HLA-DR Protein Expression Correlates with Non-Neoplastic T-Cell Infiltration and Predicts Survival in Patients with Primary Mediastinal Large B Cell Lymphoma (PMBCL) Treated with CHOP Chemotherapy.

Abstract 133 Background: PMBCL represents a unique lymphoma type in the WHO classification and shares overlapping histological and gene expression features with both diffuse large B-cell (DLBCL) and classical Hodgkin lymphoma. Similar to DLBCL, PMBCL may show loss of expression of the major histocompatibility class II (MHC II) antigens, which correlates with inferior patient survival (Rimsza, Blood 2006). MHC II proteins are expressed on antigen-presenting cells and are important in eliciting immune responses. In DLBCL, decreased expression of HLA-DR is related to loss of immunosurveillence. Our aim was to validate HLA-DR as an important prognostic biomarker in PMBCL and correlate it with immune response in a cohort of patients treated with multi-agent chemotherapy. Methods: A tissue microarray block was built with duplicate 0.6mm cores of paraffin embedded diagnostic biopsies from 103 patients treated with CHOP/CHOP-like chemotherapy at the BC Cancer Agency (1980-2005). Standard immunohistochemistry was performed with CD20, HLA-DR (IgG2b), CD3, CD4, CD8, CD57, CD68 and cytotoxic markers (TIA1 & Granzyme B). HLA-DR expression was evaluated using a histoscore (intensity of staining and % of positive malignant cells) and correlated with the content (%) of different non-neoplastic infiltrating T-cell subsets and macrophages. Univariate & multivariate analyses were used to characterize overall survival (OS) and progression free survival (PFS). Results: The median follow-up of living patients was 10 years. The IPI predicted OS (p=0.042) but not PFS. Of the 92 cases with interpretable staining, 32 were positive for HLA-DR and 60 negative, with 10-year OS of 86% vs. 61% (p=0.006) and 2-year PFS of 78% vs. 53% (p=0.018), respectively. A Cox multivariate model established both HLA-DR status and IPI as independent predictors of OS (RR=0.3, 95%CI=0.12–0.75, p=0.01; RR=2.9, 95%CI=1.2–6.9, p=0.06, respectively). HLA-DR expression correlated significantly with increased content of all analyzed T cell markers, especially CD3, CD8 and TIA1 (x2, p<0.001), but not with macrophage content (CD68). Of all non-malignant markers, only TIA1+ cell content significantly correlated with survival. Of the 83 cases with interpretable staining, 43 had more than 10% of infiltrating TIA1+ cells and 40 had less, with 10-year OS of 83% vs. 57% (p=0.0014) and 2-year PFS of 76% vs. 50% (p=0.014), respectively. In multivariate analysis, including IPI and HLA-DR and cytotoxic markers, only TIA1 status was an independent predictor of OS (RR=0.3, 95%CI=0.11–0.63, p=0.003). Conclusions: We validated the negative prognostic importance of loss of HLA-DR expression by neoplastic B cells in PMBCL patients treated with multi-agent chemotherapy in a single institution experience. Loss of HLA-DR expression correlated with decreased numbers of infiltrating benign T cell populations, especially CD8+ and TIA1+ cells, where decreased cytotoxic T cell content correlated independently with inferior survival. This study shows loss of immunogenicity and immunosurveillance are key mechanisms in the response to treatment of PMBCL patients and suggests that specific therapies focused on this pathway may benefit patients. Disclosures: Connors: Roche Canada (F Hoffmann-La Roche): Research Funding. Gascoyne:Roche Canada, Genentech, Lilly, Millennium : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Somatic PRAME Deletions Are Associated with Decreased Immunogenicity, Apoptosis Resistance and Poor Outcomes in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-113516 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 667-667

Author(s):

Katsuyoshi Takata ◽

Daisuke Ennishi ◽

Ali Bashashati ◽

Saeed Saberi ◽

Elena Viganò ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Memory T Cell ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

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Outcomes of Large B-Cell Lymphoma Patients By Post CAR-T Salvage Regimen at a Single Institution

Blood ◽

10.1182/blood-2021-148751 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3851-3851

Author(s):

