Positive Chronic Lymphocytic Leukemia Research Articles

ZAP-70 Integrates Tonic B Cell Receptor and Chemokine Signaling in IGHV unmutated Chronic Lymphocytic Leukemia

The importance of B cell receptor (BCR) signaling for the progression of Chronic Lymphocytic Leukemia (CLL) has impressively been demonstrated through the clinical success of small molecule inhibitors interfering with this pathway, providing effective treatment options for a vast majority of patients. The scientific evidence which supported the development of drugs like BTK and PI3K inhibitors largely originate from studies of antigen-induced BCR-signaling. Notably, the presence of a constitutive, so-called tonic BCR-signal was long postulated to co-exist besides antigen-driven activation and is based on the identification of constitutively activated signaling molecules down-stream of the BCR. We here provide direct evidence for the presence of a tonic BCR-signal, which critically relies on the expression of the tyrosine kinase ZAP-70. We have previously established a method to inhibit ZAP-70 expression in primary CLL cells usingRNA-interference in combination with an extended co-culture system. Our previous work demonstrated that ZAP-70 is required for the constitutive gene expression of T cell chemokines CCL3 and CCL4 and contributes to the dysregulation of global protein synthesis and MYC expression in CLL ( Blood, 2021). Applying this method, we here provide evidence for the presence of a constitutive tonic BCR signal, which is dependent on ZAP-70 expression and exclusively found in IGHV unmutated CLL, but not in IGHV mutated cases discordantly expressing ZAP-70. We demonstrate that ZAP-70 is directly involved in the constitutive activation of AKT and GSK-3beta. Furthermore, ZAP-70 expression increases steady state activation of CD19 and the downstream kinase SRC, providing direct evidence for the existence of a ZAP-70 dependent tonic BCR signal in I GHV unmutated CLL only. To identify the underlying signaling events driving this signal, we performed mass spectrometry in combination with proximity ligation assays on CLL cells in the absence of an induced BCR-signal. Unexpectedly, we identified that the majority of ZAP-70 binding proteins do not related to BCR-signaling components, but to cytoskeleton organizing proteins involved in cell migration. Indeed, migration assays demonstrated that the loss of ZAP-70 directly and significantly impaired cell migration induced by CCL19 and CCL21, in keeping with previous data indication an association between ZAP-70 and cell migration. These results were confirmed using a switchable model of ZAP-70, which allowed us to toggle ZAP-70 expression on and off at any given time. To further define the mechanisms of ZAP-70 mediated cell migration, we studied CCL19 and CCL21 signaling in ZAP-70 proficient and deficient cells. Unexpectedly, we identified that ZAP-70 is required for chemokine-mediated activation of AKT. In summary, we here demonstrate that a tonic BCR-signal is present only in IGHV-unmutated CLL and dependent on the expression of ZAP-70. Furthermore, ZAP-70 converges two seemingly independent signaling events, namely chemokine- and tonic BCR signaling. Our data provide further explanations for the more aggressive clinical course of ZAP-70 positive CLL.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Chronic lymphocytic leukemia (CLL) patients with hepatitis B virus (HBV) infection have a poor prognosis, underlying mechanism remains unclear. NOTCH mutations are frequent in CLL and associated with disease progression and drug resistance. It is also reported to be associated with hepatitis infection in lymphoid malignancies. In order to investigate the relation between NOTCH pathway and HBV-associated CLL, we studied 98 previously untreated HBV positive CLL patients and 244 HBV-negative CLL. A total of 342 treatment naïve patients diagnosed with CLL at the First Affiliated Hospital of Nanjing Medical University from 1 January 2010 to 31 October 2021 were enrolled in our study. The mutation hotspots of NOTCH pathway genes were analyzed by Next Generation Sequencing (NGS) of DNA that used PCR for the identification of genomic mutation. Fisher exact and χ2 tests were used to assess the correlation between HBV infection and clinical, demographic factors in CLL patients. Survival curves were generated by Graphpad 9.5. NOTCH mutations were more frequent in HBV positive CLL subgroup (17.3% vs. 7.4%, p = 0.033). By survival analysis, HBV infection was associated with disease progression and poor survival (p = 0.0099 for overall survival [OS] and p = 0.0446 for time-to-treatment [TTT]). Any lesions of the NOTCH pathway (NOTCH1, NOTCH2 and SPEN) aggravated prognosis. In multivariate analysis, NOTCH mutation retained an independent significance for HBV-infected patients (p = 0.016 for OS and p = 0.023 for TTT). However, HBV positive with NOTCH unmutated had no statistical difference in prognosis compared with HBV negative patients (p = 0.1706 for OS and p = 0.2387 for TTT), which indicated that NOTCH pathway mutation contributed to inferior prognosis in HBV-infected CLL. In conclusion, a cohort of CLL patients with HBV positive displayed a worse clinical outcome and the status of NOTCH signaling pathway might play a crucial role. Keywords: chronic lymphocytic leukemia (CLL), diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Antibodies Produced by CLL Phenotype B Cells in Patients With Myasthenia Gravis Are Not Directed Against Neuromuscular Endplates.

Myasthenia gravis (MG) can in rare cases be an autoimmune phenomenon associated with hematologic malignancies such as chronic lymphocytic leukemia (CLL). It is unclear whether in patients with MG and CLL, the leukemic B cells are the ones directly driving the autoimmune response against neuromuscular endplates. We identified patients with acetylcholine receptor antibody-positive (AChR+) MG and CLL or monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, and described their clinical features, including treatment responses. We generated recombinant monoclonal antibodies (mAbs) corresponding to the B-cell receptors of the CLL phenotype B cells and screened them for autoantigen binding. A computational immune cell screen revealed a subgroup of 5/38 patients with MG and 0/21 healthy controls who displayed a CLL-like B-cell phenotype. In follow-up hematologic flow cytometry, 2 of these 5 patients were diagnosed with an MBL. An additional patient with AChR+ MG as a complication of manifest CLL presented at our neuromuscular clinic and was successfully treated with the anti-CD20 therapy obinutuzumab plus chlorambucil. We investigated the specificities of expanding CLL-like B-cell clones to assess a direct causal link between the 2 diseases. However, we observed no reactivity of the clones against the AChR, antigens at the neuromuscular junction, or other common autoantigens. Our study suggests that AChR autoantibodies are produced by nonmalignant, polyclonal B cells The new anti-CD20 treatment obinutuzumab might be considered in effectively treating AChR+ MG. This is a single case study and provides Class IV evidence that obinutuzumab is safe to use in patients with MG.

