High CD38 Expression Research Articles

Targeting the CD47-SIRPα pathway promotes macrophage-mediated trogocytosis of ovarian cancer cells

Targeting the CD47-SIRPα pathway promotes macrophage-mediated trogocytosis of ovarian cancer cells

Monocyte Infiltration and Differentiation in 3D Multicellular Spheroid Cancer Models.

Tumor-associated macrophages often correlate with tumor progression, and therapies targeting immune cells in tumors have emerged as promising treatments. To select effective therapies, we established an in vitro 3D multicellular spheroid model including cancer cells, fibroblasts, and monocytes. We analyzed monocyte infiltration and differentiation in spheroids generated from fibroblasts and either of the cancer cell lines MCF-7, HT-29, PANC-1, or MIA PaCa-2. Monocytes rapidly infiltrated spheroids and differentiated into mature macrophages with diverse phenotypes in a cancer cell line-dependent manner. MIA PaCa-2 spheroids polarized infiltrating monocytes to M2-like macrophages with high CD206 and CD14 expression, whereas monocytes polarized by MCF-7 spheroids displayed an M1-like phenotype. Monocytes in HT-29 and PANC-1 primarily obtained an M2-like phenotype but also showed upregulation of M1 markers. Analysis of the secretion of 43 soluble factors demonstrated that the cytokine profile between spheroid cultures differed considerably depending on the cancer cell line. Secretion of most of the cytokines increased upon the addition of monocytes resulting in a more inflammatory and pro-tumorigenic environment. These multicellular spheroids can be used to recapitulate the tumor microenvironment and the phenotype of tumor-associated macrophages in vitro and provide more realistic 3D cancer models allowing the in vitro screening of immunotherapeutic compounds.

CD38 expression is an important prognostic marker indiffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases with variable outcomes. Although several prognostic markers have been developed, specific biomarkers for stratifying treatment strategies have not been fully investigated. This study aimed to analyze the clinical impact of the expression of cluster of differentiation (CD) 38, which is associated with cellular proliferation and disease progression, in patients with de-novo DLBCL. Using flow cytometry analysis, 137 cases with DLBCL were investigated for surface expression of CD38. Based on the cut-off value by the survival classification and regression tree analysis, the patients were categorized into a CD38HIGH group (n=37) and CD38LOW group (n=100). The 4-years progression-free survival (PFS) was 31.6% in the CD38HIGH group and 60.7% in the CD38LOW group (p<0.001). Multivariate analysis showed the CD38HIGH group to be associated with significantly worse PFS (adjusted hazard ratio [aHR], 2.15, 95% CI: 1.26-3.68, p=0.005) and poor overall survival (OS) (aHR, 2.54, 95% CI: 1.25-5.19, p=0.010) than the CD38LOW group. In conclusion, we demonstrated that high CD38 expression is an independent adverse prognostic factor associated with poor clinical outcomes compared to low CD38 expression. CD38 expression in DLBCL cells might be useful for predicting outcomes and designing risk-adapted therapies for patients with de-novo DLBCL.

Clinicopathologic and Prognostic Significance of CD47 Expression and Tumor-associated Macrophages in Endometrial Carcinoma.

Tumor-associated macrophages (TAMs) influence cancer progression. CD47 is an antiphagocytic molecule aiding tumor resistance against host immune surveillance. The relationship between CD47 expression and TAM-related microenvironment in endometrial carcinoma (EC) is poorly understood. The expression and prognostic significance of CD47 and CD163-labeled TAMs in 165 EC cases was assessed with CD47 and CD163 immunohistochemical studies. CD47 expression was found in 156/165 (94.6%) cases. CD47 expression was significantly higher in nonendometrioid carcinomas. CD47 overexpression was associated with histologic grade. High epithelial and stromal TAMs counts were also associated with high tumoral CD47 expression. High epithelial, stromal, and margin TAMs counts were associated with higher histologic grade and lymphovascular invasion. Epithelial TAMs counts were higher in patients with nonendometrioid carcinomas (P=0.0001) and cases with recurrence (P=0.018). High stromal TAMs counts were associated with deeper myometrial invasion (P=0.017) and the presence of distant metastasis (P=0.024). The counts of margin TAMs was significantly correlated with the depth of myometrial invasion, lymphovascular invasion, FIGO stage, lymph node metastases, distant metastasis, and recurrence (P=0.0001, 0.0001, 0.004, 0.005, 0.014, and 0.04, respectively). CD47 expression was not associated with overall survival (OS) and progression-free survival. However, high epithelial and stromal TAM counts were associated with shorter OS. Besides, high epithelial and margin TAM counts were associated with shorter progression-free survival. Furthermore, increased stromal and margin TAM counts were determined to be an independent prognostic marker of reduced OS. TAM count is, therefore, a significant prognostic factor in EC and the CD47 assessment has potential benefit for future clinical use.

CD70 antibody-drug conjugate: A potential novel therapeutic agent for ovarian cancer.

