Kimberly J. Henderson

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.

Appraisal of immunoglobulin free light chain as a marker of response

The immunoglobulin free light chain (FLC) assay is an invaluable tool for following patients with oligosecretory plasma cell dyscrasia. Baseline values have also been shown to be prognostic in all plasma cell disorders tested. A looming question, however, is the role it should play in following myeloma patients with disease that is measurable using serum and urine electrophoresis. We used the data and stored samples from a mature Eastern Cooperative Oncology Group clinical trial (E9486) to assess serum levels of FLC at baseline and after 2 months of alkylator-based therapy. For serial determinations, the absolute level of involved serum FLC or the difference of the involved and uninvolved FLC is preferred over the ratio of involved to uninvolved FLC. FLC response after 2 months of therapy was superior to early M-protein measurement to predict overall response. The ideal cut-point for FLC change appears to be between 40% and 50% reduction. The correlation between serial measurements of serum FLC and urine M-protein is inadequate to abolish the serial 24-hour urine protein. Although baseline values of FLC are prognostic in newly diagnosed myeloma patients, serial measurements do not appear to have added value in patients who have M-proteins measurable by electrophoresis.

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.

Identification of Copy Number Abnormalities and Inactivating Mutations in Two Negative Regulators of Nuclear Factor-κB Signaling Pathways in Waldenström's Macroglobulinemia

Waldenströms macroglobulinemia (WM) is a distinct clinicobiological entity defined as a B-cell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow (BM) and IgM paraprotein production. Cytogenetic analyses were historically limited by difficulty in obtaining tumor metaphases, and the genetic basis of the disease remains poorly defined. Here, we performed a comprehensive analysis in 42 WM patients by using a high-resolution, array-based comparative genomic hybridization approach to unravel the genetic mechanisms associated with WM pathogenesis. Overall, 83% of cases have chromosomal abnormalities, with a median of three abnormalities per patient. Gain of 6p was the second most common abnormality (17%), and its presence was always concomitant with 6q loss. A minimal deleted region, including MIRN15A and MIRN16-1, was delineated on 13q14 in 10% of patients. Of interest, we reported biallelic deletions and/or inactivating mutations with uniparental disomy in tumor necrosis factor (TNF) receptor-associated factor 3 and TNFalpha-induced protein 3, two negative regulators of the nuclear factor-kappaB (NF-kappaB) signaling pathway. Furthermore, we confirmed the association between TRAF3 inactivation and increased transcriptional activity of NF-kappaB target genes. Mutational activation of the NF-kappaB pathway, which is normally activated by ligand receptor interactions within the BM microenvironment, highlights its biological importance, and suggests a therapeutic role for inhibitors of NF-kappaB pathway activation in the treatment of WM.

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

Impact of gene expression profiling-based risk stratification in patients with myeloma receiving initial therapy with lenalidomide and dexamethasone

Detection of specific chromosomal abnormalities by FISH and metaphase cytogenetics allows risk stratification in multiple myeloma; however, gene expression profiling (GEP) based signatures may enable more specific risk categorization. We examined the utility of 2 GEP-based risk stratification systems among patients undergoing initial therapy with lenalidomide in the context of a phase 3 trial. Among 45 patients studied at baseline, 7 (16%) and 10 (22%), respectively, were high-risk using the GEP70 and GEP15 signatures. The median overall survival for the GEP70 high-risk group was 19 months versus not reached for the rest (hazard ratio = 14.1). Although the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk patients. The C-statistic for the GEP70, GEP15, and FISH based risk stratification systems was 0.74, 0.7, and 0.7, respectively. Here we demonstrate the prognostic value for GEP risk stratification in a group of patients primarily treated with novel agents. This trial was registered at www.clinicaltrials.gov as #NCT00098475.

Impact of high-risk classification by FISH: An Eastern Cooperative Oncology Group (ECOG) study E4A03

Lenalidomide with dexamethasone is a standard induction treatment regimen for newly diagnosed myeloma (although a Federal Drug Administration indication is still absent). In the context of the Phase 3 clinical trial E4A03 (lenalidomide plus dexamethasone in low or high doses), we queried whether a fluorescence in situ hybridization (FISH)‐based genetic classification into high risk (HR) and standard risk (SR) multiple myeloma (MM) would remain clinically significant. Of 445 E4A03 patients, 126 had FISH analysis; 21 were classified HR with t(4;14), t(14;16), or 17p13 deletions. Median survival follow‐up approached 3 years. Patients with FISH data tended to be younger and healthier compared to the rest of the study population and, consequently, had superior overall survival (OS) results. Within the FISH cohort, shorter OS in the HR versus SR group (P = 0·004) corresponded to a hazard ratio of 3·48 [95% confidence interval: (1·42–8·53)], an effect also observed in multivariate analysis. Two‐year OS rates were 91% for SR MM and 76% for HR MM. There was also evidence of interaction between risk status and treatment (P = 0·026). HR patients were less likely to attain good partial response (SR 46% and HR 30%, Odds Ratio = 2·0 [0·7–5·6]), but overall response rates were not different. FISH‐based risk classification retained prognostic significance in patients receiving lenalidomide‐based induction.