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.

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.

Plasma-only circulating tumor DNA analysis detects minimal residual disease and predicts early relapse in hepatocellular carcinoma patients undergoing curative resection.

Background: Minimal residual disease (MRD) is considered an essential factor leading to relapse within 2 years (early relapse) after radical surgery, which is challenging to be detected by conventional imaging. Circulating tumor DNA (ctDNA) provides a novel approach for detecting MRD and predicting clinical outcomes. Here, we tried to construct a fixed panel for plasma-only ctDNA NGS to enable tumor-uninformed MRD detection in hepatocellular carcinoma (HCC). Methods: Here, we performed the followings: (i) profiling genomic alteration spectrum of ctDNA from the Chinese HCC cohort consisting of 493 individuals by NGS; (ii) screening of MRD monitoring genes; and (iii) performance evaluation of MRD monitoring genes in predicting early relapse in the ZJZS2020 cohort comprising 20 HCC patients who underwent curative resection. Results: A total of 493 plasma samples from the Chinese HCC cohort were detected using a 381/733-gene NGS panel to characterize the mutational spectrum of ctDNA. Most patients (94.1%, 464/493) had at least one mutation in ctDNA. The variants fell most frequently in TP53 (45.1%), LRP1B (20.2%), TERT (20.2%), FAT1 (16.2%), and CTNNB1 (13.4%). By customized filtering strategy, 13 MRD monitoring genes were identified, and any plasma sample with one or more MRD monitoring gene mutations was considered MRD-positive. In the ZJZS2020 cohort, MRD positivity presented a sensitivity of 75% (6/8) and a specificity of 100% (6/6) in identifying early postoperative relapse. The Kaplan-Meier analysis revealed a significantly short relapse-free survival (RFS; median RFS, 4.2 months vs. NR, P=0.002) in the MRD-positive patients versus those with MRD negativity. Cox regression analyses revealed MRD positivity as an independent predictor of poor RFS (HR 13.00, 95% CI 2.60-69.00, P=0.002). Conclusions: We successfully developed a 13-gene panel for plasma-only MRD detection, which was effective and convenient for predicting the risk of early postoperative relapse in HCC.

A durable response to savolitinib in a patient with lung adenocarcinoma harboring two novel MET exon 14 skipping sites.

MET exon 14 ( METex14 ) skipping variants are oncogenic drivers in non-small-cell lung cancer. Several METex14 skipping alterations have been identified, but different mesenchymal-epithelial transition (MET) exon splicing variants tend to present different clinical outcomes. Here, we reported that a patient with lung adenocarcinoma harbored two novel METex14 skipping mutations (c.2888-35_2888-16del and c.2888-4T>G) identified by the tissue-based next-generation sequencing (NGS) and received savolitinib treatment after chemotherapy failed with brain metastasis. The patient responded well to savolitinib until disease progression in brain lesions and achieved a progress-free survival (PFS) of over 19.7 months. Considering the durable response for extracranial lesions and the same METex14 skipping sites identified by circulating tumor DNA-based NGS, the patient was still given savolitinib combined with stereotactic body radiation therapy for brain lesions. An extracranial PFS of 28 months was achieved. This is the first report of a patient with lung adenocarcinoma harboring two novel METex14 skipping mutations that responded to the MET inhibitor savolitinib. Our case may provide evidence for the treatment of patients with two novel METex14 skipping variants and offer a therapy regimen for those with intracranial progression.

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.

Identification of BIK as an unfavorable prognostic marker and novel therapeutic target in microsatellite stable colorectal cancer harboring KRAS mutations.

KRAS mutations lead to persistent activation of multiple downstream effectors that drive the cancer phenotype. Approximately 30%-50% of colorectal cancer (CRC) patients harbor KRAS mutations, which confer more aggressive tumor biology and shorter overall survival (OS), especially in microsatellite stable (MSS) metastatic CRC. Given that KRAS mutant protein has been proven difficult to target directly, identifying genes that function closely with KRAS and targeting these genes seems to be a promising therapeutic strategy for KRAS-mutated MSS CRC. Here, KRAS function-sensitive genes were identified by assessing the correlation between gene dependency scores from CRISPR knockout screens and KRAS mRNA expression in KRAS-mutated MSS CRC cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. If the correlation coefficient was ≥ 0.6, the gene was considered a KRAS function-sensitive gene. Then KRAS function-sensitive genes related to prognosis were screened out in The Cancer Genome Atlas (TCGA) cohort, and the prognostic value was validated in the Gene Expression Omnibus (GEO) cohort. Single-sample gene set enrichment analysis (ssGSEA) was performed to investigate the potential mechanisms. PockDrug-Server was used to predict the druggability of candidate genes. The results showed that in 20 KRAS-mutated MSS CRC cell lines, 13 genes were identified as KRAS function-sensitive genes. Of these 13 genes, only BIK expression was significantly associated with progression-free survival (PFS) and OS, and the BIK-high patients had significantly poorer PFS (HR=3.18, P=0.020) and OS (HR=4.74, P=0.030) than the BIK-low patients. Multivariate Cox regression analysis revealed high BIK expression as an independent predictor for poorer prognosis in KRAS-mutated MSS CRC. The prognostic value of BIK was also successfully validated in a GEO cohort. The results of ssGSEA showed that the BIK-high group was more prone to strong metastasis activity than the BIK-low group. Pocket druggability prediction analysis presented that BIK had three druggable pockets, and their druggability scores were above 0.8. These findings suggested that BIK is a promising prognostic marker and therapeutic target in KRAS-mutated MSS CRC.

