Reversed Helper/Suppressor T-Lymphocyte Ratio in Bronchoalveolar Lavage Fluid from Patients with Breast Cancer and Pneumocystis carinii Pneumonla':'
JAMES SIMINSKI, PAMELA KIDD, GRETCHEN D. PHILLIPS, CAROLYN COLLINS, and GANESH RAGHU
Introduction
Pneumocystis carinii pneumonia (PCP) is an important consideration in the etiology of pulmonary infiltrates in the immunocompromised patient (1-5). PCP most commonly occurs in patients with acquired immunodeficiency syndrome (AIDS), malignant and nonmalignant diseases of the lymphoreticular tissues, congenital immunodeficiency disorders, in premature or malnourished infants, and in patients treated with corticosteroids and immuosuppressive agents (6-10). In contrast, PCP has been rarely reported in patients with solid tumors (11-13). Fiberoptic bronchoscopy examination is often done to confirm the diagnosis of PCP in specimens retrieved from bronchoalveolar lavage (BAL) and/or transbronchiallung biopsy (TBL) (14). HAL is a useful technique to sample cells and secretions from the lower respiratory tract in interstitial lung diseases (15-17). Cellular profiles have been useful in characterizing several types of interstitial pneumonitis; increased proportions of lymphocytes are common findings in BAL from patients with hypersensitivity pneumonitis and sarcoid. Altered T-Iymphocyte subsets in BAL are commonly believed to differentiate these diseases: decreased CD 4:CD s ratio and increased CD 4:CD s ratio of HAL lymphocyte subsets are considered typical findings in hypersensitivity pneumonitis and sarcoidosis, respectively (18-20). Recent reports in patients with AIDS have also demonstrated decreased CD 4:CDs ratio in BAL lymphocytes (21). We describe HAL findings in three cases of PCP in patients treated for breast cancer at our institution (UWM C) over a 1.5-yr period. These patients had no serologic evidence of infection with human immunodeficiency virus (HIV). The findings of peripheral and HAL lymphocyte count and T-cellsubsets are discussed.
Case Reports Patient 1 A previously healthy 24-yr-old woman presented to our institution with a right breast mass. Lumpectomy and axillary node dissectionrevealeda high grade medullary carcinoma with 12/12 nodes negative for malignancy. The tumor was negative for estrogen and progesterone receptors. The patient wastreated with 5,040cGy tangential external beam irradiation over a 48-day period followedby a 2,000 cGy boost with an iridium-192implant. One month later a right internal mammary node was found
SUMMARY Pneumocysfls pneumonia (PCP) usually occurs In patients with hematologic malignancies and acquired Immunodeficiency syndrome (AIDS). Patients with solid tumors represent a very small fraction of the reported cases of PCP.Over an 18-month period, PCP was diagnosed In three patients who had received radiation and chemotherapy for breast cancer. In all three patients, there was no serologic or clinical evidence of AIDS. Direct staining of bronchoalveolar lavage fluid (BAL) revealed Pneumocystls carlnll, and cellular analysis of BAL revealed an Increased percentage of lymphocytes with reversed helper/lnducer:suppressor/cytotoxlc T-cell (CD4:CD a) ratio. Beca~se decreased CD4:CD a ratio In BAL Is commonly accepted as findings consistent with hypersensitivity pneumonitis and AIDS, we conclude that similar findings In patients without AIDS are not specific for hypersensitivity pneumonitis, and If carlnllshould be ruled out In the appropriate clinical setting. AM REV RESPIR DIS 1991; 143:437-440
to be positive for poorly differentiated carcinoma. She received an additional 5,040 cGy to the right internal mammary lymphatic chain and right supraclavicular fossa over the next 41 days. Vinblastine and fluorouracil (5FU) were administered once weeklyfor 4 wk during radiation therapy. This was followed by 12 months of combination chemotherapy consisting of cyclophosphamide, 5FU, and doxorubicin. A 10-mg dose of dexamethasone was administered orally prior to chemotherapy. Fifteen days after the last dose of antineoplastic drugs, she presented with dyspnea on exertion, nonproductivecough, and fever.Physical examination revealed a temperature of 38.4 0 C, tachypnea, tachycardia, orthostatic hypotension, and bibasilar inspiratory crackles. Fiberoptic bronchoscopy and analysis of BAL fluid wereperformed as described below.BAL fluid and TBL specimen disclosed P. carinii on GramWeigert and methanamine silver strains. Viral cultures, fungal stain and culture, Legionella by direct fluorescent antibody stain (DFA) and culture, and acid-fast bacilli (AFB) stain and culture were all negative. The patient was treated with trimethoprim/ sulfamethoxazole, with subsequent resolution of symptoms and pulmonary infiltrates. The patient's serum was negative for HIV antibody by ELISA on two separate occasions. Ten months after the pneumonia the absolute CD 4 ( + ) lymphocyte count increased to 480/mm3 , and she has remained well.
