College of American Pathologists
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  Lymphoma microarrays
  stir up debate


cap today

March 2005
Cover Story

William Check, PhD

To jump-start a debate on molecular classification of lymphoma at the annual meeting of the Association for Molecular Pathology last November, Howard Ratech, MD, who had organized the session, read the contents of a letter which, he said, he "was able to retrieve from the trash of a large urban medical center." The purported letter, addressed to the chairman of pathology, began, "Please accept my resignation as director of hematopathology in your department." It continued in part: "I base my decision on moral grounds. I find it highly unethical to continue the practice of pathology, recklessly using antiquated light microscopy, living under the constant fear of misdiagnosing neoplastic lymphoid lesions, when gene chips have made lymphoma subclassification highly accurate and reproducible."

After reading the letter, Dr. Ratech, who is professor of pathology, director of hematopathology, and director of the molecular pathology laboratory at Albert Einstein College of Medicine-Montefiore Medical Center, asked the panel members: "When do you anticipate that you will write your own letter of resignation as a classical hematopathologist?"

Clearly, Dr. Ratech intended to be provocative with this "letter," just as he had been in formulating the session as a debate and in stating the proposition that molecular classification should be adopted for the "primary diagnosis" of lymphoma. He calls the letter "an educational tool to start people talking and thinking about the issues of molecular diagnosis of lymphomas." His goal in the session: "to raise the issue to a public debate rather than private handwringing."

For the purpose of the debate, Dr. Ratech alphabetically assigned panelists to pro or con. "Many of the panelists did not want to be on the pro side," he says. "Some had written contrary opinions in the literature." Even though all the panelists were prominent molecular pathologists, Dr. Ratech says, "I don’t believe any of them at this moment would leap to do lymphoma diagnosis without recourse to the microscope or protein expression by flow cytometry or immunohistochemistry."

Subsequent interviews with the panelists verify Dr. Ratechs assessment. "In my career we are always going to be using morphology as our starting point for classifying lymphomas," says Daniel A. Arber, MD, director of clinical hematology and flow cytometry, associate director of molecular pathology, and professor of pathology at Stanford University School of Medicine.

Adam Bagg, MD, director of hematology and director of the minimal residual disease resource laboratory in the Department of Pathology and Laboratory Medicine at the University of Pennsylvania, acknowledges that there’s been a lot of "hype that pathologists will become redundant and so-called traditional technologies will fall by the wayside. But," he says, "even those of us who practice molecular pathology on a daily basis and who are very sensitive to the exciting data from microarrays and even apply some of these data in our practice know that it is not going to supplant what we now do in the workup of lymphomas." Dr. Bagg feels that all the panelists came away in large part with a sense that gene expression analysis has a role—"but just a role," he says. "It’s not the final answer. Microscopy will certainly have a role in the future, maybe a lesser role but still a central one."

Rita M. Braziel, MD, professor of pathology and director of hematopathology, flow cytometry, and molecular pathology at Oregon Health Sciences University, agrees. "We certainly aren’t ready to use molecular techniques alone as a subclassification tool for lymphomas. But they are an important adjunct to establishing an accurate diagnosis in some cases and in determining prognosis."

In her opinion, microscopy will never be obsolete. "That is always the starting point," she says. Nor does she think pathologists will become obsolete. "Pathologists are very flexible creatures and adapt to new technologies pretty well."

Microscopy is the "entry point" to diagnosis of lymphoma, in the words of Kojo S.J. Elenitoba-Johnson, MD, associate professor of pathology, director of molecular hematopathology and proteomics, and director of the molecular genetic pathology program at the University of Utah. "Histology informs the nature of subsequent tests that you order."

Dan Jones, MD, PhD, medical director of the molecular diagnostics laboratory at the University of Texas MD Anderson Cancer Center, says, "People will always do histology because it is cheap. And histology tells you the whole outcome of the genetic program in the tumor. When you do morphologic evaluation, you are looking at the end stage."

Moreover, Dr. Jones says, "At the moment there are a lot of publications with microarray data. But in terms of day-to-day practice, we are still in the phase where molecular techniques have not made an impact in lymphoma. We are lagging a bit behind diagnostics in leukemias."

