NK cell and IFN signatures are positive prognostic biomarkers for resectable pancreatic cancer

doi:10.1016/j.bbrc.2017.12.083

Biochemical and Biophysical Research Communications

Volume 495, Issue 2, 8 January 2018, Pages 2058-2065

https://doi.org/10.1016/j.bbrc.2017.12.083 Get rights and content

Highlights

•
Integrative immunomonitoring was undertaken in resected pancreatic cancer patients.
•
Immunomonitoring employed flow cytometry, multiplex-bead assays and RMA-Seq.
•
Higher NK cell % in peripheral blood are associated with better prognosis.
•
Type I and II IFN signatures in the tumor are associated with better prognosis.
•
Epithelial mesenchymal transition is associated with early recurrence.

Abstract

To establish prognostic biomarkers and to identify potential novel therapeutic targets, we performed integrative immunomonitoring of blood and tumor in patients with resectable pancreatic cancer. Flow cytometry (FC) was employed for phenotyping immune cells, multiplex bead assays for plasma cytokine and chemokine determination, and RNA-Seq for the analysis of gene expression in the tumor. Nineteen pancreatic cancer patients were stratified into those with longer or shorter than median recurrence-free survival after surgery (median, 426 days). There were no significant differences between the two groups for clinical parameters including age, sex, surgical procedure, stage, or postoperative adjuvant therapy. However, we found that the percentages of NK cells as assessed by FC in peripheral blood mononuclear cells were higher in patients with late recurrence (P = .037). RNA-Seq data indicated no differences in the amount of immune cells or stromal cells between the two groups, although NK cells in the tumor did tend to be higher in patients with late recurrence (P = .058). Type I and II IFN signatures were enriched in late-recurring tumors (FDR q-value <0.001), while genes related to KRAS signaling and the epithelial mesenchymal transition (EMT) were enriched in early recurrence. We conclude that tumor-intrinsic properties of metastasis and recurrence influence prognosis, whereas NK cells that might contribute to prevent metastasis are associated with longer recurrence-free survival. Therefore, enhancement of NK cell activity and inhibition of the EMT and KRAS signaling might represent appropriate therapeutic targets following surgical resection of pancreatic cancer.

Graphical abstract

Introduction

The outlook for pancreatic cancer patients after surgery remains highly unsatisfactory; approximately 23,000 Japanese die of this disease per year, making it the 4th leading cause of cancer-related deaths in the country. Recent studies have documented that adjuvant chemotherapy using gemcitabine (GEM) or S-1 can improve recurrence-free and overall survival [[1], [2], [3]]. However, the recurrence rate is >80% even after potentially curative surgery. Therefore, development of more effective therapies is urgently needed.

Recently, immune checkpoint inhibitors have shown major clinical benefit, especially in melanoma and lung cancer. However, pancreatic cancer remains poorly responsive to checkpoint blockade [4]. Marked infiltration of immunosuppressive cells into the dense desmoplastic tumor stroma is observed in most pancreatic cancer patients, and intratumoral infiltration of effector cells is commonly very poor. It has been shown that prognosis is better for resectable pancreatic cancer patients whose tumors have more T-cell infiltration. However, the immunological profile and determinants of immune activation in pancreatic cancers are still poorly understood. Novel immunotherapeutic approaches could emerge from a better understanding of these issues.

Here, we performed integrative immunomonitoring of peripheral blood and tumor from resectable pancreatic cancer patients. We sought immunological factors for predicting the prognosis after surgery, which might reveal novel potential targets for pancreatic cancer immunotherapy.

Section snippets

Patient selection

From August 2012 to December 2013, 45 patients with pancreatic cancers underwent surgical resections at the Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery at the University of Tokyo Hospital. Of these, patients with R0 or R1 resections were selected. Patients who were receiving systemic steroid therapy which affects host immune system were excluded. Double cancer cases were also excluded from the study.

