Pathologists receive a hefty number of biopsies from the esophagus with nothing more than, "Barrett's esophagus; rule out dysplasia" provided in the clinical history. In this post, I will go through the process pathologists use to fulfill this "simple request" from the clinician.
Making a Diagnosis of Barrett's Esophagus
If you haven't already read my previous post that discussed how gastroesophageal reflux (GERD) can progress to intestinal metaplasia, dysplasia, and eventually carcinoma, take some time to read that before continuing.
A quick refresher, making a diagnosis of "Barrett's Esophagus" includes looking for the presence of glandular cells, then determining whether there are any "true" goblet cells around (blue, mucinous amphophilic cytoplasm). In my previous post, I described how PAS-alcian blue stain could sometimes be used to determine whether a "goblet-oid cell" was a true GC (stains blue) or some gastric cardiac/foveolar mucinous gland that is a "wannabe goblet cell" (stains magenta). However, that is not often done in clinical practice as H&E should be sufficient for a diagnosis.
If goblet cells (GC) are seen in the esophagus, this is referred to as "intestinal metaplasia" (IM). Just seeing goblet cells, however, is insufficient for the diagnosis of BE. The exact requirements for a diagnosis of "Barrett's esophagus" vary on which country you are in and which societies' guidelines you use. In the US, we typically require the presence of goblet cells at least 1 cm ABOVE the GEJ/top of the gastric fold (aka, the biopsy needs to be from the DISTAL esophagus- or above), and the endoscopist must describe seeing an abnormality at that site (often described as "salmon colored plaques") in order to make this diagnosis.
One thing that I did not mention in the last post (to limit confusion) was how "Intestinal metaplasia" could still be called in the absence of goblet cells. This is because definition of intestinal metaplasia could also include the presence of Paneth cells in the glandular epithelium (cells that contain dense pink, coarse granules in their cytoplasm), although this is a much less commonly encountered scenario.
Ok... moving on. Now that a diagnosis of "Barrett's Esophagus" is made, what's next?!
Fulfilling the clinician's request:
"RULE OUT DYSPLASIA"
In order to address the clinician's request, the pathologist must look at every piece of tissue on the slide to determine whether there is any dysplastic or invasive glands lurking around. In this post, I will be discussing the criterion for the diagnosis of low/high grade dysplasia, clues to suggest an invasive carcinoma, and lastly, how p53 and/or Ki-67 stains could be helpful during the workup.
Before we start, it is important to recognize how a pathologist grades the degree of dysplasia affects patient management:
A Systematic Approach to Evaluating for Dysplasia
When trying to figure out whether a gland is "benign" or "dysplastic", always evaluate these four key features:
Surface maturation compared to underlying glands
Inflammation, erosions or ulceration in the background
1. Is there surface maturation present?
This is best assessed at LOW POWER. As glands "mature" the nucleus gets smaller and the cytoplasm gets filled with more mucin as you go from the base (bottom) of a gland up to the luminal surface (top) of a gland
The nuclei at the base of the crypt/gland are larger and darker (more hyperchromatic) & can have a few mitotic figures
The nuclei at the "top"/luminal surface of the gland appear lighter and more "open" due to their inc mucin (clear-blue-light pink cytoplasm) & smaller nuclei
Mitotic figures and nuclei should NOT extend to the surface
Atypical nuclei extending all the way up to the surface, giving it a "dark" appearance at low power
Loss of mucinous (clear) epithelium at surface
Retention of the darker, larger nuclei +/- mitoses at the surface
2. Is the glandular architecture ok?
"Glandular architecture describes the relationships between glands and the lamina propria and also encompasses the shapes of the glands."
CIRCULAR/ ROUND GLANDS (cross section)/ 'STRAIGHT TEST TUBES' (longitudinal section)
All of the cells are lined up perpendicularly to the basement membrane
Only one cell layer thick
There is no budding of glands
Glands should be surrounded by abundant lamina propria (there should be space between each gland)
Increased numbers of glands that appear "crowded" or "back-to-back"
Angulated, abnormally shaped glands
Cribriform glands (looks like someone took a cookie cutter and "cut out spaces" from the clustered glands)
Necrotic luminal debris
3. Is there cytologic atypia- Do the cells look "good" or "bad"?
This is perhaps the number 1 feature to consider whether to call something dysplastic vs. reactive (or invasive).
Allow some "funky cells" in the basal zone nuclei or in columnar epithelium adjacent to squamous mucosa
Nuclear polarity is maintained (long axis of the nucleus remains perpindicular to the basement membrane, nuclei are aligned parallel to each other)
Variation in size/shape when compared to "neighbors"
Nuclear membrane irregularities (jagged, or elongated "pencillate" nuclei)
Loss of nuclear polarity (loss of perpendicular orientation, randomly arranged nuclei in relation to the basement membrane & to it's neighbor, overlapping, jumbled up appearance)
4. Background Inflammation?
Inflammation causes the epithelium to "react", which can be can be ugly. In the presence of increased background inflammation, you should really back-off on calling something dysplastic unless it is just "entirely too funky".
I like to say, "If there is inflammation in the back-ground, BACK DOWN!"
In the presence of abundant inflammation, consider "downgrading" your call by 1 grade.
