RAGNI ET AL.

493

Pneumocystis Infection of the Thyroid Pneumocystis in patients with AIDS. This observation suggests that aerosolized pentamidine should be recommended as only part of an effective prophylaxis regimen to prevent Pneumocystis infection in such patients. The combination of intermittent oral trimethoprim-sulfamethoxazole (Bactrim) or intermittent intravenous pentamidine or Bactrim, for example, with aerosolized pentamidine might be considered; unfortunately, the minimal effective dose of such drugs, when used in combination, is not known. In summary, this case illustrates three major points12: extrapulmonary Pneumocystis infection may present as thyroiditis with hypothyroidism5; "hematomas" in unusual sites in HIV-infected hemophiliacs may represent opportunistic infection; and1 aerosolized pentamidine prophylaxis alone may be insufficient to prevent extrapulmonary Pneumocystis infection. Acknowledgments. The authors acknowledge Dr. Julio A. Martinez for performing electron microscopic studies, Dr. Janet Amico for helpful advice, and Michele Macom for preparation of the manuscript. REFERENCES 1. Centers for Disease Control. HIV/AIDS surveillance report- -United States. October 1989:1-16.

2. Hopewell PC. Pneumocystis carinii pneumonia: diagnosis. J Infect Dis 1988;157:1115-1119. 3. Raviglione MC, Mariuz P, Sugar J, Mullen MP. Extrapulmonary Pneumocystis infection. Ann Intern Med 1989; 111:339. 4. Richie TL, Yamaguchi E, Virani NA, Quinn BD, Chaisson RE. Extrapulmonary Pneumocystis infection. Ann Intern Med 1989;111:339-340. 5. Case records of the Massachusetts General Hospital (case 9—1989). N Engl J Med 1989;320:582-587. 6. Girard PM, Landman R, Gaudebout C, et al. Prevention of Pneumocystis carinii pneumonia relapse by pentamidine aerosol in zidovudine-treated AIDS patients. Lancet 1989;1:1348-1353. 7. Conte JE, Hollander H, Golden JA. Inhaled or reduced dose of intravenous pentamidine for Pneumocystis carinii pneumonia: a pilot study. Ann Intern Med 1987;107:495-498. 8. Gallant JE, Enriquez RE, Cohen K.L, Hammers LW. Pneumocystis carinii thyroiditis. Am J Med 1988;84:303-306. 9. Lopresti JS, Fried JC, Spencer CA, Nicoloff JT. Unique alternations of thyroid hormone indices in the acquired immunodeficiency syndrome (AIDS). Ann Intern Med 1989; 110:970-975. 10. Kapadia SB, Dekker A, Cheng VS, Desai UMA, Watson CG. Malignant lymphoma of the thyroid gland: a clinicopathologic study. Head Neck Surg 1982;4:270-280. 11. Ragni MV, Lewis JH, Bontempo FA, Spero JA. Lymphoma presenting as a traumatic hematoma in an HTLV-II1 antibody positive hemophiliac. N Engl J Med 1985;313:640. 12. Campbell WG. Ultrastructure of Pneumocystis in human beings. Arch Pathol 1972;93:312-324. 13. Cupples JB, Blackie SP, Road JD. Granulomatous Pneumocystis carinii pneumonia mimicking tuberculosis. Arch Pathol Lab Med 1989:113:1281.

Malignant Peritoneal Mesothelioma in Childhood with Long-term Survival WILLIAM A. GEARY, M.D., P H . D . , STACEY E. MILLS, M.D., HENRY F. FRIERSON, J R . , M.D., AND THOMAS L. POPE, M.D.

