Immunohistochemistry

Slide 1 · Immunohistochemistry

Tumor lineage, metastatic origin, and therapeutic biomarkers

From a difficult H&E diagnosis to a clinically useful protein-expression result

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Why morphology alone may be insufficient

Routine hematoxylin and eosin sections establish the initial architecture and cytology.

Severely anaplastic tumors may share overlapping appearances despite different lineages.

A correct lineage diagnosis matters because prognosis and treatment differ.

Immunohistochemistry complements—not replaces—morphologic assessment.

Slide 3 · Learning pathway

Define

What is measured?

Classify

Which lineage?

Localize

Where did the metastasis arise?

Predict

Which protein may guide treatment?

Keep one clinical question attached to every immunostain.

Slide 4 · What immunohistochemistry measures

Immunohistochemistry (IHC) applies antigen-specific antibodies to tissue sections.

The reaction demonstrates expression of a selected protein within its tissue context.

Markers can indicate differentiation, organ of origin, or a therapeutic target.

The measured feature is protein expression in a histologic section.

Slide 5 · The antigen-to-interpretation chain

Tissue

Tumor cells in section

Antibody

Recognizes selected protein

Signal

Stain is visualized

Interpretation

Combine with histology

An antibody reaction becomes a diagnosis only after contextual interpretation.

Slide 6 · Three distinct questions for one technique

Classification

What lineage does this poorly differentiated tumor resemble?

Examples: Cytokeratin, CD45, desmin, SOX10

Clinical stratification

Which organ or molecular target does the tumor suggest?

Examples: PSA, thyroglobulin, ER, PR, HER2, ALK

Marker selection follows the diagnostic question.

Slide 7 · A positive stain is not a complete diagnosis

A marker demonstrates a feature of a tumor—not its full identity.

The clinical presentation, H&E appearance, and other marker results remain relevant.

The best interpretation explains what the staining supports and what remains unresolved.

Use support-language rather than unsupported certainty.

Slide 8 · Lineage markers versus organ-specific markers

Lineage-oriented

Resolve broad differentiation categories.

Cytokeratin → epithelial; CD45 → hematopoietic; Desmin → muscle; SOX10 → melanocytic context

Organ-oriented

Refine a metastatic tumor’s likely origin.

PSA → prostatic origin; Thyroglobulin → thyroid follicular origin

Lineage and anatomic site are different levels of diagnostic inference.

Slide 9 · The diagnostic path begins with H&E

H&E review

Form initial differential

Targeted IHC

Test plausible lineages

Synthesis

Revisit morphology and clinical data

Do not design a panel detached from the histologic differential.

Slide 10 · Why undifferentiated tumors create a problem

Loss of differentiation can erase characteristic tissue morphology.

Carcinoma, lymphoma, melanoma, and sarcoma may be difficult to separate on H&E alone.

These represent fundamentally different clinicopathologic groups.

The first useful IHC question is often broad lineage, not exact subtype.

Slide 11 · Anaplastic tumor: Broad differential

Malignant neoplasm with limited differentiation

Carcinoma

Epithelial

Lymphoma

Hematopoietic

Melanoma

Melanocytic

Sarcoma

Mesenchymal / muscle

Initial categories direct the first-line protein-expression questions.

Slide 12 · Why intermediate filaments are useful

Intermediate-filament composition reflects cellular differentiation.

Certain solid-tumor cells retain filaments associated with their cell of origin.

IHC detection of these proteins can uncover otherwise invisible lineage information.

Protein phenotype can persist when microscopic differentiation is inconspicuous.

Slide 13 · Cytokeratin: Evidence of epithelial lineage

Target

Cytokeratins are intermediate-filament proteins of epithelial cells.

Inference

Tumor-cell staining supports carcinoma in an appropriate morphologic setting.

Cytokeratin is a lineage clue, not an automatic site-of-origin diagnosis.

Slide 14 · Desmin: A muscle-differentiation clue

Target

Desmin is an intermediate-filament protein associated with muscle differentiation.

Inference

Tumor-cell expression supports the muscle branch of a differential.

Interpret desmin as part of the complete phenotype and morphology.

Slide 15 · CD45: Hematopoietic differentiation

Target

CD45 is a major marker of hematopoietic cells and neoplasms.

Inference

Staining in an anaplastic tumor supports a hematolymphoid category.

A hematolymphoid result changes the subsequent diagnostic work-up.

Slide 16 · SOX10: A melanocytic clue

Target

SOX10 is a transcription factor expressed by most melanomas.