EX: If something appears "high grade" but there is a lot of inflammation, "downgrade" it to a "Low Grade Dysplasia"
EX: If it looks low grade but there is a lot of inflammation, call it "Reactive Atypia"
"Normal" Glands- a.k.a. "NEGATIVE FOR DYSPLASIA"
I love this "tip" from Drs. Voltaggio & Montgomery (see below).
Does it pass the "4 lines/tiers" test?
Starting at the surface:
First tier - formed by the gastric foveolar type mucin droplet
Second tier- formed by the base of the foveolar mucin vacuole
Third tier - formed by the cytoplasm below the mucin vacuole
Fourth tier- formed by the fourth by the row of nuclei
Finding "4 lines/tiers" is normal- this includes reactive epithelium in the gastric cardiac mucosa or non-dysplastic Barrett mucosa.
If you see the 4 distinct zones, then it is NOT DYSPLASTIC! If the lines are blurred & all jumbled up, then it's DYSPLASTIC!
The example below demonstrates reactive changes from an inflammatory response. Although the nuclei are elongated and contain prominent nucleoli, there are still 4 distinct lines made by these cells. If this was dysplastic, these zones would not be easily distinguished.
LOW GRADE DYSPLASIA
Basically, think of all the changes you see in a typical tubular adenoma of the colon.
Surface maturation= The surface can look similar to the underlying glands at low power or show only slight maturation. There will be a loss of mucin at the dysplastic area where the nuclei instead extend up to the surface
There is a LOSS of the organized 4 lines/tiers seen at the surface of normal or reactive epithelium in the higher magnification of this low grade dysplastic lesion below.
Architecture= Mildly to markedly distorted (Crowded glands); There still is some degree of stratification.
Lamina propria should still be present between glands!
Cytology= Should extend at least focally to the surface. Nuclei at the surface are irregular, hyperchromatic, & mildly enlarged.
**CLUE** Look for an abrupt transition between "normal" and "dysplastic" epithelium
Some cases may be polypoid in which case "POLYPOID LOW GRADE COLUMNAR EPITHELIAL DYSPLASIA" is an appropriate way to sign out the case.
HIGH GRADE DYSPLASIA
Similar to high grade lesions in the colon, look for:
loss of surface maturation- large, dark, ugly nuclei extend up to the surface- loss of mucin
crowded, distorted glandular architecture
back-to-back glands with little lamina propria between them
cribriforming of glands
round nuclei (instead of the pencillate, elongated nuclei in LGD)
Look for markedly enlarged, round, dark nuclei that extend up to the surface with a loss of nuclear polarity.
NOTE: Usually there are NOT prominent nucleoli in high grade dysplastic lesions. If present, this suggests either reactive changes or an invasive carcinoma. In these cases, there is often evidence of erosion or ulceration that are causing these plump nucleoli, which can be a helpful clue!
I will delve into the details of diagnosing an intramucosal carcinoma or invasive carcinoma in a separate post.
Use of p53 in Evaluation of Dysplasia
p53 is a transcription factor that mediates cell cycle arrest or apoptosis in the setting of cellular stresses, including DNA damage.
TP53 inactivation is among the most frequent molecular genetic events in neoplasia
Missense mutations in p53 DNA binding core domain --> change in shape of protein --> longer half life --> increased expression of p53 mutant protein
Loss of the wild-type allele from deletion of or truncation of gene= Complete loss of p53 expression
According to Dr. Andrew M. Bellizzi,
"I occasionally use p53 IHC in the assessment of atypical BE foci, particularly when I am considering a differential of indefinite for dysplasia versus low-grade dysplasia, including cases in which atypia is confined to the crypt compartment"
"In evaluating the p53 immunostain, I am seeking to colocalize a histologically atypical focus with patterns of expression in keeping with TP53 inactivation."
How does "Normal" tissue stain with p53?
Given an inflammatory milieu, increased expression of wild-type p53 in nondysplastic BE is not surprising.
This takes the form of diffuse expression of weak intensity, often punctuated by scattered more darkly staining nuclei.
The wild-type pattern is often misinterpreted as a positive result
Remember, just because something "turns brown", does NOT mean it is a "POSITIVE" result!!
What is the staining pattern for TP53 mutation (inactivation)?
Missense mutations in p53 cause an accumulation/over-expression of an abnormal (non-functional) p53 protein and will show diffuse strong, nuclear staining pattern. This is the most common situation.
DIFFUSE, STRONG, NUCLEAR STAINING (most common)
or it could show a...
COMPLETE ABSENCE OF STAINING (null pattern) in a background of wild-type pattern staining
The null staining pattern arises when there is a large deletion or truncation of the p53 gene. This results in complete loss of the p53 gene and is referred to as having a "null pattern" (complete loss of staining in a background of "Wild-type" patchy staining pattern of "normal glands" in the background). This less familiar null pattern may be misinterpreted as a negative result.
The finding of a pattern of p53 expression characteristic of TP53 inactivation supports a diagnosis of low-grade dysplasia over indefinite for dysplasia.
Strongly + or Completely - ---> LOW GRADE DYSPLASIA
Abnormal p53 staining is typical of high-grade dysplasia and carcinoma, and, thus, not considered as useful for dysplasia grading. You should be able to diagnose high grade based on findings on H&E.
These are EXTREMELY USEFUL... Give them a read for yourself!