Editor's Note : From June 8th to 11th, the 28th European Hematology Association (EHA) Annual Congress was held in a combined online and offline format in Frankfurt, Germany. Top global experts in hematology shared various scientific themes, clinical and basic research, discussing cutting-edge achievements. Dr. Wei Jia’s team, currently stationed at Tongji Shanxi Hospital, Director of the Hematology Department at Shanxi Baiqiu'en Hospital, China, had three research results selected for presentation. Oncology Frontier specially invited Dr. Kui to present the findings.01
P1522 PERIPHERAL BLOOD SMEARS DISTINGUISH INFECTIVE FEVER AFTER CAR-T THERAPY
Background:
Chimeric Antigen Receptor T-cell (CAR-T) therapy provides an effective treatment for refractory or relapsed B-cell non-Hodgkin lymphoma (r/r B-NHL) and B-cell acute lymphoblastic leukemia (r/r B-ALL) patients. Cytokine Release Syndrome (CRS) is the most common toxicity associated with CAR-T therapy, often challenging to differentiate from infections. Early identification of CRS and infections in patients with fever after CAR-T infusion is crucial for clinical management. Recently, studies have suggested that the morphology of peripheral blood smears (PBS) can provide important clues for the dynamic assessment after CAR-T infusion. This study focuses on the characteristics of CAR-T cells in the PBS of patients who experience recurrent fever after CAR-T infusion, combining conventional pathogen detection methods, metagenomic next-generation sequencing (mNGS), and relevant clinical signs and symptoms to explore the causes of fever.
Methods:
From May 2019 to November 2022, 27 patients (R/R B-ALL, n=6, R/R T-ALL, n=1, R/R DLBCL, n=20) who experienced recurrent fever within one month after receiving CAR-T therapy at Tongji Hospital, Huazhong University of Science and Technology, were included. Recurrent fever was defined as a body temperature exceeding 38°C on two or more occasions within one month after CAR-T infusion. Infections were defined as confirmed pathogen carriers through culture or non-culture methods (including pathogen nucleic acid testing, mNGS, or conventional serological antigen/antibody testing).
Results:
Among the 27 patients who received CAR-T infusion in this study, 10 received CD19 CAR-T therapy, 1 received CD22 CAR-T therapy, 10 received CAR19/22 “cocktail” therapy, 5 received CD19-CD22 dual-target CAR-T therapy, and 1 received CD7 CAR-T therapy. Twenty out of 27 patients received CAR-T infusion after ASCT. All 27 patients experienced recurrent fever within one month after CAR-T cell infusion, with a median of 9.4 days (average 9.3 days; range 4-19) for the first PBS. After CAR-T cell infusion, the lymphoblast cells of all patients decreased to 0% in the first PBS, while atypical lymphocytes increased from 2% to 85%. We found a positive correlation between the percentage of atypical lymphocytes in PBS and CAR transgene copy number, as well as absolute lymphocyte count, suggesting that expanded lymphocytes consisted of CAR-T cells. Additionally, the proportion of atypical lymphocytes in PBS was significantly higher in the non-infection group than in the infection group (P<0.05), and the IL-6 levels were significantly higher in the infection group than in the non-infection group (P<0.05).
Furthermore, we observed similar morphological features among different CAR-T cells, including CD7, CD19, and CD22 CAR-T cells. They exhibited larger cell bodies (approximately 4-5 times the size of red blood cells) and irregular mononuclear cell-like shapes. The cell nuclei were mostly round or irregular, concave, unfolded, with loose chromatin, uneven distribution, small clumps, indistinct nucleoli, and visible pseudo-nucleoli. The cytoplasm was rich, with outer cytoplasm appearing dark blue, inner cytoplasm appearing light blue, significantly basophilic, and containing some neutrophilic granules, but no vacuoles were observed.
Conclusion:
In summary, our study results suggest that PBS can serve as an indicator of CAR-T cell expansion and can also be used to differentiate early fever causes after CAR-T therapy.
02
P712 SOLUBLE PD-L1 PREDICT POOR OVERALL SURVIVAL AND DISEASE PROGRESSION IN PATIENTS WITH DE NOVO MYELODYSPLASTIC SYNDROME
Background:
Myelodysplastic syndrome (MDS) is a group of myeloid tumors characterized by clonal proliferation of hematopoietic stem cells, ineffective hematopoiesis, peripheral blood cell reduction, and a high risk of progressing to acute myeloid leukemia (AML). Increasing evidence suggests an association between dysregulation of the immune checkpoint pathways and the pathogenesis of MDS. Soluble immune checkpoints (sICs) are the soluble forms of immune checkpoints that can be measured in peripheral blood from patients. In solid tumors, several studies have found that sICs play an important role in predicting prognosis and mediating resistance to immune checkpoint inhibitors (ICIs). However, it’s unclear whether sIC expression is dysregulated in MDS and its impact on patient prognosis. This study analyzed the expression levels of sICs in newly diagnosed MDS patients, cytokines potentially mediating PD-1/PD-L1 expression, and PD-1 protein expression on T cells, examining their potential correlations with clinical characteristics and patient prognosis.
Methods:
From June 2020 to April 2022, 79 patients newly diagnosed with MDS/sAML were included in this prospective study and clinically assessed for prognosis risk using the revised International Prognostic Scoring System (IPSS-R). Concurrently, 55 healthy donors (HD) were included as a control group. ELISA was used to detect the expression levels of various cytokines (sPD-1, sPD-L1, sCTLA-4, sLAG-3, sTIM-3, sGITR, sOX40, s4-1BB, sST-2) and cytokines (IFN-γ, IFN-α2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α). Flow cytometry (FCM) was used to measure PD-1 expression levels on T cells (CD3+, CD4+, CD8+, DPT, DNT).
