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Abstract Hyperdiploid (>48 chromosomes) multiple myeloma (H-MM) and high hyperdiploid (>50 chromosomes) acute lymphoblastic leukemia (H-ALL) are characterized by aneuploidy and multiple recurrent trisomies (chromosome 3,5,7,9,11,15,19 for H-MM and chromosomes X,4,6,10,14,17,18,21 for H-ALL). Little is known about the oncogenic events, consequences of the trisomies and reasons for the different recurrent trisomies. In an attempt to answer these questions, we undertook a combined gene expression and network/pathway analysis approach. Gene expression data was generated using the Affymetrix U133A chip (Affymetrix, Santa Clara, Ca) for 53 H-MM and 37 non-hyperdiploid MM (NH-MM) cases using CD138-enriched plasma-cell RNA. Gene expression data using the same chip for ALL was obtained from previous published data (Ross ME et al Blood2004; 104: 3679–3687). Analysis was performed using Genespring 7 (Agilent Technologies, Palo Alto, CA). By comparing the median expression of all genes on each chromosome between H-MM/H-ALL and their non-hyperdiploid counterparts (NH-MM and NH-ALL) for the 23 chromosomes (excluding Y), one can clearly identify the commonly trisomic chromosomes in H-ALL and H-MM. However, the relationship of gene expression was highly variable for H-MM and NH-MM as compared to H-ALL and NH-ALL which tended to have expression ratios close to 1 for the non-trisomic chromosomes. Sixty-nine percent of the differentially expressed genes generated by ANOVA analysis (p<0.001) in H-ALL were on the commonly trisomic chromosomes and were upregulated whereas the corresponding figure in H-MM is 40%. These similarities and differences probably reflect both an overall gene dosage effect and the different complexities of the karyotypes of H-MM and H-ALL compared to NH-MM and NH-ALL respectively (MM karyotypes are more complex, hence difference between H and NH-MM is greater and less confined to the trisomic chromosomes). We next performed network analysis using a curated web-based software (MetaCore, GeneGo Inc, St Joseph, MI) using the 2 sets of differentially expressed genes. Majority of genes differentially expressed in H-MM are involved in mRNA translation/protein synthesis whereas the genes differentially expressed in H-ALL were mainly involved in signal transduction. Therefore the transcriptional program that characterize the difference between H and NH-MM/ALL seem to recapitulate normal cellular function: protein synthesis in the mature secretory plasma cells and signal transduction in response to cytokines in a differentiating early-B cell. However, due to the concurrent deregulation of many genes on these trisomic chromosomes, these and other cellular programs are deregulated resulting in malignant transformation. We also intersected the 2 lists of differentially expressed genes to find genes that are up- or downregulated in both H-MM and H-ALL relative to the NH tumors. Thirteen genes including interferon response genes (TNFSF10, MX1, ZNF185) and transcription factors like RUNX1 were upregulated, whereas 13 genes including a cancer testis antigen gene (MAGED4) were downregulated in both H-MM and H-ALL. These genes may point to common oncogenic mechanisms.

Abstract There is a high background risk of venous thrombotic events (VTE) in patients with Multiple Myeloma (MM) and this is significantly increased following treatment with Thalidomide. The risk may be mediated by the production of a prothrombotic state, via an angiopathic mechanism, or alternatively it may arise from the release of prothrombotic factors from dying myeloma cells targeted by the treatment. No unifying evidence of a prothrombotic state has been demonstrated to date and it remains important to understand the mechanism underlying VTEs in order to prevent them effectively. Inherited genetic variants in the form of SNPs can affect the behaviour of clotting factors. These SNPs are functional and well characterised in the coagulation system and we have used them to analyse the mechanism underlying thalidomide induced VTE. As part of the Bank On A Cure TM project we have carried out an analysis of SNPs in 11 genes: GPIIA, ITGA, MTHFR, MTR, PPARG2, TFP1, VCAM, factor V, HABP2, prothrombin and THDB in a case series of 90 patients treated with thalidomide, with and without DVTs. This study showed a significant association with MTHFR and factor V polymorphisms. In order to validate and extend these initial observations we carried out an analysis of a second nested case control study using 131 Myeloma patients from within the MRC Myeloma IX trial, comprising 47 cases with a VTE and 94 age and sex matched controls exposed to the same treatment. We used a SNPlex panel containing 16 functional venous thrombosis associated SNPs, all of these functional polymorphisms have reported mechanisms and biology. We looked at polymorphisms in the genes: fibrinogen A (Ala331Thr), B (-455G>A, Lys478Arg) and G polypeptides (His140Tyr); Coagulation factors V(Arg534Gln), VIII (Glu1260Asp), IX(Ala194Thr), XI (Phe339Cys) and XII(-4G>T); PROCR(Gly219Ser); prothrombin(19911A>G ,20210G>A); MTHFR(Glu429Ala, Ala222Val); Serpine1(-844G>A) and TFPI(Met292Val). We have typed our selected SNPs using the SNPlex™ Genotyping System, which is based on the oligonucleotide ligation/PCR assay with a universal fluorescently labeled ZipChute™ probe detection. The probes are hybridized to complementary ZipCode™ sequences that are part of genotype-specific amplicons. Probes are eluted and detected on a Applied Biosystems 3130x DNA Analyser. This approach is an attractive alternative to existing genotyping methodologies, as it requires only three unlabeled probes per SNP, and consumes very little gDNA. The data analysis on the polymorphisms frequencies is ongoing to examine associations of thalidomide treatment with VTE events and will be presented. These datasets will allow a better definition of the pathogenesis of thalidomide related VTE, so that we can intervene therapeutically in a rational fashion.

