Genomic and transcriptomic profiling of combined small-cell lung cancer through microdissection: unveiling the transformational pathway of mixed subtype.

BACKGROUND: Combined small-cell lung carcinoma (cSCLC) represents a rare subtype of SCLC, the mechanisms governing the evolution of cancer genomes and their impact on the tumor immune microenvironment (TIME) within distinct components of cSCLC remain elusive. METHODS: Here, we conducted whole-exome and RNA sequencing on 32 samples from 16 cSCLC cases. RESULTS: We found striking similarities between two components of cSCLC-LCC/LCNEC (SCLC combined with large-cell carcinoma/neuroendocrine) in terms of tumor mutation burden (TMB), tumor neoantigen burden (TNB), clonality structure, chromosomal instability (CIN), and low levels of immune cell infiltration. In contrast, the two components of cSCLC-ADC/SCC (SCLC combined with adenocarcinoma/squamous-cell carcinoma) exhibited a high level of tumor heterogeneity. Our investigation revealed that cSCLC originated from a monoclonal source, with two potential transformation modes: from SCLC to SCC (mode 1) and from ADC to SCLC (mode 2). Therefore, cSCLC might represent an intermediate state, potentially evolving into another histological tumor morphology through interactions between tumor and TIME surrounding it. Intriguingly, RB1 inactivation emerged as a factor influencing TIME heterogeneity in cSCLC, possibly through neoantigen depletion. CONCLUSIONS: Together, these findings delved into the clonal origin and TIME heterogeneity of different components in cSCLC, shedding new light on the evolutionary processes underlying this enigmatic subtype.

Dynamic circulating tumor DNA during chemoradiotherapy predicts clinical outcomes for locally advanced non-small cell lung cancer patients.

The value of circulating tumor DNA (ctDNA) during chemoradiotherapy (CRT) remains unclear but is critical for detecting molecular residual disease (MRD). In this prospective study, we sequenced 761 blood samples from 139 patients with locally advanced non-small cell lung cancer treated with definitive radiation therapy (RT). ctDNA concentrations showed a significantly declining trend as CRT progressed at on-RT and after-RT time points versus baseline. Thirty-eight (27.3%) patients with early undetectable ctDNA at both on-RT (RT reached 40 Gy) and after-RT time points, indicating early response to CRT, had better survival outcomes for both with or without consolidation immune checkpoint inhibitors. Longitudinal undetectable MRD was found in 20.1% patients. The 2-year cancer-specific progression-free survival of these patients was 88.4%, corresponding to a potentially cured population. Further analysis revealed that pretreatment ctDNA variants serve as an essential MRD informed source. These data provide clinical insights for ctDNA-MRD detection.

Immune characteristics associated with lymph node metastasis in early-stage NSCLC.

PURPOSE: Tumor metastasis significantly impacts the prognosis of non-small cell lung cancer (NSCLC) patients, with lymph node (LN) metastasis being the most common and early form of spread. With the development of adjuvant immunotherapy, increasing attention has been paid to the tumor-draining lymph nodes（TDLN) in early-stage NSCLC, especially tumor-metastatic lymph nodes, which provides poor prognostic information but has potential benefits in adjuvant treatment. METHODS: We showed the remodeled immune environment in TDLNs through using TCR-seq to analyse 24 primary lung cancer tissues and 134 LNs from 24 lung cancer patients with or without LN metastasis. Additionally, we characterized the spatial profiling of immunocytes and tumor cells in TDLNs and primary tumor sites through using multi-IHC. RESULTS: We found the remodeled immune environment in TDLNs through analyzing primary lung cancer tissues and LNs from NSCLC patients with or without LN metastasis. Considering the intricate communication between tumor and immunocytes, we further subdivided TDLNs, revealing that metastasis-negative LNs from LN-metastatic patients (MNLN) exhibited greater immune activation, exhaustion, and memory in comparison to both metastasis-positive LNs (MPLN) and TDLNs from non-LN-metastatic patients (NMLN). CONCLUSIONS: Our data indicate that LN metastasis facilitated tumor-specific antigen presentation in TDLNs and induces T cell priming, while existing tumor cells generate an immune-suppressive environment in MPLNs through multiple mechanisms. These findings contribute to a comprehensive understanding of the immunological mechanisms through which LN metastasis influences tumor progression and plays a role in immunotherapy for NSCLC patients.

