Betty Perr's strange symptoms had begun just two weeks earlier, when the 67-year-old St. Louis resident thought she heard two of her grandchildren, Josephine, then 5, and Alexandria, 4, talking and giggling outside her house. The children lived just down the road with her daughter and son-in-law, Miriam and Robert Butler, and often dropped in to see their Mama. But when she opened the door, they weren't there. That weekend, she heard the children's voices again—only this time she and her husband, Hugo, were 120 miles away at a wedding in Jefferson City. On the way home and over the following days, she complained about hearing a turkey gobbling, a dog barking, and the ghostly sound of a child's footsteps running through a house where she worked every week as a housecleaner.
After this last incident, Miriam Butler and her sister, Kathy Kramer, both nurses, urged their mother to see a doctor. A few days later Perr was admitted to the St. Louis University Health Sciences Center, where Miriam worked. A magnetic resonance imaging (mri) scan revealed the cause of the phantom voices and noises to be a tumor in the right temporal lobe of her brain, in an area just above her ear. The location of the tumor, in a part of the brain thought to be involved in our ability to remember voices, songs, and other familiar sounds, accounted for her symptoms. From their sudden onset and the look of the mri, Kenneth Smith, the hospital's chief of neurosurgery, suspected a fast-growing tumor called a glioblastoma. Glioblastomas, which afflict about 10,000 Americans each year, grow so rapidly—some doubling in size every ten days—that they cut off their own blood supply, leaving an area of dead tissue that appears dark against the illuminated mass on the mri scan.
If it is a glioblastoma, Perr's prognosis is grave. Surgery and radiation, the standard course of treatment, can give her a year or two at best. But this type of tumor inevitably returns. Glioblastomas literally crawl across the brain—the cells can move—along the star-shaped glial cells that support and insulate networks of neurons. By the time a glioblastoma is diagnosed, it has usually spread so widely that even the most skillful surgeon cannot remove all of it.
However, Smith can offer Perr a ray of hope beyond surgery—an experimental treatment called gli-328, which is now being tested at St. Louis University and 43 other hospitals in the United States and abroad. gli-328 is neither radiation nor chemotherapy; it is a form of gene therapy, one that may offer victims of glioblastomas a chance to beat the odds against them.
The procedure will be performed in two parts by a team of three neurosurgeons. First, Smith and Lynn Bartel will remove as much of the tumor as possible. Then, if the diagnosis of glioblastoma is confirmed, Richard Bucholz will implant suicide genes into the remaining cancer cells to get them to self-destruct. Specifically, he will inject mouse skin cells carrying a genetically altered mouse leukemia virus into Perr's brain. The mouse virus contains a gene from the herpes simplex virus that causes cold sores, called the herpes thymidine kinase-1, or the herpes tk gene. Its manufacturer, Genetic Therapy, Inc., a Gaithersburg, Maryland, biotechnology company owned by Novartis Pharmaceuticals, has removed other bits of dna to cripple the virus, so that it can infect but not reproduce in human cells. In particular, the virus can infect only dividing cells, which means that the only brain cells infected will be tumor cells, because normal brain cells do not divide. Once infected by the virus, the tumor cells will produce thymidine kinase, making them vulnerable to the antiviral drug ganciclovir.
Surgery and radiation alone can't get all of the tumor cells. We need a smarter weapon, explains Bucholz, who developed a computerized-image guidance system that will play a crucial role in the surgery and the gene implantation. The surgical instruments used during the operation are equipped with infrared-light-emitting diodes, similar to those on a television remote control, and are continuously tracked by two overhead cameras. The information is then fed into a computer, which compares the position of the instruments with data collected and stored during the planning of the operation: a few days before, the team performed ct and mri scans on Perr to visualize the tumor, distinguish the exact borders between malignant and normal tissue, and locate neighboring blood vessels. In the operating room a three-dimensional display based on this information will provide the surgical team with a map to help plan the safest and most effective path to the tumor—and to deliver the gene therapy precisely to those areas where it should be needed most.
At 8:30 in the morning, the operation is under way. Bartel uses a small air-powered drill to remove a small rectangular section of Perr's skull. Then, with Smith holding open gelatinous flaps of brain tissue, she isolates and cauterizes blood vessels to cut off blood flow to the area of the tumor. Working quickly, guided by the computer images, the surgeons are able to remove the main portion of the tumor in less than an hour. A sample is rushed to the pathology lab for microscopic examination. Minutes later, the doctors' worst suspicions are confirmed: the tumor is indeed a glioblastoma.
In a lab on the floor below, medical technologist Tim Kilcoyne at once begins warming and preparing the deep-frozen virus-producing cells (vpcs) that will deliver the herpes tk gene to the tumor cells. The thawing process is delicate and slow. The cells are easily damaged. You can't just pop them in the microwave, says Kilcoyne. His task is to count and test the viability of the cells over the next hour and a half. To produce enough herpes tk gene to combat the tumor requires 5 cubic centimeters (about a billion cells), enough to fill the bottom of a small plastic Ziploc bag. While the cells are being prepared, Smith and Bartel try to remove a portion of the tumor that extends deeper into Perr's brain. By 11 o'clock, they have gone as far as they safely can.
