Dispenzieri, Angela

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 Background: Patients with SMM/IMM are at high risk for progression to active MM and are appropriate candidates for chemoprevention trials. High IL-1beta levels are a useful surrogate marker for progression from SMM/IMM to active myeloma. Methods: We carried out a Phase II clinical trial of IL-1Ra (Anakinra) in patients with SMM/IMM to determine the biologic activity, progression-free rate, and toxicity of IL-1Ra. Since IL-1beta induces paracrine IL-6, we hypothesized that IL-1Ra would inhibit IL-6 production and myeloma cell growth. 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. Patients received 100 mg of Anakinra (IL-1Ra) SQ qd for a total duration of 6 months. Non-progressors were allowed to continue on therapy with IL-1Ra until they converted to active myeloma. IL-1beta levels were measured in a bioassay with a read out of IL-1 inducible IL-6 production. CRP levels served as a surrogate for IL-6 production. Results: Thirty-six patients are included for analysis based on intent to treat at diagnosis. At baseline, twenty-nine patients had elevated functional IL-1beta levels consistent with progressive disease and 12 patients had radiologic evidence of disease on bone survey. The median TTP for the entire group was 1 year and 2 patients exhibited a minor response based on M-protein reduction. A proportional hazards analysis was performed and the variables examined were: % bone marrow plasma cells, plasma cell labeling index (PCLI), circulating plasma cells, albumin, M-protein level, IgA subtype, CRP, CRP % reduction ≥ one-third, beta-2 microglobulin, creatinine, calcium, hemoglobin, urine total protein, presence of lytic bone disease, and IL-1 level. Univariate Cox model results showed that % BMPC, PCLI, circulating plasma cells, CRP % reduction, presence of lytic disease, and IL-1 level were all statistically significant (p < .05) variables. However, only the CRP % reduction from baseline remained significant in the multivariate analysis (p < 0.01). The median TTP for patients without (n=18) and with (n=18) a decrease in CRP was 6 months and > 2 years, respectively (p < .0001). Toxicities included injection site reactions during the first month of therapy and asymptomatic neutropenia necessitating a reduction in therapy to every other day. Five patients in whom the CRP decreased and the baseline PCLI was > 0 also demonstrated a parallel decrease in the PCLI. Several of the patients with progressive disease on IL-1Ra alone could later be rescued with the addition of low dose dexamethasone (20 mg once a week) suggesting that the endogenous IL-1beta levels were too high to inhibit with IL-1Ra alone. Conclusion: In summary, IL-1Ra can prolong the TTP to active myeloma in responsive SMM/IMM patients through inhibition of IL-6 production (decrease in CRP) and myeloma cell growth. Figure Figure

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 Background: Primary systemic amyloidosis (AL) is an incurable plasma cell disorder. For selected patients high dose chemotherapy with peripheral blood stem cell support is effective, but even in those patients, only 50% derive organ responses. Other patients are too sick to undergo that therapy. Thalidomide has limited utility in this disease, largely because of its toxicity profile in patients with AL. Lenalidomide, Celgene’s lead clinical compound in a new group of drugs called IMiDs®, is highly active in patients with multiple myeloma, especially in conjunction with dexamethasone. We sought to determine the toxicity and efficacy of lenalidomide in patients with AL. Methods: Patients with symptomatic AL who had a measurable plasma cell disorder (defined as serum M-spike ≥ g/dL urine M-spike >200 mg/24 hours; or involved immunoglobulin free light chain (FLC) ≥10 mg/dL and an abnormal FLC ratio) and adequate organ reserve defined as a creatinine ≤3 mg/dL, absolute neutrophil count ≥1000, platelet count ≥75000, and a hemoglobin ≥8 g/dL were eligible. Patients were started on lenalidomide 25 mg/day for 21 days followed by a 7 day rest (1 cycle). Dose modifications were made based on toxicity. If there was evidence of progression before 3 months or no evidence of hematologic response after 3 cycles, dexamethasone 40 mg p.o. days 1–4 and 15–18 was added. Results: Twenty-three patients were enrolled between 10/28/04 and 4/7/05; 14 were previously treated. Patient characteristics are shown in Table. Organ involvement was as follows: cardiac (61%); renal (70%), hepatic (22%); nerve (13%). Nine patients withdrew from study before completing 3 months of therapy. The reasons were: cancel (1); death (4); adverse events (2); and progression (2). At the time of our preliminary analysis in July 2005, an additional 3 patients have withdrawn from study: death (1) and patient refusal (2). Of the 5 patients who died, 4 had severe cardiac involvement and at least 3 organs involved by amyloid. The median follow-up for the eleven patients remaining on study is 6.2 months. Of the 12 patients who have crossed the 3 month treatment landmark, there are 7 hematologic partial responses (4 confirmed and 3 unconfirmed), two renal responses and one liver response. Of these same 12 patients, all but one has had dexamethasone added to their treatment program. The most common grade 3–4 adverse advents at least possibly attributable to lenalidomide were neutropenia (43%), thrombocytopenia (26%), rash (17%), dyspnea (9%), fatigue (9%), and edema (4%). Expansion of the trial is planned to accrue an additional 10 evaluable patients. Conclusions: Early results suggest that lenalidomide ± dexamethasone has activity in patients with primary systemic amyloidosis. Supported in part by the Caliguiri Fund for Amyloidosis Research, Robert A. Kyle Hematologic Malignancies Program, and Celgene. Patient Characteristics Median Range Age 64 44–88 Major Organs 2 0–3 Involved FLC, mg/dl 23 4.1–278 Serum alb, g/dl 2.8 1.2–3.7 Urine protein, g/24 hrs 3.7 0.02–14.3 Creatinine, mg/dl 1.3 0.7–2.6 Alkaline phos IU/l (nml <115) 99 57–1729 NY Heart Class 1 1–3 Troponin T, mcg/l 0.03 0.01–0.55 NT-proBNP, ng/l 1419 109–42844 LV Septum, mm 13 9–24 LV EF, % 63 22–72

Abstract Background: High dose therapy with autologous stem cell rescue has been shown to prolong survival in patients with multiple myeloma in randomized controlled trials. However, most of the prospective studies have included younger patients, usually 65 or less. It is important to have a better understanding of the outcome of transplantation in the older patients given the median age of onset of myeloma of 65 years. We retrospectively reviewed our institutions experience with high dose therapy for patients over 70 years. Methods: We identified 35 patients with multiple myeloma, from the transplant database, who were at or over the age of 70 at the time of their high dose therapy. We matched these patients to 70 patients (two matches for each patient), based on stage at transplant (primary refractory, plateau phase, relapse off therapy, or relapse on therapy), Durie Salmon stage, high or low labeling index, conventional cytogenetics (abnormal vs normal), presence or absence of circulating plasma cells at time of transplant, and whether cyclophosphamide was used as part of mobilization in that order of priority. Results: The median age of the two groups were 55.3 (Range 37.3–64.8) and 71.7 (Range 70–75.8) years at the time of transplant. The median time to transplant from diagnosis was similar (6.4 for the older patients compared to 6.9 months for the other, P = NS). Ten of the 35 older patients received reduced dose melphalan (140 mg/2)) compared to 3 patients in the control group; P < 0.01. The median follow up from transplant was 10.1 months for the older patients compared to 18 months for the control group. The overall response rate was similar for the two groups (97.1% for the older patients compared to 95.5 for the control group). Eleven (31%) of the older patients and 17 (24%) of the control patients achieved a CR (P = NS). The post transplant progression free survival estimate at 1 year post transplant was 65.3% for the older patients compared to 66% for the control group (P = 0.3)The two year estimated overall survival from transplant was similar in the two groups; 58% for the older patients compared to 67% for the control group. The overall survival from diagnosis was similar for the two groups (P = 0.6). The median number of days hospitalized was 9 days for the older population compared to 5 days for the control group (P = 0.37). Four patients died within the first one hundred days, one (3%) among the older patient group and 3 (4.3%) in the control group. Conclusions: High dose therapy and autologous stem cell transplantation is feasible in selected patients with multiple myeloma over 70 years. It is likely that these older patients were selected based on their overall performance status, a factor that is difficult to analyze in this retrospective review. Nearly 70% of the elderly patients received full dose melphalan for conditioning (200 mg/m2). The toxicity of transplant as well as the outcome appears to be very similar to the younger patients. Patients with multiple myeloma should not be excluded from high dose therapy solely on the basis of their chronological age.

