Analysis Of EphA3 Expression In Hematologic Malignancies By Quantitative PCR

Abstract EphA3 is a receptor tyrosine kinase that plays an important role in cell positioning and tissue organization during fetal development, but is not thought to play a significant role in healthy adults. However, aberrant EphA3 expression has been reported in a variety of hematologic and solid tumors. KaloBios is conducting a clinical study (KB004-01) of the anti-EphA3 monoclonal antibody KB004 in patients with hematologic malignancies. To gain further insight into the expression of EphA3 in human cancer, we are analyzing EphA3 mRNA expression in a broad range of hematologic diseases using quantitative PCR analysis. The goal of this research is to determine EphA3 expression in hematologic diseases, to identify cytogenetic and/or molecular mutations associated with aberrant EphA3 expression, and to identify EphA3 positive patient populations for inclusion in the ongoing KB004-01 clinical trial. To date, a total of 45 bone marrow aspirate samples have been analyzed from a cohort of patients with AML, MDS, multiple myeloma (PCM), PMF, CML, ALL, or CLL and the study is ongoing. Relative quantification of EphA3 mRNA was determined by normalization to GAPDH and B2M control genes, and expression calculated relative to the level of EphA3 found in normal bone marrow. Patients were designated EphA3+ if the relative EphA3 expression was >2-fold above normal bone marrow. In AML, 8 out of 14 (57.1%) were EphA3+ with expression levels ranging from 2 - 203 fold. The two AML samples with the highest EphA3 expression (43 and 203 fold) were from refractory AMLs. In MDS, 7 out of 15 (46.7%) were EphA3+ with expression ranging from 2 - 33 fold. In PCM, 2 out of 3 (66.6%) were EphA3+ with expression ranging from 10 - 55 fold. The sample with high EphA3 expression of 55 fold had involvement of both PCM and PMF disease. In PMF, 2 out of 3 (66.6%) were EphA3+ with expression levels ranging from 8 - 977 fold. The PMF sample with 977 fold EphA3 expression is the highest level of EphA3 measured in the study to date. The remaining samples analyzed consisted of 1 unspecified MPN, 3 CML, 2 ALL, and 4 CLL all of which had EphA3 mRNA levels equal to or lower than normal bone marrow. In summary, elevated levels of EphA3 mRNA were detected in a number of bone marrow aspirates from AML, MDS, PCM, and PMF patients suggesting these diseases as possible candidates for anti-EphA3 targeted therapy. Disclosures: Woronicz: KaloBios: Employment. Cortes:KaloBios: Research Funding. Jorgensen:KaloBios: Research Funding. Challagundla:KaloBios: Research Funding. Walling:Amgen: Equity Ownership; KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity’s Board of Directors or advisory committees. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.

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The Targeted Histone Deacetylase Inhibitor Tefinostat (CHR-2845) Shows Selective In Vitro Efficacy In Monocytoid-Lineage Acute Myeloid Leukaemia (AML)

Blood ◽

10.1182/blood.v122.21.1297.1297 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1297-1297 ◽

Cited By ~ 1

Author(s):

Joanna Zabkiewicz ◽

Marie Gilmour ◽

Robert K. Hills ◽

Elizabeth Bone ◽

Alan Davidson ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Bone Marrow Cells ◽

