1 Breast biopsy or excision
2 Fibrocystic breast changes
- 2.1 Microscopic examination
  - 2.1.1 Differential diagnosis
3 Fibroepithelial tumor
- 3.1 Gross examination
- 3.2 Microscopic evaluation
4 Atypical ductal hyperplasia
- 4.1 Gross examination
- 4.2 Microscopic evaluation
  - 4.2.1 Differential diagnoses
5 Invasive ductal carcinoma
6 Invasive lobular carcinoma
- 6.1 Gross examination
- 6.2 Microscopic evaluation
7 Lobular carcinoma in situ

Breast biopsy or excision

Fresh breast specimens should be put in formalin within one hour.^{[note 2]} Breast specimens should be immersed in formalin for 6-72 hours.^[1]

Gross processing

Selection and trimming

For excision (also called lumpectomy):

Determine total specimen size. ((Weight the specimen^[2]))
Ink margins. If sample orientations are marked, use different colors for different directions.^[2]
Palpate the specimen for masses. If felt, estimate the greatest dimension.^{[note 3]} Compare with radiography if available.^[2] Confirm the presence of any known biopsy clips, either visibly or by post-operative radiography (in order to confirm that the specimen includes the region of interest).

Cut a lumpectomy in the direction that gives the shortest distance from margin to margin. If a lumpectomy is uneven as shown, cut so that the first and last slice become wedge shaped (because these will later be cut further, perpendicularly to the margin), avoiding a wedge-shape for remaining slices.

Serially section the specimen.

When performing triage of fresh lumpectomies, generally make slices 1.1 to 1.5 cm thick. When laid down and flattened, this is generally thin enough to allow for fixation over at least 6 hours, and thick enough to be further cut into 2 or 3 slices after fixation; Note the direction of up/down on each such thick slice so that the relative orientation of the thinner slices thereof is maintained. Slices with solid tumors are cut somewhat thinner since they won't flatten as much when laid down.
When selecting tissue for submission after fixation, make 3-4 mm thick slices.^[2]

Inspect for any grossly visible lesions. If found, measure at least the greatest dimension of the lesion on the slice where it appears largest. (Also compare the greatest gross measurement (including any palpated one) with the greatest measurement at previous imaging, and note if it confers a staging discrepancy between imaging and gross dimensions, with cutoffs at 0.1 cm, 0.5 cm, 1.0 cm, 2.0 cm, and 5 cm.)

Invasive ductal carcinomas generally appear grossly as tan-white firm areas, here seen (white arrow) on a slice with inked margins. A radioactive seed (black arrow) was inserted by radiology to locate the tumor.

Re-excisions

For breast re-excisions, ink the new margin (in this case marked with a suture).
Re-excisions are subsequently serially sectioned perpendicularly to the new margin (to be able to measure distance from any tumor to the new margin). For large specimens, every other section may be submitted.

Tissue selection

Further serially section each of the two end sections into perpendicular sections, so that the distance between the surgical margin and any tumor involvement can be measured. If they are visibly involved as pictured, sections can be selectively taken from there. If end sections are not grossly involved, submit one perpendicular section, (every other perpendicular section), ((or the entire end sections)).

For lumpectomies, submit:^[2]

Entire specimen if it can fit in 3-5 slices.
If larger:

For relatively well demarcated tumors: 1 slice per cm of tumor (minimum of 3 slices of tumor), including both center and periphery of tumor, including closest distance to the surgical margin if possible.
For diffuse or even invisible tumors such as often is the case for DCIS and ILC, either still submit whole, or use X-ray to guide what tissue to submit.

Additional suspicious areas, including those indicated by radiography.
((Representative sections of margins in each direction.))

Breast specimens where breast cancer is possible should generally not be decalcified even when containing small calcifications, to preserve the ability to perform immunohistochemistry.

