chapter 5 Peritoneal washing cytology (PWC) was introduced in the 1950s as a way to identify microscopic spread of cancer not visible by gross inspection of the peritoneal surface.1 In some cancer patients, positive PWC may be the only evidence of metastatic disease to the peritoneum. Because positive results correlate with a poorer prognosis,2,3 cytologic findings are included in the staging systems for ovarian and fallopian tube cancers.4,5 The yield, however, is low: Positive washings by themselves change the surgical stage of only 3% to 5% of women with gynecologic cancers.6,7 PWC is also used to exclude an occult cancer in patients who undergo laparoscopy or laparotomy for presumed benign gynecologic conditions, such as endometriosis or leiomyomata, and in women with BRCA1 and BRCA2 mutations undergoing risk-reducing salpingo-oopherectomy.8 PWC can be employed to monitor a patient’s response to treatment for advanced ovarian cancer and other malignancies (the “second-look” procedure), but this is usually limited to patients in research protocols.9–13 In some instances, PWC is used to detect peritoneal spread of nongynecologic malignancies like pancreatic and gastric cancer. On entering the peritoneal cavity, the surgeon evacuates any preexisting peritoneal fluid and submits it separately for cytologic examination. Washings are obtained by instilling 50 to 200 mL of sterile saline or other physiologic solution into several different areas, usually the pelvis, the right and left paracolic gutters, and the undersurface of the diaphragm. The fluid is aspirated, and heparin is added to prevent clotting. Segregating washings from different sites does not have any advantage over combining them into a single specimen.14 The specimen should be delivered to the laboratory unfixed and refrigerated at 4° C until slides can be prepared. If a significant delay before cytopreparation is anticipated, an equal volume of 50% ethanol can be added. To prepare slides, the specimen is thoroughly mixed, and an aliquot (often 50 mL) is centrifuged to a cell sediment/pellet. From this sediment one can prepare smears, cytocentrifuge preparations, or thinlayer preparations, depending on the resources and preferences of the laboratory.15 The remaining (or separately centrifuged) cell sediment can also be fixed in 10% formalin, embedded in paraffin, and processed as a histologic specimen (“cell block”). Cell block sections are very useful, especially for morphologic comparison to the patient’s resected neoplasm.16 Results of PWC are commonly reported as “negative,” “atypical,” “suspicious,” or “positive.”17 “Atypical” (connoting a low degree of suspicion for malignancy) and “suspicious” (connoting a high degree of suspicion) interpretations should be avoided whenever possible, because they are not helpful to a physician faced with making a treatment decision.15,18 In most cases, only an unequivocally positive diagnosis is used for staging purposes—anything less is treated as a negative result. Side-by-side comparison with the corresponding resection specimen often helps resolve an equivocal case. Criteria for specimen adequacy have not been established, but it seems reasonable to require some benign mesothelial cells before considering a peritoneal washing specimen adequate and negative for malignant cells.19 A specimen with malignant cells is always adequate. Not all patients with metastases to the peritoneum have positive PWC results. In fact, 23% to 52% of patients with biopsy-proven peritoneal involvement have negative results.17,20–23 When peritoneal washings are examined as part of a second-look procedure, the false-negative rate is even higher, ranging from 31% to 86% of patients with biopsy-proven metastases.9,11,21,22,24–26 The higher false-negative rate of second-look procedures may be due to the poor distribution of fluid when the abdominal cavity is altered by adhesions. False-positive diagnoses are uncommon but well documented,2,3,21,27–29 occurring in less than 5% of cases.2,23,30 Causes include reactive mesothelial proliferation with psammoma bodies23,27,28 and endometriosis.22,29 In particular, eosinophilic metaplastic atypia in endometriosis can be a source of atypical (but benign) cells that might be misconstrued as malignant.31 In one instance, ectopic pancreatic tissue was misinterpreted as adenocarcinoma in a patient with a mucinous ovarian cancer.32 Mesothelial cells in peritoneal washings are arranged predominantly in flat sheets, often large and occasionally folded (Fig. 5.1). In cell block sections the sheets are transected and appear as long, thin ribbons (Fig. 5.2). The cells are evenly spaced, with a moderate amount of cytoplasm. Nuclear membranes are thin, and the chromatin is pale and evenly dispersed; small nucleoli are often present. Although usually