Pathology Tests and Immunohistochemistry: Section 6.b.

CONTENTS:

6.4 Histochemistry (‘Special Stains’)
6.5 What is Immunohistochemistry (IHC)?
6.6 ‘Diagnostic’ Breast Immunohistochemistry (IHC)
6.6.1 Diagnostic Breast Immunohistochemistry Markers
6.6.2 Examples of the Use of IHC in Breast Pathology Problem Solving
i. Epithelial Hyperplasia versus Epithelial Atypia versus DCIS
ii. Is Micro-invasive Carcinoma Present?
iii. Radial Scar versus Tubular Carcinoma
iv. Papillary Lesions
6.6.3 Which immunohistohemistry Markers do a Diagnostic Breast Panel make?
6.7 ‘Prognostic’ Breast Immunohistochemistry (IHC)
6.7.1 Basal IHC Markers
6.7.2 Proliferation IHC Markers
6.7.3 Apoptotic IHC Markers
6.7.4 Other Markers

Forward to section 6c on ER, PR and HER2. Back to 6A introducing pathology tests.

6.4 Histochemistry (‘Special Stains’)

The Pathologist must firstly stain the breast tissue sections with special dyes before he/she examines them down the light microscope.

The pathologist uses histochemical stains or dyes on the thin tissue sections to identify the cells and their structures and to identify the non-cellular tissue components under microscopic magnification.

Pathologists probably really enjoyed the microscope they got for Christmas, when they were kids.

Microscopes, Stains and Other things …

Light microscopy is the usual way of examining the stained sections of breast tissue for diagnosis. For breast cancer diagnosis, the most common histochemical stains are:-

Hematoxylin: stains cell nuclei blue/black.

Eosin: stains protein and cell cytoplasm deep pink.

When used together, hematoxylin and eosin is termed ‘H&E.’

Periodic acid Schiff (PAS): for mucin and glycogen (purple).

PAS with diastase (PASD): digests glycogen and detects mucin. (pink/purple)

Oil-red-O (ORO): stains lipid deep orange/red.

Want to have some fun? Ask any medical student, what does the H & E stain of ____ disease show? They will think you are smarter than them.

But don’t tease med students because they are usually VERY tired and sensitive.

Figure 6.6 The hematoxylin & eosin (H&E) stained slide

Photomicrograph of a low-grade breast cancer shows purple-black
nuclear staining (N) with intra-nuclear nucleoli (n)
and pink cytoplasm (C)

Figure 6.7 The Varied Appearance
of Nuclear Mitoses

I recognize the word ‘Mitoses’ from high school biology.

It’s what my brother usually yells when he stubs his toe.

Figure 6.8 Apoptotic Figures in
a High Grade Breast Cancer (*)

6.5 What is Immunohistochemistry (IHC)?

The Pathologist, who looks at tissue sections using a microscope, can distinguish normal from abnormal tissue components by examining their microscopic appearance (morphology). Most diagnoses of benign and malignant conditions in breast pathology can be made using the H&E (hematoxylin & eosin) stained tissue section.

The technique of immunohistochemistry (IHC) adds to morphological identification of cells and tissues and uses antibodies made to ‘stick’ to known antigens that are specific for cell types or cell components. The location of these antibodies is visible down the light microscope because a visual label tags them.

The first immunohistochemical techniques used fluorescent dyes attached to antibodies that required specialized fluorescence microscopy to see where they localized to.

In the past 30 years, specialists have refined immunohistochemistry techniques to develop more specific and reliable antibodies (monoclonal antibodies) and enzymatic labels that are visual using the light microscope (commonly brown or red in color). Radioactive elements, peroxidase, alkaline phosphatase and fluorescein are all used for special immune-localization techniques.

This is a bit too much like chemistry. Who’s for coffee?

Yes bring coffee and a donut…put them on my desk…back away slowly…and we all might survive this!

The Uses and Limitations of Immunohistochemistry

There may be limitations in the use of some antibodies in IHC, so specialists tend to use ‘panels’ of antibodies. (Walker et al., 2012). Diagnostics implement quality control for immunohistochemistry staining methods and interpretation in pathology laboratories. Also, it is important that IHC is only used when combined with an analysis of breast tissue morphology on the H&E stained tissue section.