Therapeutic Window ◽

Normal Bone Marrow ◽

Inhibitory Effects ◽

Normal Bone ◽

Growth Inhibitory ◽

Deacetylase Inhibitor

Abstract Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase inhibitor (HDACi) that is cleaved to an active acid, CHR-2847, by an intracellular esterase (human carboxylesterase-1, hCE-1), found only in cells of monocytoid lineage and hepatocytes. The clinical uptake of HDAC inhibition to date has been restricted by systemic toxicities including gastrointestinal disturbance, thrombocytopenia and fatigue. Accumulation of CHR-2847 in hCE-1-expressing cells results in a 20-100-fold increase in targeted anti-proliferative potency, considerably widening the potential therapeutic window in malignancies involving cells of monocytoid lineage (AML-M4, AML-M5 and CMML) by sparing the systemic toxicological effects associated with non-selective HDAC inhibition. The in vitro efficacy of tefinostat was assessed in primary AMLs using stored mononuclear cells obtained at diagnosis from 70 AML patients. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in M4 /M5 AMLs (median IC50; 1.1µM+/-1.8) compared to non-M4/M5 FAB types (median IC50 5.1µM +/-4.7) (p=0.007). This potency and monocytoid specificity was not reproduced when using an alternative HDACi, tefinostat analogue CHR-8185 which is not cleaved by hCE-1. hCE-1 protein expression in patient samples was measured by both intracellular flow cytometry and immunoblotting, with highest levels seen in M4/M5 patients. This observation was validated by microarray analysis of hCE-1 mRNA in a further 130 AML samples with M4/M5 AMLs showing significant overexpression compared to normal bone marrow CD34+ cells (p=0.009). High levels of hCE-1 expression were found to drive a significant increase in tefinostat efficacy as measured by growth inhibition assays (p=0.001), and also strongly correlated with expression of the mature monocytoid marker CD14+. Sub-population analysis by flow cytometry revealed variable sensitivity to tefinostat within AML blasts, with CD14+ expressing cells showing maximum growth inhibition. This CD14+ response was accompanied by an induction of intracellular protein acetylation at nanomolar concentrations in tefinostat-responsive samples. Tefinostat-sensitive samples also showed strong induction of the cell cycle arrest and DNA damage sensor protein pH2AX, which is a potential biomarker of patient responsiveness. Importantly, no growth inhibitory effects were seen in normal bone marrow cells (n=5) exposed to AML-toxic doses of tefinostat while, in comparison, equivalent concentrations of the non-hCE-1-dependent analogue CHR-8185 caused considerable cytotoxicity, again emphasising the potential for expansion of the clinical therapeutic window using an hCE-1-dependent agent. In vitro synergy was demonstrated in combination experiments with tefinostat and cytarabine (median Combination Index value=0.68) which is likely to be a logical combination for future clinical evaluation. In summary, monocytoid targeting of HDACi activity was achieved using tefinostat in primary AML samples of monocytoid lineage, with minimal toxicity to normal bone marrow cells at equimolar concentrations. Given the absence of significant toxicity seen in a recently-published phase 1 study of tefinostat in patients with advanced haematological malignancies, further larger scale clinical evaluation of this compound is warranted in haematological malignancies involving cells of monocytoid lineage. Disclosures: Zabkiewicz: Chroma Therapeutics: Research Funding. Gilmour:Chroma Therapeutics: Research Funding. Hills:Chroma Therapeutics: Research Funding. Bone:Chroma Therapeutics: Employment. Davidson:Chroma Therapeutics: Employment. Burnett:Chroma Therapeutics: Research Funding. Knapper:Chroma Therapeutics: Research Funding.

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The Proliferation Inhibitor CDKN1C (P57KIP2) Is Over Expressed in CD34+ Cells of Patients with MDS and Determines a Worse Prognosis Independently of IPSS Score Factors

Blood ◽

10.1182/blood.v120.21.3820.3820 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3820-3820

Author(s):

Sascha Dietrich ◽

Mindaugas Andrulis ◽

Andrea Pellagatti ◽

Aleksandar Radujkovic ◽

Ulrich Germing ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Protein Expression ◽

