ALBERT

Description: IL-6 signaling can be enhanced through transsignaling by the soluble IL-6 receptor (sIL-6r), allowing for the pleiotropic cytokine to affect cells it would not ordinarily have an effect on. Serum levels of sIL-6r can be used as an independent prognostic indicator and further stratify the GEP 70-gene low-risk group to identify an intermediate-risk group in multiple myeloma (MM). By analyzing more than 600 MM patients with ELISA, genotyping, and gene expression profiling tools, we show how the combination of 2 independent molecular genetic events is related to synergistic increases in sIL-6r levels. We also show that the rs2228145 minor allele is related to increased expression levels of an IL-6r splice variant that purportedly codes exclusively for a sIL-6r isoform. Together, the SNP rs2228145 minor allele C and amplification of chromosome 1q21 are significantly correlated to an increase in sIL-6r levels, which are associated with lower overall survival in 70-gene low-risk disease, and aid in identification of the intermediate-risk MM group.

Description: To molecularly define high-risk disease, we performed microarray analysis on tumor cells from 532 newly diagnosed patients with multiple myeloma (MM) treated on 2 separate protocols. Using log-rank tests of expression quartiles, 70 genes, 30% mapping to chromosome 1 (P 〈 .001), were linked to early disease-related death. Importantly, most up-regulated genes mapped to chromosome 1q, and down-regulated genes mapped to chromosome 1p. The ratio of mean expression levels of up-regulated to down-regulated genes defined a high-risk score present in 13% of patients with shorter durations of complete remission, event-free survival, and overall survival (training set: hazard ratio [HR], 5.16; P 〈 .001; test cohort: HR, 4.75; P 〈 .001). The high-risk score also was an independent predictor of outcome endpoints in multivariate analysis (P 〈 .001) that included the International Staging System and high-risk translocations. In a comparison of paired baseline and relapse samples, the high-risk score frequency rose to 76% at relapse and predicted short postrelapse survival (P 〈 .05). Multivariate discriminant analysis revealed that a 17-gene subset could predict outcome as well as the 70-gene model. Our data suggest that altered transcriptional regulation of genes mapping to chromosome 1 may contribute to disease progression, and that expression profiling can be used to identify high-risk disease and guide therapeutic interventions.

Description: In addition to its role in diagnosis and assessing the prognosis of numerous malignancies, immunohistochemistry (IHC) is an important tool for the analysis of protein biomarker expression in tissue sections. The combination of bone marrow (BM) biopsy and IHC is frequently used to study histopathological alterations in diseases such as multiple myeloma (MM). However, due to the harsh decalcification process generally used for processing of bone marrow biopsies, protein epitopes are occasionally rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin-clot matrix that allows for IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Briefly, bone marrow aspirates drawn from MM patients were subjected to a brief processing step to isolate the marrow spicules. Once isolated, the spicules were admixed with thromboplastin and pooled normal human plasma to create a clot. The clot was allowed to harden at 37°C after which it was placed into an embedding cassette for fixation and histological processing. The result was a formalin-fixed, paraffin-embedded clot suitable for IHC studies, including the construction of TMAs. Using patient paired spicule-clot and core biopsies from patients diagnosed with myeloma, we studied several antibodies including, but not limited to, kappa and lambda immunoglobulin light chains, CD138 and Cyr61, a member of the CCN family of extracellular matrix-associated signaling proteins. Results demonstrated that IHC staining for all antibodies was comparable in both the spicules and in the cores. While BM core biopsies had areas of non specific staining for several antibodies, this was not generally observed in the BM spicule samples with the exception of immunoglobulin light chains which displayed an element of non-specific staining with both samples. Moreover, we observed a consistently superior morphology in the BM spicule samples than in matched bone marrow core biopsies. In conclusion, our study demonstrates that BM spicule samples processed from MM patients using this novel fibrin-clot matrix technique were suitable for IHC and had generally lower background and non-specific staining coupled with better morphology when compared to matched core biopsies.

