Chromosome 1p21 deletion is a novel prognostic marker in patients with multiple myeloma

The combination of fluorescence in situ hybridization with cytoplasmic light chain detection identified chromosome 1p21 deletion in 18 (20%) of 87 patients with multiple myeloma. 1p21 deletion was associated with higher serum calcium level, 13q deletion, and t(4;14). Patients with 1p21 deletions had a significantly shorter progression-free survival (PFS) (median 10.5 vs. 22.3 months, P = 0.0002) and shorter overall survival (OS) (median 33.9 months vs. not reached, P = 0.002) than those without 1p21 deletions. On multivariate analysis, which included deletions of 13q, TP53, t(4;14) and CKS1B amplification, 1p21 deletion remained as an independent risk factor for PFS (P = 0.01) and OS (P = 0.04).     

Cyclin D type does not influence cell cycle response to DNA damage caused by ionizing radiation in multiple myeloma tumours

Multiple myeloma (MM) is characterized by over‐expression of cyclin D1 (CCND1) or D2 (CCND2), which control G1 phase cell‐cycle progression. Proteolytic degradation of CCND1 (but not CCND2), resulting in G1 arrest, is reported in non‐MM cells post‐DNA damage, affecting DNA repair and survival. We examined the effect of ionizing radiation (IR) on D‐cyclin levels and cell‐cycle kinetics of MM cells, exploring differences based on D‐cyclin expression. We showed that CCND1 is downregulated, whereas CCND2 is not, following IR. This did not lead to hypo‐phosphorylation of retinoblastoma protein or G1 arrest. Both CCND1‐ and CCND2‐expressing MM cells arrested in S/G2/M, and did not differ in other cell‐cycle proteins or sensitivity to IR. When treated with a CDK4/6 inhibitor, both CCND1 and CCND2 MM cells arrested in G1 and therefore are subject to physiological regulation at this checkpoint. Immunoprecipitation showed that, despite CCND1 degradation following IR, sufficient protein remains bound to CDK4/6 to prevent G1 arrest. Aberrant expression of CCND1 driven from the IGH promoter in t(11;14) MM cells maintains progression through G1 to arrest in S/G2/M. Differential expression of D‐cyclin does not appear to affect cell‐cycle response to IR, and is unlikely to underlie differential sensitivity to DNA damage.     

Cyclin D3 is a target gene of t(6;14)(p21.1;q32.3) of mature B-cell malignancies.

Chromosomal translocation t(6;14)(p21.1;q32.3) has been reported as a rare but recurrent event not only in myeloma and plasma cell leukemia but also in diffuse large B-cell non-Hodgkin lymphoma (B-NHL) (diffuse large B-cell lymphoma [DLBCL]) and splenic lymphoma with villous lymphocytes (SLVL); however, the nature of the target gene(s) has not been determined. This study identified t(6;14)(p21.1;q32.3) in 3 cases of transformed extranodal marginal zone B-NHL, in 1 case of SLVL, and in 1 case of a low-grade B-cell lymphoproliferative disorder. In a sixth case, a CD5(+) DLBCL, the translocation was identified by molecular cloning in the absence of cytogenetically detectable change. Two chromosomal translocation breakpoints were cloned by using long-distance inverse polymerase chain reaction methods. Comparison with the genomic sequence for chromosome 6p21.1 showed breakpoints approximately 59 and 73.5 kilobases 5' of the cyclin D3 (CCND3) gene with no other identifiable transcribed sequences in the intervening region. Although Southern blotting with derived genomic 6p21.1 probes failed to detect other rearrangements, fluorescent in situ hybridization assays, using BAC (bacterial artificial chromosome) clones spanning and flanking the CCND3 locus, along with probes for IGH confirmed localization of 6p21.1 breakpoints within the same region, as well as fusion of the CCND3 and IGH loci. Furthermore, in all cases, high-level expression of CCND3 was demonstrated at RNA and/or protein levels by Northern and Western blotting and by immunohistochemistry. These data implicate CCND3 as a dominant oncogene in the pathogenesis and transformation in several histologic subtypes of mature B-cell malignancies with t(6;14)(p21.1;q32.3) and suggest that CCND3 overexpression seen in about 10% of DLBCL cases may have a genetic basis.     

Fusion of PDGFRB to two distinct loci at 3p21 and a third at 12q13 in imatinib-responsive myeloproliferative neoplasms

We identified four patients who presented with BCR-ABL1 negative myeloproliferative neoplasms and cytogenetically visible abnormalities of chromosome band 5q31-35. Fluorescence in situ hybridization indicated that the platelet-derived growth factor receptor β gene ( PDGFRB ) was disrupted in all four cases and 5' rapid amplification of cDNA ends identified in-frame mRNA fusions between PDGFRB and WDR48 (3p21), GOLGA4 (3p21) and BIN2 (12q13). Strikingly, all three genes encode proteins involving intracellular trafficking. Imatinib, a known inhibitor of PDGFRβ, selectively blocked the growth of t(3;5) myeloid colonies and produced clinically significant responses in all patients. We conclude that PDGFRB fuses to diverse partner genes in atypical myeloproliferative neoplasms (MPNs). Although very rare, identification of these fusions is critical for proper management of affected individuals.     

