Application of next-generation sequencing technology to precision medicine in cancer: joint consensus of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology

Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology(CSCO) and the China Actionable Genome Consortium(CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians,pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure(SOP), data analysis, report, and NGS platform certification and validation.

Predictive value of co-expression patterns of immune checkpoint molecules for clinical outcomes of hematological malignancies

Co-expression of immune checkpoint(IC)molecules can exacerbate T cell exhaustion in patients with hematological malignancies(HMs)and contribute to the immune escape of tumor cells,which is related to poor clinical outcome.It is worth establishing and optimizing an ideal prediction model based on the co-expression patterns of IC molecules to evaluate the immune status of HM patients and predict their clinical outcome.In this perspective,we summarize the co-expression patterns of IC molecules and their importance as biomarkers that predict the prognosis of patients with different HMs,providing new insights for designing dual IC blockades(ICBs).

Stimulation of staphylococcal enterotoxin A combined with PML-RARα peptide on the specifical T-cells against NB4 cell line

Objective： To investigate the effects of staphyococcal enterotoxin A （SEA） on the cytotoxicity of T cells stimulated by PML-RARa peptide in vitro. Methods： Peripheral blood mononuclear cells （MNC） from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARa peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 （CCK-8）. The CD4 and CD8 surface markers on the harvested CD3^＋ T cells were detected by flow cytometry （FCM）. Results： The cytotoxicity of T cells induced by PML-RARa peptide with SEA was higher than that of T cells induced only by PML-RARa peptide against NB4 cells. The FCM assay showed that the ratio of CD4^＋/CD8^＋ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARe peptide with SEA. Conclusion： The specific cytotoxicity of T cells induced by PML-RARa peptide against NB4 cells could be enhanced with superantigen SEA.

Higher PD-1 expression concurrent with exhausted CD8+ T cells in patients with de novo acute myeloid leukemia

Objective： To investigate the association between the T cell inhibitory receptor programmed death 1（PD-1）and T cell exhaustion status in T cells from patients with de novo acute myeloid leukemia（AML） and AML in complete remission（CR）.Methods：Surface expression of PD-1 and the exhaustion and immunosenescence markers CD244 and CD57 on CD3＋,CD4＋ and CD8＋ T cells from peripheral blood samples from 20 newly diagnosed,untreated AML patients and 10 cases with AML in CR was analyzed by flow cytometry.Twenty-three healthy individuals served as control.Results：A significantly higher percentage of PD-1＋ cells were found for CD3＋ T cells in the de novo AML group compared with healthy controls.In addition,an increased level of PD-1＋ CD8＋ T cells,but not PD-1＋ CD4＋,was found for CD3＋ T cells in the de novo AML and AML-CR samples.A higher percentage of CD244＋ CD4＋,CD244＋ CD8＋,CD57＋ CD4＋ and CD57＋ CD8＋ T cells was found in CD3＋ T cells in samples from those with de novo AML compared with those from healthy controls.Strong increased PD-1＋ CD244＋ and PD-1＋ CD57＋ coexpression was found for CD4＋ and CD8＋ T cells in the de novo AML group compared with healthy controls.Conclusions：We characterized the major T cell defects,including co-expression of PD-1 and CD244,CD57-exhausted T cells in patients with de novo AML,and found a particular influence on CD8＋ T cells,suggesting a poor anti-leukemia immune response in these patients.

Extensive exploration of T cell heterogeneity in cancers by single cell sequencing

Human T cells are a highly heterogeneous population and can recognize a wide variety of antigens by their T cell receptors(TCRs). Tumor cells display a large repertoire of antigens that serve as potential targets for recognition,thus making T cells in the tumor micro-environment more complicated. Making a connection between TCRs and the transcriptional information of individual T cells will be interesting for investigating clonal expansion within T cell populations under pathologic conditions. Advances in single cell RNA-sequencing(scRNA-seq) have allowed for comprehensive analysis of T cells. In this review, we briefly describe the research progress on tumor microenvironment T cells using single cell RNA sequencing, and then discuss how scRNA-seq can be used to resolve immune system heterogeneity in health and disease. Finally, we point out future directions in this field and potential for immunotherapy.