This study aimed to investigate the cytotoxicity of a cluster of differentiation 70 antibody‐drug conjugate (CD70‐ADC) against ovarian cancer in in vitro and in vivo xenograft models. CD70 expression was assessed in clinical samples by immunohistochemical analysis. Western blotting and fluorescence‐activated cell sorting analyses were used to determine CD70 expression in the ovarian cancer cell lines A2780 and SKOV3, and in the cisplatin‐resistant ovarian cancer cell lines A2780cisR and SKOV3cisR. CD70 expression after cisplatin exposure was determined in A2780 cells transfected with mock‐ or nuclear factor (NF)‐κB‐p65‐small interfering RNA. We developed an ADC with an anti‐CD70 monoclonal antibody linked to monomethyl auristatin F and investigated its cytotoxic effect. We examined 63 ovarian cancer clinical samples; 43 (68.3%) of them expressed CD70. Among patients with advanced stage disease (n = 50), those who received neoadjuvant chemotherapy were more likely to exhibit high CD70 expression compared to those who did not (55.6% [15/27] vs 17.4% [4/23], P < .01). CD70 expression was confirmed in A2780cisR, SKOV3, and SKOV3cisR cells. Notably, CD70 expression was induced after cisplatin treatment in A2780 mock cells but not in A2780‐NF‐κB‐p65‐silenced cells. CD70‐ADC was cytotoxic to A2780cisR, SKOV3, and SKOV3cisR cells, with IC50 values ranging from 0.104 to 0.341 nmol/L. In A2780cisR and SKOV3cisR xenograft models, tumor growth in CD70‐ADC treated mice was significantly inhibited compared to that in the control‐ADC treated mice (A2780cisR: 32.0 vs 1639.0 mm3, P < .01; SKOV3cisR: 232.2 vs 584.9 mm3, P < .01). Platinum treatment induced CD70 expression in ovarian cancer cells. CD70‐ADC may have potential therapeutic implications in the treatment of CD70 expressing ovarian cancer.

Preclinical Studies Of SL-401, a Targeted Therapy Directed To The Interleukin-3 Receptor (IL3-R), In Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Potent Activity In BPDCN Cell Lines, Primary Tumor, and In An In Vivo Model

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), an aggressive malignancy derived from plasmacytoid dendritic cells (PDCs), typically presents as cutaneous lesions, can also involve lymph nodes and spleen, and invariably terminates in an acute leukemic phase. Its diagnosis is based on this clinical presentation, as well as the unique pathological and immunophenotypic features of a cutaneous PDC neoplasm, with high expression of CD123 (interleukin-3 receptor [IL-3R]), CD304, and/or CD303 (Garnache-Ottou F et al., 2009); CD56 and CD4 are also commonly expressed. There is no accepted standard of care to treat BPDCN and therapeutic strategies have never been prospectively evaluated. Treatment approaches include: symptomatic therapy, intensive multi-agent chemotherapy, and allogenic hematopoietic cell transplantation. Although chemotherapy-naïve patients often respond to chemotherapy, most subjects experience recurrent disease shortly thereafter; median overall survival ranges from 9 to 13 months. Since BPDCN cells express high levels of IL-3R, the antitumor effects of SL-401, a novel targeted therapy directed to IL-3R, are being evaluated in preclinical and clinical studies. SL-401, which consists of IL-3 fused to a truncated diphtheria toxin payload, potently inhibits protein synthesis following IL-3R-mediated binding and internalization. Since normal hematopoietic cells express negligible levels of IL-3R, myelosuppression is negligible. To better understand the antitumor effects of SL-401 against BPDCN, the cytotoxicity of SL-401 was assessed in BPDCN patient-derived cell lines (CAL-1 and GEN2.2) and primary BPDCN cells isolated directly from patients (n = 12). Cytotoxicity was measured using flow cytometry (AV/7AAD staining) and an MTT assay after SL-401 treatment for 18 or 48 hours, respectively, as a single agent or compared with a range of cytotoxic agents. We confirmed the robust single agent activity of SL-401 against BPDCN cell lines (Angelot-Delettre F et al., ASH 2011) and found that treatment with SL-401 at concentrations substantially lower than peak concentrations achieved in patients induced a percent decrease in viability of BPDCN cell lines of 92% (62 ± 7% to 5 ± 2%) (n = 9 experiments) and primary BPDCN cells of 80% (50 ± 5% to 10 ± 1%) (n = 12 experiments) (Fig. A and B). Moreover, SL-401 was more effective than 7 out of 8 chemotherapeutic agents tested (Fig. C). SL-401 was also evaluated in a NOD-SCID IL2RγcKO (NSG) BPDCN mouse model. After 2 Gy irradiation, mice were injected with GEN 2.2 cells (1 x 106, IV) and then treated with SL-401 at clinically relevant doses or PBS (IP daily for 5 days) initiated on day 7 post-implantation. The median overall survival (OS) was significantly improved in mice receiving a single cycle of SL-401 compared to untreated controls (53 d vs. 15 d, respectively) (Figs. D and E). These findings provide strong in vitro and in vivo evidence that BPDCN cells are highly sensitive to SL-401. Since BPDCN patients express high levels of IL-3R (Garnache Ottou et al, 2009), these results also indicate that SL-401 may provide a clinical benefit for BPDCN patients. A Phase 2 multi-cycle trial in this orphan indication is currently being planned in North America and Europe. [Display omitted] (A) BPDCN cell lines CAL-1 and GEN 2.2 (n= 9) and (B) primary cells (12 patients) are sensitive to SL-401 (2.7 fM) treatment (18 h) in vitro. **p<0.001. (C) BPDCN cells were cultured alone or with SL-401 (2.7 fM) or ERW (erwinase, 10 UI/ml), KID (kidrolase, 10UI/ml), MTX (methotrexate, 4.5µg/ml), CYC (cyclophosphamide, 100 µmM), CYT (cytarabine, 80 µg/ml), DEX (dexamethasone, 250 µg/ml), VIN (vincristine, 20 ng/ml), or IDA (idarubicin, 0.078 µg/ml). The viability was evaluated by cytometry using AV/7AAD staining after 18 h of culture. Results (mean + SEM) of 4 experiments.*p<0.05. (D) Survival rate of 2 Gy irradiated NSG mice injected IV with 1 x 106 GEN 2.2 cells on day 0. SL-401 treatment IP (2 µg) daily for 5 days (pink area) was initiated on day 7. The solid and dotted lines show saline-treated controls (n = 3) and SL-401 treated animals (SL-401, n=4), respectively. Results from one representative experiment out of 3. (E) GEN 2.2 cells detected in a node 2 weeks post-injection. Cells were stained with standard MGG solution and show a blastic morphology. Disclosures:Frankel:Stemline Therapeutics: Patents & Royalties, Research Funding. Brooks:Stemline Therapeutics: Employment, Equity Ownership. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership.