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy involving complex cytogenetic dysregulation of genes and heterogeneous expression of multiple B cell and plasma cell (PC) surface antigens. 60% of MM patients have cytogenetic translocations of the IGH locus. In 16% of myeloma patients, the partner oncogene is cyclin D1 on 11q13 causing t(11;14)(q13;q32) (Fonseca et al. Blood. 2002). This translocation is more common in patients with a lymphoplasmacytic morphology and confers a more favorable prognosis (Fonseca et al. Blood. 2003). A subset of MM patients (20%) harbor a clonotypic population of CD20+ bone marrow plasma cells (BMPC). These patients have a shortened survival time (SanMiguel et al. Br J Haematol. 1991). There is a strong association with CD20+ surface expression and t(11;14)(Robillard et al. Blood. 2003) and CD20+ PC has been suggested to be clonogenic and important in the pathogenesis of MM. A small Phase II study showed only a modest response to the humanized anti-CD20 monoclonal antibody rituximab in MM patients with CD20+PC (1 PR and 5SD) (Treon et al. J. Immunother. 2002). We report a response to treatment with rituximab in a MM patient with CD20+ BMPC and t(11;14)(q13;q32) by FISH. Case: The patient is a 68 year old male with a 1.5 year history of MM. Initial diagnosis of ISS stage I disease was made after a three year history of IgA kappa monoclonal gammopathy of undetermined significance (MGUS). At diagnosis of MM, he had symptomatic anemia Hgb10.5g/dL, rising serum IgA of 3950 mg/dL, serum m-spike of 4.1 g/dL (IgA kappa), PCLI (plasma cell labeling index) of 1%, and a small monoclonal IgA kappa with kappa fragment in the urine. BM showed 44% monoclonal kappa plasma cells with 90% CD20+,clonal PC on flow cytometry. Karyotype was normal; 46, XY, but FISH showed evidence of an IGH translocation t(11;14)(q13;q32) in 60% of PC. Immunohistochemical staining was positive for cyclin D1. Given the marked elevation of CD20+ PC and early stage of disease, initial treatment was employed with four weekly cycles of rituximab (375mg/m2). IgA fell from 4750 mg/dL to a nadir of 2990 mg/dL, (m-spike; 4.5g/dL to 3.2 g/dL) five weeks after completion of treatment. The monoclonal protein (MP) in the urine disappeared. Hgb improved to 12.5g, with erythropoietin. Three months later, IgA rose to 3260mg/dL (m-spike 3.2 g/dL) and urinary MP returned. The patient remained asymptomatic. A second course of weekly rituximab was started. IgA level and serum m-spike remained fairly stable over the next 7 months. All treatment was tolerated well except for initial transient rigors, fever, and nausea. Despite bi-monthly maintenance rituximab, the patient devloped a rising IgA of 4940 mg/dL, m-spike of 4.7, falling Hgb and a rising PCLI of 1.5%, 15 months after his original diagnosis of MM. Of particular concern is the development of new, unfavorable, cytogenetic aberrations by FISH with BMPC now showing deletion 13 in 93% and mutation of p53 (17p13) in 98%.100% show fusion of CCND1 and IGH and PC now only partially express CD20. Conclusion: This report suggests that MM patients with CD20+ BMPC and t(11;14)(q13;q32) may represent a target population for anti-CD20 therapy. Our report supports the concept proposed that targeting this clonotypic subset of B cells possibly interrupts a critical oncogenic pathway that is important in the pathogenesis of the clonogenic development of MM.

