Malignant Lymphocytes Research Articles

Abstract 2943: MYC functions as a master switch for natural killer cell-mediated immune surveillance of lymphoid malignancies

Abstract The MYC oncogene drives the pathogenesis of many hematopoietic malignancies, including Burkitt’s lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). These malignancies are often “oncogene-addicted” to MYC. Using mass cytometry (CyTOF), we demonstrate that MYC-addicted T-ALL excludes specific immune subsets from the tumor microenvironment implicated in immune surveillance, including natural killer (NK) cells. MYC inhibition clears malignant lymphocytes from the spleen and restores the normal splenic NK composition. Concordantly, peripheral blood of T-ALL patients have reduced percentages of activated NK cells as compared to healthy individuals. We show that MYC excludes activated NK cells from sites of lymphomagenesis by suppressing ERK1/2-STAT1/2-Type I IFN signaling, in both murine and human lymphomas. Furthermore, MYC-associated BL patients with higher than median expression of STAT1/2, and cytotoxic NK cell genes, PRF1 and Granzymes, have favorable clinical outcomes. Hence, oncogenic MYC appears to causally and reversibly suppress NK-mediated immune surveillance during lymphomagenesis. Citation Format: Srividya Swaminathan, Adriane Mosley, Crista Horton, Daniel F. Liefwalker, Anja Deutzmann, Renumathy Dhanasekaran, Arvin Gouw, Andrew Gentles, Martin Eilers, Holden T. Maecker, Dean W. Felsher. MYC functions as a master switch for natural killer cell-mediated immune surveillance of lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2943. doi:10.1158/1538-7445.AM2017-2943

Assessment of HTLV-1 proviral load, LAT, BIM, c-FOS and RAD51 gene expression in adult T cell leukemia/lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is a life-threatening malignancy of HTLV-1 infected Th lymphocytes. In the present study host-virus interactions were investigated by assessment of HTLV-1 proviral load (PVL) and host gene expression. A cross-sectional study was carried out on 18 ATLL, 10 HAM/TSP patients and 18 HTLV-1 asymptomatic carriers (ACs). DNA and mRNA of the peripheral blood mononuclear cells were extracted for PVL and LAT, BIM, c-FOS and RAD51 gene expression measurement using qRT-PCR. The mean PVL in ATLL patients was 11,430±3770copies/104 which was statistically higher than ACs, 530±119copies/104, (p<0.001). The expression of BIM, and c-FOS in ATLL patients were higher than HTLV-1 ACs; however, there were no statistically significant differences. The expression of RAD51 as an essential player on DNA repair showed around 160 times increase in ATLL group (166±95) compared to ACs (1.04±0.34) which is statistically significant (p<0.001). Interestingly, there was a positive correlation between RAD51 expression and HTLV-PVL. The expression of LAT as a central adaptor in TCR signaling interestingly was around 36 times higher in ATLL group than ACs (ATLL; 41.33±19.91 vs. ACs; 1.15±0.22, p<0.001). This finding showed that TCR signaling pathway mainly provides the growth factors for transformed cells. Furthermore, the overexpression of RAD51 which has been induced in HTLV-1 infected cells as a consequence of virus replication is not able to overcome the DNA damage toward cell transformation.

Circulating tumour DNA reflects treatment response and clonal evolution in chronic lymphocytic leukaemia

Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter’s syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.

RAG1/2 induces genomic insertions by mobilizing DNA into RAG1/2-independent breaks.

The RAG recombinase (RAG1/2) plays an essential role in adaptive immunity by mediating V(D)J recombination in developing lymphocytes. In contrast, aberrant RAG1/2 activity promotes lymphocyte malignancies by causing chromosomal translocations and DNA deletions at cancer genes. RAG1/2 can also induce genomic DNA insertions by transposition and trans-V(D)J recombination, but only few such putative events have been documented in vivo. We used next-generation sequencing techniques to examine chromosomal rearrangements in primary murine B cells and discovered that RAG1/2 causes aberrant insertions by releasing cleaved antibody gene fragments that subsequently reintegrate into DNA breaks induced on a heterologous chromosome. We confirmed that RAG1/2 also mobilizes genomic DNA into independent physiological breaks by identifying similar insertions in human lymphoma and leukemia. Our findings reveal a novel RAG1/2-mediated insertion pathway distinct from DNA transposition and trans-V(D)J recombination that destabilizes the genome and shares features with reported oncogenic DNA insertions.

Plasma matrix metalloprotease 9 correlates with blood lymphocytosis, leukemic cell invasiveness, and prognosis in B-cell chronic lymphocytic leukemia

The complex biology underlying chronic lymphocytic leukemia cell migration and tissue invasiveness is not yet completely understood and might provide novel predictive markers and therapeutic targets. A total of 36 patients out of treatment from at least 3 months were enrolled and followed up for a median period of 44.2 months (range: 4.4-99.2). Matrix metalloprotease 9 and tissue inhibitor of metalloproteases 1 plasma levels and production/release from lymphoid cells were measured by zymography and enzyme-linked immunosorbent assay (ELISA) analysis. Malignant and normal lymphocyte mobility and matrix-degradation capability were studied using a Boyden chamber system, with and without autologous plasma. Free matrix metalloprotease 9 plasma levels were related with blood lymphocytosis, especially in more advanced stages (p = 0.003), and higher concentrations were associated with an increased disease progression risk (hazard ratio = 9.0, 95% confidence interval = 1.5-13.8). Leukemic cells expressed and secreted very little matrix metalloprotease 9. On the contrary, normal lymphocytes derived from the same leukemic patients showed matrix metalloprotease 9 intracellular levels that were lower in subjects with higher blood lymphocytosis (p = 0.024) and more advanced stages (p = 0.03); the released quantities were inversely associated with matrix metalloprotease 9 plasma concentrations (p = 0.035). Leukemic cells had a reduced spontaneous mobility and matrix-degradation capability that were stimulated by autologous plasma (p = 0.001) and normal lymphocytes (p = 0.005), respectively. Matrix metalloprotease 9 affected cell invasiveness depending on concentration and disease stage. In conclusion, chronic lymphocytic leukemia cells have a reduced mobility, matrix-degradation capability, and matrix metalloprotease 9 production compared to their own autologous normal lymphocytes. They are exposed to matrix metalloprotease 9 of prevalently systemic origin whose higher levels are associated with both leukemic and normal lymphocyte accumulation in the peripheral blood and have a negative prognostic value.