Abstract 5432: Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL

Abstract Introduction: CD37 has shown promise as a new therapeutic target for B-cell malignancies. CD37 is highly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Alpha-emitting radionuclides have demonstrated potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm) causing localized and irreparable DNA damage while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-generating radioisotope 212Pb for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Approximately 90,000 cases of CLL and NHL are expected annually in the US. Standard of care includes chemotherapy combined with anti-CD20 antibodies (obinutuzumab and rituximab) and Bruton’s tyrosine kinase inhibitor ibrutinib. While these therapies are initially effective, most patients inevitably relapse. Materials and methods: The efficacy and tolerability of a single dose 212Pb-NNV003 treatment were evaluated in disseminated models using CD37-expressing human CLL (MEC-2) and Burkitt’s lymphoma (Daudi) cells. Comparison to therapeutic doses of ibrutinib was investigated in the MEC-2 model. R2G2 or CB17-SCID mice were injected intravenously with 2.5×106 MEC-2 or 10×106 Daudi cells on day 0, followed by intravenous injection of 212Pb-NNV003 (2.5, 5 or 7.5 µCi in Daudi model and 5, 10, 15 or 20 µCi in MEC-2 model) or daily oral administration of ibrutinib (12.5 or 25 mg/kg) starting on day 2 (n=10-12 per group). 212Pb-cetuximab (unspecific isotype control), unlabelled NNV003 and NaCl were used as controls. Results: In vitro testing of the antibody showed that MEC-2 cells were 5 times more resistant to ibrutinib than Daudi cells. Treatment of mice injected intravenously with Daudi cells with 2.5 μCi 212Pb-NNV003 resulted in survival of 100% of the mice 100 days after start of treatment, while 100 days after start of treatment with 100 μg obinutuzumab only 40% of the mice were still alive. Control mice had a median survival of 7-8 weeks. Treatment of mice injected intravenously with MEC-2 cells with 15 μCi 212Pb-NNV003 resulted in survival of 90-100% for more than 25 weeks in two independent experiments. Ibrutinib treated mice with intravenous MEC-2 cells presented a median survival of only 4.3-4.4 weeks, comparable to the unlabelled NNV003 and saline groups (4.1 and 4.4 weeks). Mice receiving non-specific 212Pb-cetuximab presented a median survival of 7-9 weeks. Conclusion: The study shows that a single injection 212Pb-NNV003 is safe and effective for the treatment of CD37 positive CLL and NHL in preclinical models, with promising efficacy in both ibrutinib-resistant and sensitive CLL models. Treatment with 212Pb-NNV003 was superior to both ibrutinib and obinutuzumab. Further clinical testing is warranted. Citation Format: Jostein Dahle, Amal Saidi, Tania Stallons, Helen Heyerdahl, Ada Repetto-Llamazares, Julien Torgue. Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5432.

Evaluation of ROR1 Targeted Antibody Drug Conjugates in Acute and Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is a developmentally restricted receptor tyrosine kinase that is expressed on the surface of multiple hematological and solid malignancies but not on normal adult tissues. Recently, there has been an increased interest in ROR1 as a potential therapeutic target in multiple ROR1 positive leukemia and lymphomas. In this study, we present novel anti-ROR1 monoclonal antibody drug conjugates (ADCs) based on chimeric rabbit/human mAb “XBR1-402” or its humanized version “huXBR1-402”, that show both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) leukemic cells.Methods: From our studies, we saw no difference in activity between XBR1-402-PNU and huXBR1-402-PNU. XBR1-402-PNU was used for our studies in pre B cell ALL and huXBR1-402-PNU was used for our studies in MCL and CLL. For ALL in vitro experiments, we monitored cytotoxicity in Kasumi2 and 697 cell lines. For MCL in vitro experiments we monitored cytotoxicity in Jeko and Mino cell lines. For CLL in vitro experiments, we monitored cytotoxicity in primary CLL samples. For ALL in vivo experiments we monitored overall survival in a murine disseminated 697 model. For CLL in vivo experiments we monitored ROR1+ leukemic cell depletion in human ROR1 transgenic mice engrafted with human ROR1+TCL1 leukemia.Results: We observed a decrease in viability for all XBR1-402-PNU and huXBR1-402-PNU treated ROR1 positive leukemic cell lines and primary cells in vitro . In the in vitro model of pre B cell ALL we observed a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU(EC50 = 77 and 1 ng/mL, respectively) when compared to treatment with the isotype-matched control ADC Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL, respectively). In the in vitro model of MCL, we observed a decrease in viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 and Jurkat cell lines when compared to the unconjugated huXBR1-402 antibody or the control Tras-PNU. Additionally, we tested primary CLL samples confirmed to be positive for ROR1 and found a significant decrease in viability over 96 hours when treated with the antibody-drug-conjugate huXBR1-402 (78.6 + 6.1%)as compared to the unconjugated antibody (100% + 2.4; p=0.022, N=7) and the control Tras-PNU (87.4% +/- 6.1; p=0.038). We have also detected promising in vivo activity with XBR1-402-PNU and huXBR1-402-PNU in ALL and CLL models, respectively. In vivo studies on a murine disseminated 697 ALL model showed that treatment with XBR1-402-PNU increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days after implantation respectively, p=0.021, n=6). In addition, in preliminarily experiments, we have tested depletion of human ROR1+ leukemic cells and normal B cells in vivo using human ROR1 transgenic mice engrafted with human ROR+TCL1 leukemia and observed a preferential depletion of ROR1+TCL1 leukemic cells upon treatment with huXBR1-402-PNU compared to control Tras-PNU.Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration in patients with ROR1 positive leukemia and lymphomas. Current long-term studies are ongoing to evaluate the in vivo cytotoxic effects of huXBR1-402-PNU on total tumor burden, disease progression, and overall survival.(EH is supported by Graduate Pelotonia Fellowship) DisclosuresWaldmeier:NBE-Therapeutics Ltd: Employment. Beerli:NBE-Therapeutics Ltd: Employment. Grawunder:NBE-Therapeutics Ltd: Employment. Byrd:The Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding.

