Greipp, Philip R.

Abstract Background: Patients with early stage myeloma are typically observed without therapy until symptomatic disease occurs. We have reported early results of therapy with single-agent thalidomide as initial therapy for early stage multiple myeloma in an attempt to delay progression to symptomatic disease (Leukemia2003;17:775–9). We now report the long-term results of this phase II trial. Methods: Thirty-one patients with smoldering or indolent multiple myeloma were enrolled between April 1999–March 2002. All pts had bone marrow (BM) plasma cells >=10% and measurable disease defined as serum monoclonal (M) protein >=2gm/dL and/or urine M protein >=400mg/24 hours. Two patients were deemed ineligible because they were found to have received prior therapy for myeloma and were excluded from analysis. Thalidomide was initiated at a dose of 200 mg/day and increased as tolerated by 200 mg/day every 2 weeks to a maximum dose of 800 mg/day. The dose of thalidomide was decreased as needed to minimize toxicity. Treatment was continued until disease progression or serious toxicity, except for patients achieving less than a minor response (<25% paraprotein reduction) to therapy in whom therapy was stopped at one year. Response was defined as a decrease in serum and urine monoclonal (M) protein by 50% or higher. If the serum M protein was unmeasurable, a 90% or higher decrease in urine M protein was required. Responses need to be confirmed at least 4 weeks apart. Results: Ten patients (34%) had indolent myeloma, including 4 patients with lytic bone lesions, 3 patients with anemia (Hb <11gm/dL), and 3 patients with both anemia and lytic bone lesions. Median follow up of living patients is 5.6 years (range, 4.0–7.1 years). Of the twenty-nine eligible patients, ten (34%) had a partial response to therapy with at least 50% or greater reduction in serum and urine monoclonal (M) protein. Three patients are still on study and receiving therapy. The remaining have gone off-study: seven patients (27%)had progressive disease while on therapy; other reasons for ending study treatment included completed therapy per protocol (2 pts), patient refusal (2pts), toxicity (8 pts), alternate treatment (3 pts), other medical problems (1 pt), died on study (1 pt), and other (2 pts). The median time to progression (TTP) was 3.2 years (95% CI 2.1–5.6 yrs). Median progression free survival (PFS) was 3.0 yrs (95% CI 2.0–5.1 yrs). Median overall survival has not been reached; 5 year survival rate by the Kaplan Meier method is 60%. Major grade 3–4 non-hematologic adverse events at any time during therapy were noted in 34% of patients; this includes 14% with grade 4 toxicity. Grade 3–4 toxicities occurring in more than one patient include neuropathy (2 pts) and sinus bradycardia (2 pts). Conclusions: Thalidomide has activity in asymptomatic myeloma. This approach must be further tested in randomized trials to determine if the survival estimates are superior to observation. Given the activity of thalidomide, trials are also needed with lenalidomide.

Abstract The t[4;14] simultaneously deregulates fibroblast growth factor receptor 3 (FGFR3) and MMSET in 15% of patients (pts) with Multiple Myeloma (MM). Although oncogenic, FGFR3 expression is lost in ~25% of t[4;14] pts on gene expression profiling while MMSET is always retained. The t[4;14] is highly associated with poor prognosis, furthermore FGFR3 kinase inhibitors are in Phase I trials, thus detection of t[4;14] pts is increasingly important. The Multiple Myeloma Research Consortium (MMRC) therefore evaluated 4 competing methodologies for the diagnostic detection of t[4;14]. Bone marrow (BM) samples were collected in uniform fashion from 85 pts. FGFR3 immunocytochemistry (ICC) and flow cytometry (FC) were performed at the retrieval site, while split samples were shipped to the MMRC tissue bank and processed under GLP conditions. Processed samples were analyzed by cIg-FISH and quantitative IgH-MMSET RT-PCR (QPCR) on unsorted BM and blood. Of 60 samples with sufficient plasma cells for analysis, 8 were FISH +ve [13.3%]. Low % BM plasma cells prevented successful FISH in 25% of samples reflecting large volume harvests and hemodilution. Analysis was conducted in a blinded