谢谢张主任告知会诊结果。

形态学是不能除外该诊断，但IHC标记结果将如何解释？会诊时是否重新做过IHC？

感谢Dr.djdnx提供 Dr.Miller 2010-5在美国颌面病理学术年会上的“未分化恶性肿瘤IHC鉴别诊断”讲座！

现将小结部分简译， 供参考。

IMMUNOHISTOCHEMICAL APPROACH TO UNDIFFERENTIATED MALIGNANT TUMOR.

Initial considerations（首先考虑）:

1. Value of looking at H&E first without looking at patient history first.（不看临床病史，先观察HE切片很有价值）

2. Clinical information can guide your differential diagnosis (age, history, location, etc.) 临床资料可指导鉴别诊断（年龄、病史、病变部位等）。If prior pertinent pathology exists – try to look at that first.（如有前病理诊断，尽可能先看前病理切片）

3. How much tissue do we have to work with, and are clinicians in a big rush?（明确供诊断的组织有多少？临床医生是否急需病理诊断？）

- If lots of tissue and no rush – sequential rounds of stains OK（如组织多、临床不急需，沿常规染色步骤）

- If minimal tissue or big rush – better to “blast away” up front (cost of more stains is usually < cost of re-biopsy^{)（如组织少、临床急需病理报告，最好紧急处理，标记更多抗体比再次活检的花费少）}

4. If minimal tissue – order stains in “order of importance”, and have 10 or more extra sections cut and held, in case more stains are needed (rather than having to re-face the paraffin block another time) 如果组织稀少，要根据重要性选择抗体，多切10张以上白片待用，根据需要再次标记（不必花时间再次切蜡块）

5. Use lists to help jog your memory（运用标记菜单来帮助您的记忆）

Useful Initial Panel 运用一线抗体菜单 (if you don’t have a clue what the tumor is): （如果你对肿瘤毫无诊断线索）

CK-LMW Sox10 CD45 (LCA) CD43 SYN

CK-HMW MART-1 CD30 (Ki-1) SALL4 CHG

SUMMARY AND CONCLUSIONS

1. Appearances can be deceiving.（形态学表现可有欺骗性）

2. Importance of good clinical information and review of previous pathology.（完整的临床资料和复习以前病理切片非常重要）

3. Importance of panel approach – often need >1 marker per diagnostic category.（诊断套路很重要，对一个诊断系列要选择一种以上的抗体）

4. Be mindful of what you have to work with, and alter your approach accordingly.（记住你要鉴别的病变，根据鉴别诊断调整抗体。

5. Use lists so that you won’t forget things as easily. 运用鉴别菜单以避免遗漏。

It must be kept in mind that there are exceptions to everything, and not all tumors will necessarily react as I have indicated. This underscores the importance of a carefully performed H&E examination by a skilled anatomic pathologist in arriving at the correct diagnosis.（必须牢记：任何事物都有例外，并非所有的肿瘤都会像上述的免疫标记。这就强调对一个有经验的解剖病理医师，在做出正确的病理诊断前仔细观察HE切片的重要性。

If the immunostain findings don't make sense when viewing the H&E, then the immunostain findings should be considered suspect (especially if the immunostains were performed using so-called "Predilute Ready-To-Use" antibodies).如果IHC标记结果难以解释镜下HE形态，此时IHC标记结果必须认为可疑（尤其是用预先稀释的即用型抗体）

看到Dr.XLJin的详尽诊断思路和鉴别要点后，早上很幸运查到了Dr.Rodney T. Miller的一个较新的对所谓未分化肿瘤的详实讲解和记录，和大家分享。

里面提到很多较新而全面的形态学和免疫组化内容，真的是宝贝。对本例诊断思路和后续工作可能会有帮助。在Google里是一定可以查打这篇原文的，是免费的。

对于本例：在临床未提供相应转移瘤可能的前提下，可能还是要考虑胸腺上皮起源的恶性肿瘤，期待楼主的后续报道。

Immunohistochemical Approach

to “Undifferentiated” Tumors

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 – Tucson, AZ

Sunday, May 16, 2010

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 1

INTRODUCTION

As we all know, immunohistochemistry (IHC) is an extremely valuable adjunct to

standard morphologic diagnosis in diagnostic pathology. It has rapidly become the

standard of care, particularly when dealing with a number of common diagnostic

problems, including "undifferentiated malignant tumor", metastatic carcinoma of

unknown origin, lymphoma classification, and similar problems. In this portion of the

presentation, I will discuss an approach to the diagnosis of malignant tumors that appear

"undifferentiated" by light microscopy. I should say at this point that the approach

discussed includes my personal biases, and other pathologists may advocate a different

approach or employ different antibodies (Abs) in certain types of cases. However, the

approach I will discuss has served me well in the past, and I look forward to improving

these approaches as new and more specific markers become available. We also need to

keep in mind that there are exceptions to everything, and not all tumors will necessarily

react as they are "supposed" to (since "tumors don't read textbooks"). This underscores

the importance of a thorough background in standard morphologic diagnosis employing

the time-honored and venerable H&E section.

