ALBERT

Description: Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Description: Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

Description: Lymphomas represent nearly 70 distinct diseases with unique clinical presentations, therapeutic responses and underlying biology. There is a pressing shortage of publically available cell line and in vivo models of nearly all of these diseases, which has severely hampered efforts to understand and target their biology. To address this issue, we have established a repository of patient-derived xenografts (PDX) of lymphomas by engrafting human tumors into immunodeficient NOD/SCID/IL2rgnull (NSG) mice. These lymphomas, along with a spectrum of other PDXs of hematologic malignancies, are available to collaborators through the online portal PRoXe (Public Repository of Xenografts) at http://PRoXe.org. Blood and bone marrow specimens involved with tumor are injected by tail vein (IV) injection. Lymph node and extranodal biopsy specimens are implanted under the renal capsule as a 1x1x2mm tumor seed (renal), which maintains the in situ microarchitecture. A full description of xenografted lymphomas is included in the Table. Table 1.DiseaseType of implant# in 1st passage# in 2nd passage or higherT-cell prolymphocytic leukemiaIV1Angioimmunoblastic T-cell lymphomaIV11Mantle cell lymphomaIV12Double-hit DLBCLIV2Sézary SyndromeIV1Adult T-cell Leukemia/LymphomaIV1Diffuse large B cell lymphomaIV2Diffuse large B cell lymphomarenal2Marginal zone lymphomarenal11NK/T-cell lymphomarenal1Peripheral T-cell lymphoma-NOSrenal1Breast implant-associated anaplastic large cell lymphomarenal1 Engrafted PDXs have been extensively characterized by immunohistochemistry, flow cytometry, transcriptome sequencing and targeted DNA sequencing. Flow cytometric analysis of patient tumors and their respective xenografts consistently revealed highly concordant immunophenotypes compared to the original tumors. Similarly, immunohistochemistry of involved tissues confirmed retention of tumor immunophenotypes, architecture, and even tissue tropism in the PDXs. Examples include a Sézary syndrome PDX that was injected by tail vein and trafficked to spleen, bone marrow, blood and skin, a diffuse large B-cell lymphoma (DLBCL) PDX that infiltrated the CNS, and a second DLBCL PDX that was implanted into the renal capsule of the left kidney and progressed within 8 weeks to bilateral renal involvement. Other notable models include a breast implant-associated, ALK-negative anaplastic large cell lymphoma implanted under the renal capsule that metastasized to the liver and spleen while uniformly retaining CD30 positivity. Two double-hit lymphoma (DHL) PDXs maintained their CD20-negative phenotype through serial passage to P1. A peripheral T-cell lymphoma-NOS (PTCL) specimen implanted under the renal capsule engrafted in the spleen, with a notable admixture of nonmalignant T cells and scattered EBV-positive B cells. T-cell receptor gene rearrangement PCR performed on this PTCL demonstrated an identical rearrangement pattern in the primary tumor and the PDX. Luciferized mantle cell lymphoma and DHL PDXs clearly home to bone marrow, lymph nodes, spleen, and liver as early as two weeks after injection. These findings support the utility of these PDX lines as in vivo models that more accurately recapitulate the human disease than commonly used subcutaneous cell line models. In addition to generating PDXs that remain faithful to their source tumors, we have witnessed interesting examples of in vivo histologic transformation, opening the door to studies of disease progression. One primary follicular lymphoma specimen injected into a cohort of mice transformed to DLBCL in one mouse and a lymphoblastic lymphoma-like disease in another mouse, as confirmed by IHC and flow cytometry. Further xenografting of primary tumors is underway with the goal of establishing a large repository of lymphoma PDXs useful for biologic interrogation and preclinical trials. Disclosures Davids: Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees.

