Lessons from a consultation practice

(1)

Pitfalls in the

Application of Immunohistochemistry

in

Diagnostic Pathology

Kevin O. Leslie, MD

Professor and Consultant

Mayo Clinic Arizona

Scottsdale, Arizona

(2)

Presenter Disclosures

• Personal financial relationships with commercial

interests relevant to this presentation during the past 12 months:

Kevin O. Leslie, MD

• Personal financial relationships with non-commercial interests (e.g., government or other nonprofit funding)

relevant to this presentation, within past 12 months:

• Relevant institutional financial interests

• Personal financial relationships with tobacco industry entities within the past 3 years:

(3)

(4)

(5)

(6)

The Territory Ahead…

• Introduction

• The critical role of sample size and quality

• Tumors can be “triaged” by pattern

• Not all antibodies are created equal

• Top 10 Pitfalls you can avoid

(7)

Why is this presentation useful?

• Today, immunohistochemistry (IHC) is fully

integrated into contemporary diagnostic pathology • Technical challenges of the past have been largely

eliminated by commercialization and automation • The expertise of application is highly variable in

(8)

• Problems in selection of antibodies and

interpretation of staining results play a major role in the consultation practice

• A limited set of recurrent pitfalls emerge

• Examining these in detail may help us hone our individual mastery of this broad and complex

ancillary technique in our daily practice

(9)

A word about cost containment…

• The reality of contemporary IHC is that multiple antibody determinations are the rule. The trick is to avoid wasteful avenues of investigation.

• Today, it is becoming progressively difficult to

identify proliferative histopathological lesions that rely on routine morphology alone!

(10)

AAH AdenoCa+BAC Metaplasia PB AdenoCa BAC AAH Metaplasia PB Metaplasia PB BAC AdenoCa AdenoCa+BAC AdenoCa+BAC

(11)

A word about cost containment…

• The reality of contemporary IHC is that multiple antibody determinations are the rule. The trick is to avoid wasteful avenues of investigation.

• Today, it is becoming progressively difficult to identify proliferative histopathological lesions that rely on routine morphology alone!

• So, for all of those cases where we must rely on IHC, we should try to use a consistent strategy

(12)

IHC is most helpful when it…

• Clarifies the phenotype of a neoplasm or pathogen

• Clarifies the origin of a metastatic tumor

• Clarifies tumor behavior, and possibly therapy

• Clarifies patient prognosis

(13)

Case example

Case courtesy of Dr. Fuad Al Dayal, Saudi Arabia

A 10 year old boy presented with a hemorrhagic left pleural effusion. The child is hearing and

speech impaired.

The past medical history is significant for

recurrent lung infections, recurrent hemorrhagic pleural effusions and a cerebrovascular accident.

(14)

(15)

A surgical wedge lung biopsy

was performed…

(16)

(17)

(18)

(19)

(20)

KOL03

(21)

(22)

(23)

(24)

Diagnosis

Diffuse Pulmonary

Lymphangiomatosis

(25)

The Territory Ahead…

• Introduction

(26)

The critical role of sample size

• There is a natural tendency for clinicians to

expect more and more “information”

from smaller and smaller biopsy samples.

Case example:

A 66 year old woman is found to have a 3 cm

lung mass. A transbronchial biopsy is

(27)

You decide the included cell group is

malignant, and “nonsmall” cell….

You attempt IHC to confirm lung origin.

Results: TTF-1 neg, CK 7 pos

CK20, synapto, chromogranin, and

P63 …insufficient tumor in the recuts

After signing the case out as “nonsmall

cell carcinoma”, the clinician calls to

ask if it could be from her breast

cancer…

…and if it is please send for Her2

neu.

