Clinical Cancer Research CR Balducci Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 14, 1438, March 1, 2008. doi: 10.1158/1078-0432.CCR-07-4178
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zimmerer, J. M.
Right arrow Articles by Carson, W. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zimmerer, J. M.
Right arrow Articles by Carson, W. E., III

Cancer Therapy: Clinical

IFN-{alpha}-2b–Induced Signal Transduction and Gene Regulation in Patient Peripheral Blood Mononuclear Cells Is Not Enhanced by a Dose Increase from 5 to 10 Megaunits/m2

Jason M. Zimmerer1,2, Amy M. Lehman3, Amy S. Ruppert3, Carl W. Noble2, Thomas Olencki4, Michael J. Walker4, Kari Kendra4 and William E. Carson, III2,4

Authors' Affiliations: 1 Integrated Biological Sciences Graduate Program, 2 Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, 3 Center for Biostatistics, and 4 Department of Surgery, and the Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, Ohio

Requests for reprints: William E. Carson III, Department of Surgery, The Ohio State University, N924 Doan Hall, 410 West 10th Avenue, Columbus, OH 43210. Phone: 614-293-6306; Fax: 614-293-3465; E-mail: William.carson{at}osumc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The precise molecular targets of IFN-{alpha} therapy of melanoma are unknown but likely involve signal transducer and activator of transcription (STAT) 1 signal transduction within host immune effector cells. We hypothesized that intermediate and high doses of IFN-{alpha} would be equally effective in activating patient immune cells.

Experimental Design: Eleven metastatic melanoma patients who were enrolled in a clinical trial of bevacizumab in combination with escalating doses of IFN-{alpha}-2b (5 megaunits/m2 and then 10 megaunits/m2) were included in the study. Peripheral blood mononuclear cells (PBMC) were procured from patient blood just before therapy and again 1 h after each dose of IFN-{alpha}-2b and analyzed for the presence of phosphorylated STAT1, phosphorylated STAT2, and the induction of IFN-stimulated gene (ISG) transcripts.

Results: Phosphorylated STAT1 was significantly greater at the 5 megaunits/m2 dose compared with the 10 megaunits/m2 dose of IFN-{alpha}-2b (P = 0.02). In contrast, no significant difference in phosphorylated STAT2 was observed at a dose of 5 megaunits/m2 compared with 10 megaunits/m2 (P = 0.20). There were also no significant differences in the induction of ISGs within PBMCs between the two doses (P > 0.4 for all ISGs). Suppressor of cytokine signaling 1 and 3 (two inhibitors of IFN-{alpha} signaling) transcripts were significantly higher among patient PBMCs following the 10 megaunits/m2 dose of IFN-{alpha} (P < 0.001).

Conclusion: These results suggest that lower doses of IFN-{alpha}-2b are as effective as higher doses with respect to the induction of Janus-activated kinase-STAT signal transduction and the transcription of ISGs within immune effector cells.

The IFN-{alpha} receptor is widely expressed on both tumor cells and immune effector cells (1, 2). Binding of IFN-{alpha} to its receptor activates Janus-activated kinase 1 (JAK1) and tyrosine kinase 2, which in turn phosphorylate tyrosine residues within the cytoplasmic region of the IFN-{alpha} receptor. These phosphotyrosine residues provide docking sites for signal transducer and activator of transcription (STAT) 1 and STAT2, latent cytoplasmic transcription factors that are phosphorylated by the JAKs (3). The prototypical IFN-{alpha} signaling reaction results in the formation of IFN-stimulated gene (ISG) factor 3, a DNA-binding complex that consists of STAT1{alpha} (or STAT1β) and STAT2, and a chaperon protein known as IFN regulatory factor 9 (4). ISG factor 3 translocates to the nucleus and binds to IFN-stimulated response elements located in the promoter regions of IFN-responsive genes (5). This signaling event induces the expression of several immunoregulatory genes and largely determines the pattern of immune cell activation following exposure to IFN-{alpha} (6–8).

