Novel AI Combining CNN and SVM to Predict Colorectal Cancer Prognosis and Mutational Signatures from HE Images.

Reducing recurrence following radical resection of colon cancer without over- or under-treatment remains a challenge. Postoperative adjuvant chemotherapy (Adj) is currently administered based solely on pathological tumor, node, and metastasis (TNM) stage. However, prognosis can vary significantly among patients with the same disease stage. Therefore, novel classification systems in addition to the TNM are necessary to inform decision-making regarding postoperative treatment strategies, especially stage II and III disease, and to minimize overtreatment and undertreatment with Adj. We developed a prognostic prediction system for colorectal cancer by using a combined convolutional neural network (CNN) and support vector machine (SVM) approach to extract features from hematoxyling and eosin staining (HE) images. We combined the TNM and our AI-based classification system into a TNM-AI (mTNM-AI) classification system with high discriminative power for recurrence-free survival (RFS). Furthermore, the cancer cell population recognized by this system as low risk of recurrence exhibited the mutational signature SBS87 as a genetic phenotype. The novel AI-based classification system developed here is expected to play an important role in prognostic prediction and personalized treatment selection in oncology.

High serum proteinase-3 levels predict poor progression-free survival and lower efficacy of bevacizumab in metastatic colorectal cancer.

BACKGROUND: To improve the prognosis of patients with metastatic colorectal cancer (mCRC), investigating predictive biomarkers of their prognosis and chemotherapeutic responsiveness is necessary. This study aimed to analyze the clinical significance of serum proteinase-3 (PRTN3) as a predictor for prognosis and chemosensitivity, especially to bevacizumab therapy, in mCRC. METHODS: This single-center retrospective observational study enrolled 79 patients with mCRC in our hospital and 353 patients with colorectal cancer in the TCGA database. Preoperative serum PRTN3 levels were measured using an enzyme-linked immunosorbent assay. The clinicopathological characteristics and prognosis according to serum PRTN3 levels were then evaluated. PRTN3 expression in tumor and stromal cells was evaluated immunohistochemically. The impact of PRTN3 levels on angiogenesis and bevacizumab sensitivity was evaluated using the tube formation assay. RESULTS: Serum PRTN3 levels were an independent poor prognostic factor for progression-free survival (PFS) (hazard ratio, 2.082; 95% confidence interval, 1.118-3.647; P=0.010) in patients with mCRC. Similarly, prognostic analysis with TCGA data sets showed poorer overall survival in patients with PRTN3 expression than that in patients without PRTN3 expression, especially in patients with stage IV. Immunohistochemical analysis of resected specimens revealed that stromal neutrophils expressed PRTN3, and their expression level was significantly correlated with serum PRTN3 levels. Interestingly, the effectiveness of first-line chemotherapy was significantly poorer in the high serum PRTN3 level group. High serum PRTN3 was significantly associated with poor PFS (hazard ratio, 3.027; 95% confidence interval, 1.175-7.793; P=0.0161) in patients treated with bevacizumab, an anti-angiogenic inhibitor. The tube formation assay revealed that PRTN3 administration notably augmented angiogenesis while simultaneously attenuating the anti-angiogenic influence exerted by bevacizumab therapy. CONCLUSIONS: Serum PRTN3 levels could be a novel predictive biomarker of PFS of first-line chemotherapy, especially for bevacizumab therapy, in patients with mCRC.

HLA Class I Analysis Provides Insight Into the Genetic and Epigenetic Background of Immune Evasion in Colorectal Cancer With High Microsatellite Instability.

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

IL-6 Levels Correlate with Prognosis and Immunosuppressive Stromal Cells in Patients with Colorectal Cancer.

