ONCOIMMUNOLOGY 2016, VOL. 5, NO. 2, e1078058 (2 pages) http://dx.doi.org/10.1080/2162402X.2015.1078058
AUTHOR’S VIEW
The colorectal cancer immune paradox revisited Mihaela Angelova, Pornpimol Charoentong, Hubert Hackl, and Z. Trajanoski Biocenter, Division of Bioinformatics, Medical University of Innsbruck Innrain 80, Innsbruck, Austria
ABSTRACT
ARTICLE HISTORY
Tumor infiltrating lymphocytes (TILs) represent a strong independent predictor of relapse and overall survival in colorectal cancer (CRC). However, it appears that a majority of CRCs, i.e., microsatellite stable (MSS) tumors, are refractory to immune checkpoint blockers. The results of recent comprehensive analyses of genomic data provide possible answers.
Received 24 July 2015 Accepted 24 July 2015
Currently we are witnessing major breakthroughs in cancer therapy by the development of effective immunotherapeutic approaches. Specifically, strategies that use antibodies to block immune checkpoint molecules like CTLA-4, PD-1, and PD-L1 are showing impressive results in a number of cancers including not only melanoma, but also cancers like lung, head and neck, or bladder cancers among others.1 Paradoxically, patients with CRC, a cancer for which tumor infiltrating lymphocytes (TILs) represent a strong independent predictor of relapse and overall survival,2 do not benefit from the administration of a PD-1 targeting antibody.3 A notable exception represents patients with microsatellite-instable (MSI) phenotype.4 We have recently provided a high-resolution genomic view on the immunophenotypes and antigenomes (repertoire of tumor antigens) 5 and provide here explanations for this paradox. We developed an analytical strategy and examined genomic data sets from The Cancer Genome Atlas (TCGA) (n D 598).6 Briefly, we used RNA- and whole-exome NGS data to chart the antigenome comprising two major classes: cancer-germline antigens and neo-antigens. Additionally, we defined a compendium of genes using expression data from purified immune cells in order to derive immune signatures related to specific subpopulations, and then used RNA-sequencing data from the TCGA cohort to identify subpopulations of TILs.5 The cohort was analyzed with respect to the three distinct molecular phenotypes: (1) mutational status (hypermutated and non-hypermutated), (2) microsatellite status (MSS and MSI), and (3) methylation status. The quantification of the immune subpopulations showed that TILs were associated with distinct molecular phenotypes or the combinations thereof. As expected, MSI tumors were characterized by enrichments of TILs mostly related to adaptive immunity. A small group of MSS tumors, which were hypermutated were characterized by lower enrichment of effector memory and central memory CD4C and CD8C cells. The non-hypermutated MSS tumors CONTACT Z. Trajanoski
KEYWORDS
Immune escape; immunophenotype; tumor heterogeneity
were also enriched with TILs, but the levels were reduced compared to the hypermutated tumors. The genetic and the immunophenotypic variability of the CRC tumors imposed the question if different tumors use different mechanisms of tumor escape. We first analyzed the genetic heterogeneity of the tumors using exome sequencing data and SNP-array data. Based on the cancer cell fractions, the cohort was divided into six groups: MSI group (n D 69), hypermutated MSS group (n D 19), and four groups of MSS tumors with low (n D 63), two intermediate (n D 123, and n D 96) and high heterogeneity (n D 105). We then examined the infiltration of immunosuppressive cell types (myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs)), and the expression of five classes of immunomodulating molecules: immunoinhibitory genes, immunostimulatory genes, MHC class I, MHC class II, and non-classical MHC molecules. One striking observation was how the genetic basis of the tumors determines the tumor escape mechanisms. Hypermutated tumors (MSI and hypermutated MSS tumors) showed higher intratumor heterogeneity, suggesting that the greater mutational load in these tumors results in a higher load of neoantigens (635 § 308 and 1,651 § 1,455, respectively), and likely promotes T-cell activation and infiltration. Furthermore, in these tumors, Tregs and MDSCs were depleted whereas activated CD8C and CD4C cells were enriched. This strong immunological response was counterbalanced by an increased expression of several immunoinhibitors including CTLA-4, PD-1, and IDO1, which might explain why are these tumors progressing. Interestingly, similar effective immune responses were observed also in the group of non-hypermutated tumors with low genetic heterogeneity. This inverse association of the tumor heterogeneity and the immune responses in MSS tumors was evident at several levels: enrichment of immunosuppressive cells, expression of immunostimulators, and expression of MHC class I and MHC class II molecules. The data suggests that in the three groups of MSS tumor with intermediate and
Email: [email protected]
Published with license by Taylor & Francis Group, LLC © Mihaela Angelova, Pornpimol Charoentong, Hubert Hackl, and Z. Trajanoski This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
e1078058-2
M. ANGELOVA ET AL.
Our model is in concordance with recently suggested ones 9,10 and extends our understanding of the tumor-immune interaction in CRC. We advocate that for the treatment of CRC patients with checkpoint inhibitors not only the characterization of the genetic status (i.e., mutational load and tumor heterogeneity), but also of the immunophenotype (TILs and immunomodulatory molecules) of the tumor is required.