Audrey M. Sigmund ◽

Nathan Denlinger ◽

Amneet Bajwa ◽

Patrick Elder ◽

David A. Bond ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Salvage Regimen ◽

Large B Cell Lymphoma ◽

Car T ◽

Large B Cell

Abstract Introduction: Outcomes of patients with large B-cell lymphoma that relapse after frontline anthracycline based chemotherapy are typically poor, with a 3-year event-free survival of approximately 30% (Gisselbrecht JCO 2010). Chimeric antigen receptor T-cell (CAR-T) therapy represents a breakthrough therapy for these patients, with an overall response rate (ORR) of 83% and a complete response (CR) rate of 58% for axicabtagene ciloleucel (axi-cel) seen in the ZUMA-1 trial, with similar rates for tisagenlecleucel in the JULIET trial (Locke Lancet Oncol 2018; Schuster NEJM 2018). Unfortunately, the majority of patients treated with CAR-T therapy experience disease progression. There is limited data evaluating the best salvage regimen for these patients. Thus, we sought to assess outcomes in large B-cell lymphoma patients with progressive disease post CAR-T cell therapy with the goal of identifying those therapies with optimal outcomes. Methods: A retrospective study was performed on all patients with large B-cell lymphoma undergoing leukapheresis for CAR-T therapy (tisagenlecleucel or axi-cel) at the Ohio State University from December 2017 to January 2021. Patients who died prior to CAR-T infusion were excluded from analysis. Demographics and disease characteristics as well as best response to CAR-T and date of relapse or progression following therapy were collected. First salvage therapy at relapse or progression and response to therapy were also collected, with choice of therapy driven by the treating physician. Patients were divided by salvage regimen into five groups for analysis: checkpoint inhibitor based, lenalidomide based, Bruton Tyrosine Kinase inhibitor (BTKi), chemoimmunotherapy, and other (including small molecular inhibitor, radiation, allogeneic stem cell transplant, antibody drug conjugates, and bispecific antibodies). The primary endpoint was overall survival (OS), which was calculated using Kaplan Meier Curves. Rates of CR and ORR were also assessed. Results: A total of 144 patients underwent leukapheresis for CAR-T cell therapy during the time period; of these patients, 13 died prior to undergoing CAR-T cell infusion and were excluded from analysis. The primary cohort included 131 patients. Median age at the time of T-cell collection was 62 years old (range 23-85) and 61% were male. The majority (50%) had germinal center (GCB) subtype, with 42% non-GCB and subtype unavailable for 8%. A small number (3%) had primary mediastinal B-cell lymphoma. The majority had high-risk disease, with 45% having primary refractory disease, 14.5% double or triple hit, and 81% Ann Arbor stage III or IV at diagnosis. Median prior lines of therapy was 3 (range 0-10). Sixty-six patients received axi-cel and 65 patients received tisagenlecleucel. Forty percent of patients attained a CR, 18% partial response (PR), 3% stable disease (SD), 33% progressive disease (PD), and 6% of patients died prior to disease assessment. For those 76 patients that received a CR or PR to therapy, 43% relapsed post CAR-T and 57% remained in CR at last follow-up. Of those patients who relapsed or progressed post CAR-T, 69% (54/78) patients received additional therapy. The most common therapies utilized were lenalidomide based (35%), BTKi (22%), chemoimmunotherapy (13%), and checkpoint inhibitor based (15%). Other therapies represented 15% of cases and included small molecular inhibitor, radiation, allogeneic stem cell transplant, antibody drug conjugates, and bispecific antibodies (Table 1). Overall response rates and median OS for the groups were 50% and 2.25 years for BTKi, 13% and 0.96 years for checkpoint inhibitor based, 71% and not reached (NR) for chemoimmunotherapy, 47% and 2.4 years for lenalidomide based, and 75% and NR for other (Table 1; Figure 1). Median OS for those patients who did not receive any salvage therapy was 0.19 years. Conclusion: Consistent with prior studies, median OS following relapse post CAR-T therapy was poor, with median OS 0.19 years for patients who did not receive therapy and ranging from 0.96 years to NR for those that did. Rates of CR were highest in patients treated with BTKis. In our series, ORR rate to checkpoint inhibitors was relatively low in contrast to other recently published retrospective reports. Future prospective studies are needed to further assess the optimal therapy for patients who relapse post CAR-T therapy. Figure 1 Figure 1. Disclosures Bond: Kite/Gilead: Honoraria. Brammer: Seattle Genetics: Speakers Bureau; Kymera Therapeutics: Consultancy; Celgene: Research Funding. de Lima: Miltenyi Biotec: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Jaglowski: Novartis: Consultancy, Research Funding; Takeda: Consultancy; Juno: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; CRISPR Therapeutics: Consultancy. Kittai: Bristol-Meyers Squibb: Consultancy; Janssen: Consultancy; Abbvie: Consultancy.