Abstract Background: Multiple myeloma (MM) is an incurable disease with a anticipated overall survival (OS) ranging from months to decades. Modest improvements in OS have been made with high-dose chemotherapy with peripheral blood stem cell transplant (PBSCT), but to date prognostic factors have a greater impact on OS than do individual therapies. Patients with adverse risk factors such as elevated beta-2 microglobulin (B2M), plasma cell labeling index, deletions of the long arm of chromosome 13 by metaphase cytogenetics (del 13q) require innovative new treatment strategies. Bortezomib has significant activity in patients with both newly diagnosed and relapsed/refractory MM, but its specific role in patients with adverse features has not yet been defined. Methods: Patients with newly diagnosed “high-risk” myeloma (B2M ≥ 5.5., PCLI ≥ 1, or del 13q) and adequate organ and functional status were eligible. Patients were treated with bortezomib 1.3 mg/m2 day 1, 4, 8, and 11 every 21 days for 8 cycles as induction. After induction, patients were scheduled to receive bortezomib 1.3 mg/m2 every other week indefinitely. Elective peripheral stem cell mobilization (growth factor alone) was allowed after 4 cycles of bortezomib. Patients relapsing on maintenance schedule were to have the full induction schedule resumed. Responses were by the EBMT criteria but a very good partial response category was included. The primary end-point was the response rate in these high-risk patients (90% power to detect a response rate of 50% or higher). The study decision rule requires that 16 or more responses, among 39 eligible patients, are seen in order to declare this treatment effective. Results: Between March 15, 2004 and March 10, 2005, 44 patients enrolled on study. Among the 43 eligible patients, median age was 63; 51% were male. All patients had high risk disease: del 13q (6/41); plasma cell labeling index ≥1% (16/34); and B2M≥5.5 (34/43). Preliminary response data are available for 18 of the 44 cases enrolled, of which 7 had partial response, 1 had minimal response, 1 had no response, 2 had progressive disease, and 5 were unevaluable. Among those patients completing induction therapy and with response information, the median number of cycles of therapy administered is 5, range (0,8). The most common non-hematologic adverse events (AEs) of grade 3 or higher included hyponatremia (9 patients) and diarrhea (6 patients). Mild sensory peripheral neuropathy was common: grade 1, 16 patients; grade 2, 2 patients. Only 1 patient had grade 3 peripheral neuropathy. One patient died after receiving 2 doses bortezomib due to heart block and asystole. Two patients had a grade 4, 25 patients had grade 3, and 13 had grade 1 or 2 as the worst grade non-hematologic adverse event. Based on data received by August 1, 2005, 18 patients have gone off study: AEs (2); death (1); progressive disease (9); and other reasons (6). Updated results on the full study population along with FISH data for IgH translocations and deletions of 13q and 17p will be presented at the meeting. Conclusions: Preliminary results suggest that upfront bortezomib has activity in patients with high-risk MM, but further follow-up is required.

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 Introduction: The Multiple Myeloma Research Consortium (MMRC) has established a tissue bank for the deposition of bone marrow (BM) samples from patients with multiple myeloma (MM) to be mailed and processed under good laboratory practices (GLP). To date over 300 samples have been collected. The ability for multiple sites to utilize the same GLP protocols for isolation and storage of BM for batch testing can be cost prohibitive and can introduce derived variability. Currently, limited information is available on shipped BM aspirates in regards to cell viability, cell yield, cell purity, and subsequent RNA yield and quality. Materials and methods: To test these determinants we performed a pilot study on behalf of the MMRC where samples were drawn at Mayo Clinic Rochester (MCR) pooled and split into 2 equal aliquots. One-half of each sample was processed following the provided GLP compliant standard operating procedures (SOP’s), immediately after sample procurement, at MCR. The CD138+ cells were stored in TRIZOLTM and the RNA was isolated and analyzed in a single batch. The other half of the aspirate was sent overnight to Mayo Clinic Scottsdale (MCS) where they were processed using an identical protocol. At both locations samples were tested for the following quality determinants; cell yield, RNA yield and integrity, and viability using a 3 color flow cytometric method (CD45, CD38 