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
rodney.miller@propath.com
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
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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
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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
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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
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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.
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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 (710). 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
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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
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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-
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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. TTF1 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.
endometrial
adenocarcinoma,
Some authors have described TTF-1 in 17% of
and
indeed
I
have
adenocarcinomas with rather striking TTF-1 reactivity.
seen
several
endometrial
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).
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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 (TTF1 was positive in 73% of cases). There were 13 Napsin A positive, TTF-1 negative cases,
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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.
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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
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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
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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
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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
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IHC for Carcinoma of Unknown Primary
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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.
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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
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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,
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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%
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(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
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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
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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
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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
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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
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“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.
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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
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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
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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 clearcell 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
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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,
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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 cordstromal tumors of the genital tract, and calretinin is well known as a mesotheliomaassociated 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 nonrenal 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.
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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,
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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 (7090%), 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
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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 noncarcinomas 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
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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.
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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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HepPar1
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222. Lin F, Shi J, Liu H et al: Immunohistochemical detection of the von Hippel-Lindau gene
product (pVHL) in human tissues and tumors. Am J Clin Pathol 129(4): 529-605, Apr 2008.
223. Lin F, Shi J, Liu H et al: Diagnostic utility of S100P and von Hippel-Lindau gene product
(pVHL) in pancreatic adenocarcinoma – with implication of their roles in early tumorigenesis.
Am J Surg Pathol 32(1): 78-91, Jan 2008.
SALL4
224. Cao D, Guo S, Allan RW et al: SALL4 is a novel sensitive and specific marker of ovarian
primitive germ cell tumors and is particularly useful in establishing yoke sac tumor from clear cell
carcinoma. Am J Surg Pathol 33(6): 894-904, Jun 2009 .
225. Cao D, Li J, Guo S et al: SALL4 is a novel diagnostic marker for testicular germ cell tumors.
Am J Surg Pathol 33(7): 1065-1077, Jul 2009 .
226. Ushiku T, Shinozaki A, Shibahara J et al: SALL4 represents fetal gut differentiation of
gastric cancer, and is diagnostically useful in the distinguishing hepatoid gastric carcinoma from
hepatocellular carcinoma. Am J Surg Pathol 34(4): 533-540, Apr 2010
Arginase-1
227. Yan BC, Gong C, Song J et al: Arginase-1. A new immunohistochemical marker of
hepatocytes and hepatocellular neoplasms. Am J Surg Pathol 34(8):1147-1154, Aug 2010.
Rodney T. Miller, M.D.
ProPath Laboratory
January 2011
Miller
IHC for Carcinoma of Unknown Primary
Immunohistochemical
Approach to Metastatic
Carcinoma of Unknown
Primary Origin
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)
Rodney T. Miller, M.D.
Director of Immunohistochemistry
ProPath Laboratory, Inc.
Dallas, Texas
Metastatic Carcinoma
of 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.
Page 47
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 Abs
B. 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.
Met Ca: Organ-Related Abs
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
Miller
IHC for Carcinoma of Unknown Primary
Met Ca: Organ-Related Abs
Met Ca: Organ-Related Abs: Breast
PSMA, prostate ca
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
Page 48
PSMA on RCC
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 Breast ca to endomet
Mammaglobin
P501S, prostate ca
- Pos in 50-60% Breast ca, also
Endometrial ca (40-70%), Endocervical ca (30%), Ovarian (17%),
Salivary ca, Sweat Gland ca,
NKX3.1 – Great nuclear marker: 98.6%
sensitive (68/69), 99.7% specific (1/349
cases, a lobular breast ca)
Mammaglob, endomet ca
NKX3.1, prostate ca
Met Ca: Organ-Related Abs
Met Ca: Organ-related Abs
Thyroglobulin
TTF-1 (Thyroid Transcription Factor-1)
- Pos: 75% Lung Non-Small Cell ca,(75%
Pos: Follicular and Papillary ca
thyroid, sometimes focal
pos in Anaplastic (less
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
sensitive for thyroid than TTF-1)
Neg: Medullary ca thyroid,
Non-thyroid carcinomas
76F, pleural bx
Cytoplasmic in hepatoma, gastric ca,
clone 8G7G3/1 only (not SPT24)
Pax8 also a good thyroid marker, discussed later
Met Ca: Organ-related Abs
Napsin A
Lung CA
Hepatoma
Met Ca: Organ-related Abs
Estrogen Receptor (SP1 or 1D5)
Breast Ca
Lung ca in liver bx
- Pos: 70-80% Lung Adenocarcinoma,
Kidney: 79% papillary, 34% clear cell
<5% lung squamous, pancreaticobiliary,
thyroid, bladder, colon, female genital tract
Pos: Breast, Female Genital Tract,
Thyroid, Salivary, Sweat Gland,
Chordoma, 10-15% Lung ca,
few Urothelial, Hepatoma (rare)
TTF-1
Neg: GI tract, Kidney, Pancreas, Bile Ducts
TRAP: Papillary RCC (79%)
Clear cell RCC (34%)
Alveolar macrophages
ER+ lung Ca
ER+ Normal Liver
Miller
IHC for Carcinoma of Unknown Primary
Met Ca: Organ-related Abs
Page 49
Met Ca: Organ-related Abs
clear cell hepatoma
HepPar 1 (Hepatocyte Paraffin 1)
Arginase-1 (Arg-1)
- Pos in 50-90% hepatomas,
also significant # gastric ca’s
and “hepatoid” carcinomas
- Pos in 96% hepatomas
(also neutrophils, histiocytes)
- Pos in only 2/557 non-HCC
- Better than HepPar1
Reactivity tends to occur along with
strong granular cytoplasmic
reactivity with TTF-1 (8G7G3/1)
Cytoplasmic and nuclear, but
cytoplasmic reactivity required
for a “positive” stain
HepPar 1+ Gastric Ca
Met Ca: Organ-related Abs
Met Ca: Organ-related Abs
WT1 (Wilms Tumor Gene)
Pax8
Nuclear positivity in:
Arg-1, pancreatic bx
with metastatic HCC
Pleural fluid – Met RCC
1: Kidney tumors
2: Female genital tract adenoca
3: Thyroid tumors (better than
TTF-1 and TG in anaplastic ca)
Nuclear positivity in
mesothelioma and serous
carcinoma of ovary, tube, and
peritoneum (+/- in uterine serous)
WT1 on mesothelioma
(Also in endometrial stromal sarcoma,
granulosa cell tumor, thecoma,
uterine smooth muscle).
also: mesonephric things, some
NE tumors, some lymphoid cells
(Cytoplasmic reactivity nonspecific)
LN – Met Serous Ca
WT1 on serous adenoca
Case: 62F with ascites and L axillary
adenopathy undergoes L ax LN bx
GCDFP-15
Mammaglobin
ER
Miller
IHC for Carcinoma of Unknown Primary
Page 50
Met Ca: Organ-related Abs
