Tschumper, Renee C.

Abstract Background: Monoclonal B-cell lymphocytosis (MBL) is characterized by the presence of a circulating population of clonal B-cells where the total B-cell count is <5 × 109/L and criteria for the diagnosis of other lymphoproliferative disorders are not present. In most patients the immunophenotype is similar to that of chronic lymphocytic leukemia with co-expression of CD19 and CD5. The natural history of MBL is unclear. We used the Mayo Clinic hematopathology database to identify patients with MBL and abstracted clinical information on natural history and prognostic parameters from clinical and research records. Methods: After IRB review and approval, we reviewed the hematopathology records of 2292 patients seen at Mayo Clinic between August 1998 and May 2006 who had a clonal B-cell population on flow cytometry phenotypically consistent with CLL and fewer than <5 × 109/L B-cells. Clinical records were then reviewed to identify patients who fulfilled the criteria for MBL (no evidence of other lymphoproliferative disorders, no adenopathy on exam). Patient characteristics, prognostic parameters, and clinical outcomes were abstracted from the clinical and research records of these patients. Cox regression analysis was used to evaluate association between prognostic markers and time to treatment. Results: A total of 118 patients meeting the criteria for MBL were identified. Median age at the time of MBL diagnosis was 69.4 years (range 39.0 – 91.4). A slight male preponderance was observed (54% male). CD38 status was available for 106 (90%) patients of whom 20% were CD38 positive using the 30% threshold to classify CD38. Cytogenetic analysis by fluorescent in situ hybridization (FISH) was available in 58 (49%) patients. Using the hierarchical classification system of Dohner and colleagues, 26 (44.8%) were 13q-, 10 (17.2%) were trisomy 12, 2 (3.4%) were 11q-, 1 (1.7%) was 17p-, and 19 were (32.8%) no defect was detected. ZAP-70 status was available for 32 patients of whom 22% were ZAP-70+ positive. IgVH gene mutation status was only available for 15 (12.7%) patients of whom 40% were unmutated. Time from MBL diagnosis to treatment is shown in Figure 1. Survival free of treatment was 92.7% (95%CI: 86.8–99.1%) at 1 year, 90.9% (95%CI: 84.2–98.2%) at 2 years, and 87.5% (95% CI: 78.7–97.4%) at 5 years. While age (p=0.68) and gender (p=0.14) were not found to be associated with time to treatment (TTT), CD38 status showed a strong relationship with TTT (p<0.001, HR: 22.7, 95% CI: 3.4–239.6). Conclusion: The distribution of prognostic parameters (e.g. CD38, FISH) in patients with MBL appears similar to that of patients with early stage CLL. We observed a low rate of progression, with 5 year survival free of treatment greater than 87%. TTT among newly diagnosed MBL patients was associated with the CD38 status of clonal B-cells. Figure Figure

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Abstract Despite the remarkable outcomes and recent FDA approval of CD19 directed chimeric antigen receptor T (CART19) cell therapy in B cell malignancies, the durable responses in diffuse large B cell lymphoma are less than 40% and CART activity in chronic lymphocytic leukemia (CLL) is further limited. This is thought to be related to loss of CART persistence, poor trafficking to lymph nodes and inhibition by the leukemic microenvironment. Therefore, strategies to enhance CART cell function to overcome these limitations are needed. Recent studies have shown that abnormal expression of the receptor tyrosine kinase (RTK) AXL is associated with poor prognosis in human cancers. AXL signaling is associated with tumor proliferation, survival, metastasis, and drug resistance. Inhibition of AXL RTK with TP-0903, a high affinity AXL inhibitor has been found to induce robust apoptosis of CLL B cells. Based on the significant modulation of T cell functions observed with BTK inhibitor, we examined the role of AXL RTK inhibition with TP-0903 on T cell function in CLL and other B cell malignancies. First, we investigated the effect of AXL inhibition on T cell phenotype in normal donors. When naïve T cells were stimulated with PMA/Ionomycin and cultured with low dose TP0903, cytokine production was favorably altered through the promotion of Th1 and reduction of Th2 cytokines. This was associated with a significant reduction of inhibitory receptors (Fig 1a). Western blot of T cell lysates suggests low dose TP-0903 results in inhibition of LCK. When effector T cells and regulatory T cells (Treg) were treated with TP-0903 for 3 days, there was a preferential reduction of Treg (Fig 1b). Next, we investigated the influence of TP-0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. Similar to our findings on naïve T cells, TP-0903 treatment led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP-0903 treatment also significantly downregulated inhibitory receptors on activated CART cells, including a reduction of canonical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1c). The combination of CART19 cells and TP-0903 yielded a synergistic antitumor activity against JeKo in vitro, at low E:T ratios (Fig 1d). Western blot of T cell lysates revealed phosphorylation of LCK was remarkably reduced in the presence of TP-0903, suggesting a mechanism for the observed Th1 polarization. We compared the transcriptome of activated CART