Sample preparation, RNA sequencing and data processing

Pancreatic tumors and peripheral blood from the same patients were

Patients

A total of 19 consenting pancreatic cancer patients who received surgery (R0/R1 resections) were analyzed in this study. The patients were 14 males and 5 females, with a median age of 70 years (range, 44–80 years) (Table 1). Pancreaticoduodenectomy was performed in 9 patients, distal pancreatectomy in 9, and total pancreatectomy in the remaining patient. As postoperative adjuvant therapy, 3 patients received GEM alone and 6 a combination of GEM plus γδ T cells in a phase I clinical trial at our

Discussion

In the present study, we performed flow cytometry for the relative quantification of different subsets of immune cells and multiplex bead assays for plasma cytokines and chemokines in the peripheral blood of patients with resectable pancreatic cancer. Furthermore, RNA-Seq gene expression profiling of the tumor was undertaken. By integrating these data into a comprehensive immunomonitoring paradigm, we identified certain immunological features in the periphery and the tumor microenvironment as

Financial support

Funding: This study was supported in part by JSPS KAKENHI (Grants-in-Aid for Scientific Research) Grant Numbers 16H04708 (to K.K.) and 16K07162 (to H.M.) and Pancreas Research Foundation of Japan (to T.A.).

Acknowledgements

We thank Mr. Atsushi Kondo and Tamaki Iino (Department of Immunotherapeutics, The University of Tokyo Hospital) for their wonderful technical assistance.

References (27)

K. Uesaka et al.
Adjuvant chemotherapy of S-1 versus gemcitabine for resected pancreatic cancer: a phase 3, open-label, randomised, non-inferiority trial (JASPAC 01)
Lancet
(2016)
A. Liberzon et al.
The Molecular Signatures Database (MSigDB) hallmark gene set collection
Cell Syst
(2015)
T. Aoki et al.
Adjuvant combination therapy with gemcitabine and autologous gammadelta T-cell transfer in patients with curatively resected pancreatic cancer
Cytotherapy
(2017)
D. Shafer et al.
Low NK cell counts in peripheral blood are associated with inferior overall survival in patients with follicular lymphoma
Leuk. Res.
(2013)
B. Perussia
Lymphokine-activated killer cells, natural killer cells and cytokines
Curr. Opin. Immunol.
(1991)
S.L. Topalian et al.
Immune checkpoint blockade: a common denominator approach to cancer therapy
Cancer Cell
(2015)
H. Oettle et al.
Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial
J. Am. Med. Assoc.
(2007)
H. Oettle et al.
Adjuvant chemotherapy with gemcitabine and long-term outcomes among patients with resected pancreatic cancer: the CONKO-001 randomized trial
J. Am. Med. Assoc.
(2013)
J.R. Brahmer et al.
Safety and activity of anti-PD-L1 antibody in patients with advanced cancer
N. Engl. J. Med.
(2012)
D. Kim et al.
TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions
Genome Biol.
(2013)

C. Trapnell et al.

Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation

Nat. Biotechnol.

(2010)

E. Becht et al.

Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression

Genome Biol.

(2016)

A. Subramanian et al.

Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

Proc Natl Acad Sci U S A

(2005)

Cited by (33)