CD4 T-cell cytokines synergize to induce proliferation of malignant and nonmalignant innate intraepithelial lymphocytes

Refractory celiac disease type II (RCDII) is a severe complication of celiac disease (CD) characterized by the presence of an enlarged clonal population of innate intraepithelial lymphocytes (IELs) lacking classical B-, T-, and natural killer (NK)-cell lineage markers (Lin-IELs) in the duodenum. In ∼50% of patients with RCDII, these Lin-IELs develop into a lymphoma for which no effective treatment is available. Current evidence indicates that the survival and expansion of these malignant Lin-IELs is driven by epithelial cell-derived IL-15. Like CD, RCDII is strongly associated with HLA-DQ2, suggesting the involvement of HLA-DQ2-restricted gluten-specific CD4+ T cells. We now show that gluten-specific CD4+ T cells isolated from CD duodenal biopsy specimens produce cytokines able to trigger proliferation of malignant Lin-IEL lines as powerfully as IL-15. Furthermore, we identify TNF, IL-2, and IL-21 as CD4+ T-cell cytokines that synergistically mediate this effect. Like IL-15, these cytokines were found to increase the phosphorylation of STAT5 and Akt and transcription of antiapoptotic mediator bcl-xL Several small-molecule inhibitors targeting the JAK/STAT pathway blocked proliferation elicited by IL-2 and IL-15, but only an inhibitor targeting the PI3K/Akt/mTOR pathway blocked proliferation induced by IL-15 as well as the CD4+ T-cell cytokines. Confirming and extending these findings, TNF, IL-2, and IL-21 also synergistically triggered the proliferation of freshly isolated Lin-IELs and CD3-CD56+ IELs (NK-IELs) from RCDII as well as non-RCDII duodenal biopsy specimens. These data provide evidence implicating CD4+ T-cell cytokines in the pathogenesis of RCDII. More broadly, they suggest that adaptive immune responses can contribute to innate IEL activation during mucosal inflammation.

CpG-ODN Stimulation in Population Screening Monoclonal B-Cell Lymphocytosis

Synthetic CpG-oligodeoxynucleotides (CpG-ODN) induce proliferation in normal and malignant lymphocytes B (LyB). This effect is widely exploited in CLL conventional chromosome banding analysis (CBA), which has become reliable only after the cultivation of CLL LyB using CpG-ODN stimulation in combination with IL-2. Monoclonal B-cell lymphocytosis (MBL) differs from CLL mainly in clone size. Only cytogenetic data on recurrent chromosomal aberrations analyzed by fluorescence in situ hybridization (FISH) are available for population screening MBL (sMBL). In sMBL the clone of malignant LyB is typically below 10/µL. We compared CpGODN stimulation in healthy donors and in individuals with sMBL. LyB and MBL LyB count were determined by flow cytometry in 15 samples from healthy subjects and 12 MBL cases. Mitotic indices were determined and CBA was done after cultivation of samples by CpG-ODN + IL2. In MBL samples, FISH analysis was performed on isolated LyB. MBL LyB clones in sMBL cases presented less than 1% of WBC and up to 33% of LyB. The MBL group was therefore compared to the group of healthy donors. Although normal and MBL group did not differ in WBC, overall LyB, and normal LyB count, a significantly higher mitotic index was observed in MBL samples (p = 0.0139). We were able to accomplish CBA in all samples which revealed a normal karyotype in all but one case. In this particular sMBL case FISH performed on isolated LyB showed 5% trisomy 12 which was later confirmed by CBA on CpG stimulated blood sample in 15% of metaphases. Our study, which was done on MBL cases obtained by population screening, confirmed that CpGODN preferentially induced proliferation in MBL LyB over normal LyB. Therefore, CBA can also be successfully accomplished in sMBL and can be used to additionally confirm clonality as well as to improve sensitivity of FISH analysis. Due to coexistence of comparable size of normal and malignant LyB, MBL can serve as a model for exvivo studying of LyB stimulation by CpG-ODN.

Tracking B-Cell Repertoires and Clonal Histories in Normal and Malignant Lymphocytes.

Methods for tracking B-cell repertoires and clonal history in normal and malignant B-cells based on immunoglobulin variable region (IGV) gene analysis have developed rapidly with the advent of massive parallel next-generation sequencing (mpNGS) protocols. mpNGS permits a depth of analysis of IGV genes not hitherto feasible, and presents challenges of bioinformatics analysis, which can be readily met by current pipelines. This strategy offers a potential resolution of B-cell usage at a depth that may capture fully the natural state, in a given biological setting. Conventional methods based on RT-PCR amplification and Sanger sequencing are also available where mpNGS is not accessible. Each method offers distinct advantages. Conventional methods for IGV gene sequencing are readily adaptable to most laboratories and provide an ease of analysis to capture salient features of B-cell use. This chapter describes two methods in detail for analysis of IGV genes, mpNGS and conventional RT-PCR with Sanger sequencing.