Abstract 801: Expression of BCL2 alternative proteins and association with outcome in CLL patients with venetoclax

Abstract Introduction: Venetoclax is an oral inhibitor of the anti-apoptotic protein BCL-2, and is highly effective for the treatment of patients with chronic lymphocytic leukemia (CLL). Dependence of CLL cells on alternative anti-apoptotic proteins may result in intrinsic resistance to BCL-2 inhibition. The predictive and prognostic role of the expression of BCL-2 alternative proteins in CLL patients treated with venetoclax has not been yet explored. Methods: Patients with CLL treated with single agent venetoclax at MD Anderson Cancer Center between 05/2012 and 01/2016, and with available pre-treatment bone marrow samples, were included in the study. All samples were analyzed by immunohistochemistry (IHC) for BCL-W, BCL-XL, BCL2-A1 and MCL-1, and >1 positive CLL cells per high power field (40X) was considered positive. Response was assessed according to 2008 iwCLL guidelines, using the clinical and imaging criteria. Results: Twenty-seven patients were included in the study: 74.1% were male, 50% aged > 65 years, and 63% had Rai stage III-IV; 26% had mutated IGHV, 48% had del17p/del11q by FISH, and 40% had a complex karyotype by metaphase cytogenetics; 96% had been previously treated, 37% with a BTK inhibitor. Overall, 15% had pre-treatment bone marrow samples positive for BCL-W, and 7% for BCL-2A1 (Figure), while none of the samples was positive for BCL-XL or MCL-1. Overall response rate was 93%, and 14 (52%) patients achieved CR. A higher CR rate was observed in patients with positive BCL-W (75% vs 48%, p=0.60), positive BCL-2A1 (100% vs 48%, p=0.60), and double positive patients (80% vs 45%, p=0.33), though not statistically significant After a median follow-up of 50 months (95%CI, 46-54 months), 13 (48%) patients progressed, and median progression-free survival (PFS) was 51 months (95% CI, 27-75 months). A longer median PFS was observed for patients with positive BCL-W (not reached [NR] vs 36 months, p=0.46), positive BCL-2A1 (NR vs 35 months, p=0.29), and double positive patients (NR vs 36 months, p=0.20)(Figure 2), though not statistically significant. At most recent follow-up, 9 (33%) patients died, all of progressive disease, and median overall survival (OS) was 65 months (range, 2-65 months). A longer median OS was observed for patients with positive BCL-W (NR vs 65 months, p=0.19), positive BCL-2A1 (NR vs 65 months, p=0.36), and double positive patients (NR vs 65 months, p=0.12) (Figure), though not statistically significant. Conclusion: Pre-treatment expression of BCL-2 alternative proteins by IHC does not predict response to venetoclax in patients with CLL. More sensitive techniques, such as gene expression profiling and BH3 profiling, are needed to explore the predictive role of alternative anti-apoptotic pathways in these patients. Dependence on alternative BCL-2 proteins may associate with a better outcome for CLL patients, and larger dataset are needed to confirm this finding. Citation Format: Fateeha Furqan, Ellen J. Schlette, Francisco Vega, Ignacio I. Wistuba, Luisa M. Soto, Mario L. Piubelli, William G. Wierda, Alessandra Ferrajoli, Paolo Strati. Expression of BCL2 alternative proteins and association with outcome in CLL patients with venetoclax [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 801.

Refractory and 17p-deleted chronic lymphocytic leukemia: improving survival with pathway inhibitors and allogeneic stem cell transplantation.

Refractory/early relapsed and 17p deletion/p53 mutation (del(17p)/TP53mut)-positive chronic lymphocytic leukemia (CLL) has been conventionally considered a high-risk disease, potentially eligible for treatment with allogeneic stem cell transplantation (alloSCT). In this multicenter retrospective analysis of 157 patients, we compared the outcomes of patients with high-risk CLL treated with alloSCT, a B-cell receptor pathway inhibitor (BCRi), and both. Seventy-one patients were treated with BCRis, 67 patients underwent reduced-intensity conditioning alloSCT, and 19 received alloSCT with a BCRi before and/or after transplantation. Inverse probability of treatment weighting analyses were performed to compare the alloSCT and no-alloSCT groups; in the 2 groups, 5-year OS, PFS, and cumulative incidence of nonrelapse mortality (NRM) and relapse were 40% versus 60% (P = .096), 34% versus 17% (P = .638), 28% versus 5% (P = .016), and 38% versus 83% (P = .005), respectively. Patients treated with alloSCT plus BCRi had a 3-year OS of 83%. The 3-year OS and NRM by year of alloSCT, including patients treated with BCRi, were 53% and 17% in 2000 to 2007, 55% and 30% in 2008 to 2012, and 72% and 18% in 2013 to 2018. In conclusion, the combination of pathway inhibitors and alloSCT is feasible and may further improve the outcome of high-risk CLL patients.

CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression

AbstractCD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d− subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d− cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

Targeted alpha therapy for chronic lymphocytic leukaemia and non-Hodgkin's lymphoma with the anti-CD37 radioimmunoconjugate 212Pb-NNV003.

