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PurposeAnalysis of circulating tumor DNA (ctDNA) in blood could allow noninvasive genetic analysis of primary tumors. Although there have been unmet needs for noninvasive methods in patients with primary central nervous system lymphoma (PCNSL), it is still not determined whether plasma ctDNA analysis could be useful for patients with PCNSL. Materials and MethodsTargeted deep sequencing of 54 genes was performed in cell-free DNA isolated from plasma samples collected pretreatment, during treatment, and at the end of treatment in 42 consecutively diagnosed PCNSL patients between January 2017 and December 2018.ResultsTargeted sequencing of plasma cell-free DNA detected somatic mutations representing ctDNA in 11 cases (11/41, 27%). The detection of ctDNA was not related to the concentration of cell-free DNA or tumor volume. The mutation profiles of these 11 cases varied between patients. The most frequently mutated gene was PIM1 (4/11, 36.4%), whereas KMT2D, PIK3CA, and MYD88 were each observed in three patients (3/11, 27%). The mutations of 13 genes were concordantly found in primary tumor tissue and plasma ctDNA, giving a detection sensitivity of 45%. During the serial tracking of seven patients with complete response, the disappearance of ctDNA mutations was found in four patients, whereas three patients had detected ctDNA mutation at the end of treatment. ConclusionThe plasma ctDNA mutation analysis still has limited value for surveillance and predicting treatment outcomes of PCNSL because the detection efficiency was lower than other systemic lymphomas. Thus, analytical platforms should be improved to overcome anatomical hurdles associated with PCNSL.

Background: Palbociclib plus endocrine therapy (ET) demonstrated significant progression-free survival (PFS) benefit in Young Pearl, a randomized phase ll trial comparing palbociclib + ET versus capecitabine in premenopausal women with hormone receptor positive, HER2 negative metastatic breast cancer (MBC). This exploratory analysis investigated potential biomarkers of palbociclib plus ET on PFS. Patients and methods: Of 178 patients randomized (92 palbociclib plus ET; 86 capecitabine), we performed targeted sequencing (141 patients) and whole transcriptome sequencing (165 patients) using baseline tumor samples to examine genomic alteration in relation to drug response on PFS. Hazard ratios (HRs) were estimated using unstratified Cox proportional hazards models. Results: PIK3CA (41%) and TP53 (33%) mutations and CCND1 copy number variation (29%) were found most frequently in targeted sequencing of 141 patients. ESR1 mutations were found only in 3.5% of patients of this population. Luminal type showed better prognosis in palbociclib + ET arm but no impact on PFS difference in capecitabine arm. High TMB, TP53 mutation, PTEN loss of function mutation and RB1 pathway alteration showed worse prognosis in palbociclib plus ET arm. Patients with BRCA2 pathogenic mutations showed worse prognosis regardless of PAM50 subtypes. AURKA mutation/amplification, BRIP1/MYC/RAD51C amplification were significantly associated to the patients with short PFS

Cone-beam computed tomography (CBCT) is a promising imaging modality for sinonasal evaluation, with advantages of relatively low radiation dose, low cost, and quick outpatient imaging. Our study aimed to compare the diagnostic performance and image quality of CBCT with those of multi-detector computed tomography (MDCT) with different slice thickness.We retrospectively reviewed 60 consecutive patients who had undergone both CBCT and MDCT. MDCT images was reconstructed with 1 and 3 mm slice thickness. The quantitative image quality parameters (image noise, signal-to-noise ratio [SNR], and contrast-to noise ratio [CNR] were calculated and compared between the two imaging modalities. Two observers (ENT surgeon and neuroradiologist) evaluated the presence of seven sinonasal anatomic variations in each patient and interobserver agreements were analyzed. The diagnostic performance of CBCT (0.3 mm) and MDCT (3 mm) was assessed and compared with that of high resolution MDCT (1 mm), which is considered as the gold standard.The image noise was significantly higher and SNR and CNR values were lower in the CBCT (0.3 mm) group than in the MDCT groups (1 and 3 mm). The diagnostic performance of CBCT (0.3 mm) was similar to that of MDCT (1 mm) and superior to that of MDCT (3 mm). The highest interobserver agreement was for high resolution MDCT (1 mm), followed by CBCT (0.3 mm), and MDCT (3 mm).Considering its low radiation dose, low cost, and ease of clinical access, CBCT may be a useful imaging modality for as first line sinonasal evaluation and repeated follow up.Study design: Retrospective study in a tertiary referral university center.Level of evidence: NA.