A diffuse, well-differentiated, malignant peritoneal mesothelioma (MPM) developed in a nine-year-old girl. She received limited chemotherapy and radiation therapy and is alive and well without clinical evidence of disease 109 months after diagnosis. The neoplastic cells stained immunohistochemically for cytokeratin and epithelial membrane antigen but were unreactive with B72.3, anti-carcinoembryonic antigen, and anti-Leu-Ml. Ultrastructurally, the tumor cells had abundant desmosomes, numerous tonofilament bundles, and variable-length microvilli. These findings confirm the mesothelial nature of the cells. Features con-

sistent with malignancy included DNA aneuploidy by flow cytometric analysis and diffuse peritoneal involvement. The three previously described survivors with MPM were also premenarchal girls. Some MPMs in premenarchal girls have an indolent biologic behavior similar to that of low-grade peritoneal serous neoplasia or well-differentiated papillary mesothelioma in adult women. (Key words: Mesothelioma; Childhood; Immunohistochemistry; Flow cytometry; DNA analysis; Peritoneum; B72.3; Leu-Ml; Carcinoembryonic antigen; Cytokeratin; Epithelial membrane antigen) Am J Clin Pathol 1991;95:493-498

Most malignant peritoneal mesotheliomas (MPMs) occur men, many of whom have a history of asbestos exposure.12 Long-term survival in adults with MPMs is distinctly uncommon. The rare MPMs occurring in childhood have several important clinical differences from their adult counterparts.3"7 In children, MPMs have no clearcut association with asbestos exposure and no male predilection. Furthermore, isolated examples of survival in

From the Departments of Pathology and Radiology. University of Virin ginia Health Sciences Center, Charlottesville, Virginia. Received April 19, 1990; received revised manuscript and accepted for publication July 12, 1990. Dr. Pope is currently a member of the Department of Radiology, Bowrri£.:i Gray Medical Center, Winston-Salem, North Carolina. Address reprint requests to Dr. Mills: Department of Pathology, Box 214, University ofVirginia Health Sciences Center, Charlottesville, Virginia 22908.

Vol. 95 • No. 4

494

ANATOMIC PATHOLOGY

Single Case Reports TABLE 1. ANTIBODIES USED FOR IMMUNOHISTOCHEMICAL STAINING Antibody

Specificity

Dilution

Source

Anti-Leu-Ml B72.3 CAM 5.2 E29/EP1 MA009A*

Lacto-N-fucopentose III Human tumor-associated glycoprotein-72 Cytokeratins: 39 kD, 43 kD, 50 kD Epithelial membrane antigen Carcinoembryonic antigen

1:10 1:100 Predil 1:250 Predil

Becton Dickinson, Mountainview, CA Biomedical Technologies, Stoughton, MA Becton Dickinson DAKO, Santa Barbara, CA BioGenex, Dublin, CA

Predil •= prediluted.

* Control number, as designated by BioGenex.

children with MPM have been reported. 248 It is interesting that all survivors have been female.2,4,8 We describe a welldifferentiated MPM that arose in a nine-year-old girl who has been followed for more than nine years. A review of the literature relating to MPM in childhood is also included. Our observations suggest that well-differentiated MPM occurring in young girls who are survivors is a distinct entity analogous to both well-differentiated papillary peritoneal mesothelioma and low-grade peritoneal serous neoplasia of adult women. MATERIALS AND METHODS Tissue for standard light microscopic examination, histochemical studies, and immunohistochemical studies was fixed in 10% formalin and embedded in paraffin. Slides were stained with hematoxylin and eosin, colloidal iron with and without hyaluronidase predigestion, and periodic acid-Schiff (PAS) with and without diastase pretreatment. Immunohistochemistry with an avidin-biotinperoxidase complex technique was used in accordance with standard procedures.9 Antibodies used included B72.3 and those to Leu-Ml antigen, cytokeratin (CAM 5.2), epithelial membrane antigen (EMA), and carcinoembryonic antigen (CEA) (Table 1). Visualization of the antigen-antibody reaction was achieved with 3,3' diaminobenzidine. Appropriate positive and negative controls were used. For electron microscopic examination, fresh tissue was fixed in 2% glutaraldehyde and prepared for analysis with a Zeiss EM-9® (Carl Zeiss, Thornwood, NY) electron microscope. For ploidy analysis, a 50-jum section from the paraffin block of the biopsy specimen was deparaffinized according to the technique of Hedley and colleagues.10 The cells, stored frozen in a dimethylsulfoxide (DMSO)-citrate buffer, were subsequently prepared for cytometric analysis with the use of the detergent-trypsin method of Vindelov and colleagues.'' Nuclei were stained with propidium iodide and evaluated with an EPICS C® flow cytometer (Coulter Electronics, Hialeah, FL) equipped with a 5-W argon laser. A minimum of 10,000 nuclei were analyzed. Nonneoplastic nuclei present in the paraffin blocks served

