Randomized comparative trial of pegylated liposomal doxorubicin versus bleomycin and vincristine in the treatment of AIDS-related Kaposi's sarcoma. International Pegylated Liposomal Doxorubicin Study Group

PURPOSE: Cytotoxic chemotherapy is frequently required for the more severe manifestations of human immunodeficiency virus (HIV)-related Kaposi's sarcoma. Combinations of bleomycin and vincristine (BV) or BV with the addition of doxorubicin (ABV) are the most commonly used regimens against which new treatments may be compared. We report a multicenter phase III study that compared pegylated liposomal doxorubicin (PLD) to the BV combination. PATIENTS AND METHODS: We conducted a randomized study that compared PLD 20 mg/m2 with a combination of bleomycin 15 IU/m2 and vincristine 2 mg in 241 patients with HIV-related Kaposi's sarcoma. Both regimens were administered by intravenous infusion every 3 weeks for six cycles. RESULTS: A total of 121 patients received PLD and 120 patients the BV combination. The response to PLD was superior to BV: 58.7% versus 23.3% (P < .001). Patients who were randomized to receive BV, however, were more likely to terminate treatment early because of an adverse event (26.7% v 10.7%), and fewer completed the full six cycles of treatment (30.8% v 55.4%). Treatment with BV was associated with a significantly higher incidence of peripheral neuropathy (P < .001), whereas PLD treatment was more commonly associated with neutropenia and delays in receiving treatment (P < or = .001). CONCLUSION: Pegylated liposomal doxorubicin is an effective treatment for HIV-related Kaposi's sarcoma with a higher response rate than the BV combination. It is well tolerated but more myelosuppressive.     

Kaposi's sarcoma (author's transl)

Kaposi's sarcoma of the skin and an aplastic syndrome occurred together in a 51-year-old patient. Macroscopically livid papules and nodules were observed. Histomorphologically endotheliomatous cell proliferation with signs of infiltrative growth was found. Because of the aplastic pancytopenia cytostatic treatment of the Kaposi sarcoma was contraindicated. The patient finally died of vascular failure with haemorrhagic diathesis being manifest. Syntropy of Kaposi's sarcoma with malignant haematological diseases is known. However, association with aplastic anaemia has not been observed so far. The pathogenesis of Kaposi's sarcoma is unknown.     

Treatment of early AIDS-related Kaposi's sarcoma with oral all-trans-retinoic acid: results of a sequential non-randomized phase II trial. Kaposi's Sarcoma ANRS Study Group. Agence Nationale de Recherches sur le SIDA

OBJECTIVE: To assess the efficacy and safety of all-trans-retinoic acid (ATRA), a retinoid with antitumour activity that inhibits in vitro the growth of Kaposi's sarcoma cells, in patients with low-risk AIDS-associated Kaposi's sarcoma. DESIGN: Non-randomized phase II study, using a group sequential procedure to determine whether the response rate to ATRA was above 10%. SETTING: Nine referral French centres. PATIENTS: Twenty HIV-seropositive men with CD4 cells > or = 200 x 10(6)/l, low-risk Kaposi's sarcoma [T0I0S0 according to the classification of AIDS Clinical Trials Group (ACTG)] not previously treated with systemic anti-Kaposi's sarcoma agents, and with at least four measurable lesions were included. INTERVENTIONS: ATRA was given orally 45 mg/m2 daily for 12 weeks. MAIN OUTCOME MEASURE: Tumour response evaluated according to ACTG criteria. RESULTS: Nineteen patients were evaluated for response: partial response, stabilization and progression were found in eight (42%), seven (37%), and four (21%) patients, respectively. Gradual flattening and lightening of lesions was observed in responders after at least 2 months of ATRA. All patients with partial response at week 12 pursued ATRA for another 15+/-7 weeks. Further improvement was observed in six patients. Median duration of response was 332 days. Cheilitis, transient headaches and skin dryness were the main toxicities noted. No significant changes in HIV viral burden or serum interleukin-6 pathways were observed. CONCLUSIONS: ATRA is well tolerated and effective enough in Kaposi's sarcoma patients to warrant further evaluation.     