Daniel E. Sabath, MD, PhD, associate professor of laboratory medicine, head of hematology, and director of the clinical hematology laboratory at the University of Washington, says much progress is being made in characterizing lymphomas with gene expression profiles. "But we are not yet at the point where they can replace current diagnostic modalities—histology, flow, and immunohistochemistry."

And Janina A. Longtine, MD, chief of molecular diagnostics in the Department of Pathology at Brigham and Women’s Hospital and associate professor of pathology at Harvard Medical School, adds this consideration: Therapeutic options lag behind the ability at this point to classify lymphomas with microarrays. "Right now we can say whether something is a good or bad kind of chronic lymphocytic leukemia," she says, "but we lack drug options to treat those two types."

Debating the role of molecular methods in diagnosing lymphomas is, to some extent, moot, since molecular methods are already ensconced in the hematopathology laboratory. "In some ways we have molecular classification now," Dr. Arber says. "We think of some types of lymphomas in a more molecular sense." Gene amplification methods and fluorescence in situ hybridization, or FISH, are already used in diagnosis, such as in looking for t(14;18) in follicular lymphoma and t(11;14) or its surrogate cyclin D1 gene expression in mantle cell lymphoma. In Burkitt’s lymphoma, t(8;14) in the c-myc gene is an important marker.

In addition, genetic testing for somatic hypermutation informs prognosis in chronic lymphocytic leukemia and its cousin, small lymphocytic lymphoma. By morphology and standard immunophenotyping CLL appears homogeneous, but the presence or absence of heavy chain gene rearrangement divides patients into good- and bad-prognosis categories.

"The nice thing about IgH somatic hypermutation analysis in CLL," Dr. Jones says, "is that enough people have decided they want to do that test that we have a marker we can evaluate across the country in large numbers to ask how it is affecting treatment and outcome a few years down the line." Hypermutation is a "nice story," he says, showing that a molecular test can "make it through all the hurdles such as getting samples and clinicians agreeing they want it." Dr. Jones is starting a parallel evaluation of clinical hypermutation testing in small lymphocytic lymphoma.

While molecular methods such as gene amplification and FISH are already part of the hematopathology laboratory’s toolbox, it is still controversial whether microarrays should be added as well. Enthusiasm for doing so has arisen largely from research by two consortia on diffuse large B-cell lymphoma, which is curable in less than 50 percent of patients (Shipp MA, et al. Nat Med. 2002;8:68-74; Rosenwald A, et al. N Engl J Med. 2002;346:1937-1947). Both groups used gene expression profiles to identify prognostic subtypes of this malignancy. One group segregated patients into two groups with divergent five-year survival rates in response to standard chemotherapy 70 percent versus 12 percent. The other group found three prognostically significant histological subtypes: germinal center B-cell-like, activated B-cell-like, and type 3 diffuse large B-cell lymphoma.

Whether and how to incorporate these analyses into clinical practice remains the big question. "I am not aware of any groups routinely doing this kind of analysis on large B-cell lymphomas," Dr. Jones says.

Dr. Braziel has worked on some studies done by one of the research consortia, the Lymphoma/ Leukemia Molecular Profiling Project, or LLMPP. She took part in two of the project’s studies trying to use the gene expression data for diffuse large B-cell lymphoma to devise simpler tests to identify the subgroups (Iqbal J, et al. Am J Pathol. 2004;165:159-166; Hans CP, et al. Blood. 2004;103:275-282). In one study researchers set out to develop a small chip called a tissue array that could rapidly identify lymphoma patients with particular genetic profiles.

A standard expression microarray chip contains thousands to tens of thousands of genes, to which cDNA or cRNA from a patient’s tissue is hybridized. For a tissue array, on the other hand, two to three cores from each patient’s biopsy are placed on a slide and stained with antibodies to proteins coded by genes that discriminate the subgroups. Eighty cases can be analyzed on a standard microscope slide. Using such a tissue array, which Dr. Braziel calls "basically complex IHC," the group could identify the same clinically relevant subsets as were identified on full microarrays. Some version of this technique may well become feasible over the next five to 10 years, in Dr. Braziel’s view. "It may actually change our algorithm somewhat," she says. "We wouldn’t have to test for so many specific genetic factors like we do with targeted PCR. But we are certainly not there yet."

