat shock, anticancer drugs, radiation or tumor necrosis factors [47]. In sarcoma cell lines, hsp72 has been shown to be selectively expressed on their cell surface thereby overcoming protection and acting as the target for natural killer cells [48]. Hsps are frequently overexpressed in different human cancers and correlation of expression with prognosis and resistance or response to chemotherapy is not uniform in all tumors. It has been reported that hsp27 overexpression in human osteosarcomas is related with poor prognosis [34]. The authors group has shown that hsp72 is de-novo expressed in human osteosarcoma [49]. This hsp72 de-novo expression correlates with a good response to neoadjuvant chemotherapy.
8. Antibodies to heat shock proteins in sera
Fig. 1.
Autoantibodies against heat shock protein 90 in breast cancer have been reported to correlate with survival, the presence of involved lymph nodes and metastatic occurrence [50,51]. Recent results
K. Trieb, R. Kotz / The International Journal of Biochemistry & Cell Biology 33 (2001) 11–17
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Table 1 Association of proteins and antibodies with prognosis and response to neoadjuvant chemotherapy in osteosarcomaa Marker
Abb expr
Prognosis
Response
Reference
HER-2/cerbB-2 p53 p-Glycoprotein Metallothinein Chromosomal hsp 27 hsp72 Anti-hsp90 antibody Anti-hsp60 antibody
+ + +
+ − + − − +
+
[22–24] [25–27] [28–31] [33,34] [35–38] [34] [49] [52] [53]
a
+ + + +
− − − + + −
+, correlation; −, no correlation; abb exp, abberant expression.
suggest that the humoral immune response to hsp90 might be of predictive value in human osteosarcoma, because the presence of anti-hsp90 antibodies correlates with a good response to neoadjuvant chemotherapy and their absence correlates with the occurrence of metastases [52] (Table 1). The mechanism of the protective effect of hsp90 is not yet clarified, it might be accompanied by an immune response to the associated hsp or by a direct protective effect of anti-hsp90 antibodies. Antibody titers against hsp60 have been reported to be increased in patients with osteosarcoma, but there was no correlation with clinical parameters [53].
10. Inhibitors of apoptotic proteins The family of antiapoptotic proteins is growing, ranging from bcl-2, discovered first to newly detected ones like survivin. Apoptosis is a programmed cell death ending — in contrast to necrosis—in typical cell fragmentation. Proteins inducing (like APO-1 or bax) or inhibiting apoptosis have been discovered in the recent years and regulation of apoptosis is important in cancer cell growth. Their expression and prognostic value has been evaluated in many tumors, but so far, although promising canditates, they are not investigated in human osteosarcoma. Preliminary data suggest a poor prognosis for survivin negative high-grade osteosarcomas (Trieb et al., unpublished observations).
9. Serological markers Reports on the prognostic value of the alkaline phosphatase (AP) and lactate dehydrogenase (LDH) serum levels are controversial. Some groups describe a correlation of pretreatment levels of the two enzymes within the normal range with a better outcome [54], whereas other could find such a correlation only for LDH, but not for AP [55]. Such a correlation has been reported only for patients without metastases, but not for those with metastases [56]. In the COSS-80 and COSS-86 studies a correlation of a high AP serum level with a worse outcome was reported. Other serum factors like erythrocyte sedimentation rate were found to be of no prognostic value.
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