It is helpful to note the sensation obtained when the needle enters the lesion, largely because this can help inform the operator that the lesion has been successfully targeted. Details may be included in the report for subsequent audit. The character of the lesion can be described and correlated in the following way, according to the modified United Kingdom National Health Service Breast Screening Programme (NHSBSP) guidelines.⁹

In order to obtain an optimal or near optimal cell yield, aspirator skill is critical for success of FNA.^4,19

Results from the UK National Health Service Breast Screening Programme (NHSBSP) have shown that a low sensitivity to a large degree is due to a high inadequacy rate, most often due to the lack of aspirator skill.²⁰ Overall, results are better when cytopathologists perform their own aspirations.²¹ In a rapid one-stop clinic setting, a cytopathologist can further reduce the incidence of non-diagnostic or equivocal results by taking a new sample if the initial one is suboptimal.

FNA of palpable lesions

• Fix the lump between the second and the third finger of the non-dominant hand

• Insert the needle ideally at a 90° angle (Fig. 4.1), but adapting this to take account of structures that are normally avoided, such as pleura and areola. This angle will minimise missing small lesions. For central lesions, it is usually possible to sample without going through the areola either by approaching from the side, or asking the patient to pull the skin gently so that the lesion is no longer under the areola

• Apply negative pressure when the needle tip is in the lesion

• Move the needle to and fro and sample from as many parts of the lump as feasible. Sampling is then a result of both aspiration/suction and/or capillary action. Avoid holding the needle still and only applying suction, as this will result in more blood and less tissue and cells. Soft cellular lesions will tend to need fewer movements of the needle than hard sclerotic ones. The aim is to move the needle sufficiently to notice the sample or blood in the hub of the needle at which point it is best to stop

• Unless a cyst is being drained, stop when material appears in the needle hub or if there is excessive bleeding

• Release the negative pressure before the needle is withdrawn.

Fig. 4.1 Aspiration technique: it is important to insert the needle vertically. Small lesions may be missed if the needle is inserted obliquely.

Bloodstained nipple secretion is an indication for cytological examination of the nipple fluid. However, it is recognised that cytology of nipple discharge has a low sensitivity for identifying underlying malignancy and a negative result should not preclude further investigation. Nipple discharge fluid is best prepared by placing a glass slide tangentially to the nipple in such a way as to touch the drop of secretion, making sure not to touch the surrounding skin in order to avoid contamination. After the secretion is transferred to the glass, a conventional smear is made.

Ulceration or eczematous changes on the nipple likewise warrant further investigation and scrape material may be examined. The debris covering the ulceration is carefully removed and the non-cutting edge of a scalpel blade or the edge of a clean slide is used to scrape cells from the surface. Preparation of both nipple fluid and scrape material can be handled in the same way as FNA material.

Breast cytological material is usually stained with conventional cytological stains, such as Papanicolaou (PAP) for alcohol-fixed slides and May-Grünwald Giemsa (MGG) for air-dried slides. MGG is ideal as a rapid stain (Diff-Quik® or equivalent), and hence is the stain of choice in one-stop clinics. It can be done in less than 2 min on air dried direct smears. The smears can be examined without a cover glass, which is particularly useful for checking adequacy when the patient is still on the couch and the cytologist is considering whether to take a second sample. If a Diff-Quik® smear is not adequately stained, it may be restained with MGG in the laboratory.

Stain precipitates may be observed in MGG. Its appearance should prompt a change of staining solutions. Starch granules from gloves may seriously impair the evaluation of MGG-stained smears. To avoid this problem the aspirator should not use starched gloves. Air drying artefact causing ‘exploded nuclei’ is the most serious artefact besetting MGG air-dried smears. If widespread, it may render a specimen inadequate for assessment. Furthermore, the novice may try to interpret the less affected cells that appear artificially large. The appearance, sometimes called ‘the osmotic effect’, is that of a cell whose nucleus has expanded and ruptured with spillage of chromatin at the cell border, forming a flare. This artefact has been attributed to the effects of saline, local anaesthetic fluid and autolysis from slow drying, as when the smear is placed directly into an airtight box. Over-heating when an electric hairdryer is used to ensure rapid drying and the use of excessive pressure when making the smear, causing the crush artefact, are also possible culprits.

Crushed nuclei (Fig. 4.2) are distinct from the phenomenon of exploded nuclei described above as the spilt chromatin is spread in the direction of the smear. It is due to the use of excessive pressure, but some types of malignant tumours are particularly prone to this problem and the artefact can be a clue to the diagnosis.

Fig. 4.2 Crushing artefact. These breast carcinoma cells show typical crushing and spreading of the chromatin. The tendency for this artefact to develop is a function of the fragility of the cells and the pressure used to make the smear.

A few red blood cells provide a scale for comparison with the size of the nucleated cells. The presence of a large amount of blood, however, will obscure other cellular or extracellular material and in air-dried aspirates the stains often do not penetrate a thick film of blood.

As ultrasound localisation of breast lumps is adopted more widely, cell lysis by contamination by ultrasound transmission jelly poses a threat to cytological diagnosis.³¹ The effect of the jelly on cell morphology is dramatic and, in experiments in vitro, varies with the length of exposure before fixation. The phenomenon is seen even in rapidly air-dried samples, presumably from the mixing of the jelly with the sample within the needle rather than on the slide. Initially, there is some swelling of the cell cytoplasm and nucleus (Fig. 4.3). This can make benign cells more worrying when compared with uncontaminated controls. Cell swelling is closely followed by leakage of nuclear chromatin, then complete dissolution of cell structure to form granular basophilic material and, eventually, a basophilic ‘soup’. The jelly may be misinterpreted as necrotic material or mucin. Radiologists regard ultrasound jelly as a bland substance. If made aware of its detrimental effects on aspirated cells, they can modify their technique to lessen the risk of contamination.