Nonrandom chromosomal rearrangements of 14q32.3 and 19p13.3 and preferential deletion of 1p in 21 patients with multiple myeloma and plasma cell leukemia

Structural chromosomal abnormalities and their break-points were characterized in 17 patients with multiple myeloma (MM) and 4 with plasma cell leukemia by banding. Chromosome 14q32 translocations with a variety of partners were detected in 13 patients, and a variant translocation t(8;22)(q24.1;q11) was detected in 1. Three recurrent 14q32 translocations have been identified: t(6;14)(p21.1;q32.3) occurring in 3 cases, and t(11;14)(q13;q32.3) and t(14;18) (q32.3;q21.3) each occurring in 2 cases. Translocations t(1;14)(q21;q32.3), t(3;14)(p11;q32),t(7;14)(q11.2;q32.3), and t(11;14)(q23;q32.3) were found in each patient, whereas in the remaining 2 patients, partner chromosomes could not be determined. The band 19p13.3 was newly delineated as a recurrent breakpoint involved in translocations in MM. Chromosomes 1 and 6 were also commonly involved in structural abnormalities (14 and 10 patients, respectively), although no particular bands were noted. However, the short arm of chromosome 1 was preferentially involved in deletion, suggesting a certain antioncogene on 1p associated with the development of myeloma. In addition; fluorescence in situ hybridization was successfully applied to determine the nature of the structural abnormalities in a patient with t(8;22) translocation. The present findings suggest that there may be subsets of 14q32 translocations specific to MM.     

A gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme.

The chromosomal region 3p21 is thought to be the site of a lung tumor suppressor gene. We recently cloned a gene from this region that has greatly reduced expression in almost all lung tumor cell lines examined, in spite of being widely expressed in a variety of other tumor and nontumor cell types. We report here the sequence of this gene and show that it has significant homology to the genes encoding the ubiquitin-activating enzymes of three species, including humans. This suggests it is a second, autosomal member of this gene family in humans and may play a role in the ubiquitin conjugation pathway, which is of central importance in all eukaryotes.     

Development of a criterion for response to therapy at 6 months in multiple myeloma

Abstract: To investigate the prognostic value of therapy at 6 months on survival in multiple myeloma, to develop a new criterion assessing response to initial therapy at 6 months, and to compare it to a current criterion. The types of initial and 6-month therapy were considered in a prognostic factor analysis in 70 patients treated in routine practice. Using the response to initial therapy defined by the clinician's decision as grouping variable in this group, a discriminant analysis identified the characteristics of responder patients. The validity of the resulting criterion was tested in another test group. Its prognostic ability was compared to the CLMTF criterion (50% M-component reduction from baseline). The therapy at 6 months, reflecting the clinician's appraisal of response to initial therapy, predicted survival significantly. A criterion combining two variables (M-component change and haemoglobin level at 6 months) classified 70% and 72.4% of patients correctly regarding response status in the training and test groups respectively. This criterion was shown to perform better than the CLMTF criterion in predicting survival. Conclusion: A new criterion for response to therapy at 6 months, also presented in a nomogram, combines M-component change and haemoglobin level at 6 months.     

The kinetics of immunoglobulin synthesis by pokeweed mitogen stimulated peripheral blood lymphocytes in multiple myeloma.

As a means of investigating the immunoglobulin (Ig) deficiency in multiple myeloma, the effects of pokeweed mitogen (PWM) on morphologic transformation and concomitant immunoglobulin synthesis and secretion kinetics were studied in peripheral blood lymphocytes (PBL) of normals and patients with myeloma. Lymphocyte Ig production kinetics, both in the unstimulated state and after five days of culture with PWM, were evaluated by 3H-leucine incorporation. B-lymphocytes were found to be decreased by EC3 rosetting in the myeloma patients studied. In addition their lymphocytes had a mean of 30% transformation compared to 53% for normals (P less than 0.005). After five days of mitogen stimulation in culture, myeloma PBL failed to show the rise in intracellular Ig production seen in normal PBL under similar conditions. Thus, myeloma patients were found to have decreased numbers of EC3 rosetting B-cells, decreased morphologic transformation rate in response to mitogenic stimulation, and failure to increase intracellular Ig after mitogenic stimulation. Possible etiologic mechanisms for the decreased Ig production are discussed.     

Effect of interferon-α on immunoglobulin production by peripheral blood mononuclear cells in multiple myeloma

Abstract: To test a hypothesis that interferon-α (IFN) treatment might restore normal immunoglobulin (Ig) production in multiple myeloma (MM), the effect of IFN on Ig isotype (IgG and IgA) production by peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) in MM patients was analyzed by ELISA. IFN at a concentration of 1000 U/ml was found to enhance IgA production by PB MNCs in IgA-MM and had a trend to stimulate IgG production in IgG-MM. The effect of IFN on nonparaprotein Ig isotype production was more variable, with mostly neutral or inhibitory effects being seen in both the MM subtypes. In contrast to the influences observed in MM patients, IFN at the same concentration inhibited both IgG and IgA production by PB MNCs in healthy controls. In studying BM cells, IFN was found to reduce IgA production in IgA-MM, but had a neutral effect on IgG production in IgG-MM. In the controls, the production of both the IgG and the IgA isotypes by BM MNCs was decreased by IFN. On the basis of these results it seems that the disease itself somehow affects the Ig-producing cells in MM, when measured as different responses of the cells to exogenous IFN in vitro. The results do not support the hypothesis that IFN treatment could restore normal Ig production in MM patients.     