The therapeutic effect of intralesional interferon in classical Kaposi's sarcoma

Interferon alpha-2a, which has antineoplastic, antiviral, immunomodulatory, and antiangiogenic effects, was evaluated in a prospective study, treating 10 lesions of patients with classical Kaposi's sarcoma. Ten patients received injections of interferon alpha-2a intralesionally in a dose of 3 million units three times weekly for 4 weeks, and in a variable dose for 4 more weeks. Two of the patients had a complete response, and in one of these, distant lesions also responded. Seven had a partial response, and one did not respond. The treatment was generally well tolerated. The results of our study support the use of interferon in the therapy of classical Kaposi's sarcoma, although it would appear that to achieve maximum efficacy, a longer period of treatment is needed.     

Chest discomfort associated with liposomal amphotericin B: report of three cases and review of the literature

Liposomal formulations of amphotericin B are designed to maintain therapeutic efficacy of amphotericin B deoxycholate while reducing its associated toxicities. In three patients chest discomfort occurred during planned 1-hour infusions of liposomal amphotericin B (AmBisome) 3 mg/kg/day during an open-label trial. The first patient experienced chest tightness and difficulty breathing and the second had dyspnea and acute hypoxia, both within 10 minutes of the start of the infusion. The third patient complained of chest pain 5 minutes after the start of two infusions. All symptoms resolved on terminating therapy. Two patients were later rechallenged with slower infusions and tolerated the drug well. A review of the English-language literature revealed only two other case reports of infusion-related chest or pulmonary reactions with the drug, although similar reactions were noted in several reports of clinical trials. Further review of the literature revealed reports of chest and pulmonary adverse events with other liposomal formulations of amphotericin B, liposomal daunorubicin, liposomal doxorubicin, and liposomes. The pathophysiology of such reactions remains unclear, and premedication with diphenhydramine did not completely prevent this reaction in one of our patients. We recommend infusing liposomal amphotericin B over at least 2 hours with careful monitoring for adverse reactions.     

Kinetics and toxicity of liposomal and conventional amikacin in a patient with multidrug-resistant tuberculosis

The pharmacokinetics and toxicity of liposomal amikacin in a patient treated for advanced pulmonary multidrug-resistant tuberculosis are described. A dose of 20 mg/kg of liposomal amikacin was given on alternate days for 14 days and weekly thereafter for 9 weeks, for a total dose of 20.1 g in 17 divided doses. Accumulation occurred with alternate-day, but not weekly, dosing. The serum levels of amikacin obtained with the liposomal preparation were considerably greater than those obtained with the conventional preparation (range, 81-457 mg/l vs. 4.1-37.7 mg/l). The liposomal amikacin was well tolerated and led to clinical improvement, but it failed to achieve a microbiological response. The patient's sputum remained smear- and culture-positive during the treatment period with liposomal amikacin and for 9 months afterward.     

Diagnosis of pulmonary Kaposi's sarcoma by detection of human herpes virus 8 in bronchoalveolar lavage

Human herpes virus 8 (HHV8) DNA has recently been detected in sarcoma tissue of patients with Kaposi's sarcoma. HHV8 DNA could also be found in bronchoalveolar lavage (BAL) fluid of patients with tracheobronchial Kaposi's sarcoma. To determine the specificity, sensitivity and predictive values of HHV8 DNA detection in the BAL for the diagnosis of pulmonary Kaposi's sarcoma, 100 consecutive BAL were prospectively analyzed for the presence of HHV8 DNA using a nested PCR assay. In addition, 19 BAL samples of 14 AIDS patients with cutaneous or visceral Kaposi's sarcoma were retrospectively investigated. The prospective group consisted of 79 BAL performed in immunocompromised and of 21 BAL in nonimmunocompromised patients. Four patients of the prospectively analyzed group undergoing six BAL showed tracheobronchial Kaposi's sarcoma at five bronchoscopies. All of the five BAL samples performed in these patients with endoscopically visible Kaposi's sarcoma were positive for HHV8 DNA. Following chemotherapy and antiretroviral treatment tracheobronchial Kaposi's sarcoma was no longer detectable at a subsequent bronchoscopy and HHV8 DNA in BAL became negative in one patient. One BAL sample of a HIV-positive patient with no evidence of Kaposi's sarcoma was HHV8 DNA-positive. 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. Twelve of 19 BAL samples of the retrospective group were HHV8 DNA-positive. In this group, 10 patients undergoing a total of 14 BAL suffered from pulmonary Kaposi's sarcoma. HHV8 DNA was documented in 10 of these 14 BAL samples. In three BAL of this group HHV8 DNA was positive, but pulmonary Kaposi's sarcoma was diagnosed at a later stage. In conclusion, the detection of HHV8 DNA in BAL is restricted to patients with Kaposi's sarcoma and is highly sensitive and specific for pulmonary involvement of Kaposi's sarcoma.     

