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What's New in Research - January 29, 2003
Circulating peripheral blood plasma cells as a prognostic indicator in patients with primary systemic amyloidosis
Animesh Pardanani, Thomas E. Witzig, Georgene Schroeder, Edwin A. McElroy, Rafael Fonseca, Angela Dispenzieri, Martha Q. Lacy, John A. Lust, IMF Scientific Advisory Board Chair Robert A. Kyle, Philip R. Greipp, IMF Scientific Advisor Morie A. Gertz, and S. Vincent Rajkumar
Division of Hematology and Internal Medicine and the Division of Biostatistics, Mayo Clinic, Rochester, MN.
Blood, 1 February 2003, Vol. 101, No. 3, pp. 827-830

ABSTRACT - This study examined the prognostic value of circulating peripheral blood plasma cells (PBPCs) in patients with primary systemic amyloidosis (AL). A sensitive slide-based immunofluorescence technique was used to assess 147 patients for circulating PBPCs. Circulating monoclonal plasma cells were quantified as a percentage of circulating cytoplasmic immunoglobulin-positive cells (PBPC%). The absolute circulating plasma cell count was also determined. When analyzed retrospectively, 24 (16%) of 147 patients were found to have detectable circulating PBPCs. Overall survival for patients with high PBPC%'s (> 1%) was poorer (median survival, 10 vs 29 months; P = .002). Similarly, overall survival for patients with high PBPC counts (> 0.5 × 106/L) was significantly poorer (median, 13 vs 31 months; P = .003). Increased percentages of bone marrow plasma cells (BMPC%; P = .0004), increased levels of serum 2-microglobulin (P = .04), and dominant cardiac amyloid involvement (P = .03) also predicted poorer survival. The combined consideration of circulating PBPCs and BMPC% identified low-, intermediate-, and high-risk groups with median survivals of 37.5, 15.5, and 10 months, respectively (P = .0003). Multivariate analysis revealed circulating PBPCs and BMPC% to be independent prognostic factors for survival. Patients with PBPC%'s of 2% or higher were significantly more likely to have a coexisting clinical diagnosis of multiple myeloma (50% vs 12%, P = .008). The prognostic value of circulating PBPCs may help select treatment for patients with AL.

Gene expression profiling of human plasma cell differentiation and classification of multiple myeloma based on similarities to distinct stages of late-stage B-cell development
Fenghuang Zhan, Erming Tian, Klaus Bumm, Ruston Smith, Bart Barlogie, and John Shaughnessy, Jr
Blood 2003 101: 1128-1140. Prepublished online September 26, 2002; DOI 10.1182/blood-2002-06-1737

ABSTRACT: To identify genes linked to normal plasma cell (PC) differentiation and to classify multiple myeloma (MM) with respect to the expression patterns of these genes, we analyzed global mRNA expression in CD19-enriched B cells (BCs) from 7 tonsils, CD138-enriched PCs from 11 tonsils, 31 normal bone marrow samples, and 74 MM bone marrow samples using microarrays interrogating 6800 genes. Hierarchical clustering analyses with 3288 genes clearly segregated the 4 cell types, and chi-square and Wilcoxin rank sum tests (P < .0005) identified 359 and 500 previously defined and novel genes that distinguish tonsil BCs from tonsil PCs (early differentiation genes [EDGs]), and tonsil PCs from bone marrow PCs (late differentiation genes [LDGs]), respectively. MM as a whole was found to have dramatically variable expression of EDGs and LDGs, and one-way analysis of variance (ANOVA) was used to identify the most variable EDGs (vEDGs) and LDGs (v1LDG and v2LDG). Hierarchical cluster analysis with these genes revealed that previously defined MM gene expression subgroups (MM1-MM4) could be linked to one of the 3 normal cell types. Clustering with 30 vEDGs revealed that 13 of 18 MM4 cases clustered with tonsil BCs (P = .000 05), whereas 14 of 15 MM3 cases clustered with tonsil PCs when using 50 v1LDG (P = .000 008), and 14 of 20 MM2 cases clustered with bone marrow PCs when using 50 v2LDG (P = .000 09). MM1 showed no significant linkage with normal cell types studied. Thus, genes whose expression is linked to distinct transitions in late-stage B-cell differentiation can be used to classify MM.

