Graphical abstract graphic file with name fx1.jpg [45]Open in a new tab Highlights * • The study evaluates anti-PD-L1-based triple combination in advanced G/GEJ cancer * • Favorable efficacy and safety profiles with this combination * • Patients achieve encouraging ORR, OS, and PFS * • Differences in the PBIC profile may be a predictor of treatment outcomes __________________________________________________________________ Liangyu Bie et al. show that the triple combination of benmelstobart, anlotinib, and chemotherapy has favorable efficacy and safety in HER2-negative advanced gastric or gastroesophageal junction adenocarcinoma. PBIC profile analysis and omics exploration may provide insights on prognostic value. Introduction Gastric or gastroesophageal junction (G/GEJ) adenocarcinoma is the fifth most prevalent and the fifth most lethal cancer worldwide.[46]^1 Despite the availability of early screening and local resection, most diagnoses occur at an advanced stage, and the prognosis of patients with G/GEJ cancer remains poor.[47]^2 Notably, pivotal clinical trials, such as CheckMate 649[48]^3 and ORIENT-16,[49]^4 have underscored the efficacy of programmed death-1 (PD-1) inhibitors in combination with chemotherapy, establishing them as a standard first-line treatment for patients with HER2-negative advanced G/GEJ cancer. However, immunochemotherapy revealed limited survival advantage in the cohorts with programmed death-ligand 1 (PD-L1) combined positive score (CPS) <5.[50]^4^,[51]^5 Therefore, there is a pressing need to explore novel approaches to enhance long-term survival outcomes for these patients. Strategically combining anti-angiogenic agents with immunochemotherapy may represent an attractive therapeutic approach for human epidermal growth factor receptor 2 (HER2)-negative advanced G/GEJ cancer in the first-line setting, as demonstrated by feasibility from lenvatinib plus pembrolizumab and chemotherapy,[52]^6 regorafenib plus nivolumab and chemotherapy,[53]^7 and apatinib plus camrelizumab and chemotherapy[54]^8 ([55]Table S1). These triple combination therapies improved patient prognosis by enhancing the immune response, reducing resistance to a single drug, and exerting multiple synergistic mechanisms.[56]^9^,[57]^10 Nevertheless, the efficacy and safety of such combinations are preliminary, and optimal combinations remain unclear. Anlotinib is an oral novel small-molecule multitargeted tyrosine kinase inhibitor that targets receptors on vascular endothelial cells to inhibit tumor angiogenesis.[58]^11 It has exhibited promising efficacy and favorable safety profiles when combined with chemotherapy or immune checkpoint inhibitors in many advanced solid tumors, including G/GEJ cancer.[59]^12^,[60]^13 Benmelstobart (TQB2450), a humanized IgG1 anti-PD-L1 antibody, has shown significant sequence divergence in complementarity-determining regions from other PD-L1 inhibitors and blockade of the interaction of PD-L1 with PD-1 and CD80 receptors.[61]^14 Besides, the modified Fc domain could minimize FcγR binding and eliminate antibody-dependent cell-mediated cytotoxicity/complement-dependent cytotoxicity activities, contributing to the favorable safety (serious adverse events, 27.5% vs. 33.3%–54%) of benmelstobart compared to other PD-L1 inhibitors.[62]^14^,[63]^15^,[64]^16^,[65]^17 Consequently, benmelstobart and anlotinib may represent the promising immune and antiangiogenic components in our triple combination, under the additional rationale indicated by the impressive clinical benefits of benmelstobart plus anlotinib and chemotherapy in first-line therapy for small-cell lung cancer.[66]^18 As such, we conducted this phase 2 study to assess the efficacy and safety of benmelstobart plus anlotinib and chemotherapy as first-line treatment in patients with HER2-negative advanced G/GEJ cancer. In doing so, we also explored the predictive role of peripheral blood immune cells (PBICs) in the tumor microenvironment and PD-L1 expressions on clinical benefits. Results Patient characteristics and treatment A total of 25 patients were enrolled between May 2021 and June 2022 ([67]Figure 1). All 25 patients received at least one dose of protocol treatment (full analysis set [FAS] population). However, one patient died from COVID-19 after one cycle of treatment. A total of 24 patients underwent at least one efficacy assessment (per-protocol set [PPS] population). Fifteen (60.0%) patients completed 6 cycles of induction