—  SPECIALTY CONFERENCE  —

Cytopathology

Case 6 - Malignant Mesothelioma

Sana O. Tabbara
The George Washington University
Washington, DC


Click on each slide thumbnail image for an enlarged view
Clinical History
A 56 year-old man presented with a history of intermittent fever, flushing, nausea, vomiting, and abdominal pain and distention. A colonoscopic exam performed in the past year was reportedly normal. An abdominal CT scan was performed and revealed a 12.0x5.0x 13.0 cm intra-abdominal mass involving the small bowel. A core biopsy was obtained with ultrasound guidance but did not provide adequate tissue for diagnosis. A PET scan confirmed the presence of a hypermetabolic, ill-defined soft tissue mass in the lower abdomen arising from small bowel or mesenteric wall. Additional small coalescent masses or adenopathy were also noted as well as increased marrow activity. An open biopsy with intraoperative consultation was performed. Diff-Quik and H&E-stained smears were prepared. (Figures 1-3)


Case 6 - Figure 1 - Highly cellular smears show a monotonous population of polygonal cells organized in loose clusters, sheets and numerous single cells. The cells have large, round, or slightly irregular centrally placed nuclei and macronucleoli as well as abundant dense cytoplasm. Although monotonous some nuclear size variability is easily observed and intercellular gaps are evident. Scattered cells show single large cytoplasmic vacuoles containing eosinophilic material. (Diff-Quik stain, medium power)
Case 6 - Figure 2 - Numerous cells with abundant cytoplasm and large round vesicular nuclei with prominent nucleoli are present in flat sheets and singly. Intercellular spaces or windows are apparent. Despite some degree of monotony, nuclear pleomorphism is present with some larger, hyperchromatic nuclei. Bi-and multinucleation is illustrated as well as the presence of mitotic figures. (H&E stain, medium power)
Case 6 - Figure 3 - In addition to the cytologic features described above, some cells show small punched out vacuoles in a perinuclear location and sometimes overlapping the nucleus. The abundant dense cytoplasm appears more delicate at the periphery in some cells and intercellular spaces or windows are also seen. (Diff-Quik stain, high power)

Diagnosis: Malignant Mesothelioma

Cytologic findings
Smears were highly cellular and showed a monotonous population of polygonal cells organized in loose clusters, sheets and numerous single cells. Occasional tissue fragments were present and had fine capillaries coursing in between cellular groups, without any characteristic architectural pattern. The cells had large, round, or slightly irregular centrally placed nuclei. Chromatin was vesicular and prominent single or multiple nucleoli were present. Although monotonous, variability in nuclear size was easily observed. Bi- and multinucleation was also present. The cells had abundant dense cytoplasm with hazy cell borders. Frequently, on the Diff-Quik smear, cells showed small punched out vacuoles in a perinuclear location and sometimes overlapping the nucleus. A light eosinophilic fuzzy cytoplasmic border was discernable in some cells and intercellular spaces or windows were also seen on the H&E-stained smear in areas where the cells are present in sheets. Scattered cells show single large cytoplasmic vacuoles that contained eosinophilic material on Diff-Quik. Occasional mitoses were found. The cytologic features were not compatible with the diagnosis of lymphoma. The overall appearance of the cells and specific cellular features were suggestive of mesothelial origin.

Histology and Clinical Follow up
The clinical findings based on the results of the CT and PET favored a diagnosis of malignant lymphoma, followed by metastatic carcinoma. A preoperative bone marrow aspirate was negative for malignancy. At the time of surgery the tumor was identified in the inferior half of the abdomen. The bowels were frozen by tumor however no single point of definite obstruction was identified. Ascitic fluid was present. The intraoperative consultation ruled out the possibility of lymphoma. A working diagnosis of carcinoma vs. mesothelioma was provided and the final diagnosis was deferred. Following histologic evaluation a diagnosis of malignant mesothelioma was established. Histologic sections revealed large polygonal neoplastic cells growing predominantly in a solid with focal areas of tubulopapillary architecture. Prominent vascularity, multifocal necrosis and high mitotic activity were also present. The malignant cells had deeply eosinophilic cytoplasm and distinct cytoplasmic borders. They had central or sometimes eccentrically placed nuclei with prominent, mostly single nucleoli. The malignant cells were diffusely immunoreactive with pancytokeratins AE-1/AE3, Calretinin (both nuclear and cytoplasmic), WT-1, vimentin and EMA. B72.3, CEA, LeuM-1, CD10, anti-human Hepatocyte and AFP were all negative. Bile and mucicarmine stains were also negative. Electron microscopic examination revealed features of mesothelioma including the characteristic long branching wavy microvilli. The patient was placed on a chemotherapeutic regimen.