p63
Pax8
WT1
Dx: Met. Serous
Adenocarcinoma
Pos in myoepithelial cells,
prostatic (& other) basal
cells, squamous ca’s,
urothelial ca’s, thymoma
p63 on squamous ca
- Good for sarcomatoid ca
(Scattered pos cells common in many
tumors)
Pax2
p63 on urothelial ca
Case: 49M with a Hx of L neck FNA showing
adenoca of unknown primary, developed pericardial
effusion. Cell block obtained
Met Ca: Organ-related Abs
RCC
Renal cell, breast (33%), colon (38%),
prostate 27%, Lung 14%, Ovary 12% RCC on met RCC to pleura
(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)
EMA
pVHL on met RCC to pleura
Calretinin
OCT3/4 and SALL4 for Germ cell Tumors
TTF-1
Napsin A
pVHL
Pax8
Highly sensitive and specific
OCT3/4: Seminoma, Embryonal ca
SALL4: Above plus Yolk Sac ca and others
Vim
Dx: Met. Pap. Renal Cell Ca
Embryonal ca in LN
in situ germ cell neoplasia
Miller
IHC for Carcinoma of Unknown Primary
Ab for Met ca:
EMA
Page 51
Met Ca: CK 7 and 20 (Wang 1995)
Pos in many epithelial tumors,
some lymphoid tumors
(ALCL, myeloma)
- Neg in Adrenal, Hepatoma
(dots), Germ Cell tumors
CK 7 pos, CK 20 pos:
CK 7 pos, CK 20 neg:
Urothelial, Pancreatic, Ovarian Mucinous, Stomach
Lung, Breast, Ovary (non-mucinous), Endometrial,
Mesothelial, Pancreaticobiliary, Stomach, Small bowel,
Thyroid
Hepatomas – EMA dots
(except Chorioca, Teratoma)
CK 7 neg, CK 20 pos:
CK 7 neg, CK 20 neg:
Colon, Duodenal / Ampullary Ca, Stomach
Lung squamous, Hepatoma, Kidney, Prostate, Stomach
Mesothelioma
Metastatic Carcinoma
Met Ca: Useful Abs
Use of CK7, CK20, and Villin
Colon ca
VILLIN (actin binding protein)
Pos: Brush border: GI, Pancreas, Bile
ducts, Gallbladder, some Lung ca
Cytoplasmic: 68% Lung
Canalicular: 50% Hepatoma
Membrane: Carcinoid, other NE
Lung ca
Carcinoid
Neg: Breast, Mesothelioma
(weak in some cases)
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)
Hepatoma
Metastatic Carcinoma
Metastatic Carcinoma
Use of CK7, CK20, and Villin
Use of CK7, CK20, and Villin
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)
Villin Negative
Hepatoma
Some prostate ca’s
Rare: breast, lung
Rare: breast (3%), lung
(rare), bladder, fem
(rare), bladder, fem
genital, mesothelioma
genital, mesothelioma
Miller
IHC for Carcinoma of Unknown Primary
Metastatic Carcinoma
Page 52
Case: 86M with a PSA of 37 with multiple
bone & lung lesions undergoes lung bx.
Use of CK7, CK20, and Villin
CK7 Negative, CK20 Negative
Villin Positive
Stomach, Renal cell,
Lung Squamous,
Hepatoma (canalicular)
Prostate? (33% villin pos)
Villin Negative
Mesothelioma, Renal
Cell, Lung Squam.,
Hepatoma, Prostate,
(Breast)
Neuroendocrine
Rare: stomach, ov.,
pancreas, urothel.
Rare: mesothelioma,
breast, ov., urothel.,
pancreas.
Additional IHC on Liver Biopsy
CK 7
CK 20
Villin
CEA
PSA
PSAP
CK7 Negative, CK20 Positive
Villin Positive
Stomach, Duodenal
Ampullary, Colon
Hepatoma (canalicular)
Villin Negative
Hepatoma
Some prostate ca’s
Rare: breast, lung,
Rare: breast (3%), lung,
bladder, fem genital, bladder, fem genital,
mesothelioma
mesothelioma
Dx: Metastatic Colonic Ca
(confirmed on colon bx)
Doctor reacts to the news
Met Ca: Useful Abs
Cytokeratin (AE1/AE3)
Imperfect “first line” epithelial screen:
not a true “Pankeratin”
Neg: Hepatoma, Seminoma, some
Renal cell, Adrenal, Prostate,
some Carcinoid & Islet Cell
AE1/AE3 neg Hepatoma
AE1/AE3 pos Lung ca
AE1/AE3 neg Hepatoma
Hepatoma, foc+
Miller
IHC for Carcinoma of Unknown Primary
Met Ca: Useful Abs
Page 53
Met Ca: Useful Abs
Cytokeratin LMW (8,18)
Merkel
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)
AE1/AE3
Paget’s Disease
Hepatoma
Merkel
Pos: Squamous carcinoma
Basaloid carcinoma,
Basal cell carcinoma,
Epithelial Mesothelioma
Thymoma, Myoepithelial
Neg: Adenocarcinomas
Urothelial carcinoma
(scattered pos cells or
clusters common)
Paget’s Disease
Mesothelioma
Small cell ca
Use of Cytokeratin LMW & HMW
Cytokeratin 5 or (5/6)
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
Met Ca: Useful Abs
Vimentin
Pos: Kidney (usual type),
Thyroid, Endometrioid,
Paraganglioma, Melanoma
Neg: Chromophobe kidney, Hepatoma,
Neuroblastoma, GI, Urothelial,
Pancreas, Prostate, Germ cell
variable
Colon
Hepatoma
Met Ca: Useful Abs
Met Ca: Useful Abs
CDX2
CEA (clone COL1)
Pos: Many Adenocarcinomas, Canalicluar CEAm (Rare)
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)
Hepatoma
Strong pos: Colonic & duodenal,
Muc ovarian, morules, yolk sac,
some NE, esp. midgut carcinoids
Heterogeneous: Panc/BD, Stomach
Carcinoid
TCC
TCC Bladder
Bladder
Neg: Lung, GU, Hepatoma (r+?), Breast (2.4%,
13/546 cases), Fem Gen (non-mucinous), ENT
(At ProPath, occas lung
and TCC’s &1 hepatoma)
Hepatoma
Lung
Lung
Miller
IHC for Carcinoma of Unknown Primary
Met Ca: Useful Abs
N Cadherin
Pos:
Approach to the individual case
Liver
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:
Page 54
Clinical findings & history?