therapy, and 19 (75.0%) patients received planned maintenance therapy. The primary reason for induction therapy discontinuation was COVID-19 (n = 3) and disease progression (n = 2). At the final analysis for overall survival (OS) (the data cutoff, 1 May 2024), one patient (4.0%) is still undergoing the assigned treatment. Figure 1. [68]Figure 1 [69]Open in a new tab Trial profile Baseline characteristics of the FAS population are listed in [70]Table 1. The median age was 59 years (range, 19–69), and 21 (84.0%) patients were male. Most patients had Eastern Cooperative Oncology Group performance status 1 (80.0%) and primary tumors located in the stomach (72.0%). All patients had at least 1 metastatic site, of which 7 (28%) had 2 metastatic sites and 1 (4%) had 3 metastatic sites, with the most common metastatic sites being lymph node (60%) and liver (40%). Among 20 patients with available PD-L1 assessments, 10 (50%) patients had CPS ≥1, 8 (40%) had CPS ≥5, and 5 (25%) had CPS ≥10. Table 1. Baseline characteristics Characteristics FAS population (n = 25) Age, years (median, range) 59 (19–69) __________________________________________________________________ Gender __________________________________________________________________ Female 4 (16.0) Male 21 (84.0) __________________________________________________________________ ECOG performance status __________________________________________________________________ 0 5 (20.0) 1 20 (80.0) __________________________________________________________________ Primary tumor location __________________________________________________________________ Gastric 18 (72.0) Gastric esophageal junction 7 (28.0) __________________________________________________________________ Histologic grade __________________________________________________________________ Highly differentiated 1 (4.0) Moderately differentiated 10 (40.0) Poorly differentiated 5 (20.0) Unknown 9 (36.0) __________________________________________________________________ Metastatic site __________________________________________________________________ Lymph node 15 (60.0) Live 10 (40.0) Lung 2 (8.0) Peritoneal 2 (8.0) Bone 1 (4.0) Ovarian 1 (4.0) __________________________________________________________________ Number of metastatic sites __________________________________________________________________ 1 17 (68.0) 2 7 (28.0) ≥3 1 (4.0) MSS 25 (100.0) __________________________________________________________________ PD-L1 CPS score[71]^a __________________________________________________________________  ≥1 10 (50.0) ≥5 8 (40.0) ≥10 5 (25.0) Missed 5 (25.0) [72]Open in a new tab Data are n (%). ECOG, Eastern Cooperative Oncology Group; MSS, microsatellite stable; PD-L1, programmed death-ligand 1; CPS, combined positive score; FAS, full analysis set. ^a Samples evaluable for PD-L1 CPS were available in 20 patients. Efficacy In the FAS population, the objective response rate (ORR) was 72.0% (95% confidence interval [CI], 50.6%–87.9%), and the disease control rate (DCR) was 96.0% (95% CI, 79.6%–99.9%). Of 24 PPS patients, 18 (75.0%, 95% CI, 53.3%–90.2%) patients achieved an objective response. Six (25%) patients had stable disease (SD), and the DCR was 100.0% (95% CI, 85.8%–100.0%; [73]Table 2). All these 24 patients achieved a reduction of target lesions from the baseline ([74]Figure 2A). The treatment duration for all patients is shown in [75]Figure 2B. The median duration of response (DoR) was 10.9 (95% CI, 9.7–12.1) months in the 18 responders. Subgroup analyses showed consistent ORR across all patient subgroups including age, gender, PD-L1 CPS, metastasis, or location of the tumor ([76]Figure S1). Table 2. Summary of response outcomes FAS population (n = 25) PPS population (n = 24) Best response __________________________________________________________________ Partial response 18 (72, 50.6–87.9) 18 (75, 53.3–90.2) Stable disease 6 (24, 9.4–45.1) 6 (25, 9.8–46.7) Progressive disease 0 0 Not evaluable 1 (4, 0.1–20.4)[77]^a – Objective response rate 18 (72, 50.6–87.9) 18 (75, 53.3–90.2) Disease control rate 24 (96, 79.6–99.9) 24 (100, 85.8–100.0) [78]Open in a new tab Data are n (%) or n (%, 95% confidence interval). FAS, full analysis set; PPS, per-protocol set. ^a One patient could not be evaluated for tumor response because of the unavailable post-treatment imaging. Figure 2. [79]Figure 2 [80]Open in a new tab Tumor response and survival (A) Waterfall plot of tumor size change from baseline to maximum percentage in