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Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short
discussion of current topics and may be helpful to you in your practice.
Our speaker for this program is Dr. Dora Lam-Himlin, Senior Associate Consultant, Division
of Anatomic Pathology and Assistant Professor of Laboratory Medicine and Pathology at
Mayo Clinic Arizona. Dr. Lam-Himlin describes the histologic and clinical features of Graft
Versus Host Disease (GVHD) and considerations in diagnosis.
Thank you for the kind introduction. It is a privilege to speak to you about the histologic
diagnosis of graft versus host disease and other diagnostic considerations in apoptotic
pattern of injury. The content of this lecture is intended for pathologists in practice and in
training, and the scope will cover diagnosis of graft versus host disease as well as the
differential diagnoses.
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I have no conflicts of interest to disclose.
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There are 4 primary objectives that viewers should achieve at the end of this presentation.
The first is the ability to outline the histologic features of graft versus host disease (GVHD).
Secondly, viewers should be able to list the differential diagnoses for apoptotic pattern of
injury.
The third objective is to recognize that GVHD is a clinical correlation diagnosis.
In doing so, by the end of this presentation, viewers will be able to list the clinical and
medication considerations in the diagnosis of GVHD.
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This is the overview, or road map, for our discussion over the next 20 minutes.
I will start with a basic review of GVHD, including a discussion of the pathophysiology,
clinical features, endoscopic findings, and histology and grading. Following this, we will
discuss apoptotic pattern of injury through a series of 4 case examples.
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To whet your appetite for the upcoming discussion, and to introduce the concept of
apoptosis as a nonspecific pattern of injury, observe these four examples of colonic mucosa
with abundant apoptoses. These examples each show colonic crypts with markedly
increased apoptotic bodies, and look remarkably similar. However, each of these 4 cases
has distinctly different etiologies. Keep this in mind as we move into our discussion of
GVHD.
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Graft versus host disease is the donor T-lymphocyte mediated destruction of host tissues
that occurs following allogeneic transplant. Hematopoietic transplant is the most likely to
cause GVHD, with a frequency of 40-80% in transplant patients. Donor lymphocyte
transfusions, such as might be seen in whole blood transfusion, can also cause GVHD. Rare
reports of GVHD secondary to solid organ transplantation have been recorded, although
this is exceptionally rare.
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Patients present with gastrointestinal tract symptoms, the most common of which include
nausea, vomiting, and diarrhea. Abdominal pain may or may not be a significant symptom.
Some patients present with gastrointestinal bleeding, which can be significant enough to
require transfusion support. More commonly affected than the gastrointestinal tract is the
skin, which can show a sunburn-like rash, particularly in the face and chest areas. The
luminal gastrointestinal tract is the next most common site of involvement, followed by the
liver.
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Endoscopic findings are essentially nonspecific, and can range from showing patchy
erythema and ulceration in severe cases to normal in more than 40% of cases of GVHD. The
best diagnostic material for evaluation of GVHD are biopsies of the duodenum and colon.
These sites are the most sensitive for diagnosis. It is worthy to note that severity of GVHD
can vary from site to site and the effects of GVHD can be patchy. As a result, concordance
rates of GVHD grading between upper and lower GI biopsies are poor. As such, some
institutions include both upper and lower gastrointestinal biopsies in their GVHD protocol.
However, it is not uncommon to receive only upper or only lower GI biopsies for the
evaluation of GVHD.
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Histologically, apoptosis is the hallmark of this disease, and historically pathologists have graded
GVHD on a 4-tiered grading system. I will first describe these criteria, and in the next few slides we
can review some photomicrograph examples.
The mildest form, grade 1 GVHD, is defined as the presence of increased apoptoses in the crypt
epithelium. While the normal number of apoptoses has been reported as 1 per 10 crypts in the
colon and up to 1 per 4 crypts in the duodenum, a practical strategy to keep in mind is that
apoptoses in the setting of high clinical suspicion are likely to represent GVHD. Note, for example,
that the Stanford criteria for GVHD require only that a single apoptotic body is present in each
tissue fragment. When struggling with an equivocal case of low grade GVHD, investigation into the
patient’s clinical symptoms may be helpful; for example, the presence of a skin rash is a useful
indicator that the patient has some degree of GVHD. In addition, it is also helpful to keep in mind
that low grade GVHD, such as grade 1 GVHD, is not always treated. Rather, clinicians may choose to
watch and wait.
