carcinoma her2 non mammary handout

Immunohistochemistry in the

Diagnosis of Metastatic Carcinoma

of Unknown Primary Origin

Rodney T. Miller, M.D. Director of Immunohistochemistry

ProPath Laboratory 1355 River Bend Drive

Dallas, TX 75247-4915

[email protected]

www.propath.com

American Academy of Oral and

Maxillofacial Pathology Annual Meeting

San Juan, Puerto Rico

Saturday, April 30, 2011, 8:30-11:30 am

Miller IHC for Carcinoma of Unknown Primary Page 1

INTRODUCTION

Carcinomas are undoubtedly the most frequent type of malignancy seen by diagnostic

surgical pathologists. Making the diagnosis of carcinoma is often very simple, but

determining its origin can be very challenging. Occasionally prior medical history, clinical

findings, or x-ray findings may make the origin obvious, but as we all know there are many

cases where the primary site remains a mystery. A valid question that may arise is whether

finding the primary really matters at all. For some patients with widespread metastatic

disease and a virtually hopeless prognosis, the answer to this question may be “no”, and

resources would be better spent at providing palliative and comfort care. However, we all

know that clinicians, patients, and their families frequently want to know where the cancer

is coming from, and immunohistochemistry (IHC) is well suited to address this problem.

There are some who criticize the use of immunohistochemistry in this situation because it

is "expensive". However, from my standpoint, I know that IHC frequently obviates the

need for certain far more expensive diagnostic procedures that would be considered during

the search for a primary. Although a complete battery of immunostains may generate a

sizable bill, in the grand scheme of patient care it is well worth the cost and frequently

saves the patient and the health-care system a great deal of money if performed well and

early in the patient’s course. Furthermore, the cost of a misdiagnosis is far greater than the

cost of an appropriate battery of immunostains.

Principles of Immunophenotyping

Before discussing specifics, it is important to keep certain principles in mind at all

times, which will help to keep us out of trouble when using immunostains to assist with

diagnostic problems.

I. Immunostains must be of high quality. My late father, a very wise, talented and

practical man, told me many times when I was growing up that "You need good tools if

you want to do a good job", words that are particularly pertinent to IHC and diagnostic


pathology in general. If the immunostains employed are of substandard quality and suffer

from poor sensitivity, poor specificity, lack of reproducibility, or are performed in a

laboratory with lax quality control procedures that does not validate the methodology and

markers used in-house, it is a waste of time (and overtly dangerous) to embark on

immunophenotyping. Either fix the problems in the lab, or send the stains to a lab that can

do them right. (Subliminal message: Send all your technical immunostaining work to Miller’s Lab). Another

dangerous mindset is to assume that "automation = quality”. Using a machine to do your

immunostains does not free you from the responsibility of optimizing (including

determination of optimal titers) and validating your immunostains, something that should

not be abdicated to a vendor or manufacturer (Cardinal Rule 1: There is no such thing as a

"predilute ready to use" antibody.) (Cardinal Rule 2: If you use an avidin-biotin based

detection system, you must take steps to block endogenous biotin activity with every stain

you perform, or you are asking for trouble). (Cardinal Rule 3: You should use some type

of multitumor or multitissue positive control material, and it should be mounted on the

same slide as the patient tissue). Cardinal Rule 4: Treat your tissue right (garbage in,

garbage out).

II. You must be able to generate the appropriate differential diagnosis based on

H&E, know the spectrum of reactivity of the markers used, and know the expected

immunophenotypes of the tumors in the differential diagnosis. This point may be an

obvious one (duh…..), but it is worth stressing, since incomplete knowledge of the

spectrum of immunoreactivity with the various markers used has been responsible for

many diagnostic errors when employing IHC in diagnosis. For example, many people

employ cytokeratin AE1/AE3 as a “pan-cytokeratin” – NOT!! (see discussion in the

:undifferentiated malignant tumor” handout). If you plan on using these markers in your

practice, you must make a commitment to become knowledgeable about the tools that you

will be using. I can think of several genius-level pathologists who can remember all of this

important information, but I am certainly not one of them, so I rely heavily on a

comprehensive series of notes that I call my “IHC peripheral brain”. My personal “IHC

peripheral brain” is in spreadsheet format (which is readily searchable), and has a number


of different “sheets” in the complete “workbook”, organized to rapidly assist in answering

the following recurrent questions:

A. Given the clinical findings and H&E morphology of this tumor, what entities

should I be thinking about in the differential diagnosis? This sheet in the "brain" is

broken into several categories (such as “pleomorphic large cell tumors”, “small blue cell

tumors”, “epithelioid tumors”, “spindle cell tumors”, etc.), with tumors that may show

those morphologic features listed under the appropriate category. Going through this list

when I see cases has helped me on many occasions to stumble onto the correct diagnosis,

or at least point my way toward the correct path.

B. What type of tumors would be expected to stain (or not stain) with antibody X?

This sheet in the "brain" consists of a list of antibodies, with expected positive and negative

tumors listed below each antibody, along with pertinent notes and references to both the

pertinent literature and to prior personally-studied cases that can be retrieved for review if

needed.

C. What is the expected immunophenotype of tumor X? (i.e., does the

immunophenotype that I see in this case fit for tumor X?) This sheet in the workbook

consists of a list of tumors with expected immunophenotypes listed under the particular

tumors, along with pertinent notes and references as above.

III. There are no perfect markers. Or, to put it another way, there are no (or virtually

no) markers that are 100% sensitive and 100% specific. Generate a logical differential

diagnosis based on the clinical and morphologic findings, and USE PANELS of antibodies

to narrow the differential diagnosis. Don’t succumb to “immunohistochemical guilt”

when ordering panels of antibodies for difficult cases. Unfortunately, some pathologists

suffer from intense guilt every time they order an immunostain, and their level of guilt rises

(sometimes exponentially) with each immunostain that is ordered. I have seen many cases

where this guilt has directly contributed to a misdiagnosis, secondary to insufficient and


incomplete analysis of the case. My advice to these pathologists is to “Get over it” (the

“tough love” approach), or see a therapist if needed, since as mentioned previously, the

cost of an erroneous diagnosis is far greater than the cost of an appropriate panel of

immunostains.

IV. Tumors do not read textbooks (and sometimes the textbooks are wrong or

outdated). This is an important point to keep in mind, because you will undoubtedly see

cases that exhibit immunophenotypes that they are not “supposed” to have (particularly if

the immunostains are not of high-quality or if you are using so-called "predilute ready to

use" antibodies). Don’t allow an aberrant immunophenotype to sway you into making an

insane diagnosis, and before accepting an aberrant phenotype, make sure you look at the

positive control material (which ideally should consist of a multitumor sandwich with

expected positive and expected negative cases, and be on the same slide as the patient

tissue), to make certain that the correct antibody has been placed on that slide, and to make

certain that the slide bears the appropriate label corresponding to the actual stain that was

performed. The immunophenotype is only one piece of evidence (along with the H&E

morphology, clinical findings, and laboratory findings) that you should consider before

making a diagnosis. In addition to good immunostains, it takes common sense to be a good

pathologist.

V. You will get some cases wrong (hopefully not very many). Anyone who has not

gotten a case wrong has probably not been in practice for very long, or has a personality

disorder that prevents them from seeing (or admitting) their mistakes (i.e., those that

trained at the “It is what I say it is because I say so” school of pathology). Unfortunately,

we live in an imperfect world.


ANTIBODIES FOR METASTATIC CARCINOMA

OF UNKNOWN PRIMARY

“ORGAN-SPECIFIC” OR “ORGAN-RELATED” MARKERS”

PROSTATE MARKERS: Prostate-Specific Antigen (PSA), Prostatic Acid

Phosphatase (PSAP), Prostate-Specific Membrane Antigen (PSMA), and P501S

(prostein) (all cytoplasmic reactivity(1-12); NKX3.1(nuclear reactivity) (12a): PSA and

PSAP antibodies have been around a long time, and they are very useful for detecting

prostate adenocarcinoma, although they may also be found in some other types of tumors

(see below). PSA is very specific for prostate carcinoma, although it will also stain a small

number of breast carcinomas and some salivary gland tumors. Rectal carcinoid tumors

(and less frequently non-rectal carcinoids) may show reactivity with PSAP, so that

potential trap is important to know. Parenthetically, some primary prostatic tumors may

express significant chromogranin and synaptophysin, and may look just like carcinoid

tumors (4), another potential pitfall. It is not at all unusual for PSA to be negative in

poorly-differentiated prostate tumors, and in my experience PSAP is more sensitive than

PSA. PSAP reactivity has also been described in periurethral glands in females, sweat

glands, breast carcinoma, rare islet cell tumors, some salivary gland tumors, and rare renal

cell carcinomas. Some authors report that PSA and PSAP are negative in about 5% of high

grade prostate cancers, and others report that treatment of prostate cancer may be

associated with loss of reactivity to PSA and PSAP. In these situations, the more recently

available markers PSMA and P501S (prostein) may be of particular utility. PSMA has

been shown to be positive in many prostate cancers that are negative for PSA and PSAP (7-

10). It is a transmembrane glycoprotein that functions as a folate hydrolase, and it is

expressed at low levels in benign prostate epithelium, but shows marked upregulation in

prostate carcinoma. The degree of expression of this marker seems to be inversely

correlated with the degree of differentiation of the tumor. As such, high grade prostate

carcinomas tend to express this marker in a very high percentage of tumor cells, whereas

lower grade tumors show more heterogeneous expression. Expression of PSMA has also

been reported in the brain (weak), salivary glands, a subpopulation of proximal renal


tubules, duodenal mucosa, and a subpopulation of neuroendocrine cells in colonic mucosa.

Interestingly, endothelial cells within tumors of many different types express PSMA,

although most normal endothelial cells are negative for PSMA (although I have seen

normal liver sinusoidal endothelial cells express PSMA on many occasions). Epstein (12)

found expression of P501S in 68 of 69 (99%) of cases of prostate cancer, and it has a

characteristic perinuclear cytoplasmic Golgi pattern of reactivity. In addition to prostate

cancers, I have seen P501S reactivity in a few breast carcinomas (not a big problem in male

patients), and in a rare lung carcinoma and an acinic cell carcinoma of the parotid.

NKX3.1 is the newest prostate-related marker on the scene (and has become commercially

available only since November 2010). Unlike the other prostate markers, it is localized to

the nucleus of the cells. Gurel et al (12a) studied tissue microarrays (TMAs) of 69 prostate

carcinomas and 349 non-prostate carcinomas, and found that nuclear reactivity with

NKX3.1 had excellent sensitivity for prostate carcinoma, staining 68 of the 69 cases,

(98.6%). Only 1 of the 349 cases of non-prostate carcinoma was positive, a case of lobular

breast carcinoma.

From a practical standpoint, if I am “screening” a poorly differentiated tumor for

prostate origin, I typically order NKX3.1. If that is negative and prostate origin is still

strongly expected, I will then order PSMA, P501S, PSA, and PSAP.

