Kyle, Robert A.

Abstract Background: The p53 tumor suppressor gene has a common sequence polymorphism that results in either proline or arginine at amino-acid position 72. The Pro72 and Arg72 variants have been reported to differ in its functional activity. The Arg72 variant induces apoptosis more efficiently than Pro72 variant by its greater ability to localize to the mitochondria and increase the release of cytochrome c into the cytoplasm. In addition, recent studies have demonstrated that Arg72 variant was a better suppressor of cellular transformation, an activity believed to rely on the pro-apoptotic function of p53. Moreover, the Pro72 variant has been associated with a higher incidence of tumors. Aim: To evaluate the effect of the polymorphic variants of codon 72 and clinical outcome in patients with plasma cell malignancies. Patients and Methods: We analyzed 79 patients with plasma cell malignancies seen at Mayo Clinic between 2000 and 2003. Genomic DNA was extracted from CD138+ sorted plasma cell from bone marrow aspirates. PCR amplification of codon 72 and posterior sequencing was used for the analysis. Results: A total of 79 patients were analyzed. Thirty-four patients (34%) were heterozygous for p53, 57 (59%) homozygous for Arginine and 6 (6%) homozygous for Proline. The distribution among disorders was as follow, 52 patients had MM, 14 patients with SMM and 13 with MGUS. Among patient with MM 25% were Pro/Arg heterozygous, 71 % were Arg/Arg homozygous and 3.8% Pro/Pro homozygous. Survival data among MM patients and genotype were analyzed to Kaplan- Meier method. No significant differences were found among patients who carried homozygous (72R) or heterozygous genotype and overall survival. We exclude the analysis in MGUS and SMM for a low number of patients available. Conclusions: These findings indicate that codon 72 arginine p53 may not be associated with a prolonged survival in patients with Multiple Myeloma, but further study is needed to assess whether this polymorphism is associated with progression of MGUS to MM, age of onset, clinical manifestations, response to treatment etc.

Abstract Background: We reported a high response rate with the combination of lenalidomide plus dexamethasone (Rev/Dex) as initial therapy for newly diagnosed multiple myeloma (Blood2005;106:4050–3). We now present new data on time to progression (TTP), progression free survival (PFS), and overall survival (OS) from this phase II trial, and also include updated response data. Patients and Methods: 34 patients (23 male and 11 female) were enrolled. Lenalidomide was given orally 25 mg daily on days 1–21 of a 28-day cycle. Dexamethasone was given orally at a dose of 40 mg daily on days 1–4, 9–12, 17–20 of each cycle. Patients were allowed to go off treatment after 4 cycles of therapy to pursue autologous stem cell transplantation (SCT), but treatment beyond four cycles was permitted at the physician’s discretion. For patients continuing therapy beyond 4 months, the dose of dexamethasone was reduced to 40 mg on days 1–4 of each cycle. Response was assessed by modified EBMT/International Myeloma Working Group Uniform Response criteria. All patients received aspirin (81 mg or 325 mg daily) as prophylaxis against DVT. Results: The median age was 64 years (range, 32–78). All patients were evaluable for response and toxicity. Median follow up is 21 months. Thirteen patients proceeded to SCT following initial therapy with Rev/Dex and were censored at that time point for purposes of calculation of response, TTP and PFS. Patients who discontinued therapy to proceed to SCT received a median of 4 cycles of therapy (range, 4–13), while those staying on Rev/Dex (n=21) received a median of 19 cycles of therapy (range, 2–30). Thirty-one of 34 patients (91%) achieved an objective response to therapy; including 6 patients (18%) achieving a complete response (CR) and 13 patients (38%) achieving very good partial response for a CR+VGPR rate of 56%. The CR+VGPR rate among the 21 patients staying on Rev/Dex as primary therapy without SCT was 67% (CR 24%, VGPR 43%). Median TTP, PFS, and OS have not been reached (Figure). By Kaplan-Meier method, the estimated 2 year progression rate was 18%. The 2-year PFS rate and OS rate were 74% and 91%, respectively. Fifty-five percent of patients experienced grade 3 or higher non-hematologic toxicity at any point during therapy, most commonly fatigue (21%), neutropenia (21%), anxiety (6%), pneumonitis (6%), muscle weakness (6%), and rash (6%). Two patients died on study: one attributed to infection unrelated to therapy, the patient had stopped all therapy for over a month before the fatal infection occurred; the other death was due to infection felt possibly related to therapy. One patient developed a pulmonary embolism (grade 4 toxicity), but recovered with therapy; no other patient developed deep vein thrombosis or pulmonary embolism. Conclusion: Rev/Dex is highly active and well tolerated in the treatment of newly diagnosed multiple myeloma with a high CR+VGPR rate of 56% for the trial, and 67% among the subset of patients receiving this regimen as primary therapy. Responses are durable with a low progression rate at 2 years.