As GVHD progresses in severity to grade 2, one can observe the presence of crypt abscesses and
individual crypt damage. When crypts begin to drop out and focal mucosal necrosis is present, this
is considered grade 3 GVHD. Finally, grade 4 GVHD is defined by the presences of diffuse mucosal
necrosis and denudation of the mucosa. Remember that these histologic findings exist along a
spectrum, and by necessity some degree of subjectivity in interpretation will be present.
While most pathologists are accustomed to the 4-tiered grading system for GVHD, a separate
National Institute of Health consensus recommendation for the reporting of GVHD exists. This
reporting mechanism utilizes the degree of confidence that a pathologist feels in diagnosing the
presence or absence of GVHD by reporting absent, possible, probable, and unequivocal GVHD.
Although this method of reporting has been adopted in some centers, many clinicians prefer to
receive the numerical grading system described previously.
Finally, before we move on to histologic examples of GVHD, it is important to note that
longstanding low-grade GVHD can cause crypt distortion which can be mistaken as chronicity
changes of inflammatory bowel disease. Before diagnosing a bone marrow transplant patient with
new onset inflammatory bowel disease, investigate their history of GVHD.
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The following several slides include examples of histologic findings in GVHD. It is not
possible to identify apoptotic bodies at this low power view of the colonic mucosa.
However, at this scanning magnification, one can note the slight hyperchromasia of the
deep crypt epithelium –the area which we know is the proliferative compartment. This is a
helpful clue at low power and further investigation of these hyperchromatic areas will be
the highest yield for apoptotic bodies.
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At higher magnification, one can appreciate the presence of multiple apoptotic bodies in
close proximity to one another. These clusters of dying epithelial cells contain numerous
minute fragments of nuclear debris that are deeply basophilic. There is no significant crypt
damage or destruction in this example, which is sufficient for a diagnosis of grade 1 GVHD
in the proper clinical context.
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Another example of apoptotic bodies with preservation of crypt architecture is seen here.
This example shows abundant apoptotic bodies, which are seen as small round dark bodies
surrounded by a white halo. A common question from pathologists is, “How can one
differentiate apoptotic bodies from lymphocytes, which may also appear as small dark
round bodies with white halos?” This is an important question to ask, and has a relatively
simple solution. One can search for a known lymphocyte in the lamina propria. Take, for
example, the 2 small lymphocytes found in the lamina propria of this photo at the far left.
Use the nuclei of these inflammatory cells as the benchmark size, which should be about
10 microns in diameter. Compare this nuclear size to the body in question. Apoptotic
bodies will be smaller in size by comparison. Numerous examples exist within this photo
and show a wide range of sizes for apoptotic bodies.
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Although I frequently emphasize that apoptotic bodies are found in clusters in GVHD, single
cell apoptosis is also seen. These 2 examples highlight the subtlety of single cell apoptoses.
Again, note the presence of small dark round nuclear debris surrounded by a white halo.
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This example of GVHD grade 2 shows a crypt abscess and early crypt damage with early
crypt dropout.
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Higher power magnification of the previous photo confirms the presence of abundant
apoptotic bodies.
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Grade 3 GVHD is characterized by loss of crypts, as seen here. Note the architectural
distortion that is present, with areas of crypt dropout.
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This example of severe grade 4 GVHD shows complete loss of crypts and epithelium. Only
granulation-type tissue is present.
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With the key features of GVHD in mind, let’s take a look at our 4 example cases.
Remember, these four cases all demonstrate a pattern of injury that exhibits prominent
apoptotic bodies, but all 4 have different etiologies.
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Case 1. This photograph shows a colonic crypt with abundant apoptotic bodies. In the
absence of any clinical information, a pathologist can only comment that an apoptotic
pattern of injury is present and list the differential diagnoses. However, further information
is available which reveals that this biopsy is from a 32-year-old man who is status post bone
marrow transplant and presents with diarrhea. The clinicians ask pathology to rule out
GVHD. While the finding of apoptoses is compatible with a diagnosis of GVHD, further
examination of the biopsy reveals enlarged cells with densely eosinophilic nuclear
inclusions, such as seen in the bottom photo.
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The diagnosis in this case is cytomegalovirus infection (CMV). It is the most common GI
pathogen in immunocompromised patients who often present with complaints of
dysphagia, odynophagia, and diarrhea. The endoscopic finding is of punched-out ulcers,
while histology shows a neutrophilic infiltrate, ulceration, and nuclear and cytoplasmic
inclusions. The virus preferentially infects endothelium, stromal cells, and macrophages.
Treatment is with antiviral therapy and the key to diagnosis is identification of characteristic
viral inclusions. Immunohistochemistry may also be necessary in some cases, and is often
an up-front immunostain in GVHD protocols.
