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Purpose:The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19+ B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy.Patients and Methods:Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array.Results:Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14+CD33+HLA−DR−) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2.Conclusions:Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.

Figure S3. Baseline Risks as a Function of Year of Diagnosis

Detailed description of the patients and infused cells

Interaction Analysis of RT by DS

Manufactured CAR batches were analyzed for the presence of CAR T cells and phenotyped for different markers as indicated in the Figure

A biologic signature for breast ductal carcinoma in situ
 to predict radiation therapy (RT) benefit and assess recurrence risk

Purpose:Ductal carcinoma in situ (DCIS) patients and their physicians currently face challenging treatment decisions with limited information about the individual's subsequent breast cancer risk or treatment benefit. The DCISionRT biological signature developed in this study provides recurrence risk and predicts radiotherapy (RT) benefit for DCIS patients following breast-conserving surgery (BCS).Experimental Design:A biological signature that calculates an individualized Decision Score (DS) was developed and cross-validated in 526 DCIS patients treated with BCS ± RT. The relationship was assessed between DS and 10-year risk of invasive breast cancer (IBC) or any ipsilateral breast event (IBE), including IBC or DCIS. RT benefit was evaluated by risk group and as a function of DS.Results:The DS was significantly associated with IBC and IBE risk, HR (per 5 units) of 4.2 and 3.1, respectively. For patients treated without RT, DS identified a Low Group with 10-year IBC risk of 4% (7% IBE) and an Elevated Risk Group with IBC risk of 15% (23% IBE). In analysis of DS and RT by group, the Elevated Risk Group received significant RT benefit, HR of 0.3 for IBC and IBE. In a clinicopathologically low-risk subset, DS reclassified 42% of patients into the Elevated Risk Group. In an interaction analysis of DS and RT, patients with elevated DS had significant RT benefit over baseline.Conclusions:The DS was prognostic for risk and predicted RT benefit for DCIS patients. DS identified a clinically meaningful low-risk group and a group with elevated 10-year risks that received substantial RT benefit over baseline.

Simple Summary Females show a favorable sex difference in incidence as well as in survival for many cancer types, so also in lymphoma. The reasons for this are unknown. We have therefore analyzed global gene expression in a large cohort of the most common lymphoma type, diffuse large B-cell lymphoma. We show that many genes are differentially expressed between males and females. Furthermore, the results demonstrate sex-dependent differences in gene expression between DLBCL subtypes. In addition, gene expression differs in pre- vs. postmenopausal women suggesting that estrogen regulation of genes is involved. Thus, estrogens may contribute to the sex and female age differences in incidence and prognosis observed. For most lymphomas, including diffuse large B-cell lymphoma (DLBCL), the male incidence is higher, and the prognosis is worse compared to females. The reasons are unclear; however, epidemiological and experimental data suggest that estrogens are involved. With this in mind, we analyzed gene expression data from a publicly available cohort (EGAD00001003600) of 746 DLBCL samples based on RNA sequencing. We found 1293 genes to be differentially expressed between males and females (adj. p-value < 0.05). Few autosomal genes and pathways showed common sex-regulated expression between germinal center B-cell (GCB) and activated B-cell lymphoma (ABC) DLBCL. Analysis of differentially expressed genes between pre- vs. postmenopausal females identified 208 GCB and 345 ABC genes, with only 5 being shared. When combining the differentially expressed genes between females vs. males and pre- vs. postmenopausal females, nine putative estrogen-regulated genes were identified in ABC DLBCL. Two of them, NR4A2 and MUC5B, showed induced and repressed expression, respectively. Interestingly, NR4A2 has been reported as a tumor suppressor in lymphoma. We show that ABC DLBCL females with a high NR4A2 expression showed better survival. Inversely, MUC5B expression causes a more malignant phenotype in several cancers. NR4A2 and MUC5B were confirmed to be estrogen-regulated when the ABC cell line U2932 was grafted to mice. The results demonstrate sex- and female reproductive age-dependent differences in gene expression between DLBCL subtypes, likely due to estrogens. This may contribute to the sex differences in incidence and prognosis.

