College of American Pathologists
Printable Version

  High hopes, hard work await
  gene tests


cap today

December 2001
Cover Story

Karen Titus

The magic number in genetic testing is anybody’s guess.

When researchers discovered the delta F508 gene mutation in the late 1980s, linking it to cystic fibrosis, the critical figure appeared to be one. One gene, one mutation, one diagnosis.

With the more recent appearance of cDNA microarrays, the number grew larger. Thousands of genes, thousands of mutations, and thousands of diagnostic possibilities.

These days, the critical number appears to waver somewhere in between. Anywhere from a handful to perhaps several hundred genes may lie at the root of illnesses ranging from rare hereditary disorders to common cancers to cardiovascular disease.

Which leaves laboratories—where, exactly?

In a position that’s enviable and daunting, astonishing yet mundane.

The images emerging from the mapping of the human genome are as exhilarating as the glimpses of space being sent back by the Hubble telescope, which peers at nothing less than the origins of time. As genetics and proteomics research likewise unravel heretofore unfathomable mysteries, current limits on diagnosing and treating disease would seem on the verge of lifting.

"Genetics is becoming important to every specialty," says Wayne Grody, MD, PhD, professor in the departments of Pathology, Pediatrics, and Human Genetics at the University of California, Los Angeles, and director of UCLA Medical Center’s DNA diagnostic laboratory.

"I tell med students that there’s a lot in their basic science years that they’ll think is arcane, and that they’ll never use in their practice. But with DNA testing, I tell them, ’You’re all going to use it. All the time. It doesn’t matter what specialty you go into, from surgery to psychiatry. You are going to be ordering these tests,’" adds Dr. Grody, who also chairs the CAP Molecular Pathology Committee.

In cancer alone, the potential is breathtaking, says Mahul Amin, MD, associate professor of pathology and urology and director of surgical pathology at Emory University Hospital, Atlanta, and vice chair of the CAP Cancer Committee. Using cDNA microarrays, pathologists could classify tumors according to their molecular profile and spot adverse prognostic genes; single out markers to predict therapeutic response and uncover new targets for therapy; and identify novel diagnostic markers.

"We’re performing studies we never could have imagined in our wildest dreams," he says.

But even as genetics advances the medical frontier, laboratories are finding that their best strategies lie closer to home. Bringing gene-based testing onboard is more than a matter of adopting sophisticated technologies. It means sorting through, once again, the quotidian details of running a lab.

That may seem a bit odd, given that genetic testing is not a new phenomenon. "We’ve been doing genetic tests for quite some time now to diagnose lymphomas, leukemias, neuroblastomas, and more recently sarcomas," says Marc Ladanyi, MD, associate attending pathologist and director of the Laboratory of Diagnostic Molecular Pathology in the Department of Pathology at Memorial Sloan-Kettering Cancer Center, New York. "But certainly it’s accelerating as more and more clinically relevant genetic diagnostic markers and prognostic markers are identified."

Yet the number of labs doing genetic testing is not keeping pace, says Ron McGlennen, MD, who is a member of the CAP/ACMG Biochemical and Molecular Genetics Resource Committee (which is chaired by Dr. Grody). "In the last three years, a time in which we have cloned and sequenced the human genome, the number of labs participating in the molecular pathology proficiency Surveys has hardly increased—only by 20 or 30 labs," he says. Fewer than 200 laboratories are enrolled, and "only a tiny subset of them participate in all the markers," he says. You can practically hear him shake his head in amazement.

Dr. McGlennen echoes many with his observation that the field is held back by its lack of standardization in protocols and controls. As with any test, gene-based assays will gain wider acceptance only if they’re clinically as well as analytically valid. Nothing new here. Certainly there’s no shortage of discussions about the challenges of optimizing hybridization of DNA probes, or identifying key mutations for which to develop probes. And one would be hard-pressed to identify areas where therapeutics has outpaced the discovery of genetic markers.

Then there’s the little matter of money.

"Molecular genetics grew up in an era where the people who would pay for these tests were not going to give us carte blanche and say, ’Here’s $200 to do a factor V Leiden,’" says Dr. McGlennen, associate professor and medical director of the molecular diagnostics laboratory at the University of Minnesota Medical School. "I think the last point of reference, if you will, when reimbursement was much more liberal, was in the era when flow cytometry was first being introduced in the clinical lab."