Analysis of genomic and immune intratumor heterogeneity in linitis plastica via multiregional exome and T-cell receptor sequencing.

The molecular landscape and the intratumor heterogeneity (ITH) architecture of gastric linitis plastica (LP) are poorly understood. We performed whole-exome sequencing (WES) and T-cell receptor (TCR) sequencing on 40 tumor regions from four LP patients. The landscape and ITH at the genomic and immunological levels in LP tumors were compared with multiple cancers that have previously been reported. The lymphocyte infiltration was further assessed by immunohistochemistry (IHC) in LP tumors. In total, we identified 6339 non-silent mutations from multi-samples, with a median tumor mutation burden (TMB) of 3.30 mutations per Mb, comparable to gastric adenocarcinoma from the Cancer Genome Atlas (TCGA) cohort (P = 0.53). An extremely high level of genomic ITH was observed, with only 12.42%, 5.37%, 5.35%, and 30.67% of mutations detectable across 10 regions within the same tumors of each patient, respectively. TCR sequencing revealed that TCR clonality was substantially lower in LP than in multi-cancers. IHC using antibodies against CD4, CD8, and PD-L1 demonstrated scant T-cell infiltration in the four LP tumors. Furthermore, profound TCR ITH was observed in all LP tumors, with no T-cell clones shared across tumor regions in any of the patients, while over 94% of T-cell clones were restricted to individual tumor regions. The Morisita overlap index (MOI) ranged from 0.21 to 0.66 among multi-regions within the same tumors, significantly lower than that of lung cancer (P = 0.002). Our results show that LP harbored extremely high genomic and TCR ITH and suppressed T-cell infiltration, suggesting a potential contribution to the frequent recurrence and poor therapeutic response of this adenocarcinoma.

CD8+ T cell/cancer-associated fibroblast ratio stratifies prognostic and predictive responses to immunotherapy across multiple cancer types.

Background: Cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) are critical for immune suppression by restricting immune cell infiltration in the tumor stromal zones from penetrating tumor islands and changing their function status, particularly for CD8+ T cells. However, assessing and quantifying the impact of CAFs on immune cells and investigating how this impact is related to clinical outcomes, especially the efficacy of immunotherapy, remain unclear. Materials and methods: The TME was characterized using immunohistochemical (IHC) analysis using a large-scale sample size of gene expression profiles. The CD8+ T cell/CAF ratio (CFR) association with survival was investigated in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) lung cancer cohorts. The correlation between CFR and immunotherapeutic efficacy was computed in five independent cohorts. The correlation between CFR and objective response rates (ORRs) following pembrolizumab monotherapy was investigated in 20 solid tumor types. To facilitate clinical translation, the IHC-detected CD8/α-SMA ratio was applied as an immunotherapeutic predictive biomarker in a real-world lung cancer cohort. Results: Compared with normal tissue, CAFs were enriched in cancer tissue, and the amount of CAFs was overwhelmingly higher than that in other immune cells. CAFs are positively correlated with the extent of immune infiltration. A higher CFR was strongly associated with improved survival in lung cancer, melanoma, and urothelial cancer immunotherapy cohorts. Within most cohorts, there was no clear evidence for an association between CFR and programmed death-ligand 1 (PD-L1) or tumor mutational burden (TMB). Compared with TMB and PD-L1, a higher correlation coefficient was observed between CFR and the ORR following pembrolizumab monotherapy in 20 solid tumor types (Spearman's r = 0.69 vs. 0.44 and 0.21). In a real-world cohort, patients with a high CFR detected by IHC benefited considerably from immunotherapy as compared with those with a low CFR (hazard ratio, 0.37; 95% confidence interval, 0.19-0.75; p < 0.001). Conclusions: CFR is a newly found and simple parameter that can be used for identifying patients unlikely to benefit from immunotherapy. Future studies are needed to confirm this finding.

Heterogeneity of tumor immune microenvironment and real-world analysis of immunotherapy efficacy in lung adenosquamous carcinoma.