Discussion
Malignant mesothelioma is most frequently seen in the pleural cavities where it affects predominantly older patients (50-70 years). It has a strong association with asbestos, however 50% of men and 95% of women with malignant mesothelioma have no history of asbestos exposure. Patients present with chest pain, shortness of breath, weight loss and pleural effusions. The right side is about twice as often involved as the left side. The pleura is usually diffusely thickened and nodular especially at the base and posteriorly, with tumor eventually encasing the lung, producing thickened interlobular fissure and obliterating the pleural cavity. The key feature for malignancy is the production of an invasive mass, however, an intraparenchymal lesion in the lung and regional lymph node metastases are more compatible with carcinoma than mesothelioma. Malignant mesothelioma also occurs in the peritoneum, pericardium and tunica vaginalis.

Malignant mesothelioma in the peritoneum usually occurs in patients over 40 years old, with a male predominance. About half of the patients have a history of heavy exposure to asbestos with a latency period of approximately 2-4 decades. Some cases are associated with Thorotrast, a contrast medium previously used in radiology, or with repeated mesothelial irritation and some coexist with pleural mesotheliomas. Patients with peritoneal mesothelioma present with recurrent ascites, abdominal cramps and intermittent partial bowel obstruction. The tumor can manifest itself initially in a hernia sac, umbilicus, ovary or bowel wall, as inguinal or cervical lynphadenopathy from metastases, or as an inflammatory disease. The tumor usually appears as multiple plaques and nodules on the visceral and parietal peritoneum, with adhesions producing shortening of the mesentery. Associated fibrous plaques are more common than in pleural mesothelioma. Occasionally malignant mesothelioma may present as an isolated mass.

Histologically, the microscopic pattern is variable with epithelial tumor displaying tubular, papillary, microcystic, and solid areas composed of atypical mesothelial cells. In papillary areas, the fibrovascular stroma may contain psammoma bodies. In biphasic tumors, sarcomatoid spindle cells alternate with mesothelial-like cells. Pure sarcomatoid mesotheliomas can be distinguished from other sarcomas occurring in the pleural/peritoneal cavity based on cytogenetic studies. Desmoplastic mesothelioma may pose a diagnostic problem especially in cases associated with inflammation. The presence of nuclear atypia, necrosis, mitoses, storiform and fascicular patterns and infiltration of adjacent tissues are markers for malignancy. Lymphohistiocytoid mesothelioma, deciduoid and small cell variants have also been described. Well-differentiated papillary mesothelioma another variant show a great predilection to women and is usually multifocal. In some cases the mesothelial cells may have prominent vacuoles and may appear clear as a result of hydropic change or lipid accumulation may give the cells a foamy cytoplasm. Multinucleation is not uncommon. Stromal metaplasia can be encountered.

Typically, the diagnosis of mesothelioma is first suggested in body cavity fluid specimens rather than on a FNA sample. The effusion at presentation is bloody, or yellow viscous or gelatinous. The characteristic cytologic appearance is that of numerous large clusters of cells with irregular, scalloped or knobby edges each containing several hundreds mesothelial cells. However, in the presence of a large mass, fine needle aspiration may be the appropriate approach to diagnosis. The FNA smears are cellular and contain numerous clusters and sheets, some showing branching papillae. Some cases consist predominantly of single cells. An occasional spindle cell may suggest a biphasic component and help in arriving at the correct diagnosis.

