Imatinib-Induced Regression of AIDS-Related Kaposi's Sarcoma
http://www.100md.com
《临床肿瘤学》
the Beth Israel Deaconess Medical Center
PerkinElmer Life Sciences, Boston
Cell Signaling Technology, Beverly, MA
University of Pittsburgh Cancer Institute, Pittsburgh, PA
ABSTRACT
PATIENTS AND METHODS: Ten male patients with AIDS-related cutaneous KS, which progressed despite chemotherapy and/or highly active antiretroviral therapy, received imatinib mesylate administered orally, 300 mg twice daily. Clinical response was determined by serial tumor measurements. To determine biologic and histologic response, skin lesion biopsies were obtained at baseline and following 4 weeks of therapy.
RESULTS: Five of 10 participants had a partial response by tumor measurements. Biopsies after 4 weeks of therapy demonstrated histologic regression in four of six patients. Four patients' tumor biopsies were assessable for immunohistochemistry end points pre- and post-therapy. These demonstrated inhibition of PDGFR and its downstream effector, extracellular receptor kinase, which is a member of the mitogen-activated protein kinase family. The most common adverse event was diarrhea, which led to dose reduction in six patients.
CONCLUSION: Imatinib mesylate administered orally twice daily for AIDS-related KS results in clinical and histologic regression of cutaneous KS lesions within 4 weeks. These promising results demonstrate that inhibition of the c-kit and/or PDGF receptors may represent an effective strategy for treating KS.
INTRODUCTION
Both in vivo and in vitro studies have suggested platelet-derived growth factor (PDGF) signaling has a role in KS formation. KS spindle cells have been shown by in situ hybridization and immunohistochemistry to express both {alpha} and PDGF receptors (PDGFRs).8 PDGF, the ligand for PDGFR, is expressed by a distinct subpopulation of KS spindle cells.8,10 Studies have demonstrated that the growth of KS spindle cells can be arrested by placing them in PDGF-depleted medium.11 This arrest can be mitigated by the addition of recombinant PDGF.8 Additionally, PDGF has been shown to induce the expression of vascular endothelial growth factor (VEGF) by KS spindle cells.9 These data suggest that the PDGFR is involved in two critical pathways in KS development: (1) induction of KS spindle cell growth and (2) induction of angiogenesis through VEGF.
In addition to PDGFR, another receptor tyrosine kinase that has been implicated in KS formation is c-kit. KSHV infection of dermal microvasculature endothelial cells (DMVEC) results in the upregulation of the c-kit receptor.12 Furthermore, KSHV-infected DMVEC proliferate in response to c-kit ligand (stem-cell factor), which is constitutively expressed by DMVEC.12 Expression of c-kit in HIV-related KS in vivo has been demonstrated by immunohistochemistry.13 Activating mutations of c-kit have not been reported in KS lesions. These observations suggest that c-kit may play an important role in the development of KS.
Activated PDGFR and c-kit stimulate multiple signaling pathways through downstream effectors. Both receptors have been shown to activate the serine/threonine kinase AKT via phosphoinositide 3-kinase (PI3K).14,15 AKT may mediate cell cycle progression via cyclin D and block apoptosis through pathways that induce Bcl-XL, an anti-apoptotic Bcl-2 family member, as well as phosphorylate and inhibit Bad, a pro-apoptotic Bcl-2 family member.16-18 PDGFR and c-kit may also activate the serine/threonine kinase extracellular receptor kinase (ERK), which is a member of the mitogen-activated protein kinase family.19,20 ERK plays a critical role in the regulation of a number of transcription factors associated with cell proliferation. ERK, in conjunction with AKT signaling, activates nuclear factor kappa B, a transcription factor that has proliferative as well as anti-apoptotic effects on cells.21 ERK can also influence the cell cycle by regulating Kip1/p27 and cyclin D.22 Activation of these pro-growth and/or anti-apoptotic pathways by PDGFR and c-kit via AKT and/or ERK could contribute to the development of the KS lesion.
Imatinib mesylate (Gleevec; Novartis Pharma AG, Basel, Switzerland) selectively inhibits the tyrosine kinases Abl and c-kit, which result in its activity in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors, respectively.23,24 Imatinib has been used successfully to treat dermatofibrosarcoma protuberans and hypereosinophilic syndrome, which are dependent on the PDGF pathway.25,26 Because of the role of PDGFR and c-kit in KS, we investigated the response of AIDS-related KS to imatinib. In CML, the recommended imatinib dose is 400 to 600 mg per day for chronic phase and 600 to 800 mg per day for accelerated phase. Based on these data we chose a dose of 600 mg per day to optimize the chance for response. We also investigated the effect of therapy on the activation of the PDGFR and c-kit receptors; their downstream signaling intermediates, ERK and AKT; as well as the effect on plasma VEGF concentrations.
PATIENTS AND METHODS
Study Design
In this open-label pilot study, imatinib was administered orally at a dose of 300 mg twice daily (600 mg/d) for 4 weeks. At the end of the 4-week period, patients without evidence of progression were allowed to continue on the drug until there was evidence of disease progression. Imatinib was provided by Novartis Pharma AG and was formulated as 100-mg hard gelatin capsules.
