S. Van Wier

Genetic aberrations and survival in plasma cell leukemia

Plasma cell leukemia (PCL) is an aggressive and rare hematological malignancy that originates either as primary disease (pPCL) or as a secondary leukemic transformation (sPCL) of multiple myeloma (MM). We report here the genetic aberrations and survival of 80 patients with pPCL or sPCL and make comparisons with 439 cases of MM. pPCL presents a decade earlier than sPCL (54.7 vs 65.3 years) and is associated with longer median overall survival (11.1 vs 1.3 months; P<0.001). 14q32 (IgH) translocations are highly prevalent in both sPCL and pPCL (82–87%); in pPCL IgH translocations almost exclusively involve 11q13 (CCND1), supporting a central etiological role, while in sPCL multiple partner oncogenes are involved, including 11q13, 4p16 (FGFR3/MMSET) and 16q23 (MAF), recapitulating MM. Both show ubiquitous inactivation of TP53 (pPCL 56%; sPCL 83%) by coding mutation or 17p13 deletion; complemented by p14ARF epigenetic silencing in sPCL (29%). Both show frequent N-RAS or K-RAS mutation. Poor survival in pPCL was predicted by MYC translocation (P=0.006). Survival in sPCL was consistently short. Overall pPCL and sPCL are different disorders with distinct natural histories, genetics and survival.

Prognostic value of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma

A specific role for increased level of expression of CKS1B, as a consequence of chromosome 1q21 copy number gain, has been postulated as both pathogenic, as well as a powerful clinical prognostic factor in multiple myeloma (MM). The purpose of this study is to determine the clinical associations and prognostic impact of copy number gain at chromosome 1q21 (with a bacteria artificial chromosome clone containing CKS1B) and CKS1B gene level of expression in MM. We studied the chromosome region 1q21 for copy number change in a cohort of myeloma patients treated by high-dose therapy with stem-cell rescue (HDT) (n=159). A separate cohort of patients, treated by HDT was studied for CKS1B messenger RNA expression by gene expression profiling (n=67). 1q21 gain was then correlated with clinical parameters and survival. Gain of 1q21 copy number was detected in about a third of MM and was associated with more proliferative disease and poor-risk cytogenetic categories such as t(4;14), and chromosome 13 deletion. Both 1q21 gain and increase gene expression level were significantly associated with reduced survival. However, neither is an independent prognostic marker in MM on multivariate Cox proportional hazard analysis.

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.

Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations

Chromosomal hyperdiploidy is the defining genetic signature in 40–50% of myeloma (MM) patients. We characterize hyperdiploid-MM (H-MM) in terms of its clinical and prognostic features in a cohort of 220 H-MM patients entered into clinical trials. Hyperdiploid-myeloma is associated with male sex, kappa immunoglobulin subtype, symptomatic bone disease and better survival compared to nonhyperdiploid-MM (median overall survival 48 vs 35 months, log-rank P=0.023), despite similar response to treatment. Among 108 H-MM cases with FISH studies for common genetic abnormalities, survival is negatively affected by the existence of immunoglobulin heavy chain (IgH) translocations, especially those involving unknown partners, while the presence of chromosome 13 deletion by FISH did not significantly affect survival (median overall survival 50 vs 47 months, log-rank P=0.47). Hyperdiploid-myeloma is therefore a unique genetic subtype of MM associated with improved outcome with distinct clinical features. The existence of IgH translocations but not chromosome 13 deletion by FISH negatively impacts survival and may allow further risk stratification of this population of MM patients.

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.

Promiscuous MYC locus rearrangements hijack enhancers but mostly super-enhancers to dysregulate MYC expression in multiple myeloma.

MYC locus rearrangements—often complex combinations of translocations, insertions, deletions and inversions—in multiple myeloma (MM) were thought to be a late progression event, which often did not involve immunoglobulin genes. Yet, germinal center activation of MYC expression has been reported to cause progression to MM in an MGUS (monoclonal gammopathy of undetermined significance)-prone mouse strain. Although previously detected in 16% of MM, we find MYC rearrangements in nearly 50% of MM, including smoldering MM, and they are heterogeneous in some cases. Rearrangements reposition MYC near a limited number of genes associated with conventional enhancers, but mostly with super-enhancers (e.g., IGH, IGL, IGK, NSMCE2, TXNDC5, FAM46C, FOXO3, IGJ, PRDM1). MYC rearrangements are associated with a significant increase of MYC expression that is monoallelic, but MM tumors lacking a rearrangement have biallelic MYC expression at significantly higher levels than in MGUS. We also have shown that germinal center activation of MYC does not cause MM in a mouse strain that rarely develops spontaneous MGUS. It appears that increased MYC expression at the MGUS/MM transition usually is biallelic, but sometimes can be monoallelic if there is an MYC rearrangement. Our data suggest that MYC rearrangements, regardless of when they occur during MM pathogenesis, provide one event that contributes to tumor autonomy.

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

6q deletion in Waldenström macroglobulinemia is associated with features of adverse prognosis

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenström macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q−) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M‐FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of β2‐microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q− among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series.

Correlation between array-comparative genomic hybridization-defined genomic gains and losses and survival: identification of 1p31-32 deletion as a prognostic factor in myeloma

In this study, we correlated array-comparative genomic hybridization-defined abnormalities with survival in two different cohorts of patients treated with therapy based on high-dose melphalan with autologous stem-cell transplantation (64 from the Mayo Clinic and 67 from the University of Arkansas Medical School) and identified that several regions of genomic gains and losses were significantly associated with poorer survival. Three noncontiguous survival relevant regions covering 1p31-33 and two noncontiguous regions covering 20p12.3-12.1 were common between the two datasets. The prognostic relevance of these hotspots was validated in an independent cohort using fluorescent in situ hybridization, which showed that 1p31-32 loss is significantly associated with shorter survival (24.5 months versus 40 months, log-rank P-value=0.01), whereas 20p12 loss has a trend toward shorter survival (26.3 months versus 40 months, log-rank P-value=0.06). On multivariate analysis, 1p31-32 loss is an independent prognostic factor. On further analysis, the prognostic impact of 1p31-32 loss is due to shortening of post-relapse survival as there is no impact on complete response rates and progression-free survival.

RELATIONSHIP BETWEEN ELEVATED IMMUNOGLOBULIN FREE LIGHT CHAIN AND THE PRESENCE OF IgH TRANSLOCATIONS IN MULTIPLE MYELOMA

Elevated immunoglobulin free light chain (FLC) level and abnormal FLC ratio are commonly seen in multiple myeloma (MM) and have prognostic implications. We hypothesized that presence of immunoglobin heavy chain (IgH) translocations leads to unbalanced production of light chains and more extreme abnormalities of FLC, and may explain the prognostic value of FLC. We studied 314 patients with newly diagnosed MM enrolled in a phase III trial, in whom results of fluorescence in situ hybridization testing and data on serum FLC levels were available. Cytogenetic analyses and FLC estimates were performed on stored samples and results were correlated with clinical data. The median ratio (FLC ratio) and the absolute difference (FLC diff) between the involved and uninvolved FLC were higher among those with IgH translocations, especially t(14;16). In multivariate analysis, the prognostic value of FLC estimates on progression-free and overall survival were independent of high-risk IgH translocations t(4;14) and t(14;16). A combination of the risk factors; either abnormal FLC estimate and/or the presence of high-risk IgH translocation, achieved better prognostic stratification. We conclude that patients with IgH translocations have higher FLC levels and abnormal ratios, but the prognostic effect of FLC is only partially explained by translocation status. A system including both these risk factors allows better prediction of outcome.