CD33 Is Expressed in a Significant Subset of Multiple Myeloma Patients in the US and May Represent a Viable Therapeutic Target

Background: CD33 (sialic acid binding Ig-like lectin 3; SIGLEC3) is a type I transmembrane receptor glycoprotein that may function as a sialic acid-dependent cell adhesion molecule. CD33 is generally viewed as a marker of myeloid lineage, and its expression on lymphoid lineage cells is thought to be aberrant and rare, reported to be present in only 1% - 8% of non-myeloid samples, with estimated similarly low expression in multiple myeloma plasmocytes specifically. Some data indicate that CD33 may be expressed on multiple myeloma (MM) plasmocytes in a much larger proportion of MM patients than previously thought, and that when expressed it may confer worse prognosis. However, these data were obtained from established MM cell lines and small series of patient samples. For the first time, we have attempted to obtain data on CD33 expression on MM plasmocytes from patient samples in a large US library.Methods: Data from the IA9 release of the MMRF CoMMpass study was downloaded from the MMRF Researcher Gateway. The per patient visit clinical table was filtered to include only patient visits with numerical calls in the “CD33_detected” field, which indicates whether CD33 was detected on the surface of MM plasmocytes by flow cytometry. Data in the per patient visit clinical table was further filtered to include only “Baseline” and “Confirm Progression” visits. Any patient with a baseline visit was considered in the analysis of baseline, newly-diagnosed MM patients. Only patients with both a baseline and at least one confirm progression visit were considered in the analysis of patients with CD33 data at two or more timepoints. For each visit, the percent CD33+ plasma cell calls (CD33_PC_Percent) were assigned a bin as follows: <20%, 20-39%, 40-59%, 60-79%, 80-100% CD33 positive plasma cells. High expression of CD33+ was defined as CD33 detected in ≥40% of MM plasmocytes whereas very high expression was defined as CD33 detected in ≥60% of MM plasmocytes.Results: The IA9 CoMMpass release includes 995 samples that have corresponding CD33 data, representing 865 unique patients. Samples were collected at baseline for newly-diagnosed MM patients (n=865) and at relapse when patients had progression events (n=59 patients, 66 samples). Among newly-diagnosed MM patients at baseline 25% (n=219) of patient tumors expressed CD33 based on flow cytometry analysis, whereas 75% (n=646) did not. Among patients with CD33+ MM, the proportion of CD33+ plasmocytes detected were as follows: <20% cells: 11.0% of samples (n=24); 20%-39% of cells: 27.4% of samples (n=60); 40%-59% of cells: 20.5% of samples (n=45); 60%-79% of cells: 10.5% of samples (n=23); 80-100% of cells: 30.6% of samples (n=67). At baseline, 61.6% (n=135) of patients exhibited a high percentage of CD33+ plasmocytes (≥40% of MM plasmocytes expressing CD33) and 41% (n=90) of patients exhibited a very high percentage of CD33+ plasmocytes (≥60% of MM plasmocytes expressing CD33).Similarly to newly diagnosed patients, among the 59 patients that had samples available at baseline and at least one progression visit, 27.1% (n=16) were CD33+ at relapse. Plasmocytes from 62.7% (n=37) were CD33- at both baseline and progression, 20.3% (n=12) were CD33+ at both baseline and progression, 10.2% (n=6) were CD33+ at baseline and CD33- at progression, and 6.8% (n=4) were CD33- at baseline and CD33+ at progression. Among the 12 patients that were CD33+ at both baseline and progression, 58.3% (n=7) had very high CD33 at both timepoints. Of the other 5 patients, 25% (n=3) exhibited an increase in the percent of CD33+ plasmocytes, going from low to high (n=1), low to very high (n=1), and high to very high (n=1), whereas 16.7% (n=2) of patients exhibited a decrease in the percent of CD33+ plasmocytes, going from very high to high (n=1) or very high to low (n=1) at baseline versus relapse.Conclusions: These findings indicate that a substantial proportion of MM patients express CD33 on their myeloma plasmocytes, including relapsed patients. Therefore, CD33 may represent a viable target in multiple myeloma. Relevance of this pattern of CD33 expression may be further enhanced by the presence of a number of CD33 targeting agents already in clinical trials in acute myeloid leukemia, which could allow for a rapid translation into the clinical settings in multiple myeloma. One trial in relapsed and refractory multiple myeloma with a CD33 targeting radiolabeled monoclonal antibody is underway. DisclosuresLevy:Actinium Pharmaceuticals: Equity Ownership; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; Takeda: Consultancy, Speakers Bureau. Cicic:Actinium Pharmaceuticals: Employment, Equity Ownership.

