scholarly journals Minimal Hematology Analyzer Plus Blood Smear Digital Imaging/ Analysis Provides Better Clinical Hematology Results Than a Complex Hematology Analyzer Alone

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4731-4731
Author(s):  
H. Elizabeth Broome ◽  
Han-Inge Bengtsson ◽  
Laura Stephens ◽  
Lisa Palm
Keyword(s):  
Blood Count ◽  
Blood Smear ◽  
Excellent Correlation ◽  
False Negatives ◽  
Employment Equity ◽  
Cell Counts ◽  
Hematology Analyzer ◽  
Abnormal Cells ◽  
Equity Ownership ◽  
Hematology Analyzers

Abstract Introduction: Devices such as the CellaVision® DM96 (CellaVision AB, Lund, Sweden) locate and image nucleated cells on blood smears. Using image recognition software, the DM96 also pre-classifies those cells into differential categories similar to the most complex hematology analyzers. We compared the cell counts, differential counts and flagging information gained from a complex hematology analyzer, the XE5000 (Sysmex, Kobe, Japan), with information from a minimal hematology analyzer (Sysmex PocHi) plus the DM96. We found that the cell counts, differential and flagging capabilities are similar, but the PocHi plus DM96 advantages include allowing remote review of the blood smear. Methods: 210 blood samples, selected for various abnormalities, had complete blood counts with automated differentials produced by a Sysmex XE5000 hematology analyzer. These results were compared with cell counts from the Sysmex PocHi hematology analyzer, their 100-cell DM96 post reclassification differentials, and with DM96 pre-classification differentials using standard regression analyses and Rumke 95% confidence intervals (CI) as calculated using the Clopper-Pearson method. Flagging by the XE5000 for immature granulocytes (IG's) and for blasts/abnormal cells was compared to the DM96 pre-classification using truth tables with the DM96 post reclassification as the gold standard. The following translations were used to compare flagging: IG's > 2 for either post reclassification DM96 differential, XE5000 or the DM96 pre-classification differential; Any blast cells on the manual differential were compared to XE5000 flags WBC Abn Scg; NRBC Abn Scg; Blasts?; Atyp LY?; Abn Ly/ L_Bl? and to DM96 pre-classification % Blasts > 0%; unidentified cells >3%. . Results: Non-differential blood count parameters including white blood count, red blood count, hemoglobin, mean corpuscular volume (MCV), and platelet count showed excellent correlation between the PocHi and the XE5000 with R2>0.95. Differential-dependent blood count parameters including neutrophils, lymphocytes, monocytes, eosinophils, basophils and immature granulocytes showed excellent correlation between the XE5000 and pre-classification DM96 with R2>0.95. Nucleated red cells also showed excellent correlation between the XE5000 and the DM96 with R2>0.85. For blasts/abnormal cells, the DM96 showed 100% sensitivity and 40% specificity with 0% false negatives. The XE5000 showed 93% sensitivity and 19% specificity with 3% false negative. Two of the false negatives were shared by both instruments and were 1% blasts. Of the three false negatives with the XE5000 that were true positives with the DM96, two had 1% blasts while one had 2% blasts. For immature granulocytes (IG's), the XE5000 showed 94% sensitivity and 79% specificity with 2% false negatives. The DM96 showed 85% sensitivity and 95% specificity with 4% false negatives. All of the false negatives were for IG's < 5% Conclusions: Pairing the DM96 or a similar imaging instrument with a relatively inexpensive hematology analyzer, such as those commonly used in physician office laboratories, would provide all of the information available from expensive, complex hematology analyzers in high throughput laboratories AND allow remote review of the blood smear findings by experts. Disclosures Broome: CellaVision: Consultancy, Research Funding. Bengtsson:CellaVision AB: Employment, Equity Ownership. Palm:CellaVision: Employment, Equity Ownership.

scholarly journals Evaluation of the Flagging Performance of the Hematology Analyzer Sysmex XN Series on the Basis of “Q Values”

2017 ◽  
Vol 142 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Oh Joo Kweon ◽  
Mi-Kyung Lee ◽  
Hye Ryoun Kim
Keyword(s):  
Diagnostic Accuracy ◽  
Sensitivity And Specificity ◽  
Area Under The Curve ◽  
Q Value ◽  
Cell Counts ◽  
Related Cell ◽  
Hematology Analyzer ◽  
Abnormal Cells ◽  
Q Values ◽  
Hematology Analyzers