Fig. 4.3 Ultrasound jelly artefact. These breast carcinoma cells show and intermediate degree of damage as a result of the presence of ultrasound jelly. The nuclei show loss of the chromatin detail and the constituent of the jelly is seen as a purple granular precipitate. The inset shows the cells at a slightly more advanced stage of dissolution resulting from a greater concentration of the jelly (MGG).

Cytological material is well suited for immunocytochemistry (ICC), in situ hybridisation (ISH) and other molecular diagnostic or research procedures. Oestrogen receptor (ER) (Fig. 4.4), progesterone receptor (PgR) and HER-2/neu status are routine markers that are determined in all breast carcinomas. Although usually undertaken on histological material, with a careful technique they can be assessed using cytological specimens. ER and PgR are steroid receptors which are highly vulnerable to suboptimal handling of both cells and tissues. In cytological procedures, the critical point seems to be air drying of the smears at any point, both pre- and post-fixation. Air drying starts immediately after smearing and can be recognised on smears stained with antibodies to ER and/or PgR as a rim of negative tumour cell nuclei in the periphery of the smears. Optimal fixation of cytological material depends on the type of fixative. The most common is 4% buffered formalin, as in histology, or a three-step fixation with methanol, acetone and formalin. Some centres use 96% ethanol with acceptable results. The literature results favour liquid-based fixation.^32–37 Subject to adequate fixation and the absence of air drying, ER/PgR ICC results correlate closely with the histological findings.^38–44 Positive results should be expressed as the percentage of tumour cells showing positive nuclear staining⁴⁵ and can be used reliably in patient management, that is if >50% of cell nuclei are ER positive. If ER/PgR are either negative or weakly positive (<50% of the cells positive), the receptor status should be repeated on the histological specimen. Most institutions will perform ER/PgR primarily on the histology. However, in metastases and recurrences, resection is not always indicated and ER/PgR ICC is a valuable procedure in such cases. The advantage of using liquid-based cytology techniques is that the known positive cases can be used as controls. Cells in suspensions (stored at room temperature) and smears made from such suspensions may be stored for several months (at −20°C or −80°C) without losing their reactivity.

Fig. 4.4 (ER) Liquid-based preparation. Oestrogen receptor positivity in breast carcinoma cells.

HER-2/neu is a transmembrane growth factor receptor. Both protein overexpression and gene amplification are associated with poor prognosis. HER-2/neu status is a routine marker and is at present being evaluated on all breast carcinomas in order to predict responsiveness to anthracycline and trastuzumab (Herceptin®). Protein expression may be determined by ICC, but all commercial kits and procedures are standardised for histological rather than cytological specimens. Reports in the literature indicate a high correlation of cytological results with histological results and in situ hybridisation (ISH).^46–49

ISH, with fluorescent (FISH) chromogenic (CISH) or silver (SISH) visualisation, can be applied on cytological material,^45,50,51 both direct smears (Figs 4.5, 4.6) and preparations from liquid suspensions as well as on cell blocks (Fig. 4.7). Different fixations may require some modifications of the pre-treatment (demasking), but in general, cytological material requires little or no pre-treatment at all.^{46–50,52–54}

Fig. 4.5 CISH on direct smear: HER-2 gene amplification in breast carcinoma cells (not the same case as Fig. 4.6).

Fig. 4.6 CISH on direct smear: Chromosome 17 centromere probe in sheet of breast carcinoma cells showing polysomy (not the same case as Fig. 4.5).

Fig. 4.7 CISH on cell block: chromosome 17 centromere probe.

HER-2/neu status may occasionally vary between the primary tumour and the metastases. Recurrences and metastases should therefore, as a rule, have a re-determination of their HER-2 status. If resection or biopsy is not indicated, ISH on cytological material is a valid option. ISH signal counts on cytological material will be higher than on histological sections, because the nuclei are not truncated. The HER-2/CEP17 (chromosome 17) ratio will be the same and the cut-offs will be the same as for histology.⁵⁵

A number of other prognostic markers have been investigated using cytological preparations. Proliferation markers such as Ki-67, tumour suppressor gene TP53, EGFR, topoisomerase IIα as well as ploidy/DNA measurements have all been investigated, but in most institutions, these results have no therapeutic implications at present (see Ch. 34).^{46,47,56–58}

Complications are few and seldom serious. The only common problem encountered clinically is the formation of a haematoma. This is unpredictable and can be minimised by prolonged pressure following aspiration, but sometimes the bleeding occurs very rapidly, even before the needle is withdrawn. This can obscure the lesion, making a second attempt difficult or impossible. Haemorrhage following FNA may cause interpretive problems for mammograms and ultrasound images, and as a rule, the radiological investigation should be completed before FNA is done.

Pneumothorax has been reported as a very rare complication,^59,60 the most risk probably being in the axillary tail of a thin patient. Strict adherence to the method outlined earlier in this chapter should minimise this risk.

