Identification of RNF213 as a Potential Suppressor of Local Invasion in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a lethal cancer with poor survival especially when it spreads. The histopathology of its rare intraductal papillary neoplasm of the bile duct type (IPNB) characteristically shows cancer cells originating within the confined bile duct space. These cells eventually invade and infiltrate the nearby liver tissues, making it a good model to study the mechanism of local invasion, which is the earliest step of metastasis. To discover potential suppressor genes of local invasion in ICC, we analyzed the somatic mutation profiles and performed clonal evolution analyses of the 11 pairs of macrodissected locally invasive IPNB tissues (LI-IPNB) and IPNB tissues without local invasion from the same patients. We identified a protein-truncating variant in an E3 ubiquitin ligase, RNF213 (c.6967C>T; p.Gln2323X; chr17: 78,319,102 [hg19], exon 29), as the most common protein-truncating variant event in LI-IPNB samples (4/11 patients). Knockdown of RNF213 in HuCCT1 and YSCCC cells showed increased migration and invasion, and reduced vasculogenic mimicry but maintained normal proliferation. Transcriptomic analysis of the RNF213-knockdown vs control cells was then performed in the HuCCT1, YSCCC, and KKU-100 cells. Gene ontology enrichment analysis of the common differentially expressed genes revealed significantly altered cytokine and oxidoreductase-oxidizing metal ion activities, as confirmed by Western blotting. Gene Set Enrichment Analysis identified the most enriched pathways being oxidative phosphorylation, fatty acid metabolism, reactive oxygen species, adipogenesis, and angiogenesis. In sum, loss-of-function mutation of RNF213 is a common genetic alteration in LI-IPNB tissues. RNF213 knockdown leads to increased migration and invasion of ICC cells, potentially through malfunctions of the pathways related to inflammation and energy metabolisms.

Availability of genome-matched therapy based on clinical practice.

BACKGROUND: Comprehensive genomic profiling (CGP) provides new opportunities for patients with advanced cancer to receive genome-matched therapies, but the availability rate of these remains low. We reviewed our CGP cases and suggested possible strategies to improve the current status from a clinical perspective. METHODS: Druggable genomic alterations and barriers to accessing genome-matched therapies were investigated in 653 patients with 30 various types of cancers who underwent CGP. RESULTS: While the availability rate of genome-matched therapies as a whole was 9.5%, CGP was useful in some cancer types. Patients with thyroid cancer and lung cancer harbored druggable genomic alterations at high rates, while sarcoma rarely harbored these alterations (100%, 76%, and 15.2%, respectively). In contrast, the availability rate of genome-matched therapies was highest in patients with sarcoma and head and neck cancer (HNC) (60% and 40%, respectively). One hundred thirteen patients (63.5%) had multiple barriers to accessing genome-matched therapy. Of 178 patients, 21 patients (11.8%) could not be considered for genome-matched therapies solely because of the deterioration of their performance status. CONCLUSION: This study demonstrated the usefulness of CGP for patients with sarcoma and HNC in addition to lung cancer in clinical practice. Performing CGP at the front line has the potential to improve the availability of genome-matched therapy.

Prognostic impact of cancer genomic profile testing for advanced or metastatic solid tumors in clinical practice.

Cancer genomic profile (CGP) testing, which is covered by the national health insurance system in Japan, has been introduced as a routine clinical practice. However, the effects of CGP testing on prognoses remain unclear. Drug accessibility rates and prognoses after CGP testing were retrospectively investigated in 713 patients who underwent CGP testing examined by our molecular tumor board between November 2019 and October 2022,. Overall survival (OS) was examined using the log-rank test and the Kaplan-Meier method. The median age of patients (326 males and 387 females) was 58 years (12-85 years). CGP testing revealed one or more gene mutations in 681 cases (95.5%), among which actionable gene mutations were detected in 439 (61.6%). Although treatment options were recommended for 285 cases (40.0%) by the molecular tumor board, only 45 received treatment based on their gene mutations. During the median observation period of 8.6 months, 351 (49.2%) patients died of the exacerbation of existing diseases. No significant differences were observed in OS between patients treated with and without genomically matched therapy (p = 0.285). According to clinical responses to treatment based on gene mutations, median OS was significantly longer in patients who achieved partial response and stable disease (26.5 months; 95% CI 14.4-38.6) than in those with progressive disease and not evaluated (9.8 months; 95% CI 5.8-13.8, p = 0.013). Responses to treatment based on gene mutations may improve prognoses, and it is important to increase the drug accessibility rate after CGP testing.

