Search

Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTKC481 , we identified BTKT316A , a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts. Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

The BTK inhibitor ibrutinib has demonstrated a remarkable therapeutic effect in mantle cell lymphoma (MCL). However, approximately one-third of patients do not respond to the drug initially. To identify the mechanisms underlying primary ibrutinib resistance in MCL, we analyzed the transcriptome changes in ibrutinib-sensitive and ibrutinib-resistant cell lines on ibrutinib treatment. We found that MYC gene signature was suppressed by ibrutinib in sensitive but not resistant cell lines. We demonstrated that MYC gene was structurally abnormal and MYC protein was overexpressed in MCL cells. Further, MYC knockdown with RNA interference inhibited cell growth in ibrutinib-sensitive as well as ibrutinib-resistant cells. We explored the possibility of inhibiting MYC through HSP90 inhibition. The chaperon protein is overexpressed in both cell lines and primary MCL cells from the patients. We demonstrated that MYC is a bona fide client of HSP90 in the context of MCL by both immunoprecipitation and chemical precipitation. Furthermore, inhibition of HSP90 using PU-H71 induced apoptosis and caused cell cycle arrest. PU-H71 also demonstrates strong and relatively specific inhibition of the MYC transcriptional program compared with other oncogenic pathways. In a MCL patient-derived xenograft model, the HSP90 inhibitor retards tumor growth and prolongs survival. Last, we showed that PU-H71 induced apoptosis and downregulated MYC protein in MCL cells derived from patients who were clinically resistant to ibrutinib. In conclusion, MYC activity underlies intrinsic resistance to ibrutinib in MCL. As a client protein of HSP90, MYC can be inhibited via PU-H71 to overcome primary ibrutinib resistance.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Introduction Ibrutinib (ibr), a first-in-class BTK inhibitor, has high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL) independent of high-risk FISH abnormalities (NEJM. 2015; 373:2425-37, NEJM. 2013; 369:32-42). However, about 25% of patients discontinue ibr therapy at a median of 20 months treatment and ~40% patients stop ibr due to disease progression (JAMA Oncol 2015; 1:80-7, Blood 2015; 125:2062-67). Among progressed patients, at least half developed Richter's transformation (RT). Treatment options following progression are limited, with mortality rates exceeding 75% and a short median overall survival of 3 months. As the use of ibr becomes more prevalent in CLL and other types of non-Hodgkin lymphoma (NHL), more patients are at risk to develop resistance (BJH 2015; 170:445-56). Strategies to prevent and treat ibr-relapsed patients by understanding mechanisms of resistance are critically needed and will support rational drug development and therapeutic approaches. Recent studies including ours have provided some insights into ibr-resistance. Both BTK and PLCG2 mutations have been shown to confer ibr-resistance (NEJM 2014; 370:2352-54, NEJM 2014; 370:2286-94). Additionally, TRAIL-R has also been associated with the drug resistance in several ibr-relapsed patients (Nat Comm 2016). However, there is still limited understanding in the setting of disease progression during ibr treatment, such as what risk factors predispose patients to relapse, whether other molecular or cytogenetic lesions are associated with disease progression, and how RT is related to CLL tumor cells in the blood. Materials & Methods To address these questions, we analyzed 9 CLL patients treated with ibr and relapsed at the University of Chicago between 2008-2016. Among 9 patients, 6 developed RT at progression. The median age was 66.3 yrs (range, 52-88) and the median number of therapies prior to ibr initiation was 2 (range, 1- 4). The median duration of response to ibr was 16 months (range, 2-30). Eight patients discontinued ibr therapy due to CLL progression or RT. Longitudinal samples including peripheral blood (PB), bone marrow (BM) and tissue were collected from each patient at time points prior to ibr initiation (pre-ibr), post-relapse and at the time of RT. When possible, samples were also collected during the responding phase. Samples were analyzed using both UCM-OncoPlus, a hybrid capture 1,212 cancer-associated Next generation sequencing (NGS) gene panel and Affymetrix SNP arrays (CytoScan and OncoScan) to assess mutations, indels, copy number variations (CNVs) and loss of heterozygosity. A custom algorithm was developed to calculate mutant clonal frequencies (MCFs) using an integrative approach combining allelic frequencies, tumor purity and CNV data. K-means clustering was performed to identify gene mutations belonging to the same clonal populations. An amplicon-based 17-gene CLL panel was further used to sequence BTK in greater depth (~10,000x) to confirm the presence of minor clones (1-5%) in some samples. Results & Conclusions To determine the risk factors associated with relapse, we compared all pre-ibr samples for recurrent molecular and cytogenetic abnormalities. Eight of nine patients were found to have TP53 mutations and/or del (17p), several other genetic lesions also seem to be enriched in our cohort compared to larger populations of treated or untreated CLL patients. The mutation profiles of matched pre-ibr and relapsed CLL PB/BM for each patient were then compared to identify relapse-specific alterations that might drive CLL progression. As expected, BTK mutations were found in 5 of 9 patients including previously reported C481S/R as well as a structurally novel T316A located in the SH2 domain. The findings confirm that mutated BTK is the most common mechanism responsible for disease progression on ibr. Furthermore, the emergence of minor BTK clones was detected in progressed patients. Analyses are ongoing to determine the clonal relationship between CLL leukemia in PB/BM and large cell transformation at the tissue site. So far, IGH gene rearrangement assay on three of five patients demonstrated that the CLL and RT are of the same B-cell origin. We are also in the process of performing cluster analyses to understand mutations that travel in the same malignant clones or sub-clones in each patient and updated results will be presented. Disclosures Smith: Celgene: Consultancy; Amgen: Other: Educational lecture to sales force; Genentech: Consultancy, Other: on a DSMB for two trials ; AbbVie: Consultancy; TGTX: Consultancy; Gilead: Consultancy; Portola: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy. Wang:Portola Pharmaceuticals: Honoraria, Research Funding.

Abnormal upregulation of NFκB activity is observed in a variety of B cell malignancies, resulting in proliferative and survival signals that contribute to tumor progression. Under normal resting conditions, NFκB is negatively regulated principally via its physical association with IκB (inhibitor of NFκB) family members, thereby inhibiting nuclear transport or access to DNA. In B cells, NFκB is typically activated via various external stimuli (e.g., ligation of the B cell antigen receptor (BCR), toll-like receptors, cytokine receptors, CD40), leading to IκB kinase complex-dependent phosphorylation of IκB members and targeting them for ubiquitination and degradation. In some cases, the need for external stimuli is diminished or completely circumvented by mutations to critical regulators of NFκB, as has been described in the context of activating mutations to CD79A/B, MYD88, and CARD11, as well as inactivation of negative regulators such as A20 and IκB family members (reviewed by Staudt, 2010). Each of these mutations has been observed clinically in patients with B cell malignancies (Wilson et al, 2012; Norenberg et al, 2015; Mansouri et al, 2015), and can impact the anti-tumor activity of selective BCR pathway inhibition (Davis et al, 2010; Wilson et al, 2012) in part via induction of autocrine cytokine stimulation leading to JAK/STAT-dependent up-regulation of MCL1 (Lam et al, 2008). We previously reported that cerdulatinib, a small molecule kinase inhibitor that dually targets SYK and the JAK family members JAK1, JAK3, and TYK2, maintained anti-tumor activity in DLBCL cell lines bearing mutations to CARD11, MYD88, and A20 (Ma et al, 2015). The majority of DLBCL cell lines exhibit various degrees of reliance on SYK and JAK signaling for survival, however in a screen of 15 DLBCL cell lines we found 3 that were completely resistant to cerdulatinib and are described here. In one of the cerdulatinib-resistant cell lines, RCK8, next generation sequencing revealed bi-allelic inactivation of the IκBα gene. One allele carries a frameshift mutation in exon 1 resulting in the generation of a premature stop codon, and the