Exploiting antitumor immunity to overcome relapse and improve remission duration
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- Chen, L.L., Chen, X., Choi, H. et al. Cancer Immunol Immunother (2012) 61: 1113. doi:10.1007/s00262-011-1185-1
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Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient’s lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient’s own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib’s effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response. Interim analysis of eight patients demonstrated significant induction of IFN-γ-producing-CD8+, -CD4+, -NK cell, and IFN-γ-producing-tumor-infiltrating-lymphocytes, signifying significant Th1 response and NK cell activation. After a median follow-up of 3.6 years, complete response (CR) + partial response (PR) = 100%, overall survival = 100%, one patient died of unrelated illness while in remission, six of seven evaluable patients are either in continuing PR/CR (5 patients) or have progression-free survival (PFS, 1 patient) exceeding the upper limit of the 95% confidence level of the genotype-specific-PFS of the phase III imatinib-monotherapy (CALGB150105/SWOGS0033), demonstrating highly promising clinical outcomes. The current trial is closed in preparation for a larger future trial. We conclude that combination of targeted therapy and immunotherapy is safe and induced significant Th1 response and NK cell activation and demonstrated highly promising clinical efficacy in GIST, thus warranting development in other tumor types.
KeywordsIFN-γ IFN-α Peginterferon α-2b GIST Imatinib Immunotherapy
Despite effective treatments achieving remission, cancer survivors often relapse, after which interventions become largely unsuccessful. One culprit is drug-resistant clones—pre-existing and evolving continuously—and another is tumor stem cells—repopulating, resilient, and poorly understood. With their unique features, once outgrowth has occurred, both culprits can evade standard therapies and prevail. The innate and adaptive immunity have been shown to play important roles in protecting the host through tumor immunosurveillance [1, 2, 3, 4, 5, 6]. Unfortunately, mechanisms of tumor-induced tolerance enable tumors to escape immunosurveillance . However, the delicate balance can be restored if we can design novel treatment that can break tolerance while promote innate and adaptive antitumor immunity.
Dendritic cells (DCs) capture, process, and cross-present antigens in the context of MHC class-I and costimulatory molecules to subsets of T-lymphocytes, and play critical roles in the regulation and development of distinct immune responses [8, 9, 10] including (1) Th1 adaptive cell-mediated immunity (Th1 response) signified by interferon-γ (IFN-γ) secretion and play major roles in protection against pathogens and tumors [1, 2, 3, 4, 5, 6], (2) Th2, (3) Th17, and (4) T-regulatory responses (tolerance).
IFN-α is a type-1 IFN and a physiological danger signal [11, 12] can upregulate expression of MHC class-I molecules and costimulatory molecules on DCs, activate innate immunity, modulate DCs, promote Th1 response, help clonal expansion/survival and memory differentiation of T-lymphocytes [9, 10, 11, 12, 13], and has been shown to be an effective vaccine adjuvant in animal models  and clinical trials [15, 16]. The immunological consequences of tumor cell death induced by individual chemotherapy agents  and the subsequent differentiation of DCs and T-lymphocytes in the ensuing 2-week span are of pivotal importance in influencing the development toward the distinct immune responses (Th1, Th2, Th17, or tolerance). Cytotoxic chemotherapy often results in prolonged severe leukopenia depriving DCs of proper maturation (differentiation), thus often resulting in a tolerant/dysfunctional immune state.
With recognition of the IFN-α qualities [9, 10, 11, 12, 13, 14], the pivotal role of DCs in the development of distinct immune responses [8, 9, 10] and support from our preclinical data, we hypothesize that (1) combining IFN-α with effective non-marrow-suppressive antitumor agent(s) could induce innate immunity and Th1 response; (2) the antitumor immunity can help eradicating tumor cells including the drug-resistant clones and tumor stem cells upfront thus improve response rate; (3) most importantly, antitumor immunity can monitor continuously and eradicate the various continuing-evolving drug-resistant clones and the resilient tumor stem cells when they first emerge at the cellular/subclinical level prior to outgrowth, and this would delay/prevent relapse, ultimately leading to the improvements in progression-free survival (PFS) and overall survival (OS).
