Characterization of lymphocyte-rich hepatocellular carcinoma and the prognostic role of tertiary lymphoid structures.

BACKGROUND & AIMS: Lymphocyte-rich hepatocellular carcinoma (LR-HCC) is largely unknown and a rare subtype of HCC with immune-rich stroma. Tertiary lymphoid structures (TLS), frequently observed in LR-HCC, are known to be prognostically significant in various malignancies; however, their significance in HCC remains unevaluated. METHODS: Clinicopathologic data of 191 cases of surgically resected conventional HCC (C-HCC, n = 160) and LR-HCC (n = 31) were retrieved. Immunohistochemistry, multiplex immunofluorescence staining, RNA sequencing and proteomic analysis were conducted. Differences between the subtypes were statistically evaluated. RESULTS: LR-HCC was significantly correlated to larger tumour size, higher Edmondson-Steiner grade, presence of TLS and higher CD3-, CD8- and FOXP3-positive T cell, high PD-1 and PD-L1 expression (p < .001 for all) compared to C-HCC. Patients with LR-HCC exhibited significantly better overall survival (OS) (p = .044) and recurrence-free survival (RFS) (p = .025) than C-HCC. LR-HCC demonstrated TLS signatures with significantly higher proteomic-based immune scores in 14 of 17 types of tumour-infiltrating immune cells. Furthermore, C-HCC with secondary follicles, the most mature form of TLS, exhibited significantly better OS (p = .031) and RFS (p = .033) than those without. Across the global proteome, LR-HCC was well-differentiated from C-HCC and a map of protein-protein interactions between tumour-infiltrating lymphocytes and HCC in tumour microenvironment was completed. CONCLUSION: LR-HCC is clinicopathologically and molecularly distinct and shows better prognosis compared to C-HCC. Also, the presence of secondary follicle can be an important prognostic marker for better prognosis in both LR-HCC and C-HCC.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

PURPOSE: Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. MATERIALS AND METHODS: To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. RESULTS: Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. CONCLUSIONS: These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

Revolutionizing Non-Small Cell Lung Cancer Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-term Stored Plasma.

PURPOSE: Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non-small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). MATERIALS AND METHODS: Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. RESULTS: The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). CONCLUSION: Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.

BRCA-mutated gastric adenocarcinomas are associated with chromosomal instability and responsiveness to platinum-based chemotherapy.

BACKGROUND: Homologous recombination defect is an important biomarker of chemotherapy in certain tumor types, and the presence of pathogenic or likely pathogenic mutations involving BRCA1 or BRCA2 (p-BRCA) mutations is the most well-established marker for the homologous recombination defect. Gastric cancer, one of the most prevalent tumor types in Asia, also harbors p-BRCA mutations. METHODS: To investigate the clinical significance of p-BRCA mutations, we analyzed 366 gastric cancer cases through next-generation sequencing. We determined the zygosity of p-BRCA mutations based on the calculated tumor purity through variant allelic fraction patterns and investigated whether the presence of p-BRCA mutations is associated with platinum-based chemotherapy and a certain molecular subtype. RESULTS: Biallelic p-BRCA mutation was associated with better response to platinum-based chemotherapy than heterozygous p-BRCA mutation or wild type BRCA genes. The biallelic p-BRCA mutations was observed only in the chromosomal instability subtype, while all p-BRCA mutations were heterozygous in microsatellite instability subtype. CONCLUSIONS: In conclusion, patients with gastric cancer harboring biallelic p-BRCA mutations were associated with a good initial response to platinum-based chemotherapy and those tumors were exclusively chromosomal instability subtype. Further investigation for potential association with homologous recombination defect is warranted.

Glutathione dynamics is a potential predictive and therapeutic trait for neoadjuvant chemotherapy response in bladder cancer.

Radical cystectomy with preoperative cisplatin-based neoadjuvant chemotherapy (NAC) is the standard care for muscle-invasive bladder cancers (MIBCs). However, the complete response rate to this modality remains relatively low, and current clinicopathologic and molecular classifications are inadequate to predict NAC response in patients with MIBC. Here, we demonstrate that dysregulation of the glutathione (GSH) pathway is fundamental for MIBC NAC resistance. Comprehensive analysis of the multicohort transcriptomes reveals that GSH metabolism and immune-response genes are enriched in NAC-resistant and NAC-sensitive MIBCs, respectively. A machine-learning-based tumor/stroma classifier is applied for high-throughput digitalized immunohistochemistry analysis, finding that GSH dynamics proteins, including glutaminase-1, are associated with NAC resistance. GSH dynamics is activated in cisplatin-resistant MIBC cells, and combination treatment with a GSH dynamics modulator and cisplatin significantly suppresses tumor growth in an orthotopic xenograft animal model. Collectively, these findings demonstrate the predictive and therapeutic values of GSH dynamics in determining the NAC response in MIBCs.