Lung adenosquamous carcinoma (ASC) is an uncommon histological subtype. We aimed to characterize the tumor immune microenvironment (TIME) in lung ASC and estimate patient response to immune checkpoint inhibitors (ICIs), which have never been systematically investigated. In cohort I, we collected 30 ASCs from a single center for analysis of TIME characteristics, including immuno-phenotyping, tumor mutation burden (TMB), T-cell receptor (TCR) repertoires, tumor-infiltrating lymphocytes (TILs), and immune checkpoint expression. Twenty-two (73.3%) patients were EGFR-positive. The TIME was defined by immune-excluded (60%) and immune-desert phenotype (40%). Strikingly, programmed cell death-ligand 1 (PD-L1) and programmed cell death-1 (PD-1) were predominantly expressed in squamous cell carcinoma components (SCCCs) versus adenocarcinoma components (ACCs), where enhanced CD4+ FOXP3+ regulatory T cell and attenuated CD57+ natural killer cell infiltration were present, consistent with a landscape of fewer innate immune cells, more immunosuppressive cells. SCCCs had higher TMB, higher TCR clonality, and lower TCR diversity than ACC. In cohort III, the efficacy of ICI-based therapy was estimated using a real-world data of 46 ASCs from 11 centers. Majority of 46 patients were driver genes negative and unknown mutation status, 18 (39%) and 18 (39%), respectively. The overall objective response rate of 28%, median progression-free survival of 6.0 months (95% confidence interval [CI] 4.3-7.7), and median overall survival of 24.7 months (95% CI 7.2-42.2) were observed in the ICI-based treatment. This work ascertains suppressive TIME in lung ASC and genetic and immuno-heterogeneity between ACCs and SCCCs. Lung ASC patients have a moderate response to ICI-based immunotherapy.

Somatic Mutation of FAT Family Genes Implicated Superior Prognosis in Patients With Stomach Adenocarcinoma.

FAT family genes encode protocadherin, which regulates tumor cell proliferation and migration. Although transcriptional levels of FAT family members had been reported in multiple malignant tumors, the association between mutation and prognosis of the FAT family in stomach adenocarcinoma (STAD) has not been investigated. Herein, we performed a multi-omics integrative bioinformatics analysis using genomic and mRNA expression data to explore the role of gene mutations across the FAT family on clinical outcomes of STAD. The results showed that FAT mutations occurred in 174 of 435 (40%) of the samples. Patients with FAT mutations possessed significantly better progression-free survival (P = 0.019) and overall survival (P = 0.034) than those with non-FAT mutations, and FAT mutations exhibited significantly higher tumor mutational burden (TMB) and microsatellite instability. Notably, FAT mutations had a greater effect on somatic single-nucleotide variation than copy number variation and resulted in more abundant DNA damage repair (DDR) mutations. Further investigation demonstrated that FAT mutations contributed to an inflammatory tumor microenvironment (TME), as indicated by significantly increased numbers of activated CD4 and CD8 T cells, and significantly decreased numbers of mast cell, plasmacytoid dendritic cell, type 2 T helper cell, and high expression of immune-promoting genes. Moreover, biological process antigen processing and presentation, DNA replication, and DDR-related pathways were significantly upregulated in patients with FAT mutations. Collectively, FAT mutations significantly improved the survival of patients with STAD by enhancing tumor immunogenicity (e.g., TMB and DDR mutations) and an inflamed TME, indicating that the FAT family might be a potential prognostic and therapeutic biomarker for STAD.

Co-occurrence of CDKN2A/B and IFN-I homozygous deletions correlates with an immunosuppressive phenotype and poor prognosis in lung adenocarcinoma.

