Hematopoietic stem and progenitor cells (HSPCs) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow ...niches on controlling the behavior of leukemic cells; however, whether normal hematopoietic stem cell (HSC) and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the mixed lineage leukemia‐AF9 (MLL‐AF9) murine acute myeloid leukemia (AML) in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL‐AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to antileukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation. Stem Cells 2015;33:3635–3642
Lymphoid aggregates (LA) are occasionally seen in bone marrow biopsies (BMB) of myelodysplastic syndromes (MDS) patients. Our aim was to evaluate their incidence and association with prognosis.
We ...compared BMB reports of MDS patients treated at the Tel Aviv Sourasky Medical Center (2011-2018), and controls (2015-2017, normal BMB), and examined the charts of the MDS patients (LA+ and LA-). Categorical, normally and non-normally distributed continuous variables were compared using Fisher's exact, independent t and Mann-Whitney tests respectively. Adjusted age, gender, lymphocytes, white blood cells (WBC) and diabetes mellitus (DM) Cox proportional hazard model examined survival at 12 and 24 months.
MDS patients (N=140) were older than controls (N=38; 74.1 vs 69.2 years, p=0.005); 34 MDS (24.3%) and 5 controls (13.2%) had LA+ (P=0.141). CD20/CD3 staining suggested LA polyclonality. MDS/LA+ (vs MDS/LA-) patients were younger, with a trend (not statistically significant) towards poor prognostic parameters: lower Hb, WBC, and platelets, higher LDH, BM cellularity, and IPSS-R score. The incidence of cardiovascular disease was similar, but MDS/LA+ had twice the incidence of DM (38.2% vs 19.0%, p=0.022). Similar trend for cancer (26.5% vs 14.3%, p=0.102). Twelve-month survival: 24/34 (70.6%) MDS/LA+; 88/106 (83.0%) MDS/LA- (p=0.140). This trend, seen in Kaplan-Meier curves, disappeared at 24 months. The hazard ratio for LA was 2.283 (p=0.055) for 12 months.
These preliminary data suggest LA are relatively common (24%) in MDS BMB, and might indicate poor prognosis. This may reflect involvement of the immune system in MDS. Future studies will examine larger groups, to clarify the incidence, significance and the pathophysiology.
Data regarding efficacy and toxicity of chimeric antigen receptor T (CAR-T) cell therapy in the elderly, geriatric population are insufficient. In 2019, tisagenlecleucel and axicabtagene-ciloleucel ...were commercially approved for relapsed/refractory diffuse large B-cell lymphoma. From May 2019 onwards, 47 relapsed/refractory diffuse large Bcell lymphoma patients, ≥70 years underwent lymphopharesis in three Israeli centers. Elderly (n=41, mean age 76.2 years) and young (n=41, mean age 55.4 years) patients were matched based on ECOG performance status and lactose dehydrogenase levels. There were no differences in CD4/CD8 ratio (P=0.94), %CD4 naive (P=0.92), %CD8 naive (P=0.44) and exhaustion markers (both HLA-DR and PD-1) between CAR-T cell products in both cohorts. Forty-one elderly patients (87%) received CAR-T cell infusion. There were no differences in the incidence of grade ≥3 cytokine-release-syndrome (P=0.29), grade≥3 neurotoxicity (P=0.54), and duration of hospitalization (P=0.55) between elderly and younger patients. There was no difference in median D7-CAR-T cell expansion (P=0.145). Response rates were similar between the two groups (complete response 46% and partial response 17% in the elderly group, P=0.337). Non-relapse mortality at 1 and 3 months was 0 in both groups. With a median follow-up of 7 months (range, 1.3-17.2 months), 6- and 12-months progression-free and overall survival in elderly patients were 39% and 32%, and 74% and 69%, respectively. EORTC QLQ-C30 questionnaires, obtained at 1 month, showed worsening of disability and cancer-related-symptoms in elderly versus younger patients. We conclude that outcomes of CAR-T cell therapy are comparable between elderly, geriatric and younger patients, indicating that age as per se should not preclude CAR-T cell administration. Longer rehabilitation therapy is essential to improve disabilities and long-term symptoms.