as an internal diploid standard. DNA aneuploidy was considered to be present when another G 0 /Gi peak was present in addition to the diploid peak. The flow cytometric results for this case were included in a large series of malignant mesotheliomas that was reported previously.12 REPORT OF CASE A nine-year-old white girl initially presented in January 1981 with malaise, irritability, abdominal cramps, diarrhea, and arthralgias. Roentgenograms of the ileum were compatible with Crohn's disease, and she was started on prednisone and sulfasalazine. She continued to lose weight and two months later had shortness of breath and a rightsided pleural effusion. She was treated with antibiotics without improvement and then was admitted to the University of Virginia Health Sciences Center. On admission, decreased breath sounds were noted at the right lung base. The laboratory parameters were remarkable for a white blood cell count of 13.2 X 109/L. A chest roentgenogram showed a large rightsided pleural effusion. An abdominal ultrasonogram revealed ascites and a questionable mass in the right lower quadrant. Cytologic examination of ascitic fluid yielded negative results for malignancy. An abdominal computed tomography (CT) scan showed a large amount of pelvic soft tissue consistent with inflammation or a neoplasm. At exploratory laparotomy, there were 2 L of straw-colored ascites. The external surfaces of the ovaries, fallopian tubes, and uterus were normal. A diffuse, soft pelvic mass was present. Multiple "sand-like" nodules were present on the serosal surfaces of liver and spleen. A normalappearing appendix was excised. Biopsies were performed of the greater omentum, pelvic peritoneum, small bowel serosa, and liver surface. After a diagnosis of malignant mesothelioma was rendered, chemotherapy was initiated with vincristine, actinomycin D, Adriamycin8 (doxorubicin; Adria Laboratories, Dublin, OH), cyclophosphamide, and prednisone. One month later, chest roentgenograms showed an increased right pleural effusion with pleural thickening. Chemotherapy was continued, and one month later she was admitted with complaints of left anterior neck pain. Recurrent ascites was observed on abdominal examination. A chest roentgenogram showed a large, right-sided pleural effusion with a mass in the soft tissue of the left side of the neck. Venography documented a left subclavian vein thrombus. A CT scan showed a soft tissue density in the manubrium that displaced the innominate vein posteriorly. Additionally, there was an increase in soft tissue density in the left apical area, upper left chest wall, and left neck. Because of the clinical suspicion of tumor, 1,600 rads was delivered to the left supraclavicular area. This resulted in reduction of the size of the mass and pain relief. Three months after the original diagnosis, chemotherapy and radiation therapy were discontinued.

A.J.C.P. 'April 1991

GEARY ET Ah. Malignant Peritoneal Mesothelioma

495

FlG. 1 (left). Neoplastic mesothelial cells occur singly, in small clumps, and form rare papillary structures. A lymphoid infiltrate also is prominent. Hematoxylin and eosin (X100). FIG. 2 (right). The cells of malignant mesothelioma contain multiple cytoplasmic vacuoles. The larger vacuoles displace the nucleus in a "signetring" configuration. Hematoxylin and eosin (X400).

One year later the patient was admitted with recurrent headaches. Physical examination showed bilateral papilledema but no visual field defects. A CT scan of the head was unremarkable. She has now been followed at the University of Virginia Health Sciences Center for a total of 109 months. At the time of her most recent examination, there was no evidence of malignancy or recurrent effusions.

cells were seen involving the capsule of the liver, serosa of the small bowel and appendix, and pelvic peritoneum. In some areas the proliferating mesothelial cells were associated with a prominent lymphoid infiltrate. Histochemistry and