BACKGROUND: The prognosis for patients with colorectal cancer (CRC) is determined by tumor characteristics as well as the host immune response. This study investigated the relationship between an immunosuppressive state and patient prognosis by evaluating the systemic and tumor microenvironment (TME) interleukin (IL)-6 levels. METHODS: Preoperative serum IL-6 levels were measured using an electrochemiluminescence assay. Expression of IL-6 in tumor and stromal cells was evaluated immunohistochemically in 209 patients with resected CRC. Single-cell analysis of tumor-infiltrating immune cells was performed using mass cytometry in 10 additional cases. RESULTS: Elevated serum IL-6 levels were associated with elevated stromal IL-6 levels and a poor prognosis for patients with CRC. High IL-6 expression in stromal cells was associated with low-density subsets of CD3+ and CD4+ T cells as well as FOXP3+ cells. Mass cytometry analysis showed that IL-6+ cells among tumor-infiltrating immune cells were composed primarily of myeloid cells and rarely of lymphoid cells. In the high-IL-6-expression group, the percentages of myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3highCD45RA- effector regulatory T cells (eTreg) were significantly higher than in the low-IL-6-expression group. Furthermore, the proportion of IL-10+ cells in MDSCs and that of IL-10+ or CTLA-4+ cells in eTregs correlated with IL-6 levels. CONCLUSION: Elevated serum IL-6 levels were associated with stromal IL-6 levels in CRC. High IL-6 expression in tumor-infiltrating immune cells also was associated with accumulation of immunosuppressive cells in the TME.

Phase I study of a novel therapeutic vaccine as perioperative treatment for patients with surgically resectable hepatocellular carcinoma: The YCP02 trial.

AIM: Developing effective adjuvant therapies is essential for improving the surgical outcomes in patients with hepatocellular carcinoma (HCC). Immunotherapy against HCC has become a promising strategy; however, only approximately 30% of all HCC patients respond to immunotherapy. Previously, we generated the novel therapeutic vaccine comprising multi-human leukocyte antigen-binding heat shock protein 70/glypican-3 peptides with a novel adjuvant combination of hLAG-3Ig and poly-ICLC. We also confirmed the safety of this vaccination therapy, as well as its capacity for the effective induction of immune responses in a previous clinical trial. METHODS: In this phase I study, we administered this vaccine intradermally six times before surgery, and 10 times after surgery to patients with untreated, surgically resectable HCC (stage II to IVa). The primary end-points of this study were the safety and feasibility of this treatment. We also analyzed the resected tumor specimens pathologically using hematoxylin-eosin staining and immunohistochemistry for heat shock protein 70, glypican 3, CD8 and programmed death-1. RESULTS: A total of 20 human leukocyte antigen-matched patients received this vaccination therapy with an acceptable side-effect profile. All patients underwent planned surgery without vaccination-related delay. Immunohistochemical analyses revealed that potent infiltration of CD8+ T cells into tumors with target antigen expression was observed in 12 of 20 (60%) patients. CONCLUSIONS: This novel therapeutic vaccine was safe as perioperative immunotherapy for patients with HCC, and has the potential to strongly induce CD8+ T cells infiltration into tumors.

[A Recurrent Colon Cancer Successfully Treated by Nivolumab plus Ipilimumab with On and Off for Preventing Exacerbation of Skin Disorders].

Here, we report a case in which nivolumab plus ipilimumab combination therapy was significantly effective for MSI-high recurrent colon cancer with acute exacerbation after 5-FU/L-OHP/CPT-11 treatment. At the end of 4 cycles of combination therapy, clinical CR was obtained on diagnostic imaging. At the end of the 2 cycles of transition from combination therapy to monotherapy, eosinophilia was observed in a quadratic function, and exacerbation of skin disorders was observed. Eosinophil counts normalized promptly after discontinuation of treatment, and skin disorders gradually improved. Two months after the discontinuation of treatment, monotherapy was restarted. After the resumption of treatment, an increase in eosinophils and worsening of skin symptoms were observed again, and stopped treatment. We report an interesting case in which immune checkpoint inhibiter were turned on and off according to eosinophil counts for preventing exacerbation of skin disorders, and for maintaining cancer remission by continuing immune checkpoint inhibitor treatment.

