B. Van Ness

International Myeloma Working Group molecular classification of multiple myeloma: spotlight review

Myeloma is a malignant proliferation of monoclonal plasma cells. Although morphologically similar, several subtypes of the disease have been identified at the genetic and molecular level. These genetic subtypes are associated with unique clinicopathological features and dissimilar outcome. At the top hierarchical level, myeloma can be divided into hyperdiploid and non-hyperdiploid subtypes. The latter is mainly composed of cases harboring IgH translocations, generally associated with more aggressive clinical features and shorter survival. The three main IgH translocations in myeloma are the t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Trisomies and a more indolent form of the disease characterize hyperdiploid myeloma. A number of genetic progression factors have been identified including deletions of chromosomes 13 and 17 and abnormalities of chromosome 1 (1p deletion and 1q amplification). Other key drivers of cell survival and proliferation have also been identified such as nuclear factor- B-activating mutations and other deregulation factors for the cyclin-dependent pathways regulators. Further understanding of the biological subtypes of the disease has come from the application of novel techniques such as gene expression profiling and array-based comparative genomic hybridization. The combination of data arising from these studies and that previously elucidated through other mechanisms allows for most myeloma cases to be classified under one of several genetic subtypes. This paper proposes a framework for the classification of myeloma subtypes and provides recommendations for genetic testing. This group proposes that genetic testing needs to be incorporated into daily clinical practice and also as an essential component of all ongoing and future clinical trials.

Deletions of chromosome 13 in multiple myeloma identified by interphase FISH usually denote large deletions of the q arm or monosomy

Deletions of the long arm of chromosome 13 (13q−) are observed in patients with multiple myeloma (MM), are rarely observed in the monoclonal gammopathy of undetermined significance (MGUS) and have been associated with a worsened prognosis in MM. However, no minimally deleted region in the13q arm has been defined at 13q, and consequently no tumor suppressor genes have yet been identified that are important for disease pathogenesis. We attempted to characterize these chromosome 13q deletions at the molecular cytogenetic level. We studied 351 newly diagnosed patients, entered into the E9486/E9487 clinical study of the Eastern Cooperative Oncology Group. Fluorescent in situ hybridization (FISH) combined with immune fluorescent detection (cIg-FISH) of clonal plasma cells (PC) and cytomorphology were used to analyze interphase, bone marrow (BM) cell, cytospin slides. We simultaneously used DNA probes for the following locus specific probes (LSI); LSI 13 (Rb) and D13S319, which hybridize to 13q14. We subsequently studied distal deletions using the D13S25 probe (13q14.3) and a subtelomeric probe (13qSTP) for the 13q-arm (D13S327) in 40 cases with documented LSI 13 (Rb)/D13S319 deletion and 40 without deletion of these loci. Of 325 evaluable patients, we found 13q deletions in 176 (54%) using LSI 13 (Rb) and D13S319 probes. Of 40 patients with LSI 13 (Rb)/D13S319 deletions, 34 (85%) had coexistent deletion of both D13S25/13qSTP. These results indicate that chromosome 13 deletions in MM involve loss of most if not all of the 13q arm perhaps even indicating monosomy. In six cases the 13qSTP signal was conserved, but D13S25 was lost indicating large interstitial deletions involving 13q14. In 39 of the 40 cases without LSI 13 (Rb)/D13S319 deletions, the normal pattern of two pairs of signals was observed for D13S25/13qSTP. Deletions involving 13q14 are very common in MM as detected by cIg-FISH. These deletions appear to predominantly involve loss of large segments of the 13q arm or monosomy 13, and only occasionally represent an interstitial deletion.

Clinical and biological significance of RAS mutations in multiple myeloma

Primary genetic abnormalities in myeloma (MM) such as trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21 associated with hyperdiploid MM and translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 and three main recurrent partners: MMSET/FGFR3, CCND1 and c-MAF are already present in the pre-malignant monoclonal gammopathy of undetermined significance (MGUS) stage.1 Some patients with these genetic abnormalities may remain as MGUS for many years without transforming to MM, suggesting that they are involved in clonal initiation but do not mediate malignant transformation.

