Dorfman, David M.

Context.— In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program—PCNEO—to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. Objective.— To report the results from the first 4 years of the PCNEO program. Design.— Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. Results.— A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%–100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%–97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic κ and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%–5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91%–96% of participants). Conclusions.— Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm.

Abstract Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 1545 Introduction: The diagnosis of peripheral T-cell lymphoma (PTCL) is difficult and accurate diagnosis and subclassification relies on correlating histologic, immunophenotypic, molecular genetic, and clinical data. Evidence-based guidelines for the appropriate diagnostic work-up of PTCL are lacking. The objective of this study was to evaluate a large series of PTCLs by experienced hematopathologists with a tiered approach to immunohistochemistry (IHC) and molecular genetic characterization to document overall diagnostic accuracy and clinical relevance using this approach. Methods: 7 experienced hematopathologists from 5 institutions reviewed 374 cases of peripheral T and NK cell lymphomas (referred to collectively as PTCL). 6 cases of cutaneous T-cell lymphoma were excluded after final review in addition to 29 non-PTCL cases submitted as control cases to mimic diagnostic practice during the review. Cases received tier 0, 1 and 2 diagnoses by 3 independent pathologists, based on review of hematoxylin and eosin (HE) stain with basic demographic data, panel 1 IHC (CD3, CD5, CD10, CD20, CD21, CD30, CD45, PAX5), and panel 2 IHC (CD2, CD4, CD7, CD8, CD23, PD1, CD56, EBER, ALK, TIA1, TCRg, TCRbF1), respectively. A tier 2b diagnosis was then rendered after gene rearrangement data were available. A final consensus diagnosis was rendered after discussion of each case by the 3 reviewers with all available clinical data. Overall survival (OS) was assessed using Kaplan-Meier (KM) curves and Cox proportional hazards models. Results: 1122 individual diagnoses leading to 339 final consensus PTCL diagnoses were rendered. 341 (91%) cases had complete phenotypic data and 241 had gene rearrangement data. There was no bias of missing data according to final diagnosis subtype. Reviewer diagnoses using specific WHO subclassification were 16.3%, 36.9%, 82.7%, and 85.9% for tier 0, 1, 2, and 2b, demonstrating a significant increase in diagnostic certainty after a complete IHC panel. Gene rearrangement only contributed to a change in diagnosis in 51/650 (8%) individual reviews. Across all 374 cases, a small number of cases (n=28, 7.5%) showed no agreement among the 3 independent reviewers after tier 2b and required debate. These generally represented refinement of the subclassification of a PTCL. The most common disagreements were between PTCL, not otherwise specified (nos) vs. unclassifiable T-cell lymphoma, and PTCL, nos vs. angioimmunoblastic T-cell lymphoma (AITL). Currently, OS data was available in 198 cases and 52% have died (median age follow-up of 15 months for those still alive, range 0–160 mo). Figure 1 shows the KM survival curves for types with more than 10 cases. Of note, unclassifiable PTCL cases had poor OS, comparable to PTCL nos; a trend was seen for CD30+ ALK- ALCL to have a longer OS compared to PTCL nos (HR=0.5, 95% CI: 0.21–1.19, P=.09). EBER positivity in tumor cells was associated with poor OS in the cohort of all PTCL patients (HR=2.32, 95% CI: 1.28–4.23, p=0.006, Figure 2); the association was similar when nasal NK/T-cell lymphoma cases were excluded (HR=2.50, 95% CI: 1.00–6.26, p=0.05). Trends for poor prognosis were also seen for TIA-1 expression in PTCL, nos (HR=1.9, 95% CI 0.91–3.96, P=.09) and PD1 expression in AITL (HR=6.25, 95% CI 0.85–46.09, P=.07). Clinical data collection is still ongoing and will be updated. Conclusions: We demonstrate the diagnostic accuracy among experienced hematopathologists of a defined IHC panel, showing an overall ability to reach consensus diagnosis of 93% in PTCL cases. The resulting consensus diagnostic subtypes showed expected outcomes relative to other large series of PTCLs, and EBER positivity is a poor prognostic marker among T and NK cell lymphomas. A tiered approach to IHC is recommended when a PTCL is under differential diagnostic consideration since the first tier can often resolve the main question of whether a lesion is reactive or lymphoma. This can lead to more efficient use of the expanded tier 2 panel that will enable diagnosis and specific subclassification of PTCL. Gene rearrangement studies are not required in the great majority of cases. This evidence-based approach to the diagnosis of PTCL should inform practicing pathologists, clinical trial design, and policy makers regarding required ancillary studies in this group of diseases. Disclosures: Hsi: Allos: Research Funding. Said:Allos: Research Funding. Macon:Allos: Research Funding. Rodig:Allos: Research Funding. Gascoyne:Seattle Genetics: Research Funding. Ondrejka:Allos: Research Funding. Dorfman:Allos: Research Funding. Maurer:Allos: Research Funding. Dogan:Allos: Research Funding.