The use of immunohistochemistry is now a ‘routine’ diagnostic technique in most Pathology laboratories. Indeed IHC is an extra refinement in diagnosis.

Immunohistochemistry in breast histopathology has three roles:-

Diagnosis
Prognosis
Predictive

Figure 6.9 Immunohistochemistry (IHC)

Huh? Lets replace this picture with a kitty or puppy.

You promise, really? A puppy would be good.

6.6 Diagnostic Breast Immunohistochemistry (IHC)

When the Pathologist examines a breast biopsy sample using microscopy, there are two main diagnostic challenges:-

The identification of tumor cells (ductal, lobular or other)
The confirmation or exclusion of tumor cell invasion.

6.6.1 Diagnostic Breast IHC Markers

Over time, antibody technology has developed and refined antibodies that can identify antigens with a high degree of specificity and sensitivity. The following immunohistochemistry markers are now commonly in use to add precision to breast histopathology:-

Cytokeratins:-

CK5, CK10, CK14 and CK17: are basal layer cytokeratins, expressed by myoepithelial cells.
CK5/6: or ‘basal cytokeratins’ in normal breast detect both myoepithelial cells and luminal epithelial cells.
34β12: is an antibody that recognizes CKs 1, 5, 10 and 14.

It’s not procrastinating if you’re drinking coffee, it’s ‘procaffinating’.

Smooth Muscle-Related Markers:

H-caldesmon: a smooth muscle actin (SMA)-binding protein. Only found in myoepithelial cells of breast ducts, so highly specific.
Smooth muscle actin (SMA): these antibodies detect actin microfilaments; poor specificity since SMA also detects myofibroblasts (found in tissue repair).
Smooth muscle myosin heavy chain (SMMHC): structural component of smooth muscle myosin with the SM2 isoform expressed in breast myoepithelial cells. High sensitivity and specificity as SMMHC is present in peri-acinar and peri-ductal myoepithelial cells, but not stromal myofibroblasts.
Calponin: a 34 kD polypeptide, modulates actomyosin ATPase activity. Good sensitivity but present in a subset of stromal myofibroblasts.
p63-p53 homologue: a nuclear marker, highly sensitive for myoepithelial cells.
CALLA: an endo-peptide that is expressed in myoepithelial cells.

P-cadherin:

A cell adhesion molecule with high sensitivity for myoepithelial cells in normal breast and no reactivity with myofibroblasts.

GCDFP:

Gross Cystic Disease Fluid Protein

E-cadherin:

a cell adhesion molecule used to distinguish between DCIS and LCIS: DCIS typically shows membrane staining and most LCIS is negative.

Melanoma markers:

S100, melan-A, HMB45

Lymphoid markers:

CD20, LCA, CD15, CD30

Vascular endothelial markers:

CD31, CD34, Factor VIII, d2-40 (podoplanin)

Cell proliferation markers:

Ki67, mib-1, PPH3

Apoptosis markers:

bcl-2m bax, bcl-x, survivin

Wow. I skipped that whole section.

Me too.

Samies!

6.6.2 Examples of the Use of IHC in Breast Pathology Problem Solving

There are many situations in which immunohistochemistry can support the light microscopic opinion in breast pathology.

i. E-cadherin membrane staining for lobular neoplasia (ALH) and LCIS

Loss of cell membrane staining supports a lobular neoplastic lesion.

Figure 6.10 E-Cadherin in Lobular Neoplasia

IHC staining (brown) shows loss of cell membrane staining of the surface
of neoplastic lobular cells in atypical lobular hyperplasia
(ALH) ( E-cadherin x 60)

This picture reminds me of a painting by….whats-his-name?

Picasso?

No, a guy who likes to fry a lot of eggs.

ii. Epithelial Hyperplasia versus Epithelial Atypia versus DCIS

Benign hyperplasia of usual type and hyperplasia in papillomas show reactivity for CK5/6 and CK14 whereas atypical hyperplasia and DCIS do not.

iii. Is Micro-invasive Carcinoma Present?

Markers for myoepithelial cells are of value in assessing whether or not micro-invasion is present in a breast in-situ carcinoma. Myofibroblast reactivity, discontinuity of staining of myoepithelial cells and staining of vascular smooth muscle cells can cause difficulties in interpretation. The use of two myoepithelial cell markers is recommended, for example, p63 and SMMHC or ASMA or calponin.

