Lenvatinib plus anti-PD-1 antibodies as conversion therapy for patients with unresectable intermediate-advanced hepatocellular carcinoma: a single-arm, phase II trial.

BACKGROUND: Over 70% of the patients with hepatocellular carcinoma (HCC) are diagnosed at an advanced stage and lose the opportunity for radical surgery. Combination therapy of tyrosine kinase inhibitors (TKIs) and anti-programmed cell death protein-1 (PD-1) antibodies has achieved a high tumor response rate in both the first-line and second-line treatment of advanced HCC. However, few studies have prospectively evaluated whether TKIs plus anti-PD-1 antibodies could convert unresectable intermediate-advanced HCC into resectable disease. METHODS: This single-arm, phase II study enrolled systemic therapy-naïve adult patients with unresectable Barcelona Clinic Liver Cancer stage B or C HCC. Patients received oral lenvatinib one time per day plus intravenous anti-PD-1 agents every 3 weeks (one cycle). Tumor response and resectability were evaluated before the fourth cycle, then every two cycles. The primary endpoint was conversion success rate by investigator assessment. Secondary endpoints included objective response rate (ORR) by independent imaging review (IIR) assessment per modified RECIST (mRECIST) and Response Evaluation Criteria in Solid Tumors, V.1.1 (RECIST 1.1), progression-free survival (PFS) and 12-month recurrence-free survival (RFS) rate by IIR per mRECIST, R0 resection rate, overall survival (OS), and safety. Biomarkers were assessed as exploratory objectives. RESULTS: Of the 56 eligible patients enrolled, 53 (94.6%) had macrovascular invasion, and 16 (28.6%) had extrahepatic metastasis. The median follow-up was 23.5 months. The primary endpoint showed a conversion success rate of 55.4% (31/56). ORR was 53.6% per mRECIST and 44.6% per RECIST 1.1. Median PFS was 8.9 months, and median OS was 23.9 months. Among the 31 successful conversion patients, 21 underwent surgery with an R0 resection rate of 85.7%, a pathological complete response rate of 38.1%, and a 12-month RFS rate of 47.6%. Grade ≥3 treatment-related adverse events were observed in 42.9% of patients. Tumor immune microenvironment analysis of pretreatment samples displayed significant enrichment of CD8+ T cells (p=0.03) in responders versus non-responders. CONCLUSION: Lenvatinib plus anti-PD-1 antibodies demonstrate promising efficacy and tolerable safety as conversion therapy in unresectable HCC. Pre-existing CD8+ cells are identified as a promising biomarker for response to this regimen. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry, ChiCTR1900023914.

ANO1-Mediated Inhibition of Cancer Ferroptosis Confers Immunotherapeutic Resistance through Recruiting Cancer-Associated Fibroblasts.

The application of immunotherapy in gastrointestinal (GI) cancers remains challenging because of the limited response rate and emerging therapeutic resistance. Combining clinical cohorts, multi-omics study, and functional/molecular experiments, it is found that ANO1 amplification or high-expression predicts poor outcomes and resistance to immunotherapy for GI cancer patients. Knocking-down or inhibiting ANO1 suppresses the growth/metastasis/invasion of multiple GI cancer cell lines, cell-derived xenograft, and patient-derived xenograft models. ANO1 contributes to an immune-suppressive tumor microenvironment and induces acquired resistance to anti-PD-1 immunotherapy, while ANO1 knockdown or inhibition enhances immunotherapeutic effectiveness and overcomes resistance to immunotherapy. Mechanistically, through inhibiting cancer ferroptosis in a PI3K-Akt signaling-dependent manner, ANO1 enhances tumor progression and facilitates cancer-associated fibroblast recruitment by promoting TGF-ß release, thus crippling CD8+ T cell-mediated anti-tumor immunity and generating resistance to immunotherapy. This work highlights ANO1's role in mediating tumor immune microenvironment remodeling and immunotherapeutic resistance, and introduces ANO1 as a promising target for GI cancers' precision treatment.

Intensive cycles of neoadjuvant camrelizumab combined with chemotherapy in locally advanced esophageal squamous cell carcinoma: a single-arm, phase II trial.

