Section 1 -

New Techniques of Immunohistochemistry (IHC) and in situ Hybridization (ISH) for Lymphoma Diagnosis Thomas M. Grogan

I. Objective: To emphasize the importance of new IHC/ISH technologies in improving the diagnosis and treatment of lymphoma. II. Historic Perspective: As illustrated in Figure 1, new technologies have had a profound effect over time on the diagnosis of non-Hodgkin's lymphomas. As shown, the dominant technology over time has been the microscope. It was the tool that Rudolf Virchow used to establish the cellular basis of pathology and to establish the first lymphoma diagnostic category: lymphosarcoma [1]. Histology, namely the microscopist's H&E stain, remains to this day the basis of lymphoma diagnosis. Microscopy now, as then, gives morphologic, microanatomic context critical to diagnosis. While morphology and the microscope remain foundational, it was the advent of a second technology: immunologic methods which lead to an explosion of lymphoma biology and new categorizations. Starting in 1976 with the addition of immunologic categories like lymphoblastic lymphoma [2] and new immunologic-based classification schemes by Lukes & Butler [3], there was a proliferation of new lymphoma biology knowledge. In the 1980's came the advent of the Nobel prize winning concept of monoclonal antibodies and new enzyme detection systems (e.g., DAB) leading to the birth of immunohistochemistry (IHC). Within the decade came an explosion of new molecular techniques (PCR, FISH, ISH) which combined with IHC data spawned a new synthesis of lymphoma diagnosis - one based on the laboratory-biologic properties of lymphomas. This scheme of some 28 lymphoma categories was promulgated by an international group known as the International Lymphoma Study Group and was known as the Revised European - American Lymphoma Classification [4]. This became global with the addition of more international co-authors and the now 30 lymphoma categories were published as the WHO Classification in 2001 [5]. You may say that Figure 1 is showing us a classic technology "S" curve [6]. As shown, beginning with one technology (microscopy) progress is very slow. Then, as a second and third technology are added (e.g., the 1980's IHC and molecular techniques) there is an explosion (1990's) followed by then gradual maturation of the process. On another technologic front, also shown in Figure 1, there was substantial progress regarding the chemical treatment of lymphoma beginning with the advent of modern combination chemotherapy (e.g., MOPP) in 1965, the advent of curative-Adriamycin therapy 1979, and the more recent targeted antibody therapy (Rituxamab, 1998). These technical treatment advances have asked of the pathologist new questions: besides the diagnosis, what are the targets of therapy (e.g., CD20) and what predicts response to therapy (e.g., BCL2, BCL6) [7, 8]. There is another way to benchmark technologic progress over time. That is not from the academic proliferation of diagnoses, but rather from the point of view of the patient. Before the advent of IHC and ISH technologies, the diagnostic category of "undifferentiated malignancy" was an every day event in surgical pathology practice. In the late 1960's when curative-intent chemotherapy began, but diagnostic testing was not sophisticated, it was common to use the category of "undifferentiated malignancy cannot exclude lymphoma". The reasoning being that a patient even suspected to have lymphoma would benefit from combination chemotherapy. This primitive state of diagnosis was quickly ameliorated with IHC markers [9]. As shown, CD45 (LCA) positive "undifferentiated tumors" had a much better survival than CD45-negative tumors. So improved diagnosis from new technologies measurably led to improved patient survival. III. Current State of the Art: Current technologies have given us a world of diagnostic capabilities. [10, 11, 12] While the focus for the past 150 years has been on diagnosis, the last 20 years with the advent of slide-based chemistry (IHC, ISH, FISH) have gone beyond diagnosis to inform us about pathogenesis, etiology and prognosis. In particular, there are IHC assays to the infectious agents associated with lymphoma, for example: 1) Helicobacter pylori in Mucosa-Associated lymphoma (MALTOMA); 2) Epstein-Barr virus in Burkitt's lymphoma; 3) HHV8 in HIV associated lymphoma [10]. Regarding genetic events, FISH has delineated relevant chromosomal translocations (e.g., t(8;14) Burkitt's, t(14;18) follicular lymphoma, t(11;14) mantle cell lymphoma) [11]. Beyond diagnosis and pathogenesis, panels of assays have revealed prognostic markers (e.g., BCL2 in DLBCL, B versus T cell lineage in DLBCL, ALK1+ in ALCL, HLADR in DLBCL) [12]. In the last twenty years new technologies have taken us from the generic diagnosis (e.g., do I have lymphoma) to which of 30 fully articulated types do I have and how will I do? (prognosis). So now that we are at the peak of the diagnostic "S" curve in the year 2006, are we done? IV. Future Developments: While a few new rarities may emerge, the new emphasis has switched from diagnosis to treatment. We have gone from: what do I have? To how will I be treated? And how will I respond? To improve, as a profession, we will need not only better diagnostic ability but also more treatment relevant assays and finally we will need to better communicate these more complex results with both the oncologist and with the patients. These new demands on the pathologist will require new technologies. These new technologies will require new chemistries and new instrumentation. The new chemistries include a need for more monoclonal antibodies, more molecular probes, better detection, and more multiparameter and multiplexing capability. The new monoclonals will include newer higher affinity rabbit monoclonals (e.g., Anti-Cyclin D1), antibodies to phosphorylated cell signaling molecules (e.g., mTOR) and monoclonals to targets of therapy (e.g., CD20), and antibodies to indicators of response to therapy (e.g., BCL2, BCL6) [8]. We will require more molecular probes including cocktails of oligionucleotides (e.g., detection of Ig mRNA monoclonality via ISH). As we seek to detect cell signaling events, we will require more sensitive detection utilizing polymers and better heterobifunctional conjugates. To get at genetic events we will need to detect single gene events, requiring new detection technologies including the next generation fluorochromes (e.g., Q-Dots) and the next generation chromogens (e.g., enzyme metallography) [13]. The value the next generation fluorochromes is they allow discreet, non-quenching signals which facilitate multiplexing, quantitation and morphometrics. The combination of these three could facilitate the ability to do multiple antigens simultaneously resulting in "flow-cytometry-on-a-glass-slide." Besides improved chemistry, we will also require improved instrumentation including: 1) immunostainers 2) imaging and 3) information technology. The next generation of immunostainers need to give us total control of temperature, stringency, Ph, reagent dosing, and washing. It needs to deliver "baking through cover slipping" and random access with "same day" results of both IHC and ISH. The latter combined IHC/ISH assays give a "multiparameter" "gene plus protein" result now proven useful (e.g., Her2neu) which should soon apply to hematopathology [13]. The imaging will eventually prove pivotal as the multiplexing and quantitation will require multispectral and morphometric analytic capabilities. Lastly to write a report to satisfy both medical professional, patient, and administrative needs will require new information technology (the "so-called" third instrument). The goal is through LIS connectivity to produce an integrative report of clinical, laboratory and radiologic findings. The report then may be communicated to the physician (Physician-centric report) and to the patient (Patient-centric report). V. The Role of the Pathologist: As indicated above, the ultimate role of the pathologist is to serve: 1) as the interpreter of the data (e.g., what does the H&E show?; what do the immunostains contribute?), 2) as the integrator of the data (how do the molecular, phenotypic and morphologic findings combine?), and 3) as the communicator of the data in a report. We have discussed how technology will help us, but will it replace us? We wonder will the pathologist be replaced by a "chip"? Will multiplexed DNA arrays, mass spectroscopy, RT-PCR, spiral CT, or PET scans replace pathologists. Possibly, as recently shown with the diagnosis of certain rarities (e.g., Cyclin D1 negative Mantle Cell Lymphoma, Burkitt's lymphoma without C-myc expression and DLBCL with C-myc expression) by DNA array assay, the new molecular methods may give superior results [14, 15]. They are already spawning new categories resulting in a new technology "S" curve [6]. To avoid being replaced, we must remember the pathologists role as integrator. Firstly, the pathologist by microscopy provides microanatomic context which is often pivotal. Often the issue is: what is the chemistry of the lesional tissue? This the pathologist is uniquely able to provide by combining microscopy and chemistry. Secondly, if the pathologist continues to improve his diagnostic armamentarium (e.g., multiparameter combined "gene-protein" assays, and multiplexing assays). Then, he can "match-in-kind" the molecular result with the advantage of cellular context. Finally, it is a medical mind that has to meld all this and communicate a meaningful, actionable medical result … and presuming we continue to embrace new technologies that will best be a pathologist. That would be the technologically advanced pathologist of the future with better probes and antibodies, better detection chemistry, better instruments and better tools of communication. References Virchow R. 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