Homozygous deletion (HD) of CDKN2A and CDKN2B (CDKN2A/BHD ) is the most frequent copy-number variation (CNV) in lung adenocarcinoma (LUAD). CDKN2A/BHD has been associated with poor outcomes in LUAD; however, the mechanisms of its prognostic effect remain unknown. We analyzed genome, transcriptome, and clinical data from 517 patients with LUAD from the Cancer Genome Atlas (TCGA) and from 788 primary LUAD tumor and matched control samples from the MSK-IMPACT clinical cohort. CDKN2A/BHD was present in 19.1% of the TCGA-LUAD cohort and in 5.7% of the MSK-IMPACT cohort. CDKN2A/BHD patients had shorter disease-free survival and overall survival compared with CDKN2A/BWT individuals in both cohorts. Differences in clinical features did not influence the outcomes in the CDKN2A/BHD population. Mutation analyses showed that overall tumor mutational burden and mutations in classical drivers such as EGFR and RB1 were not associated with CDKN2A/BHD . In contrast, homozygous deletion of type I interferons (IFN-IHD ) frequently co-occurred with CDKN2A/BHD . CDKN2A/B and IFN-I are co-located in the same p21.3 region of chromosome 9. The co-occurrence of CDKN2A/BHD and IFN-IHD was not related to whole-genome doubling, chromosome instability, or aneuploidy. Patients with co-occurring CDKN2A/BHD and IFN-IHD had shorter disease-free survival and overall survival compared with CDKN2A/BWT patients. CDKN2A/BHD IFN-IHD had downregulated several key immune response pathways, suggesting that poor prognosis in CDKN2A/BHD LUAD could potentially be attributed to an immunosuppressive tumor microenvironment as a result of IFN-I depletion.

Safety and efficacy of mutant neoantigen-specific T-cell treatment combined anti-PD-1 therapy in stage IV solid tumors.

Aims: This trial explored the safety and efficacy of neoantigen-specific T cells (Nas-Ts) combined with anti-PD-1 (Nas-T + anti-PD-1). Patients & methods: This non-randomized trial recruited participants with solid tumors treated with at least two prior systemic treatment lines. For comparison, 1:1-matched controls who received anti-PD-1 alone were recruited. The primary end point was safety. Results: 15 participants were enrolled in the Nas-T + anti-PD-1 group, the objective response rate was 33.3%, and the disease control rate was 93.3%. The median progression-free survival was significantly different between the Nas-T + anti-PD-1 and control groups (13.8 vs 4.2 months; p = 0.024), but no difference in overall survival was found (p = 0.126). The most common adverse events were maculopapular skin reaction (53.3%), rash (53.3%), hepatotoxicity (53.3%) and fever (53.3%) in the Nas-T + anti-PD-1 group. No serious safety issues were experienced. Conclusion: Nas-Ts combined with anti-PD-1 could be more effective than anti-PD-1 alone in prolonging progression-free survival, with good safety.

Cancer immune escape is a major mechanism allowing cancer cells to avoid treatments, and PD-1 is one of those mechanisms. Nevertheless, therapies targeting PD-1 are still somewhat unsatisfactory. In this trial, we explored the safety and efficacy of mutant neoantigen-specific T cells (Nas-Ts) as adoptive cell immunotherapy individualized for each tumor, combined with an anti-PD-1 regimen (Nas-T + anti-PD-1). We recruited participants with solid tumors treated with at least two prior systemic treatment lines: 15 participants were enrolled in the Nas-T + anti-PD-1 group and 15 more in the control group. After the last follow-up, the percentage of patients on whom a therapy had some defined effect as well as the percentage of patients with advanced and metastatic cancer who achieved complete response was significantly higher for those who received Nas-T + anti-PD-1. No serious safety issues were experienced. This study confirmed that Nas-Ts combined with anti-PD-1 could be more effective than anti-PD-1 alone in delaying progression, with good safety.

Activation of PI3K/AKT Pathway Is a Potential Mechanism of Treatment Resistance in Small Cell Lung Cancer.

