1 Anatomic Pathology / PRIMARY PERITONEAL MESOTHELIOMA Cytomorphologic Features of Primary Peritoneal Mesothelioma in Effusion, Washing, and Fine-Needle Aspiration Biopsy Specimens Examination of 49 Cases at One Institution, Including Post Intraperitoneal Hyperthermic Chemotherapy Findings Nilesh P. Patel, MD, 1 Cullen A. Taylor, MD, 1* Edward A. Levine, MD, 2 Jacqueline K. Trupiano, MD, 1* and Kim R. Geisinger, MD 1 Key Words: Peritoneal; Mesothelioma; Cytology; Chemotherapy; Cytoreductive surgery DOI: /DV1JYBL8LLYYT4J5 Abstract Primary peritoneal mesotheliomas (PPMs) are rare tumors of adults. At our institution, PPMs are treated with a combination of cytoreductive surgery and intraperitoneal hyperthermic chemotherapy (IPHC) in appropriate patients. We present a summary of cytologic features of PPM in 49 positive (malignant) specimens during a 15-year period at 1 institution. Of the corresponding 49 PPM histologic specimens, 46 were epithelial, 2 sarcomatoid, and 1 multicystic mesothelioma. This includes our experience with washing specimens obtained from patients with PPM following treatment with cytoreductive surgery combined with IPHC. The rarity of PPM makes this neoplasm unfamiliar to most pathologists. However, cytologic features can be diagnostic in a majority of cases. We present a summary of cytologic features that, in our experience, we find to be most useful in making or excluding a diagnosis of PPM. To our knowledge, this is the first large series reporting the cytomorphologic features of PPM in peritoneal effusions, pelvic washing specimens, and infradiaphragmatic fine-needle aspiration biopsy specimens. Primary peritoneal mesotheliomas (PPMs) are rare tumors of adults The cytomorphologic features of PPM have not been extensively described, although it is reported to be indistinguishable from mesothelioma occurring within the thorax. We present a summary of the cytologic features of PPM in 49 positive (malignant) cytologic specimens from 43 patients during a 15-year period at 1 institution. We also describe our experience with washing specimens obtained from patients with PPM following treatment with cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (IPHC), as our institution serves as a major referral center for this treatment modality. Our aim is to provide a concise, practical reference on the cytomorphologic features of PPM with an emphasis on cytologic features most helpful in distinguishing mesothelial neoplasia from reactive or hyperplastic mesothelial proliferations. Materials and Methods The electronic pathology files at Wake Forest University Baptist Medical Center, Winston-Salem, NC, were searched for all cases of PPM from January 1990 to December Only cases with a definitive diagnosis of PPM were included; any case favoring mesothelioma was excluded unless a definitive diagnosis of mesothelioma was established on a concurrent specimen. A total of 49 PPM specimens from 43 patients were recovered that had smeared glass slides available for review. This included 15 ascitic fluid specimens, 27 washing specimens (including 7 post-iphc specimens), and 7 fine-needle aspiration biopsy specimens (FNABs). We also reviewed 6 post-iphc washings interpreted as negative for 414 Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5
2 Anatomic Pathology / ORIGINAL ARTICLE residual PPM. Cytoreductive surgery and IPHC have been used at our institution for PPM for more than a decade. Our technique and results have been described elsewhere. 12,13 Briefly, after aggressive resection of gross disease, the peritoneum is perfused with chemotherapy (mitomycin C, mitoxantrone and/or cisplatin) for 2 hours with mild hyperthermia (40 C). When this is completed, peritoneal washing specimens are obtained in selected cases before definitive surgical closure. Results The mean patient age was 53 years (range, years) with a male/female ratio of 2.5:1. All specimens had alcoholfixed Papanicolaou (Pap)-stained smeared slides available for review; in addition, paired air-dried smears stained by a rapid Romanowsky (Diff-Quik) method were available for most effusion specimens (9/15) and all FNABs. Histologically, the majority of mesotheliomas were epithelial (tubulopapillary) (46/49). Mesothelioma variants included 2 sarcomatoid cases and 1 multicystic case. Cytologic Features of Effusion Specimens We reviewed 15 cases of PPM diagnosed by effusion cytologic examination, including cases that were initially diagnosed as PPM and others that were initially thought to be suspicious for PPM; all cases were confirmed to be mesothelioma by histologic evaluation. The volume of ascitic fluid varied greatly from 15 ml to substantially more than 1,000 ml. The majority of cases demonstrated a moderate to high number