Inference

SOX10 expression supports consideration of melanoma in the right differential.

“Most” is not “all,” and expression is not equivalent to a complete diagnosis.

Slide 17 · First-line lineage comparison

Marker

Protein / role

Main inference

Cytokeratin

Epithelial intermediate filaments

Carcinoma lineage

Desmin

Muscle intermediate filament

Muscle differentiation

CD45

Hematopoietic antigen

Hematolymphoid lineage

SOX10

Transcription factor

Melanoma in context

Slide 18 · Marker interpretation: Three questions

Identity

Which cells show the reaction?

Meaning

What differentiation does it support?

Concordance

Does it fit H&E and clinical data?

Tumor-cell identity, inference, and concordance are separate checks.

Slide 19 · Broad lineage and tumor subtyping

First decision

Resolve carcinoma versus hematolymphoid, melanocytic, or other lineage.

Use broad markers aligned to the H&E differential.

Later decisions

Refine tumor type, tissue of origin, and therapeutic phenotype.

Use narrower markers only after the broad differential is organized.

A staged work-up preserves the purpose of every test.

Slide 20 · Illustrative case: Undifferentiated mass

Illustrative case — constructed teaching example

A biopsy shows a malignant tumor with no convincing epithelial, lymphoid, or melanocytic features on H&E.

What should the first IHC panel aim to resolve?

Broad cellular lineage

Exact primary site immediately

HER2 status before lineage

Reasoning

Lineage separation as a central application of IHC in anaplastic tumors.

Slide 21 · Case reasoning: Broad lineage panel

Morphology

Anaplastic neoplasm

Alternatives

Carcinoma / lymphoma / melanoma / sarcoma

Markers

Cytokeratin / CD45 / SOX10 / desmin

Next step

Integrate pattern with H&E

This is an illustrative decision sequence, not a rigid universal panel.

Slide 22 · Illustrative case: Cytokeratin-positive tumor

Illustrative case — constructed teaching example

A poorly differentiated malignant tumor shows convincing tumor-cell cytokeratin expression.

Which initial conclusion is justified?

Supports epithelial differentiation

Proves a specific anatomic primary

Proves sensitivity to anti-HER2 therapy

Reasoning

Cytokeratin is used to infer epithelial lineage; Organ and treatment are separate questions.

Slide 23 · Illustrative case: CD45-positive mass

Illustrative case — constructed teaching example

A malignant tumor of uncertain type shows CD45 expression in the tumor-cell population.

What diagnostic branch becomes particularly important?

Hematolymphoid neoplasm

Thyroid follicular carcinoma

HER2-amplified breast carcinoma

Reasoning

CD45 expression supports hematopoietic lineage; Further classification may require additional tests.

Slide 24 · Illustrative case: SOX10-positive tumor

Illustrative case — constructed teaching example

An anaplastic tumor has a protein-expression pattern that includes SOX10.

What is the appropriately qualified interpretation?

Melanoma enters the favored differential

Every SOX10-positive tumor is melanoma

Carcinoma is proven

Reasoning

SOX10 expression in most melanomas, but interpretation remains contextual.

Slide 25 · Illustrative case: Desmin-positive tumor

Illustrative case — constructed teaching example

A malignant spindle-cell tumor expresses desmin in the neoplastic cells.

Which differentiation is being suggested?

Muscle differentiation

Hematopoietic origin

HER2 amplification

Reasoning

Desmin provides an intermediate-filament clue to muscle origin.

Slide 26 · Why lineage identification matters clinically

The potential tumor groups have different biological behavior.

Management is not interchangeable across carcinoma, lymphoma, melanoma, and sarcoma.

IHC can make a morphologically ambiguous specimen clinically actionable.

The value of a lineage stain depends on whether it changes the classification.

Slide 27 · The metastatic lesion may arrive first

Patients can present with metastatic cancer before the primary tumor is known.

Clinical assessment and imaging may identify the origin, but sometimes remain inconclusive.

IHC can search for tissue-associated antigens within the metastatic biopsy.

The metastatic biopsy can carry clues to the tumor’s original tissue.

Slide 28 · Unknown-primary reasoning sequence

Presentation

Metastatic deposit

Clinical work-up

History, examination, imaging

Tissue question

Which organ-specific markers fit?

Integrated result

Refine likely origin

IHC contributes to—but does not replace—the clinical search.

Slide 29 · PSA: A clue to prostatic origin

Protein

Prostate-specific antigen (PSA) can be detected in tumor tissue by IHC.