Context.— In the XN series of hematology analyzers (Sysmex, Kobe, Japan), the probability of the presence of abnormal cells is indicated by flags based on Q values. Objective.— To evaluate the Q value performance of the Sysmex XN-20 modular analyzer. Design.— The interinstrumental concordance, intrainstrumental precision, and diagnostic accuracy of Q values, with tested flags of “blasts/abnormal lymphocytes,” “atypical lymphocytes,” and “blasts,” were investigated. Results.— Absolute concordance rates in flagging between 2 analyzers ranged from 69.8% to 80.8%, and κ values ranged from 0.43 to 0.61. In samples with absolute related cell counts lower than 100/μL, the values ranged from 0.31 to 0.52. For intrainstrumental precision, standard deviations ranged from 4.8 to 23.9 for the blasts/abnormal lymphocytes, from 18.7 to 59.1 for the blasts, and from 11.0 to 23.0 for the atypical lymphocytes. Using a default Q value cutoff, diagnostic accuracy values based on the area under the curve, sensitivity, and specificity were, respectively, 0.910, 90.9%, and 72.2% for blasts/abnormal lymphocytes; 0.927, 84.9%, and 89.8% for blasts; and 0.865, 74.4%, and 84.9% for atypical lymphocytes. The diagnostic accuracy of Q values was much lower in samples with absolute related cell counts lower than 100/μL than in those 100/μL or higher. Conclusions.— Q values of the Sysmex XN-20 analyzer were found to be imprecise and irreproducible, especially with samples containing a small number of pathologic cells. Adjustments in the Q value threshold may help in the detection of these cells.

Occurrence of T-Lymphocytes with Aberrant Immunophenotypes Suggestive of Mature T-Cell Neoplasms In Patients with Hairy Cell Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4165-4165
Author(s):  
Wolfgang Kern ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Torsten Haferlach
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Hairy Cell Leukemia ◽  
Initial Diagnosis ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Abstract Abstract 4165 Mature T-Cell Neoplasms (MTN) comprise a heterogeneous group of diseases with largely varying clinical courses ranging from indolent cases which are asymptomatic for years to aggressive cases requiring immediate therapy. The incidence of MTN is increasing with age, however, particularly due to the large group of cases with very indolent clinical course MTN are considered underdiagnosed to a significant portion. Published data indicates that T-lymphocytes with aberrant immunophenotype, i.e. double-positive cell expressing both CD4 and CD8, are present in healthy subjects, however, their frequency in general amounts to clearly less than one percent of total leukocytes. With increasing age, these T-lymphocytes with aberrant immunophenotype are detected in more subjects and at higher frequencies suggesting a higher incidence of mature T-cell neoplasms or at least of pre-malignant conditions in these cases. The diagnosis of hairy cell leukemia has not yet been linked to a higher frequency of these T-lymphocytes with aberrant immunophenotype or of MTN. Following the identification of various cases with both a diagnosis of hairy cell leukemia and the presence of T-lymphocytes with aberrant immunophenotype in our laboratory we hypothesized that both of these conditions co-occur at a higher rate than would be expected by chance. We therefore retrospectively evaluated multiparameter immunophenotyping results of 338 patients diagnosed with hairy cell leukemia (newly diagnosed or during follow-up) between August 2005 and July 2010 for the presence of an increased percentage (more than 1%) of T-lymphocytes with an aberrant immunophenotype. We identified 31 such patients, i.e. 9.2% of all patients with hairy cell leukemia. 17 were identified at initial diagnosis and 14 during follow-up after therapy for hairy cell leukemia. The patients` ages ranged from 43.4 to 89.3 years (median, 62.1 years), 21 were male. The aberrant immunophenotype comprised the coexpression of CD3, CD4, and CD8 in all cases and in addition of CD56 in 25/31 (80.6%) cases. The median values and ranges for blood cell counts amounted to: WBC, 2.6 ×10e9/l, 1.0–24.6 ×10e9/l; hemoglobin, 12.7 g/dl, 7.3–16.5 g/dl; thrombocytes, 116 ×10e9/l, 24–258 ×10e9/l. The percentage of T-lymphocytes with an aberrant immunophenotype (compared to all leukocytes) ranged from 1% to 22% (median, 4%); the respective concentrations ranged from 0.013 ×10e9/l to 0.984 ×10e9/l (median, 0.122 ×10e9/l). The concentrations of T-lymphocytes with an aberrant immunophenotype tended to be higher in cases at follow-up as compared to those at initial diagnosis, although this difference was not significant (mean±SD, 0.266±0.275 ×10e9/l vs. 0.144±0.119 ×10e9/l). In four of the 31 patients with T-lymphocytes with an aberrant immunophenotype molecular genetic analysis of T-cell receptor (TCR) rearrangement was performed. In 3/4 patients both TCR beta and gamma were found rearranged and in one of these also TCR delta was rearranged, however, in 1/4 patient no TCR rearrangement was present. This data indicates that T-lymphocytes with aberrant immunophenotypes are present in patients with hairy cell leukemia much more often and at higher concentrations than in the general population. This data therefore suggests that the incidence of mature T-cell neoplasms may be higher in hairy cell leukemia patients. Clinical symptoms and findings like cytopenia and splenomegaly therefore may not be attributable to hairy cell leukemia alone which may have significant therapeutic implications. It is suggested to monitor for T-lymphocytes with aberrant immunophenotypes in patients with hairy cell leukemia and to perform analysis for TCR rearrangements in positive cases. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals An All Antibody Approach for Conditioning Bone Marrow for Hematopoietic Stem Cell Transplantation with Anti-cKIT and Anti-CD47 in Non-Human Primates