BACKGROUND: Two cycles of neoadjuvant PD-1 blockade plus chemotherapy induced favorable pathological response and tolerant toxicity in patients with locally advanced esophageal squamous cell carcinoma (ESCC). However, approximately 25% of patients relapsed within 1 year after surgery, indicating that a short course of treatment may not be sufficient. Therefore, exploring the effects of intensive treatment is needed for optimal clinical outcomes. METHODS: Locally advanced ESCC patients were administered three cycles of camrelizumab plus nab-paclitaxel and capecitabine, followed by thoracoscopic esophagectomy. The primary endpoint was pathologic response. Secondary endpoints included safety, feasibility, radiologic response, survival outcomes, and immunologic/genomic correlates of efficacy. RESULTS: Forty-seven patients were enrolled in the study. Forty-two patients received surgery, and R0 resection was achieved in all cases. The complete and major pathological response rates were 33.3% and 64.3%, respectively, and the objective response rate was 80.0%. Three cycles of treatment significantly improved T down-staging compared to two cycles (P = 0.03). The most common treatment-related adverse events were grades 1-2, and no surgical delay was reported. With a median follow-up of 24.3 months, the 1-year disease-free survival and overall survival rates were both 97.6%, and the 2-year disease-free survival and overall survival rates were 92.3% and 97.6%, respectively. Three patients experienced disease recurrence or metastasis ranging from 12.5 to 25.8 months after surgery, and one patient died 6 months after surgery due to cardiovascular disease. Neither programmed death-ligand 1 expression nor tumor mutational burden was associated with pathological response. An increased infiltration of CD56dim natural killer cells in the pretreatment tumor was correlated with better pathological response in the primary tumor. CONCLUSIONS: It seems probable that intensive cycles of neoadjuvant camrelizumab plus nab-paclitaxel and capecitabine increased tumor regression and improved survival outcomes. Randomized controlled trials with larger sample sizes and longer follow-up periods are needed to validate these findings. Trial registration Chinese Clinical Trial Registry, ChiCTR2000029807, Registered February 14, 2020, https://www.chictr.org.cn/showproj.aspx?proj=49459 .

Chromosome 11q13 amplification correlates with poor response and prognosis to PD-1 blockade in unresectable hepatocellular carcinoma.

Background & aims: Little is known about molecular biomarkers that predict the response and prognosis in unresectable hepatocellular carcinoma (HCC) treated with programmed death (PD)-1 inhibitors. Methods: A total of 62 HCC patients who underwent next-generation sequencing were retrospectively included in our department for this study. Patients with unresectable disease were subjected to systemic therapy. PD-1 inhibitors intervention (PD-1Ab) group and nonPD-1Ab group included 20 and 13 patients, respectively. Primary resistance was defined as initial on-treatment progression or progression with an initial stable disease of less than 6 months. Results: Chromosome 11q13 amplification (Amp11q13) was the most common copy number variation in our cohort. Fifteen (24.2%) patients harbored Amp11q13 in our dataset. Patients with Amp11q13 showed higher level of Des-γ-carboxy-prothrombin (DCP), tumor number and were more prone to be combined with portal vein tumor thrombosis (PVTT). In the PD-1Ab group, the proportion of progressive disease (PD) in patients with Amp11q13 was significantly higher than that in patients with nonAmp11q13 (100% vs 33.3%, P=0.03). In the nonPD-1Ab group, the proportion of PD in patients with Amp11q13 and nonAmp11q13 had no significant difference (0% vs 11.1%, P>0.99). In the PD-1Ab group, the median progression-free survival (PFS) was 1.5 months in Amp11q13 patients vs 16.2 months in non-Amp11q13 patients (HR, 0.05; 95% CI 0.01-0.45; P = 0.0003). No significant difference was observed in the nonPD-1Ab group. Notably, we found that hyperprogressive disease (HPD) might be associated with Amp11q13. The increased density of Foxp3+ Treg cells in HCC patients with Amp11q13 might be one of potential mechanisms. Conclusion: HCC patients with Amp11q13 are less likely to benefit from PD-1 blockade therapies. These findings may help guide the use of immunotherapy for HCC in routine clinical practice.

China special issue on gastrointestinal tumors-NTRK fusion in a large real-world population and clinical utility of circulating tumor DNA genotyping to guide TRK inhibitor treatment.

Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are rare oncogenic drivers and targets of TRK inhibitors in solid tumors. Little is known about NTRK fusion in Chinese patients with pan-cancer. Our study investigated the prevalence and genomic features of NTRK1/2/3 gene fusions in 67 883 Chinese patients with pan-cancer using next-generation sequencing (NGS) data and circulating tumor DNA (ctDNA) NGS to guide TRK inhibitor treatment and resistance monitoring. The prevalence of NTRK fusion (tissue NGS) in the pan-cancer population was 0.18%, with 46 unique NTRK-fusion partner pairs, of which 33 were not previously reported. NTRK2 breakpoint occurred more frequently in intron 15 than intron 12. In colorectal cancers (CRCs), compared to NTRK-negative tumors, NTRK-positive tumors displayed higher tumor mutational burden (TMB) levels (54.6 vs 17.7 mut/Mb, P < .0001). In microsatellite instability-high (MSI-H) CRC, patients with NTRK fusion had a significantly lower TMB than NTRK-negative cases (69.3 vs 79.9 mut/Mb, P = .012). The frequency of NTRK fusion in a ctDNA NGS cohort of 20 954 patients with cancer was similar to that of the tissue NGS cohort. In eight NTRK fusion ctDNA-positive patients, larotrectinib induced objective response in 75% of patients and median progression-free survival was 16.3 months. Blood samples collected from a patient with disease progression after larotrectinib treatment revealed NTRK3 G623R as the potential resistance mechanism. Our study revealed previously unreported NTRK fusion partners, associations of NTRK fusion with MSI and TMB, and the potential utility of ctDNA to screen candidates for TRK inhibitors and monitor drug resistance.