Abstract Background: Multiple myeloma (MM) is a plasma cell malignancy that is incurable with current approaches. The median survival for patients with MM is around four years and a significant proportion of patients experience a course characterized by multiple relapses treated with different therapies. The median survival for patients relapsing after the initial therapy is nearly 18 months and successive treatment strategies result in decreasing response durations, likely reflecting acquired drug resistance. In order to better understand the biological changes associated with advanced, relapsed, refractory MM, we compared gene expression profiles (GEP) of malignant plasma cells isolated from patients with relapsed refractory MM and compared them to plasma cells from patients with newly diagnosed MM. Methods: In order to obtain two relatively homogenous group of patients, we compared samples from 44 patient with newly diagnosed MM enrolled in the ECOG E1A00 clinical trial (comparing thalidomide and dexamethasone to dexamethasone alone) to 44 patients with relapsed refractory MM enrolled in a phase II trial of Velcade (SUMMIT), where most patients had four or more previous relapses. Plasma cells from bone marrow aspirates were separated by magnetic bead selection of CD138 positive cells and studied using Affymetrix HG-U133A chips using standard methodology. The arrays were analyzed using Genespring 7.2 software following GCRMA normalization and genes with differential expression between the two datasets were examined. Differentially expressed genes were further analyzed using Ingenuity Pathways Analysis program. Results: A total of 864 genes were identified which were at least two fold and significantly different between the newly diagnosed and relapsed patients. Using Ingenuity software, 437 of these genes were mapped to different biological networks. Examination of the canonical pathways demonstrated several important cellular pathways differentially regulated between the two groups. Several important mediators of the cytokines, receptors and respective signaling pathways appear to be down regulated in the relapsed group and included IGF-1, HGF, SDF-1 alpha, gp130 and importantly the MEK/ERK pathway. Additionally expression of adhesion molecules such as VCAM1 and PECAM was decreased in the relapsed group compared to newly diagnosed pts. There appear to increased tissue hypoxia in the relapsed marrow as indicated by up regulation of HIF-1 alpha as well increased levels of Placental growth factor. Myeloma cells from relapsed disease were characterized by decreased expression of mcl1, FLIP1, and bcl-xL and increased caspase 8 relative to newly diagnosed group. Also seen was decreased expression of the glucocorticoid and interferon receptors in the relapsed setting. Conclusion: Comparison of the GEP between MM cells from newly diagnosed and relapsed pts demonstrates important differences that have potential biological relevance. The plasma cell in the relapsed setting appears to be more independent of the tumor microenvironment. Additionally, differential expression of some of the genes provides clues to mechanisms of drug resistance commonly observed in the relapsed pts. We are in the process of validating some of the key findings from these analyses.

Abstract Background: Multiple Myeloma (MM) is characterized by accumulation of malignant plasma cells with relatively low proliferative and apoptotic rates. The proliferative rate of plasma cells, measured in terms of % plasma cells labeled by bromodeoxyuridine (BrdU) using slide based fluorescence microscopy (plasma cell labeling index or PCLI) has been shown to be a powerful prognostic factor in MM. We have previously shown that the % bone marrow plasma cells in apoptosis (PCAP) determined by a flow cytometry method that uses Annexin V staining or 7-amino Actinomycin D (7-AAD) correlates with disease stage in MM. The goal of this study was to examine the prognostic value of the PCAP. Methods: We studied 145 patients with MM including 74 with newly diagnosed MM, 40 with relapsed MM, and 31 with MM on treatment in whom simultaneous measurements of % plasma cell apoptosis and PCLI were available. The PCLI is expressed as the percentage of cytoplasmic immunoglobulin positive plasma cells that have taken up BrdU. The PCLI was characterized as high when >= 1%. Mononuclear cells isolated from bone marrow aspirate, following red cell lysis with ACK solution, are incubated with CD38-PE and either CD138-FITC or CD45-FITC. This is followed by incubation with 7-AAD and the plasma cells are gated using the CD38/CD45 staining pattern. The Annexin method used a similar strategy, with Annexin V in place of 7-AAD. The percentage of plasma cells in apoptosis (PCAP) was categorized as high when >5%, the median value. Results: The median follow up for the entire group was 34 months (range, 0.1 mo to 9 years) and 112 (77%) had died at the time of analysis. Forty seven patients (32%) had a high PCLI (>=1%). The median overall survival for the patients with high PCLI was 14.6 months versus 42.3 months for those with a low PCLI; P = 0.0005 (log rank test). Seventy-five (51.7%) patients had a high PCAP. The median overall survival for those patients was 28.9 months versus 40.2 months for those with a low PCAP; P = 0.078. We next examined if there was any correlation between a high PCAP and high PCLI and found none; P = NS by (Fisher’s exact test). Additionally in a multivariate Cox model using both PCLI and PCAP, both were independently prognostic for overall survival. We then developed an index by adding PCLI to log transformed PCAP (PCLI + log PCAP). Using a cutoff of 1.5 for the new index we were able identify a group of patients with poor survival. The 59 patients with a high value for the new index had a median survival of 19.3 months vs 46.1 months for the rest (P < 0.0001, log rank test). Conclusion: The measurement of plasma cell apoptosis has prognostic value in patients with MM. More importantly, we have shown that by combining measures of two important biological characteristics of the plasma cell, namely proliferation and apoptosis, patients with a poor prognosis can be identified. Such a prognostication strategy can help stratify patients in clinical trials as well as shed light on the disease biology.

