Epor Is Not Expressed or Functional in Primary Tumor Cells Isolated Directly From Human Primary and Metastatic Epithelial Tumors Including Breast, Non-Small Cell Lung, Colorectal, and Ovarian Tissues

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4781-4781
Author(s):  
Ian McCaffery ◽  
John M Rossi ◽  
Katherine L Paweletz ◽  
Yanyan Tudor ◽  
Steve Elliott ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Tumor Cells ◽  
Human Tumor ◽  
Small Cell ◽  
Small Cell Lung ◽  
Employment Equity ◽  
Equity Ownership ◽  
And Function ◽  
Epor Expression

Abstract Abstract 4781 Several clinical trials have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents (ESA) in patients with cancer. One hypothesis proposes that ESAs stimulate tumor proliferation and or survival directly through binding and activating cell-surface receptors (EpoR). Expression and function of cell surface EpoR in human tumor tissue is controversial and has not been extensively investigated using validated reagents. Methods EpoR expression and function was analysed in disaggregated primary tumor cells obtained from surgically resected human tumor tissues from 186 patients: colorectal (n=46), breast (n=34, including 8 Her2+ tumors defined by HercepTest™), non small cell lung (n=41), ovarian (n=35), Head and Neck (n=5) and other tumor types (n=8). Metastatic tissues were also analysed: colon (n=2), ovary (n=3), lung (n=2), pancreatic (n=1) as well as tumors from pre-treated patients: colorectal (oxaliplatin, bevacizumab; n=1), breast (cyclophosphamide, methotrexate, fluorouracil; n=2), breast (radiation, docetaxel cyclophosphamide, Paclitaxel; n=1), non small cell lung (radiation; n=2), ovarian (carboplatin and Paclitaxel; n=2). To examine if cell surface EpoR is expressed in the tumor cell compartment, flow cytometry analysis was carried out using a validated, EpoR-specific antibody (mAb 307). To evaluate if tumor cells respond functionally to recombinant human Epo (rHuEpo), levels of phosphorylated downstream signaling proteins (Akt, Erk1/2 and STAT5) were analysed by intracellular flow cytometry using phospho-specific antibodies following ex vivo stimulation with a titration of rHuEpo (0U [vehicle] to 300U/mL for 5 and 30 min). Expression of known tumor growth factor receptors (IGF-1R, c-Met and EGFR) was analyzed as positive controls. Additionally, the response of Akt, Erk and STAT pathways to stimulation with a tumor growth factor cocktail (“GF”: EGF, HGF and IGF-1), was used as a control for pathway integrity. The sensitivity of all flow based assays was validated through the analysis of EpoR expression and function in erythroid precursor cells (EPCs) across a time course of differentiation from primary human bone marrow samples. Results Though tumor samples exhibited cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface EpoR was detected in tumor cells from any of the 186 tumors examined by flow cytometry. Mean expression relative to negative control: EGFR = 2.80 (95% CI, 1.63–3.97), c-Met = 5.28 (95% CI, 4.39–6.17), IGF-1R = 8.03 (95% CI, 6.81–9.24) and EpoR (0.69 (95% CI, 0.64–0.73). No EpoR was detected by Western blot analysis of tumor lysates. As shown in Table 1, treatment with the GF cocktail stimulated phosphorylation of Akt and Erk in all cohorts. However, no activation of these pathways (or STAT5) by rHuEpo was observed in any of tumors even at high supra-pharmacological concentrations of rHuEpo (up to 300U/mL) and extended stimulation times (up to 30 mins). Analysis of EpoR expression and function during differentiation in-vitro of EPCs revealed detectable EpoR cell surface expression (2.13-fold above baseline) and function (e.g. pSTAT5: 1.39-fold relative to vehicle at 300 U/mL rHuEpo) as early as day 1 when EPCs (CD36+/CD34-) comprised only 6.9% of the population. By day 8, (>90% EPCs) EpoR expression was detectable at 6.36-fold relative to baseline and pSTAT5 activation was observed 5.6-fold relative to vehicle at 300U/mL rHuEpo. Conclusion Using sensitive methods no evidence of EpoR expression or function in primary human tumor cells isolated directly from primary and metastatic tumors was observed in multiple epithelial tumor types. Expression and functional activation of EGFR, c-Met and IGF1R was readily detectable. Therefore, this study suggests that it is unlikely that rHuEpo acts as a growth factor for primary human tumor cells. Disclosures: McCaffery: Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Elliott:Amgen Inc: Employment. Fitzpatrick:Amgen Inc.: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership.

