October 2011
Feature Story

William Check, PhD

All matter is composed of elementary particles called quarks, according to the Standard Model of particle physics. Quarks make up protons and neutrons, which, with electrons, make up atoms. In anatomic pathology, too, diagnoses are made up of a few standard elements: clinical history, H&E stain, and, in many cases, immunohistochemistry. More recently, nucleic acid technologies such as FISH, PCR, and gene sequencing have entered the picture. Whole genome sequencing may be invoked in exceptional cases.

Like quarks in particle physics, immunostains are a foundation stone in many areas of pathology. “These days immunohistochemistry is indispensable for gynecologic pathologists,” Charles J. Zaloudek, MD, professor of pathology at the University of California, San Francisco, says. “There are areas where molecular pathology has become important, such as soft tissue tumors and hematopathology. But it is not used so much in gynecologic pathology practice. For these tumors, immunohistochemistry is the main tool we use to supplement H&E morphology.”

What immunohistochemistry has done is to bring a higher level of objectivity to surgical pathology, says Mahul B. Amin, MD, professor and chair of the Department of Pathology and Laboratory Medicine at Cedars-Sinai Medical Center, Los Angeles. As a specific application, Dr. Amin cites the diagnostic utility of IHC in prostate carcinoma, for which he co-authored a best practices article (Paner GP, et al. Arch Pathol Lab Med. 2008;132: 1388–1396).

For optimal results, IHC must be used with knowledge and restraint, Dr. Amin notes. ”Over the last decade or two, immunohistochemistry has become increasingly used and pathologists are ordering large numbers of antibodies. This is good and encouraging,” he says. Yet there may be drawbacks: “People may overutilize stains without adequate knowledge about sensitivity and specificity of the markers, which can lead to confusion. We need a middle approach.” That is the focus of the best practices series.

“We have seen an explosion in the use of immunohistochemistry,” says Rajan Dewar, MD, PhD, staff hematopathologist and molecular genetic pathologist at Beth Israel Deaconess Medical Center and Harvard Medical School. In hematopathology, IHC is most important for the diagnosis of lymphoma.

“Knowing more about markers many times avoids overuse,” Dr. Dewar says, echoing Dr. Amin’s remarks. He’s taught in India extensively in the past five years. Even where IHC is available in India, panels are narrow. “I wondered, Are we [in the U.S.] doing a lot of unnecessary staining?” Dr. Dewar’s questioning led to a 2011 ASCP annual meeting course, which he is teaching this month in Las Vegas with pathologist Dennis O’Malley, MD, who practices at Clarient Inc. and is adjunct faculty at MD Anderson Cancer Center, and oncologist Ranjana Advani, MD, of Stanford University School of Medicine. Dr. Dewar’s mentor during his fellowships at Stanford, hematopathologist Daniel Arber, MD, triggered Dr. Dewar’s early interest in the efficient use of immunostains. “In a small subset of cases,” Dr. Dewar recalls, “Dr. Arber consistently challenged us by asking, ‘If you could order only four immunostains, what would they be?’”

“If you do a morphological exam and have a short list of differential diagnoses, immunostains can help you in narrowing down from that list and making a more accurate diagnosis. That is how I think immunostains should be used,” Dr. Dewar says. He offers the example of a lymph node with nodular proliferations. “My primary thinking might be follicular lymphoma. I also want to exclude mantle cell lymphoma. It would take three or four immunostains to do that. On the other hand, if I am working to exclude other random possibilities, I might use 20 immuno-stains and still not get a diagnosis.” The bottom line: “Get the most you can out of H&E. Morphology is the key.”

In last month’s article on IHC, the focus was on breast and hepatocellular carcinomas and melanocytic lesions. This month, we look at lymphoma, prostate and ovarian cancers, and spindle cell neoplasms of the GI tract.

Dr. Dewar’s approach to the diagnosis of lymphoma illustrates his conservative approach to the use of IHC. Flow cytometry is the first test he orders after morphology. “With the information from flow cytometry, along with H&E, you practically don’t need immunostains in a good proportion of solid tumors of the lymph node,” he says. “You are almost to a diagnosis, with maybe a few immunostains.” But a small proportion of lymphomas are challenging and need a more extensive workup— for example, the grey zone or intermediates in lymphoma. Also, Dr. Dewar says, “T-cell disorders are still challenging, especially entities such as angio-immunoblastic T-cell lymphoma (aberrantly expressing CXCL-13 and CD10).”