Abstract Chromosome 13 (Δ13) abnormalities are found in greater than 50% of patients with Multiple Myeloma (MM). MM is most commonly defined by chromosome 13 monosomy or 13q loss (85%). Interstitial deletions comprise the remaining 15%. Many studies have revealed that Δ13 in MM are associated with poor survival and reduced response to therapy. Genes mapping to chromosome 13 may be involved in pathogenesis and/or progression of the disease due to loss of function from gene mutation or from epigenetic effects such as haploinsufficiency. In this study, array-based comparative genomic hybridization coupled with microarray technology (aCGH) is used to detect gene copy number loss on chromosome 13 from nine MM patient samples. Whole genome long-oligo microarrays constructed by Agilent Technologies were used which contain 40,000 genes that span the human genome with an average spatial resolution of ~75 kb. Using genomic DNA isolated from MM patients with interstitial deletions on chromosome 13, DNA was amplified, labeled and hybridized with a differentially labeled normal DNA reference to determine gene/genomic copy number changes. Arrays were analyzed to search for the minimum region of loss based upon single copy loss for a series of nearby mapping transcripts. A common region of loss of 2.2 Mb, at 13q14.2 was detected. Additionally, we investigated the correlation between genomic copy number change and the expression level for MM patients in the13q region. From an independent gene expression data set whose expression measurements were conducted with Affymetrix HG-U133A v2 microarrays, data was selected that corresponded to the samples used for current aCGH. Expression values from MM samples were divided by the mean expression values from 12 normal bone marrow samples for each gene and the resulting values were treated as surrogate ratios between MM and normal samples. Probes from both microarrays were then aligned according to their chromosomal positions and merged if their chromosomal positions overlapped. Composite chromosomal maps were generated that displayed the expression levels and copy number changes. The maps were used to differentiate chromosomal regions in 13q where copy number changes and expression levels show high correlation and regions where such correlation was not observed. Although the number of probes sampled in the expression microarray was much smaller than those in aCGH microarray, a chromosomal region of great interest, that encompasses 13q14.2, arose naturally from this analysis. Although the mechanism by which loss of 13q effects tumorigenesis in MM could be a haploinsufficiency model, we are not ruling out the presence of a tumor suppressor gene in this region. We are evaluating candidate tumor suppressor genes in the region for loss of function by mutational analysis and hypermethylation studies.