For grade 3/4 toxicity, imatinib was withheld until the toxicity resolved to ≤ grade 1. However, for grade 3/4 hematologic toxicity, defined as an absolute neutrophil count < 1,000/μL, or a platelet count < 50,000/μL, imatinib was withheld until the toxicity had resolved to ≤ grade 2. After resolution, the dose of imatinib was reduced to 200 mg twice daily. If the grade 3/4 toxicity recurred, imatinib was held until the toxicity resolved to ≤ grade 1 (≤ grade 2 for neutropenia and thrombocytopenia), and the daily dose of imatinib was reduced to 100 mg twice daily. If the grade 3/4 toxicity recurred yet again, therapy was discontinued. No dose reductions were performed for grade 1 to 4 anemia. Once on study, patients could be transfused packed red blood cells at the discretion of the investigators.
Schedule of Events
Clinical assessments, including a history and physical examination that focused on HIV- and KS-related signs and symptoms, evaluation of KS lesions, a complete blood count with differential, serum electrolytes, renal and liver function tests, amylase, lactate dehydrogenase, total protein, and albumin were performed on days 1, 8, 15, and 29 and then every 4 weeks after therapy was initiated. A 4-mm punch biopsy of a representative KS lesion at baseline and at 4 weeks was performed on all patients who consented and when the biopsy would not interfere with measuring tumor response. Plasma samples for cytokine concentrations were collected before imatinib administration and then 4 weeks after treatment was started. KS tumor assessments were based on measurement of cutaneous marker lesions and on the overall number and characteristics of cutaneous lesions. For patients with ≤ 50 total skin and oral lesions, all lesions were evaluated for flattening and change in number. For patients with > 50 lesions, three representative anatomic areas were chosen for ongoing evaluation of lesion number and characteristics.
Plasma Imatinib Concentrations
Blood samples were collected for pharmacokinetic analysis before imatinib administration and then 4 weeks after the first dose. Samples were collected in heparinized tubes and centrifuged at 2,400 xg for 5 minutes. Plasma was aliquoted and stored at –80°C until the time of analysis. Plasma concentrations of imatinib and its main metabolite (CGP-74588) were determined with a previously described and validated liquid chromatographic-mass spectrometric assay.27
KS Biopsy/Immunohistochemistry
Tumor biopsies were immediately placed in formalin for fixation. After paraffin embedding, tissue sections of 5-μm thickness were prepared and stained using the antibodies anti-PDGFR, anti-c-kit, anti-phosphorylated (phospho)-PDGFR (tyrosine 751), anti-phospho-AKT (serine 473), and anti-phospho-ERK (threonine 202/tyrosine 204; Cell Signaling, Beverly, MA). Slides were de-paraffinized in xylene and ethanol. Antigen retrieval was performed by boiling the slides for 10 min in pH 6 citrate buffer. The slides stained for phospho-AKT and phospho-ERK were cooled for 30 minutes. The slides stained for phospho-PDGFR were cooled for 15 minutes in 1mM EDTA, pH 8.0. After 10 minutes in 3% H2O2, slides were incubated overnight with the primary antibody at a 1/250 dilution for phospho-AKT, 1/100 dilution for phospho-ERK, and 1/50 dilution for phospho-PDGFR at 4°C. This was followed by a 30-minute incubation with 100 μL of biotinylated anti-rabbit secondary antibody, then 30 minutes with ABC avidin/biotin (Vector Labs, Burlingame, CA). Slides were incubated in ethanol and xylene before mounting coverslips. Immunohistochemistry stains were then scored on a scale of 0 to 4+ by a dermatopathologist (S.R.T.).
VEGF Concentrations
The PerkinElmer Cytokine Chip (PerkinElmer, Cambridge, MA) was used to analyze VEGF concentrations. The assays rely on "sandwich" fluorescent detection, employing two antibodies per cytokine. A capture antibody that was specific for each cytokine was spotted in quadruplicate (one time in each subarray, four subarrays per gel pad) on the HydroGel-coated slide (PerkinElmer). The sample (75 μL) was pipetted onto one of the pads on the HydroGel-coated slide and allowed to incubate for 2 hours, during which time any cytokine present bound to its capture antibody. Unbound material was washed away and a biotinylated detector antibody mixture (75 μL) was added and incubated for 1 hour. This mixture contained detector antibodies that were specific for each cytokine and bound to the cytokine that had been retained by the appropriate capture antibody, thus forming a sandwich. The detector antibody, in turn, was bound by cyanine 3–conjugated streptavidin (75 μL for 30 min). The intensity of the fluorescent signal was proportional to the amount of cytokine protein in the sample.
After running the assay, slides were imaged using a ScanArray Confocal Laser Scanner (PerkinElmer). After imaging, the resulting tif files were quantitated using QuantArray software (PerkinElmer). Replicate spots were averaged and tested for outliers using the extreme studentized deviate method (n = 4; critical Z value, 1.48). Four-point standard curves were used to estimate the concentration of each cytokine in the samples. Standards were analyzed by point-to-point linear regression, and concentrations of experimental samples were estimated from these standards.