To test the hypothesis, we designed a new strategy aiming at developing innate immunity and Th1 response concomitant with partial response (PR) or complete response (CR) achieved by effective non-marrow-suppressive drug therapy. Gastrointestinal stromal tumor (GIST), a sarcoma with incidence of 5000/year in US, represents an excellent model to test our hypothesis for the following reasons. First, imatinib mesylate (IM, Gleevec®, Glivec®) , a selective inhibitor of ABL, KIT, PDGFRA/B, is highly effective, induces swift apoptosis/necrosis of GIST within 3–7 days , and is non-marrow-suppressive, allowing proper DC and cytotoxic T-lymphocyte differentiation toward Th1 response. Second, GIST cell alterations include cancer/testis antigens , tumor-antigens created by activating mutations in KIT (c-kit) or PDGFRA [21, 22, 23, 24] and new mutation(s) responsible for IM resistance [25, 26, 27]. Third, IM-monotherapy trials in GIST patients have reported response rates (PR + CR) of 54% , 52% [29, 30], and 48% [22, 30]. The median PFS remains ≤ 2 years [22, 29, 30] mainly due to the development of IM resistance [25, 26, 27]. Discontinuing IM resulted in high rate of relapse due to repopulating stem cells . Thus, better therapies for GIST are needed.
IM was reported to induce DC-mediated natural killer (NK) cell IFN-γ production [32, 33] and potentiate adaptive immunity through IM-off-target inhibition of KIT on DCs  and inhibition of Ido ; both IM-off-target immunological anti-GIST effects plus IM-inhibition of KIT/PDGFRA signaling contribute to the IM-monotherapy efficacy [22, 28, 29, 30] as described above and is less than satisfactory. We intend to bring out the full potential of anti-GIST immunity by a new strategy of combining peginterferon α-2b (PegIFNa2b, Peg-Intron®)  with IM and have demonstrated significant Th1 response, innate immunity, and highly promising clinical outcome comparing to IM-monotherapy , strongly support all three parts of our hypothesis.
Materials and methods
Specimens were collected under MD Anderson Institutional Review Board (IRB) protocols LAB_00143. Primary tumor cells were isolated after digesting fresh tumor with collagenase. The chimeric SYN-SSX was sequenced . Peripheral blood mononuclear cell (PBMC)-derived DCs were isolated by plastic adherence and culture supplemented with GM-CSF and IL-4. Cytokine cocktail consisted of TNF-α (R&D), IL-1β (R&D), IL-6 (R&D), and PGE-2 (Sigma) . IL-12-p70 was analyzed using ELISA (Biosources, Camarillo, CA.) and read with UV-900 microplate reader (Bio-Tek Instruments, Winooski, VT). The plastic non-adherent cells were used to positively select CD8+ T-lymphocytes using anti-CD8 monoclonal antibody (mAb) coupled to magnetic microbeads (Miltenyi Biotec, Auburn, CA).
IFN-γ-enzyme-linked immunosorbent spot (IFN-γ-ELISPOT) assay
CD8+ T-lymphocytes were cultured in AIM-V medium supplemented with IL-2 and IL-7 and stimulated with various antigen preparations twice, total 14 days, to generate CTLs. The 96-well ELISPOT plate (Millipore, Billerica, MA) was precoated with anti-IFN-γ antibody, incubated at 4°C overnight, plated with CD8+ T-lymphocytes at 2 × 105 cells/well, and stimulated with 4 × 104 irradiated primary tumor cells for 40 h at 37°C. Biotinylated IFN-γ antibody was added, followed by streptavidin peroxidase. IFN-γ spots were counted using an ELISPOT reader.
Cryopreserved primary tumor cells were used as targets and K562 cells as control. We labeled 2 × 106 target cells with 100 μCi of Na251CrO4 (ICN Biomedicals, Irvine, CA) and distributed 3,000 target cells in each well. Blocking experiments were performed using anti-HLA-A.B.C antibody and isotype control (Dako, Carpinteria, CA).
Refer to "Results".
As described previously .
PBMCs were cultured with phorbol ester PMA (5 ng/ml) plus ionomycin (745 ng/ml) for 1 h, add brefeldin A (5 mcg/ml) and cultured for additional 4 h. After surface staining with CD4-PerCP, CD8-APC, or CD3-FITC (BD Biosciences), cells were fixed and stained with anti-human IFN-γ-PE (BioLegend, San Diego, CA) . Data were acquired on a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc., Ashland).
