Cryopreservation of stem cells after collection from peripheral blood or bone marrow for autologous transplantation necessitates protection with dimethyl sulfoxide (DMSO). Unfortunately, DMSO, when infused with the thawed cell suspension, may induce serious complications and side effects. To assess whether depletion of DMSO before autografting affects safety and efficacy, 56 consenting consecutive patients treated with high-dose chemotherapy and autologous blood stem cell transplantation were assigned to obtain either an untreated or DMSO-depleted autograft. On the day of transplantation, the cryopreserved cells were thawed and infused to the patient either immediately or after washing 3 times in normal saline supplemented with 6% anticoagulant citrate dextrose solution. Cell count with viability, clonogenic assay, and phenotyping were performed before and after thawing and after washing. Hematologic recovery, side effects, and complications were recorded. The in vitro and clinical data on 56 patients show that the depletion of DMSO in vitro before autografting does not induce a significant loss of cell number, viability, colony-forming unit-granulocyte-macrophage activity, or number of CD34(+) cells. Furthermore, it leads to a safe and sustained engraftment. The complications and side effects, as recorded by continuous monitoring, were substantially less; however, the procedure takes 3 to 4 hours of laboratory work per patient.

Summary:C-erbB-2/HER-2 (designated HER-2) is overexpressed in both primary and metastatic breast cancer and predicts poor prognosis. We investigated the expression of HER-2 in patients with metastatic breast cancer undergoing high-dose chemotherapy (HDCT) with autologous blood stem cell (ABSC) support and correlated the presence (positive) or absence (negative) of HER-2 overexpression in these patients with response to treatment, progression-free survival (PFS) and overall survival (OS). The level of HER-2 expression was analyzed in 57 patients with metastatic breast cancer undergoing HDCT with ABSC support. Plasma from peripheral blood was taken at three different time points during the course of treatment and was analyzed using an enzyme immunoassay (EIA) to detect circulating levels of the extracellular portion of HER-2. HER-2 levels were elevated (Ͼ0.2 U/mg protein) in 27/57 (47.4%) patients at one or more time points during treatment. The level of HER-2 varied during the course of treatment. Following induction chemotherapy (ICT), five patients who were negative initially, showed overexpression of HER-2. Three patients overexpressed HER-2 only after HDCT/ABSC. Response to treatment was similar in patients independent of plasma HER-2 levels. Overexpression of HER-2 was associated with a significantly shorter PFS (P = 0.004, log rank) and OS (P = 0.003, log rank) after HDCT/ABSC. HER-2 overexpression, patient age, estrogen receptor status, progesterone receptor status, and previous hormone treatment were assessed by univariate and multivariate analysis. Univariate analysis determined that only HER-2 overexpression correlated significantly with decreases in progression free survival (P = 0.005, Cox regression). Decreased overall survival correlated significantly with HER-2 overexpression (P = 0.004) and decreased expression of both estrogen receptor (P = 0.032) and progesterone receptor (P = 0.039). In multivariate analysis of these variables, only HER-2 expression levels proved to be of independent statistical significance in predicting outcome for both PFS (P = 0.007) and OS Correspondence: Dr S Glück, Depts Oncology, Medicine, and Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Tom Baker Cancer Centre, 1331, 29th Street NW, Calgary, Alberta, Canada Received 18 August 1998; accepted 5 May 1999 (P = 0.002). These results suggest that overexpression of HER-2, measured by EIA in plasma may predict a shorter PFS and OS in patients with metastatic breast cancer treated with HDCT and ABSC support.

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P-selectin glycoprotein ligand-1 (PSGL-1) is a type I transmembrane protein expressed on the surface of most hematopoietic cells. PSGL-1 can engage multiple ligands (e.g., selectins, VISTA, Siglec-5, versican, CCL19, and CCL21). Apart from being a key adhesion molecule involved in immune cell trafficking, PSGL-1 has been shown to function as a negative immune checkpoint receptor in both T cells and macrophages. PSGL-1 is highly expressed in tumor-infiltrating T cells (TILs) and in tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). PSGL-1 signaling in TILs induces development of T-cell exhaustion and, in TAMs, it promotes immunosuppressive activity of macrophages. In the TME, PSGL-1 is also highly expressed on the surface of myeloid-derived suppressor cells, regulatory T cells, and some types of cancer cells. We hypothesized that PSGL-1 may also act as a ligand to modulate the effector functions of human T cells and macrophages in the TME. In the present study, flow cytometry analysis showed that monocytes and granulocytes had a higher PSGL-1 expression than CD4+ and CD8+ T cells isolated from normal human blood cells. To mimic the cell-expressing PSGL-1, a recombinant human PSGL-1-Fc protein (rhPSGL-1-Fc, R&D Systems) was coated onto 96-well plates overnight. Human peripheral blood mononuclear cells (PBMCs), purified human CD4+ T cells, or CD8+ T cells were cultured in the PSGL-1-coated plates for 3 days in the presence of anti-CD3 antibody. In some experiments, a commercial tool PSGL-1 monoclonal antibody (mAb) with the ability to block PSGL-1/P-selectin interaction was added into the culture system. Cytokine secretion in the culture supernatants was measured using Meso Scale Discovery assays. Similar experiments were also conducted with commercially supplied human M1 and M2 macrophages. The results demonstrated that plate-bound rhPSGL-1-Fc dose-dependently inhibited IFN-γ, IL-2, and TNF-α cytokine production in human PBMCs, CD4+ T cells, and in CD8+ T cells following T-cell receptor stimulation. The tool PSGL-1 mAb was able to partially rescue the suppression of cytokine production. Plate-bound rhPSGL-1-Fc also inhibited TNF-α secretion from human M1 macrophage, and the PSGL-1 mAb significantly enhanced TNF-α production in both human M1 and M2 macrophages treated with plate-bound PSGL-1-Fc. Together, these data indicate that PSGL-1 expressed on immune cells in TME may act as both an inhibitory ligand and receptor to impact on T-cell and macrophage function. Acknowledgements: Thanks to Patrick Mayes and Ricardo Macarron for scientific input and thanks to Qian Wang Yao and Lynn Leffet for technical assistance. Trial Registration: N/A Ethics Approval: N/A Citation Format: Jun Guan, Sundee Dees, Tony Chadderton, Alejandro Amador Arjona. P-selectin glycoprotein ligand-1 modulates the functions of human T cells and macrophages in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6373.