Relapse of chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma after standard of care treatment is common and new therapies are needed. The targeted alpha therapy with 212Pb-NNV003 presented in this study combines cytotoxic α-particles from 212Pb, with the anti-CD37 antibody NNV003, targeting B-cell malignancies. The goal of this study was to explore 212Pb-NNV003 for treatment of CD37 positive chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma in preclinical mouse models.An anti-proliferative effect of 212Pb-NNV003 was observed in both chronic lymphocytic leukaemia (MEC-2) and Burkitt’s lymphoma (Daudi) cells in vitro. In biodistribution experiments, accumulation of 212Pb-NNV003 was 23%ID/g and 16%ID/g in Daudi and MEC-2 tumours 24 h post injection. In two intravenous animal models 90% of the mice treated with a single injection of 212Pb-NNV003 were alive 28 weeks post cell injection. Median survival times of control groups were 5–9 weeks. There was no significant difference between different specific activities of 212Pb-NNV003 with regards to therapeutic effect or toxicity. For therapeutically effective activities, a transient haematological toxicity was observed. This study shows that 212Pb-NNV003 is effective and safe in preclinical models of CD37 positive chronic lymphocytic leukaemia and non-Hodgkin’s lymphoma, warranting future clinical testing.

212Pb-NNV003 as a Novel Targeted Alpha Therapy for CD37 Positive B-cell Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL)

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in western countries, accounting for approximately one quarter of all leukemias and Non-Hodgkin lymphoma (NHL) caused an estimated 200,000 cancer deaths worldwide in 2014. More than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US each year. Immuno-chemotherapy using anti-CD20 monoclonal antibodies (mAb) in combination with DNA alkylating agents is the front-line therapy of CLL and NHL. Despite promising initial results, relapses after repeated administration of immuno-chemotherapy are frequent and relapsed/refractory patients show poor prognosis. As CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies, the development of new therapies targeting CD37 expressing cells may prove useful for relapsed or refractory patients as an alternative to CD20 targeting agents. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the in-vivo alpha-particle-generator, 212Pb. When treated with alpha radiation, targeted cancer cells are exposed to high linear energy transfer (LET). LET acts over a short range (50–100 μm), causing double strand breaks in the DNA of targeted cells while sparing adjacent normal tissues. The efficacy of a single escalating 212Pb-NNV003 administration was evaluated on disseminated models of human Burkitt’s lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. Dose range finding and tolerability studies were performed in CB17-SCID and R2G2 tumor-free mice to define the maximum tolerated dose prior to the efficacy studies. 212Pb-NNV003 displays a favorable toxicity profile after a single intravenous injection in tumor-free mice. No acute hematological toxicity was observed, and animals presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered 4-weeks after injection. A single intravenous dose of 10, 15 or 20 μCi of 212Pb-NNV003 led to 70%, 90% and 100% of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection. Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively. A single intravenous dose of 2.5, 5 and 7.5 μCi 212Pb-NNV003 led to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection. Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively. The results of preclinical studies suggest that TAT using 212Pb-NNV003 may have positive clinical implication for the treatment of CD37 positive CLL and NHL.

In Human Visualization of Ibrutinib-Induced CLL Compartment Shift

BACKGROUND. B-cell receptor (BCR) signaling is a central driver for chronic lymphocytic leukemia (CLL), and its targeting through irreversible inhibition of Bruton's tyrosine kinase (BTK) by ibrutinib has significantly improved the prognosis of CLL patients. Ibrutinib treatment has become standard of care, and, recently, has advanced to the first-line setting. A treatment-induced, sometimes dramatic increase of peripheral lymphocytosis, has emerged as a class effect of BTK inhibitors in CLL. Mechanistically, BTK blocking by ibrutinib might affect adhesion molecules and chemokine receptors, such as CXCR4 and CXCR5, thus interfering with the protective tissue microenvironment and mobilizing tissue-resident CLL cells from the lymph nodes and bone marrow into the peripheral blood. This hypostasized mechanism of a "compartment shift," however, has not yet been demonstrated experimentally or visually. Positron emission tomography (PET) with [68Ga]Pentixafor, a radiotracer that specifically targets the CXCR4 receptor, was recently established as a sensitive approach with which to detect CLL in vivo. As a proof-of-concept, we here present three CLL patients to demonstrate the potential of [68Ga]Pentixafor-PET/MR imaging to functionally track CLL cells along the redistribution induced by ibrutinib. METHODS. Three CLL patients were included: patient 1 was a 74-year-old treatment-naïve male, with unmutated IGHV status and high-risk cytogenetics, including del17p13 and TP-53 mutation; patient 2 was a 53-year-old female with late relapse (12 years after allogeneic stem cell transplantation), IGHV was unmutated, and cytogenetic abnormalities included del11q22 and del13q14; and patient 3 was a 59-year-old treatment-naïve male with unmutated IGHV and del11q22 and del13q14. Whole-body PET/MRI with injection of 150 MBq of [68Ga]Pentixafor was performed pre and on ibrutinib treatment (patient 1: one week; patient 2: two weeks; and patient 3: three weeks after start of the first therapy cycle, respectively). Treatment consisted of continuous oral administration of 420 mg of ibrutinib. Mean standardized [68Ga]Pentixafor uptake values (SUVmean) of involved lymph nodes, the bone marrow, and the spleen were measured. Isolated peripheral blood mononuclear cells (PBMCs) were stained with fluorescence-labeled antibodies, and measurements were performed on flow cytometer. RESULTS. In all three cases - at one week, two weeks, and three weeks on ibrutinib treatment - CXCR4 density, as measured on [68Ga]Pentixafor-PET, shifted from the bone marrow and lymph nodes toward the spleen (Figs. 1-3). In patient 1, the SUVmean decreased in the bone marrow (-9.8%) and lymph nodes (-12.0%), whereas it increased markedly in the spleen (+29.8%). At flow cytometry, this patient exhibited an increase of CXCR4-high (tissue-resident) CLL cells upon ibrutinib treatment (+56.7% relative to baseline). In patient 2, the SUVmean decreased in the bone marrow (-14.7%) and lymph nodes (-27.3%), whereas it more than doubled in the spleen (+133.0%). At flow cytometry, individual CLL cells demonstrated a relative increase (+39.1%) of CXCR4 positivity. In patients 3, the SUVmean decreased in the bone marrow (-27.6% %) and lymph nodes (-41.9%), whereas it increased markedly in the spleen (+26.1%). At flow cytometry, the relative increase of CXCR4-high CLL cells was +58.3%. CONCLUSIONS. We here provide the first pictures of the early functional treatment effects of ibrutinib. While our analyses confirmed a shift of CXCR4 positive CLL cells from lymph nodes to peripheral blood, they also revealed that ibrutinib rapidly released CLL cells from the bone marrow. Also, unexpectedly, CLL cells redistributed to the orthotopic splenic cavernous system. Visualization of CLL on ibrutinib supports the pre-existing clinical hypothesis of a "compartment shift", however it also modified and refined the mechanistic model by describing early clearing of the bone marrow and re-distribution to the peripheral blood and the spleen. Disclosures Jaeger: Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria. Wester:Scintomics: Other: Spouse CEO of Company; CXCR4-targeted radiopharmaceuticals: Other: Inventor; Scintomics GmbH, Germany: Other: Shareholder. Staber:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria, Speakers Bureau; Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract 1541: Evaluation of ROR1 targeted antibody drug conjugate in ROR1 positive leukemia