Spatiotemporal commonality of the TCR repertoire in a T-cell memory murine model and in metastatic human colorectal cancer.

Immune checkpoint inhibitors (ICIs) have shown superior clinical responses and significantly prolong overall survival (OS) for many types of cancer. However, some patients exhibit long-term OS, whereas others do not respond to ICI therapy at all. To develop more effective and long-lasting ICI therapy, understanding the host immune response to tumors and the development of biomarkers are imperative. In this study, we established an MC38 immunological memory mouse model by administering an anti-PD-L1 antibody and evaluating the detailed characteristics of the immune microenvironment including the T cell receptor (TCR) repertoire. In addition, we found that the memory mouse can be established by surgical resection of residual tumor following anti-PD-L1 antibody treatment with a success rate of > 40%. In this model, specific depletion of CD8 T cells revealed that they were responsible for the rejection of reinoculated MC38 cells. Analysis of the tumor microenvironment (TME) of memory mice using RNA-seq and flow cytometry revealed that memory mice had a quick and robust immune response to MC38 cells compared with naïve mice. A TCR repertoire analysis indicated that T cells with a specific TCR repertoire were expanded in the TME, systemically distributed, and preserved in the host for a long time period. We also identified shared TCR clonotypes between serially resected tumors in patients with colorectal cancer (CRC). Our results suggest that memory T cells are widely preserved in patients with CRC, and the MC38 memory model is potentially useful for the analysis of systemic memory T-cell behavior.

Perioperative circulating tumor DNA enables the identification of patients with poor prognosis in upper tract urothelial carcinoma.

Perioperative systemic chemotherapy improves the prognosis of upper tract urothelial carcinoma (UTUC). The first objective of this study was to verify whether perioperative circulating tumor DNA (ctDNA) analysis using a pan-cancer gene panel and next-generation sequencing could identify patients with poor prognosis who require perioperative chemotherapy. Second, we investigated whether ctDNA is useful for minimal residual disease (MRD) detection and treatment monitoring in UTUC. This study included 50 patients with untreated UTUC, including 43 cases of localized UTUC. We performed targeted ultradeep sequencing of plasma cell-free DNA (cfDNA) and buffy coat DNA and whole-exome sequencing of cancer tissues, allowing exclusion of possible false positives. We attempted to stratify the prognosis according to the perioperative ctDNA levels in patients with localized UTUC. In patients with metastatic UTUC, ctDNA was evaluated before, during, and after systemic treatment. In total, 23 (46%) of 50 patients with untreated UTUC were ctDNA positive, and 17 (40%) of 43 patients with localized UTUC were ctDNA positive. Of the detected TP53 mutations, 19% were false positives due to clonal hematopoiesis of indeterminate potential. Among preoperative risk factors, only the preoperative ctDNA fraction>2% was a significant and independent risk factor associated with worse recurrence-free survival (RFS). Furthermore, the existence of ctDNA early points after the operation was significantly associated with worse RFS, suggesting the presence of MRD. ctDNA also showed a potential as a real-time marker for systemic therapy in patients with metastatic UTUC. Detection of ctDNA may indicate potential metastasis and guide decisions on perioperative chemotherapy.

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy.

BACKGROUND: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy. METHODS: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs). RESULTS: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells. CONCLUSION: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT.

Genomic determinants impacting the clinical outcome of mogamulizumab treatment for adult T-cell leukemia/lymphoma.