An exploration of gastric cancer with heterogeneous mismatch repair status.

Survival benefits or symptom alleviation from immune checkpoint blockade therapy can be seen in microsatellite instability-high (MSI-H) cases. However, genetic heterogeneity within a specific subgroup of MSI-H tumors may be associated with poor response and prognosis. We investigated the molecular changes and microsatellite status of the cases with heterogeneous MMR protein staining by polymerase chain reaction (PCR) and next-generation sequencing (NGS). Data from 3723 patients with gastric cancer were retrospectively analyzed to determine the mismatch repair (MMR) status by performing immunohistochemical staining of four major MMR proteins (MLH1, PMS2, MSH2, and MSH6). When heterogeneous MMR protein staining result was positive, PCR and NGS were performed. Heterogeneous MMR protein staining was observed in 12 cases. In microsatellite stable (MSS) cases, TP53 mutation appeared to accompany heterogeneous staining (HS) of MLH1. However, TP53 variation was not observed with MSI-H occurrence. Cases showing heterogeneous MSH6 protein staining revealed MSH6 mutations. Some cases with the same MMR protein staining set had varying MSI results. In one case whose primary and metastatic foci presented MLH1-HS and PMS2-HS, the microsatellite status was classified as MSS and MSI-H, respectively. Moreover, HS was also found in multiple biopsies and surgical specimens. This study found a preliminary relationship between heterogeneously stained MSH6 or MLH1 proteins and their gene mutations, as well as between MSI-H/TP53 - and MSS/TP53 + tumors. The microsatellite status of patients with heterogeneous MMR protein staining is unpredictable. Given the heterogeneity of mismatch repair, microsatellite status should be assessed for all specimens if sufficient specimens can be obtained.

Development and validation of postoperative circulating tumor DNA combined with clinicopathological risk factors for recurrence prediction in patients with stage I-III colorectal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) detection following curative-intent surgery could directly reflect the presence of minimal residual disease, the ultimate cause of clinical recurrence. However, ctDNA is not postoperatively detected in ≥ 50% of patients with stage I-III colorectal cancer (CRC) who ultimately recur. Herein we sought to improve recurrence risk prediction by combining ctDNA with clinicopathological risk factors in stage I-III CRC. METHODS: Two independent cohorts, both consisting of early-stage CRC patients who underwent curative surgery, were included: (i) the discovery cohort (N = 124) with tumor tissues and postoperative plasmas for ctDNA determination; and (ii) the external validation cohort (N = 125) with available ctDNA results. In the discovery cohort, somatic variations in tumor tissues and plasmas were determined via a 733-gene and 127-gene next-generation sequencing panel, respectively. RESULTS: In the discovery cohort, 17 of 108 (15.7%) patients had detectable ctDNA. ctDNA-positive patients had a significantly high recurrence rate (76.5% vs. 16.5%, P < 0.001) and short recurrence-free survival (RFS; P < 0.001) versus ctDNA-negative patients. In addition to ctDNA status, the univariate Cox model identified pathologic stage, lymphovascular invasion, nerve invasion, and preoperative carcinoembryonic antigen level associated with RFS. We combined the ctDNA and clinicopathological risk factors (CTCP) to construct a model for recurrence prediction. A significantly higher recurrence rate (64.7% vs. 8.1%, P < 0.001) and worse RFS (P < 0.001) were seen in the high-risk patients classified by the CTCP model versus those in the low-risk patients. Receiver operating characteristic analysis demonstrated that the CTCP model outperformed ctDNA alone at recurrence prediction, which increased the sensitivity of 2 year RFS from 49.6% by ctDNA alone to 87.5%. Harrell's concordance index, calibration curve, and decision curve analysis also suggested that the CTCP model had good discrimination, consistency, and clinical utility. These results were reproduced in the validation cohort. CONCLUSION: Combining postoperative ctDNA and clinical risk may better predict recurrence than ctDNA alone for developing a personalized postoperative management strategy for CRC.

Special issue "The advance of solid tumor research in China": FGFR4 alterations predict efficacy of immune checkpoint inhibitors in nonsmall cell lung cancer.

