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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.

Formalin-fixed, paraffin-embedded (FFPE) specimens are an underutilized resource in medical research, particularly in the setting of transcriptome sequencing, as RNA from these samples is often degraded. We took advantage of an exome capture-based RNA-sequencing protocol to explore global gene expression in paired fresh-frozen (FF) and FFPE samples from 16 diffuse large B-cell lymphoma (DLBCL) patients. While FFPE samples generated fewer mapped reads compared to their FF counterparts, these reads captured the same library complexity and had a similar number of genes expressed on average. Furthermore, gene expression demonstrated a high correlation when comparing housekeeping genes only or across the entire transcriptome (r = 0.99 for both comparisons). Differences in gene expression were primarily seen in lowly expressed genes and genes with small or large coding sequences. Using cell-of-origin classifiers and clinically relevant gene expression signatures for DLBCL, FF, and FFPE samples from the same biopsy paired nearly perfectly in clustering analysis. This was further confirmed in a validation cohort of 50 FFPE DLBCL samples. In summary, we found the biological differences between tumors to be far greater than artifacts created as a result of degraded RNA. We conclude that exome capture transcriptome sequencing data from archival samples can confidently be used for cell-of-origin classification of DLBCL samples.

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.

Cancer treatment has been transformed by checkpoint blockade therapies, with the highest anti-tumor activity of anti-programmed death 1 (PD-1) antibody therapy seen in Hodgkin lymphoma. Disappointingly, response rates have been low in the non-Hodgkin lymphomas, with no activity seen in relapsed/refractory chronic lymphocytic leukemia (CLL) with PD-1 blockade. Thus, identifying more powerful combination therapy is required for these patients. Here, we preclinically demonstrate enhanced anti-CLL activity following combinational therapy with anti-PD-1 or anti-PD-1 ligand (PD-L1) and avadomide, a cereblon E3 ligase modulator (CELMoD). Avadomide induced type I and II interferon (IFN) signaling in patient T cells, triggering a feedforward cascade of reinvigorated T-cell responses. Immune modeling assays demonstrated that avadomide stimulated T-cell activation, chemokine expression, motility and lytic synapses with CLL cells, as well as IFN-inducible feedback inhibition through upregulation of PD-L1. Patient-derived xenograft tumors treated with avadomide were converted to CD8+ T cell-inflamed tumor microenvironments that responded to anti-PD-L1/PD-1-based combination therapy. Notably, clinical analyses showed increased PD-L1 expression on T cells, as well as intratumoral expression of chemokine signaling genes in B-cell malignancy patients receiving avadomide-based therapy. These data illustrate the importance of overcoming a low inflammatory T-cell state to successfully sensitize CLL to checkpoint blockade-based combination therapy.

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

The abundant hepatocyte-expressed plasma protein histidine-rich glycoprotein (HRG) enhances antitumor immunity by polarizing inflammatory and immune cells in several mouse models, however, the clinical relevance of HRG in human cancer is poorly explored. The expression and role of HRG in human B-cell lymphomas was investigated in order to find new tools for prognosis and treatment.Immunohistochemical (IHC) analysis and RNA hybridization of tissue microarrays showed that (i) HRG was expressed by tumor cells in marginal zone lymphoma (MZL), in 36% of 59 cases. Expression was also detected in follicular lymphoma (22%), mantle cell lymphoma (19%), and indiffuse large B-cell lymphoma (DLBCL;5%) while primary CNS lymphoma (PCNSL) lacked expression of HRG. (ii) MZL patients positive for HRG showed a superior overall survival outcome (HR = 0.086, 95% CI = 0.014-0.518,HRG expression by MZL tumor cells correlates with an altered transcription profile and improved overall survival.

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.