fashion. With FISH as the gold standard for detection of t[4;14] the sensitivity and specificity of the other diagnostic tests are: BM QPCR is most sensitive (7 of 8 FISH positive detected) and specific (42/43 negatives correctly identified). QPCR on peripheral blood is ongoing. FC is more sensitive to the detection of FGFR3 protein than ICC. Correlation between FC and ICC was only 0.46. Interestingly, 6 of 7 evaluable pts here and 13/14 (92%) in an expanded analysis of FISH +ve patients also expressed the FGFR3 protein. Given the discrepancy between this finding and previously reported loss of FGFR3 expression in 25% of pts we further explored t[4;14] stability. Clonal selection/heterogeneity was determined by the % plasma cells in each pt. with an unbalanced translocation (loss of one der chromosome). In 42 t[4;14] pts, 13 (31%) had a balanced translocation(>75% of cells with a double fusion), 14 (33%) had an unbalanced translocation (>75% cells with only one signal), and 15 (36%) had a chimeric picture. This heterogeneous pattern is suggestive of an evolution towards an unbalanced translocation. Despite this survival did not differ between pts with balanced or unbalanced translocation. Conclusions: These results indicate that the t[4;14] is highly heterogeneous with respect to balanced versus unbalanced translocations, which likely evolve over time. For detection, cIg-FISH continues to be the gold standard diagnostic methodology but may be limited by low plasma cell numbers in dilute BM or during MM remission. QPCR and FC appear most sensitive and specific for presence of t[4;14] and FGFR3 protein expression respectively. Surprisingly (given prior evidence of loss of FGFR3 expression in 25% of pts) 92% of t[4;14] pts in this series expressed FGFR3 protein. # Analyzed % Positive Sensitivity Specificity FISH 60 13 - - QPCR 65 14 87.5 97.6 Flow 82 15 85.7 91 ICC 85 15 62.5 92

Abstract Plasma cell (PC) proliferative disorders vary in behavior ranging from MGUS, a usually indolent disorder, to multiple myeloma, an aggressive disease with poor long-term survival. Detecting PC immunoglobulin (Ig) light chain restriction is commonly used to demonstrate PC clonality and establish the diagnosis. Furthermore, greater degrees of peripheral blood (PB) and/or bone marrow (BM) involvement are associated with more aggressive disease types and, therefore, are adverse prognostic indicators. Flow cytometric immunophenotyping (FCIP) is often used for detecting PC Ig light chain restriction. Shortcomings of FCIP as traditionally performed include its relative insensitivity and its underestimation of PC number. For these reasons, the FCIP PC clonality assessment in our laboratory is currently supplemented with a slide-based immunofluorescence (IF) technique. Recent advances in flow cytometry have led to the development of powerful instruments and reagents, potentially enhancing the ability of FCIP to analyze PC clones. In this study a newly available single tube, 6-color FCIP approach for detecting and enumerating monotypic PCs was compared to the current combined FCIP/IF assay. 94 PB and 124 BM samples were analyzed by combined FCIP/IF and single tube 6-color FCIP assays. For PB, FCIP/IF used 2-color CD38-PE/ CD45-PerCP FCIP to enumerate PCs and slide based IF to detect Ig light chain restriction. For BM FCIP/IF used two 4-color FCIP tubes (Tube1: cytoplasmic (cy)-kappa Ig-FITC/CD20-PE/CD45-PerCP/CD38-APC; Tube 2: cy-lambda Ig-FITC/CD56-PE/CD45-PerCP/CD38 APC) and slide-based IF to confirm clonality. These FCIP assays were performed on a FACSCaliber. The single tube, 6-color FCIP assay used the following fluorochrome-antibody combination: cy-kappa Ig-FITC/cy-lambda Ig-PE/CD138-PerCP-Cy5.5/CD38-APC/CD19-PE-Cy7/CD45-APC-Cy7 with data acquired on a FACSCanto. The results are summarized in Tables 1 & 2, the concordance between the methods was high. All false-negative 6-color FCIP results were attributable to the use of isotonic lysis for cell isolation. When limiting the comparison to the ficolled cells alsoused for FCIP/IF, 6-color FCIP was consistently more sensitive. The 6-color assay also allowed for the discriminition between CD19-positive polytypic PCs and CD19-negative monotypic PCs and the detection of CD45-positive monotypic PC subsets which were often present and which were not identified in the combined FCIP/IF approach. Estimates of PB disease burden generated from the 6-color technique were similar to those determined from the FCIP/IF data which have previously been shown to be of prognositic significance. 