Cabozantinib (s)-Malate

In the U.S., FDA approval establishes Opdivo in combination with doxorubicin, vinblastine and dacarbazine (AVD) as the first immunotherapy combination approved for adult and pediatric patients 12 years and older with previously untreated, Stage III or IV classical Hodgkin Lymphoma (cHL) With approval in the EU, Opdivo in combination with brentuximab vedotin is now the first immunotherapy combination approved to treat certain pediatric and adult patients with relapsed or refractory cHL PRINCETON, N.J.--(BUSINESS WIRE)-- Bristol Myers Squibb (NYSE: BMY) today announced that Opdivo® (nivolumab) has received approval for two new classical Hodgkin Lymphoma (cHL) indications in the U.S. and the European Union (EU). The U.S. Food and Drug Administration (FDA) granted approval of Opdivo in combination with doxorubicin, vinblastine and dacarbazine (AVD) for the treatment of adult and pediatric patients 12 years and older with previously untreated, Stage III or IV cHL.1 In the EU, the European Commission (EC) approved Opdivo in combination with brentuximab vedotin for the treatment of children 5 years of age and older, adolescents, and adults up to 30 years of age with relapsed or refractory cHL after one prior line of therapy.2 “These approvals represent a defining moment for people living with classical Hodgkin Lymphoma,” said Monica Shaw, MD, Senior Vice President of Oncology Commercialization. “In the U.S., we are particularly proud that Opdivo in combination with AVD now stands as an immunotherapy combination available for adults and pediatric patients, ages 12 and older, with previously untreated advanced disease.1 Concurrently, in the EU, Opdivo in combination with brentuximab vedotin has also achieved a milestone as the first immunotherapy combination for certain relapsed or refractory patients.2 These milestones reflect our continued commitment to advancing science that meaningfully improves the lives of patients and families worldwide.” The U.S. approval is based on the Phase 3 SWOG 1826 (CA2098UT) study, evaluating Opdivo in combination with AVD for adult and pediatric (12 years and older) patients with previously untreated Stage III or IV cHL.3 A submission based on SWOG 1826 study is also currently under evaluation by the European Medicines Agency (EMA). Opdivo and Yervoy are associated with the following Warnings and Precautions: severe and fatal immune-mediated adverse reactions including pneumonitis, colitis, hepatitis and hepatotoxicity, endocrinopathies, nephritis with renal dysfunction, dermatologic adverse reactions, other immune-mediated adverse reactions; infusion-related reactions; complications of allogeneic hematopoietic stem cell transplantation (HSCT); embryo-fetal toxicity; and increased mortality in patients with multiple myeloma when Opdivo is added to a thalidomide analogue and dexamethasone, which is not recommended outside of controlled clinical trials. Please see the Important Safety Information section below. The EU approval is based on the Phase 2 CheckMate -744 (CA209744) study, evaluating Opdivo in combination with brentuximab vedotin for the treatment of children 5 years of age and older, adolescents and adults up to 30 years of age with relapsed or refractory cHL after one prior line of therapy.4 “For decades, treatment approaches in classical Hodgkin Lymphoma have presented significant challenges, both for newly diagnosed patients and those facing relapse,”5,6 said Alex Herrera, M.D., Chief of Division of Lymphoma, Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center. “In the U.S., the nivolumab-based combination for patients with previously untreated Stage III or IV cHL demonstrated improved progression-free survival compared with standard of care, BV-AVD. The SWOG 1826 study provides data for frontline use of this immunotherapy-based regimen.”5 “The availability of another treatment option for people living with certain types of Hodgkin lymphoma can make a real difference,” says Gwen Nichols, M.D., Chief Medical Officer of Blood Cancer United. “Each new FDA-approved therapy brings renewed hope for patients and their families, and advances like this one signal meaningful progress in improving outcomes for people facing this disease.”5 SWOG 1826 (Study CA209-8UT) demonstrated a 58% reduction in the risk of disease progression or death as determined per investigator (Hazard Ratio [HR] 0.42; 95% Confidence Interval [CI] 0.27–0.67; P=<0.0001). The trial demonstrated a statistically significant improvement in the primary endpoint of progression-free survival (PFS) for patients who received Opdivo in combination with AVD, which reflect a median follow-up of 13.7 months in the intention to treat population. After a median follow-up of 36.7 months, the median overall survival (OS) had not been reached in either treatment arm with a total of 26 deaths: 9 (1.8%) deaths in the Opdivo in combination with AVD arm and 17 (3.4%) deaths in the BV plus AVD arm.7 Select Safety Profile from SWOG 1826 (CA2098UT) Serious adverse reactions occurred in 39% of patients receiving Opdivo in combination with doxorubicin, vinblastine, and dacarbazine (AVD) (n=490). The most frequent serious adverse reactions reported in ≥5% patients who received Opdivo in combination with AVD were peripheral neuropathy (41%), neutropenia (7%), pyrexia (7%), febrile neutropenia (6%), and nausea (6%). Fatal adverse reactions occurred in 3 patients (0.6%), all from sepsis. The most common adverse reactions were nausea (70%), neutropenia (61%), fatigue (59%), anemia (51%), constipation (49%), leukopenia (44%), musculoskeletal pain (42%), transaminases increase (41%), vomiting (33%), and stomatitis (30%). About SWOG 1826 (CA2098UT) SWOG 1826, also known as CA2098UT, is a randomized, multicenter, Phase 3 study evaluating Opdivo® (nivolumab) in combination with doxorubicin, vinblastine and dacarbazine (AVD) for adult and pediatric (12 years and older) patients with previously untreated Stage III or IV classical Hodgkin Lymphoma (cHL).3 The study is designed to assess progression-free survival as the primary endpoint, with key secondary endpoints that include overall survival and other measures of efficacy and safety.3 The SWOG 1826 study is sponsored by the National Cancer Institute (NCI), part of the National Institutes of Health (NIH) under a Cooperative Research and Development Agreement with Bristol Myers Squibb and conducted in the NCI National Clinical Trials Network (NCTN) led by the SWOG Cancer Research Network in collaboration with the Children’s Oncology Group (COG).3 It is the largest cHL study conducted in the NCTN.3 Bristol Myers Squibb co-sponsored the study and supplied Opdivo to the NCI through a Cooperative Research and Development agreement.3 About CheckMate -744 (CA209744) CheckMate -744, also known as CA209744, is a risk-based, response-adapted, open-label, Phase 2 study investigating Opdivo® (nivolumab) in combination with brentuximab vedotin for children, adolescents, and young adults (between 5 and 30 years old) with CD30+ classical Hodgkin lymphoma (cHL) that has relapsed or is refractory after first-line treatment.4 The study aimed to determine the safety and efficacy of nivolumab plus brentuximab vedotin, with a subsequent treatment arm of brentuximab vedotin plus bendamustine for patients with a suboptimal response.4 The trial evaluated the overall effectiveness and tolerability of these regimens in this younger, relapsed/refractory patient population.4 Data from the Phase 2 CheckMate -744 study were presented at the American Society of Clinical Oncology (ASCO) Annual Meeting in 2023 and demonstrated that Opdivo in combination with brentuximab vedotin achieved high complete metabolic response rates in children, adolescents, and young adults with relapsed or refractory cHL after one prior line of therapy.6 The response-adapted regimen enabled the majority of patients to proceed to consolidation while maintaining a manageable safety profile.6 Responses were durable at follow-up, supporting the potential of Opdivo-based, chemotherapy-sparing approaches in this population.6 About Classical Hodgkin Lymphoma Hodgkin lymphoma (HL), also known as Hodgkin disease, is a cancer that starts in white blood cells called lymphocytes, which are part of the body’s immune system.8 HL is the most common cancer diagnosed in adolescents (ages 15-19).9 It is most often diagnosed in early adulthood (ages 20-39) and late adulthood (older than 55 years of age).10 Classical Hodgkin lymphoma is the most common type of HL, accounting for 95% of cases.11 Despite progress in frontline therapy, advanced-stage HL still carries a substantial risk of relapse, highlighting the need for innovative approaches.10 About Opdivo Opdivo is a programmed death-1 (PD-1) immune checkpoint inhibitor that is designed to uniquely harness the body’s own immune system to help restore anti-tumor immune response. By harnessing the body’s own immune system to fight cancer, Opdivo has become an important treatment option across multiple cancers. Opdivo’s leading global development program is based on Bristol Myers Squibb’s scientific expertise in the field of Immuno-Oncology and includes a broad range of clinical trials across all phases, including Phase 3, in a variety of tumor types. To date, the Opdivo clinical development program has treated more than 35,000 patients. The Opdivo trials have contributed to gaining a deeper understanding of the potential role of biomarkers in patient care, particularly regarding how patients may benefit from Opdivo across the continuum of PD-L1 expression. In July 2014, Opdivo was the first PD-1 immune checkpoint inhibitor to receive regulatory approval anywhere in the world. Opdivo is currently approved in more than 65 countries, including the United States, the European Union, Japan and China. In October 2015, the Company’s Opdivo and Yervoy combination regimen was the first Immuno-Oncology combination to receive regulatory approval for the treatment of metastatic melanoma and is currently approved in more than 50 countries, including the United States and the European Union. INDICATIONS OPDIVO® (nivolumab), as a single agent, is indicated for the treatment of adult and pediatric patients 12 years and older with unresectable or metastatic melanoma. OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the treatment of adult and pediatric patients 12 years and older with unresectable or metastatic melanoma. OPDIVO® is indicated