Background Systemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells. Methods Fluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential. Findings We found that in SM 60–99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FceRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. Interpretation The KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright (c) 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Programmed cell death 1 (PD-1) and its ligands PD-L1 and PD-L2, as well as Indoleamine 2,3-deoxygenase (IDO1) can be expressed both by tumor and microenvironmental cells and are crucial for tumor immune escape. We aimed to evaluate the role of PD-1, its ligands and IDO1 in a cohort of patients with primary diffuse large B-cell lymphoma of the CNS (PCNSL). Material and methods Tissue microarrays (TMAs) were constructed in 45 PCNSL cases. RNA extraction from whole tissue sections and RNA sequencing were successfully performed in 33 cases. Immunohistochemical stainings for PD-1, PD-L1/paired box protein 5 (PAX-5), PD-L2/PAX-5 and IDO1, and Epstein-Barr virus encoding RNA (EBER) in situ hybridization were analyzed. Results High proportions of PD-L1 and PD-L2 positive tumor cells were observed in 11% and 9% of cases, respectively. High proportions of PD-L1 and PD-L2 positive leukocytes were observed in 55% and 51% of cases, respectively. RNA sequencing revealed that gene expression of IDO1 was high in patients with high proportion of PD-L1 positive leukocytes (p = .01). Protein expression of IDO1 in leukocytes was detected in 14/45 cases, in 79% of these cases a high proportion of PD-L1 positive leukocytes was observed. Gene expression of IDO1 was high in EBER-positive cases (p = .0009) and protein expression of IDO1 was detected in five of six EBER-positive cases. Conclusion Our study shows a significant association between gene and protein expression of IDO1 and protein expression of PD-L1 in the tumor microenvironment of PCNSL, possibly of importance for prediction of response to immunotherapies. Title in Thesis: PD-L1 and IDO1 are important immunosuppressive molecules in primary diffuse large B-cell lymphoma of the CNS

Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoma where associations with viral hepatitis and autoimmune and inflammatory diseases (AID) have been indicated. We aimed at assessing the prevalence of viral hepatitis and AID at SMZL diagnosis and outcome by treatment in a Swedish population-based study. A total of 277 SMZL patients registered in the Swedish Lymphoma Register in 2007-2017 were included. A history of viral hepatitis was reported in five (2%) patients and AID prior to SMZL in 72/240 (30%) patients. Treatment was given up front for 207 (75%) patients. Splenectomy with or without systemic treatment was performed in 119 (57%) and was associated with statistically significantly better overall survival [hazard ratio, HR = 0 center dot 47 (95% confidence interval, CI: 0 center dot 23-0 center dot 93), P = 0 center dot 03] and progression-free survival (HR = 0 center dot 55, 95% CI: 0 center dot 35-0 center dot 86, P = 0 center dot 008) compared to non-splenectomised patients in multivariable analyses. The up-front splenectomised group was younger and generally had a lower Ann Arbor stage, but also more frequently B symptoms and high lactate dehydrogenase than the non-splenectomised group. Viral hepatitis and AID history did not affect SMZL outcome. We report high incidence of AIDs and low incidence of viral hepatitis in this population-based study of SMZL. Splenectomy up front was associated with a favourable outcome.

We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex-determining region Y-box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T-box transcription factor TBX21 (T-bet), programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1) and CD163 were investigated for all-cause mortality in 282 patients with MCL and time-to-progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3(+) T lymphocytes was seen. T-cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki-67, morphology and frequency of tumour cells. The all-cause mortality was higher in patients with PD-L1-expression above cut-off [hazard ratio (HR) 1 center dot 97, 95% confidence interval (CI) 1 center dot 18-3 center dot 25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163(+) cells (continuously, HR 1 center dot 51, 95% CI 1 center dot 03-2 center dot 23, adjusting for age, sex, morphology, Ki-67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3(+) cells (HR 3 center dot 22, 95% CI 1 center dot 40-7 center dot 43) and CD163(+) cells (HR 6 center dot 09, 95% CI 1 center dot 84-20 center dot 21), independent of sex and MIPI. When combined a higher frequency of CD163(+) macrophages and PD-L1(+) cells or high CD163(+) macrophages and FoxP3(+) regulatory T cells indicated worse outcome independent of established risk factors. The T-cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age.

