Normal Volunteer Donors Research Articles

Deep Peripheral T Cell Immune-Profiling in Relapsed/Refractory Non-Hodgkin Lymphoma: Evaluation of Baseline Samples from the Epcoritamab Epcore NHL-1 Trial

Background: Lymphomas account for approximately 4% of cancer diagnoses worldwide, of which 90% are non-Hodgkin lymphomas (NHL). NHLs encompass a group of heterogeneous hematological malignancies with over 60 different subtypes; the most frequently diagnosed subtypes are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Treatment regimens incorporating α-CD20 monoclonal antibodies have become the standard-of-care for DLBCL and FL; however, intrinsic or acquired resistance will occur in the majority of patients and therefore newer therapeutic interventions are needed. Epcoritamab is a CD3 redirecting, CD20 targeting bispecific antibody that redirects patient tumor and peripheral T cells to lymphoma cells expressing CD20. Therefore understanding both the baseline T cell immune status and T cell fitness in patients with relapsed/refractory (R/R) disease is essential. Here we utilized a peripheral immune profiling multi-omics approach to enable deep characterization of the T cell immune landscape at baseline in R/R DLBCL and FL patient populations from EPCORE NHL-1, a phase 1/2, open-label, multi-center clinical trial of epcoritamab (NCT03625037; EudraCT 2017-001748-36). Methods:Samples and immune-phenotyping: Peripheral blood mononuclear cells (PBMCs) were prospectively collected from the EPCORE NHL-1 trial from patient whole blood and frozen for subsequent immune profiling. PBMCs were also collected from normal healthy volunteer (NHV) donors as age-matched controls. Multiplexed flow cytometry-based immune-phenotyping panels were developed and validated for clinical exploratory use to profile the major T cell subsets and their activation states, including CD4/CD8 naïve T cells (Tnaive), memory T cells (Tmem) and T-regulatory cells (Tregs). Eighty-seven DLBCL, 78 FL patient samples and 24 NHV controls were included in the study. Transcriptomics and TCRseq: PBMCs were also lysed for HTG EdgeSeq transcriptomic profiling and TCR repertoire sequencing (TCRseq). Normal healthy tissue gene expression was established by profiling NHV tonsils and lymph nodes as controls. FFPE DLBCL resections and core needle biopsies (n=99), FL (n=19) and normal lymph nodes (n=8) were sourced commercially. FFPE sections were processed into lysates and hybridized with HTG's Human Transcriptome Panel (HTP) probes for mRNA quantification. Results: Immune profiling of R/R DLBCL and FL patients uncovered distinct patient clusters and inter-disease specific differences in the Tnaive and Tmem cell subsets. Overall R/R FL patients showed elevated levels of CD19+ B cells at baseline compared to R/R DLBCL. DLBCL patients had increased levels of CD8+CD107a+ Tnaive /Tmem cells and PD-1+ Tregs compared to FL patients and NHV donors. In the CD4 T cell compartment, FL patients showed elevated levels of activated CD4+ T cells compared to DLBCL. Intriguingly, overall peripheral Tregs were higher in FL patients when compared to NHV donors and DLBCL patients. In good agreement with the EPCORE NHL-1 patient immune-phenotyping data in the periphery, transcriptomic profiling of commercially sourced NHL tumors showed an elevated Treg mRNA signature in FL when compared to DLBCL and NHV tonsil. Finally, the immune repertoire profiling by TCRseq in the periphery demonstrated that NHL patients generally exhibited lower clonal diversity than age-matched NHV donors. Conclusions: Comprehensive immune profiling of prospectively collected baseline PBMC samples from the EPCORE NHL-1 clinical trial using a multi-omics approach uncovered distinct inter- and intra-disease immune states in NHL patients. DLBCL patients had elevated expression of the degranulation and cytotoxic-specific marker CD107a in the periphery, suggesting an active immune state in these R/R patients. Notably, FL patients showed increased Tregs in the periphery and these data correlated with non-matched commercially sourced tumor when compared to DLBCL patients, while DLBCL patient Tregs had elevated T cell activation markers including PD-1. Finally, both DLBCL and FL patients had lower clonal diversity than NHV donors which may be a function of prior treatment. Ongoing studies include measuring the patient immune landscape over time and correlating immune pharmacodynamic/biomarkers with clinical outcomes.

Exceptionally Potent Cytotherapy Using T Cells Armed with Novel Tetravalent Recombinant Bispecific Antibodies Specific for GD2 and HER2