Adding anti-PD1 antibodies to 5-FU/platinum chemotherapy improves survival in a subset of advanced gastroesophageal adenocarcinoma (GEA) patients. Beyond PD-L1 expression and mismatch repair status we have limited insight into molecular predictors of response, or the relative contribution of PD-1 blockade. We conducted an investigator sponsored phase II trial (n = 47) sequentially adding pembrolizumab to standard 5-FU/platinum in previously untreated advanced GEA (ClinicalTrials.gov: NCT04249739). With an overall response rate of 67% the activity paralleled phase III chemoimmunotherapy trials. To understand on-treatment tumor and immune adaptations patients underwent serial biopsy of the primary tumor, including baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab. We leveraged transcriptional profiling from 358,067 cells to identify multicellular networks of malignant, stromal, and immune cells after chemotherapy and concurrent chemoimmunotherapy. The relative usage of pro-tumor and anti-tumor interaction hubs differed between fast and slow progressing patients. Chemotherapy induced early on-treatment formation of hubs centered on tumor-reactive T-cell and M1-oriented macrophage interactions with pro-inflammatory cytokines in slow progressors. Faster progression was characterized by increased MUC5A and MSLN containing programs in tumor cells and M2-oriented macrophages with immunosuppressive stromal interactions. After adding pembrolizumab we observed increased CD8 T-cell infiltration by scRNAseq and multiplex immunofluorescence and development of an immunity hub involving co-variation of the tumor-reactive CXCL13 program and epithelial interferon-stimulated gene programs enriched in slow progressors. Together this data provides prospective evidence of differential early on-treatment evolution of the gastric immune microenvironment and nominates candidate cellular interactions for clinical targeting.

Chemotherapy is ubiquitous in first-line treatment of advanced gastric cancer, yet responses are heterogeneous, and little is known about mediators of chemotherapy response. To move forward, an understanding of the effects of standard chemotherapy on the tumor–immune microenvironment (TME) is needed. Coupling whole-exome sequencing, bulk RNA and single-cell transcriptomics from paired pretreatment and on-treatment samples in treatment-naïve patients with HER2-positive and HER2-negative gastric cancer, we define features associated with response to platinum-based chemotherapy. Response was associated with on-treatment TME remodeling including natural killer (NK) cell recruitment, decreased tumor-associated macrophages, M1-macrophage repolarization, and increased effector T-cell infiltration. Among chemotherapy nonresponders, we observed low/absent PD-L1 expression or modulation, on-treatment increases in Wnt signaling, B-cell infiltration, and LAG3-expressing T cells coupled to an exodus of dendritic cells. We did not observe significant genomic changes in early on-treatment sampling. We provide a map of on-treatment TME modulation with standard chemotherapy and nominate candidate future approaches.Significance:Using paired pretreatment and on-treatment samples during standard first-line chemotherapy, we identify chemotherapy-induced NK-cell infiltration, macrophage repolarization, and increased antigen presentation among responders. Increased LAG3 expression and decreased dendritic cell abundance were seen in nonresponders, emphasizing remodeling of the TME during chemotherapy response and resistance.This article is highlighted in the In This Issue feature, p. 873

Supplementary Figure from Early Tumor–Immune Microenvironmental Remodeling and Response to First-Line Fluoropyrimidine and Platinum Chemotherapy in Advanced Gastric Cancer

Adding anti-PD1 antibodies to 5-FU/platinum chemotherapy improves survival in a subset of advanced gastroesophageal adenocarcinoma (GEA) patients. Beyond PD-L1 expression and mismatch repair status we have limited insight into molecular predictors of response or the relative contribution of PD-1 blockade. We conducted an investigator sponsored phase II trial (n = 47) sequentially adding pembrolizumab to standard 5-FU/platinum in previously untreated advanced GEA (ClinicalTrials.gov:NCT04249739). With an overall response rate of 67% the activity paralleled phase III chemoimmunotherapy trials. To understand on-treatment tumor and immune adaptations patients underwent serial biopsy of the primary tumor, including baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab. We leveraged transcriptional profiling from 358,067 cells to identify multicellular networks of malignant, stromal, and immune cells after chemotherapy and concurrent chemoimmunotherapy. The relative usage of pro-tumor and anti-tumor interaction hubs differed between fast and slow progressing patients. Chemotherapy induced early on-treatment formation of hubs centered on tumor-reactive T-cell and M1-oriented macrophage interactions with pro-inflammatory cytokines in slow progressors. Faster progression was characterized by increased MUC5A and MSLN containing programs in tumor cells and M2-oriented macrophages with immunosuppressive stromal interactions. After adding pembrolizumab we observed increased CD8 T-cell infiltration by scRNAseq and multiplex immunofluorescence and development of an immunity hub involving co-variation of the tumor-reactive CXCL13 program and epithelial interferon-stimulated gene programs enriched in slow progressors. Together this data provides prospective evidence of differential early on-treatment evolution of the gastric immune microenvironment and nominates candidate cellular interactions for clinical targeting.