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Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 As a Therapeutic Target for Restoring MHC Expression in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-112010 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1560-1560

Author(s):

Daisuke Ennishi ◽

Katsuyoshi Takata ◽

Wendy Béguelin ◽

Gerben Duns ◽

Anja Mottok ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Mhc I ◽

Mhc Ii ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Introduction: Among the tumor immune escape mechanisms described to date, alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Although the frequency of loss of MHC expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, distinct genetic alterations and molecular features that affect MHC expression and the composition of immune cells in the tumor microenvironment remain ill-defined. Here, we aimed to uncover the biologic and genomic basis underlying acquired loss of MHC expression. Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP in British Columbia. We performed targeted resequencing, SNP6.0 array and RNAseq for genetic analyses. Immunohistochemical (IHC) staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by the Lymph2Cx assay in 323 cases (183 GCB, 104 ABC and 36 unclassifiable). Immune cell composition was assessed by IHC, flow cytometry and gene expression profiling (GEP)-based deconvolution of cellular signatures. To experimentally confirm decreased MHC expression induced by EZH2 mutation, we measured surface MHC-I and -II expression on tumor B cells using EZH2Y641/BCL2 mouse model which was previously established (Beguelin et al, Cancer Cell 2013). We also treated human DLBCL cells harboring EZH2 mutation and wild type using EZH2 inhibitor (EPZ-6438), and evaluated their surface MHC-I and -II expression. Results: Loss of MHC-I and -II expression was observed in 43% and 28% of DLBCL cases, respectively. MHC-II loss of expression was significantly associated with the reduction of tumor-infiltrating lymphocytes (TILs), especially CD4 positive T-cells (FOXP3+ cells, PD-1+ cells, and CD4+ naïve and memory T-cells), and cytolytic activity (GZMB and PRF1 mRNA expression) in GCB-DLBCL (all; p<0.001), but not in ABC-DLBCL. MHC-II-negativity was associated with unfavorable prognosis only in GCB-DLBCL (5-year time-to-progression; 59% vs 79%, p=0.007), whereas there was no prognostic impact of MHC-I expression in either subtype, suggesting a link between loss of MHC-II expression and reduced immune surveillance leading to poor prognosis, specifically in GCB-DLBCL. We next performed GEP using RNAseq separately in each COO subtype. Interestingly, only four genes (HLA-DMA, DRA, DPA1 and CD74) were differentially expressed according to MHC-II expression (FDR<0.001) in ABC-DLBCL. By contrast, a total of 641 genes were differentially expressed in GCB-DLBCL. Of importance, a dark zone (DZ) B-cell signature was strongly enriched in MHC-II-negative GCB-DLBCL cases (FDR<0.001), suggesting that MHC-II deficiency defines the tumor originated from DZ of the germinal center. Correlative genetic analysis revealed that, as expected, mutations of CIITA and RFXAP were detected more frequently in MHC-II-negative GCB-DLBCL (p=0.01 and 0.003, respectively). Strikingly, CD83 mutations, which elevate and stabilize MHC-II expression in centrocytes of the light zone (LZ), were significantly enriched in MHC-II positive GCB-DLBCL (p= 0.008), suggesting that these mutations affecting the antigen presentation machinery are selectively acquired in GCB-DLBCL tumors to further reduce and increase the surface MHC-II expression. Genetic analysis also highlighted that EZH2 mutations were most significantly enriched in MHC-II-negative as well as MHC-I-negative GCB-DLBCL cases (both, p<0.001). Indeed, 77% of EZH2 mutated cases demonstrated loss of either MHC-I and/or MHC-II expression on the tumor cells. Notably, we found significantly lower MHC-I and MHC-II expression in high-grade lymphomas of EZH2 mutant Vav-BCL2 transgenic mice compared to EZH2 wildtype control tumors. Furthermore, of potential clinical relevance, in-vitro EZH2 inhibition significantly restored MHC-I and MHC-II gene expression as well as protein expression in EZH2-mutated human DLBCL cells, but not EZH2 wild type tumor cells. Conclusion: Our findings provide important implications for understanding the cancer biology underlying acquired loss of MHC expression. The restoration of MHC expression by EZH2 inhibitors suggests a novel approach of epigenetically enhancing tumor recognition and eradication in combination with immune therapies. Disclosures Sehn: Abbvie: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Connors:Roche Canada: Research Funding; Takeda: Research Funding; Merck: Research Funding; F Hoffmann-La Roche: Research Funding; Cephalon: Research Funding; Seattle Genetics: Honoraria, Research Funding; Amgen: Research Funding; Bayer Healthcare: Research Funding; Bristol Myers-Squibb: Research Funding; Lilly: Research Funding; NanoString Technologies: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding; Genentech: Research Funding. Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Roche: Research Funding; Janssen: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding.