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4428-4428
Author(s):  
Kristopher D Marjon ◽  
James Y Chen ◽  
Jiaqi Duan ◽  
Timothy S Choi ◽  
Kavitha Sompalli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Mast Cell Degranulation ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Updated Results from an Open-Label, Multicenter, Expanded Treatment Protocol (ETP) Phase (Ph) 3b Study of Ruxolitinib (RUX) in Patients (Pts) with Polycythemia Vera (PV) Who Were Hydroxyurea (HU) Resistant or Intolerant and for Whom No Alternative Treatment (Tx) Was Available

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1774-1774 ◽  
Author(s):  
Lynda Foltz ◽  
Gian-Matteo Pica ◽  
Hacene Zerazhi ◽  
Jan Van Droogenbroeck ◽  
Sorin Visanica ◽  
...  
Keyword(s):  
Cell Carcinoma ◽  
Board Of Directors ◽  
Research Funding ◽  
Blood Count ◽  
Study Drug ◽  
Employment Equity ◽  
Advisory Committees ◽  
Medical Need ◽  
Equity Ownership ◽  
Grade 3

Abstract BACKGROUND Few Tx options are available for pts with inadequately controlled PV. European LeukemiaNet defined resistance/intolerance was seen in ≈25% pts treated with HU (Alvarez-Larran et al, 2012). In the HU-resistant/intolerant PV pts evaluated in the pivotal RESPONSE study (week [wk] 208), RUX was well tolerated and superior to standard therapy in achieving durable hematocrit (HCT) control, hematologic response, and spleen size and symptom reductions. This Ph 3b ETP study was planned to provide RUX Tx to HU-resistant/intolerant PV pts, who have no alternative standard Tx, and are not eligible for any ongoing clinical studies. Results from wk 24 data cutoff of this study (Devos et al) were presented at ASH 2017. Here, we report consolidated findings from the ETP study at wk 96 data cutoff (Dec 29, 2017 [final database lock]) to further support the use of RUX in this pt population with an unmet medical need. METHODS RUX Tx was initiated at a starting dose of 10 mg bid (could be titrated to a maximum of 25 mg bid). Visits were scheduled every 4 wks until wk 24 and every 12 wks thereafter; final analysis was done when all pts had been followed for 30 days after discontinuation of Tx or completion of Tx per protocol (transitioned to commercial RUX or until Dec 31, 2017, whichever date occurred first). The primary endpoint was to assess the safety of RUX. Secondary endpoints included change in HCT level, change in spleen length, and pt-reported outcomes (change in MPN-SAF TSS score). HCT control at wk 24 was defined by absence of phlebotomy (PBT) eligibility starting at wk 8 and continuing through wk 24, with no more than 1 PBT eligibility occurring after first dose date and prior to wk 8. PBT eligibility was defined by confirmed HCT >45% (at least 3 percentage points higher than HCT at baseline [BL]), or confirmed HCT >48%. Blood count remission at wk 24 was defined by HCT control, and white blood cell count <10 × 109/L, and platelet count ≤400 × 109/L. RESULTS At data cutoff, 161 pts with PV were enrolled (BL characteristics similar to that presented at ASH 2017). End of Tx was reported for all 161 pts: Tx duration completed per protocol (141 pts), adverse event (AE [12 pts]), consent withdrawal (3 pts), pt decision (2 pts), disease progression (2 pts), and death (due to accident [1 pt]). The median exposure was 25.1 wks (range, 0.4-104.7), and median dose intensity of RUX was 20.0 mg/day (range, 6.7-47.7). AEs (regardless of study drug relationship) led to dose adjustment/interruption in 37.9% pts and study drug discontinuation in 8.7% pts. The most common hematologic AEs (rate=number of events per 100 pt-year exposure [pt-year exposure=110.2]; all grades]) included anemia (31.8) and thrombocytosis (10.0), while headache (24.5), diarrhea (14.5), constipation (12.7), and fatigue (12.7) were the most frequent non-hematologic AEs. For all reported grade 3/4 AEs, exposure-adjusted rate was less than 3. Thromboembolic events (all grade; Standardized MedDRA Query) were reported in 3 pts. Disease progression was reported in 4 pts (myelofibrosis=3 pts; acute myeloid leukemia=1 pt). The incidence of other neoplasms (regardless of study drug relationship) was low (leiomyoma, malignant melanoma, marginal zone lymphoma, renal cancer [1 pt each]; squamous cell carcinoma [2 pts]; basal cell carcinoma [3 pts]). Infections (all grades) were reported in 57 pts (grade 3/4 in 5 pts). At wk 24, 73 pts (45.3% [95% CI, 37.5%-53.4%]) achieved HCT control; hematologic remission was seen in 29 pts (18% [95% CI 12.4%-24.8%]). Changes in blood count parameters over time are shown in Fig. 1. In evaluable pts (N=105), use of PBT decreased from BL (39 PBTs between screening and BL) to end of Tx (5 PBTs in 12 wks prior). Best spleen response from BL for each pt by wk 96 is shown in Fig. 2. At least 50% spleen length reduction was seen in 86.7% (78/90) of pts from BL at any time in the study. Overall, 33.8% (46/136) of pts had ≥50% reduction in MPN-SAF TSS from BL at the end of Tx. CONCLUSION The observed safety profile of RUX in the ETP study was consistent with that of the RESPONSE studies. Efficacy results were close to the observed values in the RESPONSE studies. RUX Tx resulted in HCT control, hematologic remission, spleen response, and symptom reduction in this HU-resistant/intolerant pt population in need of a viable Tx option. Safety and efficacy findings from this ETP study support the use of RUX for pts with inadequately controlled PV, an unmet medical need. Disclosures Foltz: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Promedior: Research Funding; Gilead: Research Funding. Leber:Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees. de Almeida:Celgene: Speakers Bureau; Novartis: Speakers Bureau. Ranta:Novartis: Consultancy. Cartes:Novartis: Honoraria. Kiladjian:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Chrit:Novartis: Employment, Equity Ownership. Yin:Novartis: Employment. Morando:Novartis: Employment, Equity Ownership. Devos:Celgene: Consultancy; Novartis: Consultancy; Takeda: Consultancy.