Needle tract seeding after FNA has been reported in various tumours but is rare in breast cancer.⁶¹

Histologically, the breast is composed of regularly arranged, radially disposed, independent glandular units forming a bush-like structure, with ducts as branches and lobules as berries. These 15–25 separate units end at the collecting ducts. The collecting ducts lead to lactiferous sinuses, distensible structures that act as a temporary reservoir for milk, which then form the lactiferous ducts that open onto the nipple. The glandular units contain the terminal duct-lobular units (TDLU). The TDLU, the most hormone sensitive part of the breast, is the main functional component, and therefore not surprisingly is the major site of origin for most of the pathological processes. There are tens of thousands of lobules in each breast. Subgross stereo-microscopy of cleared breast tissue reveals that the lobules overlap and mingle with those of adjacent segments.

All the glandular elements are surrounded by connective tissue. The undistinguished interlobular connective tissue contains a very variable amount of adipose tissue.

Accessory and ectopic breast tissue as well as intramammary lymph nodes are normal clinical variants. The breast is not a well demarcated or encapsulated organ and mammary lobules may be found beyond the normal anatomical boundaries. Most commonly, this is proved by the presentation of ectopic axillary breast tissue as a lump either in pregnancy or because of increased ‘breast awareness’. Aspiration of such a lump reveals normal breast tissue components only.

Lymph nodes from the lower axillary group may be found in the upper outer quadrant or even the lower outer quadrant of the breast where they may present as a lump. The combination of the clinical features and an aspirate of normal or reactive lymphoid tissue permits diagnosis and reassurance.

Cytological findings: the normal, mature breast

FNA from normal, adult breast tissue will contain variable amounts of ductal epithelial cell groups, small stromal fragments and fatty tissue. The epithelial cell yield is usually higher in younger age groups.

• Normal epithelial cells (Figs 4.8–4.10)

• Small cohesive groups

• Monolayer sheets

• Occasional complete TDLU

• Oval nuclei with regular outlines, 8–10 μm in diameter

• Inconspicuous nucleolus

• Evenly distributed chromatin

• Scanty cytoplasm

• Myoepithelial cells appear as ovoid, dense nuclei at the periphery of ductal sheets and groups

• Naked, bipolar (myoepithelial cell) nuclei in the background.

Fig. 4.8 Normal breast. A group of benign ductal cells surrounded by single bipolar nuclei in the background (PAP).

(Courtesy of Dr G McKee, Boston).

Fig. 4.9 The histology of normal breast lobules (H&E).

Fig. 4.10 The typical benign pattern (C2) of a sheet of cohesive epithelial cells with bipolar nuclei in the background (H&E).

Fatty tissue may be the sole component of breast aspirates and is a common additional finding in both benign and malignant aspirates. Fatty aspirates contain balloon-like fat cells in clusters of variable sizes, sometimes associated with strands of fibrocollagenous tissue or occasional capillaries. The stromal fragments are usually small and few. Occasional naked, spindled fibrocyte nuclei may be found. The number of naked myoepithelial cell nuclei will mirror the amount of epithelial cell groups and sheets. Platelets appear as small granular amphophilic aggregates. Skeletal muscle fibres are seen rarely, particularly if the correct aspiration technique is used. They appear as distinctive elongated cylinders with basophilic cytoplasm, on air-dried smears, orange on PAP-stained aspirates, with cross striations and peripherally located nuclei, intense blue on MGG. Organising haematoma may contain degenerate material, large spindle cells, large cells containing pigment and prominent nucleoli giving a potential for misdiagnosis as metastatic melanoma.

During the 6th–20th weeks of pregnancy, the breast undergoes intensive growth. At the cellular level, there is enlargement of both the nuclei and the cytoplasm of duct and lobular epithelial cells. This growth slows dramatically in the third trimester when secretory changes become more prominent. During lactation the secretory cells become smaller and flattened with vacuolation of their cytoplasm. Following the cessation of lactation, the breast undergoes an involutionary process over several months, until the TDLUs return to their resting state. The development of a mass during pregnancy may be due to an uneven response to hormonal stimulation or enlargement of a pre-existing lesion such as a fibroadenoma. Other masses, such as lactating adenoma and galactocele arise de novo in pregnancy. Most galactoceles are so characteristic clinically that no further investigation is undertaken and the majority resolve spontaneously. The commonest cause of a mass during lactation is the development of an abscess. The accompanying pain and erythema usually make the diagnosis obvious but anxiety over the possibility of an inflammatory carcinoma may lead to a request for FNA cytology diagnosis.

Tumours and tumour-like lesions that occur during pregnancy and lactation are usually benign. However, carcinomas do occur, and tend to be high grade and oestrogen receptor negative. Some are reluctant to do mammography during pregnancy. Evaluation of mammograms during pregnancy and lactation may be difficult because the breast tissue is usually very dense. Ultrasound and FNA cytology are the two most important tools in work-up of tumours and tumour-like lesions during pregnancy and lactation.

Cytological features of pregnancy-related changes

Acinar cells have abundant granular or vacuolated cytoplasm that is unusually fragile, frequently stripping away leaving naked nuclei in a granular ‘dirty’ background. The nuclei are large and round with active vesicular chromatin and a distinct large nucleolus. The nucleolus in lactating epithelial cells is larger than in most malignant breast tumours (Figs 4.11, 4.12). However, the presence of lipid-laden secretory material in the background is a helpful feature.

Fig. 4.11 Typical lactation pattern with a granular-vacuolated background due to cytoplasmic rupture of the fragile epithelial cells. Loose cellular sheets. Nucleoli are large, larger than in most breast carcinomas (MGG).

Fig. 4.12 Typical lactation pattern with a granular-vacuolated background due to cytoplasmic rupture of the fragile epithelial cells. Loose cellular sheets. Nucleoli are large, larger than in most breast carcinomas (PAP).

