Pathology Student

I received this very good question by email yesterday. Please feel free to send in your questions, and I’ll post the answers here.

Q. When a squamous cell carcinoma is designated as “poorly differentiated”, what other parameters/tests are performed to determine the tissue of origin?

A. Differentiation, for those of you who have just joined us, is a quality of tumors that has to do with how  much the tumor cells resemble their tissue of origin. Well-differentiated tumors are composed of cells that closely resemble their tissue of origin, whereas poorly-differentiated tumors are composed of cells that have little resemblance to their tissue of origin. Anaplastic tumors are the least differentiated of all: they show no resemblance to their tissue of origin.

This concept is important for a couple reasons. First, the degree of differentiation of a tumor often has a bearing on prognosis. Well-differentiated tumors generally carry a better prognosis than poorly differentiated tumors. Second, when a tumor is totally undifferentiated (anaplastic), you have to resort to special tests in order to figure out its origin (is it a squamous cell carcinoma, an adenocarcinoma, a lymphoma, a sarcoma, etc.).

Back to our question: when you have a poorly-differentiated squamous cell carcinoma, how do you know it’s a squamous cell carcinoma (as opposed to an adenocarcinoma, for example)? If the tumor is poorly-differentiated, that means there are still some morphologic features (albeit few) that reveal the squamous nature of the tumor. If you look carefully, you should be able to find some of these features, which would then point you towards the diagnosis of squamous cell carcinoma.

Two clear-cut features of squamous cell carcinoma are intercellular bridging and keratin pearls (there are other, “softer” features indicating a squamous cell origin, but we’ll focus on the more definitive features). Intercellular bridging is a term describing the special connection between the epithelial cells of squamous epithelium (it’s not present in glandular epithelium). By light microscopy, you can see little horizontal hair-like connections between the epithelial cells in both normal squamous epithelium and in malignant squamous epithelium. Look closely between the epithelial cells in the above image of a squamous cell carcinoma. See the little connections between the cells (they look like little zippers connecting the cells)? Those are intercellular bridges.

Keratin pearls are whorl-shaped accumulations of keratin made by malignant squamous cells. In normal squamous epithelium, keratin lies in a nice flat layer on the epithelial surface. In malignant squamous epithelium, the tumor cells can grow in any direction they want, and so the keratin they produce often gets trapped inside the tumor, forming pink, glassy, spherical masses. There’s a nice keratin pearl at about 2 o’clock in the image above.

So, if you have a tumor that you think might be a squamous cell carcinoma, but the cells aren’t showing clear squamous cell differentiation, look closely for epithelial bridging and keratin pearls. If you find either of these, it’s a good bet that you’re dealing with a squamous cell carcinoma. There are other little clues that point towards other types of tumors (like adenocarcinoma, or melanoma, or sarcoma), but that’s for another post.

Sometimes, you’ll get a tumor that shows no defining morphologic features whatsoever – no interepithelial bridging, no keratin pearls, no signs of differentiation along any other cell line. In these tumors (which would be described as “anaplastic”), you need to use a secret weapon to figure out what the cells are: immunohistochemistry. In this technique, you use a reagent consisting of antibodies against specific components of cells (there are lots of these specific components: squamous cells have cytokeratin in them, muscle cells have actin in them, etc.). These antibodies are bound to a substance that appears brown under the microscope. The concept is simple: you apply the reagent to the tissue in question, allow it to bind to the cells, then wash off the excess reagent and look at it under the microscope. If the tumor cells appear brown, that means they possess whatever antigen the antibodies in your reagent are directed against, and that information can help you figure out what type of cells your tumor contains.

So, if you have an anaplastic tumor, you might choose to apply immunohistochemical stains for cytokeratin, actin, and CD45 (an antigen present on lymphoid cells). If the cytokeratin stain comes back positive (brown), and the actin and CD45 stains come back negative, the tumor is most likely a squamous cell carcinoma. There are tons of immunohistochemical stains for all different types of cells. Generally, these stains are pretty specific for one cell line, but it’s not always totally straightforward. Some tumors stain positive for markers from cell lines other than their cell of origin, and some tumors show only weak staining with the stains that are supposed to be nice and positive. So you really need to use a panel of a bunch of different stains to make the best diagnosis.

The bottom line, then, if you have an undifferentiated appearing tumor: look for little morphologic clues (like keratin pearls) first. If you see few or no clues, then your next step is immunohistochemical staining, which will almost always reveal the origin of the tumor. If that doesn’t work, there are still other tests you can do – like cytogenetics or molecular diagnostics – and we’ll talk about those in future posts.

Photo credit: AFIP
http://commons.wikimedia.org/wiki/File:Well_differentiated_squamous_cell_carcinoma.jpg

Next in our little series on hereditary hemolytic anemias is glucose 6 phosphate dehydrogenase (or G6PD) deficiency.