For a nodule composed of small cells proliferating in a lymph node, Dr. Dewar says, “One of the clinically most relevant entities in the differential is mantle cell lymphoma [MCL]. In this situation immunohistochemistry for Bcl-1 can make a huge and significant clinical difference.” Bcl-1 is fairly specific for MCL, which, Dr. Dewar says, “has a completely different treatment regimen from follicular lymphoma or marginal zone lymphoma.”

To confirm Hodgkin lymphoma and differentiate it from non-Hodgkin lymphoma is also important, he says. “For this purpose, staining for CD30, CD15, and CD20 is very useful and perhaps essential. Staining for PAX-5, on the other hand, is often less useful.” Dr. Dewar also says that IHC for Oct-2 or Bob-1 upfront “is questionable. Whether we need immunostains for subtypes of Hodgkin lymphoma is also questionable.”

Different T-cell disorders can be difficult to distinguish. “Fortunately or unfortunately,” Dr. Dewar says, “clinically they are all treated the same way. If I suspect a T-cell lymphoma, as long as I’m convinced, my next step is to prove a clonal T-cell disorder. I stop there, even though there is an explosion of immunostains for T-cell lymphoma. Their utility is unproven. We don’t use them in our practice.”

Some of the new entities in the 2008 WHO classification of lymphoma can also be challenging. However, Dr. Dewar notes, “If a subclassification is not clinically relevant, we should not do this workup. Our practicing colleagues, the oncologists, make a huge difference by telling us which entities lead to different treatment.” That’s why the third speaker in the ASCP course is oncologist Dr. Advani, he says.

Dr. Dewar seeks to discourage one more activity: ordering up-front IHC panels. “Some private practice settings or diagnostic companies get immunostains before H&E. In our consults we see quite a few cases like this. It is not a good practice or strategy.”

For prostate carcinoma, there are several settings in which IHC is needed to confirm a diagnosis. “Its most prominent use is in an atypical small focus of proliferation,” Dr. Amin says. “Proving a small focus as adenocarcinoma probably makes up more than 95 percent of our use of IHC in the prostate gland.” Less common uses are to investigate an atypical large focus of proliferation in the gland, to confirm a benign diagnosis in an unusual or atypical focus, and to confirm cancer with an unusual pattern. For example, making a diagnosis of cancer in the setting of prior treatment can be difficult because treatment can alter tissue histology.

Widespread screening with prostate-specific antigen and greater reliance on needle core biopsies are two major reasons for the increased use of IHC in prostate specimens. Taken together, these trends have resulted in greater detection of prostatic carcinoma and smaller-volume samples for the pathologist to work with.

Absence of the basal cell layer is a feature of invasive prostate carcinoma, so one way to establish a diagnosis of malignancy is to stain with a combination of basal cell layer biomarkers plus a marker that characterizes malignancy. Markers of the basal cell layer include high-molecular-weight cytokeratin 34βE12 or p5/6 and p63. A “cancer-associated” marker, one that preferentially stains malignancies, is P504S—a monoclonal antibody to α-methylacyl coenzyme A racemase (AMACR). Polyclonal antibody to AMACR may also be used. In the best practices article, Dr. Amin and his colleagues call the difference in staining between monoclonal and polyclonal antibodies “clinically insignificant.” AMACR is present in 75 percent to 90 percent of Gleason grade five to 10 prostate carcinomas. Dr. Amin uses a cocktail consisting of antibodies to AMACR, p63, and high-molecular-weight cytokeratin 34βE12 or p5/6, all done on the same slide. Used together, these immunostains neutralize possible false-negativity for the basal cell layer markers and, importantly, prevent loss of representation of the focus on three slides that would be necessary for IHC if the markers were stained separately.

“In the posttreatment setting, we can use cytokeratin stains to differentiate carcinoma from xanthomatous inflammation,” Dr. Amin says. Xanthomatous inflammation is one of the mimics of prostate carcinoma. Dr. Amin says the triple-stain cocktail plus CK AE1/AE3 “brings out cancer in tricky patterns.”

“Some pathologists perform proliferation markers, such as Ki-67 (MiB-1), for prognostic purposes,” he adds. “However these are not standard of practice in prostate carcinoma.”

