Originally published as JCO Early Release 10.1200/JCO.2005.12.147 on August 1 2005

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Correale, P. Articles by Francini, G. Search for Related Content PubMed PubMed Citation Articles by Correale, P. Articles by Francini, G. Social Bookmarking                            What's this?

Chemo-Immunotherapy of Metastatic Colorectal Carcinoma With Gemcitabine Plus FOLFOX 4 Followed by Subcutaneous Granulocyte Macrophage Colony-Stimulating Factor and Interleukin-2 Induces Strong Immunologic and Antitumor Activity in Metastatic Colon Cancer Patients

Pierpaolo Correale, Maria Grazia Cusi, Kwong Yok Tsang, Maria Teresa Del Vecchio, Stefania Marsili, Marco La Placa, Chiara Intrivici, Angelo Aquino, Lucia Micheli, Cristina Nencini, Francesco Ferrari, Giorgio Giorgi, Enzo Bonmassar, Guido Francini

From the Section of Medical Oncology; Section of Pathology, Department of Human Pathology and Oncology; Section of Virology, Department of Molecular Biology; Giorgio Segre Department of Pharmacology; Department of Imaging and Radiology; Interdepartmental Oncopharmacological Centre, Siena University School of Medicine, Siena; Medical Oncology and Pharmacology Section, Department of Neuroscience, Tor Vergata University, Rome, Italy; and the Experimental Oncology Section, Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, MD

Address reprint requests to Prof. Guido Francini, Director of the Oncology Section, Department of Human Pathology and Oncology, Siena University School of Medicine, Viale Bracci 11, 53100 Siena, Italy; e-mail: francini{at}unisi.it.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Tumor cell killing by anticancer drugs may be supported by their immuno- and pharmacologic effects. Chemotherapy is in fact able to (A) upregulate tumor-associated antigen expression, including carcinoembryonic antigen (CEA) or other target molecules such as thymidylate synthase (TS); and (B) downregulate tumor cell resistance to the death signals induced by tumor antigen–specific cytotoxic T lymphocytes. This provides the rationale for combining chemo- and immunotherapy.

MATERIALS AND METHODS: We describe the results of a translational phase II trial designed to evaluate the toxicity, antitumor activity and immunologic effects of gemcitabine + FOLFOX-4 (oxaliplatin, fluorouracil, and folinic acid) polychemotherapy followed by the subcutaneous administration of granulocyte macrophage colony-stimulating factor and low-dose interleukin-2 in colorectal carcinoma patients. The study involved 29 patients (16 males and 13 females with a mean age of 69 years), 21 of whom had received a previous line of treatment, and 19 had liver involvement.

RESULTS: The treatment was well tolerated and induced very high objective response (68.9%) and disease control rates (96.5%), with an average time to progression of 12.5 months. An immunologic study of peripheral blood mononuclear cells (PBMCs) taken from 20 patients showed an enhanced proliferative response to colon carcinoma antigen and a significant reduction in suppressive regulatory T lymphocytes (CD4⁺CD25_T-reg⁺). A cytofluorimetric study of the PBMCs of five HLA-A()02.01⁺ patients who achieved an objective response showed an increased frequency of cytolytic T lymphocyte precursors specific for known CEA- and TS-derived epitopes.

CONCLUSION: The results show that our regimen has strong immunologic and antitumor activity in colorectal cancer patients and deserves to be investigated in phase III trials.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Colorectal carcinoma is the second leading cause of cancer deaths; almost 50% of the patients die because of problems related to disease progression.¹

Over the last few years, higher response rates have been achieved using the latest poly-chemotherapy regimens combining fluorouracil (FU) ± levofolinic acid (LF) with irinotecan (FOLFIRI) or oxaliplatin (FOLFOX), alone or together with monoclonal antibodies such as bevacizumab or cetuximab.^2-6 However, patients with metastatic colorectal cancer still have a poor prognosis, with an average overall survival of 20 months.^2-6

Recent progress in human immunobiotechnology has opened up new perspectives in cancer treatment: active specific immunotherapy or vaccine therapy has become a new operative treatment modality that is under large-scale investigation worldwide also for colorectal cancer.⁷ Specific immunization against a target antigen has been achieved in some patients with a number of different anticancer vaccines,^8-16 but they have been unsuccessful so far in controlling cancer progression for various reasons.^9,16,17 In order to circumvent some of these difficulties, attempts have been made to combine cancer vaccines with biologic agents or cytotoxic drugs, and to test new immunization strategies.^18-22

In this context, we have previously shown that a novel, highly cytotoxic and pro-apoptotic multidrug GOLF regimen (gemcitabine [GEM] plus oxaliplatin, LF, and FU) can induce molecular and structural changes in human colon cancer cell lines. These antigenic changes make malignant cells capable of priming an efficient multi-antigenic cytotoxic T-cell (CTL) response with anti tumor activity. We have generated different CTL lines from HLA-A()02.01⁺ donors (normal or colon cancer patients) by stimulating their peripheral blood mononuclear cells (PBMCs) in vitro with low-dose interleukin (IL) -2 and autologous dendritic cells (DCs) loaded with the mixed cell lysate of two colon cancer cell lines (WiDr and HT-29) previously exposed to different multidrug treatments including FOLFOX and GOLF. We found that the CTL lines generated using GOLF-treated colon cancer cells (GOLF-CTL lines) were strongly and specifically cytotoxic against class I HLA-matching colon cancer cells in vitro, whereas those generated using the lysate of colon cancer cells (untreated or exposed individually to each of the four drugs in the combination) were much less efficient in killing the same targets. The GOLF-CTL lines also showed a greater frequency of CTL precursors recognizing carcinoembryonic antigen (CEA) and thymidylate synthase (TS) epitope peptides, and were capable of specifically killing class I HLA-matching target cells (CIR-A2 cells) transfected with the CEA or TS gene (manuscript submitted for publication).²³