Abstract Background: Plasma cell leukemia (PCL) is a rare plasma cell (PC) dyscrasia which may be designated as primary (PPCL) or de novo PCL when recognized at the time of diagnosis and secondary (SPCL) when there is leukemic transformation of a previously recognized multiple myeloma (MM). PCL has been reported to exhibit distinct clinical and immunophenotypic features that distinguish it from MM, but little is known about the specific genetics of this disease. To better understand the genetic features of the clonal PC from PPCL and SCPL we assessed in these patients the molecular and cytogenetic abnormalities most commonly found in MM. Patients and Methods: In our study we analyzed 3 groups of patients; 18 with PPCL, 23 with SPCL and a control group of 345 newly diagnosed MM previously published. Diagnostic criteria for PCL followed the presence of >2 x103/L PC in PB (Kyle et al. Arch Intern Med1974; 133–813–8). Cytogenetic abnormalities were assessed by the c-Ig FISH method; mutational analysis was performed using Conformational-sensitive gel electrophoresis (CSGE). Methylation was analyzed by methyl specific PCR (MSP) after bisulfite genomic DNA modification. Results: Translocation of immunoglobulin heavy chain with Cyclin D1 (t (11; 14)) was present in 76.9% (10 of 13) of PPCL, 71% (5 of 7) of SPCL, in contrast to 15.8% (53 of 336) of MM cases. Translocations’ involving the FGFR3-MMSET genes (4p16.3 locus) and c-MAF (16q32) genes were not observed among 10 patients studied with PPCL, but were present in 12.7 % each in SPCL. The t (4;14)(p16;q32) was present in 12.7% cases of MM and the t(14;16)(q32;q23) was present in 4.6 % of MM cases. Deletion of 17p13.1 (p53 locus) was found in 10% (37 of 345) of MM cases, 53.8% (7 of 13) for PPCL and 42.8% (3 of 7) for SPCL. Deletion of 13q was present in 54.2% (176 of 325) of MM, 76.9 % (10 of 13) of PPCL and 57.1% of SPCL. Ras mutations were found in 15% (2/13) of PPCL, located in codon 1 and 2 of the K-ras gene. In addition, 23% for SPCL presented mutations in the N-ras gene. Two mutations were located in codon 2 of N-ras and 1 patients in showed a mutation in codon 1 of K-ras gene. Ras mutations were present in 27% of MM cases. Mutations for p53 gene were present in 30.7% (4/13) of PPCL, 17% (3/17) for SPCL and 5% for MM. Methylation specific PCR for p16 gene was found in 23% (3/13) of PPCL, 29% of SPCL and 33.9% (149/439) of MM. Hypermethylation of p14 was not detected in PPCL but a 23 % was found in SPCL. Conclusions: In this study we demonstrate that PCs from PPCL more frequently carry the t (11; 14). Comparing SPCL and MM, PC’s from de novo PPCL do not show t (4; 14) or (16; 14). Deletion of 13q was more prevalent among patients with PPCL. Ras mutations appear to be as frequent in MM as in PPCL and SPCL. Mutations in p53 appeared to be more prevalent in PPCL than in SPCL and MM. Hypermethylation of p16 does not differ while hypermethylation of p14 is more frequent in SPCL than PPCL. These characteristics may lead to a different onset or disease evolution of PPCL compared to MM and secondary plasma cell leukemia and this may be important for diagnostic and treatment.

