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Journal of Clinical Oncology, Vol 26, No 6 (February 20), 2008: pp. 830-832 © 2008 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.14.9062
Of Mice and (Wo)Men: Is This Any Way to Test a New Drug?University of Minnesota Cancer Center, Minneapolis, MN
The best laid schemes of mice and men oft go awry. Mouse models are undeniably vital to our understanding of the molecular basis and pathogenesis of breast cancer. In addition, they provide valuable information for the development of novel antineoplastic agents with regard to basic pharmacokinetics, pharmacogenomics, and toxicity analysis. Their role in evaluating cancer drug efficacy, however, has been limited by inconsistencies in the translation of basic science into effective clinical treatments.1 Ideally, mimicking the intricacies of human tumorigenesis and metastasis in model systems would simplify the development of targeted therapeutics; however, modeling such interplay between the tumor and its surrounding stroma, matrix proteins, immune cells, endothelia, and lymphatics in mice is a seemingly insurmountable task. Xenograft models employ human tumor cells isolated from metastatic deposits that may have been passaged in vitro hundreds of times before injection into an orthotopic site of an immunodeficient mouse host. Although some of the original molecular and cellular pathways of the tumor cells are preserved in cell lines,2 the macro- and microenvironment surrounding such tumors are artificial, and the true heterogeneity within the tumor is likely compromised. Their value in the historical development of cytotoxic chemotherapeutic agents, however, is supported by an extensive retrospective analysis from the National Cancer Institute.3 This study compared drug activity between xenograft model systems and phase II human participants. It demonstrated that clinical activity was absent in all six chemotherapy drugs in which xenograft activity was observed in less than one third of the xenografts tested, whereas 45% of the drugs that were active in more than one third of the xenografts were also active in clinical subjects. Although this is the best evidence to support the correlation between drug efficacy in preclinical xenograft mouse and early phase clinical studies in humans, it is tempered by the fact that only 17% of all cancer therapies that are evaluated in human studies will survive phase II evaluation.1,4 More recently, genetically engineered mouse models have become a promising alternative to traditional xenograft models in that they provide in situ tumor development in an immunocompetent setting. Their major limitation is their inability to replicate advanced cancer states and metastases—characteristics shared by participants in early-phase human clinical trials. Thus, their role in drug development yet remains to be defined. This can be particularly troublesome for monoclonal antibodies directed against human targets. Unless there is substantial cross-reactivity between mouse and human epitopes, host/antibody interactions cannot be modeled. Orthotopic transplantation of human tumor xenografts, on the other hand, promotes the establishment of high tumor volume and distant metastases. Although this seems preferable to other assessable systems, the technology is labor intensive, expensive, requires small-animal imaging to monitor response to therapy, and may not be easily reproducible. Although no one mouse model system can perfectly simulate human cancer biology, the ability of scientists to humanize these models will likely require a combination of such methodologies. As contemporary drug development takes aim at specific molecular targets, compared with their cytotoxic chemotherapeutic predecessors, it is fair to question whether mouse models are even appropriate for evaluating their efficacy. Despite such flaws, imperfect mouse models have provided important insights into breast cancer therapy. Cultured human estrogen receptor (ER)-positive breast cancer MCF-7 cells were found to be estrogen-dependent in vitro and in xenograft models.5 This model system was important in discovering the mechanisms of action and in vivo effects of antiestrogen therapy. Indeed, early studies of tamoxifen demonstrated its capacity to induce regression of ER-positive human breast cancer cell lines in nude mice.5 Such growth inhibition, however, was not observed in the ER-negative cell lines. These results were consistent with those of subsequent human clinical trials in which women with advanced, ER-positive, metastatic disease were found to have a significantly higher response rate to tamoxifen than did those with ER-negative tumors.6 Similar results were also observed in the adjuvant setting, and it is clear that tamoxifen significantly reduces the odds of recurrence and death resulting from breast cancer in women with ER-positive, but not ER-negative, disease.7 Studies in mice have also been