Clinical significance of TP53 mutation in myeloma

The p53 tumor suppressor is a critical regulator of tissue homeostasis, and its inactivation at the gene or protein level confers cellular properties conducive for oncogenesis and cancer progression. Furthermore, p53 inactivation has been associated with resistance to therapy. Indeed, the p53 response is deficient in 450% of cancers mainly through gene mutation. In contrast to other solid tumors and carcinomas, TP53 mutations are rare in multiple myeloma (MM), a malignancy characterized by clonal plasma cells secreting monoclonal immunoglobulin. Previous studies of TP53 mutations in MM were hampered by clinical heterogeneity in the study cohorts and the relatively small sample size (all with o100 patients). These studies reported a prevalence of TP53 mutation ranging from 0 to 20%. However, it is not always obvious whether the study cohorts consist of newly diagnosed or relapsed patients. This is important as the prevalence of TP53 mutations increases with more advance disease (even this is not clearly defined and seemed to include Durie–Salmon stage III and plasma cell leukemia) and is very prevalent in HMCLs. Furthermore, these studies generally limit their investigation to exons 5–9, whereas several studies in other cancers have shown that mutations can occur in other exons. At present, the prognostic importance of TP53 mutations in myeloma is unknown. In this study, we comprehensively define the prevalence of TP53 mutations in newly diagnosed myeloma patients by screening genomic DNA from unsorted whole bone marrow from a large cohort of patients entered into an Eastern Cooperative Oncology Group clinical trial E9486/E9487 (n1⁄4 561) using conformation sensitive gel electrophoresis (CSGE). A total of 268 patients, based on sample availability, were included in our current study. These patients had extensive follow-up information with median follow-up of survivors 4.8 years and only 4.5% (n1⁄4 25) of the cohort alive at the time of our analysis, resulting in negligible censoring. Fluorescent in situ hybridization (FISH) studies using the cytoplasmic immunoglobulin-FISH technique in this cohort of patients has been previously reported. Polymerase chain reaction primers were designed to amplify 11 DNA fragments from exon 1 to 11 (exon 1: CCA TGT GCT CAA GAC TGG C, CGA GCT GAA AAT ACA CGG AG; exon 2: CAG GAG TGC TTG GGT TGT, CCC ACA GGT CTC TGC TAG G; exon 3: CTG TGG GAA GCG AAA AT, GAT GGG TGA AAA GAG CAG TCA; exon 4: GGG CTG AGG ACC TGG T, ACA GGA AGC CTA AGG GTG AAG; exon 5: TTG CTG CCG TGT TCC A, CAA CCA GCC CTG TCG TCT CT; exon 6: GGC TGG AGA GAC GAC AGG G, ATC TCA TGG GGT TAT AGG GAG; exon 7: TTG CCA CAG GTC TCC C, ATG GAA GAA ATC GGT AAG AG; exon 8: TTT AAA TGG GAC AGG TAG GAC, CTT ACC TCG CTT AGT GCT; exon 9: GGG AGC ACT AAG CGA GGT A, CAA CCA GGA GCC ATT GTC TTT; exon 10: TTG CTT TTG TAC CGT CAT AA, ACA GCT GCC TTT GAC CAT; exon 11: GCA CAG ACC CTC TCA CTC ATG TGA, AGA CCC AAA ACC CAA AAT G). The primers were optimized and grouped into three multiplex reactions. These groups had to be compatible according to primer length, MgCl2 concentration and annealing temperature. Multiplex one contained exons 4, 7, 8 and 10; multiplex two contained exons 1, 3, 5 and 9; and multiplex three contained exons 2, 6, 7 and 11. The radiolabeled amplicons were run through 15% mild denaturing 0.4 mm polyacrylamide gel for 4 h at 40 W. The gel was dried and placed on a photoimager screen for analysis. Abnormal banding patterns were subsequently directly sequenced. It appears that TP53 mutations are relatively rare in newly diagnosed patients as only nine of the 268 samples (3%) tested were positive for mutations. The actual prevalence may be slightly higher if one considers the sensitivity of CSGE (10%) and the fact that non-purified bone marrow samples are used (bone marrow samples were all collected before the availability of practical CD138þ cell sorting). However, the median plasma cell infiltration of the cohort is 40% (range 20–95%) with 70% of patients having more than 30% of plasma cells in the bone marrow. Only three of the mutations are point mutations, and majority resulted in premature termination and a predicted truncated protein product (Table 1). Seven of the nine mutations occurred in the DNA-binding domain, where the majority of reported TP53 mutations occur. However, we did not see any mutations in codons 175, 245, 248, 249, 273 and 282 that accounts for 28% of all TP53 mutations in cancers. In addition, we found several mutations outside exons 5–9 (all previous studies only examined exons, 5–9). None of the mutations are known polymorphism and all of them are predicted to alter protein structure and function. Therefore, the spectrum of TP53 mutations is broad and not typical of other malignancies. We also examined the association between the presence of TP53 mutations and other clinical features and common genetic abnormalities using the Fisher’s exact test. The presence of TP53 mutations was associated with presence of soft tissue plasmacytoma (37 versus 7%, P1⁄4 0.018). Unlike previous studies that found TP53 mutation to be more common in advance stage MM, we could not confirm this because the distribution of International Staging System (ISS) stage is relatively even, although clearly the total number of patients with an abnormality is quite small to draw firm conclusions. As our analysis was conducted in newly diagnosed and pretreatment samples, it provides a better baseline for examining the relation between TP53 mutation and disease stage. The presence of TP53 mutations was significantly associated with 17p13 deletions as five of the nine patients (56%) with mutation also had 17p13 hemizygous loss (versus 10%, P1⁄4 0.01). This is consistent with previous observations in other malignancies that many tumors that harbor TP53 mutations also show loss of heterozygosity. In those patients with only 17p13 hemizygous loss, it would be important to assess how the p53 pathway is affected and whether the other allele of p53 is suppressed by other means such as epigenetic mechanisms. These studies are currently being conducted in our laboratory. Patients with TP53 mutations were also enriched for primary translocations, such as t(11;14), t(4;14) and t(14;16) (67 versus 24%, P1⁄4 0.014). In contrast, there was no association with D13 or hyperdiploid status. We also report for the first time the prognostic significance of TP53 mutations. The presence of TP53 mutations was associated with very poor survival of only one and a half years (Figure 1). We have previously reported the short survival associated with 17p13 deletions in this cohort of patients. We did not report on the difference in outcome for those with 17p13 deletions and Letters to the Editor