▪Introduction: T-cell based therapies have emerged as one of the major breakthroughs in anticancer treatment: Immune checkpoint inhibitors, chimeric antigen receptor gene-modified T-cells (CAR-T-cells), and T-cell engaging bispecific antibodies (BsAb) are leading the advances. In the era of personalized medicine, T-cells offer alternative strategies to overcome resistance to chemotherapy or small molecules. Yet, hurdles for such therapy can be crippling, such as inability of T cells to infiltrate “cold tumors”, cytokine release syndrome following T cell-based therapies, neurologic toxicity, and on-target off-tumor effects. To address these hurdles, polyclonal T-cells armed with GD2xCD3 or HER2xCD3 BsAb for cytotherapy hold promise. Ganglioside GD2 and HER2 are tumor associated surface antigens expressed in a broad spectrum of aggressive malignancies, while being restricted in normal tissues. Phase I trials of T-cells armed with a chemical conjugate of hu3F8 x mouse OKT3 (NCT02173093) or trastuzumab x mouse OKT3 (NCT00027807) demonstrated the safety of 160 x 106/kg/injection x 8 doses (or 1.28 x 109/cycle) with suggestion of clinical benefit. Here, we report the safety and efficacy of adoptive T-cell therapy armed with the recombinant forms of these BsAb for the treatment of GD2(+) and/or HER2(+) tumors in preclinical models.Methods: Recombinant anti-GD2 BsAb and anti-HER2 BsAb were made using the IgG(L)-scFv platform (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from normal volunteer donors were isolated, activated and expanded by CD3/CD28 beads in the presence of 100 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T cells (ATCs) were harvested and armed for 20 minutes at room temperature with -GD2-BsAb or HER2-BsAb. After washing, armed ATCs were tested for cell surface density of BsAb and antibody dependent T cell mediated cytotoxicity (ADTC) in vitro. In vivo anti-tumor potencies of armed T cells were tested against GD2(+) or HER2(+) cell lines or patient derived xenografts (PDXs) in BALB-Rag2-/-IL-2R-γϲ-KO (DKO) mice.Results: GD2-BsAb of the IgG(L)-scFv form showed superior potency over other bispecific platforms in vitro and in vivo. GD2-BsAb or HER2-BsAb armed ATCs showed potent antigen-specific cytotoxicity against GD2 or HER2 positive tumors such as neuroblastoma, melanoma and osteosarcoma in vitro over a range of antibody dose (5 to 500 ng/106 cells). Optimal arming per T cell required 25,000 to 45,000 idiotype(+) molecules. There was no evidence of activation induced cell death when confronted by antigen or tumor target. In vivo GD2-BsAb or HER2-BsAb armed ATCs could ablate neuroblastoma, malignant melanoma, and osteosarcoma tumors over a range of cell doses (10x106, 20x106 and 40x106 per injection, one to three times a week for 2 to 4 weeks) with a range of BsAb doses (5 ng to 500 ng/million of T-cells) without significant toxicities in DKO mice. By immunohistochemistry, the frequency of tumor infiltrating CD3(+) T-cells strongly correlated with tumor response.Conclusions:Using the IgG(L)-scFv format, GD2-BsAb or HER2-BsAb armed ATCs could provide a potent and economical cytotherapy platform against GD2(+) or HER2(+) tumors without the complexity of gene modification (as in chimeric antigen receptor modified T cells). At such low arming doses, where BsAb is T cell bound, where ADTC is not induced, and T cell expansion is not required for anti-tumor effect, clinical toxicity is expected to be low. DisclosuresCheung:Ymabs: Patents & Royalties.

Utilization of Peripheral Blood Cell-Free DNA in Myelodysplastic Syndromes: Clinical and Molecular Characteristics and Utilization for Genetic Analyses Using Conventional and Next-Generation Strategies

Abstract Background: Genetic mutations are detected in over 90% of patients with myelodysplastic syndromes (MDS). Some mutations may be critical not only for the pathogenesis of MDS, but also for disease progression to acute myelocytic leukemia and the acquirement of drug resistance; however, the detailed functions of these mutations remain unclear. To determine the roles of these mutations, genetic analyses using serially harvested tumor DNA are required. To date, genomic DNA from bone marrow (BM) cells has been utilized for genetic analyses of MDS patients, but bone marrow aspiration cannot be performed repeatedly because it causes physical pain in patients. Recently, peripheral blood cell-free DNA (PB-cfDNA) obtained from plasma and/or serum has received much attention as an alternative tumor DNA source, especially for solid tumors. Here, we demonstrate that PB-cfDNA may be used as an alternative DNA source instead of BM cells, that it reflects MDS disease status, and that the mutations in MDS can be successfully detected from PB-cfDNA by Sanger sequencing and next-generation targeted sequencing analyses. Aims: 1) Confirmation of the molecular and clinical basis of PB-cfDNA in MDS; 2) detection of genetic mutations using PB-cfDNA with conventional and next-generation sequencing strategies; and 3) optimization of sequencing conditions using PB-cfDNA. Methods: PB-cfDNA from patients with MDS and other related diseases (N = 33) and normal volunteer donors (N = 19) was analyzed. The concentration of PB-cfDNA was measured and correlations between the PB-cfDNAconcentration and clinical data were analyzed. DNA sequencing was performed using Sanger sequencing and targeted sequencing was performed using a TruSight Myeloid Sequencing Panel (Ilumina). Results: The plasma PB-cfDNA concentration was significantly higher in MDS patients than in normal donors (p = 0.0405), and was significantly higher in those with a higher International Prognostic Scoring System (IPSS) score than in those with a lower score (p = 0.0339). The concentration of plasma and serum PB-cfDNA was significantly correlated with the serum lactate dehydrogenase (LDH)level (both p < 0.0001) and the blast cell count in PB (plasma, p = 0.0373; serum, p = 0.0274). Since PB-cfDNA showed a fragmented pattern reflecting its oligonucleosomal structure, amplification using primers for PCR-based sequencing analyses were optimized when the size of the PCR products were approximately 160 bp. For Sanger and targeted sequencing, 1 and 50 ng of PB-cfDNA, respectively, were required for each assay. Almost all genetic mutations detected by Sanger and targeted sequencing using BM cells from MDS patients (TET2, IDH2, SETBP1, U2AF1, SRSF2, NRAS, TP53) were also detected using the PB-cfDNA, but they were not detected in the germline controls. Serially harvested PB-cfDNA samples from a patient with refractory anemia with excess blasts-1 (RAEB-1) who underwent cord blood transplantation (CBT) after 5-azacytidine treatment were utilized for genetic analyses; they showed a correlation between PB-cfDNA concentration and the clinical course, and that the U2AF1 and SETBP1 mutations detected before CBT were no longer detectable 150 days after CBT. Discussion: These data suggest that PB-cfDNA likely originates from MDS clones, which reflect their disease status, and that they can be utilized as a biomarker and an alternative promising source of tumor DNA instead of BM cells for genetic analyses; including not only conventional Sanger sequencing, but also next-generation targeted sequencing. However, since the PB-cfDNA concentration of some MDS patients was too low (less than 10 ng/1 mL plasma) to be used for targeted sequencing and/or whole exome sequencing, optimization of the sensitivity of sequencing analyses is still required. Disclosures Tomita: Janssen Pharmaceutical K.K.: Consultancy. Ishikawa:GlaxoSmithKline K.K.: Research Funding. Kiyoi:Alexion Pharmaceuticals: Research Funding; Eisai Co., Ltd.: Research Funding; Japan Blood Products Organization: Research Funding; FUJIFILM RI Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Bristol-Myers Squibb: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding.