round or oval (Fig. 5.3), the nuclei are sometimes scalloped, wrinkled, or grooved,33 possibly due to fixation artifact. Isolated mesothelial cells, similar to those seen in ascites, are also encountered. Figure 5.1 Mesothelial cells. Figure 5.2 Mesothelial cells (cell block). Figure 5.3 Mesothelial cells. Spherical masses of collagen surrounded by benign, flattened mesothelial cells, known as collagen balls, are seen in up to 50% of peritoneal washings.33,34 Aqua in color with the Papanicolaou stain, they have round or bosselated contours (Fig. 5.4). They are usually few in number, but occasionally they can be abundant. They have no known significance (and therefore do not deserve mention on a cytology report). It has been suggested that they result from a pinching off of mesothelial-lined stromal projections known as micropapillomatosis on the surface of the ovaries.34,35 Figure 5.4 Collagen ball. Numerous histiocytes, scattered either as isolated cells or in variably sized aggregates, are often present (Fig. 5.5). Because they look different from mesothelial cells and are often haphazardly aggregated, they might be misinterpreted as metastatic cancer cells. Attention to the typical nuclear and cytoplasmic features of histiocytes (oval, folded, and kidney-shaped nuclei; pale chromatin; granular and microvacuolated cytoplasm) is helpful in correctly identifying them. Skeletal muscle and adipose tissue fragments are sometimes seen. (They fall into the peritoneal cavity when the abdominal incision is made and are suctioned along with the fluid.) Detached ciliary tufts, presumably of fallopian tube origin, are a relatively common incidental finding, especially when the washings are obtained during the secretory (luteal) phase of the menstrual cycle.36 Figure 5.5 Histiocytes. A group of benign conditions involving the peritoneal and ovarian surfaces can result in a proliferation of fallopian tubal-type epithelial cells or mesothelial cells, often with psammoma bodies. These conditions mimic peritoneal involvement by a serous borderline tumor and serous adenocarcinoma; familiarity with them is thus important. Endosalpingiosis is a proliferation of benign glands and cysts lined by ciliated, fallopian tube-like epithelium. Common locations include the ovarian cortex, uterine serosa, peritoneal surface, and omentum. Psammoma bodies are often seen. Some authors reserve the term endosalpingiosis for proliferations that include tubal-type stroma as well as glands, and use the term müllerian inclusion cysts for cases that lack tubal-type stroma.35 Both endosalpingiosis and müllerian inclusion cysts, however, are usually incidental, microscopic findings in a patient undergoing surgery for something else. Histologically, they raise the possibility of metastatic disease but are identified as benign proliferations because the cells are uniform and bland and lack mitotic activity. Prior surgery, pelvic inflammatory disease, a ruptured cyst, and other conditions cause florid mesothelial hyperplasia, sometimes accompanied by psammoma body formation.15 The conditions described above all have a similar appearance in peritoneal washings. Clusters of cuboidal mesothelial-like cells (Fig. 5.6), some arranged around a psammoma body (Fig. 5.7 and Fig. 5.8) or nondescript calcification, are present and can be abundant.28 Cilia may be present, but often they are absent or impossible to identify. Nuclei are round or oval, with a pale chromatin pattern and small nucleoli. Nuclear atypia is mild, and mitoses are very uncommon. Figure 5.6 Reactive mesothelial cells (ovarian torsion). Figure 5.7 Endosalpingiosis. Figure 5.8 Serous adenofibroma of the ovary. These benign conditions are potential causes of a false-positive interpretation of involvement by a serous adenocarcinoma or borderline tumor.27 To avoid a false-positive interpretation, a diagnosis of malignancy should not be based on the presence of psammoma bodies alone.22,27 Correlation with the concurrent histologic material is helpful in most cases. Certainly, if the patient does not have an adenocarcinoma or borderline tumor, one should avoid rendering such a diagnosis on the basis of the peritoneal washings alone. Some patients with endosalpingiosis or benign mesothelial proliferations, however, can also have a serous borderline tumor confined to the ovary.18 Comparison of the peritoneal washings, particularly cell block preparations, with the histologic sections from the tumor often resolves equivocal cases, but the final interpretation is not always straightforward, and the pathologist is sometimes required to make the best judgment possible. There are few helpful morphologic clues, particularly because many serous borderline tumors have only mild cytologic atypia, equivalent to what can be seen with florid mesothelial hyperplasia