Clinical interest in the measurement of Cereblon (CRBN), the primary target of the IMiDs immunomodulatory drugs lenalidomide and pomalidomide, has been fueled by its essential requirement for antitumor or immunomodulatory activity of both drugs in multiple myeloma (MM). However, limited analyses of clinical samples for CRBN gene expression or protein levels have utilized unvalidated reagents and assays, raising uncertainty about the interpretation of these results. We previously described a highly specific rabbit monoclonal antibody CRBN65 against 65-76 AA of human Cereblon. Here we describe a validated dual color bright-field Cereblon/CD138 immunohistochemical (IHC) assay utilizing CRBN65 and a commercial mouse monoclonal CD138 antibody. Sensitivity and specificity of the assay was determined and assay precision was shown for both cytoplasmic and nuclear Cereblon in MM bone marrow samples with coefficient of variation values of 5% and 2%, respectively. The dual IHC assay was effective for detecting a continuous range of Cereblon levels in 22 MM patient bone marrow core biopsies and aspirate clots, as shown by average cytoplasmic H-scores ranging from 63 to 267 and nuclear H-scores ranging from 17 to 250. Interpathologist comparison of MM sample H-scores by 3 pathologists demonstrated good concordance (R 2=0.73). This dual assay demonstrated superior Cereblon IHC measurement in MM samples compared with the single IHC assay using a published commercial rabbit polyclonal Cereblon antibody and could be used to explore the potential utility of Cereblon as a biomarker in the clinic.

Background: Cereblon (CRBN) is required for the antiproliferative activity of the IMiDs® immunomodulatory drugs lenalidomide (LEN) and pomalidomide (POM) in multiple myeloma (MM), but its value as a biomarker of IMiD response remains unknown. We have shown a disconnect between CRBN mRNA and protein regulation, indicating that measurement of CRBN protein is critical. However, accurate quantification of CRBN protein levels is challenging due to scarcity of standardized and validated reagents and assays. Several publications have suggested that CRBN mRNA and/or protein levels detected with commercial reagents and assays correlate with LEN and/or POM treatment outcomes. In an effort to establish a standardized and validated approach for CRBN protein measurement in MM biopsies, we developed a dual bright-field immunohistochemistry (IHC) assay to measure CRBN levels in CD138+ MM cells.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Abstract Background CNS manifestations of aggressive non-Hodgkin Lymphoma are associated with serious morbidity and adverse prognosis. Primary CNS lymphomas (PCNSL) exhibit a dichomatous growth pattern, either dissemination within brain, typical at presentation, and/or leptomeningeal spread, common at relapse. Elucidation of the mechanistic basis of CNS lymphoma progression as well as drug resistance requires preclinical models that recapitulate their pathogenesis. Methods We developed a novel method to derive cell lines of CNS lymphoma that recapitulate disease phenotypes upon intracranial implantation into mice. We are applying genomics, in vitrochemotaxis, preclinical testing of targeted therapies and neuroimaging to evaluate mechanisms of invasion and resistance. Results We developed 7 CNS lymphoma cell lines; 6 DLBCL (all ABC-type), 1 Burkitt; 5 from secondary CNS lymphoma (SCNSL), and 2 from PCNSL, of which 1 was treatment naïve. Intracranial implantation of lymphoma cells from these tumours within NSG mice provides a reproducible model to dissect the pathogenesis of CNS lymphomas. PCNSL specimens were 10X more efficient in CNS dissemination than SCNSL. High resolution array-CGH demonstrated that intracranial tumour growth was associated with retention of genomic aberrations of the original tumours (e.g. del 6q, gains on 12, etc) and that these were maintained with serial passage in vivo. CNS-infiltrative lymphomas expressed significantly increased levels of MMP-7 and RGS-13 transcripts compared to lymphomas that did not infiltrate brain, while osteopontin and cathepsin D expression by lymphoma cells did not correlate with CNS invasion. Targeted shRNA-mediated knockdown of RGS-13 was performed using lentiviral infection and resulted in significant delay of CNS lymphoma growth in vivo in a xenograft model but had no effect on lymphoma proliferation in culture. Therapeutic response to lenalidomide, minus and plus rituximab, was recapitulated in RAG-/- mice, despite deficient T-cell function and