The topics that I will be covering today are very broad, so I will not be able to cover

everything in the degree of detail that I would like. However, I have tried to make the

handout materials as useful as possible, by condensing much of the information into lists

and tables. I have found these types of lists to be very helpful in jogging my memory, and

I hope the lists find themselves within easy reach of your microscope as you deal with

difficult cases.

IMPORTANCE OF A PANEL APPROACH TO DIAGNOSTIC PROBLEMS

The importance of a panel approach to diagnostic problems cannot be

overemphasized. Many of the most egregious errors that can occur will happen because

the pathologist has not ordered enough stains in his or her diagnostic battery to

appropriately address the differential diagnostic possibilities in a particular case. We use

IHC because the cases are by nature difficult, and skimping on stains may save the patient

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 2

a bit of money, but may also provide the patient with an incorrect or otherwise

suboptimal diagnosis.

The Concept of “Undifferentiated Tumors

"Undifferentiated" typically refers to tumor that is composed of a proliferation of

“primitive cells” that do not show any recognizable line of cellular differentiation. It is

well known however, that most tumors that appear "undifferentiated" by light microscopy

do express evidence of a specific line of cellular differentiation when studied using other

techniques, commonly IHC. As such, in the particular context of our role as diagnostic

pathologists, the term "undifferentiated" is more accurately defined as "I don't know what

this tumor is based on the H&E appearance".

As diagnostic pathologists, our role is to precisely define the nature of the tumors that

we see, so that the clinicians who treat the patients can offer them the best and most

appropriate therapy. Most of the time that can be done on a standard H&E section, but

we are all too familiar with the fact that tumors of widely varying histogenesis can look

identical on an H&E stain. Fortunately, there are only a limited number of lines of

cellular differentiation that need to be considered, and if we are aware of the most useful

markers to screen for each of these pathways of cellular differentiation, a logical approach

to diagnosis can be formulated. The main lines cellular differentiation (histogenetic

categories) can be listed as follows:

1. Epithelial Tumors (carcinoma)

2. Germ cell – Sex Cord Stromal Tumors

3. Neuroendocrine Tumors

4. Melanocytic Tumors

5. Lymphohematopoetic Tumors (lymphoma, leukemia, histiocytic)

6. Mesenchymal Tumors (sarcoma)

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 3

When confronted by a tumor that appears undifferentiated by light microscopy, it can

be useful to try to place a tumor into a particular morphologic category of tumor, listed

below:

1. Pleomorphic Large Cell Tumors

2. Epithelioid Large Cell Tumors

3. Spindle Cell (Sarcomatoid) Tumors

4. Small Blue Cell Tumors (Round or Spindled)

In my day-to-day practice, I rely heavily on the use of sets of computerized notes to

assist my imperfect memory and to make certain that I do my best to consider all

reasonable possible diagnoses in my working differential diagnosis. These notes (which I

maintain in Excel spreadsheet format so that they are highly portable and readily

searchable), have enabled me to make a correct diagnosis in numerous difficult cases that

I otherwise would have missed. For those of you can, I encourage you to make use of

similar computerized notes and lists.

EPITHELIAL MARKERS

Cytokeratins: Cytokeratins (CK’s) are the most frequently used epithelial markers,

and I would estimate that 97% to 98% of carcinomas will stain with CK, provided an

appropriate CK Ab is employed.

What about “Pan-Cytokeratin”? Before discussing cytokeratins further, it is

worthwhile to discuss so called "pan-cytokeratin" or "pan-keratin" stains. To my

knowledge, there are no particular clones of antibodies that truly recognize all types of

cytokeratin, and for that reason, I have banished the term "pan-keratin" from my

consultation reports (and from my laboratory). Additionally, when I see the term "pankeratin"

on a pathology report, I do not really know what this means, particularly if the

clones used in this "pan-keratin" are not specified. Actually, I think the use of the term

"pan-keratin" can be misleading and sometimes dangerous, since some antibodies that are

frequently employed in this fashion (particularly AE1/AE3) are far from ideal "pankeratin"

reagents, and a number of pathologists who have employed "pan-keratin" stains

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 4

and have obtained negative results have erroneously concluded that the tumor they are

studying is not epithelial.