Supplementary Table from Early Tumor–Immune Microenvironmental Remodeling and Response to First-Line Fluoropyrimidine and Platinum Chemotherapy in Advanced Gastric Cancer

Figure S8. Volcano plot of GSVA analyses showing the difference between (A) merged gBRCA1/2 mutation vs. wild type (WT), (B) gBRCA mutations vs. WT in triple negative patients, (C) gBRCA2 mutations vs. WT in estrogen receptor-positive patients.

Table S1. List of 381 genes from CancerSCANTM Table S2. gBRCA1/2 mutation profile of study population. Table S3. Raw data for differentially expressed genes found by comparing gBRCA1 and gBRCA2 mutants. Table S4. Raw GSVA data derived by comparing gBRCA1 and gBRCA2 mutants. Table S5. Raw data for differentially expressed genes found by comparing patients with mutated and wild-type gBRCA1/2. Table S6. Raw GSVA data derived by comparing patients with mutated and wild-type gBRCA1/2. Table S7. Raw data for differentially expressed genes found by comparing mutated and wild-type gBRCA1 in triple negative patients. Table S8. Raw GSVA data derived by comparing mutated and wild-type gBRCA1 in triple negative patients. Table S9. Raw data for differentially expressed genes found by comparing mutated and wild-type gBRCA2 in estrogen receptor-positive patients. Table S10. Raw GSVA data derived by comparing mutated and wild-type gBRCA2 in estrogen receptor-positive patients. Table S11. Loss of heterozygosity status and the allele frequency of samples available for the target sequencing.

Purpose:Although programmed death 1/programmed death ligand 1 (PD-1/PD-L1) inhibitors are promising agents for recurrent or metastatic nasopharyngeal carcinoma (NPC), PD-1/PD-L1 inhibitor monotherapy has shown modest efficacy. This study evaluated the efficacy and safety of nivolumab plus gemcitabine in patients with NPC who failed prior platinum-based chemotherapy.Patients and Methods:This is a phase II, multicenter, open-label, single-arm study. Patients with recurrent or metastatic NPC received nivolumab 3 mg/kg and gemcitabine 1,250 mg/m2 every 2 weeks until disease progression or intolerable toxicity. The primary endpoint was progression-free survival (PFS). The secondary endpoints included objective response rate (ORR), overall survival (OS), and safety. To identify potential biomarkers, whole-exome sequencing, whole-transcriptome sequencing, and immune phenotype analysis based on Lunit SCOPE IO, an artificial intelligence–powered spatial tumor-infiltrating lymphocyte analyzer, were performed.Results:Thirty-six patients were enrolled between June 2018 and June 2019. The ORR was 36.1% and disease control rate was 97.2%. With median follow-up of 22.0 months, median PFS was 13.8 months [95% confidence interval (CI), 8.6–16.8 months]. Median OS was not reached, and OS rate at 6 months was 97.0% (95% CI, 80.4%–99.6%). The grade ≥3 treatment-related adverse events were hypertension (2.8%) and anemia (2.8%). In multivariate analysis of mutation of chromatin modifier gene, tumor mutational burden (≥ 2.1 mut/Mb), and somatic copy-number alteration (SCNA) level, the group with high SCNA (> 3 points; HR, 7.0; 95% CI, 1.3–37.9; P = 0.02) had independently associated with poor PFS. Immune phenotype analysis showed that tumors with high proportion of immune-excluded immune phenotype was significantly correlated with poor PFS (HR, 4.4; 95% CI, 1.2–16.2; P = 0.018).Conclusions:Nivolumab plus gemcitabine showed promising efficacy with favorable toxicity profiles in patients with advanced NPC in whom platinum-based combination chemotherapy failed.

Supplementary Data from A Phase II Study of Nivolumab plus Gemcitabine in Patients with Recurrent or Metastatic Nasopharyngeal Carcinoma (KCSG HN17–11)

Figure S1. Somatic mutations of each tumor. Missense, nonsense and splicing identified by whole-exome sequencing analysis

Supplementary Figure S1. Histopathology of NSCLC brain metastases and their corresponding PDXs. Histological subtype and differentiation status was indicated for each case. ADC = adenocarcinoma, LCC = large cell carcinoma, SQCC = squamous cell carcinoma.

Supplementary Table S1. Read mapping information of CRC samples

Supplementary Figure 1. Survival plot using Kaplan-Meier survival analysis between two patient groups with high- and low-heterogeneity.

Visualization of immune cell landscape for multiple myeloma and healthy bone marrow.

Gene expression features of myeloma cells.