Phase II trial of 13-cis-retinoic acid for poor risk HIV-associated Kaposi's sarcoma

Fifteen men with HIV-associated Kaposi's sarcoma (KS) and poor risk disease according to the TIS staging were enrolled in a phase II trial of oral 13-cis-retinoic acid. The median CD4 cell count was 95 cells/microl (range 7-260) and 6 had prior AIDS-defining opportunistic infections. One patient was withdrawn on account of cutaneous toxicity. Evaluation was by AIDS Clinical Trials Group (ACTG)1 defined assessment. One patient achieved a partial response and remains on treatment in partial remission. Thus the overall response rate is 7% (95% confidence interval 0-23%). A further 5 patients had stable disease (38%: 95% confidence interval 7-64%). The overall low activity, considerable toxicity and limited cosmetic benefit even in responding patients limits the value of this approach in KS. However, this treatment strategy may be more rewarding in early good risk KS.     

Liposomal doxorubicin in the treatment of AIDS-associated Kaposi's sarcoma: clinical, histological and cell biological evaluation

AIDS-associated Kaposi's sarcoma (KS) in eight patients was treated with the systemic application of liposomal doxorubicin (20 mg/m2 per cycle). After six cycles of treatment a significant regression of KS was observed in all patients. Tumour volume was reduced from 556 +/- 635 mm3 before therapy to 42 +/- 134 mm3 after therapy as determined by ultrasonography of selected tumours. Histological examination revealed a reduction of tumour-like structures and the absence of KS spindle cells in involved areas after therapy. In vitro experiments with KS-derived cell cultures, which most likely represent the KS spindle cells, suggested that liposomal doxorubicin may cause regression of KS via two different mechanisms: (i) by highly specific inhibition of KS spindle cell proliferation and (ii) by induction of monocyte chemoattractant protein-1 expression in KS spindle cells, which may result in increased recruitment of phagocytic cells (monocytes/macrophages) into the lesions. A cooperative action of both mechanisms may explain the high efficacy of liposomal doxorubicin in the treatment of AIDS-KS.     

Kaposi's sarcoma following long-term immunosuppressive therapy: clinical, histologic, and ultrastructural study

Multifocal Kaposi's sarcoma in a patient with chronic myeloid leukemia treated with busulfan, a cytostatic and suppressive drug, is reviewed. After five years of treatment, during which temporary remissions occurred, the patient experienced a relapse of leukemia and a considerable immune deficiency. This was expressed by a decrease in the ratio of CD4/CD8 lymphocytes in the peripheral blood. The relation of Kaposi's sarcoma with leukemia, as well as with the state of immunity in this case, does not evoke any doubts. Verification of oncologic treatment brought about a remission of leukemia, an improvement in the patient's immune state, as well as an inhibition of new foci of the Kaposi's sarcoma in the skin in the course of a few months of follow-up evaluation.     

Detection of human herpesvirus 8 (HHV 8) in Kaposi sarcomas of the gastrointestinal tract

Involvement of Kaposi's sarcoma in the gastrointestinal tract is common in AIDS patients. The disease is, however, usually asymptomatic and, due to the tumor growth primarily in the submucosa, biopsy diagnosis is possible in under 25%. The recently described human herpes virus 8 (HHV8) is closely associated with all forms of Kaposi's sarcoma. Detection of HHV8 in the tissue samples may therefore improve the diagnosis of gastrointestinal Kaposi's sarcoma. In the present study we analyze autopsy samples of tumor and non-tumor tissue from the gastrointestinal tract in patients with and without Kaposi's sarcoma for the presence of HHV8 DNA using a nested polymerase chain reaction (PCR) assay. HHV8 DNA was present in all 15 tissues with Kaposi's sarcoma. In contrast, HHV8 DNA was present only in 3 (18.8%) of 16 gastrointestinal tissues of patients with Kaposi's sarcoma but without histologically detectable tumor. No HHV8 DNA was present in 15 tissue samples of AIDS patients without Kaposi's sarcoma. Our data show that detection of HHV8 DNA using a nested PCR assay is a highly sensitive and specific diagnostic test for Kaposi's sarcoma in autopsy tissue samples from the gastrointestinal tract. It should therefore be possible to use detection of HHV8 DNA in biopsy material as an assay for the diagnosis of Kaposi's sarcoma.     

Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is invariably present in Kaposi's sarcoma lesions. KSHV contains several viral oncogenes and serological evidence suggests that KSHV infection is necessary for the development of Kaposi's sarcoma, but cellular transformation by this virus has not so far been demonstrated. KSHV is found in the microvascular endothelial cells in Kaposi's sarcoma lesions and in the spindle 'tumour' cells, which are also thought to be of endothelial origin. Here we investigate the biological consequences of infecting human primary endothelial cells with purified KSHV particles. We find that infection causes long-term proliferation and survival of these cells, which are associated with the acquisition of telomerase activity and anchorage-independent growth. KSHV was present in only a subset of cells, and paracrine mechanisms were found to be responsible for the survival of uninfected cells. Their survival may have been mediated by upregulation of a receptor for vascular endothelial growth factor. Our results indicate that transformation of endothelial cells by KSHV, as well as paracrine mechanisms that are induced by this virus, may be critical in the pathogenesis of Kaposi's sarcoma.     

Laryngeal Kaposi's sarcoma in patients with AIDS

Over a period of 10 years 17 human immunodeficiency virus(HIV)-infected patients with laryngeal Kaposi's sarcoma were seen and treated at University College London Hospitals. All patients had advanced HIV disease. Their presentation was with symptoms of upper airway obstruction in the majority of cases and the diagnosis was made by fibreoptic examination of the larynx. Biopsy was associated with brisk haemorrhage in one patient, who required a temporary tracheostomy, and was not performed in the other 16 cases. The commonest site of laryngeal involvement was the supraglottis in 11 patients, with glottic lesions noted in eight patients: subglottic lesions were seen in only three. Treatment of laryngeal Kaposi's sarcoma was, in general, conservative, five patients received low dose radiotherapy to the larynx and 10 were treated with systemic chemotherapy for disseminated Kaposi's sarcoma. Laryngeal Kaposi's sarcoma did not contribute to patient mortality.     

Laser therapy of eyelid and conjunctival tumors, especially in AIDS patients

We treated patients with Kaposi's sarcoma of the lid or conjunctiva, molluscum contagiosum and papilloma of the lid with dye laser photocoagulation. This therapeutic device is extremely efficient as the chosen wavelength of 577 nm is absorbed selectively by haemoglobin. Since Kaposi's sarcoma is a haemoglobin-containing tumour composed of endothelial cells and papilloma is a benign tumour composed of connective tissue and branching vessels, dye laser photocoagulation causes selective damage to abnormal vessels and surrounding connective tissue. After local anaesthesia a small incision was made at the margin of the lesion five patients each with papilloma and with molluscum contagiosum. In six patients with Kaposi's sarcoma no incision had to be done. Starting from the incision or the margin of the Kaposi's sarcoma 50-100 shots of 0.3-0.5 mm diameter and 1 s duration were applied until the lesion was completely gone. We used a dye laser with 577 nm wavelength and 1 W energy. In patients with papilloma, the tumour basis was coagulated and the tumour itself underwent histological examination. There was a marked decrease of the size of the lesion or even complete disappearance. Since Kaposi's sarcoma in AIDS patients can be relatively fast growing, resulting in cosmetically irritating manifestations and, rarely, haemorrhages, treatment may become necessary. AIDS patients with molluscum contagiosum may also benefit from treatment, as spreading of DNA virus particles may result in viral follicular conjunctivitis. Dye laser photocoagulation, however, cannot protect against relapse.     

