What Happens to Your Breast Biopsy/Lumpectomy/Mastectomy Specimen in Pathology
Accessioning | Gross Examination | Fixation | Processing | Embedding | Making Slides | Review of Slides | Immunohistochemical Stains | Prognostic Factors in Cancer
It is difficult for patients and other physicians to understand what happens to a specimen that the surgeon sends to pathology, like a breast biopsy or mastectomy. One reason is that not all pathology laboratories at different hospitals process specimens the same way, and in the same time period. To clarify our process, we’ll take you through the steps that will occur assuming that your biopsy is done here at The Johns Hopkins Hospital. For simplicity, we will assume that it was done on a Monday morning.
As
soon as your specimen arrives in pathology, your specimen is assigned a pathology
number so we can keep track of it. This number is different from your
medical record number or hospital identification number. The number begins
with the letter S (for Surgical Pathology, as opposed to a C for cytology),
followed by the last two numbers of the year. All of the specimens that
arrive here in a given year are numbered consecutively starting from 1 (last
year we received over 60,000 different specimens). If only one specimen is
sent, it is assigned part 1. If the surgeon has sent other specimens from you
separately (for example, the lumpectomy and the axillary lymph node
dissection arrive in two different containers), the first specimen is
designated part 1 while the second is assigned part 2. Therefore, your specimen might be assigned the number S01-12367, with part 1 being the lumpectomy and part 2 being an axillary dissection.
If you have had specimens taken before at our hospital, they will have been given different pathology numbers. For example, a skin biopsy taken in 1984 might have the number S84-112. A pap smear (cytology) from this year might have the number C01-12854. A simple search of our computer system will let us know that you have had other specimens taken here before. This is important because we may need to review the slides from those specimens (which are kept permanently on file) when we evaluate the current specimen.
Gross Examination is done within a few hours of receiving your
specimen. A physician (pathologist 1, a resident) will examine your specimen,
measuring its size, describing its color and texture, and determining if any
masses are present. If so, the size of the mass and its distance to the
specimens’ edge (the margin-where the surgeon cut) will be recorded. The pathologist then sections the specimen into thin slices with a scalpel and further examines the slices. The pathologist then takes small pieces of the slices (about the area of a dime and about twice as thick) and puts them into small containers called cassettes. Cassettes are essentially small, 2cmX2cmX0.5cm, rectangular boxes with multiple small openings in their walls which let the formalin fixative in to bathe the tissue. Once fixed and processed (see below), these pieces of tissue will be the blocks of the case.
 Open Cassette |
 Closed Cassette Note the specimen and block number. |
Multiple cassettes in a container of formalin |
If the specimen comes late in the day, it may require overnight fixation, which will delay the diagnosis by one day. While we try to avoid delay, we feel that it is better to have a small delay than to proceed with inadequately fixed tissue. Inadequately fixed tissue can lead to
- inability to make an adequate slide,
particularly if the breast tissue is fatty. This make it impossible to
assess margins, or determine how large the tumor is, both of which
affect prognosis.
- unreliable immunohistochemical stains for prognostic markers like estrogen and progesterone receptor, Her2/neu, and Ki-67. If these proteins are not preserved properly by fixation, they degrade and become impossible to detect.
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Tissue Blocks |
Tissue Block File |
A thin (5 micron) section of the tissue is cut from the block using a microtome (a cutting instrument), and stained with Hematoxylin and Eosin (H and E). The slides are now ready to be examined under the microscope, and are delivered to pathologist 1, who examined the fresh specimen.
 Microtome |
 Staining Machine |
 Slide |
Pathologist 1 receives all of the slides
corresponding to blocks (derived from cassettes) that he/she made the day
before along with a typed dictation of his/her gross findings, and a code as
to from where each section was taken. This pathologist reviews the slides,
correlates the microscopic findings with the gross findings, and makes a
provisional pathology report.