PURPOSE: Here, we have investigated treatment resistance mechanisms in small cell lung cancer (SCLC) by focusing on comparing the genotype and phenotype in tumor samples of treatment-resistant and treatment-sensitive SCLC. EXPERIMENTAL DESIGN: We conducted whole-exome sequencing on paired tumor samples at diagnosis and relapse from 11 patients with limited-stage (LS)-SCLC and targeted sequencing of 1,021 cancer-related genes on cell-free DNA at baseline and paired relapsed samples from 9 additional patients with LS-SCLC. Furthermore, we performed label-free mass spectrometry-based proteomics on tumor samples from 28 chemo-resistant and 23 chemo-sensitive patients with extensive-stage (ES)-SCLC. The main findings were validated in vitro in chemo-sensitive versus chemo-resistant SCLC cell lines and analyses of transcriptomic data of SCLC cell lines from a public database. RESULTS: Genomic analyses demonstrated that at relapse of LS-SCLC, genes in the PI3K/AKT signaling pathway were enriched for acquired somatic mutations or high-frequency acquired copy-number variants. Pathway analysis on differentially upregulated proteins from ES-SCLC cohort revealed enrichment in the HIF-1 signaling pathway. Importantly, 7 of 62 PI3K/AKT pathway genes containing acquired somatic copy-number amplifications were enriched in HIF-1 pathway. Analyses of transcriptomic data of SCLC cell lines from public databases confirmed upregulation of PI3K/AKT and HIF-1 pathways in chemo-resistant SCLC cell lines. Furthermore, chemotherapy-resistant cell lines could be sensitive to PI3K inhibitors in vitro. CONCLUSIONS: PI3K/AKT pathway activation may be one potential mechanism underlying therapeutic resistance of SCLC. This finding warrants further investigation and provides a possible approach to reverse resistance to chemo/radiotherapy.

T-Cell Receptor Profiling and Prognosis After Stereotactic Body Radiation Therapy For Stage I Non-Small-Cell Lung Cancer.

Radiotherapy is known to influence immune function, including T cell receptor (TCR) repertoire. We evaluated the TCR repertoire before and after stereotactic body radiotherapy (SBRT) for stage I non-small-cell lung cancer (NSCLC) and explored correlations between TCR indexes and distant failure after SBRT. TCR repertoires were analyzed in peripheral blood mononuclear cells (PBMCs) collected before and after SBRT from 19 patients. TCR combinational diversity in V and J genes was assessed with multiplex PCR of genomic DNA from PBMCs and tested for associations with clinical response. All patients received definitive SBRT to a biologically effective dose of >=100 Gy. The number of unique TCR clones was decreased after SBRT versus before, but clonality and the Shannon Entropy did not change. Four patients (21%) developed distant metastases after SBRT (median 7 months); those patients had lower Shannon Entropy in post-SBRT samples than patients without metastasis. Patients with a low change in Shannon Entropy from before to after SBRT [(post-SBRT Shannon Entropy minus baseline Shannon)/(baseline Shannon) * 100] had poorer metastasis-free survival than those with high change in Shannon Entropy (P<0.001). Frequencies in V/J gene fragment expression in the TCR ß chain were also different for patients with or without metastases (two V fragments in baseline samples and 2 J and 9 V fragments in post-treatment samples). This comprehensive analysis of immune status before and after SBRT showed that quantitative assessments of TCRs can help evaluate prognosis in early-stage NSCLC.

Genomic comparison between cerebrospinal fluid and primary tumor revealed the genetic events associated with brain metastasis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is most common pathological type of lung cancer. LUAD with brain metastases (BMs) usually have poor prognosis. To identify the potential genetic factors associated with BM, a genomic comparison for BM cerebrospinal fluid (CSF) and primary lung tumor samples obtained from 1082 early- and late-stage LUAD patients was performed. We found that single nucleotide variation (SNV) of EGFR was highly enriched in CSF (87% of samples). Compared with the other primary lung tissues, copy number gain of EGFR (27%), CDK4 (11%), PMS2 (11%), MET (10%), IL7R (8%), RICTOR (7%), FLT4 (5%), and FGFR4 (4%), and copy number loss of CDKN2A (28%) and CDKN2B (18%) were remarkably more frequent in CSF samples. CSF had significantly lower tumor mutation burden (TMB) level but more abundant copy number variant. It was also found that the relationships among co-occurrent and mutually exclusive genes were dynamically changing with LUAD development. Additionally, CSF (97% of samples) harbored more abundant targeted drugs related driver and fusion genes. The signature 15 associated with defective DNA mismatch repair (dMMR) was only identified in the CSF group. Cancer associated pathway analysis further revealed that ErbB (95%) and cell cycle (84%) were unique pathways in CSF samples. The tumor evolution analysis showed that CSF carried significantly fewer clusters, but subclonal proportion of EGFR was remarkably increased with tumor progression. Collectively, CSF sequencing showed unique genomic characteristics and the intense copy number instability associated with cell cycle disorder and dMMR might be the crucial genetic factors in BM of LUAD.

