Lust, John A.

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Abstract Background: Abnormally increased tumor associated neovasculature plays an important role in tumor progression in solid tumors and hematological malignancies. In multiple myeloma (MM) bone marrow angiogenesis, measured in terms of microvessel density (MVD), has prognostic value, and appear to increase with disease progression from monoclonal gammopathy of undetermined significance to relapsed MM. We have shown that MVD has prognostic value in patients with newly diagnosed MM including patients undergoing upfront high dose therapy and stem cell transplantation, but comparison with other known prognostic factors has been limited by sample size. It is also not known whether the MVD adds prognostic value once the recently described International Staging System (ISS) is applied. The goal of this study was to determine the prognostic effect of bone marrow MVD in newly diagnosed MM relative to the ISS and other known prognostic factors. Methods: We studied 400 patients with newly diagnosed MM seen at the Mayo Clinic between March of 1988 and December 2001. Sections from bone marrow biopsy blocks from the time of initial diagnosis were studied by immunohistochemistry using antibodies against CD34 antigen to high light the endothelial cells. Under low power (100X); three areas with maximum number of microvessels (hotspots) were identified. Each of these areas was further studied at 400X magnification and the number of microvessels per high power field counted. The average of the three readings was taken as the MVD for the sample. In addition, the samples were graded as low, intermediate or high by using a visual estimate as previously described. Additional clinical data was extracted from medical records. Some of the patients have been included in previous studies related to angiogenesis. Results: A total of 400 patients in whom a bone marrow biopsy from within 30 days of diagnosis was available were studied. The pts were followed for a median of 37 months (Range: 1 month to 16.5 years) and 318 pts (80%) had died at the time of this analysis. The median MVD for the entire group was 14.7 (Range 0–168). The median overall survival for the three groups according to the MVD grade was lower for the high grade group (31.9 months) compared to the intermediate grade group (37.2 months) and the low grade group (not reached); P <0.029, log rank test. We examined this group of patients for other factors prognostic for overall survival. Factors significant on univariate analysis included ISS stage, platelet count (<150 vs. >= 150 X 106/L), and plasma cell labeling index (<1 vs >=1). In a multivariate analysis using these variables and MVD as a continuous variable, high MVD and the ISS staging system were significantly associated with poorer survival (Table). Conclusion: In this large group of pts with newly diagnosed MM, we confirm the prognostic value of increased bone marrow angiogenesis. We examined the MVD as a continuous variable in the multivariate analysis for a closer evaluation of this measure in this comparison. More importantly, the prognostic value appears to be independent of the ISS and other major prognostic factors. The resultsof this study reinforces the biological relevance of this finding in MM. HR 95% CI P value MVD 1.006 (1.001, 1.011) 0.0279 ISS Stage I 0.37 (0.25, 0.56) <0.001 ISS Stage II 0.58 (0.41, 0.83) 0.0025

Abstract Introduction: Increased bone marrow angiogenesis is a characteristic feature of multiple myeloma (MM) and correlates with disease progression. Increased bone marrow angiogenesis at the time of diagnosis, measured in terms of microvessel density (MVD), is a powerful adverse prognostic factor for patients with MM. To better understand the biological basis of this phenomenon and to understand the mechanisms responsible for increased MVD in MM we compared the gene expression profiles of plasma cells from newly diagnosed MM patients with low and high MVD. Methods: Bone marrow biopsy sections from pts with newly diagnosed MM were studied using CD34 immunostaining and graded as low, intermediate, or high by MVD as previously described. 