Immune Evasion of Hepatoma Cancer Stem-Like Cells from Natural Killer Cells.

BACKGROUND: Poor prognosis in liver cancer is due to its high frequency of intrahepatic metastasis. Cancer stem-like cells (CSLCs), which possess the properties of stemness, tumor initiation capability, and resistance to therapy, also exhibit metastatic potential. Immune surveillance plays an important role in the accomplishment of metastasis. Herein, the property of immune evasion in CSLCs was investigated. METHODS: Sphere cells were induced as CSLCs using a sphere induction medium containing neural survival factor-1. The expression of genes involved in immune evasion was determined using RNA-sequencing for sphere and parental cells followed by validation using flow cytometric analysis and ELISA. Susceptibility to natural killer (NK) cell-mediated cytotoxicity was examined by a chromium release assay. A xenograft model using BALB/c nu/nu mice was used to assess tumor growth. Gene set enrichment analysis was performed for interpreting RNA sequencing. RESULTS: The cell surface expressions of PD-L1, PD-L2, and CEACAM1 were upregulated and those of ULBP1 and MICA/MICB were downregulated in SK-sphere, CSLCs derived from SK-HEP-1, compared with that in parental cells. Levels of soluble MICA were elevated in conditioned medium from SK-sphere. Expression of HLA class I was not downregulated in SK-sphere. The susceptibilities to NK cell-mediated killing and secreted perforin were significantly lower in both CSLCs derived from SK-HEP-1 and HLE than in parental cells. Tumors formed upon inoculation of SK-sphere in immunodeficient mice harboring NK cells were larger than those formed upon inoculation of parental cells. CONCLUSION: Human hepatoma cell line-derived CSLCs may possess immune evasion properties, especially from NK cell-mediated immunity.

Elevated expression of RAB3B plays important roles in chemoresistance and metastatic potential of hepatoma cells.

BACKGROUND: Cancer stem cells (CSCs) are thought to play important roles in carcinogenesis, recurrence, metastasis, and therapy-resistance. We have successfully induced cancer stem-like sphere cells (CSLCs) which possess enhanced chemoresistance and metastatic potential. To enable the development of targeted therapy against CSLCs, we identified a gene responsible for this phenotype in CSLC. METHODS: Human hepatoma cell line SK-HEP-1 was used for CSLC induction with a unique sphere inducing medium, and HuH-7 cells were used as non-sphere forming cells in the same condition. RNA-sequencing was performed followed by validation with quantitative RT-PCR and western blotting. Knockdown experiments were done by using CRISPR-Cas9 genome-editing, and the rescue experiments were performed using the expressing plasmid vector. Chemoresistance and liver metastasis of the cells, was studied following the splenic injection of cells to severely immune deficient mice and evaluated using the MTS assay. Quantification of exosomes in the medium was done using ELISA. RESULTS: RAB3B was identified as an up-regulated gene in both CSLCs and prognostically poor hepatocellular carcinoma (HCC) by RNA-sequencing. RAB3B-KD cells showed altered CSLC phenotypes such as sphere formation, chemoresistance, and metastatic potentials, and those were rescued by RAB3B complementation. Increased exosome secretion was observed in CSLCs, and it was not observed in the RAB3B-KD cells. In addition, the RAB3B expression correlated with the expression of ABCG2, APOE, LEPR, LXN, and TSPAN13. CONCLUSION: The up regulation of RAB3B may play an important role in the chemoresistance and metastatic potential of CSLCs.

CD4 and FOXP3 as predictive markers for the recurrence of T3/T4a stage II colorectal cancer: applying a novel discrete Bayes decision rule.

