Jimenez-Zepeda, Victor Hugo

Abstract Waldenström’s macroglobulinemia (WM) is an incurable low-grade lymphoproliferative disorder characterized by bone marrow (BM) infiltration of a clonal population of small B-lymphocytes, plasmacytoid lymphocytes and plasma cells that secrete monoclonal IgM antibody. Because it is difficult to obtain tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported and the genetic basis of the disease remains poorly defined. Therefore, we performed a comprehensive high-resolution study to identify genomic abnormalities involved in WM pathogenesis. Fifty-seven WM patients were analyzed using a combination of array-based comparative genomic hybridization (aCGH)(N=42); gene expression profiling (GEP)(N=22), DNA sequencing (N=24) and cIgM-FISH (N=57). Agilent 244A and Affymetrix U133 platforms were used in the aCGH and GEP experiments, respectively. An NF-κB index was calculated from GEP data, as a surrogate marker of NF-κB transcription activity. NFKB2 sub cellular localization was determined by immunofluorescence staining. Overall, 83% of samples have aCGH defined chromosomal abnormalities, with a median of 3 abnormalities per patient (range 0 to 27). We identified 16 recurrent regions of copy number change found in >5% (3 or more patients); 10 deleted and 6 amplified regions. The most common abnormality was the entire or partial deletion of 6q, identified in 40% of cases. Four nonoverlapped minimal deleted regions were identified on 6q (MDR1 to MDR4), each of them being present in at least 14 of 17 patients with the abnormality. Gain of 6p was the second most common abnormality (17%) and its presence was always concomitant with 6q loss. Other recurrent deletions were 13q14 (10%) and 7q22, 8p, 11q22-q23, 11q23-q24 and 17p11-p13 (7% each). Copy gains were identified as partial or entire gains of chromosomes 18 (17%), 4 (12%) and 3 (10%), 8q (10%) and Xq27.1-q28 (10%). Three patients had either homozygous deletions or LOH with mutations affecting both alleles of TRAF3 on 14q32. TRAF3 inactivation was correlated with an increased NF-kB transcriptional signature and it was associated with activation of the non-canonical NFkB pathway. Additionally, we identified the inactivation of TNFAIP3, another negative regulator of the NF-kB pathways, in one of 24 patients. Finally, interstitial deletions identified in 4 patients at 13q14 identified a 1.1 Mb MDR, including MIRN15A and MIRN16-1. These findings confirm that focal 13q14 deletion is not restrict to B-CLL and that the MIRN15A/MIRN16-1 abnormalities might be common to several indolent B-cell diseases. Overall, we identified TRAF3 and TNFAIP3 inactivation in 5.3% and 2.4%, respectively. To our knowledge, this is the first study showing the inactivation of bona fide tumor suppressor genes in WM patients. Furthermore, these genes are negative regulators of NF-kB signaling pathway, highlighting its biologic importance in sustaining and limiting growth in WM. Mutational activation of this pathway, which is normally activated by ligand-receptor interactions within the BM microenvironment, suggest a therapeutic role for inhibitors of NF-KB pathway activation in the treatment of WM.