Expression pattern of GATA-1,-2 and-3 genes in leukemic bone marrow microenvironment

Objective： The aim of the study was to investigate the expression pattern of hematopoietic transcription factor GATA-1, -2 and -3 genes in leukemic bone marrow （BM） micreenvironment [including bone marrow stremal cells （BMSCs） and BM hematopoietic cells]. Methods： Mononuclear cells were isolated from BM of patients with acute myeloid leukemia （AML）, chronic myelogenous leukemia （CML）, or acute lymphoblasUc leukemia （ALL）. Adherent cells （BMSCs） and nonadherent ceils （BM hematopoietic cells） were collected after long-term culture in vitro. The semi-quantitative expression levels of GATA genes in the BMSCs or BM hematopoietic cells from patients with leukemia were analyzed by using RT-PCR-ELISA and com- pared with normal controls. Results： The expression level of GATA-1 gene in the BMSCs from CML group was significantly lower than that of the normal controls. The expression level of GATA-3 gene in the BMSCs from ALL was higher than that of the normal controls, but that from CML was lower than the normal controls. Dominant expression of GATA-3 gene was found in the normal BM hematopoietic cells. The dominant expression of GATA-2 gene was found in the normal BMSCs and the BMSCs from CML, whereas the dominant expression of GATA-3 gene was detected in the BMSCs from AML. Conclusion： GATA-1, -2 and -3 genes might play a role in hematopoiesis regulation in leukemia, and the changes of expression pattern of GATA genes might influence the hematopoiesis in BM microenvironment and relate to the pathogenesis and development of leukemia.

Expression pattern of BIM,BCL-6,and c-MYC in adult B-cell acute lymphoblastic leukemia

Objective We aimed to evaluate the expression pattern of the genes BIM, BCL-6, and c-MYC in adult patients at initial diagnosis of B-cell acute lymphoblastic leukemia(B-ALL).Methods Relative m RNA levels of BIM, BCL-6, and c-MYC in peripheral blood mononuclear cells(PBMCs) from B-ALL patients were determined by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) using SYBR Green dye. PBMCs from healthy volunteers served as a control. GAPDH was used as a reference gene.Results Relative expression of BIM, BCL-6, and c-MYC m RNA in B-ALL patients was significantly lower than in healthy controls(P < 0.05). Furthermore, this result was observed for both newly diagnosed B-ALL patients and those incomplete remission(CR)(P < 0.05). There were no statistically significant differences in the expression levels of BIM, BCL-6, and c-MYC between these B-ALL patient groups(P > 0.05). Spearman's rank correlation analyses revealed the expression level of BIM to be positively correlated with that of BCL-6 in B-ALL patients.Conclusion Expression of the genes BIM, BCL-6, and c-MYC is decreased in adult B-ALL patients. Moreover, the expression pattern of these genes may be similar in such patients at initial diagnosis and following CR. The expression characteristics of BIM, BCL-6, and c-MYC may constitute useful markers for the diagnosis of adult B-ALL.

High percentage of bone marrow CD8^(+)tissue-resident-like memory T cells predicts inferior survival in patients with acute myeloid leukemia