Patient 2 A previously healthy 44-yr-old woman presented with a left breast mass. Lumpectomy and axillary node dissection revealed infiltrating ductal carcinoma with 3/26 nodes involved. The tumor was negative for estrogen and progesterone receptors. She was treated with cyclophosphamide, 5FU, and doxorubicin for 6 months. After this, tangential external beam irradiation (5,040 cGy) was delivered to the left breast and supraclavicular region over a 44-day period, and an iridium-192 implant wasplaced (1,500 cGy). Concomitant cyclophospha-
mide and 5FU were administered weekly during radiation therapy. A IO-mgdose of dexamethasone was administered orally prior to chemotherapy. Two weeks after completing radiation therapy, the patient was hospitalized with shortness of breath, fever, night sweats, and nonproductive cough. Physical examination was significant for a temperature of 37.8° C, tachypnea, and tachycardia, and lung sounds were normal to auscultation (table I). Fiberoptic bronchoscopy was performed, and analysis of BAL fluid was done as described below. R cariniion Gram-Weigert and methanamine silver stains were identified in the BAL fluid and TBL specimen. Viral cultures, fungal stain and culture, Legionella stain and culture, and AFB stain and culture were all negative. The patient was treated with trimethoprim/sulfamethoxazole with subsequent resolution of symptoms and infiltrate. The patient's serum was negative for, ntv antibody by ELISA on two separate occasions,Tenmonths after the pneumonia the absolute peripheral CD 4 ( +) lymphocyte count increased to 450/mm3 , and she has remained well.
Patient 3 A previously healthy 48-yr-old woman presented
(Received in original form April 27, 1990 and in revised form August 23, 1990) 1 From the Divisions of Pulmonary and Critical Care Medicine and of Oncology, Departments of Medicine and Laboratory Medicine, University of Washington Medical Center (UWMC), Seattle, Washington. 2 Correspondence and requests for reprints should be addressed to Ganesh Raghu, M.D., Division of Pulmonary and Critical Care Medicine, Universityof Washington Mail Stop RM-12,University of Washington Medical Center, Seattle, WA 98195.
437
438
CASE REPORT
TABLE 1 PERTINENT CLINICAL AND LABORATORY FINDINGS IN THE THREE PATIENTS
Age, yr Fever Nonproductive cough Dyspnea Chest roentgenograph Interstitial pattern Arterial blood gas determinations F102 pH Paco2 Pao2 Peripheral blood WBC Lymphocytes CD.. (normal = 400-1,500) CD, (normal = 300-800) CD..:CD, (normal = 0.9-3.0) Serum LDH (normal < 304 U/L) Bronchoalveolar lavage Macrophages, % (normal> 85%) Lymphocytes, % (normal < 150/0) PMNs, % (normal, 0-1) Eosinophils, % (normal, 0-1) Monocytes, % (normal, 0-1) Epithelial cells, 0/0 Basophils, % (normal, 0-1) T-lymphocyte subsets CD.., 0/0 CD" 0/0 CD..:CD, (normal, 1.5-2) Microbiology/virology/stains TBUhistopathology
Patient 1
Patient 2
Patient 3
24
44
48
+ + +
+ + +
+ +
Diffuse, bilateral
Left upper lobe
Diffuse, bilateral
0.21 7.49 33 mm Hg 51 mm Hg
2 Ipm O2 7.43 36 mm Hg 99 mm Hg
0.21 7.56 21 mm Hg 71 mm Hg
5,000/mm3
3,800/mm 3
7,500/mm 3
440/mm3 128/mm3 320/mm3 0.42 638 U/L
800/mm3 180/mm3 560/mm3 0.32 442 U/L
1,390/mm 3 930/mm 3 355/mm 3 2.64 631 U/L
42 34 12 1 4 6 1
24 21 22 20 9 3 1
17 75 7 1 0 0
14 66 0.21 P. carinii+ P. carinii
28 68 0.4 P. carinii+ P. carinii
47 48 0.98 P. carinii+
* Values in parentheses are normal for UWMC laboratory.
with a left breast mass. Left modified radical mastectomy and axillary node dissection revealed infiltrating ductal carcinoma with 4/7 nodes involved. The tumor was positive for estrogen and progesterone receptors. The patient wastreated with diethylstilbesterol and received six cycles of cyclophosphamide, methotrexate, and fluorouracil followed by 5,000 cGy of external beam irradiation to the left chest, supraclavicular area, and a boost to the axilla. Approximately 2 yr later she was found to have metastases in bone and liver. She received 3 months of cyclophosphamide, 5FU, adriamycin, and subsequently methotrexate and Velban because of an increasein sizeof livermetastases. A lo-mg dose of dexamethasone was administered orally prior to chemotherapy. Approximately six weeks after the last dose of antineoplastic drugs, she presented to the hospital with dyspnea on exertion, low grade fever, and 4 days of nonproductive cough. Physical examination was significant for a temperature of 36.00 C and tachypnea, and lung sounds were normal on auscultation. Fiberoptic bronchoscopy and analysis of BAL of fluid were performed as described below. BAL fluid disclosed R cariniiby Gram-Weigert stain. Viral cultures, fungal stain and culture, Legionella DFA and culture, and AFB stain and culture were negative. The patient was initially treated with trimethoprim/sulfamethoxazole, but after several days she was switched to trimethoprim/dapsone secondary to nausea and vomiting. The patient's symptoms and pulmonary infiltrates subsequently resolved. Her serum was tested for HIV antibody by ELISA and was negative.
