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Mantle Cell Lymphoma (MCL) is a relatively uncommon, aggressive form of B-cell non-Hodgkin’s lymphoma (NHL), defining approximately 5-8% of adult NHL in the United States. Although individual survival times have increased in recent years, prognosis remains among the poorest of NHL, with a median survival of 5 to 7 years. Despite an initial response to chemotherapy, MCL cells remain residually in the marrow and digestive tract, making MCL prone to recur leading to short survival prognosis. The characteristic re-emergence of MCL is likely related to drug resistance generated from the tumor microenvironment (TME). In particular, stromal cells found in bone marrow and secondary lymphoid sites promote the movement and proliferation and possibly drug resistance from cross-talk between stromal and lymphoma cells. While studies show a correlation between stromal cells and tumor survival in various forms of NHL, the mechanisms are poorly understood and its role in MCL has not extensively explored. Based on our experimental studies on an extensive spectrum of representative patient samples, we have shown that in vitro culturing of primary leukemic phase MCL cells (15 cases) usually leads to the spontaneous formation of activated CD68+ monocytic macrophages and stromal cells. The MCL tumor cells tended to form rosettes, clustering around and adhering to these cultured lymphoma-associated macrophages (LAM). The LAM formed in culture coexisted with rosetted MCL tumor cells for up to 4 months. During this period, a small subset of MCL cells showed limited mitotic figures, suggesting that these cells were proliferating and possibly becoming incipient immortalized MCL cells. When these cells were weaned from the adherent LAM, they began to expand autonomously and proliferate as an autonomous cell line. Using this methodology, we have developed 3 MCL cell lines (PF-1, PF-2, and PF-3) from 15 leukemic phase MCL cases. PF-1 MCL cell line represents classic typical MCL cells, while PF-2 and PF-3 MCL cell lines represent the in situ indolent MCL cells that are CD200 positive. When these cells were xeno-transplanted into SCID mice, lymphoma cell uptakes were disseminated throughout the lymphatic system, including the spleen, lymph nodes, and GI tract, representing excellent in vivo models for MCL. Next, we delineate the biologic functions of LAM in the MCL microenvironment milieu. Our data showed that when we initially removed the accessory cells (monocytes, etc.) from the MCL primary culture, no LAM were formed and the purified MCL cells died out in Disclosures Pham: Onyx/Amgen: Research Funding.

Background: The gain/amplification of the CKS1B gene that is located on chromosome 1q21 is a predictor of poor outcome in multiple myeloma (MM). However, there are little data in the literature on the characteristics and outcomes of patients with this cytogenomic marker. Historically, median PFS after auto-HCT for all patients with multiple myeloma is 24-30 months in patients transplanted in 1st remission and 12-16 months in patients transplanted for relapsed disease. We wanted to compare the outcomes of patients with CKS1B amplification with the historical data. Methods: This is a single-center retrospective analysis of patients with MM, who received high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto-HCT) at our institution between 2011 and 2014. We identified 64 patients, 8.2% of 777 auto-HCT performed during this period, who had CKS1B gain/amplification on FISH studies, or chromosome 1q21 gain on conventional cytogenetics. The primary objective was to describe the clinical characteristics and outcome of these patients, including progression-free (PFS) and overall survival (OS). Results: Median age at auto-HCT was 60.4 years (range 34-79). Median interval between diagnosis and auto-HCT was 7.4 months (range 3.7-143). The ISS stage at diagnosis was available in 54 patients, 24 (39%) of whom had stage 3 disease. Ten out of 44 patients (23%) with available lactic dehydrogenase (LDH) levels at diagnosis had higher than normal levels. Besides the CKS1B gain/amplification, concurrent high-risk cytogenomic markers detected were: monosomy/deletion 13 in 32 (50%), del 17/17p in 8 (12.5%) and del 1p in 7 patients (11%). Fifty-six (88%) patients had received bortezomib-based induction or salvage therapy before auto-HCT. Thirty-five (55%) patients were in 1st remission while 29 (45%) patients had relapsed disease at auto-HCT. Prior to auto-HCT 3 (5%) patients were in complete remission (CR), 17 (27%) in very good partial remission (VGPR) and 29 (45%) were in partial remission (PR). Seven patients (11%) had progressive disease at auto-HCT. After at least 90 days post auto-HCT, 7 (11%) patients achieved a CR, 24 (37.5%) a VGPR, 20 (31%) a PR, with an overall response rate of 80%. One-hundred-day and 1-year treatment-related mortality were both 0%. After a median follow up of 5.7 moths, 18 (28%) pts had started maintenance treatment, 15 (83%) of these with lenalidomide. Median PFS was 10.7 months and median OS has not yet been reached. Median PFS was 13.3 months for patients transplanted in 1st remission and 8.3 months for patients transplanted after a relapse (p=0.005). Median OS has not yet been reached whether the patients were transplanted in 1st remission or after a relapse. Conclusion: This analysis confirms CKS1B gene gain/amplification as a marker for poor PFS outcomes after stem cell transplantation. Patients with CKS1B gain/amplification are likely to have additional high-risk chromosomal abnormalities, and a shorter PFS whether transplanted in 1st remission or for relapsed disease. Disclosures No relevant conflicts of interest to declare.