Immune checkpoint inhibitors (ICIs), which represent the new standard of care for advanced nonsmall cell lung cancer (NCSLC), are not effective in many patients. Biomarkers are needed to guide treatment. Sequencing data of an ICI-treated cohort were analyzed to identify genomic signatures predicting ICI efficacy, followed by validation using multiple independent cohorts. Their predictive mechanism was explored by evaluating the tumor immune microenvironment and tumor mutational burden (TMB). In the discovery cohort, patients carrying FGFR4 alterations (FGFR4altered ) had a better objective response rate (ORR) (50.0% vs 19.4%; P = .057) and improved median progression-free survival (mPFS) (13.17 vs 3.17 months; HR 0.37; 95% CI 0.14-1; P = .04) than wild-type patients (FGFR4wt ). In the publicly available validation cohorts, FGFR4 alterations correlated with higher ORR (100% vs 31%; P = .028), longer median overall survival (mOS) (not reached [NR] vs 11 months; HR 0.28, 95% CI 0.09-0.89, P = .02), and mPFS (NR vs 6.07 months; HR 0.05, 95% CI 0-3.94, P = .039). FGFR4 alterations were confirmed as an independent predictor of superior PFS (P = .014) and OS (P = .005). FGFR4altered patients also exhibited a significantly improved disease control rate (100% vs 60%, P = .045) and prolonged mPFS (9.70 vs 3.16 months; P = .095) compared to FGFR4wt patients in our Shanghai Pulmonary Hospital cohort. FGFR4 alterations associated with a higher TMB levels, more CD8+ T cells in the tumor stroma, and a higher M1/M2 ratio for tumor-associated macrophages in the tumor center and stroma. Thus, FGFR4 alterations may serve as a potential independent predictor of ICI efficacy in NSCLC.

Trastuzumab Combined with Irinotecan in Patients with HER2-Positive Metastatic Colorectal Cancer: A Phase II Single-Arm Study and Exploratory Biomarker Analysis.

PURPOSE: The human epidermal growth factor receptor 2 (HER2) is an established therapeutic target for various kinds of solid tumors. HER2 amplification occurs in approximately 1% to 6% of colorectal cancer. In this study, we aimed to assess the efficacy and safety of trastuzumab in combination with chemotherapy in HER2-positive metastatic colorectal cancer (mCRC). Materials and Methods: An open-label, phase II trial (Clinicaltrials.gov: NCT03185988) was designed to evaluate the antitumor activity of trastuzumab and chemotherapy in HER2-positive digestive cancers excluding gastric cancer in 2017. Patients from this trial with HER2-positive, KRAS/BRAF wild-type, unresectable mCRC were analyzed in this manuscript. Eligible patients were treated with trastuzumab (8 mg/kg loading dose and then 6 mg/kg every 3 weeks) and irinotecan (120 mg/m2 days 1 and 8 every 3 weeks). The primary endpoint was the objective response rate. RESULTS: Twenty-one HER2-positive mCRC patients were enrolled in this study. Seven patients (33.3%) achieved an objective res-ponse, and 11 patients (52.4%) had stable disease as their best response. The median progression-free survival (PFS) was 4.3 months (95% confidence interval, 2.7 to 5.9). Four of the 21 patients (19.0%) had grade 3 adverse events, including leukopenia, neutropenia, urinary tract infection, and diarrhea. No treatment-related death was reported. Exploratory analyses revealed that high tumor tissue HER2 copy number was associated with better therapeutic response and PFS. Alterations in the mitogen-activated protein kinase pathway, HER2 gene, phosphoinositide 3-kinase/AKT pathway, and cell cycle control genes were potential drivers of trastuzumab resistance in mCRC. CONCLUSION: Trastuzumab combined with chemotherapy is a potentially effective and well-tolerated therapeutic regimen in mCRC with a high HER2 copy number.

Next-generation sequencing based detection of BRCA1 and BRCA2 large genomic rearrangements in Chinese cancer patients.

BRCA1/2 mutation is a biomarker for guiding multiple solid tumor treatment. However, the prevalence of BRCA1/2 large genomic rearrangements (LGRs) in Chinese cancer patients has not been well revealed partially due to technical difficulties in LGR detection. This study utilized next-generation sequencing (NGS) to analyze the BRCA1/2 mutation profile, including LGR, in 56126 Chinese cancer patients. We also reported that two ovarian and breast cancer patients with NGS-determined BRCA1/2 LGR benefited from PARP inhibitors (PARPi). DNA sequencing identified BRCA1/2 variants (including LGR, pathogenic and likely-pathogenic variants) in 2108 individuals. Seventy patients were discovered to harbor germline LGRs in BRCA1 and 14 had germline LGRs in BRCA2. Among the LGRs detected, exon 1-2 deletion was the predominant LGR (14/70) in BRCA1, and exon 22-24 deletion was the most frequent LGR (3/14) in BRCA2. Notably, the BRCA1 exon 7 deletion was a novel LGR and was identified in six patients, suggesting a specific LGR in Chinese cancer patients. The prevalence analysis of BRCA1 and BRCA2 LGRs across multiple cancers revealed that BRCA1 LGR more frequently occurred in ovarian cancer (1.31%, 33/2526), and BRCA2 LGR was more commonly seen in cholangiocarcinoma (0.47%, 2/425). Two ovarian and breast cancer patients with BRCA1/2 LGR benefited from PARPi therapy. This is the first study to reveal the BRCA1/2 LGR profile of a Chinese pan-cancer cohort by using an NGS-based assay. Two breast and ovarian cancer patients harboring NGS-determined BRCA1/2 LGR benefited from PARPi, indicating that NGS-based detection of BRCA1/2 LGR has the potential to guide PARPi treatment.