Inference

In a metastatic carcinoma, an appropriate positive result may identify a prostatic source.

Tissue PSA staining is not the same task as serum PSA monitoring.

Slide 30 · Thyroglobulin: A clue to thyroid origin

Protein

Thyroglobulin is a tissue-associated protein used for thyroid-origin assessment.

Inference

Staining in a metastatic carcinoma can support thyroid follicular origin.

Choose markers according to the plausible organs and morphology.

Slide 31 · From lineage to organ of origin

Clinical question

Representative marker

Interpretive level

Is the tumor epithelial?

Cytokeratins

Broad epithelial lineage

Is prostate an origin?

PSA

Prostate-associated antigen

Is thyroid an origin?

Thyroglobulin

Thyroid-associated antigen

Slide 32 · Illustrative metastasis: PSA expression

Illustrative case — constructed teaching example

A metastatic carcinoma has an uncertain origin. The tumor cells show PSA expression.

Which statement is best supported?

A prostatic primary is suggested

This proves a thyroid primary

Tumor lineage is hematopoietic

Reasoning

PSA staining as a means of identifying prostatic carcinoma among metastases.

Slide 33 · Illustrative metastasis: Thyroglobulin

Illustrative case — constructed teaching example

A metastatic carcinoma is being evaluated for a possible thyroid primary. Tumor cells stain for thyroglobulin.

How should the result be used?

Support a thyroid source in context

Treat as a measure of ALK activity

Ignore the histologic appearance

Reasoning

Thyroglobulin is the source example of an organ-associated antigen in metastatic diagnosis.

Slide 34 · Histology and IHC answer different questions

Histology

Recognizes architecture, cytologic features, and tissue relationships.

May suggest malignancy even without recognizable lineage.

IHC

Shows selected proteins within the examined tissue.

May clarify lineage, likely primary, or clinical target.

A coherent final diagnosis synthesizes both evidence streams.

Slide 35 · Clinical biomarkers: Three distinct roles

Diagnostic: Helps define tumor identity or origin.

Prognostic: Associated with expected disease behavior or outcome.

Predictive: Identifies potential response to a particular therapy.

A marker may serve more than one role, but the meanings must not be conflated.

Slide 36 · ER and PR expression in breast cancer

Test

IHC detects estrogen receptor (ER) and progesterone receptor (PR) expression in tumor cells.

Clinical use

The hormone-receptor phenotype has prognostic and therapeutic relevance.

This example moves beyond lineage into a treatment-linked phenotype.

Slide 37 · Hormone receptors and prognosis

Receptor-positive breast cancers generally have a better prognosis than receptor-negative cancers.

“Generally” describes a broad association rather than an individual patient prediction.

Clinical interpretation remains integrated with the remainder of tumor assessment.

Prognostic information refers to expected disease behavior.

Slide 38 · Hormone receptors and therapeutic reasoning

IHC finding

ER/PR expression

Biologic context

Hormone-linked phenotype

Therapeutic relevance

Consider antiestrogen treatment

A predictive biomarker links a biological feature to a treatment option.

Slide 39 · Prognostic versus predictive: ER/PR

Prognostic statement

Receptor-positive breast cancers generally show more favorable outcomes than receptor-negative tumors.

Predictive statement

Hormone-receptor expression provides a rationale for antiestrogen therapy.

One result can inform both categories without making them synonymous.

Slide 40 · HER2 and the ERBB2 gene

ERBB2 is the gene encoding the HER2 receptor tyrosine kinase.

In some breast carcinomas, ERBB2 amplification produces high HER2 protein expression.

IHC assesses the protein, whereas in situ hybridization can assess gene amplification.

The protein assay and genomic assay address related but distinct analytes.

Slide 41 · HER2: Gene-to-protein-to-treatment

Genomic event

ERBB2 amplification

Protein phenotype

HER2 overexpression

IHC evidence

Strong HER2 staining

Targeted therapy

HER2-directed antibodies

This is the biologic chain described for a subset of breast cancers.

Slide 42 · HER2 IHC and FISH are complementary

Immunohistochemistry

What is assessed? HER2 protein expression in tumor tissue.

Output: Protein-staining interpretation.

Fluorescence in situ hybridization

What is assessed? ERBB2 gene amplification.

Output: Gene-copy evidence.

FISH as an adjunct when confirmation is needed.

Slide 43 · Why FISH may add information

HER2 protein overexpression and ERBB2 amplification are related biological events.

A protein-expression result is not identical to a direct gene assay.

The two techniques can therefore provide complementary evidence.