Description: Myeloma is intimately associated with osteolytic bone disease, in part by inhibiting mesenchymal stem cell (MSC) differentiation into osteoblasts via blocking Wnt signaling (Tian, N Engl J Med2003;349:2483). Stimulation of osteoblast activity by bortezomib is associated with anti myeloma effects in patients (Zangari, Br J Haematol2005;131:71) and by MSC infusion in a model system (Yaccoby, Haematologica2006;91:192). To further evaluate the potential therapeutic utility of MSCs, we investigated the effects of MSCs on myeloma cell survival and the genomic consequences of MSC-myeloma cell interactions. CD138-purified myeloma (MM) cells from 16 consented patients were cocultured with MSCs derived from the bone marrow of 3 healthy donors (MSC 1, 2, &3, provided by Dr. D. Prockop, Tulane University). In 7 co-culture experiments, MSCs supported MM cell survival for 6–8 days compared to MM cells cultured alone, with a median viable cell count increasing by 2.3 fold v controls (1.2–4.1, p=0.003); in 4, coculture suppressed MM survival (median=0.5, range 0.3–0.7, p=0.016); and in 5 there was no effect (median=0.9, range 0.9–1.0). In 3 experiments, the effects of the different MSCs on MM cell survival varied inconsistently among the 3 MSCs, from 0.5 to 2.5 fold surviving cells. In order to understand this heterogeneity, we investigated the genomic consequences of MM-MSC interactions. RNA was extracted immediately after mixing (t=0) and following 18 hours co-culture (t=18), and changes in gene expression analyzed using the Affymetrix microarray system. Since myeloma cells adhere tightly to MSCs, we analyzed expression changes in 1,708 genes expressed only in MM cells and 4862 expressed only in MSCs. At t=18, 250 MM cell genes were changed by 〉2 fold compared with t=0 (222 up and 28 down regulated), and 1,036 MSC genes were changed 〉2 fold (1018 up and 18 downregulated). Each of the 3 MSCs also had unique genes expressed (40, 85 and 162 for MSC 1, 2, &3, respectively), of which expression of 12, 30, and 39, respectively, was changed by 〉2 fold. Analysis of 776 MM- and 2398 MSC-related genes after a signal cutoff of 500 with Ingenuity Pathways Analysis software showed changes in the expression of several groups of interrelated genes following co-culture. For MM, these included ERKs, AKT, NFKB, E2F1, and FOS. The transcription regulators BTG2, ACTN2, CALR, E2F1, E2F5, and ATF7 were up regulated, while FOS and FOXM1 were down regulated. Groups changed in MSCs included IL1B, CCND1, MYC, CTBP1, EGFR, RUNX1, HRAS, and SMAD3. There were minor differences in changes of the expression patterns of some of the probesets between the three MSCs, likely related to alternative splicing or to variations in the 3′-UTR. These studies continue in order to discern possible differences in the interactions between MM cells and MSCs, and to identify whether MM cells or MSC determine the outcome of these differences.

Description: Monoclonal gammopathy of undetermined significance (MGUS) can progress to multiple myeloma (MM). Although these diseases share many of the same genetic features, it is still unclear whether global gene-expression profiling might identify prior genomic signatures that distinguish them. Through significance analysis of microarrays, 52 genes involved in important pathways related to cancer were differentially expressed in the plasma cells of healthy subjects (normal plasma-cell [NPC]; n = 22) and patients with stringently defined MGUS/smoldering MM (n = 24) and symptomatic MM (n = 351) (P 〈 .001). Unsupervised hierarchical clustering of 351 patients with MM, 44 with MGUS (24 + 20), and 16 with MM from MGUS created 2 major cluster branches, one containing 82% of the MGUS patients and the other containing 28% of the MM patients, termed MGUS-like MM (MGUS-L MM). Using the same clustering approach on an independent cohort of 214 patients with MM, 27% were found to be MGUS-L. This molecular signature, despite its association with a lower incidence of complete remission (P = .006), was associated with low-risk clinical and molecular features and superior survival (P 〈 .01). The MGUS-L signature was also seen in plasma cells from 15 of 20 patients surviving more than 10 years after autotransplantation. These data provide insight into the molecular mechanisms of plasma-cell dyscrasias.