6-color FCIP allows for the rapid, sensitive detection of monotypic PCs. The ability of 6-color FCIP to detect phenotypically distinct clonal PC subsets enhances the sensitivity and may provide prognositically significant data through both more complete cell enumeration and charactarization. Given these advantages, 6-color FCIP may eventually supplant traditional FCIP methods of PC analysis. Method Comparsion in BM Bone Marrow 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF Positive 81(65%) 3(2%) 84 FCIP/IF Negative 5(4%) 35(28%) 40 Total 86 38 124 Method Comparsion in PB Peripheral Blood 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF positive 24(26%) 7(7%) 31 FCIP/IF negative 9(10%) 54(57%) 63 total 33 61 94

Abstract Background: Myeloma remains incurable due to a stem cell/proliferative component that responds poorly to standard treatments. We have shown that aberrant IL-1beta stimulates the generation of paracrine IL-6, a central myeloma growth factor. A Phase II trial was completed using IL-1 receptor antagonist (IL-1Ra/Anakinra), which inhibits paracrine IL-6 production, and low dose Dexamethasone (Dex), which decreases IL-1 levels through myeloma cell apoptosis, in patients with SMM/IMM. SMM/IMM patients are most likely to benefit from anti-cytokine therapy in order to delay/prevent the development of active myeloma. Methods: Patients that had ≥ 10% bone marrow plasma cells and/or an IgG or IgA M-spike ≥ 3 g/dL and did not require immediate chemotherapy were eligible. All patients received 100 mg of IL-1Ra SQ qd for 6 months unless clinical progression occurred. Non-progressors were allowed to continue on therapy with IL-1Ra alone. Low dose Dex (20 mg qweek) was added after 6 months of IL-1Ra or for evidence of clinical progression; the dose was adjusted based on response/toxicity. Results: To investigate the effects of IL-1Ra and Dex alone and in combination in vitro, we co-cultured IL-1beta transduced +/− myeloma cells with stromal cells +/− dexamethasone, IL-1Ra, or both for 48 hours and quantitated the percent apoptotic cells by flow cytometry and IL-6 production by ELISA. The results showed that: 1) IL-1Ra was superior to Dex at inhibition of IL-6 but caused no myeloma apoptosis; 2) Dex induced apoptosis was inhibited by IL-6 3) Dex and IL-1Ra combined induced maximal IL-6 inhibition and apoptosis of myeloma cells. In the clinical trial, data were available on 47 patients; smoldering (79%) / indolent (21%). All patients received IL-1Ra initially and 20/47 subsequently received IL-1Ra/Dex. Seven patients had a decrease in the plasma cell labeling index (PCLI), a marker of cell proliferation, on IL-1Ra alone which paralleled a decrease in the high sensitivity C-reactive protein (CRP). Interestingly, in one patient treated with Dex alone for 6 months, Dex induced a decrease in the M-protein and IL-1 levels, however, the PCLI and CRP values increased. The IL-1Ra/Dex combination has resulted in stability of disease in the majority of the 47 patients; 28 continue on therapy with a median overall progression-free survival (PFS) of 37.5 months. Four of the 47 pts achieved a minor response (MR) with IL-1Ra alone, and an additional 6 pts achieved a MR/PR after addition of dex. The continuous measure of % reduction in CRP was significantly associated with time to progression (p=0.02). Similarly, those patients who had a >5% decrease in serum M protein also had a longer PFS (p<0.001). Of note, 2 patients with high PCLI values of 6.4 and 3.0, characteristic of poor prognosis, responded with a reduction in the PCLI values and continue on combination therapy for 3 and 1.5 years, respectively with stable disease. Conclusion: Agents such as IL-1Ra that specifically inhibit paracrine IL-6 production are more effective at targeting the proliferative component than apoptosis inducing agents such as Dex; the combination may be especially useful in patients that have an elevated PCLI at presentation.

