Faculty Bibliography

The majority of patients with multiple myeloma have translocations involving the immunoglobulin heavy chain switch regions on chromosome 14q32 and a promiscuous range of partner chromosomes. We describe a patient with an insertion of 132 bp of chromosome 22q12 sequence into the 5' region flanking S(mu) on chromosome 14q32. The 132 bp region from chromosome 22q12 contains the whole of exon 3 from a novel gene of unknown function in man. The significance of such insertional events remains unclear. The description of insertional events occurring as a result of abnormal switch recombination suggests that, in myeloma, dysregulation of oncogenes may occur by a mechanism other than chromosomal translocation.

Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5(-) "immature" plasma cells showed the highest levels of CD126 expression, but "mature" VLA-5(+) myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS. (Blood. 2000;96:3880-3886)

Ku is a heterodimer of Ku70 and Ku86 that binds to double-stranded DNA breaks (DSBs), activates the catalytic subunit (DNA-PKcs) when DNA is bound, and is essential in DSB repair and V(D)J recombination. Given that abnormalities in Ig gene rearrangement and DNA damage repair are hallmarks of multiple myeloma (MM) cells, we have characterized Ku expression and function in human MM cells. Tumor cells (CD38(+)CD45RA(-)) from 12 of 14 (86%) patients preferentially express a 69-kDa variant of Ku86 (Ku86v). Immunoblotting of whole cell extracts (WCE) from MM patients shows reactivity with Abs targeting Ku86 N terminus (S10B1) but no reactivity with Abs targeting Ku86 C terminus (111), suggesting that Ku86v has a truncated C terminus. EMSA confirmed a truncated C terminus in Ku86v and further demonstrated that Ku86v in MM cells had decreased Ku-DNA end binding activity. Ku86 forms complexes with DNA-PKcs and activates kinase activity, but Ku86v neither binds DNA-PKcs nor activates kinase activity. Furthermore, MM cells with Ku86v have increased sensitivity to irradiation, mitomycin C, and bleomycin compared with patient MM cells or normal bone marrow donor cells with Ku86. Therefore, this study suggests that Ku86v in MM cells may account for decreased DNA repair and increased sensitivity to radiation and chemotherapeutic agents, whereas Ku86 in MM cells confers resistance to DNA damaging agents. Coupled with a recent report that Ku86 activity correlates with resistance to radiation and chemotherapy, these results have implications for the potential role of Ku86 as a novel therapeutic target.

Although thalidomide (Thal) was initially used to treat multiple myeloma (MM) because of its known antiangiogenic effects, the mechanism of its anti-MM activity is unclear. These studies demonstrate clinical activity of Thal against MM that is refractory to conventional therapy and delineate mechanisms of anti-tumor activity of Thal and its potent analogs (immunomodulatory drugs [IMiDs]). Importantly, these agents act directly, by inducing apoptosis or G1 growth arrest, in MM cell lines and in patient MM cells that are resistant to melphalan, doxorubicin, and dexamethasone (Dex). Moreover, Thal and the IMiDs enhance the anti-MM activity of Dex and, conversely, are inhibited by interleukin 6. As for Dex, apoptotic signaling triggered by Thal and the IMiDs is associated with activation of related adhesion focal tyrosine kinase. These studies establish the framework for the development and testing of Thal and the IMiDs in a new treatment paradigm to target both the tumor cell and the microenvironment, overcome classical drug resistance, and achieve improved outcome in this presently incurable disease.

Our previous studies have shown that activation of a related adhesion focal tyrosine kinase (RAFTK) (also known as Pyk2) is required for dexamethasone (Dex)-induced apoptosis in multiple myeloma (MM) cells and that human interleukin-6 (IL-6), a known growth and survival factor for MM cells, blocks both RAFTK activation and apoptosis induced by Dex. However, the mechanism whereby IL-6 inhibits Dex-induced apoptosis is undefined. In this study, we demonstrate that protein-tyrosine phosphatase SHP2 mediates this protective effect. We show that IL-6 triggers selective activation of SHP2 and its association with RAFTK in Dex-treated MM cells. SHP2 interacts with RAFTK through a region other than its Src homology 2 domains. We demonstrate that RAFTK is a direct substrate of SHP2 both in vitro and in vivo, and that Tyr(906) in the C-terminal domain of RAFTK mediates its interaction with SHP2. Moreover, overexpression of dominant negative SHP2 blocked the protective effect of IL-6 against Dex-induced apoptosis. These findings demonstrate that SHP2 mediates the anti-apoptotic effect of IL-6 and suggest SHP2 as a novel therapeutic target in MM.