Induction of myeloma-specific cytotoxic T cells using dendritic cells transfected with tumor-derived RNA
Caterina Milazzo, Volker L. Reichardt, Martin R. Müller, Frank Grünebach, and Peter Brossart
Department of Hematology, Oncology, Immunology, and Rheumatology, University of Tübingen, Germany.
Blood, 1 February 2003, Vol. 101, No. 3, pp. 977-982

ABSTRACT: Current immunotherapeutic trials for patients with multiple myeloma (MM) focus on the idiotype (Id) as a tumor-specific antigen for active immunization. To bypass the need for the identification of shared MM-associated antigens and the characterization of possible immunogenic T-cell epitopes in a human leukocyte antigen (HLA) type-restricted manner, we focused on myeloma RNA transfection of dendritic cells (DCs). Total RNA encodes the whole antigen content of tumor cells, therefore allowing the transfected DCs to process and present the most relevant peptides and to induce a possible polyclonal cytotoxic T lymphocyte (CTL) response against different immunogenic antigens. We transfected monocyte-derived DCs with total RNA from the myeloma cell lines LP-1 and U266 by electroporation and investigated the potential of these DCs to induce myeloma-specific CTLs. We show that RNA-transfected DCs induce CTLs that lyse the LP-1 and U266 myeloma cells in an antigen-specific and major histocompatibility complex (MHC) class I-restricted manner, as demonstrated by cold-target inhibition and antibody-blocking studies. Interestingly, LP-1-specific CTLs showed no specificity for the idiotype. Consistent with studies demonstrating mucin 1 (MUC1) as a myeloma-associated antigen, we found MUC1 specificity of the CTLs induced with U266-derived RNA. As corresponding epitopes, we tested the described peptides M1.1 and M1.2 and found a striking fine specificity for M1.2, assuming a possible immunodominance of this peptide. This is the first report on the induction of myeloma-specific CTLs by RNA transfection of DCs.

Gene expression and immunologic consequence of SPAN-Xb in myeloma and other hematologic malignancies
Zhiqing Wang, Yana Zhang, Haichao Liu, Emanuela Salati, Maurizio Chiriva-Internati, and Seah H. Lim
Division of Hematology and Oncology, Texas Tech University Health Sciences Center, Amarillo; and Biotherapy and Stem Cell Transplant Program, Don and Sybil Harrington Cancer Center, Amarillo, TX.
Blood, 1 February 2003, Vol. 101, No. 3, pp. 955-960

ABSTRACT: Recent studies in tumor immunology indicate that malignant cells frequently express normal testicular-specific proteins. Because these proteins show restricted normal tissue distribution, they are usually highly immunogenic and may be potential targets for immunotherapy. In the present study, we have used a pair of sequence-specific primers in reverse transcription-polymerase chain reaction (RT-PCR) and sequence analysis to demonstrate that the X-linked gene encoding SPAN-Xb is expressed in multiple myeloma and other hematologic malignancies such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and acute myeloid leukemia (AML). RT-PCR analysis demonstrates that SPAN-Xb is a cancer/testis antigen and shows a restricted normal tissue expression. It is not expressed in any normal tissue except testis. SPAN-Xb recombinant protein was produced and used in enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. High-titer immunoglobulin G (IgG) antibodies, of IgG3 or IgG2 subclass, against SPAN-Xb were detectable in the sera of these patients. In contrast, SPAN-Xb mRNA or antibodies could not be detected in any of the healthy donors. There was a good correlation between SPAN-Xb gene expression and B-cell immune responses. These results suggest the in vivo immunogenicity of the SPAN-Xb protein. The presence of high-titer IgG responses suggests that the B-cell responses are likely to have been generated with CD4 T-cell cognitive help. Based on these data, we conclude that SPAN-Xb is a novel member of the family of cancer/testis antigens aberrantly expressed by, and capable of inducing, immune responses in patients with multiple myeloma and other hematologic malignancies.