second allele is a nonsense mutation in exon 3 at Gln154, also leading to a stop codon. In accord with a previous report (Kalaitzidis et al, 2002), the cell line lacks IκBα protein expression. We therefore proceeded to explore the possibility that loss of IκBα was responsible for resistance to cerdulatinib. Consistent with the loss of IκBα, the RCK8 cell line exhibited enhanced basal NFκB activity. Genetic re-introduction of wild type IκBα led to rapid suppression of NFκB, and ultimately cell cycle arrest and cell death, indicating that the cell line was dependent upon loss of this gene for survival. Associated with suppression of NFκB was decreased phosphorylation of cellular pAKT S473 and pERK Y202, but not of pSTAT3 Y705. We then attempted to knock down IκBα in cerdulatinib-sensitive cell lines using siRNA to determine if resistance to SYK/JAK inhibition could be generated. None of the DLBCL cell lines tested (n=4) could tolerate IκBα gene knock down, suggesting an additional mutation in RCK8 enables survival under conditions of homozygous loss of IκBα. Ligation of CD40 leads to a transient down-regulation of IκBα at the protein level (Oeckinghaus and Ghosh, 2009). We therefore examined the effect of CD40L on multiple DLBCL cell lines and found that IκBα was maximally suppressed within 30-60 minutes post CD40 stimulation, returning to pre-treatment levels by 2-4 hours. In contrast, the impact on NFκB activation was much longer, and 5 of 7 cerdulatinib-sensitive cell lines tested were made resistant by incubation with CD40L. Associated with this resistance was not only induction of NFκB, but also pERK Y204, pAKT S473, and pSTAT3 Y705. Interestingly, whereas the CD40L-induced NFκB activation was not inhibited by cerdulatinib, the other signaling events were, despite the generation of resistance. Loss of IkB family members has been described in the context of Hodgkin's lymphoma, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia (Cabannes et al, 1999; Norenberg et al, 2015; Mansouri et al, 2015). Here we demonstrate that loss of IκBα in multiple DLBCL cell lines generates resistance to cerdulatinib. We will be exploring the clinical relevance of these in vitro observations in cell lines as part of an ongoing phase II trial of cerdulatinib in patients with relapsed/refractory B cell malignancies. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Feng:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Wang:Portola Pharmaceuticals: Honoraria, Research Funding. Michelson:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Curnutte:3-V Biosciences: Equity Ownership; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding; Sea Lane Biotechnologies: Consultancy. Conley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

The Bruton tyrosine kinase (BTK) inhibitor, ibrutinib has produced durable remissions in chronic lymphocytic leukemia (CLL). We describe a CLL patient who progressed while receiving ibrutinib following 20 months of once daily dosing. A cysteine-to-serine amino acid replacement was identified in BTK at position 481 that disrupts the covalent, but not non-covalent, binding of ibrutinib to BTK in silico structural modeling1. The mutation was present in relapsed samples while absent in the pre-treatment and responding samples. Following the mutation, the B cell receptor (BCR) pathway was reactivated as evidenced by increased cell signaling activities and gene expression profiles. Comparing the relapsed samples with the pre-treatment and responding samples, at the cellular level, mutated CLL cells displayed higher levels of the cell proliferation marker Ki67 in vivo and higher levels of ex-vivo BrdU incorporation. Transfection of the C481S mutant construct into a sensitive lymphoma cell line rendered it much more resistant to ibrutinib treatment demonstrating the cellular impact of the mutation (see attached graph). Interestingly, the ibrutinib-resistant CLL cells remained sensitive to other BCR inhibitors such as dasatinib and SYK inhibitors. These results confirm BTK as an important pharmacologic target of ibrutinib. Further, a mechanism of resistance was revealed, and alternative therapeutic options for ibrutinib resistance were explored. (First three authors with equal contribution) Disclosures: Furman: Genentech: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Gilead: Consultancy. Chang:Pharmacyclics: Employment, Equity Ownership.