The Optimal Therapy after Progression on Immune Checkpoint Inhibitors in MSI Metastatic Gastrointestinal Cancer Patients: A Multicenter Retrospective Cohort Study.

BACKGROUND: In microsatellite instability (MSI)/mismatch repair-deficient (dMMR) gastrointestinal cancers, the optimum therapy after the progression of immune checkpoint inhibitors (ICIs) is yet unknown. Here, we compared the efficacy of programmed death 1 (PD1)/programmed death ligand-1 (PD-L1) inhibitors plus other therapy and chemotherapy with or without targeted therapy in MSI/dMMR gastrointestinal cancer patients after progression on anti-PD1/PD-L1 monotherapy. METHODS: We retrospectively recruited MSI/dMMR gastrointestinal cancer patients who had progressed on anti-PD1/PD-L1 monotherapy. Objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and PFS ratio (PFSr) were compared between patients who received anti-PD1/PD-L1 plus other therapy (ICI-plus group) and patients who received chemotherapy with or without targeted therapy (chemo-targeted group). RESULTS: In total, 26 and 25 patients were recruited in the ICI-plus group and chemo-targeted group, respectively. Significantly better DCR (80.8% vs. 44.0%, p = 0.007), PFS (median PFS 6.9 months vs. 3.0 months, p = 0.001), OS (median OS NR vs. 14.1 months, p = 0.043), and PFSr (2.4 vs. 0.9, p = 0.021), along with a numerically higher ORR (23.1% vs. 12.0%, p = 0.503) were observed in the ICI-plus group compared with the chemo-targeted group. Multivariate analyses identified the therapy regimen as an important prognostic factor in gastrointestinal cancers. CONCLUSIONS: Compared to conventional chemotherapy with or without targeted therapy, continuing anti-PD1/PD-L1 in combination with other treatments showed better clinical outcomes in MSI/dMMR gastrointestinal cancer patients who progressed on PD1/PD-L1 blockade, which should be validated prospectively in clinical trials.

A novel 8-gene panel for prediction of early biochemical recurrence in patients with prostate cancer after radical prostatectomy.

Approximately 25% of prostate cancer (PCa) cases experience biochemical recurrence (BCR) following radical prostatectomy (RP). The patients with BCR, especially with BCR ≤2 year after RP (early BCR), are more likely to develop clinical metastasis and castration resistance. Now decision-making regarding BCR after RP relies solely on clinical parameters. We thus attempted to establish an early BCR-risk prediction model by combining a molecular signature with clinicopathological features for guiding clinical decision-making. In this study, an 8-gene signature was derived, and these eight genes were SPTBN2, LGI3, TGM3, LENG9, HAS3, SLC25A27, PCDHGA1, and ADPRHL1. The Kaplan-Meier analysis revealed a significantly prolonged BCR-free survival in the patients with low-risk scores compared to those with high-risk scores in both training and validation datasets. Harrell's concordance index and time-dependent receiver operating characteristic analysis demonstrated that this gene signature tended to outperform three commercial panels at early BCR prediction. Moreover, this signature was also proven as an independent predictor of BCR-free survival. A nomogram, incorporating the gene signature and clinicopathologic features, was constructed and excellently predicted 1-, 2- and 3-year BCR-free survival of localized PCa patients after RP. Gene set enrichment analysis, tumor immunity, and mRNA expression profiling analysis showed that the high-risk group was more prone to the immunosuppressive microenvironment and impaired DNA damage response than the low-risk group. Collectively, we successfully developed a novel 8-gene signature as a powerful predictor for early BCR after RP and created a prognostic nomogram, which may help inform the clinical management of PCa.

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.

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.

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.

Secondary myelodysplastic syndromes identified via next-generation sequencing in a non-small cell lung cancer patient.