In preparation for the gene therapy, Bucholz now places a meshwork grid made of suture threads and small metal clips across the tumor bed, inside the incision. His plan is to inject virus-producing cells 1-centimeter intervals into each of the 20 squares of the grid, for maximum coverage. The procedure stalls when Kilcoyne calls to report that there are only 700 million cells in the preparation—fewer than the billion needed. The options are: use the available cells, begin the time-consuming work of preparing a new batch of cells, or inform Perr's family and give them the choice of declining the experimental treatment. Bucholz and Bartel cover the incision with a temporary dressing and wait.
A tense half hour later, officials at Genetic Therapy affirm by telephone that the cell count, though low, is still within the acceptable range. A few minutes later a nurse arrives with a cream-colored liquid in a plastic bag. Even after processing, the cells have a tendency to clump together, so she massages them gently, patting and smoothing the plastic packet.
Bucholz quickly loads the solution into a syringe with a long, springy needle. Then, using the computer images of Perr's brain to check position, direction, and depth, he begins the first infusion. Bartel presses the plunger of the syringe while Bucholz holds it in place. Rechecking the computer data, Bucholz then moves to the next position, repeating the process until the 5 cubic centimeters of solution have been distributed evenly over the grid. The entire procedure takes only ten minutes. Bucholz then removes the grid, and Bartel completes the five-hour operation by closing the incision, sealing Perr's skull with titanium strips, and stapling her skin closed.
After three days, Perr is discharged from the hospital. She will return in two weeks to have an intravenous line put into her arm for the twice-daily infusions of ganciclovir she will receive over the next two weeks. Ganciclovir is usually used to treat a disease related to herpes called cytomegalovirus, which causes blindness in patients with advanced aids. Now she and the doctors hope the drug will slow down the tumor and possibly stop it from coming back.
Betty Perr is the seventh patient to receive gene therapy for a glioblastoma at the St. Louis University Health Sciences Center, and one of about 200 people around the world who have received gli-328 in the five years since doctors started testing it. The idea originated not as treatment but as a self-destruct mechanism for the first gene therapy experiment, explains Michael Blaese, chief of clinical gene therapy at the National Human Genome Research Institute, part of the National Institutes of Health. In 1990, Blaese was preparing to team up with gene therapy pioneer W. French Anderson in an attempt to transplant healthy genes into the blood cells of a four-year-old girl with severe combined immunodeficiency syndrome (scids), the deadly bubble boy disease. The plan was to use a virus as a vector, a microscopic truck to ferry the therapeutic genes into the girl's cells. One of the viruses we were using was a mouse leukemia virus, recalls Blaese, and there was a concern that if you inserted the genetic material into the wrong place, it could cause the cells to become cancerous. We wanted to build a self-destruct mechanism into these viruses. If the worst happened—if cancer occurred—we could pull the plug and kill the cells.
When laboratory experiments clearly demonstrated that infected cells producing the herpes tk gene could be killed by antiviral drugs, Blaese reflects, it dawned on us that we could do it deliberately. Instead of using it as a fail-safe device, we could kill cancer with it.
More important, research in Blaese's lab showed that because the herpes tk gene changed ganciclovir into a chemical that was toxic to quickly dividing cells, the drug killed not only infected tumor cells but also nearby uninfected tumor cells, which were bathed in the toxin as the target cell died. Taking this bystander effect into account, it seemed possible that as few as one in ten cancer cells would actually have to express the suicide gene for the treatment to work. Using suicide genes as a weapon against cancer is far from the 1980s concept of gene therapy as a method to correct or replace the defective or missing genes responsible for deadly hereditary diseases such as scids or cystic fibrosis. Yet applications like gli-328 dominate gene therapy in the 1990s. According to the most recent records of the nih's Recombinant dna Advisory Committee, of 106 approved gene-therapy experiments, only a small fraction were aimed at correcting defective genes, while the vast majority involved inducing specific cells, such as cancer cells or cells infected by hiv, to produce proteins that would make them vulnerable to attack by the immune system or drugs.
From the beginning, glioblastomas seemed like ideal targets for such an approach. Glioblastomas kill by wreaking havoc on target areas in the brain, not by spreading throughout the body like other cancers. Therefore, a therapy would have to work only locally, not globally, to be effective. Another advantage is that normal brain cells do not divide. Therefore, since the mouse leukemia virus carrying the gene infects only dividing cells, only tumor cells will be targeted for destruction. Finally, the brain is an immunologically privileged site. To enter the brain, outside organisms must pass directly through the membranes of capillaries rather than through small clefts in their walls, as is the case throughout the rest of the body. Because the filtering effect of this blood-brain barrier keeps most dangerous agents away, the brain's immune defenses are weak and delayed. Elsewhere in the body, an infusion of virus-producing mouse cells would provoke the immune system to attack quickly, causing an inflammation in the area. In the brain, however, large numbers of mouse cells can be infused with only a small risk of causing inflammation.