Oh, and if it is a lung cancer, we

need to know if it is squamous. If

not please send for EGFR

(28)

The Territory Ahead…

• Introduction

• The critical role of sample size and quality

(29)

A practical approach…

• Neoplasms are the largest source of targets for diagnostic IHC

• 4 general morphological categories emerge

– Neoplasms of lymphoid cells (hematolymphoid) – Neoplasms with organoid features

– Neoplasms with spindled features

(30)

Lymphoid

• The panel (s)

CD45 CD20 CD3 CD43

Kappa and lambda (if cytoplasmic)

BCL2 (if nodular)

Unstained slides (many)

CD5 CD10 Cyclin D1 CD79a CD138 CD30 CD15 Myeloperoxidase CD68 Lysozyme S100 protein CD21/CD35

(first consult a local hematopathologist!) Purpose: Confirm hematolymphoid, confirm neoplastic; subclassify

(31)

A 47 year old man presents with weightloss, malaise and abdominal pain. Imaging shows multiple enlarged lymph nodes. A needle core biopsy is performed…

LCA

LCA

CD20

CD30

DDX:

Lymphoma

Carcinoma

Melanoma

Other?

Immunopanel:

CD45 CD20 CD3 CD43

Kappa and lambda

CD30

CD3 ALK-1

Diagnosis

Anaplastic large cell lymphoma,

ALK-1 negative (see discussion)

Another H&E…

(32)

Organoid

• The panel

CK7/20 Synaptophysin TTF-1 CDX2 PSA ER/PR Calret-CK5/6-WT1

Purpose: Determine primary origin of metastasis, guide therapy

Pattern assists general localization

Neuroendocrine carcinoma, paraganglioma Lung and thyroid, other small cell

Intestinal adenos, other mucinous, endometrioid Prostate, breast, salivary, sweat gland ca,

--other adeno, melanoma

Breast, ovary, endometrium. ER in some lung, stoma and thyroid adenos

(33)

A 62 year old patient with back pain is

found to have a lytic lesion involving

T11. A core biopsy is performed…

panCK and CK7 CK20 TTF1…or ER!

panCK and CK 7

CK20

CDX2

_{TTF1 or ER!}

Diagnosis

Metastatic adenocarcinoma

consistent with lung (TTF1)

or breast (ER) origin

DDX:

Metastatic

adenocarcinoma

of unknown

(34)

Spindled cells

• The panel PanCK S100 protein Melan-A Desmin CD99 CD31/34 CD117

Purpose: Determine primary origin/ phenotype, guide therapy

Defines epithelial phenotype, spurious in sarcomas, melanoma, dendritic cells. plasma cells

Melanocytic, neural, myoepithelial, histiocytic/dendritic, Langerhans cells, liposarcoma, chondrosarcoma

Melanocytic, pre-melanosomes, adrenocortical, sex-cord ovarian

Myogenous tumors, PNETs, epithelioid sarcoma

Lymphoblastic lymphoma, synovial sarc, EWS/PNET

Vascular tumors (CD34 better for KS); LFT/SFT (CD34) GIST

Don’t forget ER/PR for metastatic

spindled cell tumors in women!

(35)

DDX:

Sarcomatoid Ca

Primary sarcoma

Metastatic sarcoma

Sarcomatoid mesothelioma

Nerve sheath tumor

Localized fibrous tumor

A 49 year old woman presents with

chest pain and cough.. A 10 cm tumor is identified and removed from the RLL.

S100, Desmin, CD117, ER

S100, Desmin, CD117, ER

CD34 + BCL2

CD34 + BCL2

Diagnosis:

(36)

Undifferentiated epithelioid

• The panel

Pan CK S100 protein Synaptophysin CD45 ER/PR (female)

Purpose: Determine primary origin of metastasis, guide therapy

Defines epithelial phenotype

Melanocytic, neural, myoepithelial, histiocytic/dendritic, Langerhans cells

Neuroendocrine cells

Hematolymphoid cells

Breast, ovary, endometrium. ER in some lung, stomach, and thyroid adenos

(37)

A 61 year old man presents with a large left axillary mass. His past medical history is remarkable for a previously

resected skin appendage tumor from the left hand (said to have been a “malignant poroma”—outside hospital).