Background: Post-transplant lymphoproliferative disorder (PTLD) is a rare but life-threatening complication of transplantation. For refractory and relapsed PTLD new therapies are needed, such as the antibody-drug conjugate brentuximab vedotin that targets CD30. There is limited knowledge of CD30 expression in various subtypes of PTLD and its correlation to clinicopathological features. Therefore, we studied the expression of CD30 in PTLD following solid organ transplantation and correlated CD30 expression to PTLD subtype, Epstein-Barr virus (EBV)-status, intratumoral regulatory T-cells (Tregs), clinical features, and outcome. Methods: We included 50 cases of PTLD from a nation-wide study of PTLDs following solid organ transplantation in Sweden. The tumor biopsies were reevaluated, and clinical data were collected. CD30 expression on tumor cells was analyzed by immunohistochemistry with the clone Ber-H2. Thirty-one cases were stained with clone 236 A/E7 for detection of forkhead box protein 3 (FoxP3, a Treg biomarker). Results: The case series consisted of 6% polymorphic, 88% monomorphic, and 6% Hodgkin lymphoma-like PTLDs and 53% of the cases were EBV+. Overall, 70% (35/50) of the PTLDs were CD30+ (>= 1% CD30+ tumor cells) and 30% (15/50) were CD30-. All polymorphic PTLDs (n = 3) and Hodgkin lymphomas (n = 3), 88% (14/16) of non-germinal center type of diffuse large B-cell lymphoma (DLBCL), and 75% (9/12) of T-cell PTLDs were CD30+ whereas all germinal center-type of DLBCL (n = 5) and Burkitt type PTLD (n = 2) were CD30-. CD30+ PTLD tended to be EBV+ more frequently (p = .07) and occurred earlier posttransplant (2.1 vs. 8.2 years, p = .01) than CD30- PTLD. Type of transplant and localization of the tumor did not differ between the groups except that CNS engagement was more common in CD30- PTLD (p = .02). CD30-status was not associated with presence of intratumoral Tregs or overall survival. Conclusion: Expression of CD30 varied with PTLD subtype. There was no association between CD30 and survival, regardless of subtype.

OBJECTIVE: The prognostic value of site of nodal involvement in diffuse large B-cell lymphomas (DLBCL) is mainly unknown. We aimed to determine the prognostic significance of nodal abdominal involvement in relation to tumour cell markers and clinical characteristics of 249 DLBCL patients in a retrospective single-centre study. METHODS: Contrast-enhanced computed tomography (CT) of the abdomen and thorax revealed pathologically enlarged abdominal lymph nodes in 156 patients, while in 93 patients there were no pathologically enlarged lymph nodes in the abdomen. In 81 cases, the diagnosis of DLBCL was verified by histopathological biopsy obtained from abdominal lymph node. RESULTS: Patients with abdominal nodal disease had inferior lymphoma-specific survival (P = .04) and presented with higher age-adjusted IPI (P

The tumor cells in diffuse large B‐cell lymphomas (DLBCL) are considered to originate from germinal center derived B‐cells (GCB) or activated B‐cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC‐based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non‐GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression‐free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression‐free survival differed significantly. Importantly, patients assigned as non‐GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.

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.

Tumor cells engage in bidirectional interactions with stroma and immune cells to promote disease progression and immune evasion. Stroma-specific gene signatures have been associated with outcome in diffuse large B-cell lymphoma (DLBCL), but their immunobiology has been understudied. To characterize the stromal landscape in lymphoma, we performed high-dimensional imaging mass cytometry analysis of the major stroma subsets and revealed a marked expansion and remodeling of the immuno-specialized fibroblastic reticular cells (FRCs) in human DLBCL biopsies (n=53). The FRC network was similarly remodeled in tumors from the IμBcl6 transgenic model of lymphoma, and aberrant fibroblasts were in close proximity to cancer cells. Modelling the interactions between murine and patient FRCs and tumor cells, using 2D and 3D cultures, showed that lymphoma drives the acquisition of an inflammatory-like, pro-tumoral (upregulation of fibroblast activating protein-α, FAP) phenotype and associated functional capabilities. Comparative bulk transcriptomic analysis revealed that lymphoma-FRCs undergo transcriptional reprogramming and activate gene pathways associated with inflammatory responses. Moreover, single-cell RNA-seq revealed an expansion of activated FRC clusters expressing B cell supporting genes, while T cell-associated FRCs were contracted. Altered chemokine signaling pathways in DLBCL-FRCs were functionally linked to reduced attraction of T cells and impeded migration along the lymphoma-reticular network. Moreover, lymphoma-FRCs upregulated expression of inhibitory PD-1 ligands that reduced the anti-tumor cytolytic activity of CD8+ T cells, a T cell bispecific antibody (CD20-TCB, glofitamab) and anti-CD19 CAR T cells in our coculture models. To overcome the immunosuppressive activity of DLBCL-FRCs, we investigated the use of CD20-TCB in combination with stroma-targeting immunocytokine fusion protein drug (FAP-IL2v, RG7461) or costimulatory fusion protein (FAP-4-1BBL, RG7827). Functional cytotoxicity assays using human and murine primary DLBCL patient samples revealed that both stroma-targeting drugs paired effectively with the CD20-TCB to enhance the cytotoxic activity of autologous CD8+ T cells. In addition, the ability of immune-/stroma- targeted combination immunotherapy to trigger anti-tumor activity and CD8+ T cell retention within the FRC-TME was demonstrated using 3D precision-cut lymph node slice-based organotypic cultures of DLBCL and other B cell malignancies. In conclusion our data reveal that lymphoma cells actively reprogram FRCs that acquire altered immunoregulatory function which prevents effective T cell motility and suppresses the anti-tumor function of cytolytic T cells. Importantly, we demonstrate that combination immunotherapy incorporating fibroblast-targeting fusion proteins could effectively recover anti-tumor T cell activity. Citation Format: Benedetta Apollonio, Nedyalko Petrov, Filomena Spada, Peter Jarvis, Domenico Cozzetto, Shichina Kannambath, David Kuo, Mansoor Saqui, Rose-Marie Amini, Gunilla Enblad, Graham Charlotte, Reuben Benjamin, Anna Vardi, Elisabeth Phillips, Jon Salisbury, Eric N. Olson, Brian Fox, Patrick Hagner, Anita Gandhi, Ruth F. Jarrett, Sylvia Herter, Marina Bacac, Christina Klaus, Christian Klein, Alexander Deutsch, Alan G. Ramsay. Stroma-immune landscape in lymphoma: new mechanisms of immunosuppression and therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3165.