of tumor cells, regardless of specimen volume. The smears typically displayed a background of blood or proteinaceous fluid with a small number of scattered chronic inflammatory cells, including lymphocytes and histiocytes; acute inflammation was not observed. In 3 cases, necrotic debris was conspicuous. One case had rare foci of psammomatous calcification. A few smears contained scattered, single, cytologically bland mesothelial cells within the background, likely representing a separate benign population. The neoplastic mesothelial cells generally were present in high numbers, forming small to medium-sized 3-dimensional groups (typically 5-30 cells per group) Image 1. The groups were tightly cohesive and formed borders that were rounded ( cannonballs ) or knobby (scalloped). The cells at the edges of the scalloped groups often had nuclei directed toward the center with homogeneous cytoplasm directed to the perimeter. Malignant mesothelial cells were also present as dispersed single cells and in couplets, as 1 case consisted exclusively of dispersed single malignant cells. This has previously been described as an epithelial noncohesive cell type of malignant mesothelioma. 4 In our experience, it is uncommon for PPM to manifest exclusively as dispersed single cells in effusions. Image 1 Peritoneal effusion specimen. Three-dimensional group of cohesive neoplastic mesothelial cells with distinct nucleoli, nuclear grooves, and a rare nuclear pseudoinclusion (Papanicolaou, original magnification 600). Cellular groups generally displayed loss of organization and polarity. There was considerable variation in cellular and nuclear size. Cell crowding was prominent, often with compression of adjacent nuclei, but cell overlapping was minimal. Cell-in-cell engulfment was readily noted in the majority of cases (9/15). Mitotic activity was observed in one third of cases (5/15); atypical mitotic figures were not recognized. For the most part, inflammatory cells did not permeate the cell groups. Individually, malignant mesothelial cells displayed central or eccentrically placed nuclei with increased nuclear/cytoplasmic (N/C) ratios, although varying greatly among cells. Many tumor cells had N/C ratios appearing to be within the normal range. Binucleation was seen in scattered tumor cells in all 15 cases. In addition, many cases contained individual tumor cells with 3 to 8 nuclei; in such cases, the nuclei showed obvious pleomorphism but had a tendency to maintain an overall central location. Prominent nucleolation was present in a large percentage of cells in almost all cases (14/15), with nucleoli ranging from 5 to 15 µm in diameter. Nuclear irregularities were present, although sometimes minimal, and were best evaluated on the Pap smears, often revealing small nuclear nicks, notches, and thickening of the nuclear envelopes, in addition to nuclear pseudoinclusions Image 2. Such nuclear irregularities were far more common than overt nuclear abnormalities, eg, nuclear convolutions and grooves (Image 1). In the majority of cases, the tumor cell nuclei were slightly hyperchromatic, although several cases contained cells with vesicular chromatin without obvious chromatin irregularities. In our experience, chromatin detail may be more Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5 415
3 Patel et al / PRIMARY PERITONEAL MESOTHELIOMA difficult to detect in effusion specimens in comparison with specimens obtained by direct sampling methods, particularly if the smears are thick or have a bloody background. On rapid Romanowsky stained smears, most mesothelioma cells contained a modest volume of homogeneous, dense, blue-green cytoplasm and had well-defined endoplasmic-ectoplasmic demarcations and ruffled cytoplasmic edges or blebs, typical of mesothelial differentiation. There were also scattered cells with large ballooning degenerative vacuoles (empty), occasionally indenting the nucleus into a pseudo signet-ring form. Some cells at the edges of balled-up groups had abundant apical cytoplasm imparting a low-columnar appearance. Rapid Romanowsky staining revealed that a minority of cases (3/15) contained scattered cells with intracytoplasmic magenta material consistent with hyaluronic acid; it is important to not mistake this finding as evidence of epithelial mucin production.9 On many of the effusion specimens (12/15), scanning low-power magnification raised the suspicion of neoplasm owing to the overall hypercellularity and 3-dimensional aggregates of cells. Closer examination of the cytologic features of individual cells enabled the examiner to conclude that the cells were, in fact, neoplastic. Table 1 summarizes the important cytologic features of PPM in effusion specimens. Cytologic Features of Washing