Fiberoptic Bronchoscopy and Bronchoalveolar Lavage Fiberoptic bronchoscopy and bronchoalveolar lavage were performed in all three patients as previously described (22). Patients were premedicated intramuscularly with 0.4 mg of atropine, and 2OJo lidocaine was delivered via an atomizer for topical anesthesia. The bronchoscope (Olympus P20D; Olympus Corp. of America, New Hyde Park, NY) was inserted transnasally or orally, and normal endobronchial anatomy was confirmed. The tip of the bronchoscope was then wedged into the subsegmental bronchus of the involvedarea determined from the roentgenographs. Sterile isotonic saline solution (room temperature) was instilled in five 3O-ml aliquots. Each aliquot was retrieved with gentle suction and collected in sterile suction traps. The aspirate from the first aliquot was collected separately and analyzed for cytology. The aspirates from the second to fifth were pooled in another trap. Approximately 50 to 6OOJo of the instilled saline was retrieved, and approximately 20 ml were submitted to microbiology and virology for appropriate stains and cultures. The BAL fluid was analyzed by the standard procedures in use in our laboratory since 1986, modified from previously established methods (16, 22). Briefly, the BAL fluid was strained through a single layer of sterile gauze and centrifuged for 10min at 1,900rpm. The cellpellet was resuspended in phosphate-buffered saline (PBS) and centrifuged for 5 min at 1,900 rpm. The PBS was decanted, and the cell pellet was resuspended in RPMI complete media (GIBCO Laboratories, Grand Island, NY) to a total volume of 1ml. A cell viability count was performed by trypan blue exclusion. The cells
were resuspended in RPMI complete to a concentration of 5 to 9 X 106 cells/ml. Cytocentrifuge preparations were made using a Shandon Cytospin 2 (Shandon Inc., Pittsburgh, PAl, and a 200-cell differential was performed on Wright's stained slides. Five hundred thousand cells wereincubated at 4 0 C for 30 min with FITC-labeled T 4 and Ts, and the percentage of CD 4 and CDs positive cells was analyzed on a Coulter EPICS Profile flow cytometer (Coulter Cytometry, Hialeah, FL), counting only the cells in the lymphoid gate as defined by the scattergram. Pertinent clinical and laboratory findings of the three patients are listed in table 1.
Discussion Pulmonary parenchymal abnormalities are a common occurrence in the immunocompromised host (1-5). Because pulmonary infiltrates in patients who have received radiation and chemotherapy for malignancy may result from radiation damage, drug-induced hypersensitivity pneumonitis and interstitial lung disease, infection, lymphangitic dissemination of neoplasm, and heart failure, accurate diagnosis is essential to direct appropriate treatment. Often, bronchoscopy is performed to retrieve secretions and tissue from the lower respiratory tract and pulmonary parenchyma to diagnose infection or tumor. PCP is an important differential diagnostic consideration in patients with certain immune defense abnormalities, especially with aberrations in T-cell-mediated immunity and hematologic malignancies and is not generally associated with breast cancer and other solid tumors (11-13). However, in recent years patients with breast cancer are being treated more intensely with prolonged combinations of chemotherapy as well as radiotherapy (23). These regimens may result in alterations of systemic immune function, and opportunistic lung infections are important concerns in these clinical settings. We could not attribute the PCP to any specific chemotherapeutic or radiotherapeutic regimen since the treatment regimens were not exactly the same in all three patients described in this report. To our knowledge, this is the first report of BAL cell populations in HIV( -) patients with PCP after treatment for breast cancer. The clinical usefulness of cellular profile in BAL is controversial. Analysis of cellular constituents of BAL in normal nonsmoking adults reveals that macrophages constitute 85 to 90070, whereas lymphocytes are the second most numerous at 7 to 12070; approximately 70070 of lymphocytes are T-cells, 40 to 60070 of T-cells are helper-inducer cells (CD 4 ) , 20 to 30070 are suppressor cytotoxic cells (CDs), and the ratio of CD 4:CDs is approximately 1.5 to 2.0 (15-17, 24, 25). Increased lymphocytes in BAL have been described in granulomatous interstitial lung diseases, and the typical finding in patients with hypersensitivity pneumonitis is a marked increase in lymphocytes with a decreased CD 4:CDs (18-20, 24, 25). The differential count of cells in the BAL fluid in all three of our patients revealed an increased percentage of lymphocytes, increased CDs cells, and decreased
439
CASE REPORT