Description: Flow cytometry for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) has been widely used in pediatric patients to quantify therapeutic response and to assess the risk of relapse. Flow cytometry for MRD provides roughly the same level of sensitivity (0.01%) as molecular methods but at lower cost and with faster turnaround time. MRD assessment in ALL currently requires an evaluation of 20 or more parameters divided among multiple tubes. In part due to the assessment complexity, the use of flow cytometry for MRD detection in adult ALL patients has been relatively limited. We developed a 6-color, single-tube, flow cytometry assay to detect MRD in bone marrow (BM) aspirate specimens from adult ALL patients. The 73 patients included 52 patients with B-ALL (71%), 19 patients with T-ALL (26%) and 2 patients with T/myeloid leukemia (3%) and were treated with one of several standard chemotherapeutic regimens or targeted therapies. Patients were tested for MRD by flow cytometry after induction or re-induction therapy and serially thereafter. The 6-marker MRD panel was customized for each patient based on the 18-20-marker diagnostic immunophenotype. Sixty-three percent of B-ALL patients (n=33) had lymphoblasts with an aberrant immunophenotype; expression of a myeloid marker (e.g., CD13, CD15 or CD33) was the most common aberrancy. The remaining 37% of B-ALL patients (n=19) had disease with a hematogone immunophenotype, which comprised surface expression of CD10, CD19, CD20, CD34, CD38 and CD45; in the majority of these cases, leukemic cells were distinguishable from normal hematogones based on the intensity of surface marker expression. Forty-seven percent of T-ALL patients (n=9) had an aberrant immunophenotype, most often characterized by CD33 expression. One-hundred forty-six consecutive specimens analyzed for MRD by flow cytometry were classified as positive (23%), negative (72%) or uncertain (5%). Of the 34 samples classified as positive, 14 (41%) showed morphologic (i.e., BM aspirate or biopsy) evidence of disease; nineteen (65%) samples did not show morphologic evidence of disease and 1 sample did not have a concurrent morphologic assessment. Of the 105 samples classified as negative by flow cytometry, 103 (98%) were also negative by morphology and 1 sample did not have a concurrent morphologic assessment. One sample that was negative by flow cytometry had morphologic evidence of disease in the biopsy (10-20% blasts) but not the aspirate, suggesting that aspirate sampling artifact was responsible for the discrepancy. None of the 7 samples classified as uncertain by flow cytometry had morphologic evidence of disease; five out of 7 uncertain classifications were in B-ALL patients with hematogone immunophenotypes. Overall, MRD flow cytometry showed 86% concordance with the results of morphologic assessment. We evaluated outcomes in all patients with negative morphologic results and any positive MRD flow cytometry result(s). Of the 73 patients in this study, 61 had morphology-negative results that were either MRD-negative (n=45) or MRD-positive (n=16). Patients in this group were at various points of treatment post-induction or re-induction. Four out of 45 patients (9%) with MRD-negative results relapsed during a median follow-up period of 22 months, and 8 out of 16 patients (50%) with an MRD-positive result relapsed during a median follow-up period of 15 months (odds ratio for relapse 10.3, 95% confidence interval 2.5-42.4, P=0.001). In addition, relapse-related and overall mortality (Figure 1) were higher in patients with MRD-positive results (P=0.0023 and P=0.0016, respectively, by the log-rank test). In summary, we present a simplified, single-tube, flow cytometry assay that can be used to detect MRD in adult ALL at relatively low cost with rapid turnaround time; our approach was applicable to cases with either hematogone or aberrant immunophenotype, yielding a definitive result in 95% of cases. Notably, the presence of MRD was associated with relapse and mortality, suggesting that our method of MRD assessment could be used to guide treatment of adult ALL. Further analysis of the correlations between MRD results, clinical management and patient outcomes is ongoing. Disclosures: No relevant conflicts of interest to declare.

Description: To expedite the translation of biologic discoveries into novel therapeutics, there is a pressing need for panels of in vivo models that capture the molecular complexity of human disease. While traditional cell lines and genetically engineered mouse models are useful tools, they are insufficient to assess the broad diversity of human tumors within a context that recapitulates in situ biology. Patient-derived xenografts (PDXs), generated by transplanting primary human tumor cells into immune-deficient NOD.Cg-Prkdcscid/Il2rgtm1Wjl/SzJ (NSG) mice, surmount some of the limitations of these traditional platforms and have been increasingly utilized as tools for preclinical investigation. However, the infrastructure required to generate, bank, and characterize PDX models limits their availability to only a few investigators. To address this issue, we established a repository of PDX models of leukemia and lymphoma, which we have named the Public Repository of Xenografts (PRoXe). At the time of this writing, PRoXe contains 213 independent lines that have been passaged through mice once (P0), 123 of which have been repassaged in a second generation (P1) or further repassaged. The repository encompasses AML, B- and T-ALL, and B- and T-cell non-Hodgkin lymphoma (NHL) across a range of cytogenetic- and molecularly-defined subtypes (Table 1). PRoXe is extensively annotated with patient-level information, including demographics, phase of treatment, prior therapies, tumor immunophenotye, cytogenetics, and molecular diagnostics. PDX lines available for distribution are characterized by immunophenotyping, whole transcriptome sequencing (RNAseq), and targeted exon sequencing of ~300 genes. To confirm fidelity of engrafted tumors to their corresponding clinical samples, lymphomas were morphologically assessed in P0 mice by H&E and, when pathologic adjudication was required, by immunohistochemistry. Xenografted leukemias were compared to their original tumors immunophenotypically. Unsupervised hierarchical clustering was performed on 132 of these lines based on transcriptome sequencing data and demonstrated 94% concordance between classification of the PDX lines by RNA expression and by the annotated clinical-pathologic diagnoses. Discordant cases highlighted unusual variants, such as B-ALL with aberrant expression of myeloid markers and a follicular lymphoma that underwent blastic transformation in the mouse. Multiple lines have been luciferized and confirmed to home to bone marrow, spleen, and liver. Existing lines from PRoXe have already been shared with more than ten academic laboratories and multiple industrial partners. All of the data referenced here are freely available through a customized web-based search application at http://proxe.org, and lines can be requested for in vitro or in vivo experiments. We are actively expanding the size of PRoXe to allow for large pre-clinical studies that are powered to detect differences across genetically defined subsets. Thus, we are happy to host additional lines from outside investigators on PRoXe and thereby expand the availability of these valuable reagents. Finally, we have made the source code for PRoXe (in R Shiny) open-access, so that other investigators can establish their own portals. Table 1. WHO diagnostic entities encompassed within PRoXe at P1 or later, or P0 or later for B-ALLs. WHO Classification - number of lines per diagnostic entity AML, Other Myeloid, and Ambiguous Lineage [n=32] ALL [n=107] AML - recurrent gene mutations 6 B-ALL - NOS 44 AML - MDS-related changes 5 B-ALL - MLL-rearranged 11 AML - NOS 4 B-ALL - BCR-ABL 10 AML - MLLT3-MLL 2 B-ALL - hyperdiploidy 9 Acute myelomonocytic leukemia 1 B-ALL - TEL-AML1 8 Acute monocytic leukemia 1 B-ALL - E2A-PBX1 3 AML unable to classify 2 B-ALL unable to classify 1 Blastic plasmacytoid dendritic cell neoplasm 8 T-ALL 21 Mixed phenotype, MLL rearranged 1 B/myeloid acute leukemia 1 Myelodysplastic syndrome 1 Mature B cell neoplasms[n=11] Mature T and NK cell neoplasms [n=4] DBLCL - NOS 4 Angioimmunoblastic T-cell lymphoma 1 Mantle cell lymphoma 3 Adult T-cell leukemia/lymphoma 1 Extranodal marginal zone lymphoma 1 Extranodal NK/T-cell lymphoma 1 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL 3 SŽzary syndrome 1 Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Etchin:Karyopharm: Research Funding. Lane:Stemline Therapeutics, Inc.: Research Funding. Stone:Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Merck: Consultancy; Karyopharm: Consultancy; Roche/Genetech: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy.