Abstract Smoldering multiple myeloma (SMM) is characterized by a serum M protein ≥ 3g/dL and/or 10% or more of plasma cells in the bone marrow. However, the definition is not standardized, and it is not known whether both serum M protein levels and bone marrow plasma cell counts are necessary for diagnosis or if one parameter is sufficient. We reviewed the medical records and bone marrows of all patients from Mayo Clinic seen within 30 days of recognition of an IgG or IgA M protein ≥ 3g/dL or a bone marrow containing ≥ 10% plasma cells from 1970 to 1995. This allows for a minimum potential follow-up of 10 years. Patients with end-organ damage at baseline from plasma cell proliferation, including active multiple myeloma (MM) and primary amyloidosis (AL) and those who had received chemotherapy were excluded. A differential of the bone marrow aspirate coupled with the bone marrow biopsy morphology and immunohistochemistry using antibodies directed against CD138, MUM-1 and Cyclin D1 were evaluated in every case in order to estimate the plasma cell content. In all, 301 patients fulfilled either of the criteria for SMM. Their median age was 64 years and only 3% were less than 40 years of age; 60% were male. The median hemoglobin value was 12.9 g/dL; 7% were less than 10 g/dL, but the anemia was unrelated to plasma cell proliferation. IgG accounted for 75%, IgA 22%, and biclonal proteins were found in 3%. The serum light-chain was κ in 67% and λ in 33%. The median serum M spike was 2.9 g/dL; 11% were at least 4.0 g/dL. Uninvolved serum immunoglobulins were reduced in 81%; only 1 immunoglobulin was reduced in 31% and both were decreased in 50%. The urine contained a monoclonal κ protein in 36% and λ in 18% and 46% were negative. The median size of the urine M spike was 0.04 g/24h; only 5 (3%) were > 1 g/24h. The median bone marrow plasma cell content was 15 – 19%; 10% had less than 10% plasma cells, while 10% had at least 50% plasma cells in the bone marrow. Cyclin D-1 was expressed in 17%. Patients were categorized into 3 groups: Group 1, serum M protein ≥ 3g/dL and bone marrow containing ≥ 10% plasma cells (n= 113, 38%); Group 2, bone marrow plasma cells ≥ 10% but serum M protein < 3g/dL (n= 158, 52%); Group 3, serum M protein ≥ 3g/dL but bone marrow plasma cells < 10% (n= 30, 10%). During 2,204 cumulative years of follow-up 85% died (median follow-up of those still living 10.8 years), 155 (51%) developed MM, while 7 (2%) developed AL. The overall rate of progression at 10 years was 62%; median time to progression was 5.5 yrs. The median time to progression was 2.4, 9.2, and 19 years in groups 1, 2, and 3 respectively; correspondingly at 10 years, progression occurred in 76%, 59%, and 32% respectively. Significant risk factors for progression with univariate analysis were serum M spike ≥ 4g/dL (p < 0.001), presence of IgA (p = 0.003), presence of urine light chain (p = 0.006), presence of λ urinary light chain (p = 0.002), bone marrow plasma cells ≥ 20% (p < 0.001) and reduction of uninvolved immunoglobulins (p < 0.001). The hemoglobin value, gender, serum albumin, and expression of cyclin D-1 were not of prognostic importance. On multivariate analysis, the percentage of bone marrow plasma cells was the only significant factor predicting progression to MM or AL.