Normal Bone Marrow ◽

Double Staining ◽

Expression Levels ◽

Cyclin Dependent Kinases ◽

Normal Bone ◽

Cd34 Cells ◽

Worse Prognosis

Abstract Abstract 3820 Purpose: Progressive cytopenias are the cause of death for the majority of patients with myelodysplastic syndromes (MDS). In order to investigate if the proliferative activity of CD34+ cells in MDS bone marrow correlates with prognosis, we measured expression levels of the proliferation inhibitor CDKN1C (P57KIP2) in two independent study cohorts. Patients and methods: Gene expression profiling data on bone marrow CD34+ cells were obtained from 183 MDS patients (55 with RA, 48 with RARS, 37 with RAEB1 and 43 with RAEB2). mRNA expression levels of CDKN1C (P57KIP2) were correlated with overall survival (OS) and proliferation markers such as PCNA, cyclins and cyclin dependent kinases. In a second independent patient cohort comprising 93 patients (17 had RA, 13 REAB1, 27 RAEB2, 29 AML arising from MDS and 7 CMML), protein expression levels of CDKN1C (P57KIP2) were evaluated by immune histochemistry (IHC) in trephine biopsies. Protein expression levels of CDKN1C (P57KIP2) were correlated with clinical outcome, OS and the proliferation marker KI67 in CD34+ cells (double staining). Results: In purified CD34+ cells, mRNA expression of CDKN1C (P57KIP2) correlated with higher risk and poorer overall survival of patients with MDS (p=0.0006, n=183, Figure1). Furthermore, increased CDKN1C (P57KIP2) expression was significantly associated with loss of CD38 (p=0.002) as well as loss of proliferating cell nuclear antigen (PCNA, p<0.001), cyclins and cyclin-dependent kinases (p<0.001) underlining the role of CDKN1C (P57KIP2) as a proliferation inhibitor. Similarly, protein expression of CDKN1C (P57KIP2) determined in trephine biopsies predicted a poor prognosis of patients with MDS (p=0.0003, HR=2.2, n=93, Figure 1). No expression of CDKN1C (P57KIP2) could be demonstrated in 10 control patients with normal bone marrow. Separate evaluation of WHO risk categories revealed that in patients with less than 10% blasts (RA, RAEB1 and CMML1), CDKN1C (P57KIP2) was not predictive for OS. In contrast, patients with high CDKN1C (P57KIP2) expression and RAEB2 (p=.0.02, HR 2.4) or AML arising from MDS (p=0.002, HR 2.9) had a significantly worse OS than patients with low CDKN1C (P57KIP2) levels. CDKN1C (P57KIP2) expression analysis within the group of allogeneic stem cell recipients showed no impact on survival after transplant. Multivariate cox regression analysis with the confounding co-variates: age, IPSS score factors (cytopenias, cytogenetic risk profile, blast count) and primary versus secondary MDS confirmed the independent impact of CDKN1C (P57KIP2) expression on OS (p=0.0002, HR 2.9). CDKN1C (P57KIP2) protein expression could also be demonstrated in sorted CD34+ cells of MDS patients by western blot analysis and was significantly higher than in CD34- cells (p=0.03, n=7). KI67 expression was evaluated in CD34+ cells by IHC double staining in 34 trephine biopsies and 10 control patients with normal bone marrow. Percentages of CD34+ and KI67 positive cells were higher in control patients and patients with low risk MDS (RA, RAEB1) than in patients with high risk MDS (RAEB2) (p<0.01). Patients with AML (>20% blasts) had significantly higher levels of KI67 (p<0.05). In patients with MDS (<20% blasts) KI67 percentages correlated inversely with CDKN1C (P57KIP2) expression (p=0.04). Conclusions: High CDKN1C (P57KIP2) expression in CD34+ cells of patients with MDS is associated with a reduced fraction of proliferative CD34+ cells and determines a worse prognosis independently of factors used to calculate the IPSS score. Allogeneic stem cell transplantation could overcome the worse prognosis of high CDKN1C (P57KIP2) expression. Further studies are warranted to determine the impact of CDKN1C (P57KIP2) expression to guide clinical decisions. Disclosures: No relevant conflicts of interest to declare.

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KB004, a Novel Non-Fucosylated Humaneered® Antibody, Targeting EphA3, Is Active and Well Tolerated in a Phase I/II Study of Advanced Hematologic Malignancies

Blood ◽

10.1182/blood.v124.21.3756.3756 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3756-3756 ◽

Cited By ~ 3

Author(s):

Ronan T Swords ◽

Andrew H Wei ◽

Simon Durrant ◽

Anjali S. Advani ◽

Mark S Hertzberg ◽

...

Keyword(s):