High-dose IFN-{alpha} is used as an adjuvant in patients who have undergone resection of high-risk lesions (nodal disease or primary tumors with Breslow thickness >4 mm; refs. 9–13). IFN-{alpha} is also used to treat individuals with metastatic melanoma and produces clinical responses in ~10% of patients (9, 10, 14). However, it remains difficult to determine the optimal dose of IFN-{alpha} for melanoma patients or devise strategies to enhance its antitumor effects because its cellular targets and mechanism of action are largely unknown. Although IFN-{alpha} can act directly on melanoma cells to inhibit proliferation and up-regulate the expression of MHC class I antigens, its stimulatory properties on effector cells of the immune system are thought to be most important for its antitumor activity (15–22). In fact, recent data have shown that the occurrence of autoimmune sequelae and the presence of tumor-infiltrating lymphocytes correlate with clinical response in patients receiving IFN-{alpha} (23, 24). Our previous analysis of IFN-{alpha}–induced JAK-STAT signal transduction in immune cells revealed reduced phosphorylation of these proteins at higher doses of IFN-{alpha} (25). We therefore hypothesized that intermediate and high doses of IFN-{alpha} might be equally effective in activating STAT proteins and inducing the expression of ISGs in patient peripheral blood mononuclear cells (PBMC).

To test this hypothesis, we analyzed JAK-STAT–mediated signal transduction and gene regulation in patients who received escalating doses of IFN-{alpha}-2b in the setting of metastatic disease. The phosphorylation of STAT1 and STAT2 was measured by intracellular flow cytometry, and the induction of ISG transcripts was evaluated by real-time PCR and microarray analysis. These studies indicated that the PBMC response to 5 megaunits/m2 IFN-{alpha} was comparable with that of 10 megaunits/m2 in the context of the present trial. Interpretation of these results must be tempered by the observation that the reduced response to the higher dose of IFN-{alpha} could be a time-dependent effect rather than a dose-dependent effect.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patient blood samples. Samples were obtained at The Ohio State University following informed consent under an Institutional Review Board–approved protocol (OSU 99H0348). Patients on this National Cancer Institute–sponsored trial received 15 mg/kg bevacizumab (anti–vascular endothelial growth factor monoclonal antibody) every 2 weeks plus high-dose IFN-{alpha}-2b thrice weekly via the s.c. route. IFN therapy was initiated at 5 megaunits/m2. If this dose was well tolerated for 2 weeks, the patient underwent a dose escalation to 10 megaunits/m2. Peripheral blood was obtained from melanoma patients (five females and six males) immediately before and 1 h following the administration of the first dose of IFN-{alpha}-2b at 5 or 10 megaunits/m2. The characteristics of the patients on this study are listed in Table 1 . PBMCs were isolated from blood (8 mL) via centrifugation with Ficoll-Paque Plus (Amersham Pharmacia Biotech) and used immediately in the assays.


View this table:
[in this window]
[in a new window]

  		Table 1. Melanoma patients' characteristics

 
Flow cytometric analysis of STAT1 and STAT2. The native and phosphorylated forms of STAT1 (Tyr701) and STAT2 (Tyr690) were measured by intracellular flow cytometry as previously described, with modifications (25, 26). Rabbit anti-human primary antibodies (Cell Signaling Technology) were used in combination with a goat anti-rabbit Alexa Fluor 488–conjugated secondary antibody (Molecular Probes).

Real-time PCR. Following Trizol extraction and RNeasy purification, 2 µg of total RNA were reverse transcribed. The resulting cDNA was used as a template to measure gene expression by real-time PCR using predesigned primer/probe sets (Assays-On-Demand, Applied Biosystems) as previously described (27). Human β-actin was used as an internal control in each reaction well (Applied Biosystems). Real-time PCRs were done in triplicate and analyzed using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems).

cRNA preparation and array hybridization. U133 Plus 2.0 Arrays (Affymetrix), which query ~47,000 human transcripts, were used in these analyses. The cRNA was synthesized as suggested by Affymetrix. Briefly, double-stranded cDNA was generated from 8 µg of total RNA using the SuperScript Choice System according to the manufacturer's instructions (Invitrogen). Biotinylated cRNA was generated by in vitro transcription using the BioArray High Yield RNA Transcript Labeling System (Enzo Life Sciences, Inc.). The cRNA was purified, fragmented, and hybridized to the arrays (28).

Microarray data analysis. Raw data were collected with a confocal laser scanner (Hewlett-Packard) and probe level data were analyzed using dChip software (29). Quantile normalization was done, and only perfect match probes were used in computing the model-based expression indices (MBEI). "Array outliers" identified by dChip at the probe set level were set to missing. The log2(MBEIs) were then calculated and used for further analysis.