Gross report

Size of original tissue sample, preferably in 3 dimensions.
Description of inking
Tumor properties, at least:

Size in 3 dimensions.^[2]
Distance from margins^[2]
Consistency^[2]

(Description of sectioning and submissions.)
((Time of procurement and time of placement in formalin.^{[note 2]}))

Example:

(The specimen is received fresh and consists of) an irregular fragment of yellow tan fibrofatty tissue measuring 4.0 x 2.8 x 2.0 cm. (The specimen is oriented with two sutures and) the surgical margins are inked as follows: superior-blue, inferior-green, medial-red, lateral-yellow, anterior-orange, and posterior-black. {{A radioactive seed is embedded in the tissue.}} The tissue is serially sectioned {{to reveal a tan-gray, spiculated, indurated mass measuring _cm in greatest dimension. The mass is located _cm from the nearest (<<superior, inferior etc>>) margin. (The specimen is entirely submitted in sequential sections from medial to lateral in 10 cassettes. The medial and lateral margins are submitted as perpendicular sections.) The specimen was procured at _AM/PM on _/_/2020. The specimen was placed in formalin at _AM/PM on _/_/2020.

Lymph nodes

When lymph nodes are submitted together with a biopsy or excision of a suspected or previously confirmed invasive lobular carcinoma (but not necessarily invasive carcinoma with lobular features), generally put the lymph node specimen through H&E processing at a relative rush. If you don't see any involvement on the H&E stain, order immunostain for CK AE1/AE3 in order to visualize otherwise occult lymph node involvement. The rushing of the lymph node samples allows you to have the immunostained slides by a similar time as the rest of the case.^[3]

Further information: Lymph node

Staining

Usually H&E staining.

Microscopic evaluation

If tumor is found, determine:

Tumor size
Malignancy
Distance from excision margin

Malignancy

The most important is to classify a sample as either of the following:

Benign
Carcinoma in situ
Invasive cancer

Most common types

Women seeking evaluation of a breast lump^[4]
Finding	Relative incidence	Histopathology
Fibrocystic breast changes	40%	Sclerosing adenosis (pictured), with an increase in glandular elements in addition to stromal proliferation that distorts and compresses glands.^[5]
		Radial scar, seen as a fibroelastic stroma and entrapped glands radiating outward. Measure the size of these.^{[note 4]}
		Usual ductal hyperplasia: Cohesive proliferation with haphazard, jumbled cell arrangement or streaming growth pattern. Cells have mild variation in cellular and nuclear size and shape.^[6]
No disease	30%
Fibroepithelial tumor	7%	Proliferation of both stromal and epithelial components.^{[note 5]} The tumor group mainly includes fibroadenoma and phyllodes tumor.
Atypical ductal hyperplasia	7%^[7]	Epithelial proliferations which are not qualitatively or quantitatively abnormal enough to be classified as ductal carcinoma in situ.^[7]
Other benign mammary dysplasias and neoplasms	5%	Including: Flat epithelial atypia: variably dilated acini lined by one to several layers of epithelial cells with low-grade cytologic atypia, usually columnar.^[8] Columnar cell change: Terminal duct lobular unit with epithelium exhibiting tall cells with oval or elongated nuclei orientated perpendicularly to the basement membrane.^[9]
		Intraductal papilloma: unremarkable epithelial cells lining fibrovascular cores.
		Pseudoangiomatous stromal hyperplasia (PASH): Complex interanastomosing vessel-like spaces in dense collagenous, keloid-like stroma.
Breast cancer (in situ or invasive)	10%	See next section.

Breast cancer types

Breast cancer types, with relative incidences and prognoses.

Cancer type	Histopathology	Image
Invasive ductal carcinoma (IDC)	Carcinomatous cells are seen below the basement membrane of lactiferous ducts. Otherwise, there are no specific histologic characteristics, essentially making it a diagnosis of exclusion.^[10]
Ductal carcinoma in situ (DCIS)	Malignant epithelial cells confined to the ductal system of the breast, without invasion through the basement membrane.^[11]
Invasive lobular carcinoma (ILC)	The "classic" pattern is round or ovoid cells with little cytoplasm in a single-file infiltrating pattern, sometimes concentrically giving a targetoid pattern.
Lobular carcinoma in situ (LCIS)	Monomorphic, loosely cohesive, slightly enlarged and evenly spaced cells that fill acini.^[12] Cells have indistinct cell borders, pale cytoplasm, and uniform small nuclei with evenly distributed chromatin and inconspicuous nucleoli.^[12]
Mucinous carcinoma	Extracellular mucin areas around tumor cells.
Medullary carcinoma	Seemingly fused tumor cells (syncytial pattern), and a prominent lymphoid infiltrate.
Solid papillary carcinoma	Larger tumor nests with fibrovascular cores.

Further information: Evaluation of tumors

Previous biopsy site

(For excisions or larger, look up past biopsies, and if in the same area, look for biopsy sites in order to confirm that the previous biopsy represents the same pathologies.)