Abstract Aim: A phase III trial of lenalidomide plus high (standard) dose dex (RD) versus lenalidomide plus low dose dex (Rd) in newly diagnosed myeloma (MM). Methods: Pts with untreated, symptomatic MM were eligible. Pts in the RD arm (Arm A) received lenalidomide 25 mg/day PO days 1–21 every 28 days plus dex 40 mg days 1–4, 9–12, and 17–20 PO every 28 days; pts in the Rd arm (Arm B) received lenalidomide at the same dose plus dex 40 mg days 1, 8, 15, and 22 PO every 28 days. The primary endpoint was response rate at 4 months, with the null hypothesis being that the response rates in the two 2 arms are equivalent. Planned sample size was 196 eligible pts per arm with one-sided 0.10 Type I and 0.05 Type II error rate. All analysis were performed on an intent to treat basis. An independent DMC recommended release of study results. Results: 445 pts (median age, 65 yrs) were accrued; 223 randomized to RD, 222 to Rd. Median follow-up time is 17 months. Arms were well balanced for age, gender, stage, bone lesions, hemoglobin, beta-2 microglobulin, performance status, bone marrow plasma cells, and M protein levels at baseline. Major grade 3 or higher toxicities, including DVT/PE and infections, were significantly higher in the high dose dex arm (see Table). Overall survival (OS) at the second pre-planned interim analysis was significantly superior with lenalidomide plus low dose dex, P<0.001; one year survival 96% (Rd) versus 87% (RD). The 18 month survival rate is 91% versus 80%, respectively. OS differences in favor of the low dose dex arm were seen in pts <65 (P=0.022; one year rate 97% vs 92%) as well as pts 65 and older (P=0.002; one year rate 94% vs 83%), respectively. Sixty-one patients have died; 42 in the RD arm and 16 in Rd arm. The cause of death has been verified by detailed chart review in 38 patients. Of 29 verified deaths in the RD arm, 13 were due to disease progression, 6 thrombosis/embolism, 3 infection, 3 cardiac ischemia, 1 stroke, and 1 respiratory failure. Of 9 verified deaths in the Rd arm, 5 were due to disease progression, 2 infection, 1 thrombosis/embolism, and 1 cardiac arrest. Response rate data are expected to be available at the time of the meeting. Conclusions: Lenalidomide plus low-dose dexamethasone (Rd) is associated with superior OS compared to lenalidomide plus high-dose dexamethasone (RD) in newly diagnosed MM. The increased mortality in Arm A is due to disease progression (myeloma deaths) as well as increased toxicity. This study has major implications for the use of high-dose dexamethasone in the treatment of newly diagnosed MM. Major Grade 3 or Higher Adverse Events Toxicity Arm A % Arm B % P value Neutropenia 10 19 0.01 DVT/PE 25 9 <0.001 Infections 16 6 <0.001 Any grade 3 or higher non-hematologic 49 32 <0.001 Any grade 4 or higher non-hematologic 20 9 <0.001 Deaths in first 4 months 5 0.5 0.006

Abstract Background: Immunomodulatory agents and proteasome inhibitors have significantly improved outcomes in the management of patients with multiple myeloma (MM). As a consequence, the role of upfront autologous stem cell transplant (ASCT) has become more controversial. The ECOG-ACRIN Cancer Research Group trial E4A03 demonstrated the superiority of lenalidomide (LEN) and low-dose dexamethasone (DEX; Ld) over LEN and high dose DEX (LD) in newly diagnosed MM (Rajkumar SV, Lancet Oncol, 2010). This led to the universal acceptance of Ld as a standard induction regimen. More recently, the FIRST study demonstrated the superiority of Ld as continuous therapy vs Ld given x 18 cycles and melphalan-thalidomide-prednisone x 12 cycles proving continuous Ld should be considered standard of care (Benboubker L, NEJM, 2014). Upon completion of four cycles of therapy, patients enrolled in E4A03 had the option of proceeding to ASCT or continuing on their assigned Ld or LD. A prior post-hoc retrospective landmark analysis (Siegel DS, Abstract, Blood 2010) comparing outcomes of early ASCT