On the basis of these findings, we planned a phase II trial involving advanced colorectal carcinoma patients with the aim of testing a novel chemo-immunotherapy regimen based on the sequential administration of the highly effective GOLF polychemotherapy regimen (which is able to induce cancer cell apoptosis and antigen remodeling and release) and a cytokine-based immunotherapy regimen using granulocyte macrophage colony-stimulating factor (GM-CSF; ie, to activate endogenous DCs)²⁴ and IL-2 (to act as a T-cell growth factor) in order to expand antigen-presenting cell–induced, antigen-specific CTL clones.²⁵ The rationale of the study is based on the following considerations: (A) chemotherapy induces apoptosis and antigen remodeling; (B) GM-CSF increases the percentage and activation of peripheral blood DCs capable of taking up, processing and presenting antigens released by chemotherapy-treated tumor cells to the effector lymphocyte precursors; (C) IL-2 sustains the immune-response by promoting the proliferation and clonal expansion of the precursors; and (D) activated antigen-specific CTLs destroy tumor cells surviving chemotherapy.

This clinical study was designed on the basis of the results of two previous phase Ib-II trials showing (A) the significant anti tumor activity of the GOLF regimen in colorectal carcinoma patients; and (B) the low level of toxicity of both the GOLF and the IG-1 regimen (a sequential combination of subcutaneous [sc] GM-CSF and low-dose IL-2).^26,27 The immunobiologic investigation accompanying the IG-1 study showed that the regimen increased the percentage of peripheral blood DCs (from 0.25% to 20%) and their antigen presenting ability. The regimen also showed the ability to increase the absolute number of lymphocytes with the induction of a Th1 cytotoxic phenotype.²⁷ These findings allowed us to design a treatment schedule combining the two approaches in a single chemo-immunotherapy regimen (GOLFIG-1).

The aim of this phase II trial was to investigate the anti tumor activity, toxicity and immunologic effects of GOLFIG-1 in patients with advanced colorectal carcinoma.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Study Design and Patient Characteristics
The inclusion criteria were a histologic diagnosis of colorectal carcinoma, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2, a life expectancy of more than 3 months, normal renal and hepatic function, a WBC of more than 2,500/mm³, hemoglobin more than 9 g/dl, a platelet cell count of more than 100,000/mm³, and normal cardiac function. The exclusion criteria were any major organ failure, CNS involvement, second tumors, active infectious disease, major autoimmune diseases (lupus erythematosous, reumatoid arthritis, sclerodermia, any systemic vasculitis), or significant immunosuppression due to AIDS or medical treatment with major immunosuppressive agents (such as cyslosporinee for organ transplantation).

The characteristics of the 29 patients enrolled between October 2002 and July 2004 are shown in Table 1. The study was authorized by the University Committee and the Italian Ministry of Health, and all of the patients gave their written informed consent. The patients received GEM 1 g/m² in a 30-minute intravenous (IV) infusion on day 1 before any other drug. They subsequently, received LF 100 mg/m² in a 30-minute IV infusion on days 1 and 2; FU 400 mg/m² in a bolus injection followed by a 22-hour continuous infusion (800 mg/m²) on days 1 and 2; and oxaliplatin 85 mg/m² in a 4- to 6-hour IV infusion before the second administration of LF and FU (FUFA) on day 2. The treatment was repeated every 15 days. GM-CSF 150 µg (Molgramostim, Schering-Plough Corp, Milan, Italy; or Sargramostim, Immunex Corp, Berlex Laboratories, Richmond, VA) was administered SC from day 3 to day 6 of the first, third, fifth, seventh, ninth and eleventh cycles. IL-2 (Aldesleukin, Chiron Corp, Emeryville, CA) 0.5 MIU SC was administered twice daily from day 7 to day 14 of the first, second, third, fifth, seventh, ninth and eleventh cycles, and from day 3 to day 14 of the second, fourth, sixth, eighth, tenth and twelfth cycles.

GM-CSF was administered during alternating cycles because our previous study showed that the activity of IG-1 (the GM-CSF/IL-2 schedule) on DCs and lymphocytes could be maintained for nearly 30 days, and we had no information concerning the toxicologic activity of GM-CSF used in a different schedule.

Standard assessments (clinical history, physical examination, hematochemical analysis, CEA and CA19.9 assays, chest x-ray and ECHO scans) were made at baseline and repeated every 4 to 6 weeks. High-definition, multi slice computed tomography scans with contrast medium were recorded every three months; only selected cases underwent positron emission tomography. All of the patients were evaluated for survival and toxicity. Response and toxicity were assessed according to the standard WHO criteria (1979).