In order to identify genomic biomarkers for the outcome of mogamulizumab-containing treatment, an integrated molecular analysis of adult T-cell leukemia/lymphoma (ATL) was conducted on 64 mogamulizumab-naïve patients. Among driver genes, CCR4 and CCR7 alterations were observed in 22% and 11% of the patients, respectively, both consisting of single nucleotide variants (SNV)/insertion-deletions (indels) in the C-terminus. Patients with CCR4 alterations or without CCR7 alterations exhibited a more favorable clinical response (complete response [CR] rate 93%, 13/14; P=0.024, and CR rate 71%, 40/56; P=0.036, respectively). Additionally, TP53, CD28, and CD274 alterations were identified in 35%, 16%, and 10% of the patients, respectively. TP53 alterations included SNV/indels or copy number variations (CNV) such as homozygous deletion; CD28 alterations included SNV, CNV such as amplification, or fusion; CD274 alterations included CNV such as amplification, or structural variants. Univariate analysis revealed that TP53, CD28 or CD274 alterations were associated with worse overall survival (OS) (hazard ratio [HR]: 2.330, 95% confidence interval [CI]: 1.183-4.589; HR: 3.191, 95% CI: 1.287- 7.911; HR: 3.301, 95% CI: 1.130-9.641, respectively) but that CCR4 alterations were associated with better OS (HR: 0.286, 95% CI: 0.087-0.933). Multivariate analysis indicated that in addition to performance status, TP53, CCR4 or CD274 alterations (HR: 2.467, 95% CI: 1.197-5.085; HR: 0.155, 95% CI: 0.031-0.778; HR: 14.393, 95% CI: 2.437-85.005, respectively) were independently and significantly associated with OS. The present study contributes to the establishment of precision medicine using mogamulizumab in ATL patients.

Monitoring epidermal growth factor receptor C797S mutation in Japanese non-small cell lung cancer patients with serial cell-free DNA evaluation using digital droplet PCR.

Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that is effective in treating both naïve and T790M-mutated EGFR-TKI-resistant non-small cell lung cancer patients. The EGFR C797S mutation is the major osimertinib resistance mechanism. The present study monitored the EGFR C797S mutation during osimertinib treatment in Japanese patients using droplet digital PCR (ddPCR). In our first cohort, C797S detection was validated with tumor specimens and/or plasma samples from 26 patients using ddPCR with custom-designed probes detecting and discriminating T790M and C797S in cis and trans positions. In our second cohort, 18 patients with EGFR-T790M who were going to start osimertinib were analyzed using ddPCR by collecting the plasma samples every month from the beginning of the course of osimertinib. In the first cohort, C797S was detected in 15.4% of patients. C797S and T790M in cis and trans positions were distinguished using ddPCR. In the second cohort, serial cfDNA evaluation revealed that the rate of EGFR mutation changes with disease state. Increases of EGFR mutation were detected, including C797S several months before the diagnosis of disease progression. As with the first cohort, C797S and T790M in cis and trans position were distinguished by ddPCR at disease progression. Coincidentally, in the first cohort, next generation sequencing detected NRAS Q61K mutation and the resistance with NRAS Q61K mutation was overcome by trametinib. In the second cohort, serial cfDNA analysis was useful for evaluating bone oligo-progression and local radiation therapy.

T-cell complexity and density are associated with sensitivity to neoadjuvant chemoradiotherapy in patients with rectal cancer.

Emerging evidence suggests that an increased density of pre-treatment CD8+ tumor-infiltrating lymphocytes (TILs) is associated with good response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer. However, the significance of T-cell complexity in the clinical setting remains unknown. High-throughput T-cell receptor (TCR) ß sequencing was applied to quantify the TCR repertoire of pre-treatment biopsies from 67 patients with advanced rectal cancer receiving preoperative CRT. Diversity index was used to represent the complexity of the TCR repertoire in a tumor. Pre-treatment CD8+ TIL densities were assessed by immunohistochemistry. Changes in TCR repertoire before and after CRT were also analysed in 23 patients. Diversity indices were significantly higher for good responders than for non-responders (P = 0.031). The multivariate analysis revealed that both CD8+ TIL density and TCR diversity index were independently associated with good response to CRT (P < 0.001 and P = 0.049, respectively). Patients who were high for both CD8+ TIL density and TCR diversity (double-high) had markedly better responses to CRT than double-low patients (84.2% vs 16.7%, P < 0.0001). Larger changes in TCR repertoires before and after CRT were correlated with better recurrence-free survival (P = 0.027). The complexity and dynamic change in the TCR repertoire might serve as a useful indicator of response to CRT in combination with CD8+ TIL density in patients with rectal cancer.