Of Mice and Men – or a Lack of Positive Effect of Enhanced Vegetative Nervous System Tonus on Mobilization of Hematopoietic Stem and Progenitor Cells – Results in Patients with Elevated Catecholamine Level in Peripheral Blood Suffering from Acute Psychotic and Anxiety Disorders

Background. It is well known that hematopoietic stem/progenitor cells (HSPCs) circulate under steady-state conditions at detectable levels in peripheral blood (PB), with their numbers increasing in response to stress, inflammation, and tissue and organ injury. Moreover, it has been demonstrated in mice that enhanced tonus of vegetative nervous system regulates mobilization of HSPCs into PB. Moreover, UDP-galactose:ceramide galactosyltransferase-deficient mice, which exhibit aberrant nerve conduction and do not release norepinephrine (NE) into the BM microenvironment, do not mobilize HSPCs in response to G-CSF. However, as recently reported modification of sympathetic output does not affect G-CSF-induced mobilization in humans, as would be predicted. Specifically, normal human HSPC volunteer donors who were receiving NE reuptake inhibitors (NRI) for depression or β2-blockers because of hypertension mobilize in a similar manner as normal controls (Leukemia 2013; 27:24-31). Mobilization in these patients was neither enhanced by NRI administration nor suppressed by β2-blockers, as one would expect based on murine data reported in the literature.Aim of the study. To address this intriguing issue and discrepancy between human and mice, we analyzed levels of circulating HSPCs in patients suffering from acute psychosis and anxiety disorders – clinical situations with elevated level of catecholamine in PB. Namely, these patients are under the influence of several neural mediators, and it is well known that the levels of NE and dopamine are elevated in peripheral tissues and blood.Material and Metods. Enrolled in this study were 30 unrelated individuals with a diagnosis of the first-episode psychosis and 30 patients suffering from acute anxiety disorders. The patients were compared with an ethnic- and gender-matched control group of 35 healthy volunteers without psychiatric disorders, which were excluded according to an examination by a specialist psychiatrist. Patients with a history of serious lifetime medical events, organic brain injuries, or drug/alcohol dependence were excluded from the study. Mobilization of HSPCs was evaluated by i) FACS to enumerate the number of CD34+, CD133+, CD34+CD45+Lin–, and CD133+CD45+Lin– cells circulating in PB, which are enriched for HSPCs, as well as by ii) functional in vitro assays to detect the number of CFU-GM and BFU-E clonogenic progenitors circulating in PB. In parallel we measured level of adrenaline, norepinephrine (NE) and dopamine in PB serum. Both cells and catecholamine levels were enumerated in acute psychotic and anxiety disorders patients before and after treatment and compared with age- and sex-matched controls.Results. We did not observe any significant differences in the numbers of circulating CD34+, CD133+, CD34+CD45+Lin–, and CD133+CD45+Lin– cells as well as clonogenic BFU-E and CFU-GM between normal controls and psychotic patients and patients with anxiety disorders. In particular number of circulating in PB HSPCs was not affected by increased level of adrenaline, norepinephrine and dopamine in PB of patients suffering from acute psychotic syndromes.Conclusions. Our data argue against an effect of enhanced vegetative nervous system tone on the number of HSPCs circulating in PB in humans. Our negative data performed on patients suffering from acute psychoses and anxiety disorders somewhat corroborate data reported for normal HSPC volunteer donors that were previously treated with NRI because of depression or with β2-blockers because of high blood pressure and mobilized with G-CSF (Leukemia 2013; 27:24-31). This finding suggests that there are some clear differences between rodents and humans in the effect of the vegetative nervous system on HSPCs mobilization. DisclosuresNo relevant conflicts of interest to declare.