A more general lesson from this work, according to Dr. Braziel: "Pathologists should not feel the least bit despondent. We can take information from microarrays and narrow it down to manageable and clinically feasible profiles."

In mantle cell lymphoma, too, the Lymphoma/Leukemia Molecular Profiling Project has reported that gene expression profiling may offer prognostic information (Rosenwald A, et al. Cancer Cell. 2003;3:185-197). In this microarray analysis, poor outcome was predicted by factors related to proliferation, or aberrant cell cycle regulation, such as cyclin D1, which is already tested for in classifying MCL. However, a small number of patients did not have overexpression of cyclin D1, but rather cyclin D2. "Those data are preliminary, so I don’t know what to make of that yet," Dr. Braziel says. "I don’t think either microarrays or other molecular testing has come up with any answers in MCL."

Dr. Elenitoba-Johnson says other investigators have to verify this work. "We have one study that is making this point [about cyclin D1-negative MCL]." Making a far-reaching conclusion based on one paper is difficult, he notes. "We can’t change our diagnostic criteria every time someone comes up with a new concept."

Dr. Braziel took part in an LLMPP study of follicular lymphoma (Dave SS, et al. N Engl J Med. 2004;351:2159-2169). "We have had no therapy that impacted survival in follicular lymphoma," she says. In addition, prognosis is heterogeneous: Some patients live 20 years and some die in one year, with no apparent difference in morphological appearance. Molecular profiling, on the other hand, revealed that profiling the expression of genes largely related to immune response and immune status allows prediction and stratification of follicular lymphoma patients, which Dr. Braziel calls "a noteworthy achievement."As a member of the Southwest Oncology Group, Dr. Braziel has seen many clinical trials of therapy for follicular lymphoma over the years. "Nothing has had much of an impact or changed practice much," she says. "So making an observation that might play a role in patient care is really exciting."

She says it hasn’t yet translated into anything that can be used as a clinical test. "But many people are looking intensely at those data to see if we can do that." One possible contribution to patient management is that a poor prognostic index obtained at the time of initial diagnosis could influence a decision to transplant earlier.

Currently, pathologists attempt to categorize follicular lymphoma as grade 1, 2, or 3 or transformed into large cell lymphoma, Dr. Jones says. But that is not a particularly reproducible or quantitative position. "This is the next frontier for molecular methods in lymphoma," he says. "Can molecular testing now step in and not only diagnose follicular lymphoma but also show how far along the path from indolent to aggressive that a particular tumor has progressed?"

While gene expression profiling by microarrays has brought new excitement into the field of lymphoma diagnosis and prognosis, and provided a deeper understanding of the genetic processes underlying development of lymphomas, molecular pathologists are skeptical for many fundamental reasons that gene expression microarrays in their current form will become routine in the workup of lymphomas. Microarray technology is far more expensive than FISH, for example. "With gene chips in particular, the investment in equipment and personnel to bring them online is substantial," Dr. Ratech says. He notes that this investment is disproportionate to the small number of diseases they would be applied to—"no more than a few hundred cases a year per institution in primary lymphoma diagnosis. Even at the busiest medical center it would be hard to justify."

Another obstacle to clinical adoption of microarrays is that different groups can get different results on the same disease entity, a situation that has occurred in studies of diffuse large B-cell lymphoma. Dr. Sabath says the main source of this discordance is that people use different gene expression platforms. "In my laboratory we have been studying gene expression profiles of lymphocytes with an eye toward developing diagnostic tools ourselves. The hardest task for us has been finding a decent platform to make a custom microarray."

Dr. Jones says nearly all are undecided now about what to do with expression microarray classification of large B-cell lymphomas. Gene expression profiles can segregate cases into prognostic groups, but, he says, "they are based on platforms that generate large amounts of data and change gene targets all the time, almost monthly." Not surprisingly, there is no consensus yet on which markers to look at. He calls this "a huge issue."

"We are still waiting for a classification of lymphomas on a molecular basis using one of these newer techniques," he says. Reaching consensus will require agreement that a particular result was very important, and it will have to be confirmed in several laboratories using different technologies. "Expression microarrays are a bit lost in the wilderness right now," Dr. Jones says.