An ANOVA model with random effects to account for repeated measures across patients was used to determine if there was evidence of a linear or nonlinear IFN-{alpha} dose effect on gene expression values. These tests were done for 143 preselected probe sets (representing 64 IFN-responsive genes; see Supplementary Table S1) identified based on preliminary studies from our group as well as the previous work of other groups (30, 31). Paired t tests were used to test for a difference in expression between the 5 and 10 megaunits/m2 dose levels. Type I error was protected by adjusting P values using Holm's method and two-sided significance levels were set at {alpha} = 0.10.

Statistical analysis. For Fig. 1 , differences in phosphorylated STAT1 (P-STAT1) and phosphorylated STAT2 (P-STAT2) levels between doses were assessed using a random-effects ANOVA model to allow for correlations between replicate observations from the same patient. For Figs. 2 and 3 , respectively, the STAT activation efficiencies (determined by calculating the ratio of activated versus native forms of STAT1 and STAT2) and fold changes were log transformed and statistical comparisons between treatments were done using random-effects ANOVA. Treatment differences and 95% confidence intervals were estimated using a two-sided {alpha} = 0.05 level of significance. All analyses were done using Statistical Analysis System v9.1.3 (SAS Institute, Inc.).


Figure 1
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Native and activated forms of STAT1 and STAT2 levels in PBMCs following in vivo dose escalation of IFN-{alpha}-2b. PBMCs from 11 melanoma patients were obtained before and 1 h following administration of IFN-{alpha} (5 megaunits/m2 s.c.) and again 2 wk later after patients had been escalated to a higher dose of IFN-{alpha} (10 megaunits/m2 s.c.). Freshly isolated PBMCs were analyzed for P-STAT1 (Tyr701; A), total STAT1 (B), P-STAT2 (Try690; C), and STAT2 (D) by flow cytometry. Y-axis, mean specific fluorescence (Fsp). Isotype control antibodies were used to determine background staining. Flow cytometric data were derived from at least 10,000 events gated on the lymphocyte populations as determined by light scatter properties.

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Ratios of native and activated forms of STAT1 and STAT2 levels in PBMCs following dose escalation of IFN-{alpha}-2b. To determine the efficiency of STAT activation, the ratio of activated STAT protein to total STAT protein was evaluated 1 h after IFN-{alpha} therapy for STAT1 (A) and STAT2 (B) protein levels.

 

Figure 3
View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Expression of ISGs and SOCS following IFN-{alpha}-2b administration. In parallel to the flow cytometric assays, PBMCs from the 11 patient samples were immediately lysed in Trizol reagent (Invitrogen). RNA was isolated, converted to cDNA, and analyzed by real-time PCR using primers specific for OAS3 (A), G1P2 (B), IFIT1 (C), IFIT2 (D), SOCS1 (E), and SOCS3 (F). Data were expressed as the mean fold increase relative to baseline levels (pretreatment). All real-time PCR data were normalized to the level of β-actin mRNA (housekeeping gene). Columns, mean of triplicate wells; bars, SD.

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Native and activated forms of STAT1 and STAT2 in PBMCs following escalation of IFN-{alpha}-2b dose from 5 to 10 megaunits/m2. We hypothesized that an intermediate dose of IFN-{alpha} would be as effective as a higher dose for the activation of immune cells. To test this hypothesis, PBMCs from 11 metastatic melanoma patients were obtained immediately before treatment, 1 h following the s.c. administration of 5 megaunits/m2 IFN-{alpha}-2b, and 1 h following the administration of 10 megaunits/m2 IFN-{alpha}-2b 2 weeks later. Ten of 11 patients received 6 injections of IFN-{alpha}-2b at a dose of 5 megaunits/m2 before dose escalation (patient no. 10 received 12 injections of 5 megaunits/m2 over 4 weeks). Following isolation from blood, PBMCs were immediately analyzed for the native and activated forms of STAT1 (activated residue Tyr701) and STAT2 (activated residue Tyr690) by flow cytometry (Fig. 1A and B, and C and D, respectively). STAT1 activation (P-STAT1) was significantly higher at the 5 megaunits/m2 dose of IFN-{alpha} compared with the 10 megaunits/m2 dose (P = 0.02; Fig. 1A). Of note, patient no. 7, who exhibited increased activation of STAT1 following administration of IFN-{alpha} at 10 megaunits/m2, experienced psychological effects, grade 3 fatigue, and diarrhea at this dose and was eventually removed from the trial (Table 1). Within individual patients, overall levels of unphosphorylated STAT1 did not seem to diminish greatly following administration of IFN-{alpha} at 10 megaunits/m2 except for patient no. 3 (Fig. 1B). Activation of STAT2 (P-STAT2) was not significantly different between a dose of 5 megaunits/m2 and a subsequent dose of 10 megaunits/m2 (P = 0.20; Fig. 1C). Within individual patients, overall levels of unphosphorylated STAT2 did not seem to diminish greatly following administration of IFN-{alpha} at 10 megaunits/m2, except for patient no. 5 (Fig. 1D). Because of the trial design, it is not known whether the reduced IFN responsiveness (at the level of STAT1 phosphorylation) would have been observed following continued administration of the lower dose of IFN-{alpha} (i.e., the drop in P-STAT1 may have been a time-dependent effect rather than a dose-dependent effect).