Immunohistochemical analysis and confocal microscopy
Antigen retrieval with preheated EDTA/Tris buffer pH 9.0 for CD8, CD56, and CD4; and citrate Buffer pH 6.0 for IFN-γ, FasL, and granzyme B. Antibodies included CD4, CD8, CD56 (Dako, Carpinteria, CA); IFN-γ (Abcam, Cambridge, MA); and FasL and granzyme B (Novus Biologicals, Littleton, CO), goat anti-rabbit IgG antibody conjugated with Texas Red, and goat anti-mouse IgG conjugated with Alexa Fluor 488 (Novus Biologicals). Images were acquired using Fluo View software on an Olympus FV1000 confocal laser scanning microscope.
Cancer patient’s anergic T-lymphocytes in vivo can be converted into effective cytotoxic T-lymphocytes capable of killing patient’s own primary tumor cells in vitro
To the best of our knowledge, this is the first report using a patient’s own post-chemotherapy residual drug-resistant primary tumor cells—a source of micrometastasis and recurrence—as targets for CTL assays and provided direct and convincing evidence that a terminal cancer patient’s T-lymphocytes can be converted from tolerant bystanders ignoring the tumor growth in vivo into tumor-specific effective CTLs in vitro (Fig. 1d, e). Our preclinical studies served two purposes. First, these encouraging results help to justify immunotherapy approach to overcome relapse especially when no other treatment option is in sight due to current poor understanding of the culprits causing relapse. Second, our preclinical study methods are readily applicable for testing the development of tumor-specific immunity during clinical trial by collecting PBMC and applying 51Cr release or IFN-γ–ELOSPOT assays using primary tumor cells (if available) as CTL targets. Without further dwelling on in vitro studies, we move toward our ultimate goal of restoring/stimulating antitumor immunity in vivo in cancer patients.
Aiming at developing antitumor immunity in parallel with achieving PR/CR by drug therapy, we initiated a new strategy of combining PegIFNa2b (3rd signal/danger signal) with IM (killing tumor cells to undermine tumor-induced tolerance and provide 1st and 2nd signals—antigen and co-stimulation) in GIST to test our hypothesis.
GIST clinical trial design is summarized in Fig. 2
Combination treatment of PegIFNa2b plus IM-induced significant IFN-γ-producing-lymphocytes
Post-combination-treatment residual tumor showed complete remission and nearly all tumor-infiltrating-lymphocytes produce IFN-γ
Strikingly, almost all of the TILs actively produce IFN-γ (Fig. 4b, s), in sharp contrast to the totally negative IFN-γ-staining in the pretreatment GIST biopsy and all three post-IM-monotherapy controls (Fig. 4a, c–f). Uninvolved adjacent lymph nodes showed rare IFN-γ-positive cells (u) suggesting that combination treatment may have induced primarily GIST-reactive TILs.
Our translational research results showed significant induction of IFN-γ-producing -CD8+, -CD4+, -NK cell, and IFN-γ-producing TILs—signifies induction of Th1 response and NK cell activation, thus strongly support part one of our hypothesis.
GIST patient characteristics, stage, genotyping, response rate, and remission duration
Pt ID (age; PSa)
Stage; mitosis/50HPF; primary site (size); metastatic sites
KIT genotyping (mutational analysis)
Continuing PR/CR or PFSe; [S0033 Study genotype-specific
median PFS;0.95UCL]f Days
III; < 5; gastric (15.7 cm)
FNA, insufficient for genotyping
PR at wk 8
PR at wk 17
PR at mo 46
>1,572 (4.3 yr);
III; < 5; gastric (6 cm)
KIT exon 11, GTT → GAT,V559D
Not FDG avid
PR at wk 8
at wk 8)
>1,488 (4.1 yr);
IV; 40; SM (5.5 cm); liver met
KIT exon 11, 558_560del
PR at wk 9
PR at wk 9
PR at mo 5
PFS = 765 (2.1 yr);
III; 20; rectal (9.3 cm)
KIT exon 11, homozygous GTT → GAT,V560D
at wk 12
at wk 12
at wk 12
>1,319 (3.6 yr);
IV, gastric (9.8 cm); liver
Wild-type KIT & PDGFRA
PR at wk 9
PR at wk 9
at mo 15
>1,298 (3.6 yr);
IV, gastric (4.5 cm, resected); liver, lung, and peritoneal implants
Wild-type KIT & PDGFRA
at wk 8
at wk 8
at wk 8
PFS = 799 (2.2 yr)h;
III; gastric (11.4 cm)
Insufficient material from FNA for genotyping
PR at wk 13;
at mo 6
wk 13; near-CR at mo 6
at wk 13; near-CR at mo 6
NA, died of unrelated cause in remission with radiographic near-CR
IV; 17; SM (16 cm); liver
KIT exon 9, A502_Y503 dup
PR at wk 10
PR at mo 20
>1,179 (3.2 yr);
Pts#1–3 initially received HD-PegIFNa2b at 4 mcg/kg/week, developed grade 3 neutropenia, and requiring dose reduction to 3 mcg/kg/week. Later the protocol was amended and the HD-PegIFNa2b was reduced to 3 mcg/kg/week (Pts#4–8), and we observed occasional grade 1 or grade 2 neutropenia with quick recovery. Two patients (Pt#1 and 8) developed grade 3 skin rash requiring short-term steroid. All patients experienced transient low-grade fever and mild flu-like symptoms as expected.