Distinct IL-6 signal transduction leads to growth arrest and death in B cells or growth promotion and cell survival in myeloma cells

In B cell development, interleukin-6 (IL-6) induces terminal maturation of B lymphocytes into antibody producing plasma cells. Terminal differentiated B cells cell cycle arrest and death follows. In contrast, IL-6 acts as a growth factor for malignant myeloma plasma cells and in some cases protects them from therapeutic treatment. In this study, we examined two cell lines that show different responses to IL-6. Lymphoblastoid CESS cells respond to IL-6 by terminally differentiating into antibody producing plasma cells, cell cycle arrest, and undergo cell death. Continuous addition of IL-6 to these cells induces transient activation of STAT3, SHP-2 phosphorylation, and does not alter bcl-XL and c-myc expression. In contrast, the myeloma line ANBL6 proliferates when stimulated with IL-6 and this correlates with prolonged STAT3 activation and up-regulation of bcl-XL and c-myc. Interestingly, gp130-associated SHP-2 phosphorylation was detected in the IL-6-induced CESS cells but not myeloma cell lines. The data show a very distinct IL-6 signal transduction and kinetics in these cell lines and the distinct molecular events correlate closely to the cell fate of the lymphoblast and myeloma cell lines.

Nonproductive kappa immunoglobulin genes: Recombinational abnormalities and other lesions affecting transcription, RNA processing, turnover, and translation

Six nonproductive kappa immunoglobulin genes (kappa- alleles) were cloned and sequenced. The structural abnormalities discerned from sequence analysis were correlated with functional lesions at the level of transcription, RNA processing, turnover, and translation. Four kappa- alleles, three containing V kappa genes and one not, are transcribed at normal or even greater than normal rates, the defects in these genes being expressed at various posttranscriptional levels. The other two kappa- alleles, both of which lacked V genes, exhibited greatly depressed yet clearly detectable transcriptional activity. These results are consistent with a hierarchical relationship between enhancer and promoter elements in which the enhancer establishes transcriptional competence at the kappa locus and the promoter (or pseudopromoter) determines the relative level of transcriptional activity. One of the structural abnormalities discovered in this study, a large deletion which removes the entire J kappa region, also provides new insight into the mechanism of VJ and VDJ recombination.

The bone marrow stromal microenvironment influences myeloma therapeutic response in vitro.

The bone marrow microenvironment supports growth and differentiation of normal hematopoietic cells and can contribute to malignant growth. Since myeloma cells localize and accumulate in bone marrow, it is important to understand the influence of the bone marrow microenvironment not only on the growth of the malignant cells, but also on the therapeutic response of myeloma cells. Growth factors such as interleukin-6 (IL-6) produced by bone marrow stromal cells can protect myeloma cells from glucocorticoid-induced apoptosis. We examined the effect of myeloma cells–bone marrow stromal cells interaction in vitro on several therapeutic treatments. An interleukin-6-dependent myeloma cell line ANBL6 was used and treated with dexamethasone, doxorubicin, and melphalan in the presence of bone marrow stromal cells. Stromal cells were able to protect ANBL6 from dexamethasone, but significantly enhanced the effect of doxorubicin and melphalan. IL-6-induced bcl-XL and cyclin D2 expression in ANBL6 cells, but dexamethasone was able to suppress both bcl-XL and cyclin D2 expression in ANBL6. Doxorubicin and melphalan were able to suppress bcl-XL expression only in the presence of IL-6. We also looked at the effect of activating mutations of N-ras in myeloma cells interacting with stromal cells on therapeutic responses. Surprisingly, ANBL6 N-ras shows significant resistance to all drugs used. Notably, the presence of stromal cells did not alter ANBL6 Nras cells’ drug resistance. These results suggest both the bone marrow microenvironment and genetic alterations of myeloma cells can independently impact on therapeutic responses.