BACKGROUND: We recently reported the relapse-free survival (RFS) significance of the combination of CD4+ and forkhead box P3+ (FOXP3) T-cell densities identified by immunohistochemistry in patients with stage I, II, and III colorectal cancer (CRC) who underwent curative resections. This study was designed to determine the optimal combination of markers that predict recurrence in patients with T factors of T3/T4a stage II CRC by applying a novel Bayes decision rule. METHODS: Using 137 cancer tissue specimens from T3/T4a stage II patients, 12 clinicopathologic and immune factors were analysed as predictive candidates for recurrence. RESULTS: Our study showed that the combination of low CD4+ and low FOXP3+ T-cell densities resulted in extremely poor RFS. CONCLUSIONS: Adjuvant chemotherapy may be considered for patients with a combination of low CD4+ and low FOXP3+ T-cell densities. The discovery of this new prognostic indicator is important for the appropriate management of patients undergoing curative resection for T3/T4a stage II CRC.

[Tumor Immunogenicity and Immune Checkpoint Inhibitors].

Cancer immunotherapy including immune checkpoint inhibitors(ICIs)have established itself as the fourth cancer therapy. However, the response rate of ICIs is still only about 20%, and tumors resistant to ICIs are often so-called"cold-tumor"with low tumor immunogenicity. Therefore, research and development is being conducted worldwide on how to convert cold- tumors into hot-tumors with high immunogenicity. In this paper, we review the relationship between tumor immunogenicity and ICI, as well as therapeutic methods to enhance tumor immunogenicity, and introduce our research about novel cancer peptide vaccination therapy.

Novel adjuvant dendritic cell therapy with transfection of heat-shock protein 70 messenger RNA for patients with hepatocellular carcinoma: a phase I/II prospective randomized controlled clinical trial.

INTRODUCTION: A proteomic analysis of hepatocellular carcinoma (HCC) has revealed that Heat Shock Protein 70 (HSP70) is among the cancer antigen proteins of HCC. Moreover, we confirmed that HSP70 was highly expressed in HCC by immunohistochemical staining. Based on these results, we developed an HSP70 mRNA-transfected dendritic cell (DC) therapy for treating unresectable or recurrent HCC, and the phase I trial was completed successfully. Thus, we aimed to investigate the safety and efficacy of this therapy as a postoperative adjuvant treatment after curative resection for HCC to prevent recurrence by conducting a phase I/II randomized controlled clinical trial. METHODS: Patients (n = 45) with resectable HCC of stages II-IVa were registered and randomly assigned into two groups (DC group: 31 patients, control group: 14 patients) before surgery. The primary endpoint was disease-free survival (DFS), and the secondary endpoints were safety and overall survival. The DC therapy was initially administered at approximately 1 week after surgery, and twice every 3-4 weeks thereafter. RESULTS: No adverse events specific to the immunotherapy were observed in the DC group. There was no difference in DFS between the DC and control groups (p = 0.666). However, in the subgroup with HSP70-expressing HCC, DFS of the DC group tended to be better (p = 0.090) and OS of the DC group was significantly longer (p = 0.003) than those of the control group. CONCLUSION: The HSP70 mRNA-transfected DC therapy was performed safely as an adjuvant therapy. The prognosis of HSP70-expressing HCC cases could be expected to improve with this therapy.

Prediction of early recurrence of hepatocellular carcinoma after resection using digital pathology images assessed by machine learning.