Megakaryocytes Do Not Express Erythropoietin Receptor (EpoR) and Do Not Respond to Recombinant Human Erythropoietin (rHuEpo) Stimulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4783-4783
Author(s):  
Robert D Loberg ◽  
John M Rossi ◽  
Raffi Manoukian ◽  
Shen-Wu Wang ◽  
Katherine L Paweletz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Negative Control ◽  
Employment Equity ◽  
Downstream Signaling ◽  
Human Erythropoietin ◽  
Equity Ownership ◽  
And Function ◽  
No Activation ◽  
Epor Expression

Abstract Abstract 4783 Recently, clinical studies were conducted in chronic kidney disease of type 2 diabetes patients that suggested an increased risk of stroke in patients receiving Erythropoiesis Stimulating Agents (ESAs) compared to placebo. One proposed explanation is that megakaryocytes and/or platelets express EpoR and respond to ESA challenge. We evaluated the underlying and untested assumption that megakaryocytes or platelets express a functional EpoR using flow cytometry assays developed to detect cell surface EpoR using validated EpoR-specific antibodies. Additionally, in order to investigate whether megakaryocytes/platelets are responsive to ESAs, the activation status of downstream signaling proteins was evaluated after stimulation with ESAs or vehicle control. Whole bone marrow aspirates were collected from healthy volunteers and bone marrow mononuclear cells (BMMCs) isolated by Ficoll separation (n=25). Megakaryocytes were isolated from BMMCs by using CD61 immuno-magnetic bead capture and analyzed by flow cytometry, gating for megakaryocytes using viability, CD41a expression and polyploidy. Platelets were analyzed in peripheral blood from healthy volunteers (n=10) using CD41a as marker. To measure EpoR expression an EpoR-specific monoclonal antibody was used for flow cytometry analysis in both platelets and megakaryocytes that were isolated and gated as described above. Megakaryocytes and platelets were stimulated with rHuEpo (1, 10, and 300 U/mL for 5 and 30 min) and induction of pSTAT5 was measured by intracellular flow cytometry using phospho-specific antibodies. In addition, platelets (CD41a+) were assessed for EpoR expression and functional response to recombinant human erythropoietin (rHuEpo) challenge. Stimulation with thrombopoietin (TPO) was used as a positive control for pSTAT5 induction in megakaryocytes and platelets. While robust EpoR expression was, as expected, observed consistently in UT7/Epo control cells and absent in CMK negative control cells (fold over control – UT7/Epo 12.78 ± 5.03; CMK 0.77 ± 0.22 mean ± 95%CI), no significant expression of EpoR was observed on CD41a+/polyploid megakaryocytes (fold over control − 1.006 ± 0.03; mean ± 95%CI). No activation of downstream signaling (pSTAT5) was detected in megakaryocytes in any of the samples stimulated with rHuEpo at any of the concentrations or timepoints analyzed, while robust pSTAT5 was detected in the UT7/Epo control cell lines. CMK cells were included as a negative control and demonstrated an absence of pSTAT5 induction in response to rHuEpo while a robust induction to TPO was observed (fold over control – UT7/Epo 12.11 ± 7.86; CMK 1.05 ± 0.03; mean ± 95%CI). Similarly, no significant EpoR expression was detected on CD41a+ platelets while the control, c-Mpl expression, was significant (fold over control – EpoR 1.11 ± 0.019; c-Mpl 2.936 ± 0.71; mean ± 95%CI) and no activation of downstream signaling (pSTAT5) was detected in platelets in any of the samples stimulated with rHuEpo at all concentrations and timepoints analyzed. The lack of EpoR expression on megakaryocytes was confirmed by laser scanning cytometric analysis in patient samples. A robust/sensitive platform was developed to profile biologically relevant EpoR expression and function in megakaryocytes and platelets. The assays for EpoR expression and function were sufficiently sensitive to allow characterization during in-vitro differentiation of erythroid progenitors. Using this platform we demonstrated a lack of EpoR expression and functional response to rHuEpo on megakaryocytes and platelets isolated from human bone marrow (CD41a+/CD61+/polyploid). Disclosures: Loberg: Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Manoukian:Amgen: Employment, Equity Ownership. Wang:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Sable:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership. McCaffery:Amgen: Employment, Equity Ownership.