Specimens We reviewed 27 available peritoneal washing specimens; all specimens were alcohol-fixed and prepared by a modified Pap method. The specimen volumes ranged from 40 to 350 ml. Seven samples positive for residual PPM were obtained Image 2 Peritoneal effusion specimen. Small cluster of cohesive neoplastic mesothelial cells with a distinct nuclear pseudoinclusion. Note the variability of nuclear sizes (Papanicolaou, original magnification 600) Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5 Table 1 Cytomorphologic Features of 35 Cases of Primary Peritoneal Mesothelioma in Effusion and Washing Specimens* No. (%) of Cases Feature Nuclear membrane irregularities Macronucleoli Three-dimensional cohesive groups, often scalloped Background blood and chronic inflammatory cells Hypercellular smear Variation in cell size and high nuclear/cytoplasmic ratios Tubulopapillary-like arrangements without fibrovascular cores Cell-in-cell engulfment * 35 (100) 33 (94) 31 (89) 30 (86) 29 (83) 28 (80) 27 (77) 21 (60) In addition, washing specimens had large, irregular sheets of neoplastic mesothelial cells (15/20 [75%]). following IPHC and are discussed in the next section. In addition, the findings on 6 post-iphc washing specimens negative for residual PPM were reviewed. Peritoneal washing specimens in cases of PPM were generally hypercellular with a background of blood, lymphocytes, and macrophages. Cohesive cell groupings similar to those described for effusion specimens were present in most cases. Many dispersed neoplastic cells also populated the background as single cells or in small clusters. In comparison with effusion specimens, washing specimens were more likely to contain broad, irregular, branching sheets, often containing more than several hundred cells per sheet (20/27 [15/20 non-iphc cases]) Image 3. These sheets were several cell layers thick and formed sharp edges, often with loosely cohesive cells Image 3 Peritoneal washing specimen. Two-dimensional sheet of cohesive neoplastic mesothelial cells in a bloody background. Note the conspicuous intercellular windows and rather bland-appearing nuclei (Papanicolaou, original magnification 600).
4 Anatomic Pathology / ORIGINAL ARTICLE exfoliating at the perimeter. Narrow intercellular spaces ( windows ) were best observed in the thinner edges of the groups at the point of cell apposition. Papillary-type configurations were also present with cells displaying high N/C ratios; however, true fibrovascular cores were generally not visualized. Such groups contrast sharply with the flat (but often folded), 2-dimensional groups formed in washing specimens by nonneoplastic mesothelial cells. Mitotic activity was more readily observed on washing specimens than on effusion specimens, predominantly within the large sheets (22/27 [16/20 non-iphc cases]). Individual cells of PPM on peritoneal washing specimens had features similar to those described for effusion specimens. Subtle nuclear contour irregularities and chromatin abnormalities were best visualized at the edges of cell groups or when examining single dispersed cells. Table 1 also summarizes the important cytologic features of washing specimens in effusions. The single case of multicystic PPM in a 41-year-old man yielded a cellular sample with individual cells and flat sheets. Many of the cells totally resembled fully benign mesothelial cells. However, a proportion had cytoplasmic vacuoles. The latter were usually solitary, large, and sharply demarcated from the more peripheral, dense cytoplasm. Often, the vacuole occupied much of the cellular volume, with or without compression of the nucleus. Thus, some cells had an appearance resembling signet-ring cells. Within the sheets, the vacuoles created a honeycomb picture, with a close resemblance to areas of adenomatoid tumors. Cytologic Features of Post-IPHC Washing Specimens We reviewed 13 post-iphc washing specimens; 7 cases were positive for PPM, and 6 were negative. The specimen A volume of post-iphc washing specimens ranged from 40 to 250 ml. Washing specimens with residual tumor had several features in common. The background was uniformly bloody, often adding to the challenge of cytologic interpretation. The smears generally contained low cellularity; all smears demonstrated a marked reduction in the volume of neoplastic cells compared with the pre-iphc washing or effusion specimen. Residual tumor cells were generally present in small sheets and clusters, as well as singly Image 4. Invariably, scattered dispersed mesothelial cells with slight atypia were present in the background; however, it was often difficult to determine whether they were part of the neoplastic population. The most helpful approach was to compare the post-iphc specimen with the