BREAST MARKERS: Gross Cystic Disease Fluid Protein-15 and Mammaglobin

(cytoplasmic reactivity) (13-23): GCDFP-15 is a very useful marker for the identification

of breast carcinomas, although it is positive in only about 50-60% of primary breast

carcinomas (8-9). It is important to note that the pattern of reactivity with this antibody is

often very focal, and only a small percentage of tumor cells may be immunoreactive. Some

sweat gland carcinomas, salivary gland tumors, and prostate carcinomas are positive,

but it is only rarely positive in carcinomas of other sites. In a case of widely metastatic

salivary duct carcinoma that I saw several years ago in an elderly male patient, GCDFP-15

was strongly positive, and provided a strong clue regarding the origin of the patient’s

tumor. I have only seen four or five lung adenocarcinomas that have expressed this


antigen, usually weak and very focal, although some authors have reported rare lung

adenocarcinomas that are strongly positive for GCDFP-15. Mammaglobin is a more

recently available antibody that is useful as a marker of breast origin. The reported

sensitivity for breast cancer varies substantially, from ~40 to >85% of cases, and in my

experience the sensitivity I observe is probably in the 50% to 60% range. However, in the

cases I have studied, its expression appears to be independent of GCDFP-15 expression, so

if breast carcinoma is being considered, I always order both GCDFP-15 and mammaglobin.

Unfortunately, mammaglobin expression is not completely specific for breast origin, and it

has been reported in skin adnexal tumors, some salivary gland tumors (particularly strong

in some pleomorphic adenomas that I have seen), normal endocervical glands, and in a

significant number of ovarian carcinomas (17% in one study), endometrial carcinomas

(40-70%), and endocervical adenocarcinomas (30% in one study).

Thyroglobulin (cytoplasmic reactivity): Thyroglobulin is useful in detecting metastatic

papillary and follicular thyroid carcinoma, although it is negative in medullary

carcinoma of the thyroid. It is also positive in some anaplastic carcinomas of the thyroid,

although it may be very focal in these tumors, and most anaplastic thyroid carcinomas are

negative. There can be difficulty interpreting the results of the stains when tumors are

invading the thyroid gland, since some authors have found that a certain amount of antigen

diffusion (from benign thyroid tissue into adjacent tumor cells) may occur, resulting in a

risk of false positive staining of tumors of non-thyroid origin that are invading the thyroid.

TTF-1 (see below) is a more sensitive marker of thyroid tumors than thyroglobulin,

although it is not as specific for thyroid origin. Pax8 is also a good marker of thyroid

neoplasms, and is discussed below.

TTF-1 (Thyroid Transcription Factor-1) (nuclear reactivity) (24-50)): TTF-1 is a

protein involved in the regulation of surfactant proteins, and it is well established as a

useful antibody for metastatic carcinoma of unknown origin. TTF-1 is normally expressed

in the brain (diencephalon), parathyroid, C-cells of the thyroid, anterior pituitary, thyroid,

and nonciliated respiratory and alveolar epithelium. Overall, it is expressed in 75% of non-


mucinous lung adenocarcinomas, 10% of mucinous lung adenocarcinomas, and 40% of

large cell carcinomas of lung. According to some authors, TTF-1 is expressed in 100% of

non-mucinous bronchoalveolar adenocarcinoma, but is essentially absent in mucinous

bronchoalveolar carcinoma (43). However, I have seen a few mucinous bronchoalveolar

carcinomas that have been TTF-1 positive. TTF-1 is negative in squamous carcinomas of

the lung (at least when using clone 8G7G3/1), although I have observed weak to moderate

staining in pulmonary squamous tumors employing the more sensitive clone SPT24. TTF-

1 has been found to be more sensitive than PE-10 (see below) for detection of pulmonary

adenocarcinomas (32), and my personal experience with this antibody agrees with this

contention. PE-10 is frequently only focally positive in lung tumors, whereas TTF-1

usually stains a much higher percentage of tumor cells, so the utility of TTF-1 is often

greater when dealing with miniscule specimens, like FNA’s. Thyroid carcinomas are also

positive with TTF-1 (including papillary carcinoma, follicular carcinoma, and medullary

carcinoma), although anaplastic carcinomas are generally negative, and between 20-75% of

Hurthle cell tumors are reported to express this marker. TTF-1 is rarely expressed in

stomach carcinoma (1.7%), breast carcinoma, prostate carcinoma, mesothelioma, renal cell

carcinoma, and colon carcinoma. Some authors have described TTF-1 in 17% of

endometrial adenocarcinoma, and indeed I have seen several endometrial

adenocarcinomas with rather striking TTF-1 reactivity. In a study of 546 breast

carcinomas, TTF-1 was expressed in 13 cases (2.4%), so TTF-1 reactivity by itself can not

completely exclude breast origin (47a). In my own experience, I have seen several focally

positive colonic carcinomas and a positive pancreatic carcinoma, and urothelial carcinomas

may have scattered positive cells in some cases, sometimes quite strong. I have also

observed positivity in a case of desmoplastic small round cell tumor and in a subset of

lymphocytes. Strong nonspecific granular cytoplasmic staining can be observed with

TTF-1 when using clone 8G7G3/1, particularly in hepatoma (45) (where it may be a

clue to this diagnosis), GI tumors, and prostate tumors, but for the purposes of use as a

pulmonary and thyroid marker, this type of reactivity should be ignored, as only nuclear

reactivity is significant with this antibody. This cytoplasmic reactivity with clone 8G7G3/1

has been found to be due to cross-reactivity with antigens in hepatocyte mitochondria (50).


TTF-1 clone SPT24 does not show cytoplasmic staining of the type seen with clone

8G7G3/1. TTF-1 clone SPT24 is substantially more sensitive for lung carcinomas than

clone 8G7G3/1, but according to some authors may also be somewhat less specific, and

may stain some colonic adenocarcinomas (48-49). In my mind, I think that SPT24 is a

better clone than 8G7G3/1, and the so-called nonspecificity of SPT24 is actually a

reflection of its superior sensitivity. At the 1999 USCAP meeting, one group reported that

TTF-1 was absent in all of 82 thymic epithelial tumors, and was positive in 1 of 25 thymic

carcinomas (31), although we see weak to moderate TTF-1 reactivity in thymic tumors

with clone SPT24. TTF-1 is negative in mesothelioma, so it has utility in the differential

diagnosis of mesothelioma vs. adenocarcinoma. TTF-1 is also present in a fair number of

neuroendocrine tumors, including 90% of small cell carcinomas of the lung according to

some authors. In my personal experience, clone 8G7G3/1 stains about 50% of small cell

carcinomas, and clone SPT24 stains about 75-85% of small cell carcinomas. 80% of

atypical carcinoids of the lung are reported to be TTF-1 positive, but only 20% of typical

carcinoids of the lung. In addition to pulmonary small cell carcinoma, expression of TTF-1

has been reported in one study in 44% of non-pulmonary small cell carcinomas (4/4

prostate, 2/4 bladder, 1/7 cervix), and this study also reported absence of expression in all

of 49 cases of gastrointestinal carcinoids, all of 15 pancreatic islet cell tumors, and all of 21

paragangliomas (35). Another study found expression of TTF-1 in 81% of 37 pulmonary

small cell carcinomas, and also in 80% of 15 non-pulmonary small cell carcinomas, so

TTF-1 does not have pulmonary-related specificity in the setting of small cell carcinoma

(36). However, TTF-1 is negative in Merkel cell tumor, which can assist in the

differential diagnosis from small cell carcinoma (often TTF-1 positive) (37). Pulmonary

sclerosing hemangiomas are positive for TTF-1 (47). Medullary carcinomas of the

thyroid are TTF-1 positive.

Napsin A (cytoplasmic reactivity) (220-221): Napsin A is similar to TTF-1 with respect to

its sensitivity and specificity for lung carcinoma. Using tissue microarrays, Bishop et al

(220) studied 95 cases of lung carcinoma, and Napsin A was positive in 83% of case (TTF-

1 was positive in 73% of cases). There were 13 Napsin A positive, TTF-1 negative cases,


and 2 TTF-1 positive, Napsin A negative cases. By using both Napsin A and TTF-1, these

authors detected 85% of the cases of lung carcinoma. All 48 squamous carcinomas, 6

neuroendocrine tumors, 5 colonic, 31 pancreatic, 17 breast, and 38 mesotheliomas were

negative. Of 118 renal cell carcinomas, 79% of the papillary renal cell carcinomas,

34% of clear cell carcinomas, and 3% of chromophobe carcinomas were positive. Of 81

thyroid tumors, only 5% of papillary carcinomas (2 cases, both with tall cell morphology)

were Napsin A positive

PE-10 (Surfactant Apoprotein A) (cytoplasmic reactivity): This antibody has good

specificity for lung adenocarcinoma and thyroid carcinoma, although its sensitivity for

pulmonary origin is poor in my experience, so we rarely use it. Langel and colleagues (26)

report that PE-10 stains about 60-70% of lung adenocarcinomas, although in my laboratory

I would estimate that we have seen it positive in 20-30% or less of lung carcinomas,

similar to other reports (32). It will also stain alveolar lining cells and alveolar

macrophages, so that must be kept in mind when interpreting the results, especially in

pleural fluids or lung FNA’s, that may show strong reactivity in background normal lung

elements or in the background tissue fluid. This antibody is reported to be absent in breast,

colon, renal cell, and endometrial carcinomas. Like GCDFP-15, reactivity with PE-10 may

be quite focal, and that is a point that must be kept in mind when forced to deal with very

tiny amounts of diagnostic material. One report described observing PE-10 reactivity in 6

of 15 (40%) of prostate carcinomas and 3 of 7 (43%) thyroid carcinomas.

Estrogen Receptor (ER) and Progesterone Receptor (PR) (nuclear reactivity) (57-71):

Estrogen receptor can be very useful in determining the origin of metastatic carcinoma. It

is common knowledge that ER is positive in many breast carcinomas and also female

genital tract tumors (both epithelial and stromal), but it can also be positive in a number

of other tumors. Tumors that may express ER include thyroid tumors, salivary gland

tumors, sweat gland carcinomas, genital angiomyofibroblastoma, and 80% of aggressive

angiomyxomas. ER also has been recently described in some cases of skull base

chordomas (63). I have seen ER expressed in normal hepatocytes and in a few hepatomas.


ER has been reported in 7% of carcinoid tumors. Most pulmonary carcinomas are ER

negative, although 4%-15% of pulmonary carcinomas may express ER, and indeed I

have this on many occasions in my consultation practice, although it is usually focal or

patchy and relatively weak, with a few exceptional cases that show strong diffuse staining.

When using the ER antibody clone 6F11 on the Ventana automated immunostainer, Dabbs

et al (69) reported ER positivity in 67% of a series of 45 primary pulmonary

adenocarcinomas, although since I do not use that clone, I cannot comment on that figure.

(They did not report any ER positive cases when using clone 1D5). ER is negative in

small cell carcinomas and gastrointestinal tract carcinomas, so ER reactivity can be

very useful in ruling out those possibilities. ER and PR reactivity is also reported in

stromal cells and rarely epithelial cells of hepatobiliary and pancreatic mucinous

cystadenocarcinomas (64), although in my experience pancreatic ductal adenocarcinomas

and the “usual” type of biliary tract carcinomas are ER negative. Interestingly, I have seen

progesterone receptor (PR) reactivity in rare pulmonary carcinomas, and rare GI tract

tumors, so I would not recommend using PR in the same fashion as ER if one is dealing

with the problem of a metastatic tumor of unknown primary. PR is also reported to be

positive in some medullary carcinomas of the thyroid, some melanomas, meningiomas, and

in 20% of carcinoid tumors and 20% of small cell carcinomas. I have also seen strong PR

reactivity in minute pulmonary meningothelial-like nodules (so-called chemodectoma).