Statistics and Response Criteria
Complete response was defined as the absence of any detectable residual disease persisting for at least 4 weeks. Partial response was defined as either a ≥ 50% decrease in the number of all previously existing lesions or complete flattening of at least 50% of all previously raised lesions, or a 50% decrease in the sum of the products of the largest perpendicular diameters of the marker lesions. A partial response was required to persist for at least 4 weeks without the development of new lesions, new visceral sites of involvement, or increased tumor-associated edema or effusions. Progressive disease was defined by any of the following: ≥ 25% increase in the sum of the perpendicular diameters of the indicator lesions; ≥ 25% increase in the total lesion count; ≥ 25% increase in the number of raised lesions; new visceral sites of involvement; and development of new tumor-associated edema or effusion that lasted at least 1 week and interfered with normal activities. Stable disease was defined as any response not meeting the criteria for complete response, partial response, or progressive disease.28
RESULTS
Clinical Events
All 10 patients remained on study for the 4 weeks specified by the protocol. The median duration of therapy was 7 weeks (range, 4 to 24 weeks). By the fourth week of treatment, all patients required dose reduction to 200 mg orally twice a day. The most commonly reported adverse event was diarrhea, for which five patients discontinued therapy (Table 2). Their diarrhea was initially grade 3 (n = 4) or grade 4 (n = 1) and typically occurred in the second or third week on therapy. Although the diarrhea recurred despite drug withdrawal and dose reductions, its severity was reduced to grade 1/2 by treating with antimotility agents. No infectious causes were found for any of these cases. Two of five patients who discontinued therapy had grade 1/2 diarrhea at baseline. One patient developed grade 4 neutropenia that recurred despite drug withdrawal and dose reductions. One patient developed grade 3 depression that recurred despite drug withdrawal and dose reductions.
Plasma Imatinib Concentrations
Because a higher-than-expected rate of gastrointestinal drug toxicity was observed, plasma samples were analyzed to determine concentrations of imatinib and its metabolite, CGP-74588 (Table 2). At steady-state, the mean imatinib concentration was 1,342 ng/mL (range, 0 to 2846 ng/mL) and CGP-74588 was 354 ng/mL (range, 38 to 519 ng/mL; n = 9). These concentrations were lower than those achieved in patients with other solid tumors maintained on the same dose schedule, 2,939 ng/mL (range, 2,009 to 3,497 ng/mL) for imatinib and 733 ng/mL (range, 459 to 1,046 ng/mL) for CGP-74588 (n = 4).29
Antitumor Effects
Five of 10 patients had a partial response by tumor measurement after only 4 weeks of imatinib therapy (Table 1). The remaining five patients, who had been developing new lesions before therapy, had stable disease at 4 weeks. Three of the patients with stable disease had biopsies after 4 weeks of therapy. Two of the three biopsies demonstrated histologic regression by the criteria previously described (Fig 1).13
PDGFR/c-kit Pathways
Four patients had baseline biopsies that were assessable for PDGFR (3+ to 4+ staining) and c-kit (all 3+ staining) expression by immunohistochemistry (Fig 2). Additionally we examined four sets of biopsies from baseline and 4 weeks for evidence of PDGFR activation by immunohistochemistry staining for phospho-PDGFR, phospho-ERK, and phospho-AKT (Table 1). Staining with phospho-c-kit was equivocal before and after treatment in these patients. Three of the four patients had a decrease in PDGFR and ERK activity by immunoreactivity (Figs 3A and B). AKT immunoreactivity was weak at baseline (0 to 1+ staining) in all patients, and we were unable to detect any change in staining after therapy (Fig 3C).
Plasma VEGF Concentrations
Plasma VEGF concentrations did not significantly differ between responders and nonresponders at baseline or 4 weeks VEGF concentrations at baseline were 363 pg/mL ± 180 for responders and 229 pg/mL ± 213 for nonresponders. After 4 weeks of imatinib therapy, VEGF concentrations were 380 pg/mL ± 187 for responders and 294 pg/mL ± 265 for nonresponders.
DISCUSSION
Imatinib, administered 300 mg orally twice daily to patients with AIDS-related KS and receiving HAART, was poorly tolerated. The incidence of diarrhea was higher than in trials with CML patients treated at the same dose. In those trials, 24% of the patients had grade 1/2 diarrhea and none had grade 3/4 diarrhea.23,30 One possible explanation for the increased frequency and severity of the diarrhea is a drug interaction with the medications involved in the HAART regimen. Imatinib is a substrate for the cytochrome p450 isoforms, CYP3A4 and CYP2D6. All of the protease inhibitors approved at the time of the study, which included amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, are CYP3A4 inhibitors, with ritonavir inhibiting CYP2D6 to a lesser extent. Diarrhea is a side effect common to all of the protease inhibitors. The other class of antiretrovirals shown to affect the p450 system is non-nucleoside reverse transcriptase inhibitors. Delavirdine is a CYP3A inhibitor, while nevirapine is a CYP3A inducer, and efavirenz is a mixed CYP3A inducer/inhibitor. No correlation between any specific antiretroviral regimen and the incidence of diarrhea was observed. There was no obvious increase in plasma steady-state concentrations of imatinib or its metabolite. Although these data suggest there was no inhibition of imatinib metabolism, the imatinib concentrations may not be truly representative of the steady-state concentration. Samples were randomly obtained on day 28 and by that time, all patients with diarrhea had developed symptoms. The presence of the diarrhea could have affected the absorption of imatinib as well as patient adherence with the therapy. Unfortunately, plasma concentrations of the antiretrovirals were not measured. It is plausible that the observed toxicities may be due to imatinib inhibition of antiretroviral metabolism because imatinib is a substrate of the CYP3A4 and CYP2D6 pathways. Investigation into the mechanisms of imatinib toxicity is the subject of a future trial.
In this pilot study, imatinib was shown to induce clinical regression in five of 10 patients within 4 weeks of therapy. Patient 3, who had only been on HAART for 4 weeks before beginning therapy, had an increase in his CD4 count after 4 weeks of therapy. This raises the question of whether his response was to imatinib or was a delayed response to HAART. Although we cannot determine the reason for his response from our limited data, it is notable that he had been developing new lesions the week before he started imatinib.