Intercellular cross-talk through lineage-specific gap junction of cancer-associated fibroblasts related to stromal fibrosis and prognosis.

Stromal fibrosis in cancer is usually associated with poor prognosis and chemotherapy resistance. It is thought to be caused by fibroblasts; however, the exact mechanism is not yet well understood. The study aimed to identify lineage-specific cancer-associated fibroblast (CAF) subgroup and their associations with extracellular matrix remodeling and clinical significances in various tumor types using single-cell and bulk RNA sequencing data. Through unsupervised clustering, six subclusters of CAFs were identified, including a cluster with exclusively high gap junction protein beta-2 (GJB2) expression. This cluster was named GJB2-positive CAF. It was found to be a unique subgroup of terminally differentiated CAFs associated with collagen gene expression and extracellular matrix remodeling. GJB2-positive CAFs showed higher communication frequency with vascular endothelial cells and cancer cells than GJB2-negative CAFs. Moreover, GJB2 was poorly expressed in normal tissues, indicating that its expression is dependent on interaction with other cells, including vascular endothelial cells and cancer cells. Finally, the study investigated the clinical significance of GJB2 signature score for GJB2-positive CAFs in cancer and found a correlation with poor prognosis. These results suggest that GJB2-positive CAF is a unique fibroblast subtype involved in extracellular matrix remodeling, with significant clinical implications in cancer.

Whole-genome sequencing reveals an association between small genomic deletions and an increased risk of developing Parkinson's disease.

Single-nucleotide variants (SNVs) associated with Parkinson's disease (PD) have been investigated mainly through genome-wide association studies. However, other genomic alterations, including copy number variations, remain less explored. In this study, we conducted whole-genome sequencing of primary (310 PD patients and 100 healthy individuals) and independent (100 PD patients and 100 healthy individuals) cohorts from the Korean population to identify high-resolution small genomic deletions, gains, and SNVs. Global small genomic deletions and gains were found to be associated with an increased and decreased risk of PD development, respectively. Thirty significant locus deletions were identified in PD, with most being associated with an increased PD risk in both cohorts. Small genomic deletions in clustered loci located in the GPR27 region had high enhancer signals and showed the closest association with PD. GPR27 was found to be expressed specifically in brain tissue, and GPR27 copy number loss was associated with upregulated SNCA expression and downregulated dopamine neurotransmitter pathways. Clustering of small genomic deletions on chr20 in exon 1 of the GNAS isoform was detected. In addition, we found several PD-associated SNVs, including one in the enhancer region of the TCF7L2 intron, which exhibited a cis-acting regulatory mode and an association with the beta-catenin signaling pathway. These findings provide a global, whole-genome view of PD and suggest that small genomic deletions in regulatory domains contribute to the risk of PD development.

Reshaping tumor immune microenvironment by Epstein-Barr virus activation in the stroma of colorectal cancer.

The formation of tumor immune microenvironment (TIM) is complicated and poorly understood. Little is known about the effect of a viral infection potentially inducing an additional immune response in the TIM. Here, we identify Epstein-Barr virus (EBV) expression in the TIM in colorectal cancer (CRC) tissue through EBV-encoded RNA in-situ hybridization and RNA sequencing data and investigate the effects of EBV on TIM composition and clinical outcomes. EBV was detected in tumor-infiltrating lymphocytes, but not in cancer cells. EBV positivity was associated with older age, male sex, and SMAD4 mutations. EBV-positive tumors were characterized by enrichment in chemokine/cytokine signaling pathways and altered immune cell composition, including plasma and CD4 T cells, as well as cancer cells intrinsically enriched pathways related to immune tolerance, leading to poor prognosis. In conclusion, we identified EBV expression in TIM and suggested its association with poor prognosis by altering the TIM in CRC.

Clinicogenomic characteristics and synthetic lethal implications of germline homologous recombination-deficient hepatocellular carcinoma.