Ratio of activated to total STAT1 and STAT2 in PBMCs following escalation of IFN-{alpha}-2b dose from 5 to 10 megaunits/m2. To determine the efficiency of STAT activation in PBMCs following IFN-{alpha} administration, the ratio of activated STAT protein to the total level of the STAT protein was evaluated. This measure is an artificial means of quantifying the ability of the cell to phosphorylate the available STAT protein within the cytoplasm. However, it does not imply a greater activation of STAT1 because the functional effect of a STAT protein is mediated by the binding of its phosphorylated form to specific DNA elements. The estimated ratio of P-STAT1 to total STAT1 at the 10 megaunits/m2 dose of IFN-{alpha} was only 40% of the ratio at the 5 megaunits/m2 dose (95% confidence interval, 21.6-67.5%; P = 0.002; Fig. 2A). Similarly, the ratio of P-STAT2 to total STAT2 at the 10 megaunits/m2 dose of IFN-{alpha} was only 58% of the ratio at the 5 megaunits/m2 dose (95% confidence interval, 32.6-94.8%; P = 0.003, Fig. 2B). These results indicate that the efficiency of STAT1 and STAT2 activation in response to exogenous IFN-{alpha} was significantly higher at the 5 megaunits/m2 compared with the 10 megaunits/m2 dose.

ISG expression following IFN-{alpha}-2b administration. To characterize the induction of ISGs following IFN-{alpha}-2b administration at the two doses, patient PBMCs were analyzed for the induction of specific ISGs by real-time PCR. As with the activation of STAT1 and STAT2, the expression of several well-characterized ISGs (OAS3, G1P2, IFIT1, and IFIT2) was not significantly enhanced following the increase in IFN-{alpha}-2b dose from 5 to 10 megaunits/m2 (P > 0.4 for all genes; Fig. 3A-D).

Suppressor of cytokine signaling gene expression following IFN-{alpha}-2b administration. Investigators have identified a family of proteins known as suppressors of cytokine signaling (SOCS) that negatively regulate JAK-STAT signal transduction (32). Our group has shown that SOCS1 and SOCS3 mediate potent inhibitory effects on IFN-{alpha}–stimulated signal transduction, gene regulation, and antitumor activity in a murine melanoma model (22, 33–36). However, the effect of exogenous IFN-{alpha} on SOCS expression in resting immune cells has yet to be defined in the context of cancer immunotherapy. For both SOCS1 and SOCS3, expression was induced to a greater degree following administration of IFN-{alpha} at 10 megaunits/m2 compared with the 5 megaunits/m2 dose (P < 0.001 for both SOCS1 and SOCS3; Fig. 3E and F). These data suggest that IFN-{alpha} may induce the transcription of genes that negatively regulate the IFN-{alpha} response.

Gene regulation in PBMCs from metastatic melanoma patients receiving increasing doses of IFN-{alpha}. PBMCs from four melanoma patients undergoing immunotherapy with escalating doses of IFN-{alpha}-2b were evaluated by microarray analysis (patient nos. 2, 5, 6, and 7 from above). Sixty-four known IFN-regulated genes represented by 143 probe sets were analyzed (Supplementary Table S1; refs. 30, 31). Of these, 34 genes showed a dose response, with a significant difference in expression after treatment with IFN-{alpha} at 10 megaunits/m2 compared with pretreatment expression (adjusted P < 0.10; Supplementary Table S1). Only 1 of the 34 genes, LGALS3BP (endothelial adhesion molecule), was induced (or suppressed) to a greater extent by IFN-{alpha} at 10 megaunits/m2 compared with 5 megaunits/m2 (1.46- and 0.99-fold inductions, respectively; adjusted P = 0.10; Table 2 , group 1; ref. 37). Of the remaining 33 genes, 13 were similarly up-regulated or down-regulated by IFN-{alpha} at the 5 and 10 megaunits/m2 dose levels. More specifically, a test for a nonlinear dose response showed that these 13 genes were more similarly up-regulated or down-regulated than expected by the two doses of IFN-{alpha} (adjusted P < 0.10; Table 2, group 2). These results were validated by real-time PCR for several genes and corresponded well with the microarray results in that the increase in levels of IFI44, OAS1, and MX1 transcript were similar following both doses of IFN-{alpha}. Likewise, LGASL3BP transcript was induced to a greater extent by the higher dose of IFN-{alpha} (Fig. 4A-D ).