diagnostic material obtained before institution of this therapy. Post-IPHC cases that were positive for residual disease contained at least a scattered population of cells with cytologic features similar to those of the previous diagnostic material. Three-dimensional aggregates of atypical mesothelial cells usually indicated residual disease and were observed in 4 of 7 cases. Table 2 summarizes the important cytologic features of PPM in post-iphc washing specimens. Post-IPHC washing specimens that were negative for residual PPM typically produced smears that were paucicellular or virtually acellular with a bloody background. When mesothelial cells were present, they were generally dispersed. Rare, cohesive groups of mesothelial cells were interpreted as benign if they were present in flat 2-dimensional aggregates without significant atypia. Individual cells were interpreted as nonneoplastic if they had low N/C ratios and round, smooth nuclei. Nucleoli, when present, were small but distinct. Macronucleoli should be interpreted with suspicion. Smears B Image 4 Post intraperitoneal hyperthermic chemotherapy peritoneal washing specimens. Three-dimensional cluster (A) and a 2-dimensional sheet (B) of cohesive neoplastic mesothelial cells with macronucleoli (A and B, Papanicolaou, original magnification 600). Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J
5 Patel et al / PRIMARY PERITONEAL MESOTHELIOMA Table 2 Cytomorphologic Features of Seven Cases of Primary Peritoneal Mesothelioma in Post Intraperitoneal Hyperthermic Chemotherapy Washing Specimens Feature Obscuring background blood Residual neoplastic mesothelial cells in small sheets, clusters, and singly Macronucleoli Hypocellular smear Variation in cell size and high nuclear/cytoplasmic ratios Rare, 3-dimensional cohesive groups No. (%) of Cases 7 (100) 7 (100) 7 (100) 6 (86) 5 (71) 4 (57) that had even rare scattered benign and/or reactive-appearing mesothelial cells were interpreted as negative for residual mesothelioma, provided that the specimen volume was adequate. Entirely acellular specimens were interpreted as nondiagnostic and unsatisfactory for evaluation owing to acellularity. Specimens of less than 20 ml that were acellular or contained benign and/or reactive-appearing mesothelial cells were interpreted as nondiagnostic and unsatisfactory for evaluation owing to insufficient volume. Cytologic Features of FNABs We examined 7 cases of PPM diagnosed by FNAB that targeted 4 abdominal masses and 3 in the omentum; 5 cases were for primary diagnosis, and 2 were for recurrence. The following types were diagnosed: epithelial type, Image 5 Omental fine-needle aspiration biopsy specimen. Three-dimensional group of cohesive neoplastic mesothelial cells with increased nuclear/cytoplasmic ratios, nuclear membrane irregularities, and macronucleoli (rapid Romanowsky, original magnification 600) Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5 4 cases; sarcomatoid type, 2 cases; and multicystic mesothelioma, 1 case. With the exception of the multicystic mesothelioma, all FNABs were suspicious for neoplasm at scanning power owing to hypercellularity and the architectural arrangement of the cell groups. FNAB of epithelial PPM generally yielded highly cellular smears populated by atypical cells arranged in variably cohesive 3-dimensional sheets Image 5. Tubulopapillary structures were common, accompanied by a large number of dispersed atypical cells in the background. The milieu varied greatly; most cases had a clean or slightly bloody background, whereas frank necrosis was seen in 3 of 7 cases. Mitotic figures were identified in all 7 cases, but the number varied greatly, and, on occasion, several mitoses were seen in a single high-power field Image 6. Rare foci of extracellular hyaluronic acid were present in 2 cases of epithelial PPM. This was best visualized on the rapid Romanowsky stained smears because the hyaluronic acid was bright magenta and formed homogeneous, laminar structures that appeared to entrap individual tumor cells Image 7. In other foci, mesothelial cells were arranged in acinar formations with hyaluronic acid present in the center of the group or in the cytoplasm of individual cells, mimicking adenocarcinoma with mucin production. As described by Wojcik and Naylor,14 peritoneal washing specimens in women may contain collagen balls. These structures are composed of collagen cores surrounded by mesothelial cells and are thought to be derived from the surface of the ovaries. They are pitfalls in the diagnosis of glandular Image 6 Omental fine-needle aspiration biopsy specimen. Three-dimensional group of cohesive neoplastic mesothelial cells with a conspicuous mitotic figure (rapid Romanowsky, original magnification 600).