Purpose: Tumors continuously evolve to maintain growth; secondary mutations facilitate this process, resulting in high tumor heterogeneity. In this study, we compared mutations in paired primary and metastatic colorectal cancer tumor samples to determine whether tumor heterogeneity can predict tumor metastasis.Experimental Design: Somatic variations in 46 pairs of matched primary-liver metastatic tumors and 42 primary tumors without metastasis were analyzed by whole-exome sequencing. Tumor clonality was estimated from single-nucleotide and copy-number variations. The correlation between clinical parameters of patients and clonal heterogeneity in liver metastasis was evaluated.Results: Tumor heterogeneity across colorectal cancer samples was highly variable; however, a high degree of tumor heterogeneity was associated with a worse disease-free survival. Highly heterogeneous primary colorectal cancer was correlated with a higher rate of liver metastasis. Recurrent somatic mutations in APC, TP53, and KRAS were frequently detected in highly heterogeneous colorectal cancer. The variant allele frequency of these mutations was high, while somatic mutations in other genes such as PIK3CA and NOTCH1 were low. The number and distribution of primary colorectal cancer subclones were preserved in metastatic tumors.Conclusions: Heterogeneity of primary colorectal cancer tumors can predict the potential for liver metastasis and thus, clinical outcome of patients. Clin Cancer Res; 23(23); 7209–16. ©2017 AACR.

Table S5. Mapping statistics of sequencing reads of RNA sequencing

Supplementary table S3. Clinicopathological characteristics and in vivo tumorigenicity of primary NSCLC cases.

Figure S1, Figure S2, Figure S3, Figure S4, Figure S5

Multivariate analysis of overall survival

Cytotoxic immune cells in the peripheral blood of healthy donors and multiple myeloma patients.

Gene expression features of myeloma cells.

Immune cell type identification using DEGs and annotation package SingleR.

Purpose:In early-stage, EGFR mutation–positive (EGFR-M+) non–small cell lung cancer (NSCLC), surgery remains the primary treatment, without personalized adjuvant treatments. We aimed to identify risk factors for recurrence-free survival (RFS) to suggest personalized adjuvant strategies in resected early-stage EGFR-M+ NSCLC.Experimental Design:From January 2008 to August 2020, a total of 2,340 patients with pathologic stage (pStage) IB–IIIA, non-squamous NSCLC underwent curative surgery. To identify clinicopathologic risk factors, 1,181 patients with pStage IB–IIIA, common EGFR-M+ NSCLC who underwent surgical resection were analyzed. To identify molecular risk factors, comprehensive genomic analysis was conducted in 56 patients with matched case–controls (pStage II and IIIA and type of EGFR mutation).Results:Median follow-up duration was 38.8 months (0.5–156.2). Among 1,181 patients, pStage IB, II, and IIIA comprised 577 (48.9%), 331 (28.0%), and 273 (23.1%) subjects, respectively. Median RFS was 73.5 months [95% confidence interval (CI), 62.1–84.9], 48.7 months (95% CI, 41.2–56.3), and 22.7 months (95% CI, 19.4–26.0) for pStage IB, II, and IIIA, respectively (P < 0.001). In multivariate analysis of clinicopathologic risk factors, pStage, micropapillary subtype, vascular invasion, and pleural invasion, and pathologic classification by cell of origin (type II pneumocyte-like tumor cell vs. bronchial surface epithelial cell–like tumor cell) were associated with RFS. As molecular risk factors, the non-terminal respiratory unit (non-TRU) of the RNA subtype (HR, 3.49; 95% CI, 1.72–7.09; P < 0.01) and TP53 mutation (HR, 2.50; 95% CI, 1.24–5.04; P = 0.01) were associated with poor RFS independent of pStage II or IIIA. Among the patients with recurrence, progression-free survival of EGFR-tyrosine kinase inhibitor (TKI) in those with the Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) mutation signature was inferior compared with that of patients without this signature (8.6 vs. 28.8 months; HR, 4.16; 95% CI, 1.28–13.46; P = 0.02).Conclusions:The low-risk group with TRU subtype and TP53 wild-type without clinicopathologic risk factors might not need adjuvant EGFR-TKIs. In the high-risk group, with non-TRU subtype and/or TP 53 mutation, or clinicopathologic risk factors, a novel adjuvant strategy of EGFR-TKI with others, e.g., chemotherapy or antiangiogenic agents needs to be investigated. Given the poor outcome to EGFR-TKIs after recurrence in patients with the APOBEC mutation signature, an alternative adjuvant strategy might be needed.