Daratumumab Efficiently Targets NK/T Cell Lymphoma with High CD38 Expression

CD38 expression can be detected on a majority of NK/T cell lymphoma (NKTCL). Based on the clinical data of 94 patients with ENKTL, high CD38 surface expression was an adverse prognostic factor for progression free survival, and these patients had significantly inferior outcomes (Wang, 2015). This corroborates with our own studies showing that relapsed and refractory NKTCL patients almost always exhibit high CD38 expression, thereby proposing that it may be worthwhile to target CD38 as a novel therapeutic option.Daratumumab is a human anti-CD38 monoclonal antibody which has shown significant anti-cancer activity in multiple myeloma. In this study, we investigated the potential cytotoxic effects and mechanisms of actions of Daratumumab in a panel of 10 NKTCL cell lines. Firstly, we identified that cell lines can be stratified into CD38Hi, CD38Mid and CD38Lo as quantified by mRNA expression and CD38 protein surface expression which was qualitatively analysed by FACS and IHC. Daratumumab is able to induce antibody dependent cellular cytotoxicity (ADCC) in a CD38-dependent manner and in a dose-dependent fashion. We also observe a greater induction of antibody dependent cellular phagocytosis (ADCP) activation in correlation with CD38 expression. Furthermore, complement-dependent cytotoxicity (CDC) was significantly triggered by Daratumumab in CD38Hi and CD38Mid cells. However, we found evidence of an additional level of regulation provided by the expression of complement inhibitory proteins(CIPs) CD46, CD55 and CD59 on NKTCL cell lines which can limit and inhibit the induction of CDC regardless of CD38 surface expression levels.All-trans retinoic acid (ATRA) is a vitamin A derivative that binds to retinoic acid receptor alpha leading to the upregulation of CD38 expression. Treatment with ATRA increases CD38 mRNA and surface protein expression in all CD38Hi-Lo NKTCL cell lines. Interestingly CD38Lo cell lines showed the greatest fold increase of CD38 mRNA and surface protein expression. Notably, addition of ATRA to SNK-10, a CD38LoCD55Hi cell line, prior to Daratumumab treatment was able to improve the induction of ADCC but not CDC. This suggests a critical role for CIPS in mediating the induction of CDC in NKTCL.Subsequent investigation for the efficacy of Daratumumab in vivo is currently being performed.We also discovered a synergistic cytotoxic effect in NKTCL CD38Hi cell lines mediated by L-Asparaginase (a component of the current chemotherapeutic regimen in NKTCL) treatment in combination with Daratumumab. Altogether,these results propose a rationale therapeutic option for single or combined Daratumumab treatment in the clinic especially for relapsed and refractory patients. DisclosuresJin:Janssen China R&D: Employment. Yu:Janssen China R&D: Employment. Chen:Janssen China R&D: Employment. Yang:Janssen China R&D: Employment. Chng:Janssen China R&D: Research Funding.

Upregulation of CD38 Expression on Multiple Myeloma Cells By the HDAC Inhibitor Ricolinostat Augments the Efficacy of Daratumumab

Background:The anti-CD38 antibody daratumumab is effective and increasingly being utilized to treat multiple myeloma. A clinical observation with daratumumab is that expression of CD38 on pretreatment myeloma cells correlates with efficacy, and patients with higher CD38 expression have a higher chance to respond. Moreover, during daratumumab therapy, there are reduced CD38 expression levels on myeloma cells, favoring immune escape and disease progression. We have recently shown that the histone deacetylase (HDAC) inhibitor panobinostat induces CD38 upregulation and augments the antibody-dependent cellular cytotoxicity (ADCC) of daratumumab (García-Guerrero E, Blood 2017). Here, we investigated the alternative HDAC inhibitor ricolinostat and all-trans retinoic acid (ATRA) to determine whether upregulation of CD38 is a class effect, and to identify the substance that works most synergistically with daratumumab.Methods:The myeloma cell lines MM1.S and OPM2 were treated with titrated doses of ricolinostat (0, 5, 10 µM), ATRA (0, 10, 25 nM) and panobinostat (0, 10, 25 nM). Expression of CD38, B-cell maturation antigen (BCMA) and SLAMF7 was analyzed by flow cytometry after 24, 48 and 72 h of exposure. ADCC against HDAC inhibitor-treated and untreated myeloma cells was analyzed after addition of PBMC at an effector to target ratio of 25:1 in the presence of daratumumab (1, 10, 50 ug/mL) or isotype control.Results:We first treated the myeloma cell line MM1.S with each of the HDAC inhibitors and analyzed their direct cytotoxic antimyeloma effect. We found the strongest effect with ricolinostat where live MM1.S myeloma cells had decreased to 50% and 20% after 48 h of exposure to 5 and 10 µM of the drug, respectively. Consistent with previous work the direct anti-myeloma effect of panobinostat (decrease of live MM1.S myeloma cells to 85% and 50% after 48 h of exposure to 10 and 25 nM, respectively) and ATRA (no cytotoxic effect) was lower compared to ricolinostat.We then analyzed expression of CD38 on residual live i.e. 7-AAD negative MM1.S cells by flow cytometry and observed a 2-fold (5 µM) and 2,5-fold (10 µM) increase in CD38 expression by mean fluorescence intensity (MFI) after treatment with ricolinostat-treated compared to untreated MM1.S cells. The increase in CD38 expression was already detectable after 24 h and further increased with longer exposure to the drug. Off note, withdrawal of ricolinostat resulted in rapid decline of CD38 expression to baseline levels but increased again to the same magnitude within 24 h of re-exposure to the drug. Overall, the increase in CD38 expression was strongest with ricolinostat followed by panobinostat and ATRA, which we confirmed with several myeloma cell lines and primary myeloma cells. We also confirmed that ricolinostat-induced upregulation of CD38 specifically occurred in myeloma cells and not in the lymphoma cell lines Raji and JeKo-1. In contrast to CD38, expression of BCMA and SLAMF7 after treatment of myeloma cells with ricolinostat had decreased.We then treated myeloma cells with ricolinostat for 48 hours to induce upregulation of CD38 and then performed ADCC assays with daratumumab in the presence of ricolinostat. We found, that the combination treatment ricolinostat plus daratumumab eliminated 95,6% of MM1.S cells after 20h; while 87,4% were eliminated with ricolinostat alone, and only 22,6% with daratumumab alone. Preliminary data show that ricolinostat does not affect expression of the complement inhibitor CD55 and induces a lower increase in CD59 expression compared to panobinostat. Experiments to evaluate complement-dependent cytotoxicity (CDC), and to confirm the synergistic antimyeloma effect of ricolinostat and daratumumab in a murine xenograft myeloma model, are ongoing.Conclusions:Our data demonstrate that the HDAC inhibitor ricolinostat induces upregulation of CD38 expression on myeloma cells. The increase in CD38 expression after treatment with ricolinostat is stronger compared to panobinostat and ATRA. Our data suggest that upregulation of CD38 on myeloma cells is a class effect of HDAC inhibitors, and identify ricolinostat as a highly synergistic combination partner for daratumumab, due to its considerable intrinsic antimyeloma activity. Our data suggest that combination treatment with ricolinostat and daratumumab could increase response rates and extend duration of responses of daratumumab therapy. DisclosuresNo relevant conflicts of interest to declare.