19 pts each, with low or high MVD were included for this study. RNA was isolated from CD138+ plasma cells of MM patients and analyzed using Affymetrix HG-U133A arrays. To examine differential expression, GC-RMA normalized data was analyzed using GeneSpring 7.2 software and genes with ≥ 2-fold differential expression between high and low MVD samples were identified. The Welch T-test was used to determine the significance of differential expression. Results and Conclusion: Expression of 42 transcripts was increased ≥ 2-fold in samples with high MVD in comparison to samples with low MVD. Of these transcripts, 14 were found to be significantly increased (P<0.05) using the Welch T-test (see Table 1). Expression of 16 transcripts was decreased ≥ 2-fold in samples with high MVD in comparison to samples with low MVD. Of these transcripts, 6 were found to be significantly decreased (P<0.05) using the Welch T-test (see Table 1). Genes differentially expressed include those involved in inhibiting apoptosis, facilitating IL-6 signaling, ion transport, extracellular matrix interaction, and regulating gene expression. Classical angiogenesis genes including VEGF, FGF, and IGF were not found to be differentially expressed (> 2-fold). In conclusion, the list of differentially expressed genes reveals many functions relevant to MM disease pathology including proliferation, apoptosis, regulation of gene expression, cytokine signaling, and adhesion. Current studies evaluating the expression and function of these genes in relation to MM may identify factors critical for angiogenesis and MM progression and provide insight for therapeutic intervention. Gene Description Fold Change COL1A2 collagen, type I, alpha 2 3.008 MCL1 myeloid cell leukemia sequence 1 (BCL2-related) 2.793 209183_s_at chromosome 10 open reading frame 10 2.541 VIL2 villin 2 2.478 IL6ST interleukin 6 signal transducer (gp130) 2.404 CLIC2 chloride intracellular channel 2 2.364 DEPDC6 DEP domain containing 6 2.35 PBXIP1 pre-B-cell leukemia transcription factor interacting protein 1 2.337 CLIC4 chloride intracellular channel 4 2.334 CYP51A1 cytochrome P450, family 51, subfamily A, polypeptide 1 2.095 LIMS1 LIM and senescent cell antigen-like domains 1 2.091 YWHAE tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide 2.042 OAS1 2′,5′-oligoadenylate synthetase 1, 40/46kDa 2.031 S100A11 S100 calcium binding protein A11 2.018 222378_at unknown transcribed sequences 0.496 SLC5A3 mitochondrial ribosomal protein S6 0.463 213089_at unknown transcribed sequences 0.406 PDE4B phosphodiesterase 4B, cAMP-specific 0.454 SVIL supervillin 0.386 RIPX rap2 interacting protein x 0.383

Abstract Background: Thalidomide and dexamethasone combination has been shown to be an effective therapy for multiple myeloma (MM). In newly diagnosed patients with MM, the combination results in overall response rates of over 70% of patients. However, a considerable number of patients do not respond and will need to proceed to other therapies. The ability to identify patients who are unlikely to respond to particular therapies will allow tailoring of therapeutic approaches and in turn will reduce unnecessary toxicity and morbidity from lack of rapid disease control. With the availability of new technology such as gene expression profiling (GEP), this has become a possibility. We employed this approach to identify genes that can reliably predict lack of response to the thalidomide dexamethasone combination. Methods: Patient samples from the Eastern Co-operative Oncology Group clinical trial (E1A00), that compared thalidomide-dexamethasone combination with dexamethasone, and patient samples from the phase II Mayo Clinic study of thalidomide and dexamethasone, both for newly diagnosed MM, were used for this study. Thirty newly diagnosed patients who were evaluable for response were included in the analysis. GEP was performed using the Affymetrix U133A microarray platform as per manufacturer’s recommendation. The Affymetrix output (CEL files) was imported into Genespring 7.2 (Agilent Technologies) microarray analysis software, normalized across chips using GCRMA followed by per gene normalization to median. For the purposes of this study, responses were defined as reduction in the serum paraprotein (or the urinary M protein in the absence of a serum M protein) of >=50% (PR), 25 to 49% (MR), increase of >=50% (PD) and stable disease for the remaining. Results: Five of the 30 patients had no response to the thalidomide Dexamethasone therapy. Using the class prediction tool available in Genespring (Support Vector Machines), we identified 25 genes that reliable predicted non responders (PD, NC) from the responders (MR, PR, and CR). See table for a list of predictive genes with identified genes. Conclusion: Using a combination of two datasets we have identified a set of genes that can be used to reliably predict lack of response to thalidomide and dexamethasone in patients with newly diagnosed MM. We think that this represents a