DDX

Inflammatory “pseudotumor”

Lymphoma

Plasma cell myeloma

Melanoma

The Panel

panCK

S100 protein

LCA

Synaptophysin

Kappa + lambda

L K

SYN, LCA. S100

panCK panCK

Diagnosis:

Metastatic

carcinoma

(38)

The Territory Ahead…

• Introduction

• The critical role of sample size and quality

• Neoplasms can be “triaged” by pattern

(39)

Who to trust….

• Sensitivity and specificity issues

– Example: synaptophysin and chromogranin

• Certain antigens in tissue are more resistant to fixation, processing, and tissue degredation

– Example: panCK versus S100 protein

• The utility and specificity of some antibodies requires “context”

– Example: CD30 in ALCL versus carcinoma, or melanoma!

(40)

A 71 year old man, smoker, is found to have a large central lung mass. A transbronchial biopsy is performed…

Small Cell Carcinoma

TTF-1 and MIB-1

(41)

The Territory Ahead…

• Introduction

• The critical role of sample size and quality

• Neoplasms can be “triaged” by pattern

• Not all antibodies are created equal

(42)

TOP TEN PITFALLS IN IHC

(43)

A 57 year old woman presents with an enlarged

groin lymph node. She has a history of node +

breast cancer. A needle core biopsy is performed…

DDX:

Carcinoma

Lymphoma

Melanoma

Paraganglioma

Sarcoma

panCK, HMB45, EMA, CK7, CK20 panCK, HMB45, EMA, CK7, CK20

LCA

LCA

S100 Protein

Diagnosis:

Metastatic melanoma, amelanotic

(44)

A 26 year old African woman presents to the emergency room with cough and chest pain, 1 month after delivering a healthy baby. Bronchoscopy yields this biopsy…

DDX Carcinoid Melanoma Sarcoma Sarcomatoid Ca Vascular Smooth muscle IMFT

PanCK

S100 PanCK Synapto, CD34, TTF1, Melan-A CD31 CD31

(45)

TOP TEN PITFALLS IN IHC

10. Incorrect panel of antibodies 9. Incomplete panel of antibodies

(46)

The surgeon finds diffuse thickening of the pleura without a definite mass in

underlying lung. He feels the changes are quite typical for mesothelioma in his

experience…

Some diagnoses require a combination of

IHC results for validity

• A 72 year old man, long time smoker, presents with right sided chest pain and breathlessness.

• Imaging reveals a right pleural

effusion. The underlying lung is not well visualized. He gives a history of asbestos exposure.

(47)

A limited battery of IHC stains is

performed, including calretinin…

Some diagnoses require a combination of IHC

results for validity

CALRET

A diagnosis of “malignant

mesothelioma, epithelial type”. The

family swears that the patient had no

asbestos exposure and requests that the

biopsy be sent out for review…

CALRET

B72.3

BerEP4

B72.3 BerEP4 CALRET CK 5/6 WT-1

(48)

TOP TEN PITFALLS IN IHC

10. Incorrect panel of antibodies 9. Incomplete panel of antibodies 8. Excessive panel of antibodies

(49)

Thyroidectomy from a young patient with Hashimoto thyroiditis…

(50)

TOP TEN PITFALLS IN IHC

(51)

The case was sent out for consultation. The lesion was recognized as epithelioid hemangioendothelioma and confirmatory IHC was performed…

TTF1

A 37 year old man presents with chest discomfort and is found to have several nodular lung lesions. A VATS biopsy is performed…

PanCK

PanCK

TTF1

CD34/CD31

DDX:

Carcinoma

Mesothelioma

Melanoma

Chordoma

Hamartoma?

(52)

TOP TEN PITFALLS IN IHC

7. Incorrect histopathological DDX

(53)

A 22 year old college student notices a

persistent swelling above her right knee.