Abstract Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is expressed on the surface of multiple blood and solid tumors. There have been increased interest in ROR1 as a potential therapeutic target in ROR1 positive leukemia and lymphomas. In this study, we present a novel first-in-class anti-ROR1 monoclonal antibody drug conjugate (ADC) based on chimeric rabbit/human mAb “XBR1-402” or its humanized version “huXBR1-402”. Conjugated to a highly potent anthracycline derived toxin, PNU-159682 (PNU) via a non-cleavable peptide/amide linker, it shows both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) cells. Results: We saw a decrease in viability for all ADC treated ROR1 positive leukemic cell lines. We saw a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU (EC50 = 77 and 1 ng/mL) when compared to treatment with isotype-matched control Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL). In in vitro models of MCL, we saw decreased viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 cell line when compared to Tras-PNU. While we were unable to observe significant direct cytotoxicity in primary CLL cells, we show that PNU ADCs mediates antibody dependent cellular cytotoxicity and phagocytosis in vitro. We also show that PNU ADCs mediates a G2/M cell cycle arrest in affected cell lines and hypothesize that this is necessary for direct cytotoxicity. As ex vivo primary CLL cells are non-proliferative, we postulate that PNU would target actively dividing cells in proliferation centers (e.g. secondary lymphoid organs). To test this hypothesis, we set up in vivo studies to test with XBR1-402-PNU and huXBR1-402-PNU in disseminated and aggressive ROR1+ ALL and CLL models, respectively. In vivo studies on a murine 697 ALL model suggested XBR1-402-PNU treatment increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days post implantation, p=0.021, n=6). In vivo CLL studies with engrafted murine human-ROR1 expressing TCL1 leukemic cells showed that huXBR1-402-PNU treatment (3 times/week for 1 week) both suppressed leukemia burden and increased overall survival when compared with Tras-PNU control (Median survival of 62 and 41 days post implantation, p=0.0028, n=4). Ongoing proliferation and cell cycling studies will confirm the mechanism of the in vivo cytotoxicity of huXBR1-402-PNU. Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration as either a single agent or combination therapy in patients with ROR1 positive leukemia and lymphomas. Citation Format: Eileen Hu, Priscilla Do, Rajeswaran Mani, Frank Frissora, Rebecca Pearson, Gerard Lozanski, Haiyong Peng, Lorenz Waldmeier, Roger Beerli, Christoph Rader, Ulf Grawunder, John Byrd, Natarajan Muthusamy. Evaluation of ROR1 targeted antibody drug conjugate in ROR1 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1541.

BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.

Efficacy of Ibrutinib-Based Regimen in Chronic Lymphocytic Leukemia: A Systematic Review.

Ibrutinib has shown to have better efficacy than standard chemoimmunotherapy in del17 positive chronic lymphocytic leukemia (CLL) patients; however its role in del17 negative patients is less clear. We aim to evaluate the efficacy of ibrutinib-based regimens in CLL. Seven databases were searched in accordance with PRISMA statement guidelines using the following keywords: chronic lymphocytic leukemia, CLL, Bruton tyrosine kinase inhibitor, BTK inhibitor, ibrutinib, and PCI-32765. Data from only prospective clinical trials was included. In a phase 3 trial (n = 136), the overall response rate (ORR) with ibrutinib was 92% whereas 18% patients had a complete response (CR). Progression free survival (PFS) and overall survival (OS) at 2 years were 89% and 95% respectively. Phase 3 trial (n = 195) with single agent ibrutinib showed ORR of 63%. PFS at 6 months and OS at 12 months were 88% and 90% respectively. In a phase 2 trial of relapsed and/or refractory (R/R) or high risk treatment naive (TN) patients, combination of ibrutinib and rituximab (n = 104) achieved an ORR of 100% (CR 28%) as compared to ORR 98% (CR 21%) with ibrutinib monotherapy (n = 102) with no significant difference in PFS. Combination of ibrutinib and ublituximab (n = 64) had an ORR of 78% (CR 7%) in a phase 3 study. In del17p negative R/R patients, combination of bendamustine/rituximab (BR) and ibrutinib (n = 289) achieved an ORR of 83% (CR/CRi 10%) and the 18 month PFS was 79%. In a phase 2 trial treated with ibrutinib (n = 145), patients with del17p R/R disease achieved an ORR of 64% and the 24 month PFS and OS was 63% and 75% respectively. In TN del17p patients (n = 35), ORR was 97% (CR-0) and the 24 month PFS and OS were 82% and 84% respectively with single agent ibrutinib. Ibrutinib is the treatment of choice for patients with del17p mutation and has good efficacy in RR/TN patients without del17p mutation. Ibrutinib is being evaluated in combination with rituximab for del17p mutations. Future prospects include combination of ibrutinib with frontline chemotherapy and other novel agents for TN and RR del17p negative patients.