Taking it a step further, Dr. Grody points to billing formalities, a considerable stumbling block in the case of large microarrays. "Do you use a separate CPT code for every probe on a chip?" he asks. "This thing would cost a million dollars per test if you did that."

The lag in genetic testing is also due to less-obvious reasons, says Dr. McGlennen, who casts a jaundiced eye on companies that have long promised to bring new gene-based technologies into the lab.

"Everybody pays lip service to the idea that they’re going to make their technology available for clinical diagnostics," says Dr. McGlennen, who serves also as medical director of Esoterix Molecular Genetics, based in Minnesota. "They take their instrument, their chip, their enzyme, and walk along this fence and say, ’We’ve demonstrated that it works, and now we’re going to show that it works in the clinical setting.’" What has happened instead, he says, is many of these technologies "have fallen off that fence onto the other side, where they’ve become largely the tools of the pharmaceutical industry and the genomics industry."

It makes sense, from a business standpoint. "The genomics industry pays cash on the barrelhead—there’s a lot of money to be made in discovering new genes and doing so faster and better and cheaper," Dr. McGlennen says. And though it’s easy to argue that there’s money to be made in diagnostics as well, "the intricacies of reimbursement and clinical utility and analytic validation often scare these technology companies away."

The goal is to find the handful of genetic tests that will serve the larger medical market.

The candidates haven’t always been obvious. Despite initial high hopes, the delta F508 mutation has not provided clear-cut answers in CF. "Six hundred point mutations later, we’re still talking about what is a reasonable, cost-effective strategy for diagnosing cystic fibrosis," says Dr. McGlennen. And since CF is a relatively easy clinical diagnosis to make, "You’re performing an expensive genetic test to confirm what is somewhat obvious."

He compares this with inherited thrombophilia, which can be diagnosed with a small cluster of mutations, including factor V Leiden. The case for a genetic diagnosis is evident. "Hundreds of thousands of people are affected by this, but you can’t look at them and say, ’I bet that guy’s going to develop a DVT today,’" Dr. McGlennen says.

That’s not to say widespread genetic testing won’t make its way into most labs eventually, he says. "We need to get gene-based testing to a larger end of requesting physicians," he says. "And we will."


Even as tests prove their prime-time readiness in terms of analytic and clinical validity, cost-effectiveness, and ease of use, doubts remain. Physicians unschooled in genetic medicine may shy away from genetic tests. "There’s still a lot of trepidation out there," says Dr. McGlennen.

So far reference laboratories have excelled at capitalizing on those fears, he says. When a small hospital laboratory is asked if it will perform a factor V Leiden test, too often the response is a timid "maybe." "The lab says, ’Why, I can’t do that kind of stuff, I don’t know enough about genetics.’ The local pathologist doesn’t know enough to do the test, the laboratory staff is afraid of the test, and the laboratory or hospital administrator is saying, ’I’ve heard we don’t get reimbursed for this sort of thing,’" he says.

Reference labs, on the other hand, have seen reimbursements stabilize, have discovered efficiencies in performing these tests, and are finding significant margins, Dr. McGlennen suggests. "That’s why, when you look at the Quests and the LabCorps, even though genetic testing represents a tiny fraction of their total, without a doubt it’s the strongest dot on their radar screen. That’s where they’re having explosive growth and very
nice returns."

Does that mean genetic testing belongs solely with the big boys—reference laboratories and large academic medical centers?

Dr. McGlennen makes it quite clear that the answer is no. "We can start talking about the potential now of molecularizing the coagulation laboratory, molecularizing the hematology laboratory," he says. "It’s time to put these tests, along with their representative technology, back in the hands of the laboratory, which can work genetics in with a CBC, or a pro time, and so on. Because it makes more sense there. And then the doctors who order these tests will better understand how all the elements fit together."

Opportunities abound for all pathologists, Dr. McGlennen says. "But they have to stop looking at genetics as one big lump, where all the complex stuff is equated with the simple stuff."


They will also need to take a hard look at how to manage the information explosion—and sometimes explosive information—that accompanies genetic testing.