Immunocyte profiling changes in patients received epidural versus intravenous analgesia after pancreatectomy: A randomized controlled trial
2024, Journal of the Formosan Medical Association
Perioperative immunosuppressants, such as surgical stress and opioid use may downregulate anti-cancer immunocytes for patients undergoing pancreatectomy. Thoracic epidural analgesia (TEA) may attenuate these negative effects and provide better anti-cancer immunocyte profile change than intravenous analgesia using opioid.
We randomly assigned 108 adult patients undergoing pancreatectomy to receive one of two 72-h postoperative analgesia protocols: one was TEA, and the other was intravenous patient-controlled analgesia (IV-PCA). The perioperative proportional changes of immunocytes relevant to anticancer immunity—namely natural killer (NK) cells, cytotoxic T cells, helper T cells, mature dendritic cells, and regulatory T (Treg) cells were determined at 1 day before surgery, at the end of surgery and on postoperative day 1,4 and 7 using flow cytometry. In addition, the progression-free survival and overall survival between the two groups were compared.
After surgery, the proportions of NK cells and cytotoxic T cells were significantly decreased; the proportion of B cells and mature dendritic cells and Treg cells were significantly increased. However, the proportions of helper T cells exhibited no significant change. These results were comparable between the two groups. Furthermore, there were no significant differences in progression-free survival (52.75 [39.96] and 57.48 [43.66] months for patients in the TEA and IV–PCA groups, respectively; p = 0.5600) and overall survival (62.71 [35.48] and 75.11 [33.10] months for patients in the TEA and IV–PCA groups, respectively; p = 0.0644).
TEA was neither associated with favorable anticancer immunity nor favorable oncological outcomes for patients undergoing pancreatectomy.
Natural killer cells for pancreatic cancer immunotherapy: Role of nanoparticles
2023, Cancer Letters
Advanced pancreatic cancer patients have a dismal prognosis despite advances in integrative therapy. The field of tumor immunology has witnessed significant advancements for cancer treatment. However, immunotherapy for pancreatic cancer is not very effective due to its highly complex tumor microenvironment (TME). Natural killer (NK) cells are lymphocytes that play an important role in the innate immune system. NK cells do not require antigen pre-sensitization, nor are they confined by the major histocompatibility complex (MHC). NK cells have the potential to eliminate cancer cells through CAR-dependent and CAR-independent pathways, demonstrating reduced levels of systemic toxicity in the process. The availability of several potential sources of NK cells is an additional benefit that contributes to meeting the therapeutic criteria. Adding nanotechnology to enhance the functions of effector NK cells is also an appealing strategy. This article primarily discusses various approaches recently been utilized to enhance the NK functions for the treatment of pancreatic cancer. In addition, new advances in boosting NK cell therapeutic efficacy by nanoparticle mediation are presented, with a focus on pancreatic cancer.
Biological characteristics of pancreatic ductal adenocarcinoma: Initiation to malignancy, intracellular to extracellular
2023, Cancer Letters
Pancreatic ductal adenocarcinoma (PDAC) is a highly life-threatening tumour with a low early-detection rate, rapid progression and a tendency to develop resistance to chemotherapy. Therefore, understanding the regulatory mechanisms underlying the initiation, development and metastasis of pancreatic cancer is necessary for enhancing therapeutic effectiveness. In this review, we summarised single-gene mutations (including KRAS, CDKN2A, TP53, SMAD4 and some other less prevalent mutations), epigenetic changes (including DNA methylation, histone modifications and RNA interference) and large chromosome alterations (such as copy number variations, chromosome rearrangements and chromothripsis) associated with PDAC. In addition, we discussed variations in signalling pathways that act as intermediate oncogenic factors in PDAC, including PI3K/AKT, MAPK/ERK, Hippo and TGF-β signalling pathways. The focus of this review was to investigate alterations in the microenvironment of PDAC, particularly the role of immunosuppressive cells, cancer-associated fibroblasts, lymphocytes, other para-cancerous cells and tumour extracellular matrix in tumour progression. Peripheral axons innervating the pancreas have been reported to play a crucial role in the development of cancer. In addition, tumour cells can influence the behaviour of neighbouring non-tumour cells by secreting certain factors, both locally and at a distance. In this review, we elucidated the alterations in intracellular molecules and the extracellular environment that occur during the progression of PDAC. Altogether, this review may enhance the understanding of the biological characteristics of PDAC and guide the development of more precise treatment strategies.