Cytokeratin AE1/AE3: This antibody cocktail if often referred to as a “pancytokeratin”,

but it is NOT a “pan-cytokeratin”, and anyone who uses this antibody in this

fashion is certainly doing some of their patients a disservice. The problem with

cytokeratin AE1/AE3 is that this cocktail does a poor job of detecting CK8, which is the

major CK in hepatocellular carcinoma (HCC) and many other carcinomas (ca’s),

particularly renal cell carcinoma (RCC), prostate carcinoma, and many other

adenocarcinomas. It is also not as good as high molecular weight CK antibody Ab (clone

34bE12) for recognizing squamous tumors. Below is a list of major problems that can

occur when CK AE1/AE3 is used as “pan-CK”:

1. The majority of HCC will be AE1/AE3 negative or very weak with AE1/AE3

2. A significant number of RCC and some prostate ca’s are negative with AE1/AE3

3. Some neuroendocrine ca’s are negative with AE1/AE3

4. A few squamous ca’s are negative with AE1/AE3

In my practice, the main utility of AE1/AE3 is in the diagnosis of HCC, since those

tumors are characteristically negative or weak with this antibody. I also use it in

situations where I want to highlight a population of cells that I know will stain positively

with it, but I NEVER USE AE1/AE3 AS A “PAN-CYTOKERATIN”!

I should say at this point that I do not have personal experience with some other socalled

“pan-CK” antibodies such as OSCAR, so I cannot address the strengths or

weaknesses of those reagents.

So-called “Pan-cytokeratin”: A better approach: In my experience, a far better

way to use CK’s to detect the greatest number of epithelial tumors is by doing 2 stains:

one stain that detects low molecular weight CK’s 8 and 18 (CK-LMW) (such as clones

CAM5.2, 5D3, Zym5.2), and a stain for high molecular weight CK (CK-HMW) using

clone 34bE12. This combination of CK-LMW and CK-HMW will enable you to

detect the broadest spectrum of epithelial tumors using CK Abs. Although these 2

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 5

reagents could be made into a cocktail, I prefer to see the results of the 2 stains separately,

as in many instances one can gain valuable information about the tumor based on the

relative intensity and pattern of staining with these 2 CK’s. Specifics of these 2 Abs

follow.

CK-LMW (CK’s 8 and 18): This CK Ab stains most adenocarcinomas, including

those that are often or may be negative with AE1/AE3, including HCC, RCC, prostate ca,

Hurthle cell ca of thyroid, and neuroendocrine ca’s. In the case of neuroendocrine ca’s,

perinuclear dots of CK-LMW may provide a clue that you are dealing with a

neuroendocrine tumor. Potential “traps” involve lack of staining of certain types of

epithelial tumors and staining of certain non-epithelial cells, as listed below:

1. CK-LMW may not stain translocation-associated RCC (TFE3+)

2. CK-LMW stains some non-epithelial cells and tumors, including the following:

- Interstitial reticulum cells in lymph nodes

- Submesothelial fibroblasts

- Epithelioid angiosarcoma (50-70%)

- Synovial sarcoma

- Epithelioid Sarcoma

- Alveolar rhabdomyosarcoma

- Some leiomyosarcomas, Ewing / PNET

- Few cases of gastrointestinal stromal tumors (GIST)

- Rare AML, myeloma, pheochromocytoma

CK-HMW, clone 34bE12 (CK’s 1, 5, 10, 14): This CK Ab has a number of useful

attributes:

1. Virtually all squamous ca’s show strong and diffuse reactivity with CK-HMW

2. If the CK-HMW stain is stronger than the CK-LMW stain, you are probably

dealing with a squamous tumor

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 6

3. CK-HMW is typically absent or weak and focal in HCC, RCC of conventional

type, and adrenocortical ca

4. Strong CK-HMW is uncommon in prostate ca

Phenotypic features of Squamous ca’s:

1. Strong diffuse expression of CK-HMW

2. Intensity of CK-HMW > CK-LMW

3. Strong diffuse co-expression of CK5 (or CK5/6) and nuclear p63

Traps: Cutaneous adnexal tumors show the above, and expression of CK5 is often

patchy in basaloid squamous ca’s

Limitations of Cytokeratins as a marker of epithelial tumors:

1. Expression of CK may be focal in poorly differentiated tumors (sampling issues)

2. A small percentage (<2%) of epithelial tumors are negative with CK’s (including

a high percentage of translocation-associated RCC’s, TFE3+)

3. Watch out for “bleeding artifacts”, which may make a non-epithelial tumors

appear to have CK

Epithelial Membrane Antigen (EMA): EMA is expressed in many epithelial

tumors, but is also expressed in many non-epithelial tumors, including certain lymphomas

and sarcomas. Therefore, in order for it to be used as a valid marker of epithelial

differentiation, mesenchymal and hematoplymphoid neoplasms must be excluded.