With increasing understanding of the genetic basis of diffuse large B cell lymphoma (DLBCL), more targeted therapies have been developed. However, most of these therapies have activities only against a subset of DLBCL, activated B-cell like subtype (ABC), in particular, where the BCR pathway is known to be chronically active. Specific single-molecule directed therapies, although effective, often induce resistance over long term treatment. Agents with broader activities are needed for the treatment of this heterogeneous disease. Since both the BCR and JAK/STAT pathways are strongly implicated in the pathogenesis of DLBCL, in the present study, we evaluated the anti-lymphoma activity of PRT062070 (PRT2070), a novel compound that dually targets both JAK and SYK signaling pathways. We analyzed a panel of DLBCL cells lines, of both ABC and germinal center B-cell like (GCB) subtypes, as well as DLBCL patient samples, for cellular and molecular events affected by PRT2070. Immunoblotting analyses showed that ABC and GCB subtype of DLBCL cells exhibit different JAK/STAT and BCR signaling profiles. For instance, AKT was highly expressed in GCB cells, whereas STAT3 was more strongly expressed in ABC cells. In GCB cell lines, PRT2070 blocked G1/S transition and caused cell cycle arrest. In contrast, in ABC cells, the drug induced apoptosis and down-regulated MCL1 protein. Furthermore, array of PRT2070-treated ABC subtype of DLBCL cells revealed that several STAT3 regulated cytokines and chemokines, including IL10, were down-regulated, confirming that PRT2070 affects ABC-DLBCL cells via JAK/STAT pathway. Genetic knockdown of both JAK and SYK reduced the growth and the survival of DLBCL cells more pronouncedly than knockdown of JAK or SYK alone, suggesting the anti-tumor activity of PRT2070 was mediated by dual inhibition of JAK and SYK signaling pathways. Importantly, JAK/STAT and BCR signaling can be blocked by PRT2070 in GCB and non-GCB primary human DLBCL cells, which led to death of these cells. Our work provided mechanistic insights into the actions of JAK/SYK dual inhibitor PRT2070 suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures: Coffey: Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment.

Abstract 5068 Busulfan, a highly effective and established drug in polycythemia vera (PV), produces lasting clinical and hematologic responses. Its use as a first and second line therapy for PV recently diminished owing to largely unsubstantiated concerns of increased leukemogenicity. However, in a pivotal phase III trial of busulfan vs. P32 conducted by the EORTC in patients with PV, busulfan sustained long term clinical and hematologic responses as well as superior 10 year overall survival (70% vs. 55%). Toxicity was minimal and a low incidence of acute leukemia was reported (1% at 8 years). Accordingly, we treated 5 PV patients with busulfan, 4 of whom were refractory to multiple drugs including hydroxyurea (HU), pegylated interferon alfa-2a (pIFN), imatinib, dasatinib, and anagrelide. Clinical and hematologic response was graded according to PVSG criteria and molecular response according to ELN criteria. JAK2V617F allele burdens were determined by pyrosequencing, which quantifies mutant alleles more than 5%. If negative by pyrosequencing, we used an ARMS-PCR assay with a sensitivity of 0. 1%. Phlebotomy was performed to maintain the hematocrit (Hct) less than 45% for men and 42% for women. Treatment with busulfan was discontinued if patients experienced adverse side effects and/or had platelet counts less than 100, 000/mL while in clinical remission. All 5 patients had complete hematologic responses (CHR) within 3 months of starting busulfan (table 1). Molecular responses (MR) were: 1 complete (CMR) after 6 months, 1 partial (PMR) after 6 months, and 3 with no response (NMR) after 3, 7, and 60 months of treatment respectively. The 2 patients who had MR were homozygous for JAK2V617F, and the 3 who did not were heterozygous. Treatment was discontinued in the patient with CHR and CMR after 7 months due to thrombocytopenia; the patient has since maintained CHR and CMR for 3 years off treatment. The remaining 4 patients have maintained CHR on low doses of busulfan (table 1). No adverse events were observed over a median treatment duration of 15 months (range: 3–60 months). The significant difference in molecular response between patients with homozygous and heterozygous JAK2 mutations receiving similar doses of busulfan is noteworthy. This suggests a susceptibility of homozygous JAK2V617F clones to busulfan, but not to other drugs including HU, IFNa, and anagrelide. In summary, our 5 patients with multidrug refractory PV had rapid and sustained hematologic responses to busulfan at low doses, with favorable short and long term toxicity profiles. Two JAK2V617F homozygous patients had the best MR. Our findings indicate the effectiveness of a safe, relatively inexpensive drug in inducing clinical outcomes not significantly different from that of costly drugs, such as JAK2 inhibitors. In addition, the high rates of MR we observed in patients with homozygous JAK2 mutations warrant further study of busulfan with respect to this parameter. Table 1: Demographics and treatment results of 5 patients treated with busulfan for PV Patient Age (yr)-Sex (M/F) Prior treatments-duration (yr) Busulfan dose Adverse effects Duration of tx (months) Hematologic response/time to response (months) Molecular response/time to response (months) Pre-busulfan JAK2 allele burden 1 75-F HU-2 yr 4mg daily thrombocytopenia 7 CHR/3 CMR/6 100% 2 70-F HU+anagrelide-1yr Imatinib-2yr Dasatinib-3 yr IFNa-1 yr 2mg 3 times a week None 15 CHR/1 PMR/6 85% 3 84-F HU-2 yr Dasatinib- 3yr Imatinib-1yr 2mg 4 times a week None 18 CHR/3 None 27% 4 81-M HU-5 yr 2mg daily None 3 CHR/2 None 23% 5 81-F None 2mg 3 times a week None 60 CHR/3 None 45% Disclosures: No relevant conflicts of interest to declare.