Calreticulin mRNA Expression Levels and Clinicopathologic Characteristics in Hematologic Malignancies

Background: Calreticulin (CARL) is a multifunctional protein that plays a role in calcium homeostasis and chaperoning molecular processes. Recently, somatic mutations in the gene CALR have been found in 20-25% of patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL and the JAK-STAT pathway appears to be activated in MPN with mutated CALR. In the other hand, CARL overexpression has been reported in a variety of solid tumors and is also associated with a more invasive and advanced malignant processes [Zamanian M et al, Pathol Oncol Res 2013]. The objective of this work was to evaluate the expression levels of the CALR and clinicopathologic characteristics in patients with a variety of hematologic malignancies. To the best of our knowledge, this is the first study to compare CALR expression levels among hematologic malignancies.Methods: The study group included 127 patients having diagnosed as malignant or benign hematologic diseases in Mokdong Hospital, Ewha Womans University School of Medicine, Seoul, Korea between 2010 and 2014 ; 44 AML, 16 acute lymphoblastic leukemia (ALL), 5 mixed phenotype acute leukemia, 35 MPN or (7 polycythemia vera, 14 ET, 4 MPN-U, 10 CML), 5 MDS/MPN, and 22 benign hematologic condition. RNA was extracted using bone marrow aspirates or peripheral blood at diagnosis. CALR mRNA levels were measured through quantitative real time-PCR targeting CRLR gene in exon 7-8 boundary (Taqman Gene Expression Assays, Life Technologies). As a reference control, G6PDH mRNA was simultaneously processed in the same samples and peripheral bloods obtained from 4 different normal volunteer donors were pooled for normal control. The relative quantification of CALR mRNA is calculated and the association between CALR expression levels and clinicopathologic parameters were reviewed based on the medical records retrospectively. All statistical analyses were performed using Analyse-it, 3.70 (Analyse-it Software, Ltd, UK).Results: Higher expression of CALR mRNA level was noted in AML patients (median:1.18, range:0.24-6.38) when compared with ALL (median:0.44, range:0.19-1.02), mixed phenotype acute leukemia (median:0.58, range:0.01-2.01), MPN (median:0.58, range:0.14-1.10), MDS/MPN (median:0.25, range:0.10-0.60) and benign-hematologic conditions (median:0.50, range:0.14-1.10) (p<0.0001) (Fig.1). Although calcium depletion is regarded as an important activator of the CALR gene promoter, we were not able to identify any significant correlation between calcium level and CARL mRNA level (r= -0.236, p= 0.0146). In addition, no correlation was detectable between CARL mRNA level and hemoglobin (r= -0.263, p= 0.0028), WBC count (r= 0.009, p= 0.9225) and platelet count (r= -0.193, p= 0.0300). No difference of CARL expression level was noted according to the WHO 2008 AML sub-classification (Fig.2). When gene expression variable was categorized in four intervals (<0.5, 0.5< <1.0, 1.0< <2.0, and >2) in AML patients, no significant differences in the gene expression level were seen in relation to the age, gender, calcium level, hemoglobin, WBC count, platelet count, blast count, relapse rate and overall survival. Of 24 MPN patients (7 polycythemia vera, 14 ET, 4 MPN-U), CALR gene expression was higher in JAK2 non-mutated patients (median:0.76, range:0.14-0.89) than JAK2 mutated patients (median:0.58, range:0.23-1.10 ), but not statistically significant, probably due to the limited number of samples studied (p=0.8019).Conclusion: This study showed that the up-regulated expression of CALR in AML compared with other hematologic malignancies was notable, but the association of CALR expression and clinicopathologic characteristics were not found. The exact roles of CALR is yet be clarified due to its diverse interactions into various cellular processes and signaling pathways and further studies are needed to investigate the clinical relevance of CALR expression in hematologic malignancies. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Plasma von Willebrand factor multimer quantitative analysis by in-gel immunostaining and infrared fluorescent imaging

Introduction Electrophoretic analysis of plasma von Willebrand factor (VWF) multimer distribution and infrastructure is essential for subtyping von Willebrand disease. To improve the sensitivity, precision and efficiency of this assay, we developed and validated a new in-gel infrared fluorescent VWF multimer imaging method to visualize and quantify VWF multimers directly in the agarose gel, thus eliminating electroblotting or autoradiographic steps. Materials/Methods VWF multimer analyses of plasma samples from 34 patients with known von Willebrand disease or acquired von Willebrand syndrome, 9 patients with acquired VWF abnormalities, 26 normal volunteer donors and 49 patient samples referred for von Willebrand factor multimer analysis were performed by both traditional autoradiographic and the new infra-red imaging methods and compared. VWF multimer image data were electronically acquired, archived and analyzed. Results The in-gel infrared method has a sensitivity of detecting VWF antigen as low as approximately 1.6 IU/dL, a reliable fluorescent intensity with intra- and inter-day variability (CV) of 5% and 6% respectively, and provides superior imaging resolution and shortened test turnaround time. Using intermediate resolution agarose gel electrophoresis, the infra-red method sensitively detects subtle loss of highest molecular weight von Willebrand factor multimers in plasmas with acquired VWF abnormalities and in commercial normal reference plasmas, and provides evidence of increased proteolysis of ultralarge multimers in some type 2 VWD plasmas. Conclusions The in-gel infrared fluorescent VWF multimer imaging method provides a sensitive, reliable, efficient and robust system to improve laboratory testing for von Willebrand disease classification.