Abstract 5090 Background: The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth, and apoptosis. c-MYC is located on the long arm of chromosome 8 (8q24.1) and its rearrangement has been reported in 15% of myeloma patients independent of the disease stage (Avet-Loiseau et al. Blood 2001). However, the clinical significance of c-MYC rearrangement is not well described. Here we report the characteristics and outcome of 7 myeloma patients with c-MYC rearrangements that were treated at our institution. Methods: Between July 2007 and May 2011, we identified 7 patients (4 males, 3 females) who had c-MYC rearrangements on fluorescence in situ hybridization (FISH) analyses at the time of diagnosis. The primary objective of this study was to describe the patient characteristics, response to therapy, time to progression (TTP), and overall survival (OS). Results: Median age at diagnosis was 58 years (49–72). There were 4 Caucasians, 2 African-Americans and 1 Asian. International Staging System stage was I, II, and III in 3, 2, and 2 patients, respectively. Two patients had serum creatinine of ≥2 mg/dL as well as Hgb ≤10 g/dL, two other patients had hypercalcemia at diagnosis. Six (85%) patients had myelomatous bone lesions at diagnosis. Plasma cell counts in bone marrows ranged from 20%-78%, with a mean of 51%. Abnormalities involving chromosome 8q24.1, the c-MYC locus, were detected on conventional cytogenetics in all 7 patients, including t(8;14)(q24.1) in 3 cases, a t(2;8)(p12;q24.1), a variant of the t(8;14), in 2 cases, and an abnormal chromosome 8 with unknown material attached to the 8q24.1 region confirmed by FISH in two cases. This is in contrast to an earlier report where only 25% of patients with c-MYC rearrangement on FISH had corresponding cytogenetic abnormalities. Three patients also had a del(13)(q14.1)/RB1, one of whom also had a del(17)(p13)/TP53, while two other patients had t(11;14)(q13;q32) involving CCND1-XT/IGH rearrangements. Six patients (85%) received induction with a bortezomib+ dexamethasone regimen, and one patient received thalidomide+ dexamethasone. Five patients achieved a partial (PR) or very good partial remission (VGPR) to induction, while 2 patients had Conclusion: This is the first report describing clinical characteristics of myeloma patients with c-MYC rearrangements. All 7 patients had concurrent translocations involving chromosome 8q24 on conventional cytogenetics, and had a shorter TTP and OS than our historical data. The role of c-MYC rearrangement in myeloma needs to be explored in prospective studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 333 Impact of Non High-Risk Chromosomal Abnormalities on the Outcome of Autologous Hematopoietic Stem Cell Transplantation in Multiple Myeloma Background: Despite novel therapeutic agents and high-dose chemotherapy with autologous hematopoietic stem cell transplantation (auto-HCT), most patients eventually progress and die of their disease. Recent advances in cytogenetic, molecular and genomic studies have led to identification of several chromosomal and molecular abnormalities. These abnormalities are important predictors of response to therapy, progression-free survival (PFS) and overall survival (OS). On conventional cytogenetic (CC) analyses, del 13, t(4;14), t(14;16) and