New Principles of Treatments in Malignant Hematological Diseases, as Acute and Chronic Lymphocytic Leukemia or Multiple Myeloma

Aim of this study is to present the latest researches in the field of molecular medicine, in terms of treatments in malignant hemopathies, emerged from the P53 gene deletion in human lymphoma genome. Method: In recent years proved that the best techniques in the investigation of malignant lymphocytes are the Flow Cytometry, Elisa, ICT and Fluorescence in Situ Hybridization (FISH). This method is used as an alternative to chromosomal banding, a conventional application in molecular medicine. Discussion: Recent, endogenous somatic gene therapy research is a basic of trial clinical and therapeutic trial. The DNA is used to treat a disease arising as a result of mutations in chromosomal regions. In the past few years, this method has been included in the treatment of CLL, acute lymphocytic leukemia, [ALL], or multiple myeloma [MM]. Conclusion: The frequencies of P53 gene mutations and deletion in CLL can be categorized as individual biomarkers in proteomic and genomic profile for this type of leukemia that can be implemented in targeted patient treatment of personalized medicine.

Foundation-OneR-Heme Next Generation Sequencing of Hairy Cell Leukemia Variant Lymphocytes

Hairy Cell Leukemia variant (HCL-V) is a rare disorder of small, mature, B-cell lymphocytes, and is characterized clinically by splenomegaly, an elevated white blood cell (WBC) count, and a hypercellular bone marrow. It accounts for 0.4% of chronic lymphoid and 10% of all cases of hairy cell leukemia (HCL) cases, respectively, and is now considered by the World Health Organization (WHO) to be a distinct provisional entity, biologically unrelated to classical HCL (HCL-C). The median survival is 9 years, with 42% of patients dying of unrelated causes. Transformation to large cell lymphoma is seen in 6% of patients. Whereas HCL-C patients are typically middle-aged males, who manifest lymphocytosis, splenomegaly, and cytopenias, with malignant lymphocytes that express CD103 and CD25, the median age of HCL-V patients is 71 years with no gender preference, and whose cells are characterized by expression of CD103, but lacking CD25. HCL-V also has a more aggressive clinical course, with far lower response rates to purine nucleoside analogs, but better responses to anti-CD20 and anti-CD22 immunotherapies. The BRAF-V600E mutation is considered the molecular hallmark of HCL-C, while 16% have recurrent inactivating mutations of the cell cycle inhibitor CDKN1B, making it the second most common mutation in HCL-C. In contrast, while del17p13 and mutations of the TP53 and MAP2K1 genes are frequent in HCL-V, there are no defining chromosomal or genetic abnormalities, and little is known about the genetic underpinnings of this rare condition.We evaluated a 50 year-old African-American male who was referred to us for an incidental asymptomatic lymphocytosis of 11,990/uL. Flow cytometry showed these monoclonal B lymphocytes to express CD103, CD11c (bright), CD20, CD22, and CD52, but they did not express CD5, CD10, or CD25, with a chronic lymphocytic leukemia (CLL) immunophenotypic (Matutes) score of 0, consistent with HCL-V. A CCND/IgVH translocation was seen in 5% of these cells, and the IgVH gene was mutated at a rate of 7.2%. The patient had a normal hemoglobin and platelet count, and a computed tomography (CT) scan with contrast showed no adenopathy or splenomegaly. Foundation One' Heme Next Generation Sequencing (NGS) was performed on this patient's peripheral blood lymphocytes, and revealed several variants of unknown significance (VUS) including ALK P1599H, BRD4 Q969_L9, CARD11 D764G, CBL L43_S44, CHEK2 D438Y, FANCC Q465R, FANCL S82R, FOX03 P138L, GNAS T398M, GSK3B R396Q, HDAC7 T161M, MET V919I, PIM1 E142D, SOX2 T232A, SYK Y348C, TRAF2 R372H, and YY1AP1 S47P. Notably, no abnormalities in the BRAF, TP53, MAP2K1, and CCND family of genes were observed.A search of multiple literature and genetic databases for the gene sequence variants listed above was performed to assess for their known correlations to lymphoproliferative malignancies, especially HCL-V and HCL-C. These databases included PubMed (http://www.ncbi.nlm.nih.gov/pubmed), Genetics Home Reference (https://ghr.nlm.nih.gov/) Cancer Genetics Web (http://www.cancer-genetics.org/), and Gene Cards (http://www.genecards.org/). Mutations or translocations in the genes ALK, CARD11, HDAC7, MET, leukemia, Burkitt's lymphoma and Hodgkin's disease leukemia, Burkitt's lymphoma and Hodgkin's disease leukemia, Burkitt's lymphoma and Hodgkin's diseasePIM1, SOX2, and SYK, were found to have clear associations with various lymphoid malignancies, including large B cell lymphoma (CARD11, PIM1), Burkitt lymphoma (HDAC7, MET), anaplastic large cell lymphoma (ALK, SOX2), Hodgkin lymphoma (MET), mantle cell lymphoma (SOX2), pro-B cell ALL (HDAC7), and rare peripheral T cell lymphomas (SYK). Alterations in CARD11 have also been associated with congenital B cell lymphocytosis. However, none of the specific variants/mutations identified by NGS of our patient's HCL-V cells have previously been described to be associated with either HCL-V or HCL-C. These results may thus help to elucidate the molecular pathogenesis of this rare hematologic malignancy, and emphasize the need to apply this technology to a greater number of HCL-V cases to assess for recurring genetic variations that may distinguish this unique disorder. DisclosuresNo relevant conflicts of interest to declare.