A final application of IHC in the prostate is to determine whether a focus of carcinoma is prostatic or nonprostatic in origin. “For high-grade cancer in the prostate gland, the differential is urothelial carcinoma or transitional cell carcinoma of the prostate,” Dr. Amin says. In this situation he recommends a panel consisting of the prostate lineage-specific markers PSA and prostatic acid phosphatase, along with p63 and high-molecular-weight cytokeratin. The latter two markers are typically negative for prostate carcinoma and positive for urothelial carcinoma. Prostate lineage-specific markers can also be used to exclude several noncancer mimics of prostatic carcinoma—Cowper’s glands, seminal vesicles, nephrogenic adenoma, and hyperplasia of mesonephric (Müllerian) remnants. Ideally, these entities should be considered in the differential diagnosis and eliminated at the morphologic stage, since, like prostate carcinoma, they can be negative for high-molecular-weight cytokeratin and confuse the diagnosis.

Dr. Zaloudek of UCSF lists three main histologic types of ovarian carcinoma: epithelial adenocarcinoma, which makes up 90 percent to 95 percent of all ovarian malignancies; germ cell tumors in (usually) younger women; and sex cord-stromal tumors, which often produce hormones and present with rather dramatic clinical symptoms. “Immunohistochemical stains are available that help with the diagnosis and classification of all three types of tumors,” Dr. Zaloudek says.

For the first category, epithelial tumors, Dr. Zaloudek says, “First we need to talk about fallopian tube tumors.” Surgeons treat women with BRCA mutations prophylactically with bilateral salpingo-oophorectomy (BSO) to reduce the risk of or prevent breast and ovarian cancers. “UCSF may be one of the medical centers where that was done to a considerable extent,” Dr. Zaloudek says.

Clinically, the most important finding was that salpingo-oophorectomy did have a beneficial effect in reducing ovarian cancer, Dr. Zaloudek says. “It also had a beneficial effect on breast cancer, perhaps because it changed the hormonal environment.”

Important information for pathologists arose from examining BSO tissue. “We thought that in most patients we wouldn’t find anything in the surgical specimens,” Dr. Zaloudek says. “The surprising result was that most of the small cancers and precancers we discovered were not in the ovaries but in the fallopian tubes.” Further work revealed that at least some high-grade serous tumors that affect the ovaries don’t originate in the ovary but in the fallopian tubes, with subsequent spread to the ovary. Fortunately, wherever the tumors originated, BSO was effective.

“Pathologists have to examine [BSO] specimens for small cancers or precancers,” Dr. Zaloudek says. “If you have a lot of experience, you can look at the fallopian tubes with H&E and figure it out. But, particularly for pathologists who don’t see a lot of these cases, immunohistochemistry is valuable for highlighting abnormal areas that we can then study in greater detail on H&E.” Areas of intraepithelial carcinoma and invasive serous carcinoma have a particular staining pattern that can support the diagnosis. “Stains for p53, p16, and Ki-67 are all positive in these carcinomas, so it is often possible to make a firm diagnosis even if you don’t see these cases very often,” Dr. Zaloudek says, adding, “Immunohistochemistry is pretty much essential to assess these tumors.”

Epithelial tumors in the ovary come in various histological types, of which high-grade serous is the most familiar. Other important histologies are mucinous, endometrioid, and clear cell. “It is becoming more important to distinguish between various histological types,” Dr. Zaloudek says, because they may have prognostic significance and may respond differently to chemotherapy.

In addition, IHC and molecular studies have shown differences between high- and low-grade tumors. For example, he says, “High- and low-grade serous carcinomas are really different tumors with different origins.” As noted, many high-grade serous carcinomas originate in the fallopian tubes, whereas low-grade tumors seem to arise from borderline serous tumors in the ovary. Prognosis and response to therapy differ between these types: high-grade serous carcinomas are very aggressive but respond to chemotherapy, while the low-grade variety is less aggressive with longer survival but has a poor response to chemotherapy. “Sometimes we can tell high- from low-grade on H&E,” Dr. Zaloudek says, “but pathologists may disagree.” He recommends using IHC: High-grade serous carcinomas stain like tumors from the fallopian tube—positive for p53 and p16. Low-grade serous tumors typically stain negative for these markers.

Immunostains can also be used to distinguish serous from endometrioid carcinomas, which Dr. Zaloudek calls “a common diagnostic problem.” Histologically, endometrioid carcinomas often present with squamous or mucinous differentiation. On IHC, serous carcinomas are positive for p53, p16, and Ki-67 (and have high-grade nuclei), as well as WT1. Endometrioid carcinomas are typically negative for all these markers. “Even just using WT1 stain could be helpful to make that distinction,” Dr. Zaloudek says.