IHC characterizes protein expression; FISH assesses ERBB2 amplification.

Slide 44 · HER2 as a predictive biomarker

Identify

HER2-associated protein phenotype

Correlate

Evaluate gene amplification if indicated

Clinical role

Select HER2-directed treatment

The central predictive link is between a molecular target and a treatment.

Slide 45 · Illustrative case: Strong HER2 staining

Illustrative case — constructed teaching example

A breast carcinoma shows strong HER2 protein staining. A request is made to evaluate ERBB2 amplification.

What is the reason for adding an in situ hybridization assay?

To assess the associated gene-level event

To detect desmin filaments

To establish hematopoietic lineage

Reasoning

FISH can confirm ERBB2 amplification as a complement to protein IHC.

Slide 46 · ALK protein as a therapeutically relevant clue

Molecular context

Some tumors express constitutively active ALK fusion proteins.

Assay and consequence

ALK IHC may identify such tumors for consideration of ALK tyrosine kinase inhibition.

A protein result can point toward an actionable kinase alteration.

Slide 47 · ALK activation and targeted therapy

Molecular change

ALK gene fusion

Oncoprotein

Constitutively active ALK

Tissue evidence

ALK protein IHC

Therapy

ALK kinase inhibitor

The source describes ALK-driven lung cancers and lymphomas.

Slide 48 · ALK-positive lung cancer context

A subset of lung carcinomas contains ALK fusion proteins.

An EML4–ALK fusion as one molecular example.

Recognition of activated ALK has therapeutic implications because ALK can be inhibited.

The relevant chain is fusion → active kinase → therapeutic target.

Slide 49 · ALK-positive lymphoma context

The textbook also identifies ALK-expressing lymphomas.

ALK expression can therefore be clinically relevant in more than one tumor lineage.

Tumor type must be established before interpreting an ALK result as a therapeutic phenotype.

One target can appear across distinct histologic categories.

Slide 50 · Clinical roles of the representative markers

Marker

Main question

Use emphasized here

Cytokeratin / CD45

What is the lineage?

Diagnostic

PSA / thyroglobulin

Where might it arise?

Diagnostic

ER / PR

Prognosis and endocrine response?

Prognostic + predictive

HER2 / ALK

Is a target expressed?

Predictive / treatment linked

Slide 51 · IHC does not directly measure every alteration

Protein-based readout

Cytokeratin, desmin, CD45, SOX10, ER/PR, HER2, ALK.

Information about expression and phenotype.

Gene-level readout

ERBB2 amplification by FISH; ALK rearrangement is a genomic event.

Molecular mechanism may require a complementary test.

Do not equate protein positivity with a specific genomic event in every case.

Slide 52 · Specimen adequacy precedes assay interpretation

The source emphasizes representative sampling and proper preservation.

An unrepresentative specimen can misdirect both morphologic and ancillary interpretation.

The tumor-cell population must be identified within the tested material.

Better marker selection cannot rescue the wrong sampled tissue.

Slide 53 · Specimen-to-report quality chain

Obtain

Representative material

Preserve

Appropriate tissue handling

Assess

Morphology and tissue context

Test

Question-driven IHC

Report

Integrated interpretation

The quality of the final inference depends on every earlier stage.

Slide 54 · What a disciplined IHC interpretation says

State which relevant tumor cells express the selected antigen.

Explain the differential diagnosis supported by that result.

Distinguish a lineage inference from an organ inference or therapy implication.

Acknowledge unresolved alternatives rather than claiming certainty from one marker.

The report should answer the clinical question, not merely list positive stains.

Slide 55 · Flow cytometry: The adjacent method

Flow cytometry measures cellular properties rapidly and quantitatively.

It analyzes viable cells maintained in suspension.

It is used particularly for leukemias and lymphomas arising from hematopoietic tissues.

Its specimen requirements differ fundamentally from tissue-section IHC.

Slide 56 · How flow cytometry builds a phenotype

Input

Viable cell suspension

Antibodies

Fluorescently labeled

Acquisition

Multiple markers per cell

Output

Quantified cell populations

The ability to measure several antigens on the same cell is a central advantage.

Slide 57 · IHC versus flow cytometry

Immunohistochemistry

Works on tissue sections.

Retains architectural context.

Flow cytometry

Requires viable cell suspension.

Quantifies several antigens on individual cells simultaneously.

Different specimen formats generate complementary information.

Slide 58 · Illustrative case: A suspected lymphoma

Illustrative case — constructed teaching example

A tumor is suspicious for a hematopoietic neoplasm, and material suitable for viable-cell suspension is available.