Analysis of cell surface erythropoietin receptor (EpoR) expression and function in human epithelial tumor tissues

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 11104-11104
Author(s):  
J. Rossi ◽  
I. McCaffery ◽  
K. Paweletz ◽  
Y. Tudor ◽  
S. Elliott ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Human Tumor ◽  
Erythropoietin Receptor ◽  
Epithelial Tumor ◽  
And Function ◽  
Tumor Types ◽  
Epor Expression ◽  
Positive Controls

11104 Background: EpoR mRNA and protein expression have been reported at low levels in human tumors implying EpoR may be functional in tumor cells with inferred implications for the use of erythropoiesis stimulating agents in the Oncology setting. However, mRNA studies use bulk tumor tissue which ignores contribution from the stroma and IHC studies have all used Abs that have since been shown to not be specific for EpoR. Therefore this important question remains unresolved. Methods: EpoR expression and function was investigated in viable, non-apoptotic primary human tumor cells (using tumor specific Abs) that were disaggregated from at least 30 patients from each of the following tumor types: breast (incl. metastases), NSC lung, colorectal, and ovarian. Additional tumor types included head and neck, GBM, pancreatic and gastric. Analysis was performed under conditions that were shown to preserve EpoR expression and function in positive control tissues (primary human erythroid progenitors from bone marrow and UT-7 cells). Cell surface EpoR expression was evaluated by flow cytometry using novel EpoR-specific Abs. EpoR function was investigated by analysis of EpoR signaling using phospho-specific Abs specific for STAT5, Erk, Akt etc following ex vivo Epo stimulation (0U- 300U/mL; for 5 and 30 min). Results: No expression of cell surface EpoR was detected in tumor cells in any of the over 130 tumors. In contrast, high levels of expression were observed in positive controls analyzed in parallel. No induction of EpoR signaling was observed in tumor cells at any [Epo], whereas activation was readily detected in tumor cells treated in parallel with a cocktail of known tumor growth factors. Positive controls treated in parallel, showed robust Epo concentration-dependent signaling. No evidence of EpoR expression or function was observed in tumor endothelial cells. Conclusions: These data demonstrate that epithelial tumor cells do not express functional cell surface EpoR and are not responsive to physiological, therapeutic or indeed very high levels of Epo (300U/mL). No significant financial relationships to disclose.

Analysis of Cell Surface Erythropoietin Receptor (EpoR) Expression and Function in Epithelial Human Tumor Tissues Reveals No Detectable Cell Surface Expression or Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5396-5396
Author(s):  
Ian McCaffery ◽  
John Rossi ◽  
Katherine Paweletz ◽  
Yanyan Tudor ◽  
Steve Elliott ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tumor Cells ◽  
Primary Tumor ◽  
Tumor Tissue ◽  
Human Tumor ◽  
Human Tumor Cells ◽  
Epithelial Tumor ◽  
Tumor Tissues ◽  
And Function ◽  
Epor Expression