Cytological findings: pregnancy-related changes

• The aspirate may be moderately or markedly cellular

• The cells are single and well dispersed in a lipid rich foamy or granular background

• The cells and their nuclei are large; there is abundant vacuolated or wispy cytoplasm

• Bare nuclei are common

• The nuclei are round and uniform with active granular or vesicular, but evenly distributed chromatin.

• Single prominent nucleoli

Diagnostic pitfalls: pregnancy-related changes

The main risk is that the low-power impression of a cellular aspirate of single, large cells is taken as evidence of malignancy when the pathologist is not aware of the pregnant or lactational state of the patient. The distinctive granular background and critical assessment of the nuclear features should prevent this error.

It is important for the cytopathologist to be informed about pregnancy or lactation when the aspirate is performed by others, to lessen the likelihood of interpretive errors; one must also remember that foci of lactational change can occur unassociated with pregnancy and generalised lactation.

Galactoceles are often easily diagnosed clinically without need for further investigation. However, occasionally a galactocele can accumulate abundant inspissated milk to form a large mass, even up to 80 mm in diameter, and this can be clinically worrying. It generally arises shortly after pregnancy, during lactation. The diagnosis may be readily made from the history, confirmed by the aspiration of milk, a procedure both diagnostic and therapeutic. The smears show abundant secretory material with scattered foamy macrophages. Epithelial cells are rarely seen. In the light of the clinical details the diagnosis should be benign, not inadequate.

Milk granuloma is thought to arise when milk leaks into the mammary stroma. The aspirates are likely to have an inflammatory appearance.⁶²

Gynaecomastia is the enlargement of the male breast (Figs 4.13–4.15) due to hypertrophy and hyperplasia of both the glandular and stromal components. When arising before the age of 25, it is usually due to pubertal hormonal changes and commonly reverses spontaneously. In later life, the most common causes are drug therapy, androgenic steroid abuse, hormone producing tumours and cirrhosis of the liver with resulting failure to metabolise endogenous oestrogen.

Fig. 4.13 A typical pattern of benign (gynecomastia) ductal sheets and aggregates and stroma fragments (MGG).

Fig. 4.14 Gynaecomastia cohesive and complex aggregate of ductal epithelial cells and a moderate number of naked nuclei (MGG).

Fig. 4.15 Larger magnification of Fig. 4.14. Hyperplastic, three-dimensional aggregate with benign ductal nuclei and scattered myoepithelial cell nuclei (MGG).

Gynaecomastia may be unilateral or bilateral. The superficial nature of the mass in gynaecomastia generally makes sampling easy but it should be noted that the procedure is particularly painful in the male breast and good technique, even combined with the use of local anaesthetic, is important.

Histologically there is dilatation of large or intermediate ducts that are filled with secretion, and to a variable extent foamy macrophages, siderophages and cholesterol crystals that form the inspissated, pasty material seen on gross examination (Fig. 4.16). Epithelial proliferation is not a feature, but reparative changes in epithelium next to areas of inflammation may give rise to a spurious impression of atypia both histologically and cytologically. Rupture of the epithelial layer and basement membrane cause chronic inflammation, fibrosis and scarring of the surrounding stroma. It is usually an incidental finding in FNA or seen in smears of nipple discharge. Smears contain secretory debris and a variable number of foamy macrophages. Macroscopically the aspirated material often appears thick, creamy and homogeneous. Much of the material appears to dissolve in methanol fixative.

Fig. 4.16 Duct ectasia. This aspirate contained numerous foamy macrophages (upper arrowhead) and amorphous debris (lower arrowhead). There is little if any epithelium. The appearances are typical of those seen in duct ectasia. The inset shows the histological appearances of the same case (PAP).

Fat necrosis of the breast (Fig. 4.17) may be associated with mammary duct ectasia and fibrocystic change when there is rupture of a duct or cyst causing extravasation of contents with secondary necrosis of adjacent fat. It may also follow surgery and radiotherapy. Traumatic fat necrosis tends to be more superficial, often occurring in the subcutaneous fat, rather than within the breast itself. There is usually a history of injury 1–2 weeks, or even longer, before the lump is noted and there may or may not be bruising. The lesion is often tender on palpation. Clinically and/or radiologically, the lump may be suspicious, even with a hint of skin tethering in healing cases. Radiological findings may show an image that is typical of fat necrosis, but often mammography and/or ultrasound imaging is equivocal and occasionally suspicious for malignancy.

Fig. 4.17 Fat necrosis. (A) At low power the pattern may be dominated by focal under-staining artefact due to impenetrable lipid, with foamy macrophages, lymphoid cells and sometimes lipid crystals visible in the better stained areas (MGG). (B) Occasionally histiocytes can demonstrate severe nuclear atypia which may mimic carcinoma (MGG).

Post-surgical, or post-traumatic fat necrosis can persist for some years. Fat necrosis may be seen in old surgical sites as lumps or thickenings which become more prominent after the menopause as the surrounding breast atrophies, and may be both a clinical and a cytological mimic of recurrent cancer.

FNA of fat necrosis tend to be thick, granular and fatty when spread and contain many foamy macrophages and variable numbers of multinucleate giant cells. Other inflammatory cells may be present, but there is usually a paucity of epithelial elements. The background contains fatty globules and fragments of fatty tissue, some showing degeneration. Occasionally, particularly in cases of old or membranous fat necrosis, an aspirate may contain only oily lipid material, with few or no cells visible.