GERM CELL AND SEX-CORD STROMAL MARKERS

In the past several years, there have been great improvements in the markers available

for the recognition of germ cell tumors, relegating some of the previous markers (such as

placental alkaline phosphatase) obsolete. The most useful markers in my experience are

the following:

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 7

Germ Cell Tumor Markers:

SALL4: Nuclear marker of Yolk Sac Tumor, Embryonal ca, and Seminoma

OCT3/4: Nuclear marker of Embryonal ca and Seminoma

CD30 (Ki-1): Embryonal ca

D2-40 (podoplanin): Seminoma, in a diffuse strong membranous pattern

Glypican-3: Yolk Sac Tumor – stains many more cells than AFP

AFP: Stains a variable minority of cells in Yolk Sac Tumor

HLA-G: Excellent marker for the intermediate trophoblastic cells of choriocarcinoma

HCG: Syncytiotrophoblasts of choriocarcainoma and syncytiotrophoblastic giant cells

that may be present in other germ cell tumors

Other new markers (which I have not yet used):

NANOG: Nuclear marker of Embryonal ca and Seminoma, also some breast ca’s

SOX2: Embryonal ca, also gliomas, with 1 report of squamous ca’s of GI tract

GDF3: Strong in Seminoma, moderate to strong in Embryonal ca, weak or neg in Yolk

Sac

Traps: With SALL4 and OCT3/4, the reactivity should be diffuse and moderate to

strong in intensity. Some non-germ cell tumors show weak focal staining with these

markers. Additionally, I have seen a small number of high grade non-germ cell

carcinomas that have shown SALL4 expression similar to that seen in germ cell tumors.

However, germ cell tumor was excluded in these cases by other means. Obviously, it

must be kept in mind that CD30 stains some lymphomas, and I have also seen strong

staining of a few sarcomas with this marker, including dedifferentiated liposarcoma.

Glypican-3 also stains HCC, Wilms tumor, and occasionally some carcinomas. HLA-G

staining should be seen in >70% of the tumor cells in order to use it as a valid marker of

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 8

intermediate trophoblasts, as focal patchy staining may be seen in some non-trophoblastic

lesions. HCG is usually very strong in choriocarcinoma, and usually demonstrates

extensive bleeding artifact.

Sex-Cord Stromal Tumors:

Markers of Sex-Cord Stromal Tumors include Inhibin, Calretinin, and MART-1,

clone A103. In addition to staining sex-cord stromal tumors, the antibodies also stain the

following:

Inhibin: adrenocortical lesions, granular cell tumors, some neuroendocrine tumors,

and occasional carcinomas

Calretinin: adrenocortical lesions, mesothelioma, about 30-40% of squamous ca’s,

neural lesions, and occasionally other carcinomas

MART-1, clone A103: adrenocortical lesions and melanocytic lesions

NEUROENDOCRINE MARKERS

Chromogranin and Synaptophysin are an excellent screen for neuroendocrine

tumors. They should almost always be ordered together, as some neuroendocrine markers

will be positive for only 1 of these 2 markers. So-called “neuron-specific enolase” is not

neuron-specific, and is best avoided.

MELANOCYTIC MARKERS

S100 protein: Positive in 97-99% of melanomas, sensitive but not very specific

- also stains some carcinomas (especially breast ca), histiocytes (Langerhans cells),

sarcomas, myoepithelial and salivary gland tumors, neural tumors, sex-cord stromal

tumors, and rare lymphomas.

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 9

Sox10: Nuclear marker, stains a higher percentage of melanomas and nevi than S100,

but does not stain breast carcinomas or histiocytes (useful for melanoma sentinel nodes).

- also stains neural lesions (schwannoma, neurofibroma, astrocytoma), granular cell

tumors, myoepithelial lesions (including salivary gland tumors). Cytoplasmic expression

may be seen in some T-cells, osteoclastic giant cells, and renal oncocytoma.

