Oesophagus and gastrointestinal tract

• Brushing under endoscopic view of the lesions is considered a good method to enhance detection of early lesions as it can represent more than the 1% of tissue obtained by biopsy alone.¹⁰ The endoscopic brush should be employed like a needle rather than as a swab and jabbed through the surface mucosa or ulcer slough. Brush samples have advantages over the usual small biopsies since they include surface mucus or exudates from wider, sometimes unrelated mucosal surfaces and inevitably include exfoliated material. This is perhaps best explained by considering the identification of Helicobacter pylori in disposable brush wash preparations. The organisms are usually best seen in free wisps or trails of mucus rather than in close association with an epithelial surface as in histological sections. They are commonplace within oesophageal and duodenal brushes as a result of the wide inclusion of a sometimes cellular exudative gastric mucus sample within an otherwise directed brush sample. This of course considerably improves the yield from such samples on direct comparison with biopsy. Similarly, brush samples are superior to biopsy for the detection of the commoner types of epithelial malignancy in the oesophagus and stomach, and certainly for ulcerated epithelial malignancy. On the other hand, biopsy is superior for the detection of submucosal spread of signet-ring cell carcinoma of the stomach, for the rare submucosal glandular or endocrine tumours, primary lymphomas and for non-epithelial stromal tumours

• Imprint or crushing of biopsy fragments in addition to brushing, as previously mentioned, are well-documented techniques to detect H. pylori^11,¹²

• Salvage cytology and washings are used much less often now. Salvage cytology is a procedure complementary to biopsies, intended to retrieve cells from the biopsy forceps and endoscopic tube

• Washings are generally badly tolerated by patients and clinicians do not feel comfortable with a morphological diagnosis that has no endoscopic correlation

• Endoscopic fine-needle aspiration biopsy with or without ultrasound-guidance (EUS-FNA) has gained relevance in the diagnosis of lesions not directly seen with the endoscope or in those where forceps biopsy failed to reach a diagnosis. It has clinical relevance because it changes diagnostic and therapeutic procedures, as for example, when staging oesophageal carcinoma. It is particularly important that cytopathologists assess the specimen adequacy and determine which ancillary studies will be needed at the time of the procedure.²

Cytological material can be prepared by direct smear with immediate fixation in alcohol or air-drying. Other authors prefer to rinse the brushes in a preservative liquid with the subsequent use of cytocentrifuge or liquid based cytology (LBC). Also cell blocks can be prepared either from the FNA needle or from other collecting devices. This methodology is particularly useful for immunocytochemistry and molecular biology studies.

Papanicolaou stain (PAP), May–Grünwald Giemsa (MGG) or similar stains and haematoxylin and eosin (H&E) are used according to local custom and which yield the best results. Ancillary techniques, including routine histochemistry for microorganisms, can also be applied, even on pre-stained slides. Immunocytochemistry, flow cytometry, molecular biology techniques and morphometry can be used in selected cases.

General principles

One should always apply the principle of getting as much information as one can to establish a good rapport with clinicians and endoscopists. This is particularly important when dealing with EUS-FNA. Smears can be evaluated on site, which improves the cell yield and avoids repeating procedures later.¹³

At the microscope, both high- and low-power views are important. On low power we evaluate the background (mucous, inflammation, necrosis), overall architecture of the cell groups and how they are arranged on the smear. With high power we apply the classical cytological criteria for the nucleus, nucleoli and cytoplasm to categorise the cells as reactive, dysplastic, benign or malignant. Interpretation and diagnosis when possible should be based on surgical pathology terminology. Although we cannot always give a specific diagnosis, a clinically relevant comment should be given.

Pitfalls

• One must bear in mind that heterotopias such as cardiac gastric glands, remnants of ciliated epithelium, sebaceous and pancreatic or endocrine cells in the oesophagus and stomach may occur and cause interpretive problems

• The cytological response to inflammation can be worrisome. Repair and malignancy can be very difficult to distinguish and criteria such as cell grouping, enlarged nuclei, prominent nucleoli and mitoses can be seen in both settings.¹ Tumour diathesis must be distinguished from ulceration slough. On the other hand, the presence of intact single cells, loss of polarity, irregular nuclear borders, irregular and multiple nucleoli and abnormal mitotic figures can be more reliable for the diagnosis of malignancy. Delicate chromatin is often seen in repair albeit with an enlarged nucleus and prominent nucleoli

• Granulation tissue from the base of an ulcer can pose a diagnostic problem with spindle cell tumours, particularly in HIV-positive patients where multiple infections and tumours occur together

• Radiation and chemotherapy can induce cytological atypia, most frequently described in the squamous epithelium of the oesophagus, as seen in the uterine cervix, but these changes can also occur in the glandular gastric lining ¹⁴

• Contaminants rarely pose a diagnostic problem, unless they obscure and entrap the diagnostic cells. Vegetable cells and other food debris, tissue fragments and talc are among the commoner ones found.