Clinically, this lesion may mimic carcinoma, as there can be retraction of the nipple associated with a well-defined lesion, usually centrally located. In a fifth of cases there is nipple discharge. It can be seen as part of the spectrum of duct ectasia. Mammography may reveal tubular, annular or linear calcifications. The aetiology is not clear but the basic abnormality is stagnation of secretion, possibly due to loss of elastin support in duct walls, leading to ectasia. There is an association with cigarette smoking.

Histologically, there is a spectrum of appearances reflecting the different stages of the disease process, but plasma cells are a characteristic finding. Macroscopically, the aspirated material may have the same appearance as simple duct ectasia.

Microscopically, there is amorphous debris with variable numbers of foamy macrophages and other inflammatory cells. Occasionally small numbers of reactive epithelial cells with degenerative and/or reparative changes are seen.

Granulomatous mastitis (Figs 4.18, 4.19)

Various systemic and local conditions can give rise to the formation of a granulomatous response in the breast. Some cases of granulomatous mastitis are part of the spectrum of duct ectasia. There is a distinct group in which the granulomatous inflammation is lobulocentric⁶³ and associated with either recent pregnancy or another cause of high serum prolactin levels, such as phenothiazine therapy. Sarcoidosis of the breast is rare. Tuberculosis (Figs 4.20, 4.21) of the breast is more common in developing countries.⁶⁴ In the developed world, tuberculous mastitis is usually found in immigrants from countries with a high prevalence of tuberculosis.

Fig. 4.18 Irregular histiocytic aggregates in a case of granulomatous mastitis in a woman with SLE (MGG).

Fig. 4.19 Silicone granuloma. (A) Loose histiocytic aggregates in a granulomatous reaction to silicone from an implant (MGG). (B) Foamy macrophages are associated with intracellular and extracellular globules of refractile, colourless silicone. The presence of other inflammatory cells is variable (MGG).

Fig. 4.20 Tuberculous mastitis. This aspirate contained numerous groups of epithelioid histiocytes and occasional giant cells. Cultures and histology confirmed a diagnosis of tuberculous mastitis (MGG).

Fig. 4.21 Tuberculous mastitis. Histological section (H&E).

Other entities that display granulomatous features on aspirates include granulomatous autoimmune disorders (Wegener’s granulomatosis,⁶⁵ rheumatoid nodule, giant cell arteritis) as well as fungal infections, foreign body reaction to implanted silicone and tumours. Clues to the most probable aetiology of the granulomatous infiltration may be had from the gestational history, previous medical history, ethnic origin or other clinical findings, but the cytological features will rarely allow a specific diagnosis.

As few, if any, of these diseases benefit from surgical intervention, the provision of a specific diagnosis on aspirated material is particularly gratifying. Additional material for special stains (Fite, Ziehl-Neelsen, Grocott, PAS) is important to identify specific organisms.

Idiopathic granulomatous mastitis^66–72 has no known aetiology. It can be seen as a clinical mass, ranging in size from <1 cm to 8 cm and may mimic carcinoma clinically, radiologically and cytologically.^73,74 The diagnosis depends on the exclusion of other causes of granulomatous inflammation in the breast.

Breast abscesses and acute mastitis (Fig. 4.22) occur most commonly, but not invariably, in the puerperium. The diagnosis is usually made clinically and effective antibiotic treatment is given without need for a cytological or tissue diagnosis. Occasionally, however, resolution does not occur or is slow and surgical drainage is planned. The possibility of an inflammatory carcinoma may then be considered and an FNA diagnosis sought.

Fig. 4.22 Breast abscess. This aspirate contains numerous macrophages and neutrophil polymorphs with little if any epithelium (PAP).

Aspirates contain neutrophil polymorphs and macrophages in considerable numbers as well as abundant cell debris. Reactive/reparative epithelial cells, derived from adjacent inflamed and possibly lactating breast tissue may also be found.

Subareolar abscess (Fig. 4.23)

This condition is thought to have some affinity with mammary duct ectasia. There is often a history of the recurrent formation of a tender mass in the subareolar region, sinus tract formation and discharge with partial healing. Histologically, excised lesions are seen to consist of an inflammatory sinus tract lined by granulation tissue but often partially by squamous epithelium. FNA consists primarily of inflammatory exudate but additionally there may be anucleate squames, multinucleate giant cells and epithelium showing reactive atypia. The cytological appearance is not dissimilar to that of an infected or ruptured epidermal cyst except that these do not contain ductal cells.

Fig. 4.23 Subareolar abscess with a mixed inflammatory infiltrate with many neutrophilic granulocytes, anuclear squames, macrophages and multinucleated histiocytic giant cells (MGG).

This lesion, also referred to as lymphocytic or diabetic mastopathy, has well-documented histopathological features of dense lobulocentric lymphoid infiltration associated with marked stromal fibrosis. There is an association with insulin-dependent diabetes mellitus, thyroiditis and arthropathy, although many cases are sporadic without known underlying disease. An overlap with fibrous disease is apparent. It can present as a lump and it might be expected to be the target of a FNA. The cytological features are not specific, merely showing a paucicellular benign pattern with lymphocytes.⁷⁵ These lesions can be clinically quite suspicious, and the condition is frequently identified in a core biopsy following scanty benign or non-diagnostic cytology in the presence of an irregular, hard mass.

Localised collections of amyloid can occur in a variety of sites including the breast.⁷⁶ These deposits have been termed amyloid tumour and present as a solitary localised mass, rarely bilateral. It occurs in older women and can be firm or hard and discrete. It can imitate carcinoma clinically and mammographically.