Immunologic Studies
Peripheral blood samples for immunologic study were drawn from all of the patients at baseline and at the end of each cycle. PBMCs were obtained by means of Ficoll-Hypaque gradient separation,²⁸ and the serum samples were prepared by means of simple centrifugation; these samples were immediately frozen at –80°C until their final examination.

Proliferation Assay
The PBMCs (10⁴ cells x 100 µL/well) were seeded in 96-well flat-bottomed sterile plates (BD Biosciences, Erembodegem-Dorp, Belgium) and incubated for 72 hours with (A) complete AIM-V culture medium containing 5% human AB serum (CTR); (B) 10 µg/mL influenza virosome containing the plasmid backbone (immune-reconstituted influenza virosomes [IRIV]); (C) 10 µg/mL influenza virosome including the TS gene (TS/IRIV); (D) 10 µg/mL influenza virosome including the CEA gene (CEA/IRIV); (E) WiDr cell lysate (colon cancer cell lines); (F) LNCaP cell lysate (a prostate carcinoma cell line); (G) allogenic PBMC lysate (data not shown); or (H) 25 µg/mL phytohemoagglutinine. ³H-thymidine (1 µCi/well) was subsequently added to each well and incubated for a further 24 hours. The cells and supernatants were then harvested through a cover-glass filter (Whatman, Cincinnati, OH), which was examined for beta particle emissions. The experiment was performed in triplicate and the results expressed as a proliferation index (experimental point/CTR).

Flow Cytometry
Standard single- and double-color flow cytometric analyses were used, as previously described.^27,29

Precursor frequency and epitope peptide–specific T-cell receptor expression per cell were evaluated using the Cytofluorimetric dimer assay.³⁰ The kit and reagents were purchased from BD Biosciences (Eremboregem-Dorp, Belgium) and the tests were performed in accordance with the manufacturer's instructions. The CEA peptides [CAP-1 (YLSGANLNL), (CEAP)-1 (IQNDTGFYT), and (CEAP)-2 (LLSVTRNDV)] and TS peptides [TS-1 (AVSEHQLLH), TS-2 (FLHHLIAEIH) and TS-3 (TSTTSLELD)] were synthesized and characterized as previously described.^8,20,21

Statistical Considerations
The study was designed to test the hypothesis that the GOLFIG-1 regimen was an active treatment for patients with advanced colorectal carcinoma. A minimum of 25 patients was required in order to maintain alpha and beta errors of 5% and 20%, respectively. Because the response rate of colorectal carcinoma patients to second line-treatment is generally less than 25%, the study was to be stopped if an objective response was not observed in three of the first 14 consecutively enrolled patients.

The between-mean differences in the immunologic results were statistically analyzed using Stat View statistical software (Abacus Concepts, Berkeley, CA). The results were expressed as the mean ± standard deviation of four determinations made in three different experiments, and the differences determined using the Student two-tailed t test for paired samples. A P value < .05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Toxicity
A total of 240 chemo-immunotherapy cycles were administered (a median of nine cycles per patient; range, 6 to 12 cycles). The most frequent adverse events were bone pain, erythema and enduration at the site of the cytokine injection, grade 1 to 2 fever, flu-like syndrome, and grade 1 to 2 hematologic toxicity with moderate anemia, neutropenia, and thrombocytopenia (Table 2). One patient died because of a sudden internal hemorrhage few days after the first treatment cycle. One patient, who experienced grade 4 gastrointestinal toxicity with diarrhea and mucositis during the first cycle, was found to have a significant deficit in dihydro-pyrimidine-dehydrogenase (DPD), an enzyme that is involved in fluoropyrimidine catabolism. DPD deficiency increases the area under the curve of the blood concentration of FU, thus increasing drug toxicity, which often becomes lethal if administered at conventional doses. This patient continued the treatment at a 75%-reduced FU dose and also achieved an objective response. A few other cases of gastroenteric toxicity (diarrhea, mucositis, and vomiting) were reported, and two patients required a 25% reduction in the FU dose, respectively, starting from the fifth and ninth treatment cycles. Seven patients manifested reversible grade 2 peripheral neurotoxicity. Three patients developed hypersensitivity to oxaliplatin, which was, therefore, withdrawn from their treatment regimen starting from the second, ninth, and 11th cycles, respectively.

View larger version (122K): [in this window] [in a new window]   Fig 1. Some of the most significant objective responses obtained using GOLFIG-1 (gemcitabine plus oxaliplatin, LF, and FU plus a sequential combination of subcutaneous granulocyte macrophage colony-stimulating factor and low-dose interleukin-2) chemo-immunotherapy. Baseline abdominal computed tomography scans of a male (patient 10; A and C) with abdominal and liver metastases and a female (patient 22; B and D) with intrapelvic lesions, recorded after six and three treatment cycles, respectively.

The proliferative response of the PBMCs to influenza virus and tumor-associated antigens was monitored by exposing them to various types of IRIV: an IRIV containing the plasmid backbone, the CEA gene plasmid (CEA-IRIV), and the TS gene plasmid (TS-IRIV).

