Prospects for high-dose chemotherapy seemed to dim last spring with the news that in only one of five breast cancer studies had patients with advanced disease or at high risk of recurrence fared significantly better on it than on standard lower-dose regimens. Despite that, the cancer research community is far from ready to give up on HDC striving instead to improve the treatment.
HDC would be highly lethal were it not for autologous bone marrow transplants which, because they contain blood-forming stem cells, can repair the damage the drugs do to the body's immune system. However, the transplants actually portions of the patient's own marrow which are removed before HDC and then returned are themselves vulnerable to injury from these drugs. HDC is, therefore, a once-only option for cancer patients and it is that limitation that some researchers are trying to address.
"Bone marrow toxicity is the major side-effect of any chemotherapy," said Charles Hesdorffer, M.D., of the Columbia University College of Physicians and Surgeons in New York. "So the thinking is that putting a gene for drug resistance into the stem cells of the transplants should prevent the toxicity. It then becomes conceivable, which it isn't now, that you could treat patients with higher than normal doses of chemotherapy repeatedly."
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A follow-up clinical study also in patients with advanced cancers is now examining whether a drug-resistance gene that is conveyed by transplant will actually serve its intended purpose in the patient, and if so, how well and for how long.
Vexing Hurdle
Efficiency, or more accurately its absence, has been a vexing issue in all of gene therapy.
Speaking from his laboratory at St. Jude Children's Hospital in Memphis, Tenn., Brian Sorrentino, M.D., traced the problem to the fact that the original vectors designed to ferry new genes into stem cells (and the only ones the U.S. Food and Drug Administration has, to date, approved for clinical use) were derived from the Moloney leukemia virus or other murine retroviruses.
Mouse-derived vectors only enter stem cells that are actively dividing, which has become a stumbling block because stem cells of primates including humans do not divide as often as those of mice. Sorrentino, however, thinks it is a stumbling block that can be overcome.
There is some evidence, he said, that primate stem cells can be made "less quiescent" without sacrificing their ability to reconstitute the immune system after it is damaged by HDC or radiation therapy. Also under development are vectors derived from lentiviruses, which, he says, may have the molecular machinery to slip into stem cells and integrate whether or not the cells are dividing.
A third alternative: a combination of drugs that Sorrentino and his colleagues have pioneered. Given post-transplant, these drugs enable stem cells that are expressing the transplanted gene to thrive, killing off those stem cells that are not.
"With this system," Sorrentino said, "we can take a mouse that starts with 5% of its stem cells transduced . . . and, with a few rounds of the post-transplant treatment, get that up to 80%. So even should it turn out that a gene for drug resistance or another purpose can be transferred to and functions in only 1% of a patient's stem cells, it may be possible to circumvent that limitation with this approach."
Testicular Cancer
At Indiana University Medical Center in Indianapolis, meanwhile, efforts to genetically fortify autologous transplants against drug toxicity have used mostly recurrent testicular cancer for the research. Why this particular cancer?
Ken Cornetta, M.D., the project's principal investigator, said one reason is that testicular cancer is highly chemoresponsive. "We think that it will help us to learn things about autologous transplants that may then be applicable to breast and other cancers," he said.
Also, the plight of patients with a metastatic recurrence of testicular cancer has itself been an incentive, Cornetta emphasized. Even those who achieve a complete remission [with HDC plus autologous transplant], he said, have a high probability of relapse.
That probability, according to Cornetta led to an idea proposed by Larry Einhorn, M.D., to lower the relapse rate by administering daily maintenance doses of oral etoposide (VP-16TM) after HDC. Like virtually all anti-tumor drugs, etoposide is bone marrow-suppressive so this idea is not a feasible strategy now. Still, a small clinical study that Cornetta, Einhorn, and their colleagues conducted may have moved it closer to becoming a reality.
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That was good news. But, in addition, the transferred gene could be detected in about 15% of the patients' stem cells a month or more later. "A rate of gene transfer that high had not previously been observed except in animals," Cornetta said.
He attributed this achievement to preparation of the vector bearing the drug-resistance gene with a genetically modified fragment of fibronectin, an extracellular protein with a somewhat gluey consistency, which seems to enhance the vector's chances of linking up with and entering target cells. "Even a year and some out," said Cornetta, "we are still seeing evidence of the gene in these patients' stem cells, though only in about 5%."
Defanging BCNU
Ireland Cancer Center at University Hospitals of Cleveland-Case Western Reserve University is another institution where there is a focus on neutralizing the marrow toxicity of a chemotherapeutic agent in this case BCNU, a nitrosourea and, by extension, its methylated analogs.
Interest in defanging this drug began in the early 1990s when Stanton Gerson, M.D., and his group discovered why some patients who were given BCNU, or a sister compound, eventually developed leukemia. The culprit was too little of a repair enzyme in their marrow called AGT: a finding that was further driven home when the Gerson group created transgenic mice that overexpressed the gene that codes for this enzyme. Despite being challenged with a nitrosourea, none of them developed secondary leukemia.
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Gerson and his colleagues selected a gene called mutant MGMT, which confers benzylguanine resistance, to do the job. Their educated guess was that marrow stem cells supplied with this gene would be unharmed not only by benzylguanine, but also by BCNU.
The approach worked at least in mice. In a study of nude mice with human tumor xenografts, those mice that had stem cells enriched with the MGMT gene tolerated higher levels of chemotherapy, had better tumor responses, and lived longer than the rest. "Here was definitive evidence," said Gerson, "that, by protecting the bone marrow, we could enhance the effect of chemotherapy."
Acccording to Gerson, a study now in press found similar effects in ordinary laboratory mice receiving a combined dose of benzylguanine and BCNU, and a stem cell infusion of a few marrow cells carrying the transplanted MGMT gene.
"If we treat the mice two or three times with the benzylguanine-BCNU combination, we can without resorting to transplants essentially completely replace the stem cells that are damaged by these drugs with new ones that take the drugs in stride," said Gerson. "Our experience, moreover, is that the changeover, once it occurs, is stable for a prolonged period of time."
Clinical Proof
The obvious question is whether these results can be duplicated in humans. Gerson, with the approval of the U.S. FDA, is gearing up to find out. Patients with advanced malignancies of many different types will, like the mice, get no high-dose chemotherapy or autologous bone marrow transplants at least not full-blown ones. What they will get is some of their own bone marrow stem cells (harvested from peripheral blood) that have been retrovirally transduced with the MGMT gene after the patients have had an initial standard dose of benzylguanine and BCNU. These standard doses will be repeated at 6-week intervals in the hope that just as this protocol made the marrow stem cells of the mice drug resistant, its effect on human marrow stem cells will be the same.
So where will this field be 5 or 10 years from now? No one, of course, can be sure. But Cynthia Dunbar, M.D., of the National Heart, Lung, and Blood Institute will be looking for solid clinical evidence of three kinds of progress: whether genetically engineered stem cells carrying the drug-resistance gene or some other gene can be counted on to engraft; whether the genes will function as intended; and whether the genetically enriched stem cells will enjoy a survival advantage over their unmanipulated counterparts.
A very tall order, researchers agree. But if it is not filled, it will not be for lack of trying.
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