CD 4:CDs ratio. In Patient 3 the lymphocytosis was markedly elevated, similar to that classically described in patients with hypersensitivity pneumonitis. In our three patients the differential diagnosis for pulmonary infiltrates and BAL findings included drug-induced hypersensitivity pneumonitis, radiation pneumonitis, and opportunistic infection. Because PCP was the diagnosis in all three patients, the differential diagnosis of cellular analysis of BAL in drug- or radiation-induced hypersensitivity pneumonitis should include PCP in a similar clinical setting. Cytotoxic agents used to treat breast carcinoma have been associated with decreased total lymphocytes, decreased mitogen responses, and impaired antibody production in the peripheral blood (26, 27). Although these agents have been associated with decreases in CD 4:CDs ratios in the peripheral blood, there has been no apparent increase in the rate of Pneumocystis infection. Radiation therapy has also been associated with decreases in peripheral T-Iymphocyte number and reactivity, and decreased CD 4:CDs ratios in the peripheral blood can be present from several months to years after treatment (28-31). Radiation and chemotherapy may have contributed to the decreased CD 4cell counts and decreased CD 4:CDs ratios in the peripheral blood and BAL in Patients 1 and 2 at presentation. However, Patient 3 had normal CD4 counts in the peripheral blood, but her BAL done on the same day revealed an increased CDs suppressor cell with decreased CD 4:CDs ratio. This suggests that PCP can occur despite normal CD4 counts in the peripheral blood, and the increased T-Iymphocytes and CD s( +) cells in BAL may be a local response to PCP in the lung. A recent report of cellular analysis of BAL in patients with radiation pneumonitis revealed an increased percentage of lymphocytes, but T-cell subsets were not done (32). Although all three patients reported herein had received radiation therapy, the clinical and radiologic presentations were highly consistent with radiation pneumonitis only in Patient 2 since the unilateral infiltrate occurred in the lung exposed to radiation. The BAL analysis revealed a remarkably high percentage of eosinophils (200/0) in Patient 2. Although not to the extent seen in this patient, increased eosinophils have been reported in patients with PCP in AIDS (33). Because peripheral eosinophilia was not found in any patients, the increased eosinophils seen in BAL may therefore represent a local recruitment secondary to PCP. Increased lymphocytes, PMNs, and, occasionally, eosinophils have been described in BAL fluid from patients with AIDS and pneumonitis with and without PCP (21, 33-35). Increased T-Iymphocytes, CD s( +) cells, and decreased CD 4:CDs ratio in BAL were typical findings in those patients. The significance of the BAL lymphocytosis in this clinical setting is unknown. It is possible that in an underlying immunosuppressed state the lung might be a site of lymphocyte sequestration
from blood. The mechanism is unknown, but specific chemotactic factors recently recognized in lymphocyte-mediated lung diseases may be responsible for this (36). An alternate explanation is the local expansion of lymphocyte populations at the sites of disease activity. Either of these mechanisms might regulate conditions that make the lung a target organ for opportunistic infection. The findings of BAL lymphocytosis in patients described in this report may therefore have represented a response to a chemotherapeutic agent, radiation therapy, R carinii, or a combination of these. A serial study of lymphocyte subsets and their functional changes in the immunosuppressed patient will help to understand the role of pulmonary lymphocytes in similar clinical settings. Our findings suggest that the lymphocytosis with a decreased CD 4:CDs ratio and the presence of eosinophils in BAL from HIV( - ) patients with interstitial lung disease who have received radiation and chemotherapy for solid tumors should also raise the suspicion of PCP. We raise caution in interpreting differential analysis of BAL in similar clinical settings and emphasize that a diagnosis of drug- or radiation-induced hypersensitivity pneumonitis should not be made unless PCP and other causes of opportunistic lung infection are excluded.
References 1. Singer C, Armstrong 0, Rosen PP, Walzer PO, YuB. Diffuse pulmonary infiltrates in immunosuppressed patients. Am J Med 1979; 66:110-20. 2. Matthay RA, Greene WHo Pulmonary infections in the immunocompromised patient. Med Clin North Am 1980; 64:529-51. 3. Fanta CH, Pennington JE. Fever and new lung infiltrates in the immunocompromised host. Clin Chest Med 1981; 2:19-31. 4. Rosenow EC, Wilson WR, Cockerill FR. Pulmonary disease in the immunocompromised host. Mayo Clin Proc 1985; 60:473-87. 5. Wilson WR, Cockerill FR, Rosenow EC. Pulmonary disease in the immunocompromised host. Mayo Clin Proc 1985; 60:610-31. 6. Murray JF, Felton CP, Caray SM, et al. Pulmonary complications of the acquired immunodeficiency syndrome. Report of a national heart, lung and blood institute workshop. N Engl J Med 1984; 310:1682-8. 7. Rubin RH, Wolfson JS, Cosimi AB, TolkoffRubin NE. Infection in the renal transplant recipient. Am J Med 1981; 70:405-11. 8. Gajdusek OS. Pneumocystiscarinii: etiologic agent of interstitial plasma cell pneumonia of premature and young infants. Pediatrics 1957; 19:543-65. 9. Godell B, Jacobs JB, Powell RD, et al. Pneumocystiscarinii: the spectrum of diffuse interstitial pneumonia in patients with neoplastic diseases. Ann Intern Med 1970; 72:337. 10. Wolff LJ, Bartlett MS, Baehner RL, et al. The causes of interstitial pneumonitis in immunocompromised children: an aggressive, systematic approach to diagnosis. Pediatrics 1977; 60:41. 11. Kovacs JA, Hiemenz JW, Macher AM. Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Ann Intern Med 1984; 100:663-71. 12. Rosen P, Armstrong 0, Ramos C. Pneumocys-