Abstract Cancer/testis antigens (CTAs), comprising the MAGE, CTAG, SSX and GAGE families, are expressed in different tumor types and restricted in normal tissues to germ cells of the testis. Many CTAs contain epitopes recognized by cytotoxic T cells and are potential targets for active immunotherapy. In MM, different CTAs have been shown to be expressed in cell lines and patient cells, and are associated with more proliferative and advance stage tumors. However, correlation with survival has never been reported. We undertook a global survey of CTA expression using gene expression profiling of CD138-enriched plasma-cell (PC) RNA in 15 normal, 101 MM, 22 MGUS and 24 SMM samples using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). 22 CTA genes were differentially expressed across the samples and unsupervised clustering using these genes clearly identifies a group of patients with high CTA expression. Most CTAs have significantly higher expression in MM as compared to SMM and especially MGUS, which has similar expression as normal PCs. As these antigens are not expressed in normal PCs, an upper limit of normal expression (ULN) was derived based on mean+3SD of expression in normal PCs for each CTA. A strict cut-off of normalized expression value greater than 2xULN was used to assign positive expression for each CTA genes. Using this method, the most commonly expressed CTAs are MAGE-A3 (73% of MM), MAGE-C1 (45%), MAGE-A6 (35%), SSX-1 (21%), GAGE-5 (18%) and NY-ESO-1 (14%). We then assigned patients with expression of more than 2 of these genes as having a CTA signature and correlated with clinical parameters. 45 patents (41 MM and 4 SMM) have the CTA signature. These patients have a significantly higher PCLI (median 0.2 v 0.7, p=0.0003). In fact, there is a significant correlation between the number of positive CTAs and PCLI (r=0.59, p<0.0001). Interestingly, there is no association between patients with a CTA signature and any of the recurrent genetic abnormalities (IgH translocations, chromosome 13 deletion and TC classes), yet MM patients with this signature have a significantly shorter survival (median 459 days vs not reached, log-rank p=0.017). A prognostic hierarchy exists amongst these CTAs. Only MAGE-A6, MAGE-A12, SSX-1 and SSX-5 expression has significant prognostic implications with the SSX family conferring the worse prognosis. Using a restricted panel of 3 CTAs (SSX-1, -5 and MAGE-A12), about 28% of MM patients expressing one or more of these genes can be identified with poor survival (median less than 1 year) that is independent of genetic subtypes. The genes differentially expressed between patients with and without the CTA signature are mainly involved in cell cycle regulation. Since the expression of this large set of CTAs appears to be under epigenetic control, we speculate that the same epigenetic processes that lead to dysregulation of the cell cycle also cause ectopic expression of CTA genes. As CTA expression is rare in MGUS, epigenetic processes may therefore also mediate transformation from MGUS to MM. These poor prognosis patients represent good candidates for novel therapies targeting the epigenetic machinery.

Abstract Hyperdiploid myeloma (H-MM) affects about 50% of MM. These patients exhibit significant karyotypic and clinical heterogeneity. In this study, we addressed possible biological heterogeneity within H-MM by gene expression profiling of CD138-enriched plasma-cell RNA from 53 H-MM patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Analysis was performed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Unsupervised hierarchical clustering performed using the top 100 most variable genes (generated by ANOVA) identifies 4 putative patient clusters. These clusters were reproduced using principal component analysis. Cluster 1 was characterized by high expression of a large group of cancer testis antigens and mitosis/proliferation related genes (TOP2A, NEK, ASPM, CENPA). Cluster 2 was characterized by high expression of genes involved in IL-6 and HGF signaling (MET, IL6R, SOCS3, SPINT1). Cluster 3 was characterized by high expression of genes involved in TNF/NF-KB signaling and anti-apoptosis (NFKB2, CFLAR, BIRC3, TNFAIP3, RRAS2). Cluster 4 was characterized by