and 7ADD). Cell counts were performed on the CD138+ fraction to determine plasma cell recovery and a slide based immunofluorescent assay used to determine purity. RNA recovery and integrity were assessed using the Agilent BioanalyzerTM. Lastly, gene expression profiles was compared to determine the “signature” emanating from the shipment of samples. Results: In aggregate, all quality determinants showed similar values when the two sets of samples were compared. Cell viability was similar in both sets of samples as was our ability to collect a highly enriched plasma cell population. The cell yield was very similar (r2=0.52) but slightly lower in the shipped samples (median 71% of locally processed, range 40–140%), probably due to some shipment-associated apoptosis with subsequent loss of cell surface CD138 antigen. The purity of the shipped samples was very similar to that of locally processed (median 94%). Subjective qualitative analysis of the RNA was similar between both groups (shipped yield being 80% of local) with no evidence of degradation in the shipped samples. Details regarding the shipment signatures using gene expression profiling will be presented at the meeting. Conclusion: Here we show that the shipment of samples is feasible with no appreciable loss in cell yield or quality of derived products. % APOPTOTIC+ DEAD PC YIELD PURITY (%) RNA Recovered MCR MCS MCR MCS MCR MCS MCR MCS 8.9 ND 15.2 6.8 93 90 11.4 12.8 6.2 ND 2 2.8 95 99 8 17.2 17.4 8 1.4 1.4 75 88 2.2 0 2.1 18.7 2.8 1.6 96 68 7.2 2 18.1 9.5 1.2 .8 100 94 3.4 .4 5 5.0 8.5 7.2 98 91 12.6 17.8 3.7 5.1 .4 .4 88 87 ND ND 19.5 10.3 1.6 1.2 88 79 .4 3.6 29.1 ND 9.2 1.2 100 56 30.2 1.4 5.6 ND 9.2 7.6 92 88 5.2 16.8 20.3 62.8 2.4 .8 84 61 4.6 0.2 4.6 3.7 10.4 2.4 100 94 29.4 8.4 26.5 13.5 5.8 2.8 ND 97 16.8 5.8 4.5 6.2 2.2 2 100 99 10 4.4 17.5 ND 19.2 12 ND ND 16.4 14

Abstract Background: Waldenström macroglobulinemia (WM) is a plasma cell dyscrasia characterized by a monoclonal IgM paraproteinemia. The most common genomic abnormality in the clonal cells of WM is loss of portions of the long arm of chromosome 6 (6q-), however a minimal area of deletion has yet to be identified. We decided to further clarify the area of deletion on the 6q-arm in WM along with its prevalence. Methods: Patients (n=41) were required to have a serum IgM paraproteinemia of 1.5 g/dl or greater and a monoclonal lymphoplasmacytic infiltrate. We used interphase fluorescence in situ hybridization (FISH) combined with cytoplasmic immunoglobulin (cIg) staining with an AMCA conjugated anti-IgM antibody along with cytomorphology for the detection of the clonal cells on cytospin slides. Ten probes were used including two commercial (CEP 6 and Telomere 6q) and eight Bacteria Artificial Chromosome (BAC). BAC clones were selected based on a specific gene encompassed in the insert and/or localization on the q arm. Genes of interest included BLIMP1, c-myb, BTF, SHPRH, LATS1 and FOP. BAC’s were grown in selective media; DNA was extracted and directly labeled with a SpectrumGreen or SpectrumOrange fluorophore by standard nick translation. Primers were designed to confirm the insert contained the gene of interest and probes were tested on normal metaphases to reaffirm localization to the correct area of the chromosome and rule out any cross hybridization. Results: Deletions of 6q were noted in 21 (51%) patients. Of the 19 abnormal patients with all probes tested 1 (5%) case had ten probes deleted, 7 (37%) cases had nine probes, 1 (5%) had eight, 4 (21%) had seven, 3 (16%) had six and 3 (16%) had four probes deleted. Only 3 cases retained the end of the q arm indicating an interstitial deletion while the remaining 16 cases indicate terminal deletions. Deletions of the BAC containing the gene SHPRH at 6q24 were seen in each of the 19 abnormal cases. The majority of patients exhibited little heterogeneity for any given deletion having greater than 80% involvement of the clone. Conclusion: This study demonstrates that large deletions of the long arm of chromosome 6 in WM are frequent and are usually terminal deletions rather than interstitial and have a common area of deletion of the q24 portion of the q arm. Using the given number of probes localizing along the q arm makes it highly unlikely of any areas of noncontiguous deletion. The presence of deletions in the majority of the clonal cells may suggest that clones with 6q- emerge early in the course of the disease and is selected because of growth advantage or apoptosis resistance. Whether 6q deletions are important for disease pathogenesis has yet to be determined. Clinical prognosis studies and microarray data analysis are pending.