Abstract Background B-cell chronic lymphocytic leukemia (B-CLL) is a well-defined clinical entity with heterogeneous molecular and cytogenetic features. Chromosome aberrations could be associated to specific CLL clinical features and outcomes and their impact on clonal evolution should have been addressed by using temporal analysis. In order to demonstrate the role of temporal analysis in CLL we conducted a comprehensive karyotypic survey of a large number of CLL cases with already known genetic aberrations to fully describe their meaning in terms of biological evolution. Methods A total of 1749 karyotypes were retrieved from the Mitelman Database of Chromosome Aberrations in Cancer. A matrix depicting the 360-band human chromosome ideogram was created. Regions that were either lost or gained in more than 3% of the cases were retained and identified as recurrent imbalances. Early and late imbalances were defined according to the appearance on the complex karyotypes. Descriptive statistics were used to summarize the genetic abnormalities. Results The median chromosome number and the median number of abnormalities per tumor (NAPT) were 46 and 1 respectively. (Figure 1 A and 1B) The most common abnormality seen was trisomy 12 which occurred in 29% (508 cases) followed by 13q14del (10.34%), 13q13del (6.63%) and add14q32 (6.63%). The temporal analysis revealed +12, 13q-, −17, 17p-, −Y and −X to be early imbalances (TO<5), followed predominately by a late loss with TO=5–10.5 (11q-). Hierarchical clustering suggested there are different groups including: trisomy 12, 13q-, 11q-, and 6q-, which at least in this clustering analysis appeared to be mutually exclusive. In summary, we can conclude that overall CLL is a neoplasia that shows remarkable chromosome stability even when only abnormal karyotypes are evaluated. Interestingly, clustering analysis suggests there are non-overlapping, unique subsets of CLL cases where trisomy 12 is the most common and along with 13q-emerged as early events on CLL clonal evolution. Figure 1A. Chromosomes distribution in chronic Lymphocytes Leukemia karyotypes Figure 1A. Chromosomes distribution in chronic Lymphocytes Leukemia karyotypes Figure 1b. Chronic Lymphocytes Leukemia and Number of Abnormalities Per Tumor (NAPT) distribution Figure 1b. Chronic Lymphocytes Leukemia and Number of Abnormalities Per Tumor (NAPT) distribution Figure Figure

Abstract Abstract 3063 Background: The combination of Cyclophosphamide, Bortezomib, and Dexamethasone (CYBOR-D) is proven to be highly active and effective in multiple myeloma. (Reeder, Leukemia 2009) We aim to evaluate the safety and efficacy of this regimen in a separate plasma cell neoplasm, AL Amyloidosis. The primary endpoint of this study is hematologic response. Complete hematologic response is defined as normalization of the free light chain ratio with no evidence of a monoclonal protein by immunofixation. Partial hematologic response is defined as a 50% reduction in M-spike or absolute light chain level (Gertz Am J Heme 2006). Methods: We report a series of patients with symptomatic AL Amyloidosis who received treatment with a combination of Bortezomib (1.3mg/m2 on days 1, 4, 8, and 11 every 28 days or 1.5 mg/m2 weekly), Cyclophosphamide (300 mg/m2 orally weekly) and Dexamethasone (40 mg weekly). We include patients in this study regardless of autologous bone marrow transplant candidacy or previous treatment history. Results: Fifteen patients with AL amyloidosis received two to six cycles of CYBOR-D. The treatment history, bone marrow transplant candidacy prior to treatment, hematological response, and status of transplant after therapy are shown in table 1. Of note, 8 (53%) had symptomatic cardiac involvement all of whom had elevated levels of both cardiac biomarkers (Troponin T and B-natriuretic peptide). Complete hematological response occurred in 11 patients (73.3%) with partial hematological response in 3 patients (20.0%). One patient (6.6%) did not achieve a partial response but did receive autologous bone marrow transplant after treatment with CYBOR-D. Median time to response was 2 months. Six patients (40%) had objective evidence of kidney response to therapy with a greater than 50% decrease in proteinuria. Conclusions: CYBOR-D produces a rapid and complete hematological response in the majority of patients with AL amyloidosis regardless of previous treatment history or bone marrow transplant candidacy. Overall, it is well tolerated with few side effects that included peripheral neuropathy and infectious complications. Two patients originally determined to be not eligible for transplant improved sufficiently on CYBOR-D to become eligible and then went on to receive autologous bone marrow transplant. The retrospective nature and small sample size limit this study, and prospective, randomized studies are needed to further elucidate the role of this regimen in AL amyloidosis. Disclosures: Stewart: Millennium Pharmaceuticals, Inc.: Honoraria, Research Funding; Celgene: Honoraria. Fonseca:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Onyx: Research Funding; Otsuka: Consultancy; Medtronic: Consultancy.

