1 Departments of Oncology and HIV Medicine, Chelsea and Westminster Hospital, London; 2 Department of Immunology, Division of Investigative Science, Faculty of Medicine, Imperial College of Science Technology and Medicine, Chelsea and Westminster Hospital, London; 3 Bristol-Myers Squibb Pharmaceuticals Limited, London, UK
Received 13 March 2003; revised 23 May 2003; accepted 12 August 2003
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Abstract |
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Murine data indicate that angiogenesis is central to the aetiopathogenesis of Kaposis sarcoma (KS). Therefore, we measured angiogenic cytokines and growth factors in patients with AIDS-related KS during treatment with both antiretrovirals and second-line paclitaxel chemotherapy. Cytokines measured included tumour necrosis factor- (TNF-
), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and the interleukins IL-2, -6 and -12.
Patients and methods:
Enzyme-linked immunosorbent assays (ELISAs) were carried out to measure plasma cytokine levels in 17 patients with AIDS-related KS who had progressed within 6 months of receiving liposomal anthracyclines and were treated with paclitaxel 100 mg/m2 every 2 weeks. Measurements were carried out before progression, at commencement and at the completion of paclitaxel.
Results:
The objective response rate to paclitaxel was 71% (95% confidence interval 60% to 81%). In 17 patients with AIDS-related KS, we observed eight partial responses and four complete responses. Patients with AIDS Clinical Trial Group stage T1 disease had higher plasma VEGF (P = 0.05) and lower plasma TNF- levels (P = 0.05) than patients with earlier stage T0 KS. There were no correlations between plasma cytokines (bFGF, VEGF, TNF-
, and IL-2,-6 and -12) and the CD4 and CD8 cell counts or HIV-1 RNA viral load. Response to paclitaxel was associated with a fall in plasma IL-6 levels (P = 0.04) but no change in other cytokines. There were no significant changes in CD4, CD8, CD16/56, CD19 cell counts and HIV-1 viral loads during chemotherapy.
Conclusions:
Angiogenic cytokines may correlate with KS disease extent but not with cellular immune function or HIV viraemia. Response to paclitaxel therapy correlates with a fall in plasma IL-6 levels and recent data indicate this may be a surrogate marker of KS-associated herpesvirus viral load. Overall, clinical response in KS correlates poorly with known angiogenic cytokines.
Key words: AIDS, angiogenesis, cytokines, human immunodeficiency virus, Kaposis sarcoma, paclitaxel
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Introduction |
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Aggressive KS is a significant cause of morbidity and mortality in those in whom HAART is failing due to antiretroviral resistance, intolerance or adherence issues, and in some patients with good serological parameters (undetectable HIV-1 viraemia and CD4 cell counts >400/mm3) who presumably have failed to reconstitute a full immunological repertoire [1012].
The optimal first-line chemotherapy for AIDS-KS is liposomal anthracyclines [1315]. Other cytotoxic agents tested include vinca alkaloids [16, 17], bleomycin [18] and etoposide [19]. Paclitaxel has been shown to have single-agent activity against KS in phase II studies that have included some patients who had previously received anthracylines [2023]. Toxicity from these agents is often significant, particularly in individuals who are immunologically suppressed due to HIV-1 infection. Indeed in the pre-HAART era, patients with KS had a high mortality predominantly from other opportunistic infections [24]. There was also a reluctance to treat KS with systemic palliative chemotherapy, since there was no evidence of sustained remissions and there was a high risk of adversely influencing the HIV disease [25, 26]. For this reason, we followed changes in lymphocyte subsets as this has implications for prophylaxis of opportunistic infection.
Recent data from experiments in mice have indicated a central role of angiogenesis and angiogenic factors in the aetiopathogenesis of KS [27]. These experiments, more than three decades after Folkman [28] showed that tumour growth was dependent on the formation of new blood vessels, found that systemic administration of the protease inhibitors (PIs) saquinavir and inidinavir to nude mice blocked the development of, and induced regression of, angioproliferative KS-like lesions promoted by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF), or bFGF and VEGF combined. These drugs were also found to block bFGF- or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to that of paclitaxel, used as a positive antiangiogenic control. We therefore wished to observe whether in vivo human measurements of a large number of cytokines implicated in tumour growth and angiogenesis change during treatment of KS.
