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PI3K/Akt/Mtor Signaling and Plasma Membrane Proteins Are Implicated in Responsiveness to Adjuvant Dendritic Cell Vaccination for Metastatic Colorectal Cancer David C

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PI3K/Akt/Mtor Signaling and Plasma Membrane Proteins Are Implicated in Responsiveness to Adjuvant Dendritic Cell Vaccination for Metastatic Colorectal Cancer David C Published OnlineFirst July 19, 2016; DOI: 10.1158/1078-0432.CCR-16-0623 Personalized Medicine and Imaging Clinical Cancer Research PI3K/Akt/mTOR Signaling and Plasma Membrane Proteins Are Implicated in Responsiveness to Adjuvant Dendritic Cell Vaccination for Metastatic Colorectal Cancer David C. Qian1, Xiangjun Xiao1, Jinyoung Byun1, Arief A. Suriawinata2, Stephanie C. Her3, Christopher I. Amos1, and Richard J. Barth Jr.4 Abstract Purpose: We have previously demonstrated that patients with her tumor genotype data and variant association effect sizes metastatic colorectal cancer who exhibit immune responses to a computed from the other 21 patients; greater weighting was dendritic cell (DC) vaccine have superior recurrence-free survival placed on gene products with cell membrane–related functions. following surgery, compared with patients in whom responses do Results: There was no correlation between vaccine response not occur. We sought to characterize the patterns of T-lymphocyte and intratumor, peritumor, or hepatic densities of T-cell sub- infiltration and somatic mutations in metastases that are associ- populations. Associated genes were found to be enriched in the ated with and predictive of response to the DC vaccine. PI3K/Akt/mTOR signaling axis (P < 0.001). Applying a consis- Experimental Design: Cytotoxic, memory, and regulatory T tent prediction score cutoff over 22 rounds of leave-one-out cells in resected metastases and surrounding normal liver tissue cross-validation correctly inferred vaccine response in 21 of 22 from 22 patients (11 responders and 11 nonresponders) were patients (95%). enumerated by immunohistochemistry prior to vaccine admin- Conclusions: Adjuvant DC vaccination has shown promise as a istration. In conjunction with tumor sequencing, the combined form of immunotherapy for patients with metastatic colorectal multivariate and collapsing method was used to identify gene cancer. Its efficacy may be influenced by somatic mutations that mutations that are associated with vaccine response. We also affect pathways involving PI3K, Akt, and mTOR, as well as tumor derived a response prediction score for each patient using his/ surface proteins. Clin Cancer Res; 23(2); 399–406. Ó2016 AACR. Introduction immune system's role in cancer progression have prompted tremendous efforts to develop immunotherapies for colorectal Colorectal cancer is the fourth leading cause of cancer death cancer (7). in the world (1). About one in three patients diagnosed with Immunotherapies aim to either enhance the antitumor colorectal cancer dies from metastatic disease (2). The major- immune response or prevent immunosurveillance suppression ity of metastases develop in the liver and lung. Although by cancer cells. Whole tumor vaccines for colorectal cancer have metastases can be completely resected in many patients, 60% not been shown to confer survival benefit. Since only a tiny to 80% of these patients eventually die due to the growth of fraction of proteins in an autologous tumor vaccine is specificto small metastases that were undetectable at the time of surgery cancer cells, dilution by self-antigens is believed to make the (3, 4). Currently, the addition of adjuvant chemotherapy vaccine poorly immunogenic (8). Specific colorectal cancer has limited impact on patient survival (5, 6). The pressing tumor-associated antigens have subsequently been identified and need for better treatments and growing recognition of the used as peptide vaccines (9–14). However, the extent of antigen presentation varies widely depending on patients' HLA type (15). 1Department of Biomedical Data Science, Dartmouth Geisel School of Medicine, Dendritic cell (DC) vaccines overcome the limitations of these ex vivo Lebanon, New Hampshire. 2Department of Pathology, Dartmouth-Hitchcock two earlier vaccine approaches. Following stimulation Medical Center, Lebanon, New Hampshire. 3Department of Computer Science, by tumor lysate, DCs are not confined to process any specific Dartmouth College, Hanover, New Hampshire. 