Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.

Neoantigen quality predicts immunoediting in survivors of pancreatic cancer.

Cancer immunoediting1 is a hallmark of cancer2 that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice1,3, whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features-'non-selfness'  based on neoantigen similarity to known antigens4,5, and 'selfness'  based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer.

Antitumor Effect of 5-Aminolevulinic Acid Through Ferroptosis in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Due to its tumor-specific metabolic pathway characteristics, 5-aminolevulinic acid (5-ALA) is a natural amino acid widely used in cancer treatment. The current study, demonstrated that 5-ALA induced ferroptosis via glutathione peroxidase 4 (GPX4) and heme oxygenase 1 (HMOX1) and had an antitumor effect in esophageal squamous cell carcinoma (ESCC). METHODS: Expression of GPX4 and HMOX1 in pathologic specimens of 97 ESCC patients was examined, and prognostic analyses were performed. Real-time polymerase chain reaction (RT-PCR), RNA microarray, and Western blotting analyses were used to evaluate the role of 5-ALA in ferroptosis in vitro. In addition, this study used ferrostatin-1, a ferroptosis inhibitor, and a lipid peroxidation reagent against cell lines treated with 5-ALA. Finally, the role of 5-ALA was confirmed by its effect on an ESCC subcutaneous xenograft mouse model. RESULTS: The study showed that upregulation of GPX4 and downregulation of HMOX1 were poor prognostic factors in ESCC. In an RNA microarray analysis of KYSE30, ferroptosis was one of the most frequently induced pathways, with GPX4 suppressed and HMOX1 overexpressed by 5-ALA treatment. These findings were verified by RT-PCR and Western blotting. Furthermore, 5-ALA led to an increase in lipid peroxidation and exerted an antitumor effect in various cancer cell lines, which was inhibited by ferrostatin-1. In vivo, 5-ALA suppressed GPX4 and overexpressed HMOX1 in tumor tissues and led to a reduction in tumor size. CONCLUSIONS: Modulation of GPX4 and HMOX1 by 5-ALA induced ferroptosis in ESCC. Thus, 5-ALA could be a promising new therapeutic agent for ESCC.

NEAT1 is Required for the Expression of the Liver Cancer Stem Cell Marker CD44.

CD44, a cancer stem cell (CSC) marker, is required for maintaining CSC properties in hepatocellular carcinoma (HCC). Nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), is an oncogenic driver in HCC. In the present study, we investigated the significance of the NEAT1 gene in association with CD44 expression in liver CSCs of human HCC cell lines. The CSC properties were evaluated by spheroid culture, CSC marker expression, and sensitivity to anti-cancer drugs. The expression of both NEAT1 variant 1 (NEAT1v1) and variant 2 (NEAT1v2) as well as CD44 was significantly increased in the spheroid culture, compared with that in monolayer culture. Overexpression of Neat1v1, but not Neat1v2, enhanced the CSC properties, while knockout of the NEAT1 gene suppressed them. CD44 expression was increased by the overexpression of Neat1v1 and abrogated by NEAT1 knockout. The overexpression of NEAT1v1 restored the CSC properties and CD44 expression in NEAT1-knockout cells. NEAT1v1 expression in HCC tissues was correlated with poor prognosis and CD44 expression. These results suggest that NEAT1v1 is required for CD44 expression. To our surprise, NEAT1v1 also restored the CSC properties even in CD44-deficient cells, suggesting that NEAT1v1 maintains the properties of CSCs in a CD44-independent manner.

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promotes immortalization by protecting telomeres in cancer cells. Mutation of the TERT promoter is one of the most common genetic alterations in hepatocellular carcinoma (HCC), indicating that TERT upregulation is a critical event in hepatocarcinogenesis. Regulators of TERT transcription are, therefore, predicted to be plausible targets for HCC treatment. We undertook a genome-wide shRNA library screen and identified C15orf55 and C7orf43 as regulators of TERT expression in HepG2 cells. Promoter assays showed that C15orf55- and C7orf43-responsive sites exist between base pairs -58 and +36 and -169 and -59 in the TERT promoter, respectively. C15orf55 upregulates TERT expression by binding to two GC motifs in the SP1 binding site of the TERT promoter. C7orf43 upregulates TERT expression through Yes-associated protein 1. The expression levels of C15orf55 and C7orf43 also correlated with that of TERT, and were significantly increased in both HCC tissues and their adjacent non-tumor tissues, compared to normal liver tissues from non-HCC patients. Analysis of 377 HCC patients in The Cancer Genome Atlas dataset showed that overall survival of patients with low levels of C15orf55 and C7orf43 expression in tumor tissues was better compared with patients with high levels of C15orf55 and/or high C7orf43 expression. These results indicate that C15orf55 and C7orf43 are involved in the incidence and progression of HCC by upregulating TERT. In conclusion, we identified C15orf55 and C7orf43 as positive regulators of TERT expression in HCC tissues. These genes are promising targets for HCC treatment.