In December 1999, TAX/22-99.002, a pan-European phase IIIb open-label protocol for previously treated AIDS-KS was initiated in our unit. The trial was prematurely closed in June 2000 as a result of poor recruitment rates at other centres. Treatment using the same protocol was continued for other patients. We describe the efficacy and safety of paclitaxel, administered at 2-week intervals to patients who had liposomal anthracycline-resistant KS, defined as progression on liposomal anthracycline treatment or progression within 6 months of treatment. We also evaluated plasma angiogenic marker and growth factor concentrations before treatment with liposomal anthracycline, at relapse and following therapy with paclitaxel.
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Methods |
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All individuals gave written informed consent and the study received ethical approval in accordance with the Helsinki Declaration. Toxicity was graded using the National Cancer Institute Common Toxicity Criteria version 2.0. Disease evaluation (including bidimensional measurements of cutaneous lesions) was recorded every other cycle using the amended AIDS Clinical Trial Group (ACTG) response assessment [31]. A Gehan two-stage design with 95% power at a 10% standard error was used. It was originally intended to recruit nine patients and if there were no objective responses or stabilisation of disease, the trial was to be terminated. There was a 5% probability that this would occur with a response or disease stabilising rate of 30%.
Immunological parameters
Total lymphocyte and subset analysis was carried out using whole blood stained with murine anti-human monoclonal antibodies to CD4, CD8, CD16/56 and CD19 (TetraOne; Beckman Coulter, High Wycombe, UK) and were evaluated on an Epics XL-MCL (Beckman Coulter) flow cytometer. Viral loads in patient plasma were measured using the Quantiplex HIV RNA 3.0 assay (Chiron bDNA; Chiron Diagnostics, Halstead, UK) with a lower limit of detection of 50 HIV-1 copies/ml. These parameters were measured before and at the end of chemotherapy and then at 1, 3 and 12 months thereafter.
Cytokines and statistical analysis
Plasma samples were collected in heparinised tubes at the start of therapy with anthracyclines, at relapse and immediately following the completion of treatment with paclitaxel. They were stored at 80°C and analysed together on single plates for each cytokine to reduce variability in results, using quantitative sandwich immunoassay kits. Enzyme-linked immunosorbent assays (ELISAs) were carried out for VEGF (Amersham, Little Chalfont, UK; lower limit of detection 8 pg/ml, upper limit of detection 2000 pg/ml), bFGF (Immunotopics, San Clemente, CA, USA; 32470 RU/ml), tumour necrosis factor- (Roche, Mannheim, Germany; 20864 pg/ml), interleukin (IL)-2 (Diaclone, Besancon, France; 101000 pg/ml), IL-6 (Diaclone; 149 pg/ml) and IL-12 (Diaclone; 101000 pg/ml).
Due to variability in plasma levels, a variance-stabilising transformation was made on these data. Non-parametric statistical methods were used for between-group comparisons and the KruskalWallis one-way analysis of variance test was used to compare the initial three results arms (pre-anthracycline, at relapse, post-paclitaxel) at each time period. All non-parametric data are presented as medians with interquartile ranges. Data were further analysed for trends over time using repeated-measures ANOVA with the MIXED procedure in SAS. Between- and within-patient adjusted slopes over time were estimated on log10-transformed plasma levels for each of the study arms and these are presented with 95% confidence intervals (CIs). All P values presented are two-sided.
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Results |
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Response
The response rate as measured by ACTG criteria on an intention-to-treat basis was 71% (95% CI 60% to 81%); in total 12 patients showed a partial or complete response (Table 2). One patient had progressive disease during treatment and this patient died 4 weeks after his second cycle of paclitaxel due to multicentric Castlemans disease. A further four patients (24%) had stable disease during treatment. Figure 1 demonstrates the time to progression in this group of patients. The median time to progression for all patients is 0.5 years and for those with a complete or partial response to treatment it is 1.4 years. This compares with a median time to progression for the same patients of 0.5 years on liposomal anthracyclines (Figure 1).
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Toxicity
There were no cases of cessation of treatment due to toxicity alone. The most common toxicities were grade I anaemia, and neuropathy (29%) and grade II nausea/vomiting (29%), fatigue (23%) and alopecia (17%) (Table 3). One patient with grade III neuropathy had had a grade II antiretroviral-related neuropathy previously. Grade III neutropenia occurred in four patients. One patient with grade IV neutropenia (the only grade IV toxicity recorded) was admitted with a fever, which resolved with antibiotics. This patient was also the only individual who received granulocyte colony-stimulating factor (G-CSF) during the study and developed nail dystrophy (another individual had paronychia). Overall, six patients had either a 1-week delay in treatment or a 25% dose reduction. No patients developed opportunistic infections during treatment and HAART was continued unchanged in 16 patients (94%).