Description: Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p

Description: Background: Despite mycosis fungoides (MF) and Sezary syndrome (SS) comprising the most common forms of cutaneous T cell lymphoma, the pathophysiology underlying these disorders remains poorly understood. Consequently, current prognostic guidelines based on disease spread exhibit wide variations in clinical outcome within each stage, underscoring an urgent need for novel approaches to MF/SS disease evaluation. A growing body of research suggests that systemic immune dysregulation represents an early, cardinal feature of MF/SS. We hypothesized that tracking this immune dysfunction in conjunction with disease spread may generate important pathophysiologic and prognostic information for patients. We focused on myeloid-derived suppressor cells (MDSCs), a recently discovered population of immunosuppressive innate immune cells related to neutrophils and monocytes, because their expansion in numerous solid tumor settings have correlated reliably with poor patient outcomes. Whether MF/SS augments circulating MDSC abundance remains unexplored, prompting us to evaluate whether this could serve as a marker for disease progression and treatment response. Methods: We used multiparametric flow cytometry to analyze the frequency and immunophenotype of MDSCs from the peripheral blood of 15 healthy donors and 30 patients with MF/SS. Patients at varying stages of MF/SS disease progression and treatment were included in the study. We defined granulocytic MDSCs (G-MDSCs) as cells positive for CD15, CD11b, and the recently discovered surface marker LOX-1, and negative for CD14. Monocytic MDSCs (M-MDSCs) were defined as cells positive for CD14 and CD11b, negative for CD15, and low/negative for HLA-DR. Each patient sample also underwent flow cytometry evaluating for circulating neoplastic T cells. These results were correlated with each participant's other hematologic parameters and clinical information through manual chart review. Results: We found that healthy donors harbored no quantifiable circulating MDSCs of either monocytic or granulocytic lineage, a result in keeping with previous studies. In contrast, MF/SS patients exhibited robust, statistically significant increases in the frequencies of both G-MDSCs and, to a lesser extent, M-MDSCs. G-MDSCs exceeded 20% of all CD15-positive cells in some patients. When patients were stratified by MF/SS clinical stage, those with more advanced disease displayed significantly higher G-MDSC abundance than early-stage patients. G-MDSC frequency was positively correlated with circulating CD4+ CD26- T cell counts often used in evaluating Sezary syndrome (R2 = 0.498; p 〈 0.0001). However, patients with early, skin-restricted disease also showed statistically significant increases in circulating G-MDSCs compared to healthy controls. This suggested that G-MDSC expansion may serve as a sensitive, blood-based disease marker even in the absence of systemic involvement by neoplasia. Patients who underwent recent treatment exhibited variable G-MDSC counts in the peripheral blood that were lower than in similar untreated patients on average. Serial measurements for two patients enrolled in a clinical trial for dual phosphoinositide 3-kinase and histone deacetylase inhibition revealed that G-MDSC frequencies markedly decreased over the course of treatment, mirroring the decrements of aberrant T cells circulating in the blood. Conclusion: These findings provide clear evidence of G-MDSC expansion in the peripheral blood of MF/SS patients that begins in early/locally restricted disease, grows with disease progression, and responds to systemic therapy. Such immunometric assays may illuminate a novel source of staging and prognostic information and may permit less invasive disease monitoring than current methods require. Disclosures No relevant conflicts of interest to declare.