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Case 2. At low magnification, this colon biopsy shows hyperchromasia at the proliferative
compartment. Higher magnification reveals the presence of abundant apoptotic bodies.
Again, in the absence of any clinical information, this pattern of apoptotic injury is
nonspecific, and only a differential diagnosis can be given. However, the clinical information
provided for this case tells us that the biopsies were taken from a 53-year-old woman who
is status post bone marrow transplant. She presented with nausea, vomiting, and diarrhea,
and the clinical team has asked pathology to rule out GVHD. Further investigation reveals
that this patient is taking Cellcept or mycophenolate mofetil.
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CellCept and Myfortic, also known as mycophenolic acid, are immunosuppressive drugs
that were originally developed for solid organ transplantation. They are now widely used
for heart and lung transplantation as well as bone marrow transplantation. Medication
reaction is dose dependent and patients present with gastrointestinal distress. The
histology of mycophenolate-induced injury is essentially apoptotic in nature, but can mimic
ischemia, GVHD, and inflammatory bowel disease. Pictured here is a severe example of
mycophenolate-induced injury, which shows complete loss of epithelium and crypts.
Pathologists should keep a low threshold for investigating a patient’s medication list if there
is any history of transplantation or signs of apoptotic injury. Treatment is simple with either
dose modification or discontinuation of the drug. The result is usually dramatic and nearimmediate resolution of symptoms. It is important to note that there are not reliable
histologic features to differentiate GVHD from mycophenolate-induced injury, and further
investigation by the clinician is necessary in cases in which both GVHD and mycophenolate
are a consideration.
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Case 3. This example shows basophilia of the colonic proliferative compartment at low
power, while high power examination shows abundant apoptotic bodies. Similar to our
other cases, clinical information is imperative for interpretation of this case. This biopsy is
from a 61-year-old man who is status post bone marrow transplantation and he presents
with diarrhea, nausea, and vomiting. The clinicians request that pathology exclude GVHD.
Further investigation reveals that this patient has a skin rash, has negative CMV serology,
and is not taking mycophenolate.
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The best diagnosis in this example is: “Colonic mucosa with increased apoptotic bodies,
consistent with mild GVHD (grade 1 of 4), in the proper clinical setting.”
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This brings us to the last example, case 4. These colon biopsies show apoptotic activity and
come from a 46-year-old woman with severe diarrhea and abdominal discomfort. Given
the nonspecific nature of the symptoms and histology, further investigation into the
patient’s history is performed and reveals that the patient is receiving intravenous Taxol
(paclitaxel) for vaginal adenocarcinoma.
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Chemotherapeutic effect can manifest as apoptotic injury in the gastrointestinal tract.
Chemotherapy results in reactive oxygen species and some medications, such as Taxol,
inhibit microtubule breakdown which results in arrested mitoses. The example here shows
abundant arrested mitoses, some exhibiting ring forms. The presentation, endoscopy, and
histology are all nonspecific, and the key to diagnosis is in the clinical history and
medication list. It is important to note that cytoreductive therapy for bone marrow
transplantation can also exhibit these same changes. As a result, chemotherapeutic effect
is a consideration if biopsies of the GI tract are taken within the first 21 days of bone
marrow transplantation.
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As you can see through these examples, the presence of apoptotic bodies in
gastrointestinal biopsies is a nonspecific pattern of injury with multiple possible etiologies.
Listed here for your reference are a number of situations associated with apoptotic injury.
These include GVHD, acute cellular rejection of transplanted organs, chemotherapy and
radiation therapy effect, zinc deficiency or any nutritional deficiency such as might be seen
in fasting, inflammatory bowel disease, AIDS enteropathy, thymoma, medications, viral
infections, cell-mediated immune response, and autoimmune enteropathy. The key to
diagnosis in all of these cases is correlation with clinical information.
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As we conclude our session today, I would like to reemphasize some of the key take-home
points. Recall, apoptosis in the GI tract is not specific for GVHD. In all cases, correlation with
clinical information is a must, but particularly when dealing with bone marrow transplant
patients. In addition, we discussed that drug and viral effects are frequent in bone marrow
transplant patients and should be kept in the forefront of your mind during signout. Finally,
modification of mycophenolate dosage should have a rapid clinical response and this
feature can help differentiate medication reaction from GVHD in some cases.
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For those interested in learning more about this entity, selected references are included.
Thank you for taking the time to join me today in our discussion of graft versus host disease
and apoptotic pattern of injury. It has been my pleasure and privilege.
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