INTRODUCTION

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Recently herpes virus-like DNA sequences have been detected in Kaposi's sarcoma defining a new herpes virus, Kaposi's sarcoma-associated virus, or human herpes virus 8 (HHV8) (1). HHV8 DNA was first described in skin biopsies of HIV-positive patients, but also in HIV-negative patients with Kaposi's sarcoma (1). HHV8 DNA can also be found in visceral Kaposi's sarcoma lesions and in the peripheral blood of patients with skin or visceral Kaposi's sarcoma involvement (9).

Intrathoracic Kaposi's sarcoma is frequent in AIDS patients and occasionally develops in transplant recipients. Features of tracheobronchial Kaposi's sarcoma are found in 8 to 15% of bronchoscopies performed in AIDS patients for respiratory symptoms and/or infiltrates on chest X-ray (10, 11). At bronchoscopy, Kaposi's sarcoma is diagnosed by visual identification of characteristic multiple red or purple flat lesions (12). Biopsy of bronchial lesions is not performed routinely for several reasons: bronchial biopsy samples have a poor diagnostic yield and cannot be repeated frequently due to the risk of significant bleeding; the diagnosis is difficult to make histologically on bronchial biopsy specimens because crush artefacts and reactive fibrous tissue have similar appearances (12, 13). In patients with mucocutaneous Kaposi's sarcoma and respiratory symptoms, the incidence of tracheobronchial lesions ranges from 20 to 40%. However, pulmonary Kaposi's sarcoma can rarely be found in absence of skin involvement (14).

We recently described the detection of HHV8 in bronchoalveolar lavage fluid (BAL) of HIV-positive patients with pulmonary Kaposi's sarcoma and in the BAL of a HIV-negative kidney transplant recipient who developed visceral Kaposi's sarcoma with tracheobronchial involvement (15). Herpes virus-like DNA was detected by nested PCR in the BAL fluid allowing us to confirm the endoscopic diagnosis of tracheobronchial Kaposi's sarcoma without the need for further biopsies. However, specificity and sensitivity of HHV8 DNA detection in the BAL for the diagnosis of pulmonary Kaposi's sarcoma is not yet known. We therefore conducted a prospective study to evaluate the specificity of HHV8 detection in bronchoalveolar lavage. HHV8 was investigated in 100 consecutive BAL performed in immunocompromised patients with fever, respiratory symptoms, and/or infiltrates on chest X-ray and in patients with antibiotic resistant infiltrates to determine the sensitivity and specificity of HHV8 for the diagnosis of pulmonary Kaposi's sarcoma. In addition, HHV8 DNA was retrospectively investigated in a group of HIV-positive patients with cutaneous or visceral Kaposi's sarcoma.

	METHODS

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Patients

One hundred bronchoalveolar lavages (BAL) performed consecutively for diagnostic purposes at the University Hospital Basel in immunocompromised patients (79 BAL) and in patients without immunosuppression (21 BAL) were prospectively investigated for the presence of HHV8 DNA. Indication for BAL were respiratory symptoms, fever and/or infiltrates on chest X-ray.

The immunocompromised group consisted of 79 BAL in 72 patients (Table 1): 38 BAL in HIV-positive patients, 14 in renal transplant recipients, nine in patients following allogenic bone marrow or peripheral stem cell transplantation, nine in neutropenic patients after chemotherapy for hematologic diseases or lung cancer and nine in patients immunosuppressed for autoimmune diseases or pulmonary fibrosis. Underlying autoimmune diseases were: two lupus erythematosis, one systemic collagenosis, one panarteriitis nodosa, one Wegener's granulomatosis. Twenty-one BAL were prospectively analyzed for HHV8 DNA detection in nonimmunocompromised patients, 17 of them with antibiotic resistant infiltrates on chest X-ray, and four patients with lung fibrosis.

In addition to the prospective study, BAL samples of AIDS patients with known cutaneous or visceral Kaposi's sarcoma were retrospectively investigated. Sixty-five BAL had been performed in 41 AIDS patients with Kaposi's sarcoma. Cytological smears of 26 BAL samples were available for further evaluation. Nineteen BAL samples of 14 patients revealed adequate DNA and could therefore be included for further HHV8 DNA evaluation.

Sample Analysis

BALs were done for diagnostic purposes in patients with fever, respiratory symptoms and/or infiltrates on chest X-ray. To obtain BAL fluid, 150-300 ml of 0.9% NaCl was instilled to the middle lobe or the segment of the most prominent radiologic infiltrate (16). BAL fluid was routinely investigated for bacterial, fungal, and viral microorganisms. Conventional cytology was performed including Grocott's stain for Pneumocystis carinii.

