Hematopathology: New Technologies
Moderators: Dr. John Wing Chan and Dr. Thomas Grogan
Section 1 -
New Techniques of Immunohistochemistry (IHC) and in situ Hybridization (ISH) for Lymphoma Diagnosis
Thomas M. Grogan
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 . 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  and new immunologic-based classification
schemes by Lukes & Butler , 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 . 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 .
You may say that Figure 1 is showing us a classic technology "S" curve . 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)
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 .
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
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 . 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
. 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
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)
. 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)
. 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
which should soon apply to hematopathology . 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
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
They are already spawning new categories resulting in a new technology "S"
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.
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