correlated with baseline relative cereblon expression, as quantified using a highly specific immunohistochemical assay. The emergence of resistance to lenalidomide in human CNS lymphoma xenografts also correlated with loss of cereblon protein expression, supporting a role for cereblon in the efficacy of lenalidomide in CNS lymphomas. Notably, significant cereblon protein expression by lymphoma cells was detected by immunohistochemistry in 12/22 diagnostic specimens of aggressive CNS lymphoma. Metabolic imaging of model CNS lymphomas using magnetic resonance spectroscopy demonstrated significant intratumoural lactate production in the microenvironment, detectable before evidence of aberrant T2 signal and reduced diffusion. Lenalidomide reduced tumour expression of lactate dehydrogenase and lactate, as well as RGS-13, consistent with anti-proliferative as well as anti-invasive effects. Conclusions To the best of our knowledge we have developed the first panel of patient-derived CNS lymphoma cell lines. We have used these to generate intracranial xenografts that provide a highly reproducible model system to dissect key elements of CNS lymphoma pathogenesis, leading to the elucidation of mechanisms of CNS lymphoma growth and invasion as well as resistance. Our results support a direct, T-cell independent effect of lenalidomide on CNS lymphoma growth and invasion which may be cereblon-dependent. Additional studies are needed to define the role of cereblon as a biomarker and mediator of lenalidomide efficacy in CNS lymphomas. In addition, we are using these models to identify novel genomic and metabolic aberrations predictive of early resistance to lenalidomide and other targeted therapies. Supported by the Lymphoma Research Foundation, Leukemia and Lymphoma Society, and by NIH R01CA139-83-01A1. Disclosures: Heise: Celgene: Employment, Equity Ownership. Rubenstein:Celgene: Research Funding; Genentech: Research Funding. Off Label Use: use of lenalidomide in CNS lymphoma.

Abstract Abstract 2053 Poster Board II-30 Activating mutations in the receptor tyrosine kinase FLT3 are present in up to 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a potential target for kinase inhibitor therapy. AC220, a potent and selective FLT3 inhibitor, is currently in Phase II clinical trials in both FLT3-ITD positive and WT patients. Similar to imatinib therapy for CML, it is possible that targeted therapies will be delivered chronically. Therefore, we examined the efficacy of AC220 in a limited versus chronic dosing regimen in the homozygous FLT3-ITD-dependent MV4-11 disseminated mouse disease model. As AML is a heterogeneous disease, we also examined the in vivo efficacy of AC220 in the MOLM-14 disease model. This cell line is heterozygous for a FLT-3 ITD mutation in addition to carrying the MLL-AF9 fusion. In the MV4-11 model, a 30 day dosing regimen was compared to chronic dosing. In the vehicle control group, median survival time following inoculation was 47 days, with mortality preceded by clinical signs of disease and detection of MV4-11 cells in the blood. No specific clinical signs or body weight loss were attributed to the study drug. AC220 demonstrated dose-dependent efficacy from 0.1 mg/kg to 10 mg/kg orally once per day for 30 days. The 0.1 mg/kg group had a marginal (10%) increase in life span (ILS) relative to vehicle control, while a significant increase of survival was observed at the 1.0 mg/kg dose (55% ILS). The 10 mg/kg dose led to 80% survival at study termination, day 172 (>250% ILS). Prolonged survival with AC220 correlated with delayed disease onset as measured by clinical signs and detection of circulating MV4-11 cells. Chronic dosing in the 1.0 mg/kg group further delayed disease onset and mortality with an ILS of 155% relative to vehicle, and 63% relative to 30-day dosing. Similar to the 30-day dosing group, chronic administration of AC220 at 10 mg/kg led to 80% survival at day 172. A separate study was conducted to examine the relationship between bone marrow engraftment, tumor burden in peripheral blood and disease onset. At day 20, engraftment was detected only in the bone marrow. At day 35, when clinical signs of disease are typically apparent, levels of tumor cells as high as 80% and 35% were detected in the bone marrow and peripheral blood, respectively. AC220 given for 28 days at 1.0mg/kg delayed median onset of disease by 24 days (63%), with tumor burden undetectable in the absence of clinical signs of disease. The 28 day dosing at 10.0mg/kg completely inhibited disease onset, with no detectable