Results:
Compared to the healthy control group, plasma sPD-L1 levels were significantly elevated in newly diagnosed MDS patients, while other sICs, including sPD-1, sLAG-3, sGITR, and s4-1BB levels, were lower. The percentages of PD-1+CD3+ cells, PD-1+CD4+ cells, and PD-1+CD8+ cells in MDS patients were significantly lower than those in the control group. The percentage of PD-1+CD4+ T cells was positively correlated with sPD-1 levels. In clinical-pathological features, lower levels of hemoglobin (<80g/L) were significantly associated with higher sPD-L1 levels. Additionally, high-risk group patients had significantly higher sPD-L1 levels than low-risk group patients. The study also analyzed the relationship between cytokines reported in previous literature to upregulate PD-1 levels on T cells and sICs levels, finding a positive correlation between TNF-α levels and sPD-L1 and sST2 levels. To further explore the relationship between sPD-L1 and prognosis in newly diagnosed MDS patients, the study determined the optimal cut-off value for sPD-L1 using ROC curve analysis as 55.81 pg/mL. Patients with higher plasma sPD-L1 levels (>55.81 pg/mL) had poorer overall survival (OS) with a median OS of 6.97 months (95% CI 5.533-9.167, P = 0.006). Multivariate Cox regression analysis further identified transfusion dependence (HR 3.151, 95% CI 1.475-13.952, P = 0.008) and higher sPD-L1 levels (HR 3.472, 95% CI 1.122-9.862, P = 0.039) as independent risk factors affecting OS in newly diagnosed MDS patients.
Conclusion:
Higher sPD-L1 levels are an independent risk factor for poorer overall survival in newly diagnosed MDS patients. Additionally, higher sPD-L1 levels are associated with lower hemoglobin concentration and MDS progression.
03
P472 SINGLE-CELL PROFILING REVEALS HETEROGENEITY OF EXPANDED PLASMACYTOID DENDRITIC CELLS IN ACUTE MYELOID LEUKEMIA
Background:
Plasmacytoid dendritic cells (pDCs) are the primary producers of type I interferon (IFN-I) and play a crucial role in immune responses. Under physiological conditions, the proportion of pDCs in nucleated cells in the bone marrow or peripheral blood is less than 1%. However, in diseases characterized by uncontrolled proliferation of pDCs, such as blastic plasmacytoid dendritic cell neoplasm (BPDCN), there’s an excessive increase in pDCs. Abnormal expansion of pDCs in myeloid tumors like MDS/AML has also been reported, potentially contributing to disease progression. This study retrospectively analyzed and employed single-cell RNA sequencing (scRNA-seq) to explore differences in pDC characteristics between BPDCN and pDC-AML/MDS, investigating the transcriptional characteristics of pDCs in these diseases and their mediated mechanisms of therapeutic escape.
Methods:
A retrospective analysis was conducted on 31 patients diagnosed with pDC-AML/MDS and BPDCN from July 2014 to September 2022 at Tongji Hospital, Huazhong University of Science and Technology (1 case of pDC-MDS, 13 cases of pDC-AML, 17 cases of BPDCN). Additionally, 2 cases of pDC-AML patients and 1 case of BPDCN patients were prospectively included for single-cell transcriptome sequencing using 10x Genomics technology on bone marrow samples. Integrated analysis was performed using scRNA-seq data from 5 BPDCN patients and 5 healthy donors (HDs) obtained from public databases utilizing the same single-cell sequencing technology.
Results:
The median proportions of pDCs in pDC-AML/MDS and BPDCN patients were 7.4% (2.7-33.6%) and 35.5% (0.2-90.0%), respectively. As of September 2022, the median survival period was 6 months for pDC-MDS/AML patients and 9 months for BPDCN patients (P > 0.05). Results revealed that pDC-AML/MDS patients were insensitive to chemotherapy, with most patients (81%, 9/11) requiring two or more cycles of chemotherapy to achieve complete remission (CR). Patients not receiving HSCT had poorer overall survival (OS) compared to those who underwent HSCT (P<0.05). To further elucidate the role of pDCs in these patients, scRNA-seq was performed, identifying 35,796 cells from all samples (2 cases of pDC-AML, 6 cases of BPDCN, and 5 healthy donors) across 18 distinct cell subtypes on t-SNE maps. Differential gene expression (DEGs) exhibited different expression patterns between HDs, pDC-AML, and BPDCN patients. Between pDC-AML and HDs, 425 DEGs were downregulated and 609 DEGs were upregulated, while between pDC-AML and BPDCN, 1117 DEGs were downregulated and 1077 DEGs were upregulated. To further elucidate the function of these DEGs, gene set enrichment analysis (GSEA) was conducted, revealing upregulation of “TGF-β signaling pathway,” “NF-κB-mediated TNF-α signaling pathway,” and “p53 pathway” in pDC-AML compared to HDs, while the “interferon-α response” pathway was downregulated. These results suggest that pDCs from pDC-AML exhibit similar characteristics to AML precursor cells, and cytokines like TGF-β and TNF-α may mediate their proliferation.
Conclusion:
pDC-AML/MDS patients are insensitive to chemotherapy, and their bone marrow pDCs demonstrate unique transcriptional features. The analysis suggests these pDCs may originate from AML precursor cells, and their proliferation may be mediated by cytokines like TGF-β and TNF-α.