Bacterial smear test of drainage fluid after pancreaticoduodenectomy can predict postoperative pancreatic fistula.

OBJECTIVES: It is widely accepted that postoperative pancreatic fistula (POPF) accompanied by bacterial infection results in a worse outcome than POPF alone. However, few studies evaluating predictive indicators of POPF have focused on bacterial infection. METHODS: A consecutive 100 patients who underwent pancreaticoduodenectomy at our institute for periampullary disease were enrolled. POPF was assessed according to the International Study Group of Pancreatic Fistula consensus guidelines; grades B and C were defined as clinically relevant POPF (CR-POPF). The patients' characteristics, perioperative surgical factors, and laboratory data including the results of culture and smear testing performed using drainage fluid on postoperative days (PODs) 1 and 3 were analyzed. RESULTS: The overall incidence of CR-POPF was 25%. Univariate analyses revealed that the factors associated with CR-POPF were male sex, soft pancreas, MPD diameter, higher serum C-reactive protein concentration and white blood cell count on POD 3, higher amylase concentration in drainage fluid, and culture and/or smear positivity of drainage fluid. Multivariate analysis newly revealed that the smear positivity of drainage fluid on POD 3 was the independent risk factors for CR-POPF (p = 0.027). CONCLUSIONS: Smear positivity of drainage fluid on POD 3 after pancreaticoduodenectomy may be a new predictor of CR-POPF.

Impact of postoperative mean arterial pressure on the incidence of postoperative complications after hepatic resection for primary liver malignancy.

PURPOSE: We conducted this study to evaluate the impact of the postoperative mean arterial pressure (MAP) on surgical complications after hepatic resection. METHODS: The subjects of this study were 199 patients who underwent hepatic resection for primary liver malignancy between 2004 and 2013. A clinically relevant postoperative complication was defined as a Clavien-Dindo grade ≥ III complication. RESULTS: Based on an MAP cut-off value of 81.1 mmHg, the patients were grouped as follows: low MAP on both postoperative days (PODs) 1 and 2 (continuously low MAP), normal MAP on both PODs 1 and 2 (normal MAP), and others (transiently low MAP). The continuously low MAP group had the highest incidence of complications and the normal MAP group had the lowest incidence of complications compared with the expected incidence for this cohort (p < 0.01 and p = 0.01, respectively). Multivariate analysis revealed that both a continuously and transiently low MAP were independent predictors of postoperative complications (p = 0.03 and p < 0.01, respectively). Among the subtypes of complications, a low MAP had a significant relationship with ascites/pleural effusion and respiratory complications (p < 0.01 and p = 0.03, respectively). CONCLUSIONS: A low MAP on POD 1 and/or 2 is an independent predictor of postoperative complications.

Combined preoperative platelet-to-lymphocyte ratio and serum carbohydrate antigen 19-9 level as a prognostic factor in patients with resected pancreatic cancer.

