Can “Negative” Be Positive?
Authors: Fintan R. Steele
Journal: Molecular Therapy (2002)

This issue of Molecular Therapy features two articles that report disappointing preclinical results from a gene therapy approach to treating cancer [1Pawliuk R. Continuous intravascular secretion of endostatin in mice from transduced hematopoietic stem cells.Mol. Ther. 2002; 5: 345-351Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 2Eisterer W. Unfulfilled promise of endostatin in a gene therapy-xenotransplant model of human acute lymphocytic leukemia.Mol. Ther. 2002; 5: 352-359Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar]. The disappointment comes not from the failure of the approach. Indeed, the transduction of hematopoietic stem cells (HSCs) with the anticancer gene worked surprisingly well in the mouse models used, and should be useful for testing other genes that show therapeutic potential for a variety of diseases. But the negative part concerns the transferred gene itself: endostatin. Contrary to prior published work, there was no antiangiogenic effect from endostatin seen in these studies, even at circulating levels beyond those previously noted to be effective. In general, it is tough to get negative results past an editor onto a journal's pages. Thus, it is fair to ask what makes these two studies of particular interest to Molecular Therapy? Obviously, the first is the molecule itself. Many patient groups and not a few researchers have, rightly or wrongly, seized on endostatin as perhaps a “magic bullet” against many forms of cancer. Phase I and now phase II clinical trials are underway. These current papers are also of interest because they are very well performed and carefully reviewed. Further, they raise a series of questions about animal models and preclinical data that go beyond endostatin to almost any other gene therapy protocol under development. And finally, these papers may help outline that fog that stubbornly refuses to lift from the space between the bench and the bedside, and which obscures a fundamental rift between basic and clinical researchers that exists in other gene therapy clinical applications. First a bit about the manuscripts themselves. The goal of both was to establish a constant therapeutic level of endostatin by gene transfer, a goal also of the clinical trials underway, but by injection of recombinant protein rather than gene transfer (see below). To this end, a group of researchers led by Philippe Leboulch of Harvard and the Massachusetts Institute of Technology transduced mouse hematopoietic stem cells (HSCs) with a retrovirus expressing a secretable form of endostatin. (Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII.) Leboulch then collaborated with Connie Eaves of the Terry Fox Laboratory in Vancover and her coworkers to test this approach further after transduction of HSCs in SCID/NOD mice. Two different cancers were evaluated: human acute lymphocytic leukemia by Eaves and her collaborators, and murine fibrosarcoma by the Leboulch group (the fibrosarcoma cells were those tested in the original endostatin papers by recombinant protein injection [3O'Reilly M. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4250) Google Scholar, 4Boehm T. Folkman J. Browder T. O'Reilly M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature. 1997; 390: 404-407Crossref PubMed Scopus (1612) Google Scholar]). Neither approach demonstrated antiangiogenic efficacy, despite prolonged and robust systemic expression of functional endostatin. The researchers were also careful to sequence the secreted recombinant protein to confirm its identity and integrity, although they could not formally rule out protein misfolding or modification. In response to an editorial demand for a “positive” control, the researchers were—surprisingly—unable to inhibit the growth of similar tumors in non-transplanted mice after repeated subcutaneous injections of recombinant endostatin protein (prepared as described in the original papers). Even more puzzling is the fact that they could not detect a significant increase in serum endostatin concentrations after repeated subcutaneous injections, as assessed by a sensitive ELISA assay, whereas mice transplanted with the transduced bone marrow stem cells had 750% greater steady-state serum endostatin levels than controls. What went “wrong”? It is not clear that anything did, but the results from these two groups certainly contradict much of what has appeared in prior publications [3O'Reilly M. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4250) Google Scholar, 4Boehm T. Folkman J. Browder T. O'Reilly M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature. 1997; 390: 404-407Crossref PubMed Scopus (1612) Google Scholar, 5Bergers G. Javaherian K. Lo K.-M. Folkman J. Hanahan D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice.Science. 1999; 284: 808-812Crossref PubMed Scopus (878) Google Scholar, 6Blezinger P. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene.Nat. Biotechnol. 1999; 17: 343-348Crossref PubMed Scopus (299) Google Scholar, 7Sauter B.V. Martinet O. Zhang W.-J. Mandeli J. Woo S.L.C. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases.Proc. Natl. Acad. Sci. USA. 2000; 97: 4802-4807Crossref PubMed Scopus (245) Google Scholar], raising some interesting questions. Perhaps the single most difficult issue is what, precisely, is endostatin? That is not as silly a question as it appears: it is very unclear what is meant by “endostatin.” The form shown to be functional, and that being tested in the clinic, is a relatively insoluble form expressed in bacteria or yeast, with various modifications in preparation methods [3O'Reilly M. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4250) Google Scholar, 8Dhanabal M. Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma.Cancer Res. 1999; 59: 189-197PubMed Google Scholar, 9Taddie L. Inhibitory effect of full-length human endostatin on in vitro angiogenesis.Biochem. Biophys. Res. Commun. 1999; 263: 340-345Crossref PubMed Scopus (90) Google Scholar, 10Huang X. Soluble recombinant endostatin purified from Escherichia coli: antiangiogenic activity and antitumor effect.Cancer Res. 2001; 61: 478-481PubMed Google Scholar]. Does this “endostatin” differ in some mysterious way from the “native” form? Endostatin raised a great deal of interest and hope, primarily because it appeared to induce tumor regression rather than just slowing growth (as seen in the current studies). If nothing else, the varied literature on this subject suggests that there is a great need for a hardy and reproducible in vitro assay so that it is clear that the “endostatin” being tested is the same thing in different labs and settings. Establishment of such an assay is handcuffed by a lack of understanding of the mechanism and/or target of the molecule, that is, sufficient basic science to understand what endostatin is and what it does. This in itself does not necessarily mean that endostatin should not be tested for clinical efficacy, presuming that there are sufficiently rigorous and suitable controls in place. But even this is being questioned by a growing number of researchers. Phase I clinical endostatin trials were initiated at three sites based on the early exciting observations in murine models. One of these trials was supported directly by the company Entremed, Inc., in Rockville, Maryland, and the other two by the National Cancer Institute. On the one hand, these dose-ranging studies demonstrated that injection of recombinant endostatin was incredibly safe, more so than any other potential antitumor agent tried so far (with the possible exception of homeopathic remedies). On the other hand, few patients demonstrated significant or even minimal clinical benefit. However, the safety results and some suggestion in a few patients of some efficacy have led to phase II trials to test endostatin further. These trials, also sponsored by EntreMed, are currently enrolling at the Dana Farber Cancer Center (neuroendocrine tumors) and the MD Anderson Cancer Center (sarcoma and melanoma). The Anderson site includes plans for a pump, similar to that used by diabetics for insulin, to provide a continuous systemic level of the protein (the goal of the gene therapy approach), based on preclinical data. Interestingly, these trial designs reflect our total ignorance of endostatin mechanism and target. The only read-out for selecting a dose for further studies is attaining plasma concentrations that are comparable to the concentrations that were active in the early successful murine models. The current Molecular Therapy papers raise the question of what really are the appropriate measurements or outcomes? For the good of both endostatin and potential patients, it is critical that this be answered: “failure” of the clinical trials (that is, lack of tumor regression despite “therapeutic” levels of circulating endostatin) may not mean much scientifically, but could have disastrous public relations effects. The current trials also highlight the divide between clinic and bench, which is always present in translational biomedical science. It is true that drug development is different from basic research. It is also true that clinical and basic researchers are, in many ways, very different creatures: progress would never be made if promising results stayed on the bench top until basic researchers squeezed out every scrap of data. Furthermore, it is clear that preclinical research, including animal models, is not always (or even often) a reliable indicator of clinical outcome. Nevertheless, when carefully tested and controlled preclinical animal models provide ambiguous or even contradictory results, it is incumbent on basic and clinical researchers, academics and industry sponsors, to step back and evaluate more closely what is going on before proceeding with human testing. And it is incumbent on editors to publish such contradictory work, even if it is not in the best political interests of the journal. Endostatin and other antiangiogenic molecules being tested in labs and clinics around the world are incredibly exciting and offer great hope for many. But high hopes can also lead to big disappointments and nasty backlash, as the gene therapy world knows too well. Open discussion of peer-reviewed and published conflicting data will ultimately take science further into the clinic than competing press releases, financial interests, and tragically dashed patient hopes. This is true not only for endostatin, but any biomedical entity. Thus, to my mind, some “negative publishing” can be a very positive thing. Errata et al.Molecular TherapySeptember, 2002In BriefMolecular Therapy 6: 137 (2002). doi:10.1006/mthe.2002.0644 Full-Text PDF Open Archive