scholarly journals Comparison of Different Small Clinical Hematology Laboratory Configurations With Focus on Remote Smear Imaging

2019 ◽  
Vol 143 (10) ◽  
pp. 1234-1245 ◽  
Author(s):  
Laura Stephens ◽  
Nicholas J. Bevins ◽  
Hans-Inge Bengtsson ◽  
H. Elizabeth Broome
Keyword(s):  
Digital Imaging ◽  
Blood Count ◽  
Complete Blood Count ◽  
Cost Savings ◽  
Reference Method ◽  
Cost Modeling ◽  
Special Focus ◽  
Imaging Device ◽  
Cell Counts ◽  
Hematology Analyzer

Context.— Stand-alone clinical sites (eg, infusion centers) are becoming increasingly common. These sites require timely hematology analysis. Here we compare performance and costs of currently available analysis configurations with special focus on a proposed alternative using a minimal hematology analyzer plus a digital imaging device, allowing for remote oversight and interpretation. Objectives.— To determine whether low-volume laboratories might realize savings while gaining function by substituting commonly used configurations with a proposed alternative. Design.— To evaluate the performance of the proposed alternative configuration, blood counts with automated differentials produced by a Sysmex XE5000 (complete blood count reference method) were compared with cell counts from the Sysmex pocH-100i, CellaVision DM96 preclassified differentials, and DM96 reclassified differentials (differential reference method) by using standard regression analyses, 95% CIs, and truth tables. Financial cost modeling used staffing practices, test volumes, and smear production rates observed at remote clinics performing on-site hematology analysis within the University of California at San Diego Health system. Results.— Differential blood count parameters showed excellent correlation between the XE5000 and preclassification DM96 with R2 &gt; 0.95. For blasts/abnormal cells, immature granulocytes, and nucleated red blood cells, the DM96 showed higher sensitivity and similar specificity to the XE5000. Cost modeling revealed that decreased personnel costs through remote monitoring of results facilitated by the DM96 would lead to lower operational costs relative to more conventional analysis configurations. Conclusions.— A digital imaging instrument with an inexpensive hematology analyzer provides similar information to a complex hematology analyzer and allows remote review of the blood smear findings by experts, leading to significant cost savings.