Before we get into G6PD deficiency, we need to discuss just what this G6PD enzyme does. If you can close your eyes and try to think back to biochemistry, see if you can recall something called glutathione (if you can’t, look on Wikipedia). It’s a tripeptide that protects cells from free-radical injury. It exists in two states: a reduced state (GSH) and an oxidized state (GSSG). In the reduced state, GSH is able to donate a hydrogen ion to dangerous toxic substances (like reactive oxygen species), making them much less nasty. You need to have this stuff in your cells, or else you won’t be able to detoxify the nasty stuff that the cell makes during metabolism – and the cell will die a mature death.

In doing its important detoxification work, glutathione moves from the reduced state (GSH) to the oxidized state (GSSG). Obviously, you need to change the GSSG back into GSH in order for this pathway to continue to work. That’s where G6PD comes in. G6PD reduces NADP to NADPH, a substance which in turn converts GSSG into GSH. Whew. That’s a lot of biochem. See the little “Pentose phosphate pathway and glutathione production” box here. So: if you don’t have G6PD, you can’t reduce your NADP, which means that you can’t reduce your GSSG. And the cell will suffer an untimely demise.

That’s what happens in patients with G6PD deficiency. Usually, patients are fine, with no perceptible anemia, until they encounter an oxidant of some sort (the list is long, and includes a wide variety of substances, from fava beans to aspirin to mothballs). The oxidant creates more free radicals in the red cell, which means that glutathione is particularly active. Without G6PD, the glutathione remains in its oxidized state, unable to act further, and the free radicals attach sulfhydryl groups and disulfide bonds, liberating heme from globin. The globin becomes denatured and forms into a little ball called a Heinz body, which sticks to the red cell membrane. Macrophages in the spleen see these Heinz bodies and bite them out of the red cells, making unusually shaped “bite cells.”

The gene for G6PD is on the X chromosome, so males are much more likely to have full expression of the disease. The disease is more common in African Americans (10% of African American males have the gene). It also has a particularly high incidence in areas in which malaria was endemic, and it is thought to confer a selective advantage against malaria infection.

If you look at a blood smear of a patient who is in an acute hemolytic episode, you’ll see cell fragments, including bite cells (yes, they’re really called that), which are caused by recent pitting of Heinz bodies. To see the actual Heinz bodies, you need to do a supravital stain (see the image above – the Heinz bodies appear as green dots).

Therapy is pretty straightforward. Obviously, the patient needs to avoid taking offending drugs or drinking hydrogen peroxide. The hemolysis in G6PD deficiency is self-limiting, usually resolving within a week. This is because it’s the older red cells (which are already pretty G6PD depleted) get killed off first; and as new ones come out into the circulation, they have a full amount of functioning G6PD, so they can handle the oxidant better. For severe or prolonged crises, red cell transfusions may be necessary.

What if you had a blood smear in which you thought the diagnosis was chronic myeloid leukemia (CML), but you didn’t have access to a cytogenetic or molecular lab (to look for the Philadelphia chromosome or the bcr-abl translocation)?

Well, first you’d look for all the morphologic clues you could. CML usually presents with a marked leukocytosis (the WBC is often over 100,000), with a left shift all the way back to myeloblasts (though there are relatively few myeloblasts around). A benign left shift usually presents with a mild to moderate leukocytosis (the neutrophil count is often just above normal; it’s generally nowhere near the magnitude often seen in CML), and the neutrophils are shifted back to the metamyelocyte or myelocyte stages (you’ll very rarely see promyelocytes, and you’ll virtually never see myeloblasts). Also, CML tends to have a “bulge” at the metamyelocyte stage, whereas a benign left shift does not (the cells are more or less present in decreasing amounts by stage of maturation, i.e., there are more segmented than band neutrophils, more bands than metamyelocytes, more metamyelocytes than myelocytes, more myelocytes than promyelocytes…and blasts are basically nonexistent). Finally, CML has a basophilia, whereas a benign left shift does not.

But if you wanted more proof that your case was CML, you could do a leukocyte (or neutrophil) alkaline phosphatase (LAP). This test is not done as much as it used to be, because now everyone goes right to cytogenetics or molecular testing in order to find the Philadelphia chromosome or the bcr-abl translocation. But it’s still a good test, and it would be a good thing to do if you couldn’t look for the Philadelphia chromosome.

Here’s the principle behind the test. LAP is an enzyme present in normal neutrophils, but absent (or present at very low concentrations) in malignant neutrophils (i.e., the ones in CML). So if you have a whole bunch of neutrophils around, and the LAP is strongly positive in those cells, as in the top image, you can be quite sure that it is a benign bunch of neutrophils. However, if the LAP is negative, or only weakly positive, as in the bottom image, that probably means that those neutrophils are malignant and that you’re dealing with a case of CML. 

You’d still want to send off a blood or bone marrow specimen to a cytogenetics and/or molecular diagnostics lab, but in the meantime, the LAP can help you quickly assess and triage your patient.