References
Figure 1. Proposed model that may predict responses to anti-PD-1 blockade. The tumor escape mechanism is determined by the genetic status, i.e., hypermutation (for MSI and MSS tumors) or tumor homogeneity (for non-hypermutated MSS tumors). Left: Tumors escape immunosurveillance by upregulation of immunoinhibitory molecules, are enriched with CD8C and CD4C cells, and hence, would benefit from therapy with anti-PD-1 antibodies. Right: tumors escape immunosurveillance by downregulation of MHC I and MHC II molecules, and upregulation of HLA-G. These tumors are enriched with immunosuppressive cells and would not respond to therapy with anti-PD-1 antibodies.
high heterogeneity the tumor escape was governed by downregulation MHC I and MHC II molecules, and upregulation of HLA-G, which is associated with worse overall survival.7 In summary, the results of our in silico analyses provide possible answer for the CRC immune paradox (Fig. 1). Patients with MSI tumors benefit from therapy with anti PD-1 antibodies due to the favorable genetic (heterogeneous tumors, high load of neo-antigens) and immune characteristics (infiltration of CD8C and CD4C T cells, depletion of Tregs and MDSCs, upregulation of MHC class I and MHC class II molecules). In contrast, a majority (84%) of the patients with MSS tumors show unfavorable features: downregulation of MHC class I and MHC class II molecules, increased expression of HLA-G, and enrichment of MDSCs. Following these arguments, there are two groups of patients, patients with hypermutated MSS and non-hypermutated MSS but homogeneous tumors (16%) that would likely benefit from anti PD-1 blockers since the immunophenotypes are similar to the MSI tumors. This is further supported by a recent study in which a small group of MSS patients indeed showed response.8
1. Topalian SL, Drake CG, Pardoll DM Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 2015; 27:450–461; PMID:25858804; http://dx.doi.org/10.1016/j.ccell.2015. 03.001 2. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, LagorcePages C, Tosolini M, Camus M, Berger A, Wind P et al Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313:1960–1964; PMID:17008531; http://dx.doi.org/10.1126/science.1129139 3. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB et al Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012; 366:2443–2454; PMID:22658127; http://dx. doi.org/10.1056/NEJMoa1200690 4. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, Skora AD, Luber BS, Azad NS, Laheru D et al PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med 2015; 372:2509–2520; PMID:26028255; http://dx.doi.org/10.1056/NEJMoa 1500596 5. Angelova M, Charoentong P, Hackl H, Fischer ML, Snajder R, Krogsdam AM, Waldner MJ, Bindea G, Mlecnik B, Galon J et al Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy. Genome Biol 2015; 16:64; PMID:25853550; http://dx.doi.org/10.1186/s13059015-0620-6 6. Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012; 487:330– 337; PMID:22810696; http://dx.doi.org/10.1038/nature11252 7. Zeestraten EC, Reimers MS, Saadatmand S, Dekker JW, Liefers GJ, van den Elsen PJ, van de Velde CJ, Kuppen PJ. Combined analysis of HLA class I, HLA-E and HLA-G predicts prognosis in colon cancer patients. Br J Cancer 2014; 110:459–468; PMID:24196788; http://dx. doi.org/10.1038/bjc.2013.696 8. Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube JM, Blosser RL, Fan H, Wang H, Luber BS et al The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov 2015; 5:43–51; PMID:25358689; http://dx.doi.org/10.1158/2159-8290.CD14-0863 9. Housseau F, Llosa NJ. Immune checkpoint blockade in microsatellite instable colorectal cancers: Back to the clinic. Oncoimmunology 2015; 4:e1008858; PMID:26155426; http://dx.doi.org/10.1080/2162402X. 2015.1008858 10. Kroemer G, Galluzzi L, Zitvogel L, Fridman WH. Colorectal cancer: the first neoplasia found to be under immunosurveillance and the last one to respond to immunotherapy? Oncoimmunology 2015; 4: e1058597; PMID:26140250; http://dx.doi.org/10.1080/2162402X.2015. 1058597
AUTHOR’S VIEW
The colorectal cancer immune paradox revisited Mihaela Angelova, Pornpimol Charoentong, Hubert Hackl, and Z. Trajanoski Biocenter, Division of Bioinformatics, Medical University of Innsbruck Innrain 80, Innsbruck, Austria
ABSTRACT
ARTICLE HISTORY
Tumor infiltrating lymphocytes (TILs) represent a strong independent predictor of relapse and overall survival in colorectal cancer (CRC). However, it appears that a majority of CRCs, i.e., microsatellite stable (MSS) tumors, are refractory to immune checkpoint blockers. The results of recent comprehensive analyses of genomic data provide possible answers.