Abstract Analysis of Cell Surface Erythropoietin Receptor (EpoR) Expression and Function in Epithelial Human Tumor Tissues Reveals No Detectable Cell Surface Expression or Function A number of studies have reported that EpoR mRNA is detectable at low levels in human epithelial tumor tissues and have raised the possibility that this surface EpoR protein expression could render human tumor cells responsive to Epo with inferred implications for the use of erythropoietic stimulating agents in the Oncology setting. To date the only data supporting this hypothesis has been generated using mRNA analysis or protein analysis using antibodies that have subsequently been shown to not be specific for EpoR. To address these possible issues more directly we have analyzed EpoR expression and function in a large panel of human primary tumor tissues isolated by surgical resection from a variety of epithelial tumor cells (including colon, non small cell lung, breast and ovarian tumors). Solid human tumor tissue was disaggregated using a treatment shown to preserve cell surface EpoR density and function in positive controls (below). Flow cytometric analysis of primary tumor cells in the disaggregated tumor population was performed using multiple markers specific for epithelial tumor cells, as well as viability dyes and apoptotic markers to exclude non-viable and or apoptotic cells from the analysis. Using anti-EpoR monoclonal antibodies with high sensitivity and specificity for EpoR, no expression of cell surface EpoR was detected in primary tumor cells in any of the more than 60 human epithelial tumor specimens analyzed. In contrast, high levels of expression were observed in a positive control cell line (UT7/Epo) analysed in parallel, as well as a physiologically relevant primary tissue (differentiated erythroid progenitor cells). Notably, in a fraction of the ovarian and breast tumor tissues cell surface EpoR expression was identified in CD45+ tumor infiltrating leukocytes. No EpoR expression was detected in non tumor cells that were not CD45+ suggesting no contribution from other stromal elements in the tumor. These observations may explain the reported detection of EpoR mRNA in a subset of breast and other solid tumor patients, as those previous analyses involved bulk tumor tissue and did not allow analysis of tumor cell specific expression. To test the possibility that low cell-surface EpoR protein density on tumor cells may be sufficient for function but undetectable by flow cytometry, we evaluated the ability of primary human tumor tissue to support an EpoR-driven signal pathway. EpoR function was analyzed in primary human tumor cells treated with a range of concentrations of recombinant human Epo (rHuEpO; 0.1–300U/mL) under conditions shown to result in productive EpoR dependent signaling in positive control cells/tissues. Analysis of possible EpoR-driven signaling was determined by intracellular flow cytometry using antibodies specific for the phosphorylated forms of STAT5, Akt, Erk1/2, p70S6, STAT3, STAT1, STAT6, JNK, and c-jun. Attribution of any detected signaling to viable tumor cells was performed via the use of a combination of tumor cell specific and viability/apoptosis markers. No evidence of downstream signaling was observed in primary tumor cells in epithelial tumors from over 60 patients at any concentration of rHuEpo, whereas UT7/Epo cells, treated in parallel, showed robust Epo concentration-dependent activation of signaling. Furthermore, activation of these signaling proteins was detected when the same primary human tumor cells were treated in parallel with a cocktail of known human tumor growth factors, confirming that these cells are capable of responding to exogenous stimuli using the same pathways as EpoR and that these signaling events can be readily detected using the platform. Taken together these data support the hypothesis that tumor cells in solid human tumors do not express functional cell surface EpoR and are not responsive to physiological or therapeutically relevant concentrations of Epo or indeed very high levels of Epo (300U/mL).

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4070-4070
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Udaya Rangaswamy ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cytokine Secretion ◽  
Employment Equity ◽  
Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

Erythropoietin and Erythropoietin Receptor Expression in Early Stage Non-Small Cell Lung Cancer: Prognostic Significance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4258-4258 ◽  
Author(s):  
Khalid Amin ◽  
Zishan A. Haroon ◽  
Seok J. Kim ◽  
Sufeng Li ◽  
Zahid N. Rabbani ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Overall Survival ◽  
Tumor Cells ◽  
Early Stage ◽  
Small Cell ◽  
Wilcoxon Test ◽  
Small Cell Lung ◽  
Primary Nsclc ◽  
Epor Expression