HepPar 1 (cytoplasmic granular reactivity) (72-78): HepPar 1 (short for “hepatocyte

paraffin 1”) is a monoclonal antibody useful in the diagnosis of hepatocellular carcinoma

(HCC), where it has been found to show 82% sensitivity and 90% specificity for

hepatocellular neoplasms in one study, although I think it is vastly overrated as a hepatoma

marker. In my own experience, I see it in only about 50% of the HCC’s that are sent to me

for immunophenotyping, although there is probably some selection bias in my figures,

since classic cases of HCC are generally not sent to us for immunophenotyping. It is far

more sensitive than AFP for hepatoma, as AFP stains only about 15% of cases. However,

it is not completely specific for hepatoma, and has been found to be strongly expressed in a

fairly high percentage of gastric adenocarcinomas, as well as occasional other non-liver


tumors, underscoring the importance of using appropriate panels of antibodies when

evaluating cases, and not relying on a single marker.

Arginase-1(Arg-1)(cytoplasmic and nuclear reactivity) (227): Argnase-1, an enzyme

involved in the urea cycle, has been found to be expressed normally in liver cells but few

others in the body. At the 2010 USCAP meeting, Yan et al (abstract #1668) reported on

their experience using Arginase-1 as a specific marker of hepatocytes and hepatocellular

neoplasms, and they subsequently published their findings in the August 2010 issue of Am

J Surg Pathol. A series of 193 hepatocellular carcinomas (HCC), Arginase-1 had a

sensitivity of 96%. Only 2 of 557 non-hepatocellular tumors expressed Arg-1 (1

cholangiocarcinoma and 1 prostate carcinoma), and the staining was focal and weak. When

compared with HepPar1, Arg-1 was clearly superior from both the standpoints of

sensitivity and specificity for HCC. Arg-1 stains both cytoplasm and nuclei, but the authors

required cytoplasmic reactivity in order to qualify for a "positive" Arg-1 stain. Arg-1 also

stains neutrophils and macrophages. I have been very impressed with this antibody, and

expect that it will relegate HepPar1 to the trash bin for the diagnosis of HCC.

Pax8 (nuclear reactivity) (217-219): Pax 8 is relatively new on the scene, but I have

found it to be one of the most useful and highly valued markers for addressing the problem

of metastatic carcinoma of unknown primary, where it can be used as a marker of thyroid

carcinomas, female genital tract carcinomas, and renal cell carcinomas. In one study

of 94 thyroid tumors (17 papillary carcinomas, 18 follicular adenomas, 16 follicular

carcinomas, 7 poorly differentiated carcinomas, 28 anaplastic carcinomas, and 8 medullary

carcinomas), Pax 8 was diffusely expressed in all of the papillary carcinomas, follicular

adenomas, and poorly differentiated carcinomas. Expression was variable in medullary

carcinomas. In contrast to TTF-1 (which stained only 18% of the anaplastic carcinomas),

Pax8 was positive to a variable degree in 79% of the anaplastic tumors.

In a study of 182 kidney tumors, Tong et al (217) found Pax8 expression in 98% of

clear cell renal cell carcinomas, 90% of papillary renal cell carcinomas, 82% of


chromophobe carcinomas, 71% of sarcomatoid renal carcinomas, and 95% of

oncocytomas. 23% of urothelial carcinomas arising from the renal pelvis were positive for

Pax8, but not those arising in the bladder.

Nonaka et al (218) found Pax8 to be very useful in distinguishing ovarian carcinomas

from breast carcinomas. Of 124 ovarian carcinomas (84 papillary serous, 18 endometrioid,

12 mucinous, 10 clear cell), Pax 8 was expressed typically in a diffuse fashion in 96% of

the papillary serous tumors, 89% of the endometrioid tumors, 100% of the clear cell

tumors, and 8% of the mucinous tumors. All 243 cases of breast cancer (178 ductal and 65

lobular) were negative for Pax8. We have also found this antibody very useful in detecting

endometrial adenocarcinomas.

Long et al (219a) reported that Pax8 is positive in a high proportion of pancreatic

endocrine tumors, in the majority of duodenal and rectal carcinoid tumors, and a minor

subset of appendiceal and gastric carcinoids and it was not expressed in the ileal and

pulmonary carcinoid tumors.

At ProPath, we see occasional non-thyroid/kidney/female genital tract tumors (e.g., lung

ca, esophageal adenoca, hepatoma, thymoma, mesothelioma) that show weak staining, and

to my knowledge this is of no significance and should be ignored. Pax 8 stains some

lymphocytes (likely B-cells), histiocytes, and also classical Hodgkin cells. I have seen

cases of Pax8 positive embryonal carcinoma and pleomorphic rhabdomyosarcoma, and a

single case of Pax8 positive breast carcinoma. A case of basaloid squamous carcinoma of

the anus was moderately positive for Pax8. Normal pancreatic islet cells and normal

adrenal cortical cells are Pax8 positive.

Wilms Tumor Gene (WT1) (nuclear or cytoplasmic reactivity) (79-90): Nuclear

reactivity with WT1 has been found to be very useful in the recognition of mesothelioma,

but it is also characteristically expressed in the nuclei of serous adenocarcinoma from the

ovary and fallopian tube (or surface serous carcinoma from the pelvic peritoneum, which


are now felt to arise from small subclinical primaries in the distal fallopian tube).

Therefore, if strong nuclear reactivity with WT1 is present in a non-mesothelial tumor, it

most likely represents a serous adenocarcinoma. Goldstein (83) has reported that WT1 is

absent in uterine papillary serous adenocarcinomas, although I have seen several cases in

my laboratory that have been clearly positive with this marker. As discussed in the prior

presentation on IHC in gynecologic lesions, there are also a number of other groups that

have identified nuclear WT1 in a certain proportion of uterine serous carcinomas. Most

authors would agree however, that the frequency of nuclear WT1 expression is lower in

uterine serous carcinoma as compared to ovarian serous carcinoma (and surface serous

carcinoma of the peritoneum). Dr. Allen Gown also reports that WT1 may be expressed in

some renal cell carcinomas and prostate carcinomas, as well as mucinous carcinoma of the

breast. Cytoplasmic reactivity is present in a large number of tumors and to my knowledge

has no particular diagnostic significance (perhaps other than the typically intense

cytoplasmic reactivity in rhabdomyosarcoma). I have also seen strong nuclear reactivity

with this marker in endometrial stromal sarcoma, granulosa cell tumor, thecoma, and

normal uterine smooth muscle cells.

p63 (nuclear reactivity) (91-100): In the past several years, p63 has found increasing

utility in a number of areas of diagnostic pathology, including its use as a marker of

myoepithelial cells in breast and elsewhere, and as a marker of prostatic basal cells that

can be used as an alternative to high molecular weight cytokeratin. In addition, it serves as

a useful marker of squamous cell carcinoma (including basaloid squamous cell carcinoma

and "lymphoepithelioma") (similar to the use of cytokeratin 5 or 5/6 for recognizing

squamous differentiation). We have also observed strong p63 in metaplastic or

sarcomatoid breast carcinoma (a finding recently observed by others as well) (99), where

it is probably a reflection of squamous or myoepithelial differentiation (considering that

these cases have also shown strong staining with cytokeratin 5 or 5/6, also typical of

squamous and myoepithelial tumors). It also stains a significant percentage of urothelial

carcinomas, so it can be of utility in the recognition of those tumors. Not surprisingly, we

have observed strong p63 expression in Brenner tumors and transitional cell


carcinomas of the ovary. Many tumors will show occasional scattered p63-positive cells,

but that pattern of reactivity has no particular diagnostic significance. Other tumors that

may express strong and diffuse p63 include thymoma, basal cell carcinoma, and

cutaneous adnexal tumors (such as syringoma, spiradenoma, etc.). Since it is a good

myoepithelial marker, p63 also stains tumors that include a population of myoepithelial

cells or show myoepithelial differentiation, including salivary gland tumors like

pleomorphic adenoma and adenoid cystic carcinoma. Interestingly, we have observed that

benign glandular inclusions in axillary lymph nodes have an associated myoepithelial cell

layer that is highlighted nicely by p63 (and also smooth muscle myosin), a feature that can

be useful in the differential diagnosis of benign glandular inclusions vs. metastatic well

differentiated ductal carcinoma in axillary lymph nodes.

Renal cell carcinoma marker (RCC) (cytoplasmic reactivity) and Pax-2 (nuclear

reactivity) (51-56). RCC (aka gp220) has been available for a number of years, but only

since clone PN-15 became available did I have much success with it, and even then not

much success. To be honest, I think both RCC and Pax2 are overrated as markers of

kidney tumors, particularly since Pax8 became available as a kidney marker. The RCC

antibody requires enzymatic digestion for optimal staining (we use pepsin), and one thing

that we discovered when using RCC is that it is important to do the stain with several

protease digestion times (we use 5 minutes and 10 minutes), to help deal with the varying

sensitivity of different cases to protease digestion. On more than one occasion, cases of

renal cell carcinoma digested for 5 minutes have been positive and the same case digested

for 10 minutes has been negative secondary to overdigestion (and vice versa). RCC is

reportedly positive in 80% or more of renal cell carcinomas of conventional type and

papillary renal cell carcinomas, but its expression can be focal, a problem when dealing

with small biopsies. RCC is negative in chromophobe carcinoma and oncocytoma. RCC

is not completely specific, as it has also been reported in some breast carcinomas, thyroid

carcinomas, and yolk sac carcinomas. It can also be seen in normal breast, thyroid,

epididymis and parathyroid, and I have seen it expressed in basal cell carcinoma of the skin

and parathyroid adenoma, as well as focally within a case of ovarian serous carcinoma.


More recently Pax2, a nuclear transcription factor involved in development of renal

epithelium, has been touted as a marker of renal cell carcinoma (54-55), where it has

reported in 60-88% of cases of conventional renal cell carcinoma, tending to be stronger in

the lower-grade tumors. It has also been reported in ovarian serous carcinomas, Wilms

tumor, and nephrogenic adenoma. In my lab, I have also seen it stain B-cells in lymph

nodes, normal distal tubules in the kidney, normal bile ducts, with faint staining in several

renal oncocytomas. I have not been impressed with the antibody, as it does not appear to

be particularly robust, and we have much better results with Pax8.

von Hippel-Lindau gene product (pVHL) (cytoplasmic reactivity)(222-223): Lin et al

studied a large series of tumors, and found pVHL in 99% of 79 clear cell renal cell

carcinomas, 100% of 57 papillary renal cell carcinomas, 100% of 26 chromophobe

carcinomas, 100% of 24 oncocytomas, and 95% of 37 metastatic renal cell carcinomas. Of

213 non-renal tumors studied, pVHL was found in 17.4% of cases, and 34 of these 37

cases were clear cell carcinomas of the ovary or uterus (there were 19 cases each of clear

cell carcinoma of the uterus and ovary, and pVHL stained 17 of 19 in each site, or 89%). 3

of 13 hepatomas showed focal or moderate staining. Pancreatic carcinomas (n=20),

hemangioblastomas (n=14), pheochromocytomas (n=12), colonic carcinoma (n=11), breast

carcinoma (n=42), lung adenocarcinoma (n=16), endometrioid carcinomas (n=12), serous

carcinomas (n=20), thyroid carcinomas (n=20; 10 follicular, 10 papillary), urothelial

carcinomas (n=10), and mesotheliomas (n=5) were negative.