crucial to the development of targeted cancer therapies against the epidermal growth factor receptor (EGFR) family, including HER-2 (human epidermal growth factor receptor 2). As the role of HER-2/neu in the pathogenesis and progression of breast cancer was defined, inhibitors against the surface membrane receptor were generated in mouse models. The 4D5 murine monoclonal antibody was shown to have specific dose-dependent antiproliferative effects in human tumor cell lines that overexpress HER-2.8 This led to the development of trastuzumab, a recombinant, humanized murine anti–HER-2 antibody that has demonstrated significant clinical benefit in the treatment of women with HER-2–overexpressing metastatic breast cancer and for adjuvant therapy in women with resected early-stage disease.9-11 Both ER and HER-2 have proven to be important prognostically and as biomarkers for treatment efficacy. Indeed, mouse models of acquired endocrine resistance have demonstrated the role for EGFR/HER-2 signaling and the cotargeting of these pathways,12 and such strategies can be tested in human clinical trials. The article by Guix et al13 in this issue of the Journal of Clinical Oncology reports a clinical study based on data obtained from a preclinical mouse xenograft study using erlotinib, an EGFR tyrosine kinase inhibitor. In the xenograft study, athymic mice were inoculated with human pulmonary adenocarcinoma cell lines with mutations in EGFR or K-ras. The hypersensitive xenograft tumors showed complete elimination of the tumor by day 14 of erlotinib treatment. In situ measurements of proliferative and apoptotic markers demonstrated maximal response to therapy as early as day 5, and they were assumed to be important biomarkers for clinical response. In the clinical trial, women with early-stage breast cancer were treated with a short course of erlotinib before their definitive surgery, and then drug activity was analyzed on the resected tumor. A 6- to 10- day course of erlotinib-induced inhibition of activated EGFR and HER-2 by examination of surgical specimens. A reduction in Ki67 expression, a surrogate marker of proliferation, was demonstrated in ER-positive tumors but not in those that overexpressed HER-2 or were HER-2, ER, and progesterone (PR) negative. In addition, the antiproliferative response was independent of EGFR status. The results of the clinical study suggest that a short course of established or experimental therapy in women with operable breast cancer may lead to the identification of biomarkers in the surgical specimen that may ultimately predict clinical response to therapy, as was observed in the mouse xenograft study. Surprisingly, measurements of the targets (EGFR or HER-2) could not identify tumors with a decrease in Ki67, and only ER status was a predictive biomarker. Much of the preclinical data have shown that ER and EGFR are inversely related, suggesting that EGFR would not be a target in ER-rich tumors. Guix et al show that erlotinib affects ER-phosphorylation on ser118, a mitogen-activated protein kinase phosphorylation site. It would be interesting to know the status of ser167, an Akt target. This study attempted to address two important questions in biomarker development: are there biomarkers that predict response to a specific drug? and are there biomarkers that serve as surrogates for clinical outcomes? The use of Ki67 as a surrogate marker for clinical benefit has been best illustrated by the results of the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) trial.14 In this study, postmenopausal women with ER-positive breast cancer were treated with a 12-week course of therapy before definitive surgery. At 2 weeks, patients underwent biopsy, and several biomarkers were measured. This study showed that anastrozole significantly suppressed tumor Ki67 expression more than tamoxifen or the combination of the two. This short-term effect on Ki67 was eventually found to be predictive of the long-term clinical outcome when the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial demonstrated a greater recurrence-free survival for women taking anastrozole for their adjuvant therapy.15 The IMPACT trial and the studies by Guix et al13 provide evidence for the use of a short course of neoadjuvant therapy to identify a surrogate marker of response (Ki67) that may best predict clinical response. Thus, mouse models that link surrogate biomarkers to clinical benefit are informative. Could we ever use these types of data in drug approval? Can robust surrogate markers of response serve as important clinical end points? In cardiology, for example, hypercholesterolemia is an established surrogate for coronary artery disease. It is now common practice for lipid-lowering agents to be prescribed for prevention of heart disease on the basis of their ability to decrease cholesterol. A similar surrogate for clinical benefit in cancer would be welcome. Guix et al13 have also demonstrated the pitfalls