In order to elucidate which signaling pathways may have been critical to imatinib-induced response, we examined tumor biopsies for evidence of decreased phosphorylation (ie, inhibition) of PDGFR and c-kit, as well as their downstream effectors AKT and ERK. Four patients had tumor biopsies that were assessable at baseline and at 4 weeks by immunohistochemistry with phosphospecific antibodies. After 4 weeks of therapy, three of these biopsies demonstrated a decrease of phosphorylation (activation) of the PDGFR and ERK pathways when compared to baseline. The fourth biopsy (patient 10) demonstrated +3 immunoreactivity for phospho-PDGFR and phospho-ERK at baseline and after 4 weeks of therapy. Interestingly, patient 10 had stable disease after 4 weeks on imatinib therapy and had low concentrations on imatinib and its metabolite at the time of biopsy. In contrast, there was minimal or no activation of AKT in pretreatment tumor specimens. Because the phospho-c-kit antibody recognized the phosphorylation of the activation motif on both PDGFR and c-kit, we were unable to determine conclusively the phosphorylation status of c-kit. Post-treatment biopsies demonstrated dramatic reduction in ERK and PDGFR phosphorylation in three patient specimens. These observations suggest that the ERK pathway, in contrast to the AKT pathway, may be critical for proliferation of KS lesions. This is of interest, as signaling from PDGFR in other cell systems is often transmitted through AKT.31
VEGF concentrations were measured in nine patients at baseline and after 4 weeks of therapy. In this study, no difference was seen in VEGF concentrations between responders and nonresponders. Although in vitro evidence suggested that inhibition of the PDGF pathway would decrease VEGF concentrations, our data would suggest that either PDGF regulation of VEGF is not the primary pathway in vivo, or there are parallel pathways that are able to compensate for PDGFR inhibition.
In conclusion, we demonstrated that imatinib may be an effective form of therapy in patients with AIDS-related KS. Unfortunately, imatinib administered at a dose of 600 mg daily was poorly tolerated in some patients. Therefore, a follow-up study planned by the AIDS Malignancy Clinical Trial Consortium is being performed at a dose of 400 mg per day. The reason for the increased toxicity in HIV patients on HAART is unclear and the mechanism of toxicity will be investigated in a follow-up study. This is the first study to demonstrate that inhibition of PDGFR and ERK correlates with regression of KS. Our findings support the need for further evaluation of imatinib as a single agent in patients with KS that is refractory to standard therapies.
Authors' Disclosures of Potential Conflicts of Interest
Acknowledgment
We thank Jerome Groopman for his support during this project.
NOTES
Supported by the AIDS Oncology Clinical Scientist Development Program (H.B.K., grant No. K12CA077846-03).
H.B.K. and G.J.B contributed equally to this work.
Funding for this study was provided by Novartis Pharma AG, Basel, Switzerland.
Presented in part at the International AIDS Malignancy Conference, Bethesda, MD, April 28-29, 2003, and 39th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, May 31-June 3, 2003.
Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
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2. Danzig JB, Brandt LJ, Reinus JF, et al: Gastrointestinal malignancy in patients with AIDS. Am J Gastroenterol 86:715-718, 1991
3. Flaitz CM, Nichols CM, Hicks MJ: Herpesviridae-associated persistent mucocutaneous ulcers in acquired immunodeficiency syndrome. A clinicopathologic study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 81:433-441, 1996
4. Masood R, Cai J, Zheng T, et al: Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc Natl Acad Sci U S A 94:979-984, 1997
5. Miles SA, Rezai AR, Salazar-Gonzalez JF, et al: AIDS Kaposi sarcoma-derived cells produce and respond to interleukin 6. Proc Natl Acad Sci U S A 87:4068-4072, 1990
6. Ensoli B, Nakamura S, Salahuddin SZ, et al: AIDS-Kaposi's sarcoma-derived cells exp ress cytokines with autocrine and paracrine growth effects. Science 243:223-226, 1989
7. Miles SA: Kaposi sarcoma: A cytokine-responsive neoplasia Cancer Treat Res 63:129-140, 1992
8. Sturzl M, Roth WK, Brockmeyer NH, et al: Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance. Proc Natl Acad Sci U S A 89:7046-7050, 1992
9. Cornali E, Zietz C, Benelli R, et al: Vascular endothelial growth factor regulates angiogenesis and vascular permeability in Kaposi's sarcoma. Am J Pathol 149:1851-1869, 1996
10. Sturzl M, Brandstetter H, Zietz C, et al: Identification of interleukin-1 and platelet-derived growth factor-B as major mitogens for the spindle cells of Kaposi's sarcoma: A combined in vitro and in vivo analysis. Oncogene 10:2007-2016, 1995
11. Roth WK, Werner S, Schirren CG, et al: Depletion of PDGF from serum inhibits growth of AIDS-related and sporadic Kaposi's sarcoma cells in culture. Oncogene 4:483-487, 1989
12. Moses AV, Jarvis MA, Raggo C, et al: Kaposi's sarcoma-associated herpesvirus-induced upregulation of the c-kit proto-oncogene, as identified by gene expression profiling, is essential for the transformation of endothelial cells. J Virol 76:8383-8399, 2002
13. Pantanowitz L, Dezube BJ, Pinkus GS, et al: Histological characterization of regression in acquired immunodeficiency syndrome-related Kaposi's sarcoma. J Cutan Pathol 31:26-34, 2004
14. Blume-Jensen P, Janknecht R, Hunter T: The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136. Curr Biol 8:779-782, 1998