Multi-region exome sequencing reveals the intratumoral heterogeneity of surgically resected small cell lung cancer.

Small cell lung cancer (SCLC) is a highly malignant tumor which is eventually refractory to any treatment. Intratumoral heterogeneity (ITH) may contribute to treatment failure. However, the extent of ITH in SCLC is still largely unknown. Here, we subject 120 tumor samples from 40 stage I-III SCLC patients to multi-regional whole-exome sequencing. The most common mutant genes are TP53 (88%) and RB1 (72%). We observe a medium level of mutational heterogeneity (0.30, range 0.0~0.98) and tumor mutational burden (TMB, 10.2 mutations/Mb, range 1.1~51.7). Our SCLC samples also exhibit somatic copy number variation (CNV) across all patients, with an average CNV ITH of 0.49 (range 0.02~0.99). In terms of mutation distribution, ITH, TMB, mutation clusters, and gene signatures, patients with combined SCLC behave roughly the same way as patients with pure SCLC. This condition also exists in smoking patients and patients with EGFR mutations. A higher TMB per cluster is associated with better disease-free survival while single-nucleotide variant ITH is linked to worse overall survival, and therefore these features may be used as prognostic biomarkers for SCLC. Together, these findings demonstrate the intratumoral genetic heterogeneity of surgically resected SCLC and provide insights into resistance to treatment.

Lung adenocarcinoma with ERBB2 exon 20 insertions: Comutations and immunogenomic features related to chemoimmunotherapy.

BACKGROUND: The genomic mutation and immune feature landscape of ERBB2 exon 20 insertion (ERBB2-ex20ins)-driven non-small cell lung cancer and the features associated with the response to chemoimmunotherapy are currently unknown. METHODS: The genomic landscape of ERBB2-ex20ins lung adenocarcinoma (LUAD) patients was characterized by next-generation sequencing (NGS) of 1021 cancer genes. The clinical outcomes of chemoimmunotherapy were evaluated among 13 patients with stage IV ERBB2-ex20ins LUAD, and potential biomarkers of the response to chemoimmunotherapy were explored using NGS and T cell receptor sequencing. RESULTS: Among 8247 LUAD patients, 207 (2.5%) had ERBB2-ex20ins, of whom 181 (87.4%) harbored more than one comutation. The most common comutations were in TP53. Patients with ERBB2-ex20ins had a low tumor mutational burden (TMB; median, 4.2 mutations/Mb), and most (66.7%) were PD-L1 negative. Thirteen of the 207 patients received chemoimmunotherapy, for whom the objective response rate, disease control rate, and median progression-free survival were 31%, 77%, and 8.0 months, respectively. Responders exhibited a higher TMB and a trend toward lower clonality in tumors compared with nonresponders (p = 0.0067 and p = 0.085, respectively). A high TMB combined with mutations in DNA damage repair pathways and SWI/SNF chromatin remodeling complexes was associated with a benefit from chemoimmunotherapy. CONCLUSIONS: The efficacy and outcome of chemoimmunotherapy were encouraging among ERBB2-ex20ins LUAD patients, who were characterized by low TMB and negative PD-L1 expression. The combination of TMB and comutations is a potential biomarker to identify patients who will benefit from chemoimmunotherapy.

Molecular subtype identification and prognosis stratification by a metabolism-related gene expression signature in colorectal cancer.