A randomized prospective trial of radiation therapy for AIDS-associated Kaposi's sarcoma

PURPOSE: The optimal dose of radiation in the treatment of AIDS-associated Kaposi's sarcoma has been controversial based on previous nonrandomized retrospective studies. METHODS AND MATERIALS: Seventy-one cutaneous AIDS-associated Kaposi's sarcoma lesions were randomly assigned to 1 of 3 radiation dose regimens--8 Gy in 1 fraction, 20 Gy in 10 fractions, and 40 Gy in 20 fractions. Lesions were measured prior to and following treatment. Complete resolution of palpable tumor was considered a complete response, regardless of residual purple pigmentation. Reduction in palpable tumor to less than 50% of pretreatment area was considered an objective response. Less than 50% reduction in tumor size was considered a nonresponse. RESULTS: Complete response was higher (p = .04) with 40 Gy (83%) and 20 Gy (79%) than with 8 Gy (50%). Absence of residual purple pigmentation was greater (p = .005) with 40 Gy (43%) than with 20 Gy (8%) or 8 Gy (8%). Lesion failure was lower (p = .03) with 40 Gy (52%) than with 20 Gy (67%) or 8 Gy (88%). Median time to failure was 43 weeks with 40 Gy, 26 weeks with 20 Gy, and 13 weeks with 8 Gy (p = .003). CONCLUSION: Fractionated radiotherapy to higher total doses resulted in improved response and control of cutaneous Kaposi's sarcoma. This dose-dependence should be considered in determining the optimal radiotherapeutic regimen for individual patients treated for epidemic Kaposi's sarcoma.     

Kaposi's sarcoma of the head and neck in patients with acquired immunodeficiency syndrome

Kaposi's sarcoma is the most common neoplastic process in patients infected with the human immunodeficiency virus. Moreover, the occurrence of Kaposi's sarcoma in human immunodeficiency virus-infected patients advances their classification to having the acquired immunodeficiency syndrome. We reviewed the medical records of 48 patients with human immunodeficiency virus infection who had Kaposi's sarcoma documented on their initial visit to the hospital. The onset of Kaposi's sarcoma occurred independent of the Centers for Disease Control and Prevention classification of human immunodeficiency virus infection (modified to exclude Kaposi's sarcoma). This neoplasm developed more frequently in patients who acquired human immunodeficiency virus infection by sexual contact (75% of cases), but manifestations were not significantly different in any of the risk populations for human immunodeficiency virus infection. Kaposi's sarcoma lesions were unpredictable and either showed progression, remained static, or occasionally, regressed spontaneously. Moreover, the lesions were usually multifocal at presentation, with the head and neck (62.5% of cases) as the primary site of involvement. In this region cutaneous lesions predominated (66.7%), followed by mucosal (56.7%) and deep structure (13.3%) involvement. The majority of patients with acquired immunodeficiency syndrome Kaposi's sarcoma involving head and neck structures were asymptomatic (80% of cases). Mucosal lesions were associated with symptoms in 29.3% of cases, whereas cutaneous lesions had symptoms in 5% of cases.     

Zenker diverticulum

BACKGROUND: Liposomal doxorubicin (Caelyx, Doxil) delivers doxorubicin to a tumor, but has a vastly altered pharmacology and attenuated acute and chronic toxicities. Therefore, its efficacy in soft tissue sarcomas is worth exploring. PATIENTS AND METHODS: Sixteen patients with recurrent or metastatic soft tissue sarcomas who had not failed prior doxorubicin were accrued into this phase II study. Patients were treated with Doxil at a dose of 50 mg/m2 every four weeks. RESULTS: No responses were seen but three patients were removed from study after only one cycle of treatment. Moreover, leiomyosarcoma was the most common histology and most patients had low grade, and bulky, disseminated tumors. Treatment was well tolerated with no episodes of grade 4 toxicity and only five episodes of grade 3 toxicities: two episodes of neutropenia and one each of stomatitis, dermatologic toxicity and nausea and vomiting. CONCLUSIONS: Doxil's lack of activity in this study of patients with adult soft tissue sarcoma may be related to the poor prognostic features of our population. We confirm its favorable toxicity profile and suggest that additional studies be done in patients with other characteristics.     