Purpose: The increasing prevalence of distant metastases from non–small cell lung cancer (NSCLC) indicates an urgent need for novel therapeutic modalities. Brain metastasis is particularly common in NSCLC, with severe adverse effects on clinical prognosis. Although the molecular heterogeneity of NSCLC and availability of various targeted agents suggest personalized therapeutic approaches for such brain metastases, further development of appropriate preclinical models is needed to validate the strategies.Experimental Design: We established patient-derived xenografts (PDX) using NSCLC brain metastasis surgical samples and elucidated their possible preclinical and clinical implications for personalized treatment.Results: NSCLC brain metastases (n = 34) showed a significantly higher successful PDX establishment rate than primary specimens (n = 64; 74% vs. 23%). PDXs derived from NSCLC brain metastases recapitulated the pathologic, genetic, and functional properties of corresponding parental tumors. Furthermore, tumor spheres established in vitro from the xenografts under serum-free conditions maintained their in vivo brain metastatic potential. Differential phenotypic and molecular responses to 20 targeted agents could subsequently be screened in vitro using these NSCLC PDXs derived from brain metastases. Although PDX establishment from primary NSCLCs was significantly influenced by histologic subtype, clinical aggressiveness, and genetic alteration status, the brain metastases exhibited consistently adequate in vivo tumor take rate and in vitro tumor sphere formation capacity, regardless of clinical and molecular conditions.Conclusions: Therefore, PDXs from NSCLC brain metastases may better represent the heterogeneous advanced NSCLC population and could be utilized as preclinical models to meet unmet clinical needs such as drug screening for personalized treatments. Clin Cancer Res; 21(5); 1172–82. ©2014 AACR.

DEGs of MPC clusters.

Supplementary Data from Clinical, Pathologic, and Molecular Prognostic Factors in Patients with Early-Stage EGFR-Mutant NSCLC

Purpose: The potential of metastasis can be predicted from clinical features like tumor size, histologic grade, and gene expression patterns. We examined the whole-genome transcriptomic profile of a xenograft model of breast cancer to understand the characteristics of brain metastasis.Experimental Design: Variants of the MDA-MB-435 cell were established from experimental brain metastases. The LvBr2 variant was isolated from lesions in a mouse injected in the left ventricle of the heart, and these cells were used for two cycles of injection into the internal carotid artery and selection of brain lesions, resulting in the Br4 variant. To characterize the different metastatic variants, we examined the gene expression profile of MDA-MB-435, LvBr2, and Br4 cells using microarrays.Results: We could identify 2,016 differentially expressed genes in Br4 by using the F test. Various metastasis-related genes and a number of genes related to angiogenesis, migration, tumorigenesis, and cell cycle were differentially expressed by the Br4 cells. Notably, the Notch signaling pathway was activated in Br4, with increased Jag2 mRNA, activated Notch intracellular domain, and Notch intracellular domain/CLS promoter-luciferase activity. Br4 cells were more migratory and invasive than MDA-MB-435 cells in collagen and Matrigel Transwell assays, and the migration and invasion of Br4 cells were significantly inhibited by inactivation of Notch signaling using DAPT, a γ-secretase inhibitor, and RNA interference–mediated knockdown of Jagged 2 and Notch1.Conclusions: Taken together, these results suggest that we have isolated variants of a human cancer cell line with enhanced brain metastatic properties, and the activation of Notch signaling might play a crucial role in brain metastasis.

Supplementary Data from Activation of Notch Signaling in a Xenograft Model of Brain Metastasis

Tumor heterogeneity influences the clinical outcome of patients with cancer, and the diagnostic method to measure the tumor heterogeneity needs to be developed. We analyzed genomic features on pairs of primary and multiple metastatic lymph nodes from six patients with lung cancer using whole-exome sequencing and RNA sequencing. Although somatic single-nucleotide variants were shared in primary lung cancer and metastases, tumor evolution predicted by the pattern of genomic alterations was matched to anatomic location of the tumors. Four of six cases exhibited a branched clonal evolution pattern. Lymph nodes with acquired somatic variants demonstrated resistance to the cancer treatment. In this study, we demonstrated that multiple biopsies and sequencing strategies for different tumor regions are required for a comprehensive understanding of the landscape of genetic alteration and for guiding targeted therapy in advanced primary lung cancer. Cancer Res; 76(22); 6568–76. ©2016 AACR.

Gene-set enrichment analysis between BM and EM

Purpose:In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites.Experimental Design:Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed.Results:Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells.Conclusions:These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion.

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BackgroundAlthough temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application.MethodsWe stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n= 29) and sensitive (n= 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy.ResultsIn vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P= 1.12e−4 for progression-free survival (PFS) and 3.63e−4 for overall survival (OS)). Moreover, we found that elevated gene expression ofEGR4,PAPPA,LRRC3, andANXA3was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration inPTEN,EGFR, andCDKN2A/Bwas more frequent in TMZ-sensitive samples (Fisher’s exactP= 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P= 4.58e−4 for PFS and 3.66e−4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage (http://www.wang-lab-hkust.com:3838/TMZEP).ConclusionsWe identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.