Targeted Alpha Therapy with 212Pb-NNV003 for the Treatment of CD37 Positive B-Cell Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL)

BackgroundEach year more than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US. The standard of care is immuno-chemotherapy with alkylating agents in combination with an anti-CD20 monoclonal antibody (mAb). While immuno-chemotherapy regimens are initially effective in inducing responses, most patients inevitably relapse and the same therapies show decreasing efficacy with repeated administration.In recent years, CD37 has been considered as a target for B-cell malignancies. CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Therapies targeting CD37 expressing cells may become a useful alternative to CD20 targeting agents. Alpha-emitting radionuclides have demonstrated good potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm). The absorbed energy cause irreparable DNA double-strand breaks and localized cytotoxicity while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-emitting radioisotope 212Pb.Materials and MethodsDose-dependent efficacy and tolerability of a single-dose 212Pb-NNV003 treatment was evaluated using escalating activity levels in human disseminated models of Burkitt's lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. A study of the tolerability of 212Pb-NNV003 was performed in R2G2 mice without tumor prior to the efficacy studies.Results212Pb-NNV003 displays a favorable toxicity profile in tumor-free mice single intravenous dose injections up to 15 µCi showing 100 % survival 4 weeks post-injection. No acute hematological toxicity was observed and animals who received 5, 10 or 15 μCi doses of 212Pb-NNV003 presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered by 20 days after injection.A single intravenous dose of 10, 15 or 20 µCi of 212Pb-NNV003 lead to 70 %, 90 % and 100 % of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection (Figure 1). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively.A single intravenous dose of 2.5, 5 and 7.5 µCi 212Pb-NNV003 lead to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection (Figure 2). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively.ConclusionThe results of preclinical studies suggest that TAT using 212Pb-NNV003 is a safe and effective method for the treatment of CD37 positive CLL and NHL. [Display omitted] DisclosuresSaidi:Orano Med: Employment. Maaland:Nordic Nanovector: Employment. Torgue:Orano Med: Employment, Membership on an entity's Board of Directors or advisory committees. Heyerdahl:Nordic Nanovector: Employment, Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties.

The VLA-4 Integrin Is Constitutively Activated in a Subset of CD49d-Expressing CLL: A Relationship with the Autonomous BCR-Mediated Signaling?

Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of tumor cell-microenvironment interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its specific ligands. In this context, VLA-4 needs to be maintained in an activated state by a continuous stream of inside-out signals from the BCR, which can be triggered by canonical antigens as well as in an autonomous antigen-independent manner, a BCR-mediated signaling recently emphasized to specifically occur in CLL cells.Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this still not described feature with the presence of signals from the BCR continuously activating VLA-4.Methods. Expression of the integrin alpha (CD49c, CD49d, CD49e) and beta1 (CD29) chains was analyzed by flow cytometry. The VLA-4 activation state was investigated by flow cytometry using the conformational sensitive anti-CD29 monoclonal antibody HUTS21, employed in conjunction with sources of VLA-4 ligands: either autologous plasma-containing fibronectin (FN) and soluble (s) VCAM-1 or increasing concentrations of exogenously added LDV-containing peptides as a VLA-4 specific ligand. In detail: i) HUTS21 expression was analyzed using whole blood (WB) samples from 727 CLL patients. Negative controls were obtained after plasma depletion through washing of WB samples; ii) the VLA-4 activation state expressed as receptor occupancy (RO) (J Biol Chem, 284,14337, 2009) was analyzed in sequential (t=0, day14, day 30) frozen/thawed samples from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=16) and from a clinical trial (NCT02827617, n=14). BCR engagement was performed using goat F(ab′)2 anti-human IgM. ELISA assays were used to quantify FN and sVCAM-1 in plasma samples.Results. According to the 30% cutoff, CD49d was expressed in 351/727 (48%), while CD29 was expressed in 676/727 (93%), and a strong correlation between CD49d and CD29 was observed (rho=0.7, p<0.0001). Among CD49d positive CLL cases, 81/351 (23%) expressed a constitutively activated form of VLA-4 identified by >20% (arbitrary cut-off) HUTS21 expression. Notably, HUTS21 positive expression was not circumscribed to VLA-4 expressing CLL, being also detected in 64/375 (17%) CD49d-, CD49c+ and/or CD49e+ CLL, thus envisioning a role of other integrins (CD49c and CD49e) in CLL cell interactions in tissues sites. HUTS21 expression did not correlate with a definite cytogenetic group, IGHV mutational status or with a specific IGHV family and stereotype. Depletion of the plasma component from the WB samples before HUTS21 staining significantly reduced the proportion of HUTS21 positive cells compared with those measured in WB samples (p=0.001, n=11), suggesting the need of plasma-borne ligands for HUTS21 epitope exposure. Consistently, both FN (mean 350 μg/ml) and sVCAM-1 (mean 4.8 μg/ml) were detected in the plasma of CLL cases, irrespective to HUTS21 positivity. According to these observations, variable constitutive VLA-4 activation states were observed in CLL cells collected at the pre-treatment stage from patients undergoing IB treatment (VLA-4 RO ranging from 0.22 to 0.40); these values significantly decreased in CLL cells collected at day 14 (p=0.03) and day 30 (p=0.02) on IB, suggesting an IB-dependent impairment of antigen-independent (autonomous) BCR signals. The variability of the constitutive VLA-4 activation levels observed at pre-treatment was paralleled by variable VLA-4 activation levels upon BCR triggering (VLA-4 RO ranging from 0.56 to 0.65). Of note, an inverse correlation between the VLA-4 constitutive level and the extent of anti-IgM induced-VLA-4 activation was found (n=19, rho=-0.5, p=0.0093), implying a competition between antigen-independent and antigen-driven BCR signalling.Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a subset of CLL which might be connected to continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling, which is currently under evaluation. DisclosuresZaja:Takeda: Honoraria; Sandoz: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria.