Abstract Erythropoietin (EPO) and its cell surface receptor EPOR have been shown to be expressed in many different types of cancer such as breast, prostate and head and neck cancer. The objectives of this study were to determine the frequency of EPO and EPOR expression in non-small cell lung cancer (NSCLC) and to characterize the association of EPO and EPOR expression with clinical and histological features of early stage NSCLC and its outcome. Seventy three patients with primary NSCLC from the Durham Veterans Affairs Hospital who underwent primary resection of their tumors were included in the study after providing informed consent in accordance with a research protocol approved by the Institutional Review Board at Durham Veterans Administration Medical Center. The median follow up period was 3.8 years. At the time of last follow-up 24 patients (33%) had recurred and 38 patients (52%) had died. Histologically, 38 primary tumors (52%) were squamous cell carcinomas, 27 (37%) were adenocarcinoma (ADC) and 8 (11%) were other types of NSCLC. Thirty-three patients (45%) had T1 lesions, 37 (51%) had T2 and 3 patients (4%) had T3 tumors. Nodal stage was N0 in 59 patients (81%) and N1 in 14 patients (19%). Fifty-six patients (77%) had pathologic stage I disease and 17 patients (23%) has stage II disease. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tumors from each patient using monoclonal antibodies against EPO and EPOR. The expression was determined using a semi-quantitative scoring scale taking into consideration both the intensity and the percentage of tumor cells staining positive. Weak expression in ≤ 5% of tumor cells was considered negative. EPO was expressed in 60 of the 73 tumors (82%) and frequently exhibited a focal pattern of staining. EPOR was expressed in 60 tumors (82%) with predominantly cytoplasmic localization except for several cases where membrane staining of EPOR was also observed. Both EPO and EPOR were expressed in 55 (75%) tumors. We also characterized the expression of hypoxia-inducible factor-1a (HIF1a), the hypoxia regulated protein CA-IX, and to determine the Ki-67 proliferation index and microvessel density (MVD) using CD31 immunostaining. Patients with adenocarcinoma had lower EPOR expression in comparison to patients with other histologic subtypes (Wilcoxon test, P=0.03). EPOR expression was not associated significantly with other clinical or histological parameters or outcome. Patients with early nodal stage (N0) and overall pathologic stage (stage I) disease had significantly higher EPO expression (Wilcoxon test, P=0.01 and P=0.02, respectively). High MVD levels were associated with high EPO expression (Wilcoxon test, P=0.01) but EPO was not associated with any other histologic variables. Interestingly, higher EPO expression was associated with significantly better recurrence-free survival (Logrank test, P=0.006) and overall survival (P=0.008). Cox proportional hazard regression analysis revealed that high EPO expression was associated with better survival (P<0.05). In multivariate analysis, EPO was not an independent prognostic factor. In conclusion, these data in our cohort of patients demonstrate that - EPO and EPOR are expressed with high frequency in primary NSCLC with the majority of tumors co-expressing both EPO and EPOR and - High EPO expression in tumor cells is associated with better recurrence-free and overall survival.

scholarly journals CD33 SNP Genotype and Splice Variation Are Associated with CD33 Cell Surface Expression and SGN-CD33A Pharmacokinetics

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3942-3942
Author(s):  
Katherine Tarlock ◽  
Zixing Wang ◽  
Rory Rohm ◽  
Travis Biechele ◽  
Rhonda E. Ries ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Drug Exposure ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Set Domain ◽  
Employment Equity ◽  
Exon 2 ◽  
Splice Variation ◽  
Equity Ownership