The PBMCs were also incubated with WiDr cell lysate and PHA (the latter used as a positive control). Stimulation with allogenic PBMCs (data not shown) and LNCaP lysates was used as further controls. A moderate proliferative response to IRIV, CEA-IRIV and TS-IRIV, and a dramatic proliferative response to colon carcinoma cell lysate was detected in the PBMCs of patients who had received at least two treatment cycles. On the other hand, very limited proliferation occurred in the baseline and control PBMCs (Fig 2). No significant differences were observed when the control (healthy donor) PBMCs, baseline PBMCs, or post-treatment PBMCs were exposed to PHA, LNCaP, or allogenic PBMC lysates.

View larger version (18K): [in this window] [in a new window]   Fig 2. Antigen-specific-proliferative response of peripheral blood mononuclear cells (PBMCs) from 20 patients enrolled in the trial, isolated at baseline, and after 15 and 30 days of treatment. PBMCs from a healthy donor were used as controls. The results are expressed as a proliferation index. () control; () baseline; () post–day 15; and () post–day 30 PBMCs.

The PBMCs of the patients who had received two treatment cycles showed a doubling in the frequency of the precursors for TS-1, TS-2, and CAP-1 in comparison with their baseline PBMCs, and a two- to six-fold increase in comparison with normal donor PBMCs (Fig 3A). We also observed much greater mean fluorescence intensity in the post-treatment CTL precursors specific for TS-1, TS-2, CEAP-1, CEAP-2, and CAP-1. This finding is consistent with the hypothesis that these CTLs express a larger number of epitope/peptide-specific T-cell receptors on the membrane than the controls (Fig 3B). No change in precursor frequency or T-cell receptor expression was observed for the control peptide.

View larger version (21K): [in this window] [in a new window]   Fig 3. Cytofluorimetric peptide-specific precursors frequency study. Antigen-peptide-specific-frequency of cytolytic T lymphocyte precursors in the peripheral blood mononuclear cells (PBMCs) of five HLA-A()0.2.01+ patients who achieved an objective response. (A) The percentage of CD8/peptide dimer double-positive T cells (peptide-specific precursors). (B) Mean fluorescence intensity (T-cell receptor expression) per cell. () healthy donor; () baseline; and () post–day 30 PBMCs.

We therefore investigated the expression of CD4⁺, CD8⁺, CD4⁺CD25⁺ T_reg, and CD95⁺ (FAS) subsets in the PBMCs of the first 20 enrolled patients, and found that two treatment cycles significantly decreased the CD4⁺CD25⁺ T _reg (from 9.4% ± 4.47% to 3.37% ± 1.92%) and CD95⁺ subsets (from 14% ± 6% to 3.3% ± 2.29%), whose values became similar to those observed in normal individuals (CD4⁺CD25⁺ T_reg, 3.1% ± 2.2%; CD95⁺, 3% ± 1.2%). These events were paralleled by the finding of an increase in the CD4/CD8 T cell ratio (from 1.27 ± 0.25 to 2.1 ± 0.24), which once again became indistinguishable from that observed in normal individuals (2.3 ± 0.3).

Taken together, these findings suggest that GOLFIG-1 chemo-immunotherapy may counter a possible state of immunosuppression.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
A number of different cancer vaccine strategies are currently being evaluated in clinical trials involving patients with various gastrointestinal neoplasms. However, although the preliminary results indicate that they are capable of inducing an effective antigen-specific cellular and humoral response in cancer patients,^9-17 no correlation has been found between successful immunization and clinical outcome.^9-17 The possible reasons for this are related to the fact that a larger tumor burden leads to more general immune anergy and a greater likelihood of generating immunoresistant clones. One of the major problems facing cancer therapy is that the extraordinary adaptability of tumor cells leads to drug- and radioresistance^16,17 as well as to acquired resistance to the effector lymphocytes possibly generated by host vaccination.^16,17 In this context, it must be remembered that common antigens such as MUC-1 or CEA are not critical for tumor cell survival, and so they can be lost under the selective pressure of a vaccine-induced antigen-specific immune response without really damaging tumor development.

However, there are various other mechanisms that may explain how neoplastic cells can avoid being recognized by the CTLs elicited by cancer vaccines.^{7,16,17,37-39} It is widely believed that, like drug- and radioresistance, immunoresistance may depend on the degree of cancer cell heterogeneity and thus on the tumor burden. One possible means of overcoming the adaptive response of tumor cells and the consequent occurrence of antigen-specific immunoresistance is the simultaneous immunization of cancer patients against multiple antigens using irradiated autologous cancer cells^23-40 or tumor cells induced to express inflammatory cytokines and co-accessory molecules by means of genetic engineering,^41,42 viral constructs,⁴³ or heat shock proteins extracted from cancer cells and containing multiple antigen-derived peptides.^44,45 A number of trials have investigated these approaches in colon carcinoma patients; some of them have led to convincing results in terms of immunologic and antitumor activity, especially when the immunologic reagents were tested in an adjuvant setting under conditions of minimal disease.^46-48

Another possible approach to overcome resistance is to reduce the tumor burden by combining immunotherapy with radio- and/or chemotherapy, which could lead to significant debulking and simultaneously affect the phenotype of tumor cells (antigen remodeling), thus making them more susceptible to vaccine-activated effectors. In an attempt to avoid the occurrence of immunoresistance, a number of empirically designed clinical studies of different malignancies have investigated the possibility of combining cytotoxic drugs with biologic agents and/or cytokines (eg, IL-2 and interferon alfa), but the results have been conflicting in terms of clinical response and survival.^49-55