MART-1, HMB-45, and Tyrosinase: These markers have good specificity for

melanoma, and in my experience MART-1 is more sensitive than HMB-45. They also

stain perivascular epithelioid tumors (PEComas), and rare (<0.5%) of carcinomas may

show significant staining (including some cases of TFE3+ RCC). As mentioned above,

MART-1 clone A103 also stains adrenocortical tumors and sex-cord stromal tumors.

My current “melanoma screen” consists of immunostains for Sox10 and MART-1.

LYMPHOHEMATOPOETIC MARKERS

CD45 (Leukocyte Common Antigen, LCA): CD45 is highly sensitive and specific

for tumors of hematopoetic origin. A small minority of lymphohematopoetic tumors lack

CD45 (including Hodgkin lymphoma, CD30+ lymphomas, some T-cell lymphomas, and

some leukemias) so its absence does not rule out a hematolymphoid tumor. However, the

presence of CD45 virtually assures that you are dealing with a hematolymphoid process

of some type. CD45 shows a membranous pattern of immunoreactivity. I have also

observed that the antibody stains the luminal surface of proximal convoluted tubules in

the kidney, and also stains the cytoplasm of renal oncocytoma (even when using a

polymer detection complex, so the staining cannot be ascribed to endogenous biotin).

CD30 (Ki-1): CD30 is needed to recognize CD30-positive anaplastic large cell

lymphomas (which may be CD45 negative) and is also important in diagnosing Hodgkin's

disease. As mentioned above, CD30 can also be useful in subclassifying germ cell

tumors, since embryonal carcinomas are CD30 positive, whereas other types of germ cell

tumors are not. CD30 immunoreactivity can be both cytoplasmic and membranous. In

many cases a cytoplasmic "glob" of immunoreactivity is noted, corresponding to the

"golgi" area of the cell.

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 10

B-cells markers: CD20, CD79a, and Pax5: These are all good markers of B-cells

tumors, although some important points to keep in mind are the following:

CD20 (L26) – Lost in patients who have received Rituximab therapy. Also negative

in some cases of B lymphoblastic leukemia / lymphoma. Staining limited to nucleoli is

nonspecific and should be ignored.

CD79a (mb1) – Also positive in acute promyelocytic leukemia (APL), plasma cells,

and some plasma cell myelomas.

Pax5 – stains nuclei of B-cells and Hodgkin cells, but also positive in some

neuroendocrine tumors (including Merkel cell tumor), mesonephric and mullerian

tumors, and alveolar rhabdomyosarcoma).

My current “screen” for B-cell lymphoma is CD20 and Pax5.

T-cell markers: CD2, CD3, CD5, CD7, TDT: It is a good idea to always employ >1

T-cell marker if T-cell lymphoma is a consideration, as T-cell lymphomas often

aberrantly lack one or more pan-T-cell markers. Some additional points to keep in mind

are as follows:

CD5 - coexpressed in some B-cells tumors (mantle cell lymphoma, CLL, and a small

percentage of diffuse large B-cell lymphoma).

CD7 - expressed in some myeloid leukemias. Ignore any artifactual nucleolar staining.

TDT - expressed in T-lymphoblastic leukemia / lymphoma, also in some myeloid

leukemias. It is also expressed in normal thymic T cells, and these cells typically show a

high Ki-67 proliferative fraction. Therefore, TDT(+) T cells with a high proliferative

fraction do not necessarily mean you are dealing with a neoplasm, since these findings

could be explained by sampling normal thymic T-cells. If you have a piece of tissue, you

can usually pick up cytokeratin-positive cells in the background, which can be a tip-off

that you are looking at thymic tissue.

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 11

Natural killer (NK cell marker: CD56. Natural killer cell neoplasms show variable

expression of T-cell markers. CD56 is also expressed in some plasma cell myelomas,

leukemias, neuroendocrine tumors, certain sarcomas, and some carcinomas.

Myeloid Leukemia Markers: Myeloperoxidase, lysozyme, CD68, CD13, and CD33

are good markers of myeloid differentiation. There is some evidence that CD68 clone

PG-M1 has specificity for monocytic differentiation.

Histiocytic / Dendritic cell markers: Lysozyme and CD68 are good markers of

histiocytic origin. Follicular dendritic cell tumors express CD21 and Clusterin, and

Langerhans cell neoplasms express S100 protein and CD1a.

CD43: One could make an argument for including CD43 in a screening lymphoma

panel, since it stains tumors that may lack other lymphohematopoetic screening markers.