each patient as per RECIST version 1.1 (n = 24). (B) Swimmer plot representing the duration of treatment (n = 24). (C) Progression-free survival in FAS population (n = 25). (D) Overall survival in FAS population (n = 25). RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; PR, partial response; ECOG, Eastern Cooperative Oncology Group; PD-L1, programmed death-ligand 1; CPS, combined positive score; NA, not assessable; PFS, progression-free survival; OS, overall survival; mo, months; No., number; FAS, full analysis set. As of the data cutoff, among 25 FAS patients, the median follow-up was 15.8 (range, 2.7–34.1) months. The median progression-free survival (PFS) was 10.3 (95% CI, 5.2–12.8) months; the 6-month and 1-year PFS rates were 64.0% (95% CI, 47.7%–85.9%) and 36.0%, respectively (95% CI, 21.3%–60.7%; [81]Figure 2C). The median OS, 1-year, and 18-month OS rates were 18.2 (95% CI, 13.3–22.0) months, 68.0% (95% CI, 52.0%–89.0%), and 52.0% (95% CI, 35.7%–75.8%; [82]Figure 2D), respectively. There were no significant correlations between clinical characteristics and survival outcomes ([83]Figure S2). PD-L1 expression Samples evaluable for PD-L1 CPS were available in 20 patients ([84]Figure S3). Irrespective of the cutoff values of CPS, PD-L1 expression levels were not significantly associated with PFS and OS. The median PFS with benmelstobart plus anlotinib and chemotherapy in patients with PD-L1 CPS ≥1 and <1 was 10.4 and 8.9 months, respectively; median OS was 17.1 and 19.1 months, respectively. The median PFS and OS for patients with a PD-L1 CPS ≥5 were 8.2 and 17.1 months, respectively, whereas the median PFS and OS of those with a PD-L1 CPS ≥10 were 5.1 and 15.7 months, respectively. PBIC profiles The PBICs were collected from patients before the first and third cycle of therapy. We divided the patients into two groups based on their PFS: the long-term response (LTR) group (PFS > 12 months) and the short-term response (STR) group (PFS ≤ 12 months). Results revealed significant differences in PBIC profiles of baseline between the LTR and STR groups in the percentages of lymphocyte (p = 0.022), T cells (p = 0.042), and CD3^+CD8^+ T cells (p = 0.02) ([85]Figure 3). Other PBIC profiles were similar between the two groups ([86]Figure S4), suggesting that these immune markers were not strongly predictive of the long-term survival benefit. All PBIC profiles did not show significant differences between the before-treatment and post-treatment of the LTR or STR groups ([87]Figures S5 and [88]S6). Figure 3. [89]Figure 3 [90]Open in a new tab Significant differences in PBIC profiles of baseline between the LTR and STR groups Differences of (A) absolute lymphocyte counts, (B) absolute T cell counts, and (C) absolute counts of CD3^+ CD8^+ T cells in pretreatment PBIC profiles between the LTR and STR groups. Points represent individual patients, boxes show median and interquartile range, and p values from Wilcoxon rank-sum tests are indicated. PBIC, peripheral blood immune cell; LTR, long-term response; STR, short-term response. We also analyzed predictive values of PBIC profiles on the treatment response and found a higher number of lymphocytes, T cells, and CD3^+CD8^+ T cells in the responders (R group) compared to non-responders (NR group), without statistical significance ([91]Figure S7). The comparative analysis of PBIC profiles before and after treatment, stratified by response, is provided in [92]Figures S8 and [93]S9. Biomarker analyses Blood samples from 12 patients were collected prior to treatment for transcriptomic profiling. The differential expression analysis between the partial response (PR) and SD groups revealed multiple significantly differentially expressed genes (false discovery rate [FDR] < 0.05), notably the upregulation of cluster of differentiation 177 (CD177) and downregulation of leukocyte-associated immunoglobulin-like receptor 2 (LAIR2) in PR patients ([94]Figure 4A). Gene set enrichment analysis using a less stringent FDR threshold (FDR < 0.1) identified several potentially enriched pathways. Three pathways showed positive enrichment in the PR group: the G2M checkpoint pathway demonstrated the strongest enrichment (normalized enrichment score [NES] = 1.73, FDR = 0.056), followed by the E2F target pathway (NES = 1.55, FDR = 0.056) and the inflammatory response pathway (NES = 1.51, FDR = 0.077). In contrast, the interferon-alpha response pathway showed negative enrichment (NES = −1.56, FDR = 