cells treated with TP-0903 and more than 100 genes were differentially expressed compared to non-treated cells. Among these genes, immune synapse related genes such as cell junction and cell migration related genes were significantly increased in activated CART cells treated with TP-0903. To investigate the effect of AXL RTK inhibition of CART cells with TP-0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of JeKo into NSG mice. A week after the injection of JeKo, mice were treated with either vehicle alone, TP-0903 (20mg/kg/day) alone, 0.5x106 of CART19 alone, or TP-0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of JeKo. Mice treated with CART19 and TP-0903 rejected the JeKo tumor challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1e). Finally, we validated our preclinical findings in a correlative analyses of Phase I clinical trial of TP-0903 for patients with solid tumors (NCT02729298). Blood T cells from 3 patients were isolated and analyzed before and a week after treatment with TP-0903. Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned Phase I clinical trial of TP-0903 in relapsed/refractory CLL (NCT03572634). In summary, we demonstrated for the first time that AXL inhibitior is capable of polarizing T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes with CART cells in B cell malignancies. These findings encourage further study of TP-0903 as an enhancer of T cell immunotherapies. Disclosures Mouritsen: Tolero Pharmaceuticals: Employment. Foulks:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria. Ding:Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Abstract Aberrant over production of monoclonal immunoglobulin is a classic feature of the plasma cell (PC) malignancy multiple myeloma (MM). At diagnosis and more frequently upon relapse, some MM patients present with increased production of monoclonal free light chain (FLC) without a corresponding increase in intact immunoglobulin. This phenomenon is commonly referred to as light chain escape (LCE). As previously described (Walters et al. 2018, Experimental Hematology, Vol 57 p42-49), we have established a monoclonal IgA kappa MM cell line, designated as MC-B11/14, from the bone marrow (BM) aspirate of a 46 year old man diagnosed with MM. This cell line is non-hyperdiploid and possesses an 11;14 chromosomal translocation. Given our long standing interest in immunoglobulin function and secretion, we used CRISPR technology to knock out IgA heavy chain production in an effort to create a model of LCE. Successful knockout (KO) of IgA expression was demonstrated by immunofluorescence, flow cytometry, and western blot. As expected, secretion of intact IgA was undetectable in the MC-B11/14IgA- cells. Notably, no significant difference in morphology, phenotypic markers, or growth patterns was observed between the MC-B11/14WT and MC-B11/14IgA- cells. Examination of drug sensitivity between the MC-B11/14WT and MC-B11/14IgA- cells revealed that the response to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, the MC-B11/14IgA- cells were found to be more resistant to treatment with bortezomib. Given that increased bortezomib resistance was the only significant difference detected between the MC-B11/14WT and MC-B11/14IgA- cell lines in our initial study, we decided to take a metabolomics approach to examine whether loss of IgA heavy chain expression may alter cellular metabolism. Metabolomics or the profiling of small molecules is a powerful tool to characterize the metabolome of cells/biological systems and has proven to be useful in identifying alterations that occur in malignancy. In this regard, we employed pathway-specific targeted LC-MS/MS protocols in order to examine over 330 metabolites in 35 metabolic pathways in MC-B11/14WT and MC-B11/14IgA- methanolmetabolite extracts using an Agilent 1290 UPLC-6490 QQQ-MS liquid chromatography mass-spectrometer. Of the 330 metabolites examined, 28 were found to be significantly more abundant in the MC-B11/14IgA- cells compared to MC-B11/14 WT cells while 79 were found to be significantly less abundant. In general, the majority of significant metabolic differences between the two cell lines were found to involve metabolites from various amino acid synthesis pathways. More specifically, the MC-B11/14 WT cell line was found to possess significantly more citraconic acid, anthranilic acid, and homovanillic acid. By contrast, further analysis revealed that the MC-B11/14IgA- cell line was found to possess an 11.5 fold increase of the metabolite 2-hydroxyglutarate (2-HG). 2-HG has previously been shown to act as a competitive antagonist of α-ketoglutarate (α-KG), which results in the inhibition of α-KG-dependent dioxygenases including the JmjC domain-containing histone demethylases (KDMs) and the ten-eleven translocation (TET) family of DNA hydroxylases. Notably, these proteins are known to play a role in histone and DNA demethylation, respectively. Taken together, these data suggest that MM cells that have undergone LCE may also possess an accumulation of 2-HG which could lead to altered patterns of histone and DNA methylation. Given that MM patients who relapse with LCE typically have a poorer prognosis, further investigation of metabolomics in light-chain only expressing cell lines and patients who have relapsed with LCE is warranted. Disclosures No relevant conflicts of interest to declare.