Background: The advance of next generation sequencing (NGS) leads to give the abundant genetic information of breast cancer (BC). We developed the prediction model of neoadjuvant chemotherapy (NAC) response and long term survival using genetic characteristics in cooperation with clinicpathologic features. Methods: Early and locally advanced BCs which would be planned to receive standard NAC (four cycles of anthracycline plus cyclophosphamide and four cycles of docetaxel or docetaxel plus trastuzumab for human epidermal growth factor receptor 2[HER2+] disease) followed by curative surgery. We prospectively collected tumor tissue and matched blood three times for each patients: at BC diagnosis (T1), three weeks after the first cycle of NAC (T2), and curative surgery (T3). Whole exome sequencing (WES) and RNASeq were performed to detect somatic mutation, mutational signature, tumor mutational burden (TMB) and PAM50 prediction was to classify intrinsic subtype. In terms of clinical variables, clinical stage and IHC subtype at diagnosis, pathologic complete response (pCR), residual cancer burden (RCB) class and distant recurrence free survival (DRFS) were used for prediction model development. Logistic regression was used for predicting RCB class and pCR with clinical and genomic variables at T1. Univariate and multivariate Cox regression were performed to identify prognostic factors for DRFS. Results: In total, 210 patients who were treated with scheduled NAC were enrolled. Finally, WES in 252 BC tissues (T1:123, T2:106 and T3:23) RNASeq in 198 BC tissues (T1:100, T2:77 and T3:21) were conducted from 123 patients. In NAC response, 52 patients achieved pCR(42.3%) and 14 patients were in RCB class 3, 39 in class 2, 14 in class 1 and 51 in class 0. Median follow up duration was 44months and distant recurrence was observed in 13 patients. TP53 mutation (60%) was the most commonly detected genetic alteration followed by TTN(31%), ERBB2(25%) and PIK3CA(18%). Eight BCL11A (3%) and 3 MPL mutations (1%) were also observed respectively. Serial tumor sequencing suggested that mutation profile have not changed during NAC. In terms of mutational signature, signature 3 was most frequently observed and no difference was observed in serial sequencing data. In addition, APOBEC (sig.2) and HRD (sig.3) were observed differently according to intrinsic subtype. In terms of intrinsic subtype, basal subtype decreased during NAC and luminal A type increased. Median TMB was 87 (range, 14-570). In prediction model, clinical N3(cN3) stage (Odds ratio [OR] of cN3 vs. not : 4.536, 95% confidence interval [CI]: 0.925, 44.016, p=0.042) and intrinsic subtype at BC diagnosis (OR of non-luminal vs. luminal types: 0.208, 95% CI: 0.062, 0.604, p=0.001) were associated with pCR. Clinical stage (OR of clinical stage III vs. II: 4.115, 95% CI: 1.094, 19.217, p=0.021), TMB (OR of TMB high vs. low: 0.255, 95% CI: 0.043,1.042, p.value=0.045), MPL genetic alteration (OR of mutation vs. wild type: 16.347, 95% CI: 0.797,1013.88, p.value=0.037), BCL11A (OR of mutation vs. wild type: 16.347, 95% CI: 0.797,1013.88, p.value=0.037) were associated with RCB class (3 vs. others). In terms of DRFS, prediction model consisted of clinical stage (Hazard ratio [HR] of stage III vs. II: 3.496, 95% confidence interval [CI]:1.337, 5.654), PIK3CA (HR of mutation vs. wild type: 1.572, 95% CI:-0.008, 3.152) TMB (HR of high vs. low: -1.999. 95% CI:-3.669, -0.329) had c-index of 0.861(95% CI:0.805, 0.916). Conclusions: During NAC, somatic mutation and mutation signature were consistently maintained while intrinsic subtype dynamically changed. In prediction model, TMB, PIK3CA mutation and clinical stage showed predictive roles on DRFS of BC in NAC setting. Citation Format: Ji-Yeon Kim, Kyunghee Park, Woong-Yang Park, Jeong Eon Lee, Seok Won Kim, Seok Jin Nam, Se Kyung Lee, Zhengyan Kan, Yeon Hee Park. Prediction model of the response of neoadjuvant chemotherapy and long term survival according to multi-omic profiling in cooperation with clinicopathologic features in patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-07.