del 17p are considered high-risk (HR). On Fluorescence in situ hybridization (FISH) analysis, all except del 13 are considered HR (Munshi, N et al. Blood 2011 117: 4696–4700). However there are a number of chromosomal abnormalities whose significance is not clearly identified (non-HR). In this study we report the impact of these non-HR chromosomal abnormalities on the outcome of patients who received high-dose chemotherapy and auto-HCT. Methods: We performed a retrospective review of patients with multiple myeloma who underwent high dose chemotherapy followed by auto-HCT at the M.D. Anderson Cancer Center. Between 10/1991 and 12/2010, 1570 patients received auto-HCT. The results of CC studies were available for 1329 patients, either at diagnosis or at relapse, but before auto-HCT. The primary objective was to study the impact of non-HR chromosomal abnormalities on PFS and OS, and to compare them to patients without chromosomal abnormalities. Results: Patient characteristics and major outcomes are summarized in the attached Table. In 1329 patients with available CC analyses before auto-HCT, chromosomal abnormalities were identified in 405 (30%) patients. One-hundred and seven (7%) patients had known HR chromosomal abnormalities, while 298 (23%) patients had non-HR chromosomal abnormalities. Fifty (17%) patients with non-HR chromosomal abnormalities and 296 patients (32%) with normal CC achieved complete or stringent complete responses (CR + sCR) (p=0.0001). Median follow up in surviving patients was 36 months. Median PFS in patients with non-HR chromosomal abnormalities and normal CC were 18.2 months (95%CI: 16–22.7) and 32.7 months (95% CI: 27.8–36.3), respectively (p= Figure 1 ). The OS in patients with non-HR chromosomal abnormalities and with normal CC were 56.5 months (95% CI: 43.2–66.9) and 87.2 months (95%CI: 80.1–102.4), respectively (p= Figure 2 ). Download : Download high-res image (40KB) Download : Download full-size image FIGURE 1 . Download : Download high-res image (42KB) Download : Download full-size image FIGURE 2 . Conclusions: In this large single center study with a long follow up, we demonstrated that non-HR chromosomal abnormalities in myeloma are associated with a lower CR rate and shorter PFS and OS after auto-HCT. Further studies are needed to better define these non-HR abnormalities and their impact on prognosis. TABLE 1 . Normal (n= 924) Non-High Risk (n= 298) Median age at auto-HCT 57 59 Age >65 at auto-HCT 137/924 (14.8%) 70/298 (23.4%) Race White = 605 (65%) Black = 148 (16%) Other = 171 (19%) W = 202 (67%) B = 44 (14%) O = 52 (19%) Gender Female = 391 (42%) Male = 533 (58%) F = 104 (34%) M = 194 (66%) Disease status at transplant First Remission = 799 (86%) Relapsed: 110 (12%) Unknown = 15 (2%) First Remission = 224 (75%) Relapsed = 72 (24%) Unknown = 2 (1%) Conditioning Regimen Melphalan or non melphalan = 198 (79%) Melphalan alone = 726 (21%) Melphalan or non melphalan = 57 (81%) Melphalan alone = 241 (19%) Final Response PD = 17 (1.8%) PD = 20 (6.7%) Progressed 447/924 (48%) 172/298 (57%) Died 278/924 (30%) 123/298 (41%) PD: Progressive Disease PR: Partial Remission CR: Complete Response VGPR: Very Good Partial Response sCR: Stringent Complete Response Disclosures: No relevant conflicts of interest to declare.