0.056), indicating its association with the SD group ([95]Figure 4B). QuanTIseq deconvolution analysis revealed complex immune cell composition patterns across patients, with T cells (both CD4^+ and CD8^+) and monocytes comprising the predominant populations ([96]Figure 4C). While no statistically significant differences in individual immune cell proportions were observed between the PR and SD groups, a consistent trend toward higher CD8^+ T cell levels in PR patients was noted ([97]Figure 4D), aligning with our PBIC profiling results. Figure 4. [98]Figure 4 [99]Open in a new tab RNA-seq-based molecular and immune profiling in PR versus SD blood samples (A) Volcano plot showing differential gene expression analysis between PR and SD response groups. The x axis shows log2 fold change, and the y axis shows −log10 FDR. Red dots indicate significantly differentially expressed genes (FDR < 0.05). Key genes are labeled. (B) Gene set enrichment analysis (GSEA) results show normalized enrichment scores (NES) for hallmark pathways (FDR < 0.1). The G2M checkpoint pathway showed the strongest positive enrichment (NES = 1.73), while the interferon-alpha response pathway showed negative enrichment (NES = −1.56). Color gradient represents FDR values, with darker colors indicating lower FDR values. The width of bars represents the NES. (C) Stacked bar plot showing immune cell composition across individual patients as determined by quanTIseq deconvolution of RNA sequencing (RNA-seq) data. Each bar represents a patient sample, with colors indicating different immune cell populations. (D) Boxplots comparing immune cell proportions between PR and SD response groups for six major immune cell types. Points represent individual patients, boxes show median and interquartile range, and p values from Wilcoxon rank-sum tests are indicated. PR, partial response; SD, stable disease; NES, normalized enrichment scores; GSEA, gene set enrichment analysis. Safety All 25 patients were evaluable for safety ([100]Table 3). Treatment-related adverse events (TRAEs) of any grade occurred in 24 (96.0%) patients, with nausea (68.0%), thrombocytopenia (64.0%), anemia (64.0%), lymphopenia (48.0%), white-cell count decreased (44.0%), and neutrophil count decreased (40.0%) being common. Grade 3 TRAEs occurred in 6 (24.0%) patients and included lymphopenia (8.0%), white-cell count decreased (4.0%), neutrophil count decreased (12.0%), and gastrointestinal hemorrhage (8.0%). One (4.0%) patient experienced grade 4 lymphopenia (4.0%). No grade 5 TRAEs occurred. Six patients (24.0%) had TRAEs leading to a dose reduction of chemotherapy. Nineteen deaths occurred in the study; of these, 18 were attributed to progressive disease, and the remaining 1 was due to COVID-19, deemed unrelated to treatment (based on the assessment of the pulmonology specialist). Table 3. Treatment-related adverse events in the safety analysis set (n = 25) Number of patients with event (%) __________________________________________________________________ All grade Grade 1 Grade 2 Grade 3 Grade 4 All TRAEs 24 (96) 24 (96) 15 (60) 6 (24) 1 (4) Nausea 17 (68) 16 (64) 1 (4) 0 0 Thrombocytopenia 16 (64) 10 (40) 6 (24) 0 0 Anemia 16 (64) 11 (44) 5 (20) 0 0 Lymphopenia 12 (48) 4 (16) 6 (24) 1 (4) 1 (4) White-cell count decreased 11 (44) 6 (24) 4 (16) 1 (4) 0 Neutrophil count decreased 10 (40) 2 (8) 5 (20) 3 (12) 0 Vomiting 8 (32) 7 (28) 1 (4) 0 0 Fatigue 7 (28) 5 (20) 2 (8) 0 0 Hypoproteinemia 6 (24) 6 (24) 0 0 0 Hyperthyroidism 5 (20) 1 (4) 4 (16) 0 0 Hypothyroidism 4 (16) 2 (8) 2 (8) 0 0 Aspartate aminotransferase increased 4 (16) 3 (12) 1 (4) 0 0 Bilirubin increased 4 (16) 4 (16) 0 0 0 Hypertension 4 (16) 1 (4) 3 (12) 0 0 Alanine aminotransferase increased 3 (12) 3 (12) 0 0 0 Peripheral neuropathy 3 (12) 2 (8) 1 (4) 0 0 Gastrointestinal hemorrhage 1 (4) 0 0 1 (4) 0 Alkaline phosphatase increased 1 (4) 1 (4) 0 0 0 Hyponatremia 1 (4) 1 (4) 0 0 0 Hypokalaemia 1 (4) 1 (4) 0 0 0 Hypercalcemia 1 (4) 1 (4) 0 0 0 Hand-foot syndrome 1 (4) 1 (4) 0 0 0 [101]Open in a new tab Data are n (%). Discussion It is noteworthy that current investigations into triple combination are underway across multiple countries, reflecting the practical value of immunotherapy plus anti-angiogenic drugs and chemotherapy for untreated HER2-negative advanced G/GEJ cancer.