"Use of oral mucosal neutrophil counts to detect the onset and resolution of profound neutropenia following high-dose myelosuppressive chemotherapy"
Gorgun Akpek 1, Robert D. Knight 2, Daniel G. Wright 1*
1Castle Hematology Research Laboratory of the Section of Hematology and Oncology, Department of Medicine, Boston University Medical Center, Boston, Massachusetts. 2Hematology-Oncology Service of the Walter Reed Army Medical Center, Washington, DC
*Correspondence to Daniel G. Wright, Boston University Medical Center, EBRC 405, 650 Albany St., Boston, MA 02118

ABSTRACT: Severe neutropenia following cytotoxic, anti-cancer chemotherapy is well-known to be associated with an increased risk of infections that may be life-threatening, particularly if not treated immediately. Consequently, serial measurements of neutrophil counts in peripheral blood are done routinely following the administration of high-dose myelosuppressive chemotherapy in order to monitor the onset, severity, and duration of iatrogenic neutropenia. We have studied a non-invasive method of quantifying neutrophils recoverable from the oral mucosa, a normal tissue site of neutrophil turnover, as an alternative approach for monitoring severe, chemotherapy-induced neutropenia. This method is based on the quantification of fluorochrome-stained neutrophils present in timed mouthwash specimens. Blood neutrophil (ANC) and mucosal neutrophil counts (MNC) were measured repeatedly in 23 patients who had been treated with dose-intensive chemotherapy for a variety of indications. All 23 patients developed profound neutropenia (ANC < 100/mm3), and 19 developed neutropenic fever (>101°F) during the 2 weeks following treatment. Nadirs of neutropenia defined by MNC were significantly less prolonged than those defined by the ANC. Furthermore, the onset and resolution of neutropenic fever coincided more precisely with nadirs of neutropenia defined by the MNC than with those defined by the ANC. Our findings indicate that oral mucosal neutrophil counts predict the timing of clinical events associated with neutropenia (e.g., the onset and resolution of fever) with significantly greater accuracy than blood neutrophil counts. Am. J. Hematol. 72:13-19, 2003. © 2002 Wiley-Liss, Inc.

Expression and function of chemokine receptors in human multiple myeloma
Moller C, Stromberg T, Juremalm M, Nilsson K, Nilsson G.
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Leukemia 2003 Jan;17(1):203-10

ABSTRACT: Multiple myeloma (MM) is a B cell tumor characterized by its selective localization in the bone marrow. The mechanisms that contribute to the multiple myeloma cell recruitment to the bone marrow microenvironment are not well understood. Chemokines play a central role for lymphocyte trafficking and homing. In this study we have investigated expression and functional importance of chemokine receptors in MM-derived cell lines and primary MM cells. We found that MM cell lines express functional CCR1, CXCR3 and CXCR4 receptors, and some also CCR6. Although only a minority of the cell lines responded by calcium mobilization after agonist stimulation, a migratory response to the CCR1 ligands RANTES and MIP-1alpha was obtained in 5/6 and 4/6, respectively, of the cell lines tested. Five out of six cell lines showed a response to the CXCR4 ligand SDF-1. In addition, 3/6 cell lines migrated in response to MIP-3alpha and IP-10, ligands for CCR6 and CXCR3, respectively. The expression of CXCR4 and CCR1 and the migration to their ligands, SDF-1, and RANTES and MIP-1alpha, respectively, were also demonstrated in primary MM cells. These findings suggest that chemokine receptor expression and the migratory capacity of MM cells to their ligands are relevant for the compartmentalization of MM cells in the bone marrow.