Although CD19-targeting CAR T cell therapy has shown remarkable success against B cell malignancies there is still room for improvement. To improve long-term responses amongst lymphoma patients, mechanisms underlying initial response and tumor relapses must be addressed. Recent studies have highlighted the importance of the quality of the starting cell material for CAR T cell production and the composition of the CAR T cell infusion product for clinical response. In our study, we have evaluated the clinical response to third generation anti-CD19 CAR T cell therapy in 23 patients with B cell lymphoma. Further, we evaluated the CAR T cell infusion products using targeted single-cell RNA sequencing and multi-color flow cytometry to find potential T cell subsets associating with clinical response. Patients were divided into responders (CR + PR) and non-responders (SD + PD) at the 1-month follow-up after treatment. The CAR T cell products of both responding and non-responding patients were dominated by CD8+ CAR T cells. When comparing cells from the two patient groups we found that the presence of effector-like CD8+ CAR T cells with a high cytotoxic and cytokine secretion and low dysfunction gene expression profile were associated with clinical response. Furthermore, CD8+ CAR T cells of responders displayed a higher polyfunctional gene expression profile, which has previously been shown to associate with clinical response. In contrast, CD8+ CAR T cells from the infusion products of non-responders displayed an elevated dysfunction gene expression profile. Our study reveals that single-cell RNA expression analysis of individual CAR T cell infusion product can identify subsets of effector cells correlating with clinical response. This can be used as a guidance in the CAR T cell production processes, to obtain CAR T cells with the desired characteristics. Citation Format: Tina Sarén, Mohanraj Ramachandran, Gustav Gammelgård, Tanja Lövgren, Kristina Wikström, Jamileh Hashemi, Eva Freyhult, Claudio Mirabello, Åsa K Björklund, Håkan Ahlström, Rose-Marie Amini, Hans Hagberg, Angelica Loskog, Gunilla Enblad, Magnus Essand. Single-cell RNA analysis reveal effector-like CD8+ CAR-T cell subpopulations associated with response in lymphoma patients [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 1793.

Outcome in PCNSL patients and its association with PD-L1+ leukocytes in the tumor microenvironment

The glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed-death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event-free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p Authors in thesis list of papers: Gholiha, A.R., Hollander, P., Hedstrom, G., Molin, D., Hjalgrim, H., Smedby, K.E., Glimelius, I., Hashemi, J., Amini, R-M., Enblad, G.Title in thesis list of papers: Checkpoint CD47 Expression Predicts Inferior Survival in classical Hodgkin Lymphoma

In classical Hodgkin Lymphoma (cHL), immunoediting via protein signaling is key to evading tumor surveillance. We aimed to identify immune-related proteins that distinguish diagnostic cHL tissues (=diagnostic tumor lysates, n = 27) from control tissues (reactive lymph node lysates, n = 30). Further, we correlated our findings with the proteome plasma profile between cHL patients (n = 26) and healthy controls (n = 27). We used the proximity extension assay (PEA) with the OlinkTM multiplex Immuno-Oncology panel, consisting of 92 proteins. Univariate, multivariate-adjusted analysis and Benjamini–Hochberg’s false discovery testing (=Padj) were performed to detect significant discrepancies. Proteins distinguishing cHL cases from controls were more numerous in plasma (30 proteins) than tissue (17 proteins), all Padj < 0.05. Eight of the identified proteins in cHL tissue (PD-L1, IL-6, CCL17, CCL3, IL-13, MMP12, TNFRS4, and LAG3) were elevated in both cHL tissues and cHL plasma compared with control samples. Six proteins distinguishing cHL tissues from controls tissues were significantly correlated to PD-L1 expression in cHL tissue (IL-6, MCP-2, CCL3, CCL4, GZMB, and IFN-gamma, all p ≤0.05). In conclusion, this study introduces a distinguishing proteomic profile in cHL tissue and potential immune-related markers of pathophysiological relevance.