Cancer genetics is an obvious example. "We’re starting to realize that the real insights come not from a single gene or a small number of gene alterations, but rather from understanding how a wide array of cancer-related genes become altered," says Sydney Finkelstein, MD, associate professor of pathology and director of molecular anatomical pathology at the University of Pittsburgh Medical Center.

The implications are staggering. In the case of cDNA microarrays, laboratories could be producing information about tens of thousands of genes.

The key is to refine the information and to translate new research insights into useful tests—a challenge Dr. Finkelstein embraces.

He’s focused his efforts on developing tests for tissue specimens, an area that he says has lagged behind other areas of cancer genetics research. Because tissue specimens tend to be small and have been subjected to fixation (which has a detrimental effect on RNA) they’re often not candidates for the molecular methods being devised for other samples. Compounding the problem, tissue specimens tend to be heterogeneous, which makes sample selection that much more difficult. "It has become a major challenge not only to know what to look for, but also to work up ways in which these tissue specimens can be made to reveal their alterations," Dr. Finkelstein says.

Dr. Finkelstein uses an approach he calls microdissection genotyping. Sophisticated microdissection techniques enable pathologists to select from the tissue section the specific, tiny area corresponding to morphologic alterations; polymerase chain reaction is then used to amplify the DNA, which can then be analyzed using molecular methods. It’s the best of both worlds: key samples taken from small amounts of material, yet yielding insights from dozens of genes.

Likewise, the data gleaned from gene expression profiling using microarrays could eventually be converted to more focused assays that evaluate the expression of perhaps only a dozen genes. "And those might be looked at by a variety of existing techniques: real-time RT-PCR, or immunohistochemistry, or in situ hybridization," says Memorial Sloan-Kettering’s Dr. Ladanyi.

He predicts the impact of microarray data on the pathologic workup of carcinomas might resemble the effects chromosomal translocation analysis has had on lymphomas, leukemias, and sarcomas. These very specific, tumor type-specific genetic changes are now recognized as powerful diagnostic markers for specific tumors, he explains. "And what has happened there is that people have perhaps begun to agonize less over making difficult distinctions by H&E histopathology." It’s not a matter of shifting to a screening strategy, he says; rather, "It’s kind of a triage that uses H&E histopathology to identify which molecular tests need to be run to confirm the diagnosis."

In the classic problem of carcinomas of unknown primary site, a similar practice may emerge. Determining the primary site based only on subtle histologic features could be a thing of the past. "The case might just be triaged and sent for some type of expression profiling that will resolve or help resolve the problem," Dr. Ladanyi says.


Implicit in these scenarios is the continued demand for anatomic pathology techniques. The timeworn fear, of course, is that traditional AP skills will become obsolete as gene-based testing hunkers down in the lab.

"A lot of people like to make that claim, yes," says Jeffrey A. Kant, MD, PhD, director, Division of Molecular Diagnostics, University of Pittsburgh Medical Center. "But then, people have been making that claim for at least 25 years. And it hasn’t happened yet, as far as I can tell."

Gene expression profiles do present a potential threat to the AP way of life, he admits, since they could reduce the subjectivity associated with traditional tumor analysis. "But tumors are so complex," he says. "There’s going to have to be a lot of data accumulation and clinical validation before we get to the point that we’ll have enough information to even consider doing that."

Dr. Kant, who also oversees the residency program, says, "I’m not telling my residents to sell their microscopes and pick up a pipette yet."

At Pittsburgh, Dr. Kant and his colleagues are centralizing testing that uses molecular-based technologies. "The majority of testing for infectious agents, cancer, and inherited diseases is in the Division of Molecular Diagnostics," he says. "And we’re collaborating closely with our anatomic pathology group here, which is developing tests for solid tumors." In addition, the division uses faculty from traditional disciplines as adjunct faculty to relay test information to clinician users.

The benefits of the centralized approach are clear in Dr. Kant’s mind. Concentrating expertise has saved money, streamlined training, and made it easier to manage the lab. And it makes it easier for the lab to design and troubleshoot the home-brew assays that are common in the field.

This strategy also enables the laboratory to route information smoothly, which is no small consideration. As Dr. Finkelstein puts it, "There’s no point in generating all this information, then having it just sit there."