Progression-free survival and overall survival
After a median follow-up of 3.6 years (3.2–4.3 years), the OS is 100% with PS = ECOG 0 for all seven evaluable patients. Pt#1 had unsuccessful genotyping and was grouped with KIT exon 11—the most favorable group—for the evaluation. Pt#7 died at age 85 of unrelated illness while in remission with a radiographic near-CR (Table 1; Fig. 5, 3rd row). The PFS of Pt#6 and the continuing PR/CR of Pts#1, 2, 4, 5, and 8—a total of 6 out of 7 evaluable patients—exceed not only the genotype-specific median PFS  but also the upper limit of the 95% confidence level of the genotype-specific PFS of S0033 IM-monotherapy trial (Table 1, last column; S0033 Study data are in brackets) [Personal communication with Dr. Michael C. Heinrich, PI of IM-monotherapy phase III S0033 Study]. Pt#3 harbored aggressive GIST with extensive liver metastasis and achieved PR at week 8 by all three criteria, but three out of the numerous liver lesions showed evidence of increased SUV on PET-CT scan without new lesions on day 765, and the genotype-specific PFS was slightly longer than that of S003 IM-monotherapy trial .
Taken together, combination treatment with IM plus PegIFNa2b was well tolerated, safe, demonstrated a 100% response rate (PR + CR), 100% OS rate, and substantially prolonged continuing PR/CR (5 patients) and PFS (2 patients) after a median follow-up of 3.6 years (3.2–4.3 years), and strongly supports part two and three of our hypothesis.
In CML, combining two active agents, peginterferon-alfa-2a (Pegasys®) and IM, demonstrated tolerability and improved efficacy over IM-monotherapy , but the immunological implications of IFN-α was not addressed. Our GIST study of adding PegIFNa2b (immunotherapy) to the current standard IM (targeted therapy) demonstrated significant induction of innate immunity and Th1 response and highly promising clinical outcome comparing to IM-monotherapy albeit small group study, and strongly supported our hypothesis. Although IM has off-target effect of activating innate [32, 33] and potentiating adaptive immunity [34, 35], the significant induction of IFN-γ-producing-lymphocytes (Fig. 3a) in this study is mainly attributed to PegIFNa2b rather than IM alone because stopping HD-PegIFNa2b (Fig. 3b, n) or switching to maintenance LD-PegIFNa2b while continuing IM resulted in a sharp decline of IFN-γ-producing-lymphocytes to barely detectable level. This new concept/strategy of combining immunotherapy with effective non-marrow-suppressive treatments might be beneficial to other cancer types as well. Combining IFN-α or peginterferon α with radiation, hormone/hormone antagonist, small molecule targeted therapies, or monoclonal antibodies in radiosensitive tumors, prostate, breast, pancreatic, melanoma, hepatocellular, colorectal, and sarcoma may help delay/prevent relapse and warrant further investigations.
National Cancer Institute Grants CA016672, U01-CA70172-01 and N0-CM-17003 to University of Texas MD Anderson Cancer Center; National Cancer Institute Award P30CA042014 to Huntsman Cancer Institute; Public Health Services research grants UL1-RR025764 and National Center for Research Resources C06-RR11234 to University of Utah. We thank Schering/Merck for supplying peginterferon α-2b and partial funding; Katie Thomas, RN and Grace Noda, PA-C, for excellent patient care; Cherry Vanover for superb clinical trial assistance; Aida B. Narvios, MD and Regan Butterfield for assistance in leukapheresis and radiology.
Conflict of interest
The authors declare that they have no conflict of interest.
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