Thalidomide Therapy For Multiple Myeloma Patients May Lengthen Survival
SOURCE: Mayo Clinic

ROCHESTER, MN -- January 14, 2003 -- Nearly one-third of patients with advanced multiple myeloma who had failed current standard therapy of chemotherapy or stem cell transplantation responded to thalidomide for a median duration of nearly one year in a Mayo Clinic study of the effects of thalidomide on myeloma. The findings are reported in the January issue of Mayo Clinic Proceedings.

Many studies in the last three years have determined that thalidomide is effective in the treatment of multiple myeloma, following the initial report by researchers at the University of Arkansas. However, information is limited on how long thalidomide therapy works and on survival rates with such therapy. The Mayo Clinic researchers report on the results of a study that looked at 32 patients with relapsed multiple myeloma.

"Thalidomide is useful in the treatment of patients with relapsed multiple myeloma," said Vincent Rajkumar, M.D., a hematologist at Mayo Clinic and an author of the study. "Our study confirms an earlier report from the University of Arkansas that among patients who respond to therapy, the benefits are not transient, but last approximately one year on average." Studies are now addressing thalidomide's role in combination with other treatments and in earlier stages of the disease.

The researchers note that an estimated 14,600 new patients were diagnosed with myeloma in the United States during 2002 and an estimated 10,800 deaths will be due to myeloma in the same period. The average survival from diagnosis among patients treated with conventional chemotherapy is three to four years. Multiple myeloma, cancer of the bone marrow, remains an incurable cancer despite advances in high-dose chemotherapy and stem cell transplantation therapy. Thalidomide is not currently approved by the U.S. Food and Drug Administration for the treatment of myeloma.

Researchers are looking to thalidomide, as well as other treatments or combinations, to find ways to lengthen the survival of patients with multiple myeloma. Another study in the same issue of the journal reviews the records of all patients in whom multiple myeloma was initially diagnosed at Mayo Clinic Rochester from Jan. 1, 1985, to Dec. 31, 1998, and found the median duration of survival among the 1,027 patients was 33 months and did not improve during this period.

The article reviews its findings of the features of the disease to aid physicians in recognizing and diagnosing it. They found that bone pain and fatigue related to anemia were common.

Despite the lack of improved survival over 13 years, Mayo Clinic researchers say there is reason to believe survival rates in the future will improve significantly because high-dose therapy with stem cell support and new agents for treatment are being introduced.

First, the use of stem cell transplantation has been shown to prolong survival significantly compared with standard-dose chemotherapy. Second, thalidomide has recently shown significant activity in relapsed myeloma, with a median response duration of approximately one year. Third, promising new drugs have shown impressive activity in patients with advanced myeloma. Supportive care has improved for patients with bony lesions, and efforts to develop oral maintenance drug regimens are ongoing.

"These advances, coupled with remarkable strides in the understanding of the biology of the disease, provide considerable hope and optimism for both patients and myeloma researchers," said Robert Kyle, M.D., of Mayo Clinic and an author of the study.

The study was supported by a grant from the National Cancer Institute. In an editorial in the same issue, Kenneth Anderson, M.D., of the Dana-Farber Cancer Institute in Boston, Mass., notes the progress in the research of multiple myeloma and lauds the research of Dr. Kyle, whose work over more than 25 years has contributed to the understanding of the history and symptoms of multiple myeloma.

The study by Dr. Rajkumar and others was supported in part by grants from the National Cancer Institute in Bethesda, Md., and by the Celgene Corporation of Warren, N.J. Drs. Raphael Fonseca, a hematologist at Mayo Clinic and a researcher on the study, and Rajkumar received Leukemia and Lymphoma Society of America Translational Research Awards, and Dr. Rajkumar is supported by the Goldman Philanthropic Partnerships of Lake Forest, Ill., and the Multiple Myeloma Research Foundation.