A pilot study of neoadjuvant combination of anti-PD-1 camrelizumab and VEGFR2 inhibitor apatinib for locally advanced resectable oral squamous cell carcinoma.

Novel neoadjuvant therapy regimens are warranted for oral squamous cell carcinoma (OSCC). In this phase I trial (NCT04393506), 20 patients with locally advanced resectable OSCC receive three cycles of camrelizumab (200 mg, q2w) and apatinib (250 mg, once daily) before surgery. The primary endpoints are safety and major pathological response (MPR, defined as ≤10% residual viable tumour cells). Secondary endpoints include 2-year survival rate and local recurrence rate (not reported due to inadequate follow-up). Exploratory endpoints are the relationships between PD-L1 combined positive score (CPS, defined as the number of PD-L1-stained cells divided by the total number of viable tumour cells, multiplied by 100) and other immunological and genomic biomarkers and response. Neoadjuvant treatment is well-tolerated, and the MPR rate is 40% (8/20), meeting the primary endpoint. All five patients with CPS ˃10 achieve MPR. Post-hoc analysis show 18-month locoregional recurrence and survival rates of 10.5% (95% CI: 0%-24.3%) and 95% (95% CI: 85.4%-100.0%), respectively. Patients achieving MPR show more CD4+ T-cell infiltration than those without MPR (P = 0.02), and decreased CD31 and ɑ-SMA expression levels are observed after neoadjuvant therapy. In conclusion, neoadjuvant camrelizumab and apatinib is safe and yields a promising MPR rate for OSCC.

Combination of AKT1 and CDH1 mutations predicts primary resistance to immunotherapy in dMMR/MSI-H gastrointestinal cancer.

BACKGROUND: Gastrointestinal (GI) cancer is the second most common cancer type with mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) phenotype that is expected to respond to immune-checkpoint inhibitors (ICIs). However, approximately half of the patients with dMMR/MSI-H GI cancer derive no benefit from ICIs. We sought to identify the predictors of primary resistance to ICIs in dMMR/MSI-H GI cancer. METHODS: Three independent cohorts were included: (1) the discovery cohort (65 patients with dMMR/MSI-H GI cancer) with ICI efficacy data and pre-ICIs tissue samples for genomic profile and tumor immune infiltration; (2) the validation cohort (22 patients with dMMR/MSI-H GI cancer) with ICI efficacy data and pre-ICIs plasma samples for genomic profile; and (3) the TCGA (The Cancer Genome Atlas) cohort not receiving ICIs (152 patients with MSI-H GI cancer) with genomic profile and survival data. RESULTS: AKT1 and CDH1 mutations were identified as independent predictors of poor progression-free survival (PFS) and primary resistance to ICIs in dMMR/MSI-H GI cancer. We combined these two genes as an immuno-oncology therapy predictor (IOpred), which could recognize 52.4% (11/21) of dMMR/MSI-H patients with primary resistance to ICIs with a positive predictive value (PPV) of 91.7% (11/12). Receiver operating characteristic analysis demonstrated IOpred with a good performance in predicting primary resistance (area under the curve 0.751). Patients with IOpred-Mut (mutant AKT1 or CDH1) GI cancer had significantly shorter PFS (HR=8.36, p<0.001) and overall survival (OS, HR=5.17, p<0.001) than IOpred-WT (wild-type for both AKT1 and CDH1) cases upon ICI treatment. The validation cohort also confirmed the correlation between IOpred-mutation and poorer prognosis (PFS, HR=4.68, p=0.004; OS, HR=15.98, p<0.001) in dMMR/MSI-H patients after ICIs. The PPV of IOpred in identifying primary resistance to ICIs was 80% (4/5) in the validation cohort. Additionally, IOpred-WT patients could be further stratified by tumor mutational burden (TMB), wherein TMB-low patients (TMB ≤26.19 mutations per megabase (Mb)) had a significantly higher primary resistance rate to ICIs (34.8% vs 6.7%, p=0.014) and poorer PFS (HR=3.46, p=0.008) and OS (HR=4.42, p=0.047) than TMB-high patients (TMB >26.19 mutations/Mb). CONCLUSIONS: IOpred was identified as a powerful predictor of primary resistance to ICIs in dMMR/MSI-H GI cancer, which might serve as a promising biomarker to help guide immunotherapy decision-making.

ctDNA-adjusted bTMB as a predictive biomarker for patients with NSCLC treated with PD-(L)1 inhibitors.