BACKGROUND: Metabolic reprograming have been associated with cancer occurrence and progression within the tumor immune microenvironment. However, the prognostic potential of metabolism-related genes in colorectal cancer (CRC) has not been comprehensively studied. Here, we investigated metabolic transcript-related CRC subtypes and relevant immune landscapes, and developed a metabolic risk score (MRS) for survival prediction. METHODS: Metabolism-related genes were collected from the Molecular Signatures Database and metabolic subtypes were identified using an unsupervised clustering algorithm based on the expression profiles of survival-related metabolic genes in GSE39582. The ssGSEA and ESTIMATE methods were applied to estimate the immune infiltration among subtypes. The MRS model was developed using LASSO Cox regression in the GSE39582 dataset and independently validated in the TCGA CRC and GSE17537 datasets. RESULTS: We identified two metabolism-related subtypes (cluster-A and cluster-B) of CRC based on the expression profiles of 539 survival-related metabolic genes with distinct immune profiles and notably different prognoses. The cluster-B subtype had a shorter OS and RFS than the cluster-A subtype. Eighteen metabolism-related genes that were mostly involved in lipid metabolism pathways were used to build the MRS in GSE39582. Patients with higher MRS had worse prognosis than those with lower MRS (HR 3.45, P < 0.001). The prognostic role of MRS was validated in the TCGA CRC (HR 2.12, P = 0.00017) and GSE17537 datasets (HR 2.67, P = 0.039). Time-dependent receiver operating characteristic curve and stratified analyses revealed the robust predictive ability of the MRS in each dataset. Multivariate Cox regression analysis indicted that the MRS could predict OS independent of TNM stage and age. CONCLUSIONS: Our study provides novel insight into metabolic heterogeneity and its relationship with immune landscape in CRC. The MRS was identified as a robust prognostic marker and may facilitate individualized therapy for CRC patients.

An epigenomic landscape of cervical intraepithelial neoplasia and cervical cancer using single-base resolution methylome and hydroxymethylome.

BACKGROUND: Cervical cancer (CC) is the second leading cause of cancer death among women worldwide. Epigenetic regulation of gene expression through DNA methylation and hydroxymethylation plays a pivotal role during tumorigenesis. In this study, to analyze the epigenomic landscape and identify potential biomarkers for CCs, we selected a series of samples from normal to cervical intra-epithelial neoplasia (CINs) to CCs and performed an integrative analysis of whole-genome bisulfite sequencing (WGBS-seq), oxidative WGBS, RNA-seq, and external histone modifications profiling data. RESULTS: In the development and progression of CC, there were genome-wide hypo-methylation and hypo-hydroxymethylation, accompanied by local hyper-methylation and hyper-hydroxymethylation. Hydroxymethylation prefers to distribute in the CpG islands and CpG shores, as displayed a trend of gradual decline from health to CIN2, while a trend of increase from CIN3 to CC. The differentially methylated and hydroxymethylated region-associated genes both enriched in Hippo and other cancer-related signaling pathways that drive cervical carcinogenesis. Furthermore, we identified eight novel differentially methylated/hydroxymethylated-associated genes (DES, MAL, MTIF2, PIP5K1A, RPS6KA6, ANGEL2, MPP, and PAPSS2) significantly correlated with the overall survival of CC. In addition, no any correlation was observed between methylation or hydroxymethylation levels and somatic copy number variations in CINs and CCs. CONCLUSION: Our current study systematically delineates the map of methylome and hydroxymethylome from CINs to CC, and some differentially methylated/hydroxymethylated-associated genes can be used as the potential epigenetic biomarkers in CC prognosis.

The Dynamic Alternation of Local and Systemic Tumor Immune Microenvironment During Concurrent Chemoradiotherapy of Cervical Cancer: A Prospective Clinical Trial.

PURPOSE: This work assessed local and systemic alternations of the tumor immune microenvironment during concurrent chemoradiation therapy (CCRT) of local advanced cervical cancer to estimate the optimal timing for immune therapy in relation to CCRT. METHODS AND MATERIALS: In this single-center prospective clinical trial, 55 patients with stage IIA through IVA cervical cancer were enrolled between December 2016 and November 2017. The median follow-up was 32.1 months. All patients received cisplatin concurrently with external beam radiation therapy combined with high-dose-rate brachytherapy. Tumor tissues and peripheral blood mononuclear cells (PBMCs) were collected before, during and after CCRT. We analyzed the changes in lymphocyte subpopulations, programmed death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, and the T cell receptor (TCR) repertoire that occurred throughout CCRT. RESULTS: The frequencies of CD4+ and PD-1+ T cells in PBMCs decreased after the start of CCRT, whereas that of inhibitory regulatory T cells increased. In the tumor tissues, CCRT decreased the numbers of CD4+ and CD8+ T cells and reduced the median percentage of positive cells expressing PD-L1 from 78.1% to 49.8%. As indicated by the numbers of unique clones, the TCRs of PBMCs exhibited greater diversity before CCRT than after CCRT. Greater TCR diversity in PBMCs before CCRT was associated with superior 30-month progression-free survival (hazard ratio [HR], 0.12; 95% confidence interval [CI], 0.04-0.39; P = .001) and overall survival (HR, 0.17; 95% CI, 0.04-0.68; P = .004). CONCLUSIONS: CCRT for cervical cancer altered the tumor immune microenvironment by reducing CD4+ and CD8+ T lymphocyte populations, PD-1/PD-L1 expression, and TCR diversity. Higher TCR diversity in PBMCs before CCRT resulted in better survival and prognosis, indicating that CCRT might inhibit immune activation. Our results suggest that it might be more effective to administer immune checkpoint inhibitors before CCRT of cervical cancer rather than during or after CCRT.