High-dose therapy followed by autologous haematopoietic stem cell transplantation in multiple myeloma."
Koh LP, Linn YC, Teoh G, Goh YT, Tan PH.
Department of Haematology, Singapore General Hospital, Outram Road, Singapore 169608.
Ann Acad Med Singapore 2002 Nov;31(6):731-7

The median survival of patients with multiple myeloma (MM) after conventional chemotherapy is 3 years or less. Previous studies have shown that high-dose therapy, supported by haematopoietic stem cell rescue, improves survival of patients with MM. We analysed the outcome of 29 myeloma patients who had autologous haematopoietic stem cell transplantation (AHSCT) in our institution over an 8-year period.

Between May 1993 and August 2001, 29 patients with MM underwent high-dose therapy followed by unpurged AHSCT. There were 16 male and 13 female patients. The median age of the patients was 52 years (range, 31 to 67 years). All patients had at least a partial remission after initial chemotherapy. The preparative regimen for the AHSCT was melphalan 200 mg/m2 in 25 patients, melphalan-total body irradiation in 1 patient, and busulphan-cyclophosphamide (BuCy) in 3 patients. Twenty-three patients received peripheral blood stem cells (PBSCs) autograft, 3 patients received bone marrow autograft and 3 patients received both. RESULTS: Treatment-related death occurred in only 2 patients (7%). The median time to neutrophil engraftment was 11 days (range, 8 to 22 days). With a median follow-up period of 18.5 months, the 5-year overall survival (OS) and event-free survival (EFS) rates were 71% and 21%, respectively. The OS was found to be superior to a group of historical controls who were treated with conventional chemotherapy without transplantation (71% vs 19%; P = 0.014).

In conclusion, high-dose therapy followed by AHSCT is safe and beneficial for patients with MM.

The clinical and prognostic significance of erythrocyte sedimentation rate (ESR), serum interleukin-6 (IL-6) and acute phase protein levels in multiple myeloma.
Alexandrakis MG, Passam FH, Ganotakis ES, Sfiridaki K, Xilouri I, Perisinakis K, Kyriakou DS.
Division of Medicine, Departments of Haematology, Internal Medicine, Medical Physics, University Hospital of Heraklion, Crete, Department of Haematology, Venizelion General Hospital, Crete, Department of Haematology, University Hospital of Larisa, Thessalia, Greece.
Clin Lab Haematol 2003 Feb;25(1):41-46

ABSTRACT: Interleukin-6 (IL-6) and acute phase proteins are commonly increased in patients with multiple myeloma. Several of these acute phase proteins are believed to predict prognosis and influence survival. We measured interleukin-6 (IL-6), C-reactive protein (CRP), alpha-1-antitrypsin (a1AT), acid alpha-1-glycoprotein (a1AG), haptoglobin (HAP), transferrin (TRF), hemoglobin (Hb), beta-2-microglobulin (beta2M) and erythrocyte sedimentation rate (ESR) in 42 newly diagnosed multiple myeloma patients and 25 normal controls. At the time of blood collection, nine patients were at stage I of disease, 14 at stage II, and 19 at stage III according to the Durie and Salmon myeloma staging system. Mean +/- SD values of IL-6, CRP, a1AT, a1AG, HAP, beta2M, and ESR were significantly higher and Hb significantly lower than those found in the controls. Univariate analysis, using the log-rank test, showed that among the acute phase proteins, serum CRP (P < 0.002), a1AT (P < 0.008) and ESR (P < 0.008) were significantly correlated with survival. However, when a multivariate Cox proportional hazard model was performed, ESR, CRP, a1AT, a1AG and beta2M were identified as independent prognostic factors, while the others were not. We conclude that ESR, a simple and easily performed marker, was found to be an independent prognostic factor for survival in patients with multiple myeloma.