Prospectively tested BAL fluids were centrifuged in 1.5 ml tubes and the cell pellet stored at 20° C if not immediately processed. For the retrospective analysis, cells were scraped from glass slides of frozen smears under appropriate conditions to avoid cross-contamination. DNA was extracted as previously described (17). In brief, the cell pellet was digested over night in proteinase K at 37° C (200 µg/ ml¹ proteinase K, 10 mmol Tris-HCl [pH 7.4], 25 mmol EDTA) followed by phenol/chloroform-extraction and ethanol precipitation. The pellet was dissolved in sterile water. Five microliters of DNA was applied to the nested polymerase chain reaction (PCR) using two outer primers (KS3: 3' ACA GCA ACA CCC AGC TAG CA and KS4: 5'-AGA TCG TCA AGC ACT GCG AG) for the first round and the internal primers KS330/233 (F' ACA GCA ACA CCC TAG CA sense; 5' AGA TCG TCA AGC ACT CGC AG antisense) for the second round (1, 8). Thirty cycles were used in each round, with the following cycling conditions: denaturation at 94° C for 1 min; reannealling at 55° C for 45 s; extension at 72° C for 45 s. All PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. The presence of appropriate DNA was verified by testing for the presence of the human beta-globin gene as an internal control by a single step PCR (8). Stringent laboratory conditions were used to avoid cross-contamination and appropriate negative controls were performed in each assay to exclude false-positive results.

	RESULTS

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Bronchoalveolar Lavage Results

Indication for bronchoscopy was fever in 53 to 67% of cases in the immunocompromised patient group (Table 1). Infiltrates on chest X-ray were present in at least half of these patients: renal transplant recipients 50%; HIV-positive patients 68%; bone marrow transplant recipients 78%; autoimmune disease 89%; high dose chemotherapy 100%.

All patients of the nonimmunocompromised group showed infiltrates on chest X-ray. In 17 of these cases there were localized infiltrates not responding to antibiotic treatment. In four patients with diffuse interstitial lung disease BAL was performed before immunosuppression was started.

Microorganisms found in BAL are summarized on Table 2. A significant bacterial growth was documented in 30 BAL. Typical and atypical mycobacteria were found in a total of five cases, two of them in the group of patients with antibiotic resistant infiltrates. Pneumocystis carinii was detected in seven BAL of HIV patients, three BAL of renal transplant recipients and in one BAL of a patient following BMT. Cytomegalovirus (CMV) was cultured from nine BAL of HIV-positive patients, three BAL of renal transplant recipients and in two BAL of patients treated with steroids and cyclophosphamide for autoimmune disease.

CMV was found in 11 and Pneumocystis carinii in three of 19 BAL of the HIV-positive patient group retrospectively analyzed. In three patients CMV pneumonitis with interstitial infiltrates and a positive CMV cytology and cell culture in the BAL was diagnosed. Atypical mycobacteria were cultured from two BAL.

Pulmonary Kaposi's Sarcoma

Characteristic features of tracheobronchial Kaposi's sarcoma with purple lesions were observed in five bronchoscopies performed in four patients of the prospectively analyzed group (Table 3). Three of these patients were HIV-positive and one of them underwent three bronchoscopies. No endoscopic features of tracheobronchial Kaposi's sarcoma were found at the third bronchoscopy following treatment with chemotherapy and under antiretroviral agents in this patient (Patient 15 on Table 3). At the time of the third bronchoscopy cutaneous Kaposi's sarcoma lesions had also disappeared. The fourth patient with tracheobronchial Kaposi's sarcoma had received a renal transplant complicated by repeated rejection episodes. In this patient, endobronchial lesions were extremely hypervascularized and partly stenotic. Visceral Kaposi's sarcoma involvement of the lung, spleen, lymph nodes, and transplanted kidney was confirmed at autopsy. All patients with pulmonary Kaposi's sarcoma also suffered from cutaneous involvement.

Tracheobronchial Kaposi's sarcoma was visible in 11 of 19 bronchoscopies of the retrospectively analyzed BAL group. Ten of the 14 patients of this group developed endoscopically of histologically (autopsy) proven pulmonary Kaposi's sarcoma. In patient 13 (Table 3) a total of four bronchoscopies were performed within two years. At the third and fourth bronchoscopy typical features of tracheobronchial Kaposi's sarcoma were observed. In the four patients who did not develop pulmonary Kaposi's sarcoma but suffered from cutaneous Kaposi's sarcoma a total of five bronchoscopies were performed. One of these patients (Patient 4) suffered from cutaneous Kaposi's sarcoma and died with body cavity-associated lymphoma.