Distinct Patterns of PD-L1 and PD-L2 Expression By Tumor and Non-Tumor Cells in Patients with MM, MDS and AML

Introduction:Programmed death-ligand 1 (PD-L1) contributes to tumor escape from immune surveillance by binding to programmed death-1 (PD-1), a negative regulator of T-cell responses. PD-L1 is expressed by both tumor cells and immune cells in the tumor microenvironment. In contrast, the role of PD-L2 in tumor immunity is unclear. To better understand the contribution of PD-L1/L2 to immune escape in marrow-based hematologic malignancies, we used multi-color flow cytometry and immunohistochemistry (IHC) to characterize cell-specific PD-L1/PD-L2 expression in patients with multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML).Results:PD-L1 was detectable (> 2% positive cells) in 100% of patients, with distinct disease-specific patterns. PD-L1 was expressed most widely in MM, predominantly by malignant plasma cells (median, 95% of plasma cells; n = 5) and lymphocytes (median, 12% of marrow lymphocytes; n = 5). In contrast, in MDS and AML, PD-L1 was more commonly expressed by non-tumor hematopoietic cells: MDS median, 36% of CD34− myeloid precursors and 47% of lymphocytes (n = 13) vs 12% of CD34+ myeloid blasts; AML median, 19% of CD34− myeloid precursors and 26% of lymphocytes (n = 7) vs 16% of CD34+ myeloid blasts were PD-L1+. A higher proportion of CD8+ T cells expressed PD-L1 compared with CD4+ T cells in all 3 malignancies: the CD8:CD4 ratio for PD-L1+ T cells was 3.02 in MM (n = 11), 1.92 in MDS (n = 10), and 1.29 in AML (n = 7). PD-L2 expression was largely absent in AML and MDS (< 2% of CD34+ blasts or lymphocytes expressed PD-L2 (n = 13 MDS and n = 7 AML) but was expressed in a subset of patients with MM on plasma cells (median, 18%; n = 5) but not lymphocytes (< 2%). Across all indications on both tumor cells and lymphocytes, PD-L1 was expressed by a larger fraction of cells than PD-L2 (AML: CD34+ blasts, P < .01 and lymphocytes, P = .005 [n = 7]; MDS: CD34+ blasts, P < .01 and lymphocytes, P = .0001 [n = 13]; MM: plasma cells, P = .03 and lymphocytes, P = .04 [n = 5]). These results were confirmed in a distinct set of 16 cases of primary AML analyzed independently, with detectable PD-L1 expression on myeloid blasts in 14 of 16 cases (88%) without co-expression of PD-L2 (0 of 16 cases). Comparisons of flow cytometry and IHC revealed that some IHC methods may underestimate the prevalence of PD-L1/PD-L2 in marrow-based hematologic malignancies, and results comparing different methods for detecting PD-L1/PD-L2 in the bone marrow will be presented.Conclusions:PD-L1 is highly prevalent in MM, MDS and AML, with significant expression by non-tumor hematopoietic cells, particularly CD8+ T cells. PD-L2 expression was largely absent in myeloid diseases but detectable in MM. Interestingly, PD-L1 expression was most common on tumor cells in MM and on non-tumor hematopoietic cells in MDS, whereas expression on non-tumor and tumor cells in AML was comparable. These data support clinical development of anti-PD-L1/PD-1 therapies in MM, MDS and AML. Future analyses will determine whether different patterns of PD-L1 expression are associated with clinical efficacy.Authors M Dail, L Yang, S Rodig, and J Venstrom contributed equally to this work. DisclosuresDail:Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Kadel:Genentech, Inc.: Employment. Koeppen:Roche: Employment, Equity Ownership. Adamkewicz:Genentech, Inc.: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Venstrom:Genentech: Employment.

Relapsed Nodular Lymphocyte-Predominant Hodgkin Lymphoma: An Analysis from the German Hodgkin Study Group (GHSG)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare entity accounting for approximately 5% of all Hodgkin lymphoma (HL) cases. Pathological and clinical features differ from classical HL (cHL). Pathologically, the malignant lymphocyte predominant (LP) cells stain consistently positive for CD20 and are negative for CD30. Clinically, NLPHL often has an indolent course. However, patients tend to develop late and multiple relapses. Since data on the treatment of relapsed NLPHL are scarce, we used the database of the German Hodgkin Study Group (GHSG) to obtain information on characteristics, treatment and outcome of NLPHL patients with disease recurrence after initial response to first-line treatment. A total of 99 patients who had their first-line treatment within 12 GHSG studies between 1993 and 2008 and subsequently relapsed with NLPHL histology were identified and considered for the present analysis. Patients with primary disease progression or transformation into aggressive B-cell non-Hodgkin lymphoma (NHL) at relapse were not included. At initial NLPHL diagnosis, the median age of the 99 eligible patients was 40 years; 66% of patients had early favorable, 14% had early unfavorable and 20% had advanced stages. First-line treatment consisted of radiotherapy (RT) alone in 22%, combined-modality treatment (CMT) in 68%, single agent anti-CD20 antibody treatment with rituximab in 7% and chemotherapy alone in 3% of patients. At a median observation of 10.8 years from initial diagnosis, relapse occurred after a median of 6.2 years. At relapse, 32% of patients received RT alone or conventional chemotherapy followed by RT, 28% received high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) and 26% had anti-CD20 antibody treatment either alone or in combination with conventional chemotherapy. In 13% of cases, patients either received no salvage treatment or the kind of salvage treatment was unknown. At a median observation of 3.8 years after disease recurrence, 11% of patients had developed a second relapse and 4% a third relapse. Five-year progression-free survival (PFS) estimates after occurrence of the first relapse were 78.6% (95%-CI: 61.6%-95.6%), 90.0% (95%-CI: 76.5%-100%) and 72.4% (95%-CI: 53.6%-91.2%) after salvage treatment with RT alone or conventional chemotherapy followed by RT, high-dose chemotherapy followed by ASCT and anti-CD20 antibody treatment optionally combined with conventional chemotherapy, respectively. Thus, there were no significant differences between the applied approaches. Only 10 patients (10%) died during observation; 5 deaths were NLPHL-related, 3 patients died due to second malignancies, and 1 patient each died from respiratory failure and infection. Hence, 90% of NLPHL patients who developed disease recurrence after initial response to first-line treatment were alive at the end of follow-up. Given these excellent survival data, it appears necessary to define patients who are sufficiently treated with non-toxic approaches associated with a potentially low risk for the development of late effects. A pragmatic approach based on the present data may consist in the division of relapsed NLPHL patients into 2 groups. Patients with a longer remission after first-line treatment who present with limited tumor mass at relapse may be treated with anti-CD20 antibodies alone or in combination with conventional chemotherapy, RT alone or conventional chemotherapy followed by RT. In contrast, patients with early disease recurrence and more extensive disease at relapse including extranodal involvement may require more aggressive treatment consisting of high-dose chemotherapy followed by ASCT. Disclosuresvon Tresckow:Novartis: Consultancy, Other: travel grants, Research Funding; Takeda: Consultancy, Other: travel grants; Millenium: Consultancy. Engert:Takeda: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.