Tissue-resident memory T(TRM)cells infiltrating solid tumors could influence tumor progression and the response to immune therapies.However,the proportion and prognostic value of TRM cells in the bone marrow(BM)of patients with acute myeloid leukemia(AML)are unclear.In this study,we used flow cytometry to assay the phenotype of 49 BM samples from patients newly diagnosed with AML(ND-AML).We found that the BM CD8^(+)effector memory(TEM)cells highly expressed CD69(CD8^(+)TRM-like T cells),and their percentage was significantly increased in patients with ND-AML compared with that in healthy individuals(HI).The high percentage of CD8^(+)TRM-like subset was associated with poor overall survival in our ND-AML cohort.The Kaplan–Meier Plotter database verified a significantly reduced survival rate among patients with high expression of CD8^(+)TRM-like T cell characteristic genes(CD8A,CD69,and TOX),especially the M4 and M5 subtypes.Phenotypic analysis revealed that the BM CD8^(+)TRM-like subpopulation exhibited exhausted T cell characteristics,but its high expression of CD27 and CD28 and low expression of CD57 suggested its high proliferative potential.The single-cell proteogenomic dataset confirmed the existence of TRM-like CD8^(+)T cells in the BM of patients with AML and verified the high expression of immune checkpoints and costimulatory molecules.In conclusion,we found that the accumulation of BM CD8^(+)TRM-like cells could be an immune-related survival prediction marker for patients with AML.

Activation of transmembrane receptor tyrosine kinase DDR1-STAT3 cascade by extracellular matrix remodeling promotes liver metastatic colonization in uveal melanoma

Colonization is believed a rate-limiting step of metastasis cascade.However,its underlying mechanism is not well understood.Uveal melanoma(UM),which is featured with single organ liver metastasis,may provide a simplified model for realizing the complicated colonization process.Because DDR1 was identified to be overexpressed in UM cell lines and specimens,and abundant pathological deposition of extracellular matrix collagen,a type of DDR1 ligand,was noted in the microenvironment of liver in metastatic patients with UM,we postulated the hypothesis that DDR1 and its ligand might ignite the interaction between UM cells and their surrounding niche of liver thereby conferring strengthened survival,proliferation,sternness and eventually promoting metastatic colonization in liver.We tested this hypothesis and found that DDR1 promoted these malignant cellular phenotypes and facilitated metastatic colonization of UM in liver.Mechanistically,UM cells secreted TGF-β1 which induced quiescent hepatic stellate cells(qHSCs)into activated HSCs(aHSCs)which secreted collagen type I.Such a remodeling of extracellular matrix,in turn,activated DDR1,strengthening survival through upregulating STAT3-dependent Mcl-1 expression,enhancing sternness via upregulating STAT3-dependent S0X2,and promoting clonogenicity in cancer cells.Targeting DDR1 by using 7rh,a specific inhibitor,repressed proliferation and survival in vitro and in vivo outgrowth.More importantly,targeting cancer cells by pharmacological inactivation of DDR1 or targeting microenvironmental TGF-β1-collagen I loop exhibited a prominent anti-metastasis effect in mice.In conclusion,targeting DDR1 signaling and TGF-β1 signaling may be a novel approach to diminish hepatic metastasis in UM.

The role of cholesterol metabolism in leukemia

Leukemia is a common hematological malignancy with overall poor prognosis.Novel therapies are needed to improve the outcome of leukemia patients.Cholesterol metabolism reprogramming is a featured alteration in leukemia.Many metabolic-related genes and metabolites are essential to the progress and drug resistance of leukemia.Exploring potential therapeutical targets related to cholesterol homeostasis is a promising area.This review summarized the functions of cholesterol and its derived intermediate metabolites,and also discussed potential agents targeting this metabolic vulnerability in leukemia.

The importance of genomic predictors for clinical outcome of hematological malignancies

Hematological malignancy is 1 of the top 10 malignant diseases with regards to cancer patient morbidity and mortality.^(1) Although hematopoietic stem cell transplantation,chemotherapy,and targeted therapy have made great progress in recent years,patients with hematological malignancies still have adverse clinical outcomes,particularly elderly patients.^(2,3) Therefore,it is necessary to explore for an optimal prediction model to evaluate the clinical outcome,which is important for devising a therapeutic strategy for hematological diseases.