T-NHL subtypes and clinical perspective

Peripheral T-cell lymphomas (PTCL) are a distinct pathological entity with clinical advancements lagging behind its B-cell lymphoma counterpart. A significant proportion (15–20%) of PTCL cases are misdiagnosed as reactive or a different lymphoma subtype. An accurate diagnosis routinely requires analysis of the immunophenotype in conjunction with cellular morphology, analysis of lymph node architecture and molecular genetic analysis. Recent major advances in genomic studies examining gene expression profiling (GEP) and the genetic landscape of T-cell and NK-cell neoplasms have resulted in diagnostic, prognostic and therapeutic implications and has consequently led to revisions in the classification of PTCLs and introduction of new provisional entities. Incorporating these molecular and genetic markers, the latest 2016 WHO classification, an update of the current fourth edition, has identified 25 definitive and 6 provisional entities and further groups these lymphomas according to their usual presentation into disseminated disease (leukaemias), predominantly nodal, predominantly extra-nodal or cutaneous lymphomas. Nodal PTCLs represent the most prevalent PTCL subtype and include peripheral T-cell lymphoma - not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), follicular T-cell lymphoma (FTCL), nodal PTCL with T follicular helper (TFH) phenotype, and systemic anaplastic large cell lymphoma (sALCL) either ALK-positive or ALK-negative. Due to better understanding of the cell of origin in T-cell lymphomas, AITL, FTCL and PTCL with TFH phenotype have been recognised as being derived from TFH cells as these neoplastic cells express at least two or three TFH cell antigens which include CD279/PD1, CD10, BCL6, CXCL13, ICOS, SAP and CCR5. In addition to the common cell of origin, these lymphomas share pathological and clinical features as well as recurrent genetic abnormalities such as mutations in TET2, IDH2, DNMT3A, RHOA and CD28, and gene fusions such as ITK-SYK and CTLA-CD28. Such features suggest that these lymphomas belong to the same spectrum of disease and are therefore now unified under a common heading. PTCL-NOS encompasses those which do not match the classification of other PTCL categories. This subtype mostly has a CD4+/CD8– phenotype and shows extreme cytological and phenotypical heterogeneity but GEP have identified at least three subgroups characterised by overexpression of GATA3, TBX21 and cytotoxic genes associated with varied clinical behaviour. Systemic ALCLs have a primarily aggressive nodal presentation and are further subdivided to ALK-positive or ALK-negative based on whether they carry the nucleoplasmin (NPM) anaplastic lymphoma kinase (ALK) translocation between chromosome 2 and 5, designated as t(2;5). We now know that a subset of ALCL, ALK-negative cases harbour rearrangement at the locus containing DUSP22 and IRF4 in chromosome 6p25, which induces down regulation of DUSP22, a dual specific phosphatase that inhibits TCR signalling. Another subset of ALK-negative ALCL has rearrangements in TP63. Clear distinction of PTCL with high CD30 expression from ALCL, ALK-negative have also been possible through GEP and as such, the former is no longer considered a provisional entity. Breast implant associated ALCL, a unique form of ALCL, ALK-negative arising in association with breast implants, is increasingly being recognised and has been included as a provisional entity. Cutaneous ALCL (cALCL), ALK-negative, the fourth subtype, is an indolent form limited to the skin. Whilst notorious to have an aggressive clinical course and an extremely poor overall prognosis, clinicians are constantly challenged with low rates of complete remissions, early relapses and failures to achieve long-term remissions despite aggressive first-line chemotherapy, in the majority of patients. More recently, improved understanding of disease biology and the molecular profile of specific PTCL subtypes has opened the door to the potential of more targeted therapies with a number of new biological therapies approved by regulatory agencies in the last few years.

A first-in-human study of AO-176, a highly differentiated anti-CD47 antibody, in patients with advanced solid tumors.