vs. no early ASCT showed that in patients <65, 65-70, and >70, the survival with early ASCT at 3 years appeared higher, supporting the role of early consolidative ASCT in newly diagnosed patients. This analysis is the long-term follow-up of the aforementioned study. Methods: A post hoc, retrospective landmark analysis was performed evaluating overall survival (OS) by early ASCT vs. no early ASCT overall and within age subgroups. The Kaplan-Meier method was used to estimate survival distributions and Cox regression for hazard ratio (HR) estimates. The landmark analysis included only patients surviving the first four cycles of therapy. Results: 21% of patients opted for early ASCT (n=90). At baseline (four cycles prior), early ASCT patients were younger (median 57.5y vs 66y), more fit (ECOG PS 0 45% vs 56%) and with less aggressive disease (ISS Stage 3 12% vs 28%). Median treatment duration was 7.6m for no early ASCT patients. The 1, 2, 3, 4, and 5-year survival probability estimates were higher for early ASCT vs. no early ASCT at 99%, 93%, 91%, 85%, and 80% vs. 94%, 84%, 75%, 65%, and 57%. The median OS in the early ASCT vs. no early ASCT was not reached vs. 5.8 years, respectively (Table 1). The survival hazard ratio (HR) for early ASCT was 0.55 [95%CI (0.38-0.80)]. In a further subgroup analysis of patients </= 65, 5-yr survival probability of those who had early ASCT vs. no early ASCT was 79% [95%CI (67-87)] vs. 62% [95%CI (54-69)], respectively. In patients </= 65, median OS in the early ASCT vs. no early ASCT was not reached vs. 7.4 years and the survival HR was 0.74 [95%CI (0.48-1.15)]. For patients >65, 5-yr survival probability of those who had early ASCT vs. no early ASCT was 83% [95%CI (56-94)] vs. 52% [95%CI (45-59)], respectively. In patients >65, median OS in the early ASCT vs. no early ASCT was not reached versus 5.2 years and the survival HR was 0.38 [95%CI (0.17-0.87)]. In all age groups, overall response rate was similar prior to the decision of proceeding with ASCT vs. no early ASCT. In a multivariable model adjusting for baseline prognostic factors, early ASCT retained marginal significance. Conclusions: The landmark analysis demonstrated that patients opting for ASCT after induction Ld/LD had a higher 1, 2, 3, 4, and 5- year survival probability and improvement in median OS regardless of DEX dose density. Results were stronger within the older age cohort and upheld in adjusted models. Limitations include lack of randomization and data on salvage therapy. At a time when lenalidomide and dexamethasone as induction is gaining worldwide acceptance in all age groups, early ASCT is safe and efficacious and should remain a standard of care for newly diagnosed MM even in the era of novel therapies. Table 1. Survival Probabilities (Time from Registration) for all Patients Included in the Landmark Analysis Median 1 year 2-year 3-year 4-year 5-year N Events Median (Yrs)(95% CI) Events Survival Probability(95% CI) Events Survival Probability(95% CI) Events Survival Probability(95% CI) Events Survival Probability(95% CI) Events Survival Probability(95% CI) No Early ASCT All 341 195 5.78(5.11 - 6.59) 22 0.94 (0.90 - 0.96) 53 0.84 (0.80-0.88) 84 0.75 (0.70-0.79) 119 0.65 (0.59-0.69) 144 0.57 (0.51 - 0.62) Early ASCT All 90 34 NR 1 0.99 (0.92 - 1.00) 6 0.93 (0.86 - 0.97) 8 0.91 (0.83 - 0.95) 13 0.85 (0.76 - 0.91) 18 0.80 (0.69 - 0.87) NR = not reached Disclosures Biran: Celgene: Speakers Bureau. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Fonseca:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Applied Biosciences: Membership on an entity's Board of Directors or advisory committees; Onyx/Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Binding Site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Vesole:Idera Pharmaceuticals: Research Funding; Celgene Corporation: Speakers Bureau. Williams:Celgene: Consultancy, Research Funding. Abonour:Celgene: Research Funding, Speakers Bureau.