15. Franke TF, Yang SI, Chan TO, et al: The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727-736, 1995
16. Brennan P, Babbage JW, Burgering BM, et al: Phosphatidylinositol 3-kinase couples the interleukin-2 receptor to the cell cycle regulator E2F. Immunity 7:679-689, 1997
17. Gesbert F, Griffin JD: Bcr/Abl activates transcription of the Bcl-X gene through STAT5. Blood 96:2269-2276, 2000
18. Rosa Santos SC, Dumon S, Mayeux P, et al: Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line. Oncogene 19:1164-1172, 2000
19. Chaudhary LR, Avioli LV: Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells. J Cell Biochem 76:354-359, 2000
20. Pearson MA, O'Farrell AM, Dexter TM, et al: Investigation of the molecular mechanisms underlying growth factor synergy: The role of ERK 2 activation in synergy. Growth Factors 15:293-306, 1998
21. Liu W, Liu Y, Lowe WL Jr: The role of phosphatidylinositol 3-kinase and the mitogen-activated protein kinases in insulin-like growth factor-I-mediated effects in vascular endothelial cells. Endocrinology 142:1710-1719, 2001
22. Weber JD, Hu W, Jefcoat SC, Jr., et al: Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27. J Biol Chem 272:32966-32971, 1997
23. Talpaz M, Silver RT, Druker BJ, et al: Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: Results of a phase 2 study. Blood 99:1928-1937, 2002
24. Demetri GD, von Mehren M, Blanke CD, et al: Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 347:472-480, 2002
25. Rubin BP, Schuetze SM, Eary JF, et al: Molecular targeting of platelet-derived growth factor B by imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans. J Clin Oncol 20:3586-3591, 2002
26. Cools J, DeAngelo DJ, Gotlib J, et al: A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med 348:1201-1214, 2003
27. Parise RA, Ramanathan RK, Hayes MJ, et al: Liquid chromatographic-mass spectrometric assay for quantitation of imatinib and its main metabolite (CGP 74588) in plasma. J Chromatogr B Analyt Technol Biomed Life Sci 791:39-44, 2003
28. Krown SE, Metroka C, Wernz JC: Kaposi's sarcoma in the acquired immune deficiency syndrome: A proposal for uniform evaluation, response, and staging criteria. AIDS Clinical Trials Group Oncology Committee. J Clin Oncol 7:1201-1207, 1989
29. Remick SC, Ramanathan RK, Mulkerin D, et al: A Phase I Pharmacokinetic Study of STI-571 in Patients with Advanced Malignancies and Varying Degrees of Renal Dysfunction. Proc Am Soc Clin Oncol 22:503a, 2003
30. Druker BJ, Talpaz M, Resta DJ, et al: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 344:1031-1037, 2001
31. Chaudhary LR, Hruska KA: The cell survival signal Akt is differentially activated by PDGF-BB, EGF, and FGF-2 in osteoblastic cells. J Cell Biochem 81:304-311, 2001(Henry B. Koon, Glenn J. B)
PerkinElmer Life Sciences, Boston
Cell Signaling Technology, Beverly, MA
University of Pittsburgh Cancer Institute, Pittsburgh, PA
ABSTRACT
PATIENTS AND METHODS: Ten male patients with AIDS-related cutaneous KS, which progressed despite chemotherapy and/or highly active antiretroviral therapy, received imatinib mesylate administered orally, 300 mg twice daily. Clinical response was determined by serial tumor measurements. To determine biologic and histologic response, skin lesion biopsies were obtained at baseline and following 4 weeks of therapy.
RESULTS: Five of 10 participants had a partial response by tumor measurements. Biopsies after 4 weeks of therapy demonstrated histologic regression in four of six patients. Four patients' tumor biopsies were assessable for immunohistochemistry end points pre- and post-therapy. These demonstrated inhibition of PDGFR and its downstream effector, extracellular receptor kinase, which is a member of the mitogen-activated protein kinase family. The most common adverse event was diarrhea, which led to dose reduction in six patients.
CONCLUSION: Imatinib mesylate administered orally twice daily for AIDS-related KS results in clinical and histologic regression of cutaneous KS lesions within 4 weeks. These promising results demonstrate that inhibition of the c-kit and/or PDGF receptors may represent an effective strategy for treating KS.
INTRODUCTION
Both in vivo and in vitro studies have suggested platelet-derived growth factor (PDGF) signaling has a role in KS formation. KS spindle cells have been shown by in situ hybridization and immunohistochemistry to express both {alpha} and PDGF receptors (PDGFRs).8 PDGF, the ligand for PDGFR, is expressed by a distinct subpopulation of KS spindle cells.8,10 Studies have demonstrated that the growth of KS spindle cells can be arrested by placing them in PDGF-depleted medium.11 This arrest can be mitigated by the addition of recombinant PDGF.8 Additionally, PDGF has been shown to induce the expression of vascular endothelial growth factor (VEGF) by KS spindle cells.9 These data suggest that the PDGFR is involved in two critical pathways in KS development: (1) induction of KS spindle cell growth and (2) induction of angiogenesis through VEGF.
In addition to PDGFR, another receptor tyrosine kinase that has been implicated in KS formation is c-kit. KSHV infection of dermal microvasculature endothelial cells (DMVEC) results in the upregulation of the c-kit receptor.12 Furthermore, KSHV-infected DMVEC proliferate in response to c-kit ligand (stem-cell factor), which is constitutively expressed by DMVEC.12 Expression of c-kit in HIV-related KS in vivo has been demonstrated by immunohistochemistry.13 Activating mutations of c-kit have not been reported in KS lesions. These observations suggest that c-kit may play an important role in the development of KS.