Abstract The cell surface antigen CD33 is expressed on the majority of AML blasts and is appropriate for immunotherapeutic targeting with antibody drug conjugates (ADCs). Expression of CD33 is in part mediated by splicing of the CD33 transcript, and has been demonstrated to be one of the factors that may mediate response to the ADC gemtuzumab ozogamicin, which results in significant benefit in some patients but lacks responses in others. Splicing of the CD33 transcript is in part regulated by a single nucleotide polymorphism (SNP) in exon 2 (e2) that causes a C>T substitution and the resultant skipping of e2. CD33 thus exists in 2 main isoforms, as either a full length (FL) transcript or a truncated version missing e2 (Δe2), which includes the IgV binding domain that is the epitope for diagnostic and therapeutic antibodies (Ab). The CC genotype encodes the FL isoform at a higher rate compared to the CT or TT, and the TT genotype encodes the short isoform at a higher rate compared to CT or CC. SGN-CD33A is a CD33-directed ADC, utilizing a pyrolobenzodiazepine (PBD) dimer. SGN-CD33A has been evaluated in multiple clinical trials as either monotherapy or in combination. We hypothesized that the patient's CD33 genotype would impact CD33 expression as well as response characteristics following treatment with SGN-CD33A. We analyzed CD33 genotype variation in bone marrow (BM) or peripheral blood (PB) samples from patients treated with SGN-33A as either monotherapy (NCT01902329; n=133) or in combination with hypomethylating agents (HMAs; NCT02785900; n=83). CD33 SNP genotyping was determined on gDNA using RFLP PCR with 2 restriction enzymes recognizing cut sites generated by the C and T polymorphisms and genotype confirmed using fragment length analysis (CC=108, CT=86, TT=22). CD33 surface expression on the AML blasts was determined by flow cytometric analysis using the human anti-CD33 monoclonal Abs HIM3-4 and H212, which bind to the membrane-proximal C2-set and V-set domain, respectively. HIM-34 measured CD33 levels independent of SNP-driven splice variation. The h2H12 epitope is within e2, thus its binding may be susceptible to splice variation. We subsequently evaluated the association of CD33 genotype with pharmacokinetic (PK), clinical and other variables using a generalized regression model. Patients classified as CC genotype had significantly higher surface CD33 expression as determined by flow cytometry in both BM and PB. In accordance with observed differences in CD33 expression, we also found drug exposure demonstrated an inverse relationship according to CC genotype in both mono and combination therapy trials. For monotherapy, compared to patients with CC and CT genotypes, patients with TT genotypes had significantly higher drug exposure following SGN-CD33A. Patients with TT had higher AUCs following the first and last doses of SGN-33A (p < 10-4 -; Fig 1). Cmax following SGN-CD33A exposure was higher in TT genotype patients compared to the CT and CC (p< 10-1.5 for Cmax following the first dose and p<10-1.6 for Cmax over all treatments;Fig 1). In combination with HMAs, the TT genotype was also associated with significantly higher SGN-CD33A AUC and Cmax (p-values ranging from 10-3.3 - 10-9.7). We next examined expression and subcellular localization of CD33 to elucidate the mechanism by CD33 variation contributes to cell surface presentation. Transfection of cDNA encoding the FL CD33 transcript resulted in increased cell surface expression, as indicated by flow cytometry with both HIM3-4 and h2H12. In contrast, both Abs failed to detect cell surface CD33 following transfection with cDNA encoding the Δe2 variant. Examination of the intracellular compartment revealed that HIM3-4, but not 2H12, binds to the Δe2 variant in a pattern localized proximal to the nucleus. Taken together, our findings suggest that the Δe2 splice CD33 variant lacks the portion of the V-set domain required for h2H12/SGN-CD33A binding and does not efficiently traffic to the cell surface. We show that CD33 SNP genotype is associated with CD33 expression, with CC patients demonstrating higher CD33 as detected by flow cytometry; and that CD33 SNP genotype affects the PK profile of SGN-CD33A, with TT patients having higher levels of drug exposure. Our findings suggest that the CD33 genotype can impact CD33 expression, PK profile, and trafficking of bound agents and thus may impact therapeutic targeting of CD33-directed agents. Disclosures Wang: Seattle Genetics: Employment, Equity Ownership. Rohm:Seattle Genetics: Employment, Equity Ownership. Biechele:Seattle Genetics: Employment, Equity Ownership. Means:Seattle Genetics: Employment, Equity Ownership. Thurman:Seattle Genetics: Employment, Equity Ownership. Arthur:Seattle Genetics: Employment, Equity Ownership.

scholarly journals Detection of disseminated tumor cells and their relationship with a population of bone marrow lymphocytes in patients with non-small cell lung cancer

2020 ◽  
Vol 22 (3) ◽  
pp. 94-99
Author(s):  
T. M. Djumanazarov ◽  
S. V. Chulkova ◽  
N. N. Tupitsyn ◽  
O. A. Chernysheva ◽  
A. K. Allakhverdiev ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Small Cell Lung Cancer ◽  
Tumor Cells ◽  
Disseminated Tumor Cells ◽  
Small Cell ◽  
Small Cell Lung ◽  
Bone Marrow Damage ◽  
Lymphocyte Populations