Immune checkpoint inhibition targeting the programmed death receptor (PD)-1 pathway is a novel treatment approach in relapsed and refractory classical Hodgkin lymphoma (cHL). Identifying patients with a high risk of treatment failure could support the use of PD-1 inhibitors as front-line treatment. Our aim was to investigate the prognostic impact of PD-1, programmed death-ligand 1 (PD-L1), and PD-L2 in the tumor microenvironment in diagnostic biopsies of patients with cHL. Patients from Denmark and Sweden, diagnosed between 1990 and 2007 and ages 15 to 86 years, were included. Tissue microarray samples were available from 387 patients. Immunohistochemistry was used to detect PD-1, PD-L1, and PD-L2, and the proportions of positive cells were calculated. Event-free survival (EFS; time to treatment failure) and overall survival (OS) were analyzed using Cox proportional hazards regression. High proportions of both PD-1

18F-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT) is used for staging classical Hodgkin lymphoma (cHL) with high sensitivity for skeletal involvement. However, it is unclear whether a single bone lesion carries the same adverse prognosis as multifocal lesions and if this is affected by type of chemotherapy [ABVD (adriamycin, bleomycin, vincristine, dacarbazine) versus BEACOPP (bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone)]. We reviewed the clinico-pathological and outcome data from 209 patients with newly diagnosed cHL staged by FDG-PET/CT. Patterns of skeletal/bone marrow uptake (BMU) were divided into ‘low’ and ‘high’ diffuse BMU (i.e. without focal lesions), and unifocal or multifocal lesions. Additional separate survival analysis was performed, taking type of chemotherapy into account. Forty patients (19·2%) had skeletal lesions (20 unifocal, 20 multifocal). The 3-year progression-free-survival (PFS) was 80% for patients with ‘low BMU’, 87% for ‘high BMU’, 69% for ‘unifocal’ and 51% for ‘multifocal’ lesions; median follow-up was 38 months. The presence of bone lesions, both uni- and multifocal, was associated with significantly inferior PFS (log rank P = 0·0001), independent of chemotherapy type. Thus, increased diffuse BMU should not be considered as a risk factor in cHL, whereas unifocal or multifocal bone lesions should be regarded as important predictors of adverse outcome, irrespective of the chemotherapy regimen used.