6 Anatomic Pathology / ORIGINAL ARTICLE Image 7 Omental fine-needle aspiration biopsy specimen. Three-dimensional group of cohesive neoplastic mesothelial cells with magenta-staining cytoplasmic hyaluronic acid (rapid Romanowsky, original magnification 600). neoplasms in peritoneal fluids. Although hyaluronic acid and collagen may display similar staining qualities with rapid Romanowsky and Pap stains, hyaluronic acid is best distinguished by its typical intracellular location and presence in individual cells and cohesive aggregates. The neoplastic cells clearly resembled mesothelial cells, even at scanning low-power magnification. This impression was largely due to the plump nature of the tumor cells; the centrally placed, round nucleus; and abundant, dense cytoplasm. On closer inspection, narrow spaces at the point of cell apposition (windows) were occasionally noted. The aspirates of epithelial PPMs were composed of pleomorphic cells with markedly elevated N/C ratios, including many cells with small rims of cytoplasm. The nuclei were generally round (even when quite large) and often centrally placed and bearing prominent macronucleoli, occasionally occupying more than one third of the diameter of the nucleus. The cytoplasm was typically dense and homogeneous, with occasional small perinuclear vacuoles. Many multinucleated tumor cells were present, usually with moderate pleomorphism with rare cells demonstrating up to 10 nuclei. Alcohol-fixed Pap-stained smears revealed moderate nuclear hyperchromasia in most of the cases with minimal chromatin irregularities. Table 3 summarizes the important cytologic features of epithelial PPM in FNABs. Sarcomatoid mesothelioma was diagnosed in 2 FNAB cases. Scanning low-power magnification revealed overt evidence of malignancy in both cases; however, unlike epithelial mesotheliomas, there was no clear resemblance to mesothelial cells. The aspirates were highly cellular with loosely cohesive cell groups and many single pleomorphic cells with elongated contours. Cells had widely variable N/C ratios with large, irregular nuclei. Nuclear outlines were generally oval or spindled with scattered convoluted and irregular forms. The nuclei were hyperchromatic with coarse, unevenly distributed chromatin. Nucleoli were variably present. The cytoplasm was dense and homogeneous with tapered ends. No intercellular windows were observed. A single case of multicystic PPM was evaluated by FNAB. The aspirate was moderately cellular with cohesive aggregates of monomorphic cells with mesothelial features. The background contained areas with thick proteinaceous material; there was no necrosis or inflammation. The sheets of tumor cells were 2-dimensional and folded with an orderly arrangement around variably sized, well-defined, cystic spaces Image 8. This architectural arrangement imparted a honeycomb appearance to the groups. Tubulopapillary structures were not present. The individual cells retained many of the cytologic features typical of nonneoplastic mesothelial cells. The cells had small, central, round to ovoid nuclei without atypia and abundant, cyanophilic cytoplasm with prominent cytoplasmic vacuoles and peripheral pallor. Windows were noted at the point of cell apposition. Nuclei were normochromatic with fine, evenly distributed chromatin. Nucleoli, when present, were small and inconspicuous. Tumor cells were focally entrapped in a bland, paucicellular, collagenous stroma. Mitoses were not seen. Discussion PPMs are uncommon tumors Their cytomorphologic features have not been extensively described, although they are reported to be indistinguishable from those occurring within the thorax. 10 In our experience, the same cytomorphologic features used to distinguish reactive mesothelium from Table 3 Cytomorphologic Features of Four Cases of Epithelial Primary Peritoneal Mesothelioma in Aspiration Specimens Feature No. (%) of Cases Hypercellular smear 4 (100) Three-dimensional cohesive groups 4 (100) Tubulopapillary-like arrangements without 4 (100) fibrovascular cores Multinucleated tumor cells 4 (100) Macronucleoli 4 (100) Mitotic figures (variable numbers) 4 (100) Slightly bloody background 3 (75) Variation in cell size and high nuclear/ 3 (75) cytoplasmic ratios Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5 419
