From DNA to doctor's office

High-tech genetic analysis helps researchers improve treatment odds for cancer patients

Genetic fingerprinting has become a commonplace technique for generating courtroom evidence to convict criminals or settle paternity disputes. What many people don't know is that the same technology also is advancing medical science and saving lives.

No longer simply a promise for future medical breakthroughs, such DNA technology is put to work every day to help patients. At the Fred Hutchinson Cancer Research Center, several new procedures harness a new generation of DNA-analysis technology. These leading-edge tools- including the Center's computerized DNA-array facility, which allows researchers to analyze thousands of genes simultaneously-has accelerated the pace of genetic research and delivered new medical procedures to patients quickly.

Many new procedures-which improve cancer diagnosis, treatment and relapse prevention-are based on research by scientists who embody the "bench-to-bedside" approach to discovery that makes the Hutchinson Center unique, said Dr. Lee Hartwell, president and director.

"Virtually all of our clinical faculty who work with patients also conduct laboratory research. Many are taking full advantage of the cutting-edge technologies that are now available to them," he said. "That immersion in research is an enormous benefit to our patients, who gain access to new discoveries as soon as they are shown to be safe and effective."

The process of tissue-typing bone-marrow and stem-cell donors is a notable area of cancer care that has been enhanced by DNA research.

Leukemia patients seeking a transplant face a critical step in their treatment: the quest for a well-matched donor of bone marrow or stem cells. A mismatch can lead to transplant rejection and other serious complications.

Traditionally, a perfect tissue-type match between patient and donor has been the goal. But finding that genetic fit can be a long and difficult process for individuals with rare tissue types and no closely matched relatives.

New research by Dr. Effie Petersdorf-using highly sensitive DNA analysis-however, shows that some tissue-type mismatches can work just as effectively as perfect matches. This discovery potentially expands the pool of donors for transplant patients with cancers of the blood and immune systems.

State-of-the-art technology is key

"This work demonstrates that transplant success can be enhanced by applying state-of-the-art technology for genetic matching of stem-cell donors," said Petersdorf, leader of the study, which was reported recently in the New England Journal of Medicine.

Petersdorf and colleagues initiated their research to determine whether subtle differences-detectable only via highly sensitive DNA technology-between tissue-type genes from a healthy donor and a leukemia patient impact the success of transplantation. Their findings show that some single mismatches detectable only by DNA typing methods are viable for patients.

"By looking carefully at the precise genetic mismatches between donor and recipient, we've learned that some mismatches are well tolerated, and others are less so," Petersdorf said. "This is information we can begin to implement immediately to help patients find appropriate donors."

DNA analysis detects subtleties

Tissue type is determined by the information specified in six genes known as HLA (human leukocyte antigen) genes. Since every individual inherits two sets of chromosomes (and thus two versions of each HLA gene), every person can have up to 12 different tissue-type proteins. Ideally, donor and recipient possess identical information in all 12 genes, but a small degree of disparity between individuals may be tolerated.

Historically, HLA typing has been performed with serological tests, in which blood serum from the patient or donor is tested with a panel of antibodies that react against HLA proteins. More recently, DNA analysis, including DNA sequencing and fingerprinting, has been used in addition to serological testing. This kind of sensitive molecular analysis can detect subtle differences that may cause no discernable serological reaction.

Because tissue type is inherited, doctors first look to a patient's brothers and sisters as potential donors. But when that option is unavailable, matched unrelated donors are sought through the National Marrow Donor Program, a nonprofit organization based in Minneapolis that facilitates marrow and blood stem-cell transplants for patients who have no matching donors in their families.

For some patients, particularly those of minority or mixed-race backgrounds as well as others with rare tissue types, the search for a close match, much less a perfect one, can be long and sometimes unsuccessful. Peterdorf's findings open more options for such patients. Thanks to new methods and careful research, Petersdorf said, the matching continues to be refined.

"The definition of a perfect match is an evolving concept," she said. "As we learn about new genes important for a successful transplant and as more sophisticated technology for analyzing them becomes available, we continually refine the matching process."

In their most recent study, Petersdorf and colleagues used DNA sequencing to determine the exact tissue type of 471 patients who received unrelated donor transplants for chronic myeloid leukemia. The transplants were performed between 1985 and 2000. Sequence information was obtained for each of the six HLA genes. With this DNA sequence information, Petersdorf correlated mismatch status with transplant outcome.

Some donor mismatches tolerated

"We found no difference in graft failure between those with perfect matches and those with a single allelic mismatch-a mismatch in the DNA sequence that has no serological difference," she said. "What this means for patients is that the overall success of transplantation is not compromised by a single allelic mismatch."

In contrast, a single antigenic mismatch - a sequence difference that does cause a serological reaction - as well as multiple DNA-sequence mismatches were associated with increased risk of transplant rejection.

Petersdorf said the Clinical Immunogenetics Lab at the Seattle Cancer Care Alliance, which performs tissue-type matching of Hutchinson Center patients and donors, already performs typing with a higher degree of discrimination than is routinely available.

"But we're always refining the process," she said. "Retrospective studies on transplant outcome provide invaluable information that can be applied to make transplantation a safe and effective therapy."