2516 Background: AO-176 is a humanized IgG2 antibody that specifically targets CD47. Expressed by multiple tumor types, CD47 binds to signal regulatory protein a (SIRPa) on phagocytes, including macrophages and dendritic cells. The CD47-SIRPa complex results in a “don’t eat me” signal that allows the tumor to escape removal by the innate immune system, disabling the generation of an adaptive immune response. The differentiated mechanisms of action of AO-176 include promotion of phagocytosis, direct tumor cell killing through programmed cell death type III and induction of damage associated molecular patterns/immunogenic cell death, preferentially binding to tumor cells vs. normal cells, and enhanced binding at an acidic pH as found in tumor microenvironments. AO-176 has negligible binding to RBCs. Methods: In a phase 1/2 first-in-human study (NCT03834948) of AO-176, pts with advanced solid tumors associated with high CD47 expression and an ECOG PS of 0-1 were enrolled into escalating dose cohorts of AO-176 given IV every 7 days. Objectives included evaluation of safety, dose-limiting toxicity (DLT) and recommended phase 2 dose (RP2D), antitumor activity, pharmacokinetic (PK) parameters and exploratory biomarkers. Results: As of 4 Jan 2021, 27 pts were enrolled (median age 64 years; 67% female; 67% ECOG PS 1; median [range] of 4 [1-7] prior therapies for metastatic disease). Dose levels of 1, 3, 10, 20 and 20 (using step-up dosing) mg/kg were evaluated in &gt;250 infusions. Most common (&gt;10%) treatment-related adverse events (TRAEs) of any grade were thrombocytopenia and infusion-related reaction (IRR) (33% each), anemia (22%) with no evidence of hemolysis, nausea (19%), and fatigue (15%). The only G3+ TRAE occurring in &gt;10% of pts was asymptomatic, brief thrombocytopenia (22%). No platelet transfusions were given. DLTs included IRRs in 2 pts dosed at 20 mg/kg, and asymptomatic thrombocytopenia and a cerebrovascular accident in 1 pt each in the 20 mg/kg step-up cohort. The RP2D was 10 mg/kg. Implementation of additional pre-medication and a 6-hr infusion duration in cycle 1 eliminated subsequent IRRs. Dexamethasone tapering and shortening of the infusion duration to 2 hrs was successful in all pts after cycle 1. Interim PK analysis of AO-176 demonstrated consistent exposure with linear PK. The T1/2 was ̃5 days. One pt with endometrial carcinoma who had not responded to any of 4 prior systemic regimens had a confirmed PR and remains on study for &gt;1 year. 7 pts had SD as a best response, with 2 pts (endometrial carcinoma, gastric cancer) on study for &gt;6 mos. Conclusions: AO-176 is a well-tolerated, differentiated anti-CD47 therapeutic. Durable anti-tumor activity was observed. Evaluations of AO-176 in combination with paclitaxel in pts with select solid tumors (NCT03834948) and as a single-agent in pts with multiple myeloma (NCT04445701) are ongoing. Clinical trial information: NCT03834948.

CD38 and MGluR1 as possible signaling molecules involved in epileptogenesis: A potential role for NAD+ homeostasis

In spite of long-term intensive scientific research efforts, there are still many issues concerning the mechanisms of epileptogenesis and epilepsy to be resolved. Temporal lobe, in particular hippocampus, is vulnerable to epileptogenic process. Herein, electrical kindling model of temporal lobe were analyzed using proteomic approach. A dramatic decrease in nicotinamide adenine dinucleotide (NAD+) level was exhibited during the kindling procedure in hippocampus. After stage 3, high CD38 expression was detected by qPCR, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and western blot analysis. An increase in expression of CD38/NADase activity was observed during the kindling procedure in hippocampus that suggest it as one of the most important NAD+ degrading enzymes during epileptogenesis. Subsequently, gene expression of CD38 metabolite related proteins (Ryr2, FKBP-12.6, Chrm1, mGluR1 and Cnx43) were examined. Among them, changes in the expression level of mGluR1 was more than other genes, which was also confirmed by LC MS/MS and western blotting analysis. These findings provided valuable information about changes in the expression of CD38/cADPR signaling pathway and suggest its crucial role during epileptogenesis.

Construction and Identification of Leukemia Cell Line Stably Expressing CD123 and CLL-1

To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1. The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry. The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×108 after quantitative PCR assay. The K562 leukemia cell line obtained positive expression cells after being infected by puromycin. The high expression of CD123 and CLL-1 was confirmed by RT-PCR, while the significantly high expression of CD123 and CLL-1 was confirmed by flow cytometry. Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.

Intensity of antigen expression reflects IGHV mutational status and Dohner-defined prognostic categories in chronic lymphocytic leukemia, monoclonal B-cell lymphocytosis, and small lymphocytic lymphoma

We demonstrate the prognostic utility of antigen quantitation in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and monoclonal B-cell lymphocytosis (MBL). Median antibody-bound-per-cell (ABC) of CD20, CD22, CD25, CD19, and %CD38(+) was determined in CLL (185/208), SLL (8/208) and MBL (15/208) cases by flow cytometry, then compared to Dohner-classification, immunoglobulin status (mutated, IGHV-M; unmutated, IGHV-U), CLL-IPI risk and time to first treatment (TTFT). Trisomy 12 cases showed increased %CD38-expression (p = .0379). Higher %CD38 was observed in IGHV-U versus IGHV-M (p = .0003). CD20ABC was increased in IGHV-U versus IGHV-M (p = .006). Del13q cases demonstrated lower CD22ABC (p = .0198). Cases without cytogenetic abnormality exhibited higher CD19ABC (p = .0295) and CD22ABC (p = .0078). Del17p cases demonstrated lower CD25ABC (p = .0097). High and very-high CLL-IPI risk groups were associated with high CD38-expression (p = .02) and low CD25ABC (p = .0004). Shortened TTFT was associated with high CD38-expression (p < .0001). Interestingly, high CD25ABC trended toward shortened TTFT (p = .07). Quantitative antigen expression reflects CLL-IPI risk groups and Dohner-classification.

High Expression of Interleukin-3 Receptor Alpha Chain (CD123) Predicts Favorable Outcome in Pediatric B-Cell Acute Lymphoblastic Leukemia Lacking Prognosis-Defining Genomic Aberrations.