View this table:
[in this window]
[in a new window]

  		Table 2. IFN-{alpha}-2b dose response

 

Figure 4
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Real-time PCR validation of microarray data from PBMCs of patients receiving escalating doses of IFN-{alpha}. The expression of representative genes was validated by real-time PCR analysis of IFI44, OAS1, MX1 (expression values at 5 and 10 megaunits/m2 were more similar than expected), and LGALS3BP (expression was significantly higher at 10 megaunits/m2 than at 5 megaunits/m2). Data were expressed as the mean fold increase relative to baseline levels (pretreatment). All real-time PCR data were normalized to the level of β-actin mRNA (housekeeping gene). Columns, mean of triplicate wells; bars, SD.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that IFN-{alpha}–induced signal transduction and gene transcription in patient PBMCs in response to IFN-{alpha} at 5 megaunits/m2 was not enhanced by an increase to a dose of 10 megaunits/m2. Specifically, levels of P-STAT1 and P-STAT2 were not induced to a greater degree with the higher dose of IFN-{alpha}, despite higher cellular levels of STAT1 and STAT2. The higher levels of total STAT1 and STAT2 after the 10 megaunits/m2 dose probably reflect the fact that STAT1 and STAT2 are IFN-inducible genes that can be up-regulated during the course of treatment with IFN-{alpha}. Thus, the higher levels of total STAT1 and STAT2 present in PBMCs at the time of the 10 megaunits/m2 dose may reflect the fact that there was more of each of these proteins in cells before this dose, as a result of their previous IFN exposure. Interestingly, two important inhibitors of IFN-{alpha} signaling, SOCS1 and SOCS3, were induced more effectively by higher doses of IFN-{alpha}.

It is important to note the order in which the two doses of IFN-{alpha} were administered. If IFN-{alpha} were first administered at a dose of 10 megaunits/m2 followed later by the 5 megaunits/m2 dose, it is possible that superior results would have been obtained for the higher dosage of IFN-{alpha}. However, the dose escalation schema used in this study is more likely to be tolerated by patients and is therefore of greater clinical utility. Because of the trial design, it is not known whether the reduced IFN responsiveness (at the level of STAT1 phosphorylation) would have been observed following continued administration of the lower dose of IFN-{alpha} (i.e., the drop in P-STAT1 was a time-dependent effect rather than a dose-dependent effect). That being stated, we currently have data from two patients that received the same 10 megaunits/m2 dose of IFN-{alpha} over a time period of 6 weeks. P-STAT1 levels were induced to a similar degree following each dose of cytokine in both patients (data not shown). Additionally, we evaluated a patient who underwent dose escalation (from 5 to 10 megaunits/m2) and then required a dose reduction back to 5 megaunits/m2. This patient exhibited lower levels of P-STAT1 following the dose escalation. However, STAT1 activation returned to previous levels following the next dose reduction (data not shown). These data are not conclusive, but these results suggest that the observed drop in the generation of P-STAT1 following the IFN-{alpha} dose escalations may not have been a time-dependent event. The use of IFN-{alpha} in patients with metastatic disease (as opposed to patients with no evidence of disease) and the concurrent administration of bevacizumab may also have influenced our results.