BACKGROUND: Carbohydrate antigen 19-9 (CA19-9) is the most frequently used tumor marker and serves as a prognostic indicator in patients with pancreatic cancer (PC). The platelet-to-lymphocyte ratio (PLR) is thought to be an inflammation-related serum marker. An elevated PLR represents increased inflammatory status and is associated with poor prognosis in patients with various cancers including PC. METHODS: This study involved 103 patients with a histopathological diagnosis of pancreatic ductal adenocarcinoma who underwent pancreatectomy. The patients were assessed to determine the prognostic significance of the combination of the PLR and CA19-9 level. RESULTS: Based on the receiver operating characteristic analysis results, the patients were divided into PLRHigh (PLR ≥ 129.1) and PLRLow (PLR < 129.1) groups and into CA19-9High (CA19-9 ≥ 74.0 U/mL) and CA19-9Low (CA19-9 < 74.0 U/mL) groups. The cumulative 5-year overall survival (OS) and disease-specific survival (DSS) rates significantly differed by both the PLR (PLRHigh group: 19.5% and 22.9%; PLRLow group: 39.1% and 45.9%) and CA19-9 (CA19-9High group: 19.1% and 25.6%; CA19-9Low group: 41.0% and 41.0%). We then divided the patients into Groups A (PLRLow/CA19-9Low), B (PLRLow/CA19-9High or PLRHigh/CA19-9Low), and C (PLRHigh/CA19-9High). The cumulative 5-year OS rates in Groups A, B, and C were 44.0%, 31.9%, and 11.9%, respectively (P = 0.002). The cumulative 5-year DSS rates in Groups A, B, and C were 47.7%, 36.4%, and 16.8%, respectively (P = 0.002). Multivariate analysis revealed that the combination of the PLR and CA19-9 was an independent prognostic factor in patients with resected PC. CONCLUSIONS: The combination of the PLR and CA19-9 is useful for predicting the prognosis of patients with resected PC.

HBx and c-MYC Cooperate to Induce URI1 Expression in HBV-Related Hepatocellular Carcinoma.

Unconventional prefoldin RNA polymerase II subunit 5 interactor (URI1) has emerged as an oncogenic driver in hepatocellular carcinoma (HCC). Although the hepatitis B virus (HBV) represents the most common etiology of HCC worldwide, it is unknown whether URI1 plays a role in HBV-related HCC (HCC-B). In the present study, we investigated URI1 expression and its underlying mechanism in HCC-B tissues and cell lines. URI1 gene-promoter activity was determined by a luciferase assay. Human HCC-B samples were used for a chromatin immunoprecipitation assay. We found that c-MYC induced URI1 expression and activated the URI1 promoter through the E-box in the promoter region while the HBx protein significantly enhanced it. The positivity of URI1 expression was significantly higher in HCC-B tumor tissues than in non-HBV-related HCC tumor tissues, suggesting that a specific mechanism underlies URI1 expression in HCC-B. In tumor tissues from HCC-B patients, a significantly higher level of c-MYC was recruited to the E-box than in non-tumor tissues. These results suggest that HBx and c-MYC are involved in URI1 expression in HCC-B. URI1 expression may play important roles in the development and progression of HCC-B because HBx and c-MYC are well-known oncogenic factors in the virus and host, respectively.

The attenuation value of preoperative computed tomography as a novel predictor for pancreatic fistula after pancreaticoduodenectomy.

PURPOSE: Pancreatic fistula (PF) is the most serious complication following pancreaticoduodenectomy (PD). This study was performed to identify new clinical factors that may predict the development of PF after PD to improve perioperative management. METHODS: Seventy-five consecutive patients who underwent PD from 2012 to 2015 were evaluated. The patients' perioperative data including the computed tomography (CT) parameters were collected. The minimum, maximum, and mean CT attenuation values (HUmin, HUmax, and HUmean, respectively) were extracted from the pancreatic parenchyma (≥ 100 pixels), and the standard deviation of these values (HUSD) was determined from the slice in which the superior mesenteric and splenic veins were merged. PF was defined as grade B or C according to the International Study Group for Pancreatic Fistula criteria. RESULTS: The PF occurrence rate (grade B or C) was 25.3% in 75 patients. A multivariate analysis identified a larger HUSD (odds ratio 3.092; 95% CI 1.018-9.394) and higher amylase concentration in drainage fluid on postoperative day 1 (odds ratio 1.0001; 95% CI 1.00001-1.00022) as significant risk factors for PF. CONCLUSIONS: The HUSD of preoperative CT attenuation values in the pancreatic parenchyma was found to be an independent predictor for PF after PD and it might therefore positively contribute to the perioperative management of PD.

Prognostic value of postoperative complication for early recurrence after curative resection of hepatocellular carcinoma.