Biopsy site at 11 days, low magnification, showing involved area consistent with a needle size
High magnification shows granulation tissue (fibroblasts, red blood cells and lymphocytes). Multinucleated giant cells may be present.
One month after biopsy, typically showing fibrosis. Foreign material is also present.

If tumor is not found

Check the imaging indication, and look for abnormalities that may explain the diagnostic findings:

Dense stromal fibrosis for radiodense areas.
Calcifications if found on X-ray.

If still not found, note their absence, since it indicates that the biopsy may have missed the target of interest.

Look extra close for invasive lobular carcinoma which may be subtle, as shown
Dense stromal fibrosis, in this case with entrapped fat cells and involuted entrapped ducts (arrows). In contrast, normal breast parenchyma has loose collagenous mammary stroma.^[13]
Conventional microcalcifications in non-neoplastic breast
If needing to look for calcifications, and no conventional ones are found, also look in cystic areas, without condenser, for calcium oxalate crystals, which are colorless and generally irregular, but may show on X-ray.^{[image 1]}^[14]
Stacking of cells on top of each may mimic microcalcifications, but will be revealed by focusing at different levels.

Microscopy report

A normal biopsy may be reported as follows

(Fibrofatty tissue with benign ducts and lobules.) Negative for (atypia and) malignancy.

More detailed reports are given at the disease-related articles.

Fibrocystic breast changes

Microscopic examination

Types:

Sclerosing adenosis, with an increase in glandular elements in addition to stromal proliferation that distorts and compresses glands.^[15]
A radial scar of the breast, seen as a fibroelastic stroma and entrapped glands radiating outward.

Differential diagnosis

Sclerosing adenosis may look similar to invasive ductal carcinoma (IDC), but IDC will:^[15]

Not be lobular at low power
Have marked cellular atypia
Have no myoepithelial cells surrounding ducts.

When unsure, perform immunohistochemistry for myoepithelial markers (preferably p63 and calponin in breast lesions):

Immunostain with a myoepithelial marker (calponin) in sclerosing adenosis, showing myoepithelial cells surrounding the ducts, thereby excluding invasive carcinoma.

Fibroepithelial tumor

Gross examination

As per:

Breast biopsy or excision

or mastectomy.

Microscopic evaluation

Fibroadenoma

Characteristics of fibroepithelial tumor

Biplastic, having proliferation of both stromal and epithelial components,^{[note 6]} arranged into either a pericanalicular pattern (stromal proliferation around epithelial structures), or an intracanalicular pattern (stromal proliferation compressing the epithelial structures into clefts).

Characteristics of fibroadenoma

Fibroadenomas characteristically display hypovascular stroma compared to malignant tumors.^[16]^[17]^[18] Furthermore, the epithelial proliferation appears in a single terminal ductal unit and has duct-like spaces surrounded by a fibroblastic stroma. The basement membrane is intact.^[19]

In fibroadenomas, glands may vary from relatively normal to completely compressed (at right in image), and possibly dilated.
Fibroadenoma with proliferation of intralobular stroma (which may be variably eosinophilic as shown), compressing and distorting the epithelium.

Characteristics of phyllodes tumor

Benign phyllodes tumor

In contrast to fibroadenomas, phyllodes tumors display a leaf-like architecture, and an increased stromal cellularity.^[20] In needle biopsy specimens, phyllodes tumors can be diagnosed in a fibroepithelial tumor if there is prominent mitotic activity of ≥3 per 10 high power fields, or the finding of 3 or more of the following characteristic histologic features:^[20]

Stromal overgrowth
Fat infiltration
Stromal fragmentation
Subepithelial stromal condensation
Stromal nuclear pleomorphism

Further workup

After diagnosing a fibroepithelial tumor, classify it as benign, borderline or malignant:^[21]

	Benign	Borderline	Malignant
Stromal atypia	Mild	Moderate	Marked
Stromal cellularity	Mildly increased, can be focal	Moderately increased, can be focal	Markedly and diffusely increased
Stromal overgrowth*	Absent	Absent or very focal	Present
Mitotic count	< 5/10 HPF or < 2.5/mm²	5 - 9/10 HPF or 2.5 - < 5/mm²	≥ 10/10 HPF or ≥ 5/mm²
Tumor border	Well defined	Well defined or focally permeative	Diffusely permeative
Malignant heterologous elements	Absent	Absent	Presence directly upgrades to malignant category**

* Stromal overgrowth can be defined as absence of epithelial elements containing stroma only in 1 low power field.