tis carinii pneumonia: a clinicopathologic study of twenty patients with neoplastic disease. Am J Med 1972; 53:428-36. 13. Burke BA, Good RA. Pneumocystis carinii infection. Medicine (Baltimore) 1973; 52:23-51. 14. Young JA, Stone JW, McGonigle RJS, Adu 0, Michael J. Diagnosing Pneumocystis carinii pneumonia by cytologic examination of bronchoalveolar lavage fluid: report of 15 cases. J Clin Pathol 1986; 39:945-9. 15. Reynolds HY, Fulmer JD, Kazmierowski JA, Roberts WC, Frank MM, Crystal RG. Analysis of cellular and protein components of bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis. J Clin Invest 1977; 59:165-75. 16. Hunninghake GW, Gadek JE, Kawanami 0, Ferrans VJ, Crystal RG. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am J Pathol 1979; 97:149-206. 17. Merrill WW, Reynolds HY. Bronchial lavage in inflammatory lung disease. Clin Chest Med 1983; 4:71-84. 18. Leatherman JW, Michael AF, Schwartz BA, Hoidal FR. Lung T-cells in hypersensitivity pneumonitis. Ann Intern Med 1984; 100:390-2. 19. Hunninghake GW, Crystal RG. Pulmonary sarcoidosis: a disorder mediated by excess helper T-Iymphocyte activity at sites of disease activity. N Engl J Med 1981; 305:429-34. 20. Pesci A, Bertorelli G, Marchioni M. T-Iymphocytes in bronchoalveolar lavage fluid in hypersensitivity pneumonitis. Chest 1985; 87:133-4. 21. Young KR, Rankin JA, Naegel GP, Paul ES, Reynolds HY. Bronchoalveolar lavage cells and proteins in patients with the acquired immunodeficiency syndrome: an immunologic analysis. Ann Intern Med 1985; 103:522-33. 22. Martin TR, Raghu G, Maunder RJ, Springmeyer SC. The effects of chronic bronchitis and chronic airflow obstruction on lung cell population recovered by bronchoalveolar lavage. Am Rev Respir Dis 1985; 132:254-60. 23. Glick JH. Meeting highlights: adjuvant therapy for breast cancer. J Nat! Cancer Inst 1988; 80:471-5. 24. Reynolds HY. Bronchoalveolar lavage. Am Rev Respir Dis 1987; 135:250-63. 25. Rankin AR. Pulmonary immunology. Clin Chest Med 1988; 9:387-93. 26. Zelinski CC, Stuller I, Dorner F, et al. Impaired primary but not secondary immune response in breast cancer under adjuvant chemotherapy. Cancer 1986; 58:1648-52. 27.. Strender L, Blomgren H, Petrini B, et a/. Immunologic monitoring in breast cancer patients receiving postoperative adjutant chemotherapy. Cancer 1981; 48:1996-2002. 28. Neuman GH, Rees GJG, Jones RSJ, et a/. Changes in helper and suppressor T-Iymphocytes following radiotherapy for breast cancer. Clin Radiol 1987; 38:191-3. 29. Petrini B, Wasserman J, Glas U, et a/. T-Iymphocytes, subpopulations in blood following radiation therapy for breast cancer. Eur J Cancer Clin Oncol 1982; 18:921-4. 30. Hoppe RT,Fuhs ZY, Strober S, et a/. The longterm effects of radiation on T- and B-Iymphocytes in the peripheral blood after regional irradiation. Cancer 1977; 40:2071-8. 31. Wasserman J, Wallgren A, Blomgren H, et a/. Prognostic relevance of postirradiation lymphocyte reactivity in breast cancer patients. Cancer 1986; 58:348-51. 32. Gibson PG, Bryant DH, Morgan GW, et a/. Radiation-induced lung injury: a hypersensitivity
440 pneumonitis? Ann Intern Med 1988; 109:288-91.
33. Fleury-Feith J, nan Van Nhieu J, Picard C, Escudier E, Bernaudin JF. Bronchoalveolar lavage eosinophilia associated with Pneumocystis carinii pneumonitis in AIDS patients. Chest 1989; 95: 1198-201. 34. Fleury J, Escudier E, Rochelle JM, Carre C,
CASE REPORT
Bernaudin JF. Cell population obtained by bronchoalveolar lavage in Pneumocystis carinii pneumonitis. Acta Cytol 1985; 29:721-6. 35. Wallace JM, Barbers RO, Hoshi JS, Price H. Cellular and T-Iymphocyte subpopulation profiles in bronchoalveolar lavage fluid from patients with acquired immunodeficiency syndrome and pneu-
monitis. Am Rev Respir Dis 1984; 130:786-90. 36. Berman JS, Beer OJ, Theodore AC, Kornfeld H, Bernado J, Center DM. Lymphocyte recruitment to the lung. Am Rev Respir Dis 1990; 142:238-57.
JAMES SIMINSKI, PAMELA KIDD, GRETCHEN D. PHILLIPS, CAROLYN COLLINS, and GANESH RAGHU
Introduction
Pneumocystis carinii pneumonia (PCP) is an important consideration in the etiology of pulmonary infiltrates in the immunocompromised patient (1-5). PCP most commonly occurs in patients with acquired immunodeficiency syndrome (AIDS), malignant and nonmalignant diseases of the lymphoreticular tissues, congenital immunodeficiency disorders, in premature or malnourished infants, and in patients treated with corticosteroids and immuosuppressive agents (6-10). In contrast, PCP has been rarely reported in patients with solid tumors (11-13). Fiberoptic bronchoscopy examination is often done to confirm the diagnosis of PCP in specimens retrieved from bronchoalveolar lavage (BAL) and/or transbronchiallung biopsy (TBL) (14). HAL is a useful technique to sample cells and secretions from the lower respiratory tract in interstitial lung diseases (15-17). Cellular profiles have been useful in characterizing several types of interstitial pneumonitis; increased proportions of lymphocytes are common findings in BAL from patients with hypersensitivity pneumonitis and sarcoid. Altered T-Iymphocyte subsets in BAL are commonly believed to differentiate these diseases: decreased CD 4:CD s ratio and increased CD 4:CD s ratio of HAL lymphocyte subsets are considered typical findings in hypersensitivity pneumonitis and sarcoidosis, respectively (18-20). Recent reports in patients with AIDS have also demonstrated decreased CD 4:CDs ratio in BAL lymphocytes (21). We describe HAL findings in three cases of PCP in patients treated for breast cancer at our institution (UWM C) over a 1.5-yr period. These patients had no serologic evidence of infection with human immunodeficiency virus (HIV). The findings of peripheral and HAL lymphocyte count and T-cellsubsets are discussed.