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In patients with aggressive and extensive mucocutaneous disease or with visceral KS, systemic chemotherapy and HAART is the treatment of choice [32]. This trial specifically investigated the use of a paclitaxel in patients who were unsuccessfully treated with optimal first-line chemotherapy. One phase II study of paclitaxel (135 mg/m2 every 3 weeks) for KS enrolled 28 patients and reported a response rate of 71%. This included four patients (14%) who had received anthracyclines, but no patients received HAART [21]. A second, larger study of 56 patients included 20 (36%) who received a PI at some stage during the study and 40 (70%) who had received prior therapy for KS, which included liposomal anthracyclines in 17 (30%). The overall objective response rate was 59% (amended ACTG criteria), and a median response duration of 10.4 months [22]. A further open-label multicentre trial enrolled 107 individuals; the response rate was 56% and PI use (77% of patients) had a significant impact on survival. The main side-effect reported in these studies was neutropenia. Our study and subgroup analysis of these previous trials demonstrate that paclitaxel is a safe and effective therapy in patients with anthracycline-resistant AIDS-KS, with a response rate of 71%.
Analysis of angiogenic marker and growth factor concentrations was confounded by marked heterogeneity in levels, a situation previously observed in other studies [33]. Levels of bFGF, VEGF, IL-6, IL-2 and IL-12 did not correlate with response to treatment, although trends towards decreased levels were observed in both responders and non-responders. Levels of IL-6 were significantly decreased by the end of therapy with paclitaxel and recent work has indicated that this may be a surrogate marker of KS-associated herpesvirus (KSHV) viral load (J. Wilkinson, personal communication). This is also consistent with other recent data showing that increased levels of IL-6 are correlated with a negative outcome in both KSHV-related [34] and -unrelated conditions [35].
The lack of differences in plasma levels in the other cytokines between responders and non-responders indicates that while paclitaxel has antiangiogenic effects in a mouse model of KS [27] and can lead to a decrease in angiogenic cytokines, there is only a tenuous relationship with tumour response in humans. Consistent with the mouse model, however, bFGF levels were significantly decreased in those individuals on HAART containing a PI. In addition, those with more aggressive KS were noted to have higher levels of VEGF. A more accurate method of studying the biological effects may involve the examination of biopsy material before and after treatment to assess the vascularity and the expression of angiogenic markers by in situ hybridisation techniques.
The mechanism of paclitaxel activity in KS has been investigated using other models of experimental KS induced by the inoculation of KS-derived spindle cells into nude mice and primary cultures of KS spindle cells [36]. Paclitaxel has been found to (i) promote regression of KS lesions in vivo, (ii) block the growth, migration and invasion of KS spindle cells in vitro, and (iii) promote apoptosis and reduction of the antiapoptotic Bcl-2 protein within these lesions. Recently, its antiangiogenic properties have been described in detail [27], while anthracyclines have not been shown to have the same effect [37]. However, the clinical importance of paclitaxel-induced changes in plasma angiogenic marker levels remains questionable.
It has been reported that the majority of affected individuals have advanced immunosuppression at the time of the initial KS diagnosis [38]. Our cohort was not profoundly immunosuppressed, with a median CD4 cell count at entry of 287/mm3 and 59% with undetectable HIV-1 RNA loads. Furthermore, there was no significant fall in lymphocyte subsets or rise in viral loads or opportunistic infections during chemotherapy or for up to 1 year after completion of paclitaxel. In contrast, Gill et al. [22] reported 51 AIDS-defining opportunistic infections in the 56 patients treated with paclitaxel (10.5/100 patient months on paclitaxel), only 36% of whom received HAART, and Welles et al. [21] reported 27 opportunistic infections (8.4/100 person months on paclitaxel) among her cohort, none of whom received HAART. Thus the concomitant use of HAART and paclitaxel appears to be safe and not detrimental to immune function, despite initial concerns over pharmacological interactions [39].
Overall, paclitaxel was well-tolerated. As previously reported for this group of patients, the major toxicity was neutropenia, although only 23% developed grade III or IV neutropenia and only one patient required G-CSF support. There were no treatment-related deaths and the only other grade III toxicity reported was peripheral neuropathy.
In conclusion, this trial has provided convincing evidence that paclitaxel has activity against anthracyline-resistant AIDS-KS and may safely be administered with HAART. Moreover, there were no significant adverse effects on immune or virological parameters.
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Acknowledgements |
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Footnotes |
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