Chimeric Antigen Receptor T-cell (CAR T) therapy has become the preferable treatment in relapsed/refractory diffuse large B-cell lymphomas (DLBCL) patients. Detection of CAR Ts in peripheral blood ...smear (PBS) is challenging due to insufficient data regarding their morphology and low sensitivity. The morphological evolution of CAR Ts along their production process, and in patients, was established by Full-Field Morphology (FFM), a novel digital microscopy approach that provides highly sensitive PBS analysis. At day 8 of production, 42.7 ± 10.8% of the CAR T transduced cells exhibited activated morphology compared with 9.3 ± 3.8% in untransduced cells. Moreover, engagement of transduced CAR Ts with target cells resulted in further morphological transformation into activated morphology (83 ± 5.6% of the cells). In patients, the average number of day 5 CAR Ts, and their sustained presence, were significantly higher in patients obtaining complete response. A high number of activated morphology CAR Ts at day 14 was associated with prolonged cytokine release storm. Overall, CAR Ts exhibited heterogeneous morphology, with the activated morphology attributed predominantly to transduced cells following engagement with target cells. Post-transfusion CAR T detection was associated with increased complete responses. FFM CAR T surveillance in PBS may serve as a simple inexpensive method to provide clinically relevant insights into this treatment modality.
The relationships between cancer cells and the microenvironment play a critical role in cancer growth and development. The bone stroma consists of mesenchymal stem cells and mature osteoblasts that ...promote cancer growth. Yet it is not completely understood what are the molecular processes guiding cancer cells progression to the bone. In this study, a coculture assay and subsequent gene profiling arrays were used to compare the gene expression profile of a pre‐osteoblastic (PO) cell line (MBA‐15) with that of a mammary adenocarcinoma (DA3) cells. After coculture, cells were separated by magnetic beads based on the expression of CD326 antigen. RNA was purified and hybridized on gene expression array. The gene expression pattern changes were followed by qRT‐PCR. We demonstrate that cocultured DA3 cells express elevated levels of genes that regulate growth and responses to both hormonal stimulus and wounding, as well as reduced expression of genes related to lipid metabolism. Also, cocultured PO cells showed reduced expression of cell junction genes. The study presents a simplified model system, composed of PO and mammary cancer cells, that potentially mimics the molecular interactions in the tumor microenvironment which contribute to tumor progression.
Abstract
Cancer cell and metastatic progression from primary site are predominantly influenced by signals that are derived from primary tumor stroma. SVEP1, a novel CAM molecule cloned in our ...laboratory, shares its expression in preosteoblastic and mammary carcinoma cells. SVEP1 was suggested to be involved in cell adhesion processes. It is crucial to learn its role in metastatic breast carcinoma, leading to bone niche, since SVEP1 can be a candidate for breast cancer cells homing to bone. We aim to understand molecular regulation of metastatic process and advance our knowledge about molecules that play a role in cell-cell interactions and homing of mammary carcinoma cells to specific sites.
Bioinformatics tools were used to reveal putative promoter region of SVEP1 gene, potential transcriptional binding sites and to expand our knowledge about SVEP1 alternative spliced forms. SVEP1 promoter was cloned upstream to reporter gene and transiently tranfected for learning its transcriptional regulation. We detect specific transcription factors binding by Chromatin immunoprecipitation (ChIP). For identification of SVEP1 as an epigenetically silenced cancer-related gene cells will be treated with 5aza2'cytidine and/or trichostatin A, then SVEP1 gene expression will be examined by RT-PCR. 800bp upstream the TSS-SVEP1 promoter will be subjected to Methylation specific PCR (MSP) following different stimuli. Differential expression of SVEP1 spliced forms will be screened by RT-PCR using specific primers.