Seroconversion for human herpesvirus 8 during HIV infection is highly predictive of Kaposi's sarcoma

BACKGROUND: The finding of antibodies against human herpesvirus 8 (HHV-8) is associated with the occurrence of Kaposi's sarcoma in persons infected with HIV. However, the predictive value of HHV-8 antibodies for Kaposi's sarcoma in HIV infection is unknown. METHODS: The Amsterdam Cohort Studies on HIV infection and AIDS started in 1984 for homosexual men and in 1985 for injecting drug users. Serum samples from 1459 homosexual men and 1167 drug users were tested for antibodies to recombinant HHV-8 lytic-phase capsid (ORF65) antigen and latent-phase nuclear (ORF73) antigen. Individuals were retrospectively identified as HHV-8-positive or HHV-8-negative at enrolment or HHV-8 seroconverter during the study. Kaposi's sarcoma-free survival time was compared between HIV-infected men who were positive for HHV-8 at enrolment and those who later seroconverted for HHV-8. Hazard ratios were estimated for Kaposi's sarcoma, lymphoma, and opportunistic infection according to the HHV-8 serostatus. RESULTS: The incidence of HHV-8 seroconversion among drugs users was 0.7 per 100 person-years based on 31 seroconversions, whereas an incidence of 3.6 was found among homosexual men based on 215 seroconversions. The hazard ratio for Kaposi's sarcoma was 3.15 (95% CI: 1.89-5.25) in HIV-infected individuals if HHV-8 antibodies were present either at enrolment or at HIV seroconversion. In HIV-infected persons who later seroconverted to HHV-8, Kaposi's sarcoma developed more rapidly: hazard ratio of 5.04 (95% CI: 2.94-8.64), an additional risk of 1.60 (95% CI: 1.01-2.53; P = 0.04). Time-dependent adjustment for CD4+ cell count and HIV RNA had no impact on the additional risk, although the CD4+ cell count was an independent risk factor for Kaposi's sarcoma. HHV-8 infection did not increase the risk of AIDS-related lymphoma or opportunistic infections. CONCLUSIONS: The incidence of HHV-8 infection is higher in homosexual men than in drug users. The presence of HHV-8 antibodies in HIV-infected persons increases the risk of Kaposi's sarcoma. Among HIV-infected persons, those who subsequently seroconvert for HHV-8 are at highest risk. These results strongly confirm the causal role of HHV-8 in Kaposi's sarcoma and emphasize the clinical relevance of HHV-8 seroconversion before and after the HIV infection.     

Bronchopulmonary Kaposi's sarcoma in 106 HIV-1 infected patients

The objectives of this study were to describe the clinical and radiological features at presentation, and the natural history of HIV-related bronchopulmonary Kaposi's sarcoma. A retrospective review of medical records and chest radiographs was performed in 106 HIV-infected homosexual men with bronchopulmonary Kaposi's sarcoma diagnosed at bronchoscopy between September 1988 and November 1994. The majority of patients had evidence of advanced HIV disease at diagnosis (median CD4 cell count was 15 x 10(6)/l, range 0-288), and 93% had had a diagnosis of cutaneous Kaposi's sarcoma for a median duration of 11 months prior to diagnosis of their bronchopulmonary disease. The most frequent symptoms at presentation were cough (92%), dyspnoea (69%), pleuritic pain (20%), haemoptysis (13%) and wheezing (10%). The most common radiological finding in 73% of our series was of poorly defined and confluent opacities, with predominant middle and lower zone involvement. Median survival was 4 months (range 0-37 months) from diagnosis and 9 months (range 1-25) from the onset of symptoms. Treatment with either chemotherapy or radiotherapy was associated with a significantly reduced risk of death (hazards ratio (HR)=0.48, 95% CI=0.26-0.87). Factors associated with a poor survival, after adjustment for treatment effect were older age (HR=1.79, 95% CI=1.22-2.84) for each 10-year increase in age; a history of pleuritic pain (HR=2.97, 95% CI=1.39-6.32); presence of pleural effusion on X-ray (HR=2.01, 95% CI=1.13-3.59) and a prior diagnosis of cutaneous Kaposi's sarcoma (HR=1.8, 95% CI=1.00, 3.24). Bronchopulmonary Kaposi's sarcoma occurs mainly in patients with advanced HIV disease and a prior history of cutaneous disease. Survival is poor, and adverse prognostic factors include older age at diagnosis and the presence of pleural disease.