BACKGROUND: In non-small cell lung cancer (NSCLC) patients receiving immune checkpoint inhibitors (ICIs), higher blood tumor mutational burden (bTMB) was usually associated with better progression-free survival (PFS) and objective response rate (ORR). However, the association between bTMB and overall survival (OS) benefit remains undefined. It has been reported that patients harboring a high level of circulating tumor DNA (ctDNA) had poor survival. We hypothesized that ctDNA-adjusted bTMB might predict OS benefit in NSCLC patients receiving ICIs. METHODS: Our study was retrospectively performed in three cohorts, including OAK and POPLAR cohort (n = 853), Shanghai and Wuhan (SH&WH) cohort (n = 44), and National Cancer Center (NCC) cohort (n = 47). Durable clinical benefit (DCB) was defined as PFS lasting ≥ 6 months. The cutoff value of ctDNA-adjusted bTMB for DCB prediction was calculated based on a receiver operating characteristic curve. Interaction between treatments and ctDNA-adjusted bTMB was assessed. RESULTS: The bTMB score was significantly associated with tumor burden, while no association was observed between ctDNA-adjusted bTMB with tumor burden. In the OAK and POPLAR cohort, significantly higher ORR (P = 0.020) and DCB (P < 0.001) were observed in patients with high ctDNA-adjusted bTMB than those with low ctDNA-adjusted bTMB. Importantly, the interactions between ctDNA-adjusted bTMB and treatments were significant for OS (interaction P = 0.019) and PFS (interaction P = 0.002). In the SH&WH cohort, the interactions between ctDNA-adjusted bTMB and treatment were marginally significant for OS (interaction P = 0.081) and PFS (interaction P = 0.062). Similar result was demonstrated in the NCC cohort. CONCLUSIONS: Our study indicated that ctDNA-adjusted bTMB might predict OS benefit in NSCLC patients receiving ICIs. The potential of ctDNA-adjusted bTMB as a noninvasive predictor for immunotherapy should be confirmed in future studies.

Mutations of PI3K-AKT-mTOR pathway as predictors for immune cell infiltration and immunotherapy efficacy in dMMR/MSI-H gastric adenocarcinoma.

BACKGROUND: A significant subset of mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) gastric adenocarcinomas (GAC) are resistant to immune checkpoint inhibitors (ICIs), yet the underlying mechanism remains largely unknown. We sought to investigate the genomic correlates of the density of tumor-infiltrating immune cells (DTICs) and primary resistance to ICI treatment. METHODS: Four independent cohorts of MSI-H GAC were included: (i) the surgery cohort (n = 175) with genomic and DTIC data, (ii) the 3DMed cohort (n = 32) with genomic and PD-L1 data, (iii) the Cancer Genome Atlas (TCGA) cohort (n = 73) with genomic, transcriptomic, and survival data, and (iv) the ICI treatment cohort (n = 36) with pre-treatment genomic profile and ICI efficacy data. RESULTS: In the dMMR/MSI-H GAC, the number of mutated genes in the PI3K-AKT-mTOR pathway (NMP) was positively correlated with tumor mutational burden (P < 0.001) and sensitivity to PI3K-AKT-mTOR inhibitors and negatively correlated with CD3+ (P < 0.001), CD4+ (P = 0.065), CD8+ (P = 0.004), and FOXP3+ cells (P = 0.033) in the central-tumor rather than invasive-margin area, and the transcription of immune-related genes. Compared to the NMP-low (NMP = 0/1) patients, the NMP-high (NMP ≥ 2) patients exhibited a poorer objective response rate (29.4% vs. 85.7%, P < 0.001), progression-free survival (HR = 3.40, P = 0.019), and overall survival (HR = 3.59, P = 0.048) upon ICI treatment. CONCLUSIONS: Higher NMP was identified as a potential predictor of lower DTICs and primary resistance to ICIs in the dMMR/MSI-H GAC. Our results highlight the possibility of using mutational data to estimate DTICs and administering the PI3K-AKT-mTOR inhibitor as an immunotherapeutic adjuvant in NMP-high subpopulation to overcome the resistance to ICIs.

Various Subtypes of EGFR Mutations in Patients With NSCLC Define Genetic, Immunologic Diversity and Possess Different Prognostic Biomarkers.

Based on data analysis of 9649 Chinese primary NSCLC patients, we calculated the exact proportion of EGFR subtypes in NSCLC and evaluated the TMB level, PD-L1 expression level and tumor immune microenvironment among different EGFR mutation subtypes. Postoperative follow-up data for 98 patients were collected and analyzed. The results showed that several uncommon EGFR mutation subtypes have a higher proportion of TMB-high or strong positive PD-L1 expression than the total EGFR mutation group. In addition, different subtypes have different characteristics related to the immune microenvironment, such as G719 mutations being associated with more CD8+ T cell infiltration into tumors; except for EGFR 19del, CD8+ T cell infiltration into tumors of other EGFR mutation subtypes were similar to that of wildtype EGFR. Moreover, follow-up results revealed that components of the immune microenvironment have prognostic value for NSCLC patients, with different prognostic biomarkers for NSCLC patients with and without EGFR mutations. These results suggest that patients with different EGFR mutations need to be treated differently. The prognosis of NSCLC patients may be assessed through components of tumor immune microenvironment, and ICIs treatment may be considered for those with some uncommon EGFR mutation subtypes.