Immunogenomics in personalized cancer treatments.

Recent advances in next-generation sequencing technologies have led to significant improvements in cancer genomic research and cancer treatment. Through the use of comprehensive cancer genome data, precision medicine has become more of a reality; albeit, at present, only ~10-15% of patients can benefit from current genomic testing practices. Improvements in cancer genome analyses have contributed to a better understanding of antitumor immunity and have provided solutions for targeting highly cancer-specific neoantigens generated from somatic mutations in individual patients. Since then, numerous studies have demonstrated the importance of neoantigens and neoantigen-reactive T cells in the tumor microenvironment and how their presence influences the beneficial responses associated with various cancer immunotherapies, including immune checkpoint inhibitor therapy. Indeed, cancer immunotherapies that explicitly target neoantigens specific to individual cancer patients would lead to the ultimate form of cancer precision medicine. For this to be realized, several issues would need to be overcome, including the accurate prediction and selection of neoantigens that can induce cytotoxic T cells in individual patients. The precise prediction of target neoantigens will likely accelerate the development of personalized immunotherapy including cancer vaccines and T-cell receptor-engineered T-cell therapy for patients with cancer.

Genomic alterations in gynecological malignancies: histotype-associated driver mutations, molecular subtyping schemes, and tumorigenic mechanisms.

There are numerous histological subtypes (histotypes) of gynecological malignancies, with each histotype considered to largely reflect a feature of the "cell of origin," and to be tightly linked with the clinical behavior and biological phenotype of the tumor. The recent advances in massive parallel sequencing technologies have provided a more complete picture of the range of the genomic alterations that can persist within individual tumors, and have highlighted the types and frequencies of driver-gene mutations and molecular subtypes often associated with these histotypes. Several large-scale genomic cohorts, including the Cancer Genome Atlas (TCGA), have been used to characterize the genomic features of a range of gynecological malignancies, including high-grade serous ovarian carcinoma, uterine corpus endometrial carcinoma, uterine cervical carcinoma, and uterine carcinosarcoma. These datasets have also been pivotal in identifying clinically relevant molecular targets and biomarkers, and in the construction of molecular subtyping schemes. In addition, the recent widespread use of clinical sequencing for the more ubiquitous types of gynecological cancer has manifested in a series of large genomic datasets that have allowed the characterization of the genomes, driver mutations, and histotypes of even rare cancer types, with sufficient statistical power. Here, we review the field of gynecological cancer, and seek to describe the genomic features by histotype. We also will demonstrate how these are linked with clinicopathological attributes and highlight the potential tumorigenic mechanisms.

Immunogenomic landscape of gynecologic carcinosarcoma.

OBJECTIVE: Carcinosarcoma (CS) of the uterus or ovary is a rare, biphasic tumor comprising epithelial and mesenchymal elements, and exhibits more aggressive clinical features than its carcinoma counterpart. Four molecular subtypes of CS were recently established based on genomic aberration profiles (POLE, MSI, CNH, and CNL) and shown to be associated with multiple clinicopathological parameters, including patient outcomes. However, the role of the immune microenvironment in CS remains unclear. Here, we investigated the influence of the immune cells that infiltrate CS to better understand the immunological status of gynecological CS. METHODS: Tumor immune microenvironmental analyses on CS samples were performed using immune cell profiling with RNA-seq, transcriptomic subtyping with microenvironmental genes, and T-cell receptor repertoire assay. Carcinoma and sarcoma elements from CS samples were also assessed separately. RESULTS: Relying on estimations of tumor-infiltrating cell types from RNA-seq data, POLE and MSI (hypermutator) tumors showed an enrichment of M1 macrophages, plasma cells and CD8+ T cells, whereas CNH and CNL (non-hypermutator) tumors had high levels of M2 macrophages. Further subclassification by immune-related, non-cancer genes identified a fraction of tumors with distinct patient outcomes, particularly those with the CNH genomic aberration subtype. T-cell heterogeneity was independently correlated with prolonged progression-free survival. Differential analysis of carcinoma and sarcoma elements identified many shared mutations but there was little overlap in the T-cell receptor repertoire between the two elements. CONCLUSIONS: Tumor immune microenvironmental analyses could offer potential clinical utility in the stratification of gynecological CS above classification by genomic aberration subtype alone.