OCT3/4 (nuclear reactivity) (101-106) and SALL4 (224-226): OCT3/4 (also known

as OCT3 or OCT4) is a nuclear transcription factor expressed in early embryonic cells,

germ cells, and stem cells. A number of studies have shown that it is a highly sensitive and

specific marker of seminoma, dysgerminoma, and embryonal carcinoma (although yolk

sac tumor is negative). Because seminoma, dygerminoma, and embryonal carcinoma can

often mimic other metastatic carcinomas, OCT3/4 is an excellent marker for screening for

these tumors. With the exception of one report of focal staining in 4 of 14 clear cell

carcinomas of the ovary (106) it stains virtually no other types of tumors, so its utility far


exceeds that of previously-used markers of germ cell tumors such as placental alkaline

phosphatase (PLAP). As might be expected, OCT3/4 is also a superb marker for detecting

in-situ germ cell neoplasia. SALL4 is a more recently described marker that stains nuclei

of seminoma, dysgerminoma, embryonal carcinoma, and yolk sac tumor, so it is an

excellent screening marker for those tumors. Ushika et al (226) report that SALL4 is also

positive in hepatoid carcinoma of the stomach, but negative in hepatoma, so it is useful

for that differential diagnosis. We have seen occasional cases of very poorly differentiated

non-germ cell carcinomas that express SALL4, and several dedifferentiated liposarcomas

have shown strong expression of SALL4.

Epithelial Membrane Antigen (membrane or cytoplasmic reactivity) (107-111):

EMA is certainly not an organ-related marker, since it is present in many carcinomas.

However, the absence of EMA does have some degree of organ-specificity. It is

characteristically absent in adrenal carcinoma, most hepatomas (although occasional

tiny intercellular dots or canalicular patterns can be seen in some hepatomas), and certain

germ cell tumors (seminoma, embryonal carcinoma, and yolk sac tumor) (although I

have seen a few EMA-positive cells in yolk sac tumor). Papillary cystic tumors of the

pancreas, ovarian granulosa cell tumors, and Sertoli-stromal tumors are also EMA

negative. I have also seen a few EMA-negative prostate carcinomas. EMA can be positive

in choriocarcinoma.

INTERMEDIATE FILAMENTS (Cytokeratins etc.)

AND RELATED MARKERS

Cytokeratins 7 and 20 (cytoplasmic or membrane reactivity) (112-140): These are very

useful antibodies in dealing with the problem of metastatic carcinomas, since the patterns

of immunoreactivity with these two antibodies can help to substantially narrow the

likelihood of various primary sites. Common tumors positive for both CK7 and CK20

include urothelial (transitional cell) carcinoma, pancreatic carcinoma, and ovarian

mucinous carcinomas. Invasive papillary carcinoma of the breast and about 1/3 of

mucinous breast carcinomas also co-express CK7 and CK20 (abstract, Am J Clin Pathol 110:517,


1998). CK7 positive, CK20 negative tumors include lung, breast, non-mucinous ovarian,

endometrial, mesothelial, pancreaticobiliary tract, gallbladder, small bowel, some

squamous carcinomas, and thyroid tumors. CK7 negative, CK20 positive reactivity is

typical of colorectal carcinoma, and tumors negative for both CK7 and CK20 include

hepatoma, renal cell carcinoma, prostate carcinoma, and some squamous carcinomas.

There are a significant number of exceptions, however, particularly in bladder, stomach

and pancreaticobiliary tract tumors, so these antibodies must be used as part of a panel

approach. Despite this limitation, these antibodies are indeed extremely useful.

Villin (cytoplasmic or membrane reactivity) (141-146): Villin is a GI-related

cytoskeletal protein associated with brush border microfilaments, and it has been found to

be useful in the workup of metastatic carcinomas. In my mind, one of the most important

aspects of villin is that breast carcinomas only very rarely (<1-2% of cases) show

strong reactivity with villin. I am not aware of any reports of villin-positive invasive

breast cancer, and in all the breast carcinomas that I have seen, I remember only 6 or 7 that

have shown moderate to strong villin reactivity. Therefore, in the large majority of

instances in which strong villin reactivity is observed (particularly with a brush border

pattern), breast carcinoma is unlikely as a potential primary site. Villin is reported to be

quite sensitive in the detection of gastrointestinal tumors (including pancreas and

biliary tract), and is reported to stain nearly 100% of colon tumors, with a “brush border”

pattern of reactivity. It is positive in about 50% of hepatomas, and will often show a

“canalicular” pattern of reactivity, similar to polyclonal CEA and CD10 in some

hepatomas. Focal positivity can be seen in 26% of renal cell carcinomas and 36% of

endometrial carcinomas. A substantial number of lung adenocarcinomas express villin

(although the percentage of positive cases is certainly less than GI carcinoma), including

some cases that show a prominent brush border pattern of accentuation. Villin can be

positive in mucinous tumors of the ovary (50%), but not in serous tumors. Membranous

reactivity can be observed in carcinoid tumors. Some authors have noted that villin is

much less frequent in pancreatic endocrine neoplasms (islet cell tumors) than carcinoid

tumors (146), where it was expressed in only 7% (1of 15) of islet cell tumors, but in 82%


(18 of 22) of GI carcinoid tumors, although I have seen it expressed in a number of

pancreatic endocrine tumors. Villin was found in only 2 of 24 (8%) lung carcinoids.

These authors also found that 4 of 4 small cell carcinomas of GI origin expressed villin,

whereas all 24 lung small cell carcinomas were villin negative, as were 11 small cell

carcinomas of other sites (3 esophagus, 3 prostate, 1 bladder, 1 thyroid, 1 nose, 1 parotid, 1

ovary). Therefore, it is possible that villin expression in a metastatic small cell carcinoma

might favor a GI primary. Villin is also common in other neuroendocrine tumors,

including large cell neuroendocrine carcinomas, medullary thyroid carcinoma, and primary

neuroendocrine carcinoma (“carcinoid-like”) of the prostate. Some authors report

cytoplasmic villin reactivity in up to 68% of lung carcinomas, although in my experience

this figure seems high. Normal pancreatic acini may show luminal staining for villin, and I

have seen strong villin in yolk sac tumors.

Often, the combination of immunostaining results with CK7, CK20, and villin can

substantially narrow the possibilities for most likely primary site of a metastatic carcinoma.

Tables of likely (and unlikely) possible primary sites based on the patterns of

immunoreactivity with CK7, CK20, and villin are included at the end of this handout.

Cytokeratin (AE1/AE3) (cytoplasmic reactivity): CK(AE1/AE3) should be present in

most carcinomas, but as discussed in the “undifferentiated tumor” talk, it is an imperfect

antibody and should NOT be viewed as a “Pankeratin. There are certain tumors

(including some cases of renal cell carcinoma, adrenal cortical carcinoma, prostatic

carcinoma, carcinoid tumors, small cell carcinomas, and pancreatic islet cell tumors)

where CK (AE1/AE3) may be weak or absent, although most of these are positive with

low molecular weight cytokeratins (i.e., cytokeratins 8 and 18). The extent and intensity of

reactivity with CK (AE1/AE3) can also be of diagnostic utility, since strong diffuse

AE1/AE3 immunoreactivity is exceedingly rare in hepatoma and seminoma (although

focal or faint reactivity may be seen in some tumors, with a perinuclear dot-like pattern in

some seminomas). (Interestingly, AE1/AE3 may be strongly expressed in ependymoma

and in some shwannomas, and this usually parallels the degree of GFAP reactivity, but


widespread staining of these tumors with other cytokeratin antibodies is very unusual)

(156). In addition, ependymoma is about the only tumor that I can think of (other than

some well-differentiated squamous tumors) that expresses strong AE1/AE3 but is often

negative with low molecular weight cytokeratin. On occasion I have observed patchy

perinuclear dot-like immunoreactivity with CK (AE1/AE3) in normal myometrial smooth

muscle cells.

Low Molecular Weight Cytokeratin (CK8 & CK18) (CK-lmw) (cytoplasmic

reactivity): For detecting CK-lmw (cytokeratins 8 and 18) a number of clones can be used,

such as Zym 5.2, CAM5.2 or 5D3. CK-lmw is useful as part of an “epithelial screen”,

since it will nearly always detect those epithelial tumors that are negative with

Cytokeratin AE1/AE3 (particularly hepatoma, and those cases of renal cell carcinoma,

prostate carcinoma, and neuroendocrine tumors that are negative with AE1/AE3). (An

exception to this is ependymomas, which are often strongly positive with AE1/AE3, but

negative or weak with CK-lmw) (156). Finally, in most cases CK-lmw is better than

AE1/AE3 for detecting epithelial differentiation in small cell carcinomas. In practice, I

think it is always a good idea to make certain that both CK-lmw and CK-hmw are negative

before excluding the possibility of an epithelial tumor when dealing with a poorly

differentiated neoplasm. A population of CK-lmw positive spindle cells exists in normal

lymph nodes, associated primarily with the lymph node sinuses and the areas around

follicles in the cortex. These represent a normal keratin-positive population of dendritic

cells in normal lymph nodes, which are part of the “accessory immune cells” (149). It is

useful to keep these “cytokeratin positive interstitial reticulum cells” (CIRC) in mind so

that they are not misinterpreted as evidence of carcinoma, particularly when using CK-lmw

for sentinel lymph node studies on breast cancer patients.

Before proceeding further, I think it is useful to mention a pattern of cytokeratin

immunoreactivity frequently seen in neuroendocrine tumors. Most pathologists are

probably familiar with the perinuclear “globs” of keratin in Merkel cell tumor, but

perhaps fewer are aware that similar but smaller perinuclear keratin “dots” are


characteristically seen in small cell carcinoma and sometimes in other neuroendocrine

tumors. In fact, it is so characteristic of small cell carcinoma that I am very hesitant to

diagnose a tumor as a small cell carcinoma unless I can see perinuclear dots on the

cytokeratin stain, highlighted to best advantage in most cases with CK-lmw. On a few

occasions I have noticed a similar pattern of reactivity in non-neuroendocrine tumors,

including granulosa cell tumors of the ovary, poorly differentiated (insular or “primordial”)

carcinoma of the thyroid gland, and microglandular adenocarcinoma of the pancreas.

Some renal oncocytomas will also show prominent cytoplasmic “globs” of reactivity on

CK-lmw stains. Suster et al (120) have reported similar findings in 80% of mediastinal

seminomas and 20% of testicular seminomas. Prominent globs of cytokeratin have been

described in paraganglioma of the cauda equina (but not those outside the spinal canal) and

in gangliocytomas of the pituitary (122). Additionally, perinuclear dots of low molecular

weight cytokeratin are a common pattern of “aberrant” reactivity observed in sarcomas that

express this marker (121). A small number of melanomas may express CK-lmw, and

indeed I have seen several melanomas express an impressive amount of CK-lmw, although

these cases have been negative with CK (AE1/AE3). In at least one of these cases,

perinuclear dots or globs of cytokeratin was observed.