of using model systems in defining predictive biomarkers for response. Although the cell lines used suggested that expression of EGFR would be an excellent predictive biomarker for benefit to erlotinib, this was not seen. Although autophosphorylation of EGFR and HER-2 was documented, the inhibition of these biochemical events was not correlated with the antiproliferative effects. It is possible that EGFR/HER-2 regulates other breast cancer phenotypes, such as motility or invasion, processes that are not easily detectable in clinical trials. In contrast, ER expression and phosphorylation were better predictors of response, a relatively unexpected finding requiring additional laboratory investigation to completely understand. To their credit, Guix et al did not restrict their study to proving the hypotheses generated from in vitro and xenograft studies. If they restricted their study to only EGFR-overexpressing tumors, they would have likely missed the relevance of ER expression in predicting erlotinib induced changes in Ki67. So did the preclinical models tell us anything about conducting breast cancer clinical trials with erlotinib? On one hand, the biochemical modulation of EGFR and immediate downregulation of Ki67 observed in animal models could also be seen in human tumors. On the other hand, the biochemical inhibition of EGFR had little to do with decreasing proliferation in the absence of ER. Guix et al13 did what every good scientist should in designing a validation study with positive and negative controls. In human clinical trials, this translates into enrolling "all comers" and not only patients with tumors that fit the hypothesis. By not preselecting patients, they provided important data necessary to design the next generation of erlotinib studies. Moreover, they showed how a small neoadjuvant study with built-in tissue acquisition could provide strategies to optimize erlotinib use in breast cancer. The idea that EGFR inhibitors should be tested in triple-negative breast cancer is supported by the preclinical data, but not by this study. The mice helped, but plans went awry, and they could not substitute for a well-designed clinical trial. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest. AUTHOR CONTRIBUTIONS Conception and design: Douglas Yee Manuscript writing: Tufia C. Haddad, Douglas Yee Final approval of manuscript: Douglas Yee REFERENCES 1. Sharpless N, DePinho R: The mighty mouse: Genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov 5:741-754, 2006[CrossRef][Medline] 2. Fejzo M, Godfrey T, Chen C, et al: Molecular cytogenetic analysis of consistent abnormalities at 8q12–q22 in breast cancer. Genes Chromosomes Cancer 22:105-113, 1998[CrossRef][Medline] 3. Johnson J, Decker S, Zaharevitz D, et al: Relationship between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer 84:1424-1431, 2001[CrossRef][Medline] 4. Kola I, Landis J: Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 3:711-715, 2004[CrossRef][Medline] 5. Osborne C, Hobbs K, Clark G: Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice. Cancer Res 45:584-590, 1985 6. Bezwoda W, Esser J, Dansey R: The value of estrogen and progesterone receptor determinations in advanced breast cancer. Cancer 68:867-872, 1991[CrossRef][Medline] 7. Group EBCTC: Tamoxifen for early breast cancer: An overview of the randomised trials. Lancet 351:1451-1467, 1998[CrossRef][Medline] 8. Lewis G, Figari I, Fendly B, et al: Differential responses of human tumor cell lines to anti-p185HER-2 monoclonal antibodies. Cancer Immunol Immunother 37:255-263, 1993[CrossRef][Medline] 9. Carter P, Presta L, Gorman C, et al: Humanization of an anti-p185HER-2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89:4285-4289, 1992 10. Piccart-Gebhart M, Procter M, Leyland-Jones B, et al: Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353:1659-1672, 2005 11. Vogel C, Cobleigh M, Tripathy D, et al: Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20:719-726, 2002 12. Massarweh S, Osborne C, Jiang S, et al: Mechanisms of tumor regression and resistance to estrogen deprivation and fulvestrant in a model of estrogen receptor-positive, HER2/neu-positive breast cancer. Cancer Res 66:8266-8273, 2006 13. Guix M, de Matos Granja N, Meszoely I: Short preoperative treatment with erlotinib inhibits tumor cell proliferation in hormone receptor-positive breast cancers. J Clin Oncol 26:897-906, 2008 14. Dowsett M, Smith I, Ebbs S, et al: Short-term Ki67 during neoadjuvant treatment of primary breast cancer with anastrozole or tamoxifen alone or combined correlate with recurrence-free survival. Clin Cancer Res 11:951s-958s, 2005 15. Baum M, Budzar A, Cuzick J, et al: Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early stage breast cancer: First results of the ATAC randomised trial. Lancet 359:2131-2139, 2002[CrossRef][Medline] Related Article
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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