7 Patel et al / PRIMARY PERITONEAL MESOTHELIOMA A B Image 8 Omental fine-needle aspiration biopsy specimens of multicystic primary peritoneal mesothelioma. Two-dimensional groups of cohesive neoplastic mesothelial cells with bland appearing nuclei and prominent cytoplasmic vacuoles, resembling an adenomatoid tumor (A and B, Papanicolaou, original magnification 400). malignant mesothelioma in the thorax can be translated to the peritoneum. Cerruto et al 15 recently examined the prognostic significance of various histomorphologic parameters in PPMs treated with surgery and IPHC and concluded that nuclear/nucleolar size was useful in assessing prognosis. 15 Because our institution is a referral center for IPHC treatment of peritoneal malignancies, we see a disproportionate number of cases. This report is intended to provide a concise, practical review of the cytologic features of PPM. We have intentionally excluded any reference to the clinical, radiographic, immunohistochemical, and molecular characteristics of mesothelioma; such topics have been well covered elsewhere. 1-3,5-8,16-18 Although PPMs are rare, the diagnosis is commonly considered when pathologists are faced with a hypercellular cytologic sample from the peritoneum. In our experience, hypercellular smears from large-volume, reactive effusions may, at times, be particularly challenging. In such cases, an understanding of the typical cytologic features of PPM is helpful in discerning mesothelial neoplasms from reactive or hyperplastic proliferations. Currently, cytopathologists must rely almost exclusively on cytologic features when making this distinction. We believe that with experience, a diagnosis of PPM can be made from examination of peritoneal fluids alone, without concurrent tissue histologic confirmation, in the appropriate clinicoradiographic context. For the purpose of our study, all 49 cases of PPM had histologic confirmation of mesothelioma. Although the clinicoradiographic findings may influence the interpretation, there are no immunohistochemical stains routinely used to distinguish benign and malignant mesothelial cells. 19 In 1998, Wolanski et al 20 examined the immunoreactivity of epithelial membrane antigen and quantification of silver-stained nucleolar organizer regions (AgNORs) for their usefulness in distinguishing benign and malignant mesothelial lesions. They concluded that a combination of positive immunostaining with epithelial membrane antigen and an increased average area of AgNOR per cell resulted in increased sensitivity in detection of epithelial malignant mesotheliomas. 20 In 2007, Kato et al 21 reported the use of GLUT-1, a member of the mammalian facilitative glucose transporter family, in helping to distinguish benign and malignant mesothelial populations of cells in the context of lung and pleural lesions. They concluded that GLUT-1 was a sensitive and specific immunohistochemical marker for distinguishing benign mesothelium and malignant mesothelioma (positive in only malignant mesothelioma) but was not helpful in distinguishing malignant mesothelioma and non small cell lung carcinoma because both had positive immunostaining. 21 In some situations, a cytologic specimen from the peritoneum may demonstrate unequivocal evidence of malignancy, and the task is to assign the appropriate histogenesis, as is often the case with FNAB of PPM. FNAB of PPM typically yields highly cellular samples with frank architectural and cytologic atypia often accompanied by a tumor diathesis. Coupled with the clinical and radiographic evidence of diffuse tumor, a definitive diagnosis of malignancy can usually be made. However, ancillary studies are generally necessary to confirm the cytologic impression that the lesion is indeed mesothelial in origin Cell-block material for immunohistochemical staining should be obtained at the time of FNAB for this valuable purpose. In general, mesothelioma cells clearly resemble their nonneoplastic counterparts, even at scanning power. This is 420 Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5
8 Anatomic Pathology / ORIGINAL ARTICLE particularly true of multicystic mesothelioma, a rare tumor with bland cytomorphologic features most often affecting women of reproductive age Excluding this rare subtype, features most helpful in establishing a diagnosis of PPM include a background tumor diathesis, large 3-dimensional groups, a high N/C ratio in a subset of tumor cells, hyperchromasia, macronucleoli, and cell-in-cell engulfment. The presence of significant nuclear pleomorphism within a single multinucleated cell was a distinct feature of malignancy; we have not observed this feature in reactive mesothelial proliferations. In effusion specimens, the cell groupings may be smaller than in FNABs, mitoses may be scant, and necrosis is usually absent. Cells suspended in ascitic fluid may begin to degenerate and lose chromatin detail, making interpretation more difficult. In addition, although most cases of PPM manifest clinically with signs and symptoms indicating malignancy or radiographically with apparent diffuse tumor, occasionally, patients may merely have ascites. Effusion specimens from such patients, with no clinical suspicion of malignancy, should be interpreted cautiously because clinical evidence of tumor is often cited as an important criterion for establishing the diagnosis. 