Cytokeratin 5 or 5/6 (cytoplasmic reactivity): Cytokeratin 5 antibodies are

diagnostically equivalent to cytokeratin 5/6 antibodies. These CK antibodies are very

useful because they typically stain squamous carcinomas strongly and diffusely. Many

different types of tumors contain scattered cytokeratin 5 positive cells (particularly if they

show focal areas of squamous differentiation). However, focal reactivity with cytokeratin

5 has relatively little diagnostic significance. However, when expressed in a strong diffuse

fashion it can be used as a marker of squamous differentiation (providing that

mesothelioma has been excluded) when trying to assess the nature of poorly differentiated

carcinoma. It also stains basaloid squamous carcinomas and papillary

“squamotransitional” carcinomas of the uterine cervix, so it can be very useful in

identifying these unusual variants of squamous carcinoma. Other tumors that

characteristically show strong staining with CK5 include cutaneous basal cell carcinoma


and “lymphoepitheliomas” of the nasopharynx and other sites (which represent poorly

differentiated variants of squamous carcinoma), and thymoma. We have also observed

strong CK 5 in a number of cases of metaplastic or sarcomatoid breast carcinoma

(similar to strong p63 staining in these tumors). The only tumor with glandular features

that may show strong diffuse staining with CK5 is epithelial mesothelioma, and this fact

can be exploited in the diagnosis of mesothelioma. (Parenthetically, it is worthwhile to

mention that the mesothelial-related marker calretinin is also commonly expressed in

pulmonary squamous carcinomas, in up to 40% or 50% of cases.) Remember that p63 also

strongly stains squamous carcinomas, but unlike p63, cytokeratin 5 does not show strong

and diffuse staining in urothelial (transitional cell) carcinomas (in the absence of overt

squamous differentiation). Therefore, in the appropriate clinical context a carcinoma

that is strongly p63 positive but negative or weak for cytokeratin 5 is likely to

represent urothelial (transitional cell) carcinoma. (Parenthetically, I should mention

that I have seen a case of squamous carcinoma in situ of the eyelid that was essentially

negative for cytokeratin 5 but was strongly positive for nuclear p63, although most

squamous proliferations express both cytokeratin 5 and p63). Some authors have utilized

cytokeratin 5 in a fashion similar to high molecular weight cytokeratin for the

interpretation of difficult prostate needle biopsies (158).

Use of High and Low Molecular Weight Cytokeratin (cytoplasmic reactivity): In

some instances, the staining results with the combination of low molecular weight

cytokeratin (keratins 8 and 18) and high molecular weight cytokeratin (employing clone

34βE12) can be useful in the problem of metastatic carcinoma of unknown origin. Certain

tumors tend to strongly express both of these keratins, including carcinomas of the breast,

ovary, pancreas, bladder, stomach, and, non-squamous non-small cell carcinomas of the

lung. However, hepatoma, "typical" clear cell carcinoma of the kidney, and

adrenocortical carcinomas characteristically lack expression of high molecular weight

cytokeratin, or express it so focally and weakly as to be forgettable. Prostate carcinoma

also typically lacks CK-HMW or expresses it only focally. Therefore, if you have a tumor

that expresses strong high molecular weight cytokeratin, you can place those possibilities

(i.e. typical renal cell carcinoma, hepatoma, adrenocrotical carcinoma, and prostate


adenocarcinoma) way down on the list of potential primary sites. Essentially all squamous

carcinomas express strong high molecular weight cytokeratin. Well-differentiated

squamous carcinomas express low molecular weight cytokeratin weakly or not at all.

However, poorly differentiated squamous carcinomas may express significant low

molecular weight cytokeratin, but it is almost always less than (in a few cases equal to) the

degree of expression of high molecular weight cytokeratin. Because of this feature of

squamous carcinoma, if you have a tumor that expresses strong low molecular weight

cytokeratin but expresses high molecular weight cytokeratin weakly or not at all, you

are not dealing with a squamous carcinoma, and other possibilities should be

considered. Conversely, if you have a tumor that expresses substantially more high

molecular weight cytokeratin than low molecular weight cytokeratin, squamous

carcinoma should be considered.

Cytokeratin 17 (cytoplasmic reactivity): CK17 is expressed in a wide variety of

carcinomas, including pancreas (58% positive), squamous carcinoma (75%),

cholangiocarcinoma (38%), ovarian serous carcinoma (73%), lung adenocarcinoma (23%),

urothelial carcinoma (70%), and endometrial carcinoma (13%) (151). This antibody can be

helpful in some situations, as it is reported to be negative (i.e., < 1% of tumor cells

positive) in stomach carcinoma, colon carcinoma, kidney carcinoma, hepatoma, prostate

carcinomas, mesotheliomas, breast lobular carcinomas, and most breast ductal carcinomas

(92%). For a number of years I have been using it to attempt to separate gastric

adenocarcinoma (ideally cytokeratin 17 negative) from pancreatic adenocarcinoma (58%

positive). However, I have seen a number of cases of gastric carcinoma that have shown

strong expression of cytokeratin 17, so I have doubts about its true utility for this

differential diagnosis.

Vimentin (cytoplasmic reactivity): In certain situations, vimentin can be a useful

marker to assess the most likely primary site of a tumor (147). Although vimentin is

widely expressed, there are certain tumors that are characteristically vimentin positive and

others that are usually vimentin negative. When dealing with tumors having


“endometrioid” morphology in a female, the differential diagnosis often includes

endometrial, ovarian, endocervical, or colonic origin. Vimentin is usually positive in

endometrial carcinomas (80%), and is positive in about 30% of ovarian endometrioid

adenocarcinomas. In contrast, vimentin is negative or only focally positive in

adenocarcinomas arising from the colon or endocervix. Hepatomas are characteristically

negative for vimentin, and embryonal carcinomas are usually negative or only focally

positive. Adenocarcinomas of the colon, pancreas, gallbladder, and prostate are usually

vimentin negative. Transitional cell carcinomas and pancreatic carcinomas are also usually

negative. Adenocarcinomas of the thyroid and kidney are almost always vimentin

positive (except for chromophobe renal cell carcinoma, which is vimentin negative).

Vimentin is variable in adenocarcinomas of breast (12% positive), stomach (33% positive),

lung (42% positive), and ovary (44% positive). Small cell carcinomas, carcinoids, and

paragangliomas can express vimentin, but islet cell tumors are reported to be generally

negative (although I cannot confirm this with my personal experience). An important

point to keep in mind is that virtually any type of spindle cell carcinoma also expresses

strong vimentin.

NEUROENDOCRINE MARKERS

Chromogranin and Synaptophysin (cytoplasmic reactivity) (160-162): These markers

of neuroendocrine differentiation should always be used together, since it is not uncommon

for a neuroendocrine tumor to lack one or the other of these markers. If adenocarcinomas

contain only occasional scattered positive cells, it is best not to label these as

“neuroendocrine carcinomas”, but rather to consider them as carcinomas that happen to

contain scattered neuroendocrine cells, which is probably of doubtful significance in most

instances. As previously mentioned, the identification of small perinuclear cytokeratin dots

may be the first hint that one is dealing with a neuroendocrine tumor, and should prompt

immunostains for neuroendocrine markers.


CD56 (NCAM) (membrane reactivity) (160-162): This marker does not have the

specificity for neuroendocrine lesions that chromogranin and synaptophysin do, but it is

more sensitive for neuroendocrine differentiation in some instances. This is particularly

true in the identification of neuroendocrine differentiation in small cell carcinoma, where it

shows strong and diffuse cytoplasmic membrane staining in nearly all cases. For this

reason, I also add this marker to the "neuroendocrine panel" in selected cases, particularly

if small cell carcinoma is in the morphologic differential diagnosis.

Neuron specific enolase (NSE) (cytoplasmic reactivity): The utility of this putative

neuroendocrine marker in the diagnosis of tumors (neuroendocrine or otherwise) can be

summarized briefly: It is worthless. Those pathologists who use this marker as a

reflection of neuroendocrine differentiation do so at their own peril.

MISCELLANEOUS MARKERS

CEA (clone COL1) (cytoplasmic or membrane reactivity); Polyclonal CEA antibodies

are to be avoided unless you are trying to demonstrate canalicular reactivity in

hepatocellular carcinoma. I prefer the COL1 clone (Zymed) of CEA, as it shows no

reactivity with non-specific cross reacting antigen (NCA), and it performs very well (and I

have never seen a mesothelioma that has shown even a single positive cell with this

clone!). Parenthetically, if you see staining of neutrophils on a CEA immunostain, that

CEA antibody is cross reacting with NCA, and you would be well served to find another

CEA antibody that does not cross-react with NCA. Although monoclonal CEA is positive

in many types of adenocarcinomas, it should be negative in renal cell carcinoma, adrenal

carcinoma, and “typical” papillary or follicular thyroid carcinoma (although it may be

positive in areas of squamous differentiation) and should be negative in the cytoplasm of

hepatomas. However, I have seen a small number of cases where CEA (COL1) has shown

a beautiful canalicular pattern in hepatoma, identical to that described for polyclonal CEA,

villin, and CD10 antibodies, so that fact should be kept in mind. Medullary thyroid

carcinomas are always strongly and diffusely positive for CEA, expressing it in nearly

100% of cells. On several occasions, this finding in a TTF-1 positive tumor has given us


the first clue to the diagnosis, and allowed us to correctly diagnose a medullary thyroid

carcinoma in the setting of a metastatic tumor of unknown primary. Most prostate

carcinomas and endometrial adenocarcinomas are CEA negative, although they may show

patchy areas of positivity. CEA can also be useful in the workup of tumors with an

“endometrioid” morphology, since CEA is positive in the vast majority of endocervical

adenocarcinomas (65-95%) and colonic adenocarcinomas with an “endometrioid”

morphology (90%), but is negative or only focally positive in the vast majority of

endometrioid adenocarcinomas arising in the ovary or endocervix . Clone Z3 of CEA has

been reported to be useful in separating the tall cell variant of papillary carcinoma of the

thyroid (CEA+ and also CD15+) from “usual” papillary carcinoma of the thyroid (CEA-,

CD15-) (although I have no personal experience with this) (173).