for the adjuvant treatment of adult and pediatric patients 12 years and older with completely resected Stage IIB, Stage IIC, Stage III, or Stage IV melanoma. OPDIVO® (nivolumab), in combination with platinum-doublet chemotherapy, is indicated as neoadjuvant treatment of adult patients with resectable (tumors ≥4 cm or node positive) non-small cell lung cancer (NSCLC). OPDIVO® (nivolumab) in combination with platinum-doublet chemotherapy, is indicated for neoadjuvant treatment of adult patients with resectable (tumors ≥4 cm or node positive) non-small cell lung cancer (NSCLC) and no known epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) rearrangements, followed by single-agent OPDIVO® as adjuvant treatment after surgery. OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the first-line treatment of adult patients with metastatic non-small cell lung cancer (NSCLC) whose tumors express PD-L1 (≥1%) as determined by an FDA-authorized test, with no EGFR or ALK genomic tumor aberrations. OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab) and 2 cycles of platinum-doublet chemotherapy, is indicated for the first-line treatment of adult patients with metastatic or recurrent non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations. OPDIVO® (nivolumab) is indicated for the treatment of adult patients with metastatic non-small cell lung cancer (NSCLC) with progression on or after platinum-based chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving OPDIVO. OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the first-line treatment of adult patients with unresectable malignant pleural mesothelioma (MPM). OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the first-line treatment of adult patients with intermediate or poor risk advanced renal cell carcinoma (RCC). OPDIVO® (nivolumab), in combination with cabozantinib, is indicated for the first-line treatment of adult patients with advanced renal cell carcinoma (RCC). OPDIVO® (nivolumab) is indicated for the treatment of adult patients with advanced renal cell carcinoma (RCC) who have received prior anti-angiogenic therapy. OPDIVO® (nivolumab), as a single agent, is indicated for the treatment of adult patients with classical Hodgkin lymphoma (cHL) that has relapsed or progressed after autologous hematopoietic stem cell transplantation (HSCT) and brentuximab vedotin or after 3 or more lines of systemic therapy that includes autologous HSCT. OPDIVO® (nivolumab), in combination with doxorubicin, vinblastine, and dacarbazine (AVD), is indicated for the treatment of adult and pediatric patients 12 years and older with previously untreated, Stage III or IV classical Hodgkin lymphoma (cHL). OPDIVO® (nivolumab) is indicated for the treatment of adult patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) with disease progression on or after platinum-based therapy. OPDIVO® (nivolumab), as a single agent, is indicated for the adjuvant treatment of adult patients with urothelial carcinoma (UC) who are at high risk of recurrence after undergoing radical resection of UC. OPDIVO® (nivolumab), in combination with cisplatin and gemcitabine, is indicated for the first-line treatment for adult patients with unresectable or metastatic urothelial carcinoma. OPDIVO® (nivolumab) is indicated for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum-containing chemotherapy or have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy. OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the treatment of adult and pediatric patients 12 years and older with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) unresectable or metastatic colorectal cancer (CRC). OPDIVO® (nivolumab), as a single agent, is indicated for the treatment of adult and pediatric (12 years and older) patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer (CRC) that has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the first-line treatment of adult patients with unresectable or metastatic hepatocellular carcinoma (HCC). OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the treatment of adult patients with unresectable or metastatic hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. OPDIVO® (nivolumab) is indicated for the adjuvant treatment of completely resected esophageal or gastroesophageal junction cancer with residual pathologic disease in adult patients who have received neoadjuvant chemoradiotherapy (CRT). OPDIVO® (nivolumab), in combination with fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of adult patients with unresectable advanced or metastatic esophageal squamous cell carcinoma (ESCC) whose tumors express PD-L1 (≥1). OPDIVO® (nivolumab), in combination with YERVOY® (ipilimumab), is indicated for the first-line treatment of adult patients with unresectable advanced or metastatic esophageal squamous cell carcinoma (ESCC) whose tumors express PD-L1 (≥1). OPDIVO® (nivolumab) is indicated for the treatment of adult patients with unresectable advanced, recurrent or metastatic esophageal squamous cell carcinoma (ESCC) after prior fluoropyrimidine- and platinum-based chemotherapy. OPDIVO® (nivolumab), in combination with fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the treatment of adult patients with advanced or metastatic gastric cancer, gastroesophageal junction cancer, and esophageal adenocarcinoma whose tumors express PD-L1 (≥1). IMPORTANT SAFETY INFORMATION Severe and Fatal Immune-Mediated Adverse Reactions Immune-mediated adverse reactions listed herein may not include all possible severe and fatal immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue. While immune-mediated adverse reactions usually manifest during treatment, they can also occur after discontinuation of OPDIVO or YERVOY. Early identification and management are essential to ensure safe use of OPDIVO and YERVOY. Monitor for signs and symptoms that may be clinical manifestations of underlying immune-mediated adverse reactions. Evaluate clinical chemistries including liver enzymes, creatinine, adrenocorticotropic hormone (ACTH) level, and thyroid function at baseline and periodically during treatment with OPDIVO and before each dose of YERVOY. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate. Withhold or permanently discontinue OPDIVO and YERVOY depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information). In general, if OPDIVO or YERVOY interruption or discontinuation is required, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose immune-mediated adverse reactions are not controlled with corticosteroid therapy. Toxicity management guidelines for adverse reactions that do not necessarily require systemic steroids (e.g., endocrinopathies and dermatologic reactions) are discussed below. Immune-Mediated Pneumonitis OPDIVO and YERVOY can cause immune-mediated pneumonitis. The incidence of pneumonitis is higher in patients who have received prior thoracic radiation. In patients receiving OPDIVO monotherapy, immune-mediated pneumonitis occurred in 3.1% (61/1994) of patients, including Grade 4 (<0.1%), Grade 3 (0.9%), and Grade 2 (2.1%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated pneumonitis occurred in 7% (31/456) of patients, including Grade 4 (0.2%), Grade 3 (2.0%), and Grade 2 (4.4%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated pneumonitis occurred in 3.9% (26/666) of patients, including Grade 3 (1.4%) and Grade 2 (2.6%). In NSCLC patients receiving OPDIVO 3 mg/kg every 2 weeks with YERVOY 1 mg/kg every 6 weeks, immune-mediated pneumonitis occurred in 9% (50/576) of patients, including Grade 4 (0.5%), Grade 3 (3.5%), and Grade 2 (4.0%). Four patients (0.7%) died due to pneumonitis. Immune-Mediated Colitis OPDIVO and YERVOY can cause immune-mediated colitis, which may be fatal. A common symptom included in the definition of colitis was diarrhea. Cytomegalovirus (CMV) infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. In patients receiving OPDIVO monotherapy, immune-mediated colitis occurred in 2.9% (58/1994) of patients, including Grade 3 (1.7%) and Grade 2 (1%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated colitis occurred in 25% (115/456) of patients, including Grade 4 (0.4%), Grade 3 (14%) and Grade 2 (8%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated colitis occurred in 9% (60/666) of patients, including Grade 3 (4.4%) and Grade 2 (3.7%). Immune-Mediated Hepatitis and Hepatotoxicity OPDIVO and YERVOY can cause immune-mediated hepatitis. In patients receiving OPDIVO monotherapy, immune-mediated hepatitis occurred in 1.8% (35/1994) of patients, including Grade 4 (0.2%), Grade 3 (1.3%), and Grade 2 (0.4%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated hepatitis occurred in 15% (70/456) of patients, including Grade 4 (2.4%), Grade 3 (11%), and Grade 2 (1.8%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated hepatitis occurred in 7% (48/666) of patients, including Grade 4 (1.2%), Grade 3 (4.9%), and Grade 2 (0.4%). OPDIVO in combination with cabozantinib can cause hepatic toxicity with higher frequencies of Grade 3 and 4 ALT and AST elevations compared to OPDIVO alone. Consider more frequent monitoring of liver enzymes as compared to when the drugs are administered as single agents. In patients receiving OPDIVO and cabozantinib, Grades 3 and 4 increased ALT or AST were seen in 11% (35/320) of patients. Immune-Mediated Endocrinopathies OPDIVO and YERVOY can cause primary or secondary adrenal insufficiency, immune-mediated hypophysitis, immune-mediated thyroid disorders, and Type 1 diabetes mellitus, which can present with diabetic ketoacidosis. Withhold OPDIVO and YERVOY depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information). For Grade 2 or higher adrenal insufficiency, initiate symptomatic treatment, including hormone replacement as clinically indicated. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism; initiate hormone replacement as clinically indicated. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism; initiate hormone replacement or medical management as clinically indicated. Monitor patients for hyperglycemia or other signs and symptoms of diabetes; initiate treatment with insulin as clinically indicated. In patients receiving OPDIVO monotherapy, adrenal insufficiency occurred in 1% (20/1994), including Grade 3 (0.4%) and Grade 2 (0.6%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, adrenal insufficiency occurred in 8% (35/456) of patients, including Grade 4 (0.2%), Grade 3 (2.4%), and Grade 2 (4.2%).In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, adrenal insufficiency occurred in 7% (48/666) of patients, including Grade 4 (0.3%), Grade 3 (2.5%), and Grade 2 (4.1%).In patients receiving OPDIVO and cabozantinib, adrenal insufficiency occurred in 4.7% (15/320) of patients, including Grade 3 (2.2%) and Grade 2 (1.9%). In patients receiving OPDIVO monotherapy, hypophysitis occurred in 0.6% (12/1994) of patients, including Grade 3 (0.2%) and Grade 2 (0.3%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, hypophysitis occurred in 9% (42/456) of patients, including Grade 3 (2.4%) and Grade 2 (6%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, hypophysitis occurred in 4.4% (29/666) of patients, including Grade 4 (0.3%), Grade 3 (2.4%), and Grade 2 (0.9%). In patients receiving OPDIVO monotherapy, thyroiditis occurred in 0.6% (12/1994) of patients, including Grade 2 (0.2%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, thyroiditis occurred in 2.7% (22/666) of patients, including Grade 3 (4.5%) and Grade 2 (2.2%). In patients receiving OPDIVO monotherapy, hyperthyroidism occurred in 2.7% (54/1994) of patients, including Grade 3 (<0.1%) and Grade 2 (1.2%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, hyperthyroidism occurred in 9% (42/456) of patients, including Grade 3 (0.9%) and Grade 2 (4.2%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, hyperthyroidism occurred in 12% (80/666) of patients, including Grade 3 (0.6%) and Grade 2 (4.5%). In patients receiving OPDIVO monotherapy, hypothyroidism occurred in 8% (163/1994) of patients, including Grade 3 (0.2%) and Grade 2 (4.8%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, hypothyroidism occurred in 20% (91/456) of patients, including Grade 3 (0.4%) and Grade 2 (11%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, hypothyroidism occurred in 18% (122/666) of patients, including Grade 3 (0.6%) and Grade 2 (11%). In patients receiving OPDIVO monotherapy, diabetes occurred in 0.9% (17/1994) of patients, including Grade 3 (0.4%) and Grade 2 (0.3%), and 2 cases of diabetic ketoacidosis. In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, diabetes occurred in 2.7% (15/666) of patients, including Grade 4 (0.6%), Grade 3 (0.3%), and Grade 2 (0.9%). Immune-Mediated Nephritis with Renal Dysfunction OPDIVO and YERVOY can cause immune-mediated nephritis. In patients receiving OPDIVO monotherapy, immune-mediated nephritis and renal dysfunction occurred in 1.2% (23/1994) of patients, including Grade 4 (<0.1%), Grade 3 (0.5%), and Grade 2 (0.6%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated nephritis with renal dysfunction occurred in 4.1% (27/666) of patients, including Grade 4 (0.6%), Grade 3 (1.1%), and Grade 2 (2.2%). Immune-Mediated Dermatologic Adverse Reactions OPDIVO and YERVOY can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug rash with eosinophilia and systemic symptoms (DRESS) has occurred with PD-1/PD-L1 blocking antibodies. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate non-exfoliative rashes. Withhold or permanently discontinue OPDIVO and YERVOY depending on severity (please see section 2 Dosage and Administration in the accompanying Full Prescribing Information). In patients receiving OPDIVO monotherapy, immune-mediated rash occurred in 9% (171/1994) of patients, including Grade 3 (1.1%) and Grade 2 (2.2%). In patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, immune-mediated rash occurred in 28% (127/456) of patients, including Grade 3 (4.8%) and Grade 2 (10%). In patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, immune-mediated rash occurred in 16% (108/666) of patients, including Grade 3 (3.5%) and Grade 2 (4.2%). Other Immune-Mediated Adverse Reactions The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received OPDIVO monotherapy or OPDIVO in combination with YERVOY or were reported with the use of other PD-1/PD-L1 blocking antibodies. Severe or fatal cases have been reported for some of these adverse reactions: cardiac/vascular: myocarditis, pericarditis, vasculitis; nervous system: meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barré syndrome, nerve paresis, autoimmune neuropathy; ocular: uveitis, iritis, and other ocular inflammatory toxicities can occur; gastrointestinal: pancreatitis to include increases in serum amylase and lipase levels, gastritis, duodenitis; musculoskeletal and connective tissue: myositis/polymyositis, rhabdomyolysis, and associated sequelae including renal failure, arthritis, polymyalgia rheumatica; endocrine: hypoparathyroidism; other (hematologic/immune): hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection, other transplant (including corneal graft) rejection. In addition to the immune-mediated adverse reactions listed above, across clinical trials of YERVOY monotherapy or in combination with OPDIVO, the following clinically significant immune-mediated adverse reactions, some with fatal outcome, occurred in <1% of patients unless otherwise specified: nervous system: autoimmune neuropathy (2%), myasthenic syndrome/myasthenia gravis, motor dysfunction; cardiovascular: angiopathy, temporal arteritis; ocular: blepharitis, episcleritis, orbital myositis, scleritis; gastrointestinal: pancreatitis (1.3%); other (hematologic/immune): conjunctivitis, cytopenias (2.5%), eosinophilia (2.1%), erythema multiforme, hypersensitivity vasculitis, neurosensory hypoacusis, psoriasis. Some ocular IMAR cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada–like syndrome, which has been observed in patients receiving OPDIVO and YERVOY, as this may require treatment with systemic corticosteroids to reduce the risk of permanent vision loss. Infusion-Related Reactions OPDIVO and YERVOY can cause severe infusion-related reactions. Discontinue OPDIVO and YERVOY in patients with severe (Grade 3) or life-threatening (Grade 4) infusion-related reactions. Interrupt or slow the rate of infusion in patients with mild (Grade 1) or moderate (Grade 2) infusion-related reactions. In patients receiving OPDIVO monotherapy as a 60-minute infusion, infusion-related reactions occurred in 6.4% (127/1994) of patients. In a separate trial in which patients received OPDIVO monotherapy as a 60-minute infusion or a 30-minute infusion, infusion-related reactions occurred in 2.2% (8/368) and 2.7% (10/369) of patients, respectively. Additionally, 0.5% (2/368) and 1.4% (5/369) of patients, respectively, experienced adverse reactions within 48 hours of infusion that led to dose delay, permanent discontinuation or withholding of OPDIVO. In melanoma patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, infusion-related reactions occurred in 2.5% (10/407) of patients. In HCC patients receiving OPDIVO 1 mg/kg with YERVOY 3 mg/kg every 3 weeks, infusion-related reactions occurred in 8% (4/49) of patients. In RCC patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, infusion-related reactions occurred in 5.1% (28/547) of patients. In MSI-H/dMMR mCRC patients receiving OPDIVO 3 mg/kg with YERVOY 1 mg/kg every 3 weeks, infusion-related reactions occurred in 4.2% (5/119) of patients. In MPM patients receiving OPDIVO 3 mg/kg every 2 weeks with YERVOY 1 mg/kg every 6 weeks, infusion-related reactions occurred in 12% (37/300) of patients. Complications of Allogeneic Hematopoietic Stem Cell Transplantation Fatal and other serious complications can occur in patients who receive allogeneic hematopoietic stem cell transplantation (HSCT) before or after being treated with OPDIVO or YERVOY. Transplant-related complications include hyperacute graft-versus-host-disease (GVHD), acute GVHD, chronic GVHD, hepatic veno-occlusive disease (VOD) after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between OPDIVO or YERVOY and allogeneic HSCT. Follow patients closely for evidence of transplant-related complications and intervene promptly. Consider the benefit versus risks of treatment with OPDIVO and YERVOY prior to or after an allogeneic HSCT. Embryo-Fetal Toxicity Based on its mechanism of action and findings from animal studies, OPDIVO and YERVOY can cause fetal harm when administered to a pregnant woman. The effects of YERVOY are likely to be greater during the second and third trimesters of pregnancy. Advise pregnant women of the potential risk to a fetus. Advise females of reproductive potential to use effective contraception during treatment with OPDIVO and YERVOY and for at least 5 months after the last dose. Increased Mortality in Patients with Multiple Myeloma when OPDIVO is Added to a Thalidomide Analogue and Dexamethasone In randomized clinical trials in patients with multiple myeloma, the addition of OPDIVO to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of patients with multiple myeloma with a PD-1 or PD-L1 blocking antibody in combination with a thalidomide analogue plus dexamethasone is not recommended outside of controlled clinical trials. Lactation There are no data on the presence of OPDIVO or YERVOY in human milk, the effects on the breastfed child, or the effects on milk production. Because of the potential for serious adverse reactions in breastfed children, advise women not to breastfeed during treatment and for 5 months after the last dose of OPDIVO or YERVOY. Serious Adverse Reactions In Checkmate 037, serious adverse reactions occurred in 41% of patients receiving OPDIVO (n=268). Grade 3 and 4 adverse reactions occurred in 42% of patients receiving OPDIVO. The most frequent Grade 3 and 4 adverse drug reactions reported in 2% to <5% of patients receiving OPDIVO were abdominal pain, hyponatremia, increased aspartate aminotransferase, and increased lipase. In Checkmate 066, serious adverse reactions occurred in 36% of patients receiving OPDIVO (n=206). Grade 3 and 4 adverse reactions occurred in 41% of patients receiving OPDIVO. The most frequent Grade 3 and 4 adverse reactions reported in ≥2% of patients receiving OPDIVO were gamma-glutamyltransferase increase (3.9%) and diarrhea (3.4%). In Checkmate 067, serious adverse reactions (74% and 44%), adverse reactions leading to permanent discontinuation (47% and 18%) or to dosing delays (58% and 36%), and Grade 3 or 4 adverse reactions (72% and 51%) all occurred more frequently in the OPDIVO plus YERVOY arm (n=313) relative to the OPDIVO arm (n=313). The most frequent (≥10%) serious adverse reactions in the OPDIVO plus YERVOY arm and the OPDIVO arm, respectively, were diarrhea (13% and 2.2%), colitis (10% and 1.9%), and pyrexia (10% and 1.0%). In Checkmate 238, serious adverse reactions occurred in 18% of patients receiving OPDIVO (n=452). Grade 3 or 4 adverse reactions occurred in 25% of OPDIVO-treated patients (n=452). The most frequent Grade 3 and 4 adverse reactions reported in ≥2% of OPDIVO-treated patients were diarrhea and increased lipase and amylase. In Checkmate 76K, serious adverse reactions occurred in 18% of patients receiving OPDIVO (n=524). Adverse reactions which resulted in permanent discontinuation of OPDIVO in >1% of patients included arthralgia (1.7%), rash (1.7%), and diarrhea (1.1%). A fatal adverse reaction occurred in 1 (0.2%) patient (heart failure and acute kidney injury). The most frequent Grade 3-4 lab abnormalities reported in ≥1% of OPDIVO-treated patients were increased lipase (2.9%), increased AST (2.2%), increased ALT (2.1%), lymphopenia (1.1%), and decreased potassium (1.0%). In Checkmate 816, serious adverse reactions occurred in 30% of patients (n=176) who were treated with OPDIVO in combination with platinum-doublet chemotherapy. Serious adverse reactions in >2% included pneumonia and vomiting. No fatal adverse reactions occurred in patients who received OPDIVO in combination with platinum-doublet chemotherapy. In Checkmate 77T, serious adverse reactions occurred in 21% of patients who received OPDIVO in combination with platinum-doublet chemotherapy as neoadjuvant treatment (n=228). The most frequent (≥2%) serious adverse reaction was pneumonia. Fatal adverse reactions occurred in 2.2% of patients, due to cerebrovascular accident, COVID-19 infection, hemoptysis, pneumonia, and pneumonitis (0.4% each). In the adjuvant phase of Checkmate 77T, 22% of patients experienced serious adverse reactions (n=142). The most frequent serious adverse reaction was pneumonitis/ILD (2.8%). One fatal adverse reaction due to COVID-19 occurred. In Checkmate 227, serious adverse reactions occurred in 58% of patients (n=576). The most frequent (≥2%) serious adverse reactions were pneumonia, diarrhea/colitis, pneumonitis, hepatitis, pulmonary embolism, adrenal insufficiency, and hypophysitis. Fatal adverse reactions occurred in 1.7% of patients; these included events of pneumonitis (4 patients), myocarditis, acute kidney injury, shock, hyperglycemia, multi-system organ failure, and renal failure. In Checkmate 9LA, serious adverse reactions occurred in 57% of patients (n=358). The most frequent (>2%) serious adverse reactions were pneumonia, diarrhea, febrile neutropenia, anemia, acute kidney injury, musculoskeletal pain, dyspnea, pneumonitis, and respiratory failure. Fatal adverse reactions occurred in 7 (2%) patients, and included hepatic toxicity, acute renal failure, sepsis, pneumonitis, diarrhea with hypokalemia, and massive hemoptysis in the setting of thrombocytopenia. In Checkmate 017 and 057, serious adverse reactions occurred in 46% of patients receiving OPDIVO (n=418). The most frequent serious adverse reactions reported in ≥2% of patients receiving OPDIVO were pneumonia, pulmonary embolism, dyspnea, pyrexia, pleural effusion, pneumonitis, and respiratory failure. In Checkmate 057, fatal adverse reactions occurred; these included events of infection (7 patients, including one case of Pneumocystis jirovecii pneumonia), pulmonary embolism (4 patients), and limbic encephalitis (1 patient). In Checkmate 743, serious adverse reactions occurred in 54% of patients receiving OPDIVO plus YERVOY. The most frequent serious adverse reactions reported in ≥2% of patients were pneumonia, pyrexia, diarrhea, pneumonitis, pleural effusion, dyspnea, acute kidney injury, infusion-related reaction, musculoskeletal pain, and pulmonary embolism. Fatal adverse reactions occurred in 4 (1.3%) patients and included pneumonitis, acute heart failure, sepsis, and encephalitis. In Checkmate 214, serious adverse reactions occurred in 59% of patients receiving OPDIVO plus YERVOY (n=547). The most frequent serious adverse reactions reported in ≥2% of patients were diarrhea, pyrexia, pneumonia, pneumonitis, hypophysitis, acute kidney injury, dyspnea, adrenal insufficiency, and colitis. In Checkmate 9ER, serious adverse reactions occurred in 48% of patients receiving OPDIVO and cabozantinib (n=320). The most frequent serious adverse reactions reported in ≥2% of patients were diarrhea, pneumonia, pneumonitis, pulmonary embolism, urinary tract infection, and hyponatremia. Fatal intestinal perforations occurred in 3 (0.9%) patients. In Checkmate 025, serious adverse reactions occurred in 47% of patients receiving OPDIVO (n=406). The most frequent serious adverse reactions reported in ≥2% of patients were acute kidney injury, pleural effusion, pneumonia, diarrhea, and hypercalcemia. In Checkmate 205 and 039, adverse reactions leading to discontinuation occurred in 7% and dose delays due to adverse reactions occurred in 34% of patients (n=266). Serious adverse reactions occurred in 26% of patients. The most frequent serious adverse reactions reported in ≥1% of patients were pneumonia, infusion-related reaction, pyrexia, colitis or diarrhea, pleural effusion, pneumonitis, and rash. Eleven patients died from causes other than disease progression: 3 from adverse reactions within 30 days of the last OPDIVO dose, 2 from infection 8 to 9 months after completing OPDIVO, and 6 from complications of allogeneic HSCT. In Study CA209-8UT (SWOG 1826), serious adverse reactions occurred in 39% of patients receiving OPDIVO in combination with doxorubicin, vinblastine, and dacarbazine (AVD) (n=490). The most frequent serious adverse reactions reported in ≥5% patients who received OPDIVO in combination with AVD were neutropenia (7%), pyrexia (7%), febrile neutropenia (6%), and nausea (6%). Fatal adverse reactions occurred in 3 patients (0.6%), all from sepsis In Checkmate 141, serious adverse reactions occurred in 49% of patients receiving OPDIVO (n=236). The most frequent serious adverse reactions reported in ≥2% of patients receiving OPDIVO were pneumonia, dyspnea, respiratory failure, respiratory tract infection, and sepsis. In Checkmate 275, serious adverse reactions occurred in 54% of patients receiving OPDIVO (n=270). The most frequent serious adverse reactions reported in ≥2% of patients receiving OPDIVO were urinary tract infection, sepsis, diarrhea, small intestine obstruction, and general physical health deterioration. In Checkmate 274, serious adverse reactions occurred in 30% of patients receiving OPDIVO (n=351). The most frequent serious adverse reaction reported in ≥2% of patients receiving OPDIVO was urinary tract infection. Fatal adverse reactions occurred in 1% of patients; these included events of pneumonitis (0.6%). In Checkmate 8HW, serious adverse reactions occurred in 46% of patients receiving OPDIVO in combination with YERVOY. The most frequent serious adverse reactions reported in ≥1% of patients who received OPDIVO with YERVOY were adrenal insufficiency (2.8%), hypophysitis (2.8%), diarrhea (2.0%), abdominal pain (2.0%), small intestinal obstruction (2.0%), pneumonia (1.7%), acute kidney injury (1.4%), immune mediated enterocolitis (1.4%), pneumonitis (1.4%), colitis (1.1%), large intestinal obstruction (1.1%), and urinary tract infection (1.1%). Fatal adverse reactions occurred in 2 (0.6%) patients who received OPDIVO in combination with YERVOY; these included myocarditis and pneumonitis (1 each). In Checkmate 8HW, serious adverse reactions occurred in 39% of patients receiving OPDIVO alone. The most frequent serious adverse reactions reported in >1% of patients who received OPDIVO as a single agent were intestinal obstruction (2.3%), acute kidney injury (1.7%), COVID-19 (1.7%), abdominal pain (1.4%), diarrhea (1.4%), ileus (1.4%), subileus (1.4%), pulmonary embolism (1.4%), adrenal insufficiency (1.1%) and pneumonia (1.1%). Fatal adverse reactions occurring in 3 (0.9%) patients who received OPDIVO as a single agent; these included pneumonitis (n=2) and myasthenia gravis. In Checkmate 901, serious adverse reactions occurred in 48% of patients receiving OPDIVO in combination with chemotherapy. The most frequent serious adverse reactions reporting in ≥2% of patients who received OPDIVO with chemotherapy were urinary tract infection (4.9%), acute kidney injury (4.3%), anemia (3%), pulmonary embolism (2.6%), sepsis (2.3%), and platelet count decreased (2.3%). Fatal adverse reactions occurred in 3.6% of patients who received OPDIVO in combination with chemotherapy; this included sepsis (1%). OPDIVO and/or chemotherapy were discontinued in 30% of patients and were delayed in 67% of patients for an adverse reaction. In Checkmate 9DW, serious adverse reactions occurred in 53% of patients receiving OPDIVO with YERVOY (n=332). The most frequent serious adverse reactions reported in ≥2% of patients who received OPDIVO with YERVOY were diarrhea/colitis (4.5%), immune-mediated hepatitis (3%), gastrointestinal hemorrhage (2.4%), and hepatic failure (2.4%). Fatal adverse reactions occurred in 12 (3.6%) patients who received OPDIVO with YERVOY; these included 4 (1.2%) subjects who died due to immune-mediated or autoimmune hepatitis. In Checkmate 040, serious adverse reactions occurred in 59% of patients receiving OPDIVO with YERVOY (n=49). Serious adverse reactions reported in ≥4% of patients were pyrexia, diarrhea, anemia, increased AST, adrenal insufficiency, ascites, esophageal varices hemorrhage, hyponatremia, increased blood bilirubin, and pneumonitis. In Attraction-3, serious adverse reactions occurred in 38% of patients receiving OPDIVO (n=209). Serious adverse reactions reported in ≥2% of patients who received OPDIVO were pneumonia, esophageal fistula, interstitial lung disease, and pyrexia. The following fatal adverse reactions occurred in patients who received OPDIVO: interstitial lung disease or pneumonitis (1.4%), pneumonia (1.0%), septic shock (0.5%), esophageal fistula (0.5%), gastrointestinal hemorrhage (0.5%), pulmonary embolism (0.5%), and sudden death (0.5%). In Checkmate 577, serious adverse reactions occurred in 33% of patients receiving OPDIVO (n=532). A serious adverse reaction reported in ≥2% of patients who received OPDIVO was pneumonitis. A fatal reaction of myocardial infarction occurred in one patient who received OPDIVO. In Checkmate 648, serious adverse reactions occurred in 62% of patients receiving OPDIVO in combination with chemotherapy (n=310). The most frequent serious adverse reactions reported in ≥2% of patients who received OPDIVO with chemotherapy