SUPREMO Pathology

This page reiterates the pathology section of the SUPREMO protocol for ease of reference and gives illustrative guidance on grading, assessing lymphatic invasion and lymph nodes for pathologists. We acknowledge that these assessments are prone to some subjective interpretation and to that end we would welcome feedback via the SUPREMO web site.

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The SUPREMO Pathology Protocol

UICC staging (6th edition) should be used.

7.1.1 The size of the primary tumour should be measured.

7.1.2 All primary tumours should be graded according to the Nottingham modification of the Bloom & Richardson grading system.

7.1.3 The adequacy of the excision margin should be measured. An adequate margin is any margin that is deep, anterior or radial. The margins are to be clear of either invasive or non-invasive disease, that is invasive carcinoma or ductal carcinoma in situ (DCIS). It does not include the presence or absence of lymphatic/vascular invasion.

7.1.4 A minimum of 8 axillary nodes should be examined in an axillary clearance.

7.1.5 All submitted axillary nodes in a axillary node sample should be examined.

7.1.6 A copy of the pathology report on the primary tumour and axillary node(s) should be sent to the trial administrator.

7.1.7 The original reported grade and lymphovascular status will be accepted for the purpose of the trial.

7.1.8 A password protected website for the trial will be provided giving examples of grading and lymphovascular invasion to facilitate standardisation of reporting between pathologists.

7.1.9 A panel of three pathologists will undertake the review of all cases entered by examining a representative H&E section taken from the block submitted to the trial central laboratory. Each pathologist will review one third of the cases, randomly allocated, and assess grade and lymphatic/vascular invasion. The pathologists will be blinded to the original pathology report. Those cases where the review grade and lymphatic/vascular invasion status is in agreement with those originally r eported will be reviewed no further. In those cases where there is disagreement between the reviewing pathologist and the original report there will be a formal review by all three reviewing pathologists to achieve consensus. Criteria for review will conform to current grading guidelines (Elston CW and Ellis IO. Pathological prognostic factors in breast cancer: experience from a large series with long term follow up. Histopathology 1991; 19:403-10).

7.2. Multifocal invasive cancer: If the tumour area comprises multiple small adjacent foci of invasive carcinoma then the overall maximum dimension should be taken and must be greater than 2cm if N0 (see Diagram F below):

Size diagram

	= Invasive tumours		= Ductal Carcinoma in situ
	= Invasive tumour measurement		= Whole tumour measurement

In E the satellite focus if invasive tumour is not included in the measurement.
In F the best estimate of the total size of the invasive components is given

Grading of Breast Cancer

Histological grade provides important prognostic and management information
The internationally accepted system is that defined by Elston and Ellis¹
Assess by evaluating acinar formation, nuclear size/pleomorphism and mitotic activity
Nuclear evaluation is the most subjective and can lead to inconsistency
Although originally designed for grading NST tumours it is recommended that it is applied to all cancers
An attempt should be made to grade the pre-operative core biopsy as there is acceptable concordance with excision grade

¹ Elston CW and Ellis IO. Pathological prognostic factors in breast cancer I. The value of histological grade in breast cancer: experience from a large study with long-term follow up. Histopathology, 1991, 19:403-10

Scoring system

Each element evaluated is given a score of 1 - 3
Acinar/tubule formation is assessed over the whole tumour and is a low/medium power assessment
Nuclear evaluation is of the worst area
Mitotic count is also in the most mitotic area

Acini

> 75%	Score 1
10 - 75%	Score 2
< 10%	Score 3

Nuclear atypia/pleomorphism

Small nuclei very similar in size to benign ductal/acinar epithelial cells Minimal pleomorphism and even chromatin pattern Nucleoli very inconspicuous This score is applied uncommonly	Score 1
Larger nuclei with mild to moderate pleomorphism Nucleoli visible but small and inconspicuous	Score 2
Vesicular nuclei often with prominent nucleoli Marked variation in size and shape	Score 3

Mitoses

figures below refer to a high power field (hpf) diameter = 0.50mm (see note below)

< or = to 7 per 10 hpf	Score 1
8-14 per 10 hpf	Score 2
> 15 per 10 hpf	Score 3

When assessing mitotic count it is essential to calibrate your microscope so that you know the diameter of your (x40) hpf. Readers are referred to the calibration table in the UK breast screening guidelines².

² Pathology Reporting of Breast Disease. NHS Cancer Screening Programmes/Royal College of Pathologists. NHS BSP Publication No 58, 2005.

A minimum a 10 hpfs should be counted at the periphery of the lesion and an attempt made to seek out mitoses. The illustrations below show examples of what are and more importantly what are not mitoses. It is especially important not to count apoptitic bodies and other dark nuclear blobs.

The following score groupings are used to define the Grade of a cancer. When reporting the Grade it is good practice to give the individual score components as well as the calculated grade. This allows for possible audits in the future and also allows for immediate comment on comparisons with core grade (if its score components have also been recorded).