Abstract 4633 Background: Golgins are proteins of the Golgi complex. Several Golgins have been implicated in apoptosis. Expression of Golgin-84, a Golgin protein, is altered in apoptotic WEHI-231, a B-cell lymphoma line, suggesting that Golgin-84 may play a role in lymphoid tumorigenesis. Here, we aimed to determine the expression levels of Golgin-84 in human primary non-Hodgkin lymphomas and plasma cell myeloma. Design: Golgin-84 expression was investigated in non-Hodgkin lymphoma cell lines by using Western blot analysis and polyclonal antibodies. Using immunohistochemical stains, Western blotting analysis and Q-PCR, Golgin-84 expression was assessed in 5 reactive lymph nodes, 149 cases of primary non-Hodgkin lymphoma and 28 cases of primary plasma cell myeloma. Results: Immunohistochemical stains, Western blotting analysis and Q-PCR on 5 reactive lymph nodes demonstrated that Golgin-84 was expressed at low levels in lymphoid cells of germinal centers, mantle cells, marginal zones, and interfollicular areas. Golgin-84 was variably expressed in non-Hodgkin lymphoma cell lines tested, with the highest levels in cells from high-grade tumors (e.g. anaplastic large cell lymphoma; ALCL, Diffuse large B-cell lymphoma (DLBCL), ALCL and peripheral T-cell lymphoma unspecified (PTCL)) and the lowest levels in mantle cell lymphoma (MCL) cells. DLBCL, ALCL and PTCL frequently showed high expression of Golgin-84. Most lymphoplasmacytic lymphomas (LPL) and plasma cell myeloma (PCM) expressed high levels of Golgin-84. Expression levels of Golgin-84 were lower in MCL and low-grade B-cell non-Hodgkin lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL). Conclusions: Golgin-84 expression levels are low in lymphoid cells of normal lymph nodes. Most (>90%) cases of LPL and PCM, and at least half of cases of DLBCL, ALCL and PTCL express high levels of Golgin-84. These findings suggest that Golgin-84 may be involved in tumorigenesis or lymphoma progression, particularly in neoplasms with plasmacytic differentiation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4156 Background: Chromosomal abnormalities detected either by conventional cytogenetics (CC) or interphase fluorescence in situ hybridization (FISH) have emerged as important prognostic markers in patients with multiple myeloma (MM) and are used to stratify patients into standard-risk (SR) or high-risk (HR) disease. HR myeloma is associated with shorter remission and survival even with novel agents and autologous hematopoietic stem cell transplantation (auto-HCT). In this report, we describe the outcome of patients with myeloma who received auto-HCT at our institution between January 2008 and December 2009, and had CC and FISH analyses available before auto-HCT to identify HR or SR disease. Methods: Primary objective was to compare the response rate, transplant-related mortality (TRM), time to progression (TTP), progression-free survival (PFS) and overall survival (OS) between HR and SR myeloma. HR myeloma was defined as having deletion of chromosomal 13 or 13q, t(4;14), and del17p by CC; or detection of t(4;14), t(14;16), or del17p by FISH in the bone marrow plasma cells (Munshi, N et al. Blood 2011 117: 4696–4700). Normal or any other CC or FISH abnormalities were considered SR. Response was assessed according to international uniform response criteria for myeloma (BGM Durie et al. Leukemia 2006 20(10):1–7). Kaplan & Meier curves were generated to calculate progression free survival (PFS) and overall survival (OS). Results: Between Jan 2008- Dec 2009, we identified 205 patients, who received auto-HCT at our institution and had pre-transplant CC and FISH analyses available. Twenty-eight patients (14%) had HR while 177 (86%) had SR myeloma. Table 1 summarizes patient characteristics and outcomes for HR and SR patients. Nine HR (30%) and 29 (16%) SR patients had international staging system (ISS) stage III at diagnosis (p=0.04). Twenty-one HR patients (70%) and 91 SR patients (51%) received induction therapy with a bortezomib-based regimen (p=0.01). Thirteen (46%) HR patients and 62 (35%) SR patients received post auto-HCT maintenance therapy (p=0.24). Nineteen HR patients (69%) and 165 SR patients (92%) achieved a CR, VGPR or PR after auto-HCT (p=0.00001). The median follow up in HR and SR patients was 14.5 months (2–30) and 12 months (1–35), respectively. Twenty-two (79%) HR and 46 (25%) SR patients had progressed at last follow up (p=0.00001). Similarly, 12 (43%) HR and 12 (7%) SR patients had died at last follow up (p Conclusion: Patients with HR myeloma had significantly worse outcome than patients with SR disease, even with novel agents and auto-HCT. In HR patients, maintenance therapy was associated with longer PFS and OS. Disclosures: No relevant conflicts of interest to declare.