Bone Marrow ◽

Phase I ◽

Dose Level ◽

Research Funding ◽

Hematologic Malignancies ◽

Clinical Activity ◽

Bone Marrow Biopsies ◽

Infusion Reactions ◽

Equity Ownership ◽

Dose Levels

Abstract Background: EphA3 is a novel drug target involved in cell positioning in fetal development. In adults it is an oncofetal antigen, that is re-expressed in hematologic malignancies (blood and bone marrow, leukemic stem cells) and solid tumors. It is also upregulated in diseases characterized by abnormal proliferation and fibrosis, such as idiopathic pulmonary fibrosis and diabetic kidney disease. KB004 is a Humaneered® high affinity antibody (KD = 610 pM) targeting EphA3 with at least 3 possible mechanisms of action: direct apoptosis in tumor cells, activation of ADCC and disruption of tumor vasculature. Objectives: The primary objectives of the Phase I study component are to determine safety and MTD for KB004 in patients with hematologic malignancies, refractory to or unfit for chemotherapy. Secondary objectives are to characterize PK, immunogenicity, and preliminary clinical activity of KB004. Exploratory objectives include evaluation of EphA3 expression on tumor, stromal, and endothelial cells. Methods: Multicenter Phase I/II study. Key eligibility criteria: unsuitable for standard of care or relapsed or refractory hematologic malignancy, ECOG PS 0-1, adequate organ function, platelets ≥ 10,000/uL (untransfused for 7 days) and normal coagulation times. KB004 was administered as a 1-2 hr intravenous infusion on days 1, 8, and 15 of each 21-day cycle, at incremental doses of 20, 40, 70, 100, 140, 190, 250 and 330 mg. At 70 mg and above infusion reaction prophylaxis included H1 and H2 blockers, acetaminophen and IV steroids. Safety and activity by IWG response criteria were assessed. Peripheral blood and bone marrow biopsies for PK analysis and EphA3 expression were also collected. Results: A total of 50 patients (AML 39, MDS 7, DLBCL 1, MF 3) received KB004 in the phase I/dose finding component of the study, which has been completed. The most common toxicities were transient grade 1 and grade 2 transient infusion reactions (IRs) in 79% of patients. IRs were characterized by chills, elevated temperature, fever, rigors, back pain, nausea, vomiting, hypotension, hypertension and transient hypoxia (in 2 cases). No other significant KB004 related toxicity was observed. Two patients discontinued KB004 due to an IR. One of these (grade 3) defined a DLT at the 330mg dose level. A second patient at 330mg had grade 2 infusion reactions associated with multiple infusion delays. These observations prompted expansion of the next lowest dose cohort, 250mg. Six evaluable patients were treated at this dose level. No clinically significant IRs or DLTs were observed. This is therefore the recommended phase 2 dose (RP2D). At all dose levels observed Cmax for KB004 was approximately dose proportional. Sustained exposure above the predicted effective concentration (1ug/mL) to cover the 7-day interval between doses was achieved above 190mg. Responses according to IWG criteria were observed in patients with AML, MF and MDS at the 20 mg, 140g and 250mg dose levels, respectively. At 20mg, a 78 yr-old patient with relapsed AML achieved CRp. Remission was sustained for over 18 months and relapse was preceded by a rise in EphA3 expression. Serial bone marrow biopsies with KB004 treatment show decreased reticulin and collagen fibrosis. At 140mg, a 67 yr old patient with JAK2 V617F mutant previously untreated myelofibrosis whose predominant clinical problem at diagnosis was anemia achieved Clinical Improvement [CI]. Transfusion independency (both RBC and platelets) has been sustained for 8+ months with improvement in constitutional symptoms and improved splenomegaly. At 250 mg an 84 yr-old patient with MDS/MPN (intermediate risk) achieved a Hematologic Improvement [HI, erythroid]. A > 50% reduction in marrow blast percentage was seen in 8 patients. Bone marrow biopsies positive for EphA3 expression with a cut-off of 10% of nucleated cells were obtained in greater than 70% of AML patients. Of 20 patients for whom EphA3 expression data exists with time, 7 (35%) had at least a 50% reduction in expression with treatment. Conclusion: KB004 is a novel agent targeted against EphA3 that is well tolerated when given as a weekly 2 hour infusion. The promising clinical activity profile is postulated to be consistent with the antifibrotic mechanism. The Phase II component of the study is ongoing in which the activity of KB004 will be characterized in disease specific cohorts including AML, MDS and MF at the RP2D of 250mg. Disclosures Durrant: KaloBios: Research Funding. Advani:KaloBios: Research Funding. Greenberg:Celgene: Research Funding; Novartis: Research Funding; GSK: Research Funding; Onconova: Research Funding; KaloBios: Research Funding. Cortes:KaloBios: Research Funding. Yarranton:KaloBios: Employment; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; Stemline Therapeutics: Equity Ownership. Walling:KaloBios, Corcept Therapeutics, Prothena, NewGen Therapeutics, Valent Technologies, LBC Pharmaceuticals: Consultancy, Equity Ownership; Amgen, BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees.

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PU.1 and C/EBPalpha Expression in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.4509.4509 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4509-4509

Author(s):

Annalisa Di Ruscio ◽

Francesco D’Alò ◽

Francesco Guidi ◽

Emiliano Fabiani ◽

Giuseppe Leone ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Transcription Factors ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