Case Reports Patient 1 A previously healthy 24-yr-old woman presented to our institution with a right breast mass. Lumpectomy and axillary node dissectionrevealeda high grade medullary carcinoma with 12/12 nodes negative for malignancy. The tumor was negative for estrogen and progesterone receptors. The patient wastreated with 5,040cGy tangential external beam irradiation over a 48-day period followedby a 2,000 cGy boost with an iridium-192implant. One month later a right internal mammary node was found
SUMMARY Pneumocysfls pneumonia (PCP) usually occurs In patients with hematologic malignancies and acquired Immunodeficiency syndrome (AIDS). Patients with solid tumors represent a very small fraction of the reported cases of PCP.Over an 18-month period, PCP was diagnosed In three patients who had received radiation and chemotherapy for breast cancer. In all three patients, there was no serologic or clinical evidence of AIDS. Direct staining of bronchoalveolar lavage fluid (BAL) revealed Pneumocystls carlnll, and cellular analysis of BAL revealed an Increased percentage of lymphocytes with reversed helper/lnducer:suppressor/cytotoxlc T-cell (CD4:CD a) ratio. Beca~se decreased CD4:CD a ratio In BAL Is commonly accepted as findings consistent with hypersensitivity pneumonitis and AIDS, we conclude that similar findings In patients without AIDS are not specific for hypersensitivity pneumonitis, and If carlnllshould be ruled out In the appropriate clinical setting. AM REV RESPIR DIS 1991; 143:437-440
to be positive for poorly differentiated carcinoma. She received an additional 5,040 cGy to the right internal mammary lymphatic chain and right supraclavicular fossa over the next 41 days. Vinblastine and fluorouracil (5FU) were administered once weeklyfor 4 wk during radiation therapy. This was followed by 12 months of combination chemotherapy consisting of cyclophosphamide, 5FU, and doxorubicin. A 10-mg dose of dexamethasone was administered orally prior to chemotherapy. Fifteen days after the last dose of antineoplastic drugs, she presented with dyspnea on exertion, nonproductivecough, and fever.Physical examination revealed a temperature of 38.4 0 C, tachypnea, tachycardia, orthostatic hypotension, and bibasilar inspiratory crackles. Fiberoptic bronchoscopy and analysis of BAL fluid wereperformed as described below.BAL fluid and TBL specimen disclosed P. carinii on GramWeigert and methanamine silver strains. Viral cultures, fungal stain and culture, Legionella by direct fluorescent antibody stain (DFA) and culture, and acid-fast bacilli (AFB) stain and culture were all negative. The patient was treated with trimethoprim/ sulfamethoxazole, with subsequent resolution of symptoms and pulmonary infiltrates. The patient's serum was negative for HIV antibody by ELISA on two separate occasions. Ten months after the pneumonia the absolute CD 4 ( + ) lymphocyte count increased to 480/mm3 , and she has remained well.
Patient 2 A previously healthy 44-yr-old woman presented with a left breast mass. Lumpectomy and axillary node dissection revealed infiltrating ductal carcinoma with 3/26 nodes involved. The tumor was negative for estrogen and progesterone receptors. She was treated with cyclophosphamide, 5FU, and doxorubicin for 6 months. After this, tangential external beam irradiation (5,040 cGy) was delivered to the left breast and supraclavicular region over a 44-day period, and an iridium-192 implant wasplaced (1,500 cGy). Concomitant cyclophospha-
mide and 5FU were administered weekly during radiation therapy. A IO-mgdose of dexamethasone was administered orally prior to chemotherapy. Two weeks after completing radiation therapy, the patient was hospitalized with shortness of breath, fever, night sweats, and nonproductive cough. Physical examination was significant for a temperature of 37.8° C, tachypnea, and tachycardia, and lung sounds were normal to auscultation (table I). Fiberoptic bronchoscopy was performed, and analysis of BAL fluid was done as described below. R cariniion Gram-Weigert and methanamine silver stains were identified in the BAL fluid and TBL specimen. Viral cultures, fungal stain and culture, Legionella stain and culture, and AFB stain and culture were all negative. The patient was treated with trimethoprim/sulfamethoxazole with subsequent resolution of symptoms and infiltrate. The patient's serum was negative for, ntv antibody by ELISA on two separate occasions,Tenmonths after the pneumonia the absolute peripheral CD 4 ( +) lymphocyte count increased to 450/mm3 , and she has remained well.
Patient 3 A previously healthy 48-yr-old woman presented
(Received in original form April 27, 1990 and in revised form August 23, 1990) 1 From the Divisions of Pulmonary and Critical Care Medicine and of Oncology, Departments of Medicine and Laboratory Medicine, University of Washington Medical Center (UWMC), Seattle, Washington. 2 Correspondence and requests for reprints should be addressed to Ganesh Raghu, M.D., Division of Pulmonary and Critical Care Medicine, Universityof Washington Mail Stop RM-12,University of Washington Medical Center, Seattle, WA 98195.