We and other authors have previously described the ability of cytotoxic drugs such as triazenes, FU, VP-16 and CPT-11 to sensitize tumor cells to the cytolytic activity of antigen-specific CTLs.^18-21,56 For example, we have shown that the exposure of various colon and breast carcinoma cell lines to sublethal doses of FU is followed by a significant increase in the expression of CEA²⁰ and TS,²¹ and a consequent immunosensitization to the cytotoxic activity of class-I-HLA-matching CTL lines specific for these antigens. We have also more recently shown that the GOLF regimen is capable of inducing a much greater level of necrosis and apoptosis in the same colon carcinoma cell lines in vitro, while still retaining the FU-induced overexpression of CEA and TS. GOLF treatment allowed the tumor cells to become a more efficient means of generating a strong multi-antigenic CTL response with antitumor activity when used to stimulate human PBMCs in vitro (manuscript submitted for publication),²³ thus providing a possible model to pursue in clinical trials of chemo-immunotherapy in colorectal cancer.

The design of combined chemo-immunotherapy approaches has been criticized on the grounds that chemotherapy is immunosuppressive. This opinion is based on the fact that most cytotoxic drugs can kill granulocyte precursors in bone marrow and thus induce leukopenia, which is associated with the occurrence of bacterial and mycotic infection; however, there is no evidence that cytotoxic chemotherapy may affect an antigen-specific CTL response.

We have found recently that the antigen-specific killing ability of human CTL lines in vitro is not affected by FU, oxaliplatin, or GEM if exposure to these drugs does not occur during the stimulation phase (the time interval during which CTL precursors come into contact with TAA-loaded DCs and start the proliferation; unpublished results),²⁰ which suggests that chemotherapy and immunotherapy should be given sequentially rather than concomitantly. In relation to this, Novak et al have shown that GEM exposure after the inoculum of tumor cells engineered to express viral neuraminidase (NE), improved the efficacy of T-cell response and the tumor rejection rate in a xenograft mouse model and significantly inhibited the humoral response.^57-59 The fact that the same effect was not observed when the tumor cells had been exposed to GEM before the inoculum, that it is improved whether GEM is administered concomitantly with an activating anti-CD40 monoclonal antibody, and that in the responsive mice there was a significant NE specific CTL response, suggests that the drug treatment somehow enhances the cross presentation of potential antigens released by drug-treated cancer cells in vivo.^57-59

In line with these considerations, the results of our clinical study clearly show that the chemo-immunotherapy regimen combining GEM + FOLFOX-4 with the sequential administration of GM-CSF and IL-2 has strong antitumor activity in advanced colorectal cancer patients. This effect is associated with a low level of toxicity. The very high clinical response rate is also associated with the occurrence of immunologic events that strongly resemble those induced by the most sophisticated cancer vaccination techniques.

The toxicity level of GOLFIG-1 was no different from that reported for FOLFOX-4 or, more recently, the GEM + FOLFOX-4 regimen in advanced colon cancer patients.²⁶ The only exception was the higher frequency of fever, malaise and flu-like syndrome associated with cytokine administration, which could be easily controlled by nonsteroidal anti-inflammatory-drugs.

Our clinical results showed a high rates of objective responses and disease control: the median time to progression of 12.5 months was far greater than that reported for any other regimen, including FOLFOX, FOLFIRI, FOLFIRI + bevacizumab or cetuximab,^2-6 and GEM + FOLFOX-4 without cytokines (OR, 41.5%; disease control rate, 75.9%; time to progression, 7 months in patients receiving second- or third-line treatment).²⁶ Furthermore, they take on even more significance if it is remembered that the majority of the patients were receiving second-line treatment, had multiple metastatic sites and, in some cases, an ECOG performance status of 2.

Our regimen is based on a rational combination of cytotoxic drugs and cytokines, and the immunologic results support the hypothesis that the treatment may lead to the generation of an antigen-specific immune reaction capable of sustaining prolonged anti-tumor activity. This is suggested by the fact that the post-treatment PBMCs of five responsive patients showed a significant increase in the proliferative response to colon cancer antigens including CEA and TS, and a 2-3 times increase in the frequency of TS- and CEA-derived epitope peptide-specific CTL precursors. In addition our cytofluorimetric analysis also showed that the cell membranes of these precursors may (at least theoretically) express more antigen epitope–specific T-cell receptors, thus increasing their ability to recognize target cells.

Finally, our results show that the GOLFIG-1 regimen significantly reduced the percentage of PBMCs containing immune-suppressive CD4⁺CD25⁺T_reg,^33-35 and the number of cells expressing the FAS receptor (CD95), and also induced the complete restoration of the CD4/CD8 T-cell ratio, which is often reduced in advanced cancer patients showing a progressively deteriorating immune response.

In conclusion, the results of this study suggest that our combined chemo-immunotherapy regimen has strong immunologic and antitumor activity in colorectal carcinoma patients, and could be an attractive strategy to investigate in future phase III comparative trials.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We thank the technical, medical, and paramedical personnel of the Section of Oncology at the Siena University School of Medicine for their dedication to the patients and their helpful contribution to this study.