Abstract Background: The serum free light chain (FLC) ratio is a readily available laboratory test that has independent prognostic significance in monoclonal gammopathy of undetermined significance (MGUS) (Blood2005;106:812–17) and solitary plasmacytoma (Blood2006;108:1979–83). The purpose of this study was to determine if the FLC ratio will be of prognostic value in multiple myeloma (MM). Methods: We used a well characterized cohort of 1027 newly diagnosed MM patients seen at Mayo Clinic from January 1, 1985 to December 31, 1998 (Mayo Clin Proc2003;78:21–33) to ensure adequate follow up. Archived serum samples from blood drawn within 30 days of initial diagnosis were available on 790 of the 1027 patients. Quantitation of serum free κ and λ concentrations was performed on the archived sera by immunonephelometry, using specific antibodies on a BNII nephelometer. FLC ratio was calculated as κ /λ, i.e., free κ concentration divided by free λ, reference range 0.26–1.65. If the FLC ratio is >1.65, κ is considered to be the “involved” FLC and λ the “uninvolved” FLC, and vice versa if the ratio is less than 0.26. Incorporation of FLC into the International Staging System (ISS) was assessed in 576 of 790 patients (73%), in whom samples or data were available to estimate the ISS. Results: 790 patients (median age 66 years) were studied. The FLC ratio was outside the reference range in 95.1% of patients. The median involved κ and λ concentrations were 37.1 and 71.3 mg/dL, respectively. A cut-point κ /λ FLC ratio of <0.03 or >32 was chosen for further modeling analysis on the basis of its separation of the cohort into two roughly comparable parts. Overall survival was significantly inferior in patients with an FLC ratio <0.03 or >32 (n=479 patients) compared to those with an FLC ratio that was 0.03–32 (n=311 patients), with median survival of 30 versus 39 months, respectively, P<0.001 (hazard ratio 1.3, 95% CI 1.12–1.54). The FLC ratio (<0.03 or >32) retained independent significance after adjusting for the International Staging System (ISS) (P=0.03). The FLC ratio made the most significant contribution to predicting prognosis in patients with ISS Stage 2 (n=265 pts), separating this category of patients into those with 5 year survival rates of 20.5% (FLC ratio <0.03 or >32; n=152 pts) versus 35.2% (FLC ratio 0.03–32; n=113 pts), P=0.02. We studied the additional prognostic value of the serum FLC to the variables used in the ISS by defining FLC ratio <0.03 or >32, Sβ2M ≥3.5 g/L, and serum albumin <3.5 g/dL as three risk factors in a risk-stratification model. Patients with 0, 1, 2, or 3 risk factors had significantly different overall survival, with median overall survival times of 51, 39, 30 and 22 months, respectively, P<0.001. Conclusions: The serum FLC ratio at initial diagnosis is an important predictor of prognosis in myeloma. We show that the serum FLC ratio can be incorporated into the ISS, providing the greatest prognostic value in ISS Stage 2 patients, as well as providing a new risk-stratification model.

Abstract Genetic variations in patient populations likely contribute to disease progression and therapeutic outcomes. We have begun a systematic approach to examine the association of genetic variations (ie. functional, single nucleotide polymorhisms, SNPs) involved in myeloma growth promotion, metabolic events, drug responses, and DNA repair on clinical outcome in the intergroup trial S9321. This trial tested a single high dose regimen with autologous stem cell support against a conventional dose regimen, with further randomization of responders to maintenance with interferon or not, in newly diagnosed patients with multiple myeloma. ECOG, CALGB and SWOG enrolled 899 patients with newly diagnosed MM to receive VAD induction x 4 cycles followed by randomization to PBSC-supported high dose therapy (HDT) versus standard dose therapy (SDT) of VBMCP, using CTX 4.5 g/m2 + G-CSF for PBSC mobilization in all patients. Responders to VBMCP or HDT were randomized to IFN or no maintenance. Specimens were distributed through ECOG for biologic correlative studies, including candidate SNP analysis. SNP assays have been developed using the Sequenom Mass-extend platform for functional SNPs in IL-6, IL-1, IL-RA, IL-10, TNF, Lta, TGFb, MDR1, MPO, CYP3A4, GST (M, P, T), ERCC2 and XRCC1, and are being evaluated on 803 DNA samples prepared from patients enrolled in S9321. Preliminary findings (n=135) demonstrate functional genetic variants of IL-10 (position -1082), IL-1 (position +3953), TGFb (postion -509), and TNFa (position -308) are showing trends associated with differences in progression free survival; and variants in IL-6 (position -174) are associated with response. Median survival of the IL-10 variants was 31 months for A/A low producer alleles versus 19 months for the G/G high producer alleles (p=.5). For TNFa, 2 cases with the high producer A/A alleles died within a year, while the median survival for the lower producer G/G alleles was 2 years (p=.04). For patients with the high producer C/C/ allele of IL-1 (n=67), median survival was 2 years, versus 5 patients with the T/T low producer alleles that had a median survival greater than 5 years (preliminary p=.29). While these preliminary results are now only suggestive of trends in genetic polymorphisms associated with clinical outcome, completion of the full SNP panel on the entire sample base should provide a extensive association study, and analysis of potential differences in therapy arms of the trial. The full panel and association studies will be presented.ααββααα