high expression of genes involve in angiogenesis (protocadherin, EDNRB). Unsupervised hierarchical clustering using the same set of genes in an independent cohort of H-MM patients (n=60) yielded the same 4 patient clusters over-expressing similar genes. The gene expression profile of these clusters were not driven by differences in trisomies (only about 25% of genes that define the clusters were located on the commonly trisomic chromosomes). Association between the 4 clusters and clinical parameters such as survival, chromosome 13 deletion (Δ13), presence of IgH translocations, PCLI, RAS mutation and p16 methylation was analyzed. Cluster 1 tumors were more proliferative (median PCLI 3.8, p<0.05). Cluster 2 was associated with presence of Δ13 (56%, p<0.05) although none of the genes that defined this cluster falls on chromosome 13. p16 methylation was also less common in cluster 2 (13%, p=0.06). Cluster 3 was associated with low PCLI (median 0.2, P<0.05). Most of the patients with p16 methylation also belonged to this cluster (67%, p=0.06). There was a complete absence of Δ13 in Cluster 4. Patients in the 4 clusters also had significantly different survival (log-rank p=0.002.). The difference in survival was independent of Δ13 since Δ13 has no impact on survival in this patient cohort. The molecular profiles of these subclasses may explain some of these associations. CTA expression is associated with proliferative tumors with poor prognosis. IL-6 is an important survival factor and HGF signaling has been associated with poor survival in MM which may explain the relative poor prognosis of this group. Cluster 3 may have better survival as it is associated with an anti-apoptotic signature rather than proliferative signature. In summary, gene expression profiling identified 4 sub-classes within H-MM (validated on an independent dataset and using different clustering techniques) with distinct clinical and biological associations. The gene signature associated with each sub-classes provide a molecular basis for the different clinical behaviour and further insights into deregulated pathways in H-MM.

Abstract Introduction: Type II Cryoglobulinemia (CG) is a heterogeneous disorder caused by a monoclonal antibody with activity against polyclonal antibodies. No standard therapy exists, but two small recent studies have suggested a role for rituximab. We describe our experience with 8 patients with Type II CG who were treated with Rituximab. Methods: The data was obtained from a prospectively held dysproteinemia database and by directly reviewing patient records. Follow up time ranged from 7 to 63 months. All patients had symptomatic disease with a monoclonal IgMκ Type II cryoglobulin and had undergone previous therapy for CG. Results: Six of the patients had an underlying lymphoproliferative disorder (LPD), with two of these also having hepatitis C, and two others having Sjogren syndrome. One patient had essential CG and one had Gaucher disease with hyperslpenism. A total of 14 courses of treatment were given, as some patients received rituximab on multiple occasions. Five patients received rituximab either alone or with low dose prednisone initially, with one of these later receiving rituximab with cyclophosphamide and prednisone. One patient was treated initially with R-CHOP and subsequently with rituximab alone, one received rituximab with chlorambucil and prednisone, and one received rituximab with plasma exchange and prednisone. The patients were treated for a variety of indications including cutaneous ulcers, progressive renal failure, the underlying LPD, and biochemical recurrence of disease. Six of 8 patients experienced an improvement in their clinical status. Of the two patients who did not improve, one had end stage liver disease at the time of rituximab and died 2 years later, and the other had a progressive chronic B cell lymphoproliferative disorder leading to sepsis and death. Though lab values were not available on one patient, 4 of the remaining 7 demonstrated improvement in cryocrit, total complement, C4, and/or rheumatoid factor. The lab values correlated well with the clinical response, as treatment failures showed no laboratory changes. Cutaneous manifestations including rash and ulceration were