	NOTES

 
Supported by a grant from the Italian Ministry of University and Technology (MIUR-2004).

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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
1. Gill S, Thomas RR, Goldberg RM: Review article: Colorectal cancer chemotherapy. Aliment Pharmacol Ther 18:683-692, 2003[CrossRef][Medline]

2. Goldberg RM, Sargent DJ, Morton RF, et al: A randomized controlled trial of fluorouracil plus leucovorin, irinotecan, and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer. J Clin Oncol 22:23-30, 2004[Abstract/Free Full Text]

3. Medinger M, Steinbild S, Mross K: Adjuvant and palliative anticancer treatment of colon carcinoma. Schweiz Rundsch Med Prax 93:1633-1644, 2004[Medline]

4. Kohne CH: Palliative therapy of colorectal cancer. Onkologie 7:41-47, 2003 (suppl)

5. Hurwitz H, Fehrenbacher L, Novotny W, et al: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350:2335-2342, 2004

6. Tournigand C, Andre T, Achille E, et al: FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: A randomized GERCOR study. J Clin Oncol 22:229-237, 2004[Abstract/Free Full Text]

7. Wang RF, Rosenberg SA: Human tumor antigens for cancer vaccine development. Immunol Rev 170:85-100, 1999[CrossRef][Medline]

8. Tsang KY, Zaremba S, Nieroda CA, et al: Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 87:982-990, 1995[Abstract/Free Full Text]

9. Dalerba P, Maccalli C, Casati C, et al: Immunology and immunotherapy of colorectal cancer. Crit Rev Oncol Hematol 46:33-57, 2003[Medline]

10. Marshall JL, Hoyer RJ, Toomey MA, et al: Phase I study in advanced cancer patients of a diversified prime boost vaccination protocol using recombinant nonreplicating avipox virus to elicit anti-carcinoembryonic antigen immune responses. J Clin Oncol 18:3964-3973, 2000[Abstract/Free Full Text]

11. Morse MA, Deng Y, Coleman D, et al: A phase I study of active immunotherapy with carcinoembryonic antigen peptide(CAP-1)-pulsed autologous human cultured dendritic cells in patients with metastatic malignacies expressing carcinoembryonic antigen. Clin Cancer Res 5:1331-1338, 1999[Abstract/Free Full Text]

12. Kantor J, Irvine K, Abrams S, et al: Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen vaccinia virus vaccine. J Natl Cancer Inst 84:1084-1091, 1992[Abstract/Free Full Text]

13. Kantor J, Irvine K, Abrams S, et al: Immunogenicity and safety of a recombinant vaccinia virus vaccine expressing the carcinoembryonic antigen gene in a nonhuman primate. Cancer Res 52:6917-6925, 1992[Abstract/Free Full Text]

14. Foon K, John WJ, Chakraborty M, et al: Clinical and immune responses in advanced colorectal cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen. Clin Cancer Res 3:1267-1276, 1997[Abstract]

15. Kass E, Schlom J, Thompson J, et al: Induction of protective host immunity to carcinoembryonic antigen (CEA), a self antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus. Cancer Res 59:676-683, 1999[Abstract/Free Full Text]

16. Evans TR, Kaye SB: Vaccine therapy for cancer—Fact or fiction? QJM 92:299-307, 1999[Free Full Text]

17. Zinkernagel RM: Immunity against solid tumors. Int J Cancer, 93:1-5, 2001[CrossRef][Medline]

18. Frost P, Ng CP, Belldegrun A, et al: Immunosensitization of prostate carcinoma cell lines for lymphocyte(CTL, TIL, LAK)-mediated apoptosis via the fas-fas-ligand pathway of cytotoxicity. Cell Immunol 180:70-83, 1997[CrossRef][Medline]

19. Bergmann-Leitner ES, Abrams SI: Treatment of human colon carcinoma cell lines with anti-neoplastic agents enhances their lytic sensitivity to antigen-specific CD8⁺ cytotoxic T lymphocytes. Cancer Immunol Immunother 50:445-455, 2001[CrossRef][Medline]

20. Correale P, Aquino A, Pellegrini M, et al: 5-Fluorouracil (5-FU) enhances the susceptibility of colon and breast carcinoma cells to human carcinoembryonic (CEA) peptide-specific cytotoxic T cells in vitro. Int J Cancer 104:437-445, 2003[CrossRef][Medline]

21. Correale P, Sabatino M, Cusi MG, et al: In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase. J Chemother 13:519-526, 2001[Medline]

22. Harshyne LA, Watkins SC, Gambotto A, et al: Dendritic cells acquire antigens from live cells for cross-presentation to CTL. J Immunol 166:3717-3723, 2001[Abstract/Free Full Text]

23. Correale P, Cusi MG, Del Vecchio MT, et al: Dendritic cell-mediated cross-presentation of antigens derived from colon carcinoma cells exposed to a highly cytotoxic multidrug regimen with gemcitabine, oxaliplatin, 5-fluorouracil, and leucovorin, elicits a powerful human antigen-specific CTL response with antitumor activity in vitro. J Immunol 175:820-828, 2005[Abstract/Free Full Text]

24. Markowicz S, Engleman EG: Granulocyte-macrophage colony stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro. J Clin Invest 85:955-961, 1990