BackgroundAberrant expression of CD123 (IL-3Rα) was observed in various hematological malignancies including acute lymphoblastic leukemia (ALL), which is the most common malignancy in childhood. Although widely used for minimal residual disease (MRD) monitoring, the prognostic value of CD123 has not been fully characterized in pediatric B-ALL. This retrospective study aims to evaluate the association between the CD123 expression of leukemic blasts and the outcomes of the pediatric B-ALL patients.MethodsA total of 976 pediatric B-ALL, including 328 treated with CCLG-ALL-2008 protocol and 648 treated with CCCG-ALL-2015 protocol, were recruited in this retrospective study. CD123 expression was evaluated by flow cytometry. Patients with >50, 20–50, or <20% of CD123 expressing blasts were grouped into CD123high, CD123low, and CD123neg, respectively. The correlation between CD123 expression and the patients’ clinical characteristics, overall survival (OS), event-free survival (EFS), and relapse-free survival (RFS) were studied statistically.ResultsOf 976 pediatric B-ALL, 53.4% from the CCLG-ALL-2008 cohort and 49.2% from the CCCG-ALL-2015 cohort were CD123high. In the CCLG-ALL-2008 cohort, CD123high was significantly associated with chromosome hyperdiploidy (p < 0.0001), risk stratification (p = 0.004), and high survival rate (p = 0.005). By comparing clinical outcomes, patients with CD123high displayed favorable prognosis, with a significantly better OS (p = 0.005), EFS (p = 0.017), and RFS (p = 0.045), as compared to patients with CD123low and CD123neg. The prognostic value of CD123 expression was subsequently confirmed in the CCCG-ALL-2015 cohort. Univariate and multivariate cox regression model analysis showed that high CD123 expression was independently associated with favorable EFS (OR: 0.528; 95% CI: 0.327 to 0.853; p = 0.009) in this cohort. In patients without prognosis-defining genomic abnormalities, high CD123 expression strongly indicated superior survival rates and was identified as an independent prognosis factor for EFS and RFS in both cohorts.ConclusionsA group of B-ALL lacks prognosis-defining genomic aberrations, which proposes a challenge in risk stratification. Our findings revealed that high CD123 expression of leukemic blasts was associated with favorable clinical outcomes in pediatric B-ALL and CD123 could serve as a promising prognosis predictor, especially in patients without prognosis-defining genetic aberrations.

Prognostic significance of CD45 antigen expression in pediatric acute lymphoblastic leukemia

ObjectivesThe treatment of pediatric acute lymphoblastic leukemias (ALL) has seen remarkable advances recently. However, relapse occurs in approximately 20% of cases which necessitates identifying additional high risk parameters for treatment intensification. The aim of this study is to assess the prognostic significance of CD45 antigen expression in pediatric ALL. MethodsWe studied 363 pediatric patients with B cell precursor-ALL (BCP-ALL) (n = 313) and T-ALL (n = 50). The ratio of median fluorescence intensity of CD45 expressed in leukemic blasts and normal lymphocytes was calculated. The 75th percentile was taken as cut-off to categorise patients into CD45 high and CD45 low groups. ResultsThe 75th percentile was 0.141 in BCP-ALL and 0.548 in T-ALL. In BCP-ALL, there was a statistically significant association of age (≥10 years) (p = 0.027) and National Cancer Institute high risk group (p = 0.001) with high CD45 expression but not in T-ALL. Worse event-free survival (EFS) was seen with high CD45 expression in BCP-ALL (42.17% versus 60.83%, p = 0.0053). In T-ALL, there was no association between CD45 expression and EFS (CD45 high 40.40% versus low 67.35%, p = 0.414). The overall survival (OS) was 70% versus 60% (p = 0.38) in BCP-ALL and the OS was 82% versus 68% (p = 0.16) in T-ALL for CD45 low versus CD45 high groups, respectively. ConclusionWe conclude that high CD45 surface expression is associated with worse EFS in pediatric BCP-ALL.

Expression and Prognostic Significance of CD47-SIRPA Macrophage Checkpoint Molecules in Colorectal Cancer.

Despite the confirmed anti-cancer effects of T-cell immune checkpoint inhibitors, in colorectal cancer (CRC) they are only effective in a small subset of patients with microsatellite-unstable tumors. Thus, therapeutics targeting other types of CRCs or tumors refractory to T-cell checkpoint inhibitors are desired. The binding of aberrantly expressed CD47 on tumor cells to signal regulatory protein-alpha (SIRPA) on macrophages allows tumor cells to evade immune destruction. Based on these observations, drugs targeting the macrophage checkpoint have been developed with the expectation of anti-cancer effects against T-cell immune checkpoint inhibitor-refractory tumors. In the present study, 269 primary CRCs were evaluated immunohistochemically for CD47, SIRPA, CD68, and CD163 expression to assess their predictive utility and the applicability of CD47–SIRPA axis-modulating drugs. Thirty-five percent of the lesions (95/269) displayed CD47 expression on the cytomembrane of CRC cells. CRCs contained various numbers of tumor-associated immune cells (TAIs) with SIRPA, CD68, or CD163 expression. The log-rank test revealed that patients with CD47-positive CRCs had significantly worse survival than CD47-negative patients. Multivariate Cox hazards regression analysis identified tubular-forming histology (hazard ratio (R) = 0.23), age < 70 years (HR = 0.48), and high SIRPA-positive TAI counts (HR = 0.55) as potential favorable factors. High tumor CD47 expression (HR = 1.75), lymph node metastasis (HR = 2.26), and peritoneal metastasis (HR = 5.80) were cited as potential independent risk factors. Based on our observations, CD47–SIRPA pathway-modulating therapies may be effective in patients with CRC.

