The Significance Of CD47-SIRPα Pathway In Diffuse Large B-cell Lymphoma

BackgroundThe association between the innate immunity and cancer immune evasion is gaining investigational interest.CD47 is a novel immune checkpoint of the innate immune system.Cancer cells evade the innate immune surveillance by upregulating CD47 which interacts with its receptor SIRP α on macrophages.This interaction signals "Do’n’t eat me" to the macrophage and subsequently inhibits its phagocytic activity.Previous studies have revealed that the CD47-SIRP α axis plays an critical role in immune escape of diffuse large B cell lymphoma(DLBCL),in which elevated CD47 expression predicts inferior patient outcomes independent of the IPI score.In a recent phase lb clinical trial,an anti-CD47 monoclonal antibody has proved to be surprisingly effective for a subset of relapsing/refractory DLBCL.Although this inhibitory pathway was found nearly 10 years ago,the mechanism of immune evasion via the CD47-SIRP a axis and its clinical implication in DLBCL management remain largely to be investigated.Several critical questions need to be answered.First,DLBCL is highly heterogeneous in nature but the expression pattern of CD47 in different DLBCL subtypes is unclear.The correlation between CD47 expression level and DLBCL patient outcomes also needs to be validated with immunohistochemistry.Second,the mechanism of CD47 upregulation in DLBCL is unknown and it is hypothesized to be related to some known cytogenetic aberrations in DLBCL,which needs to be further investigated Third,any therapeutic targeting CD47 relies on phagocytic activity of host macrophages to take effect,but the mononuclear phagocyte system is hypothesized to be functionally defective in DLBCL.Whether these DLBCL patient-derived macrophages can be similarly activated as in previous studies needs to be confirmed experimentally.Answering these questions will undoubtedly lead us to better understanding of the biology in DLBCL immune evasion as well as the anti-tumor mechanisms of most anti-CD47 antibodies currently in pipeline.Meanwhile,novel biomarkers may be discovered to improve patient evaluation and stratification regarding their immune escape status,thus providing guidance for future administration of CD47-SIRP a targeting drugs in DLBCL patients.Purposes1.To investigate CD47 expression in DLBCL cancerous tissue and its correlation with patient outcomes by immunohistochemistry staining2.To explore any potential association between CD47 locus copy number aberration and CD47 gene expression3.To study the presumed phagocytic defect in DLBCL patient-derived macrophages and their response to an anti-CD47 antibodyMethods1.The FFPE samples from 74 DLBCL patients diagnosed at Peking Union Medical College Hospital(PUMCH)were reviewed and evaluated for CD47 expression via immunohistochemistry staining.The results were further analyzed for correlation with the clinicopathological features and survival of this DLBCL cohort.2.Two independent external DLBCL cohorts were included,of which the original datasets regarding copy number variation and gene expression profiling were accessible from the NCBI GEO database.Bioinformatics was used to investigate the correlation between the copy number variation and gene expression of the CD47 locus.3.Peripheral blood samples from 6 newly diagnosed DLBCL patients from PUMCH and 6 healthy volunteers were used in the in vitro phagocytosis assay.CD 14 positive monocytes were selected with magnetic beads,then cultured and subsequently differentiated into macrophages.In vitro phagocytosis of tumors and the response to an anti-CD47 antibody were tested of these macrophages by flow cytometry.Results1.Elevated CD47 expression was detected with immunohistochemistry in 56.5%of all DLCBL tumor samples.The ABC subtype shows significantly higher CD47 expression than GCB subtype.A remarkably higher percentage of CD47 over-expression was noted in MYC-positive patients than in MYC-negative patients(78%vs 41%).Independent of the IPI score,the level of CD47 expression can successfully predict PFS of DLBCL patients treated with R-CHOP regimen.2.A total of 33(16.3%)patients and 34(23.0%)patients from the two external DLBCL cohorts were found to harbor CD47 gene copy gains(copy number≥3).The frequency is significantly higher in the ABC subgroup(35.1%and 46.9%,respectively)when compared with GCB patients.Besides,CD47 copy gain is able to predict a significant inferior overall survival in DLBCL patients,independent of the IPI score.Finally,the gene expression level is positively correlated with the copy number of the CD47 gene.3.At baseline,DLBCL patient-derived macrophages showed significantly decreased phagocytic activity against tumor cells when compared with macrophages derived from healthy controls.Administration of an anti-CD47 antibody further enhanced in vitro phagocytosis in both patient and control groups.The control group showed a significantly higher response to CD47 antibody,as measured by change in phagocytosis rate,than the patient group.Conclusion1.Immunohistochemistry is a plausible and convenient method for evaluation of CD47 expression in DLBCL FFPE samples.The ABC subtype shows a significantly higher level of CD47 expression than the GCB subtype,which is consistent with previous gene expression profiling studies.CD47 over-expression is closely related to MYC over-expression in DLBCL.Finally,elevated CD47 expression can be used as an independent predictor of PFS in DLBCL patients treated with R-CHOP.2.Around 20%of DLBCL patients may carry CD47 locus copy gains,and the frequency is much higher in the ABC subtype.Copy number gain of the CD47 gene is an underlying mechanism of CD47 upregulation in at least a subset of DLBCLs.For DLBCL patients treated with CHOP regimen,copy number gain of CD47 can independently predict an inferior outcome,implying that those harboring CD47 copy gains are most likely to benefit from anti-CD47 therapeutics.3.Macrophages derived from DLBCL patients have decreased phagocytic activity against tumor cells,suggesting an immune defect of the mononuclear phagocyte system in DLBCL patients.Anti-CD47 antibody,however,is able to enhance phagocytosis and,as a result,actively involve these macrophages in anti-tumor immune response.