The Other ◽

Mrna Levels ◽

Normal Bone Marrow ◽

Normal Bone ◽

Acute Myeloid

Abstract The myeloid transcription factors C/EBPalpha and PU.1 play a pivotal role in normal hematopoiesis and alterations of their function are involved in the pathogenesis of Acute Myeloid Leukemia (AML). So far, different mechanisms have been shown to affect their function and are important in some AML subsets. However most AML patients do not apparently show any alteration of these transcription factors. Here, we investigated C/EBPalpha and PU.1 mRNA levels by real time RT-PCR in 109 AML patients and correlated these data to morphology, FLT3 mutations and cytogenetics. C/EBPalpha and PU.1 levels were expressed as percentage of 18S. Twelve normal bone marrow mononuclear cells, four CD34+ cells isolated from normal bone marrow samples and 8 peripheral blood granulocytes and monocytes, were used as controls. Heterogeneous PU.1 expression was observed in AML patients (median 0.657, range 0.004 – 24.148), while PU.1 levels were more homogeneous in normal bone marrows (median 1.5, range 0.328 – 4.737). In particular, 55 AML patients (50.5%) had PU.1 levels similar to controls, while 37 patients (33.9%) and 17 patients (15.6%) expressed PU.1 levels at levels lower and higher, than the control range, respectively. In the same way, also C/EBPalpha mRNA expression was variable (median 0.047, range 0.0002 – 1.858 in AML and median 0.064, range 0.008 – 0.138 in normal bone marrows). Fourty-five AML patients (41.%) displayed C/EBPalpha levels similar to the normal range, while 26 patients (23.8%) had lower and 37 (33.9%) higher C/EBPalpha expression. Looking at different AML subsets, we found low C/EBPalpha mRNA in patients carrying recurrent chromosomal abnormalities, such as t(8;21) and inv16, as previously reported. On the other hand, patients carrying 11q23 rearrangements showed higher PU.1 levels than normal controls. No association was found between C/EBPalpha and PU.1 levels and therapy-related AML, AML with normal karyotype, AML with multilineage dysplasia, and AML not otherwise characterized (including previous F.A.B. categories). Although experimental models showed that FLT3 internal tandem duplications (ITD) downregulate both transcription factors, we did not find any association between the presence of FLT3 ITD and D835 mutations and C/EBPalpha and PU.1 levels, both in the whole patient group and in patients with normal karyotype. We then analyzed expression of two PU.1 and C/EBPalpha target genes, the M-CSF and G-CSF receptors, in patients expressing high and low levels of these transcription factors. A direct correlation was found between C/EBPalpha and G-CSFR levels (Spearman r = 0.5; p=0.02, 95% C.I. 0.07 – 0.78), while there was a tendency to correlation between PU.1 and M-CSFR, that did not reach the statistical significance. Since mutations and post-trascriptional events may affect C/EBPalpha and PU.1 function, we analyzed protein expression of 18 patients by Western Blotting. PU.1 protein was expressed by all patients. The functional p42 C/EBPalpha isoform was absent in 2 patients that expressed only the 30 kDa isoform, and was undetectable in 5 of 18 patients. In conclusion, down regulation of PU1 mRNA was found in one third of AML patients, consistently with the oncosuppressive role recently described. On the other side, C/EBPalpha is down-regulated in specific AML subsets, with recurrent cytogenetic abnormalities, while mutations and post-translational events could affect C/EBPalpha expression in other patients.

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BCR/ABL+ CML Stem Cells (CD34+/CD38-) Express High Levels of CD33 and Are Responsive to a CD33-Targeting Drug: a New Potential Concept for Eradication of CML Stem Cells.

Blood ◽

10.1182/blood.v116.21.3382.3382 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3382-3382

Author(s):

Harald Herrmann ◽

Sabine Cerny-Reiterer ◽

Karoline V. Gleixner ◽

Katharina Blatt ◽

Susanne Herndlhofer ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Chronic Phase ◽

Normal Bone Marrow ◽

Leukemic Stem Cells ◽

Normal Bone ◽

Advanced Phase

Abstract Abstract 3382 Siglec-3 (CD33) is an established therapeutic target in acute myeloid leukemia (AML). We and others have shown that CD33 is expressed on immature CD34+/CD38- stem cells in AML. We here report that leukemic stem cells obtained from patients with chronic myeloid leukemia (CML) display high levels of CD33, and that CD33 may serve as a potential target in CML. As assessed by multi-color flow cytometry, CD34+/CD38-/CD123+ CML progenitor cells in chronic phase (CP) were found to express significantly higher levels of CD33 compared to normal CD34+/CD38- bone marrow stem cells (figure). By contrast, similar levels of the cell surface antigen MDR1 (CD243) were detected when comparing normal and CML progenitors. In chronic phase (CP) CML, CD33 was found to be expressed homogeneously on most or all CD34+/CD38- stem cells. In patients with accelerated (AP) or myeloid blast phase (BP), CML stem cells also co-expressed CD33, but the levels of CD33 varied from donor to donor, and in one patient, most CML stem cells appeared to be CD33-negative cells. In two patients with CML, CD34+/CD38- cells were highly enriched by cell sorting (purity >98%) and found to contain CD33 mRNA in qPCR analysis. The presence of BCR/ABL and thus the leukemic origin of these cells was confirmed by FISH analysis and PCR. We then examined the effects of the CD33-targeted drug gemtuzumab/ozogamicin (GO) on growth of primary CML cells. As assessed by 3H-thymidine uptake, GO produced growth inhibition in leukemic cells in all patients tested (CP, n=13; AP, n=3). The effects of GO on leukemic cell growth were dose-dependent and occurred at relatively low concentrations, with IC50 values ranging between 1 and 100 ng/ml. GO effects were also seen in precursor-enriched Lin-negative CML cells (n=3). As assessed by Annexin-V staining, GO was found to induce apoptosis in CD34+/CD38- CML progenitor cells. Next we investigated drug combination effects. In these experiments, GO was found to synergize with nilotinib and bosutinib in producing growth inhibition in primary CML cells. In conclusion, CD33 is expressed abundantly on immature CD34+/CD38- progenitor cells in CML. Whether GO can be employed to eradicate residual leukemic stem cells in CML patients alone or in combination with BCR/ABL kinase inhibitors remains at present unknown. Figure: Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Figure:. Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