Scores and equivalent Grades

Total score 3 - 5	= Grade 1
Total score 6 or 7	= Grade 2
Total score 8 or 9	= Grade 3

It is worthwhile when trying to assess tumour grade identifying those elements about which you have no doubt and then looking at the remaining scoring element(s) which may or may not be critical depending whether the two most likely scoring options push the grade one higher or one lower. In the following example the final element may or may not be non-critical depending on whether the scoring decision is between 1 & 2 or between 2 & 3. Obviously we do not want to encourage sloppy practice, on the contrary, simply a more robust approach to the practicalities of grading:

Tubule score unequivocally 2
Mitotic score unequivocally 2
Nuclear score non-critical - if 2 v 3 but critical if 1 v 2

The following examples further illustrate this point:

Tubule score unequivocally 3
Nuclear score unequivocally 3
Mitotic score (1 v 2) critical for deciding between grade 2 and grade 3

Tubule score unequivocally 2
Mitotic score unequivocally 2
Nuclear score (1 v 2) critical for deciding between grade 1 and grade 2

Examples of Tubule Formation

When assessing tubule formation, having initially assessed the tumour at scanning power examine some representative fields more closely picking out ten epithelial units at a time and estimating how many are solid and how many have a glandular space. Very quickly you will have a very good idea whether it is < 10% or > 75%.

Tubule score = 1 (>75% tubules)

The overwhelming bulk of this carcinoma shows tubular differentiation. It is morphologically of NST type and NOT a Tubular Carcinoma

Medium power view of >75% tubule formation

Tubule score = 2 (10 - 75% tubules)

This cancer shows < 75% tubule formation - Tubule score=2

This medium power view allows critical evaluation of those structures forming tubules and those that are solid

Tubule score = 3 (< 10% tubules)

This carcinoma shows no tubule formation whatsoever - Tubule score = 3

A single acinar structure is seen in the top left hand corner (arrow). Overall, tubule formation is well under 10% - Tubule score = 3

Examples of the three nuclear grades

NOTE - All the nuclear grading photographs have been taken at identical magnification for ease of comparison

Four examples of Nuclear score = 1

Two examples of a mixture of Score 1 and Score 2 nuclei (see arrows) - this is predominantly nuclear score = 1 however the higher nuclear grade always applies when there is a mixed population

Two examples of Score 2 nuclei - lower end of the range on the left, the higher end on the right

Four examples of Score 3 nuclei from the lower end of the range (top left) to the higher end (bottom right)

Examples of mitoses (Y) and not mitoses (N)

The following examples are from a selection of nuclear grade 3 carcinomas which show brisk mitotic activity. Various nuclear changes have been marked as to whether the author believes them to be (Y) or not to be (N) mitoses. It is inevitable that different pathologists will argue over some of these!

Lymphatic/vascular invasion

Best assessed around periphery of tumour
Difficulty distinguishing from shrinkage artefact in section - do not overdiagnose!
Lymphatics tend to accompany small veins arteries and nerves - if seen improves confidence in diagnosis
Tumour in vessels tends to be 'stuck' to the sides and may be accompanied by red cells
An independent prognostic factor so do not diagnose just because the nodes are positive!!

Examples of lymphatic/vascular invasion

Two foci of lymphatic invasion by carcinoma - the focus on the right is close to a venous channel		Vascular bundle with lymphatic invasion by carcinoma
Venous invasion by carcinoma - note adjacent small artery		Invasion of dermal lymphatics and dermal connective tissue by recurrent carcinoma of breast

The images below are from the same case. On the left the arrow points to a possible focus of lymphatic invasion; on the right lymphatic invasion is unequivocal. The diagnosis is made considerably more secure by confirming the presence of a lymphatic "in company" with a vein and small artery in the right hand image. Pass the mouse over these images to change the view.

Low and medium power views of intermediate grade DCIS with adjacent invasion (circled).

Low and hgh power views of lymphatic invasion - note adjacent artery & vein

An example of probable retraction artefact and NOT lymphatic/vascular invasion

This focus was regarded as NOT being diagnostic of lymphatic invasion but more likely to be simply retraction artefact of surrounding tissues

Nodal metastases

Identification of nodal metastatic disease is the most important task facing the pathologist examining an operative lymph node specimen in a case of breast cancer
Microscopic assesment should include the size of the largest metastasis, extranodal extension & invasion of adjacent lymphatics if present
Node metastases are subclassified into - replacement type, micrometastases and isolated tumour cells (ITCs)according to TNM 6 - see below

Subclassification of nodal metastases (TNM6)

Replacement metastases	> 2mm
Micrometastases	0.2mm-2mm
Isolated tumour cells	< 0.2mm

Replacement Metastases

Two examples of replacement metastases in axillary nodes. Note extra-capsular spread into adjacent fat in the images on the right

Low power view of replacement metastasis		Tumour acini infiltrating perinodal fat
Low power view of replacement matastasis - extension into perinodal fat can be seen at this magnification		Medium power view showing infitration of perinodal fat by metastatic carcinoma

Micrometastases

Subcapsular micrometastasis - this deposit is < 2mm on the H&E but the immuno stain for pan CK was on a section taken after cutting into the block further and measures just over 0.2mm qualifying as a micrometastasis

Isolated Tumour Cells and Micrometastases

Two subtle subcapsular deposits (arrows) of metastatic carcinoma in an axillary node sample - the sentinel node was negative in this case.

The deposit in the upper left quadrant measures <0.2mm and on its own would be recorded as "isolated tumour cells" (N0)

The deposit in the lower right hand quadrant measures 0.27mm and therefore qualifies as a micrometastasis (N1)

The enlarged photomicrograph below shows more detail of the metastatic foci when you move the mouse over those areas