Abstract 1249 Poster Board I-271 Purpose B-cell receptor (BCR) signaling plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). However, blocking BCR signaling with dasatinib, an inhibitor of SRC kinase, produced variable results in pre-clinical and clinical studies. We aim to define the molecular mechanisms underlying the differential dasatinib sensitivity and to uncover more effective therapeutic targets in CLL. Experimental Design Fresh CLL B cells were treated with dasatinib and cell viability was followed. The CLL cases were then divided into good and poor responders. The cellular response was correlated with the activities of BCR signaling molecules as well as with molecular and cytogenetic prognostic factors. Results and Conclusions Among 44 CLL cases, dasatinib treatment reduced cell viability by 2-90%, with an average reduction of 48% on day four of culture. The drug induced CLL cell death via the intrinsic apoptotic pathway mediated by reactive oxygen species. Unexpectedly, phosphorylation of SRC family kinases was inhibited by dasatinib in good as well as poor responders. As opposed to SRC family kinases, activities of two downstream molecules, SYK and PLCγ2, correlate well with the apoptotic response of CLL cells to dasatinib. Thus, SYK inhibition predicts cellular response to dasatinib. SYK, together with PLCγ2, may serve as potential biomarkers to predict dasatinib therapeutic response in patients. From the pathogenic perspective, our study suggests the existence of alternative mechanisms or pathways that activate SYK independent of SRC kinase activities. The study further implicates that SYK might serve as a more effective therapeutic target in CLL treatment. Disclosures Lee: Bristol-Myers Squibb Research & Development: Employment. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.

B cell receptor (BCR) signaling plays an essential role in the pathogenesis of chronic lymphocytic leukemia. In a subset of patients with a poor clinical outcome, BCR ligation leads to increased cell metabolism and cell survival (Cancer Research66, 7158–66, 2006). Based on these findings, we tested whether targeting BCR signaling with dasatinib, an inhibitor of Src kinase, would interfere with the signaling cascade and cause death of CLL B cells. CLL leukemic cells were isolated from 34 patients and were incubated with or without dasatinib at a low dose of 128 nM. Among 34 cases, viability of leukemic cells was reduced by 2% to 90%, with an average of ~50% reduction on day 4 of ex vivo culture. Further study showed that CLL B cells undergo death by apoptosis via the intrinsic pathway which involves the generation of reactive oxygen species. Analysis of the Src family kinases showed that phosphorylation of Src, Lyn and Hck was inhibited by dasatinib not only in those cases that responded to dasatinib with apoptosis, but also in those that did not respond well ( Statistical analysis showed a significant correlation between CLL dasatinib response and their IgVH mutation and ZAP70 status. Cases with worse prognoses by these criteria have a better response to the kinase inhibitor. Lastly, we have also found that ZAP70 positive cases showed a greater degree of PLC

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear hormone receptor involved in maintaining glucose and fatty acid homeostasis. Besides its metabolic functions, the receptor has also been implicated in tumorigenesis. Ligands of PPAR gamma induce apoptosis in several types of tumor cells including lymphomas, leading to the proposal that these ligands may be used as anti-cancer agents. However, apoptosis induction requires high doses of ligands, suggesting the effect may not be receptor-dependent. Previously, we reported that PPAR gamma is expressed in human primary T cell lymphoma tissues and activation of PPAR gamma with low doses of ligands protects lymphoma cells from serum starvation-induced apoptosis. The