Study of microRNA Profile as a Molecular Biomarker in Egyptian Chronic Lymphocytic Leukemia.

MicroRNAs target mRNAs for cleavage or translational repression. They play a critical role in the progression of malignancies and leukemias including chronic lymphocytic leukemia (CLL). However, microRNA expression levels in Egyptian patients with CLL, and their prognostic value remain elusive. Our main aim was to assess the expression pattern of a panel of microRNAs in CLL patients to create an informative microRNA profile. The study subjects were 40 newly diagnosed CLL patients of both sexes and 40 age and sex matched controls. The expression levels of 12 microRNAs were evaluated by qRT-PCR, including miR-15a, 16, 23b, 24, 29a, 29c, 34a, 146a, 155, 181a, 195, and 221. Flow cytometry was used to determine the expression levels of BCL2, CD38, and ZAP-70 in CLL patients. We identified various degrees of upregulated miRNAs (miR-29a, miR-29c, miR-34a, miR-155, miR-146a, and miR-195) and down-regulated ones (miR-15a, miR-16, miR-23b, miR-24, miR-181a, and miR-221) in CLL patients relative to controls. The mean fluorescence intensity ratio (MFI-R) of BCL2 was recorded and was significantly upregulated in CLL patients compared with normal controls. In addition, inverse correlations were observed between microRNAs (miR-15a, miR-16, miR-155, and miR-195) and BCL2 MFI-R while positive correlations were observed between miR-29a and miR-29c, and BCL2 MFI-R. These findings suggest that these miRNAs regulate BCL2 levels. Moreover, we found that miR-15a, miR-16, miR-155, miR-181a, miR-195 and miR-221 were significantly upregulated, while miR-29a and miR-29c were significantly downregulated in ZAP-70 positive CLL patients. Various miRNAs may play an important role in the pathogenesis of CLL and have the potential to be used for the prognosis of patients with CLL.

Clinical analysis of 70 chronic lymphocytic leukemia patients with trisomy 12 detected by FISH

目的总结探讨FISH检测12号染色体三体（CEP12）阳性慢性淋巴细胞白血病（CLL）患者的临床特征、治疗情况及预后。方法回顾性分析2003年5月至2015年4月接受FISH检测CEP12的330例CLL患者临床资料，比较CEP12阳性患者（70例）和CEP12阴性患者（260例）临床特征、疗效及预后差异。结果与CEP12阴性患者相比，CEP12阳性患者肝肿大（13.6%对4.0%，P=0.011）及LDH>247 U/L（43.3%对18.5%，χ2=15.892，P<0.001）比例更高，而发病年龄、性别、临床分期、β2微球蛋白水平、IGHV突变比例及脾脏/淋巴结肿大等方面差异无统计学意义。CEP12阳性与阴性患者比较，FMC7阳性率更高（23.8%对12.7%，χ2=4.730，P=0.030），CD23阳性率更低（95.2%对99.6%，P=0.033）。氟达拉滨组、利妥昔单抗组、传统治疗组总有效率（ORR）分别为77.5%（31/40）、84.8%（56/66）、45.4%（50/110），差异有统计学意义，且传统治疗组ORR均低于氟达拉滨组及利妥昔单抗组；氟达拉滨组中CEP12阳性患者ORR低于CEP12阴性患者（42.9%对84.8%，P=0.034）；而利妥昔单抗组中CEP12阳性患者ORR高于CEP12阴性患者（91.7%对81.0%），但差异无统计学意义（P=0.306）。CEP12阳性患者中位诊断至起始治疗时间为22.6（95%CI 15.4~31.7）个月，中位总生存（OS）时间为118.5（95%CI 74.5~162.4）个月，5年无进展生存（PFS）及OS率分别为（52.9±7.6）%、（74.8±6.6）%。CEP12阳性与阴性患者比较，PFS（χ2=0.410，P=0.478）及OS（χ2=0.052，P=0.180）差异均无统计学意义。结论CEP12阳性CLL患者临床肝肿大及LDH增高更为常见，传统治疗疗效最差，单用氟达拉滨疗效不如CEP12阴性患者，但予含利妥昔单抗的治疗方案其疗效与CEP12阴性患者相当。

The Regulation of STAT3 and Its Role in the Adhesion and Migration of Chronic Lymphocytic Leukaemia Cells