Dr. Sabath says there are also many fundamental technical issues with gene expression profiling—how to make RNA and label it, for instance. "When we take specimens out of our freezers we see changes," he says. "How do we prove RNA is adequate for microarray analysis?" He also points out that clinical use of microarrays will require "a huge cultural change" among pathologists, from routinely fixing and embedding tissue, which is not optimal for microarray analysis of RNA, to rapid freezing. (Related article: "Producing high-quality samples,")

Dr. Jones says setting up an infrastructure to collect samples in the right way to do microarray evaluation is also an obstacle. "Even in large cancer studies it is difficult to get people to treat every biopsy like it will potentially have microarray analysis." Tissue must be frozen quickly and treated carefully to eliminate non-tumor elements, which Dr. Jones calls "very laborious."

Dr. Arber, for one, doesn’t think microarrays are sufficiently useful for applying to patients. "I see them as good for defining prognostic groups. Then the key features found in the microarray would be tested by other methods, such as PCR or an immunophenotypic method," he says, noting that others have different opinions. "Perhaps mini-arrays [with a few dozen or a hundred genes] will be made for specific diseases," he allows. "But we are still some way from that."

Dr. Bagg agrees that microarrays "still stand in the experimental realm."

"I don’t know anyone applying those new findings directly to the diagnostic scenario yet," he says. Still, he adds, "The data coming out of microarray studies have turned dogma on its head in a wonderful way. This has been a humbling but very exciting experience for us. We have all these rules and guidelines, then along comes this new technology and turns things upside down."

Drs. Arber and Bagg cite the case of chronic lymphocytic leukemia to illustrate their ideas. It was known that CLL is morphologically homogeneous but clinically heterogeneous. Moreover, it was thought to be a malignancy of naive B cells, lymphocytes that had not passed through germinal centers of lymph nodes. Then molecular studies showed that in some cases of CLL malignant cells had been through germinal centers and that they displayed somatic hypermutation, providing what Dr. Bagg calls "one of the most important prognostic variables." However, he says, "to do somatic hypermutation testing on every CLL patient is not currently feasible. It is complex, expensive, and labor-intensive."

A few years ago, when samples with somatic hypermutation were compared with unmutated samples on gene expression microarrays, overexpression of certain genes was observed in unmutated specimens. One gene most indicative of this correlation was Zap-70, which Dr. Bagg considers "as good a discriminant as somatic hypermutation." Now many laboratories are setting up Zap-70 measurement by flow cytometry or immunohistochemistry. Thus, from complicated molecular analyses has emerged a useful assay in a more traditional form.

Microarrays are a "fantastic" discovery tool, Dr. Elenitoba-Johnson concurs. "They have already shown us things we would not otherwise have seen. But there might be other ways to look at the expression of discriminant or informative genes that would probably not necessitate having to evaluate an entire array." For the most part, he predicts that is the model that will be pursued. He says quantitative PCR is particularly good, with a dynamic range superior to that of microarrays. "For clinical purposes, one might need a testing modality capable of detecting fairly small changes," he suggests.

For Dr. Longtine, envisioning when microarrays will be used in the lab isn’t easy. She, too, thinks it more likely that subsets of markers discovered on microarrays will be measured by traditional methods. Even the analysis of data obtained from microarrays is more complicated than with other methods, Dr. Longtine says, noting that bioinformatics is used to analyze microarray data. "There is a discussion within the community of bioinformatics people how best to analyze these data," she says. "So much data come out of each of these microarray studies it is hard to distill which are the key markers."

Interpreting results of tens of thousands of genes requires expertise that most pathologists don’t have, Dr. Ratech agrees. "There are software programs for this, but if you are going to use data for diagnosis with clinical implications, I think you would want to understand the principles of that interpretation."

Although molecular hematopathologists are still not convinced of the clinical value of microarrays, some are having to deal with pressure from oncologists to start using them. Says Dr. Ratech: "Basically every time there is a new technological advance presented at the American Society of Hematology meeting or at ASCO, where most hematology/ oncology clinicians go, they come back to us and say, ’Why aren’t we doing this technique?’ It can be embarrassing because a pathologist can recognize the value of the technique, but not have the resources to evaluate it." There is also an issue of "prudence," Dr. Ratech says, since the clinician is referring to research papers and the technique has not been subjected to a real trial in the clinical world to assess its reproducibility, accuracy, and sensitivity and to determine whether it adds valuable information that can’t be obtained any other way.