BACKGROUNDS AND AIMS: We performed an in-depth examination of pathogenic germline variants (PGVs) and somatic variants in DNA damage response (DDR) genes in hepatocellular carcinoma (HCC) to explore their clinical and genomic impacts. APPROACH AND RESULTS: We used a merged whole-exome or RNA sequencing data set derived from in-house ( n = 230) and The Cancer Genome Atlas ( n = 362) databases of multiethnic HCC samples. We also evaluated synthetic lethal approaches targeting mutations in homologous recombination (HR) genes using HCC cells selected from five genomic databases of cancer cell lines. A total of 110 PGVs in DDR pathways in 96 patients were selected. Of the PGV carriers, 44 were HR-altered and found to be independently associated with poorer disease-free survival after hepatectomy. The most frequently altered HR gene in both germline and somatic tissues was POLQ , and this variant was detected in 22.7% (10/44) and 23.8% (5/21) of all the corresponding carriers, respectively. PGVs in HR were significantly associated with upregulation of proliferation and replication-related genes and familial risk of HCC. Samples harboring PGVs in HR with loss of heterozygosity were most strongly correlated with the genomic footprints of deficient HR, such as mutation burden and denovoSig2 (analogous to Catalogue of Somatic Mutations in Cancer [COSMIC] 3), and poor outcome. Pharmacologic experiments with HCC cells defective in BRCA2 or POLQ suggested that tumors with this phenotype are synthetic lethal with poly(ADP-ribose) polymerase inhibitors. CONCLUSIONS: Our findings suggest that germline HR defects in HCC tend to confer a poor prognosis and result in distinctive genomic scarring. Tests of the clinical benefits of HR-directed treatments in the affected patients are needed.

Data on the evaluation of the relation between ß-arrestin 2 and YAP phosphorylation in patient-derived colon cancer organoids.

The data presented in this article is related to a rapid communication entitled "ß-arrestin 2 suppresses the activation of YAP by promoting LATS kinase activity". This article describes the correlation of ß-arrestin 2 and YAP phosphorylation in patient-derived organoid models. Here, we analyzed 45 colon cancer organoids (CCOs) selected in the related research article to investigate the role of ß-arrestin 2 in YAP phosphorylation. Hematoxylin and eosin (H&E) staining and immunohistochemistry data showed that the CCOs maintained tissue architecture and histological features of their original cancer tissues. Moreover, mutation data detected from RNA-seq (RNA-sequencing) analysis showed that these CCOs retained the genetic features of their original colon cancer tissues as well. We also confirmed at the protein level that organoids expressing ß-arrestin 2 showed high expression of phosphorylated YAP. These organoid model studies strongly support the related research article that ß-arrestin 2 suppresses the activation of YAP in colon cancer.

PRRX1 is a master transcription factor of stromal fibroblasts for myofibroblastic lineage progression.

Although stromal fibroblasts play a critical role in cancer progression, their identities remain unclear as they exhibit high heterogeneity and plasticity. Here, a master transcription factor (mTF) constructing core-regulatory circuitry, PRRX1, which determines the fibroblast lineage with a myofibroblastic phenotype, is identified for the fibroblast subgroup. PRRX1 orchestrates the functional drift of fibroblasts into myofibroblastic phenotype via TGF-ß signaling by remodeling a super-enhancer landscape. Such reprogrammed fibroblasts have myofibroblastic functions resulting in markedly enhanced tumorigenicity and aggressiveness of cancer. PRRX1 expression in cancer-associated fibroblast (CAF) has an unfavorable prognosis in multiple cancer types. Fibroblast-specific PRRX1 depletion induces long-term and sustained complete remission of chemotherapy-resistant cancer in genetically engineered mice models. This study reveals CAF subpopulations based on super-enhancer profiles including PRRX1. Therefore, mTFs, including PRRX1, provide another opportunity for establishing a hierarchical classification system of fibroblasts and cancer treatment by targeting fibroblasts.

PET-Based Radiogenomics Supports mTOR Pathway Targeting for Hepatocellular Carcinoma.