The next day, pathologist 1 brings the provisional
report and the slides to a second pathologist, one who is the attending
pathologist for the case. The two pathologists review the slides together
using a multi-headed microscope. The senior pathologist reviews and edits
pathologist 1’s diagnosis, and signs out (approves) the final diagnosis. If
the findings are unusual, the senior pathologist may choose to review the
case with other pathologists in the department at a daily quality assurance conference in order
to be sure of the diagnosis. The senior pathologist will then choose a block on which immunohistochemical stains for breast cancer markers (ER, PR, Ki-67, and Her/2neu) will be performed.
Once they have been reviewed by both pathologists and the case is signed out, the slides are kept in a file room at JHH indefinitely.
These are stains done using antibodies to specific
proteins in the cell. Proteins are composed of amino acids, and make up most
of the structure of human cells. Antibodies are a specific type of protein
which have the unique property of attaching to specific molecules. Antibodies
are part of the normal human immune system, and function to protect us all
from infection. Immunohistochemistry Laboratories take advantage of the unique attaching properties
of antibodies. We use antibodies from mice or rabbits that specifically
attach to human proteins like Estrogen receptor, Progesterone receptor, or Her2/neu,
to detect these proteins in human cancer tissues. To do this test, the laboratory will cut several (about 5) additional sections from the block that the pathologist has designated. These slides will then be treated with bound labeled antibodies to the specific proteins we want to detect, treated with enzymes that detect the bound antibody by developing a brown colored chromagen, and then stained with hematoxylin. This will allow us to review the slide and see not only is the protein in question present but where it is (in the cancer or the normal benign cells).
The proteins which we usually assess in breast cancer are:
Estrogen receptor (ER) and Progesterone Receptor (PR). Normal breast epithelium usually expresses estrogen and progesterone receptors. Binding of estrogen or progesterone to these receptors stimulate normal cells to divide. In breast cancers, these are weak favorable prognostic factors, so tumors that express estrogen and progesterone receptors generally have a better prognosis that those that do not. However, expression of estrogen and progesterone receptors are strong predictive factors, in that their presence predicts response to hormone therapy such as Tamoxifen. We report the percentage of tumor cells that stain, and how strongly they stain, and based upon these two factors interpret the tumor as positive or negative. Positivity for ER and PR indicates are greater likelihood of response to Tamoxifen than positivity for only one of these, while absence of both ER and PR predicts less likelihood of response.
Ki-67- a measure of cell proliferation. This antibody labels all cells actively participating in cell division. This includes the G1 (pre-DNA synthesis), S phase (DNA synthesis), G2 (post-synthesis), and M (mitosis) phases of the human cell cycle. It does not label cells in the resting (G0) phase of the cycle. This immunohistochemical stain correlates with the number of mitoses we count and incorporate into the grade, but is more quantitative; i.e., we can give a numerical percentage of positive cells.
Her2/neu-. This oncogene (cancer gene) is amplified (more copies of its DNA are present) and overexpressed in 25-30% of human breast carcinomas. Amplification is a poor prognostic factor, though a weak one. Patients with positive lymph nodes have a worse prognosis when her2/neu is amplified. However, amplification of Her2/neu does predict response to therapy with Herceptin, the humanized anti-her2/neu antibody. One can test for Her2/neu amplification in two ways.
The most common way is by immunohistochemistry, using an anti-her2/neu antibody, to detect the Her2/neu protein overexpression which results from gene amplification. Many different antibodies are available for laboratories to us.e, and the results are surprisingly variable when different antibodies are used, so it is important to know the result and what antibody or kit was used for the test. Scores may be graded as strong positive, weak positive, or negative.
Some laboratories test all weak positives with the other method, Fluorescence In Situ Hybridization (FISH). This is a genetic test done in specialized laboratories in which one uses a DNA probe to the Her2/neu gene to determine how many copies are present in each cell. Normal cells have 2 copies of Her2/neu. The 25-30% of breast carcinomas with Her2/neu amplification have more than 2 copies, often greater than 10 per cell.