Intranodal Administration of Neoantigen Peptide-loaded Dendritic Cell Vaccine Elicits Epitope-specific T Cell Responses and Clinical Effects in a Patient with Chemorefractory Ovarian Cancer with Malignant Ascites.

Chemorefractory ovarian cancer has limited therapeutic options. Hence, new types of treatment including neoantigen-specific immunotherapy need to be investigated. Neoantigens represent promising targets for personalized cancer immunotherapy. We here describe the clinical and immunological effects of a neoantigen peptide-loaded DC-based immunotherapy in a patient with recurrent and chemoresistant ovarian cancer. A 71-year-old female patient with chemorefractory ovarian cancer and malignant ascites received intranodal vaccination of DCs loaded with four neoantigen peptides that were predicted by our immunogenomic pipeline. Following four rounds of vaccinations with this therapy, CA-125 levels were remarkably declined and tumor cells in the ascites were also decreased. Concordantly, the tumor-related symptoms such as respiratory discomfort improved without any adverse reactions. The reactivity against one HLA-A2402-restricted neoantigen peptide derived from a mutated PPM1 F protein was detected in lymphocytes from peripheral blood by IFN-γ ELISPOT assay. Furthermore, the neoantigen (PPM1 F mutant)-specific TCRs were detected in the tumor-infiltrating T lymphocytes post-vaccination. Our results showed that vaccination with intranodal injection of neoantigen peptide-loaded DCs may have clinical and immunological impacts on cancer treatment.

Cooperation of genes in HPV16 E6/E7-dependent cervicovaginal carcinogenesis trackable by endoscopy and independent of exogenous estrogens or carcinogens.

Human papillomavirus (HPV) infection is necessary but insufficient for progression of epithelial cells from dysplasia to carcinoma-in situ (CIS) to invasive cancer. The combination of mutant cellular and viral oncogenes that regulate progression of cervical cancer (CC) remains unclear. Using combinations of HPV16 E6/E7 (E+), mutant Kras (mKras) (K+) and/or loss of Pten (P-/-), we generated autochthonous models of CC without exogenous estrogen, carcinogen or promoters. Furthermore, intravaginal instillation of adenoCre virus enabled focal activation of the oncogenes/inactivation of the tumor suppressor gene. In P+/+ mice, E6/E7 alone (P+/+E+K-) failed to cause premalignant changes, while mKras alone (P+/+E-K+) caused persistent mucosal abnormalities in about one-third of mice, but no cancers. To develop cancer, P+/+ mice needed both E6/E7 and mKras expression. Longitudinal endoscopies of P+/+E+K+ mice predicted carcinoma development by detection of mucosal lesions, found on an average of 23 weeks prior to death, unlike longitudinal quantitative PCRs of vaginal lavage samples from the same mice. Endoscopy revealed that individual mice differed widely in the time required for mucosal lesions to appear after adenoCre and in the time required for these lesions to progress to cancer. These cancers developed in the transition zone that extends, unlike in women, from the murine cervix to the distal vagina. The P-/-E+K+ genotype led to precipitous cancer development within a few weeks and E6/E7-independent cancer development occurred in the P-/-E-K+ genotype. In the P-/-E+K- genotype, mice only developed CIS. Thus, distinct combinations of viral and cellular oncogenes are involved in distinct steps in cervical carcinogenesis.

Immunoglobulin profiling identifies unique signatures in patients with Kawasaki disease during intravenous immunoglobulin treatment.

Identifying the causes of high fever syndromes such as Kawasaki disease (KD) remains challenging. To investigate pathogen exposure signatures in suspected pathogen-mediated diseases such as KD, we performed immunoglobulin (Ig) profiling using a next-generation sequencing method. After intravenous Ig (IVIG) treatment, we observed disappearance of clonally expanded IgM clonotypes, which were dominantly observed in acute-phase patients. The complementary-determining region 3 (CDR3) sequences of dominant IgM clonotypes in acute-phase patients were commonly observed in other Ig isotypes. In acute-phase KD patients, we identified 32 unique IgM CDR3 clonotypes shared in three or more cases. Furthermore, before the IVIG treatment, the sums of dominant IgM clonotypes in IVIG-resistant KD patients were significantly higher than those of IVIG-sensitive KD patients. Collectively, we demonstrate a novel approach for identifying certain Ig clonotypes for potentially interacting with pathogens involved in KD; this approach could be applied for a wide variety of fever-causing diseases of unknown origin.