Hepatocellular carcinoma (HCC) is a representative primary liver cancer caused by long-term and repetitive liver injury. Surgical resection is generally selected as the radical cure treatment. Because the early recurrence of HCC after resection is associated with low overall survival, the prediction of recurrence after resection is clinically important. However, the pathological characteristics of the early recurrence of HCC have not yet been elucidated. We attempted to predict the early recurrence of HCC after resection based on digital pathologic images of hematoxylin and eosin-stained specimens and machine learning applying a support vector machine (SVM). The 158 HCC patients meeting the Milan criteria who underwent surgical resection were included in this study. The patients were categorized into three groups: Group I, patients with HCC recurrence within 1 year after resection (16 for training and 23 for test); Group II, patients with HCC recurrence between 1 and 2 years after resection (22 and 28); and Group III, patients with no HCC recurrence within 4 years after resection (31 and 38). The SVM-based prediction method separated the three groups with 89.9% (80/89) accuracy. Prediction of Groups I was consistent for all cases, while Group II was predicted to be Group III in one case, and Group III was predicted to be Group II in 8 cases. The use of digital pathology and machine learning could be used for highly accurate prediction of HCC recurrence after surgical resection, especially that for early recurrence. Currently, in most cases after HCC resection, regular blood tests and diagnostic imaging are used for follow-up observation; however, the use of digital pathology coupled with machine learning offers potential as a method for objective postoprative follow-up observation.

Determining the protective characteristics and risk factors for the development of anastomotic leakage after low anterior resection for rectal cancer.

PURPOSE: Anastomotic leakage is one of the most serious postoperative complications associated with surgery for rectal cancer. The present study aimed to identify the protective characteristics and risk factors associated with anastomotic leakage after low anterior resection for rectal cancer. METHODS: This was a retrospective, single-center study conducted between January 2009 and December 2017 at our institution. In total, 136 rectal cancer patients who underwent low anterior resection were included in the study. We analyzed preoperative and intraoperative factors. In addition, the pelvic dimensions were measured using computed tomography in all cases. RESULTS: Among the 136 patients, anastomotic leakage occurred in 21 (15.4%), including 18 males and 3 females. The median body mass index was 21.1 kg/m2. The construction of a covering stoma was found to be a protective factor. In addition, the operation time (≥ 373 min), intraoperative blood loss (≥ 105 ml), and size of the pelvic inlet (≥ 113 mm) were identified as risk factors for anastomotic leakage. CONCLUSION: The construction of a covering stoma was a possible protective factor. However, a longer operation time, higher intraoperative blood loss, and larger pelvic inlet dimensions were possible risk factors for developing anastomotic leakage after low anterior resection in patients with rectal cancer.

[Immune-Related Factors as Prognostic Markers for Resectable Colorectal Cancer].

BACKGROUND: Tumor infiltration of CD3 and CD8-positive T cells has been reported as a good prognostic marker for patients with colorectal cancer(CRC). To clarify the significance of CD4 and FOXP3-positive T cells in CRC for prognosis of intratumoral infiltration. METHODS: CD3, CD8, CD4 and FOXP3-positive T cells were immunostained(IHC)from tissue specimens of 342 CRC patients who underwent curative resection to quantify the number of infiltrating cells in the tumor. Microsatellite instability(MSI)was also evaluated in 322 samples and the clinicopathological factors and survival were analyzed. RESULTS: Highly infiltrated groups of CD3, CD4 and FOXP3-positive T cells were associated with improved relapse-free survival(RFS). Highly infiltrated groups of CD8, CD4 and FOXP3-positive T cells were associated with improved disease- specific survival(DSS). Invasion depth, vascular infiltration, and CD4-positive T cell density were independent prognostic factors for DSS. CD4 and FOXP3-positive T cell infiltration was not associated with the high-frequency microsatellite instability group, in contrast to CD3 and CD8-positive T cell infiltration. CONCLUSIONS: Intratumoral CD4-positive T cell density and FOXP3-positive T cell densities were stronger prognostic indicators than other clinicopathological features. These results may facilitate the establishment of novel prognostic factors and therapeutic strategies for CRC.

[A Resected Case of Advanced Lower Rectal Cancer with Neoadjuvant Chemotherapy by FOLFOXIRI plus Cetuximab].