Low Rates of CNS Relapse in High Risk DLBCL Patients Treated with R-CODOX-M and R-IVAC: Results from a Phase 2 UK NCRI/Bloodwise Trial

Introduction: Central nervous system (CNS) relapse of diffuse large B-cell lymphoma (DLBCL) represents a major clinical challenge and is fatal in most patients. Recently Schmitz et al (J ClinOncol 2016), defined an effective risk model, the CNS-IPI, to identify those at highest risk of CNS relapse, based on the international prognostic index (IPI) score and presence of renal or adrenal involvement. For DLBCL patients receiving R-CHOP-like regimens +/- intrathecal methotrexate, the risk of CNS relapse for low, intermediate and high-risk patients was <1%, 3-4% and 10-12%, respectively. The optimum strategy for CNS prophylaxis, however, has yet to be defined.Aim: To assess CNS relapse rates in an intermediate-high risk cohort of patients with DLBCL treated with the R-CODOX-M R-IVAC regimen, incorporating multiple CNS-penetrating agents.Methods: Patients with newly diagnosed DLBCL and an IPI score ≥3 were enrolled in a prospective, multi-centre, phase 2 trial (McMillan et al, Hematol Oncol 2015; 31(S1), 130a) and treated with modified CODOX-M and IVAC, including high dose intravenous methotrexate, cytarabine, ifosfamide and etoposide with 8-12 intrathecal injections (Mead et al, AnnOncol 2002; 23(8):1264-74); plus 8 doses of rituximab. The primary endpoint was progression-free survival (PFS). CNS involvement was diagnosed according to neurological signs, radiological findings and/or demonstration of malignant lymphocytes within the cerebrospinal fluid. Involvement ofextranodal sites was prospectively documented at registration and at relapse. Presence of CNS, adrenal and renal involvement was confirmed using case report forms prior to this post hoc analysis.Results: 108 patients were treated at 32 UK sites between May 2008 and April 2013. Median age was 50 years (18-65 years). Eight patients (7.4%) had CNS involvement at baseline. Eighty-two patients (75.9%) received 4 cycles of treatment. At a median follow-up of 45 months, PFS and overall survival were 65.5% (95% CI: 55.5 - 73.8) and 73.7% (64.0 - 81.2), respectively. Progression or relapse within the CNS occurred in 5 patients (4.6%; Table 1) at a median of 5.5 months after registration (0.9-9.1 months). All patients died within 9 months of CNS relapse, 4 due to DLBCL and one treatment-related death.Excluding those with CNS involvement at baseline or incomplete information (n=4; 2 with missing baseline information (no CNS relapse) and 2 awaiting confirmation of CNS status at relapse), CNS-IPI was evaluable in 96 patients, of which 95% had an elevated LDH, 57% had a performance status of ≥2, and 8% were ≥60 years. All patients had stage III-IV disease, 76% had >1 extranodalsite and 27% had renal or adrenal involvement. Forty-one patients (43%) were intermediate risk (2-3 factors) and 55 (57%) were high risk (4-6 factors) for CNS relapse. 2-year CNS relapse rates were 0% for intermediate risk and 6.2% (2.0 - 18.1) for high risk patients (Figure 1). Of the 3 CNS relapses in high risk patients, 2 occurred concurrently with systemic relapse; there was only one episode of isolated CNS relapse.Of the 8 patients with CNS involvement at baseline, 2 (25%) developed CNS relapse, including 1 isolated CNS relapse. One further patient died of refractory DLBCL whilst 5 (62.5%) are alive and progression free with a minimum of 28 months follow-up.Conclusions: Inclusion of CNS-directed therapy intrinsic to the R-CODOX-M IVAC regimen resulted in very low rates of CNS relapse. Although patient numbers and low event rates make direct comparison difficult, results appear promising alongside historical results with R-CHOP chemotherapy. CNS relapse rates for both intermediate and high risk patients in this trial were below the 95% confidence intervals for CNS relapse reported in large training and validation cohorts by Schmitz et al (0% vs 2.2 - 4.4 and 2.3 - 5.5 for intermediate risk patients and 6.2% vs 6.3 - 14.1 and 7.9 - 16.1 for high risk). Of note, only 2 patients in the whole cohort progressed with isolated CNS disease, one of whom had CNS disease at diagnosis. Thus, where systemic disease was fully treated, treatment failure due to inadequate CNS penetration was rare. Reasonable outcomes were achieved in patients with CNS involvement at diagnosis but greater patient numbers are required to further evaluate this regimen in secondary CNS lymphoma. [Display omitted] [Display omitted] DisclosuresPhillips:Roche: Consultancy. Patmore:Roche: Honoraria; Janssen Cilag: Honoraria. Ardeshna:Roche: Membership on an entity's Board of Directors or advisory committees, Other: Conference Expenses, Research Funding. Montoto:Roche: Honoraria; Gilead: Research Funding.