Although melanoma cells routinely express functional IFN-{alpha} receptors and exhibit reduced proliferation and/or increased apoptosis in response to this cytokine in vitro (33, 38–41), we have found that tumor expression of STAT proteins did not correlate with the effectiveness of adjuvant IFN-{alpha} (21). This prompted us to investigate if IFN-{alpha} was acting primarily as an immune activator. Using STAT1-deficient mice and STAT1-deficient melanoma tumor cells, our group showed that the antitumor effects of IFN-{alpha} are dependent on STAT1 signaling within immune cells and not the cancer cell (42, 43). In lieu of a panel of ISGs that directly correlates with the antitumor effects of IFN-{alpha}, we have used the activation of JAK-STAT signaling intermediates as a marker of the ability of IFN-{alpha} to stimulate immune effector cells. We acknowledge that this approach relies on several assumptions (e.g., that activated STAT1 and STAT2 are the prime mediators of the antitumor activity of IFN and that induction of P-STAT1 correlates well with the induction of key ISGs); however, we believe that this technique is suitable for monitoring the effects of IFN-{alpha} on the immune system.

Our analyses showed that IFN-{alpha}–induced intracellular signaling and gene regulation within patient PBMCs was higher for the initial lower dose of IFN-{alpha} (5 megaunits/m2). However, a few patients deviated from this pattern with higher STAT activation and/or ISG transcription at 10 megaunits/m2 IFN-{alpha}. Our previous work showed that maximal activation of STAT proteins may occur at different IFN-{alpha} doses in different patients (25). Furthermore, it has previously been shown by microarray analysis that IFN-responsive genes exhibit the greatest degree of interindividual variation in gene expression within unstimulated PBMCs of healthy donors (44). The authors of these studies acknowledged that this might be attributable to subclinical or recent infection in some donors; however, the existence of similar variability for ISG transcripts was later documented in other studies (45). The existence of interpatient variation in the response to IFN-{alpha} suggests that a careful evaluation of the immune response to IFN-{alpha} is necessary to identify what might be termed an optimal dose.

IFN-{alpha}-2b is the only Food and Drug Administration–approved adjuvant therapy for melanoma patients who have undergone successful surgery for high-risk lesions. There is no alternative therapy that has shown efficacy in randomized trials. Therefore, IFN-{alpha} will continue as the standard of care for the foreseeable future. It is well documented that a year of adjuvant high-dose IFN-{alpha} at 10 megaunits/m2 leads to a significant increase in relapse-free survival for melanoma patients. Importantly, trials of very low-dose IFN-{alpha} (i.e., 1 megaunit/m2) have not shown significant efficacy in the adjuvant setting (46). Thus, if it were not for the attendant side effects of high-dose IFN-{alpha}, it is likely that this treatment regimen would be widely used in high-risk patients. The results of the present study suggest that administration of IFN-{alpha} at doses below 10 megaunits/m2 may still produce significant immune stimulation.