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (

To determine the prognostic significance of co-expression of MYC, BCL-2 and BCL-6 proteins in combination with other biomarkers and clinical characteristics within a population-based cohort of diffuse large B-cell lymphoma (DLBCL) patients uniformly treated with R-CHOP.The immunohistochemical (IHC) expression of CD10, BCL-2, BCL-6, MUM1, MYC, CD5, CD30, Ki-67 and p53 was evaluated in a retrospective, population-based study comprising 188 DLBCL patients treated with R-CHOP and diagnosed in Sweden between 2002 and 2012.Patients had a median age at diagnosis of 64 years (26-85 years) with a male:female ratio of 1.4:1. Approximately half (52%) of the patients presented with an International Prognostic Index (IPI) age adjusted (IPIaa) ≥ 2. Median follow-up time was 51 months (range 0.4-158) and the five-year lymphoma-specific survival (LSS) was 76%, five-year overall survival (OS) was 65% and five-year progression-free survival (PFS) was 61%. A high Ki-67 value was found in 59% of patients, while p53 overexpression was detected in 12% of patients and MYC, BCL-2 and BCL-6 expression were detected in 42%, 55% and 74% of patients, respectively. IPIaa ≥2 (p = 0.002), Ki-67 ≥ 70% (p = 0.04) and p53 overexpression ≥50% (p = 0.02) were associated with inferior LSS and OS. Co-expression of both MYC (40%) and BCL-2 (70%) proteins was detected in 27% of patients and correlated with a significantly inferior LSS (p = 0.0002), OS (p = 0.009) and PFS (p = 0.03). In addition, triple expression of MYC, BCL-2 and BCL-6, also correlated with a significantly inferior LSS (p = 0.02).Concurrent expression of MYC and BCL-2 proteins, as detected by IHC, was strongly associated with an inferior survival in DLBCL patients treated with R-CHOP. Other markers affecting survival were triple expression of MYC, BCL-2 and BCL-6, IPIaa, high Ki-67 and p53 overexpression.

Diffuse large B-cell lymphoma (DLBCL) shows a higher incidence in males versus females. Epidemiological studies have shown that female gender is a favorable prognostic factor, which may be explained by estrogens. Here we show that when grafting human DLBCL cells to immunocompromised mice, tumor growth in males is faster. When treating mice grafted with either germinal center or activated B-cell like DLBCL cells with the selective estrogen receptor β (ERβ) agonist diarylpropionitrile, tumor growth was significantly inhibited. Furthermore, nuclear ERβ1 expression analysis in primary DLBCL's by immunohistochemistry revealed expression in 89% of the cases. Nuclear ERβ1 expression was in a univariate and multivariate analysis, an independent prognostic factor for adverse progression-free survival in Rituximab-chemotherapy treated DLBCL (p = 0.02 and p = 0.04, respectively). These results suggest that estrogen signaling through ERβ1 is an interesting future therapeutic target for treatment of DLBCL, and that ERβ1 expression can be used as a prognostic marker.

The etiology of classical Hodgkin lymphoma (cHL) is largely unknown. High serum CD30-levels are associated with increased risk of cHL. Epstein-Barr virus (EBV) is detectable in the tumor cells in 1/3 of cHL cases in the Western world. The PD-1 pathway (T-cell inflamed immune response) might contribute to the pathogenesis by enabling pre-malignant CD30+ or EBV + cells to evade immune surveillance. We aimed to investigate if high infiltrations of CD30+, PD-1+, PD-L1+ and EBV + cells in benign lymph nodes from patients that later develop cHL (cases) (n = 15) were associated with risk of cHL compared to controls (n = 45) with benign lymph nodes from patients that did not develop cHL. Pathology registries including 3500 cH L patients were screened. Lymph nodes were stained with immunohistochemistry and in situ hybridization and the risk for cHL calculated with logistic regression. High CD30-expression by B- and T-cells was associated with a decreased risk of cHL [(OR = 0.10, 95 % CI:0.03-0.39) and (OR = 0.13, 95 % CI:0.01-0.71), respectively], which remained significant for CD30 + B-cells (OR = 0.15, 95 % CI:0.03-0.60) in multivariate analyses. Amount of PD-1+, PD-L1+ and EBV + cells were not statistically significantly associated with risk of cHL. However, the amount of PD-L1+ leukocytes tended to be higher in cases later developing cHL (OR = 2.84, 95 % CI:0.61-12.61). High proportions of potential precursors to cHL, i.e. CD30 + B-cells in benign lymph nodes are not associated with an increased risk of cHL, while a tendency for a T-cell inflamed immune response, i.e. abundant PD-L1+ cells, was observed in biopsies taken prior to the cHL diagnosis.