Rationally designed redirection of natural killer cells anchoring a cytotoxic ligand for pancreatic cancer treatment
2020, Journal of Controlled Release
Citation Excerpt :
A decrease in circulating NK cells reportedly is associated with an increased cancer risk [25]. In addition, increased infiltrative NK cells in the tumor reportedly are positively correlated with the prognosis of lung, renal cell, colorectal, and pancreatic cancer [26,27]. In contrast to T lymphocytes, NK cells recognize and directly kill tumor cells without prior specific sensitization, and they have great potential for use as “off-the-shelf” allogeneic products because of the absence of graft-versus-host disease [28].
The emergence of T-cell engineering with chimeric antigen receptors (CARs) has led to attractive therapeutics; however, autologous CAR-T cells are associated with poor clinical outcomes in solid tumors because of low safety and efficacy. Therefore, the aim of our study was to develop a CAR therapy with enhanced cytotoxicity against solid cancer using allogeneic NK cells. In this study, we engineered “off-the-shelf” NK cells to redirect them towards pancreatic ductal adenocarcinoma (PDAC) by improving their target-specific cytotoxic potential. By integrated bioinformatic and clinicopathological analyses, folate receptor alpha (FRα) and death receptor 4 (DR4) were significantly highly expressed in patient-derived tumor cells. The combined expression of FRα and DR4/5 was associated with inferior clinical outcomes, therefore indicating their use as potential targets for biomolecular treatment. Thus, FRα and DR4 expression pattern can be a strong prognostic factor as promising therapeutic targets for the treatment of PDAC. For effective PDAC treatment, allogeneic CAR-NK cells were reprogrammed to carry an apoptosis-inducing ligand and to redirect them towards FRα and initiate DR4/5-mediated cancer-selective cell death in FRα- and DR4/5-positive tumors. As a result, the redirected cytotoxic ligand-loaded NK cells led to a significantly enhanced tumor-selective apoptosis. Accordingly, use of allogeneic CAR-NK cells that respond to FRα and DR4/5 double-positive cancers might improve clinical outcomes based on personal genome profiles. Thus, therapeutic modalities based on allogeneic NK cells can potentially be used to treat large numbers of patients with optimally selective cytotoxicity.
Natural Killer Cells Suppress T Cell-Associated Tumor Immune Evasion
2019, Cell Reports
Citation Excerpt :
Encouraging results using NK cells as a target for immunotherapy have been observed over recent years (Chiossone et al., 2018; Souza-Fonseca-Guimaraes et al., 2019). Additionally, the identification of NK cell signatures and improved patient prognosis in several cancer types has spurred a variety of clinical trials investigating the potential of NK cell therapies (Ascierto et al., 2013; Cursons et al., 2019; Hoshikawa et al., 2018; Pasero et al., 2015). The CRISPR/Cas9 system has proven to be a powerful technique to identify tumor-specific genes that are important for both sensitization and resistance to killing by CD8+ T cells through a loss-of-function-based screening approach.
Despite the clinical success of cancer immunotherapies, the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here, we conducted a series of unbiased, genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-γ (IFN-γ) signaling protect tumor cells from NK cell killing. Indeed, Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-γ-driven transcriptional events that regulate MHC I expression. Importantly, tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together, we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells, unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape.
Building a Better Defense: Expanding and Improving Natural Killer Cells for Adoptive Cell Therapy
2024, Cells

View all citing articles on Scopus

View full text

NK cell and IFN signatures are positive prognostic biomarkers for resectable pancreatic cancer

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Patient selection

Sample preparation, RNA sequencing and data processing

Patients

Discussion

Financial support

Acknowledgements

Lancet

Cell Syst

Cytotherapy

Leuk. Res.

Curr. Opin. Immunol.

Cancer Cell

Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial

J. Am. Med. Assoc.

Adjuvant chemotherapy with gemcitabine and long-term outcomes among patients with resected pancreatic cancer: the CONKO-001 randomized trial

J. Am. Med. Assoc.

Safety and activity of anti-PD-L1 antibody in patients with advanced cancer

N. Engl. J. Med.

TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions

Genome Biol.

Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation

Nat. Biotechnol.

Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression

Genome Biol.

Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles

Proc Natl Acad Sci U S A