437
438
CASE REPORT
TABLE 1 PERTINENT CLINICAL AND LABORATORY FINDINGS IN THE THREE PATIENTS
Age, yr Fever Nonproductive cough Dyspnea Chest roentgenograph Interstitial pattern Arterial blood gas determinations F102 pH Paco2 Pao2 Peripheral blood WBC Lymphocytes CD.. (normal = 400-1,500) CD, (normal = 300-800) CD..:CD, (normal = 0.9-3.0) Serum LDH (normal < 304 U/L) Bronchoalveolar lavage Macrophages, % (normal> 85%) Lymphocytes, % (normal < 150/0) PMNs, % (normal, 0-1) Eosinophils, % (normal, 0-1) Monocytes, % (normal, 0-1) Epithelial cells, 0/0 Basophils, % (normal, 0-1) T-lymphocyte subsets CD.., 0/0 CD" 0/0 CD..:CD, (normal, 1.5-2) Microbiology/virology/stains TBUhistopathology
Patient 1
Patient 2
Patient 3
24
44
48
+ + +
+ + +
+ +
Diffuse, bilateral
Left upper lobe
Diffuse, bilateral
0.21 7.49 33 mm Hg 51 mm Hg
2 Ipm O2 7.43 36 mm Hg 99 mm Hg
0.21 7.56 21 mm Hg 71 mm Hg
5,000/mm3
3,800/mm 3
7,500/mm 3
440/mm3 128/mm3 320/mm3 0.42 638 U/L
800/mm3 180/mm3 560/mm3 0.32 442 U/L
1,390/mm 3 930/mm 3 355/mm 3 2.64 631 U/L
42 34 12 1 4 6 1
24 21 22 20 9 3 1
17 75 7 1 0 0
14 66 0.21 P. carinii+ P. carinii
28 68 0.4 P. carinii+ P. carinii
47 48 0.98 P. carinii+
* Values in parentheses are normal for UWMC laboratory.
with a left breast mass. Left modified radical mastectomy and axillary node dissection revealed infiltrating ductal carcinoma with 4/7 nodes involved. The tumor was positive for estrogen and progesterone receptors. The patient wastreated with diethylstilbesterol and received six cycles of cyclophosphamide, methotrexate, and fluorouracil followed by 5,000 cGy of external beam irradiation to the left chest, supraclavicular area, and a boost to the axilla. Approximately 2 yr later she was found to have metastases in bone and liver. She received 3 months of cyclophosphamide, 5FU, adriamycin, and subsequently methotrexate and Velban because of an increasein sizeof livermetastases. A lo-mg dose of dexamethasone was administered orally prior to chemotherapy. Approximately six weeks after the last dose of antineoplastic drugs, she presented to the hospital with dyspnea on exertion, low grade fever, and 4 days of nonproductive cough. Physical examination was significant for a temperature of 36.00 C and tachypnea, and lung sounds were normal on auscultation. Fiberoptic bronchoscopy and analysis of BAL of fluid were performed as described below. BAL fluid disclosed R cariniiby Gram-Weigert stain. Viral cultures, fungal stain and culture, Legionella DFA and culture, and AFB stain and culture were negative. The patient was initially treated with trimethoprim/sulfamethoxazole, but after several days she was switched to trimethoprim/dapsone secondary to nausea and vomiting. The patient's symptoms and pulmonary infiltrates subsequently resolved. Her serum was tested for HIV antibody by ELISA and was negative.
Fiberoptic Bronchoscopy and Bronchoalveolar Lavage Fiberoptic bronchoscopy and bronchoalveolar lavage were performed in all three patients as previously described (22). Patients were premedicated intramuscularly with 0.4 mg of atropine, and 2OJo lidocaine was delivered via an atomizer for topical anesthesia. The bronchoscope (Olympus P20D; Olympus Corp. of America, New Hyde Park, NY) was inserted transnasally or orally, and normal endobronchial anatomy was confirmed. The tip of the bronchoscope was then wedged into the subsegmental bronchus of the involvedarea determined from the roentgenographs. Sterile isotonic saline solution (room temperature) was instilled in five 3O-ml aliquots. Each aliquot was retrieved with gentle suction and collected in sterile suction traps. The aspirate from the first aliquot was collected separately and analyzed for cytology. The aspirates from the second to fifth were pooled in another trap. Approximately 50 to 6OOJo of the instilled saline was retrieved, and approximately 20 ml were submitted to microbiology and virology for appropriate stains and cultures. The BAL fluid was analyzed by the standard procedures in use in our laboratory since 1986, modified from previously established methods (16, 22). Briefly, the BAL fluid was strained through a single layer of sterile gauze and centrifuged for 10min at 1,900rpm. The cellpellet was resuspended in phosphate-buffered saline (PBS) and centrifuged for 5 min at 1,900 rpm. The PBS was decanted, and the cell pellet was resuspended in RPMI complete media (GIBCO Laboratories, Grand Island, NY) to a total volume of 1ml. A cell viability count was performed by trypan blue exclusion. The cells
were resuspended in RPMI complete to a concentration of 5 to 9 X 106 cells/ml. Cytocentrifuge preparations were made using a Shandon Cytospin 2 (Shandon Inc., Pittsburgh, PAl, and a 200-cell differential was performed on Wright's stained slides. Five hundred thousand cells wereincubated at 4 0 C for 30 min with FITC-labeled T 4 and Ts, and the percentage of CD 4 and CDs positive cells was analyzed on a Coulter EPICS Profile flow cytometer (Coulter Cytometry, Hialeah, FL), counting only the cells in the lymphoid gate as defined by the scattergram. Pertinent clinical and laboratory findings of the three patients are listed in table 1.