step towards creation of custom microarrays spotted with genes that are capable of predicting lack of response (or response) for the purpose of tailoring therapy to the patient. Gene Predictive strength hypothetical protein FLJ20719 1.655 Tubulin alpha 6 1.653 SEC3-like 1 (S. cerevisiae) 1.649 Ribosomal protein L18 1.546 Ribosomal protein L4 1.432 Tubulin alpha 6 1.409 cleavage stimulation factor 1.393 Tubulin, alpha, ubiquitous 1.393 CD99 antigen 1.38 hmel2 1.346 Leptin receptor 1.291 Integrin-linked kinaseSMC4 structural maintenance of chromosomes 4-like 1 1.269 Cornichon homolog 1.255 Williams-Beuren syndrome chromosome region 5 1.217 Ubiquitin specific protease 1 1.2 Sarcoma antigen NY-SAR-91 1.2

Abstract Introduction: The kidney is affected by immunoglobulin light chain amyloidosis (AL) in more than 50% of patients who present with the disease, but long-term predictors for and outcomes after renal replacement therapy have not been well described in this patient group Patients: 145 patients with AL who participated in Mayo Clinic treatment trials and were monitored for at least 11 years. No patient was lost to follow-up Results: Among patients presenting with renal AL, 42% ultimately received renal replacement therapy compared with 5% of patients who did not have an initial presentation involving the kidney. Table 1. Monoclonal Protein Studies in 145 Patients With Immunoglobulin Light Chain Amyloidosisa Patients, No. (%) Monoclonal Protein Renal Amyloid at Presentation (n=84) Nonrenal Amyloid at Presentation (n=61) a P=.02 l more likely to cause renal presentation None 7 (8) 2 (3) k 6 (7) 12 (20) l 71 (85) 47 (77) Table 2. Characteristics of All 145 Patients With Immunoglobulin Light Chain Amyloidosis Amyloid Renal (n=84) Nonrenal (n=61) P Value Abbreviations: IQR, interquartile range; NA, not applicable. a included in the definition of renal amyloidosis Patients with l immunoglobulin light chain amyloid were significantly more likely to have renal involvement and had significantly greater urinary protein loss than patients with κ or no monoclonal light chain amyloid Age, median (IQR), y 61 (52–67) 62 (55–68) .47 Sex, male/female 52/32 40/21 .72 24-hr urine protein loss, median (IQR), g/d 7.0 (4.4–11.7) 0.3 (0.1–6.4) NAa Creatinine, median (IQR), mg/dL 1.1 (0.9–2.2) 1.1 (0.9–1.3) .22 Cardiac involvement (yes/no) 35/48 48/16 .0001 Table 3 Distribution of Urinary Protein and Creatinine Based on Monoclonal Protein Monoclonal Protein Test k (n=18) l (n=118) None (n=9) P Value Abbreviation: IQR, interquartile range. Patients with renal amyloid who ultimately received dialysis support had significantly higher serum creatinine and 24-hour urine protein levels at presentation. Absence of cardiac involvement at diagnosis predicted an increased likelihood of dialysis All patients (N=145) 24-hr urine protein loss, median (IQR), g/d 0.73 (0.16–2.37) 3.61 (0.35–7.8) 5.68 (0.3–12.2) .001 Creatinine, median (IQR), mg/dL 1.1 (0.9–1.3) 1.1 (0.9–1.3) 1.5 (1.2–1.6) NS Patients with renal amyloid at presentation (n=84) 24-hr urine protein loss, median (IQR), g/d 2.93 (1.1–5.0) 7.2 (4.8–11.8) 7.8 (3.8–12.6) .04 Creatinine, median (IQR), mg/dL 1.0 (0.8–2.6) 1.1 (0.9–2.2) 1.5 (1.1–1.6) NS Table 4 Characteristics of 84 Patients Presenting With Renal Amyloidosis Dialysis Subsequently Not Required Characteristic Required (n=35) (n=49) P Value Abbreviation: IQR, interquartile range. The median time from diagnosis to dialysis was 29.5 months for those that went into renal failure.). However, the actuarial risk reached 50% at 12 years Kaplan-Meier failure curve demonstrating the actuarial time from diagnosis to the start of dialysis therapy N=145 For 38 patients who received dialysis, median survival from day 1 of dialysis was 10.4 months Kaplan-Meier survival of 38 patients from the start of dialysis therapy Age, median (IQR), y 60 (52–67) 62 (52–67) .90 Sex, male/female 21/14 31/18 .76 24-hr urine protein loss, median (IQR), g/d 7.4 (5.0–14.8) 5.9 (3.7–9.1) .03 Creatinine, median (IQR), mg/dL 1.4 (0.9–2.8) 1.1 (0.9–1.5) .01 Cardiac involvement (yes/no) 9/26 26/23 .01 Conclusion: In our study, 26% of all patients with AL ultimately received renal replacement therapy compared with 42% of patients who presented with renal AL specifically. The presenting 24-hour urine protein loss and creatinine values predict which patients will require dialysis. Median survival for patients starting dialysis is less than 1 year. Presence of l light chain amyloid predicts an increased likelihood of renal involvement and, when the kidney is affected, increased proteinuria.