Circulating tumor DNA predicts neoadjuvant immunotherapy efficacy and recurrence-free survival in surgical non-small cell lung cancer patients.

Background: There is currently a lack of effective biomarkers to evaluate efficacy of neoadjuvant therapy (NAT) for resectable non-small cell lung cancer (NSCLC) patients. Circulating tumor DNA (ctDNA) has been investigated as a non-invasive tool for the assessment of tumor burden and minimal residual disease (MRD). The utility of ctDNA profiling in reflecting NAT efficacy, however, has not been confirmed. This study explored the association of ctDNA change with treatment response to NAT and recurrence-free survival (RFS) after surgery. Methods: Eligible patients with stage IB-IIIA NSCLC were retrospectively included if they had received neoadjuvant immunotherapy combined with chemotherapy (IO+Chemo), dual immunotherapy (IO+IO), or chemotherapy alone (Chemo). We conducted ctDNA profiling before and after NAT, after surgery, and during follow-ups using an ultra-deep lung cancer-specific MRD (LC-MRD) sequencing panel. Results: A total of 22 patients who received NAT followed by surgery between August 2018 and July 2019 were included in this study. The major pathological response (MPR) rates were 58.33% (7/12) in the IO+Chemo group, 25.00% (1/4) in the IO+IO group, and 16.67% (1/6) in the Chemo group. The ctDNA dynamics during NAT were highly concordant with pathologic response, demonstrating 100% sensitivity and 83.33% specificity, for an overall accuracy of 91.67%. Pre-surgery detectable ctDNA (after NAT) trended to correlate with inferior RFS [hazard ratio (HR), 7.41; 95% confidence interval (CI): 0.91-60.22, log-rank P=0.03]. At 3-8 days after surgery, ctDNA was detectable in 31.8% of patients and was an independent risk factor for recurrence (HR, 5.37; 95% CI: 1.27-22.67; log-rank P=0.01). The presence of ctDNA at 3 months after surgery showed 83% sensitivity and 90% specificity for predicting relapse (C-index, 0.79; 95% CI: 0.62-0.95). During disease monitoring after surgery, molecular recurrence by means of ctDNA preceded radiographic relapse, with a median time of 6.83 months. Conclusions: This study investigated the potential of ctDNA in evaluating NAT efficacy in NSCLC, implying the high concordance between ctDNA and pathological response. We also set out the prognostic value of perioperative ctDNA in predicting recurrence.

Characterization of the genomic landscape in large-scale Chinese patients with pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with an extremely poor prognosis. Effective targets for anticancer therapy confirmed in PDAC are limited. However, the characteristics of genomics have not been fully elucidated in large-scale patients with PDAC from China. METHODS: We collected both blood and tissue samples from 1080 Chinese patients with pancreatic cancer and retrospectively investigated the genomic landscape using next-generation sequencing (NGS). FINDINGS: We found recurrent somatic mutations in KRAS (83.2%), TP53 (70.6%), CDKN2A (28.8%), SMAD4 (23.0%), ARID1A (12.8%) and CDKN2B (8.9%) in Chinese PDAC patients. Compared with primary pancreatic cancers, more genomic alterations accumulated especially cell cycle regulatory gene variants (45.4% vs 31.6%, P < 0.001) were observed in metastatic tumors. The most common mutation site of KRAS is p.G12D (43.6%) in pancreatic cancer. Patients with KRAS mutations were significantly associated with older age and mutations in the other three driver genes, while KRAS wild-type patients contained more fusion mutations and alternative mechanisms of RTK/Ras/MAPK pathway including a number of clinically targetable mutations. KRAS mutations in Chinese cohort were significantly lower than those in Western cohorts (all P < 0.05). A total of 252 (23.3%) patients with the core DNA damage response (DDR) gene mutations were detected. ATM (n =59, 5.5%) was the most frequent mutant DDR gene in patients with pancreatic cancer from China. Patients with germline DDR gene mutations were younger (P = 0.018), while patients with somatic DDR gene mutations were more likely to accumulate in metastatic lesions (P < 0.001) and had higher TMB levels (P < 0.001). In addition, patients with mutant DDR genes and patients carrying TP53 mutation were observed mutually exclusive (P < 0.001). INTERPRETATION: We demonstrated the real-world genomic characteristics of large-scale patients with pancreatic cancer from China which may have promising implications for further clinical significance and drug development. FUNDING: The funders are listed in the Acknowledgement.