and endosalpingiosis. One possible clue: In cell block sections, some papillary fragments of serous borderline tumors have broad stromal cores, a feature not seen in cell blocks containing only mesothelial hyperplasia or endosalpingiosis. Endometriosis, the presence of ectopic benign endometrial glands and stroma in the omentum, peritoneum, ovary, and elsewhere, is another potential pitfall in the evaluation of peritoneal washings.22,29 The purpose of peritoneal washings in this setting is to exclude an occult malignancy, not to diagnose endometriosis. A diagnosis of endometriosis, in fact, can rarely be made by examining peritoneal washings alone. Hemosiderin-laden macrophages are seen in one third of women with endometriosis.37 In a young woman, hemosiderin-laden macrophages are most likely due to endometriosis,37 but by themselves they are a nonspecific finding seen in any condition associated with intraperitoneal bleeding. Some cases of endometriosis show a population of cells morphologically similar to but distinct (in subtle ways) from mesothelial cells and macrophages; these likely represent endometrial glandular and/or stromal cells. The endometrial-like glandular and/or stromal cells may, in fact, resemble the clusters of exfoliated endometrial cells seen in menstrual-phase cervical-vaginal preparations (Fig. 5.9A), but a definitive distinction between mesothelial cells and endometrial cells is difficult.37 On rare occasions, a diagnosis can be made when tissue fragments with endometrial-type glands and stroma are identified in cell block sections (Fig. 5.9B and C). Figure 5.9 Endometriosis. Occasionally, the endometrial glandular cells are enlarged and atypical and might suggest a malignancy. Correlation with concurrent histologic material is very helpful in preventing a misdiagnosis.29,31 Ovarian cancer is the ninth most common cancer in women in the United States and accounts for 6% of cancer-related mortality.38 The median age at diagnosis is 52 years. Most women (70% to 75%) with ovarian cancer present with stage III or IV disease (tumor spread beyond the pelvis). PWC is an important staging component; a positive finding modifies stage I and II tumors to stage Ic and IIc, respectively (Table 5.1). Primary treatment for presumed ovarian cancer is surgical staging and cytoreduction. Most women (except those with stage IA and IB, grade 1 tumors) receive postoperative chemotherapy, either systemic or intraperitoneal, depending on the stage of their disease. Platinum drugs (cisplatin, carboplatin) and taxanes (paclitaxel, docetaxel) are the most efficacious and are often used in combination. Novel strategies that target key molecular alterations are showing promise in the treatment of ovarian cancer.39 TABLE 5.1 DEFINITIONS OF THE STAGES IN PRIMARY CARCINOMA OF THE OVARY Stage I Growth limited to the ovaries Ia Growth limited to one ovary; no ascites with malignant cells; no tumor on the external surface; capsule intact Ib Growth limited to both ovaries; no ascites with malignant cells; no tumor on the external surfaces; capsules intact Ic Tumor either stage Ia or Ib, but with tumor on surface of one or both ovaries; or with capsule ruptured; or with ascites present containing malignant cells or with positive peritoneal washings Stage II Growth involving one or both ovaries with pelvic extension IIa Extension and/or metastases to the uterus and/or tubes IIb Extension to other pelvic tissues IIc Tumor either stage IIa or IIb, but with tumor on surface of one or both ovaries; or with capsule(s) ruptured; or with ascites present containing malignant cells or with positive peritoneal washings Stage III Tumor involving one or both ovaries with histologically confirmed peritoneal implants outside the pelvis and/or positive regional lymph nodes; superficial liver metastasis equals stage III; tumor is limited to the true pelvis but with histologically proven malignant extension to small bowel or omentum IIIa Tumor grossly limited to the true pelvis, with negative nodes, but with histologically confirmed microscopic seeding of abdominal peritoneal surfaces or histologically proven extension to small bowel or mesentery IIIb Tumor of one or both ovaries with histologically confirmed implants, peritoneal metastasis of abdominal peritoneal surfaces, none exceeding 2 cm in diameter; nodes are negative IIIc Peritoneal metastasis beyond pelvis, greater than 2 cm in diameter, and/or positive regional lymph nodes Stage IV Growth involving one or both ovaries with distant metastases; if pleural effusion is present, there must be positive cytology to allot a case to stage IV; parenchymal liver metastases equals stage IV From: Current FIGO staging for cancer of the vagina, fallopian tube, ovary, and gestational trophoblastic neoplasia. Int J Gynaecol Obstet 2009;105(1):3-4.