Results:
In transient transfection assays, TNFα and Estradiol increased SVEP1 promoter transcriptional activity. In ChIP assays TNFα enhanced ERα binding to SVEP1 promoter. These results were coincident with elevated SVEP1 full length mRNA expression following TNFα treatment. The 800bp upstream the TSS-SVEP1 promoter are transcriptionally regulated by DNA methylation. By using MSP this region was assessed to be highly methylated in DA3 cells, but less methylated in MBA15 cells. Methylation status of that region was regulated by TNF and Estrogen treatments. SVEP1 spliced forms expression pattern was characterized in preosteoblastic (MBA15) versus mammary carcinoma cells (DA3).
Conclusions:
Cell-cell contacts require engagement of adhesion factors yet to be determined. SVEP1, a novel CAM molecule, was suggested to be involved in cell adhesion processes. It is crucial to learn its role in metastatic breast carcinoma, leading to bone, since SVEP1 can be candidate for breast cancer cells homing to bone. Characterization of SVEP1 expression that participates in initial cell adhesion process is of prime interest. SVEP1's role in bone microenvironment may be important in creating an interactive network that regulates skeletal cells. Further research of SVEP1 expression regulation will help to understand its role as a mediator of interactions between normal and tumorogenic cells within bone marrow microenvironment.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 537.
SVEP1 is a multi-domain protein recognized as a cell adhesion molecule (CAM). In this study, we focused on the activity regulation of an alternative promoter (AP) and the expression of alternative ...splice forms of mRNA from SVEP1 gene. The expression of SVEP1 isoforms was analyzed on RNA isolated from pre-osteoblastic MBA-15 and mammary adenocarcinoma DA3 cells grown alone or following co-culture between these cells. The co-culture system aimed to mimic the cellular cross talk that exists in the bone microenvironment once the mammary cells invade the bone. We demonstrated that SVEP1 isoforms were differentially expressed between these cells. The various isoforms levels were affected by co-culturing or in cells treated with TNFα or estrogen. Both cell lines exhibited an increase of message levels of a and e isoforms following the co-culture conditions.
A novel aspect presented here is related to existence of an alternative promoter (AP) in SVEP1 gene. The AP was in silico predicted and analyzed for binding by specific transcription factors (TFIIB, ERα, NF-κB, Sp1 and pcJUN) using Chromatin immunoprecipitation (ChIP) assay. The binding of these TFs results in a non uniform binding pattern when comparing between the DA3 and MBA-15 cells. Using the demethylation agent, 5′-aza-deoxycitidine and histone deacetylase inhibitor, Trichostatin-A allowed to study the methylation level of the AP and the message expression. This study provides insights into alternative splice forms of SVEP1 and their regulation that may play a role within the bone niche with invading carcinoma cells.
► SVEP1 expression is regulated via alternative splicing. ► TNFα and Estrogen regulate SVEP1 splice forms expression. ► 5’-aza-dC and TSA induced de-methylation affects the splice forms expression. ► Transcription factors bind also to an alternative promoter thereby regulates its activity.
Patients with delayed B-cell reconstitution/B-cell aplasia after cellular therapy show decreased immunogenicity to the BNT162b2 mRNA COVID-19 vaccine. We prospectively evaluated both humoral and ...cellular immune response to a third vaccine dose in patients after allogeneic HCT (n = 10) or CD19-based chimeric antigen receptor T cells (CAR-T) therapy (n = 6) with low absolute B cell numbers and who failed to mount a humeral response after 2 vaccine doses. Humoral response was documented in 40% and 17% after allogeneic HCT and CAR-T therapy, respectively. None of the patients with complete B-cell aplasia developed anti-vaccine antibodies. Cellular response was documented in all patients after allogeneic HCT and in 83% of the patients after CAR-T. T-cell subclasses levels were not predictive for response, while a longer duration from infusion of cells was associated with a better cellular response. We conclude that cellular response develops with repeated vaccine doses even in patients with B-cell aplasia or delayed B-cell reconstitution, and these patients should therefore be vaccinated. These results should be considered in future studies analyzing immunogenicity in this population. Larger and longer follow-up studies are required to confirm whether cellular immunogenicity translates into vaccine efficacy.
PDF