Activated PDGFR and c-kit stimulate multiple signaling pathways through downstream effectors. Both receptors have been shown to activate the serine/threonine kinase AKT via phosphoinositide 3-kinase (PI3K).14,15 AKT may mediate cell cycle progression via cyclin D and block apoptosis through pathways that induce Bcl-XL, an anti-apoptotic Bcl-2 family member, as well as phosphorylate and inhibit Bad, a pro-apoptotic Bcl-2 family member.16-18 PDGFR and c-kit may also activate the serine/threonine kinase extracellular receptor kinase (ERK), which is a member of the mitogen-activated protein kinase family.19,20 ERK plays a critical role in the regulation of a number of transcription factors associated with cell proliferation. ERK, in conjunction with AKT signaling, activates nuclear factor kappa B, a transcription factor that has proliferative as well as anti-apoptotic effects on cells.21 ERK can also influence the cell cycle by regulating Kip1/p27 and cyclin D.22 Activation of these pro-growth and/or anti-apoptotic pathways by PDGFR and c-kit via AKT and/or ERK could contribute to the development of the KS lesion.
Imatinib mesylate (Gleevec; Novartis Pharma AG, Basel, Switzerland) selectively inhibits the tyrosine kinases Abl and c-kit, which result in its activity in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors, respectively.23,24 Imatinib has been used successfully to treat dermatofibrosarcoma protuberans and hypereosinophilic syndrome, which are dependent on the PDGF pathway.25,26 Because of the role of PDGFR and c-kit in KS, we investigated the response of AIDS-related KS to imatinib. In CML, the recommended imatinib dose is 400 to 600 mg per day for chronic phase and 600 to 800 mg per day for accelerated phase. Based on these data we chose a dose of 600 mg per day to optimize the chance for response. We also investigated the effect of therapy on the activation of the PDGFR and c-kit receptors; their downstream signaling intermediates, ERK and AKT; as well as the effect on plasma VEGF concentrations.
PATIENTS AND METHODS
Study Design
In this open-label pilot study, imatinib was administered orally at a dose of 300 mg twice daily (600 mg/d) for 4 weeks. At the end of the 4-week period, patients without evidence of progression were allowed to continue on the drug until there was evidence of disease progression. Imatinib was provided by Novartis Pharma AG and was formulated as 100-mg hard gelatin capsules.
For grade 3/4 toxicity, imatinib was withheld until the toxicity resolved to ≤ grade 1. However, for grade 3/4 hematologic toxicity, defined as an absolute neutrophil count < 1,000/μL, or a platelet count < 50,000/μL, imatinib was withheld until the toxicity had resolved to ≤ grade 2. After resolution, the dose of imatinib was reduced to 200 mg twice daily. If the grade 3/4 toxicity recurred, imatinib was held until the toxicity resolved to ≤ grade 1 (≤ grade 2 for neutropenia and thrombocytopenia), and the daily dose of imatinib was reduced to 100 mg twice daily. If the grade 3/4 toxicity recurred yet again, therapy was discontinued. No dose reductions were performed for grade 1 to 4 anemia. Once on study, patients could be transfused packed red blood cells at the discretion of the investigators.
Schedule of Events
Clinical assessments, including a history and physical examination that focused on HIV- and KS-related signs and symptoms, evaluation of KS lesions, a complete blood count with differential, serum electrolytes, renal and liver function tests, amylase, lactate dehydrogenase, total protein, and albumin were performed on days 1, 8, 15, and 29 and then every 4 weeks after therapy was initiated. A 4-mm punch biopsy of a representative KS lesion at baseline and at 4 weeks was performed on all patients who consented and when the biopsy would not interfere with measuring tumor response. Plasma samples for cytokine concentrations were collected before imatinib administration and then 4 weeks after treatment was started. KS tumor assessments were based on measurement of cutaneous marker lesions and on the overall number and characteristics of cutaneous lesions. For patients with ≤ 50 total skin and oral lesions, all lesions were evaluated for flattening and change in number. For patients with > 50 lesions, three representative anatomic areas were chosen for ongoing evaluation of lesion number and characteristics.
Plasma Imatinib Concentrations
Blood samples were collected for pharmacokinetic analysis before imatinib administration and then 4 weeks after the first dose. Samples were collected in heparinized tubes and centrifuged at 2,400 xg for 5 minutes. Plasma was aliquoted and stored at –80°C until the time of analysis. Plasma concentrations of imatinib and its main metabolite (CGP-74588) were determined with a previously described and validated liquid chromatographic-mass spectrometric assay.27
KS Biopsy/Immunohistochemistry
Tumor biopsies were immediately placed in formalin for fixation. After paraffin embedding, tissue sections of 5-μm thickness were prepared and stained using the antibodies anti-PDGFR, anti-c-kit, anti-phosphorylated (phospho)-PDGFR (tyrosine 751), anti-phospho-AKT (serine 473), and anti-phospho-ERK (threonine 202/tyrosine 204; Cell Signaling, Beverly, MA). Slides were de-paraffinized in xylene and ethanol. Antigen retrieval was performed by boiling the slides for 10 min in pH 6 citrate buffer. The slides stained for phospho-AKT and phospho-ERK were cooled for 30 minutes. The slides stained for phospho-PDGFR were cooled for 15 minutes in 1mM EDTA, pH 8.0. After 10 minutes in 3% H2O2, slides were incubated overnight with the primary antibody at a 1/250 dilution for phospho-AKT, 1/100 dilution for phospho-ERK, and 1/50 dilution for phospho-PDGFR at 4°C. This was followed by a 30-minute incubation with 100 μL of biotinylated anti-rabbit secondary antibody, then 30 minutes with ABC avidin/biotin (Vector Labs, Burlingame, CA). Slides were incubated in ethanol and xylene before mounting coverslips. Immunohistochemistry stains were then scored on a scale of 0 to 4+ by a dermatopathologist (S.R.T.).