Cytological preparations show amorphous translucent material similar to thick thyroid colloid. This material stains violet with May-Grünwald Giemsa and pale pink with the Papanicolaou method. Scanty bland spindle cells are seen, and more rarely, reactive giant cells are a feature. Faced with this picture, the pathologist can take further aspirates for Congo red staining to confirm the diagnosis.

Silicone implants quite frequently leak, which may give rise to a local mass and to axillary lymphadenopathy (Fig. 4.19).⁷⁷ Aspirates from the breast or lymph node contain numerous macrophages containing large cytoplasmic vacuoles of refringent silicone as well as giant cells of foreign body type, and often free silicone. Silicone spreads as a very sticky translucent, colourless material similar to DPX mountant. Silicone granuloma can persist for many years after removal of an implant.

Cytopathologists should be aware of the risks of sampling breast lesions adjacent to mammary prostheses. If possible, these lesions should be aspirated under ultrasound guidance to avoid penetration of the implant capsule.

Parasitic lesions in the breast are rare. The cytological appearance of cysticercosis of the breast⁷⁸ has been described. Myospherulosis is a pseudomycotic condition that can occur following the subcutaneous injection of penicillin and presents an intriguing histological appearance. Altered erythrocytes coated with lipid are deposited in the tissues within spherules. In smears they stain brown with MGG staining, but are red in PAP preparations and are negative with PAS and silver stains.

In parts of Asia, some women have injections of foreign material to increase their breast size. This gives rise to extraordinary mammographic and ultrasound findings. Cytologically the most common finding is a foreign body reaction with multinucleated giant cells and lymphocytes. Remnants of the foreign material may be aspirated and may appear oily on the smears. Dystrophic calcified debris may be found.

Fibrocystic change (Figs 4.27–4.31)

Clinically, the appearance of this, the most common cause of a palpable breast lump, has certain characteristic findings. The typical patient is between 30 and 50 years old, but an age range of 25–70 has been quoted. Often, there is a convincing history of change of the breast lump with the menstrual cycle and fibrocystic lesions are more commonly tender or painful than malignant ones. The palpable lesion is not always well-defined and may range in size from a few millimetres to a change occupying the whole breast.

Fig. 4.27 Fibrocystic change with a benign pattern (C2) of ductal cell sheets, bipolar nuclei, cyst debris and macrophages in the background (H&E).

Fig. 4.28 Foamy macrophages in benign fibrocystic change (MGG).

Fig. 4.29 Foamy macrophages in benign fibrocystic change (PAP).

Fig. 4.30 Benign breast epithelium. This field illustrates the typical appearance of benign breast epithelium. The apparent anisonucleosis is spurious and due to the presence of a number of interspersed myoepithelial cells (arrowheads) (MGG).

Fig. 4.31 Hyperplasia. The typical appearances of benign hyperplastic breast epithelium. Occasional bipolar nuclei are seen, bottom right (PAP).

The condition may be synchronously bilateral, but often the first presentation is as a solitary lesion. Patients are prone to developing multiple sequential lumps and thus may be less anxious about the appearance of further masses than women presenting with the first breast lump. Each lesion must be assessed on its own merit. Most fibrocystic lesions are radiologically benign or equivocal. Some fibrocystic lumps can present appearances that are worrying clinically, radiologically and histologically.

The spectrum of histological appearances generally included under the heading of ‘fibrocystic change’ is very wide. The basic histological elements are:

Following examination, the aspirator gains a further important diagnostic clue when the needle enters the lump. The fibrous elements frequently present in this condition will often have a distinctly rubbery or leathery feel to the needle. The cellularity of the sample usually bears a relation to the ease with which the specimen is obtained. When the needle is gripped by leathery fibrous tissue, a poorly cellular aspirate may be anticipated and a little more vigour can be applied in the movement of the needle. In other cases, the aspirate will often appear watery allowing it to be spread over the whole of one or two slides with ease.

The amount of material for microscopy varies considerably, depending on whether the lesion is from the fibrous or proliferative end of the spectrum seen in this condition. The basic pattern is benign, but there may be several cell types present. The most reassuring component is the presence of obviously benign apocrine cells. These may be in large cohesive sheets or dispersed singly or in small groups. The nuclei are large, round and relatively hyperchromatic. The nucleoli are large and prominent. The cytoplasm is abundant and usually granular. Cell borders should be well-defined in contrast to the cells of a low-grade apocrine carcinoma, which have wispy poorly defined cytoplasmic margins. Further reassurance is obtained from the presence of bipolar bare myoepithelial nuclei.

The typical clinical presentation of fibroadenoma (Figs 4.32–4.34) is a firm, discrete and highly mobile lump in a young woman.⁷⁹ Fibroadenoma most commonly presents in women between the ages of 20 and 35 years, but can come to the attention of the patient for the first time in later life, sometimes after unrelated weight loss. The use of mammography has increased the diagnosis of longstanding fibroadenomas in older women. These lesions are often of the poorly cellular ‘ancient’ type. The size may vary from a few millimeters to several centimeters, but is usually within the range of 5–30 mm. On mammograms, fibroadenomas typically present as round, well-defined lesions. On ultrasonography they appear solid, round or round-oval, with a distinct margin all around. Particularly in older women, the margins may become less distinct leading to recall and biopsy in mammographic screening.

Fig. 4.32 Fibroadenoma. Pink stromal fragment, epithelial groups and naked nuclei in the background. Typical of FA (MGG).