BACKGROUND: Postoperative complications may adversely affect oncological outcomes. The aim of this study was to evaluate the impact of postoperative complications on early-phase recurrence after curative resection for hepatocellular carcinoma (HCC). METHODS: We included 145 HCC patients who underwent initial and curative resection between January 2004 and December 2013. Postoperative complications of grade III or higher based on Clavien-Dindo classification were defined as clinically relevant postoperative complications. Recurrence within two years after hepatectomy was defined as early-phase recurrence. RESULTS: Thirty-eight patients (26%) developed postoperative complications. The only predictive factor for postoperative complication was longer operative duration (P = 0.037). The disease-specific survival rate of patients with complication was lower than that of patients without complications (P = 0.015). Early-phase recurrence was observed in 20/38 (53%) patients who suffered postoperative complications and 36/107 (34%) patients with no complications, which was statistically significant (P = 0.039). Multivariate analysis identified four factors contributing to early-phase recurrence: high serum AFP level (P = 0.042), multiple tumors (P < 0.001), poor differentiation (P = 0.036) and presence of postoperative complication (P = 0.039). CONCLUSIONS: Postoperative complication is an independent prognostic factor for early-phase recurrence after curative resection of HCC. Close observation of patients with postoperative complications may be a necessary treatment strategy for HCC.

Post-operative albumin-bilirubin grade predicts long-term outcomes among Child-Pugh grade A patients with hepatocellular carcinoma after curative resection.

BACKGROUND: Although Child-Pugh grade A patients with hepatocellular carcinoma (HCC) are candidates for curative resection, some may have a poor prognosis. The albumin-bilirubin (ALBI) grade, a measure of liver function based on albumin and bilirubin, has the potential to detect Child-Pugh grade A HCC patients with poor prognosis. Because components of the ALBI grade can be measured easily even after surgery, we explored the predictive values of ALBI in patient prognosis after HCC resection. METHODS: In this retrospective case-control study, we included 136 HCC patients who underwent curative resection between January 2004 and December 2013 at our hospital. ALBI grade was calculated from laboratory data recorded the day before surgery and at post-operative day 5. RESULTS: Pre- and post-operative ALBI grade predicted patients' long-term outcomes (P = 0.020 and P < 0.001, respectively, for overall survival, and P = 0.012 and P = 0.015, respectively, for recurrence-free survival). Post-operative ALBI grade was associated with patients' surgical factors of repeated hepatic resection (P = 0.012), intra-operative bleeding (P = 0.006), and surgery duration (P = 0.033). Furthermore, post-operative ALBI grade, rather than pre-operative ALBI grade, was an independent predictive factor of long-term outcome of Child-Pugh grade A patients with HCC. CONCLUSIONS: Post-operative ALBI grade is useful to predict the prognosis in patients after HCC resection.

Nicotine enhances the malignant potential of human pancreatic cancer cells via activation of atypical protein kinase C.

BACKGROUND: Pancreatic cancer (PC) is the most lethal malignancy among solid tumors, and the most common risk factor for its development is cigarette smoking. Atypical protein kinase C (aPKC) isozymes function in cell polarity, proliferation, and survival, and have also been implicated in carcinogenesis. However, the involvement of aPKC in PC progression and the effect of nicotine, a major component of cigarette smoke, on the biological activities of aPKC remain to be fully elucidated. METHODS: We investigated the effects of nicotine on the proliferation, migration and invasion of the human PC cell lines Panc1 and BxPC3. We analyzed aPKC localization and activity by immunohistochemistry and in vitro kinase assays, respectively, to assess their involvement in the regulation of PC progression. Moreover, we examined the effect of nicotine on implanted peritoneal tumors of PC cells in mice. RESULTS: Nicotine enhanced cell proliferation, migration and invasion in Panc1 and BxPC3 cells. In nicotine-treated PC cells, the aPKC was significantly activated. We also found that nicotine induced phosphatidylinositol 3-kinase (PI3K) signal activation, and a specific inhibitor of the nicotine acetylcholine receptor (nAChR) as well as knockdown of nAChR prevented nicotine-mediated Akt phosphorylation and aPKC activation. In a peritoneal dissemination model of PC, nicotine-treated mice had larger tumors and increased numbers of nodules. Immunohistochemistry showed enhanced expression levels of aPKC and phosphorylated Akt in nodules from nicotine-treated mice. CONCLUSIONS AND GENERAL SIGNIFICANCE: Nicotine induces aberrant activation of aPKC via nAChR/PI3K signaling in PC cells, resulting in enhancement of cellular proliferation, migration and invasion.