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Hypoxia-Inducible Factor-1 Alpha (HIF-1α) and HIF-2α Are Independent Prognostic Factors for Normal Karyotype Acute Myeloid Leukemia (NK AML)

Blood ◽

10.1182/blood.v112.11.2553.2553 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2553-2553

Author(s):

Kellie M. Demock ◽

Joseph Marinaro ◽

Ian McInnis ◽

Laurie Ann Ford ◽

Meir Wetzler ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hypoxia Inducible Factor ◽

Normal Karyotype ◽

Mrna Levels ◽

Normal Bone Marrow ◽

Normal Bone ◽

Acute Myeloid

Abstract Emerging data has shown that severe hypoxia in vitro selects for highly immature or therapy-resistant leukemia clones and may be a crucial component of leukemia stem cell niches. One mechanism utilized by normal cells to survive hypoxia is upregulation of hypoxia inducible factor-1α (HIF-1α), a master transcription factor that directly transactivates genes important for cellular responses to hypoxia including angiogenesis and anaerobic metabolic pathways. HIF-1α is rapidly degraded under normoxia but is stabilized and synthesized under hypoxia or following malignant transformation. Overexpression of HIF-1α protein is associated with increased patient (pt) mortality in multiple solid cancer types, and with worse clinical outcome in pediatric acute lymphoblastic leukemia; however, its role in acute myeloid leukemia (AML) is unknown. First, we examined the response of human AML cells in vitro to hypoxic stress. We found that in vitro exposure of human AML cell lines (HEL, HL60/VCR) with low baseline HIF-1α levels to hypoxia (≤1% O2, 5%CO2) resulted in significantly increased HIF-1α mRNA levels after 4 hours followed by increased HIF-1α protein and elevated VEGF-A and VEGFR-1 mRNA levels peaking at 8 hours. We then examined expression of HIF-1α and a related hypoxia factor, HIF-2α, in diagnostic marrow samples from 91 consecutive AML pts (46% male, 54% female) treated at our institute from 1995–2005. As karyotype is the most important prognostic factor in AML, we examined only normal karotype AML samples associated with intermediate prognosis. Median patient age was 66 years (range 21–87). Less than half (n=46, 49%) achieved complete remission (CR) following induction chemotherapy. Median overall survival (OS) was 9.6 months. HIF-1α and HIF-2α levels were measured by Q-PCR and expressed relative to normal bone marrow controls (level of mRNA expression=1). HIF-1α protein expression was also evaluated by immunohistochemistry (IHC) and qualified as nuclear vs. cytoplasmic. We found that HIF-1α mRNA levels were consistently higher in AML cells than normal bone marrow controls (median fold change 2.78; range 0.48–22.89), although HIF-1α protein levels was increased in only a minority of samples. In contrast, HIF-2α mRNA levels were consistently lower in AML samples than normal bone marrow (median 0.14; range 0.7–0.42). Univariate analysis demonstrated that age, CR, and nuclear HIF-1α protein expression by IHC (p=0.0081) impacted OS. Significant factors for event free survival (EFS) were age, CR, and cytoplasmic HIF-1α IHC expression (p=0.0422). Multivariate analysis demonstrated that age, CR, cytoplasmic HIF-1α IHC expression (p=0.0056; HR=0.22; 95% CI=0.07–0.64) and HIF-2α mRNA expression (p=0.0101; HR=0.16; 95% CI=0.04–0.65) were independent predictors for OS. Similarly, age, CR, cytoplasmic HIF-1α IHC expression (p=0.0302; HR=0.26; 95% CI=0.08–0.88), and HIF-2α mRNA levels (p=0.0016, HR 0.08, 95% CI= 0.02–0.39) were also independent factors for EFS. Conclusions: Our results are the first to demonstrate that overexpression of HIF-1α and HIF-2α are independent prognostic factors in normal karyotype AML (constituting 40–49% of all adult AML diagnoses). Given the fact that HIF expression can be upregulated by oncogenes and tumor suppressors, additional studies examining potential correlations between HIF-1/2α expression and FLT-3/NPM-1 gene mutations in NK-AML; and in other prognostic AML subgroups (i.e. AML with recurrent or complex cytogenetics) are warranted. Based on these data, inhibition of HIF-1/2α mediated pathways with targeted agents may represent a future means to improve clinical outcome for subsets of AML patients.