Background: Systemic mastocytosis (SM) is a hematological disease characterized by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ hematopoietic progenitors can carry the mutation, however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single hematopoietic stem and progenitor cells. Methods: Fluorescence-activated single-cell index sorting and D816V mutation assessment were applied to analyze mast cells and more than 10,000 CD34+ bone marrow progenitors across 10 hematopoietic progenitor subsets. In vitro assays verified cell-forming potential. Findings: We found that in SM 60-99% of the mast cells harbored the D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the hematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the hematopoietic landscape of SM patients, from hematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. Interpretation: The KIT D816V mutation arises in early hematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

Plasma cells are important prognostic actors in different malignancies. The tumour microenvironmental composition in classic Hodgkin lymphoma (cHL) is a major prognostic key element; however, clinicopathological studies regarding plasma cells in cHL are lacking. The aim of this study was to investigate CD138+ (also termed SDC1+) plasma cell and IgG4 producing (IgG4+) plasma cells infiltration in the microenvironment of cHL. Immunohistochemistry with anti-CD138 and IgG4 antibodies was performed on diagnostic tumour biopsies from 124 patients with cHL, on tissue micro array (TMA). In 120 cases, CD138+ plasma cell-infiltration was associated with the presence of B-symptoms (P = 0·028) and advanced stage, IIB-IVB (P = 0·009). In multivariate analysis, CD138+ plasma cells correlated with eosinophil infiltration (P = 0·013). The subgroup of IgG4+ plasma cells was analysed in 122 cases and only correlated to CD138+ plasma cells (P = 0·004). Patients with high proportion of tumour infiltrating CD138+ plasma cells (defined as ≥10%), had a more inferior event-free survival (P = 0·007) and overall survival (P = 0·004) than patients with a low proportion of infiltrating CD138+ plasma cells (

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.

High expression of programmed death receptor 1 (PD-1) and its ligand (PD-L1) by leukocytes in primary classical Hodgkin lymphoma (cHL) is associated with inferior outcome. However, it is unclear how expression varies during disease progression, and in the event of relapse. Our aim was to study PD-1 and PD-L1 in consecutive biopsies from untreated and treated cHL patients. We screened pathology registries from 3500 cHL patients. Eleven patients had a diagnostic cHL biopsy and a previous benign lymph node biopsy reclassified as cHL when reviewed and designated as the untreated. Thirty patients had a primary and a relapse biopsy, designated as the treated. Biopsies were immunostained to detect PD-1+ and PD-L1+ leukocytes, and PD-L1+ tumor cells. In the untreated, none of the markers were statistically significantly different when biopsies 1 and 2 were compared. In the treated, 19, 22, and 18 of 30 cases had increased proportions of PD-1+ leukocytes, PD-L1+ leukocytes and PD-L1+ tumor cells, respectively, and were all statistically significantly increased when primary and relapse biopsies were compared. PD-1 and PD-L1 most likely increase due to primary treatment with chemotherapy and radiotherapy, which could have implications regarding treatment with PD-1 inhibitors.

Results from previous investigations have shown associations between the risk of Hodgkin lymphoma (HL) and a history of autoimmune and atopic diseases, but it remains unknown whether these associations apply to all types of HL or only to specific subtypes. We investigated immune diseases and the risk of classical HL in a population-based case-control study that included 585 patients and 3,187 controls recruited from October 1999 through August 2002. We collected information on immune diseases through telephone interviews and performed serological analyses of specific immunoglobulin E reactivity. Tumor Epstein-Barr virus (EBV) status was determined for 498 patients. Odds ratios with 95% confidence intervals were calculated using logistic regression analysis. Rheumatoid arthritis was associated with a higher risk of HL (odds ratio (OR) = 2.63; 95% confidence interval (CI): 1.47, 4.70), especially EBV-positive HL (OR = 3.18; 95% CI: 1.23, 8.17), and with mixed-cellularity HL (OR = 4.25; 95% CI: 1.66, 10.90). HL risk was higher when we used proxies of severe rheumatoid arthritis, such as ever having received daily rheumatoid arthritis medication (OR = 3.98; 95% CI: 2.08, 7.62), rheumatoid arthritis duration of 6-20 years (OR = 3.80; 95% CI: 1.72, 8.41), or ever having been hospitalized for rheumatoid arthritis (OR = 7.36; 95% CI: 2.95, 18.38). Atopic diseases were not associated with the risk of HL. EBV replication induced by chronic inflammation in patients with autoimmune diseases might explain the higher risk of EBV-positive HL.