27 Evaluating post-iphc specimens for the presence of residual PPM is certainly a difficult task. Because the washing specimens are often quite bloody from the extensive cytoreductive surgery, it is important to apply a limited amount of material to the glass slide to ensure proper fixation and preservation of morphologic detail during the preparation process. Applying excess material will produce a suboptimal smear that is too thick with poor preservation. Given well-prepared material, the interpretation may still present a challenge. An inexperienced pathologist may be uncertain what degree of cytologic atypia may be secondary to the direct toxic effects of the IPHC. In our experience, reactive mesothelial cells in this setting manifest the typical reactive changes seen in other contexts, including maintenance of low N/C ratios with round, normochromatic nuclei. Nucleoli, if present, are small. We have generally not observed dramatic chemotherapy-induced changes in post-iphc mesothelial cells. This may be due to the specific protocol used at our institution that has a short duration of IPHC (about 2 hours) and the subsequent posttherapy washing. However, it is likely that the cytologic findings may vary depending on the particular treatment method used. 6,12,13,28 In 1988, Geisinger et al 29 reported findings regarding Tenckhoff catheter fluid cytology in the context of patients receiving intraperitoneal chemotherapy for ovarian cancers. They found that nuclear irregularities and hyperchromasia may be prominent in benign mesothelial cells from Tenckhoff catheters exposed intraperitoneally to cytotoxic chemotherapy. This is in contrast with our current observations regarding the presence of only typical reactive changes in post-iphc mesothelial cells. The presence of hypercellular, 3-dimensional groupings of cells indicates residual disease in post-iphc washing specimens. If the population is dispersed, it is especially helpful to compare the current cytologic features with those of the previous material. Wide variation in cell and nuclear size, scattered cells with high N/C ratios, and macronucleoli are evidence of residual tumor. There will be cases with insufficient evidence to conclude whether residual tumor is present. For example, if only rare, scattered, atypical cells are identified, we recommend using the term rare atypical mesothelial cells present and stating the significance this might represent, such as suspicious for residual mesothelioma. However, in our experience, a definitive diagnosis can be made in the majority of cases. Post-IPHC smears that are essentially acellular, but with at least rare scattered benign and/or reactive-appearing mesothelial cells, should be interpreted as negative for residual mesothelioma rather than as inadequate or nondiagnostic, provided that the specimen volume was adequate. Low-volume specimens (<20 ml) with only blood present on the smears are insufficient for interpretation. The rarity of PPM makes these neoplasms unfamiliar to most pathologists. However, cytologic features can be diagnostic in a majority of cases. We present features of PPM in peritoneal effusion specimens, washing specimens, post- IPHC washing specimens, and FNABs that, in our experience, we find to be the most useful in helping to make or exclude a diagnosis of PPM. The cytomorphologic features listed in Tables 1 through 3 are intended to be used in combination to allow for a confident diagnosis of PPM in the appropriate clinicoradiographic context. From the Departments of 1 Pathology and 2 General Surgery, Wake Forest University School of Medicine, Winston-Salem, NC. Address reprint requests to Dr Geisinger: Dept of Pathology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC * Drs Taylor and Trupiano are now with the Departments of Pathology, Forsyth Medical Center, Winston-Salem, and William Beaumont Hospital, Royal Oak, MI, respectively. References 1. Kannerstein M, Churg J. Peritoneal mesothelioma. Hum Pathol. 1977;8: Antman K, Shemin R, Ryan L, et al. Malignant mesothelioma: prognostic variables in registry of 180 patients, the Dana-Farber Cancer Institute and Brigham and Women s Hospital experience over two decades, J Clin Oncol. 1988;6: Goldblum J, Hart WR. Localized and diffuse mesotheliomas of the genital tract and peritoneum in women: a clinicopathologic study of nineteen true mesothelial neoplasms, other than adenomatoid tumors, multicystic mesotheliomas, and localized fibrous tumors. Am J Surg Pathol. 1995;19: Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5 421