Fig. 4.33 Fibroadenoma. Stromal fragment and cohesive sheet of epithelial cells and a few naked nuclei in the background (MGG).

Fig. 4.34 Fibroadenoma. Monolayer sheet with some smaller, darker myoepithelial cell nuclei and naked nuclei (MGG).

The risk of developing carcinoma within a fibroadenoma or in a breast previously treated for fibroadenoma is not significantly increased, although, as both carcinoma and fibroadenoma are relatively common lesions, they may occur together. Fibroadenomas may also be colonised by in situ carcinoma.

The cytological features of fibroadenoma closely reflect the histological features. When the cytopathologist has the opportunity to obtain the aspirate, the clinical features and cytolog-ical appearances allow a specific diagnosis in many cases.

Microscopically, the diagnosis is often obvious at low power with characteristic large frond-like epithelial groups with peripheral finger-like projections. These are sometimes likened to the antlers of stags. At high power, this epithelium is composed of closely packed, uniform cells with an irregular honeycomb appearance, best visualised on the Papanicolaou-stained smear. In air-dried preparations, sometimes it is only at the edge of the epithelial groups that the cells will be sufficiently flattened to allow satisfactory close examination. The nuclei are approximately the size of one or two erythrocytes and are round or slightly ovoid, having one or two small nucleoli and finely granular chromatin. Myoepithelial cells are seen scattered over the surface of the sheets of ductal epithelial cells.

The other essential feature is the presence of an often generous population of bare nuclei, which include stripped epithelial and myoepithelial nuclei, and some stromal cells. The bipolar cell nuclei have condensed chromatin packed into a small elongated nucleus. Although some of the nuclei are truly naked, probably representing myoepithelial cells, many are stromal cells displaying more spindly nuclei and a strand of pale blue cytoplasm at each pole, seen best under high power. In some fibroadenomas, a scattering of foamy macrophages or apocrine cells is seen. Classically aspirates from fibroadenomas are described as containing large fragments of stroma, coloured magenta by Giemsa stains. In clinical practice, the amount of stroma is variable, reflecting the histological spectrum of fibroadenomas. More fibrotic fibroadenomas may yield no stroma at all and have a rather non-specific benign pattern on FNA. At the other end of the spectrum fibroadenomas with abundant cellular stroma merge with phyllodes tumours both histologically and cytologically.

There are circumstances in which aspirates from fibroadenomas can pose diagnostic traps leading to false positive diagnosis of carcinoma: The very high cellularity of fibroadenoma aspirates can cause concern particularly if an aspirate is over-spread, leading to artefactual dissociation, or if a smear is poorly prepared and subject to, for example, slow drying artefact which makes the cells appear large and atypical.

A few FA smears may contain cells with cytological atypia such as enlarged nuclei with more prominent nucleolus, irregular nuclear margin, but uniform chromatin pattern. The cytoplasm is often dense. These cells may occur in small irregular groups or as single cells and may appear suspicious of malignancy. Some of these are probably myoepithelial hyperplastic cells. In histological sections, they usually appear rather inconspicuous, but in a few cases there may be intraductal hyperplasia with or without atypia. It is important that the general low power pattern is fully appreciated before looking at the aspirate at higher power when the spuriously worrying features may be apparent. The recognition of bipolar cells and stromal fragments is also particularly helpful in these cases.

Some fibroadenomas have a stroma that is so highly myxoid that on smears, the pattern can mimic mucinous carcinoma.

Histological variants that may lead to special cytological features include the occurrence of apocrine metaplasia, haemorrhagic infarction, especially during pregnancy, squamous metaplasia (although this is more common in phyllodes tumour) and stromal metaplasias, including the formation of smooth muscle, cartilage, bone or dystrophic calcification. The formation of bone and calcification is commoner in older women and is likely to become apparent on taking the aspirate, as the needle may refuse to enter the mass or may give a very gritty sensation. These lesions typically reveal scant cellularity. A few stromal fragments are usually present, but epithelial cell groups may not always be seen.

There is a histological and cytological overlap with phyllodes tumours at the benign end of the phyllodes spectrum. In aspirates from a clinical fibroadenoma showing highly cellular stromal fragments, very large numbers of plump, naked bipolar nuclei with hyperplastic duct cells, giant cells and an absence of apocrine cells, a diagnosis of phyllodes tumour should be considered. This is more likely in older women.

The clinical features of this lesion are indistinguishable from fibroadenoma and occasional examples show areas resembling classical fibroadenoma.⁸⁰ This suggests some homology between these conditions. Histologically, these lesions consist of a mass of densely packed benign tubular structures with a double layer of epithelial and myoepithelial cells but with very little stroma between the tubules. They represent the part of the spectrum of fibroepithelial neoplasms where the stromal component is minimal. Tubular adenomas tend to be easy to aspirate, being softer than the average fibroadenoma.

The cytological features are similar to those of a fibroadenoma but there are fewer bipolar cells and the larger complex epithelial sheets are not a feature. The relative rarity of these lesions makes it unlikely that a specific diagnosis will be made by FNA cytology and a preoperative diagnosis of fibroadenoma, fibroadenosis or non-specific benign lesion is likely.