Detection of t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma by double-color fluorescent in situ hybridization.

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), variable chromosome partners have been identified by conventional cytogenetics, including the 11q13, 8q24, 18q21, and 6p21 loci. We and others have recently reported a novel, karyotypically undetectable chromosomal translocation t(4;14)(p16. 3;q32) in MM-derived cell lines, as well as in primary tumors. The 4p16.3 breakpoints are relatively scattered and located less than 100 kb centromeric of the fibroblast growth factor receptor 3 (FGFR3) gene or within the recently identified WHSC1 gene, both of which are apparently deregulated by the translocation. To assess the frequency of the t(4;14)(p16.3;q32) translocation in MM, we performed a double-color fluorescent in situ hybridization (FISH) analysis of interphase nuclei with differently labeled probes specific for the IGH locus (a pool of plasmid clones specific for the IGH constant regions) or 4p16.3 (yeast artificial chromosome (YAC) 764-H1 spanning the region involved in breakpoints). Thirty MM patients, the MM-derived cell lines KMS-11 and OPM2, and six normal controls were examined. The identification of a t(4;14) translocation, evaluated as the presence of a der(14) chromosome, was based on the colocalization of signals specific for the two probes; a cutoff value of 15% (mean + 3 standard deviation [SD]) derived from the interphase FISH of the normal controls (range, 5% to 11%; mean +/- SD, 8.16 +/- 2.2) was used for the quantification analysis. In interphase FISH, five patients (one in clinical stage I, two in stage II, one in stage III, and a plasma cell leukemia) were found to be positive (approximately 15%). FISH metaphases with split or colocalized signals were detected in only two of the translocated cases and confirmed the pattern found in the interphase nuclei. Furthermore, in three of the five cases with the translocation, FISH analysis with the IGH joining probe (JH) showed the presence of the reciprocal product of the translocation [der(4) chromosome]. Overall, our study indicates that the t(4;14)(p16. 3;q32) chromosomal translocation is a recurrent event in MM tumors and may contribute towards the detection of this lesion and our understanding of its pathogenetic and clinical implications in MM.     

Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21.

PTPG, the gene for protein-tyrosine phosphatase gamma (PTP gamma), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP gamma allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP gamma allele was lost in three lung tumors that had not lost flanking loci. PTP gamma mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP gamma gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.     

Pamidronate is superior to ibandronate in decreasing bone resorption,interleukin-6 and beta 2-microglobulin in multiple myeloma

OBJECTIVES: Bisphosphonates have been found to reduce skeletal events in patients with multiple myeloma (MM). This is the first randomised trial to compare the efficacy of pamidronate and ibandronate, a third-generation aminobisphosphonate, in bone turnover and disease activity in MM patients. METHODS: Patients with MM, stage II or III, were randomly assigned to receive either pamidronate 90 mg (group I: 23 patients) or ibandronate 4 mg (group II: 21 patients) as a monthly intravenous infusion in addition to conventional chemotherapy. Skeletal events, such as pathologic fractures, hypercalcaemia, and bone radiotherapy were analysed. Bone resorption markers [N-terminal cross-linking telopeptide of type-I collagen (NTX) and tartrate-resistant acid phosphatase type 5b (TRACP-5b)], bone formation markers (bone alkaline phosphatase and osteocalcin), markers of disease activity (paraprotein, CRP, beta 2-microglobulin), and interleukin-6 (IL-6) were also studied. RESULTS: In both groups, the combination of chemotherapy with either pamidronate or ibandronate produced a reduction in bone resorption and tumour burden as measured by NTX, IL-6, paraprotein, CRP, and beta 2-microglobulin from the second month of treatment, having no effect on bone formation. TRACP-5b also had a significant reduction in the pamidronate group from the second month of treatment and in the ibandronate group from the sixth month. However, there was a greater reduction of NTX, IL-6, and beta 2-microglobulin in group I than in group II, starting at the second month of treatment (P = 0.002, 0.001, and 0.004, respectively) and of TRACP-5b, starting at the fourth month (P = 0.014), that being continued throughout the 10-month follow-up of this study. There was no difference in skeletal events during this period. A significant correlation was observed between changes of NTX and changes of TRACP-5b, IL-6, and beta 2-microglobulin from the second month for patients of both groups. CONCLUSIONS: These results suggest that a monthly dose of 90 mg of pamidronate is more effective than 4 mg of ibandronate in reducing osteoclast activity, bone resorption, IL-6, and possibly tumour burden in MM. TRACP-5b has also proved to be a useful new marker for monitoring bisphosphonates treatment in MM.