Genomic profiles and tumor immune microenvironment of primary lung carcinoma and brain oligo-metastasis.

Brain metastasis (BM) is a common malignant event in lung cancer. Here, we recruited 33 lung cancer patients with brain oligo-metastasis to explore the genomic features and tumor immune microenvironment (TIME) of the lung and BM independently. For genomic profiling, targeted sequencing was performed. We found that high-frequent ZFHX3 occurred in the lung (40%) and brain tumor (28%), which might relate to brain metastasis event; the vast majority of patients had lesions-shared mutations in primary tumor and BM, confirming the common clonal events; and EGFR was the most frequently clonal gene in both lung and BM, indicating its driver capability. To characterize TIME status, we also sequenced the T cell receptor (TCR) repertoires and performed immunohistochemistry (IHC) on CD8+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in 28 patients who had paired samples. Through the comparison, the TCR clonality of BM was higher than lung tumor, indicating the distinct pattern of the stronger oligoclonal T cell expansion in BM; the primary tumor had a higher TMB than oligo-BM (13.9 vs 8.7 mutations, p = 0.019); CD8 + TILs of BM were significantly lower than lung tumor (10% vs 30%, p = 0.015), revealing the lower level of cytotoxic T cell infiltration; BM showed statistically equivalent level of PD-L1 compared with lung tumor (p = 0.722). We further investigated the potential biomarkers associated with overall survival (OS) after brain surgery. We found that higher TCR clonality was related to prolonged OS in EGFR-treated patients (HR 0.175, p < 0.001) but the worse outcomes in non-EGFR-treated (HR 2.623, p = 0.034). More CD8+ TILs were an independently positive indicator for OS, in EGFR-treated (HR 0.160, p = 0.001) and non-EGFR-treated patients (HR 0.308, p = 0.009). These findings provide a meaningful molecular and clinical understanding of lung carcinoma and brain oligo-metastasis.

Post-radiation circulating tumor DNA as a prognostic factor in locally advanced esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant and deadly tumor. Radiation therapy is one of the primary treatments for locally advanced ESCC. However, the biomarkers for prognosis of definitive radiation remain undefined. Peripheral blood circulating tumor (ct)DNA provides information of tumor genetic alterations and has been confirmed as a potential non-invasive biomarker for several types of cancer. The present study investigated the clinical implications of ctDNA detection in patients with ESCC and receiving definitive radiation therapy. Patients with locally advanced ESCC were retrospectively recruited. Plasma samples were collected before, during and following radiation therapy. Next-generation sequencing was performed to identify somatic mutations in 180 genes. A total of 69 baseline and post-radiation plasma samples were collected from 25 patients. A total of 59 non-silent single nucleotide variants were present in 33 genes. All pre-radiation and 58.3% (14/24) of post-radiation samples had at least one mutation. Patients with lymph node metastases (LNM) exhibited a higher number of pre-radiation mutations compared with those without LNM. The variables, progression-free survival (PFS) and overall survival (OS) of the patients with one baseline mutation were not significantly different compared with that in patients with more than one baseline mutation. Patients with initial ctDNA-positive post-radiation samples exhibited significantly reduced PFS (P=0.047) and OS (P=0.005) compared with that in patients with ctDNA-negative samples. The post-radiation plasma ctDNA status was an independent prognostic factor from univariate and multivariate analyses. Dynamic monitoring of ctDNA during follow-up was examined. The results indicated that ctDNA was a predictive and prognostic marker in patients with ESCC and receiving definitive radiation therapy, which may guide subsequent treatment.