CDX2 (nuclear reactivity (163-170): This marker has been found to be positive in a

very high percentage of gastrointestinal adenocarcinomas, particularly those from the

colon and duodenum. In one study, (169), CDX2 was present in 188 of 189 (99%) of

colonic and duodenal adenocarcinomas. Gastric and pancreatic carcinomas showed

heterogeneous expression, but no reactivity was noted in hepatocellular carcinomas or in

carcinomas from the urinary tract (except urinary bladder adenocarcinoma), female genital

tract (except for mucinous ovarian tumors), breast, lung, and head and neck. Barbareschi

et al noted similar findings (165), with CDX2 staining 98% (88/90) of colorectal

adenocarcinomas, as well as 55% (11/20) of gastric carcinomas, 60% (3/5) of pancreatic

carcinomas, 60% (3/5) of gallbladder carcinomas, and 100% (5/5) of mucinous ovarian

tumors. They did not observe CDX2 in 117 lung cancers of different types, nor in cancers

of the breast (n=30), prostate (n=8), mesothelioma (n=5), thyroid carcinoma (n=4), kidney

carcinoma (n=5), and ovarian serous carcinoma (n=5). In a tissue microarray study (166)

where a “positive” CDX2 stain was defined as reactivity in >10% of nuclei, positivity was

found in 84% of 1288 colonic adenocarcinomas, 29% of 45 gastric carcinomas of intestinal

type, 12% of 26 gastric carcinomas of diffuse type, 10.5% of 19 mucinous ovarian

carcinomas, 9.3% of 43 endometrial carcinomas, 2% of 49 serous carcinomas, 2% of 48

lung squamous carcinomas, and 2% of 89 bladder carcinomas. Negative tumors included


50 pancreatic carcinomas, 27 cholangiocarcinomas, 48 hepatocellular carcinomas, 49

pulmonary adenocarcinomas, 48 pulmonary large cell carcinomas, 48 pulmonary small cell

carcinomas, 112 renal cell carcinomas, 93 prostate carcinomas, 153 breast carcinomas, 94

oral cancers, 103 thyroid carcinomas, and 42 carcinomas of the uterine cervix. Another

recent study (167) reported CDX2 reactivity in 13/13 colonic adenocarcinomas, 2/10

pancreatic carcinomas, 9/12 gastric carcinomas, 9/11 mucinous ovarian carcinomas, 0/5

non-mucinous ovarian carcinomas, 4/5 esophageal adenocarcinomas, 1/10 endometrial

carcinomas, 2/12 pulmonary adenocarcinomas, and 0/22 breast carcinomas. At ProPath,

our findings have been similar to those reported above. Like the last study mentioned, we

have identified significant CDX2 immunoreactivity in several unequivocal pulmonary

carcinomas, and also some neuroendocrine tumors (GI carcinoids, particularly those of

midgut origin, islet cell tumor of pancreas, and large cell neuroendocrine carcinoma), and

some urothelial carcinomas of bladder. Morules that may be present in endometrioid

adenocarcinomas also stain strongly with CDX2. In a sense, it seems reasonable to employ

it in a fashion similar to villin for the identification of GI primary tumors, although I

suspect as more laboratories use this antibody, we will find its expression in other non-GI

adenocarcinomas.

N-Cadherin (membrane reactivity): N-cadherin is a protein involved in intercellular

adhesion. Antibodies to the protein are useful in the workup of metastatic carcinomas in

females, since they have been found to stain a high percentage of serous and

endometrioid carcinomas of the female genital tract, although mucinous ovarian

carcinomas are negative (178). Mesotheliomas also frequently express this marker, and it is

not unusual for lung carcinoma to show expression. The literature on this marker is rather

scant, so its complete spectrum or reactivity is not well defined. I have personally

observed this marker in a number of other tumors (and non-neoplastic tissues), including

hepatocellular carcinoma, renal cell carcinoma (both papillary and conventional clear-

cell types), seminoma, yolk sac tumor, thymoma, melanoma (focal), liver adenoma,

ganglioneuroma, glial tissue, thymic carcinoid, medullary carcinoma of thyroid,

extraskeletal myxoid chondrosarcoma, thyroid papillary carcinoma, thyroid follicular


adenoma, solitary fibrous tumor, endometrial stromal sarcoma, schwannoma, adrenal

adenoma, desmoplastic small cell tumor, MFH, PNET, chordoma, and nerve fibers in the

myenteric plexus of the bowel wall. In addition to being strongly expressed by normal

hepatocytes, N-cadherin is also expressed by normal benign bile ducts. As such, I think

that this marker may have some potential utility in distinguishing cholangiocarcinoma

(which might be expected to express strong N-cadherin) from some of its mimics, although

at the time of this writing I am not aware of any published literature that addresses this

issue. Parenthetically, there are a few papers that have touted parathyroid hormone-related

protein (PTH-rP) as a marker of cholangiocarcinoma, but after using that marker for this

purpose for a number of years, I abandoned it because of my perception of poor sensitivity

in the recognition of cholangiocarcinoma. To my knowledge there are no good markers

available at this time that are specific for cholangiocarcinoma.

HMBE-1 (membrane or cytoplasmic reactivity): HBME-1 is an antibody to a

mesothelioma cell line. I have not found it to be useful in the diagnosis of mesothelioma,

but before the advent of Pax8, I employed it for attempting to distinguish breast carcinomas

(HMBE-1 negative in 90% of breast cancers) versus female genital tract carcinomas

(often HBME-1 positive) (134). HMBE-1 is reported to be consistently positive in

ovarian, endometrioid, and thyroid tumors, but is only rarely reported in tumors of the

colon, bladder, and kidney. Also, HBME-1 can be very useful in the recognition of the

follicular variant of papillary thyroid carcinoma, since it generally stains those tumors quite

strongly (similar to cytokeratin 19). HBME-1 has also been reported to stain almost all

chordomas, which can be useful in their differential diagnosis with chondrosarcomas,

which are HBME-1 negative (177).

BCL-2 (cytoplasmic or membrane reactivity): Alsabeh and colleagues (175) published

a paper in 1996 that studied the use of BCL-2 to aid in the distinction of breast carcinoma

(79% positive) from lung carcinoma (5.6% positive) and gastric carcinoma (8.3%

positive). In addition, there were also significant differences in the intensity of staining

with this marker. 70% of the breast carcinomas were moderately to intensely positive,


whereas only 1.9% of the lung carcinomas and only 0.9% of the gastric carcinomas showed

moderate to intense reactivity for BCL-2. I have seen BCL-2 positivity in female genital

tract tumors, thyroid tumors, neuroendocrine tumors, renal oncocytomas, and melanomas.

In lung carcinoma, some authors have noted an association of BCL-2 positivity with

neuroendocrine differentiation, in that most small cell carcinomas appear to be BCL-2

positive (137). BCL-2 may also have a role in helping to separate basal cell carcinoma of

the skin (BCL-2 positive) from squamous tumors (BCL-2 negative) (172), although

personally I have not been particularly impressed with its utility in this situation.

Inhibin, Calretinin, and A103 for Adrenal Tumors (cytoplasmic reactivity) (179-190):

These 3 markers have found utility in the recognition of adrenal cortical tumors,

including adrenal cortical carcinoma. They are also commonly expressed in sex cord-

stromal tumors of the genital tract, and calretinin is well known as a mesothelioma-

associated marker. Some endometrioid adenocarcinomas of the ovary may resemble

Sertoli cell tumors quite closely, and since inhibin stains the tubules of Sertoli cell tumors

(and not the glands of endometrioid adenocarcinomas), it can be very useful in this

differential diagnostic problem. (Parenthetically, it is worthwhile mentioning that

calretinin is commonly expressed in squamous carcinomas in addition to

mesothelioma.)

CD10 (CALLA) (cytoplasmic or membrane reactivity): CD10 (CALLA) has been

found to be expressed in a high percentage of renal cell carcinoma, and since it is

typically absent in adrenal carcinoma, it can be of use in the distinction of those two

tumors. However, its specificity is poor, since it stains a significant percentage of non-

renal tumors as well (191). CD10 is also useful in the diagnosis of hepatocellular

carcinoma, since it is one of several antibodies (along with polyclonal CEA and villin)

that may highlight a diagnostically useful canalicular pattern of reactivity in hepatoma.

Recent studies (192) have shown that CD10 is positive in mesonephric remnants and

tumors, but is negative in clear cell carcinomas of gynecologic origin, so this finding

can assist in the differentiation from clear cell carcinomas of renal origin.


Thrombomodulin (membrane reactivity): Collins et al (193) have studied the

expression of thrombomodulin, a cell surface glycoprotein, in a variety of tumors. They

found that a high percentage (91%) of transitional cell carcinomas of the urinary tract

expressed this marker, as did a high percentage (87%) of squamous carcinomas.

However, expression of thrombomodulin in other types of adenocarcinoma was much less

common, being found in 15% of lung adenocarcinoma, 17% of bladder adenocarcinoma,

19% of breast carcinoma, and 8% of ovarian carcinoma. An exception was pancreatic

carcinoma, which stained 1-25% of cells in 2 of 4 cases studied. There was no staining of

prostate carcinoma, endometrial adenocarcinoma, renal cell carcinoma, or colon

adenocarcinoma. This marker will also stain some histiocytes, normal endothelial cells,

some angiosarcomas, some trophoblastic tumors, and between 40-100% of epithelial

mesotheliomas. I have also observed this marker in yolk sac tumor, basal cell carcinoma,

PNET, and in basement membranes of seminiferous tubules. Personally, in my experience

p63 is better than thrombomodulin as a marker of urothelial carcinoma, and I do not find

thrombomodulin to be of great utility in my practice.

Uroplakin (cytoplasmic or membrane reactivity) (194-197): This marker has been

touted as useful for recognition of transitional cell tumors. Although its sensitivity is

modest, it is reported to be highly specific, and was not found in non-urothelial

carcinomas. I obtained this antibody several years ago, and in my experience its sensitivity

is low (and the vendor’s tech support was highly unpleasant, to say the least!), so I have not

found this particular antibody to be of great utility in my practice. Parker et al (197)

reported that uroplakin III was expressed in 57% of 112 urothelial carcinomas, but in none

of 498 non-urothelial carcinomas present in a tissue microarray.

CA-125 (cytoplasmic reactivity or membrane): According to Dr. Mark Wick, CA-125

is positive in Mullerian tumors and about 50% of biliary tract and pancreatic tumors. It is

also reported to be positive in clear cell carcinoma of the bladder (203). In my laboratory, I

have also seen CA-125 in amnionic epithelium, several cases of lung adenocarcinoma,


focal staining in colonic adenocarcinoma, and strong reactivity in normal reactive

mesothelial cells. I also observed rare positive cells in a case of high-grade transitional cell

carcinoma. Goldstein (204) reports that CA-125 is negative in prostate carcinoma. I have

not found this marker to be particularly useful in the types of cases that I see in my

consultation practice.

Mesothelin (clone 5B2) (cytoplasmic or membrane reactivity): This antibody was

originally raised to a mesothelioma cell line, and it stains about 55% of mesotheliomas.

Dr. Allen Gown found this marker also stains a very high percentage (approaching 100%)

of serous adenocarcinomas of female genital tract origin (unpublished observations) but

few other carcinomas, except for squamous carcinoma. A large study by Dr. Nelson

Ordonez (205) using clone 5B2 described the expression of this marker in a wide variety of

tumors. I have not found it to be particularly useful in my practice. There is some data to

suggest that expression of mesothelin favors pancreatic carcinoma when the differential

diagnosis is pancreatic carcinoma vs. reactive atypia in pancreatic epithelial cells.

However, the combination of Placental S100 (S100P) and pVHL stains are better suited to

that differential diagnosis.