Oesophagus

Normal cytological findings

The normal components of oesophageal cytology are the superficial and intermediate cells of the multilayered squamous epithelium. Oesophageal brush smears in the absence of disease are usually paucicellular. Superficial cells are eosinophilic while intermediate cells are more basophilic, both having voluminous delicate cytoplasm. The nucleus is pyknotic in the more mature cells while in the intermediate type it is slightly larger and the cytoplasm is finely granular. On vigorous sampling, parabasal cells can be present, having larger darker nuclei and basophilic cytoplasm. Sheets, clusters and pearl-like arrangements can be seen. Glandular cells from the gastric epithelium are often seen and should not raise the possibility of Barrett’s oesophagus (Fig. 7.1). Oral bacteria, sometimes overlying superficial squamous cells from the oral mucosa, food contaminants and respiratory cells can also be seen (Box 7.2).

Fig. 7.1 Normal oesophageal cytology. Observe squamous cells and normal glandular cells from gastric epithelium. Their presence does not imply a diagnosis of Barrett’s epithelium (PAP).

Box 7.2 Normal components of oesophageal brushings

• Superficial and intermediate squamous cells

• Gastric epithelium

• Respiratory cells, macrophages

• Contaminants, food particles

Inflammatory conditions

Inflammation induces a cellular response that can be morphologically worrisome. Cells from the basal layer along with the more superficial layers can appear isolated or in small groups reflecting regenerative epithelium (Fig. 7.2). Injury can be induced by infections, local action of chemicals and radiation.

Fig. 7.2 Oesophageal brush smear showing regenerative epithelium from basal layers. Observe the presence of nucleoli and cytoplasmic vacuolation (PAP).

Reflux (peptic) oesophagitis

An incompetent sphincter mechanism, either transient or chronic, may lead to reflux of acid gastric contents into the lower oesophagus. A variety of conditions including sliding hiatus hernia, excessive alcohol consumption, pyloric stenosis, diabetic autonomic neuropathy, scleroderma and previous surgery, among others, may result in persistence of the reflux. The non-keratinised stratified squamous mucosa appears less resistant to acid or bile than the mucus and alkali-protected specialised columnar gastric glandular mucosa. This results in acute inflammation, regenerative squamous hyperplasia, keratosis and subsequent healing, gastric/intestinal type metaplasia or chronic ulceration.

Cytological findings: reflux (peptic) oesophagitis

Typical brushing appearances in reflux oesophagitis will include some or all of the following features depending on a combination of the intensity or extent of the changes:

• Increased squamous cellularity and discohesiveness (squamous hyperplasia)

• Clumps of irregular parabasal type squamous cells with enlarged reactive hyperchromatic nuclei and multiple nucleoli

• Increased polymorphonuclear neutrophils and mucus admixed with the sheets of squamous cells, sometimes also associated with lymphocytes, plasma cells, macrophages and cell debris (Fig. 7.3)

• Eosinophils, which are associated with acid reflux and chemical inflammation

• Ulcer slough consisting of clumps of mixed inflammatory cells, macrophages, degenerate epithelial cells, fibroblasts, smooth muscle cells, red blood cells and small capillary or vein walls.

Fig. 7.3 Oesophageal brush smear from a patient with reflux oesophagitis. Note the high proportion of polymorphonuclear neutrophils (PAP).

Barrett’s oesophagus

Barrett’s oesophagus, a complication of chronic gastro-oesophageal reflux disease, is a pre-malignant condition for adenocarcinoma of the oesophagus and the oesophago-gastric junction. In the definition of the American College of Gastroenterology, Barrett’s oesophagus is a change in the oesophageal epithelium of any length that can be recognised at endoscopy and is confirmed to have intestinal metaplasia by biopsy (Fig. 7.4). Three types of epithelium are recognised in Barrett’s metaplasia: oxyntico-cardiac, cardiac and intestinal. Oxyntico-cardiac and cardiac types are identical to the corresponding gastric epithelial regions and one should be cautious in rendering a diagnosis of Barrett’s metaplasia in lower oesophagus samples.

Fig. 7.4 Intestinal type metaplasia in Barrett’s oesophagus identified when goblet cells are present along with columnar cells, sometimes with an identifiable brush border (H&E).

(Courtesy of Professor Paula Chaves, Instituto Português de Oncologia Francisco Gentil, Lisboa.)

Cytological findings: Barrett’s oesophagus

• Intestinal type metaplasia is identified when goblet cells are present along with columnar or cuboidal cells. A considerable amount of epithelium folded or in sheets, is necessary for recognition of the ‘portholes’ which are mucus cells seen from above/below (Fig. 7.5). The villous border is more difficult to see in cytology preparations than in sections

• Gastro-oesophageal reflux is the major cause for Barrett’s metaplasia so one can expect to have some degree of inflammatory background

• There is debate about the feasibility of using cytology to diagnose glandular dysplasia in this setting.^15,¹⁶ While there is a good agreement concerning high-grade lesions and neoplasia, in low-grade lesions it is more difficult to reach good inter-observer concordance. Meanwhile, in most centres both cytology and histology are used to improve the sensitivity and specificity of the diagnosis (Fig. 7.6)

• The criteria used favouring dysplasia are, as described above, enlarged nuclei and apparent nucleoli with delicate chromatin (Fig. 7.7).