25. Gillis S, Watson J: Interleukin-2 dependent culture of cytolytic T cell lines. Immunol Rev 54:81-109, 1981[CrossRef][Medline]

26. Correale P, Messinese S, Caraglia M, et al: A novel biweekly multi-drug regimen of gemcitabine, oxaliplatin, 5-fluorouracil (5-FU), and folinic acid (FA) in pre-treated patients with advanced colorectal carcinoma Br J cancer 90:1710-1714, 2004[Medline]

27. Correale P, Campoccia G, Tsang KY, et al: Recruitment of dendritic cells and enhanced antigen-specific immune reactivity in cancer patients treated with hrGM-CSF (molgramostim) and hr IL-2: Results from a Phase Ib Clinical Trial. Eur J Cancer 37:892-902, 2001

28. Boyum A: A one-stage procedure for isolation of granulocytes and lymphocytes from human blood: General sedimentation properties of white blood cells in a 1g gravity field. Scand J Clin Lab Invest 97:51-76, 1968

29. Guadagni F, Witt PL, Robbins PF, et al: Regulation of carcinoembryonic antigen expression in different human colorectal tumor cells by interferon-. Cancer Res 50:6248-6255, 1990[Abstract/Free Full Text]

30. Peters GJ, Jansen G: Resistance to antimetabolites, in Schilsky RL, Milano GA, Ratain MJ (eds): Principles of Antineoplastic Drug Development and Pharmacology. New York, NY, Marcel Dekker Inc, 1996, pp 543-585,

31. Landis DM, Loeb LA: Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases. J Biol Chem 273:25809-25817, 1998[Abstract/Free Full Text]

32. Francini G, Scardino A, Kosmatopoulos K, et al: High affinity HLA-A()02.01 peptides from parathyroid-hormone related protein generate in vitro and in vivo antitumor CTL response without autoimmune side effects. J Immunol 169:4840-4849, 2002[Abstract/Free Full Text]

33. Schneck JO, Slansky JE, O'Herrin SM, et al: Monitoring antigen-specific T cells using MHC-Ig dimmers, in Coligan J, Kruisbeek AM, Margulies D, et al (eds): Current Protocols in Immunology Inc, New York, NY, John Wiley & Sons, 2000, pp 17.2.1-17.2.17

34. Dubois B, Chapat L, Kaiserlian D: CD4+CD25+ T cells as key regulators of immune responses. Eur J Dermatol 13:111-116, 2003[Medline]

35. Wolf AM, Grubeck-Loebenstein B: Increase of regulatory T cells in the peripheral blood of cancer patients. J Immunother 25:202-206, 2002

36. Kukreja A, Maclaren N: Multiple immuno-regulatory defects in type-1 diabetes. J Clin Invest 109:131-140, 2002[CrossRef][Medline]

37. Carrabba M, Parmiani G, Rivoltini L: Cancer vaccines. Tumori 86:S33-37, 2000 (suppl 2)

38. Van den Eynde BJ, van der Bruggen P: T cell defined tumor antigens. Curr Opin Immunol 9:684-693, 1997[CrossRef][Medline]

39. Dorothee G, Ameyar M, Bettaieb A, et al: Role of Fas and granule exocytosis pathways in tumor-infiltrating T lymphocyte-induced apoptosis of autologous human lung carcinoma cells. Int J Cancer 91:772-777, 2001[CrossRef][Medline]

40. Nestle FO: Dendritic cell vaccination for cancer therapy. Oncogene 19:6673-6679, 2000[CrossRef][Medline]

41. Parmiani G, Rodolfo M, Melani C: Immunological gene therapy with ex vivo gene modified tumor cells: A critique and a reappraisal. Hum Gene Ther 11:1269-1275, 2000[CrossRef][Medline]

42. Parmiani G, Colombo MP, Melani C, et al: Cytokine gene trasduction in the immunotherapy of cancer. Adv Pharmacol 40:259-307, 1997

43. Aarts WM, Schlom J, Hodge JW: Vector based vaccine/cytokine combination therapy to enhance induction of immune response to a self-antigen and anti-tumor activity. Cancer Res 62:5770-5777, 2002[Abstract/Free Full Text]

44. Srivastava PK, Menoret A, Basu S, et al: Heat shock proteins come of age: Primitive functions acquire new roles in an adaptive world. Immunity 8:657-665, 1998[CrossRef][Medline]

45. Basu S, Binder RJ, Ramalingam T, et al: CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70 and calreticulin. Immunity 14:303-313, 2001[CrossRef][Medline]

46. Baars A, Buter J, Pinedo HM: Making use of the primary tumor. Bioessays 25:79-86, 2002[CrossRef]

47. Vermorken JB, Claessen AM, van Tinteren H, et al: Active specific immunotherapy for stage II and stage III human colon cancer: a randomized trial. Lancet 353:345-350, 1999[CrossRef][Medline]

48. van den Eertwegh AJ, Baars A, Pinedo HM: Adjuvant treatment of colorectal cancer: Toward tumor specific immunotherapies. Cancer and Met Rev 20:101-108, 2001[CrossRef]

49. Mavligit G, Gutterman JU, Burgess MA, et al: Adjuvant immunotherapy and chemo-immunotherapy in colorectal cancer of the Dukes' C classification: Preliminary clinical results. Cancer 36:2421-2427, 1975[CrossRef][Medline]