Figure 6.11 Alpha Smooth Muscle Actin (ASMA) in Lobular Neoplasia.

A. Breast lobules are filled with neoplastic cells. This is lobular neoplasia
or lobular carcinoma in-situ (LCIS). (H&E x 20) B. Myoepithelial cells are
intact (brown) and surround breast lobules filled with neoplastic cells.
(ASMA IHC x 20)

Simplified, the pathologist says: “There’s dark purple that’s too thick. There’s a cancer. (but just ‘in-situ’, not spreading.)”

iv. Radial Scar versus Tubular Carcinoma

In the center of radial scars (complex sclerosing lesion), there may be myofibroblasts as part of the scar; scarring can also distort the myoepithelial cell layers of beast ducts and so very sensitive myoepithelial cell markers are needed. In the situation of a radial scar, SMMHC and p63 or SMA and calponin will be present in myofibroblasts, thus excluding tubular carcinoma.

Chocolate doesn’t demand anything or ask silly questions. Chocolate understands.

Figure 6.12 Alpha Smooth Muscle Actin (ASMA) and Cytokeratin (CK) in Radial Scar

A. Irregular glands show intact myoepithelial cell layer (brown) typical of radial scar.
There is no invasive carcinoma. (ASMA IHC x 20) B. Cytokeratin shows cytoplasmic
staining (brown) of the entrapped ductal epithelial cells. (Cam5.2 IHC x 40)

v. Papillary Lesions

Core needle biopsies (CNB) of papillary lesions are particularly challenging, so immunohistochemistry is often used.

a) Intraduct papillomas have a complete layer of myoepithelial cells that may be detected by SMMHC, calponin, p63 and CK5/6; or by SMA, p63, CD10 and CK14.

b) Intracystic papillary carcinomas lack myoepithelial cells or have a few that are scattered and discontinuous.

6.6.3 Which IHC Markers May be Used in a Diagnostic Breast Panel?

Walker and colleagues (2012) have made the following recommendations for diagnostic IHC panels in breast histopathology:

Smooth muscle myosin heavy chain (SMMHC), or calponin are sensitive and specific cytoplasmic myoepithelial markers. Smooth muscle actin can be used, but care in interpretation should be given when in the presence of myofibroblasts (inflammation and healing).

P63 is a sensitive and specific nuclear myoepithelial cell marker, but staining can be discontinuous; use of a cytoplasmic myoepithelial cell marker such as SMMHC or calponin is also advised to aid interpretation.

When trying to identify the presence/absence of myoepithelial cells, combined use of p63 and SMMHC/calponin is recommended.

Figure 6.13 Paget’s Disease of the Breast

A. Enlarged atypical cells are in clusters in the epidermis of the nipple.
The differential is between intra-epithelial spread of ductal cells or
melanoma. (H&E x 60) B. IHC using a cytokeratin marker (brown)
confirms Paget’s disease. (IHC CK x 60)

6.7 ‘Prognostic’ Breast Immunohistochemistry (IHC)

A ‘prognostic‘ factor provides information on clinical outcome at the time of diagnosis and is independent of therapy.

In contrast, a ‘predictive‘ factor provides information on the likelihood of response to a given therapy.

Although prognostic and predictive factors can be separately classified, in diagnosing and classifying breast cancer some factors are both prognostic and predictive (e.g., the presence of over-expression of HER2).

Potential prognostic markers must demonstrate three factors to be of clinical use:

analytical validity
clinical validity
clinical utility

In addition, a prognostic marker should:-

provide significant and independent value
be reproducible
widely available
be readily interpretable, and
must not consume tissue needed for other tests.

6.7.1 Basal IHC Markers

Gene expression profiling has identified subgroups of breast cancers expressing genes that are characteristic for basal or myoepithelial cells of normal breast. These ‘basal markers’ are associated with poor outcome. Most of these ‘basal’ tumors are high-grade, and lack ER, PR and HER2 and have a higher risk of brain and lung metastases.