Another application of IHC in epithelial ovarian tumors is to determine whether a tumor is primary or metastatic and, if metastatic, to suggest the histological type of the primary. “Mucinous tumors are not very common in the ovary, but when they are present they pose a major problem,” Dr. Zaloudek says. With a mucinous tumor, the pathologist has to decide whether it is primary or metastatic from another site, such as the colon. “It is not so difficult if a patient has a history of colon cancer or if the surgeon finds colon cancer when removing the tumor,” Dr. Zaloudek says. If neither of those is the case, the pathologist could be the one to figure out the patient has colon cancer. In this context, IHC can be useful. “All primary ovarian tumors stain strongly for CK7 but not as strongly, more patchy, for CK20 and for a marker found in tumors of the large intestine, Cdx2,” Dr. Zaloudek says. Metastases from the colon, on the other hand, usually stain very strongly for Cdx2 and CK20, and not so strongly or negative for CK7. “When the results of these three stains suggest metastasis from the colon, in almost every case workup by the surgeon finds a primary colon tumor, which has a very different prognosis and treatment,” Dr. Zaloudek says.

Germ cell tumors make up the second main histologic type of ovarian tumor. The good news about germ cell tumors, he says, is that “this is an area where chemotherapy has become exceptionally effective, so the correct diagnosis will almost always lead to cure.” Some tumors are less aggressive, such as dysgerminomas, which are treated by surgery alone. Others, including yolk sac tumors or embryonal carcinomas, require immediate chemotherapy.

Diagnosis is mainly based on H&E morphology. However, germ cell tumors are uncommon and most pathologists see few of them, so IHC can be useful either to confirm the diagnosis or even to suggest a subtype of germ cell tumor—for instance, dysgerminoma, the counterpart in women of seminoma in men. “For each germ cell tumor an IHC profile has been or is being developed,” Dr. Zaloudek says. Probably one of the first immunostains developed for a germ cell tumor was for dysgerminoma—placental alkaline phosphatase (PLAP), which is similar to germ cell phosphatase. As a result, antibodies to PLAP also stain germ cells. Better stains have now been introduced. “Probably some of the best right now are Oct4 and Sall4, which stain tumor cell nuclei, and CD-117 and D2-40, which stain tumor cell cytoplasm,” Dr. Zaloudek says. IHC is particularly valuable in a specimen that has only a small area of viable tumor in the midst of a necrotic area, where one may not see enough to be confident of the diagnosis. Or the surgeon may take a needle biopsy of a lymph node and the histologic type may not be clear on a small sample on H&E. In these situations, Dr. Zaloudek says, “Immunostains are commonly extremely helpful.”

IHC can be important in ovarian tumors because they are often not pure tumors but a mixture of tumor types. Teratoma, yolk sac tumor, and dysgerminoma may all be present in the same lesion. With IHC it is possible to delineate the various components. “I’ve learned a lot about these mixed tumors by doing stains,” Dr. Zaloudek says. Classification may be less important with embryonal carcinoma and yolk sac tumors or choriocarcinoma, all of which will almost certainly be treated with chemotherapy after surgery. Low- or intermediate-grade teratomas or dysgerminomas, on the other hand, may be followed after surgery and are treated with chemotherapy only for recurrence. “Immunostains can spare some people having to have chemotherapy,” Dr. Zaloudek suggests.

As with the other two categories of ovarian tumor, the third group, sex cord-stromal tumors (SCSTs), offers many diagnostic possibilities. To complicate diagnosis, none of the SCSTs are very common. “Fortunately,” Dr. Zaloudek says, “many immunostains are positive in tumors in this category. α-inhibin is the most helpful one at this time.” Other IHC markers that are commonly used in SCSTs are calretinin, which Dr. Zaloudek calls a “terrific” marker for SCSTs, and CD56. Steroidogenic factor 1 (SF-1) is a newer marker.

Most recently, scientists in Vancouver published impressive results with FOXL2 in SCSTs (Al-Agha OM, et al. Am J Surg Pathol. 2011;35: 484–494). Initially they found this protein to be positive in almost every case of granulosa cell tumor, the most common type of SCST. Subsequently, they found it to be “a relatively sensitive and highly specific marker” for SCSTs in general. One exception was Sertoli-Leydig cell tumors, only half of which were positive. Expression of FOXL2 protein on IHC was more sensitive for SCSTs than mutation in the FOXL2 gene.