prosurvival effect of PPAR gamma is linked to its actions on mitochondria. In serum-deprived cells, PPAR gamma attenuates the decline in ATP, reduces mitochondrial hyperpolarization and limits the amount of reactive oxygen species (ROS) in favor of cell survival (Yang et al, Am J Pathol, 170, 722–32, 2007). In the current study, we investigated the molecular mechanisms by which PPAR gamma maintains mitochondria homeostasis. We demonstrated that PPAR gamma modulates the activities of two key components of Wnt signaling pathway, Fzd4 and GSK-3 beta. The receptor up-regulates the mRNA expression of Fzd4, a cell surface receptor of Wnt, through a conserved PPAR-response element (PPRE) in the promoter region of the Fzd4 gene. Moreover, PPAR gamma suppresses the activation of GSK-3 beta, a downstream inhibitory serine/threonine kinase, by maintaining its phosphorylation at serine 9 and decreasing its association with mitochondria. Downregulation of GSK-3 beta activity results in maintenance of mitochondrial membrance potential and suppression of ROS production in growth factor-deprived cells. Our studies revealed a novel interaction between PPAR gamma and Wnt signaling in lymphocyte survival. Since Wnt signaling plays an important role in tumorigenesis, our studies highlight the need for further investigation into the role of PPAR gamma in cancer prior to widespread use of its agonists as anticancer therapeutics.

Somatic hypermutation of immunoglobulin genes (SHM) is a physiological process that helps generate high affinity antibodies in germinal center B cells, and error-prone DNA polymerases are key enzymes involved in this process. Unlike polymerases that are responsible for DNA replication, error-prone DNA polymerases copy DNA with low fidelity leading to variations in immunoglobulin DNA sequences. Chronic lymphocytic leukemia (CLL) has been classified into mutated and unmutated subtypes based on the mutation status of the IGH variable region (IgVH). The latter has been associated with rapid disease progression and unfavorable outcome. Since determination of the IgVH mutations rely on direct sequencing of multiple PCR products and complex data analysis, the assay, although clinically useful, is not provided by many clinical laboratories. To determine whether error-prone DNA polymerases can be used as surrogate markers for IgVH mutation analysis, we examined the levels of several of them to see whether higher levels are associated with higher degree of IgVH mutation. We analyzed 30 CLL samples, of which 18 were unmutated and 12 were mutated. Quantitative PCR was performed to determine the expression levels of error-prone DNA polymerases mu, eta, iota, lambda and zeta. as well as DNA editing enzymes, terminal deoxynucleotidyl transferase (TdT) and activation-induced cytidine deaminase (AID). Statistical analysis revealed no differences in the levels of mRNA expression of any of these seven enzymes between mutated and unmutated cases of CLL. In addition, no differences were found between ZAP-70 positive and negative subgroups. The expression levels of the seven enzymes were then compared among CLL, follicular lymphomas (FL, n=8) and diffuse large B cell lymphomas (DLBCL, n=8). FL and DLBCL, being post-germinal center lymphomas that have undergone SHM, are expected to express higher levels of these enzymes than CLL. However, the statistical analysis revealed that CLL has significantly higher amount of mu, eta, iota, lambda, zeta and TdT expression than DLBCL. CLL also has significantly higher expression of iota, lambda, zeta and TdT than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.than FL. In conclusion, our results suggest that DNA polymerases studied cannot be used as surrogate markers for IgVH analysis. Contrary to the common belief, the mRNA levels of DNA polymerases/DNA editing enzymes do not reflect the SHM activity in different B cell malignancies.