Although the current standard of care for patients with lower rectal cancer in Japan includes total mesorectal resection with lateral lymph node dissection, postoperative local and distant recurrence rates are high. Multidisciplinary treatment is important to improve the prognosis. A man in his 30s was diagnosed with lower rectal cancer due to bloody stool and referred to our department. He was diagnosed as cT3N3M0, cStage Ⅲc with right obturator lymph node metastasis. Four courses of neoadjuvant chemotherapy(NAC)with FOLFOXIRI plus cetuximab were performed. Because Grade 3 neutropenia was observed in the first cycle(CTCAE v5.0), pegfilgrastim was administered in the second and subsequent cycles, and NAC was completed without dose reduction. The patient underwent laparoscopy-assisted intersphincteric rectal resection and D3+rtLD2 dissection. Histopathological resection margins were negative, and the resection was R0. Lymph node metastasis was found only in No. 263d-rt, and the pathological diagnosis was ypT3N3M0, pStage Ⅲc. Histological evaluation of response to treatment was Grade 2. The postoperative course was good and the patient was discharged on postoperative day 15. The patient received 8 courses of adjuvant chemotherapy with mFOLFOX6 from the 7th postoperative week and is alive and recurrence-free 6 months after surgery.

[A Case of Pseudo-Meigs' Syndrome Caused by Ovarian Metastasis from Colon Cancer].

This case pertains to a female patient in her 60s who was diagnosed with carcinoma in the cecum with lung, ovarian, and peritoneal metastases. She complained of abdominal distension and poor feeding because her ascites and ovarian metastasis worsened 18 months after chemotherapy initiation. Repeated cytologic examination of the ascitic fluid revealed no malignant cells. Therefore, Pseudo-Meigs' syndrome was suspected. Bilateral salpingo-oophorectomy was performed as palliative surgery because of the patient's reduced capacity to perform activities of daily living(ADL)due to ascites. After palliative surgery, her ascites disappeared, and she was able to better perform ADL. Further, chemotherapy was resumed. The patient remains well 10 months after surgery. This case highlights the importance of considering Pseudo-Meigs' syndrome in patients with massive ascites and ovarian metastasis, because surgical resection can improve their quality of life.

[A Case of Anastomotic Recurrence of Rectal Cancer Treated by Laparoscopic Total Pelvic Exenteration after Neoadjuvant Chemoradiation].

Patient is 69-year-old man, who underwent a high anterior resection with laparoscopic support for rectal cancer. The patient was diagnosed with anastomotic recurrent rectal cancer after 14 months after surgery. The pelvic MRI scan showed invasion of the prostate and seminal vesicles, so NACRT was performed. Tumors were found to have decreased in size, although there was still some residual invasion of the prostate and seminal vesicle. Laparoscopic total pelvic exenteration (Lap-TPE), and combined excision of the anal elevator muscle and bladder were performed. Preoperative diagnosis was ycT4b, N0, M0, ycStage Ⅱ, and pathological diagnosis was pT4b (prostate and seminal vesicles), INF b, Ly2, v2, Pn1b, pPM0, pDM0, pRM0, and pN0. Laparoscopic surgery allowed to operate safely, with minimal blood loss and a good field of vision. After postoperative adjuvant chemotherapy, lung and liver metastasis appeared after 6 months after surgery, but there was no local recurrence. The patient is treated with chemotherapy, and the metastases are under control. The patient is survive 17 months after Lap-TPE.

[A Case of Gastrointestinal Stromal Tumor of the Lower Rectum That Enabled Minimally Invasive Surgery with Imatinib Mesylate as Neoadjuvant Chemotherapy].