Dr. Zaloudek describes a hypothetical case in which use of IHC “would have huge clinical significance.” Two types of SCST are Sertoli cell and Sertoli-Leydig cell tumors. Endometrioid carcinoma, a type of epithelial tumor, has a Sertoli form variant that is easy to mistake for Sertoli or Sertoli-Leydig cell tumors. This would be a serious mistake, since endometrioid tumors are highly malignant, whereas Sertoli and Sertoli- Leydig cell tumors are less likely to spread. “While these tumors are difficult to separate by morphology,” Dr. Zaloudek says, “with IHC it is very easy to make that distinction.” He uses a panel composed of α-inhibin and calretinin, which will be positive in Sertoli and Sertoli-Leydig cell tumors, along with epithelial markers such as CK7 and epithelial membrane antigen, which will be positive in endometrioid carcinoma.

Jeffrey D. Goldsmith, MD, chair of the CAP’s Immunohistochemistry Committee, co-authored a best practices article on IHC for spindle cell neoplasms of the gastrointestinal tract (Turner MS, Goldsmith JD. Arch Pathol Lab Med. 2009; 133: 1370–1374).

Because spindle cell neoplasms of the GI tract show considerable morphologic overlap, Dr. Goldsmith says, “All lesions in the wall of the GI tract with spindle cell morphology require immunohistochemistry to characterize them.” Dr. Goldsmith, director of the surgical pathology laboratory in the Department of Pathology and Laboratory Medicine at Beth Israel Deaconess Medical Center and assistant professor of pathology at Harvard Medical School, says that, for a spindle cell lesion of the GI tract, “The main differential diagnosis is gastrointestinal stromal tumor, or GIST, which is the most common spindle cell lesion in the wall of hollow viscera.” Other less likely conditions include schwannomas and smooth muscle lesions like leiomyosarcomas, as well as desmoid-type fibromatoses.

Diagnosing GIST correctly is important because this neoplasm responds to tyrosine kinase receptor inhibitors such as imatinib. For this purpose, the mainstay of IHC is the antibody for kit (CD117). “Upwards of 95 percent of GIST stain positive with kit,” Dr. Goldsmith says. “Most other spindle cell neoplasms of the GI tract are negative.” One minor exception is desmoid tumors. However, with an optimally diluted antibody, only about five percent of desmoid tumors stain positive for kit; this minority of cases show only focal kit expression.

For nerve sheath tumors (schwan-nomas), S100 is useful. “Muscle markers like smooth muscle actin [SMA] and desmin stain smooth muscle tumors; both of these tumor types are kit-negative,” Dr. Goldsmith says. “α-catenin stains desmoid-type fibromatosis in a nuclear pattern.”

Whether a panel of immunostains is indicated depends on the tumor’s appearance on H&E and where it is in the bowel wall. “Many times you have a suspicion of GIST based on H&E,” Dr. Goldsmith says. “Then you do IHC for kit. Most times kit is strongly and uniformly positive and you don’t need another stain.” If kit is negative, a panel of other markers can be helpful.

Dr. Goldsmith notes two pitfalls the pathologist must keep in mind with spindle cell lesions of the GI wall. First, between one percent and five percent of GIST are negative for kit. “A newer marker, DOG1 [Discovered on GIST-1], can be helpful in diagnosis of GIST that are kit negative,” Dr. Goldsmith says. DOG1 is more likely to stain kit-negative GIST. “Many labs are now bringing up DOG1,” Dr. Goldsmith says. “We don’t use it routinely but we do use it as part of our panel.” A second caveat when a suspected GIST is negative and a panel is brought into play is that GIST can sometimes be positive for SMA or desmin. However, a GIST that is negative for kit and positive for either SMA or desmin is a statistically rare beast.

Dr. Goldsmith predicts that IHC will remain an important component in the diagnosis of spindle cell neoplasms of the GI tract. Almost all GISTs have a mutation in the KIT gene that can potentially help in diagnosis. However, he says, “It is rare for us to order a molecular test for a KIT mutation for diagnosis. The molecular test is more commonly done for therapy, since some KIT mutations give information about how the tumor will respond to a TK inhibitor.”

It is clear that IHC is a valuable adjunct to morphology across a broad spectrum of tumors. Understanding the building blocks of matter won’t permit anyone to build an atom, but understanding the optimal use of immmunostains will allow a pathology service to construct an enhanced program for detecting and differentiating neoplasia.

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