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Immunohistochemistry in Breast Pathology - Part 2




Tumour type




Classical pattern invasive lobular carcinoma
Medium power H&E of invasive lobular carcinoma showing single file runs of tumour cells High power H&E of same tumour - note the squared of interface between cells with an apparent clear space. Also note cytoplasmic eosinophilia and occasional vacuoles
It is good practice to pair an anticipated negative stain with a positive marker. In this case we have used a pan cytokeratin stain which is positive for any carcinoma to compare with the negative E Cadherin stain (right) Negative E cadherin stain - some classical pattern ilcs show weak positive staining - note the positive internal control of an entrapped duct



In the following example E Cadherin confirms a carcinoma as NST when the possibility of invasive lobular carcinoma or mixed lobular/NST was considered seriously on the H&E appearances:



Use of E Cadherin to help decide whether a tumour is NST or Lobular
Medium power H&E of invasive carcinoma showing single file runs of tumour cells - ?lobular ?NST High power H&E of same tumour - note cytoplasmic eosinophilia and occasional vacuoles
Medium power - positive E Cadherin High power - positive E Cadherin


Subtle foci of invasion



The set of four photographs below illustrate the use of CK 7 (left) and CK 5/6 (right) in helping to pick out a small focus of invasive carcinoma in an area of DCIS.

It is good practice in immunohistochemistry to pair a stain which you anticipate to be negative with one that would be expected to be positive. Here confirming that one area is in situ (with CK 5/6) and another that is invasive (CK 7). On occasions you will find the "opposite" stain revealing something that you had not appreciated on the H & E.



In situ and invasive carcinoma emphasised by CK 5/6 and CK 7 immunostaining.
These are almost identical fields from sequential sections from the same block.
Low power H&E showing in situ and invasive elements Medium power H&E of invasive focus (centre and right) and in situ focus (on left)
CK 7 staining picking out an invasive focus in an area of DCIS CK 5/6 confirms the in situ element. It is important to check the H&E and the CK 7 sections to make sure that the cells that you think lack a CK 5/6 positive bounding layer are the tumour cells you originally observed!


Variable CK 5/6 immunostaining in a core biopsy showing intermediate grade DCIS and
a focus of mucinous carcinoma. Notice the central duct which is certainly in situ but shows almost absent CK 5/6 staining.
In the light of this the absent CK 5/6 staining around the suspicious focus (lower left) should be interpreted with caution. Blue arrows indicate positive myoepithelial cells.
Low power H&E showing in situ and invasive elements Medium power H&E of invasive focus (centre and right) and in situ focus (on left)
CK 7 staining picking out an invasive focus in an area of DCIS CK 5/6 confirms the in situ element. It is important to check the H&E and the CK 7 sections to make sure that the cells that you think lack a CK 5/6 positive bounding layer are the tumour cells you originally observed!
Low power H&E showing in situ and invasive elements Medium power H&E of invasive focus (centre and right) and in situ focus (on left)


In the next sequence of photomicrographs pan cytokeratin is used to pick out a subtle focus of invasive carcinoma in a core biopsy with widespread involvement by high grade DCIS. Although a CK 5/6 is shown to confirm that the invasive focus has no myoepithelial boundary it is noteworthy that the "internal control" positive staining around ducts with DCIS is very weak but nevertheless positive.



In situ and invasive carcinoma emphasised by pan CK immunostaining
Low power H&E showing in situ (Left) and invasive elements (Right)
H&E of area of DCIS H&E of area of invasion
Weak CK 5/6 staining of myoepithelial layer in area of DCIS Negative CK 5/6 staining in invasive area
Pan CK staining, low power - DCIS on left with smoothly demarcated peripheral contours and ragged appearance of invasive foci on right Pan CK staining of invasive focus

Status of Margins



In the following images the precise extent of the leading edge of invasive carcinoma is demonstrated with clarity using a pan cytokeratin stain. In this situation it is essential to be certain (from scrutiny of the H&E) that the cells in question are genuinely tumour cells because benign epithelial groups will also stain with pan cytokeratin.




Wide local excision with tumour close to an inked margin
'Mouse over' - immunostain for CK pan highlights tumour cells


Wide local excision with tumour close to an inked margin - 'Mouse over' - immunostain for CK pan highlights tumour cells


Subtle lymph node metastases


The following sequence of images demonstrate the use of immunohistochemistry to confirm the presence of subtle lymph node tumour metastases. Current practice guidelines do not recommend the routine use of immuno to detect metastases - they should first be visible on an H&E section.