50. Bedikian AY, Valdivieso M, Mavligit G, et al: Sequential chemo-immunotherapy of colorectal cancer. evaluation of methotrexate, Baker's Antifol and levamisole. Cancer 42:2169-2176, 1978[CrossRef][Medline]

51. Richards F, 2nd, Muss HB, Cooper RM, et al: Chemotherapy versus chemo-immunotherapy in advanced adenocarcinoma of the colon and rectum: A prospective randomized study. Cancer 43:91-96, 1979[CrossRef][Medline]

52. Heinzer H, Huland E, Huland H: Systemic chemotherapy and chemo-immunotherapy for metastatic renal cell cancer. World J Urol 19:111-119, 2001[CrossRef][Medline]

53. Elias L, Binder M, Mangalik A, et al: Pilot trial of infusional 5-fluorouracil, interleukin-2, and subcutaneous interferon-alpha for advanced renal cell carcinoma. Am J Clin Oncol 22:156-161, 1999[CrossRef][Medline]

54. Lauro S, Bordin F, Trasatti L, et al: Concurrent chemo-immunotherapy in metastatic clear cell sarcoma: A case report. Tumori 85:512-514, 1999[Medline]

55. Lau WY, Leung TW, Lai BS, et al: Preoperative systemic chemo-immunotherapy and sequential resection of unresectable hepatocellular carcinoma. Ann Surg 233:236-241, 2001[CrossRef][Medline]

56. Bonmassar E, Bonmassar A, Vadlamudi S, et al: Antigenic change of L1210 leukemia in mice treated with 5-(3,3-dimethyl-1triazeno)-imidazole-4-carboxamide. Cancer Res 32:1446-1450, 1972[Abstract/Free Full Text]

57. Nowak AK, Robinson BW, Lake RA: Synergy between chemotherapy and immunotherapy in the treatment of established murine solid tumors. Cancer Res 63:4490-4496, 2003[Abstract/Free Full Text]

58. Nowak AK, Lake RA, Marzo AL, et al: Induction of tumor cell apoptosis in vivo increases tumor antigen cross-tolerizing host tumor specific CD8 T cells. J Immunol 170:4905-4913, 2003[Abstract/Free Full Text]

59. Nowak AK, Robinson BW, Lake RA: Gemcitabine exerts a selective effect on the humoral immune-response: implications for combination chemo-immunotherapy. Cancer Res 62:2353-2358, 2002[Abstract/Free Full Text]

Submitted December 19, 2003; accepted March 16, 2005.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				L. de la Cruz-Merino, E. Grande-Pulido, A. Albero-Tamarit, and M. E. Codes-Manuel de Villena Cancer and Immune Response: Old and New Evidence for Future Challenges Oncologist, December 1, 2008; 13(12): 1246 - 1254. [Abstract] [Full Text] [PDF]

				P. Correale, P. Tagliaferri, A. Fioravanti, M. T. Del Vecchio, C. Remondo, F. Montagnani, M. S. Rotundo, C. Ginanneschi, I. Martellucci, E. Francini, et al. Immunity Feedback and Clinical Outcome in Colon Cancer Patients Undergoing Chemoimmunotherapy with Gemcitabine + FOLFOX followed by Subcutaneous Granulocyte Macrophage Colony-Stimulating Factor and Aldesleukin (GOLFIG-1 Trial) Clin. Cancer Res., July 1, 2008; 14(13): 4192 - 4199. [Abstract] [Full Text] [PDF]

				C Bauer, F Bauernfeind, A Sterzik, M Orban, M Schnurr, H A Lehr, S Endres, A Eigler, and M Dauer Dendritic cell-based vaccination combined with gemcitabine increases survival in a murine pancreatic carcinoma model Gut, September 1, 2007; 56(9): 1275 - 1282. [Abstract] [Full Text] [PDF]

				D. Hoffmann, W. Bayer, T. Grunwald, and O. Wildner Intratumoral expression of respiratory syncytial virus fusion protein in combination with cytokines encoded by adenoviral vectors as in situ tumor vaccine for colorectal cancer Mol. Cancer Ther., July 1, 2007; 6(7): 1942 - 1950. [Abstract] [Full Text] [PDF]

				N. J. Nesslinger, R. A. Sahota, B. Stone, K. Johnson, N. Chima, C. King, D. Rasmussen, D. Bishop, P. S. Rennie, M. Gleave, et al. Standard Treatments Induce Antigen-Specific Immune Responses in Prostate Cancer Clin. Cancer Res., March 1, 2007; 13(5): 1493 - 1502. [Abstract] [Full Text] [PDF]

				I. Melero, A. Arina, O. Murillo, J. Dubrot, C. Alfaro, J. L. Perez-Gracia, M. Bendandi, and S. Hervas-Stubbs Immunogenic Cell Death and Cross-Priming Are Reaching the Clinical Immunotherapy Arena Clin. Cancer Res., April 15, 2006; 12(8): 2385 - 2389. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Correale, P. Articles by Francini, G. Search for Related Content PubMed PubMed Citation Articles by Correale, P. Articles by Francini, G. Social Bookmarking                            What's this?