Received 24 July 2015 Accepted 24 July 2015
Currently we are witnessing major breakthroughs in cancer therapy by the development of effective immunotherapeutic approaches. Specifically, strategies that use antibodies to block immune checkpoint molecules like CTLA-4, PD-1, and PD-L1 are showing impressive results in a number of cancers including not only melanoma, but also cancers like lung, head and neck, or bladder cancers among others.1 Paradoxically, patients with CRC, a cancer for which tumor infiltrating lymphocytes (TILs) represent a strong independent predictor of relapse and overall survival,2 do not benefit from the administration of a PD-1 targeting antibody.3 A notable exception represents patients with microsatellite-instable (MSI) phenotype.4 We have recently provided a high-resolution genomic view on the immunophenotypes and antigenomes (repertoire of tumor antigens) 5 and provide here explanations for this paradox. We developed an analytical strategy and examined genomic data sets from The Cancer Genome Atlas (TCGA) (n D 598).6 Briefly, we used RNA- and whole-exome NGS data to chart the antigenome comprising two major classes: cancer-germline antigens and neo-antigens. Additionally, we defined a compendium of genes using expression data from purified immune cells in order to derive immune signatures related to specific subpopulations, and then used RNA-sequencing data from the TCGA cohort to identify subpopulations of TILs.5 The cohort was analyzed with respect to the three distinct molecular phenotypes: (1) mutational status (hypermutated and non-hypermutated), (2) microsatellite status (MSS and MSI), and (3) methylation status. The quantification of the immune subpopulations showed that TILs were associated with distinct molecular phenotypes or the combinations thereof. As expected, MSI tumors were characterized by enrichments of TILs mostly related to adaptive immunity. A small group of MSS tumors, which were hypermutated were characterized by lower enrichment of effector memory and central memory CD4C and CD8C cells. The non-hypermutated MSS tumors CONTACT Z. Trajanoski
KEYWORDS
Immune escape; immunophenotype; tumor heterogeneity
were also enriched with TILs, but the levels were reduced compared to the hypermutated tumors. The genetic and the immunophenotypic variability of the CRC tumors imposed the question if different tumors use different mechanisms of tumor escape. We first analyzed the genetic heterogeneity of the tumors using exome sequencing data and SNP-array data. Based on the cancer cell fractions, the cohort was divided into six groups: MSI group (n D 69), hypermutated MSS group (n D 19), and four groups of MSS tumors with low (n D 63), two intermediate (n D 123, and n D 96) and high heterogeneity (n D 105). We then examined the infiltration of immunosuppressive cell types (myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs)), and the expression of five classes of immunomodulating molecules: immunoinhibitory genes, immunostimulatory genes, MHC class I, MHC class II, and non-classical MHC molecules. One striking observation was how the genetic basis of the tumors determines the tumor escape mechanisms. Hypermutated tumors (MSI and hypermutated MSS tumors) showed higher intratumor heterogeneity, suggesting that the greater mutational load in these tumors results in a higher load of neoantigens (635 § 308 and 1,651 § 1,455, respectively), and likely promotes T-cell activation and infiltration. Furthermore, in these tumors, Tregs and MDSCs were depleted whereas activated CD8C and CD4C cells were enriched. This strong immunological response was counterbalanced by an increased expression of several immunoinhibitors including CTLA-4, PD-1, and IDO1, which might explain why are these tumors progressing. Interestingly, similar effective immune responses were observed also in the group of non-hypermutated tumors with low genetic heterogeneity. This inverse association of the tumor heterogeneity and the immune responses in MSS tumors was evident at several levels: enrichment of immunosuppressive cells, expression of immunostimulators, and expression of MHC class I and MHC class II molecules. The data suggests that in the three groups of MSS tumor with intermediate and
Email: [email protected]
Published with license by Taylor & Francis Group, LLC © Mihaela Angelova, Pornpimol Charoentong, Hubert Hackl, and Z. Trajanoski This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
e1078058-2
M. ANGELOVA ET AL.
Our model is in concordance with recently suggested ones 9,10 and extends our understanding of the tumor-immune interaction in CRC. We advocate that for the treatment of CRC patients with checkpoint inhibitors not only the characterization of the genetic status (i.e., mutational load and tumor heterogeneity), but also of the immunophenotype (TILs and immunomodulatory molecules) of the tumor is required.