SRSF2 is Mutated in 47.2% (77/163) of Chronic Myelomonocytic Leukemia (CMML) and Prognostically Favorable in Cases with Concomitant RUNX1 mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 274-274 ◽  
Author(s):  
Susanne Schnittger ◽  
Manja Meggendorfer ◽  
Alexander Kohlmann ◽  
Vera Grossmann ◽  
Kenichi Yoshida ◽  
...  
Keyword(s):  
Hot Spot ◽  
Myeloproliferative Neoplasm ◽  
Gene Mutations ◽  
Chronic Myelomonocytic Leukemia ◽  
Structural Basis ◽  
Employment Equity ◽  
Cell Counts ◽  
Number Of Genes ◽  
Equity Ownership ◽  
Myelomonocytic Leukemia

Abstract Abstract 274 Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. We previously investigated 81 CMML cases and detected a number of genes frequently mutated (TET2 44.4%, CBL 22.2%, NRAS 22.2%, KRAS 12.3%, JAK2 9.8%, RUNX1 8.7%, EZH2 12.3% (Kohlmann et al., JCO, 2010; Grossmann et al., Leukemia, 2011). Recently, we detected a new candidate gene, SRSF2 (serine/arginine-rich splicing factor 2, also known as SC35) that is a component of the RNA splicing machinery and found it to be frequently mutated in MDS. Aim: As CMML has been increasingly characterized by a growing number of genes during the last years we here analyzed both the frequency of SRSF2 mutations in this entity and the relevance in the context of other previously described gene mutations, as well as to look for a potential prognostic implication. Patients and Methods: In total, 163 cases with CMML (CMML-1 n=105, CMML-2 n=58) were included. The cohort comprised 115 males and 48 females with a median age of 72.8 yrs (range: 21.9 – 88.8 yrs) including all 81 pts that have been published previously. 112 cases (69%) had a normal karyotype and 51 (31%) showed aberrant karyotypes. The mutational hot spot region of SRSF2 around Proline codon 95 (P95) was analyzed by Sanger sequencing in all cases. Data on further mutations were available in respective subcohorts: ASXL1 (n=128), CBL (n=162), EZH2 (n=134), JAK2V617F (n=162), KRAS (n=140), NRAS (n=79), RUNX1 (n=156), TET2 (n=143), TP53 (n=80). Results:SRSF2 mutations of P95 were detected in 77/163 (47.2%) of all cases (49/105, 46.7% in CMML-1, and 28/58, 48.3% in CMML-2). In detail, 74 cases had a missense mutation leading to a change of P95 to P95H (n=33), P95L (n=24), P95R (n=16) or P95A (n=1). In further 3 cases a newly described 24 bp (8 amino acids) deletion starting at P95 was observed. All cases had a mutation load of approximately 50%. The mutations were correlated with higher age (73.3 yrs vs 68.7 yrs in the SRSF2wt cases, p=0.010) and higher hemoglobin levels (11.4 vs 10.5 g/dl in the SRSF2wt cases, p=0.019) whereas white blood cell counts were not different. Further, SRSF2 mutations were mutually exclusive of EZH2 mutations (0/12, 0% vs. 66/122, 54.1% in the EZH2wt, p<0.001) whereas a high coincidence occurred with RUNX1 mutations (22/35, 62.9% vs 52/121, 43% in the RUNX1wt, p=0.054) and TET2 mutations (50/82, 61% vs 18/61, 29.5% in the TET2wt, p<0.001). With respect to associations with all other gene mutations investigated and karyotype no specific pattern was observed. In the total cohort no impact of SRSF2 on survival was observed. Because of the high coincidence of SRSF2mut with RUNX1mut and TET2mut, we performed an analysis in these specific subcohorts. No impact of SRSF2mut in the TET2mut subcohort was found. Whereas in the RUNX1mut subcohort SRSF2mut had a favorable impact on overall survival compared to SRSF2wt (median OS: 108.0 months vs 41.8 months, p=0.05). Conclusions:SRSF2 has recently been described as a new marker in CMML and demonstrated to be useful to delineate further the genetic defects of this disease. This very frequent new mutation is characterized by higher age, higher hemoglobin levels and a high coincidence with TET2 and RUNX1 mutations. It is mutually exclusive of EZH2 mutations. In the subset of RUNX1 mutated CMML SRSF2 mutations demonstrated a favorable impact on outcome. Furthermore, for the first time a 24 bp deletion was observed in three cases that may provide further insight into the structural basis for the abnormal function of SRSF2. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