Diagnostic utility of STAT6YE361 expression in classical Hodgkin lymphoma and related entities.

Although the distinction of classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma using morphology and immunostains is straightforward in most instances, occasional cases pose diagnostic challenge. We sought to determine the utility of the novel YE361 STAT6 rabbit monoclonal antibody in Hodgkin lymphoma and diagnostically challenging B- and T-cell non-Hodgkin lymphoma entities with Hodgkin-like features. Cases from seven institutions included: 57 classical Hodgkin lymphomas (31% EBV+), 34 nodular lymphocyte predominant Hodgkin lymphomas, 34 mimicking B- and T-cell non-Hodgkin lymphomas, and 7 reactive lymphoproliferations. After review of histology, STAT6YE361 immunostaining was performed. The intensity and spatial localization of immunopositivity was assessed in neoplastic cells. Additional FISH for programmed death ligand-1 (PD-L1) was performed in one patient in paired treatment-naive and relapse biopsy tissues. Two STAT6YE361 immunopositive cases were examined by whole-exome sequencing after flow sorting to assess mutations in STAT6 pathway genes. Most classical Hodgkin lymphomas showed nuclear staining for STAT6YE361 [46/57 cases (80%)] on Hodgkin cells. Staining was exclusively nuclear in a minority [12/46 (26%)], while dual nuclear and cytoplasmic localization was more common [34/46 (74%)]. In contrast, all nodular lymphocyte predominant Hodgkin lymphomas [0/34 (0%)] were negative for nuclear STAT6YE361 staining on the lymphocyte predominant cells. Within B- and T-cell non-Hodgkin lymphomas, nuclear STAT6YE361 was seen in: B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and in primary mediastinal large B-cell lymphoma. Strong PD-L1 gene amplification was noted in the paired cHL and relapse B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, although STAT6YE361 was negative in both biopsies. Whole-exome sequencing identified mutations in B2M, XPO1, and ITPKB as well CISHP213L (in the STAT pathway) in one classical Hodgkin lymphoma patient positive for nuclear STAT6YE361 although no underlying STAT6 mutations were observed in either sample examined. STAT6YE361 nuclear staining has 100% positive predictive value and 85.7% negative predictive value in confirming or excluding classical Hodgkin lymphoma diagnosis in the distinction from nodular lymphocyte predominant Hodgkin lymphoma and other benign and malignant entities.

SARS-CoV-2 genomic variations associated with mortality rate of COVID-19.

The coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly expanded to a global pandemic. However, numbers of infected cases, deaths, and mortality rates related to COVID-19 vary from country to country. Although many studies were conducted, the reasons of these differences have not been clarified. In this study, we comprehensively investigated 12,343 SARS-CoV-2 genome sequences isolated from patients/individuals in six geographic areas and identified a total of 1234 mutations by comparing with the reference SARS-CoV-2 sequence. Through a hierarchical clustering based on the mutant frequencies, we classified the 28 countries into three clusters showing different fatality rates of COVID-19. In correlation analyses, we identified that ORF1ab 4715L and S protein 614G variants, which are in a strong linkage disequilibrium, showed significant positive correlations with fatality rates (r = 0.41, P = 0.029 and r = 0.43, P = 0.022, respectively). We found that BCG-vaccination status significantly associated with the fatality rates as well as number of infected cases. In BCG-vaccinated countries, the frequency of the S 614G variant had a trend of association with the higher fatality rate. We also found that the frequency of several HLA alleles, including HLA-A*11:01, were significantly associated with the fatality rates, although these factors were associated with number of infected cases and not an independent factor to affect fatality rate in each country. Our findings suggest that SARS-CoV-2 mutations as well as BCG-vaccination status and a host genetic factor, HLA genotypes might affect the susceptibility to SARS-CoV-2 infection or severity of COVID-19.

Bioinformatic prediction of potential T cell epitopes for SARS-Cov-2.