In mice, gli-328 completely kills tumors, without side effects. But while the results of the two human trials conducted so far are intriguing, they are also controversial. In 1992, Edward Oldfield and Zvi Ram, two neurosurgeons at the National Institute of Neurological Disorders and Stroke in Bethesda, Maryland, conducted a pilot study with their colleagues on ten patients who had undergone surgery for glioblastomas and whose tumors had come back. These patients received no repeat surgery to remove the regrowth. Instead the vpcs were delivered to the tumor site through a thin tube called a cannula, guided by ct-scan images. The patients then received follow-up treatments with intravenous ganciclovir.
In this small study gli-328 was safe and effective in some cases, producing decreases in tumor size by 50 percent or more in four of the ten patients. The gene therapy helped, but not enough to produce any impact on survival. Because the delivery system limited the therapy to an area just a few cell layers deep (even with the bystander effect), only very small tumors responded. And while the therapy reduced parts of the tumors, distant portions progressed rapidly.
However, although nine of the ten glioblastoma patients died within three months to a year—about the same number expected from radiation or repeat surgery—one of the original patients, Kevin Klug, now 43, is defying the odds and is still alive five years later.
Results of this trial have also been mixed, according to neurosurgeon Mitchel Berger of the University of California at San Francisco, who was the lead investigator of the Phase II study. The average survival of the 31 patients was seven months—no improvement over repeat surgery and chemotherapy. But surprisingly, six of the patients—one out of five—are still alive three to four years later. About one in a hundred people with glioblastomas gets better for some reason. But this is not some freak phenomenon. Twenty percent achieved a major benefit, Berger asserts. We've never seen anything like it with any other therapy.
However, Edward Oldfield urges caution. Despite the very small size of these tumors, we saw very little evidence of gene distribution—and even if we can reduce the size of the tumors by 50 percent, that's not enough to produce a lasting benefit, he points out. There could be other explanations for the longevity of the handful of long-term survivors, adds Oldfield, including sheer luck.
Is the gene therapy helping these patients beat impossible odds? The study now under way in St. Louis and elsewhere is trying to answer that question. During this Phase III trial, 250 patients in ten countries will receive either treatment with surgery, radiation, and gene therapy, or surgery and radiation alone. Unlike patients in earlier trials, those enrolled in the current study are receiving gene therapy at the time of their initial surgery, not at recurrence. They will be evaluated to see if the gene therapy reduces the time to recurrence, or increases survival compared with standard therapy. This is the first time any gene therapy has been evaluated in a large, randomized trial. If therapy with the herpes tk gene shows a benefit, it could be approved for widespread use as early as the year 2000.
The stakes are high. If successful, suicide genes could be applied to other forms of cancer, or to stop restenosis, the rapid cell growth responsible for reclosing heart vessels after balloon angioplasty. Researchers are also considering using suicide gene strategies to control graft-versus-host disease after bone marrow transplantation and to kill off the proliferating cells that cause inflamed arthritic joints.
Elsewhere, doctors are testing similar techniques to fight disease. Recently, Jeffrey Isner of St. Elizabeth's Medical Center in Boston reported dramatic success in ten patients who had such severe peripheral artery disease that they were in danger of having their legs amputated. After Isner implanted genes for a hormone that stimulates the growth of blood vessels, most of the patients sprouted collateral arteries that bypassed their blocked arteries. Nine out of ten patients improved so markedly that amputations were avoided or limited to a toe.
Results with these therapies will have to overcome serious doubts about whether gene therapy will fulfill its seemingly vast potential. According to a widely discussed review of the field commissioned two years ago by nih director Harold Varmus, after more than 100 clinical gene therapy trials at a cost of well over $200 million a year, there was still no unambiguous evidence that genetic treatment has produced therapeutic benefits.
Will GLI-328 be an exception, the first gene therapy for cancer to demonstrate clear-cut results? For Richard Bucholz the treatment represents an exciting step forward, whether or not it produces dramatic benefits against brain tumors. We're just starting on the road to establishing the techniques to get the maximum response out of gene therapy. If we are successful, this approach represents a major shift in the way we treat disease, he reflects.
The gene therapy approach is very exciting because it bypasses the natural biology of these tumor cells, which are very difficult to understand and treat, adds ucsf neuro-oncologist Michael Prados, who tested gli-328 in the Phase II trial. It allows you to create a tumor cell that's the way you want it to be—killable.
Editor's note: However promising gli-328 may be for other glioblastoma patients, it was unable to save Betty Perr from her cancer. Sadly, just as we were about to go to press, we learned that this courageous woman had died. We extend to her family our condolences and our gratitude for their generous cooperation in preparing this article.