Wee J. Chng

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.

Molecular Dissection of Hyperdiploid Multiple Myeloma by Gene Expression Profiling

Hyperdiploid multiple myeloma (H-MM) is the most common form of myeloma. In this gene expression profiling study, we show that H-MM is defined by a protein biosynthesis signature that is primarily driven by a gene dosage mechanism as a result of trisomic chromosomes. Within H-MM, four independently validated patient clusters overexpressing nonoverlapping sets of genes that form cognate pathways/networks that have potential biological importance in multiple myeloma were identified. One prominent cluster, cluster 1, is characterized by high expression of cancer testis antigen and proliferation-associated genes. Tumors from these patients were more proliferative than tumors in other clusters (median plasma cell labeling index, 3.8; P < 0.05). Another cluster, cluster 3, is characterized by genes involved in tumor necrosis factor/nuclear factor-kappaB signaling and antiapoptosis. These patients have better response to bortezomib as compared with patients within other clusters (70% versus 29%; P = 0.02). Furthermore, for a group of patients generally thought to have better prognosis, a cluster of patients with short survival (cluster 1; median survival, 27 months) could be identified. This analysis illustrates the heterogeneity within H-MM and the importance of defining specific cytogenetic prognostic factors. Furthermore, the signatures that defined these clusters may provide a basis for tailoring treatment to individual patients.

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.

Clinical and Biological Implications of MYC Activation: A common difference between MGUS and newly diagnosed multiple myeloma

Events mediating transformation from the pre-malignant monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) are unknown. We analyzed gene expression data sets generated on the Affymetrix U133 platform from 22 MGUS and 101 MM patients using gene-set enrichment analysis. Genes overexpressed in MM were enriched for cell cycle, proliferation and MYC activation gene sets. Upon dissecting the relationship between MYC and cell-cycle gene sets, we identified and validated an MYC activation signature dissociated from proliferation. Applying this signature, MYC is activated in 67% of myeloma, but not in MGUS. This was further confirmed by immunohistochemistry (IHC) using membrane CD138 and nuclear MYC double staining. We also showed that almost all tumors with RAS mutations expressed the MYC activation signature, and multiple mechanisms may be involved in activating MYC. MYC activation, whether assessed by gene-expression signature or IHC, is associated with hyperdiploid MM and shorter survival even in tumors that are not proliferative. Bortezomib treatment is able to overcome the survival disadvantage in patients with MYC activation.

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 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

IGF-1R is overexpressed in poor-prognostic subtypes of multiple myeloma.

Insulin growth factor-1 (IGF-1) is an important survival and growth factor in multiple myeloma (MM) and various other malignancies, and its receptor (IGF-1R) has been recently shown to be an ideal therapeutic target. Few studies have examined IGF-1R and IGF-1 expression in MM patients. To better understand the distribution of the receptor in myeloma, we decided to perform a detailed study of its expression using gene expression profiling. We investigated the mRNA expression of IGF-1 and IGF-1R in normal and malignant plasma cells (PC) as part of gene expression profiling studies in PC from 15 normal individuals and 147 patients with various PC neoplasm (22 monoclonal gammopathy of undetermined significance (MGUS), 24 smoldering MM (SMM) and 101 symptomatic MM). Of the 101 MM cases, 72 were newly diagnosed and 29 were relapsed cases. The samples were all collected under informed consent and the study was approved by the Mayo Clinic Institutional Review Board. Bone marrow aspirates were enriched for PCs using CD138þ immunomagnetic bead selection. Total RNA was extracted, converted to biotinylated cRNA and hybridized to

Type II EATL (epitheliotropic intestinal T-cell lymphoma): a neoplasm of intra-epithelial T-cells with predominant CD8αα phenotype

In this multicentre study, we examined 60 cases of Type II enteropathy-associated T-cell lymphoma (EATL) from the Asia-Pacific region by histological review, immunohistochemistry and molecular techniques. Patients were mostly adult males (median age: 58 years, male:female 2.6:1), presenting with abdominal pain (60%), intestinal perforation (40%) and weight loss (28%). None had a history of coeliac disease and the median survival was only 7 months. Histologically, these tumours could be divided into (i) central tumour zone comprising a monotonous population of neoplastic lymphocytes, (ii) peripheral zone featuring stunted villi and morphologically atypical lymphocytes showing epitheliotropism, and (iii) distant mucosa with normal villous architecture and cytologically normal intra-epithelial lymphocytes (IELs). Characterized by extensive nuclear expression of Megakaryocyte-associated tyrosine kinase (MATK) (87%) and usually a CD8+CD56+ (88%) cytotoxic phenotype, there was frequent aberrant expression of CD20 (24%). T-cell receptor (TCR) expression was silent or not evaluable in 40% but of the remainder, there was predominant expression of TCRαβ over TCRγδ (1.6:1). In keeping with the normal ratio of IEL subsets, CD8+ cases showed predominant CD8αα homodimer expression (77%), regardless of TCR lineage. These tumours constitute a distinct entity from classical EATL, and the pathology may reflect tumour progression from IEL precursors, remnants of which are often seen in the distant mucosa.

Translocation t(4;14) retains prognostic significance even in the setting of high-risk molecular signature

Translocation t(4;14) retains prognostic significance even in the setting of high-risk molecular signature

Analysis of genetic abnormalities provides insights into genetic evolution of hyperdiploid myeloma

Aneuploidy is ubiquitous in human cancer and is seen as whole chromosome gains and losses, unbalanced translocations and inversions, duplications, deletions and loss of heterozygosity. Within this complexity, some subgroups of aneuploid tumors emerge as distinct biological and clinical entities. Hyperdiploid myeloma (H‐MM), characterized by hyperdiploid chromosome numbers because of nonrandom trisomies, is one such example. We undertook a comprehensive survey of the karyotypes of a large number of H‐MM (n = 469) to describe fully genomic instability in these tumors, to dissect pathways of genetic evolution, and identify distinct subgroups based on their genetic changes. While selective pressure apparently favors the emergence of clones with gains of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, a background of ongoing genomic instability results in gains of other chromosomes, albeit at a much lower prevalence. A deduced temporal analysis of these karyotypes indicates that selected gains are early events. Other events occurring later in the course of the disease include secondary chromosome translocations and monosomies. The development of these genetic aberrations is thus highly ordered and undoubtedly of biological relevance. Within this framework, we propose a model of genetic evolution in H‐MM.