Coordinate transcription and V(D)J recombination of the kappa immunoglobulin light-chain locus: NF-kappaB-dependent and -independent pathways of activation.

To further elucidate the potential role of mitogens and cytokines in regulation of the kappa immunoglobulin light-chain locus, we have characterized the activation of transcription factor binding, kappa germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination upon induction of model pre-B-cell lines. We find that both lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) are capable of activating germ line transcription, DNase I hypersensitivity, and recombination of the kappa locus. We also find that transforming growth factor beta is capable of completely inhibiting LPS activation of transcription and recombination but has no apparent effect on activation of transcription factor binding, including activation of NF-kappaB. To address the functional role of NF-kappaB in LPS and IFN-gamma induction of these events, we blocked the nuclear translocation of NF-kappaB by overexpression of a dominant negative mutant of IkappaB-alpha (IkappaB deltaN). Overexpression of the IkappaB deltaN protein results in an inhibition of LPS but not IFN-gamma activation of germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination. Our results demonstrate that activation of NF-kappaB is necessary but not sufficient for LPS activation of transcription and recombination at kappa. These results also suggest that NF-kappaB is not required for IFN-gamma activation of transcription or recombination. These results are important in establishing that there are multiple independent pathways of activation of both transcription and recombination.

Is flow cytometric DNA content hypodiploidy prognostic in multiple myeloma

Hypodiploid multiple myeloma is uncommon when assessed by DNA content flow cytometry, having been reported in less than 6% of patients with newly diagnosed multiple myeloma. Previous studies have shown these patients to be unresponsive to therapy and to have short survival. To address this further, we studied 349 of 504 patients eligible for Eastern Cooperative Oncology Group (ECOG) treatment trial E9486 and laboratory correlative study E9487 who had marrow mononuclear cells available for ploidy analysis. Marrow samples were studied by dual channel flow cytometry, using propidium iodide to measure the DNA content and kappa and lambda light chain antisera to identify the clonal cells. A DNA index < 0.95 was considered hypodiploid. Five patients (1.4%) were found to have hypodiploid DNA content in their marrow plasma cells. Three of the 5 patients with hypodiploid myeloma had a partial objective response to chemotherapy, which is not different from the overall objective response rate for all patients enrolled on E9486. All five patients with hypodiploid multiple myeloma died within 4 years from diagnosis, but these patients had a similar overall median survival (2.6 years) compared to the patients with diploid DNA content. Our studies confirm the poorer survival of patients with diploid versus hyperdiploid myeloma; we cannot confirm, however, the previously reported very poor outcome associated with hypodiploid myeloma using DNA content flow cytometry. Hypodiploid DNA content of plasma cells by flow cytometry may not be as ominous a factor as previously reported.

Interferon-α protects myeloma cell lines from dexamethasone-induced apoptosis

Because of the increasing use of IFN-αin both induction and maintenance therapy for multiple myeloma (MM), its effect on growth and apoptosis of myeloma cells is important to consider. To investigate the role of IFN-αon the growth of myeloma cells, we have studied its effects on the response of interleukin 6 (IL-6)-dependent myeloma cell line (ANBL6) and IL-6-independent myeloma cell line (C2E3) in the presence of IL-6 and dexamethasone (Dex). We found that although IFN-αis a potent inhibitor of proliferation, it has only a minimal effect on induction of apoptosis. Moreover, we found IFN-α as well as IL-6 can significantly suppress dexamethasone-induced apoptosis. The suppression of apoptosis is concurrent with the induction of both AP-1 and STAT binding activity. We also found that IL-6 but not IFN-αup-regulates Bcl-XL expression. However, IL-6-mediated Bcl-XL expression is suppressed in the presence of Dex. Therefore, the expression of Bcl-XL does not account for the protection of Dex-induced apoptosis by IFN-αand IL-6. Taken together, our results suggest that IFN-αmay counteract the beneficial effects of corticosteroids or perhaps other apoptosis inducing agents in the treatment of myeloma.