were pneumonia (11%), dysphagia (7%), esophageal stenosis (2.9%), acute kidney injury (2.9%), and pyrexia (2.3%). Fatal adverse reactions occurred in 5 (1.6%) patients who received OPDIVO in combination with chemotherapy; these included pneumonitis, pneumatosis intestinalis, pneumonia, and acute kidney injury. In Checkmate 648, serious adverse reactions occurred in 69% of patients receiving OPDIVO in combination with YERVOY (n=322). The most frequent serious adverse reactions reported in ≥2% who received OPDIVO in combination with YERVOY were pneumonia (10%), pyrexia (4.3%), pneumonitis (4.0%), aspiration pneumonia (3.7%), dysphagia (3.7%), hepatic function abnormal (2.8%), decreased appetite (2.8%), adrenal insufficiency (2.5%), and dehydration (2.5%). Fatal adverse reactions occurred in 5 (1.6%) patients who received OPDIVO in combination with YERVOY; these included pneumonitis, interstitial lung disease, pulmonary embolism, and acute respiratory distress syndrome. In Checkmate 649, serious adverse reactions occurred in 52% of patients treated with OPDIVO in combination with chemotherapy (n=782). The most frequent serious adverse reactions reported in ≥2% of patients treated with OPDIVO in combination with chemotherapy were vomiting (3.7%), pneumonia (3.6%), anemia (3.6%), pyrexia (2.8%), diarrhea (2.7%), febrile neutropenia (2.6%), and pneumonitis (2.4%). Fatal adverse reactions occurred in 16 (2.0%) patients who were treated with OPDIVO in combination with chemotherapy; these included pneumonitis (4 patients), febrile neutropenia (2 patients), stroke (2 patients), gastrointestinal toxicity, intestinal mucositis, septic shock, pneumonia, infection, gastrointestinal bleeding, mesenteric vessel thrombosis, and disseminated intravascular coagulation. Common Adverse Reactions In Checkmate 037, the most common adverse reaction (≥20%) reported with OPDIVO (n=268) was rash (21%). In Checkmate 066, the most common adverse reactions (≥20%) reported with OPDIVO (n=206) vs dacarbazine (n=205) were fatigue (49% vs 39%), musculoskeletal pain (32% vs 25%), rash (28% vs 12%), and pruritus (23% vs 12%). In Checkmate 067, the most common (≥20%) adverse reactions in the OPDIVO plus YERVOY arm (n=313) were fatigue (62%), diarrhea (54%), rash (53%), nausea (44%), pyrexia (40%), pruritus (39%), musculoskeletal pain (32%), vomiting (31%), decreased appetite (29%), cough (27%), headache (26%), dyspnea (24%), upper respiratory tract infection (23%), arthralgia (21%), and increased transaminases (25%). In Checkmate 067, the most common (≥20%) adverse reactions in the OPDIVO arm (n=313) were fatigue (59%), rash (40%), musculoskeletal pain (42%), diarrhea (36%), nausea (30%), cough (28%), pruritus (27%), upper respiratory tract infection (22%), decreased appetite (22%), headache (22%), constipation (21%), arthralgia (21%), and vomiting (20%). In Checkmate 238, the most common adverse reactions (≥20%) reported in OPDIVO-treated patients (n=452) vs ipilimumab-treated patients (n=453) were fatigue (57% vs 55%), diarrhea (37% vs 55%), rash (35% vs 47%), musculoskeletal pain (32% vs 27%), pruritus (28% vs 37%), headache (23% vs 31%), nausea (23% vs 28%), upper respiratory infection (22% vs 15%), and abdominal pain (21% vs 23%). The most common immune-mediated adverse reactions were rash (16%), diarrhea/colitis (6%), and hepatitis (3%). In Checkmate 76K, the most common adverse reactions (≥20%) reported with OPDIVO (n=524) were fatigue (36%), musculoskeletal pain (30%), rash (28%), diarrhea (23%) and pruritis (20%). In Checkmate 816, the most common (>20%) adverse reactions in the OPDIVO plus chemotherapy arm (n=176) were nausea (38%), constipation (34%), fatigue (26%), decreased appetite (20%), and rash (20%). In Checkmate 77T, the most common adverse reactions (reported in ≥20%) in patients receiving OPDIVO in combination with chemotherapy (n= 228) were anemia (39.5%), constipation (32.0%), nausea (28.9%), fatigue (28.1%), alopecia (25.9%), and cough (21.9%). In Checkmate 227, the most common (≥20%) adverse reactions were fatigue (44%), rash (34%), decreased appetite (31%), musculoskeletal pain (27%), diarrhea/colitis (26%), dyspnea (26%), cough (23%), hepatitis (21%), nausea (21%), and pruritus (21%). In Checkmate 9LA, the most common (>20%) adverse reactions were fatigue (49%), musculoskeletal pain (39%), nausea (32%), diarrhea (31%), rash (30%), decreased appetite (28%), constipation (21%), and pruritus (21%). In Checkmate 017 and 057, the most common adverse reactions (≥20%) in patients receiving OPDIVO (n=418) were fatigue, musculoskeletal pain, cough, dyspnea, and decreased appetite. In Checkmate 743, the most common adverse reactions (≥20%) in patients receiving OPDIVO plus YERVOY were fatigue (43%), musculoskeletal pain (38%), rash (34%), diarrhea (32%), dyspnea (27%), nausea (24%), decreased appetite (24%), cough (23%), and pruritus (21%). In Checkmate 214, the most common adverse reactions (≥20%) reported in patients treated with OPDIVO plus YERVOY (n=547) were fatigue (58%), rash (39%), diarrhea (38%), musculoskeletal pain (37%), pruritus (33%), nausea (30%), cough (28%), pyrexia (25%), arthralgia (23%), decreased appetite (21%), dyspnea (20%), and vomiting (20%). In Checkmate 9ER, the most common adverse reactions (≥20%) in patients receiving OPDIVO and cabozantinib (n=320) were diarrhea (64%), fatigue (51%), hepatotoxicity (44%), palmar-plantar erythrodysaesthesia syndrome (40%), stomatitis (37%), rash (36%), hypertension (36%), hypothyroidism (34%), musculoskeletal pain (33%), decreased appetite (28%), nausea (27%), dysgeusia (24%), abdominal pain (22%), cough (20%) and upper respiratory tract infection (20%). In Checkmate 025, the most common adverse reactions (≥20%) reported in patients receiving OPDIVO (n=406) vs everolimus (n=397) were fatigue (56% vs 57%), cough (34% vs 38%), nausea (28% vs 29%), rash (28% vs 36%), dyspnea (27% vs 31%), diarrhea (25% vs 32%), constipation (23% vs 18%), decreased appetite (23% vs 30%), back pain (21% vs 16%), and arthralgia (20% vs 14%). In Checkmate 205 and 039, the most common adverse reactions (≥20%) reported in patients receiving OPDIVO (n=266) were upper respiratory tract infection (44%), fatigue (39%), cough (36%), diarrhea (33%), pyrexia (29%), musculoskeletal pain (26%), rash (24%), nausea (20%) and pruritus (20%). In Study CA209-8UT (SWOG 1826), the most common adverse reactions (≥30%) in the OPDIVO plus AVD arm (n=490) were nausea (70%), neutropenia (61%), fatigue (59%), anemia (51%), constipation (49%), leukopenia (44%), musculoskeletal pain (42%), peripheral neuropathy (41%), transaminases increase (41%), vomiting (33%), and stomatitis (30%). In Checkmate 141, the most common adverse reactions (≥10%) in patients receiving OPDIVO (n=236) were cough (14%) and dyspnea (14%) at a higher incidence than investigator’s choice. In Checkmate 275, the most common adverse reactions (≥20%) reported in patients receiving OPDIVO (n=270) were fatigue (46%), musculoskeletal pain (30%), nausea (22%), and decreased appetite (22%). In Checkmate 274, the most common adverse reactions (≥20%) reported in patients receiving OPDIVO (n=351) were rash (36%), fatigue (36%), diarrhea (30%), pruritus (30%), musculoskeletal pain (28%), and urinary tract infection (22%). In Checkmate 8HW, the most common adverse reactions reported in ≥20% of patients treated with OPDIVO in combination with YERVOY were fatigue, diarrhea, pruritus, abdominal pain, musculoskeletal pain, and nausea. In Checkmate 8HW, the most common adverse reactions reported in ≥20% of patients treated with OPDIVO as a single agent, were fatigue, diarrhea, abdominal pain, pruritus, and musculoskeletal pain. In Checkmate 901, the most common adverse reactions (≥20%) were nausea, fatigue, musculoskeletal pain, constipation, decreased appetite, rash, vomiting, and peripheral neuropathy. In Checkmate 9DW, the most common adverse reactions (≥20%) in patients receiving OPDIVO with YERVOY (n=332) were rash (36%), pruritus (34%), fatigue (33%), and diarrhea (22%). In Checkmate 040, the most common adverse reactions (≥20%) in patients receiving OPDIVO with YERVOY (n=49), were rash (53%), pruritus (53%), musculoskeletal pain (41%), diarrhea (39%), cough (37%), decreased appetite (35%), fatigue (27%), pyrexia (27%), abdominal pain (22%), headache (22%), nausea (20%), dizziness (20%), hypothyroidism (20%), and weight decreased (20%). In Attraction-3, the most common adverse reactions (≥20%) in OPDIVO-treated patients (n=209) were rash (22%) and decreased appetite (21%). In Checkmate 577, the most common adverse reactions (≥20%) in patients receiving OPDIVO (n=532) were fatigue (34%), diarrhea (29%), nausea (23%), rash (21%), musculoskeletal pain (21%), and cough (20%). In Checkmate 648, the most common adverse reactions (≥20%) in patients treated with OPDIVO in combination with chemotherapy (n=310) were nausea (65%), decreased appetite (51%), fatigue (47%), constipation (44%), stomatitis (44%), diarrhea (29%), and vomiting (23%). In Checkmate 648, the most common adverse reactions reported in ≥20% of patients treated with OPDIVO in combination with YERVOY (n=322) were rash (31%), fatigue (28%), pyrexia (23%), nausea (22%), diarrhea (22%), and constipation (20%). In Checkmate 649, the most common adverse reactions (≥20%) in patients treated with OPDIVO in combination with chemotherapy (n=782) were peripheral neuropathy (53%), nausea (48%), fatigue (44%), diarrhea (39%), vomiting (31%), decreased appetite (29%), abdominal pain (27%), constipation (25%), and musculoskeletal pain (20%). Surgery Related Adverse Reactions In Checkmate 77T, 5.3% (n=12) of the OPDIVO-treated patients who received neoadjuvant treatment, did not receive surgery due to adverse reactions. The adverse reactions that led to cancellation of surgery in OPDIVO-treated patients were cerebrovascular accident, pneumonia, and colitis/diarrhea (2 patients each) and acute coronary syndrome, myocarditis, hemoptysis, pneumonitis, COVID-19, and myositis (1 patient each). Please see US Full Prescribing Information for OPDIVO and YERVOY. Clinical Trials and Patient Populations Checkmate 037–previously treated metastatic melanoma; Checkmate 066—previously untreated metastatic melanoma; Checkmate 067–previously untreated metastatic melanoma, as a single agent or in combination with YERVOY; Checkmate 238–adjuvant treatment of patients with completely resected Stage III or Stage IV melanoma; Checkmate 76K–adjuvant treatment of patients 