Abstract Intercellular communication between multiple myeloma (MM) cells and the normal bone marrow stroma leads to a modification of the bone marrow microenvironment, which favors tumor progression. New developments in extracellular vesicle (EV) research suggest that this diverse population of vesicles, released by cancer cells including MM cells, express transmembrane proteins and carry cargo that can modify recipient cells in myriad ways. In particular, tumor-derived EVs have been shown to create permissive microenvironments that lead to metastatic colonization by circulating tumor cells. As we have previously shown that MM EVs can enhance proliferation of recipient MM cells, we hypothesize that MM EVs can potentially play a much larger role in MM development and progression. In order to understand the scope of potential EV roles in MM, we executed a comprehensive proteomic analysis of the cargo of MM EVs. We isolated EVs from patient derived MM cell lines that represent the most common genetic variants of this tumor, and used in vitro generated plasma cells (IVPCs) as a reference population. In a first pass analysis, we selected proteins that were expressed ≥3 fold higher in MM EVs than in IVPC EVs, and this analysis identified 306 proteins. Of interest, included in the 306 differentially proteins were several involved in the regulation of cell adhesion such as members of the a disintegrin and metalloprotease domain (ADAM8, 9, 10, 13, 15 and 22) family, which are also associated with inhibition of cell proliferation. Moreover, CEACAM1, PTPRK, and CDH2, which are also linked to cellular adhesion, were also expressed at a higher level in MM EVs. Various proteins linked to myeloma cell biology were likewise found to be over-represented in MM EVs, and these include BCMA, ITGAV, ITGB5, IL6ST (gp130), CD276 (B7-H3), and CD28. As we are most interested in biologically relevant and actionable proteins present uniquely in MM derived EVs, we filtered from our data proteins that were also present in EVs from IVPCs. We further filtered out proteins known to be present in EVs from normal cells. This filtering strategy reduced the number of interesting candidates to 8, which included CD28, MET, TRKC, and ADAM15. Thus far, we have confirmed the presence of these 4 proteins in a panel of MM EVs, and we are currently in the process of validating additional candidate cargo proteins for their biological role in enhancing tumor cell proliferation and/or protection from apoptosis. In summary, EV proteomic analysis of cell lines representing MM genetic subtypes can lead to the identification of biologically relevant proteins transported systemically by EVs as well as suggest novel biomarkers that are easily detectable in plasma and may permit earlier recognition of disease progression in patients with MM. Disclosures No relevant conflicts of interest to declare.