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A Recombinant Antibody to EphA3 for the Treatment of Hematologic Malignancies: Research Update and Interim Phase 1 Study Results

Blood ◽

10.1182/blood.v118.21.4893.4893 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4893-4893 ◽

Cited By ~ 3

Author(s):

Anne Hagey ◽

Jeffrey E. Lancet ◽

Varghese Palath ◽

Andrew H Wei ◽

Martin Lackmann ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Recombinant Antibody ◽

Hematologic Malignancies ◽

Pharmacokinetic Data ◽

Employment Equity ◽

Bone Marrow Biopsies ◽

Dose Cohort ◽

Dosing Interval ◽

Equity Ownership

Abstract Abstract 4893 EphA3 is a receptor tyrosine kinase important in fetal development but apparently not in healthy adults. It is expressed in hematologic malignancies including AML, CML, MDS, MPN and Multiple Myeloma and in a range of solid tumors in particular in the stromal and vascular tissue. KB004 is a high-affinity non-fucosylated, recombinant antibody to EphA3 that triggers apoptosis and has potent antibody-dependent cellular cytotoxicity (ADCC) activity against EphA3+ cells. Direct apoptosis of EphA3+ leukemic cells ex vivo was demonstrated at antibody concentrations of 10μg/mL or greater. Expression on a CD34+ CD38− CD123+ cell population in primary AML patient bone marrow, and inhibition of long-term culture initiating cells by KB004, indicates that EphA3 may be present on leukemic stem cells (Palath et al 2010). EphA3 expression in bone marrow biopsies from AML patients was characterized by immunohistochemistry (IHC). EphA3 was detected on tumor cells by IHC in 4/10 samples analyzed. EphA3 was also present on the vasculature in AML bone marrow (6/10 samples) but not in similar tissues in bone marrow biopsies (n=2) from non-leukemic individuals. This suggests tumor vasculature as a potential further therapeutic target for KB004 in AML in addition to targeting leukemia cells directly. We are further characterizing the expression level of EphA3 in different hematologic malignancies, tumor microenvironment, and at various stages of disease progression from primary human samples. The Cynomolgus macaque was selected as a relevant species for toxicity testing since KB004 binds with equivalent affinity to human and Cynomolgus EphA3 and CD16. IHC studies demonstrated the absence of cellular expression of EphA3 in normal human or Cynomolgus monkey tissues. KB004 administered to Cynomolgus monkeys (n=44) twice weekly for 13 weeks at doses up to 100 mg/kg was well tolerated and there were no clinical or pathology adverse findings. Cardiovascular, respiratory and central nervous system functions were measured in primates after KB004 dosing 5 times over 15 days to reach steady-state exposure at doses of 10mg/kg (n=4) or 100 mg/kg (n=4). There were no KB004-related effects on body weight, food consumption, mean arterial pressure, heart rate, body temperature, neurological parameters, respiration rate, oxygen saturation, or blood gas parameters, or changes in serum concentrations of Troponin I. Qualitative evaluation of the electrocardiogram (ECG) did not reveal any electrocardiographic abnormalities and there were no effects on measured ECG intervals (i.e., duration of the QT and RR intervals, derived QTc values). The effect of KB004 on wound healing was tested in a primate incisional wound healing model in the Cynomolgus monkey. No statistically significant differences in healing were detected between control and KB004 (2×10 or 2×100mg/kg) treated animals (n=24). A Phase I clinical study has been initiated in subjects with hematologic malignancies including AML, CML, ALL, MDS and MPN. Subjects are being assessed for EphA3 protein expression at study entry and at various time points throughout the treatment period. Study objectives are to determine a maximum tolerated dose, examine the safety and tolerability profile of KB004, obtain pharmacokinetic data, describe the immunogenicity profile, and explore cell subpopulations and pharmacodynamic effects of treatments with KB004. This is an open-label, repeat administration study of weekly IV dosing of up to 17 cycles (3 doses per 21-day cycle). Dosing levels are scheduled for 20 mg (∼0.3 mg/kg), 70 mg (∼1 mg/kg), 200 mg (∼3 mg/kg) and 700 mg (∼10 mg/kg). Therapeutic antibody levels (target level 10μg/mL) are predicted to be achieved from the first dose cohort for a portion of the dosing interval. The higher doses should provide blood levels in excess of 10 μg/mL for the entire one-week dosing interval. The first cohort (n=3) has been successfully completed, and a subject with AML remains on study having received 8 doses to date. Recruitment of the next dose cohort is ongoing. Pharmacokinetic data and tumor expression profiles will be presented. Disclosures: Hagey: KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Lancet:KaloBios Pharmaceuticals, Inc.: Research Funding. Palath:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Wei:KaloBios Pharmaceuticals, Inc.: Research Funding. Lackmann:KaloBios Pharmaceuticals, Inc.: Research Funding. Cortes:KaloBios Pharmaceuticals, Inc.: Research Funding. Boyd:KaloBios Pharmaceuticals, Inc.: Research Funding. Shochat:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yarranton:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bebbington:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Leff:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership.