Abstract Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo. Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension. Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3. Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.

Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. Lenalidomide, especially in conjunction with dexamethasone, has been shown to be highly active in patients with multiple myeloma. We previously reported a hematologic response rate of 41% and an organ response rate of 23% among 22 AL patients treated with lenalidomide and on-demand dexamethasone (Dispenzieri et al, Blood 2007). Long term outcomes of AL patients treated with lenalidomide have not yet been described. Methods: Thirty-seven patients were treated on an IRB approved treatment trial with single agent lenalidomide, starting doses of 25 mg in 22 and reduced dose of 15 mg in the last 15. If progression within or no evidence of hematologic response after 3 cycles, dexamethasone was added. Patients were classified according to cardiac biomarker stage (cut-offs for serum troponin T <0.035 ng/ml and NT-proBNP <332 pg/ml--Stage I neither above cutoff; Stage III, both above cut-off; and Stage II, any one above cut-off). Survival estimates were done according to the methods of Kaplan and Meier. Results: Median age was 64 years (range 44–88), with 70% male. Sixty-five percent of patients were previously treated, and the median time from diagnosis to study entry was 8.9 months (range 0.4–237). The biomarker staging breakdown was: I, 16%; II, 43%; and III, 41%. As of 7/11/08, 5 patients remain on active therapy. The overall hematologic response rate was 43% (1 CR and 15 PR), with only one patient responding to single agent lenalidomide, and the overall organ response rate was 20%. If one excludes the 13 patients who did not complete 3 months of therapy and have the opportunity of having dexamethasone added to their program, the respective hematologic and organ response rates were 62% and 32%. With a median follow-up of 33.6 months (range 14.4–42), twenty-two patients (59%) have died, but only 2 had deaths possibly related to treatment. Median duration of response, progression free survival and overall survival were 31 months, 14.8 months (95%CI: 5.7–28.1), and 18.8 months (95%CI: 11.7-not reached), respectively. Overall survival from diagnosis was 48 months (95%CI: 21.6–153.6). As shown in Table below, outcomes were dependent on underlying risk stratification and ability to stay on therapy. High risk patients were more likely to drop out early and less likely to respond. High risk patients had shorter OS All Patients Outcome Stage I Stage II Stage III p N 6 16 15 Survival from enrollment, mo NR 18.8 (11.7–33.6) 5.7 (3.2–18.3) 0.01 Survival from Diagnosis, mo NR 48.4 (20.4–62) 21.1 (11.2–31.7) 0.005 Progression Free Survival, mo 34.7 (33.7–NR) 18.3 (6.2–28.1) 4.4 (3–8.6) NS Drop out by cycle 4, % 17 31 60 NS Median # of cycles administered 31 6 3 0.04 Time to response, mo 6.2 (3.6–9.3) 6.4 (5.2–NR) 4.7 (3.8–NR) NS Hematologic response rate, % 83 38 27 0.05 Organ response rate, % 40 20 13 NS Duration of Response, mo NR 27.2 (13.6–NR) NR NS Subgroup analysis of patients receiving >3 cycles of therapy (i.e getting Len and Dex) N 5 12 7 Time on therapy, mo 35.2 8 10.5 Hematologic Response Rate, % 100 50 56 Organ response rate, % 50 27 25 Conclusions: Based on these data, single agent lenalidomide is not recommended for patients with AL; however, lenalidomide in combination with dexamethasone can be associated with good progression free and overall survival rates, even among patients with relapsed or refractory disease.