The only non-lymphohematopoetic tumor that I am aware of that expresses CD43 is

adenoid cystic carcinoma. Occasionally one will encounter a tumor where CD43 is the

only marker that stains the neoplastic cells, a situation that I refer to as the "CD43-only

syndrome”. The differential diagnosis of this condition is as follows:

CD43-only Syndrome:

1. Anaplastic large cell lymphoma

2. Leukemias (AML, AMML, erythroleukemia)

3. NK-cell tumors, including plasmacytoid dendritic

cell tumor, CD4+ hematodermic tumor

4. Plasma cell neoplasms

5. Langerhans cell histiocytosis

Traps with lymphohematopoetic markers:

1. Ordering too few markers. If the tumor is poorly differentiated or if you have little

tissue, it is wise to use >1 marker for each lineage (e.g., CD20 and Pax5 for B-cells, CD3

and CD5 for T cells, CD43 for leukemias and NK cells).

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 12

2. Pax5 expression in neuroendocrine tumors, Hodgkin lymphoma, and alveolar

rhabdomyosarcoma

3. CD79a in acute promyelocytic leukemia (APL) and myelomas

3. TDT in thymoma and in Merkel cell tumor

4. Focal CK expression in rare lymphohematopoetic tumors

MESENCHYMAL MARKERS

Rhabdomyosarcoma: Desmin is sensitive, Myogenin (nuclear) specific, although

regenerating skeletal muscle fibers may also express Myogenin. Pax5 can be expressed

in alveolar rhabdomyosarcoma.

Leiomyosarcoma: Smooth muscle myosin and Caldesmon are specific. Smooth

muscle actin is sensitive but also stains myofibroblastic lesions. Muscle specific actin

(MSA) is a bit more specific than SMA. All of these markers also will label

myoepithelial cells.

Angiosarcoma: CD31 and CD34 offer a reasonably good screen for vascular

neoplasms. Factor VIII has good specificity (although this marker also stains

megakaryocytes and megakaryocytic tumors). D2-40 (podoplanin) is a good marker of

lymphatic differentiation, but also stains a number of other tumors including seminoma

and mesothelioma. FLI-1 can be an endothelial marker but I have not been particularly

impressed with its performance in my laboratory. LMO2 is a relatively recently-described

endothelial marker.

Synovial sarcoma: TLE-1 often shows strong diffuse nuclear staining, and these

tumors also typically express focal cytokeratin and/or EMA. They are also CD56+ and

often CD99+. A substantial minority expresses S100. FISH for SYT translocation is

very helpful in rendering a definitive diagnosis of this tumor.

Alveolar soft part sarcoma: Strong nuclear staining with TFE3 is characteristic of

this tumor.

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 13

Gastrointestinal Stromal Tumors (GIST): DOG1 is a sensitive and specific marker

for these tumors. CD117 (c-kit) stains most GIST, and many stain with CD34. They lack

Sox10, S100, and usually lack muscle markers.

Liposarcoma: Ki-67 clone K2 can be helpful in recognizing lipoblasts, as it stains

membranes of viable fat cells. MDM2 and CDK4 have been found to be useful in the

recognition of well differentiated liposarcoma and in dedifferentiated liposarcoma.

Epithelioid sarcoma: The tumors express cytokeratins and EMA, and often CD34.

Lack of nuclear staining with INI1 (BAF47) can be extremely helpful.

Malignant peripheral nerve sheath tumor (MPNST): Sox10 and S100 protein are

expressed in a subpopulation of tumors, and some also lack nuclear staining with INI1

(BAF47). Some authors state that the tumors are negative for microphthalmia

transcription factor (MiTF), which can be useful in their differential diagnosis with

melanoma, which expresses MITF (as noted above however, MiTF is not specific for

melanoma). These tumors are typically CD56+ and CD99-.

Traps with mesenchymal lesions:

1. Expression of cytokeratins and/or EMA by sarcomas

2. Spindle cell carcinoma can express SMA or MSA, and some can show occasional

desmin positive cells.

3. Misinterpretation of background cell reactivity as tumor cell reactivity

- CK reactivity in submesothelial fibroblasts

- Desmin reactivity by mesothelial cells.

- Expression of S100 by histiocytes

- Myogenin reactivity in regenerating skeletal muscle cells

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 14

IMMUNOHISTOCHEMICAL APPROACH TO

UNDIFFERENTIATED MALIGNANT TUMOR.

Initial considerations:

1. Value of looking at H&E first without looking at patient history first.

2. Clinical information can guide your differential diagnosis (age, history, location, etc.)

If prior pertinent pathology exists – try to look at that first.