12 years of age and older with completely resected Stage IIB or Stage IIC melanoma; Checkmate 816–neoadjuvant non-small cell lung cancer, in combination with platinum-doublet chemotherapy; Checkmate 77T–Neoadjuvant treatment with platinum-doublet chemotherapy for non-small cell lung cancer followed by single-agent OPDIVO as adjuvant treatment after surgery; Checkmate 227—previously untreated metastatic non-small cell lung cancer, in combination with YERVOY; Checkmate 9LA–previously untreated recurrent or metastatic non-small cell lung cancer in combination with YERVOY and 2 cycles of platinum-doublet chemotherapy by histology; Checkmate 017–second-line treatment of metastatic squamous non-small cell lung cancer; Checkmate 057–second-line treatment of metastatic non-squamous non-small cell lung cancer; Checkmate 743–previously untreated unresectable malignant pleural mesothelioma, in combination with YERVOY; Checkmate 214–previously untreated renal cell carcinoma, in combination with YERVOY; Checkmate 9ER–previously untreated renal cell carcinoma, in combination with cabozantinib; Checkmate 025–previously treated renal cell carcinoma; Checkmate 205/039–relapsed or refractory Classical Hodgkin Lymphoma; Study CA209-8UT (SWOG 1826)—previously untreated Classical Hodgkin Lymphoma; Checkmate 141–recurrent or metastatic squamous cell carcinoma of the head and neck; Checkmate 275–previously treated advanced or metastatic urothelial carcinoma; Checkmate 274–adjuvant treatment of urothelial carcinoma; Checkmate 8HW–MSI-H or dMMR metastatic colorectal cancer in combination with YERVOY; Checkmate 8HW–MSI-H or dMMR metastatic colorectal cancer, as a single agent; Checkmate 901–Adult patients with unresectable or metastatic urothelial carcinoma; Checkmate 9DW–hepatocellular carcinoma, in combination with YERVOY; Checkmate 040–hepatocellular carcinoma, in combination with YERVOY, after prior treatment with sorafenib; Attraction-3–esophageal squamous cell carcinoma; Checkmate 577–adjuvant treatment of esophageal or gastroesophageal junction cancer; Checkmate 648—previously untreated, unresectable advanced recurrent or metastatic esophageal squamous cell carcinoma in combination with chemotherapy; Checkmate 648—previously untreated, unresectable advanced recurrent or metastatic esophageal squamous cell carcinoma combination with YERVOY; Checkmate 649–previously untreated advanced or metastatic gastric cancer, gastroesophageal junction and esophageal adenocarcinoma Bristol Myers Squibb: Creating a Better Future for People with Cancer Bristol Myers Squibb is inspired by a single vision — transforming patients’ lives through science. The goal of the company’s cancer research is to deliver medicines that offer each patient a better, healthier life and to make cure a possibility. Building on a legacy across a broad range of cancers that have changed survival expectations for many, Bristol Myers Squibb researchers are exploring new frontiers in personalized medicine and, through innovative digital platforms, are turning data into insights that sharpen their focus. Deep understanding of causal human biology, cutting-edge capabilities and differentiated research programs uniquely position the company to approach cancer from every angle. Cancer can have a relentless grasp on many parts of a patient’s life, and Bristol Myers Squibb is committed to taking actions to address all aspects of care, from diagnosis to survivorship. As a leader in cancer care, Bristol Myers Squibb is working to empower all people with cancer to have a better future. About Bristol Myers Squibb’s Patient Access Support Bristol Myers Squibb remains committed to providing assistance so that cancer patients who need our medicines can access them and expedite time to therapy. BMS Access Support®, the Bristol Myers Squibb patient access and reimbursement program, is designed to help appropriate patients initiate and maintain access to BMS medicines during their treatment journey. BMS Access Support offers benefit investigation, prior authorization assistance, as well as co-pay assistance for eligible, commercially insured patients. More information about our access and reimbursement support can be obtained by calling BMS Access Support at 1-800-861-0048 or by visiting www.bmsaccesssupport.com. About the Bristol Myers Squibb and Ono Pharmaceutical Collaboration In 2011, through a collaboration agreement with Ono Pharmaceutical Co., Bristol Myers Squibb expanded its territorial rights to develop and commercialize Opdivo globally, except in Japan, South Korea and Taiwan, where Ono had retained all rights to the compound at the time. On July 23, 2014, Ono and Bristol Myers Squibb further expanded the companies’ strategic collaboration agreement to jointly develop and commercialize multiple immunotherapies – as single agents and combination regimens – for patients with cancer in Japan, South Korea and Taiwan. About Bristol Myers Squibb Bristol Myers Squibb is a global biopharmaceutical company whose mission is to discover, develop and deliver innovative medicines that help patients prevail over serious diseases. For more information about Bristol Myers Squibb, visit us at BMS.com or follow us on LinkedIn, X, YouTube, Facebook and Instagram. Cautionary Statement Regarding Forward-Looking Statements This press release contains “forward-looking statements” within the meaning of the Private Securities Litigation Reform Act of 1995 regarding, among other things, the research, development and commercialization of pharmaceutical products. All statements that are not statements of historical facts are, or may be deemed to be, forward-looking statements. Such forward-looking statements are based on current expectations and projections about our future financial results, goals, plans and objectives and involve inherent risks, assumptions and uncertainties, including internal or external factors that could delay, divert or change any of them in the next several years, that are difficult to predict, may be beyond our control and could cause our future financial results, goals, plans and objectives to differ materially from those expressed in, or implied by, the statements. These risks, assumptions, uncertainties and other factors include, among others, whether the Opdivo-based combinations for the additional indications described in this release will be commercially successful, the outcome of pricing and reimbursement negotiations in individual countries in Europe may delay or limit the commercial potential of such combinations treatments for such additional indications, any marketing approvals, if granted, may have significant limitations on their use, and that continued approval of such combination treatments for such additional indications may be contingent upon verification and description of clinical benefit in confirmatory trials. No forward-looking statement can be guaranteed. 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点击上方蓝色字体关注我们 新时代数字养虾人，千万次动心，不及你低头浅笑的样子——益栈信 一、研究的背景与核心科学问题 研究背景 肝细胞癌（HCC）是全球致死率最高的恶性肿瘤之一，其显著的瘤内与肿瘤微环境（TME）异质性是临床治疗的核心屏障。这种异质性体现在基因组、表观遗传、转录组及微环境调控等多个生物学维度，直接导致治疗耐药、肿瘤远处转移与侵袭，并最终造成患者预后极差。尽管近年来免疫检查点抑制剂、抗血管生成联合疗法等手段为HCC治疗带来突破，但临床客观缓解率仍未达到预期，且针对不同进展阶段HCC的治疗策略缺乏精准的分子依据，其核心原因在于对HCC从发生到转移全过程中，恶性细胞与TME异质性的动态演变规律解析不足。 现有HCC单细胞转录组研究多聚焦于单一患者队列或疾病某一特定阶段，且多孤立分析恶性细胞或TME单个组分，缺乏跨正常肝组织、原发肿瘤、门脉癌栓（PVTT）、淋巴结转移（MLN）的全进展阶段整合分析；同时，对恶性细胞转录特征与TME细胞亚群的空间互作关系解析较为匮乏，无法构建“分子-空间”关联的HCC进展调控模型，难以挖掘具有广谱临床价值的治疗靶点。 核心科学问题 本研究紧扣领域内的核心科学空白，旨在明确两个层层递进的核心问题：其一，HCC从正常肝组织发生，到原发瘤进展、PVTT形成及MLN远处转移的全过程中，恶性肝细胞的转录特征如何演变？TME中髓系、内皮、成纤维细胞的功能亚群发生了怎样的动态重塑？二者的互作关系如何调控HCC的进展与转移？其二，基于HCC异质性的动态演变规律，能否挖掘出跨恶性细胞与TME、兼具癌症必需性与临床预后价值的核心治疗靶点，为克服HCC治疗耐药、实现精准治疗提供新的分子方向？ 研究意义 本研究通过整合单细胞转录组与空间转录组技术，首次构建了覆盖HCC全进展阶段的细胞生态与空间分子整合图谱，填补了领域内缺乏跨阶段、多维度整合分析的空白，系统阐明了HCC恶性细胞与TME异质性的动态演变规律及互作调控机制。同时，本研究建立了“计算机模拟扰动+癌症依赖图谱+多临床队列验证”的靶点筛选体系，将基础研究发现与临床治疗需求深度结合，锁定了具有明确转化潜力的核心治疗靶点HSP90B1，为HCC精准肿瘤学的发展提供了全新的分子与空间框架，也为其他异质性显著的恶性肿瘤研究提供了可借鉴的分析范式，推动了肿瘤基础研究向临床转化的落地。 二、整体研究设计与逻辑框架 本研究针对HCC异质性解析与临床靶点挖掘的核心需求，设计了“单细胞解构—空间验证—计算机模拟靶点筛选”的三步走整合研究策略，该策略以“从临床问题出发，回归临床应用”为核心导向，实现了从现象描述到机制解析，再到靶点挖掘的完整研究闭环。 第一步通过整合正常肝、原发HCC、PVTT、MLN的大规模单细胞转录组数据，经严格质控、批次校正与细胞注释，系统解构HCC全进展阶段的细胞动力学特征，鉴定恶性肝细胞的核心转录元程序与TME中具有预后价值的功能异质性亚群，解析细胞间的通讯调控网络，完成对HCC异质性的全景式描绘（广度）；第二步利用10x Visium空间转录组技术，结合去卷积、空间通讯分析等工具，将单细胞层面发现的细胞亚群、分子互作网络映射至肿瘤组织切片，验证并构建具有明确空间结构的促肿瘤生态位，明确分子特征的空间分布规律与调控机制，实现对HCC异质性的机制性深挖（深度）；第三步基于Geneformer开展跨恶性细胞、免疫细胞、基质细胞的计算机模拟敲除筛选，结合DepMap癌症依赖图谱鉴定癌症必需基因，再通过多个临床治疗队列与预后队列进行多维度验证，最终锁定具有临床转化价值的核心治疗靶点，完成从基础研究到临床应用的回归（靶点）。 整体研究设计形成了“现象-机制-靶点”的完整逻辑闭环，各研究环节层层递进、相互印证，所有分析均基于HCC全进展阶段的样本数据，确保研究结果能真实反映HCC的生物学特征，同时将单细胞分子特征与空间组织结构、临床预后与治疗响应紧密结合，让研究发现兼具理论深度与临床应用价值。 三、数据与方法论总览 本研究采用多类型、多来源的组学数据，覆盖单细胞转录组、空间转录组与批量转录组，同时整合了前沿的生物信息学分析工具，实现了从原始数据处理、细胞异质性解析到靶点筛选与临床验证的全流程分析，以下为数据来源与核心分析方法的系统汇总。 数据来源 本研究的数据均来自公共数据库与已发表研究，其中单细胞转录组数据为核心分析数据，空间转录组数据用于分子特征的空间验证，批量转录组数据用于临床预后与治疗响应的验证，具体信息见表1。 表1 本研究使用的组学数据来源汇总 核心分析方法 本研究使用的分析工具覆盖了原始数据处理、恶性细胞解析、转录调控分析、细胞间通讯、空间解析、靶点筛选及临床验证等全流程，按研究功能对工具进行分类，各工具的核心研究角色见表2。 表2 本研究使用的核心生物信息学工具及研究角色 四、核心研究发现 1）肝细胞癌进展过程中细胞动力学的单细胞分析 研究出发点与目标 现有HCC单细胞研究多聚焦单一疾病阶段，缺乏跨正常肝→原发瘤→门脉癌栓（PVTT）→淋巴结转移（MLN）全进展阶段的细胞全景图谱，无法明确不同阶段细胞组成的动态变化规律，也难以确定HCC进展的核心细胞特征。本部分研究的核心目标是：构建HCC全进展阶段的单细胞转录组全景图谱，通过质控与批次校正排除技术偏倚，完成细胞类型注释，并解析各细胞类型的组织分布与相关性特征，为后续深入研究奠定基础。 核心研究过程与结果 1.样本处理与质控：整合6个公共数据库的scRNA-seq原始数据（183例样本），先经病理筛选排除肝内胆管癌、混合型HCC-ICC样本，再通过严格质量过滤（去除nCount_RNA<200/>50000、检测基因<200/>5000、线粒体基因表达>15%的低质量细胞），并利用decontX去除环境RNA污染，最终保留115例纯HCC样本用于后续分析。 2.批次效应校正：采用Harmony对多来源数据进行批次效应校正，通过kBET验证校正效果，数据拒绝率从0.827降至0.328，且细胞簇分布跨患者与数据集，排除了技术偏倚与患者特异性影响。 3.细胞类型注释：基于谱系特异性标记基因，成功注释出7个主要细胞亚群，各亚群核心标记基因明确： 1.肝细胞：ALB、APOA2、KRT18（增殖型肝细胞额外表达TOP2A、MKI67）； 2.内皮细胞：VWF、PECAM1； 3.成纤维细胞：COL1A1、COL1A2、ACTA2； 4.浆细胞：MZB1、IGHG1；B细胞：CD79A、MS4A1； 5.NK/T细胞：CD3D、CD3E、KLRD1、NKG7； 6.髓系细胞：LYZ、CD68、CD163、CD14。 4.细胞组成动态分析：明确各细胞类型在HCC不同阶段的分布规律，核心特征为： 1.肝细胞比例从正常肝→原发瘤→PVTT→MLN逐步递增，为转移灶的核心细胞类型； 2.髓系细胞在PVTT与MLN中显著扩增，成为转移灶主要免疫细胞； 3.NK/T细胞、B细胞富集于正常肝组织，在肿瘤与转移灶中占比大幅降低； 4.成纤维细胞特异性富集于原发肿瘤组织，为原发瘤基质核心组分。 5.细胞相关性分析：发现两个关键相关性特征：①内皮细胞与成纤维细胞呈显著正相关，提示二者在TME中存在潜在协同作用；②肝细胞、基质细胞、髓系细胞与NK/T细胞呈显著负相关，提示基质-髓系细胞的富集可能导致淋巴细胞排斥，形成免疫抑制性TME。 图 1：HCC 进展过程中细胞动力学的单细胞分析 A研究设计示意图（由 biorender 制作）。B主要细胞群中谱系特异性标记表达的散点图。C UMAP投影图，显示 7 个主要细胞群。D细胞类型在不同组织类型（正常、肿瘤、门静脉癌栓、肠系膜淋巴结）中的分布。E各组织中细胞类型的比例组成。F各样本中细胞类型比例的相关性热图。 互动问答 问：研究中通过哪些手段排除技术偏倚，确保构建的单细胞图谱能真实反映HCC的细胞生物学特征？ 答：研究通过三重手段排除技术偏倚：①严格的质量过滤+decontX去污染，去除低质量细胞与环境RNA干扰；②Harmony进行批次效应校正，并用kBET验证校正效果，大幅降低数据拒绝率；③验证细胞簇分布跨患者与数据集，且回归RNA计数后UMAP投影无明显变化，排除了患者特异性与技术变量的影响。 问：从细胞组成的动态变化，能初步判断HCC进展与转移的核心细胞特征是什么？ 答：可初步判断两大核心特征：一是恶性肝细胞的逐步富集，成为肿瘤进展与转移的核心细胞基础；二是TME的免疫抑制重塑，表现为免疫活化的NK/T、B细胞减少，免疫抑制的髓系细胞扩增，同时基质细胞（成纤维、内皮）与髓系细胞协同形成排斥淋巴细胞的微环境。 