To control and prevent the current COVID-19 pandemic, the development of novel vaccines is an emergent issue. In addition, we need to develop tools that can measure/monitor T-cell and B-cell responses to know how our immune system is responding to this deleterious virus. However, little information is currently available about the immune target epitopes of novel coronavirus (SARS-CoV-2) to induce host immune responses. Through a comprehensive bioinformatic screening of potential epitopes derived from the SARS-CoV-2 sequences for HLAs commonly present in the Japanese population, we identified 2013 and 1399 possible peptide epitopes that are likely to have the high affinity (<0.5%- and 2%-rank, respectively) to HLA class I and II molecules, respectively, that may induce CD8+ and CD4+ T-cell responses. These epitopes distributed across the structural (spike, envelope, membrane, and nucleocapsid proteins) and the nonstructural proteins (proteins corresponding to six open reading frames); however, we found several regions where high-affinity epitopes were significantly enriched. By comparing the sequences of these predicted T cell epitopes to the other coronaviruses, we identified 781 HLA-class I and 418 HLA-class II epitopes that have high homologies to SARS-CoV. To further select commonly-available epitopes that would be applicable to larger populations, we calculated population coverages based on the allele frequencies of HLA molecules, and found 2 HLA-class I epitopes covering 83.8% of the Japanese population. The findings in the current study provide us valuable information to design widely-available vaccine epitopes against SARS-CoV-2 and also provide the useful information for monitoring T-cell responses.

Significant differences in T cell receptor repertoires in lung adenocarcinomas with and without epidermal growth factor receptor mutations.

Recent clinical trials of non-small cell lung cancer with immune checkpoint inhibitors revealed that patients with epidermal growth factor receptor (EGFR) mutations had more unfavorable outcomes compared with those with wild-type EGFR. However, the underlying mechanism for the link between EGFR mutations and immune resistance remains unclear. We performed T cell receptor (TCR) repertoire analysis of resected lung adenocarcinoma tissues with and without EGFR mutations to investigate the characteristics of TCR repertoires. We collected a total of 39 paired (normal and tumor) lung tissue samples (20 had EGFR mutations) and conducted TCR repertoire analysis as well as whole-exome sequencing (WES) and transcriptome analysis. The TCR diversity index in EGFR-mutant tumors was significantly higher than that in EGFR-wild-type tumors (median [range] 552 [162-1,135] vs 230 [30-764]; P < .01), suggesting higher T cell clonal expansion in EGFR-wild-type tumors than in EGFR-mutant tumors. In WES, EGFR-mutant tumors showed lower numbers of non-synonymous mutations and predicted neoantigens than EGFR-wild-type tumors (P < .01, P = .03, respectively). The number of non-synonymous mutations revealed a positive correlation with the sum of frequencies of the TCRß clonotypes of 1% or higher in tumors (r = .52, P = .04). The present study demonstrates significant differences in TCR repertoires and the number of predicted neoantigens between EGFR-mutant and wild-type lung tumors. Our findings provide important information for understanding the molecular mechanism behind EGFR-mutant patients showing unfavorable responses to immune checkpoint inhibitors.

Immunopharmacogenomics towards personalized cancer immunotherapy targeting neoantigens.

Utilizing the host immune system to eradicate cancer cells has been the most investigated subject in the cancer research field in recent years. However, most of the studies have focused on highly variable responses from immunotherapies such as immune checkpoint inhibitors, from which the majority of patients experienced no or minimum clinical benefit. Advances in genomic sequencing technologies have improved our understanding of immunopharmacogenomics and allowed us to identify novel cancer-specific immune targets. Highly tumor-specific antigens, neoantigens, are generated by somatic mutations that are not present in normal cells. It is plausible that by targeting antigens with high tumor-specificity, such as neoantigens, the likelihood of toxic effects is very limited. However, understanding the interaction between neoantigens and the host immune system remains a significant challenge. This review focuses on the potential use of neoantigen-targeted immunotherapies in cancer treatment and the recent progress of different strategies in predicting, identifying, and validating neoantigens. Successful identification of highly tumor-specific antigens accelerates the development of personalized immunotherapy with no or minimum adverse effects and with a broader coverage of patients.