CD4 cell count was below 100/ml at the time of bronchoscopy in 16 of 19 cases. Every patient in the retrospective patient group except one had a history of homosexual contacts.

Detection of Herpes Virus 8 DNA

Herpes virus 8 DNA was detected in six of the 100 prospectively investigated BAL fluids and 94 BAL samples were negative for HHV8 DNA. The six HHV8 DNA-positive BAL were performed in four patients undergoing five bronchoscopies with tracheobronchial Kaposi's sarcoma and in one patient with no evidence of Kaposi's sarcoma of the lung or of the skin. All five of six BAL samples of patients with endoscopic tracheobronchial Kaposi's sarcoma revealed HHV8 DNA. The sensitivity, specificity, positive and negative predictive values of HHV8 detection for the diagnosis of tracheobronchial Kaposi's sarcoma were 100%, 98.9%, 83.3%, and 100%, respectively (Table 4). As described above, the patient group with tracheobronchial Kaposi's sarcoma included three HIV-positive patients (Patients 5, 14, and 15 on Table 3) and a renal transplant recipient (Patient 16). In Patient 15, tracheobronchial Kaposi's sarcoma disappeared following chemotherapy and under antiretroviral therapy and HHV8 DNA became negative in the third BAL. In two patients of the prospective group (Patients 5 and 14) three additional BAL samples could be analyzed retrospectively showing HHV8 DNA detection in one case (see Table 3).

Twelve of 19 BAL performed in 14 HIV-positive patients of the retrospective group with pulmonary or cutaneous Kaposi's sarcoma were HHV8 DNA-positive. HHV8 DNA could be detected in 71.4% (10/14) BAL samples of the 10 patients with pulmonary Kaposi's sarcoma. Two of five BAL from four patients with cutaneous Kaposi's sarcoma and no endoscopic evidence of tracheobronchial involvement were HHV8 DNA positive. Two BAL samples of the patient with body cavity-associated lymphoma and cutaneous Kaposi's sarcoma, but no evidence of pulmonary Kaposi's sarcoma at autopsy, were HHV8 negative.

Combining the results of the prospective and retrospective analysis, HHV8 DNA could be detected in 75% (15/20) of BAL samples from patients with pulmonary Kaposi's sarcoma. HHV8 DNA was positive in BAL of three patients with no evidence of pulmonary Kaposi's sarcoma; two of these three patients, however, had cutaneous Kaposi's sarcoma. Only one BAL sample of a HIV-positive patient with no evidence of Kaposi's sarcoma was HHV8 positive. Taken together, the sensitivity and specificity for the diagnosis of pulmonary Kaposi's sarcoma by detecting HHV8 DNA in the BAL was 75.0% and 97.0%, respectively. The positive predictive value of HHV8 DNA detection in the BAL for diagnosis of pulmonary Kaposi's sarcoma was 83.3% and the negative predictive value of 95.1%, respectively.

	DISCUSSION

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We have previously shown that HHV8 DNA sequences can be detected in bronchoalveolar lavage fluid of patients with pulmonary Kaposi's sarcoma using a nested PCR technique (15). In the present study 100 consecutive BAL were prospectively analyzed for the presence of HHV8 DNA to determine the specificity of this test for the diagnosis of pulmonary Kaposi's sarcoma. In addition, 19 BAL samples of AIDS patients with known cutaneous Kaposi's sarcoma were retrospectively studied to further determine the sensitivity of HHV8 DNA detection for the diagnosis of pulmonary Kaposi's sarcoma. The detection of HHV8 DNA in the BAL fluid was highly specific and sensitive for the diagnosis of pulmonary Kaposi's sarcoma.

Symptoms of pulmonary Kaposi's sarcoma include cough, dyspnea, hemoptysis and pleural pain due to pleural effusion. These symptoms are nonspecific and can also be caused by opportunistic infections (12). Lung function tests and pulmonary capillary volume alone are not useful for identifying patients with pulmonary Kaposi's sarcoma (18). The most common findings on chest X-ray are perihilar linear densities, nodular opacities and pleural effusions. Computer-tomography might be more characteristic than the conventional X-ray showing nodules, tumor masses, bronchovascular pathway thickening, and pleural effusions (19). However, because opportunistic infections are common in immunocompromised patients bronchoscopy with BAL is indicated (14). Up to now, bronchoscopic diagnosis of Kaposi's sarcoma has been based on the macroscopic appearance of characteristic tracheobronchial lesions. For the histological diagnosis of Kaposi's sarcoma larger tissue samples than those usually obtained by mucosal or transbronchial biopsy are necessary. In addition, there is a considerable risk of major bleeding after biopsy of these heavily vascularized tumor. Open lung biopsy and video-assisted thoracoscopic surgery to obtain large tissue samples are invasive procedures and rarely performed for this indication. The detection of HHV8 DNA is therefore a very useful noninvasive test to confirm endoscopically suggested Kaposi's sarcoma.