RESULTS Gross and Light Microscopic Examination The abdominal biopsy specimens ranged from 0.7 to 5.5 cm in maximum dimension. They were soft and tan to yellow-gray. Microscopically, the omental biopsy specimen contained sheets of atypical mesothelial cells that encircled islands of fat. The neoplastic cells were polygonal, with abundant granular, eosinophilic cytoplasm, and formed only a few tubular and papillary structures (Fig. 1). Many cells had single or, rarely, multiple cytoplasmic vacuoles (Fig. 2). Nuclei were round or oval, showed mild pleomorphism, and contained single nucleoli. There were rare, normal-appearing mitotic figures. Identical sheets of

Immunohistochemistry

The cytoplasm of the mesothelial cells stained with colloidal iron, and this was reduced by hyaluronidase predigestion. There were PAS-positive, diastase-digestible cytoplasmic granules, but diastase-resistant granules were not seen. Immunohistochemical stains were positive for cytokeratin and EMA. No reactivity was observed with B72.3 and antibodies to CEA and Leu-Ml. Electron Microscopic Examination The neoplastic cells were polygonal and had abundant desmosomes. Variable-length microvilli protruded from most cell surfaces, including those between adjacent cells (Fig. 3). The cytoplasm contained numerous tonofilament

Vol. 95 • No. 4

496

ANATOMIC PATHOLOGY Single Case Reports

FIG. 3. Cells of malignant mesothelioma have prominent microvilli at their junction with the underlying stroma (XI 7,000).

bundles, mitochondria, and glycogen. Occasional lipid vacuoles were seen. Flow Cytometry The histogram showed a DNA diploid peak and a DNA aneuploid peak (Fig. 4). The latter had a DNA index of 1.67. The coefficients of variation (at half maximum-peak height) were 4.5% for the DNA diploid Go/G, peak and 5.1% for the DNA aneuploid G 0 /G| peak. DISCUSSION Based on multiple lines of evidence, we believe that the lesion described in this report is a well-differentiated malignant mesothelioma. The tumor diffusely involved the pelvic peritoneum and consisted of epithelial meso-

DNA CONTENT FIG. 4. Flow cytometry histogram shows two peaks corresponding to diploid (peak 1) and aneuploid (peak 2) nuclei. The DNA index is 1.67.

thelial cells that had mild nuclear pleomorphism. The results of the histochemical stains were typical of mesothelial cells. 1314 Immunohistochemical reactivity for cytokeratin and EMA, absence of staining for CEA, and lack of reactivity with anti-Leu-M 1 and B72.3 are also typical of mesothelial cells. Bollinger and associates studied 46 serous adenocarcinomas and found that 74% stained for Leu-Ml, 72% were reactive with B72.3, and 13% were positive for CEA.9 Conversely, diffuse cytoplasmic reactivity with anti-Leu-M 1, B72.3, or anti-CEA excluded the diagnosis of malignant mesothelioma.9 Ultrastructural analysis of our case demonstrated features characteristic of mesothelial cells, including microvilli, numerous desmosomes, abundant tonofilaments, and the absence of glandular differentiation or cilia. The malignant nature of the tumor was supported by the demonstration of DNA aneuploidy by flow cytometry. In a study that examined the DNA content of reactive mesothelial cells in fresh effusion specimens, Frierson and associates found that such cells were uniformly DNA diploid.12 In contrast, 18 of 49 malignant epithelial mesotheliomas were found to be DNA aneuploid by flow cytometry.12-15 The MPMs occurring in children have usually been epithelial or biphasic neoplasms. We are aware of only two apparent fibrous MPMs in children. One occurred in a 14-year-old girl, but follow-up information was not provided.16 The other occurred in a 16-year-old girl who died after 21 months despite surgery, chemotherapy, and radiation therapy.17 Talerman and associates reported an epithelial MPM in a 13-year-old girl, and their review of the literature yielded eight additional MPMs in young or adolescent children.18 We were able to identify 14 other

Vol. 95 • No. 4

-X

G E A R Y ET

497

AL.

Malignant Peritoneal Mesothelioma TABLE 2 . REPORTED PERITONEAL MESOTHELIOMAS IN CHILDHOOD Reference

Age

Sex

Tumor Type

Therapy

19 5 5 16 22 28 24 25 7 7 7 3 20 21 23 17 18 26 27 8 4 6 Present case

13 yr 12 yr 16 yr 14 yr 2 yr 3.0 yr