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Influence of CD27- CD28- T-Cells on the Therapeutic Outcome in Adult Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma after CART-Infusion

Blood ◽

10.1182/blood-2019-126191 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1935-1935 ◽

Cited By ~ 2

Author(s):

Nina Worel ◽

Katharina Pfistershammer ◽

Winfried Pickl ◽

Marion Heinz ◽

Martina Schlager ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Peripheral Blood ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Expression Status ◽

Large B Cell

Background: Therapies involving adoptive transfer of chimeric antigen receptor-modified T-cells (CARTs) targeting CD19-expressing B-cells have shown remarkable efficacy in patients with relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL). We hypothesized, that a higher fraction of more differentiated, senescent and exhausted T-cells affects negatively ex vivo T-cell expansion in the manufacturing phase of CARTs and the in vivo function of CARTs after infusion. Of note, naïve CD4+ T-cells were shown to uniformly express the co-stimulatory receptors CD27 and CD28, while repeated cycles of activation led to their progressive loss (van Leewen et al, J. Immunol, 2004), accompanied by telomere erosion and replicative senescence (van Baarle et al., Immunol. Lett. 2005; Effros RB et al., Dev Comp Immunol 1997). CD28 expression on T-cells within the tumor environment was shown to be a decisive factor for the efficacy of anti-PD-1 therapy (Kamphorst RO et al, Science 2017). We have therefore analyzed the CD27 and CD28 expression status of CD3+ T-cells from the peripheral blood and apheresis products of adult r/r DLBCL patients at the day of leukapheresis. Methods: Peripheral blood and apheresis samples of 22 consecutive r/r DLBCL patients scheduled for CART therapy were analyzed by flow cytometry to assess their CD27 and CD28 expression status on CD3+ T-cells. Samples were stained with fluorochrome conjugated antibodies (anti-CD3 PerCP, anti-CD27 FITC, CD28 PE, anti-CD4 FITC, BioLegend) and analyzed using a FACScalibur flow cytometer supported by CELLQUEST software (Becton Dickinson, BD, Palo Alto, CA). Results: To rule out an apheresis-related bias within cell populations we analyzed peripheral blood and apheresis samples for each patient. No differences in the distribution of CD27-, CD28-, CD27-/CD28- or CD27+/CD28+ T-cells between peripheral blood and apheresis product were detected. Mean CD3+ cell count in blood samples before apheresis was 624±399/µl (range, 75-1853cells/µL) with only about 25% of the patients presenting with CD3+ cell counts within the normal range (690-3320/µL) and 70% of the patients showed an inverse CD4/CD8 ratio (<1.0). We observed a considerable heterogeneity within the T-memory cell compartment. In all samples high percentages of CD27- (39.7±18.1%), CD28- (40.2±19.0%) and CD27-/CD28- (30.7±19.8%) T-cells were seen when compared to healthy controls (CD27-: 15.2±9.9%; CD28-: 18.4±8.9%; CD27-/CD28-: 9.9±8.7%; p<0.05). Patients receiving CARTs were embedded within three different CART-trials (NCT02445248, NCT03630159, NCT03484702). A significantly lower frequency of CD27-/CD28-T-cells (20.8±18.8 vs 42.4±13.7%; p=0.045 Mann Whitney U) was found in responders (n=8) compared to non-responders (n=5; Figure 1a). We also noticed a higher frequency of CD27+/CD28+ T-cells (57.5±22.7% vs 44.3±11.7%; Figure 1b) and a trend towards a normal (>1.0) CD4/CD8 ratio (Figure 1c) in responding patients. Furthermore, we did not observe significant differences in CD27-and CD28- expression in samples derived from patients who died prior to receiving CART therapy (n=6) when compared to patients responding or progressing after CART therapy. Three patients have not been infused yet. Conclusion: We demonstrate in this small patient cohort that individuals with a lower percentage of more differentiated, senescent or exhausted T-cells are more likely to respond to CART therapy. Our observation underscores the importance of T-cells with normal replicative capacity in the apheresis material for consecutive CART production to achive therapeutic success. Further analysis is needed to determine the effect of cytotoxic pretreatment on the fraction of immunosenescent/exhausted T-cells. However, to confirm our findings additional investigations including the T-cell status of manufactured cells are warranted. Disclosures Worel: Sanofi Genzyme, Malinckrodt Therakos: Speakers Bureau; Sanofi Genzyme, Malinckrodt Therakos: Research Funding; Jazz, Sanofi, Celgene, Novartis, Malinckrodt Therakos: Honoraria. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria. Hopfinger:Celgene, Gilead, GlaxoSmithKline, Janssen, Novartis, Roche, Takeda,: Honoraria; Gilead: Research Funding.