[102]^6^,[103]^7^,[104]^8^,[105]^19 However, current data on these triple combinations remain preliminary and are primarily focused on regimens involving PD-1 blockade combined with anti-angiogenic agents and chemotherapy. Our study represents one investigation of a triple combination based on anti-PD-L1 in the first-line setting, with a favorable safety (grade ≥3 TRAEs, 28.0%) and the longest OS (18.2 months) to date. Besides, benmelstobart combined with anlotinib and chemotherapy also demonstrated a deep and durable response (ORR, 75.0%; DCR, 100.0%; DoR, 10.9 months) and an encouraging PFS of 10.3 months in this population, irrespective of PD-L1 status. Importantly, our study’s ORR, OS, and PFS outcomes surpassed numerically the results from the pivotal trials involving the first-line immunochemotherapy ([106]Table S1), under lower patient proportion with positive PD-L1 expression.[107]^3^,[108]^4^,[109]^5 A deeper understanding of the synergistic mechanisms importantly explained the clinical benefits derived from strategically combining anti-angiogenic agents with immunochemotherapy.[110]^20^,[111]^21^,[112]^22^,[113]^23 The rationale was also supported by the encouraging outcomes from the SPACE study, in a context of low percentages of patients with high CPS.[114]^24 Additionally, several studies in the preliminary analysis have also demonstrated the feasibility of triple combination regimens.[115]^6^,[116]^7^,[117]^8 Of note, benmelstobart plus anlotinib and chemotherapy has revealed superior survival benefits and durable response compared with the available triple studies.[118]^6^,[119]^7^,[120]^8^,[121]^19 Although cross-trial comparisons should be interpreted with caution due to the lack of statistical power, our better efficacy data may be explained by the biological differences between benmelstobart (IgG1 PD-L1 inhibitor) in our regimen and IgG4 PD-1 inhibitors used in first-line series. In contrast to significant plasticity of PD-1, binding of anti-PD-L1 antibodies induced minor conformational changes of PD-L1, potentially resulting in the high affinity of interactions between therapeutic antibodies and PD-L1.[122]^24^,[123]^25^,[124]^26 However, the therapeutic out-competition between PD-1 and PD-L1 inhibitors remains inconclusive in the clinical settings. Thus, the biological explanations for the effectiveness of our regimen must be considered speculative because the differences in study design may also result in differential therapeutic outcomes. As such, further evaluation in larger scale randomized trials is warranted to determine the benefits of benmelstobart plus anlotinib and chemotherapy. Numerous studies previously documented that the improvements in survival benefits and tumor response may be associated with higher PD-L1 CPS cutoffs.[125]^3^,[126]^4^,[127]^5^,[128]^27 Despite no significant correlation between PD-L1 expression and survival in our study, we cannot draw a conclusion with robustness because the small sample size may lead to insufficient power to detect such differences. Besides, we also observed similar tumor response and survival outcomes across subgroups stratified by age, metastasis, and cardiac cancer, potentially enabling benmelstobart with anlotinib and chemotherapy as a promising treatment option irrespective of patients’ baseline characteristics. Our blood transcriptomic profiling revealed key molecular signatures that differentiate immunotherapy response groups. The significant upregulation of CD177 (log2 fold change [log2FC] = 5.84) in PR patients, a known enhancer of neutrophil-mediated immune remodeling,[129]^28 alongside the downregulation of LAIR2 (log2FC = −2.17), which may potentiate natural killer cell function,[130]^29 suggests coordinated immune microenvironment reprogramming that favors treatment response. Pathway enrichment analysis further highlighted strong positive enrichment of the G2M checkpoint (NES = 1.73) and E2F targets (NES = 1.55) in the PR group, consistent with mechanisms linking cell cycle dysregulation to PD-L1 upregulation.[131]^30 These peripheral blood transcriptional signatures, alongside their biological implications, emphasize the potential of these markers for predicting immunotherapy response and warrant further exploration in larger cohorts. Notably, despite no significant differences in bulk immune cell proportions between the PR and SD groups, a consistent trend of elevated CD8^+ T cells in PR patients was observed, suggesting that immune cell functional state, rather than just abundance, may play a critical role in determining treatment efficacy. Additionally, we observed distinct differences in the PBIC profile between the LTR and STR. Specifically, the LTR group showed higher absolute counts of lymphocytes, T cells, and CD3^+CD8^+ T cells, aligning with our earlier finding that a stronger immune baseline is associated with better treatment outcomes.