There is no consensus on the immunohistochemical profile that defines these ‘basal-like’ cancers. These poor prognosis cancers are ‘triple negative breast cancers’ (TNBC), lacking ER, PR and HER2, but express basal cytokeratins 5/6 and/or 14, epidermal growth factor receptor (EGFR) and c-KIT. They may also express P-cadherin and p63 more frequently.

There are similarities between ‘basal-like’ breast cancers and breast cancers that occur in women with BRCA1 mutations.

EGFR is expressed at a high frequency in ‘basal-like’ cancers, so these patients may benefit from treatment with EGFR inhibitors.

I’m really sorry everybody. I’ve got to go and get myself a donut….or four.

Do you ever think to yourself, there’s more to life than eating, Moose?

What, like sleeping, you mean?

6.7.2 Proliferation Immunohistochemistry Markers

The ‘mitotic count’ or percentage of cells in mitosis within a cancer has long been a morphological ‘biomarker’ of cancer grade and prognosis. There are also a number of IHC markers that demonstrate cell proliferation:

Ki-67: is an antigen is expressed in the nucleus of cells in all phases of the cell cycle and is a useful marker of cell proliferation.
MIB1: is the name of an antibody that is reactive against Ki-67 in formalin-fixed, paraffin-embedded (FFPE) tissue.
PPH3: Phosphohistone-H3

The relationship between Ki67 status and prognosis in early breast cancer has been studied (Luporsi et al., 2012). Despite heterogeneity in Ki67 assessment methods used, the results of two large meta-analyses are consistent with the independent prognostic value of Ki-67. As an example, one meta-analysis that included 46 studies (and over 12,000 patients) reported that high Ki-67 levels in the primary breast cancer was associated with a higher risk of relapse in both node-positive and node-negative patients.

One problem with the use of these proliferation markers has been in the lack of an agreed ‘scoring’ system.

When everything’s going your way, you’re probably in the wrong lane.

6.7.3 Apoptosis IHC Markers

Apoptosis is also known as ‘programed cell death’ and is a biomarker of more aggressive tumors.

Apoptotic bodies can be counted in tissue sections as can mitotic figures. Apoptotic proteins can be identified using immunohistochemistry.

bcl-2
bax
bcl-x
survivin

These are not suitable as routine ‘predictive’ markers.

I know the voices in my head aren’t real..but sometimes their ideas are absolutely awesome.

6.7.4 Other ‘Prognostic’ Markers

i. p53: There have been multiple studies to investigate the prognostic role of p53 in breast cancer. The gene variations in p53 have been compiled in the International Agency for Research on Cancer (IARC) database (Petitjean et al., 2007). Mutations in the p53 tumor suppressor gene (TP53) occur in 20 to 30 % of human breast cancers. These gene mutations are seen more often in patients withhttps://www.breastcancer.org/risk/factors/genetics than in those with sporadic breast cancer. For patients with a history suggestive of a genetic predisposition to breast cancer, p53 testing may be necessary. However, routine testing is not indicated.

p53 has been considered as a potential predictor of response of breast cancers to chemotherapy, but much of the data comes from mutation analysis. IHC detects p53 protein, but this may not reflect the p53 mutation status.

ii. Topoisomerase II alpha: This is a target for the action of anthracycline a chemotherapeutic drug that is frequently used in the management of breast cancer.

Okay Moose, just for you.

References

Walker, R.A. (2008). Immunohistochemical markers as predictive tools for breast cancer. J Clin. Pathol. 61, 689–696. (Retrieved November 20^th 2014): https://www.ncbi.nlm.nih.gov/pubmed/18037665

Luporsi, E., André, F., Spyratos, F., et al. (2012). Ki-67: level of evidence and methodological considerations for its role in the clinical management of breast cancer: analytical and critical review. Breast Cancer Res Treat 132(3), 895-915. (Retrieved November 19^th 2014): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3332349/

More references for this section are on this page.

Patient Information

Breast Cancer Org. Genetics. (Retrieved February 13^th 2015): https://www.breastcancer.org/risk/factors/genetics

National Cancer Institute BRCA1 and BRCA2: Cancer Risk and Genetic Testing. (Retrieved February 13^th 2015):http://www.cancer.gov/cancertopics/factsheet/Risk/BRCA

More patient information for this section is on this page.

Forward to section 6c on ER, PR and HER2. Back to 6A introducing pathology tests.