tumor burden in either blood or bone marrow through the end of the study (terminated on day 130, >200% ILS). In the MOLM-14 model, median survival time of untreated or vehicle treated animals was 22 days. In a 21 day dosing regimen, AC220 demonstrated dose dependent efficacy, providing 9%, 64% and 127% ILS at 0.1, 1.0 and 10 mg/kg respectively. Prolonged survival correlated with a delay in both disease onset and detection of circulating MOLM-14 cells. Similar to the MV4-11 model, temporal analysis confirmed bone marrow engraftment prior to detectable levels in the circulation. At 13 days post-transplant, tumor cells are only detectable in the bone marrow, while by day 19, when clinical signs are apparent, tumor burden in the bone marrow and periphery of vehicle treated animals are as high as 35% and 6%, respectively. Unlike the MV4-11 model, dosing at 10 mg/kg for 21 days did not prevent disease onset after cessation of dosing, indicating that tumor cells were not completely eliminated, although detectable levels are not present in bone marrow or circulation until days 47 and 52, respectively. These data suggest that AC220 is efficacious against both FLT3-ITD homozygous and heterozygous genotypes, and that a chronic dosing regimen may provide greater disease protection than a limited course of therapy. This study is consistent with observed efficacy in AML patients treated continuously with AC220. Disclosures: Brigham: Ambit Biosciences: Employment. Belli:Ambit Biosciences: Employment. Breider:Ambit Biosciences: Employment. Bhagwat:Ambit Biosciences: Consultancy. Wierenga:Ambit Biosciences: Employment. Armstrong:Ambit Biosciences: Employment.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a potential target for kinase inhibitor therapy. The efficacy of AC220, a potent and selective FLT3 inhibitor, was determined in a SCID mouse model where intravenously inoculated FLT3-ITD-dependent MV4-11 cells disseminate to the bone marrow, comparing a 30 day dosing regimen to continuous dosing. In the vehicle control group, median survival time following inoculation was 47 days, with mortality preceded by clinical signs of disease and detection of MV4-11 cells in the blood. No specific clinical signs or body weight loss were attributed to the study drug . AC220 demonstrated dose-dependent efficacy from 0.1 mg/kg to 10 mg/kg orally once per day for 30 days. The 0.1 mg/kg group had a marginal (10%) increase in life span (ILS) relative to vehicle control, while a significant increase of survival was observed at the 1.0 mg/kg dose (55% ILS). The 10 mg/kg dose led to 80% survival of the animals at day 172 when the study was terminated (>250% ILS). Prolonged survival with AC220 correlated with delayed disease onset as measured by clinical signs and detection of circulating MV4-11 cells. Continuous dosing in the 1.0 mg/kg group further delayed disease onset and mortality with an ILS of 155% relative to vehicle, and 63% relative to 30-day dosing. Continuous dosing with 10 mg/kg AC220 provided similar benefit to the 30 day dosing paradigm. A 28 day dosing study was conducted to examine the relationship between bone marrow engraftment, tumor burden in peripheral blood and disease onset. At day 20, engraftment was detected only in the bone marrow. At day 35, when clinical signs of disease are typically apparent, levels of tumor cells as high as 80% and 35% were detected in the bone marrow and peripheral blood respectively. AC220 dosed at 1.0mg/kg delayed median onset of disease by 24 days (63%), with tumor burden undetectable in the absence of clinical signs of disease. AC220 dosed at 10.0mg/kg completely inhibited disease onset, with minimal tumor burden detected in either blood or bone marrow in only 1 of 8 animals through the end of the study (terminated on day 130, >200% ILS). Anti-leukemic effects of traditional chemotherapy are commonly accompanied by significant myelosuppression. Although 30-day and continuous dosing of AC220 suppressed myeloid leukemic cell counts at all doses, there was no generalized myelosuppression at the 1 mg/kg dose as indicated by absolute neutrophil counts. Continuous 10 mg/kg dosing led to approximately a 2-fold reduction in neutrophils. These data correlate with observations in an MV4-11 solid tumor model where AC220 showed substantial and dose dependent efficacy when dosed orally once a day for 28 days (Cortes et al .: Blood 2007). AC220 is currently under evaluation in a phase I clinical trial in relapsed or refractory AML patients. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2335.