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With the advent of the molecular techniques in the past ten years, there has been a steady increase in the utility of the diagnostic and predictive biomarkers in human tumors. In this educational exercise we will first examine what is the nature of these markers and how they were discovered. We will then systematically review their current practical application in diagnostic immunohistochemistry by illustrating a number of case examples. Our discussion will be limited to the “diagnostic” markers as the field of “predictive” biomarkers is rather vast and requires a separate session. The presentation will conclude by summarizing the advantages and limitations of the application of genomically-derived antibodies in diagnostic immunohistochemistry.
- The origin of genomically-derived protein markers and the methods of their discovery.
- Their practical application to diagnostic tumor immunohistochemistry.
- The advantages and potential limitations of their utilization in histopathology.
MEHRDAD NADJI, MD:
The topic of presentation, as you can see, is genomically derived antibodies in diagnostic immunohistochemistry. And the reason for that is that molecular techniques in the past 10 to 15 years have increased the utility of genomically derived diagnostic and predictive biomarkers, and today, particularly our discussion will be limited to the diagnostic markers as the field of predictive biomarkers in rather vast and requires a separate session.
Alright, this is the presentation outline. The outline is how, what, and when. And by that I mean how these proteins were discovered, what are we detecting by using this technology, and when do we use them in our daily practice. And as an example of that I'm going to show you illustrative cases, some of the common, and common tumors where these markers are used, and I will finish the talk with the values and limitations of all these technologies at this time. This is my disclaimer, and that’s that I have no interest, financial or otherwise, in the content and material presented in this educational activity.
The immunohistochemistry that we use with genomically derived antibodies we have to know how proteins are discovered first. As opposed to classic methods of producing antibodies, which normal cell and tissue proteins are isolated, an antibody was produced against them, the genomically derived antibodies take advantage of molecular abnormality detected, and those molecular abnormalities may or may not result in expression of an abnormal protein, or an abnormal expression of a protein. That’s where we go from here.
These all discovered by expression profiling, either gene amplification, gene translocation and other genetic abnormalities, gene fusion and gene mutations, which produce proteins that we can take advantage of them and use them in immunohistochemistry. So what are we detecting is basically very simple, is protein overexpression, and protein loss, or under expression, or the product of the abnormal protein, such as fusion and mutated protein products. Where do we use them, a large number of these genomically derived antibodies are used as actually predictive markers. In fact, it was started in the area of predictive medicine, and they were presented as the so called companion test for a number of targeted drugs. They are occasionally also used for prognostication. What we’ll be discussing today is diagnostic use of these markers for either tumor diagnosis or tumor classification. And I have to mention to you that the diagnostic markers, that means tumor diagnosis versus a benign or normal lesion, is very uncommonly use for immunohistochemistry of these markers. These markers, with a rare exception, are basically only used for tumor classification and cell lineage identification.
Before I start to give you examples of these usages I would like to share with you how I'm going to approach with these case examples. First, on the left upper corner is the name of that particular marker, and its available the abbreviation for that name, where this name is coming from. And then I'll take you into the mentioning it's a type of protein product, how it was produced, whether or not it is expressed in normal tissue, and the main practical application, at least in my experience, and other potential uses, and sometimes abuses.
Let's start case examples with those markers that show protein over expression, and there are a whole bunch of them. By the way, the examples I give you today are just the tip of the iceberg. There are hundreds of these particular markers that are available, and use them on a daily practice of diagnostic pathology, what I can just share with you a few of them. And what a better place to start than with DOG1, which is known to a lot of us.
It was discovered by gene expression profiling. And the staining is in cytoplasmic and membrane staining, which is unusual. Usually most of these, as we can see later, are nuclear antigens. But this particular one is actually cytoplasmic, and cytoplasmic membrane antigen.
It is expressed in a large of number gastrointestinal stromal tumors, more than 95%. The importance of this marker is that it is expressed in KIT negative gastrointestinal stromal tumor, the ones which are related to PDGF-alpha abnormality. Occasionally other tumors also could be positive, such as a chondroblastomas, and a few epithelial tumors which may be focal positive for this particular marker. An example of the staining, these are hematoxylin using photomicrographs of the gastrointestinal tumor, with some epithelioid features, low and high power. And on the right side you can see the staining for DOG1 in the cytoplasm and cytoplasmic membrane. Similar to KIT, CD117, the reaction for DOG1 is usually diffuse and the so-called all or none phenomenon. Focal staining of these tumors is very uncommon.
I'll give you an example of how it may prove to be very useful, here is a biopsy of a gastric ulcer. The patient was bleeding and the clinician felt that there was an underlying large mass protruding through the mucosa and causing this ulcer and bleeding. And this biopsy, it's very difficult to see anything different except the inflammatory cells and so forth, but paying attention to this particular area in the middle of the center of this particular biopsy, there are some nondescript cells which could potentially be an epithelioid gastrointestinal stromal tumor; and hence, with that clinical differential diagnosis we performed a KIT, or CD117. In that area you can see is clearly negative. And the epithelial nature of that area is much better seen on this KIT staining. However, the staining for DOG1 clearly demonstrates that the tumor is actually an epithelioid gastrointestinal stromal tumor, which proved on the resection of the tumor to be the case.
I'm going to go to the next category of these markers, and the one that I found very useful many times is SATB2, this is special AT-rich sequence binding protein 2. This also was discovered by protein expression and profiling. It is expressed normally in epithelial lower-GI tract, colon or occasionally appendix. The great of majority of colorectal carcinoma is a stain positive, and the marker, of course, is a nuclear marker. Unrelated, but interestingly, it is also expressed a number of osteosarcomas. As you know, these carcinomas normally don’t have too many markers. So for our part it's going to be useful for us. A particular use of this SATB2 is in those rare cases of colorectal carcinoma which are negative for CK20, or sometimes even for CDX2. And that includes the rare medullary type of colorectal carcinoma, and occasional high-grade neuroendocrine colorectal carcinoma, which look like a small-cell carcinomas. These two may be CK20 negative, but in order to prove their colonic origin, SATB2 would be very helpful.
Here on the top you can see two low and high-power magnification of this particular distal colon tumor. It looks like a small-cell tumor. On the bottom left you can see cytokeratin staining, which has a typical staining pattern for a small-cell malignant tumor, a pointy type of a staining. Next to that in the middle, synaptophysin, it shows that this is indeed a neuroendocrine tumor; however, cytokeratin 20 next to that is completely negative. Now the question, is this a metastatic tumor, is actually colorectal in origin. So here is SATB2 low power and high power showing a disuse nuclear staining universally for that particular marker, extremely helpful in these kinds of occasions.
This next slide shows an interesting case of sarcomatous carcinoma of the lung, or the so-called carcinosarcoma of lung, also known as metaplastic carcinoma of the lung. The epithelial component of the tumor not shown here was a fully differentiated squamous cell carcinoma. The mesenchymal component was a sarcoma with focal evidence of osteoid formation, as you can see with the red arrow there. And that area made us to believe that maybe the rest of the sarcomatous elements are also an osteosarcoma. And using this marker you can see that almost all the tumor cells are positive for nuclear expression of SATB2, including that area which looked like an osteoid. So not having too many markers for osteosarcoma, this may be very helpful for that differential diagnosis, unrelated to colorectal cancer.
The next protein is SF-1, it is a steroidogenic factor 1. This is a member of nuclear hormone receptor family, just like the other that we know, like ER/PR,