The excess production of monoclonal immunoglobulin is the hallmark of multiple myeloma (MM) diagnosis and is an essential element of the determination of disease response to therapy. The development of new monoclonal immunoglobulins that are distinct from the baseline diagnostic paraprotein during the course of MM therapy can therefore pose a challenge in assessing disease response or potential relapse. There are prior reports of the development of abnormal protein banding (APB) after autologous stem cell transplantation for MM comprised of transient non-myeloma related mono- or oligoclonal protein bands that have been correlated with higher rates of event-free and overall survival. We now report the development of atypical serum immunofixation patterns (ASIPs) for the first time outside of the setting of stem cell transplant during the course of MM induction therapy with the BiRD (Biaxin® [clarithromycin], Revlimid® [lenalidomide], Dexamethasone) regimen. 72 patients with newly diagnosed Stage II and III MM (Salmon-Durie Criteria) were treated in a phase 2 clinical study of BiRD. The BiRD treatment regimen was given in 28-day cycles as follows: Clarithromycin 500mg po BID for days 1 – 28, Lenalidomide 25mg po daily for days 1 – 21, and Dexamethasone 40mg po weekly on days 1, 8, 15, and 21. The median age of the patients was 63 (range 36–83), and the baseline monoclonal immunoglobulins detected on serum immunofixation were as follows: 61% IgG, 22% IgA, 17% light chain only. 24 patients (33%) developed one or more ASIPs with either a mono- or oligoclonal banding pattern during the course of BiRD therapy. The new protein bandsvaried widely in duration of appearance (range 26–315 days) as well as isotype with IgM-κ, IgM-λ, IgG-κ, IgG-λ, IgA-λ, free λ light chain, and free IgM heavy chain all observed in one or more instances. The extent of therapy prior to first ASIP development was variable as well, (range 27– 724 days, median of 178 days). Patients with ASIPs had significantly better response to BiRD vs. non-ASIP patients (p = .00002), with CR+sCR rate of 71% vs. 23%, and a VGPR or better rate of 96% vs. 61%. The extent of BiRD therapy prior to development of first ASIP did not correlate with response rate (p = .7284). Bone marrow examination by histology, karyotype, FISH, and PCR IgH / IgK clonality analysis confirmed the disease response and furthermore showed no evidence for newly emergent plasma cell or other B-cell malignancy to account for ASIP generation suggesting molecular remission in some of the patients. Peripheral blood analysis by flow cytometry also showed no evidence for a circulating B-cell neoplasm to account for the new immunoglobulin production. We propose that ASIP development during myeloma therapy with a lenalidomide-based regimen heralds a robust tumor reduction with a hitherto unprecedented rate of complete remission. The immune phenomena that contribute to ASIP generation are unclear at present, however it is neither due to a new clonal B-cell population or transformation of the original MM plasma cell clone.

Different methods using peripheral blood RNA (Vannucchi AM, et al. Leukemia. 2007,1–8), or archival bone marrow DNA (Tefferi A, et al. Leukemia. 2007,1–2) have yielded varied results correlating allele burden with severity and duration of disease. We therefore aimed to determine whether JAK2V617F allele burden correlated with certain parameters of disease. At our institution, 105 patients were diagnosed according to the criteria of the Polycythemia Vera Study Group. We grouped their JAK2V617F allele burdens into quintiles. DNA from peripheral blood was analyzed using pyrosequencing. For those patients whose allele burden was 9 cm). Thrombotic events were recorded within 5 years of JAK2V617F determination. There were 52 men and 53 women. The patients ranged in age from 35 to 88 years, median 60 years. The median duration of disease was 7.4 years (range: 0.2 - 36.6 years), and the median duration of follow-up after JAK2V617F determination was 12 months year (range: 1 - 43 months). The mean mutant allele burden was 46.0% (s.d. ± 29.7%). The fifth, and highest quintile had a mean mutant allele burden of 90.2% (s.d. ± 5.8%); the lowest quintile had a mean mutant allele burden of 9.9% (s.d. ± 6.3%). JAK2V617F did not correlate with age, gender, hematocrit and platelet count at diagnosis, or rate of phlebotomy prior to cytoreductive therapy. Increasing JAK2V617F burden did correlate with higher WBC at diagnosis (P=0.02), degree of splenomegaly (P