VEGF Concentrations
The PerkinElmer Cytokine Chip (PerkinElmer, Cambridge, MA) was used to analyze VEGF concentrations. The assays rely on "sandwich" fluorescent detection, employing two antibodies per cytokine. A capture antibody that was specific for each cytokine was spotted in quadruplicate (one time in each subarray, four subarrays per gel pad) on the HydroGel-coated slide (PerkinElmer). The sample (75 μL) was pipetted onto one of the pads on the HydroGel-coated slide and allowed to incubate for 2 hours, during which time any cytokine present bound to its capture antibody. Unbound material was washed away and a biotinylated detector antibody mixture (75 μL) was added and incubated for 1 hour. This mixture contained detector antibodies that were specific for each cytokine and bound to the cytokine that had been retained by the appropriate capture antibody, thus forming a sandwich. The detector antibody, in turn, was bound by cyanine 3–conjugated streptavidin (75 μL for 30 min). The intensity of the fluorescent signal was proportional to the amount of cytokine protein in the sample.
After running the assay, slides were imaged using a ScanArray Confocal Laser Scanner (PerkinElmer). After imaging, the resulting tif files were quantitated using QuantArray software (PerkinElmer). Replicate spots were averaged and tested for outliers using the extreme studentized deviate method (n = 4; critical Z value, 1.48). Four-point standard curves were used to estimate the concentration of each cytokine in the samples. Standards were analyzed by point-to-point linear regression, and concentrations of experimental samples were estimated from these standards.
Statistics and Response Criteria
Complete response was defined as the absence of any detectable residual disease persisting for at least 4 weeks. Partial response was defined as either a ≥ 50% decrease in the number of all previously existing lesions or complete flattening of at least 50% of all previously raised lesions, or a 50% decrease in the sum of the products of the largest perpendicular diameters of the marker lesions. A partial response was required to persist for at least 4 weeks without the development of new lesions, new visceral sites of involvement, or increased tumor-associated edema or effusions. Progressive disease was defined by any of the following: ≥ 25% increase in the sum of the perpendicular diameters of the indicator lesions; ≥ 25% increase in the total lesion count; ≥ 25% increase in the number of raised lesions; new visceral sites of involvement; and development of new tumor-associated edema or effusion that lasted at least 1 week and interfered with normal activities. Stable disease was defined as any response not meeting the criteria for complete response, partial response, or progressive disease.28
RESULTS
Clinical Events
All 10 patients remained on study for the 4 weeks specified by the protocol. The median duration of therapy was 7 weeks (range, 4 to 24 weeks). By the fourth week of treatment, all patients required dose reduction to 200 mg orally twice a day. The most commonly reported adverse event was diarrhea, for which five patients discontinued therapy (Table 2). Their diarrhea was initially grade 3 (n = 4) or grade 4 (n = 1) and typically occurred in the second or third week on therapy. Although the diarrhea recurred despite drug withdrawal and dose reductions, its severity was reduced to grade 1/2 by treating with antimotility agents. No infectious causes were found for any of these cases. Two of five patients who discontinued therapy had grade 1/2 diarrhea at baseline. One patient developed grade 4 neutropenia that recurred despite drug withdrawal and dose reductions. One patient developed grade 3 depression that recurred despite drug withdrawal and dose reductions.
Plasma Imatinib Concentrations
Because a higher-than-expected rate of gastrointestinal drug toxicity was observed, plasma samples were analyzed to determine concentrations of imatinib and its metabolite, CGP-74588 (Table 2). At steady-state, the mean imatinib concentration was 1,342 ng/mL (range, 0 to 2846 ng/mL) and CGP-74588 was 354 ng/mL (range, 38 to 519 ng/mL; n = 9). These concentrations were lower than those achieved in patients with other solid tumors maintained on the same dose schedule, 2,939 ng/mL (range, 2,009 to 3,497 ng/mL) for imatinib and 733 ng/mL (range, 459 to 1,046 ng/mL) for CGP-74588 (n = 4).29
Antitumor Effects
Five of 10 patients had a partial response by tumor measurement after only 4 weeks of imatinib therapy (Table 1). The remaining five patients, who had been developing new lesions before therapy, had stable disease at 4 weeks. Three of the patients with stable disease had biopsies after 4 weeks of therapy. Two of the three biopsies demonstrated histologic regression by the criteria previously described (Fig 1).13
PDGFR/c-kit Pathways
Four patients had baseline biopsies that were assessable for PDGFR (3+ to 4+ staining) and c-kit (all 3+ staining) expression by immunohistochemistry (Fig 2). Additionally we examined four sets of biopsies from baseline and 4 weeks for evidence of PDGFR activation by immunohistochemistry staining for phospho-PDGFR, phospho-ERK, and phospho-AKT (Table 1). Staining with phospho-c-kit was equivocal before and after treatment in these patients. Three of the four patients had a decrease in PDGFR and ERK activity by immunoreactivity (Figs 3A and B). AKT immunoreactivity was weak at baseline (0 to 1+ staining) in all patients, and we were unable to detect any change in staining after therapy (Fig 3C).