9 Patel et al / PRIMARY PERITONEAL MESOTHELIOMA 4. Kho-Duffin J, Tao LC, Cramer H, et al. Cytologic diagnosis of malignant mesothelioma, with particular emphasis on the epithelial noncohesive cell type. Diagn Cytopathol. 1999;20: Taub RN, Keohan ML, Chabot JC, et al. Peritoneal mesothelioma. Curr Treat Options Oncol. 2000;1: Loggie BW. Malignant peritoneal mesothelioma. Curr Treat Options Oncol. 2001;2: Kerrigan SA, Turnnir RT, Clement PB, et al. Diffuse malignant epithelial mesotheliomas of the peritoneum in women: a clinicopathologic study of 25 patients. Cancer. 2002;15: Trupiano JK, Geisinger KR, Willingham MC, et al. Diffuse malignant mesothelioma of the peritoneum and pleura, analysis of markers. Mod Pathol. 2004;17: Geisinger KR, Stanley MW, Raab SS, et al. Modern Cytopathology. Philadelphia, PA: Churchill Livingstone; 2004: Geisinger KR, Stanley MW, Raab SS, et al. Modern Cytopathology. Philadelphia, PA: Churchill Livingstone; 2004: Baker PM, Clement PB, Young RH. Malignant peritoneal mesothelioma in women: a study of 75 cases with emphasis on their morphologic spectrum and differential diagnosis. Am J Clin Pathol. 2005;123: Loggie BW, Fleming RA, McQuellon RP, et al. Prospective trial for the treatment of malignant peritoneal mesothelioma. Am Surg. 2001;67: Loggie BW, Fleming RA, Geisinger KR. Cytologic assessment before and after intraperitoneal hyperthermic chemotherapy for peritoneal carcinomatosis. Acta Cytol. 1996;40: Wojcik EM, Naylor B. Collagen balls in peritoneal washings: prevalence, morphology, origin and significance. Acta Cytol. 1992;36: Cerruto CA, Brun EA, Chang D, et al. Prognostic significance of histomorphologic parameters in diffuse malignant peritoneal mesothelioma. Arch Pathol Lab Med. 2006;130: Friedman MT, Gentile P, Tarectecan A, et al. Malignant mesothelioma: immunohistochemistry and DNA ploidy analysis as methods to differentiate mesothelioma from benign reactive mesothelial cell proliferation and adenocarcinoma in pleural and peritoneal effusions. Arch Pathol Lab Med. 1996;120: Ordonez NG. Role of immunohistochemistry in distinguishing epithelial peritoneal mesotheliomas from peritoneal and ovarian serous carcinomas. Am J Surg Pathol. 1998;22: Ordonez NG. The immunohistochemical diagnosis of epithelial mesothelioma. Hum Pathol. 1999;30: Churg A, Colby TV, Cagle P, et al. The separation of benign and malignant mesothelial proliferations. Am J Surg Pathol. 2000;24: Wolanski KD, Whitaker D, Shilkin KB, et al. The use of epithelial membrane antigen and silver-stained nucleolar organizer regions testing in the differential diagnosis of mesothelioma from benign reactive mesothelioses. Cancer. 1998;82: Kato Y, Tsuta K, Seki K, et al. Immunohistochemical detection of GLUT-1 can discriminate between reactive mesothelium and malignant mesothelioma. Mod Pathol. 2007;20: Weiss SW, Tavassoli FA. Multicystic mesothelioma: an analysis of pathologic findings and biologic behavior in 37 cases. Am J Surg Pathol. 1988;12: Ross MJ, Welch WR, Scully RE. Multilocular peritoneal inclusion cysts (so-called cystic mesotheliomas). Cancer. 1989;64: Devaney K, Kragel PJ, Devaney EJ. Fine-needle aspiration cytology of multicystic mesothelioma. Diagn Cytopathol. 1992;8: Silverman JF, Geisinger KR. Fine Needle Aspiration Biopsy of the Thorax and Abdomen. New York, NY: Churchill Livingstone; 1996: Sawh RN, Malpica A, Deavers MT, et al. Benign cystic mesothelioma of the peritoneum: a clinicopathologic study of 17 cases and immunohistochemical analysis of estrogen and progesterone receptor status. Hum Pathol. 2003;34: Kerrigan SA, Cagle P, Churg A. Malignant mesothelioma of the peritoneum presenting as an inflammatory lesion: a report of four cases. Am J Surg Pathol. 2003;27: Park BJ, Alexander HR, Libutti SK, et al. Treatment of primary peritoneal mesothelioma by continuous hyperthermic peritoneal perfusion (CHPP). Ann Surg Oncol. 1999;6: Geisinger KR, Ng LW, Hopkins MB, et al. Tenckhoff catheter cytology in patients with ovarian cancer. Cancer. 1988;62: Am J Clin Pathol 2007;128: DOI: /DV1JYBL8LLYYT4J5