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Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15.Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

This file contains the following figures: Supplemental Figure 1. The return of host lymphocytes is affected by the type of donor CD8+ T cells. Supplemental Figure 2. Return of myeloid cells after CTX leads to more rapid host cell reconstitution than TBI-containing regimens. Supplemental Figure 3. CTX and TBI differentially affect host T cells and NK cells. Supplemental Figure 4. Donor CD8+ T cells transferred into a TBI-conditioned host maintain functional efficacy. Supplemental Figure 5. ALT-803 (IL-15/IL-15Ra complexes) augment the persistence of Tc1 cells in irradiated hosts.

Background: Acute lymphocytic leukemia (ALL) is biologically and clinically considered as a heterogeneous neoplasm of lymphoid progenitor cells in the bone marrow (BM). 15- 20 % of children with ALL who achieve an initial remission, will show relapse. One potential mechanism behind this relapse could be the emergence of cancer stem cells (CSCs), which are considered the driving force of tumorigenesis due to their ability of self-renewal as well as the emergence of immune regulatory cells including myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Treg). Aim: The main aim of this study was to analyze the numbers of CSCs and correlate these numbers with the numbers of blasT-cells, MDSCs and Treg cells in children with B-ALL before and after induction of chemotherapy. Materials and Methods: CSCs were defined as CD45dimCD19+CD10+CD34+CD38-, MDSCs were defined as Lin-HLA-DR-CD33+CD11b+ and Treg cells were defined as CD4+CD25+CD127-. The frequencies of these cells were analyzed in the peripheral blood of B-ALL patients before (n= 10) and after (n= 10) induction of chemotherapy using flow cytometry. Results: Significant increases in the numbers of CSCs were shown in B-ALL patients after induction of chemotherapy as compared to newly diagnosed patients (7.6± 8.3 vs. 2.7± 2.4, P Citation Format: Mohamed Labib Salem, Mohamed Attia, Said Abdou, Abdel-Aziz A. Zidan, Mona F. Zidan. Higher numbers of cancer stem cells in the peripheral blood of children with B-ALL after chemotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A106.

EpCAM (epithelial cell adhesion molecule) is a cell surface molecule that is known to be highly expressed in colon and other epithelial carcinomas. EpCAM is involved in cell-to-cell adhesion and has been the target of antibody therapy in several clinical trials. To assess the value of EpCAM as a novel target for breast cancer gene therapy, we performed real-time reverse transcription-PCR to quantify the level of EpCAM mRNA expression in normal breast tissue and primary and metastatic breast cancers. We found that EpCAM is overexpressed 100- to 1000-fold in primary and metastatic breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a 35–80% decrease in the rate of cell proliferation in four different breast cancer cell lines. EpCAM siRNA treatment decreased cell migration by 91.8% and cell invasion by 96.4% in the breast cancer cell line MDA-MB-231 in vitro. EpCAM siRNA treatment was also associated with an increase in the detergent-insoluble protein fraction of E-cadherin, α-catenin, and β-catenin, consistent with the known biology of EpCAM as a regulator of cell adhesion. Our hypothesis is that modulation of EpCAM expression can affect cell migration, invasion, and proliferation by enhancing E-cadherin-mediated cell-to-cell adhesion. These data provide compelling evidence that EpCAM is a potential novel target for breast cancer gene therapy and offer insights into the mechanisms associated with EpCAM gene silencing.

Background: Acute lymphocytic leukemia (ALL) is biologically and clinically considered as a heterogeneous neoplasm of lymphoid progenitor cells in the bone marrow (BM). 15- 20 % of children with ALL who achieve an initial remission, will show relapse. One potential mechanism behind this relapse could be the emergence of cancer stem cells (CSCs) which are considered the driving force of tumourigenesis due to their ability of self-renewal as well as the emergence of immune regulatory cells including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg). Aim: the main aim of this study was to analyze the numbers of CSCs and correlate these numbers with the numbers of blast cells, MDSCs and Treg cells in children with B-ALL before and after induction of chemotherapy. Materials and Methods: CSCs were defined as CD45dimCD19+CD10+CD34+CD38-, MDSCs were defined as Lin-HLA-DR-CD33+CD11b+ and Treg cells were defined as CD4+CD25+CD127-. The frequencies of these cells were analyzed in the peripheral blood of B-ALL patients before (n= 10) and after (n= 10) induction of chemotherapy using flow cytometry. Results: Significant increases in the numbers of CSCs were shown in B-ALL patients after induction of chemotherapy as compared to newly diagnosed patients (7.6± 8.3 vs. 2.7± 2.4, P Conclusion: Our results indicate that chemotherapy of B-ALL patients results in emergence of high numbers of CSCs and MDSCs which might be contributing, respectively, to tumor relapse and creation of systemic immune suppression. This pilot study opens a new avenue to investigate the mechanism mediating the emergence of these cells on larger population of B-ALL patients at different treatment stages. Note: This abstract was not presented at the meeting. Citation Format: Mohamed L. Salem. Higher numbers of cancer stem cells in the peripheral blood of children with B-ALL upon conventional chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3713. doi:10.1158/1538-7445.AM2017-3713

Background: Acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia is an acute form of leukemia. Although the mechanism behind the relapse in ALL is not clearly known, one possible mechanism could be the accumulation of immune regulatory cells, in particular myeloid-derived suppressor cells (MDSCs). We and others have reported that MDSCs cause suppression of host immune responses through several mechanisms. Aim: The main goal of this study was to determine whether these cells accumulate in ALL patients and whether they are affected by chemotherapy. Materials and Methods: Upon informed consent, patients with B-Linage Acute Lymphoblastic Leukemia “B-ALL” (n = 20) were recruited in this study along with healthy volunteers (n = 20). Patients included in this study were risk stratified according to risk classification system and treated according to treatment protocols including high risk pre B-ALL protocol, standard risk pre B-ALL. MDSCs were characterized as HLA-DR-Lin-CD33+CD11b+ cells. Results: Our preliminary results showed significant increases in the numbers of myeloid population with MDSC-like phenotype in B-ALL patients at different stages of chemotherapy treatment when compared with healthy control volunteers (Mean = 1.01%). The increases in the numbers of these cells were higher after treatment of the patients with induction chemotherapy. Further, the increased numbers of MDSCs associated with high numbers of circulating leukemic stem cells (LSCs). Conclusion: Our preliminary data suggest that cells with MDSCs like-phenotype emerge in ALL patients and possibly play a critical role in establishing an immune suppressive environment in children with ALL. Further research is needed to determine if circulating MDSCs like-phenotype populations are a immunosuppressive and whether they can be modulated. Acknowledgment: This work has been supported by a grant (ID# 5245) funded from the Science and Technology Development Fund (STDF), Ministry of Scientific Research and Technology, Egypt to Mohamed L. Salem, the Principal investigator of this project. Note: This abstract was not presented at the meeting. Citation Format: Mohamed L. Salem. Increases in the numbers of myeloid-derived suppressor cells in Egyptian children with acute lymphoid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1297. doi:10.1158/1538-7445.AM2015-1297