Effects of imatinib mesylate on normal bone marrow cells from chronic myeloid leukemia patients in complete cytogenetic response

Information on the effects of imatinib mesylate (IM) on the non-clonal bone marrow (BM) cell compartment is scanty. We have analyzed the gene expression profile of BM hematopoietic cells after IM therapy in 20 patients with chronic myeloid leukaemia (CML) in complete cytogenetic response (CCyR) and compared it with that of normal volunteer donors by oligonucleotide microarrays. In CCyR CML samples, IM induces a decrease in proliferation as well as increase in apoptosis and ubiquitination in residual non-clonal BM cells. In addition, IM diminishes cell-to-cell adhesion and downregulates the expression of the erythropoietin (EPO) receptor gene. The latter was confirmed by RT-PCR.

Immunophenotyping by flow-cyotmetry in MDS and its potential role in remission assessment post treatment

18008 Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms with abnormal maturation and differentiation of one or more cell lineages. Diagnosis is complex and attained by evaluation of peripheral blood, bone marrow biopsy and cytogenetics/FISH. Immunophenotyping (IP) has recently been recommended as a co-criterion for diagnosis in a recent consensus publication by Valent et al. Its role in remission assessment is yet to be defined. Our aim was to evaluate the role of IP in a prospective MDS cohort treated with lenalidomide and stem cell factor and correlate morphological response with a modified MDS flow-cytometric scoring system by Wells et al. This scoring system encompasses aberrancy of myelomonocytic populations ranging from 0 in normals to 8 if multiple myelomonocytic aberrancy is determined. Methods: Four colour flow-cytometric analysis was performed to assess blast, granulocytic and monocytic populations. Antibody combinations were compared against isotype controls. Patterns of antibody expression were assessed on marrow samples from 5 normal volunteer donors and 16 haematology patients with a normal peripheral blood and bone marrow evaluation. Sequential assessment was performed using Cheson 2006 morphologic response criteria and modified MDS flow-cytometric scoring system. Results: Normal volunteers included 3 males, median age 27 (range 25–43). The haematology cohort included 16 lymphoma patients, median age 46 (range 19–79). 8 MDS patients were evaluable, median age 57 (range 43–89), including 6 males. MDS WHO subtypes included 3 cases 5q- syndrome, 2 RCMD ± (RS), 2 MDS-U, 1 RAEB type I. 5q- syndrome patients had isolated karyotypic anomalies, 5 patients had a normal karyotype. MDS flow scores pretreatment ranged from 3–5. Of the 6 patients who responded (4 CR, 2 PR) 4 demonstrated substantial improvement in their MDS flow scores with one attaining a normal score. 1 patient did not have sequential immunophenotyping after early withdrawal from the trial. Conclusions: This early cohort demonstrates potential utility of the MDS scoring system not only as a diagnostic tool but in post treatment response assessment. Larger numbers of patients are required in a prospective setting for confirmation of these findings. No significant financial relationships to disclose.

Abnormally High Levels of Circulating CD34+CD38−, CD34+CD38+, and CD10+ Hematopoietic Stem and Progenitor Cells in the Peripheral Blood of B-Cell Chronic Lymphocytic Leukemia (B-CLL) Patients Suggest the Presence of Additional Therapeutic Target Cells.

Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disorder manifested by a clonal expansion of mature long-lived functionally defective lymphocytes that are predominantly of B-cell origin (>95% of cases). CLL cells typically express the pan-B-cell antigens CD19 and CD20 as wells as CD5, an antigen of mature T-cells, which is present in 95% of B-CLL patients and is helpful for diagnosis. The disease has a median survival of 8–10 years. Asymptomatic patients (RAI stages 0–2) are often not treated. RAI stages 3 & 4 are generally treated. Current therapies for CLL included a combination of monoclonal antibodies (anti-CD20 monoclonal antibody, rituximab) with purine analogs (fludarabine or pentostatin) and alkylating agents (cyclophosphamide). Current therapies to manage B-cell CLL are not curative and therefore suggest that the current strategy of targeting therapy against B-cells may leave behind residual disease that contributes to progression or relapse. The importance of more primitive Hematopoietic Stem and Progenitor cell populations in B-CLL patients has not been considered. We hypothesized that circulating cells may be spontaneously mobilized into the periphery of CLL patients and would therefore contribute to the peripheral blood microenvironment. To test this hypothesis we examined existing CLL cell populations from un-treated “watch-and-wait” B-CLL patients as compared to normal volunteer donor peripheral blood cell populations utilizing multivariate flow cytometric analysis. We determined CD34+CD38−, CD34+CD38+, and CD10 levels in normal peripheral blood to be 81.0±14.5, 497.3±78.33, and 83.6±17.5 cells/ml respectively. We report here the presence of abnormally high levels of these stem and progenitor cell populations in B-CLL patients (see table). Although the mechanism behind the release of these cells into the periphery in B-CLL patients is unknown, it is reasonable to propose that they may be contributing to the manifestation of the disease and are therefore potential novel target cell populations for future therapies.B-CLL PatientsWBCβ2mCD34+CD38−CD34+CD38+CD10+7.932.42,52614.623,11911.86U3,14558.21,44912.5U30711.721,77316.93.76,4502513,77117.243.0285.798,02419.371.84,0861808,24719.9U1,79018.63,09919.92U52934.116,03925.811.82,9371944,61731.212.920,75330424,34043.131.51,577260.49281,97673.983.21,0991,76777,525105.532.23,26622024,070White Blood Cell (WBC) counts are expressed as 1x106 cells/ml. All other counts are expressed as cells/ml

Flow Cytometry Analysis of Peripheral Blood CD4+/CD25+/FOXP3+ T Regulator Cells from 11 Patients Treated with Ipilimumab Following Relapse of Malignancy after Allogeneic Hematopoietic Stem Cell Transplantation.