Plasma VEGF Concentrations
Plasma VEGF concentrations did not significantly differ between responders and nonresponders at baseline or 4 weeks VEGF concentrations at baseline were 363 pg/mL ± 180 for responders and 229 pg/mL ± 213 for nonresponders. After 4 weeks of imatinib therapy, VEGF concentrations were 380 pg/mL ± 187 for responders and 294 pg/mL ± 265 for nonresponders.
DISCUSSION
Imatinib, administered 300 mg orally twice daily to patients with AIDS-related KS and receiving HAART, was poorly tolerated. The incidence of diarrhea was higher than in trials with CML patients treated at the same dose. In those trials, 24% of the patients had grade 1/2 diarrhea and none had grade 3/4 diarrhea.23,30 One possible explanation for the increased frequency and severity of the diarrhea is a drug interaction with the medications involved in the HAART regimen. Imatinib is a substrate for the cytochrome p450 isoforms, CYP3A4 and CYP2D6. All of the protease inhibitors approved at the time of the study, which included amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, are CYP3A4 inhibitors, with ritonavir inhibiting CYP2D6 to a lesser extent. Diarrhea is a side effect common to all of the protease inhibitors. The other class of antiretrovirals shown to affect the p450 system is non-nucleoside reverse transcriptase inhibitors. Delavirdine is a CYP3A inhibitor, while nevirapine is a CYP3A inducer, and efavirenz is a mixed CYP3A inducer/inhibitor. No correlation between any specific antiretroviral regimen and the incidence of diarrhea was observed. There was no obvious increase in plasma steady-state concentrations of imatinib or its metabolite. Although these data suggest there was no inhibition of imatinib metabolism, the imatinib concentrations may not be truly representative of the steady-state concentration. Samples were randomly obtained on day 28 and by that time, all patients with diarrhea had developed symptoms. The presence of the diarrhea could have affected the absorption of imatinib as well as patient adherence with the therapy. Unfortunately, plasma concentrations of the antiretrovirals were not measured. It is plausible that the observed toxicities may be due to imatinib inhibition of antiretroviral metabolism because imatinib is a substrate of the CYP3A4 and CYP2D6 pathways. Investigation into the mechanisms of imatinib toxicity is the subject of a future trial.
In this pilot study, imatinib was shown to induce clinical regression in five of 10 patients within 4 weeks of therapy. Patient 3, who had only been on HAART for 4 weeks before beginning therapy, had an increase in his CD4 count after 4 weeks of therapy. This raises the question of whether his response was to imatinib or was a delayed response to HAART. Although we cannot determine the reason for his response from our limited data, it is notable that he had been developing new lesions the week before he started imatinib.
In order to elucidate which signaling pathways may have been critical to imatinib-induced response, we examined tumor biopsies for evidence of decreased phosphorylation (ie, inhibition) of PDGFR and c-kit, as well as their downstream effectors AKT and ERK. Four patients had tumor biopsies that were assessable at baseline and at 4 weeks by immunohistochemistry with phosphospecific antibodies. After 4 weeks of therapy, three of these biopsies demonstrated a decrease of phosphorylation (activation) of the PDGFR and ERK pathways when compared to baseline. The fourth biopsy (patient 10) demonstrated +3 immunoreactivity for phospho-PDGFR and phospho-ERK at baseline and after 4 weeks of therapy. Interestingly, patient 10 had stable disease after 4 weeks on imatinib therapy and had low concentrations on imatinib and its metabolite at the time of biopsy. In contrast, there was minimal or no activation of AKT in pretreatment tumor specimens. Because the phospho-c-kit antibody recognized the phosphorylation of the activation motif on both PDGFR and c-kit, we were unable to determine conclusively the phosphorylation status of c-kit. Post-treatment biopsies demonstrated dramatic reduction in ERK and PDGFR phosphorylation in three patient specimens. These observations suggest that the ERK pathway, in contrast to the AKT pathway, may be critical for proliferation of KS lesions. This is of interest, as signaling from PDGFR in other cell systems is often transmitted through AKT.31
VEGF concentrations were measured in nine patients at baseline and after 4 weeks of therapy. In this study, no difference was seen in VEGF concentrations between responders and nonresponders. Although in vitro evidence suggested that inhibition of the PDGF pathway would decrease VEGF concentrations, our data would suggest that either PDGF regulation of VEGF is not the primary pathway in vivo, or there are parallel pathways that are able to compensate for PDGFR inhibition.
In conclusion, we demonstrated that imatinib may be an effective form of therapy in patients with AIDS-related KS. Unfortunately, imatinib administered at a dose of 600 mg daily was poorly tolerated in some patients. Therefore, a follow-up study planned by the AIDS Malignancy Clinical Trial Consortium is being performed at a dose of 400 mg per day. The reason for the increased toxicity in HIV patients on HAART is unclear and the mechanism of toxicity will be investigated in a follow-up study. This is the first study to demonstrate that inhibition of PDGFR and ERK correlates with regression of KS. Our findings support the need for further evaluation of imatinib as a single agent in patients with KS that is refractory to standard therapies.
Authors' Disclosures of Potential Conflicts of Interest
Acknowledgment
We thank Jerome Groopman for his support during this project.
NOTES
Supported by the AIDS Oncology Clinical Scientist Development Program (H.B.K., grant No. K12CA077846-03).
H.B.K. and G.J.B contributed equally to this work.
Funding for this study was provided by Novartis Pharma AG, Basel, Switzerland.
Presented in part at the International AIDS Malignancy Conference, Bethesda, MD, April 28-29, 2003, and 39th Annual Meeting of the American Society of Clinical Oncology, Chicago, IL, May 31-June 3, 2003.
Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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