Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States (Siegel et al., 2021). Surgical resection is the only curative treatment for PDAC. However, the poor postoperative prognosis is usually common for most patients because of the locoregional recurrence and distant metastasis (Mizrahi et al., 2020). Several studies have investigated recurrence after curative resection of PDAC. And more than half of patients with pancreatic cancer develop hepatic recurrences after surgery, according to the literature (Sperti et al., 1997; Van den Broeck et al., 2009). In another study, which evaluated 692 patients with comprehensive and detailed follow-up data, more than a quarter of patients had only liver metastasis after surgery, with a median duration of 6.9 months. Meanwhile, the author also found that the lung-only recurrence occurred later, with a median time of 18.6 months (Groot et al., 2018b). Similar results were confirmed in another study, suggesting that early liver metastasis after surgery is a risk factor for shorter survival in pancreatic cancer patients (Kolbeinsson et al., 2020). Thus, it indicated that postoperative liver metastasis (especially liver-only recurrence) is one of the reasons for the poor prognosis of pancreatic cancer patients after surgery. Moreover, the molecular phenotype and biological behavior of tumors may vary significantly among different types of metastases.

As a result, accurate prediction of the risk of postoperative liver metastasis and identification of therapeutic targets are of great significance for guiding the postoperative treatment of pancreatic cancer, and gene-level analysis can be the most profound interpretation of the molecular characteristics associated with liver metastasis. Therefore, the purpose of this study was to investigate the possible molecular mechanisms of postoperative liver metastasis of pancreatic cancer to find potential molecular targets.

Pancreatic cancer is a malignant tumor of the digestive system. Due to local recurrence and distant metastasis, the postoperative prognosis of most patients is poor. Distinct locations of the first recurrence have unique independent predictive values for OS, which is helpful for stratification of prognosis and treatment decision after diagnosis of recurrence (Groot et al., 2018a). Several studies have reported the recurrence patterns after pancreatectomy for PDAC. Most postoperative patients with pancreatic cancer recurred at isolated distant sites, and liver recurrence tended to occur earlier (Groot et al., 2018b). The patterns of postoperative liver recurrence could be used as a basis for specific surveillance and treatment of patients with pancreatic cancer (Tanaka et al., 2019), which was verified by a meta-analysis including 89 studies with 17,313 patients undergoing PDAC resection. In this study, we presented an integrated view of hepatic recurrence after pancreatectomy at the molecular level.

We examined TCGA gene expression profile to identify potential biomarkers for postoperative liver metastasis of pancreatic cancer. Based on the KEGG analysis of DEGs, we found that DGEs were enriched highly in cytokine–cytokine receptor interaction. GO enrichment analysis showed that DEGs were significantly enriched in immune response and adaptive immune response. Furthermore, the KEGG analysis results revealed that key genes in the PPI network regarding DGEs were mostly enriched in the chemokine signaling pathway. In GO analysis, the enrichment of these genes mainly functioned to G-protein coupled receptor signaling pathway, inflammatory response, and immune response. Muller et al. (2001) validated that chemokines and their receptors play a vital role in determining the direction of tumor cell metastasis. G-protein coupled receptor kinases and receptors are also involved in tumorigenesis and cancer aggressiveness (O’Hayre et al., 2013; Wang et al., 2017; Nogues et al., 2018; Brown and Ganapathy, 2020). The immune response is implicated in tumor progression and metastasis in various ways (Berraondo et al., 2016; O’Sullivan et al., 2019; Riera-Domingo et al., 2020). It can protect the host from virus-induced tumors (Reiche et al., 2004), inhabit the inflammatory environment of tumorigenesis (Ebbin, 1989; Elinav et al., 2013), and identify specific antigens of the tumor to eliminate cancer cells (Medzhitov, 2007; Bommareddy et al., 2018). However, as T cells (Chang et al., 2015), NKT (Keating et al., 2016; Cong et al., 2018), macrophages, and dendritic cells (O’Neill and Pearce, 2016) use glucose to support their effector functions, malignant cells can deprive tumor microenvironment (TME) of glucose, thus blocking effective anticancer immunity (Badur and Metallo, 2018). Pancreatic cancer is characterized by dense fibrotic stroma, which promotes the generation of the immunosuppressive, hypoxic, and nutrient-poor TME (Kamphorst et al., 2015).