We conducted a multi-center dose-escalation trial to assess whether CTLA-4 blockade is safe and efficacious as immunotherapy in patients with relapse of malignancy following allogeneic hematopoietic stem cell transplantation (allo-HCT). We hypothesized that since CTLA-4 is a negative regulator of effector T-cell activation, inhibition of CTLA-4 ligation with a blocking human monoclonal antibody (ipilimumab) will augment graft-versus-malignancy. Here we report on the effects of a single dose of ipilimumab on in vivo T cell subsets from 11 patients treated at the highest dose level (3 mg/kg) and compare these findings with those in normal volunteer donor lymphocytes. We analyzed the expression of intracellular CTLA-4 and FOXP3 on CD4+/CD25+ Treg cells, intracellular cytokines and surface markers for T-cell activation on peripheral T cells from 11 patients and 9 normal donors. Peripheral blood mononuclear cells (PBMC) from patients before (day 0) and after the treatment at day 1, 3, 7, 14 and 30 were stained with a panel of antibodies and analyzed by flow cytometry. Lymphocytes from normal donors at time zero and 3 days after culture in IL-2 (200u/ml) were used as controls. The pre-treatment expression of intracellular CTLA-4 was significantly higher in CD4+ T cells from patients than normal controls and was increased further after antibody treatment from 7.1±3.8% at day 0 to 18.2±7.1% at day 30 (p=0.02). Although the expression of FOXP3 in CD4+/CD25+ T cells was higher in patients than in normal donors (6.3±4.8% compared with 2.0±1.6%, p=0.02), there was no significant change in the levels following ipilimumab infusion. The expression of CD4+/CD25high in the patients was 7.7±2.8%, higher than the normal donors (2.3±1.1%). However, 6/11 cases had increased expression while others had decrease or no change, overall there was no statistically significant change. CD4+/CD25low activated T cells were elevated in 10/11 patients before ipilimumab (42.1±8.5%). Their levels were not affected by CTLA-4 blockade. CD8+/CD69+ activated T-cells were significantly increased in 8/11 patients within the 30 days after ipilimumab treatment but typically returned to baseline values on longer follow-up. CD4+/CD69+ and CD4+/HLA-DR+ T-cells were unchanged following ipilimumab treatment. These data show that a single dose of ipilimumab enhances levels of some subsets of activated T cells without a significant effect on cells with a T-regulatory phenotype.

Anti-thrombomodulin antibodies and venous thrombosis.

Thrombomodulin (TM) is a cell surface endothelial glycoprotein having anticoagulant properties. It inhibits thrombin, and activates protein C, leading to the inhibition of activated factors V and VIII. TM autoantibodies could theoretically predispose to thrombosis. We have tested 83 unselected patients with deep venous thrombosis, 36 males and 47 females aged from 1 to 70 years [mean +/- standard deviation (SD), 34.2 +/- 14.5 years] for the presence of IgG anti-TM in their plasmas. Tests were performed by enzyme-linked immunosorbent assay (ELISA) using recombinant human TM kindly provided by PAION GmbH (Aachen, Germany). Results are expressed as the optical density (OD) differences between coated and un-coated wells. Plasmas from 83 normal volunteer donors were used to define the cut-off value as the mean of absorbance of the control group + 3 SDs. The median OD of normal controls group was 0.024 (mean, 0.034; SD, 0.066; range, -0.048 to 0.309). The median OD obtained with plasmas from patients was 0.048 (mean, 0.114; SD, 0.215; range, -0.039 to 1.312) and was significantly higher than that of the control group (P < 0.0001). Choosing a cut-off value of 0.232 (mean OD of the control group + 3 SDs), 11 patients are considered as positive for IgG autoantibodies to TM and three normal controls are weakly positive. Selected plasma with IgG anti-TM and purified IgG were further tested by dot blot using recombinant purified TM and were found positive. Purified IgG of positive plasmas inhibits protein C activation by TM and thrombin, suggesting that anti-TM antibodies have a procoagulant effect. Interestingly, in our study, anti-TM antibodies are found in three of six patients with Budd-Chiari syndrome and four of eight patients with cerebral venous thrombosis. In conclusion, thrombomodulin autoantibodies could be a new interesting marker of thrombophilia easily detected by ELISA.

WT1 gene expression: useful marker for minimal residual disease in childhood myelodysplastic syndromes and juvenile myelo-monocytic leukemia?