At Pittsburgh, most of the infectious and genetics tests are ordered by clinicians, says Dr. Kant, though a significant number of oncology tests—particularly in the hematology area—are ordered by a combination of clinicians and hematopathologists. In the first instance, test results are usually reported back to the ordering clinician, who serves as the referring physician.

When cases require adjunct testing—a lymph node requiring a clonality assay, for example—they are accessioned to a case pathologist. "We use the same accession number, and the molecular studies are ordered as a special procedure," Dr. Kant explains. The same holds true for flow studies and cytogenetics—"Everything related to that sample is a special procedure." All results are reported under that one accession number, and the case pathologist is informed as results become available. In most cases, that pathologist then integrates the molecular testing with other studies, such as histology, immunology, and cytogenetics, funneling them all into a broader interpretation.

The laboratory also offers a number of microdissection-based tests. Gliomas, which represent the most common primary brain tumor, are especially responsive to this methodology, Dr. Finkelstein reports. When gliomas contain deletions of chromosome 1p, chromosome 19q, or both, they’re more responsive to a particular chemotherapy, he says. Since these alterations cannot be routinely observed on a histologic slide—and because glioma tissue specimens typically are small and quite variable in their cellular makeup, microdissection genotyping makes sense. "So what we do is microdissect, according to the neuropathologist’s cues," Dr. Finkelstein says. Information about mutations is delivered back to the neuropathologist, who then integrates the genetic test results with the histologic features.

Though these tests will likely continue to be ordered by pathologists, as they are now, "many of these may be at the request of treating physicians. So pathologists will need to become much more responsive to how molecular alterations can impact therapy," Dr. Finkelstein says. They’ll also need to supplement their diagnoses with an understanding of how tumors evolve.

Pittsburgh’s experiences should serve to calm surgical pathologists fearful about their role in the molecular future. Basic surgical pathology skills will always be needed as sort of the first pass on any disease, most agree.

"Ideally, I think the surgical pathologist who’s in charge of the case should decide what to farm out for various techniques, including molecular methods," UCLA’s Dr. Grody says. "In some cases, it will be appropriate for the clinician to order the molecular tests right away; other times, it would be more cost-effective to have it go through the pathologist first. Because it may be that you don’t need this expensive test—maybe you can get your answer by more routine types of pathology study."

This will hold true even as genomics moves into proteomics, predicts Dr. Finkelstein. "Everything has to be congruent with the fact that tissues must be analyzed first to know what they are, to know where the best areas for sampling are, and how the areas interact with each other."

In short, says Dr. Finkelstein, the new methods won’t truncate pathology, but simply take it to another level, where pathologists will be able to characterize genetic alterations in a comprehensive and accurate fashion.

Emory’s Dr. Amin agrees. "We grade and stage tumors—that’s where the pathologist’s role ends today. But these methods will give us additional parameters for managing patients. And that will strengthen our role as surgical pathologists, not weaken it."


As diagnostic methods transition from research labs to everyday use, pathologists will have to make other adjustments.

There’s no doubt that the shift toward molecular diagnostics means a surge in data. "It’s a real problem," Dr. Kant says.

What it all comes down to in the end, he says, is this: judgment and restraint. "This is what makes a good pathologist, and a good clinician."

"What we owe the public as physicians is to make sure, when we offer a test, that we know its advantages, its limitations, the circumstances in which it’s good to order it, and the circumstances in which maybe it would be wise not to order it—when it could be confusing or misleading," he says.

That’s what makes the partnership between pathologists so important, he says—and so thorny. "I know at some places it’s been a bit of a bumpy road as labs coordinate things between those who do one aspect of the specimen analysis and those who do the so-called molecular tests," Dr. Kant says.

"Sometimes the people doing the molecular studies overinterpret the biological significance," he allows, "which could conflict with what’s well known from other clinical pathological studies on other types of tumors. So you have to be sure that you’re not sending to clinicians mixed or confusing messages."

The hope is that pathologists will eventually develop expert systems to alleviate those problems. Eventually, too, laboratory information systems may be able to smooth the flow of information, though most concede such systems are only a distant dream at this point.


That’s why many advocate the use of genetic counselors.