High CD200 Expression on T CD4+ and T CD8+ Lymphocytes as a Non-Invasive Marker of Idiopathic Pulmonary Hypertension–Preliminary Study

Pulmonary arterial hypertension (PAH) can develop subsequently to disorganized endothelial cell proliferation within the pulmonary arteriolar layers that provide mechanical limits to the pulmonary vascular bed. Although the actual factor triggering vascular endothelial proliferation remains unknown to date, genetic susceptibility, hypoxia, inflammation, as well as response to drugs and toxins have been proposed as possible contributors. Since inflammation contributes to vascular remodeling, the changed immune response is increasingly considered a plausible cause of this cardiovascular disease. The interaction of a membrane glycoprotein cluster of differentiation 200 (CD200) and its structurally similar receptor (CD200R) plays a crucial role in the modulation of the inflammatory response. Our previous studies have shown that the overexpression of the other negative co-stimulatory molecule (programmed death cell-PD-1) and its ligand-1 (PD-L1) is closely related to iPAH and the presence of Epstein-Barr virus (EBV) reactivation markers. Therefore, we considered it necessary to analyze the different types of PAH in terms of CD200 and CD200R expression and to correlate CD200/CD200R pathway expression with important clinical and laboratory parameters. The CD200/C200R-signaling pathway has not been subject to much research. We included 70 treatment-naïve, newly diagnosed patients with PAH in our study. They were further divided into subsets according to the pulmonary hypertension classification: chronic thromboembolic pulmonary hypertension (CTEPH) subset, pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH), pulmonary arterial hypertension associated with connective tissue disease (CTD-PAH), and idiopathic pulmonary arterial hypertension (iPAH). The control group consisted of 20 healthy volunteers matched for sex and age. The highest percentages of T CD200+CD4+ and T CD200+CD8+ lymphocytes were observed in the group of patients with iPAH and this finding was associated with the presence of EBV DNA in the peripheral blood. Our assessment of the peripheral blood lymphocytes expression of CD200 and CD200R indicates that these molecules act as negative co-stimulators in the induction and persistence of PAH-associated inflammation, especially that of iPAH. Similar results imply that the dysregulation of the CD200/CD200R axis may be involved in the pathogenesis of several immune diseases. Our work suggests that CD200 and CD200R expression may serve to distinguish between PAH cases. Thus, CD200 and CD200R might be useful as markers in managing PAH and should be further investigated.

SR-B1 and CD10 combined immunoprofile for differential diagnosis of metastatic clear cell renal cell carcinoma and clear cell carcinoma of the ovary

Both clear cell renal carcinoma (ccRCC) and clear cell carcinoma of the ovary (CCOC) have a clear cytoplasmic morphological feature, hence it is difficult to identify metastatic ccRCC and CCOC by morphology alone. At present, there are no effective immunohistochemical markers to distinguish between these two tumors. Studies have shown that the clear cytoplasm of ccRCC is mainly caused by cholesterol-rich lipids in the cytoplasm, while that of CCOC is due to the accumulation of cytoplasmic glycogen. Objective: to hypothesize that the scavenger receptor class B-type 1 (SR-B1) protein responsible for HDL cholesterol uptake may be differentially expressed in ccRCC and CCOC, and high CD10 expression in the renal tubular epithelium may assist in distinguishing between ccRCC and CCOC. Methods: effective immunohistochemical markers were applied in 90 cases of renal clear cell carcinoma and 31 cases of ovarian cancer to distinguish between the two types of tumors.Result: SR-B1 and CD10 expression is significantly higher in ccRCC than CCOC. Both SR-B1 and CD10 exhibited focal weak-medium intensity staining in CCOC, and their staining extent and intensity were significantly lower than ccRCC. The sensitivity and specificity of SR-B1 for identifying ccRCC were 74.4% and 83.9%, respectively. The sensitivity and specificity of CD10 for identifying CCOC were 93.3% and 80.6%, respectively. The combined SR-B1( +) CD10( +) immunoprofile supports the diagnosis of ccRCC with a specificity of 93.5%. The combined SR-B1(-) CD10(-) immunoprofile supports the diagnosis of CCOC with a specificity of 93.3%. Conclusions: our findings demonstrate that the combination of SR-B1 and CD10 immunoprofiling is a valuable tool for differential diagnosis of ccRCC and CCOC.

The contribution of CD200 to the diagnostic accuracy of Matutes score in the diagnosis of chronic lymphocytic leukemia in limited resources laboratories.

Flow cytometry immunophenotyping has an essential role in distinguishing chronic lymphocytic leukemia from other B-chronic lymphoproliferative disorders. Recently, CD200 is considered as a relatively consistent marker in chronic lymphocytic leukemia. We retrospectively assessed CD200 expression in 252 patients with B chronic lymphoproliferative disorders with four-color flow cytometry. CD200 expression estimation included the proportion of positive cells (≥30%) and the mean fluorescence intensity ratio. Additionally, we have incorporated CD200 into Matutes score, also replaced FMC7 and CD79b in an attempt to improve the score discriminative power. Of 252 patients enrolled, 199(79%) patients were classified as chronic lymphocytic leukemia and 53 (21%) as other B-chronic lymphoproliferative disorders. All chronic lymphocytic leukemia cases and 20 of 53 (37.7%) of other B-chronic lymphoproliferative disorders demonstrated high CD200 expression (≥30%). Further, CD200 (≥30%) revealed a higher accuracy in comparison to other markers in Matutes score (range: 51%–92.5%). Also, CD200 addition to the Matutes score has correctly recognized all 199 chronic lymphocytic leukemia cases including 10 atypical chronic lymphocytic leukemia cases. As for non-CLL cases, 20 of 53 attained a higher score, yet keeping the original diagnosis. Moreover, CD200 enhanced the diagnostic accuracy of Matutes score to 100%, and when included in a simplified 4-markers score, showed an accuracy of 99.8% compared to 99.4% of Matutes score. In conclusion, CD200 is an accurate diagnostic marker for chronic lymphocytic leukemia, and can refine the modified Matutes score accuracy when added with other markers.