The WT1 gene is considered to be highly expressed in patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia and is thought to play a key role in maintaining the viability of leukemia cells. However, little is known about the WT1 gene expression levels in pediatric patients with juvenile myelo-monocytic leukemia (JMML) and myelodysplastic syndromes (MDS). We studied WT1 expression in diagnostic bone marrow (BM) and peripheral blood (PB) samples of 90 patients with JMML, low grade MDS, advanced MDS and myelodysplasia-related AML in BM (n = 20) and PB (n = 18) samples of normal healthy volunteer donors.

In vitro effects of interferon-γ and tumor necrosis factor-α on CD34+ bone marrow progenitor cells from aplastic anemia patients and normal donors

Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.

Granulocyte colony-stimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells

Objective Because the effect of granulocyte colony-stimulating factor (G-CSF), which is widely used for allogeneic stem cell transplantation, on DNA function and stability has not yet been unequivocally elucidated, the aim of this study was to determine whether G-CSF leads to epigenetic and/or genetic modifications. Materials and methods Molecular cytogenetic techniques based on fluorescence in situ hybridization technology were used. Results Lymphocytes of G-CSF mobilized donors displayed epigenetic (altered replication timing of alleles) and genetic (aneuploidy) alterations similar to those observed in lymphocytes of cancer patients. Specifically, in the donors' lymphocytes, biallelically expressed genes ( TP53 and AML1) and a repetitive noncoding DNA sequence associated with chromosome segregation ( CEN17) showed loss of synchrony in allelic replication timing (allele-specific replication). Each displayed a highly asynchronous pattern of allelic replication similar to that characterizing monoallelic expressed genes. This non–locus-specific epigenetic phenomenon, which also affects DNA sequences associated with chromosome segregation, was accompanied by aneuploidy. Although the loss of replication synchrony in the lymphocytes of G-CSF mobilized donors was a transient epigenetic modification, aneuploidy remained unchanged. The G-CSF effect also was observed after G-CSF administration in vitro. 5-Azacytidine, a DNA methylation blocking agent, inhibited G-CSF in vitro induction of allele-specific replication. Conclusion G-CSF, probably via changes in DNA methylation capacity, leads to cancer-characteristic DNA modifications in lymphocytes of normal mobilized donors.

Response of human CD34+ cells to CXC, CC, and CX3C chemokines: implications for cell migration and activation.

Ultrastructural studies of marrow and examination of the in vivo processes of stem cell homing and mobilization show that multipotential hematopoietic progenitors are able to traverse endothelial cells. The regulation of this process by various classes of chemokines was studied in this report, using an in vitro model of transendothelial migration. Human umbilical vein endothelial cells (HUVECs) or bone marrow-derived endothelial cells (BMECs) were grown to confluence on 3-microm microporous membrane inserts and placed in 24-well culture plates. CD34(+) cells isolated from normal volunteer donor marrow by immunoadsorption or magnetic bead selection techniques were added to the inserts and various individual chemokines were added to the lower chamber of the culture plates in serum-free conditions. After 24 h, the percentage of transmigrated cells was determined. A mean of 8.5% of unfractionated marrow CD34(+) populations migrated, and all chemokines tested, with the exception of macrophage inflammatory protein-1alpha (MIP-1alpha), had some positive effect on this migration. The greatest effects were seen with stroma-derived factor-1alpha (SDF-1alpha) and stroma-derived factor-1beta (SDF-1beta), with lesser effects noted for other chemokines and cytokines. When the CD34(+) population was subselected for expression of CD38, a greater fraction of the CD38(-) cells migrated as compared to the CD38(+) fraction. CD34(+) cells isolated from mobilized peripheral blood and cord blood also migrated in response to chemokines. Chemokines of the CC, CXC, and CX(3)C classes as well as other hematopoietic cytokines may modulate the process of stem cell transmigration of endothelial cells. Further understanding of this process may help elucidate the mechanism of stem cell mobilization and homing.

PSA is a candidate self-antigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome.

Previous studies demonstrated that recognition of seminal plasma antigens can occur in patients with chronic prostatitis/chronic pelvic pain syndrome. This suggests that an autoimmune component may contribute to symptoms in some men. To determine if any of the principal secretory proteins of the prostate could be candidate antigens in autoimmune prostatitis, we examined the recall proliferative response of purified CD4 T cells in patients with chronic prostatitis and in normal volunteers using purified seminal plasma antigens and autologous dendritic cells. Peripheral blood mononuclear cells were harvested from 14 patients with chronic prostatitis and 12 normal volunteers by density gradient centrifugation. The stimulating cells were irradiated autologous dendritic cells produced by culture of monocyte-enriched fractions with IL-4 and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF). Purified CD4 T cells were the responding population. Recall proliferation assays were performed, using purified seminal plasma proteins as antigens. In 14 patients with chronic prostatitis, we detected a greater than 2-fold increase in proliferative response to PSA compared to control in 5 patients (36%). No response to Prostatic Acid Phosphatase (PAP) or beta-microseminoprotein was observed in these 14 patients. In 12 normal volunteer donors with no history of genitourinary disease or symptoms, no proliferative response above background was observed for any prostatic antigen. The data suggest that some men with symptoms of chronic prostatitis have evidence of a proliferative CD4 T-cell response to PSA. PSA is a candidate antigen in chronic prostatitis/chronic pelvic pain syndrome and may be an appropriate target for immunotherapy for prostatic cancer.