Let’s hear what Francis M. Giardiello, MD, has to say on the subject. Dr. Giardiello, a professor of medicine and oncology and director, Division of Gastroenterology at Johns Hopkins University, authored a well-known study (Giardiello FM, et al. N Engl J Med. 1997;336:823-827) looking at the use and interpretation of APC gene testing for familial adenomatous polyposis. The researchers found that nearly one-third of the physicians who referred patients for testing misinterpreted the test results; particularly disturbing, the researchers noted, was that some patients at risk for the disease would have been given a false negative result. Less than 20 percent of the 177 patients in the study received genetic counseling beforehand, and less than 17 percent provided written informed consent for the test.

Their conclusion: "...physicians who order these tests must be prepared to offer their patients genetic counseling, either personally or through referral."

Granted, that view emerged nearly five years ago. Have things improved since then? "Not much," says Dr. Giardiello.

Laboratories can get the ball rolling, he says, but they’ll need the assistance of genetic counselors.

"The lab’s role is twofold," he told CAP TODAY. "Before any blood is drawn, before any samples are sent, the lab needs to require informed consent from the patient. And they have to be able to adequately explain the test." Genetic counselors can assist with both jobs, and should, Dr. Giardiello says. "They’re critical, vital, to the process of doing gene testing. Period."

Dr. McGlennen, of the University of Minnesota, welcomes genetic counselors with open arms.

"I’m one of the believers that laboratories should invest in genetic counseling, and should use them in ways that not only engage them in genetics, and with the patients, but with the laboratory aspects," he says. "They should be familiar with why a lab test works or doesn’t work—they shouldn’t be kept in the dark about what we do."

They can also help pathologists with the onerous, and sometimes tricky, business of reporting results. "I don’t have any trouble in making an analytic diagnosis, but I do need the constant input as to what the literature is saying," Dr. McGlennen admits. "And they are key in confessing that which we do not know.

"Many times in gene-based testing, you find yourself saying, ’It’s not this mutation, and it’s not that mutation, but it still could be this disease,’" he explains. "So you use genetic counseling techniques and verbiage to help guide you when you come up with a lot of negative answers."

Not having a genetic counselor does not give the laboratory an out, Dr. Grody warns. Most primary care physicians aren’t adequately prepared to address genetic testing issues, he says, and until that changes, "It’s going to fall to the pathologist to be kind of the resident expert in genetics in their own institution, especially if it’s a smaller community hospital with
no geneticist."

That’s actually a pretty likely scenario, says Jill D. Trimbath, MS, a genetic counselor at Johns Hopkins. More precisely, she’s the genetic counselor for that institution’s hereditary colorectal cancer program, and one of nearly 20 genetic counselors on staff at the hospital. "We can afford to be specialized," she says with a laugh.

Most institutions aren’t Johns Hopkins, unfortunately. While practice opportunities have expanded in the last two years, most of the country’s approximately 2,000 genetic counselors practice at large, university-type hospitals, Trimbath says.

There’s obviously plenty of room—and need—for growth. "It’s always confirmed in my mind when I talk to patients who clearly have a family history of cancer, and none of their physicians have ever addressed it with them," Trimbath says.

Like Dr. McGlennen, Trimbath wants to see genetic counselors working alongside pathologists in the laboratory. "We can see when tests are being ordered inappropriately, and can obtain patient information when
it’s missing."

In addition, they can procure signed patient consent forms. Although not every laboratory requires them, Trimbath says they’re crucial to protecting the lab as well as the patient. Consent forms can do more than explain to patients the meaning of positive, negative, and inconclusive tests results; they can also inform them the test may uncover other clinically relevant information. "The reality is, gene tests may uncover things you’re not even looking for, and labs need to address that," she says.


So many, many things for labs to address. This is only the tip of the iceberg. Submerged far below are other issues, all of them critical, none of them easy: how to report test results clearly, in English as well as scientific language; how to deal with recalcitrant colleagues; how to decide what tests to send out, and which to do in-house. The number of challenges is exceeded only by the number of new tests heading toward the lab.

Perhaps, when all is said and done, there is no magic number. The only "number" to bear in mind might be this: The days when labs could safely ignore gene-based testing are numbered. Sooner or later, everyone will need to make room for the inevitable.

"It’s going to happen," says Dr. Amin. "The promise is tremendous. Whether it takes two years, or 10, or 20, we really don’t know. But it’s on our doorstep already."

Karen Titus is CAP TODAY contributing editor and co-managing editor.