HH-002 Effectively Suppresses Myeloproliferative Neoplasm Phenotypes in JAK2V617F Transgenic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4095-4095
Author(s):  
Wan-Ting Ho ◽  
Wanke Zhao ◽  
Paul Hallenbeck ◽  
Frank Rong ◽  
Zhizhuang Joe Zhao
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Blood Cells ◽  
Hematopoietic Cells ◽  
Mutant Form ◽  
Dependent Manner ◽  
Combination Drug ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership

Abstract Ph-negative myeloproliferative neoplasms (MPNs) represent a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). A major molecular defect in MPNs is JAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2 found in the majority of MPN patients. In earlier studies, we generated JAK2V617F transgenic mice by using the vav promoter which drives gene expression in all hematopoietic cells. These mice develop MPN-like phenotypes in a transgene dose-dependent manner. The objective of this study is to use this animal MPN model to test the efficacy of HH-002, a derivative of natural plant alkaloid homoharringtonine. Treatment of JAK2V617F transgenic mice with a daily dose of 1 mg/kg HH-002 through subcutaneous injection reduced blood cell counts to the normal range or slightly below the normal level. HH-002 causes a preferential reduction of myeloid cells in JAK2V617F transgenic mice since percentages of lymphocytes (CD3e+ T cells and CD19+ B cells) were increased (despite somewhat decreases in absolute numbers) while the percentage of Gr-1+/CD11b+ granulocytes sharply declined. HH-002 also effectively reduced the spleen size of JAK2V617F transgenic mice and prevented development of myelofibrosis. With a JAK2V617F bone marrow transplant mouse model, HH-002 showed a similar effect. Although it did not increase the ratio of JAK2V617F-negative cells to JAK2V617F-positive, it stopped further expansion of JAK2V617F-containing malignant cells in JAK2V617F bone marrow recipient mice. In vitro experiments with primary hematopoietic cells demonstrated that HH-002 potently inhibited formation of erythroid and myeloid colonies with an IC50 value of 1-3 nM. However, it does not show a preferential inhibition of JAK2V617F-containing cells. This is not unexpected since homoharringtonine is known to inhibit protein synthesis. Taken together, HH-002 is a promising candidate for development of therapeutic drugs to treat MPNs and related diseases. Combination drug therapies with JAK2 inhibitors need to be explored. Disclosures Hallenbeck: Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Rong:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Employment, Equity Ownership. Zhao:Hangzhou Bensheng Pharmaceutical Corp. Ltd.: Research Funding.

The ALK-2 Inhibitor, TP-0184, Demonstrates High Distribution to the Liver Contributing to Significant Preclinical Efficacy in Mouse Models of Anemia of Chronic Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 263-263 ◽  
Author(s):  
Peter Peterson ◽  
Wontak Kim ◽  
Hillary Haws ◽  
Clifford J. Whatcott ◽  
Adam Siddiqui-Jain ◽  
...  
Keyword(s):  
Chronic Disease ◽  
Kinase Activity ◽  
Therapeutic Window ◽  
Employment Equity ◽  
Anemia Of Chronic Disease ◽  
Bmp Receptor ◽  
Cell Counts ◽  
Equity Ownership ◽  
Preclinical Efficacy ◽  
Receptor Family

Abstract In individuals with chronic inflammatory diseases, such as cancer or rheumatoid arthritis, constitutive signaling through ALK2, a member of the bone morphogenetic protein (TGFβ/BMP) receptor family, leads to debilitating anemia, commonly referred to as anemia of chronic disease (ACD). Activation of ALK2, like other members of the BMP receptor family, leads to the phosphorylation and activation of SMAD family transcription factors via signal transduction and subsequent activation of gene expression. Activation of ALK2 in the liver induces the SMAD-driven transcription of the peptide hormone hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Lowering constitutively elevated hepcidin levels by inhibiting ALK2 kinase activity is a potentially viable therapeutic strategy for ACD. Current therapeutic approaches for ACD rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which address the underlying pathological deficit of functionally low iron levels. TP-0184 is a small-molecule, selective inhibitor of ALK2 kinase activity (IC50 = 5 nM). TP-0184 has demonstrated profound preclinical activity in three mouse efficacy models for ACD. In model 1, TP-0184 reversed hepcidin induction in mice treated with turpentine oil. In model 2, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection with heat-inactivated Brucella abortus. In model 3, TP-0184 reversed elevated hepcidin levels in TC-1 tumor bearing mice. Plasma and liver pharmacokinetics in mice revealed that TP-0184 has a high volume of distribution (Vd = 30.8) and accumulates at high concentrations in the liver (Cmax of 292 mM following a single oral dose of at 20 mg/kg). In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Collectively these studies support the clinical evaluation of TP-0184 as an alternative treatment for ACD. Disclosures Peterson: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Temporal Profiles of Lymphocyte Subsets and the Correlation with Infectious Events in Idelalisib-Treated Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5583-5583 ◽  
Author(s):  
Jeffrey P. Sharman ◽  
Gilles A. Salles ◽  
Wojciech Jurczak ◽  
Jeffrey Jones ◽  
Carolyn J. Owen ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Lymphocyte Subsets ◽  
Cd8 T Cell ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership ◽  
Grade 3