Peripheral blood progenitor cells used during high dose treatments for malignancy may be contaminated with tumour cells that could later contribute to recurrence. CD34+ selected harvests still contain tumour cells and an additional negative selection may be capable of reducing this contamination. We have assessed a two-stage technique in which a CD34+ selection is followed by a tumour specific depletion stage using a B cell or breast cancer specific antibody panel. Initial small-scale selections on 11 patients with NHL and breast cancer showed that cell loss was greatest following the CD34+ selection with a median yield of 38.8 per cent (range 17. 2-56.4 per cent). The addition of the depletion stage resulted in a minimal loss of CD34+ cells with a yield for this step of 94.2 per cent (range 77.5-99.3 per cent). Clinical scale selections were performed on seven patients with CLL and a median of 2.8x10(6)/kg CD34+ cells (range 1.5-6.1x10(6)/kg) were collected. Cell recovery was 53.3 per cent following CD34+ selection and 76.9 per cent following the tumour specific depletion stage, resulting in a final product containing a median of 1.0x10(6)/kg CD34+ cells (range 0. 55-2.0x10(6)/kg). All unmanipulated harvests were heavily contaminated with tumour cells (median contamination 10.2 per cent, range 2.0-83.1 per cent) as measured by flow cytometry and a median 4.7 log (range 3-5 log) tumour cell purge was produced following two-stage selection. Six of the patients have received cells manipulated in this way with median engraftment times of neutrophils>0.5x10(9)/l=16 days (range 13-20 days) and platelets>20x10(9)/l=16.5 days (range 11-42 days). At a median follow-up of 25 months, these transplanted patients remain well and in molecular complete remission.

PURPOSE/OBJECTIVE:To determine the effect of polymorphic variations in the tumor necrosis factor alpha (TNFalpha) and lymphotoxin alpha (LTalpha) genes on the predisposition to myeloma and the effect of these polymorphisms on response to treatment and overall survival. PATIENTS AND METHODS/METHODS:Genotype distribution was determined in 63 patients with monoclonal gammopathy of uncertain significance (MGUS) and 198 patients with myeloma and compared with that in 250 age- and sex-matched population-based controls. The effect on treatment response and survival was determined in 171 myeloma patients treated with either conventional or high-dose chemotherapy. RESULTS:Comparison of the extended TNFalpha/LTalpha haplotype in the myeloma cases and controls showed a significant excess of high-producer alleles in the cases. The double heterozygotes TNF1/2 and LT10.5/5.5 were present in 35.8% of cases but in only 18% of the controls; this presence was associated with a significant increased risk of myeloma (odds ratio, 2.05; 95% confidence interval, 1.26 to 3.35). A similar odds ratio was seen in the MGUS cases, suggesting that this genotype is associated with the initiation of plasma-cell disorders rather than the progression of MGUS to myeloma. The median overall survival time of myeloma patients was 53.8 months and showed no difference with regard to TNFalpha/LTalpha polymorphic status. A trend toward an improved progression-free survival was apparent in cases with a high-producer haplotype, although this effect was seen only in patients receiving high-dose chemotherapy. CONCLUSION/CONCLUSIONS:Individuals with polymorphisms associated with a high production of TNFalpha/LTalpha are at a significantly increased risk of developing MGUS and myeloma. The impact of polymorphic status on overall survival is minimal, although there is a trend toward an increased progression-free survival in the high-producer group.

Primary amyloidosis (AL), like multiple myeloma (MM), results from a clonal proliferation of plasma cells. Recent detection of Kaposi's sarcoma-associated herpesvirus (KSHV) gene sequences in MM patients, although controversial, suggested that KSHV may also be present in AL. In the present study, we assayed for KSHV gene sequences in patients with primary AL independently in 2 laboratories. Nested polymerase chain reaction (PCR) was performed on DNA isolated from 21 bone marrow (BM) core biopsy samples to amplify orf26 and orf72, 2 regions of the KSHV genome. Eighteen of 21 (86%) BM core biopsy samples were KSHV PCR positive. BM aspirates from 16 of these 21 AL patients were cultured for 4-6 weeks to generate long term bone marrow stromal cells (LT-BMSCs), and 13 of 16 (81%) LT-BMSCs were also KSHV PCR positive. Results in all but 1 sample were consistent in the 2 laboratories. Sequencing of the PCR products in the 2 laboratories confirmed 94-98% and 95-98% homology to the published orf 26 and orf 72 KSHV gene sequences respectively, with interpatient base pair differences. Despite the presence of KSHV gene sequences, only 4/18 (22%) KSHV PCR positive patients demonstrated KSHV lytic antibodies by immunoblot assay. A sensitive assay performed on the BCBL-1 cell line confirmed the presence of KSHV at a very low copy number in AL. PCR using patient specific light chain gene primers also amplified DNA isolated from 2 AL BM core biopsies and 3 AL LT-BMSCs which were KSHV PCR positive, suggesting the presence of clonotypic cells. Our results therefore demonstrate KSHV gene sequences albeit at a very low copy number in the majority of BM core biopsies and LT-BMSCs from AL patients, and serological responses in only a minority of cases. Ongoing studies to identify viral transcripts and gene products will determine the biological relevance of KSHV in AL disease pathogenesis.