The present study shows a high sensitivity and specificity of HHV8 DNA detection in BAL for the diagnosis of pulmonary Kaposi's sarcoma. In the prospectively analyzed BAL group the sensitivity of HHV8 DNA detection for the diagnosis of tracheobronchial Kaposi's sarcoma was 100% and higher than in the retrospectively analyzed group. Although only samples with a positive DNA control assay have been included in the study, a loss of sensitivity in the retrospectively tested samples due to DNA degradation during processing and storage cannot be excluded. HHV8 DNA was not only found in HIV-positive patients but also in the BAL of a renal transplant recipient with visceral Kaposi's sarcoma. As recently investigated by our group, tumor tissue samples of transplant-associated malignomas other than Kaposi's sarcoma were negative for HHV8 DNA detection (20). HHV8 DNA detection is therefore specific for transplant and HIV-associated Kaposi's sarcoma. HHV8 DNA has also been found in AIDS-related body cavity-based lymphomas (21). One of our patients suffered from cutaneous Kaposi's sarcoma and concomittent body cavity-associated lymphoma. This patient underwent two bronchoscopies with no evidence of tracheobronchial Kaposi's sarcoma. Both BAL samples were negative for HHV8 DNA. In contrast, HHV8 DNA could be found in lymphatic tissue at autopsy. Only one patient of the prospectively analyzed group without clinical evidence of Kaposi's sarcoma was HHV8 DNA-positive in the BAL. This patient came from central Africa, which is a region of endemic HHV8 infection and Kaposi's sarcoma (22). The clinical importance of the positive HHV8 test in this patient remains unclear. Interestingly, tracheobronchial Kaposi's sarcoma was absent in the third bronchoscopy of a patient receiving antiretroviral treatment and chemotherapy for cutaneous and endobronchial Kaposi's sarcoma (Patient 15 in Table 3). Following the complete clinical remission of Kaposi's sarcoma, HHV8 DNA could no longer be found in the patient's BAL. This suggests that the detection of HHV8 DNA may also be useful as a marker for the therapeutic response.

The specificity of HHV8 DNA detection in BAL for the diagnosis of pulmonary Kaposi's sarcoma was 97% in all patients analyzed. In two patients with cutaneous Kaposi's sarcoma and no evidence of tracheobronchial involvement at bronchoscopy, HHV8 DNA could be detected in BAL. Both patients died but no autopsy was performed. Therefore, pulmonary Kaposi's sarcoma at a later stage of disease could not be excluded. In a recently published study, HHV8 DNA was detected in the supernatant of two of 10 BAL samples but not in the cell fraction of patients with Kaposi's sarcoma restricted to the skin (23). An association of HHV8 with cellular elements is therefore possible. In one patient (Patient 3 on Table 3), HHV8 DNA was present in four consecutive BAL but only in the last two bronchoscopies were endobronchial Kaposi's sarcoma lesions observed. It can therefore be speculated that HHV8 DNA may be detected prior to clinically visible Kaposi's sarcoma and that patients with a positive HHV8 test may be at risk to develop pulmonary Kaposi's sarcoma. Therefore, detection of HHV8 DNA in the BAL may be an indication for preemptive therapy in the absence of endoscopically visible lesions.

There is increasing evidence of a causative role of HHV8 in the development of Kaposi's sarcoma. In most patients with Kaposi's sarcoma HHV8 seroconversion occurs before the clinical onset of Kaposi's sarcoma (24). The causative role of HHV8 for development of Kaposi's sarcoma is also supported by a study showing a high incidence of HHV8 in the peripheral blood of AIDS patients with Kaposi's sarcoma (9). Patients infected with HHV8 have a major risk of developing Kaposi's sarcoma; the presence of anti-HHV8 antibodies indicates those patients at risk for Kaposi's sarcoma (9, 25, 26). In addition, molecular analysis revealed viral analogs of cellular genes involved in the pathogenesis of Kaposi's sarcoma, including interleukin 6 and Cyclin D (27).

In summary, this study shows a high specificity of the HHV8 DNA detection in bronchoalveolar lavage of immunocompromised patients for the diagnosis of pulmonary Kaposi's sarcoma. Furthermore, the detection of HHV8 DNA may be useful marker for pre-emptive therapy, as well as for the therapeutic response in patients with Kaposi's sarcoma.