"More than 20 years ago, clinicians came to me and said, ’Why aren’t we doing FISH to detect chromosomal translocations?’ At that time it was a cutting-edge technique. I didn’t have—and most hematopathologists didn’t have—training in FISH. Even classical cytogenetics was a separate discipline." Yet today FISH is a standard part of making an accurate diagnosis in hematopathology. On the other hand, many commercial tests have appeared that Dr. Ratech calls "crazy" and that no pathologist would ever endorse. For instance, at one time laboratories claimed to be able to take a piece of fresh tumor, expose it to drugs in vitro, and calculate the best combination drug profile. "I don’t think any of those panned out," he says. "They were the negative expression of the desire to get a new test on line."

While not feeling pressure to adopt microarrays for lymphoma at this time, Dr. Longtine does acknowledge sometimes being pressured to set up assays she considers not ready for prime time. "Handling these situations requires close communication and discussion," she says. "Clinicians are feeling pressure from patients to have the most current tools." For example, clinicians are now being pressured to send breast cancer samples to for-profit companies for microarray expression analysis. "It is unclear how that information will be used," she says, "but patients sometimes demand these tests." As a result, oncologists may pressure pathologists to perform certain procedures before they are ready. "Part comes from patients and some from oncologists who just came back from meetings and want those tools available at their institution," Dr. Longtine says.

Dr. Arber finds it easy now to resist pressure to bring microarrays into clinical use because "none are up to standard for clinical care. They are still investigational," he says. "You can’t use gene arrays, in my opinion, to guide treatment since they are not FDA approved or validated."

At the same time that they act prudently about introducing microarrays into routine diagnosis, pathologists must become acquainted with this technology and be prepared to work with it should the clinical demand arise. Dr. Elenitoba-Johnson calls pathologists "the integrators of all of these technologies."

"Historically, we acquire new tools and investigate their potential and utility. We would be well advised to get comfortable with microarrays," he says.

He calls pathology itself "a game of serial acquisition of tools." To his mind, progress in medicine occurs via a multiparameter approach. "The more discriminating modalities you introduce, the easier it is to get data relevant to finding distinct pathological, clinical, and molecular entities," he says. "Now microarray-based data are being validated. That will add to the complexity." Sometime in the next several years the clinical utility of microarrays will be determined, with a timeline that differs among pathological entities. For diffuse large B-cell lymphoma, for instance, that determination may come as soon as three years.

"It would be a very radical change for pathologists to begin at the molecular level," Dr. Arber says. "There is always the fear, too, that if you can do that you don’t need a pathologist." That’s why he feels strongly that pathologists should take the lead in investigating molecular methods. If pathologists don’t get interested, other specialties will take it over. "Then we will have a more limited role," he warns.

Dr. Bagg emphasizes the many variables associated with good performance of microarrays and the complexity of obtaining usable clinical information from them. "That is where pathologists come in," he says. "We are best attuned to evaluate those parameters. We amongst all physicians are most skilled at QC issues." Since many publications on microarrays have emanated from nonpathology research groups, one concern has been that nonpathologists would take over diagnosis of lymphomas. "But I don’t think they are as skilled as pathologists at QC and standardization," Dr. Bagg says. "That is where we as laboratorians are going to play a central role," in taking clinically useful microarrays from the research setting to the clinical milieu.

Dr. Braziel says younger pathologists can learn about microarrays in their training. For those already in practice, says Dr. Braziel, "CAP puts on courses in molecular areas aimed to be practical enough to update people." And every journal today, she notes, even the most anatomically oriented, has papers on molecular applications.

Also, pathologists in practice can identify a person to whom they can refer people who have questions. "That is one of the roles I serve," Dr. Braziel says, "to be willing to talk to people—both clinicians and patients. Some oncologists give my name to patients if they can’t explain why the physician is ordering a particular test. I end up talking to a fair number of patients."

One of Dr. Ratech’s mentors from 20 years ago attended the Association for Molecular Pathology session on lymphoma. "He has nothing to do with molecular diagnosis," Dr. Ratech says. "I asked him why he came." He replied, "I want to see if I am out of a job, obsolete." Afterward Dr. Ratech asked his mentor what he had decided. "He said he was still worried—but he felt better."

William Check is a medical writer in Wilmette, Ill.