**Malignant categories include chondrosarcoma, liposarcoma (except well differentiated liposarcoma), osteosarcoma, rhabdomyosarcoma, angiosarcoma and leiomyosarcoma

Also, exclude another type of breast cancer, which may arise within it,^[22] possibly being:

For borderline and malignant phyllodes tumors, measure the distance from the tumor to the closest surgical margin. However, such measure is not necessary for benign phyllodes tumor.^[23]

Microscopic report

If, even after consultation, it is not clear whether a tumor is a fibroadenoma or phyllodes tumor, then it can be reported as a cellular fibroepithelial lesion or a fibroepithelial neoplasm. A benign lesion that resembles but does not clearly show a fibroadenoma can be called a fibroadenomatoid lesion.

A phyllodes tumor warrants a comment whether margins are positive or negative for tumor, whereas fibroadenoma does not need such comment.

Example report of fibroadenoma:

Right breast, lumpectomy: Fibroadenoma. Negative for atypia or malignancy.

Atypical ductal hyperplasia

Gross examination

As per:

Breast biopsy or excision

or mastectomy.

Microscopic evaluation

Histological appearance of atypical ductal hyperplasia (ADH) and immunohistochemical phenotype:^[24]
- A - One focus (< 2 mm) of two architecturally disarranged cross sections of tubuli showing a monotonous intraductal proliferation with secondary intraluminal architecture.
- B - One area of an ADH with associated intraluminal calcifications.
- C - Higher magnification of ADH shows low-grade nuclear atypia and monotonous cell proliferation along with secondary intraluminal architecture.
- D - Strong and uniform expression of estrogen receptors (ER). ER immunohistochemistry.
- E - Lack of basal cytokeratins (CK5/6). CK5/6 immunohistochemistry.

Atypical ductal hyperplasia shows epithelial proliferations which are not qualitatively or quantitatively abnormal enough to be classified as ductal carcinoma in situ.^[7]

Differential diagnoses

Ductal carcinoma in situ

There is no single definite cutoff that separates atypical ductal hyperplasia from ductal carcinoma in situ, but the following are important distinctive features of atypical ductal hyperplasia, with suggested cutoffs:^[25]

Size less than 2 mm.
Not involving more than one duct.
The atypical epithelial proliferation is admixed with a second population of proliferative cells without atypia.
The proliferation completely involves the terminal ductal lobular unit(s), to a limited extent.

Also, ADH tends to have rounded lacunae between cells, in contrast to more crescent-shaped (compressed) lucunae in DCIS.

Usual ductal hyperplasia (UDH)

Usual ductal hyperplasia

edit
In contrast to usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH) displays:^[6]

Increased amounts of pale eosinophilic to amphophilic cytoplasm with conspicuous cell borders
Atypical architectural patterns, including cribriform spaces, Roman arches, trabecular bars and micropapillae

If uncertain, immunohistochemistry can be performed:^[6]

	Usual ductal hyperplasia (UDH)	Atypical ductal hyperplasia (ADH)
High molecular weight keratins	Mosaic to occasionally diffuse	Negative
Estrogen receptor	Heterogenous staining	Diffusely positive

Invasive ductal carcinoma

Gross examination

As per:

Breast biopsy or excision

or mastectomy.

Microscopic evaluation

DCIS.

Malignant epithelial cells confined to the ductal system of the breast.^[11] The cells are cohesive and have high grade atypia.^[26]

Differential diagnoses

Invasive ductal carcinoma: Has invasion through the basement membrane of over 1 mm in size.^[27]

DCIS with microinvasion, defined as DCIS with focus of invasive cancer measuring up to 1.0 mm in size.^[28]

In uncertain cases, use immunohistochemistry stain for calponin (has the highest sensitivity) and p63 (has the highest specificity).

Immunohistochemistry for the myoepithelial marker^{[note 7]} calponin in ductal carcinoma in situ, highlighting myoepithelial cells around all tumor cells, thereby ruling out invasive ductal carcinoma.
Invasive ductal carcinoma with tubular features can look like benign tubules, but calponin and p63 shows no surrounding myoepithelial cells.