The bone marrow and lymph node microenvironments are important in promoting cell proliferation, survival and protection from drug induced apoptosis in chronic lymphocytic leukaemia (CLL). Chemokine networks, such as the CXCR4/CXCL12 axis, in combination with selectins, such as CD62L and integrins allow the migration of CLL cells to these protective niches. The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival. Further, it has been shown to interact with the signal transducer and activator of transcription 3 (STAT3) signalling pathway. The role of the STAT3 in CLL pathogenesis is unclear; however, it is constitutively phosphorylated on serine residue 727 (serine pSTAT3) in CLL cells.Here, we investigate the role of STAT3 in CLL cell survival and migration, using pharmacological inhibition and siRNA knockdown. Phospho-tyrosine and phospho-serine STAT3 were assessed by flow cytometry and western blotting. Apoptosis was assessed by Annexin V/Propidium Iodide staining by flow cytometry. The expression of cell surface markers involved in cell adhesion and homing was determined by multicolour flow cytometry.Stimulation of the BCR using immunoglobulin F(ab´)2 fragments induced tyrosine phosphorylation of STAT3 in CLL cells with unmutated immunoglobulin (IgVH) genes (n=7) but not mutated IgVH genes (n=5). This induced tyrosine phosphorylation was abrogated by pre-treatment with the Janus kinase (JAK) inhibitor Ruxolitinib and the BCR inhibitors Ibrutinib and Idelalisib (p<0.05, n=5). Gene expression studies using Taqman Assays showed BCR stimulation resulted in an upregulation of STAT3 regulated genes in CLL cells with unmutated IgVH genes. Interestingly, stimulation of BCR resulted in a significant increase in CD62L expression, which was inhibited by pre-treatment with Ibrutinib and Ruxolitinib (p<0.05, n=5).STAT3 inhibition was shown to have a divergent effect on CLL cell survival: In patient samples with >70% positive serine pSTAT3 cells, the STAT3 inhibitor cucurbitacin I induced apoptosis with a concurrent downregulation in serine phosphorylation (n=3); while in patient samples with <70% positive serine pSTAT3 cells, treatment with cucurbitacin I resulted in a decrease in apoptosis and a concurrent increase in serine phosphorylation (n=3). The STAT3 inhibitor S3I-201 had a similar effect but the upstream JAK inhibitor Ruxolitinib had no effect on serine phosphorylation and no effect on the apoptosis of CLL cells. In addition, siRNA mediated STAT3 knockdown and treatment with cucurbitacin I and S31-201, resulted in a significant decrease in CD62L positive CLL cells (p<0.0001, n=29).The role of STAT3 in CLL cell adhesion under shear flow conditions was investigated using a microfluidics system including a neMESYS Low Pressure syringe pump system and Human Umbilical Vein Endothelial Cells (HUVEC) coated biochips. Treatment of CLL cells with cucurbitacin I resulted in a significant decrease in adhesion to endothelial cells (p<0.001, n=4). The effect of STAT3 inhibition on the chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with cucurbitacin I resulted in a significant decrease in CLL cells migrated in response to the chemokine CXCL12 compared to control (p=0.0001, n=8). In addition, treatment of CLL cells with CXCL12 resulted in an increase in serine pSTAT3 that was downregulated by pretreatment with cucurbitacin I.This study has shown:1. Activation of STAT3 by BCR stimulation occurs in poor prognostic unmutated IgVH genes2. Serine pSTAT3 has a role in cell survival in response to STAT3 inhibition3. A role for STAT3 in CLL cell adhesion and migration, in particular in the regulation of the expression of CD62L.In conclusion, this study shows a role for the STAT3 pathway in cell survival and CLL cell-microenvironment crosstalk, suggesting therapeutic potential by interfering with the migration and homing of CLL cells to the lymph node and bone marrow microenvironments. DisclosuresNo relevant conflicts of interest to declare.

Surface CD5 Protein Risk Stratifies Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is a malignancy characterized by B-lymphocytes with aberrant expression of CD5. In normal T-lymphocytes, CD5 is an important regulator of T-cell receptor signaling. In CLL, CD5 acts as a repressor of B-cell receptor (BCR) signaling, whereby phosphorylated CD5 anchors SHP-1 at the cell membrane, resulting in inhibition of BCR-mediated second messenger signaling. The significance of CD5 on outcomes of CLL patients has not been evaluated. Because BCR signaling is an oncogenic stimulus in CLL, we hypothesized that higher levels of CD5 may be associated with superior clinical outcomes.Methods: Median fluorescence intensity (MFI) of CD5 on CLL cells was determined by flow cytometry. CLL samples and clinical data were obtained from patients enrolled in IRB-approved protocols at the Duke University and Durham VA Medical Centers. Molecular prognostic markers were measured as described previously. Descriptive and time to event (Cox proportional hazard and Kaplan-Meier) statistical analyses were performed in the statistical environment, R.Results: Between March 2011 and April 2015, 961 CLL samples were evaluated from 352 unique CLL patients. One to 10 samples were evaluated per CLL patient. The mean CD5 MFI was calculated for each unique patient, with a median of 206.59 (range 0.50 - 768.31) and median standard deviation of 33.02 (range 0.09 - 315.46) for CLL patients with multiple samples collected. 150 patients received therapy (44%), and there was no significant difference in CD5 MFI mean or standard deviation between patients who received and did not receive therapy. CD5 MFI values did not significantly differ among CLL demographic or prognostic groups. Cox proportional hazard ratio analyses showed that higher CD5 MFI is associated with longer time to therapy (TTT, p = 0.037), but not overall survival. Based on the distribution of CD5 MFI, we divided the CLL cohort into thirds, with low CD5 defined as MFI < 137, high CD5 defined as MFI > 283.5, and medium CD5 with MFI between these values. Kaplan-Meier analyses demonstrated a significant difference in TTT between these three groups (p = 0.004), with higher CD5 MFI associated with longer TTT. Moreover, CD5 MFI added to established molecular prognostic markers to risk stratify CLL patients (p < 0.0001 for CD38 and IGHV, p = 0.0003 for FISH, and p = 0.004 for ZAP70). Importantly, for those with good prognostic markers such as IGHV mutation or low CD38 expression, lower CD5 identified patients with shorter TTT (p = 0.01 for IGHV mutated; p = 0.0009 for CD38 negative). However, TTT was not significantly different in unmutated IGHV CLL patients or CD38 positive CLL patients with varying degrees of CD5 expression.Conclusions: Surface CD5 expression varies among CLL patients. For most individual patients, there appears to be a low level of variability in CD5 expression, regardless of CLL-directed therapy. Higher CD5 expression is associated with superior clinical outcomes in CLL, consistent with prior in vitro determination of CD5 as a negative regulator of BCR signaling. The relevance of CD5 in risk-stratifying IGHV mutated and CD38 negative CLL patients is of particular relevance, since these subgroups of CLL are particularly dependent on BCR-signaling as an oncogenic process. These findings are important both for prognostication and for development of therapies for this group of CLL patients. DisclosuresNo relevant conflicts of interest to declare.