We have shown that increasing doses of IFN-{alpha} did not enhanced signal transduction and gene stimulation in melanoma patient immune cells. These results suggest that peak activation of immune cells by IFN-{alpha} may occur at doses below 10 megaunits/m2. Individualized optimization of IFN-{alpha} dosing in the setting of melanoma is currently under investigation.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 9/12/07; revised 11/26/07; accepted 11/27/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Navarro S, Colamonici OR, Llombart-Bosch A. Immunohistochemical detection of the type I interferon receptor in human fetal, adult, and neoplastic tissues. Mod Pathol 1996;9:150–6.[Medline]
  2. von Stamm U, Brocker EB, von Depka Prondzinski M, et al. Effects of systemic interferon-{alpha} (IFN-{alpha}) on the antigenic phenotype of melanoma metastases. EORTC Melanoma Group Cooperative Study No. 18852. Melanoma Res 1993;3:173–80.[Medline]
  3. Haque SJ, Williams BR. Signal transduction in the interferon system. Semin Oncol 1998;25:14–22.[Medline]
  4. Darnell JE, Jr., Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264:1415–21.[Abstract/Free Full Text]
  5. Levy D, Reich N, Kessler D, Pine R, Darnell JE, Jr. Transcriptional regulation of interferon-stimulated genes: a DNA response element and induced proteins that recognize it. Cold Spring Harb Symp Quant Biol 1988;53 Pt 2:799–802.[Abstract/Free Full Text]
  6. Meraro D, Gleit-Kielmanowicz M, Hauser H, Levi BZ. IFN-stimulated gene 15 is synergistically activated through interactions between the myelocyte/lymphocyte-specific transcription factors, PU.1, IFN regulatory factor-8/IFN consensus sequence binding protein, and IFN regulatory factor-4: characterization of a new subtype of IFN-stimulated response element. J Immunol 2002;168:6224–31.[Abstract/Free Full Text]
  7. Hatina VJ, Kralova J, Jansa P. Identification of an intragenic interferon-stimulated response element sequence of the mouse class I major histocompatibility complex H-2Kb gene. Exp Clin Immunogenet 1996;13:55–60.[Medline]
  8. Ohmori Y, Hamilton TA. The interferon-stimulated response element and a {kappa}B site mediate synergistic induction of murine IP-10 gene transcription by IFN-{gamma} and TNF-{alpha}. J Immunol 1995;154:5235–44.[Abstract]
  9. Kirkwood JM, Strawderman MH, Ernstoff MS, et al. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group Trial EST 1684. J Clin Oncol 1996;14:7–17.[Abstract]
  10. Lens MB, Dawes M. Interferon alfa therapy for malignant melanoma: a systematic review of randomized controlled trials. J Clin Oncol 2002;20:1818–25.[Abstract/Free Full Text]
  11. Kirkwood JM, Ibrahim JG, Sondak VK, et al. High- and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. J Clin Oncol 2000;18:2444–58.[Abstract/Free Full Text]
  12. Creagan ET, Dalton RJ, Ahmann DL, et al. Randomized, surgical adjuvant clinical trial of recombinant interferon alfa-2a in selected patients with malignant melanoma. J Clin Oncol 1995;13:2776–83.[Abstract]
  13. Kirkwood JM, Ibrahim JG, Sosman JA, et al. High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801. J Clin Oncol 2001;19:2370–80.[Abstract/Free Full Text]
  14. Legha SS. Interferons in the treatment of malignant melanoma. A review of recent trials. Cancer 1986;57:1675–7.[CrossRef][Medline]
  15. Belardelli F, Ferrantini M, Proietti E, Kirkwood JM. Interferon-{alpha} in tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002;13:119–34.[CrossRef][Medline]
  16. Belardelli F, Gresser I, Maury C, Maunoury MT. Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. I. Int J Cancer 1982;30:813–20.[Medline]
  17. Belardelli F, Gresser I, Maury C, Maunoury MT. Antitumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. II. Role of host mechanisms. Int J Cancer 1982;30:821–5.[CrossRef][Medline]
  18. Gresser I, Maury C, Carnaud C, et al. Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend erythroleukemia cells. VIII. Role of the immune system in the inhibition of visceral metastases. Int J Cancer 1990;46:468–74.[Medline]
  19. Gresser I, Kaido T, Maury C, et al. Interaction of IFN {alpha}/β with host cells essential to the early inhibition of Friend erythroleukemia visceral metastases in mice. Int J Cancer 1994;57:604–11.[Medline]
  20. Fallarino F, Gajewski TF. Cutting edge: differentiation of antitumor CTL in vivo requires host expression of Stat1. J Immunol 1999;163:4109–13.[Abstract/Free Full Text]
  21. Lesinski GB, Valentino D, Hade EM, et al. Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-{alpha} adjuvant therapy. Cancer Immunol Immunother 2005;54:815–25.[CrossRef][Medline]
  22. Zimmerer JM, Lesinski GB, Kondadasula SV, et al. IFN-{alpha}-induced signal transduction, gene expression, and antitumor activity of immune effector cells are negatively regulated by suppressor of cytokine signaling proteins. J Immunol 2007;178:4832–45.[Abstract/Free Full Text]