Abstract Introduction: Idelalisib (IDELA) is a targeted PI3Kd inhibitor approved as monotherapy in relapsed follicular lymphoma and in combination with rituximab in relapsed chronic lymphocytic leukemia (CLL). Increased rates of adverse events (AEs) were recently observed in the IDELA vs placebo arms of randomized controlled trials (RCT) evaluating IDELA added to standard therapies in front-line CLL and early-line indolent non-Hodgkin lymphoma (iNHL). AEs leading to death were mainly infectious and included pneumocystis jirovecii pneumonia (PJP) and cytomegalovirus (CMV). This analysis across trials in the relapsed population evaluated whether quantitative changes in lymphocyte subsets may have contributed to these AEs. Methods: Peripheral blood immunophenotypic data available for analysis from patients (pts) (n = 1,480) treated in 5 IDELA RCTs were analyzed. Three studies (n = 787) included pts with relapsed CLL (NCT01569295: IDELA + bendamustine-rituximab [BR] vs placebo + BR; NCT01539512: IDELA + R vs placebo + R and NCT0165902: IDELA + ofatumumab [O] vs placebo + O) and 2 studies (n = 693) included R/R iNHL pts (NCT01732913: IDELA + R vs placebo + R and NCT01732926: IDELA +BR vs placebo + BR). Absolute numbers of T (CD4+ and CD8+), B (CD19+) and NK (CD16+/CD56+) cells were analyzed longitudinally in both IDELA and placebo pts across the 5 studies. Lymphocyte subsets were analyzed separately in those who died and then correlated with specific grade ≥3 AEs including infections, febrile neutropenia, and respiratory (acute respiratory failure, pneumonitis) events. Analysis was conducted within individual study and for all studies combined. Of note, samples were collected more frequently during the first 6 months (during combination therapy) and collection times varied among the 5 studies. Results: There was no specific trend noted with the CD8+ T-cells between treatment groups across the studies. Generally, NK-cells were decreased to a similar degree in both IDELA and placebo pts at weeks 10 to 12 with recovery starting around week 24. There were no differences in median NK- and CD8+ T-cell counts between pts with grade ≥3 AEs and no AEs within either group. In both BR trials, CD4+ T-cells nadir to <200 cells/µl occurred at week 22 in both groups. Recovery of CD4 to ≥200 cells/µL occurred at week 30 in CLL pts and at week 72 in iNHL pts. Median CD4+ T-cells in pts on the BR trials were lower in groups both with and without AEs, compared with non-BR trials (Table 1). There were a total of 31 cases of PJP (13 in the BR trials) and 32 cases of CMV infection (28 in the BR trials). Analysis of PJP and CMV pts with available immunophenotypic data (n = 46) revealed that 33 pts had CD4 <200 cells/µL; 31 of these were treated with IDELA plus combination therapy (Figure 1). Finally, while there were more grade ≥3 AEs within IDELA arms, these did not occur at any specific CD4 level and, in fact, grade ≥3 AEs were noted even in pts with CD4 >900 cells/µL. Conclusion: Within 5 RCTs evaluating IDELA vs placebo in combination with an anti-CD20 mAb or BR in R/R CLL or iNHL, there was no correlation between grade ≥3 AEs and NK- or CD8+ T-cell counts. Median CD4+ T-cells in pts on the BR trials were lower in both groups in those with and without AEs, compared with non-BR trials. In addition, pts with PJP and CMV infections were noted to have CD4+ T-cells <200 cells/ µL, and this was more common in IDELA-treated patients, especially when combined with BR, suggesting that the lymphosuppressive effect of IDELA may augment the myelosuppressive effect of bendamustine. While this current study involves the quantitative analysis of various immune cell subsets, it may be the qualitative function of these cells that contributed to infections. Assays evaluating the qualitative function of these cells are being investigated. All IDELA trials have been amended to include PJP prophylaxis and CMV monitoring. Figure 1 Figure 1. Incidence of PJP and CMV Infections and Correlation with CD4 Count. Disclosures Sharman: Gilead Sciences, Inc.: Honoraria, Research Funding. Salles:Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Jurczak:Celltrion, Inc: Research Funding; Janssen: Research Funding; Gilead Sciences: Research Funding; Acerta: Research Funding; Bayer: Research Funding. Jones:AbbVie: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Research Funding; PCYC: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Owen:Janssen: Honoraria; Gilead: Honoraria, Research Funding; Pharmacyclics: Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Lundbeck: Honoraria, Research Funding; Novartis: Honoraria; Roche: Honoraria, Research Funding. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Dreiling:Gilead Sciences: Employment, Equity Ownership. Xiao:Gilead Sciences: Employment, Equity Ownership. Rao:Gilead Sciences: Employment, Equity Ownership. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria.