Abstract Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract The introduction of novel immunomodulatory drugs (IMiDs), such as lenalidomide, has drastically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib-based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis and apoptosis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). Additionally, we examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its putative downstream target interferon regulatory factor 4 (IRF4), both of which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that response to lenalidomide did not correlate with specific cytogenetic translocations. There were, however, distinct groups of lenalidomide-responsive and non-responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC-1 (IC50 = 2.6 μM versus 0.005 μM, p=0.004), but did not have an effect on the IC50 of the non-responsive HMCL DP-6 (p>0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN or IRF4 expression levels. In summary, our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. Additionally, while CRBN and IRF-4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment. These results suggest that the development of a new treatment paradigm based on cytogenetic subtype of MM alone may be insufficient, and that additional markers may need to be incorporated. Furthermore, the disconnect between our findings of expression of CRBN and IRF-4 in lenalidomide-treated HMCLs and patient data highlights the need for further investigation into the role of the bone marrow microenvironment on the regulation of these previously demonstrated markers of lenalidomide response. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2838 The combination of novel agents and chemo-immunotherapy (CIT) has led to a significant increase in both overall and complete response rates for chronic lymphocytic leukemia (CLL). There is, however, significant clinical heterogeneity in the overall effectiveness of CIT. Specifically, some patients are long-term responders who experience durable clinical remission while others, even though responding well initially, have less durable remissions. It is unclear if this heterogeneity is a reflection of an incomplete cytotoxic effect or whether certain leukemia sub-populations (i.e. subclones) are resistant to the effects of CIT. Comprehensive analysis of tumor cells has provided, in some malignancies, evidence of extraordinary genomic complexity and intraclonal genetic diversity. Conversely, little is known of the genetic architecture of the leukemic clone in CLL patients and the extent to which CLL tumor cells differentiate genetically at the subclonal level. In order to gain insight into the genetic architecture and clonal evolution underlying disease progression/relapse, we performed a sequential, longitudinal whole genome analysis by array-based comparative genomic hybridization (aCGH) in 22 CLL cases with evidence of clinical progression and/or clinical relapse post therapy. These patients received standard purine analogue, cyclophosphamide and rituximab (PCR) for progressive disease. For the purposes of this study, we classified samples as; time point 1 (TP1) collected >6 months before starting first-line CIT for disease progression; time point 2 (TP2) consisting of tumor samples at the time of initial progression requiring treatment; and time point 3 (TP3) consisting of tumor samples collected >6 months after initial treatment but prior to subsequent treatments. Overall, aCGH assay was performed in 54 samples from 22 cases (2 to 4 time points analyzed per patient): 6 cases were analyzed at TP1 and TP2, 8 at TP2 and TP3, 2 at TP1 and TP3, and the remaining 6 cases were analyzed at TP1, TP2 and TP3. In cases showing genetic changes between time points, FISH analyses were performed in 32 additional samples to assess intra-clonal diversity and provide a quantification of the clone size. There was a small increase in genomic complexity in later time points with a mean of 4 (median 4, range 0–12) CNAs detected at the first time point for each patient, increasing to a mean of 5.3 (median 4.5, range 0–21) CNAs at the last time point analyzed. Diverse patterns of changes in genetic architecture were observed. The simplest change in genetic architectures was the linear sequence, found in 2 cases (9%) and characterized by the maintenance of the initial abnormalities and the subsequent acquisition of additional CNAs in the same subclone. The pattern of changes in genetic architecture in another 4 patients (18%) demonstrated marked heterogeneity including multiple genetic subclones evolving either in succession or in parallel. In all 4 cases with multiple subclones, a marked shift in the dominant subclone was observed at the time of disease relapse, with the minor subclones at TP1 and TP2 becoming the dominant subclones at TP3. In addition, the subclonal evolution continued following secondary therapies, and in all 4 cases the emerging subclone showed resistance to multiple treatments. This argues strongly in favor of subclones with specific genetic structures becoming dominant with the selective evolutionary pressure imposed by therapy. In summary, this sequential genetic study reveals that CLL leukemic B cells exhibit an unanticipated level of heterogeneity including a variable number of genetically distinct subclones, which display dynamic behavior in disease progression. The characterization of this intraclonal architecture is of critical importance to better understand the mechanisms of disease progression and relapse as well as to ultimately inform us of individualized treatment strategies. Disclosures: Fonseca: Consulting :Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