Abstract Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by the expansion of a plasma cell clone that localizes to the bone marrow. Stromal cells residing in the bone marrow respond to signals from MM cells and other cell types by producing cytokines and other proteins that stimulate tumor cell growth, survival, adhesion, migration, and drug resistance. We have examined the proteins produced by stromal cells in response to stimulation by bone marrow from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. Furthermore, we have begun analyzing the properties of one of these proteins, the pro-angiogenic chemokine IL-8, in MM. Methods: Bone marrow aspirates from patients with MGUS (n=3), SMM (n=7), and MM (n=6) were cultured for 48 hours, and the culture supernatants were incubated with stromal cells for an additional 48 hours. Protein levels were analyzed using antibody array and ELISA. Microvessel density (MVD) was determined as a measure of angiogenesis in patient bone marrow samples using CD34 staining. Flow cytometry analysis of MM cell lines and patient bone marrow samples was performed using monoclonal antibodies against IL-8 receptors CXCR1 and CXCR2. Results and Conclusion: We observed a significant increase in stromal cell IL-8 production stimulated by bone marrow cells from patients with active myeloma and a subset of SMM patients (16.67 ± 9.82 ng/ml) in comparison to bone marrow of patients with MGUS and all other SMM patients (0.55 ± 0.17 ng/ml; P=0.0004). Use of an IL-1 inhibitor and recombinant IL-1β demonstrated that IL-8 production was dependent upon IL-1β signaling. Increased BM microvessel density correlated with stimulation of stromal cell IL-8 production (P=0.0005). Furthermore, the majority of MM cell lines (7/9) and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. In addition to its function as a pro-angiogenic factor, IL-8 may directly influence MM cells through its CXCR1 and CXCR2 receptors. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1β induced, correlates with bone marrow angiogenesis, and may influence MM disease via impact upon both the microenvironment and tumor cells.

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. Methods: We studied the toxicity and efficacy of lenalidomide in patients with symptomatic AL. Patients received single agent lenalidomide. If progression by 3 months or no evidence of hematologic response after 3 cycles, dexamethasone was added. Originally, twenty-three patients (Cohort 1) were enrolled according to study design. Because of a significant early drop out rate and notable activity of the regimen, the trial was modified to include an additional 15 patients (Cohort 2). Baseline characteristics and adverse events are available for all enrolled patients, but at the time of this writing, response data are available for Cohort 1 patients due to short follow-up of Cohort 2, but will be updated by the time of the meeting. Results: Median age was 64 years, with 69% male. Twenty-three were previously treated. Organ involvement was cardiac (67%), renal (64%), hepatic (17%), nerve (17%). Thirty-three, twenty-two, and forty-four percent of patients were cardiac biomarker stage 1, 2, and, 3 respectively. Of the 37 patients, one was a cancel, and 6 have not yet made it through 3 months of protocol treatment and event monitoring. The respective median follow-ups for Cohorts 1 and 2 are 17 and 3.4 months. Of the remaining, 30 patients, within the first 3 cycles of therapy fifteen patients discontinued treatment: 7 early deaths and 8 adverse events or other causes. Three additional patients died 0.5 to 2 months after stopping treatment. The best predictor for early withdrawal and/or death was baseline NT-proBNP and cardiac biomarker staging system (cut-offs for serum troponin T <0.035 ng/ml and NT-proBNP <332 pg/ml--Stage I neither above cut-off; Stage III, both above cut-off; and Stage II, one above cut-off). Figure1 Figure1. Figure 2 Figure 2. Of the twenty 22 patients assessed for response ten patients responded to treatment for an overall response rate of 45%, including 23% organ responders. Among the patients with organ responses, there were four renal responses, two cardiac responses and two liver responses. All but one of the responders had dexamethasone added to their treatment program. The most common grade 3−4 adverse events at least possibly attributable to lenalidomide were neutropenia (38%), thrombocytopenia (21%), fatigue (15%), and rash (12%). Conclusions: Lenalidomide and dexamethasone has significant activity in patients with AL with an overall response rate of 45%. Baseline NT−proBNP may be an effective eligibility screening strategy for subsequent trials.