Increased Expression of Myc in Hairy Cell Leukemia, and Cell-Sensitivity to JQ1

Classic hairy cell leukemia (HCL), comprising 2% of leukemias, is an indolent B-cell malignancy with malignant lymphocytes expressing B-cell antigens CD20 and CD22, CD103, CD11c, CD25, Annexin A1, BRAF V600E mutation, and monoclonal immunoglobulin (Ig) rearrangement. HCL variant (HCLv), which is ~10% as common as HCL, has much poorer response to therapy and more aggressive course, lacks CD25 and Annexin A1, and is wild-type for BRAF. HCLv is considered separate from HCL by the World Health Organization in the unclassifiable splenic B-cell leukemia/lymphomas. Another poor-prognosis group overlaps HCL and HCLv in which unmutated IGHV4-34 Ig rearrangement is expressed. IGHV4-34+ leukemic cells can resemble classic HCL with CD25 and Annexin A1 expression, but are BRAF wild-type. No uniform mutation has been identified for HCLv and IGHV4-34+ HCL, although MAP2K1 (MEK1) mutations have recently been identified in half of cases. Thus HCLv and IGHV4-34+ HCL are less indolent leukemias with few therapy options and no known molecular target.To study HCLv and IGHV4-34+ HCL, leukemic samples were purified by negative B-cell isolation followed by positive CD11c sorting. Following extraction (Qiagene, AllPrep DNA/RNA Kit), RNA samples from patients were analyzed in microarray studies (Human HT-12 v4 BeadChips, Illumina, Inc.). Expression data were compared by unpaired nonparametric analysis using Mann-Whitney. MYC expression using one probe (log2 values, mean +/- standard deviation) was 7.30 +/- 1.51 for 37 HCL vs 9.77 +/- 1.15 for 32 HCLv or IGHV4-34+ HCL (2-sided p<0.0001). For the other probe, expression was 7.07 +/- 1.51 vs 9.44 +/- 1.17 (p<0.0001). Expression data for MYC had previously been submitted for 31 chronic lymphocytic leukemia (CLL) and 16 HCL samples (Dataset GSE2350, Basso et al, Nat Genet, 37:382, 2005). By 1 probe for MYC, expression was 8.07 +/- 0.55 for CLL vs 9.31 +/- 1.19 for HCL (p=0.0023). By another MYC probe, expression was 9.28 +/- 0.47 for CLL vs 10.22 +/- 0.98 for HCL (p=0.0032).To investigate potential therapeutic relevance of aberrant MYC expression in HCL, HCLv and IGHV4-34+ HCL, the bromodomain and extra terminal (BET) protein inhibitor JQ1, which has been associated with down-regulation of c-Myc via Brd4, was incubated with primary leukemic cells and ATP incorporation was measured. JQ1 inhibited 12 samples of HCL (IC50s 214 +/- 217 nM) more potently than 14 samples of CLL (IC50s 1.77uM +/-2.62 uM, p=0.020), and also inhibited 14 samples of HCLv or IGHV4-34+ HCL (IC50s 221 +/- 234 nM) more potently than the 14 CLL samples (p=0.0079). However, JQ1 inhibition was similar comparing HCL and HCLv or IGHV4-34+ HCL (p=0.89). To exclude non-specific inhibition of the cells, the inactive control molecule JQ1R was tested and was only 6.0% +/- 4.0% as active as JQ1 toward HCL or HCLv or IGHV4-34+ HCL samples. Normal peripheral blood mononuclear cells were resistant (IC50 > 20 uM).In conclusion, our results show that MYC expression is higher in HCLv and IGHV4-34+ HCL than in classic HCL and higher in classic HCL than CLL. Moreover, JQ1 inhibits HCL or its variants more potently than CLL, although the inhibition assay used does not detect a difference between the variants and classic HCL. Further experiments with other inhibitors will be needed to determine if the increased expression of MYC in HCL and its poor-prognosis variants can be exploited for treatment. DisclosuresNo relevant conflicts of interest to declare.

Novel α-substituted tropolones promote potent and selective caspase-dependent leukemia cell apoptosis

Tropolones, such as β-thujaplicin, are small lead-like natural products that possess a variety of biological activities. While the β-substituted natural products and their synthetic analogs are potent inhibitors of human cancer cell growth, less is known about their α-substituted counterparts. Recently, we synthesized a series of α-substituted tropolones including 2-hydroxy-7-(naphthalen-2-yl)cyclohepta-2,4,6-trien-1-one (α-naphthyl tropolone). Here, we evaluate the antiproliferative mechanisms of α-naphthyl tropolone and the related α-benzodioxinyl analog. The α-substituted tropolones inhibit growth of lymphocytic leukemia cells, but not healthy blood cells, with nanomolar potency. Treatment of leukemia cell lines with the tropolone dose-dependently induces apoptosis as judged by staining with annexin V and propidium iodide and Western blot analysis of cleaved caspase 3 and 7. Moreover, pre-treatment of cells with the caspase inhibitor Z-VAD-FMK inhibited the apoptotic effects of the tropolone in two lymphocytic lines. Caspase inhibition also blocked elevated histone acetylation caused by the tropolone, indicating that its effects on histone acetylation are potentiated by caspases. In contrast, α-naphthyl tropolone upregulated p53 expression and phosphorylation of Akt and mTOR in a manner that was not rescued by caspase inhibition. The effects of tropolone were blocked by co-incubation with high levels of free extracellular iron but not by pre-loading with iron. Additionally, dose and time dependent reduction in ex vivo viability of cells from leukemia patients was observed. Taken together, we demonstrate that α-substituted tropolones upregulate DNA damage repair pathways leading to caspase-dependent apoptosis in malignant lymphocytes.