Lactating adenoma, contrary to what the name might suggest, occurs more commonly during rather than after pregnancy. Its origin is controversial, but many regard it as a fibroadenoma or tubular adenoma modified by the hormonal influences of pregnancy. Foci of lactational change with identical histological and cytological features may occasionally be seen within fibroadenomas and other benign breast changes outside of the context of pregnancy. It is in that unexpected setting (or indeed in the context of known pregnancy that is not included in the clinical details provided to the cytologist) that failure to appreciate lactational changes in an aspirate from a clinical mass has a serious risk of diagnostic error. Aspirates show moderate numbers of cells singly or in groups, including intact lobules and acini. Most cells, however, are dispersed. There is a characteristic granular or foamy background consisting of cell cytoplasm fragments and lipid. The intact cells show obvious cytoplasmic vacuolation and have large round or ovoid nuclei with a smooth nuclear membrane and fine chromatin with a prominent nucleolus. The appearances are thus similar to those of the pregnancy-related changes. Cytometric analysis reveals no statistically significant difference between the mean nuclear areas of lactating adenoma cells and those of well-differentiated ductal or lobular carcinoma. There is therefore the risk that these lesions will result in a false positive diagnosis.

Phyllodes tumours⁸¹ (Figs 4.35, 4.36) form a spectrum of fibroepithelial tumours from benign with a strong resemblance to fibroadenomas, through borderline with notable stromal overgrowth and proliferation to malignant, in which the stroma is frankly sarcomatous. Benign phyllodes tumours have a propensity for local recurrence following surgery, and the borderline or low-grade malignant ones may recur at a higher grade. Phyllodes tumour is, therefore, an important lesion to consider when assessing an aspirate from a fibroepithelial tumour.

Fig. 4.35 Benign phyllodes tumour. Loose fragment of spindle cells with small, but recognisable nucleoli. Some stromal cells are more polygonal and have ample cytoplasm (MGG).

Fig. 4.36 Benign phyllodes tumour. Large fragment of moderately cellular stroma (MGG).

Benign phyllodes tumour is seen in women on average 20 years older than those with fibroadenoma. There is, however, considerable overlap and, while phyllodes tumours tend to be larger on presentation, they may present at any stage of development. Conversely, fibroadenomas may reach 100 mm or more in diameter before the patient seeks help. The differentiation between giant fibroadenoma and benign phyllodes tumour depends entirely on the histological appearance of the stroma and the distinction is frequently impossible cytologically. Late presentation, particularly in a large breast, has resulted in some very large specimens of phyllodes tumour.

On examination, these tumours are softer, less mobile and less well-defined than fibroadenomas. There is usually little resistance to the needle, unless the rare occurrence of metaplastic bone or cartilage is encountered. The aspirate is generally blood free and appears glairy when spread.

The smears are usually very cellular and the differentiation from a cellular fibroadenoma can be impossible. The clinical features are of assistance in balancing the probabilities. Additionally, the cellularity of the stromal fragments and the size and possible atypia of the many dispersed stromal cells is important in suggesting the diagnosis. MGG-stained preparations may reveal pink or purple staining ground substance within the stromal fragments. In an aspirate from a probable phyllodes tumour, the most critical cytological feature in deciding whether the lesion may be benign, borderline or malignant, is the number of and the degree of atypia of the stromal cells. They may occasionally be frankly malignant cytologically; more commonly the atypia is of a lesser degree suggesting a borderline lesion (see p. 204). Numerous mitoses might be an indication of a malignant lesion, buy are usually not so prominent in cytological material as in histological sections. Even histological assessment is difficult in this regard. There is a distinct risk of making a false positive diagnosis of malignancy when assessing a benign phyllodes tumour. Occasional phyllodes tumours contain keratin cysts leading to further diagnostic problems.

Although difficult to achieve with certainty, there is considerable value in making a correct preoperative diagnosis of phyllodes tumour as the enucleation that would be adequate for a fibroadenoma may result in a local recurrence if applied to a phyllodes tumour. Most surgeons would wish to take a margin of normal tissue around a known or suspected phyllodes tumour.

The histological features of these uncommon lesions are well described, consisting of varying amounts of breast parenchyma and adipose tissue. Cytologically this lesion cannot be reliably distinguished from normal breast tissue, fibroadenoma or other cellular benign lesions.

The clinical picture is usually benign, but can be highly suspicious.

Histologically, the basic appearances described as fibrocystic changes frequently include epithelial proliferative changes of various types. Cytologically, this may be suspected when an aspirate, otherwise typical of ‘simple’ fibrocystic change, is cellular with the additional cells being non-apocrine type. These proliferative changes also commonly occur without more conventional fibrocystic change. The histological changes include adenosis (sclerosing, microglandular), hyperplasias (intraductal hyperplasia, columnar cell lesions, duct papillomatosis), atypical ductal hyperplasia, adenomyoepithelial hyperplasia, adenosis tumour, apocrine adenosis with and without sclerosis, radial scar and complex sclerosing lesions.

One rare variant of benign epithelial hyperplasia that does have a distinctive cytological appearance is collagenous spherulosis. In May-Grünwald Giemsa-stained preparations the collagenous spherules are very conspicuous and appear magenta in colour. Collagenous spherulosis is of no clinical significance, but cytologically it may be confused with adenoid cystic carcinoma. It should be excluded when a diagnosis of adenoid cystic carcinoma is being considered.

Microcalcifications can be recognised in cytological preparations when present. They are of relatively little assistance in microscopic diagnosis as they look morphologically similar in both benign and malignant lesions. However, they are useful in confirming that a mammographic lesion containing microcalcifications has been sampled.

Cytological findings: epithelial hyperplasia

• Low or moderate cellularity with small epithelial groups or high cellularity with large flat or folded sheets and three-dimensional aggregates of cohesive regular cells

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