What does flow cytometry add beyond routine section IHC?

Simultaneous multiparameter antigen analysis

Microscopic tissue architecture

Direct measurement of ERBB2 amplification

Reasoning

The source specifically contrasts flow cytometry’s multiparameter per-cell readout with IHC.

Slide 59 · When tissue architecture is essential

Section-based IHC

Locate an antigen in the tumor and adjacent tissue.

Correlate stain with the morphological region under review.

Cell suspension

Characterize populations without preserving the original tissue architecture.

Simultaneously measure multiple fluorescence-labeled antigens.

Architecture and multidimensional cellular phenotype answer different questions.

Slide 60 · Combined testing for a difficult neoplasm

H&E

Build differential

IHC

Assign lineage / target phenotype

Flow or FISH

Address remaining specific questions

Synthesis

Clinically meaningful diagnosis

Ancillary methods are chosen for a stated unresolved question.

Slide 61 · Integrated case: Lineage then treatment

Illustrative case — constructed teaching example

An anaplastic neoplasm expresses cytokeratin. It is classified as carcinoma, and an organ-specific biomarker is then considered.

Why is the order of these decisions useful?

Classification and target assessment are different tasks

Any cytokeratin-positive tumor must express HER2

Imaging is never relevant

Reasoning

The workflow avoids conflating lineage, organ origin, and predictive phenotype.

Slide 62 · Integrated case: Uncertain primary

Illustrative case — constructed teaching example

A metastatic carcinoma is morphologically ambiguous and the clinical search has not established a primary. PSA and thyroglobulin are considered.

What determines the meaning of the results?

Tumor staining plus clinical and morphologic context

The antibody name alone

Only whether the lesion is large

Reasoning

The source describes site inference from tissue-specific antigens in biopsies of metastases.

Slide 63 · Integrated case: Breast biomarkers

Illustrative case — constructed teaching example

A breast carcinoma is evaluated with ER/PR and HER2 IHC as part of a clinically relevant protein-expression assessment.

Which statement best distinguishes the assays?

ER/PR informs endocrine relevance; HER2 informs receptor-targeted relevance

Both markers prove a prostate primary

Neither can affect treatment planning

Reasoning

Robbins assigns therapeutic relevance to both hormone receptors and HER2 through different treatment mechanisms.

Slide 64 · Five interpretive errors to avoid

Calling a broad lineage marker an organ-specific proof.

Equating a biomarker’s prognostic role with its predictive role.

Confusing HER2 protein expression with direct ERBB2 amplification measurement.

Ignoring specimen quality or discordant morphology.

Using a single illustrative marker as a universal diagnostic rule.

IHC is most reliable as a hypothesis-driven component of diagnosis.

Slide 65 · A practical IHC decision framework

1 · Question

Lineage, origin, or target?

2 · Sample

Representative tumor present?

3 · Panel

Markers relevant to differential

4 · Interpret

Stain + morphology + context

5 · Report

State evidence and limits

One workflow spans all three clinical applications emphasized.

Slide 66 · Concept synthesis: Marker to inference

Observed feature

Best initial inference

Do not conclude alone

Cytokeratin

Epithelial differentiation

Specific primary site

CD45

Hematopoietic lineage

Exact lymphoma subtype

PSA

Possible prostate source

Complete origin work-up

ER/PR

Hormone-receptor phenotype

Guaranteed treatment response

HER2

Target-related protein expression

Direct gene-copy count

Slide 67 · Core messages

Morphology establishes the problem; IHC tests a specific diagnostic or therapeutic hypothesis.

Broad lineage markers help classify anaplastic tumors; Organ-associated markers can suggest an occult primary.

ER/PR, HER2, and ALK show how tumor protein expression can guide clinical decisions.

Flow cytometry and in situ hybridization contribute different, complementary information.

Interpret staining as evidence—with its scope and limits stated.

Slide 68 · Source and scope

Robbins, Cotran & Kumar. Pathologic Basis of Disease. 11th ed. Chapter 7: Neoplasia, “Immunohistochemistry,” p. 297.

Adjacent Chapter 7: “Histologic and Cytologic Methods”; “Flow Cytometry”; “Molecular Diagnostics and Cytogenetics,” pp. 296–298.

Related Robbins context: Chapter 7 (oncogenic growth-factor receptors, p. 253); Chapter 23 (breast cancer biomarkers, pp. 959–960).

Constructed cases and visual diagrams are teaching schematics; no case represents an actual specimen or patient.