Abstract Abstract 2834 Poster Board II-810 Background: Monoclonal gammopathy of undetermined significance (MGUS) results arises from a clonal proliferation of differentiated plasma cells and is a necessary precursor to development of multiple myeloma and some other B cell malignancies. Despite a high incidence in the general population (1-2% of adults in the 6th and 7th decades) and increased relative risk for later malignancy, there are few reports about the clinical course and prognosis of MGUS in long-term immunosuppressed patients. We aimed to evaluate the significance of MGUS in patients undergoing solid transplants in terms of clinical outcomes and rate of transformation to secondary B cell malignancy. Material and methods: We reviewed the medical records of 1493 patients on which kidney (living donor related, living donor-unrelated and deceased donor), liver and pancreatic transplants were performed at Mayo Clinic Arizona, between January 1, 1999 and July 31, 2009 to determine the incidence of MGUS and to further evaluate long-term follow up. At MCA patients are routinely screened using SPEP and for those where an M-spike is detected or suspected immunofixation is required for confirmation. MGUS was defined as previously reported. Patients who had anemia unrelated to their PCD were included. Statistical Methods: Patients were observed until death or, for those still alive, until July 2009. The major goal of the study was to assess the impact of MGUS on transplant outcomes and progression to active myeloma or related disorders. Patients who did not experience progression were censored at the time of last follow-up. Nominal variables were compared using Fisher's exact test. A two-tailed P value less than .05 was considered significant. Results In this series, SPEP was performed in 1199 out of 1593 cases (75%) before transplantation. The most common abnormalities detected by SPEP included a normal pattern (70%), polyclonal pattern (16.8%), hypoalbuminemia (8%), hypogammaglobulinemia (2.4%) and the presence of M spike (2.8%). Immunofixation revealed the presence of a monoclonal immunoglobulin in 34 patients: IgG 30 cases (88%), IgM in 2 (6%) and biclonal disease in 2 (6%). The median age at diagnosis of MGUS was 60 years (range, 46 to 84 years), and included 63.7% of males. Clinical characteristics of the MGUS cases are seen in Table 1. Two MGUS patients were diagnosed only after the transplant was performed (338 and 1082 days respectively). There was no association between post-transplant lymphoproliferative (PTLPD) and MGUS and even when some MGUS patients died (7/34, 20%) death was not associated with progression of the monoclonal clone or development of a PTLPD or other malignancy. MGUS does not impact transplant outcomes. PTLPD disorders developed in 6 cases and none was associated with a MGUS. After a median of 88 months no MGUS patient developed transformation to active myeloma or a related disorder. Overall survival after transplantation was not significantly different among MGUS and Non- MGUS cases (6.48 vs 8.36 years p>0.05). Discussion Despite the frequency of MGUS in the general population there are few studies which have assessed MGUS in solid organ transplant patients. An increased incidence of monoclonal gammopathy in patients with autoimmune disease and in older non-transplant populations suggests that impaired immunity might lead to development of MGUS and later monoclonal B-cell malignancies. Interestingly, we identified two cases where MGUS appeared after transplant. In this series, we report on the incidence and follow/up of MGUS in patients screened before transplantation. The primary outcome for transplant recipients is unaffected by the presence of MGUS and incidence is in accordance with the expected frequency (2.8%). Patients with MGUS do not appear to be at risk for progression due to chronic immunosuppression. Testing for MGUS before transplantation is not cost-efficient and therefore would not be recommended. To our knowledge this is the largest series of MGUS and transplant outcome yet reported. Disclosures: Stewart: Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy; Takeda, Millenium: Research Funding; Genzyme, Celgene, Millenium, Proteolix: Honoraria.

Abstract Abstract 2403 Immunoglobulin light chain amyloidosis (AL) is a monoclonal plasma cell disorder characterized by the accumulation of monoclonal light chain fragments that have undergone a conformational transformation and deposit as amyloid fibrils in different tissues. High-dose melphalan (HDM) with autologous hematopoietic stem-cell transplant (ASCT) is increasingly used to treat patients (pts) with AL Amyloidosis. However, transplant-related mortality (TRM) is high, ranging between 13% to 43%. The survival benefit of HDM has been attributed to a patient-selection bias. Recently, a randomized clinical trial by Jaccard A et al, showed longer survival in the group assigned to receive melphalan plus dexamethasone (Mel/Dex) than in the group assigned to receive