Whether treated successfully with a transplant or chemotherapy, every patient's worst fear is that their cancer might come back.

"Patients come to the Hutchinson Center to be cured," said Dr. Jerry Radich. "But they live in absolute dread of relapse. When it happens, it is a very dark, devastating moment."

But defining relapse is a tricky business. Does the presence of any level of cancerous cells foretell impending recurrence of cancer? Or does the level need to exceed a certain threshold to determine whether a person's cancer has returned?

Radich focuses his work on redefining the meanings of remission, relapse and cure. To do that, he has developed molecular techniques for detecting extremely rare leukemia cells-a condition called minimal residual disease (MRD)-in patients who appear cured, enabling identification of patients who have a high risk of relapse. The long-term goal is to initiate secondary treatment at the earliest signs of relapse.

Using a DNA fingerprinting technique known as polymerase chain reaction (PCR), Radich's lab team can detect a single leukemic cell among 100,000 to 1 million normal cells. That contrasts with most conventional methods of determining remission or relapse, in which a pathologist uses a microscope to see when the number of cancerous cells in the blood reaches about one per 20 cells.

Radich initially developed the technique to identify MRD in patients with chronic myelogenous leukemia (CML), a disease in which virtually all patients have leukemic cells with a very specific genetic abnormality known as the Philadelphia chromosome. More recently, his lab has shown that molecular detection of MRD can be applied successfully to another blood cancer, acute lymphoblastic leukemia (ALL). Cells from some 5 percent to 20 percent of patients with ALL contain the Philadelphia chromosome.

Improving outcomes with new tools

Dr. Derek Stirewalt, a medical-oncology research associate in Radich's laboratory, used DNA fingerprinting to develop highly sensitive tests that help predict the success or failure of treatment for acute myelogenous leukemia (AML). The tests, which identify genetic abnormalities that indicate an aggressive form of the leukemia, could allow physicians to tailor therapies for patients and provide early clues about the likelihood of relapse.

With conventional treatment - chemotherapy - about 25 percent to 45 percent of AML patients can be cured. Patients who relapse may have benefited most from a bone-marrow or stem-cell transplant, a procedure that carries its own degree of risk. Doctors have been at a loss to predict which individuals will fail standard chemotherapy and which will be cured.

Because no visible clues can answer this, Stirewalt turned to DNA-based methods to detect subtle genetic abnormalities that correlate with disease severity and response to treatment.

Pinpointing patient prognosis

Mutations in a gene called FLT3 are found in 30 percent to 40 percent of patients with AML. In pediatric and adult leukemia patients, these mutations spell a poor prognosis, Stirewalt said.

"With these tests for the FLT3 abnormalities, patients with AML could have genetic analysis for mutations in the gene prior to treatment," he said. "If we detect a mutation, we could use that information to stratify patients into risk groups and select the most appropriate treatment for individual patients.

"We also have developed tests for FLT3 mutations that will allow us to follow patients prospectively throughout treatment. These tests for minimal residual disease may be able to predict the likelihood of relapse after treatment."

Two clinical trials using this method soon will be under way. Stirewalt said use of the FLT3 test will impact other research on AML treatment.

"There is a lot of excitement about new drugs that inhibit the FLT3 protein in mice," he said. "We and others are pursuing ways to block the activity of the mutated FLT3 gene. If these targeted therapies against FLT3 prove to be useful in humans, we could use our molecular test to identify those patients who would respond to the drug."

Such lifesaving molecular advances as these have been pioneered in leukemia-a disease that is highly amenable to DNA-based research-but doctors are confident that the approaches will soon be applied to many other malignancies.

"Genetic markers similar to those found in leukemia are bound to be discovered in solid tumors," Radich said. "Similar studies for detecting and treating residual disease will be part of the therapeutic strategy for these diseases as well."

Barbara Berg, Ph.D., is a science writer for the Fred Hutchinson Cancer Research Center.

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Advanced DNA technology points to tailored treatments for leukemia patients

Cancer develops when cells accumulate a handful of mutations that cause unregulated cell division. But for virtually all cancers, the identity and chronology of these genetic changes remain unknown.

Now, research from Dr. Jerry Radich's lab is unraveling this puzzle for chronic myelogenous leukemia (CML). A National Cancer Institute grant supports his work to identify the molecular changes that occur during CML progression. Already, his lab has identified genes that may ultimately serve as markers that enable doctors to tailor treatment strategies to a patient's molecular profile.

Radich and his colleagues use DNA-array technology-a method that allows them to monitor the expression of thousands of genes simultaneously-to help develop genetic "fingerprints" of CML cells.

CML goes through predictable stages from a relatively indolent condition to an aggressive and often fatal stage.

"CML is a great model disease for this approach," Radich said. "It clearly goes through these different phases. We know that without treatment, every patient will go through the same stages of progression. It's like a molecular clock. While everyone talks about tailoring treatment for cancer patients, CML is one of the few diseases right now for which there really are several different treatment options."

Using a similar approach, doctors can determine the molecular fingerprint of a patient's response to treatment and make appropriate adjustments to their therapy. Radich also uses DNA arrays to identify new genes that may be markers for trace amounts of leukemia cells, which provide the earliest clues of cancer relapse (see main story).

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