Abstract Although multiple myeloma is clinically defined as the accumulation of clonal, malignant plasma cells in the bone marrow, the disease shows significant heterogeneity with regard to morphology, progression and therapeutic response. Our hypothesis is that this heterogeneity likely is due, in part, to germline genetic polymorphisms that contribute significantly to an individual’s disease course and response. In particular, individuals deficient in the repair of DNA damage are not only at high risk for developing cancer, but also could show DNA repair dependent responses associated with DNA damaging therapeutic agents. In this study we examined the association of functional genetic polymorphisms in the DNA repair genes, XRCC1 (SNP positions 280, 399) and ERCC2 (SNP positions 312, 751), with toxicities and clinical outcomes in the ECOG, 3 arm chemotherapeutic trial, E9486 [A-VBMCP, B-VBMCP+Cylophosphamide, C- VBMCP+interferon]. DNA from 359 patients was genotyped and examined for association with response, toxicities, blood counts, bone pathology, and survival parameters. Notably, in the interferon arm of the trial significant associations were observed in progression free survival and polymorphisms of XRCC399 (AA median survival 51 months versus AG/GG median survival of 33 months; p=,008), ERCC751 (AA/AC median survival 33 months versus CC median survival of 48 months; p=.05), and ERCC312 (AA median survival 49 months versus AG/GG median survival of 34 months; p=.02). Hazard ratios ranged form 1.7 to 2.05 for survival differences associated with DNA repair genes in this arm. Interestingly, T cell counts are known to be impacted by interferon; and DNA repair polymorphisms in this arm were also associated with CD8+ T cell counts. While this represents a single arm of one clinical trial, it is intriguing to consider the impact of genetic polymorphisms on DNA repair in light of chemotherapeutic agents that may affect cells of the immune system. Previous studies from the ECOG Myeloma Committee of the E9486 trial show highly significant associations of survival and immune status in myeloma patients. Our results suggest genetic polymorphisms in DNA repair genes may influence clinical outcome in certain therapeutic regimens. Genetic polymorphisms in these DNA repair genes are currently being completed in 800 patients of the SW9321 SWOG/ECOG intergroup trial and additional association studies will be included in the presentation.

AbstractRecent analyses of circulating blood B cells in myeloma have generated controversy concerning the exact levels of these cells and whether they may represent circulating clonal tumor B cells. Previous reports suggested that CD19+ B cells are markedly increased in myeloma patients and that this population shares clonotypic rearrangements with the malignant plasma cell. We studied the numbers of CD19+ B cells by flow cytometry in previously untreated newly diagnosed myeloma patients in Eastern Cooperative Oncology Group (ECOG) phase III trial E9486. There were 628 patients who were eligible for the clinical protocol E9486, but of these 521 were also entered on the companion laboratory study (E9487) and had CD19 data. In comparison with normal controls, the myeloma patients exhibited a marked heterogeneity in the number of circulating CD19+ B cells as detected by flow cytometry. Approximately 20% of patients had significantly increased levels of circulating CD19+ B cells. However, the total CD19+ blood population from myeloma was not significantly different from the median of age-matched, normal controls. Analysis of CD19+ blood cells in relationship to circulating clonal cells was done in 13 myeloma patients using a clonotypic, quantitative allele-specific oligonucleotide-polymerase chain reaction (PCR) assay. No correlation was found between the numbers of CD19+ B cells (range, 5% to 51%) and PCR estimates of the number of clonal cells in the peripheral blood (range, .009% to 3.6%). Low CD19+ B-cell level (&lt;125 μL) was associated with clinical stage III (P = .033). A significant relationship exists between higher levels (≥125/μL) of CD19 cells and longer overall survival (P &lt; .0001). In addition, high CD19 levels also predicted a clinical response and longer event-free survival. There was a strong inverse association between the level of CD19 values at diagnosis and infections within the first 2 months of diagnosis. Importantly, the number of deaths related to infections was significantly greater in the low versus high CD19 group (P &lt; .0202). Also, CD19 is an independent prognostic factor in addition to plasma cell labeling indices, β2 -microglobulin, hemoglobin, and plasmablastic morphology. Patients with infections were more likely to have low levels of CD19+ cells. In summary, higher CD19+ cell levels are a favorable prognostic sign with no apparent relationship to circulating tumor cells. In addition, this analysis strongly suggests that low peripheral blood levels of CD19+ cells are an adverse prognostic sign in myeloma. The CD19+ cell levels in myeloma patients is an important parameter in the overall assessment of these patients.