PD-L1表达指导的信迪利单抗与帕博利珠单抗联合或不联合含铂双药化疗治疗未经治晚期非小细胞肺癌患者:一项2期随机对照试验(CTONG1901)

No direct comparison has been performed between different programmed cell death-1(PD-1)inhibitors for first-line treatment in patients with advanced non-small cell lung cancer(NSCLC).The feasibility of using PD-L1-expression-guided immunotherapy remains unknown.In this open-label,phase 2 study(NCT04252365),patients with advanced NSCLC without EGFR or ALK alterations were randomized(1:1)to receive sintilimab or pembrolizumab monotherapy(PD-L1 expression≥50%),or sintilimab or pembrolizumab plus platinum-based chemotherapy(PD-L1 expression<50%).The sample size was calculated by optimal two-stage design.The primary endpoint was the objective response rate(ORR).The study included 71 patients(sintilimab arms,n=35;pembrolizumab arms,n=36)and met its primary endpoint,with a confirmed ORR of 51.4%(18/35)in the sintilimab arms.The confirmed ORR(95%confidence interval)was 46.2%(19.2%,74.9%)and 42.9%(17.7%,71.1%)for patients treated with sintilimab and pembrolizumab monotherapy;and 54.5%(32.2%,75.6%)and 45.4%(24.4%,67.8%)for those treated with sintilimab-and pembrolizumab-based combination therapies.The median progression-free survival was6.9 versus 8.1 months for all sintilimab-treated versus all pembrolizumab-treated patients,respectively,in which it was 7.6 versus 11.0 months in monotherapy and 7.4 versus 7.1 months in combination therapies.The median overall survival was 14.9 versus 21.3 months for all sintilimab-treated versus all pembrolizumab-treated patients,respectively,in which it was 14.9 versus 22.6 months in monotherapy and 14.7 versus 17.3 months in combination therapies.Treatment-related adverse events were consistent with safety outcomes of monotherapy and combination therapy in previous phase III studies.However,the incidence of rash was higher with sintilimab than pembrolizumab monotherapy.This is the first prospective phase 2 study to directly compare two anti-PD-1 antibodies as first-line treatment in advanced NSCLC.Sintilimab was efficacious and well-tolerated irrespective of PD-L1 expression level in patients with advanced NSCLC and had similar efficacy and safety to pembrolizumab.

Disulfiram, an aldehyde dehydrogenase inhibitor, works as a potent drug against sepsis and cancer via NETosis, pyroptosis, apoptosis, ferroptosis, and cuproptosis

Regulated cell death(RCD)is essential for maintaining cell homeostasis and preventing diseases.Besides classical apoptosis,several novel nonapoptotic forms of RCD including NETosis,pyroptosis,ferroptosis,and cuproptosis have been reported and are increasingly being implicated in various cancers and inflammation.Disulfiram(DSF),an aldehyde dehydrogenase inhibitor,has been used clinically for decades as an anti-alcoholic drug.New studies have shown that DSF possesses potent anti-inflammatory and anti-cancer effects by regulating these new types of RCD.Here,we summarize the mechanisms and discuss the potential application of DSF in the treatment of cancers and inflammatory diseases.

The disruption of hematopoiesis in tumor progression

Human adult hematopoiesis maintains homeostasis by replacing depleted progeny pools and inducing a primary immune response to infectious diseases and tumors.Recent studies have shown that tumor progression is associated with profound perturbations in hematopoiesis.Scientists have sought to clarify the complex mechanisms underlying the developmental fate of hematopoiesis by assessing hematopoietic stem and progenitor cells in various tumors.Results have shown that tumors disrupt normal hematopoiesis,resulting in extramedullary hematopoiesis and myeloid skewing.The key regulatory roles played by myeloid-derived suppressor cells induce immune suppression.Here,we summarize recent findings and discuss mechanisms underlying the disruption of hematopoiesis in solid tumors.