2）cNMF分析揭示了HCC恶性细胞异质性背后的元程序 研究出发点与目标 传统细胞聚类分析仅能根据基因表达水平将恶性细胞分为不同簇，难以识别跨患者、保守的核心转录特征，也无法解释HCC恶性细胞异质性的本质来源；且前期仅完成了细胞类型的全景注释，尚未解析恶性肝细胞的核心转录规律。本部分研究的核心目标是：鉴定HCC恶性细胞中保守的转录基因表达程序（GEP），并归纳为更高层级的转录元程序（MP），明确各元程序的功能特征、组织分布规律，为后续解析其临床价值与调控机制提供分子框架。 核心研究过程与结果 1.恶性细胞鉴定与CNA分析：利用CopyKAT工具基于拷贝数变异（CNA）图谱区分恶性肝细胞与正常肝细胞，发现CNA存在显著患者间异质性，但8号染色体的扩增与局灶性缺失为跨患者的复发改变，是HCC恶性细胞的共性基因组特征。 2.GEP鉴定与验证：对鉴定出的恶性细胞采用cNMF进行基因表达程序分解，得到15个GEPs；通过患者混合熵分析验证，发现15个GEPs均跨患者共享，且分布独立于患者身份与批次效应，排除了个体特异性影响。 3.转录元程序（MP）归纳：对15个GEPs进行层次聚类，归纳出4个具有保守特征的转录元程序，且MPs的患者混合熵显著高于单个GEPs，证实其为患者独立、可复现的核心转录状态。 4.4个MPs的功能与组织分布特征：通过Metascape富集分析、GSEA分析明确各MP的功能特征，并结合组织分布分析其与疾病进展的关联，核心特征如下： 1.MP1（Diff-Metabolic，分化代谢型）：富集肝特异性代谢通路（胆汁酸、脂肪酸、异生物代谢、解毒），保留肝组织特异性功能，与良好预后相关； 2.MP2（Prolif-Stress，增殖应激型）：富集活性氧、内质网应激等应激通路，同时激活MYC、E2F靶点及G2M检查点，代表增殖状态下的细胞应激特征； 3.MP3（MYC-Biosynth-Immune，MYC合成免疫型）：以MYC通路活化、核糖体生物合成、翻译调控为核心，同时富集中性粒细胞脱颗粒等先天免疫过程，抑制适应性免疫与肝代谢通路； 4.MP4（EMT-Inflammatory，EMT炎症型）：富集细胞运动、EMT、TGFβ/NFκB/TNFα炎症通路，抑制代谢通路，代表侵袭性炎症表型。 5.MPs的组织分布规律：原发瘤中4个MPs均衡分布；PVTT中MP2与MP4显著富集，体现转移灶的增殖与侵袭特征；MLN中则以MP2为主，提示远处转移灶的恶性细胞存在高增殖型的选择性定植特征。 图 2：cNMF 分析识别 HCC 恶性细胞异质性的潜在元程序 A.15个基因表达谱(GEP)的层次聚类热图，揭示了4个元程序(MP)。B . UMAP图显示了四个元程序的表达评分：MP1（差异代谢）、MP2（增殖应激）、MP3（MYC-生物合成-免疫）和MP4（EMT-炎症）。颜色梯度表示评分强度。C .根据元程序着色的恶性细胞UMAP投影。D .元程序在不同组织类型（肿瘤、门静脉癌栓、肠系膜淋巴结）中的分布。E .每个元程序的标记基因散点图。F .验证跨元程序功能特征的标志性基因集评分。伪时间轨迹分析：G.轨迹叠加图显示了伪时间起始处的EMT-炎症以及分叉；H.元程序沿伪时间的分布。I .余弦相似性热图比较了肝细胞癌(HCC)元程序与Gavish等人提出的泛癌程序。J热图展示了 MPs 与 Hoshida 分子亚类（S1–S3）之间的相关性。K热图展示了 MPs 在 Boyault 转录组亚类（G1–G6）中的富集情况及其相关的基因特征。 互动问答 问：研究为何选择cNMF而非传统的细胞聚类方法分析恶性细胞的转录特征？ 答：传统细胞聚类仅能根据基因表达相似度将细胞分簇，易受患者特异性、技术偏倚影响，难以识别跨患者的保守规律；而cNMF可分解出基因表达程序（GEP），能捕捉恶性细胞中协同表达的基因集合，再通过层次聚类得到元程序（MP），更能反映HCC恶性细胞异质性的核心转录本质，且经患者混合熵验证，MPs具有跨患者的保守性，为后续研究提供了稳定的分子框架。 问：4个元程序的组织分布规律，反映了HCC从原发到转移的过程中，恶性细胞的转录状态发生了怎样的转变？ 答：反映了恶性细胞从“多状态均衡”向“高增殖、高侵袭”的定向转变：原发瘤中恶性细胞仍保留部分分化代谢特征（MP1），同时存在增殖、炎症、合成等多种状态；而进入脉管转移（PVTT）后，恶性细胞向增殖应激（MP2）和EMT炎症（MP4）两个促转移状态富集；到远处淋巴结转移（MLN）时，进一步筛选为高增殖型（MP2），提示高增殖能力是恶性肝细胞实现远处定植的关键特征。 3）恶性细胞的转录异质性和状态特异性治疗脆弱性 研究出发点与目标 已鉴定的4个转录元程序（MP）的转录调控网络尚未解析，无法明确其表型维持的分子机制；且MPs的临床预后价值、状态间的演化关系，以及不同MP对应的治疗响应特征与药物脆弱性均未明确，无法将分子特征与临床治疗应用挂钩。本部分研究的核心目标是：解析各MP的核心转录因子调控网络，明确MPs的演化轨迹与临床预后价值，挖掘不同MP的状态特异性治疗脆弱性，为HCC个体化治疗提供分子依据。 核心研究过程与结果 1.转录因子（TF）调控网络分析：采用pySCENIC工具分析各MP的核心TF活性，明确其转录调控特征： 1.MP1由NR1I3、SOX4、TP73等肝系谱系TF主导调控，维持其分化代谢的肝组织特征； 2.MP2高表达HOXA7、ELF3等细胞周期调控TF；MP3由HMGA1、SOX2、NEUROD1调控，强化MYC活化特征； 3.MP4由CREB5、SOX9、JUNB等间质型TF调控，介导EMT与炎症表型； 4.关键发现：TF存在跨MP共享特征（MP2/MP4共享ATF3、FOSB；MP1/MP4共享JUNB），提示共享TF可能介导恶性细胞的表型可塑性，为治疗耐药提供分子基础。 2.MPs的演化轨迹分析：采用Monocle2结合GeneSwitches进行拟时序轨迹分析，明确恶性细胞的状态转换规律：MP4（EMT-Inflammatory）为恶性细胞的起始状态，随后分叉向MP1（Diff-Metabolic）和MP2（Prolif-Stress）分化，MP3则处于中间过渡状态，同时鉴定出各阶段的时序调控基因（如干/祖细胞标记EPCAM为起始标记，肝代谢酶CYP2D6为分化后标记）。 3.临床预后价值验证：将4个MP的特征基因签名通过ssGSEA映射至TCGA-LIHC队列，进行Kaplan-Meier生存分析：MP1高评分与患者更长的总生存期显著相关，为预后良好的分子特征；而MP2、MP3、MP4高评分均与不良预后显著相关，是HCC进展的不良分子标志物。 4.状态特异性治疗脆弱性分析：利用scTherapy基于单细胞转录组特征预测药物敏感性，得到两大核心结果： 1.广谱有效药物：蛋白酶体抑制剂（硼替佐米）、DNA损伤剂（柔红霉素）对4个MP均有显著疗效，为HCC的通用治疗候选药物； 2.状态特异性敏感药物：MP2对HSP90抑制剂ganetespib、MEK抑制剂TAK-733敏感；MP4对MEK抑制剂（AS-703026）、BCL-2抑制剂navitoclax敏感；MP3对紫杉醇、MEK抑制剂trametinib敏感；HDAC抑制剂仅对MP2/3/4有效，对MP1无作用。 5.索拉非尼耐药的核心发现：在GSE109211索拉非尼治疗队列中，4个MP在非响应者中评分均显著升高，其中MP1的富集最为明显；结合MP1的代谢特征，提示其虽预后良好，但保留的肝特异性代谢通路可将索拉非尼代谢灭活，介导代谢性耐药。 图 3：恶性细胞的转录异质性和特定状态的治疗脆弱性 A.利用pySCENIC绘制的四种恶性细胞状态下转录因子活性的热图。恶性细胞分化轨迹分析：B. Monocle轨迹状态；C. GeneSwitches分析显示时间调控的基因转换。TCGA-LIHC队列中按元程序评分分层的Kaplan-Meier生存曲线：D. Diff-Metabolic（有利），E. Prolif-Stress（不利），F. MYC-Biosynth-Immune（不利），G. EMT-Inflammatory（不利）。H .箱线图比较索拉非尼应答者和非应答者之间的元程序评分（GSE109211）。I 、J.元程序特异性药物敏感性预测。I .每个元程序的选择性药物数量（生物渲染器创建的示意图）。J .不同元程序状态下的药物选择性模式。 互动问答 问：跨MP的共享转录因子（如ATF3、FOSB）对HCC治疗有何警示意义？ 答：共享TF的存在提示恶性细胞具有表型可塑性——在治疗压力下，恶性细胞可通过调控共享TF实现不同转录状态的转换，例如从对药物敏感的MP2转换为耐药的MP4，这是HCC产生治疗耐药的重要分子机制；这也警示临床治疗中，单一靶向某一MP的药物易引发耐药，需考虑联合靶向共享TF或多靶点联合治疗。 问：MP1作为预后良好的恶性细胞状态，为何会成为索拉非尼非响应者的核心特征？ 答：核心原因是MP1保留了完整的肝特异性代谢功能，其高表达的肝代谢通路（如胆汁酸、异生物代谢）包含大量药物代谢酶，可将索拉非尼这类外源性药物代谢为无活性形式，从而降低药物疗效，介导代谢性耐药；这一发现打破了“预后良好的细胞状态对治疗更敏感”的固有认知，为HCC个体化治疗提供了新的思路。 益栈信talks 公众号，点个赞，加个关注 这里总有些你没有而又恰好需要的，加工作人员微信 (备注单位+名字），领资料和免费分析咨询服务，提供方案设计和生信分析服务，提供linux系统环境配置，软件安装，更多技术服务，请联系工作人员。 4）髓系细胞图谱揭示了功能不同的巨噬细胞亚群 研究出发点与目标 髓系细胞是HCC TME的核心免疫组分，且前期研究发现髓系细胞在转移灶中显著扩增，但HCC中髓系细胞的亚群分类、各亚群的功能异质性尚未系统解析；同时，巨噬细胞作为髓系细胞的优势亚群，其在HCC进展中的功能状态转变、组织分布规律仍不明确，无法揭示其在TME免疫调控中的作用。本部分研究的核心目标是：系统解析髓系细胞的亚群分类，明确核心巨噬细胞亚群的标记基因、功能特征与组织分布规律，揭示巨噬细胞在HCC进展中的免疫调控作用。 核心研究过程与结果 1.髓系细胞整体亚群鉴定：对前期注释的髓系细胞进行亚聚类，鉴定出6个主要髓系亚群：巨噬细胞、经典单核细胞、非经典单核细胞、cDC1、cDC2、肥大细胞，各亚群由经典标记基因明确区分，其中巨噬细胞为髓系细胞的优势亚群。 2.巨噬细胞亚群精细分型：对巨噬细胞进一步亚聚类，得到10个巨噬细胞亚群，结合标记基因表达与功能富集分析（GSEA/Metascape），将其归纳为三大功能亚型，核心特征如下： 1.免疫活化型：包括Macro-CXCL9（高表达CXCL9、CXCL10）和Macro-FCN1（高表达IL1A、IL1B、IRF1、IRF8）；富集IFN-γ响应、抗原提呈、先天免疫等通路，高表达促炎细胞因子与趋化因子，具有抗瘤免疫功能； 2.免疫抑制型：包括Macro-SPP1（高表达SPP1）和Macro-TREM2（高表达TREM2）；Macro-SPP1富集糖酵解、血管生成、EMT、缺氧等促肿瘤通路，IFN-γ响应显著减弱；Macro-TREM2富集脂质定位、免疫效应调控，免疫抑制作用较Macro-SPP1温和； 3.组织驻留型：Macro-MARCO（高表达MARCO、VSIG4、IL10）；特异性富集于正常肝组织，富集脂肪酸代谢、脂质分解、解毒通路，为肝组织固有的巨噬细胞亚群。 3.巨噬细胞亚群的组织分布规律：组织驻留型Macro-MARCO仅在正常肝中富集，在肿瘤与转移灶中几乎消失；免疫抑制型Macro-SPP1和Macro-TREM2在肿瘤、PVTT与MLN中显著扩增，成为肿瘤与转移灶的主要巨噬细胞亚群；且Macro-SPP1在PVTT/MLN中更具优势，提示其在HCC转移中的免疫抑制作用更强。 4.巨噬细胞的功能对比分析：GSEA分析证实，与Macro-TREM2相比，Macro-SPP1富集更强的促肿瘤通路（血管生成、EMT、mTORC1信号），且IFN-γ响应显著减弱，是HCC TME中核心的强免疫抑制性巨噬细胞亚群。 图 4：髓系细胞图谱揭示了功能不同的巨噬细胞亚群 A.六种主要髓系细胞类型的UMAP投影：巨噬细胞、经典单核细胞、非经典单核细胞、cDC1、cDC2和肥大细胞。B .髓系细胞类型在不同组织中的分布。C .巨噬细胞亚群的UMAP投影。D .细胞比例显示组织特异性巨噬细胞亚群分布。E .髓系和巨噬细胞亚群的通路富集热图。F .巨噬细胞亚群中抗肿瘤基因表达的热图。G. 库普弗细胞标志物的表达以及Macro-MARCO中脂肪酸代谢和解毒通路的富集。H . 巨噬细胞亚群的功能基因表达和通路富集，包括Macro-CXCL9（I）、Macro-FCN1（J）、Macro-FOLR2（K）、Macro-SPP1（L）和Macro-TREM2（M）。N GSEA 比较 Macro-SPP1 和 Macro-TREM2，结果显示 Macro-SPP1 中血管生成、EMT、糖酵解、mTORC1 信号传导富集，而 IFN-γ 反应耗竭。 互动问答 问：研究中如何区分巨噬细胞的不同功能亚型，而非仅依靠基因表达进行简单分簇？ 答：研究采用**“标记基因+功能富集+组织分布”**的三维分型策略：首先通过标记基因明确各巨噬细胞亚群的基因表达特征，再通过GSEA、Metascape进行功能通路富集分析，判断其为活化/抑制/驻留功能；最后结合组织分布规律，验证功能特征与疾病阶段的关联（如驻留型仅在正常肝富集，抑制型在转移灶扩增），从而实现巨噬细胞的功能分型，而非单纯的基因表达分簇。 问：HCC进展过程中，巨噬细胞的功能状态发生了怎样的核心转变？这一转变对肿瘤进展有何意义？ 答：核心转变为从“组织驻留型”向“免疫抑制型”的定向转换，正常肝中的组织驻留型巨噬细胞（Macro-MARCO）在肿瘤进展中逐渐消失，免疫抑制型巨噬细胞（Macro-SPP1/TREM2）持续扩增，且转移灶中以强抑制型的Macro-SPP1为主；这一转变导致TME的免疫微环境从“稳态”向“免疫抑制”重塑，抑制抗瘤免疫反应，同时Macro-SPP1富集的促血管、EMT通路还能直接促进肿瘤增殖与转移，成为HCC进展与转移的重要免疫基础。 5）不同的内皮细胞和成纤维细胞亚群定义了预后和功能异质性 研究出发点与目标 基质细胞（内皮细胞、成纤维细胞）是TME的核心结构组分，前期研究发现内皮-成纤维细胞呈正相关且为转移灶的核心基质细胞，但HCC中内皮细胞与成纤维细胞的亚群异质性尚未系统解析；同时，缺乏对基质细胞亚群临床预后价值的验证，无法区分与HCC不良预后相关的“高危亚群”和具有保护作用的“良性亚群”，也不明确各亚群的功能特征与组织分布规律。本部分研究的核心目标是：系统解析内皮细胞、成纤维细胞的精细亚群分类，筛选与不良预后相关的高危基质亚群，明确各亚群的功能特征、组织分布及状态转变规律。 核心研究过程与结果 内皮细胞：鉴定13个亚群，锁定4个高危亚群 1.亚群鉴定与预后分析：对内皮细胞亚聚类得到13个转录学不同的亚群；将各亚群特征基因映射至TCGA-LIHC队列，进行单变量Cox回归分析，鉴定出4个高危亚群和4个保护性亚群，其中高危亚群的风险比（HR）均显著大于1：Endo-PTTG1（HR=2.56）、Endo-FCN2（HR=1.74）、Endo-ESM1（HR=1.69）、Endo-CXCL2（HR=1.65）。 2.核心高危亚群（Endo-ESM1）特征：①预后价值：Endo-ESM1高表达与患者更差的总生存期显著相关；②组织分布：特异性富集于肿瘤与转移灶（PVTT/MLN），在正常肝中几乎无分布；③功能特征：富集血管生成、细胞外基质（ECM）组织、层粘连蛋白相互作用、细胞迁移调控、缺氧相关通路，是促肿瘤血管生成的核心内皮亚群。 3.其他高危亚群功能：Endo-PTTG1富集细胞周期、G2/M转换通路；Endo-CXCL2富集TNF/NFκB炎症、EMT通路；Endo-FCN2富集ATP合成、补体激活通路，均通过不同机制促进肿瘤进展。 成纤维细胞：鉴定7个亚群，明确促肿瘤亚群特征 1.亚群鉴定与组织分布：对成纤维细胞亚聚类得到7个转录学不同的亚群；组织分布分析显示：Fib-CXCL12、Fib-BTG2主要存在于正常肝组织；Fib-POSTN、Fib-CD36在肿瘤、PVTT与MLN中显著扩增，为肿瘤与转移灶的核心成纤维亚群。 2.状态转变规律：采用拟时序轨迹分析发现，成纤维细胞存在从稳态向激活状态的连续转变，Fib-RACK1为激活过程中的独立分支，提示成纤维细胞的激活是一个渐进的过程。 3.促肿瘤成纤维亚群功能：对Fib-POSTN、Fib-CD36、Fib-RACK1进行Hallmark通路评分，发现三者均富集EMT、血管生成、TGF-β信号、缺氧、糖酵解等促肿瘤通路；且各亚群具有特异性功能：Fib-POSTN富集ECM降解，Fib-CD36富集PPAR信号与脂质代谢，Fib-RACK1富集血管发育。 4.特殊成纤维亚群（Fib-CXCL12）：该亚群高表达HLA家族基因和CD74，功能富集体液免疫、免疫响应调控通路，被定义为抗原呈递型癌症相关成纤维细胞（apCAF），是正常肝中具有免疫调节功能的成纤维亚群。 图 5：HCC 中的内皮细胞和成纤维细胞异质性 A.内皮细胞亚群的UMAP图，识别出13个不同的亚群。B .内皮细胞亚群中标记基因表达的散点图。C .细胞比例图，显示组织特异性内皮细胞亚群分布。D .在TCGA-LIHC队列中进行单变量Cox回归分析，识别出高风险内皮细胞亚群（Endo-PTTG1、Endo-FCN2、Endo-ESM1、Endo-CXCL2）和保护性亚群（Endo-EDNRB、Endo-RGCC、Endo-ACKR1、Endo-GJA4），并计算风险比。E . Kaplan-Meier生存曲线，比较TCGA-LIHC队列中Endo-ESM1高表达组和低表达组。F . Endo-ESM1在正常肝脏、肿瘤和门静脉癌栓/肠系膜淋巴结中的组织分布。G. Endo-ESM1的通路富集分析，包括血管形态发生、细胞外基质组织、层粘连蛋白相互作用、细胞迁移调控和缺氧通路。H.成纤维细胞亚群的UMAP图，识别出七个不同的亚群。I .细胞比例显示组织特异性成纤维细胞亚群分布。成纤维细胞亚群的伪时间轨迹分析（J），其中Fib-RACK1形成一个独特的分支（K）。成纤维细胞亚群的通路富集分析，包括Fib-RACK1（L）、Fib-CD36（M）和Fib-POSTN（N）。 互动问答 问：为何Endo-ESM1能成为HCC中最具临床意义的高危内皮亚群？ 答：因为Endo-ESM1同时具备预后价值、组织特异性和明确的促肿瘤功能：其一，其高表达与患者不良总生存期显著相关，是独立的预后不良标志物；其二，其特异性富集于肿瘤与转移灶，与HCC进展和转移密切关联；其三，其富集血管生成、ECM组织、细胞迁移等核心促肿瘤通路，是肿瘤血管生成的关键调控者，直接推动肿瘤增殖与转移，因此成为最具临床意义的高危内皮亚群。 问：成纤维细胞的“稳态-激活”连续转变规律，对理解HCC TME的重塑有何意义？ 答：这一规律提示，HCC中促肿瘤成纤维细胞（Fib-POSTN/CD36）并非肿瘤发生时突然出现的，而是由正常肝中的稳态成纤维细胞（Fib-CXCL12）经渐进的转录重编程激活而来；这一转变过程是HCC TME基质重塑的核心环节，且激活后的成纤维细胞通过富集促肿瘤通路，与内皮细胞、恶性细胞协同形成促肿瘤微环境；这也为临床治疗提供了思路——可通过靶向成纤维细胞的激活过程，阻断其从稳态向促肿瘤状态的转换，从而抑制TME的基质重塑。 6）内皮细胞-成纤维细胞相互作用网络重塑肿瘤微环境 研究出发点与目标 已鉴定出HCC中与不良预后相关的高危内皮亚群（如Endo-ESM1）和促肿瘤成纤维亚群（如Fib-POSTN/CD36），但这些核心基质亚群之间的通讯机制、与其他细胞的相互作用规律尚未明确；同时，从原发瘤到转移灶，基质细胞的通讯网络发生了怎样的动态变化，以及这种变化如何推动HCC转移，仍为科学空白。本部分研究的核心目标是：利用细胞间通讯分析工具，解析HCC TME的整体通讯网络，明确内皮-成纤维细胞的核心通讯作用，揭示基质亚群在转移灶中的通讯变化规律及分子机制。 核心研究过程与结果 本研究采用CellChat工具，从整体层面、差异层面、亚群层面三级解析HCC TME的细胞间通讯网络，核心结果如下： 1.整体层面：内皮-成纤维细胞为TME的通讯核心枢纽：全局相互作用映射显示，内皮细胞和成纤维细胞在原发瘤、PVTT/MLN中均具有最高的信号接收与发送数量及强度，远高于其他细胞类型，是HCC TME中细胞间通讯的核心枢纽，主导TME的信号调控。 2.差异层面：转移灶中基质细胞通讯显著增强：对比原发瘤与PVTT/MLN的通讯网络，发现内皮细胞和成纤维细胞与其他细胞群的相互作用强度和频率在PVTT/MLN中显著增加，提示转移灶中基质细胞的通讯调控更活跃，为肿瘤转移提供了更紧密的信号支持。 3.亚群层面：核心基质亚群形成双向正反馈通讯网络：聚焦高危内皮亚群与促肿瘤成纤维亚群，发现Endo-ESM1、Fib-POSTN、Fib-CD36三者间形成了密集的双向正反馈通讯网络，其核心通讯分子为： 1.细胞外基质（ECM）分子：胶原、纤连蛋白（FN1）、层粘连蛋白（LAMA4/LAMB2）、玻连蛋白（VTN）； 2.促血管生成因子：ANGPT家族、JAG、MDK、VEGF家族。 4.基质-免疫-恶性细胞的通讯特征：核心基质亚群（Endo-ESM1/Fib-POSTN/Fib-CD36）与免疫抑制型巨噬细胞（Macro-SPP1/TREM2）、Prolif-Stress恶性细胞的通讯显著增强；而与NK/T细胞、B细胞等适应性免疫细胞的通讯则明显减少，提示基质细胞通过增强与促肿瘤细胞的通讯、减弱与抗瘤免疫细胞的通讯，构建促血管、促ECM重塑、免疫抑制的微环境，为肿瘤进展与转移提供支持。 图 6：HCC 肿瘤微环境中的细胞间通讯 A.全局相互作用图谱显示，内皮细胞和成纤维细胞在原发肿瘤和门静脉癌栓/肠系膜淋巴结（PVTT/MLN）中均表现出较高的传入和传出相互作用强度。B . 差异通讯分析揭示，与原发肿瘤相比，PVTT/MLN中内皮细胞和成纤维细胞亚群与其他细胞群的相互作用强度和频率均有所增加。C .亚群水平相互作用分析比较了PVTT/MLN与原发肿瘤。D .配体-受体分析揭示了Endo-ESM1与成纤维细胞亚群之间存在密集的双向基质相互作用，该相互作用由……介导。细胞外基质PVTT/MLN 中的成分（胶原蛋白、FN1、层粘连蛋白、VTN）和促血管生成信号（ANGPT、JAG、MDK、VEGF）。E PVTT/MLN 中 Endo-ESM1 与髓系亚群（Macro-SPP1、Macro-TREM2）或增殖应激肝细胞之间的配体-受体相互作用。 互动问答 问：从原发瘤到转移灶，内皮-成纤维细胞的通讯强度显著增加，这一变化的生物学本质是什么？ 答：这一变化的生物学本质是TME为适应肿瘤转移而发生的通讯网络重塑：肿瘤细胞从原发瘤进入脉管（PVTT）并实现远处定植（MLN），需要更紧密的信号调控来支持血管生成、ECM重塑和免疫抑制，而内皮-成纤维细胞作为通讯核心，通过增强相互作用及与促肿瘤细胞的通讯，为肿瘤细胞的侵袭、迁移、定植提供了必要的微环境条件，是HCC转移的重要微环境基础。 问：核心基质亚群与免疫抑制型巨噬细胞、Prolif-Stress恶性细胞的通讯增强，体现了TME中怎样的细胞协同作用？ 答：体现了基质-免疫-恶性细胞的三方协同促肿瘤作用：核心基质亚群通过ECM分子和促血管因子构建促肿瘤的结构微环境，免疫抑制型巨噬细胞则为其提供免疫抑制的环境，Prolif-Stress恶性细胞在这种微环境中实现高增殖与侵袭，三者形成“基质构建框架、免疫抑制保护、恶性细胞增殖侵袭”的协同模式，共同推动HCC的进展与转移。 7）空间分辨分析揭示了肝细胞癌的有序细胞微环境 研究出发点与目标 前期在单细胞层面解析了HCC的细胞亚群、转录特征与细胞间通讯网络，但这些分子特征缺乏组织空间定位，无法明确各细胞亚群在肿瘤组织中的实际分布与物理互作模式；且单细胞层面发现的通讯通路与信号调控，尚未在组织空间中验证其真实性，无法构建“分子-空间”关联的促肿瘤微环境模型。本部分研究的核心目标是：将单细胞分子特征与组织空间结构结合，验证促肿瘤空间生态位的存在，明确其空间结构特征、核心调控通路与转录因子调控，为靶点筛选提供空间上下文。 核心研究过程与结果 本研究采用10x Visium空间转录组技术，结合Cell2location、COMMOT、MISTy、PROGENy等工具，从细胞分布、通讯验证、通路分析、TF定位多维度解析HCC的空间微环境，核心结果如下： 1.