4Department of Surgery, Dart- antigen chosen in advance and can also activate a tumor-specific mouth-Hitchcock Medical Center, Lebanon, New Hampshire. T-lymphocyte response (16). Note: Supplementary data for this article are available at Clinical Cancer We have previously demonstrated that patients with metastatic Research Online (http://clincancerres.aacrjournals.org/). colorectal cancer who exhibit immune responses to a DC vaccine Corresponding Authors: Richard J. Barth Jr. Dartmouth-Hitchcock Medical have superior 5-year recurrence-free survival rate following sur- Center, One Medical Center Drive. Lebanon, NH 03756. Phone: 603-650- gical resection of liver metastases compared with counterparts in 9479; Fax: 603-650-8608; E-mail: [email protected]; and whom immune responses do not occur (63% vs. 18%, P ¼ 0.037; Christopher I. Amos. Phone: 603-650-1972; Fax: 603-650-1966; E-mail: ref. 17). Since T-cell densities in the tumor microenvironment are [email protected] well-known to correlate with survival of patients who have either doi: 10.1158/1078-0432.CCR-16-0623 primary or metastatic colorectal cancer (18, 19), we now inves- Ó2016 American Association for Cancer Research. tigated potential relationships between DC vaccine response www.aacrjournals.org 399 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst July 19, 2016; DOI: 10.1158/1078-0432.CCR-16-0623 Qian et al. Translational Relevance Materials and Methods It has been well established that the immune system can Details on the preparation of DC vaccines, detection of effectively and specifically destroy cancer cells. Immunothera- immune response, and approval for the study of human subjects fl pies are intended to augment this natural aptitude for targeting have been previously reported (17). Brie y, 22 patients with tumor antigens. Dendritic cells serve as one of the body's most colorectal cancer underwent surgical resection of liver metastases important antigen presenters that stimulate the adaptive (Table 1) within 1 to 2 months of diagnosis at the Norris Cotton immune system. Among patients with metastatic colorectal Cancer Center. Tumor lysates were derived from resected samples. cancer, approximately half respond to a dendritic cell vaccine These lysates were then added to immature DC cultures differ- in the adjuvant setting following surgical resection of metas- entiated from autologous leukopheresed monocytes that had tases. These patients have better recurrence-free survival com- been supplemented with recombinant human granulocyte mac- pared with patients who do not respond. In the present study, rophage colony-stimulating factor (GM-CSF) and recombinant resected colorectal cancer liver metastases were sequenced to human IL4. At 4, 7, and 10 weeks after surgery, tumor lysate- Â 6 identify likely mechanisms driven by somatic mutations that pulsed DCs (5 10 cells in 0.5 mL) were injected into two fi influence response to the vaccine. Our bioinformatic impli- inguinal lymph nodes of each patient. All patients were con rmed cation of PI3K/Akt/mTOR signaling and various plasma mem- to have no detectable residual disease by computed tomography brane proteins provides both a useful model for vaccine (CT) scan prior to the initial vaccine injection. Development of fi fi response inference in precision medicine (95% accurate) and immune response was de ned as observing signi cantly greater new experimental directions for exploring cancer resistance secretion of IFNg (22) or induced proliferation of T lymphocytes against immunologic elimination. (23, 24) by peripheral blood mononuclear cells 1 week following the third autologous tumor lysate-pulsed DC vaccination, com- pared with unpulsed DC stimulation. fi and densities of cytotoxic, memory, and regulatory T cells in the Inspection of T-cell in ltration fi fi context of metastatic colorectal cancer. Furthermore, we pursued The resected specimens were formalin- xed, paraf n-pre- fi genomic analyses. A recent phase II study of pembrolizumab, an served, serially sectioned, and con rmed to contain metastatic anti-programmed death 1 immune checkpoint inhibitor, for tumor by hematoxylin and eosin staining. We performed immu- colorectal cancer showed that clinical benefit is more likely to nohistochemical analysis on tumor-containing slides using the be observed in patients with tumors carrying higher mutation Leica BOND-MAX automated IHC system (Leica Microsystems) burdens due to deficiency in DNA mismatch repair (MMR; ref. 20); along with primary antibodies to human cytotoxic T cells (CD8, however, the prevalence of MMR deficiency among metastatic Novacastra #NCL-CD8-4B11), human memory T cells (CD45RO, colorectal cancer cases is only 3.5% (21). Therefore, DC vaccina- Novacastra #NCL-L-UCHL1), and human regulatory T cells tion may be an effective therapy for a larger portion (17) of such (FoxP3, BioLegend #623802). These subpopulations were spe- fi patients. To evaluate the feasibility of precision immunotherapy ci cally chosen to examine both immune-promoting (cytotoxic using this approach, we examined mutation patterns and con- and memory) and immunosuppressive (regulatory) T-cell roles structed a vaccine response prediction model on the basis of in the tumor microenvironment. Blinded to the clinical charac- colorectal cancer metastasis sequencing. teristics of patients, an experienced gastrointestinal pathologist Table 1. Characteristics
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