A key feature in making the diagnosis of malignant mesothelioma is in recognizing the resemblance of the neoplastic cells to ordinary mesothelial cells. A continuum from bland to malignant appearing cells is present and two distinct populations cannot be recognized. The cells are cytomegalic but also variable in size, however the N/C remains relatively constant imparting a monotonous appearance to the malignant cells. Cells are round to polygonal with well-defined, abundant, dense, squamoid but sometimes delicate vacuolated and foamy cytoplasm with a peripheral halo or pale rim reflecting the presence of glycogen. Two types of vacuoles can be appreciated; degenerative vacuoles that are small, uniform and perinuclear, and glycogen vacuoles that are peripheral more variable and PAS positive, located particularly in peripheral cytoplasmic blebs. Vacuolated cells forming signet ring-like forms may be present and may contain metachromatic mesenchymal mucin. Nuclei are round, centrally placed with chromatin varying from fine to coarse and prominent nucleoli or macronucleoli. Binucleation and multinucleation is common. Intercellular spaces or windows are characteristic of mesothelial cells. Psammoma bodies and flocculent background material reflecting the presence of hyaluronic acid may be seen in the background. In cell block, mesothelioma shows either solid masses or papillae containing a collagenous core. Rarely malignant cells form luminal spaces.

In patients with malignant mesothelioma, approximately two thirds of initial effusions are negative and the overall diagnostic yield of fluid cytology is about 60% indicating that diagnostic pitfalls exist. Difficulties in arriving at a diagnosis are encountered in fluids with sparse cellularity and/or predominance of lymphocytes or in cases where highly vacuolated mesothelial cells resembling histiocytes represent the majority of the cells. In those instances a malignant diagnosis cannot be made and a diagnostic clue may be the presence of multinucleated giant cell histiocytes. Some cases that consist predominantly of single cells with only rare clusters may be hard to diagnose. Problems arise in some instances where the neoplastic cells are bland resembling benign mesothelial cells and mimicking a reactive effusion with mesothelial hyperplasia, or highly pleomorphic and not recognizable as mesothelial in origin. Identification of malignant spindle cells is a helpful clue and may suggest a biphasic mesothelioma. The cytologic differential diagnosis includes reactive effusions with mesothelial hyperplasia. Those specimens are less cellular and mesothelial cells tend to occur in flat sheets and smaller less complex groups. Nuclei may be atypical but clear-cut malignant features are not present and nucleoli although prominent are usually single and not macronucleoli.

Metastatic adenocarcinoma represents the main differential diagnostic challenge due to its overlapping features with mesothelioma and its higher incidence. In the pleural cavity, mesothelioma should be differentiated from lung carcinoma whereas in the peritoneal cavity the differential is with serous carcinoma of mullerian type (primary in the ovary, endometrium or peritoneal cavity) a carcinoma of modified mesothelial cells and pancreatic carcinoma. The identification of two-cell population is always helpful, however cases wit a pure population of carcinoma cells are not uncommon. Metastatic carcinoma occurs in clusters with smooth community borders and may display acinar or gland-like formation rather than the windows associated with mesothelioma. True papillary architecture may be associated with bronchioloalveolar adenocarcinoma of the lung or papillary thyroid carcinoma., however adenocarcinomas do not usually display collagen cores within the groups. Additionally cells of adenocarcinoma have overt features of malignancy including nuclear pleomorphism and irregularity, hyperchromasia, irregular nucleoli, increased N/C ratio. The cytoplasmic borders are often better defined than in mesothelial type cells and secretory vacuoles indenting the nucleus and intracellular mucin may be present.

Features that favor mesothelioma are prominent tubulopapillary pattern with polygonal cells and absence of marked nuclear pleomorphism or high N/C ratio, multinucleation with atypia and windows. Cell in cell pattern or clasping and long chain of cells favor mesothelioma. Unequivoval identification of mesothelioma is difficult based on cytologic grounds alone. Several modalities exist to support or confirm the diagnosis. Most commonly a panel of immunohistochemical stains helps in the final determination. Stains may be performed on cell block material or on liquid based preparations.

The following antibodies are commonly used:

Pancytokeratin: positive in both with different staining patterns; cytoplasmic and perinuclear in mesothelioma, peripheral in adenocarcinoma
CK5/6: positive in mesothelioma, negative in adenocarcinoma
EMA: positive in both with different staining patterns; thick membranous in mesothelioma, cytoplasmic in adenocarcinoma
CEA, LeuM-1, B72.3, and Ber-Ep-4: negative in mesothelioma, positive in adenocarcinoma
Calretinin: positive nuclear and cytoplasmic staining in mesothelioma, negative in most adenocarcinomas
WT1: positive nuclear staining in mesothelioma and in serous adenocarcinomas
Mesothelin: positive in mesothelioma, negative in adenocarcinoma
TTF-1: negative in mesothelioma, positive in most lung and thyroid adencarcinoma