Efficacy and predictive biomarkers of immunotherapy in Epstein-Barr virus-associated gastric cancer.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer (GC) (EBVaGC) is a distinct molecular subtype of GC with a favorable prognosis. However, the exact effects and potential mechanisms of EBV infection on immune checkpoint blockade (ICB) efficacy in GC remain to be clarified. Additionally, EBV-encoded RNA (EBER) in situ hybridization (ISH), the traditional method to detect EBV, could cause false-positive/false-negative results and not allow for characterizing other molecular biomarkers recommended by standard treatment guidelines for GC. Herein, we sought to investigate the efficacy and potential biomarkers of ICB in EBVaGC identified by next-generation sequencing (NGS). DESIGN: An NGS-based algorithm for detecting EBV was established and validated using two independent GC cohorts (124 in the training cohort and 76 in the validation cohort). The value of EBV infection for predicting ICB efficacy was evaluated among 95 patients with advanced or metastatic GC receiving ICB. The molecular predictive biomarkers for ICB efficacy were identified to improve the prediction accuracy of ICB efficacy in 22 patients with EBVaGC. RESULTS: Compared with orthogonal assay (EBER-ISH) results, the NGS-based algorithm achieved high performance with a sensitivity of 95.7% (22/23) and a specificity of 100% (53/53). EBV status was identified as an independent predictive factor for overall survival and progression-free survival in patients with DNA mismatch repair proficient (pMMR) GC following ICB. Moreover, the patients with EBV+/pMMR and EBV-/MMR deficient (dMMR) had comparable and favorable survival following ICB. Twenty-two patients with EBV+/pMMR achieved an objective response rate of 54.5% (12/22) on immunotherapy. Patients with EBVaGC with a high cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) level were less responsive to anti-programmed death-1/ligand 1 (PD-1/L1) monotherapy, and the combination of anti-CTLA-4 plus anti-PD-1/L1 checkpoint blockade benefited patients with EBVaGC more than anti-PD-1/L1 monotherapy with a trend close to significance (p=0.074). There were nearly significant differences in tumor mutational burden (TMB) level and SMARCA4 mutation frequency between the ICB response and non-response group. CONCLUSIONS: We developed an efficient NGS-based EBV detection strategy, and this strategy-identified EBV infection was as effective as dMMR in predicting ICB efficacy in GC. Additionally, we identified CTLA-4, TMB, and SMARCA4 mutation as potential predictive biomarkers of ICB efficacy in EBVaGC, which might better inform ICB treatment for EBVaGC.

DNA damage repair gene mutations predict the efficacy of platinum-based chemotherapy and immunotherapy plus platinum-based chemotherapy in advanced non-small cell lung cancer: a retrospective Chinese cohort study.

Background: Platinum-based chemotherapy (PC) and immunotherapy plus platinum-based chemotherapy (IPC) remain the first-line treatment for advanced NSCLC. But only a minority patients benefit from PC, and existing biomarkers, such as PD-L1, have been shown to be defective in predicting the efficacy of IPC. Highlighting the need to identify novel biomarkers for the efficacy of PC and IPC. DNA damage repair (DDR) mutations are known to predict response to PC in solid tumors. However, the predictive value of DDR in PC and IPC of NSCLC remains unclear. Methods: Patients diagnosed with advanced or metastatic NSCLC were retrospectively included if they underwent next generation sequencing prior to starting treatment. Primary endpoints were to explore whether DDR mutations (DDRmut) are associated with clinical outcomes of PC and IPC. Secondary end point were to explore the association between DDRmut and the choice to add immunotherapy to chemotherapy, and the impact of different DDR pathways on efficacy in PC and IPC. Results: DDRmut showed a strong association with tumor mutation burden-high (TMB-H) versus DDR wild-type (DDRwt) and higher rates of PD-L1 TPS ≥50% positivity. In 63 patients treated with PC, ORRs were 15.38% and 2.86% for DDRmut and DDRwt subgroup (P=0.1536), and DCRs were 88.46% and 45.72% (P=0.00097) at 6 months after PC. The DDRmut patients had significantly improved median PFS (mPFS) and median overall survival (mOS) than DDRwt group (mPFS: 7.6 vs. 3.9 months, HR =1.93, 95% CI: 1.09 to 3.14, P=0.0220. mOS: 29.9 vs. 20.7 months, HR =2.31, 95% CI: 1.09 to 4.9, P=0.0250). Moreover, among 37 patients treated with IPC, ORRs were 45% and 11.76% for DDRmut and DDRwt patients (P=0.0365), and the DCRs were 95% and 70.58% (P=0.0752), respectively at 6 months after IPC. The DDRmut patients had significantly improved mPFS compared to the DDRwt group (19.5 vs. 4.5 months, HR =3.28, 95% CI: 1.53 to 9.56, P=0.0022). In DDRmut group, mPFS of IPC recipients was significantly better than that of PC recipients (19.5 vs. 7.6 months, HR =2.09, 95% CI: 0.98 to 4.42, P=0.050). Conclusions: There is potential for DDR to serve as a positive predictor of PC and IPC in advanced NSCLC patients.