Introduction.Detection of disseminated tumor cells (DTC) in solid tumors is an important component of the assessment of disease prognosis. Bone marrow damage is common. There is evidence indicating an important role for bone marrow lymphocyte subpopulations in hematogenous metastasis. Aim.To evaluate the frequency of bone marrow damage in patients with non-small cell lung cancer (NSCLC) based on the detection of DTC by flow cytometry, as well as their effect on the population of bone marrow lymphocytes. Materials and methods.62 bone marrow samples of patients with a verified diagnosis of NSCLC: adenocarcinoma (33), squamous cell carcinoma (27), other types (2). Methods: morphological, multicolor flow cytometry. Studied DTC, lymphocyte populations CD3, CD4, CD8, CD19, CD20, CD16, CD27. Collection and analysis: FACS Canto II, USA, Kaluza Analysis v2.1. Results.In bone marrow, DTC (EPCAM+CD45-) were found in 43.5% of patients with NSCLC (1 cell per 10 million myelocariоcytes was taken as the threshold value). The presence of DTC did not correlate with the size of the tumor, the status of the lymph nodes, and the stage of the tumor process. DTC was more often observed in more differentiated tumors (p=0.023). A significant increase in the level of subpopulations of CD16+CD4-NK-cells (p=0.002), CD27+CD3+T-cells (p=0.015) with bone marrow damage was revealed. Conclusion.The possibility of detecting DTC in the bone marrow of patients with NSCLC was established, in 43.5% of patients with NSCLC in the bone marrow DTC was detected, and their presence was established even with a localized tumor process. More frequent bone marrow damage was observed with well-differentiated tumors. The relationship between DTC and bone marrow lymphocyte populations was revealed: subpopulations of CD16+CD4-, CD27+CD3+.

Correlation of circulating tumor enumeration and immune checkpoint marker expression on CD103+, CD4+ T cells in non-small cell lung cancer tissue.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 109-109
Author(s):  
Xiaoyang WANG ◽  
Pin-I Chen ◽  
Maria Jaimes ◽  
Humin Gu ◽  
Keith Shults ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Tumor Cells ◽  
Immune Checkpoint ◽  
Clustering Analysis ◽  
Nsclc Patient ◽  
Small Cell ◽  
Small Cell Lung ◽  
Cancer Markers

109 Background: Non-small cell lung cancer (NSCLC) has a poor prognosis as most patients are at advanced stage when diagnosed. Targeted therapy and immunotherapy in recent years has significantly improved NSCLC patient outcome. In this study, we employed cell-by-cell immune and cancer marker profiling of the primary tumor cells to investigate possible signatures that might predict the presence or absence of circulating tumor cells (CTCs). Methods: We performed a comprehensive study on 10 NSCLC patient tissue samples with paired blood samples. The solid tissue biopsy samples were dissociated into single cells by non-enzymatic tissue homogenization. The single cell suspensions were stained with a total 25 immune, cancer markers and a DNA content dye and analyzed with advanced, high-parameter flow cytometry. CTCs were isolated and analyzed from the paired peripheral blood. Results: Out of the 26 unique cell markers stained, we investigated a total of 72 biomarkers for their correlation with CTC number. Strong correlations were observed between CTC number and the frequency of immune checkpoint marker expressing lymphocytes, especially with the immune checkpoint marker expressing CD103+CD4+ T lymphocytes. CTC number is also correlated with the frequency of PD-L1 expressing cancer cells and cancer cell DNA content. In contrast, CTC number inversely correlated to the frequency of CD44+E-cadherin- cancer cells. Unsupervised clustering analysis based on the biomarker analysis separated the CTC negative patients from the CTC positive patients. Conclusions: Profiling multiple immune and cancer markers on cancer samples with multi-parametric flow cytometry allowed us to obtain protein expression information at the single cell level. Clustering analysis of the proteomic data revealed a signature driven by checkpoint marker expression on CD103+CD4+ T cells that could potentially be predictive of CTCs.

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