Abstract Background: The combination of lenalidomide and dexamethasone (Rev/Dex) has been shown to be a highly effective therapy for multiple myeloma (MM). Over half of the patients with newly diagnosed MM achieve a complete response with prolonged therapy. More recently, a phase III trial demonstrated improved survival with use of lenalidomide and low dose dexamethasone with one year survival in excess of 90%. We report the initial results from a phase II trial combining lenalidomide and low dose dexamethasone with cyclophosphamide (CRd) as initial therapy of newly diagnosed MM. Methods: The trial was initiated in July 2006 and completed the target accrual of 33 patients by July 2007. The treatment protocol consisted of lenalidomide given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dexamethasone (dex) was given orally at a dose of 40 mg on days 1, 8, 15, and 22 of each cycle. Cyclophosphamide at a dose of 300 mg/m2 was given on days 1, 8, and 15 of each cycle. Patients also received an aspirin once daily as thromboprophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours, confirmed at least 4 weeks apart. Results: The median age was 63 years (range, 44–79). Eight patients (24%) had ISS stage III disease. At this time, 19 of the 33 patients are evaluable for confirmed responses (i.e., off-study or completed at least 4 cycles of therapy). Of these, 2 achieved VGPR and 13 had a partial response giving an overall response rate of 79%. The response rate was affected by 5 of 19 patients who went off study within three cycles due to toxicities [interstitial nephritis (1 pt), multiple grade 3 toxicities including infection (1 pt) atrial fibrillation and infection (1 pt)] or alternative treatment [no response and possible renal toxicity (1 pt) and progression at 4 cycles (1 pt)]. Overall, hematological toxicity was the most common with grade 4 toxicity seen in 6 patients (20%). Non-hematological grade 3 or higher toxicities included fatigue (4 pts), thrombosis (3 pts) and renal failure (2 pts). One patient with thrombosis came off study for other toxicities, and the other two continued on study with anticoagulation. Thirteen patients (43%) had dose reductions of both cyclophosphamide and lenalidomide, most commonly due to hematological toxicity. So far, 12 patients have gone off study, 6 went to stem cell transplant, 3 due to adverse events, 1 due to disease progression and 2 patients went to alternate treatment. Conclusions: CRd has excellent activity in newly diagnosed myeloma with an estimated overall response rate of 79%. Response evaluation of the entire 33 patients will be presented at the meeting. We believe that the initial use of a 300 mg/m2 dose of cyclophosphamide resulted in 5 of the first 19 pts experiencing early toxicity and withdrawal. As a result, an expansion of the current trial is evaluating lower doses of cyclophosphamide (300 mg fixed dose). Full analysis of this study will be needed to determine if CRd can improve outcome compared to Rd.