BACKGROUND: Myelodysplastic syndrome (MDS) is a group of clonal disorders characterized by ineffective and dysplastic hematopoiesis in the bone marrow with a high risk of progression to leukemia. Many studies have demonstrated that chemo-radiotherapy for cancer patients and exposure to certain chemicals may increase the risk of secondary MDS, which is characterized by specific chromosomal abnormalities and genomic alterations. Since next-generation sequencing (NGS) has been widely used for the diagnosis of cancer patients, advanced analysis of the sequencing data may provide supplementary information for secondary MDS. CASE PRESENTATION: A male patient with non-small cell lung cancer (NSCLC) and bone metastases has presented distal obstructive inflammation, the enlargement of the left hilar, mediastinal lymph node metastases, and multiple bone metastases. This patient has undergone long-term exposures to certain chemicals. Moreover, the deletion of chromosome 7 and 5q is detected in his peripheral blood sequencing, indicating secondary MDS, subsequently confirmed by bone marrow examination. CONCLUSION: In this case, an NSCLC patient was diagnosed with secondary MDS via NGS analysis, indicating that the NGS analysis may serve as supplementary for diagnosis of secondary MDS and provide useful information of therapeutic regimens for subsequent-line treatment of EGFR-mutated lung cancer. To the best of our knowledge, this is the first report of acquired MDS in a lung adenocarcinoma patient.

Prevalence and spectrum of germline cancer susceptibility gene variants and somatic second hits in colorectal cancer.

Colorectal cancer (CRC) is one of the most heritable cancers, and genetic factors play an important role in the increased CRC risk. However, the well-established CRC-risk genes were limited for explaining the increased risk of CRC individuals. Germline mutations in DNA damage repair (DDR) genes have also been reported to be implicated in CRC heritability. Here, we aimed to determine the prevalence and significance of germline DDR and well-established CRC-risk gene variants in CRCs with paired somatic analyses. Next-generation sequencing (NGS) was performed on tumor tissues and paired white blood cells collected from 2160 Chinese patients with CRC using well-designed 381- or 733-cancer gene panel. Germline/somatic variations were identified and assessed for pathogenicity and likely pathogenicity. Of 2160 CRCs, 136 pathogenic germline mutations in 133 patients (133/2160, 6.1%) were identified in 21 genes, including 19 out of 32 examined DDR genes. Compared with non-carriers, individuals with germline variants were prone to a higher level of microsatellite instability (MSI) and tumor mutational burden (TMB), and an earlier age of onset. Somatic sequencing identified second hits in 24/133 (18%) patients with germline variants. Among the mismatch repair (MMR) genes with germline mutations, the second hit significantly increased MSI and TMB, particularly apparent in MSH6. All MMR germline variation carriers further with a second hit were all MSI-H and had an extraordinarily high level of TMB. Collectively, approximately 6.1% of CRC patients carried pathogenic germline variants, and additional somatic second hit increases the genomic instability in CRC, whereas the more clinical significance warrants further study.

Different formins restrict localization of distinct tropomyosins on dorsal stress fibers in osteosarcoma cells.

Formins and tropomyosins (Tpms) are two central components of the microfilaments. Formins are involved in the nucleation and polymerization of actin filaments, and Tpms form along the actin stress fibers to regulate their dynamics. However, the correlation between formins and Tpms remains unclear. Here, we elucidated the function of distinct formins and their specific regulation to the subcellular-localization of Tpm isoforms on dorsal stress fibers in human osteosarcoma cells. Knockdown of individual formin isoform led to varied defects in actin stress fiber network, but did not affect the expression level of other formin isoforms and Tpms. Further investigation showed that different formins regulated distinct Tpm isoforms in decorating dorsal stress fibers. Specifically, DAAM1 and FHOD1 restricted the distal end expression of Tpm3.1; INF2 controlled the approximate localization of Tpm4.2; and Dia1 partially modulated the dorsal localization of Tpm1.6. Taken together, these data provide microscopy experimental evidence that different formins restrict the localization of distinct Tpm isoforms on dorsal actin stress fibers.

Active FHOD1 promotes the formation of functional actin stress fibers.

The formin FHOD1 acts as a nucleating, capping and bundling protein of actin filaments. In cells, release from the C-terminal diaphanous autoregulatory domain (DAD) of FHOD1 stimulates the protein into the active form. However, the cellular physiological relevance of active form FHOD1 and the phenotypic regulation by FHOD1 depletion are not completely understood. Here, we show that in contrast with the cytosolic diffused expression of auto-inhibited FHOD1, active FHOD1 by C-terminal truncation was recruited into all three types of actin stress fibers in human osteosarcoma cells. Notably, the recruited active FHOD1 was more incorporated with myosin II than α-actinin, and associated with both naïve and mature focal adhesions. Active FHOD1 displayed faster turnover than actin molecules on ventral stress fibers. Moreover, we witnessed the emergence of active FHOD1 from the cell periphery, which subsequently moved centripetally together with transverse arcs. Furthermore, FHOD1 knockdown resulted in defective maturation of actomyosin bundles and subsequently longer non-contractile dorsal stress fibers, whereas the turnover of both actin and myosin II were maintained normally. Importantly, the loss of FHOD1 led to slower actin centripetal flow, resulting in abnormal cell spreading and migration defects. Taken together, these results reveal a critical role of FHOD1 in temporal- and spatial- control of the morphology and dynamics of functional actin stress fibers during variable cell behavior.