Salivary Immunoglobulin A Assessment in Lymphoma Patients before and after Chemotherapy

Background: Lymphomas are group of diseases caused by malignant lymphocytes that accumulate in lymph nodes and caused the characteristics lymphadenopathy. Occasionally, they may spill over into blood or infiltrate organs outside the lymphoid tissue. The major subdivision of lymphomas is into Hodgkin lymphoma and non–Hodgkin lymphoma and this is based on the histologic presence of Reed-Sternberg cells in Hodgkin lymphoma. Salivary immunoglobulin A is the prominent immunoglobulin and is considered to be the main specific defense mechanism in oral cavity. The aim of this study was to determine the level of salivary immunoglobulin A in lymphoma patients before and after chemotherapy treatment. Subjects, materials and methods: The study included 25 patients (15 male and 10 female) with non–Hodgkin lymphoma(B-cell type) , 25 patients( 16 male and 9 female ) with Hodgkin lymphoma and 25 (15 male and 10 female) healthy control group. Whole un-stimulated saliva was collected to determine the level of salivary immunoglobulin A, which measured by Enzyme Link Immunosorbent Assay. Results: The level of salivary immunoglobulin A was significantly higher in pre-treatment patients in comparison with control group, and there was a significant decrease after chemotherapy treatment when compared to their base line levels in both study groups. Conclusion: The salivary immunoglobulin A was higher in lymphoma patients than control, then its level showed obvious decrease after chemotherapy treatment.

Abstract A06: Development of a drug discovery platform using clinically relevant patient-derived xenograft models for cutaneous T cell lymphomas

Abstract Introduction: Cutaneous T cell lymphomas (CTCL) represent a heterogeneous group of diseases. Mycosis fungoides (MF) and Sezary syndrome (SS) are the most common subtypes of CTCL. Discovery of new therapeutic targets in T cell lymphomas has been hampered by lack of appropriate discovery platforms. Few T cell lymphoma cell lines are available; and it is likely that they have departed significantly from the original disease through long-term in vitro cultivation. Objectives: To develop patient-derived T cell lymphoma mouse xenograft models (PDX) that recapitulate the clinical presentation of human diseases and to establish a drug discovery platform using these PDX models. Materials and Methods: To establish PDX models for MF and SS, we injected lymphocytes obtained from patients into an NSG mouse (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) either subcutaneously or intravenously. Next, we investigated whether tumor derived plasma cell-free DNA can serve as a surrogate for disease burden in PDX. We first established this method in xenografts bearing a subcutaneous tumor derived from a MF cell line. We measured tumor volume by manual caliper methods twice weekly and simultaneously, we collected plasma from these mice. We measured human-actin-specific cell-free DNA concentrations in the plasma by qPCR and examined whether they can correlate with the tumor volume measured by the caliber method. After validated this method in xenografts established with a MF cell line, we examined this method in PDX. To this end, we measured human-actin-specific cell-free DNA concentrations in PDX plasma at various time points post-inoculation and investigated whether they correlated with tumor progression in PDX. Results: Three weeks after inoculation of patient-derived malignant lymphocytes, the MF PDX mouse developed clinical syndrome of MF with erythematous, scaly skin lesions and alopecia. Necropsy revealed lymphocyte infiltration in the skin, spleen, liver and lungs, resembling the disease distribution of MF in humans. The erythematous skin lesions showed atypical lymphocyte infiltration in the epidermis, a diagnostic hallmark for MF. Additionally, immunohistochemistry staining reveled malignant cells in the epidermis expressed CD3 and CD4 without co-expression of CD7, which is identical to those of the donor patient's. Furthermore, we analyzed T cell receptor (TCR) clonality by PCR and demonstrated a clonal T-cell population in both patient and mouse specimens with similar amplified peaks, consistent with successful xenograft. Similarly, the PDX mouse for SS developed the clinical syndromes with skin rashes, alopecia and was found to have Sezary cells in the blood, a diagnostic hallmark for SS. Malignant cells isolated from the spleen of a SS PDX animal had the same immunophenotype and TCR clonality as that of the donor patients, indicating successful xenografts. Importantly, we were able to passage malignant cells from one generation of PDX to the next while maintaining the clinical presentation of the disease. Finally, we were able to demonstrate that in cell-line derived xenograft mice bearing subcutaneous tumors, human-actin-specific cell-free DNA concentrations in the plasma correlated with tumor volume assessed by manual caliber measurements. Next, we extended these results to MF/SS PDX, and were able to correlate human-actin cell-free DNA concentrations with tumor progression in in these mice. Conclusion: We established PDX models that recapitulate the clinic syndrome of MF and SS with characteristic skin, nodes and blood involvement by the malignant cells. Additionally, we demonstrated that tumor-specific cell-free DNA concentration can be used as a surrogate for tumor burden in PDX. Together, our results demonstrated that we can establish a drug discovery platform using clinically relevant PDX models for T cell lymphomas. Citation Format: Wei Ai, Yen-Chen Yang Yang, Shervin Afghani, Huaxin Gao, Laura Pincus Pincus, Linlin Wang Wang, Taha Rakhshandhroo, Ronald Balassanian, James Rubenstein, Ryan Gill, Frank McCormick. Development of a drug discovery platform using clinically relevant patient-derived xenograft models for cutaneous T cell lymphomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A06.

Survival control of malignant lymphocytes by anti-apoptotic MCL-1.

Programmed apoptotic cell death is critical to maintain tissue homeostasis and cellular integrity in the lymphatic system. Accordingly, the evasion of apoptosis is a critical milestone for the transformation of lymphocytes on their way to becoming overt lymphomas. The anti-apoptotic BCL-2 family proteins are pivotal regulators of the mitochondrial apoptotic pathway and genetic aberrations in these genes are associated with lymphomagenesis and chemotherapeutic resistance. Pharmacological targeting of BCL-2 is highly effective in certain indolent B-cell lymphomas; however, recent evidence highlights a critical role for the BCL-2 family member MCL-1 in several lymphoma subtypes. MCL-1 is recurrently highly expressed in various kinds of cancer including non-Hodgkin's lymphoma of B- and T-cell origin. Moreover, both indolent and aggressive forms of lymphoma require MCL-1 for lymphomagenesis and for their continued survival. This review summarizes the role of MCL-1 in B- and T-cell lymphoma and discusses its potential as a therapeutic target.