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Detection of NFKB2 3’end Loos By Quantitative PCR (QPCR) or Detection of NFKB2 Rearrangements Correlate with Bortezomib Response in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2138.2138 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2138-2138

Author(s):

Sampath Ramachandiran ◽

Jean L. Koff ◽

Laurent Garderet ◽

Debra Saxe ◽

Souhila Ikhlef ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Quantitative Pcr ◽

Research Funding ◽

Cell Cancer ◽

Proteasome Inhibitors ◽

Structural Rearrangement ◽

Mrna Levels ◽

Irreversible Inhibitor ◽

Mixed Cell

Abstract Background: Multiple myeloma is a malignant plasma cell cancer that is increasing in frequency in today’s aging population. While proteasome inhibitors such as bortezomib have improved the outcome of treatment, many patients fail to achieve a complete remission and ultimately the disease progresses. With these new treatments come higher associated toxicities and significant healthcare costs for the drugs. Several new cancer therapies with companion diagnostics have been approved in parallel with the drug to guide physicians in selecting those patients who would specifically benefit from the drug. No such test exists for the use of bortezomib in multiple myeloma. We have previously demonstrated that the 3’ end of the nuclear factor kappa-B 2 (NFKB2/p100) gene, a member of the NFKB/Rel gene family, functions as a mediator of bortezomib apoptotic effect through the induction of caspase 8 activation. Methodology: We have investigated the loss of NF-kB2 3’end loss by quantitative PCR (QPCR) in 56 MM patients and by break-apart Fluorescent In-Situ Hybridization in the whole bone marrow of 31 patients as well as in sorted cells of 24 MM patients treated with bortezomib containing regimens. Quality of response was assessed after 4 cycles of treatment initiation. QPCR was measured in peripheral blood lymphocyte and CD138 positive cells from the bone marrow to generate a QPCR ratio. Results: 37 of the 56 newly diagnosed patient with MM achieved >partial response (PR), while 16 remained stable or progress to treatment. The mean QPCR ratio in responders was 1.69 ± 0.13, while in non-responder was 0.43 ± 0.1 (p<0.001). Based on this finding, we assessed the influence of the loss of NF-kB 3’end on the clinical response. Univariable analysis demonstrated that low CD38(+) NF-kB2 3’end mRNA levels (odds ratio [OR] of 10.8; 95% CI 1.99 to 58.16, p=0.0057) and history of MGUS (OR: 7.6; 95% CI of 1.2 to 47.6, p<0.05) were significantly associated with poor outcome. However, multivariate analysis indicated that low CD38(+) NF-kB2 3’end levels was an independent risk factor for poor response to bortezomib and dexamethasone (VD, HR:21.47, 95% CI: 2.99 to 154.399, p <0.01, supplemental Table 2). To further evaluate for the presence of a structural rearrangement, we designed a NF-kB2 break-apart FISH assay. We first performed FISH on whole bone marrow for 31 patients treated with VD. As expected the percentage of broken signals in unsorted samples was low, but when we considered the proportion of plasma cells in each sample, the total percentage of broken signals was statistically significantly higher in the nonresponder (nonresonder: 9.12± 4.2 vs Responder:2.37 ± 0.7, p<0.035). Using a cut off detected by AUC curve, we identified NF-kB2 structural rearrangement in 9 patients (30%) patients of which 6/10 were present in VD-non responders and 3/21 of VD-responders. To validate these findings we performed FISH on CD38+ sorted cell from 24 new patients treated with VD or carfilzomib, an irreversible inhibitor of the proteasome, and found consistency with our previous results, in which 7 patients demonstrated a NFKB2 positive break signal. Six out of 7 samples of non-responders patients demonstrated a positive break signal while 1/16 were positive in the responders, indicating a chromosome translocation or inversion had disrupted the NFKB2 locus and that this disruption predispose tumors to respond poorly to proteasome inhibitors. We tested multiple cell lines carrying NF-kB2 rearrangement and identified by target sequencing that HUT88 presents a balance translocation between chromosomes 10 and 3. Taking advantage of this finding we compared the sensitivity of detection between 3’end NFKB2 QPCR and the break-apart FISH. To this end, we mixed cell line with normal NF-kB2 as it is in MM1S with titrating doses of HUT88. Quantitative PCR was able to detect a minimum of 3% HUT88 cells in the mix, while NFKB2 break-apart FISH was able to detect when the percent of HUT88 reach 10%. The correlation between both test was r2:0.98, P<0.02. Conclusion: loss of NF-kB2 3’ end is a potential companion diagnostic test for bortezomib. QPCR is more sensitive to detect NFKB2 3’end abnormalities. Disclosures Kelkar: Empire Genomics: Employment. Kaplan:Empire Genomics: Employment. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

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