the most responsive to treatment. Renal disease generally failed to improve, and the response of LPDs were variable. No adverse events associated with rituximab were reported, including no flares of hepatitis. Conclusion: The high prevalence of LPD associated cryoglobulinemia distinguishes our results from the previous studies which had focused on patients with hepatitis C. Given the lack of any cryoglobulinemia specific therapy, and the potential toxicity of other immunosuppressive or anti viral therapies, rituximab remains a reasonable, safe, and effective therapeutic choice. Treatment Regimens and Responses Patient Comorbid Condition Symptom Regimen Response: Clinical/Lab LPD= lymphoproliferative disorder; HCV= Hepatitis C Virus; ESRD= End Stage Renal Disease; Y=Yes; N= No; U= Unknown 1 B Cell LPD Leg Ulcers Rituxan Prednisone Y/Y 1 Leg Ulcers Rituxan Prednisone Y/U 2 None Rash, Paresthesia Rituxan Prednisone Y/Y 2 Pupura Rituxan Prednisone Y/Y 2 Pupura Rituxan Prednisone Y/Y 2 Pupura Rituxan Prednisone Cytoxan Y/Y 3 HCV, low grade NHL Renal Failure Rituxan N/N 4 MALToma, Sjogren Disease MALToma R-CHOP Y/U 4 MALToma Rituxan N/U 5 Gaucher with hypersplenia Renal Failure Rituxan Y/Y 6 Lymphoplasmacytic Lymphoma, HCV, ESRD Lymphoma Rituxan Y/U 6 Lymphoma Rituxan N/N 7 B Cell LPD Renal Failure, ulcers, rash, LPD Rituxan chlorambucil prednisone N/N 8 MALToma Sjogren Disease Rash, Fatigue Rituxan Prednisone Y/Y

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 MM has a median age at presentation of 65–68 years, rarely occurring in pts before age 40. Here, we present clinical, laboratory and survival data in the largest ever-analyzed cohort of young pts compared with pts aged ≥ 40 years. 259 pts aged < 40 and 8,296 aged ≥ 40 years were enrolled. These 8.555 pts were part of the cohort analyzed for the ISS staging project. Previously untreated pts entered into randomized trials with standard (SDT) or high dose treatment (HDT). Clinical data were available for all pts, 526 pts had karyotypic analysis and 454 pts testing for Del 13 testing by FISH. The Chi-Square test was used to identify significant prognostic factors. A maximum likelihood model was used to estimate relative excess risk (RER) for younger versus older pts treated with SDT or HDT. Observed survival was adjusted by life expectancy depending on age, gender, and nationality. Young pts had significantly lower ISS stage I than older pts (stage I; 39% vs. 28%, stage II: 28 vs. 39%, stage III 33% vs. 33%, p<. 001, p<001, p=0.863, respectively) due primarily to a lower prevalence of high ß2m levels and low albumin levels in the younger age group. Paradoxically, the younger pts more frequently presented with higher M-component levels and Durie Salmon stage III (65% vs. 54%, p<0.001). Prevalence of other important prognostic parameters like Hb, platelet count, creatinine, calcium, CRP, LDH and bone marrow PC infiltration was similar in both groups. Younger pts had more frequently good ECOG performance status (PS) (0–2), (87% vs. 81%, p<0.01). Karyotypic abnormalities and Del 13 were similarly distributed between both cohorts. Median OS was significantly longer in young pts (median: 4.8 years vs. 3.7 years; hazard ratio (HR): 1.46; 95%, CI (1.27, 1.68); p=<0.001). Better outcome in the younger cohort was seen in all three ISS stages, independent of gender. Median OS survival was significantly longer after SDT in the young cohort (median: 49 months (mos) vs. 39 mos; HR 1.49; CI (1.27, 1.75); p<0.001). Median survival: 86 mos in young and 68 mos in older pts on HDT; this did not translate into a statistical difference (HR 1.8; CI (0.81,1.44); p=0.6). Risk estimates in a maximum likelihood analysis revealed that, apart from ISS Stage, young age followed by female sex was statistical significant for a good outcome for pts treated with SDT (Age RER: 1.43, CI (1.20, 1.70), p<0.001; Sex RER: 1.07, CI (1.02, 1.14); p < 0.013). For pts treated with HDT, only ISS Stage was found to be a risk factor, but not age or gender. Conclusions: Patients < 40 have better prognosis and survival than older patients. The most important features of young patients are low SßM, normal S. Alb, and good performance status.

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.