Two subtle subcapsular nodal tumour deposits

Two subtle subcapsular nodal tumour deposits (arrows)


Pan CK highlights tumour deposits
'Mouse over' for high power image


Lymph node with subtle subcapsular and extra-capsular metastatic tumour deposits - 'Mouse over' - immunostain for CK pan highlights tumour cells


Demonstrating epithelial cells in necrotic material


In this example pan cytokeratin staining demonstrates clearly epithelial cells in a necrotic lymph node. This patient was suspected clinically and radiologically of having metastatic carcinoma in an axillary node. The immuno supports strongly this diagnosis.


Use of cytokeratin stains to demonstrate epithelial cells in necrotic material
- click on each thumbnail to see the full sized images.


Lymph node biopsy - necrotic material only Lymph node biopsy - pan cytokeratin stain reveals epithelial cells in necrotic material
Lymph node biopsy - necrotic material only Lymph node biopsy - pan cytokeratin stain reveals epithelial cells in necrotic material

For a further example of use of immunohistochemistry in this diagnostic area click here:


Receptor studies - ER, PGR & Her-2


  • Endocrine receptors:

    • Receptor analyses are made in almost all invasive breast cancers
    • Oestrogen Receptor (ER) is the most helpful and is positive in 80% of cancers
    • ER positive tumours often respond to hormone therapy
    • Progesterone (PGR) receptors are rarely positive if ER is negative
    • There is some evidence that ER positive / PGR negative tumours behave differently
    • Androgen receptors (AR) are rarely measured but are commonly positive in apocrine carcinomas which are usually ER and PGR negative
    • ER, PGR & AR receptors are located in the nucleus

  • Her-2 receptors:

    • Her2 testing is now carried out on all newly diagnosed breast cancers in the UK - around 20% are positive
    • Her2 posiive cancers may respond to targeted immunotherapy (Trastuzumab)
    • Recent trials have shown benefits from adjuvant treatment with Trastuzumab in Her2 positive cancers
    • The Her2 receptor is on the cell membrane
    • Fluoresence in situ hybridisation (FISH) is used to evaluate Her-2 gene copy numbers when immunohistochemistry produces equivocal results. See below for images
    • Approximately 50% of cases of high grade DCIS are positive for Her-2 - the therapeutic significance of this observation is not clear at the present time


Patterns of ER staining



Left hand image shows intense nuclear staining in the vast majority of cells - Allred score Category 8; right hand image shows weak nuclear staining of approximately 15% of tumour cells - Allred score Category 4
Strong nuclear staining of tumour cells for ER - this is maximally positive category score 8 Weak patchy nuclear staining of tumour cells for ER - this is mildly positive category score 4


Patterns of Her-2 staining



Positive (3+) - left, negative (1+) - centre, edge artefact - right
click on each thumbnail to see a larger image


Strong complete membrane staining with an antibody to the Her2 receptor - this would score 3+ and is a positive result Weak incomplete membrane staining with an antibody to the Her2 receptor - this would score 1+ and is a negative result Edge artefact staining with an antibody to the Her2 receptor - this is often seen in core biopsies and should be diregarded

In the following example different staining patterns are seen in two separate tumours from the same patient. On the left there is crisp, continuous, strong membrane staining - a good example of a 3+ positive tumour. On the right there is a moderately strong cytoplasmic staining pattern often seen in tumours showing polysomy rather than true ampification. In this case both tumours were FISH tested to confirm the respective diagnoses.



Positive (3+) - left, indicative of polysomy - right
click on each thumbnail to see a larger image


Strong complete membrane staining with an antibody to the Her2 receptor - this scores 3+ and is a positive result Cytoplasmic staining with an antibody to the Her2 receptor - this is often seen in tumours showing polysomy rather than true gene amplification



Principles of FISH testing for Her-2 status

  • Trastuzumab or Herceptin® has been shown to be of benefit in the treatment of advanced breast cancers that show Her-2 gene amplification
  • The benefit for those patients who over-express the gene due to polysomy as opposed to amplification is less certain
  • Significant polysomy is relatively uncommon (<5% of breast cancers)
  • Immuno-based tests for Her-2 have an equivocal range which necessitates further testing to determine gene amplification status
  • Also, immuno-based tests cannot discriminate reliably between gene ampification and increased expression due to polysomy
  • In my practice I use a dual probe fluorescence system with a red marker for the Her-2 gene and a green marker for the Chromosome 17 centromere so that gene amplification can be distinguished from polysomy - see below:

Examples of FISH testing for Her-2
Left - Negative, Centre - positive, Right - polysomy
Click on each thumbnail to see a larger image


FISH testing for Her-2 - negative result FISH testing for Her-2 - positive result FISH testing for Her-2 - polysomy



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