PURPOSE: This work aimed to explore in depth the genomic and molecular underpinnings of hepatocellular carcinoma (HCC) with increased 2[18F]fluoro-2-deoxy-d-glucose (FDG) uptake in PET and to identify therapeutic targets based on this imaging-genomic surrogate. EXPERIMENTAL DESIGN: We used RNA sequencing and whole-exome sequencing data obtained from 117 patients with HCC who underwent hepatic resection with preoperative FDG-PET/CT imaging as a discovery cohort. The primary radiogenomic results were validated with transcriptomes from a second cohort of 81 patients with more advanced tumors. All patients were allocated to an FDG-avid or FDG-non-avid group according to the PET findings. We also screened potential drug candidates targeting FDG-avid HCCs in vitro and in vivo. RESULTS: High FDG avidity conferred worse recurrence-free survival after HCC resection. Whole transcriptome analysis revealed upregulation of mTOR pathway signals in the FDG-avid tumors, together with higher abundance of associated mutations. These clinical and genomic findings were replicated in the validation set. A molecular signature of FDG-avid HCCs identified in the discovery set consistently predicted poor prognoses in the public-access datasets of two cohorts. Treatment with an mTOR inhibitor resulted in decreased FDG uptake followed by effective tumor control in both the hyperglycolytic HCC cell lines and xenograft mouse models. CONCLUSIONS: Our PET-based radiogenomic analysis indicates that mTOR pathway genes are markedly activated and altered in HCCs with high FDG retention. This nuclear imaging biomarker may stimulate umbrella trials and tailored treatments in precision care of patients with HCC.

Histopathologic and Molecular Biomarkers of PD-1/PD-L1 Inhibitor Treatment Response among Patients with Microsatellite Instability‒High Colon Cancer.

PURPOSE: Recent clinical trials have reported response rates < 50% among patients treated with programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors for microsatellite instability‒high (MSI-H) colorectal cancer (CRC), and factors predicting treatment response have not been fully identified. This study aimed to identify potential biomarkers of PD-1/PD-L1 inhibitor treatment response among patients with MSI-H CRC. MATERIALS AND METHODS: MSI-H CRC patients enrolled in three clinical trials of PD-1/PD-L1 blockade at Asan Medical Center (Seoul, Republic of Korea) were screened and classified into two groups according to treatment response. Their histopathologic features and expression of 730 immune-related genes from the NanoString platform were evaluated, and a machine learning-based classification model was built to predict treatment response among MSI-H CRCs patients. RESULTS: A total of 27 patients (15 responders, 12 non-responders) were included. A high degree of lymphocytic/neutrophilic infiltration and an expansile tumor border were associated with treatment response and prolonged progression-free survival (PFS), while mucinous/signet-ring cell carcinoma was associated with a lack of treatment response and short PFS. Gene expression profiles revealed that the interferon-γ response pathway was enriched in the responder group. Of the top eight differentially expressed immune-related genes, PRAME had the highest fold change in the responder group. Higher expression of PRAME was independently associated with better PFS along with histologic subtypes in the multivariate analysis. The classification model using these genes showed good performance for predicting treatment response. CONCLUSION: We identified histologic and immune-related gene expression characteristics associated with treatment response in MSI-H CRC, which may contribute to optimal patient stratification.

Comprehensive characterization of viral integrations and genomic aberrations in HBV-infected intrahepatic cholangiocarcinomas.

BACKGROUND AND AIMS: Despite the epidemiological association between intrahepatic cholangiocarcinoma (iCCA) and HBV infection, little is known about the relevant oncogenic effects. We sought to identify the landscape and mechanism of HBV integration, along with the genomic architecture of HBV-infected iCCA (HBV-iCCA) tumors. APPROACH AND RESULTS: We profiled a cohort of 108 HBV-iCCAs using whole-genome sequencing, deep sequencing, and RNA sequencing, together with preconstructed data sets of HBV-infected HCC (HBV-HCC; n = 167) and combined hepatocellular cholangiocarcinoma (HBV-cHCC/CCA; n = 59), and conventional (n = 154) and fluke-related iCCAs (n = 16). Platforms based on primary iCCA cell lines to evaluate the functional effects of chimeric transcripts were also used. We found that HBV had inserted at multiple sites in the iCCA genomes in 45 (41.7%) of the tumors. Recurrent viral integration breakpoints were found at nine different sites. The most common insertional hotspot (7 tumors) was in the TERT (telomerase reverse transcriptase) promoter, where insertions and mutations (11 tumors) were mutually exclusive, and were accompanied by promoter hyperactivity. Recurrent HBV integration events (5 tumors) were also detected in FAT2 (FAT atypical cadherin 2), and were associated with enrichment of epithelial-mesenchymal transition-related genes. A distinctive intergenic insertion (chr9p21.3), between DMRTA1 (DMRT like family A1) and LINC01239 (long intergenic non-protein coding RNA 1239), had oncogenic effects through activation of the mammalian target of rapamycin (mTOR)/4EBP/S6K pathway. Regarding the mutational profiles of primary liver cancers, the overall landscape of HBV-iCCA was closer to that of nonviral conventional iCCA, than to HBV-HCC and HBV-cHCC/CCA. CONCLUSIONS: Our findings provide insight into the behavior of iCCAs driven by various pathogenic mechanisms involving HBV integration events and associated genomic aberrations. This knowledge should be of use in managing HBV carriers.