A 67-year-old woman was admitted with melena. A colonoscopy detected a 50 mm submucosal tumor close to the dentate line. We diagnosed the rectal gastrointestinal stromal tumor by EUS-FNA. With the expectation of tumor shrinkage and strong hope of the patient, we started imatinib mesylate as neoadjuvant chemotherapy. A CT scan after 3 months after administration of imatinib mesylate showed the reduction of the size to 35 mm. We operated transanal endoscopic surgery considering the localization of the tumor. From histopathological findings, the tumor was low risk in the modified-Fletcher classification, and low risk in the Miettinen classification. Eight months after the operation, no recurrence was observed without further adjuvant chemotherapy. In this case, we were able to resect the tumor without injuring the film of tumor by operating transanal endoscopic surgery, because of tumor shrinkage with imatinib mesylate as neoadjuvant chemotherapy. I considered that using imatinib mesylate preoperatively was contributed to minimally invasive surgery.

A phase I study of multi-HLA-binding peptides derived from heat shock protein 70/glypican-3 and a novel combination adjuvant of hLAG-3Ig and Poly-ICLC for patients with metastatic gastrointestinal cancers: YNP01 trial.

BACKGROUND: This phase I study aimed to evaluate the safety, peptide-specific immune responses, and anti-tumor effects of a novel vaccination therapy comprising multi-HLA-binding heat shock protein (HSP) 70/glypican-3 (GPC3) peptides and a novel adjuvant combination of hLAG-3Ig and Poly-ICLC against metastatic gastrointestinal cancers. METHODS: HSP70/GPC3 peptides with high binding affinities for three HLA types (A*24:02, A*02:01, and A*02:06) were identified with our peptide prediction system. The peptides were intradermally administered with combined adjuvants on a weekly basis. This study was a phase I dose escalation clinical trial, which was carried out in a three patients' cohort; in total, 11 patients were enrolled for the recommended dose. RESULTS: Seventeen patients received this vaccination therapy without dose-limiting toxicity. All treatment-related adverse events were of grades 1 to 2. Peptide-specific CTL induction by HSP70 and GPC3 proteins was observed in 11 (64.7%) and 13 (76.5%) cases, respectively, regardless of the HLA type. Serum tumor marker levels were decreased in 10 cases (58.8%). Immunological analysis using PBMCs indicated that patients receiving dose level 3 presented with significantly reduced T cell immunoglobulin and mucin-domain containing-3 (TIM3)-expressing CD4 + T cells after one course of treatment. PD-1 or TIM3-expressing CD4 + T cells and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT)-expressing CD8 + T cells in PBMCs before vaccination were negative predictive factors for survival. CONCLUSIONS: This novel peptide vaccination therapy was safe for patients with metastatic gastrointestinal cancers.

Serum LOX-1 is a novel prognostic biomarker of colorectal cancer.

BACKGROUND: Colorectal cancer is the third most common cancer worldwide. If biomarkers can be identified in liquid biopsy, diagnosis and treatment can be optimized even when cancerous tissues are not available. The purpose of this study was to identify proteins from liquid biopsy that would be useful as markers of poor prognosis. METHODS: First, we comprehensively analyzed serum proteins to identify potential biomarkers and focused on serum lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). The relationship between LOX-1 and the prognosis of patients with colorectal cancer has not been reported. Next, we validated this marker using serum samples from 238 patients with colorectal cancer by ELISA and 100 tissue samples by immunohistochemical staining. RESULTS: The optimal cut-off value of serum LOX-1 was 538.7 pg/mL according to time-dependent receiver operating characteristics curve analysis. The overall survival of patients with high levels of serum LOX-1 was significantly poorer than that of individuals with low levels of LOX-1 in the training and test datasets. In multivariate analysis for overall survival, serum LOX-1 was an independent prognostic factor identified in liquid biopsy (hazard ratio = 1.729, p = 0.027). The prognosis of patients with high LOX-1 expression in tumor tissues was significantly poorer than that of individuals with low expression (p =0.047 ). Additionally, inflammatory factors such as white blood cell count, C-reactive protein level, neutrophil/lymphocyte ratio, and monocyte/lymphocyte ratio were significantly higher in the group with high serum LOX-1 levels. CONCLUSIONS: Serum LOX-1 might be a useful biomarker of poor prognosis in colorectal cancer.