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Use of immune repertoire sequencing to resolve discordant microscopic and immunochemical findings in a case of T cell-rich large B cell lymphoma in a young dog

BMC Veterinary Research ◽

10.1186/s12917-021-02783-3 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Gary Kwok Cheong Lee ◽

Dorothee Bienzle ◽

Stefan Matthias Keller ◽

Mei-Hua Hwang ◽

Nikos Darzentas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Immune Repertoire ◽

Large B Cell Lymphoma ◽

Repertoire Sequencing ◽

Reactive Lymphocytes ◽

Large B Cell

Abstract Background Lymphocytic neoplasms with frequent reactive lymphocytes are uncommonly reported in dogs, and can pose a diagnostic challenge. Different diagnostic modalities such as cytology, flow cytometry, histopathology, immunohistochemistry, and clonality testing, are sometimes required for a diagnosis. This report illustrates the value of using a multi-modal diagnostic approach to decipher a complex lymphocytic tumor, and introduces immune repertoire sequencing as a diagnostic adjunct. Case presentation A 10-month-old Great Dane was referred for marked ascites. Cytologic analysis of abdominal fluid and hepatic aspirates revealed a mixed lymphocyte population including numerous large lymphocytes, yielding a diagnosis of lymphoma. Flow cytometrically, abdominal fluid lymphocytes were highly positive for CD4, CD5, CD18, CD45, and MHC II, consistent with T cell lymphoma. Due to a rapidly deteriorating clinical condition, the dog was euthanized. Post mortem histologic evaluation showed effacement of the liver by aggregates of B cells surrounded by T cells, suggestive of hepatic T cell-rich large B cell lymphoma. Immune repertoire sequencing confirmed the presence of clonal B cells in the liver but not the abdominal fluid, whereas reactive T cells with shared, polyclonal immune repertoires were found in both locations. Conclusions T cell-rich large B cell lymphoma is a rare neoplasm in dogs that may be challenging to diagnose and classify due to mixed lymphocyte populations. In this case, the results of histopathology, immunohistochemistry and immune repertoire sequencing were most consistent with a hepatic B cell neoplasm and reactive T cells exfoliating into the abdominal fluid. Immune repertoire sequencing was helpful in delineating neoplastic from reactive lymphocytes and characterizing repertoire overlap in both compartments. The potential pitfalls of equating atypical cytomorphology and monotypic marker expression in neoplasia are highlighted.

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