Comparison of five biochemical markers of bone resorption in multiple myeloma: elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for early progression of the bone disease during standard chemotherapy.
Abildgaard N, Brixen K, Kristensen JE, Eriksen EF, Nielsen JL, Heickendorff L.
Departments of Haematology and Medicine and Endocrinology, Aarhus University Hospital, Aarhus, Department of Medicine and Endocrinology, Odense University Hospital, Odense, and Departments of Diagnostic Radiology and Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
Br J Haematol 2003 Jan;120(2):235-242

ABSTRACT: Increased osteoclastic bone resorption is the major causal factor of bone disease in multiple myeloma. Recently, non-invasive methods have been developed for the estimation of bone resorptive activity. To evaluate the biological sensitivity and clinical usefulness of five biochemical assays for measuring the C-terminal telopeptide of collagen I (ICTP) in serum (beta-Crosslaps ELISA and ICTP radioimmunoassay) and urinary creatinine-adjusted excretions of pyridinoline (PYR), deoxypyridinoline (DPD) and N-terminal telopeptide of collagen I (Ntx), we performed a study of 34 consecutive newly diagnosed myeloma patients. Serum and morning-fasting, second-void urine samples were taken before the start of treatment. In total, 40 age- and sex-adjusted healthy individuals served as controls. Results were expressed as Z-scores. All test variables were highly significantly elevated in the patients (P < 0.001). Serum (S)-ICTP was elevated (Z-score > 2) in most patients (85%) and showed significantly higher Z-score values than the other markers. S-ICTP remained more sensitive than the urinary assays when patients with impaired renal function were excluded from analysis. S-ICTP and the urinary metabolites correlated significantly with skeletal morbidity. S-beta-Crosslaps correlated with the bone morbidity only when patients with renal insufficiency were excluded from the analysis. High levels of S-ICTP and urinary (U)-Ntx correlated with an increased risk for early progression of bone lesions during standard melphalan-prednisolone treatment. U-Ntx and S-ICTP are sensitive tools for estimating the increased bone resorption in multiple myeloma and are clinically useful for identifying patients with increased risk of early progression of bone disease.

Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase 8 or caspase 9 and synergy with APO2/TRAIL
Qun Liu, Susan Hilsenbeck, and Yair Gazitt*
Department of Medicine, University of Texas Health Science Center, San Antonio, TX, USA
Department of Breast Center, Baylor College of Medicine, Houston, TX, USA
* Corresponding author; email: gazitt@uthscsa.edu.
Blood First Edition Paper prepublished online January 16, 2003; DOI 10.1182/blood-2002-10-3231

ABSTRACT: Arsenic trioxide (ATO) has been shown to induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells concomitant with down regulation of the PML-RAR fusion protein, a product of the t(15:17) translocation, characteristic to APL leukemic cells. However, ATO is also a potent inducer of apoptosis in a number of other cancer cells lacking the t(15:17) translocation. The exact mechanism of ATO-induced apoptosis in these cells is not yet clear. We tested the effect of ATO on 7 myeloma cell lines with varying p53 status and report that in cells with mutated p53, ATO induced rapid and extensive (> 90%) apoptosis in a time/dose dependent manner concomitant with arrest of the cells in G2/M phase of the cell cycle. Myeloma cells with w.t. p53 were relatively resistant to ATO with maximal apoptosis of about 40% concomitant with partial arrest of cells in G1 and upregulation of p21. The Use of caspase blocking peptides, fluorescence-tagged caspase-specific substrate peptides and Western immunoblotting confirmed the involvement of primarily caspase 8 and 3 in ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase 9 and 3 in cells expressing w.t. p53. We also observed upregulation by ATO of R1 and R2, APO2/TRAIL receptors. Most importantly, however, we observed a synergy between ATO and APO2/TRAIL in the induction of apoptosis in the partially resistant myeloma cell lines and in myeloma cells freshly isolated from myeloma patients. Our results justify the use of the combination of these 2 drugs in clinical setting in myeloma patients.

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