Panels should include positive and negative identifiers for both mesothelioma and adenocarcinoma. Some suggested panels include calretinin, CK5/6 (or WT1) and CEA, B72.3 (or Ber-EP4), Ber-EP4, CK5/6 and calretinin, Ber-EP4, CK 5/6 and CEA. The immuno- panel selection should be done based on availability and convenience. Some markers such as EMA, vimentin, HBME-1 and S-100 protein have no utility in the arriving at the correct diagnosis. WT-1 is of limited usefulness in the presence of a history of serous carcinoma or when a primary peritoneal carcinoma is considered.. Mesothelioma commonly over-expresses p53 however mutation are only rarely observed. In addition markers may be lost in sarcomatoid areas of mesothelioma.

Histochemical stains may still play a role in the diagnosis of mesothelioma. Demonstrating intracytoplasmic neutral mucin (mucicarmine positive or PAS positive –diastase resistant) supports the diagnosis of adenocarcinoma. However in the peritoneum this is may not helpful and cases of mucin positive mesothelioma have been described. Mucicarmine can also stain hyaluronic acid in about 5-10% of cases. This staining can be abolished by hyaluronidase Malignant mesothelioma contains extracellular mucosubstance that are PAS negative and can be demonstrated with colloidal iron and alcian blue that are removed by hyaluronidase digestion.

In the presence of a dominant mass that is approached by fine needle aspiration, the primary entity to be considered in the differential diagnosis is again adenocarcinoma. In the peritoneal cavity, consideration has to be given to tumors comprised of polygonal cells. Serous carcinoma, renal cell carcinoma, hepatocellular carcinoma and melanoma are some considerations. Renal cell carcinoma is composed of large vacuolated or granular cells with abundant cytoplasm and may display papillary architecture. Renal cell carcinomas are immunoreactive for EMA, low molecular weight cytokeratin and CD10 and negative for high molecular weight cytokeratin. Hepatocellular carcinoma are also composed of large cells that may have abundant granular cytoplasm and large round nuclei, prominent nucleoli and intranuclear pseudoinclusion. The hallmark of hepatocellular carcinoma is the presence of a spindle cell population around cords and nests of malignant hepatocytes and a positive bile stain in addition to immunoreactivity to anti-human Hepatocyte marker and AFP. Malignant melanoma also presents with large cells with dense cytoplasm, binucleation, and prominent nucleoli mostly in single cells with loosely cohesive clusters. Nuclear pseudoinclusions and melanin pigment may be present and the malignant cells are immunoreactive to S-100 protein, HMB45 and Melanin A, and negative for keratin and calretinin.

Mesothelial cells have squamoid features therefore the possibility of a nonkeratinizing squamous cell carcinoma may be considered. Involvement of body cavity fluid by metastatic squamous cell carcinoma is uncommon and will present with a discrete population of malignant cells. Cells will have a denser cytoplasm with better-defined cell borders and nuclear pleomorphism will be more marked. Bizarre cells and pearls may be seen and are not a feature of mesothelioma.

Angiosarcoma/ hemangioendothelioma, are rare in the lung and may spread to the pleura mimicking malignant mesothelioma. These tumor will stain positively CD31, and CD34 and will be negative for mesothelial or epithelial markers.

Confirmation by electron microscopy is the next choice. Transmission electron microscopy typically demonstrate abundant bushy branching microvilli covered with fuzzy material, extracellular and intracellular neolumina, glycogen granules, junctional structures, abundant intermediate filaments condensed into tonofilaments in a perinuclear location and basal lamina.

Additional diagnostic help can be provided by computer assisted morphometry or cytophotometric quantitation of DNA. Flow cytometry has not proven a very sensitive modality but will detect aneuploidy in 50% of cases. Clonal cytogenetic aberrations that include the most common deletion of 1p, 3p, and 22q can be evaluated by FISH on liquid based preparations and may be helpful indicators of malignancy.

The pattern of spread of malignant mesothelioma is local leading to complete obliteration of the pleural/peritoneal cavity. In advanced stages tumor may invade intrabdominal organs, retroperitoneum and abdominal wall and metastasize to regional lymph nodes. Distant metastases are rare. Prognosis is extremely poor with most patient dying of disease within 2 years from the time of diagnosis. Treatment consists of debulking, combination chemotherapy and whole abdomen irradiation.

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