Atypical ductal hyperplasia

There is no single definite cutoff, but the following are suggested cutoffs defining a ductal carcinoma in situ:^[25]

Size over 2 mm.
Involving more than one duct.

Lobular carcinoma in situ (LCIS)

Lobular carcinoma in situ (LCIS) displays discohesive cells, often a feathery clear space between cells, solid growth pattern, intracytoplasmic vacuoles, and lack of polarization around luminal spaces.^[29]
LCIS typically fills smaller lobules rather than ducts, but DCIS can display lobular cancerization as shown at bottom of image.^{[image 1]}

When unsure, perform immunohistochemistry for E-cadherin and p120. Both E-cadherin (left image below) and p120 (right) have a membranous staining pattern in ductal carcinoma in situ:

In contrast:

E-cadherin is negative in lobular carcinoma in situ.
p120 has a cytoplasmic staining in lobular carcinoma in situ.

Grading

At least a low/intermediate/high grading (by Van Nuys criteria) as follows:^[30]

Low grade DCIS

Nuclei 10-15 microns (2-3 times the size of a red blood cell)
Nuclei oval, round, regular, evenly dispersed chromatin up to mildly irregular and minimally pleomorphic
Nucleoi, if present, are small and indistinct

Intermediate grade DCIS

Same nuclear features as low grade

Ductal carcinoma in situ with comedo necrosis spanning 30% of its diameter, which is generally regarded as the minimal size to classify it as comedo.^[31]

Substantial tumor cell (comedo) necrosis is present

High-grade DCIS. H&E stain.
RBC = red blood cell.^[32]

High grade DCIS

Nuclei >15 microns (over 2.5^[33] times the size of a red blood cell)
Nuclei are pleomorphic with clumped chromatin
Nucleoli are prominent, enlarged
Necrosis is almost universal and lumenal

(Numerical grading

Use the low/intermediate/high grade to give a numerical grading as follows:^[34]

Feature	Points
Feature	1	2	3
Nuclear grade	Low	Intermediate	High
Glands/papillae	>75%	10% - 75%	<10%
Mitotic rate (per 10 HPF)	<1	1 - 2	>2
Central necrosis	<10%	10% - 50%	>50%

The points for each feature are added together, giving the following result:^[34]

4 - 7 points: Grade 1
8 - 9 points: Grade 2
10 - 12 points: Grade 3

)

Extent

In biopsies, do not state the extent of DCIS.
In excisions, state the distance to the closest margin. (Additional extent specification is optional. If there is invasive tumor in addition to DCIS, you may give the DCIS extent in terms of the number of blocks containing DCIS. If an excision has DCIS without any invasive component, consider stating the total size in terms of the greatest distance between any DCIS foci.)

Histopathology of dystrophic microcalcifications in DCIS.

Microcalcifications

Microcalcifications of non-neoplastic breast glands.^{[image 1]}

If invasive ductal carcinoma is seen, make at least a low power screening for microcalcifications (to correlate with imaging), but there's no need to look carefully (as tiny microcalcifications would unlikely correlate with imaging anyways).

Estrogen and progesterone receptors

In DCIS with necrosis, only use the areas of viable DCIS for the calculation of hormone receptors on immunohistochemistry.

Generally perform immunohistochemistry for estrogen and progesterone receptors and calculate the percentage of positive tumor cells.

HER2

((HER2 testing is not necessary, but can be done for prognostic profiling.^[35])) See invasive ductal carcinoma for how to evaluate HER2.

Reporting

Architectural patterns of DCIS.^[36]

Example:

Left breast mass, 2:00, 1 cm from nipple, ultrasound-guided vacuum assisted core needle biopsy: Ductal carcinoma in situ. Negative for invasive carcinoma.

For cancers, generally include a synoptic report, such as per College of American Pathologists (CAP) protocols at cap.org/protocols-and-guidelines.

See also: General notes on reporting

Invasive lobular carcinoma

Invasive lobular carcinoma (ILC):

Gross examination

As per:

Breast biopsy or excision

or mastectomy.

Microscopic evaluation

Invasive lobular carcinoma.

ILC may be subtle on low magnification (left). Higher magnification (right) shows invasive growth pattern and vesicular nuclei with prominent nucleoli.

Characteristics

The "classic" pattern is round or ovoid cells with little cytoplasm in a single-file infiltrating pattern, sometimes concentrically giving a targetoid pattern.