Abstract Proteomics with great sensitivity and specificity effectively analyzes proteins by prior tryptic digestion and subsequent analysis by LC-MS/MS of the tryptic digest. We have utilized this approach in the development of a LC-MS/MS method to characterize minimum residual disease in multiple myeloma. The abundant antibodies produced by multiple myeloma plasma cells are identical and appear as a spike (M-spike) upon protein electrophoresis. The M-spike and histological examination of the bone marrow constitute part of the myeloma diagnostic repertoire. Upon treatment, myeloma cells and the antibodies they produce are reduced in number and amount and become increasingly hard to detect. The bone marrow biopsy serves as the “gold standard” test for clinical remission. We have developed a sensitive test for the presence of the monoclonal antibody produced by the plasma cells which may serve as a substitute for invasive bone marrow biopsy. We have focused on tryptic peptides comprising the variable CDR regions of the Ig light chains that are unique to each patients antibody clone. Utilizing 2-5 µL of patient plasma/serum from the initial M-spike sample we have utilized SDS-PAGE to yield a crudely purified immunoglobulin light chain. The light chain band is isolated, reduced, alkylated and trypsin digested with subsequent LC-MS/MS analysis to identify CDR specific tryptic peptide(s). These can be identified as variable peaks (elution time and mass) in a base peak ion chromatogram where constant region peptides of either lambda or kappa light chains (clone dependent) serve as “internal standards” for identifying the CDR tryptic peptides. The peptide’s mass and its corresponding MS/MS spectra are unique to this CDR tryptic peptide and this patient’s clone. The unique diagnostic peptide is isolated in subsequent samples by immunoaffinity purification of the target kappa/lambda clone, SDS-PAGE separation and LC-MS/MS analysis of the light chain gel band. An extracted ion chromatogram is generated based upon the CDR peptides identified in the initial analysis of the M-spike sample. In patients in clinical remission the presence of a significant signal from the targeted peptides indicates that targeting a CDR peptide from the M-spike protein is more sensitive than currently available diagnostic tools including immunofixation. Comparison of this test to the gold standard bone marrow biopsy will be examined. Disclosures: Barnidge: Mayo Foundation: provisional patent application for technology, provisional patent application for technology Patents & Royalties.

Abstract The nucleoside analogue pentostatin has clinical activity in B-Cell Chronic Lymphocytic Leukemia (CLL) and has shown significant activity and minimal toxicity when combined with cyclophosphamide for previously treated CLL. Building on this, we initiated a trial in 2002 of combined pentostatin (2mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375mg/m2). Of the 33 enrolled eligible patients included in these analyses, seventeen patients were in the high Rai risk group, 25 were male, and median age for this cohort was 62 yrs (range: 40–79); 17 were non-mutated for the immunoglobulin heavy chain variable region gene, and the majority (67%) were CD38 negative. Only 5 patients had no detectable chromosomal abnormalities by FISH at baseline, 20 had a single FISH anomaly, and 8 had 2 or more FISH anomalies. Of all 28 pts with any anomaly, the following specific abnormalities were detected: 13q- (n=17), +12 (n=8), 11q- (n=7), 17p- (n=2), t(14;18) (n=1), 6q- (n=1), and MDM2 (n=1). Of the 33 patients, 22 had grade 3+ toxicity; 16 patients had non-hematologic toxicity wtih the most common symptoms being nausea (6) and vomiting (4). One patient died on study of grade 5 hypoxia as well as hypotension and this was deemed possibly related to treatment. Almost all patients (32/33; 97%) had a best response of PR or better. Including review of bone marrows done 2 months post-treatment, there were 11 CRs (complete response), 7 nPRs (nodal partial response) and 13 PRs. No differences were observed between type of response and mutation status or CD38+ status. Post-treatment FISH analyses were available on 27 patients; results for 25 patients became normal after treatment. Of the remaining 2, one patient was 13q- x1 and went from 94% to 27.5% abnormal nuclei after treatment; the other is the patient who died on study. To establish minimal residual disease (MRD) post-response, we used three color flow cytometry to detect CD5+/CD19+/CD79b− B cells. This approach found all patients had a reduction in CLL B cells with a median reduction of 91% (range: 5 – 100%). Of interest, all 3 response groups (CR vs. nPR vs. PR) had patients with significant reductions in CLL B cells (i.e., >90%). The nPR group was most variable in terms of MRD (median 47%; range: 5–93%) compared to the CR (median: 91%; range: 42–99.9%) and PR (median: 97%; range: 46–100%) groups. In conclusion this novel regimen of pentostatin, cyclophosphamide and rituximab has demonstrated significant clinical activity irrespective of risk stratification parameters with rapid induction of responses, achievement of minimal residual disease in some, and modest toxicity. Patients continue to be accrued to further explore correlative measures of response to treatment.