[132]^31 These observations highlight the importance of immune cell counts at baseline as potential predictive biomarkers for treatment efficacy. The enrichment of cytotoxic T cells in responders further supports the role of CD8^+ T cells in mediating immune responses, particularly in immunotherapy contexts.[133]^32 Taken together, these findings suggest that immune parameters, especially those related to T cell populations, may serve as valuable biomarkers for predicting patient prognosis and guiding personalized treatment strategies. Future studies incorporating single-cell sequencing approaches will be essential to unravel cell type-specific contributions to these signatures and their precise relationship to treatment outcomes. The safety profiles identified in this study were consistent with those of individual treatments and other immunotherapies in G/GEJ cancers,[134]^3^,[135]^4^,[136]^5^,[137]^8^,[138]^19^,[139]^20^,[140]^2 1^,[141]^22 with no unexpected safety signals. Most TRAEs were grade 1–2. A meta-analysis of 125 trials has reported that PD-L1 inhibitors have a lower risk of grade 3 or higher adverse events in contrast to PD-1 inhibitors.[142]^33 Consistently, numerically lower incidence of grade 3 or higher TRAEs (28.0% vs. 53%–90%) was observed here compared to those in the first-line series of anti-PD-1 in HER2-negative G/GEJ,[143]^3^,[144]^8^,[145]^19^,[146]^20^,[147]^21^,[148]^24^,[149]^2 5^,[150]^34^,[151]^35 potentially indicating favorable tolerability of this regimen. Of note, grade ≥3 hypertension, as a class effect of anti-angiogenic therapies, has been observed in nearly all first-line studies,[152]^7^,[153]^8 but was not observed in our study. Although one patient experienced gastrointestinal hemorrhage, therapy was resumed after the improvement, with no further episodes occurring. Besides, there were no TRAEs leading to death. Together, the acceptable safety characteristics combined with improved OS suggested a favorable benefit-risk profile for first-line benmelstobart combined with anlotinib and chemotherapy for HER2-negative advanced G/GEJ cancer. In summary, benmelstobart plus anlotinib and chemotherapy demonstrated favorable efficacy and safety, and the PBIC profile analysis may provide insights on their prognostic value. This triple combination may potentially represent a promising first-line treatment option for untreated HER2-negative patients with advanced G/GEJ cancer, regardless of PD-L1 status, requiring further investigations to determine the long-term benefits. Limitations of the study This study has several limitations. First, a small sample size and single-arm design without a control group for comparison posed a challenge to decipher the benefits of combination treatment. Thus, larger scale randomized clinical trials are indispensable to further confirm the efficacy and safety of this combination therapy. Besides, all patients were Chinese, potentially limiting the generalizability of our results to other racial/ethnic groups. Third, tumor responses were not evaluated by an independent central review committee, which may introduce bias in the determination of PFS. Finally, the biomarker analyses were exploratory, and the prognostic value of some parameters for survival was not evaluated. Resource availability Lead contact For further information and requests for resources and reagents, please contact the lead contact, Ning Li (lining97@126.com). Materials availability All unique materials generated in this study are available from the [154]lead contact with a completed materials transfer agreement. Data and code availability * • The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in the National Genomic Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academic Sciences (GSA-Human: HRA010919). Data access ([155]http://ngdc.cncb.ac.cn/gsa-human) is subject to the regulations of the Ministry of Science and Technology of the People’s Republic of China. * • No custom computer codes are reported in this paper. The code for data analysis is available upon request. * • Any additional information required to reanalyze the data reported in this paper is available from the [156]lead contact upon request. Acknowledgments