HDM. The present is a retrospective study aiming to define which pts with AL are most likely to benefit from HDM. Patients and Methods We retrospectively reviewed the medical records of all the pts included in the Princess Margaret Hospital Amyloidosis Database who underwent ASCT from January 2004 to March 2010 to determine: a) TRM, b) Hematological response (HR) and Organ response (OR) and c) Overall survival (OS). We included transplanted pts who had received no more than two previous courses of any chemotherapy regimen who did not have concurrent myeloma, and who had an Eastern Cooperative Oncology Group (ECOG) performance-status score of 2 or better. As part of the ASCT the stem cells were obtained from the peripheral blood with granulocyte colony-stimulating factor (G-CSF) alone (5 μ g/kg subcutaneously, twice daily). Pts then received HDM, 200 mg/m2 given intravenously on day 0, and stem cells were infused on day 1. The HDM was reduced to 140 mg/m2 for pts 65 years of age or older and for those with an important decrease of the ejection fraction, a calculated creatinine clearance of less than 30 ml per minute, or severe liver disease. HR and OR was assessed using 2004 Amyloidosis Consensus Response Criteria (Gertz, M et al, 2004). For statistical evaluation, HR was defined as observing complete or partial remission (CR or PR); stable disease (SD) or progression was considered as no HR. All statistical tests were 2-sided X2 t test and P values less than .05 were considered to be statistically significant. Results Seventy seven pts were evaluated with a median age of 56 years (33-74). Clinical characteristics are seen in Table 1. Only 18/77 pts (23%) received induction therapy: Dexamethasone alone n=9, Cyclophosphamide and Prednisone n=2, Mel/Dex n=4, VAD n=2 and Melphalan and Prednisone n=1. TRM observed was 12% mainly due to congestive heart failure and sepsis. Complete HR was achieved in 38 cases (49%) and OR was seen in 59%. Among the most common complications to ASCT, febrile neutropenia was reported in 67%, transient renal impairment in 10% and congestive heart failure in 3%. Median OS was significantly lower for patients with B-type natriuretic peptide (BNP) >300 pg/ml (17.7 vs 64.46 months) (p0.0004), Troponin I >0.07 ng/ml (19.25 vs 97.4 months) (p0.00001) and those who achieved a complete HR (115 vs 88 months) (p0.0345). Multivariate Cox regression analysis showed that BNP>300 pg/ml and Troponin I >0.07 ng/ml (normal <0.07) were the most important factors to predict OS for patients with AL who underwent ASCT (p0.0002 and 0.04 respectively). Conclusions Based on this study of 77 patients, clinical selection is the most important factor to better improve outcomes for HDM in AL. Patients with BNP>300 pg/ml and/or abnormal levels of Troponin I (>0.07) should not be considered eligible for transplants due to a high degree of mortality. Our series showed a CR rate of 49% which is higher than 33% reported by Palladini et al with Mel/Dex. Stringent case selection may be the most important factor in decreasing mortality rate associated to ASCT leading to better outcomes. Table 1. Clinical Characteristics of patients with AL Amyloidosis undergoing ASCT (at initial diagnosis) Clinical Characteristics N Median Range % Age (years) 77 57 33–74 Hemoglobin (g/L) 77 138 104–182 Creatinine (μ mol/L) 77 129 37–700 **BNP (pg/ml) 44 180 10–1670 ***BMPC (%)– 77 7 1–30 24 Hr Proteinuria (g/d) 77 5.5 0–24.9 Intraventricular Septal Distance (mm) 77 13 9–23 Kappa/lambda ratio 53 4.4 0.001–71.2 Kidney Involvement 71 Heart Involvement 52 Liver involvement 23 GI involvement 10 3 or more organs involved by AL 16 ** B-Type Natriuretic Peptide *** BMPC: Bone marrow plasma cells Disclosures: Stewart: Millennium: Consultancy; Celgene: Honoraria. Reece: Celgene: Honoraria, Research Funding. Chen: Celgene Corporation: Consultancy, Honoraria, Research Funding. Kukreti: Celgene: Honoraria.

SummaryAutologous stem‐cell transplant has been widely used to treat patients with AL amyloidosis. However, transplant‐related mortality rates are high, and a recent randomized trial suggested that non‐transplant regimens produced comparable results with less toxicity. In order to define the role of patient selection in stem cell transplantation, we evaluated 78 consecutive AL amyloidosis patients transplanted at our centre. Transplant‐related mortality occurred in 11·5%. Complete haematological response and organ response were achieved in 56% and 60%. Median overall survival was significantly lower for patients with brain‐type natriuretic peptide (BNP) >300 pg/ml (17·5 months vs. not‐reached) (P = 0·0004), troponin‐I >0·07 ng/ml (13·5 months vs. not‐reached) (P = 0·00001) and those not achieving a complete haematological response (88 months vs. not‐reached) (P = 0·0345); high BNP and troponin‐I were the most important predictive factors in a multivariate analysis. Based on this study, patients with BNP <300 pg/ml and/or normal levels of troponin‐I should be considered transplant candidates.