Abstract Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder with a poor survival, especially in patients (pts) with multiple organ involvement. For pts with limited organ involvement, high dose chemotherapy with autologous stem cell (SCT) support is an effective approach. However, the toxicity of SCT limits its widespread use. Melphalan and dexamethasone has been previously shown to be effective in pts ineligible for SCT. Methods: We reviewed our experience with melphalan and dexamethasone for treatment of AL. Eighty one pts were treated between 2001 and 2003; median age at start of therapy was 63 years (range, 38–80) and 56 pts (69%) were males. Among these, 46 pts (57%) had 2 or more major organs (cardiac, renal, hepatic) involved. Intravenous melphalan (IV) was used in 45 pts (56%), 32 (40%) received oral melphalan (PO) and the rest received cycles of both. The most common doses were 16 mg/m2 for IV administration (every 4–6 weeks) and 0.22 mg/kg for PO administration (every 4 weeks). Dexamethasone was usually given at a dose of 40 mg for the first 4 days of each cycle. Results: Patients received a median of 5 cycles (range; 1–17). At the time of analysis, 53 (65%) pts were alive with a median follow up of 10.8 months (range, 1.4–47.1).The estimated median overall survival (OS) was 25.6 months for the entire group, with the number or type of organs involved not impacting survival. Hematological response (HR) was defined by a 50% reduction in either serum M-spike, urine M-spike, or the absolute difference between the involved and uninvolved immunoglobulin free light chains. A HR was observed in 43 pts (67%) of the 64 pts measurable for HR by conventional criteria. Achievement of HR was associated with a superior OS with a median post-therapy OS that was not reached, while non-responders had a median OS of 11.1 months (p<0.0001) (Figure 1). Among these pts, an organ response as defined by conventional criteria, was seen in 14 pts (22%), which was significantly correlated with obtaining a hematological response (33% vs. 5%, p=0.01). A 25% reduction in the difference between the involved and uninvolved FLC strongly predicted prolonged OS, with a median post-therapy OS that was not reached for responders vs. 11.3 months for non-responders (p<0.0001) (Figure 2). A decrease in post-therapy brain natriuretic peptide (BNP) also predicted for a survival improvement, with the median OS not reached for responders vs. a median OS of 17 months for non-responders (p<0.016). Conclusion: Treatment with melphalan and dexamethasone is effective and a significant survival advantage is achieved in patients with a hematological response. FLC and BNP response can help identify patients that are likely to benefit from this therapy. Figure Figure Figure Figure

Abstract Background: Primary systemic amyloidosis is an uncommon plasma cell dyscrasia characterized by organ deposition of immunoglobulin light or heavy chain fragments. It is related to the other plasma cell dyscrasias such as multiple myeloma and monoclonal gammopathy of undetermined significance. cIg-FISH on bone marrow plasma cells has become well established in the initial evaluation of myeloma as the t(4;14), t(14;16),13q-, and the 17p- abnormality are of major prognostic value. The corresponding data does not exist in amyloidosis, as there are only three small studies published to date. Methods: Using a prospectively held dysproteinemia database, we reviewed all cases of amyloidosis seen at our institution who had cIg-FISH or cytogenetics performed as part of their routine clinical testing. Three hundred thirty nine patients were identified with amyloidosis and cytogenetic testing. Of these 69 had cIg-FISH testing results. cIg-FISH was performed with fluorescent-tagged antibodies to kappa/lambda immunoglobulin and FISH probes corresponding to the following anomalies: trisomies 3, 7, 9 and 15; del17p/-17; -13/del13q; 14q32 split; and t(11;14). CCND1/IGH. If an IGH rearrangement was detected that did not involve CCNDI, we also evaluated t(4;14) or IGH/FGFR3 and t(14;16) or IGH/c-MAF. These probes constitute the standard screening panel used in MM at our institution. Results: The study population had a median age of 61 years with a male to female ratio of 1.6:1. Median follow up time was 13.5 months. By conventional cytogenetics, 88% of patients had no detectable abnormality. In contrast, only 33% were normal by cIg-FISH. The abnormalities demonstrated were 28% 13-/del13q, 42% IgH translocation, 33% t(11;14), and 3% t(14;16). No patients had a -17/del 17p- or t(4;14). Nine patients had del13q/-13 with an IgH translocation, with 7 of these having t(11;14). Both t(14;16) patients had del 13q. A trend towards increased mortality was seen in patient with any abnormality (p=0.11), but this could not be demonstrated for any single abnormality, likely due to lack of statistical power. There was no correlation between renal involvement or cardiac involvement and cIg-FISH results. Conclusion: The majority of chromosomal abnormalities in amyloidosis are undetected by conventional cytogenetics. With further study, chromosomal abnormalities may carry the same prognostic value in amyloidosis that they currently enjoy in myeloma. This effect may be independent of currently recognized risk factors. Chromosomal abnormalities in Amyloidosis as detected by cIg-FISH Abnormality Amyloidosis % (n=69) Normal 33(23) −13/del13q 28(19) IgH Abnormality 42(29) t(4;14) 0(0) t(11;14) 33(23) t(14;16) 3(2) del 17 0(0) Other 3(2)