References
Figure 1. Proposed model that may predict responses to anti-PD-1 blockade. The tumor escape mechanism is determined by the genetic status, i.e., hypermutation (for MSI and MSS tumors) or tumor homogeneity (for non-hypermutated MSS tumors). Left: Tumors escape immunosurveillance by upregulation of immunoinhibitory molecules, are enriched with CD8C and CD4C cells, and hence, would benefit from therapy with anti-PD-1 antibodies. Right: tumors escape immunosurveillance by downregulation of MHC I and MHC II molecules, and upregulation of HLA-G. These tumors are enriched with immunosuppressive cells and would not respond to therapy with anti-PD-1 antibodies.
high heterogeneity the tumor escape was governed by downregulation MHC I and MHC II molecules, and upregulation of HLA-G, which is associated with worse overall survival.7 In summary, the results of our in silico analyses provide possible answer for the CRC immune paradox (Fig. 1). Patients with MSI tumors benefit from therapy with anti PD-1 antibodies due to the favorable genetic (heterogeneous tumors, high load of neo-antigens) and immune characteristics (infiltration of CD8C and CD4C T cells, depletion of Tregs and MDSCs, upregulation of MHC class I and MHC class II molecules). In contrast, a majority (84%) of the patients with MSS tumors show unfavorable features: downregulation of MHC class I and MHC class II molecules, increased expression of HLA-G, and enrichment of MDSCs. Following these arguments, there are two groups of patients, patients with hypermutated MSS and non-hypermutated MSS but homogeneous tumors (16%) that would likely benefit from anti PD-1 blockers since the immunophenotypes are similar to the MSI tumors. This is further supported by a recent study in which a small group of MSS patients indeed showed response.8
1. Topalian SL, Drake CG, Pardoll DM Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 2015; 27:450–461; PMID:25858804; http://dx.doi.org/10.1016/j.ccell.2015. 03.001 2. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, LagorcePages C, Tosolini M, Camus M, Berger A, Wind P et al Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313:1960–1964; PMID:17008531; http://dx.doi.org/10.1126/science.1129139 3. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB et al Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012; 366:2443–2454; PMID:22658127; http://dx. doi.org/10.1056/NEJMoa1200690 4. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, Skora AD, Luber BS, Azad NS, Laheru D et al PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med 2015; 372:2509–2520; PMID:26028255; http://dx.doi.org/10.1056/NEJMoa 1500596 5. Angelova M, Charoentong P, Hackl H, Fischer ML, Snajder R, Krogsdam AM, Waldner MJ, Bindea G, Mlecnik B, Galon J et al Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy. Genome Biol 2015; 16:64; PMID:25853550; http://dx.doi.org/10.1186/s13059015-0620-6 6. Cancer Genome Atlas N. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012; 487:330– 337; PMID:22810696; http://dx.doi.org/10.1038/nature11252 7. Zeestraten EC, Reimers MS, Saadatmand S, Dekker JW, Liefers GJ, van den Elsen PJ, van de Velde CJ, Kuppen PJ. Combined analysis of HLA class I, HLA-E and HLA-G predicts prognosis in colon cancer patients. Br J Cancer 2014; 110:459–468; PMID:24196788; http://dx. doi.org/10.1038/bjc.2013.696 8. Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube JM, Blosser RL, Fan H, Wang H, Luber BS et al The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov 2015; 5:43–51; PMID:25358689; http://dx.doi.org/10.1158/2159-8290.CD14-0863 9. Housseau F, Llosa NJ. Immune checkpoint blockade in microsatellite instable colorectal cancers: Back to the clinic. Oncoimmunology 2015; 4:e1008858; PMID:26155426; http://dx.doi.org/10.1080/2162402X. 2015.1008858 10. Kroemer G, Galluzzi L, Zitvogel L, Fridman WH. Colorectal cancer: the first neoplasia found to be under immunosurveillance and the last one to respond to immunotherapy? Oncoimmunology 2015; 4: e1058597; PMID:26140250; http://dx.doi.org/10.1080/2162402X.2015. 1058597