Abstract 4645 DNA hypermethylation has been implicated in the tumorigenesis and prognosis in acute myeloid leukemia (AML). To identify and validate relevant methylated genes in AML, we have compared expression levels and methylation status of 26 candidate genes. One of the interesting candidates identified in our study is MSX2. MSX2 is a member of muscle segment homeobox gene family. MSX2 plays a role in promoting cell growth under certain conditions and may be an important target for RAS signaling pathways. However, the mechanism of transcriptional regulation and functional role of MSX2 in hematological malignancies, especially AML, are poorly understood. In our study, we determined the methylation status, and analyzed the expression levels of MSX2 in AML cell lines and primary AML cells using RT-PCR and/or Taqman real-time PCR. MSX2 mRNA expression was robust in the normal granulocytes and blasts of human bone-marrow, but was either absent or significantly diminished in 6 of 9 (66.7%) AML cell lines. The expression levels of MSX2 in those 6 AML cell lines were restored after treatment of 5-aza 2′-deoxycytidine. In addition, COBRA (Combined Bisulfite Restriction) analysis demonstrated hypermethylation of MSX2 in those AML cell lines (6 of 9, 66.7%), and partial methylation in 3 of 9 AML cell lines. The methylation status was inversely correlated with the mRNA expression levels of MSX2 in those cell lines. Furthermore, the expression levels and methylation status of MSX2 in human primary AML cells were evaluated. COBRA analysis demonstrated frequent hypermethylation of MSX2 in primary AML patient samples (19 of 32, 59.3%). Importantly, the mRNA expression levels of MSX2 as shown by Taqman real-time PCR in those 19 primary AML patient samples were inversely correlated with the methylation status of MSX2. These findings confirmed the role of frequent DNA hypermethylation in silencing MSX2 in AML. We are in the process of determining the functional role of MSX2 in the pathogenesis of AML. In addition, diagnostic and prognostic values of MSX2 in AML are being pursued. Disclosures: No relevant conflicts of interest to declare.

It has been well known that cytogenetics study plays an important role in cancer patient care, in particular in the diagnosis, treatment and prognosis of hematological malignancies. The MCIM test system, a combination of studies of cell morphology (M), cytogenetics (C), immunophenotype (I) and molecular genetics (M), has become a useful test system in hematologic disease diagnosis. It provides physicians more comprehensive and accurate diagnostic laboratory information. Deletions in the long arm of chromosome 7 are among the most common abnormalities observed in hematologic disorders. These changes were usually viewed as markers for myeloid malignancies, in particular myelodysplastic syndrome and acute myeloid leukemia. Recently, interstitial 7q deletions, a subset of the 7q deletions, have been found to be associated with B-cell lymphoproliferative disorders (LPDs). The correlation of narrow 7q deletions with specific subsets of B-cell LPDs has been extensively studied. However, the frequency of interstitial 7q deletions associated with B-cell LPDs has not been well investigated. We recently studied 7q deletions collected from our clinical cases done by the MCIM system at US Labs over the past two years. Deletions of chromosome 7q were observed in 85 of 19,483 cases. Interstitial 7q deletions were detected in 46 of the 85 cases with 7q deletions. In combination with the findings of flow cytometry, interstitial 7q deletions were found to be associated with B-cell LPDs in 10 out of the 46 cases, accounting for 21.7% of all the observed interstitial 7q deletions and 11.8% of all the 7q deletions. The B-cell LPDs associated with 7q interstitial deletion are diverse, including hairy cell leukemia, atypical chronic lymphocytic leukemia, splenic marginal zone lymphoma, and large B-cell lymphoma. Interestingly, the deleted region in the 10 cases with B-cell LPDs in this study was solely confined to 7q22-32. This is so far, to our knowledge, the largest series reported in the literature. Our investigation showed that the frequency of 7q interstitial deletions associated with B-cell LPDs is substantially high. We conclude that (1) association of B-cell LPDs must be taken into account when an interstitial 7q deletion is observed, in particular when the deleted region is confined to 7q22-32; (2) together with the flow cytometry analysis in the MCIM system within a clinical laboratory diagnostic facility, cytogenetics study can provide clinicians more accurate diagnostic information, thus strengthening patient care by improving the management of hematological malignancies. (The authors are grateful to Deanna Collins for her cooperation to collect the data in this study.)