CA19-9 (cytoplasmic reactivity): In my own experience I have found CA19-9 to have

very limited utility. Gatalica and Miettinen (198) examined a large series of tumors, and

found that it was positive in the majority of gastrointestinal and pancreatic carcinomas (70-

90%), but it also was positive in a significant proportion of other tumors as well, including

bladder tumors (64%+), lung adenocarcinomas (45%+), and up to 30% of breast ductal

carcinomas. 80% of mucoepidermoid tumors and 60% of adenoid cystic carcinomas of

salivary glands were also positive. The majority of prostate carcinomas (88%) and kidney

carcinomas (83%) were negative, and consistently negative tumors included hepatoma,

lobular breast carcinomas, GI carcinoids, islet cell tumors, mesothelioma, melanoma,

MFH, seminoma, small cell lung cancer, and squamous lung cancer. These investigators

found that CA19-9 was negative in follicular carcinoma of the thyroid, but positive in 71%

of papillary carcinomas of the thyroid, so perhaps it might be of some help in making that


distinction. However, other authors have reported up to 24% of thyroid follicular

carcinomas positive with CA19-9, and I do not have enough personal experience at the

time of this writing with this marker in that situation to render an opinion on its utility in

this circumstance.

APPROACH TO THE INDIVIDUAL CASE

When faced with an individual case, there are several questions that we should always

try to ask ourselves, and the answers to these questions may be very easy or very difficult

to come by.

1. Did the clinicians provide us with any useful history (and if we suspect we

are missing important history, have we done the appropriate things to try to obtain

this information)? Obviously, pertinent history can be very valuable to us as we approach

individual cases, but unfortunately we all know clinicians who fail to provide us with this

information. In fact, in the past when I was still practicing in a hospital lab, a clinician

once told me that he specifically withholds information from pathologists, so as to not bias

their opinion. In my unbiased opinion, such clinicians are fools who are only hurting the

interests of their patients.

2. Is the tumor really carcinoma, or could it be something else? I have been

surprised countless times by tumors that I thought were carcinoma on H&E (see the list of

"epithelioid tumors" in the accompanying IHC peripheral brain excerpt), so before we start

trying to find a primary, it is important to make sure that we really are dealing with a

carcinoma (and not melanoma, germ cell tumor, lymphoma, sarcoma etc.). In these

situations, it may be necessary to perform an appropriate screening battery to get some idea

of the true cell lineage of the neoplasm in question, before pulling out all the stops. As a

practical matter, I am personally unwilling to exclude carcinoma until I see negative stains

with CK-lmw and CK-hmw (and sometimes EMA) (although in some instances non-

carcinomas may also show reactivity with these markers, a topic beyond the scope of this

presentation). In addition, the possibility of radiation or chemotherapy effect should also


be kept in mind, as in the absence of appropriate history, it is easy to misinterpret radiation

or chemotherapy effect as malignancy.

3. If it is a carcinoma, what kind is it (adenocarcinoma vs. squamous

carcinoma vs. transitional cell carcinoma vs. hepatoma vs. neuroendocrine, etc). By

knowing the spectrum of reactivity of the markers discussed above, these questions can be

addressed in a successful fashion.

4. Where could this tumor be arising? Each case is different, so the antibody

panel that I use is not the same for every case, and obviously the gender of the patient and

the H&E morphology guide my selection of antibodies. I have never been particularly

successful with the algorithmic approach to metastatic carcinoma that has been advocated

by some authors, since I find that the published algorithms are not easily applied to the

large variety of situations that we see, and the demands for rapid turnaround time often

make the application of a sequential algorithmic approach impractical.

For most metastatic carcinomas, I find that the combination of cytokeratin 7,

cytokeratin 20, and villin generally allows me to narrow the possibilities substantially, so

that is a good place to start for nearly all cases. In my own practice, I use voice recognition

software on my PC (Dragon NaturallySpeaking) for all reports, that allows large

paragraphs or lists of antibodies to be inserted into reports by uttering only a few command

words. Therefore, when I get a carcinoma of unknown origin, I dictate my “standard huge

carcinoma panel” (which is a list of about 30-40 antibodies) into the report, and then I go

down the list and briefly think about each antibody on the list and whether or not it would

have utility in the particular case under study. Those antibodies that are of no use are

rapidly deleted from the list. Similar to using the “peripheral brain” to assist my imperfect

memory, the use of these lists has helped me on many occasions to think about differential

diagnostic possibilities that had previously escaped me, and to remember to order

important antibodies that I would have otherwise forgotten.


It is a very difficult task to adequately cover the topic of immunohistochemistry in

metastatic carcinoma in the time allotted, so it will be impossible for me to cover

everything in this handout. However, I hope that the information presented will be of use

to those in the audience who struggle with these cases as I do.

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Rodney T. Miller, M.D.

ProPath Laboratory

January 2011

Immunohistochemical Approach to Metastatic Carcinoma of Unknown

Primary Origin

Rodney T. Miller, M.D.

Director of Immunohistochemistry

ProPath Laboratory, Inc.

Dallas, Texas

IHC in Metastatic Carcinoma

of Unknown Primary: Goals

� Discuss the spectrum of reactivity of most useful antibodies (we will skip the hard-core science)

� Review illustrative cases

� Suggest an approach to workup of cases

� Provide useful written information for diagnostic pathologists (Handout)

Metastatic Carcinomaof Unknown Primary

Why Immunohistochemistry?

� Tumors of widely varying origins may look identical on standard H&E sections.

� Tumors of specific origins tend to express

certain markers or combinations of markers (immunophenotypes) that can distinguish them from other origins.

Principles of Immunophenotyping: 1

� Immunostains must be of high quality (You need good tools to do a good job).

� Generate appropriate DDx on H&E.

� You must know :A. spectrum of reactivity of AbsB. expected immunophenotypes

(Use of “IHC Peripheral Brain”).

Principles of Immunophenotyping: 2

� There are no perfect markers, so USE

PANELS and avoid “IHC Guilt Syndrome” (Immunophenotype is only part of the picture.

Correlate with other findings).

� Tumors do not read textbooks.

� You will get some cases wrong.

Prostate Specific Antigen (PSA) and

Prostate Specific Acid Phosphatase (PSAP)

PSA False +: Breast, Salivary, Anal glands, Periurethral Glands, Cystitis Cystica / Glandularis

PSAP False +: Same as above plus Rectal Carcinoid, Sweat Glands, rare Renal Cell ca, Islet Cell Tumors

PSAP+ rectal carcinoid

Met Ca: Organ-Related Abs


Prostate-Specific Membrane Ag (PSMA)

P501S (prostein), and NKX3.1

PSMA: More sensitive than PSA and PSAP in high-grade tumors. (also stains endothelial cells in many non-prostate tumors) (salivary glands, renal tubules, some GI mucosal cells,

maybe Mallory bodies??).

P501S (prostein) Characteristic perinuclear

globs in prostate ca

PSMA on RCC

PSMA, prostate ca

P501S, prostate ca


NKX3.1 – Great nuclear marker: 98.6% sensitive (68/69), 99.7% specific (1/349 cases, a lobular breast ca)

NKX3.1, prostate ca

Gross Cystic Disease Fluid Protein-15 (GCDFP-15)

- Pos in 50-60% Breast ca, also Salivary ca, Sweat Gland ca, Prostate ca, rare Lung ca

Met Ca: Organ-Related Abs: Breast

Met Breast ca to endomet

Mammaglobin

- Pos in 50-60% Breast ca, also Endometrial ca (40-70%), Endo-

cervical ca (30%), Ovarian (17%), Salivary ca, Sweat Gland ca, Mammaglob, endomet ca

Thyroglobulin

Pos: Follicular and Papillary cathyroid, sometimes focal pos in Anaplastic (less sensitive for thyroid than TTF-1)

Neg: Medullary ca thyroid, Non-thyroid carcinomas

76F, pleural bx


Pax8 also a good thyroid marker, discussed later

Met Ca: Organ-related Abs

TTF-1 (Thyroid Transcription Factor-1)

- Pos: 75% Lung Non-Small Cell ca,(75% acinar, 100% bronchoalveolar, 91% clear cell, 57% solid ca), 80% Atypical Carcinoid, 90% Small Cell ca Lung, Thyroid ca (incl Medullary) (17% endometrial ca, rare pancreas)

- Neg: Breast, Colon (r+), Gastric (1.5%+), Carcinoid Lung (20%+), Prostate, Kidney, Mesothelioma, Merkel cell

Cytoplasmic in hepatoma, gastric ca, clone 8G7G3/1 only (not SPT24)

Hepatoma

Lung CA

Napsin A

- Pos: 70-80% Lung Adenocarcinoma,

Kidney: 79% papillary, 34% clear cell

<5% lung squamous, pancreaticobiliary,

thyroid, bladder, colon, female genital tract


Alveolar macrophages

Lung ca in liver bx

TRAP: Papillary RCC (79%)Clear cell RCC (34%)

Estrogen Receptor (SP1 or 1D5)

Pos: Breast, Female Genital Tract, Thyroid, Salivary, Sweat Gland, Chordoma, 10-15% Lung ca,

few Urothelial, Hepatoma (rare)

Neg: GI tract, Kidney, Pancreas, Bile Ducts

Breast Ca

ER+ lung Ca

TTF-1

ER+ Normal Liver



HepPar 1 (Hepatocyte Paraffin 1)

- Pos in 50-90% hepatomas,

also significant # gastric ca’s and “hepatoid” carcinomas

clear cell hepatoma


HepPar 1+ Gastric Ca

Reactivity tends to occur along with strong granular cytoplasmic

reactivity with TTF-1 (8G7G3/1)

Arginase-1 (Arg-1)

- Pos in 96% hepatomas

(also neutrophils, histiocytes)

- Pos in only 2/557 non-HCC

- Better than HepPar1

Arg-1, pancreatic bx

with metastatic HCC


Cytoplasmic and nuclear, but cytoplasmic reactivity required for a “positive” stain

Pax8

Nuclear positivity in:

1: Kidney tumors

2: Female genital tract adenoca

3: Thyroid tumors (better than TTF-1 and TG in anaplastic ca)

also: mesonephric things, some NE tumors, some lymphoid cells


LN – Met Serous Ca

Pleural fluid – Met RCC

WT1 (Wilms Tumor Gene)

Nuclear positivity in mesothelioma and serous carcinoma of ovary, tube, and peritoneum (+/- in uterine serous)

(Also in endometrial stromal sarcoma, granulosa cell tumor, thecoma, uterine smooth muscle).