Effect of Antisense Oligonucleotides on CD34+ Cells from Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by a specific hybrid gene BCR-ABL (formed as a result of t(9;22)). This leads to two possible mRNA usually present in leukemic cells, either B2A2 or B3A2. Targeting these mRNA by antisense oligonucleotides (AS) might offer the opportunity to decrease leukemic growth. We have tested the ability of AS to inhibit the in vitro proliferation of CD34 positive (CD34+) blood cells from 16 patients with newly diagnosed CML. CD34+ cells were isolated by an immunomagnetic technique and incubated for 16 to 18 hours with an 18 mer AS (0.25mM). Sense oligonucleotides served as controls. The effects of AS were evaluated by clonogenic test (production of CFU-GM). Moreover, colonies were picked out and studied by RT-PCR to analyse the presence of BCR-ABL transcript. For nine patients with B3A2 transcript, the median inhibition of CFU-GM formation at day 14 was 64.0 ± 11.2% (68.0 ± 11.4% at day 21) and for the seven patients with a B2A2 transcript: 59.0 ± 11.4% (72.5 ± 12.0% at day 21). AS showed no effect on CD34+ cells from three normal volunteer donor cells. However, for every patient studied, colonies picked out remained BCR-ABL positive with the RT-PCR technique.

Early progenitor cells from human mobilized peripheral blood express low levels of the flt3 receptor, but exhibit various biological responses to flt3-L.

The biological effects of flt3-L, and the expression of its tyrosine kinase receptor (flt3, CD135) were investigated on the immature subsets of human circulating peripheral blood progenitors obtained from cancer patients or normal volunteer donors, after mobilization with rhG-CSF or chemotherapy. flt3 was expressed at low levels, and its expression increased concomitantly with expression of CD38 within the CD34+ cell population. Despite this low-level expression, flt3-L exerted synergistic effects with a combination of c-kit ligand, IL-3, IL-6, GM-CSF and G-CSF, mainly to induce proliferation of CD34+/CD38- cells. In addition, flt3-L increased the detection of HPP-CFC, both immediately after cell selection, and after 7 and 14 d of cultures. We conclude that flt3-L is active on circulating early mobilized haemopoietic progenitors, despite the low- level expression of its receptor.

Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation.

Although the adeno-associated virus type 2 (AAV) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that AAV infection of human cells is receptor mediated (S. Ponnazhagan et al., J. Gen. Virol. 77:1111-1122, 1996). In the present studies, we investigated the ability of AAV to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. Bone marrow-derived CD34+ cells from 12 hematologically normal volunteer donors were infected with a recombinant AAV containing the beta-galactosidase gene under the control of the cytomegalovirus immediate-early promoter (vCMVp-lacZ). Whereas 15 to 80% of the cells from approximately 50% of the donors showed various levels of lacZ gene expression, the expression was undetectable in cells from the remaining donors. However, if cells from both sets of donors were stimulated with various combinations of cytokines to induce differentiation into myeloid and lymphoid lineages following AAV infection, then the level of expression of the transduced gene increased up to 20-fold over a period of 14 days. The results of virus-binding assays suggested that the observed difference between the two groups was due to the differential susceptibility of CD34+ cells to AAV infection rather than to differences in transcription and translation of the transduced gene. To corroborate these results, CD34+ cells from the two donor groups, KB (human nasopharyngeal carcinoma) cells, and M07e (human megakaryocytic leukemia) cells were infected with vCMVp-lacZ. KB cells served as a positive control for AAV infection, and M07e cells served as a negative control. Whereas abundant hybridization to the single-stranded viral DNA on Southern blots was detected in KB and CD34+ cells that were positive for lacZ gene expression, little activity was detected in M07e and CD34+ cells that did not show expression of the lacZ gene. These results suggest that the levels of expression of the putative cellular receptor for AAV vary widely in CD34+ cells from different donors. These studies have implications for the potential use of AAV vectors in human gene therapy involving primary human primitive hematopoietic stem and progenitor cells.

Expression of FLT4 and its ligand VEGF-C in acute myeloid leukemia.

FLT4 represents a recently cloned member of class III receptor tyrosine kinases which include receptors for the angiogenic growth factor VEGF, namely FLT1 and KDR. The ligand of FLT4 has been identified as VEGF-C which shares sequence homology with VEGF and P1GF. In the adult FLT4 shows a restricted expression pattern that is limited to lymphatic endothelia and endothelia of some high endothelial venules (HEV). FLT4 has also been detected in some tumor cell lines including the hematopoietic line HEL. We therefore investigated expression of FLT4 and its ligand VEGF-C in fresh samples from patients with AML. Using a sensitive PCR method we detected FLT4 m-RNA in 15 of 41 patients with de novo AML at diagnosis or relapse and in three of 12 patients with secondary AML. FLT4 expression was confirmed by immunocytochemistry in a subgroup of the studied patient population. FLT4 was also found in leukemic cell line U937, but not TF-1 and KG1a. VEGF-C expression was found in leukemic samples of four of seven FLT4-positive and four of six FLT4-negative patients. U937 cells also produced VEGF-C m-RNA. Interestingly, FLT4 expression was not detected in bone marrow samples of 15 normal volunteer donors or in CD34-positive cells from three additional donors. Possible autocrine and paracrine growth stimulation of leukemic blasts by VEGF-C is currently being investigated in our laboratory.