细胞空间共定位验证：促肿瘤生态位真实存在：利用Cell2location对空间转录组数据进行去卷积分析，将单细胞亚群映射至肿瘤组织切片，证实Endo-ESM1、Fib-POSTN、Fib-CD36与Prolif-Stress/EMT-Inflammatory恶性细胞在组织中存在显著的空间共定位，形成了HCC的促肿瘤空间生态位，且该生态位在不同患者样本中均保守存在。 2.空间通讯通路验证：ANGPTL为核心通讯通路：利用COMMOT工具分析空间层面的细胞间通讯，证实单细胞层面发现的ANGPTL通路是该生态位的核心空间通讯通路，且ANGPTL4在基质细胞富集的区域呈显著高表达，验证了单细胞通讯分析结果的空间真实性。 3.核心信号通路分析：TGF-β与缺氧为生态位核心调控通路：通过MISTy空间建模和PROGENy通路评分发现，该生态位内部及周边显著富集TGF-β和缺氧通路活性；且TGF-β通路的高活性与Endo-ESM1、Fib-POSTN、Fib-CD36的空间分布高度重合，提示TGF-β是调控促肿瘤生态位的核心信号通路。 4.转录因子空间定位：共享TF介导生态位内细胞表型可塑性：将单细胞层面发现的跨MP共享TF（ATF3、FOSB、JUNB、ELF3）进行空间映射，发现：①ATF3、FOSB在Prolif-Stress/EMT-Inflammatory恶性细胞共定位的区域高表达，与单细胞层面MP2/MP4共享TF的结果相呼应；②ELF3（MP2特异性TF）主要分布于Prolif-Stress恶性细胞区域，JUNB（MP4特异性TF）主要分布于EMT-Inflammatory恶性细胞区域；提示TGF-β信号可能重编程共享TF的功能，介导恶性细胞的表型可塑性。 5.促肿瘤生态位的空间结构：明确“中心-外周”有序模式：综合细胞分布、通路活性、TF定位分析，明确了促肿瘤生态位的分层空间结构： 1.核心层：Endo-ESM1、Fib-POSTN、Fib-CD36紧密聚集，为生态位的结构核心，高表达TGFB1配体，是TGF-β信号的主要来源； 2.中间层：Macro-SPP1（免疫抑制型巨噬细胞）分布于核心层外周，形成免疫抑制屏障； 3.外层：Prolif-Stress/EMT-Inflammatory恶性细胞聚集在基质核心与巨噬细胞的周边区域，在TGF-β和缺氧的微环境中维持高增殖、高侵袭表型。 图 7：HCC 肿瘤微环境的空间组织 HCC-1T ( A ) 和 HCC-2T ( B )的细胞定位反卷积。HCC-1T (C) 和 HCC- 2T ( D ) 中关键细胞亚群的空间分布，包括增殖应激肝细胞、内皮细胞-ESM1、纤维蛋白-CD36、纤维蛋白-POSTN 和巨噬细胞-SPP1。COMMOT分析显示 HCC-2T 中 ANGPTL 信号的空间组织分布 ( E )，以及 ANGPTL2 ( F ) 和 ANGPTL4 ( G ) 的表达模式。H HCC-2T 中的MISTy空间建模显示了通路相互作用的细胞类型重要性评分。HCC-2T 中的 PROGENy 通路分析显示 TGFβ ( I ) 和缺氧 ( J ) 信号在局部富集。K基于共富集的 TGFβ 和缺氧通路特征，从 HCC-2T 中划分出感兴趣区域 (ROI)。L. ROI内肿瘤细胞亚群的空间分布。M .转录因子图谱显示ROI内ATF3、FOSB、ELF3和JUNB的空间分布模式。N .基质和免疫细胞谱分析显示ROI内Endo-ESM1、Fib-POSTN和Fib-CD36在中心区域富集，而Macro-SPP1在外周区域分布。O . ROI内TGFβ通路活性和TGFB1配体表达图谱。P . ROI内缺氧评分和HIF1A表达图谱。 互动问答 问：研究中通过哪些手段，将单细胞层面的分子发现与组织空间结构结合，实现了“分子-空间”的关联分析？ 答：研究采用了多工具协同的分析策略：①通过Cell2location将单细胞亚群映射至空间切片，实现细胞亚群的空间定位；②通过COMMOT分析空间通讯通路，验证单细胞通讯的空间真实性；③通过MISTy+PROGENy将通路活性与细胞分布结合，实现信号通路的空间定位；④通过转录因子空间映射，实现调控分子的空间定位；最终将细胞、通讯、通路、TF的空间特征整合，构建了“分子-空间”关联的促肿瘤生态位模型。 问：促肿瘤生态位的“中心-外周”分层结构，体现了空间微环境对恶性细胞表型的怎样调控作用？ 答：这种分层结构体现了空间微环境的“定向调控”作用：核心层的基质细胞分泌TGF-β并构建ECM微环境，外周的巨噬细胞提供免疫抑制保护，形成了一个具有明确信号梯度的空间微环境；恶性细胞在这种微环境中被定向调控为Prolif-Stress/EMT-Inflammatory表型，维持高增殖、高侵袭特征；这也说明，恶性细胞的表型并非仅由自身基因决定，还受空间微环境的显著调控，空间位置是决定其表型的重要因素。 8）多维度筛选揭示HSP90B1是肝细胞癌治疗的关键靶点 研究出发点与目标 前期已系统解析了HCC恶性细胞的转录异质性、TME的功能亚群、细胞间通讯网络及空间生态位的调控规律，完成了对HCC异质性演变规律的基础研究；但研究最终需从基础发现走向临床转化，挖掘可成药的核心治疗靶点，解决临床治疗耐药、缺乏有效靶点的困境。本部分研究的核心目标是：基于前期发现的核心有害细胞状态，通过多维度的靶点筛选与临床验证，锁定具有临床转化价值、跨细胞类型的HCC核心治疗靶点。 核心研究过程与结果 本研究围绕HCC中三大核心有害细胞状态转换（Macro-SPP1→免疫活化型Macro-CXCL9、恶性肝细胞→正常肝细胞、Endo-ESM1→保护性Endo-EDNRB），开展五维度的靶点筛选与验证，层层筛选后最终锁定HSP90B1为核心治疗靶点，各维度核心结果如下： 1.维度一：Geneformer计算机模拟敲除，筛选共同依赖基因：利用Geneformer对三大有害细胞状态转换进行虚拟敲除筛选，鉴定出11个跨细胞类型的共同依赖基因，这些基因是逆转有害细胞状态、恢复正常细胞功能的核心候选靶点。 2.维度二：DepMap癌细胞必需性分析，筛选核心功能基因：将11个共同依赖基因映射至DepMap癌症依赖图谱，进行CRISPR基因效应分析，筛选出在多种癌细胞系中敲除后显著抑制细胞活力的基因，最终得到3个核心候选基因：EEF1A1、HSP90B1、ACTB。 3.维度三：表达依赖性分析，筛选肿瘤特异性依赖基因：分析3个候选基因的表达水平与CRISPR基因效应评分的相关性，发现：HSP90B1的表达水平与基因效应评分呈强负相关（r=-0.874），EEF1A1为中度相关（r=-0.561），ACTB为强相关（r=-0.819）；强负相关提示高表达该基因的癌细胞对其存在“表达成瘾性”，抑制该基因对高表达的肿瘤细胞杀伤作用更强，具有肿瘤特异性。 4.维度四：临床治疗队列验证，筛选与治疗耐药相关的基因： 1.cabozantinib+nivolumab治疗队列（GSE238264）：空间转录组分析显示，非响应者中HSP90B1呈弥散性高表达，响应者中为局灶性表达，且非响应者的HSP90B1表达量显著更高；EEF1A1也呈非响应者高表达，而ACTB在非响应者中表达降低； 2.索拉非尼治疗队列（GSE109211）：差异表达分析证实，HSP90B1、EEF1A1在非响应者中显著上调，与治疗耐药密切相关。 5.维度五：临床预后队列验证，筛选与不良预后相关的基因：将3个候选基因映射至TCGA-LIHC队列进行生存分析，发现：HSP90B1高表达与患者更短的总生存期显著相关；EEF1A1的表达与患者生存期无显著关联；ACTB作为管家基因，无预后价值。 核心筛选结论 综合五维度结果，HSP90B1成为唯一符合“共同依赖+癌细胞必需+表达成瘾+治疗耐药+不良预后”的核心治疗靶点：EEF1A1虽与治疗耐药相关，但表达依赖性较弱且无预后价值；ACTB虽表达依赖性强，但为管家基因，在治疗非响应者中表达降低，无肿瘤特异性，均被排除。 图 8：HSP90B1 被确定为 HCC 的治疗靶点 A.利用Geneformer进行虚拟敲除实验，在三种细胞状态转变（Macro-SPP1到Macro-CXCL9、恶性肝细胞到正常肝细胞、Endo-ESM1到Endo-EDNRB）中鉴定出11个共有基因。B .基于DepMap数据库的CRISPR基因效应分析显示，这11个已鉴定基因在不同癌细胞系中均表现出负效应。C . CRISPR基因依赖性评分显示EEF1A1、HSP90B1和ACTB的必需性。TCGA-LIHC分析显示，肿瘤组织中EEF1A1（D）、HSP90B1（E）和ACTB（F）的表达高于正常肝组织。G .基因表达水平与CRISPR基因效应评分之间的相关性分析显示表达依赖性脆弱性。H .空间聚类分析显示，在卡博替尼联合纳武利尤单抗治疗的样本中，EEF1A1、HSP90B1和ACTB在肿瘤区域呈现一致的表达模式。I – L代表性空间图比较了应答者（HCC1R 和 HCC2R）和非应答者（HCC5NR 和 HCC6NR），显示应答者呈局灶性表达，而非应答者呈弥漫性表达。M 定量分析显示应答者和非应答者之间存在差异表达丰度，其中 EEF1A1 和 HSP90B1 在非应答者中升高。N索拉非尼治疗队列的差异表达分析。TCGA-LIHC 队列的 Kaplan-Meier 生存分析显示 EEF1A1 表达与总生存期之间无显著相关性 ( O )，而 HSP90B1 高表达与较差的总生存期相关 ( P )。 互动问答 问：研究为何选择三大有害细胞状态转换作为虚拟敲除的筛选背景，而非单一细胞类型？ 答：因为HCC的进展与转移是恶性细胞与TME共同作用的结果，单一细胞类型的靶点难以有效抑制肿瘤；选择Macro-SPP1（免疫抑制）、恶性肝细胞（核心肿瘤细胞）、Endo-ESM1（高危基质）三大核心细胞类型的有害状态转换作为筛选背景，能筛选出跨免疫、恶性、基质细胞的共同依赖基因，这类靶点可同时调控多种促肿瘤细胞类型，实现“多靶点、全方位”的肿瘤抑制，更具临床转化价值。 问：从临床转化的角度，HSP90B1作为核心治疗靶点的优势体现在哪些方面？ 答：HSP90B1作为靶点具有三大核心优势：①肿瘤特异性：高表达的HCC细胞对其存在“表达成瘾性”，抑制后对肿瘤细胞的杀伤作用更强，对正常细胞影响较小；②与治疗耐药密切相关：在索拉非尼、cabozantinib+nivolumab两种主流治疗方案的非响应者中均高表达，靶向抑制可有效逆转治疗耐药；③具有预后与疗效预测价值：其表达水平与患者生存期显著相关，空间表达模式（弥散/局灶）还可作为治疗疗效的预测标志物，为临床个体化治疗提供依据。 五、研究的逻辑链整合 本研究的所有研究发现形成了一条清晰、连续、层层递进的逻辑链，从HCC的临床治疗困境出发，通过多维度的组学分析解析生物学规律，最终回归临床靶点挖掘，每个研究节点均承接上一节点的科学空白，所有结论相互印证，形成了“临床问题—基础解析—临床转化”的完整研究逻辑，具体文字逻辑流程图如下： 研究起点：HCC临床治疗面临异质性、耐药、转移的核心困境，现有研究缺乏跨全进展阶段的整合性解析，且未明确恶性细胞与TME的互作调控机制 →节点1：整合正常肝、原发瘤、PVTT、MLN的多阶段scRNA-seq数据，经严格质控、批次校正与细胞注释，构建HCC全进展阶段的单细胞全景细胞图谱，明确核心细胞动力学特征与细胞类型相关性 →节点2：通过CopyKAT鉴定恶性肝细胞，利用cNMF解构出4个具有保守特征的转录元程序，明确各元程序的功能特征、组织分布与临床预后价值 →节点3：系统解析TME中髓系、内皮、成纤维细胞的精细亚群分类，筛选出与不良预后相关的免疫抑制型巨噬细胞亚群及高危基质细胞亚群，明确各功能亚群的分子特征与组织分布 →节点4：利用CellChat解析细胞间通讯网络，发现Endo-ESM1、Fib-POSTN、Fib-CD36形成的基质枢纽是TME的核心通讯调控者，其通过ECM分子与促血管因子构建促肿瘤通讯网络，并与免疫抑制型巨噬细胞、增殖应激型恶性细胞形成协同互作 →节点5：通过空间转录组学技术，将单细胞亚群与分子特征映射至肿瘤组织切片，验证并构建具有“中心-外周”分层结构的促肿瘤空间生态位，明确TGF-β与缺氧为该生态位的核心调控通路 →节点6：基于Geneformer针对三大有害细胞状态转换开展计算机模拟敲除，结合DepMap癌症依赖图谱，从11个共同依赖基因中筛选出3个癌症必需基因（EEF1A1、HSP90B1、ACTB） →节点7：通过表达依赖性分析、cabozantinib+nivolumab治疗队列、索拉非尼治疗队列及TCGA-LIHC预后队列进行多维度验证，排除非特异性与无预后价值的基因 →研究终点：最终锁定HSP90B1为兼具“共同依赖+癌症必需+表达成瘾+治疗耐药+不良预后”的HCC核心治疗靶点。 六、研究的方法学亮点与局限性 方法学亮点 1.研究设计的跨维度整合性：本研究首次实现了HCC全疾病进展阶段（正常肝→原发瘤→PVTT→MLN）与多技术维度（单细胞转录组+空间转录组）的深度整合分析，突破了传统研究单一阶段、单一技术的局限性，将分子层面的细胞异质性与组织层面的空间结构紧密结合，构建了“分子-空间”关联的HCC进展调控模型，更能真实反映HCC的生物学特征。 2.恶性细胞分析的策略创新性：摒弃了传统的细胞聚类分析方法，采用cNMF技术分解恶性肝细胞的基因表达程序，并归纳为更高层级的转录元程序，有效识别出跨患者、保守的核心转录状态，突破了患者特异性与技术偏倚的限制，为解读肿瘤异质性的分子本质提供了新的分析框架，该方法可广泛推广至乳腺癌、胰腺癌等异质性显著的肿瘤研究。 3.靶点筛选的体系化与高效性：建立了**“计算机模拟扰动+癌症依赖图谱+多临床队列验证”**的靶点筛选体系，将计算生物学与临床大数据深度结合，从跨细胞类型的共同依赖基因出发，通过癌症必需性、表达依赖性、治疗响应、临床预后等多维度层层筛选，大幅提升了靶点发现的效率与可靠性，减少了传统湿实验筛选的成本与时间，为肿瘤靶点挖掘提供了高效的范式。 4.TME解析的系统性与协同性：并非孤立分析TME中的免疫细胞或基质细胞，而是系统解析了髓系、内皮、成纤维细胞的功能异质性，并深入分析了三者与恶性细胞的协同互作关系，明确了“基质枢纽-免疫抑制-恶性细胞增殖侵袭”的三方协同促肿瘤模式，揭示了TME调控HCC进展的核心机制，为TME研究提供了全面、系统的分析思路。 5.研究闭环的完整性与临床导向性：本研究形成了“临床问题—全景描绘—机制解析—靶点筛选—临床验证”的完整研究闭环，所有研究环节均围绕临床治疗困境展开，所有研究发现均通过临床队列进行验证，最终挖掘的靶点直接对接HCC的治疗耐药与预后问题，让研究结果兼具理论深度与临床应用价值，实现了基础研究与临床转化的无缝衔接。 研究局限性 1.横断面的研究设计：本研究为横断面研究，仅捕捉了HCC不同进展阶段的静态细胞特征与分子规律，无法直接观察细胞状态转换与异质性演变的时间动态变化，难以明确HCC从发生到转移过程中，恶性细胞与TME互作的实时调控规律。 2.样本量与类型的限制：空间转录组的分析样本量较少，且转移性标本（PVTT/MLN）的可获得性有限；同时，研究未纳入不同病因（乙肝、丙肝、酒精性肝病）、不同种族及不同临床分期的HCC患者，可能影响研究结果的普适性，无法反映不同临床背景下HCC的异质性特征。 3.空间组学的技术分辨率限制：本研究使用的空间转录组技术为斑点水平分辨率，无法实现单细胞水平的精细解析，因此难以刻画单细胞尺度的细胞间直接互作与信号传递，对促肿瘤空间生态位的调控机制解析仍存在一定的局限性。 4.缺乏体内外的湿实验验证：本研究通过计算生物学与临床队列验证了HSP90B1的靶点价值，但尚未通过细胞系敲除/过表达、裸鼠成瘤模型、肝癌类器官等体内外湿实验，直接验证HSP90B1在HCC发生、发展中的具体功能，也未验证靶向抑制HSP90B1的治疗效果与耐药逆转作用。 5.多组学整合的不足：本研究主要基于转录组水平的数据分析，未整合代谢组、蛋白组、表观基因组、基因组等多组学数据，对HCC异质性的解析仅停留在转录层面，未涉及转录后调控、代谢重塑、表观遗传修饰、基因组变异等层面的调控机制，无法构建多维度的HCC异质性调控网络。 未来改进方向 1.开展纵向队列研究，结合HCC患者的临床随访数据与多时间点的样本检测，追踪HCC进展过程中细胞状态与分子特征的动态演变规律，明确异质性调控的时间维度特征。 2.扩大样本量，纳入不同病因、种族、临床分期与治疗方案的HCC患者，开展亚组分析，明确不同临床背景下HCC的异质性特征，提升研究结果的普适性。 3.利用更高分辨率的空间组学技术（如单细胞空间转录组、空间蛋白组、空间代谢组），解析单细胞尺度的细胞间互作与信号传递，更精准地刻画促肿瘤生态位的调控机制。 4.通过细胞系、肝癌类器官、裸鼠成瘤模型等体内外湿实验，验证HSP90B1的具体功能，并开展HSP90B1特异性抑制剂的药物筛选与联合治疗实验，验证其临床治疗效果。 5.整合代谢组、蛋白组、表观基因组等多组学数据，开展多维度的整合分析，构建HCC异质性的多层级调控网络，更全面地解析HCC的发生与转移机制。 七、对科研与临床的启示 科研启示 1.肿瘤研究的多维度数据整合范式：本研究为肿瘤异质性研究提供了**“多疾病阶段、多组学技术”**的数据整合思路，即整合从正常组织到原发、转移的全疾病阶段样本，结合单细胞转录组与空间转录组技术，实现分子特征与空间结构的深度结合，让肿瘤研究从“分子层面的现象描述”走向“组织层面的机制解析”，更能反映肿瘤的真实生物学特征。 2.恶性细胞异质性的解析思路：本研究提出的转录元程序分析框架，突破了传统细胞聚类的局限性，为解读恶性细胞的异质性提供了新的思路。该方法可有效识别肿瘤中跨患者、保守的核心转录状态，避免了患者特异性与技术偏倚的影响，更能揭示肿瘤异质性的分子本质，可广泛应用于乳腺癌、肺癌、胰腺癌等异质性显著的恶性肿瘤研究。 3.肿瘤靶点筛选的体系化思路：本研究建立的“计算机模拟扰动+癌症依赖图谱+多临床队列验证”的靶点筛选体系，为肿瘤靶点挖掘提供了计算生物学与临床大数据结合的高效范式。该体系从跨细胞类型的共同依赖基因出发，通过多维度的临床验证层层筛选，能有效提升靶点发现的效率与可靠性，为肿瘤靶向药物的研发提供了新的思路。 4.肿瘤微环境的研究方向：本研究揭示了“基质细胞为核心枢纽，基质-免疫-恶性细胞协同调控”的TME促肿瘤模式，提示后续肿瘤微环境研究应摒弃“孤立分析单一细胞类型”的思路，重点关注跨细胞类型的协同互作关系，解析不同细胞类型之间的通讯调控网络与空间互作模式，从“系统层面”解析TME的促肿瘤机制。 5.精准肿瘤学的研究导向：本研究将单细胞分子特征、空间组织结构与临床预后、治疗响应紧密结合，提示后续精准肿瘤学研究应坚持**“临床导向”**，所有的基础研究发现均需通过临床队列进行验证，所有的分子特征与靶点均需具备明确的临床预后或治疗指导价值，让基础研究真正服务于临床转化。 临床转化价值 1.基于转录元程序的HCC精准分层治疗：本研究鉴定的4个恶性肝细胞转录元程序具有明确的临床预后与药物敏感性特征，可基于此构建HCC的分子分层模型，实现患者的精准分层与个体化治疗。例如，对MP1（分化代谢型）高表达的患者，规避索拉非尼等易被肝代谢灭活的药物，选择蛋白酶体抑制剂等广谱有效药物；对MP2（增殖应激型）高表达的患者，可联合HSP90抑制剂与MEK抑制剂；对MP4（EMT炎症型）高表达的患者，可采用MEK抑制剂联合BCL-2抑制剂，提升治疗的有效性。 2.HSP90B1为核心的靶向治疗策略：HSP90B1作为本研究锁定的核心治疗靶点，兼具癌症必需性、表达成瘾性、治疗耐药相关性与不良预后价值，为HCC靶向治疗提供了新的核心方向。尤其是针对索拉非尼、cabozantinib+nivolumab等主流治疗方案的非响应者，可开发HSP90B1特异性抑制剂（如PU-WS13）进行靶向治疗，逆转治疗耐药，提升临床治疗效果。 3.基于促肿瘤生态位的联合治疗策略：基于本研究发现的“基质-免疫-恶性细胞”协同促肿瘤模式与促肿瘤空间生态位的调控机制，可设计**“靶点抑制剂+抗血管生成+免疫检查点阻断”**的三联治疗策略。以HSP90B1抑制剂靶向恶性细胞与TME的核心依赖基因，以VEGF/ANGPT抑制剂破坏内皮-成纤维细胞的促血管通讯网络，以PD-1/PD-L1抑制剂重塑肿瘤免疫微环境，从恶性细胞、TME基质、免疫调控三个维度实现多靶点的肿瘤抑制。 4.构建HCC的综合预后与疗效预测模型：本研究鉴定的Endo-ESM1、Fib-POSTN、Macro-SPP1等高危细胞亚群与HSP90B1等核心靶点，均具有明确的临床预后价值，可将这些分子标志物整合，构建HCC的综合预后评估模型，为患者的预后判断提供更精准的分子依据。同时，HSP90B1的空间表达模式（弥散性/局灶性）可作为HCC靶向治疗与免疫治疗的疗效预测标志物，为临床治疗方案的选择提供参考。 5.肿瘤微环境的靶向干预：针对本研究发现的Endo-ESM1、Fib-POSTN、Fib-CD36等高危基质细胞亚群，可开发对应的靶向抑制剂，破坏促肿瘤空间生态位的结构基础与通讯网络，从TME层面抑制肿瘤的进展与转移。同时，可通过靶向调控巨噬细胞的极化，将免疫抑制型的Macro-SPP1/TREM2重编程为免疫活化型的Macro-CXCL9/FCN1，重塑肿瘤的免疫微环境，提升免疫治疗的效果。 八、整体文章内容总结 本研究针对肝细胞癌（HCC）异质性导致的治疗耐药、转移及预后差等临床核心困境，以“临床问题出发，回归临床应用”为导向，设计了“单细胞解构—空间验证—计算机模拟靶点筛选”的三步走整合研究策略，系统整合了覆盖正常肝、原发瘤、门脉癌栓（PVTT）、淋巴结转移（MLN）的大规模单细胞转录组数据，结合10x Visium空间转录组技术与前沿的生物信息学分析工具，完成了对HCC全进展阶段细胞生态与分子调控规律的系统解析。 研究首先经严格质控与批次校正，构建了HCC全进展阶段的单细胞全景图谱，明确了肝细胞比例递增、髓系细胞扩增、NK/T细胞减少的核心细胞动力学特征，以及内皮-成纤维细胞协同、基质-髓系细胞排斥淋巴细胞的细胞相关性规律；随后通过CopyKAT鉴定恶性肝细胞，利用cNMF技术解构出4个具有保守特征的转录元程序，明确了MP1（分化代谢型）与良好预后相关，MP2/3/4与疾病进展相关的临床特征，并发现MP1虽预后良好但介导索拉非尼代谢性耐药的关键规律；同时系统解析了TME中髓系、内皮、成纤维细胞的功能异质性，筛选出免疫抑制型的Macro-SPP1/TREM2巨噬细胞亚群，以及Endo-ESM1、Fib-POSTN/Fib-CD36等高危基质细胞亚群，明确了各亚群的分子特征、组织分布与临床预后价值。 在此基础上，研究利用CellChat解析出Endo-ESM1、Fib-POSTN、Fib-CD36形成的基质枢纽是TME的核心通讯调控者，其通过ECM分子与促血管因子构建双向正反馈通讯网络，并与免疫抑制型巨噬细胞、增殖应激型恶性细胞形成协同互作；再通过空间转录组学技术，将单细胞分子特征映射至肿瘤组织切片，验证并构建了具有“基质核心-免疫外周-肿瘤细胞环绕”分层结构的促肿瘤空间生态位，明确了TGF-β与缺氧为该生态位的核心调控通路。 最终，研究基于Geneformer针对三大有害细胞状态转换开展计算机模拟敲除，结合DepMap癌症依赖图谱筛选出癌症必需基因，并通过表达依赖性分析、cabozantinib+nivolumab治疗队列、索拉非尼治疗队列及TCGA-LIHC预后队列进行多维度验证，层层筛选后锁定HSP90B1为兼具“共同依赖+癌症必需+表达成瘾+治疗耐药+不良预后”的HCC核心治疗靶点。 本研究首次构建了HCC进展的分子与空间整合图谱，填补了领域内缺乏跨疾病阶段、多维度整合分析的空白，系统阐明了HCC恶性细胞与TME异质性的动态演变规律及协同调控机制，同时建立了高效的肿瘤靶点筛选体系，为HCC的精准分层治疗、靶向药物研发与联合治疗策略设计提供了坚实的理论依据和实验线索。本研究的分析范式与研究思路也为乳腺癌、肺癌等其他异质性显著的恶性肿瘤研究提供了重要参考，推动了精准肿瘤学的发展与基础研究向临床转化的落地。 益栈信talks 公众号，点个赞，加个关注 这里总有些你没有而又恰好需要的，加工作人员微信 (备注单位+名字），领资料和免费分析咨询服务，提供方案设计和生信分析服务，提供linux系统环境配置，软件安装，更多技术服务，请联系工作人员。 参考文献： Xia P, Shuang S, Fu D, et al. Large-scale single-cell analysis and in silico perturbation reveal dynamic evolution of HCC: from initiation to therapeutic targeting[J]. npj Precision Oncology, 2026.