Background: Neutrotrophin receptor tyrosine kinase (NTRK) gene fusions (NTRK1, NTRK2, or NTRK3) are oncogenic drivers of various tumor types. The NTRK fusion was detected in less than 5% of breast, colorectal, lung or any other types of cancers. However, large scaled next generation sequencing data for NTRK fusion in breast cancer have not existed. In this study, we performed RNASeq and fusion analysis including NTRK genes. Methods: We prospectively collected BC tumor tissues from the translational research conducted in Samsung Medical Center. Fusion was predicted from RNAseq using the following seven softwares(SWs): ChimeraScan, DeFuse, MapSplice, TophatFusion, STAR.Arriba, STAR.fusion, and STAR.SEQR. To remove false-positive fusion calls, calls less than 3 left/right spanning reads and 10 total supporting reads were removed. After filtering out the blacklist fusion calls which were recurrently detected, calls commonly predicted in more than two SWs were analyzed. Results: In total 629 BC samples, 613 samples were finally analyzed after quality control (QC) of RNASeq. According to immunohistochemistry (IHC) profile, 356(58.7%) was hormone receptor (HR) positive human epidermal growth factor receptor 2 (HER2) negative BC, 42 (6.9%) of HR positive HER2 positive BC, 53 (8.7%) of HR negative, HER2 positive and 155(25.6%) of triple negative BC (TNBC). PAM50 prediction informed that 174(28.9%) of luminal A, 151(24.6%) of luminal B, 170 (27.7%) of basal-like, 85 (13.9%) of HER2-enriched and 33 (5.4%) of normal. In median number of fusion events, 12 was called by ChimeraScan (Interquartile range [IQR]: 5, 33), 57 by DeFuse (IQR: 33, 82), eight by Mapsplice (IQR: 5, 12), two by TophatFusion (IQR: 0, 4), five by STAR.Arriba (IQR: 2, 12), two by STAR.fusion (IQR:0, 5) and three by STAR.SEQR fusion caller (IQR: 1, 7) after call filtering. After initial fusion call, we excluded the results from ChimeraScan and DeFuse fusion callers because of discrepancy of number of called fusion events. In five fusion callers, median number of fusion events was eight (IQR :2,20) per BC sample. In terms of NTRK fusion, we detected NTRK2-BANCR fusion event in one TNBC patients (1/425, 0.2%). This fusion was detected in four of five SWs for fusion detection with significant number of supporting reads in RNASeq. NTRK2-BANCR fusion was the out-of-frame fusion, which C-terminal truncated protein kinase domain of NTRK2 and its partner long non-coding RNA BANCR was combined and RNA expression of this fusion was increased. Other fusion events of BCs were NCOR2-PARP4 (3.7%), BRD4-NWD1 (3.7%), ESR1-RGS17 (1.8%), FGFR1-TACC1 (0.2%) and MKRN1-BRAF (0.2%). In BC subtype according to IHC, fusion events were more frequently observed in TNBC compared with other subtypes regardless of the type of fusion filters. In terms of intrinsic subtype, fusion events were most frequently observed in basal like type and least in normal like intrinsic subtype (all ps Citation Format: Ji-Yeon Kim, Kyunghee Park, Woong-Yang Park, Jeong Eon Lee, Seok Won Kim, Seok Jin Nam, Jonghan Yu, Young-Hyuck Im, Jin Seok Ahn, Yeon Hee Park. Fusion analysis including NTRK fusion in breast cancers (BC): From RNASeq data analysis from 629 BC tissue samples [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-13-08.

CDK4/6 inhibitors such as palbociclib in combination with endocrine therapy (ET) have remarkablyimproved the outcome of patients with ER+/HER2- metastatic breast cancer (MBC). However, manypatients are intrinsically resistant to CDK4/6i therapy, and those who respond eventually acquireresistance. Although high baseline CCNE1 expression and rare alterations in RB1 and FAT1 geneshave been shown to be associated with CDK4/6i resistance, the molecular mechanisms of CDK4/6iresistance are complex and remain poorly understood. To better understand and overcome CDK4/6iresistance, we performed multi-omics profiling of paired tumor biopsies from ER+/HER2- MBCpatients treated with palbociclib combined with ET. Tumor biopsies taken at pre-treatment, on-treatment, and progressive disease (PD) from 71 patients were profiled using whole-exomesequencing (WES), whole-transcriptome sequencing (RNA-Seq) and IHC analysis. Ourcomprehensive analysis identified several tumor intrinsic molecular markers associated with worsePFS, including the Luminal B subtype (p=0.012, HR=2.593), BRCA1/2 pathogenic mutation (p=0.012,HR=2.67) and mutation signatures linked to APOBEC enzymatic activity (p=0.002, HR=3.19).Conversely, the estrogen response signature (p=0.006, HR=0.43) was associated with favorableprognosis. Unsupervised analysis revealed a cluster of tumors enriched in homologousrecombination deficiency (HRD) linked genomic scars that was associated with poor prognosis(p=0.005, HR=2.49). Of note, these HRD-high tumors responded even more poorly to treatment whenco-occurring with TP53 somatic mutations. Integrative analysis further identified three poorprognosis clusters (IC2-4) enriched in Luminal B, proliferative and HRD features when compared tothe favorable prognosis cluster (IC1).Comparing baseline vs. PD samples, we observed a pattern of post-treatment enrichment for the poorprognosis markers. In addition, breast cancer-associated genes such as BRCA1/2, TP53 and PTENharbored a higher prevalence of genomic alterations including somatic mutation, amplification,. deletion and gene fusion at PD. Cell cycle gene expression and signatures also markedly increased atPD compared to baseline whereas estrogen response signatures decreased. Upon diseaseprogression, tumors had frequently switched to molecular subtypes with aggressive and estrogenindependent characteristics, demonstrating high plasticity in response to CDK4/6i and ET treatment.These patterns of acquired resistance were validated by IHC analysis of cyclins E1 and E2, Ki67 andpRb. To investigate the genomic alterations responsible for acquired resistance, we compared 21paired baseline and PD samples. We observed that PD-specific RB1 loss-of-function events occurredwith higher prevalence than previously reported, underscoring a major role of cell cycle de-regulation in conferring resistance to CDK4/6 inhibition. In this prospective longitudinal multi-omicsstudy, we identified novel candidate biomarkers that can be used to improve prediction of responseto CDK4/6i. In addition, we derived new insights into the molecular mechanisms of drug resistanceto palbociclib plus ET that will help guide therapeutic strategies and drug development inHR+/HER2− MBC. Citation Format: Zhengyan Kan, Seock-Ah Im, Kyunghee Park, Ji Wen, Kyung-Hun Lee, Yoon-La Choi, Won-Chul Lee, Ahrum Min, Vinicius Bonato, Seri Park, Sripad Ram, Dae-Won Lee, Ji-Yeon Kim, Su Kyeong Lee, Won-Woo Lee, Jisook Lee, Miso Kim, Scott L. Weinrich, Han Suk Ryu, Tae Yong Kim, Stephen Dann, Diane Fernandez, Jiwon Koh, Song Yi Park, Shibing Deng, Eric Powell, Rupesh Kanchi Ravi, Jadwiga Bienkowska, Paul A. Rejto, Woong-Yang Park, Yeon Hee Park. Serial genomic profiling reveals molecular mechanisms of breast cancer resistance to palbociclib [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-08.