Matrix metalloproteinase 11 (MMP11) in macrophages promotes the migration of HER2-positive breast cancer cells and monocyte recruitment through CCL2-CCR2 signaling.

Matrix metalloproteinase 11 (MMP11), a member of the MMP family involved in the degradation of the extracellular matrix, has been implicated in cancer progression. Despite the stromal expression of MMP11 in breast cancer, the prognostic significance and role of MMP11 in immune or stromal cells of breast cancer remain unclear. Based on the immunohistochemical analysis of breast cancer tissues from 497 patients, we demonstrated that MMP11 expression in mononuclear inflammatory cells (predominantly macrophages) is an independent negative prognostic factor in breast cancer, whereas MMP11 expression in tumor cells and fibroblasts is not associated with patient survival. Enforced MMP11 expression in breast cancer cells did not promote cell proliferation and migration. However, MMP11-overexpressing macrophages enhanced the migration of HER2-positive (HER2+) breast cancer cells, recruitment of monocytes, and tube formation of endothelial cells. Furthermore, we found that the chemokine CCL2 secreted from MMP11-overexpressing macrophages activated the MAPK pathway via its receptor CCR2 in breast cancer cells, thereby promoting the migration of HER2+ breast cancer cells through MMP9 upregulation. We also found that MMP11 expression in macrophages was stimulated by MMP11-overepressing HER2+ breast cancer cells. Collectively, our findings provide evidence that MMP11 in macrophages may play a pro-tumoral role in HER2+ breast cancer through interaction with cancer cells, monocytes, and endothelial cells.

Application of Immunoprofiling Using Multiplexed Immunofluorescence Staining Identifies the Prognosis of Patients with High-Grade Serous Ovarian Cancer.

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420-0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.

Immuno-genomic classification of colorectal cancer organoids reveals cancer cells with intrinsic immunogenic properties associated with patient survival.

BACKGROUND: The intrinsic immuno-ge7nomic characteristics of colorectal cancer cells that affect tumor biology and shape the tumor immune microenvironment (TIM) are unclear. METHODS: We developed a patient-derived colorectal cancer organoid (CCO) model and performed pairwise analysis of 87 CCOs and their matched primary tumors. The TIM type of the primary tumor was classified as immuno-active, immuno-exhausted, or immuno-desert. RESULTS: The gene expression profiles, signaling pathways, major oncogenic mutations, and histology of the CCOs recapitulated those of the primary tumors, but not the TIM of primary tumors. Two distinct intrinsic molecular subgroups of highly proliferative and mesenchymal phenotypes with clinical significance were identified in CCOs with various cancer signaling pathways. CCOs showed variable expression of cancer-specific immune-related genes such as those encoding HLA-I and HLA-II, and molecules involved in immune checkpoint activation/inhibition. Among these genes, the expression of HLA-II in CCOs was associated with favorable patient survival. K-means clustering analysis based on HLA-II expression in CCOs revealed a subgroup of patients, in whom cancer cells exhibited Intrinsically Immunogenic Properties (Ca-IIP), and were characterized by high expression of signatures associated with HLA-I, HLA-II, antigen presentation, and immune stimulation. Patients with the Ca-IIP phenotype had an excellent prognosis, irrespective of age, disease stage, intrinsic molecular type, or TIM status. Ca-IIP was negatively correlated with intrinsic E2F/MYC signaling. Analysis of the correlation between CCO immuno-genotype and TIM phenotype revealed that the TIM phenotype was associated with microsatellite instability, Wnt/ß-catenin signaling, APC/KRAS mutations, and the unfolded protein response pathway linked to the FBXW7 mutation in cancer cells. However, Ca-IIP was not associated with the TIM phenotype. CONCLUSIONS: We identified a Ca-IIP phenotype from a large set of CCOs. Our findings may provide an unprecedented opportunity to develop new strategies for optimal patient stratification in this era of immunotherapy.