Autologous T Cells From AML Patients Can Be Effectively Recruited for In-Vitro Lysis of Blasts by a Novel CD33/CD3-Bispecific BiTE Antibody

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 14-14 ◽  
Author(s):  
Michael Aigner ◽  
Julian Feulner ◽  
Roman Kischel ◽  
Peter Kufer ◽  
Patrick A Baeuerle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Target Cells ◽  
Employment Equity ◽  
Cell Counts ◽  
Equity Ownership ◽  
Bite Antibody

Abstract Abstract 14 Bispecific T cell-engaging (BiTE®) antibodies combine in one polypeptide chain two single chain antibodies, one specific for CD3 on T cells and one for a tumor-associated antigen. The CD19/CD3-bispecific BiTE antibody blinatumomab has shown in phase 1 and 2 clinical trials very high response rates in patients with non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33, an antigen broadly expressed by myeloid cells including acute myelogenous leukemia (AML) blasts, in redirecting autologous T cells for in vitro lysis of blasts from AML patients. In a first step, the cytolytic potential of the CD33-specific BiTE (CD33 BiTE) was investigated in co-cultures of enriched resting CD8+ T cells from healthy donors and CD33+ leukemic cell lines KG-1 and U-937 as target cells. CD33 BiTE concentrations as low as 0.1 ng/ml (1.8 pM) mediated effective lysis of leukemic cell lines at effector to target (E:T) ratios of 1:1, whereas no lysis was observed with a solely CD3-binding control BiTE antibody. Peripheral CD8+ T cells that were pre-activated in cell culture or CD8+ T cell clones were even more potent in target cell lysis than previously resting T cells. Data obtained with a 51Cr release assay were comparable to those from a flow cytometry-based assay. Next, primary samples from AML patients were co-cultured with mononuclear cells (MNC) from healthy donors at an E:T ratio of 1:1. After 48 hrs of incubation in the presence of 1 ng/ml CD33 BiTE, a decrease in CD33+ AML blasts as well as of CD33+ monocytes was observed when compared to samples with control BiTE or vehicle. The CD33 BiTE induced upregulation of activation markers CD25 and CD69 on the majority of T cells. We furthermore investigated whether T cells from AML patients were capable of mediating lysis of CD33+ leukemia cells by CD33 BiTE. Resting or in vitro pre-stimulated CD8+ T cells were prepared from peripheral blood of newly diagnosed AML patients and tested for lysis of U937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls. Finally, we developed a FACS-based assay that allowed studying autologous blast lysis and T cell behaviour using cryo-preserved patient samples. Upregulation of T cell activation markers in cultures of MNC samples from AML patients was evident following addition of 1 ng/ml CD33 BiTE. Fifty five and 85% of CD4+ cells, and 57 and 65% of CD8+ cells expressed CD25 after 24 h and 48 h, respectively, but not with the control BiTE antibody (all <6%). Despite robust T cell activation, only a limited lysis of myeloid blasts was observed, presumably, due to the short incubation periods and low E:T ratios in the range of 1:5-1:21. We therefore investigated whether blast lysis is more effective after prolonged incubation. In the presence of CD33 BiTEs for 6 days, T cell numbers in AML patient samples dramatically expanded; CD8+ cell counts were up 8-fold, and CD4+ cell counts up 11-fold. This was not observed under control conditions. Up to 85% of AML blasts were now lysed. Currently, a larger collection of primary AML patient samples is being analyzed in order to determine an ex-vivo response rate for CD33 BiTE treatment and the impact of the patient samples’ E:T ratio and CD33 expression level on blasts on redirected lysis. Taken together, the novel CD33 BiTE effectively engages and activates autologous T cells for the elimination of AML blasts in vitro and may thereby constitute a novel therapeutic option for the treatment of patients with CD33-expressing myeloid leukemia. Disclosures: Aigner: Micromet Inc.: Research Funding. Kischel:Micromet Inc.: Employment, Equity Ownership. Kufer:Micromet Inc.: Employment, Equity Ownership. Baeuerle:Micromet Inc.: Employment, Equity Ownership. Mackensen:Micromet. Inc.: Research Funding. Krause:Micromet Inc.: Research Funding.

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