Analysis with ESTIMATE score also suggested that the recurrence-free group had significantly higher immune and stroma scores, which showed higher infiltration levels of B cells, CD4⁺ naive T cells, myeloid dendritic cells, and NKT cells. However, Th2 CD4⁺ T cells, common myeloid progenitor cells, were higher in the liver metastasis group. These results imply that the occurrence of liver metastasis after pancreatectomy for PDAC is closely correlated with immune cell infiltration. Solid tumors, namely, breast cancer, renal cell carcinoma, melanoma, ovarian cancer, and gastrointestinal stromal tumors, have been shown to have spontaneous T cell infiltration (Zhang et al., 2003; Kreike et al., 2007; Mahmoud et al., 2011; Azimi et al., 2012; Rusakiewicz et al., 2013). In the TME of ovarian cancer, the ratio of CD8⁺ T cells to Foxp3⁺ regulatory T cells was significantly high, which was associated with a particularly favorable prognosis (Curiel et al., 2004).

Our further WGCNA showed that the brown module was correlated with DFS and OS positively. In addition, we found that the function of key module genes was highly enriched in the chemokine signaling pathway by KEGG pathway analysis and in the inflammatory response and immune response by GO analysis. However, the turquoise module and the purple module were correlated with DFS and OS negatively. The function analysis in the purple module possessed cell cycle in KEGG pathway analysis and mitotic nuclear division, cell division, and cell proliferation in GO analysis. Furthermore, in the purple module, the top five genes (CDK1, PLK1, AURKB, CENPN, and KIF2C) showed an inverse relationship between gene expression and prognosis, and their expression was closely related to postoperative recurrence and OS.

Among the top five genes, PLK1 was reported to be closely correlated with pancreatic cancer. Polo-like kinase 1 (Plk1), a serine/threonine kinase of polo-like kinases family, plays an essential role in cell mitosis, spindle assemblies, DNA damage, and so on (Lens et al., 2010; Strebhardt, 2010). Weichert et al. (2005) have reported that PLK1 was found to be overexpressed in pancreatic neoplasia as early as in pancreatic intraepithelial neoplasia III lesions and invasive pancreatic adenocarcinomas were PLK1, strongly positive in 47.7% of cases. Additionally, a study suggested that PLK1 showed >50% downregulation in gemcitabine sensitive cases and no change in the resistant cases (Jimeno et al., 2010). Correspondingly, inhibition of PLK1 also synergized with gemcitabine in gemcitabine-refractory in vitro (Jimeno et al., 2010) and in vivo models (Li et al., 2016). Moreover, the combination of PI3K/Akt pathway inhibition and PLK1 depletion can enhance chemosensitivity to gemcitabine in pancreatic cancer (Mao et al., 2018). According to the above results, PLK1 was proposed as a promising target for the treatment of gemcitabine-resistant pancreatic cancer. In a phase I trial, rigosertib, a PLK1 inhibitor, was combined with gemcitabine to treat patients with advanced PDAC (Ma et al., 2012). This phase I study determined the recommended phase II dose (RPTD), and under this, RPTD rigosertib is well tolerated with a toxicity profile similar to the gemcitabine alone. After demonstrating the safety of rigosertib, a phase II/III randomized study was then conducted (O’Neil et al., 2015). This phase II/III randomized study was designed to compare the efficacy and safety of rigosertib plus gemcitabine vs. gemcitabine alone in patients with metastatic pancreatic cancer. However, the combination of rigosertib plus gemcitabine failed to improve prognosis or response compared with gemcitabine alone in patients with metastatic pancreatic adenocarcinoma. The failure may be owed to the inherent heterogeneity of pancreatic cancer, and more comprehensive research regarding this topic is urgently needed. Hence, if overexpression of PLK1 for patients with pancreatic cancer after pancreatectomy was detected, non-gemcitabine chemotherapy would be preferred to avoid potential chemotherapeutic resistance. If gemcitabine-based chemotherapy has to be applied, PLK1 inhibitor could be considered to combine with it, and the resulting combination is safe and theoretically helpful.

Although we found that reduced immune cell infiltration was strongly associated with postoperative liver metastasis of pancreatic cancer, the internal mechanism still needs further investigation. The direct immune killing effect on tumor cells may be the main reason. Furthermore, previous studies have shown that tumor metastasis is closely related to the epithelial mesenchymal transition (EMT) process of tumor cells rather than tumor cell proliferation. However, our study showed that the proliferation of primary tumor cells was significantly increased in patients with postoperative liver metastasis. Therefore, we hypothesized that some patients with pancreatic cancer already had preclinical liver micro-metastases, which could not be detected by current detection methods. Such micro-metastases showed liver metastasis recurrence at different times after surgery due to the different proliferation abilities of tumor cells. According to our clinical tissue samples, patients with early postoperative liver metastasis had significantly more proliferative primary tumors than patients without recurrence.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University. The patients/participants provided their written informed consent to participate in this study.

GW designed and drafted the manuscript. SD and HG conducted the analyses. YG, LY, KZ, and XH conducted the literature search. ZL and YM designed, led, supervised the study, and critically revised the manuscript. All authors approved the final draft submitted, read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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