Abstract Hyperdiploid MM (H-MM), characterized by recurrent trisomies constitute about 50% of MM, yet very little is known about its pathogenesis and oncogenic mechanisms. Studies in leukemia and solid tumors have shown gene dosage effect of aneuploidy on gene expression. To determine the possible gene dosage effect and deregulated cellular program in H-MM we undertook a gene expression study of CD138-enriched plasma-cell RNA from 53 hyperdiploid and 37 non-hyperdiploid MM (NH-MM) patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Gene expression data was analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Genes differentially expressed between H-MM and NH-MM were obtained by t-test (p<0.01). The majority of the differentially expressed genes (57%) were under-expressed in H-MM. Genes located on the commonly trisomic chromosomes were mostly (but not always) over-expressed in H-MM and constitute 76% of over-expressed genes. Chromosome 1 contained the most differentially expressed genes (17%) followed by chromosome 12 (9%), and 19 (8%). To examine the relationship of gene copy number to gene expression, we examined the expression of genes on chromosomes 9 and 15 in subjects with 2 copies (15 normal control and 20 NH-MM) and 3 copies (12 H-MM) of each chromosome as detected by interphase FISH. We then derived a ratio of the mean expression of each gene on these chromosomes between patients with 3 copies and 2 copies of the chromosome. If a simple relationship exists between gene expression and gene copy number, one would expect the ratio of expression of most genes on these two chromosomes to be about 1.5 in H-MM compared to NH-MM. However, many genes have ratios either higher than 2 or lower than 0.5. Furthermore, when the heterogeneity of cells with underlying trisomies is taken into consideration by correcting the ratio for the number of cells with trisomies, the actual ratio is always lower than the expected ratio. When the expression of genes on a chromosome was compressed to a median value, this value was always higher in the trisomic chromosomes for H-MM compared to NH-MM. The data suggests that although gene dosage influence gene expression, the relationship is complex and some genes are more gene dosage dependent than others. Amongst the differentially expressed genes with known function, 33% are involved in mRNA translation/protein synthesis. Of note, 37 of the top 100 differentially expressed genes are involved in these processes. In particular, 60 ribosomal protein (RP) genes are significantly (p<0.05) upregulated in H-MM. This signature in H-MM is not associated with increase proliferation as measured by PCLI. This predominant signature suggests that deregulated protein synthesis may be important for the biology of H-MM. Many of these RPs are involved in the synthesis of product of oncogenic pathways (e.g. MYC, NF-KB pathways) and may mediate the growth and survival of tumor cells. It is therefore possible that these tumor cells may be sensitive to the disruption of mRNA translation/protein synthesis. Targeting the mTOR pathway with rapamycin may therefore be useful for treatment of H-MM.

Abstract Background: The serum free light chain (FLC) assay is increasingly used to monitor patients (pts) with oligo-secretory or non-secretory multiple myeloma (MM) and pts with primary amyloidosis lacking measurable monoclonal protein in the serum or urine. Criteria to use this assay to assess response to therapy have recently been proposed (Rajkumar SV, Kyle RA. Best Pr Clin Haematol2005;18:585–601) but have not been validated. The goal of this study was to validate the response criteria for the FLC assay in a prospective trial of lenalidomide plus dexamethasone in newly diagnosed MM. Methods: 34 pts were enrolled in the trial; 27 pts who had serial FLC assessments were studied. FLC estimation was carried out using the serum FLC assay (FreeliteH, The Binding Site Limited, UK) performed on a Dade-Behring Nephelometer. Pts with κ /λ FLC ratio <0.26 were defined as having monoclonal λ FLC and those with ratios >1.65 as having a monoclonal κ FLC. The monoclonal light chain isotype was considered the “involved” FLC isotype, and the opposite light chain type as the “uninvolved” FLC type. Partial response (PR) required an abnormal baseline FLC ratio and any one of the two following criteria: 1) a 50% decrease in the level of the involved FLC plus a 50% decrease (or normalization) in the ratio of involved/uninvolved FLC or 2) 50% decrease in the difference between involved and uninvolved FLC levels. Complete response (CR) required normalization of FLC ratio and negative serum and urine immunofixation. Response at 4 months or earlier by the Bladé criteria was compared to FLC response criteria from the same evaluation. Results: Three pts had normal FLC levels and ratio and were not included in the analysis. 23 of the remaining 24 achieved a PR or better by Bladé criteria. A 50% decrease in the level of the involved FLC plus a 50% decrease (or normalization) in the ratio of involved/uninvolved FLC correctly classified 20 of the 22 responding pts (sensitivity 91%); 2 pts could not be evaluated since baseline FLC ratio could not be calculated. On the other hand, a 50% decrease in the mathematical difference between involved and uninvolved FLC levels correctly classified all 24 responding pts (sensitivity 100%). The one non-responding pt by Blade criteria was correctly classified by both FLC criteria. All pts were correctly classified by both criteria when only those with a baseline “involved” FLC level of at least ≥10mg/dL (≥100mg/L) were considered (15 pts). Conclusions: This study demonstrates that the serum FLC assay can be used to assess response to therapy. A 50% decrease in the difference between “involved” and “uninvolved” FLC levels will suffice as FLC criteria for PR, eliminating the need for the alternative criteria based on the involved FLC level and the ratio. We recommend that this FLC response criteria be used only in pts not having measurable levels of serum and /or urine M protein.The FLC response criteria will now enable most patients with oligo-secretory and non-secretory MM to enter trials for which they are currently ineligible due to “lack of measurable serum or urine M protein.” This study is limited by the lack of adequate non-responders to calculate specificity and lack pts who progressed to validate progression criteria and needs further validation.