Peritoneal Washings
Specimen Collection, Preparation, and Reporting Terminology
Accuracy
The Normal Peritoneal Washing
Normal mesothelial cells in peritoneal washings are often arranged in cohesive sheets. Although flat, the sheets can be folded on cytocentrifuge or thinlayer preparations. Isolated mesothelial cells are present in the background (Papanicolaou stain).
In cell block sections, a sheet of mesothelial cells is transected and looks like a string of pearls. When it curls around on itself it has a pseudoglandular appearance (hematoxylin-eosin [H & E] stain).
Usually, benign mesothelial cells have round or oval nuclei, but in some cases there are irregularities in nuclear contour. Note the spaces (“windows”) that separate adjacent cells, a common finding (Papanicolaou stain).
These spheres of collagen are surrounded by flattened mesothelial cells. A, Papanicolaou stain; B, hematoxylin-eosin (H & E)–stained cell block section.
Histiocytes are often in groups, as seen here. They have indistinct cell borders (no “windows”), granular or vacuolated cytoplasm, and occasionally folded nuclei. A short strip of mesothelial cells is also present on the cell block section. A, Papanicolaou stain; B, hematoxylin-eosin (H & E)–stained cell block.
Benign Conditions
Endosalpingiosis and Similar Benign Proliferations
The mesothelial cells are enlarged, with irregular nuclei and prominent nucleoli. Exploratory laparotomy revealed torsion of the right ovary with intraperitoneal adhesions.
A, The psammoma body has concentric lamination and is surrounded by cuboidal cells with small nucleoli and vacuolated cytoplasm. Cilia are not identified (Papanicolaou stain). B, The surface of the resected ovary shows benign ciliated glandular cells associated with psammoma bodies. There was no evidence of tumor (hematoxylin-eosin [H & E] stain).
A, A psammoma body is surrounded by evenly spaced mesothelial cells with minimal nuclear atypia (Papanicolaou stain). B, The resected ovary shows a serous adenofibroma involving the ovarian surface, with an occasional psammoma body (arrow) (hematoxylin-eosin [H & E] stain).
Endometriosis
A, A cluster of cells has scalloped borders and contains nuclear fragments. Individually, the cells resemble mesothelial cells, but the nuclear debris suggests that they are degenerating endometrial cells. Such clusters are often recognized as endometrial cells only in retrospect, after reviewing cell block sections or correlating with laparoscopic or histologic evidence (Papanicolaou stain). B, The cell block from the same case as A shows endometrial glands surrounding a core of degenerated endometrial stromal cells (hematoxylin-eosin [H & E] stain). C, Occasionally, the cell block of peritoneal washings from a patient with endometriosis shows diagnostic tissue fragments composed of intact endometrial-type glands and stroma (hematoxylin-eosin [H & E] stain).
Malignant Tumors
Ovarian Cancer
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Peritoneal Washings
Indications for peritoneal washings (summary)
Cytomorphology of benign peritoneal washings
Cytomorphology of endosalpingiosis and similar benign proliferations
Cytomorphology of endometriosis