  23. Koon H, Atkins M. Autoimmunity and immunotherapy for cancer. N Engl J Med 2006;354:758–60.[Free Full Text]
  24. Moschos SJ, Edington HD, Land SR, et al. Neoadjuvant treatment of regional stage IIIB melanoma with high-dose interferon alfa-2b induces objective tumor regression in association with modulation of tumor infiltrating host cellular immune responses. J Clin Oncol 2006;24:3164–71.[Abstract/Free Full Text]
  25. Lesinski GB, Kondadasula SV, Crespin T, et al. Multiparametric flow cytometric analysis of inter-patient variation in STAT1 phosphorylation following interferon alfa immunotherapy. J Natl Cancer Inst 2004;96:1331–42.[Abstract/Free Full Text]
  26. Fleisher TA, Dorman SE, Anderson JA, et al. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 1999;90:425–30.[CrossRef][Medline]
  27. Tokuhiro S, Yamada R, Chang X, et al. An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transporter, is associated with rheumatoid arthritis. Nat Genet 2003;35:341–8.[CrossRef][Medline]
  28. Moschella F, Bisikirska B, Maffei A, et al. Gene expression profiling and functional activity of human dendritic cells induced with IFN-{alpha}-2b: implications for cancer immunotherapy. Clin Cancer Res 2003;9:2022–31.[Abstract/Free Full Text]
  29. Li C, Wong WH. DNA-Chip Analyzer (dChip). In: Parmigiani G, Garrett ES, Irizarry R, and Zeger SL, editors. The analysis of gene expression data: methods and software. New York: Springer-Verlag; 2003.
  30. Ji X, Cheung R, Cooper S, et al. Interferon alfa regulated gene expression in patients initiating interferon treatment for chronic hepatitis C. Hepatology 2003;37:610–21.[CrossRef][Medline]
  31. Zimmerer JM, Lesinski GB, Kornacker K, et al. Gene expression profiling of the response to interferon-{alpha} immunotherapy [abstract 4679]. American Association for Cancer Research Annual Meeting, 2003, Orlando, Florida.
  32. Alexander WS. Suppressors of cytokine signalling (SOCS) in the immune system. Nat Rev Immunol 2002;2:410–6.[Medline]
  33. Brender C, Nielsen M, Kaltoft K, et al. STAT3-mediated constitutive expression of SOCS-3 in cutaneous T-cell lymphoma. Blood 2001;97:1056–62.[Abstract/Free Full Text]
  34. Sakai I, Takeuchi K, Yamauchi H, Narumi H, Fujita S. Constitutive expression of SOCS3 confers resistance to IFN-{alpha} in chronic myelogenous leukemia cells. Blood 2002;100:2926–31.[Abstract/Free Full Text]
  35. Song MM, Shuai K. The suppressor of cytokine signaling (SOCS) 1 and SOCS3 but not SOCS2 proteins inhibit interferon-mediated antiviral and antiproliferative activities. J Biol Chem 1998;273:35056–62.[Abstract/Free Full Text]
  36. Brender C, Nielsen M, Ropke C, et al. Interferon-{alpha} induces transient suppressors of cytokine signalling expression in human T cells. Exp Clin Immunogenet 2001;18:80–5.[CrossRef][Medline]
  37. Nieminen J, St-Pierre C, Sato S. Galectin-3 interacts with naive and primed neutrophils, inducing innate immune responses. J Leukoc Biol 2005;78:1127–35.[Abstract/Free Full Text]
  38. Baker PK, Pettitt AR, Slupsky JR, et al. Response of hairy cells to IFN-{alpha} involves induction of apoptosis through autocrine TNF-{alpha} and protection by adhesion. Blood 2002;100:647–53.[Abstract/Free Full Text]
  39. Chin YE, Kitagawa M, Su WC, et al. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. Science 1996;272:719–22.[Abstract]
  40. Selleri C, Sato T, Del Vecchio L, et al. Involvement of Fas-mediated apoptosis in the inhibitory effects of interferon-{alpha} in chronic myelogenous leukemia. Blood 1997;89:957–64.[Abstract/Free Full Text]
  41. Zhou Y, Wang S, Yue BG, Gobl A, Oberg K. Effects of interferon {alpha} on the expression of p21cip1/waf1 and cell cycle distribution in carcinoid tumors. Cancer Invest 2002;20:348–56.[CrossRef][Medline]
  42. Lesinski GB, Anghelina M, Zimmerer J, et al. The antitumor effects of IFN-{alpha} are abrogated in a STAT1-deficient mouse. J Clin Invest 2003;112:170–80.[CrossRef][Medline]
  43. Badgwell B, Lesinski GB, Magro C, et al. The antitumor effects of interferon-{alpha} are maintained in mice challenged with a STAT1-deficient murine melanoma cell line. J Surg Res 2004;116:129–36.[CrossRef][Medline]
  44. Whitney AR, Diehn M, Popper SJ, et al. Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci U S A 2003;100:1896–901.[Abstract/Free Full Text]
  45. Boldrick JC, Alizadeh AA, Diehn M, et al. Stereotyped and specific gene expression programs in human innate immune responses to bacteria. Proc Natl Acad Sci U S A 2002;99:972–7.[Abstract/Free Full Text]
  46. Schuchter LM. Adjuvant interferon therapy for melanoma: high-dose, low-dose, no dose, which dose? J Clin Oncol 2004;22:7–10.[Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zimmerer, J. M.
Right arrow Articles by Carson, W. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zimmerer, J. M.
Right arrow Articles by Carson, W. E., III

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online