(Cytoplasmic reactivity nonspecific)


WT1 on serous adenoca

WT1 on mesothelioma

Case: 62F with ascites and L axillary

adenopathy undergoes L ax LN bx

ERGCDFP-15 Mammaglobin


Pax8 WT1

Dx: Met. Serous Adenocarcinoma

Pax2

p63Pos in myoepithelial cells, prostatic (& other) basal cells, squamous ca’s, urothelial ca’s, thymoma- Good for sarcomatoid ca

(Scattered pos cells common in many

tumors)


p63 on squamous ca

p63 on urothelial ca

RCC Renal cell, breast (33%), colon (38%), prostate 27%, Lung 14%, Ovary 12% (overrated as a kidney marker)

pVHL

Renal cell ca, Clear cell ca of female genital tract, some cholangioca

(neg in pancreatic ca, but pos in benign pancreatic ducts, useful

in combination with Placental S100)


RCC on met RCC to pleura

pVHL on met RCC to pleura

Case: 49M with a Hx of L neck FNA showing adenoca of unknown primary, developed pericardial effusion. Cell block obtained

EMA Calretinin

TTF-1 Napsin A

Pax8 Dx: Met. Pap. Renal Cell Ca

pVHL Vim

Highly sensitive and specific

OCT3/4: Seminoma, Embryonal ca

SALL4: Above plus Yolk Sac ca and others

OCT3/4 and SALL4 for Germ cell Tumors

Embryonal ca in LN in situ germ cell neoplasia


Pos in many epithelial tumors, some lymphoid tumors

(ALCL, myeloma)

- Neg in Adrenal, Hepatoma (dots), Germ Cell tumors (except Chorioca, Teratoma)

Hepatomas – EMA dots

Ab for Met ca: EMA

Mesothelioma

� CK 7 pos, CK 20 pos:Urothelial, Pancreatic, Ovarian Mucinous, Stomach

� CK 7 pos, CK 20 neg:CK 7 pos, CK 20 neg:Lung, Breast, Ovary (non-mucinous), Endometrial, Mesothelial, Pancreaticobiliary, Stomach, Small bowel, Thyroid

� CK 7 neg, CK 20 pos:Colon, Duodenal / Ampullary Ca, Stomach

� CK 7 neg, CK 20 neg:Lung squamous, Hepatoma, Kidney, Prostate, Stomach

Met Ca: CK 7 and 20 (Wang 1995)

VILLIN (actin binding protein)

Pos: Brush border: GI, Pancreas, Bile

ducts, Gallbladder, some Lung ca

Cytoplasmic: 68% LungCanalicular: 50% HepatomaMembrane: Carcinoid, other NE

Neg: Breast, Mesothelioma

(weak in some cases)

Met Ca: Useful Abs

Colon ca

Lung ca

Carcinoid

Hepatoma

Metastatic CarcinomaUse of CK7, CK20, and Villin

CK7 Positive, CK20 Positive

Villin Positive

Stomach, Pancreas,

Bile ducts, Mucinous

Ovary, Small bowel

Rare: urothelium, breast, prostate (colon unless rectal), endomet, lung

unlikely)

Villin Negative

Mucinous Ovary,

Urothelium, Breast (1/3 of mucinous breast ca, most inv. papillary ca breast)

Rare: GI, pancreas, bile ducts (unless high grade)


CK7 Positive, CK20 Negative

Villin Positive

Lung, Pancreas/BD

Stom/SB, Endomet,

Mucinous Ovary, Sq

Rare: urothelium, breast, serous ov,

mesothel., colon

Villin Negative

Lung, Breast, Ovary

(Serous or Mucinous)

Urothelium, Endomet,

Mesothelioma, Sq

Rare: GI, pancreas,

bile ducts


CK7 Negative, CK20 Positive

Villin Positive

Stomach, Duodenal

Ampullary, Colon

Hepatoma (canalicular)

Rare: breast, lung (rare), bladder, fem

genital, mesothelioma

Villin Negative

Hepatoma

Some prostate ca’s

Rare: breast (3%), lung (rare), bladder, fem

genital, mesothelioma



CK7 Negative, CK20 Negative

Villin Positive

Stomach, Renal cell,

Lung Squamous,


Prostate? (33% villin pos)

Neuroendocrine

Rare: mesothelioma, breast, ov., urothel., pancreas.

Villin Negative

Mesothelioma, Renal

Cell, Lung Squam.,

Hepatoma, Prostate,

(Breast)

Rare: stomach, ov., pancreas, urothel.

Case: 86M with a PSA of 37 with multiple

bone & lung lesions undergoes lung bx.

PSA

PSAP

Additional IHC on Liver Biopsy

CK 7

CK 20

Villin

CEA

CK7 Negative, CK20 Positive

Villin Positive

Stomach, Duodenal

Ampullary, Colon


Rare: breast, lung,

bladder, fem genital, mesothelioma

Villin Negative

Hepatoma

Some prostate ca’s

Rare: breast (3%), lung, bladder, fem genital, mesothelioma

Dx: Metastatic Colonic Ca

(confirmed on colon bx)

Doctor reacts to the newsCytokeratin (AE1/AE3)

Imperfect “first line” epithelial screen:

not a true “Pankeratin”Neg: Hepatoma, Seminoma, some

Renal cell, Adrenal, Prostate,

some Carcinoid & Islet Cell

Met Ca: Useful Abs

AE1/AE3 neg Hepatoma

AE1/AE3 pos Lung ca AE1/AE3 neg Hepatoma Hepatoma, foc+


Cytokeratin LMW (8,18)

Compliments AE1/AE3

Pos: Hepatoma, Carcinomas neg w AE1/AE3

(Renal cell ca, Carcinoid, Prostate ca, etc.)

Better for detecting Small Cell Ca than AE1/AE3 in

nearly all cases (perinuclear dots)

Met Ca: Useful Abs

Paget’s Disease Small cell caHepatoma

AE1/AE3

Merkel

Merkel

Cytokeratin 5 or (5/6)

Pos: Squamous carcinoma Basaloid carcinoma,Basal cell carcinoma,Epithelial MesotheliomaThymoma, Myoepithelial

Neg: Adenocarcinomas Urothelial carcinoma (scattered pos cells or clusters common)

Paget’s Disease

Met Ca: Useful Abs

Mesothelioma

Use of Cytokeratin LMW & HMW

� LMW POS, HMW NEG:HCC, RCC (conv. type), Prostate

� LMW POS, HMW POS:Many Tumors (incl well diff Squamous)

� LMW NEG, HMW POS:Squamous Ca (well differentiated)

Squamous Ca’s: HMW > LMW

Strong coexpression of CK5 and p63

VimentinPos: Kidney (usual type),

Thyroid, Endometrioid,Paraganglioma, Melanoma

Neg: Chromophobe kidney, Hepatoma, Neuroblastoma, GI, Urothelial, Pancreas, Prostate, Germ cell variable

Hepatoma

Met Ca: Useful Abs

CEA (clone COL1)

Pos: Many Adenocarcinomas, Medullary ca Thyroid (~100% of cells)

Neg: Renal Cell ca, Hepatoma (rare canalicular), Thyroid, Adrenal, Mesothelioma

If your CEA Ab stains neutrophils, you should switch to another Ab (yours is cross reacting with NCA)

Canalicluar CEAm (Rare)

Hepatoma

Met Ca: Useful Abs

CDX2Strong pos: Colonic & duodenal,

Muc ovarian, morules, yolk sac,

some NE, esp. midgut carcinoids

Heterogeneous: Panc/BD, Stomach

Neg: Lung, GU, Hepatoma (r+?), Breast (2.4%,

13/546 cases), Fem Gen (non-mucinous), ENT

Met Ca: Useful Abs

Colon

Carcinoid

Lung

TCC Bladder

Lung

TCC Bladder

(At ProPath, occas lung

and TCC’s &1 hepatoma)

Hepatoma


Pos: Serous ca (mucinous neg)Endometrioid ca, Hepatoma, Renal cell ca, Bile duct ca (also Mesothelioma,Squamous ca, Thyroid, Adrenal, SFT, ESS, Thymoma, Germ cell tumors, NE tumors, Schwannoma, etc.)

Neg: Esoph-Gastric, Pancreas, Colon

Liver

Met Ca: Useful Abs

N Cadherin

Approach to the individual case

� Clinical findings & history?

� If carcinoma, what kind?

� Is it really a carcinoma?

� Where is it from?

“Standard huge carcinoma panel” “Standard huge carcinoma panel”

CK7

CK20

Villin

CK5

p63

EMA

CK AE1/AE3

CK LMW

CK HMW

CDX2

TTF-1

Napsin A

Thyroglobulin

CEA (COL-1)

PSA, PSAP

PSMA, P501S

NKX3.1

Pax8

CK15

Vimentin

N-cadherin

CD56

GCDFP-15

Mammaglob

S100

ER, PR

Arginase-1

HBME-1

WT1

CA-125

Mesothelin

MUC2

MUC5AC

b-catenin

CD10

HepPar 1

CEA (poly)

MOC-31

Chromog

Synapto

Inhibin

A103

SALL4

Case History

87F with a breast mass and multiple liver metastases. Two FNA’s of breast were negative, radiologic workup showed no other

tumor. A needle biopsy of the liver was performed, revealing adenocarcinoma (“Path

Comment: IHC can be performed if primary is

unknown”). A breast bx followed, that showed

fat necrosis but no tumor. Four weeks later, immunophenotyping of liver bx is requested by oncologist.

Liver Biopsy (S97-5632)

CK7 CK20 Villin



Villin Positive


Lung Squamous,



Neuroendocrine

Rare: mesothelioma, breast, ov., urothel.,

pancreas.

Villin Negative

Mesothelioma, Renal

Cell, Lung Squam.,

Hepatoma, Prostate, (Breast)


Liver Biopsy (S97-5632)

AE1/AE3 CEAChg - Syn

CK7 CK20 Villin


Villin Positive


Lung Squamous,



Neuroendocrine

Rare: mesothelioma,

breast, ov., urothel., pancreas.

Villin Negative

Mesothelioma, Renal

Cell, Lung Squam.,



Dx: Stomach most likely (confirmed on gastric bx)

Pictorial representation of this case

�� Breast Stomach��

�� Clinicians

Clinicians “barking up the wrong tree”

85M, FNA of L1 vertebral mass (CM02-584)

PSA, PSAP, TTF1 are neg (CM02-584)

Additional Immunostains

Chg

Syn

CK-hmw

CK20

CK7

Villin


CK7 Positive, CK20 Positive

Villin Positive

Stomach, Pancreas,

Bile ducts, Mucinous

Ovary, Small bowel

Rare: urothelium, breast, prostate (colon, endomet, lung unlikely)

Villin Negative

Mucinous Ovary,

Urothelium, Breast (1/3 of mucinous

breast ca, most inv. papillary ca breast)

Rare: GI, pancreas,

bile ducts

____________________________________________

Chg

Syn CK-hmw CK20

CK7

Villin

Dx: Metastatic Urothelial Ca

CK5p63

56M, bone & skin lesions, scalp bx.

Carcinoid vs plasmacytoid lymphoma vs

melanoma vs prostate ca??

Screening Immunostains

CD45 Chg, Syn S100, HMB45

PSA, PSAP VS38 Vim

EMA CK AE1/AE3 CK LMW

Additional Immunostains

CK 7 and 20 Villin

Villin Villin


Villin Positive


Lung Squamous,


Rare: mesothelioma, breast, ov., urothel., pancreas.

Villin Negative

Mesothelioma, Renal

Cell, Lung Squam.,



Dx: Metastatic Hepatoma


Conclusions

�

�Immunohistochemistry (IHC) plays an important role in the evaluation of

metastatic tumors of unknown origin.

�IHC can save cost and discomfort of further diagnostic procedures

(particularly with the use of tissue transfer techniques)

�IHC can allow rapid institution of

appropriate therapy.

Immunohistochemistry


carcinoma her2 non mammary handout

Documents

immunohistochemistry

diagnostic miller ihc

diagnosis of carcinoma

number miller ihc

primary site

use of immunohistochemistry

personal ihc peripheral

diagnostic problems