3. How much tissue do we have to work with, and are clinicians in a big rush?

- If lots of tissue and no rush – sequential rounds of stains OK

- If minimal tissue or big rush – better to “blast away” up front

(cost of more stains is usually < cost of re-biopsy)

4. If minimal tissue – order stains in “order of importance”, and have 10 or more extra

sections cut and held, in case more stains are needed (rather than having to re-face the

paraffin block another time)

5. Use lists to help jog your memory

Useful Initial Panel (if you don’t have a clue what the tumor is):

CK-LMW Sox10 CD45 (LCA) CD43 SYN

CK-HMW MART-1 CD30 (Ki-1) SALL4 CHG

SUMMARY AND CONCLUSIONS

1. Appearances can be deceiving.

2. Importance of good clinical information and review of previous pathology.

3. Importance of panel approach – often need >1 marker per diagnostic category.

4. Be mindful of what you have to work with, and alter your approach accordingly.

5. Use lists so that you won’t forget things as easily.

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 15

It must be kept in mind that there are exceptions to everything, and not all

tumors will necessarily react as I have indicated. This underscores the importance

of a carefully performed H&E examination by a skilled anatomic pathologist in

arriving at the correct diagnosis. If the immunostain findings don't make sense when

viewing the H&E, then the immunostain findings should be considered suspect

(especially if the immunostains were performed using so-called "Predilute Ready-To-

Use" antibodies).

Selected References

1. Stevenson AJ, Chatten J, Bertoni F et al: CD99 (p30/32MIC2)

neuroectodermal/Ewing's sarcoma antigen as an immunohistochemical marker.

Review of more than 600 tumors and the literature experience. Applied

Immunohistochemistry 2(4): 231-240, 1994.

2. Renshaw AA: O13 (CD99) in spindle cell tumors. Reactivity with

hemangiopericytoma, solitary fibrous tumor, synovial sarcoma, and meningioma, but

rarely with sarcomatoid mesothelioma. Applied Immunohistochemistry 3(4): 250-

256, 1995.

3. CD34: A review. van de Rijn M, Rouse RV: Applied Immunohistochemistry 2(2):

71-80, 1994.

4. Lumade JA, Askin FB, Perlman EJ: MIC2 analysis of small cell carcinoma.

American Journal of Clinical Pathology 102: 692-694, 1994.

5. Weiss LM, Arber DA, Chang KL: CD68: A review. Applied

Immunohistochemistry 2(1): 2-8, 1994.

6. DeYoung BR, Wick MR, Fitzgibbon JF et al: CD31: An immunospecific marker for

endothelial differentiation in human neoplasms. Applied Immunohistochemistry 1:

97-100, 1993.

7. Lloyd RV, Cano M, Rosa P et al: Distribution of chromogranin A and secretogranin I

(chromogranin B) in neuroendocrine cells and tumors. American Journal of

Pathology 130(2): 296-304, 1988.

8. Miettinen M: Synaptophysin and neurofilament proteins as markers for

neuroendocrine tumors. Archives of Pathology and Laboratory Medicine 111: 813-

818, 1987.

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 16

9. Arber DA, Kandalaft PL, Mehta P et al: Vimentin-negative epithelioid sarcoma. The

value of an immunohistochemical panel that includes CD34. The American Journal

of Surgical Pathology 17(3): 302-307, 1993.

10. Shah IA, Netto D, Schlageter MO et al: Neurofilament immunoreactivity in Merkel

cell tumors: A differentiating feature from small cell carcinoma. Modern Pathology

6(1): 3-9, 1993.

11. 11: Dorfman DM, Pinkus GS: CD99 (p30/32MIC2) immunoreactivity in the

diagnosis of thymic neoplasms and mediastinal lymphoproliferative disorders. A

study of paraffin sections using monoclonal antibody O13. Applied

Immunohistochemistry 4(1): 34-42, 1996.

12. Pinkus GS, Kurtin PJ: Epithelial membrane antigen - A diagnostic discriminant in

surgical pathology. Immunohistochemical profile in epithelial, mesenchymal, and

hematopoetic neoplasms using paraffin sections and monoclonal antibodies. Human

Pathology 16: 929-940, 1985.

13. Sloane JP, Ormerod MG: Distribution of epithelial membrane antigen in normal and

neoplastic tissues and its value in diagnostic tumor pathology. Cancer 47: 1786-

1795, 1981.

14. Lloyd RV, Jin L, Zhang S: Proconvertases 2 and 3 are specific neuroendocrine

markers. Modern Pathology 9(1):173A (abstract #1016), 1996.