• Imaging reveals involvement of the distal

femur and additional lytic bone lesions are

present.

• She is brought into the hospital for a

percutaneous needle core biopsy

• She is the only daughter of the Chief of

Surgery.

(54)

(55)

DDX:

Ewing sarcoma/PNET

Large cell lymphoma

Melanoma of soft parts

Epithelioid sarcoma

Other?

Recommended panel

panCK

LCA

S100 protein

Synaptophysin

8 unstained

Their panel

Vimentin

panCK

TdT

CD99

(56)

DDX:

Ewing sarcoma/PNET

Large cell lymphoma

Melanoma of soft parts

Epithelioid sarcoma

Other?

Recommended panel

panCK

LCA

S100 protein

Synaptophysin

8 unstained

(57)

DDX:

Ewing sarcoma/PNET

Large cell lymphoma

Melanoma of soft parts

Epithelioid sarcoma

Other?

Secondary panel

CD20

CD3

MIB1

Final Diagnosis:

Malignant lymphoma,

diffuse large B-cell

(58)

TOP TEN PITFALLS IN IHC

6. Undue pressure on speed of diagnosis 5. Overconfidence in the value of IHC

(59)

A 52 year old man has a serum PSA drawn

during a routine physical exam. This results in

sextant needle core biopsies…

(60)

TOP TEN PITFALLS IN IHC

6. Undue pressure on speed of diagnosis 5. Overconfidence in the value of IHC 4. Relying on the IHC of another lab 3. Relying on tissue from another lab

(61)

After a first round of IHC the

diagnosis remained uncertain and

the case was sent for consultation.

Lymphoid

Case in point…

A 65 year old man presents with a

soft tissue mass adjacent to his

clavicle and eroding bone. A biopsy

is performed.

VIM

LCA

(62)

Our diagnosis: Diffuse large B-cell

lymphoma, CD20 positive.

Lymphoid

Case in point…

We restained the tissue block in our

laboratory with the following

results…

panCK, S100, melanA

panCK, S100, melanA Repeat LCA

Repeat LCA

CD20

CD20

(63)

From tissue acquisition to coverslip on your

IHC slide, there are so many potential areas

for mishap that it is remarkable how

frequently IHC is successful!

Crush injury Delay in fixation Improper fixative Processing damage Overheating in paraffin Infiltration contaminants Rehydration damage Poor section adherence

Incomplete deparaffinization of sections

Antigen retrival problems IHC technical failures

-reagent sequence

-incomplete slide flooding -poor humidity control

-pipetting inaccuracy -outdated reagents -poor antibody quality

(64)

A 48 year old woman present with leg pain and is found to have a cystic lesion in the proximal tibia. Curettings of the lesion are

performed at another hospital and a frozen section is requested by the surgeon.

(65)

(66)

TOP TEN PITFALLS IN IHC

(67)

Before ordering, check the expected

positive staining reaction!

All of these are NUCLEAR stains!

ER

PR

TTF-1

CDX-2

P63

P53

PCNA

MyoD1

WT-1

TDT

Cyclin D1

Ki67

FLI-1

Myogenin

(68)

TOP TEN PITFALLS IN IHC

2. Not knowing the expected staining pattern 1. Not recognizing the histopathology

(69)

His past medical history is remarkable for left sided pneumonia 3 months earlier for which he was hospitalized and treated

empirically with broad spectrum antibiotics.

A 66 yr old man presents to the emergency room with left sided chest pain and is found to have a large left pleural effusion.

Calret, CK5/6, TTF-1. CK7. CK20, CEA all neg

Calret, CK5/6, TTF-1. CK,7, CK20, CEA all negative

PanCK

WT-1

Our diagnosis:

(70)

The Trail Behind…

• Introduction

• The critical role of sample size and quality

• Tumors can be “triaged” by pattern

• Not all antibodies are created equal

• Top 10 Pitfalls you can avoid

(71)