Is it really a carcinoma?
If carcinoma, what kind?
Where is it from?
Esoph-Gastric, Pancreas, Colon
“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
Liver Biopsy (S97-5632)
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.
CK7
CK20
Villin
Miller
IHC for Carcinoma of Unknown Primary
Page 55
Liver Biopsy (S97-5632)
CK7 Negative, CK20 Negative
Villin Positive
Stomach, Renal cell,
Lung Squamous,
Hepatoma (canalicular)
Prostate? (33% villin pos)
Villin Negative
Mesothelioma, Renal
Cell, Lung Squam.,
Hepatoma, Prostate,
(Breast)
Neuroendocrine
Rare: stomach, ov.,
pancreas, urothel.
Rare: mesothelioma,
breast, ov., urothel.,
pancreas.
CK7
AE1/AE3
CK20
Villin
Chg - Syn
CEA
Pictorial representation of this case
CK7 Negative, CK20 Negative
Villin Positive
Stomach, Renal cell,
Lung Squamous,
Hepatoma (canalicular)
Prostate? (33% villin pos)
Villin Negative
Mesothelioma, Renal
Cell, Lung Squam.,
Hepatoma, Prostate,
(Breast)
Neuroendocrine
Rare: stomach, ov.,
pancreas, urothel.
Rare: mesothelioma,
breast, ov., urothel.,
pancreas.
Stomach
Breast
Clinicians
Dx: Stomach most likely
(confirmed on gastric bx)
Clinicians “barking up the wrong tree”
85M, FNA of L1 vertebral mass (CM02-584)
Additional Immunostains
Chg
CK7
Villin
CK-hmw
Syn
PSA, PSAP, TTF1 are neg (CM02-584)
CK20
Miller
IHC for Carcinoma of Unknown Primary
Page 56
CK7
Positive, CK20 Positive
____________________________________________
CK7
Chg
Villin Positive
Stomach, Pancreas,
Bile ducts, Mucinous
Ovary, Small bowel
Rare: urothelium,
breast, prostate
(colon, endomet,
lung unlikely)
Villin Negative
Mucinous Ovary,
Urothelium, Breast
Syn
CK-hmw
CK20
Villin
(1/3 of mucinous
breast ca, most inv.
papillary ca breast)
Rare: GI, pancreas,
bile ducts
p63
CK5
Dx: Metastatic Urothelial Ca
Screening Immunostains
56M, bone & skin lesions, scalp bx.
Carcinoid vs plasmacytoid lymphoma vs
melanoma vs prostate ca??
Additional Immunostains
CK 7 and 20
Villin
Villin
Villin
CD45
Chg, Syn
S100, HMB45
PSA, PSAP
VS38
Vim
EMA
CK AE1/AE3
CK LMW
CK7 Negative, CK20 Negative
Villin Positive
Stomach, Renal cell,
Lung Squamous,
Hepatoma (canalicular)
Rare: mesothelioma,
breast, ov., urothel.,
pancreas.
Villin Negative
Mesothelioma, Renal
Cell, Lung Squam.,
Hepatoma, Prostate,
(Breast)
Rare: stomach, ov.,
pancreas, urothel.
Dx: Metastatic Hepatoma
Miller
IHC for Carcinoma of Unknown Primary
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
Page 57