15. Folpe AL, Patterson K, Gown A: Antibodies to desmin identify the blastemal

component of nephroblastoma. . Modern Pathology 10(9):895-900, 1997.

16. Nonaka D, Chiriboga L, Rubin BP: Sox10: A Pan-Schwannian and Melanocytic

Marker. The American Journal of Surgical Pathology 32(9): 1291-1298, Sep 2008.

17. 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. The American Journal of Surgical Pathology

33(6): 894-904, Jun 2009 .

18. Cao D, Li J, Guo S et al: SALL4 is a novel diagnostic marker for testicular germ cell

tumors. The American Journal of Surgical Pathology 33(7): 1065-1077, Jul 2009 .

19. Liegl B, Hornick JL, Corless CL et al: Monoclonal antibody to DOG1.1 shows higher

sensitivity than KIT in the diagnosis of gastrointestinal stromal tumors, including

unusual subtypes. The American Journal of Surgical Pathology 33(3): 437-446,

Mar 2009.

20. Singer G, Kurman RJ, McMaster MT et al: HLA-G immunoreactivity is specific for

intermediate trophoblast in gestational trophoblastic disease and cancer of as a useful

R.T. Miller, M.D. Diagnostic Immunohistochemistry Page 17

marker in differential diagnosis. The American Journal of Surgical Pathology

26(7): 914-920, Jul 2002.

21. Hornick JL, Cin PD, Fletcher CDM: Loss of INI 1 expression is characteristic of both

conventional and proximal-type epithelioid sarcoma. The American Journal of

Surgical Pathology 33(4): 542-550, Apr 2009.

22. Binh MBN, Sastre-Garau X, de Pinieux G et al: MDM2 and CDK4 immunostainings

are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma

subtypes. A comparative analysis of 559 soft tissue neoplasms with genetic data. The

American Journal of Surgical Pathology 29(10): 1340-1347, Oct 2005.

23. Terry J, Saito T, Subramanian S et al: TLE1 as a diagnostic immunohistochemical

marker for synovial sarcomas emerging from gene expression profile and studies. The

American Journal of Surgical Pathology 31(2): 240-246, Feb 2007.

24. Buresh CJ, Oliai BR, Miller RT: Reactivity with TDT in a Merkel cell carcinoma. A

potential diagnostic pitfall. The American Journal of Clinical Pathology 129 (6):

894-898. Jun 2008

25. Feldman AL, Dogan A: Diagnostic uses of Pax5 immunohistochemistry. Advances

in Anatomic Pathology 14(5):323-334, Sep 2007.

26. Zynger DL, Dimov ND, Luan C et al: Glypican-3: A novel marker in testicular germ

cell tumors. The American Journal of Surgical Pathology 30(12): 1570-1575, Dec

2006.

27. Santagat S, Ligon K, Hornick JL: Embryonic standard cell transcription factor

signatures in the diagnosis of primary and metastatic germ cell tumors (OCT3/4,

NANOG, and SOX2). The American Journal of Surgical Pathology 31(6): 836-845,

Jun 2007.

J Clin Pathol 2007;60:936-938 doi:10.1136/jcp.2006.044750

• Case report

HE 形态示多形性恶性肿瘤，鉴别诊断主要考虑：

1）淋巴造血系统恶性肿瘤，ALCL、 HL、DLBCL、组织细胞树突细胞肉瘤；

2）生殖细胞肿瘤， 精原细胞瘤、卵黄囊瘤、胚胎性癌；

3）胸腺未分化多形性癌

一、IHC标记结果CK+++、CD3-CD20-、PAX-5-、基本可除外淋巴造血系统来源。

二、CK+++、Plap+、CD117+、CD30+、AFP+提示生殖细胞肿瘤，形态是否符合精原细胞瘤？卵黄囊瘤？胚胎性癌？

三、CK+++、CD117+提示胸腺癌。

鉴别要点：1）IHC已标记的抗体结果是否可靠？

                     2）增加什么抗体能辅助鉴别 2与3 ?

成人发生的恶性生殖细胞肿瘤，很少是单一类型，多为混合性。

本例HE图片中显示肿瘤区呈网状微囊状结构，形态学信息和IHC标记的部分结果，提示倾向恶性生殖细胞肿瘤，卵黄囊瘤或许混合有其它类型的成分，需标记看有否表达HCG的成分，另外图片中画圈内的结构在高倍下是否为腺样结构（不除外含有胚胎性癌成分的可能性）。

 以上如有不妥，请指正。谢谢！

 恶性生殖细胞肿瘤，如何？

卵黄囊瘤？请见图片。

是否伴有灶性HCG阳性？