Abstract 4697: The covalent CDK7 inhibitor THZ1 can counteract apoptotic resistance and suppress the transcription of genes attributed to chronic lymphocytic leukemia malignancy

Abstract Chronic lymphocytic leukemia (CLL) is a malignancy characterized by the progressive accumulation of CD19+ B-cells capable of overcoming their regulated life cycle. Because this disease is highly heterogeneous and utilizes several mechanisms to resist apoptotic death, CLL currently remains incurable. Based on the ongoing efforts to interpret the transcriptional dynamics of CLL pathogenicity as well as prior reports demonstrating the efficacy of cyclin-dependent kinase (CDK) transcriptional inhibitors, we sought to characterize the molecular response of CLL cells treated with the covalent CDK7 inhibitor THZ1 and determine the therapeutic potential of CDK7 inhibition in CLL. We first observed that THZ1 was able to inhibit growth in malignant B-cell lines MEC1 and MEC2 in both a dose dependent (IC50: .045uM &amp; .030uM, respectively) and time dependent manner. The inhibition in cell growth corresponded with both G1-mediated cell cycle arrest as well as apoptosis in the respective cell lines. We also observed dose-dependent reduction in cell viability through apoptosis in patient-derived CLL B-cells after 24 hours. Because of the observed outcomes of THZ1-treated CLL cells, we then wanted to determine the specific transcriptional targets significantly suppressed by THZ1 treatment. To identify the THZ1-sensitive transcripts, we performed a time course RNAseq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of covered reads over a 12-hour period, we determined that the top 50 transcripts greatly diminished in both MEC1 and MEC2 contain significant enrichment in genes attributed to glycolytic metabolism (ex: ALDOC, SLC2A1, TPI1, GAPDH, ENO2). Along with the overlapping comparison between the two cell lines, we next compared the downregulated transcripts from the treated cell lines against the RNAseq expression profile of 47 CLL patients and 5 healthy donors. We observed that THZ1 was able to suppress transcripts upregulated in CLL patient samples including ENO2, FGR, WNT10A, and CBX7 in MEC1 and ENO2, MALAT1, CCR7, and PLCG1 in MEC2. Finally, we performed H3K27Ac ChIPseq in MEC1 to identify super enhancers that may overlap with THZ1-sensitive transcripts. From this comparison, we determined that super enhancers exist within reported oncogenic drivers CBX7, WNT10A, and FGR. Overall, we observe that THZ1 can effectively overcome the anti-apoptotic phenotype of CLL B-cells as well as directly suppress transcription of genes that can drive CLL malignancy. With the addition of CDK7 ChIPseq, our ultimate goal is to provide clarity regarding CDK7-mediated transcriptional regulation in CLL and demonstrate the molecular consequences of therapeutic CDK7 inhibition in CLL. Citation Format: Austin Young Shull, Jeong-Hyeon Choi, Jordan Bauer, Lirong Pei, Farrukh T. Awan, Huidong Shi. The covalent CDK7 inhibitor THZ1 can counteract apoptotic resistance and suppress the transcription of genes attributed to chronic lymphocytic leukemia malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4697.

Abstract 936: Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome

Abstract Introduction Sézary Syndrome (SS) is a rare and aggressive variant of Cutaneous-T-Cell Lymphoma (CTCL) characterized by the presence of malignant lymphocytes named Sezary (SS) cells in the skin, lymph nodes, and peripheral blood. With a poor prognosis, SS has not a specific therapy still available. As a role of skin in SS pathogenesis is not elucidated yet, here we study the contribution of this microenvironment by comparing matched skin and blood derived SS cells for tumor cell proliferation and activation levels of PI3k/AKT pathway. Using our previous SNP array data we also verified the genomic status of members belonging to this pathway in a large cohort of SS patients. Methods Expression of Ki67 proliferation marker was evaluated in perfect-paired blood and skin-paraffin biopsies obtained from eleven SS patients by flow cytometry analysis and immunohistochemistry respectively. KI67+ neoplastic cells were calculated as percentage within neoplastic CD4+ cells recognized by a co-staining with the specific TCRVb rearrangement. Phosphorylation levels of members of PI3k/AKT pathway were compared between matched circulating and skin resident SS cells proteins derived from 2 SS patients using an AKT kinase array (Cell Signaling). Affymetrix SNP6.0 arrays was used to investigate the copy number (CN) status of members of mTORC 1 pathway in a cohort in 37 SS samples derived from 23 SS patients and 3 cell lines. Results Skin derived SS cells showed a significant higher proliferation index (PI) respect to SS cells obtained from blood (12%±11 vs 1,24%+1,18; P = 0,00025). In order to identify the signals responsible for SS proliferation, we used a PI3K/AKT kinase array that revealed an enhanced phosphorylation levels of many components of this cascade, particularly of PRAS40 with a Fold change (Fc) of 6,15; GSK3a (Fc = 4.83), mTOR (Fc = 4.61) mP70S6K (Fc = 4.64) BAD (Fc = 7.09) and 4EBP1 (Fc = 5.40)in skin-SS cells respect to circulating ones. We next deepen our observations in mTORC1 signaling because of this and earlier observations made in CTCL cell lines. Using our SNP6 array data we have verified the genomic status of members of this pathway. Results obtained showed that P70S6K, the kinase downstream TORC1 that plays a crucial role in protein synthesis, showed a mono-allelic gain in 9 out 23 patients (39%) whereas PDCD4, a protein that inhibits protein translation displayed a mono-allelic loss in 10 of 23 individuals (43%). 4 of these patients showed a concomitant P70S6K gain and PDCD4 loss. Overall these genetic defects suggest that an abnormal protein synthesis can occur in these patients.. Conclusion: Our data demonstrate that skin microenvironment enhances SS cell proliferation index and activates mTORC1 signaling, an unbalanced pathway that reveals novel potential therapeutic targets for Sezary Syndrome. Citation Format: Cristina Cristofoletti, Mario Picozza, Antonella Bresin, Maria Cristina Picchio, Enrico Scala, Giuseppe A. Lombardo, Francesca Passarelli, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 936.