The Korean Society of Otorhinolaryngology-Head and Neck Surgery and Korean Rhinologic Society appointed a guideline development group (GDG) to establish a clinical practice guideline, and the GDG developed a guideline for nasal irrigation for adult patients with chronic rhinosinusitis (CRS). The guideline focuses on knowledge gaps, practice variations, and clinical concerns associated with nasal irrigation. Nasal irrigation has been recommended as the first-line treatment for CRS in various guidelines, and its clinical effectiveness has been demonstrated through a number of studies with robust evidence. However, no guidelines have presented a consistent nasal irrigation method. Several databases, including OVID Medline, Embase, the Cochrane Library, and KoreaMed, were searched to identify all relevant papers using a predefined search strategy. When insufficient evidence was found, the GDG sought expert opinions and attempted to fill the evidence gap. Evidence-based recommendations for practice were ranked according to the American College of Physicians grading system. The committee developed 11 evidence-based recommendations. This guideline focuses on the evidence-based quality improvement opportunities deemed the most important by the GDG. Moreover, the guideline addresses whether nasal lavage helps treat CRS, what type of rinsing solution should be used, and the effectiveness of using additional medications to increase the therapeutic effect.

Background & Aims Tumor necrosis factor alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn’s disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids. Methods Human enteroids derived from patients with CD and controls were treated with TNFα (30 ng/mL), and cell viability and gene expression patterns were evaluated. Results TNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations. However, expanded ISC subpopulations differed in control and CD patient-derived enteroids, with LGR5+ active ISCs in control enteroids and reserve ISCs, such as BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and cyclooxygenase-prostaglandin E2 (PGE2) activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nmol/L) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing. Conclusions TNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure), and exogenous PGE2 treatment restored the functions of LGR5+ stem cells. Therefore, PGE2 can be used to promote mucosal healing in patients with CD., Graphical abstract

Chemotherapy is ubiquitous in first-line treatment of advanced gastric cancer, yet responses are heterogeneous, and little is known about mediators of chemotherapy response. To move forward, an understanding of the effects of standard chemotherapy on the tumor–immune microenvironment (TME) is needed. Coupling whole-exome sequencing, bulk RNA and single-cell transcriptomics from paired pretreatment and on-treatment samples in treatment-naïve patients with HER2-positive and HER2-negative gastric cancer, we define features associated with response to platinum-based chemotherapy. Response was associated with on-treatment TME remodeling including natural killer (NK) cell recruitment, decreased tumor-associated macrophages, M1-macrophage repolarization, and increased effector T-cell infiltration. Among chemotherapy nonresponders, we observed low/absent PD-L1 expression or modulation, on-treatment increases in Wnt signaling, B-cell infiltration, and LAG3-expressing T cells coupled to an exodus of dendritic cells. We did not observe significant genomic changes in early on-treatment sampling. We provide a map of on-treatment TME modulation with standard chemotherapy and nominate candidate future approaches. Significance: Using paired pretreatment and on-treatment samples during standard first-line chemotherapy, we identify chemotherapy-induced NK-cell infiltration, macrophage repolarization, and increased antigen presentation among responders. Increased LAG3 expression and decreased dendritic cell abundance were seen in nonresponders, emphasizing remodeling of the TME during chemotherapy response and resistance. This article is highlighted in the In This Issue feature, p. 873