Discussion Pulmonary parenchymal abnormalities are a common occurrence in the immunocompromised host (1-5). Because pulmonary infiltrates in patients who have received radiation and chemotherapy for malignancy may result from radiation damage, drug-induced hypersensitivity pneumonitis and interstitial lung disease, infection, lymphangitic dissemination of neoplasm, and heart failure, accurate diagnosis is essential to direct appropriate treatment. Often, bronchoscopy is performed to retrieve secretions and tissue from the lower respiratory tract and pulmonary parenchyma to diagnose infection or tumor. PCP is an important differential diagnostic consideration in patients with certain immune defense abnormalities, especially with aberrations in T-cell-mediated immunity and hematologic malignancies and is not generally associated with breast cancer and other solid tumors (11-13). However, in recent years patients with breast cancer are being treated more intensely with prolonged combinations of chemotherapy as well as radiotherapy (23). These regimens may result in alterations of systemic immune function, and opportunistic lung infections are important concerns in these clinical settings. We could not attribute the PCP to any specific chemotherapeutic or radiotherapeutic regimen since the treatment regimens were not exactly the same in all three patients described in this report. To our knowledge, this is the first report of BAL cell populations in HIV( -) patients with PCP after treatment for breast cancer. The clinical usefulness of cellular profile in BAL is controversial. Analysis of cellular constituents of BAL in normal nonsmoking adults reveals that macrophages constitute 85 to 90070, whereas lymphocytes are the second most numerous at 7 to 12070; approximately 70070 of lymphocytes are T-cells, 40 to 60070 of T-cells are helper-inducer cells (CD 4 ) , 20 to 30070 are suppressor cytotoxic cells (CDs), and the ratio of CD 4:CDs is approximately 1.5 to 2.0 (15-17, 24, 25). Increased lymphocytes in BAL have been described in granulomatous interstitial lung diseases, and the typical finding in patients with hypersensitivity pneumonitis is a marked increase in lymphocytes with a decreased CD 4:CDs (18-20, 24, 25). The differential count of cells in the BAL fluid in all three of our patients revealed an increased percentage of lymphocytes, increased CDs cells, and decreased
439
CASE REPORT
CD 4:CDs ratio. In Patient 3 the lymphocytosis was markedly elevated, similar to that classically described in patients with hypersensitivity pneumonitis. In our three patients the differential diagnosis for pulmonary infiltrates and BAL findings included drug-induced hypersensitivity pneumonitis, radiation pneumonitis, and opportunistic infection. Because PCP was the diagnosis in all three patients, the differential diagnosis of cellular analysis of BAL in drug- or radiation-induced hypersensitivity pneumonitis should include PCP in a similar clinical setting. Cytotoxic agents used to treat breast carcinoma have been associated with decreased total lymphocytes, decreased mitogen responses, and impaired antibody production in the peripheral blood (26, 27). Although these agents have been associated with decreases in CD 4:CDs ratios in the peripheral blood, there has been no apparent increase in the rate of Pneumocystis infection. Radiation therapy has also been associated with decreases in peripheral T-Iymphocyte number and reactivity, and decreased CD 4:CDs ratios in the peripheral blood can be present from several months to years after treatment (28-31). Radiation and chemotherapy may have contributed to the decreased CD 4cell counts and decreased CD 4:CDs ratios in the peripheral blood and BAL in Patients 1 and 2 at presentation. However, Patient 3 had normal CD4 counts in the peripheral blood, but her BAL done on the same day revealed an increased CDs suppressor cell with decreased CD 4:CDs ratio. This suggests that PCP can occur despite normal CD4 counts in the peripheral blood, and the increased T-Iymphocytes and CD s( +) cells in BAL may be a local response to PCP in the lung. A recent report of cellular analysis of BAL in patients with radiation pneumonitis revealed an increased percentage of lymphocytes, but T-cell subsets were not done (32). Although all three patients reported herein had received radiation therapy, the clinical and radiologic presentations were highly consistent with radiation pneumonitis only in Patient 2 since the unilateral infiltrate occurred in the lung exposed to radiation. The BAL analysis revealed a remarkably high percentage of eosinophils (200/0) in Patient 2. Although not to the extent seen in this patient, increased eosinophils have been reported in patients with PCP in AIDS (33). Because peripheral eosinophilia was not found in any patients, the increased eosinophils seen in BAL may therefore represent a local recruitment secondary to PCP. Increased lymphocytes, PMNs, and, occasionally, eosinophils have been described in BAL fluid from patients with AIDS and pneumonitis with and without PCP (21, 33-35). Increased T-Iymphocytes, CD s( +) cells, and decreased CD 4:CDs ratio in BAL were typical findings in those patients. The significance of the BAL lymphocytosis in this clinical setting is unknown. It is possible that in an underlying immunosuppressed state the lung might be a site of lymphocyte sequestration
from blood. The mechanism is unknown, but specific chemotactic factors recently recognized in lymphocyte-mediated lung diseases may be responsible for this (36). An alternate explanation is the local expansion of lymphocyte populations at the sites of disease activity. Either of these mechanisms might regulate conditions that make the lung a target organ for opportunistic infection. The findings of BAL lymphocytosis in patients described in this report may therefore have represented a response to a chemotherapeutic agent, radiation therapy, R carinii, or a combination of these. A serial study of lymphocyte subsets and their functional changes in the immunosuppressed patient will help to understand the role of pulmonary lymphocytes in similar clinical settings. Our findings suggest that the lymphocytosis with a decreased CD 4:CDs ratio and the presence of eosinophils in BAL from HIV( - ) patients with interstitial lung disease who have received radiation and chemotherapy for solid tumors should also raise the suspicion of PCP. We raise caution in interpreting differential analysis of BAL in similar clinical settings and emphasize that a diagnosis of drug- or radiation-induced hypersensitivity pneumonitis should not be made unless PCP and other causes of opportunistic lung infection are excluded.
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