Comparison of single-stapling and hemi-double-stapling methods for intracorporeal esophagojejunostomy using a circular stapler after totally laparoscopic total gastrectomy.

BACKGROUND: Laparoscopic total gastrectomy is not widely performed because of the difficulty of esophagojejunal reconstruction. This study analyzed complication rates of two different methods for reconstruction by a circular stapler after totally laparoscopic total gastrectomy (TLTG). METHODS: Between 2010 and 2014, clinical data of 19 patients who underwent TLTG for gastric adenocarcinoma were collected retrospectively. There were two methods to fix the anvil of a circular stapler into the distal esophagus: In the single-stapling technique (SST) group, Endo-PSI(II) was used for purse-suturing on the distal esophagus for reconstruction, and in the hemi-double-stapling technique (hemi-DST) group, the esophagus was cut by linear stapler with the entry hole of the anvil shaft opened after inserting the anvil tail. In both groups, surgical procedures were the same, except for the reconstruction. RESULTS: All TLTGs were performed securely without mortality. Intracorporeal laparoscopic esophagojejunal anastomosis was performed successfully for all the patients. In the hemi-DST group, four patients experienced anastomotic stenosis, three of whom required endoscopic balloon dilation. In contrast, no stenosis was seen in the SST group (p = 0.033). CONCLUSIONS: Anastomosis with SST is preferred to that with hemi-DST to minimize postoperative complications.

Prognostic indicators based on inflammatory and nutritional factors after pancreaticoduodenectomy for pancreatic cancer.

PURPOSE: We evaluated prognostic indicators based on inflammatory and nutritional factors, namely, the modified Glasgow Prognostic Score (mGPS), the Prognostic Nutritional Index (PNI), the neutrophil/lymphocyte ratio (NLR), and the platelet/lymphocyte ratio (PLR), to determine their efficiency and significance after pancreaticoduodenectomy for pancreatic cancer. METHODS: The subjects of this study were 46 patients who underwent pancreaticoduodenectomy for pancreatic cancer between October 2007 and December 2014. Patients were divided into preoperative mGPS (0/1 and 2), PNI (<40 and ≥40), NLR (<2.5 and ≥2.5), and PLR (<200 and ≥200) groups, to evaluate various perioperative outcomes. RESULTS: Hemoglobin concentrations were significantly lower (P = 0.019), whereas intra-abdominal bleeding was significantly higher (P = 0.040) in the PNI (<40) group than in the PNI (≥40) group. The incidence of postoperative pneumonia was significantly higher in the mGPS (2) group (P = 0.009), and surgical complications greater than grade 3 (Clavien-Dindo classification) were significantly increased in the NLR (≥2.5) group (P = 0.041). Overall survival rates in the PNI (<40) (P = 0.019), NLR (≥2.5) (P = 0.001), and PLR (≥200) (P < 0.001) groups were significantly lower than those in the other groups. The PLR was the only independent prognostic indicator (P = 0.002) according to multivariate analysis. CONCLUSIONS: The mGPS, PNI, and NLR were effective predictive indicators of postoperative complications. The PLR was the most useful prognostic indicator for pancreatic cancer patients after pancreaticoduodenectomy.

ILC2s-Bipartisan politicians in cancer.

Group 2 innate lymphoid cells (ILC2s) are lymphocytes that both promote and suppress antitumor immunity. Jou and colleagues now report in colorectal tumorigenesis that the cytokine interleukin-25 activates ILC2s to induce myeloid cells that suppress antitumor immunity.

Reprogrammed Schwann Cells Organize into Dynamic Tracks that Promote Pancreatic Cancer Invasion.

Nerves are a component of the tumor microenvironment contributing to cancer progression, but the role of cells from nerves in facilitating cancer invasion remains poorly understood. Here we show that Schwann cells (SC) activated by cancer cells collectively function as tumor-activated Schwann cell tracks (TAST) that promote cancer cell migration and invasion. Nonmyelinating SCs form TASTs and have cell gene expression signatures that correlate with diminished survival in patients with pancreatic ductal adenocarcinoma. In TASTs, dynamic SCs form tracks that serve as cancer pathways and apply forces on cancer cells to enhance cancer motility. These SCs are activated by c-Jun, analogous to their reprogramming during nerve repair. This study reveals a mechanism of cancer cell invasion that co-opts a wound repair process and exploits the ability of SCs to collectively organize into tracks. These findings establish a novel paradigm of how cancer cells spread and reveal therapeutic opportunities. SIGNIFICANCE: How the tumor microenvironment participates in pancreatic cancer progression is not fully understood. Here, we show that SCs are activated by cancer cells and collectively organize into tracks that dynamically enable cancer invasion in a c-Jun-dependent manner. See related commentary by Amit and Maitra, p. 2240. This article is highlighted in the In This Issue feature, p. 2221.

Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions.

Transcription factors ThPOK and Runx3 regulate the differentiation of "helper" CD4+ and "cytotoxic" CD8+ T cell lineages respectively, inducing single positive (SP) T cells that enter the periphery with the expression of either the CD4 or CD8 co-receptor. Despite the expectation that these cell fates are mutually exclusive and that mature CD4+CD8+ double positive (DP) T cells are present in healthy individuals and augmented in the context of disease, yet their molecular features and pathophysiologic role are disputed. Here, we show DP T cells in murine and human tumors as a heterogenous population originating from SP T cells which re-express the opposite co-receptor and acquire features of the opposite cell type's phenotype and function following TCR stimulation. We identified distinct clonally expanded DP T cells in human melanoma and lung cancer by scRNA sequencing and demonstrated their tumor reactivity in cytotoxicity assays. Our findings indicate that antigen stimulation induces SP T cells to differentiate into DP T cell subsets gaining in polyfunctional characteristics.

AMIGO2 as a novel indicator of liver metastasis in patients with colorectal cancer.

Our previous study showed that adhesion molecule with immunoglobulin like domain 2 (AMIGO2) is a pivotal driver gene of liver metastasis via regulating tumor cell adhesion to liver endothelial cells in mouse models. The aim of the present study was to clarify the role of AMIGO2 in liver metastasis in patients the colorectal cancer (CRC). Two human CRC cell lines, Caco-2 (AMIGO2-low) and HCT116 (AMIGO2-high), were used in this study. AMIGO2-overexpressing Caco-2 and AMIGO2-knockdown HCT116 cells were generated by transfection with an AMIGO2 expression vector or AMIGO2 small interfering RNA, respectively. Cell proliferation, invasion and adhesion to human liver endothelial cells were examined in in vitro studies. Immunohistochemical analysis was also performed to evaluate the association between AMIGO2 expression and liver metastasis in patients with CRC. In vitro studies revealed that cell proliferation, invasion and adhesion to liver endothelial cells were accelerated by upregulation of AMIGO2 expression, but suppressed by downregulation of AMIGO2 expression in human CRC cells. Immunohistochemical analysis using clinical CRC specimens revealed that AMIGO2 expression was associated with the frequency of liver metastasis (P<0.01), but not that of pulmonary metastasis (P=0.611) and peritoneal dissemination (P=0.909). In addition, AMIGO2 expression levels in tumor cells were significantly higher in liver metastatic foci than primary lesions (P=0.012). In conclusion, the present results indicated that AMIGO2 expression may contribute to the formation of liver metastasis in CRC.

Clinical Significance of Serum Antithrombin III Activity After Hepatectomy for Hepatocellular Carcinoma.

BACKGROUND: As antithrombin III (AT-III) is produced in the hepatocytes, its serum activity decreases at the time of liver failure, in addition to ischemia reperfusion injury, vascular endothelial dysfunction, and disseminated intravascular coagulation (DIC). Here, we examined whether the serum AT-III value after hepatectomy could be a prognostic factor for hepatocellular carcinoma (HCC). METHODS: Of 141 patients who underwent hepatectomy for HCC, data for 101 patients in whom serum AT-III activity was measured on the first postoperative day were extracted. Patients with serum AT-III activity > 50% and ≤ 50% were assigned to high value (72 cases) and low value (29 cases) groups, respectively. We examined the clinical and prognostic differences between these two groups. RESULTS: The average age of enrolled patients (83 men and 18 women) was 68.0 years. The 5-year overall survival rate was 88% and 60% in the high and low value groups, respectively (P < 0.01). Furthermore, the 2-year relapse-free survival rate was 71% and 54% in the high and low value groups, respectively (P = 0.03). CONCLUSION: This is the first study to demonstrate that serum AT-III levels on the first postoperative day may serve as a prognostic factor in HCC patients.