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Supplementary Table 2 from Gender Disparities in the Tumor Genetics and Clinical Outcome of Multiple Myeloma

Supplementary Table 3 from Gender Disparities in the Tumor Genetics and Clinical Outcome of Multiple Myeloma

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Supplementary Methods, References, Legends for Table 1 and Figure 1 from Gender Disparities in the Tumor Genetics and Clinical Outcome of Multiple Myeloma

Purpose: Regions on 1p with recurrent deletions in presenting myeloma patients were examined with the purpose of defining the deletions and assessing their survival impact.Experimental Design: Gene mapping, gene expression, FISH, and mutation analyses were conducted on patient samples from the MRC Myeloma IX trial and correlated with clinical outcome data.Results: 1p32.3 was deleted in 11% of cases, and deletion was strongly associated with impaired overall survival (OS) in patients treated with autologous stem cell transplant (ASCT). In patients treated less intensively, del(1)(p32.3) was not associated with adverse progression-free survival (PFS) or OS. The target of homozygous deletions was CDKN2C, however its role in the adverse outcome of cases with hemizygous deletion was less certain. 1p22.1-21.2 was the most frequently deleted region and contained the candidate genes MTF2 and TMED5. No mutations were identified in these genes. 1p12 was deleted in 19% of cases, and deletion was associated with impaired OS in univariate analysis. The target of homozygous deletion was FAM46C, which was mutated in 3.4% of cases. When cases with FAM46C deletion or mutation were considered together, they were strongly associated with impaired OS in the intensive treatment setting.Conclusion: Deletion of 1p32.3 and 1p12 was associated with impaired OS in myeloma patients receiving ASCT. FAM46C was identified as a gene with potential pathogenic and prognostic significance based on the occurrence of recurrent homozygous deletions and mutations. Clin Cancer Res; 17(24); 7776–84. ©2011 AACR.

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Supplementary Table 1 from Gender Disparities in the Tumor Genetics and Clinical Outcome of Multiple Myeloma

Introduction: Molecular diagnostic testing for lymphoproliferative disorders (LPDs) includes detection of clonal immunoglobulin (IG) and/or T cell receptor (TCR) rearrangements, translocations, copy number alterations (CNA) and somatic mutations. To date, laboratories still rely on subjective and labour-intensive technologies, e.g. karyotyping/FISH to detect complex genomic alterations. Whole Genome Sequencing (WGS) can detect all genomic alterations, but factors such as cost, computation/storage, DNA requirements and poor detection of clinically relevant subclonal mutations limits the routine implementation of WGS in clinical diagnostics. We have developed "KaryoSequencing" (KS), a novel approach that combines targeted deep-sequencing, using a targeted hybridisation capture NGS panel, for rare mutation and translocation detection with shallow WGS (sWGS) for genome wide copy number analysis, in a single test. Methods: KS was validated using 138 clinical samples from patients with acute lymphoblastic leukaemia (ALL) (n=46), chronic lymphocytic leukaemia (CLL) (n=46) and plasma cell myeloma (PCM) (n=46) samples from 3 UK laboratories. Samples underwent library preparation and hybridisation using the EuroClonality-NDC assay. A KS library for each sample was prepared by combining the pre-capture and post-capture libraries at an optimised ratio to enable high coverage (>500 x) at regions covered by the targeted panel while providing 0.5-1x coverage genome-wide. Forty-six KS libraries were pooled and sequenced per NovaSeq 6000 run, using a 200-cycle SP flow cell and a 100bp paired-end strategy. For analysis of targeted regions, somatic mutation calling was performed using VarDict and structural variants (rearrangements and translocations) were detected using ARResT/Interrogate. For analysis of large chromosomal copy number variation using sWGS, a modified version of QDNASeq/ACE with a window size of 50kb was performed. For sWGS analysis, bioinformatic exclusion of all target capture regions and panel-specific off-target regions was performed using a panel of 48 DNA samples from healthy individuals ran on the same KS protocol. Results: Analysis of genome-wide copy number by sWGS was concordant in 477 of 503 (95%) evaluable FISH tests, including precise detection of hyper and hypo-diploidy and other complex karyotypes. The performance of the targeted deep-sequencing component of the KS approach was assessed to ensure comparable performance to previously validated results for the EuroClonality-NDC. The EuroClonality-NDC detected a clonal IG rearrangement in 46/46 (100%) CLL cases and in 42/46 (91.3%) PCM cases. Clonality was detected in 43/46 (93.5%) and 44/46 (95.7%) cases at an IG and TCR locus respectively in ALL, a disease entity known to exhibit cross-lineage rearrangements. Overall, 44 translocations were detected in the 138 samples by the EuroClonality-NDC. FISH results were available for 32 of the 44 translocations detected by NGS and were concordant in 29/32 (91%). In two PCM samples NGS and FISH reported a different translocation; in the ALL sample, the FISH pattern showed an additional copy but not a split signal. Across the entire cohort of 138 samples, KaryoSequencing detected 190 mutations with a variant allele frequency ranging from 4.0 - 97.6%. Of the detected mutations, 69/190 (36.3%) had a VAF Conclusions: KaryoSequencing demonstrated >95% sensitivity and specificity for detection of gross genome-wide copy number alterations while retaining the analytical performance for detection of rearrangements, translocations, and mutations for the lymphoid-specific targeted regions of the EuroClonality-NDC assay. KS is a cost-effective and high-throughput integrated alternative to current diagnostic strategies in haematological malignancies and can be implemented in routine clinical practice. Disclosures Jenner: Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy; Takeda: Consultancy; BMS/Celgene: Consultancy, Honoraria, Speakers Bureau. Gonzalez: Univ8 Genomics Ltd: Current holder of individual stocks in a privately-held company.

Risk stratification in myeloma requires an accurate assessment of the presence of a range of molecular abnormalities including the differing IGH translocations and the recurrent copy number abnormalities that can impact clinical behavior. Currently, interphase fluorescence in situ hybridization is used to detect these abnormalities. High failure rates, slow turnaround, cost, and labor intensiveness make it difficult and expensive to use in routine clinical practice. Multiplex ligation-dependent probe amplification (MLPA), a molecular approach based on a multiplex polymerase chain reaction method, offers an alternative for the assessment of copy number changes present in the myeloma genome. Here, we provide evidence showing that MLPA is a powerful tool for the efficient detection of copy number abnormalities and when combined with expression assays, MLPA can detect all of the prognostically relevant molecular events which characterize presenting myeloma. This approach opens the way for a molecular diagnostic strategy that is efficient, high throughput, and cost effective.

INTRODUCTION Features of high risk myeloma (MM) have been studied in detail but patients with longer term responses to first-line therapy are less well characterised. Identification of common features of this group may support optimised management. Here we analysed clinical and genetic characteristics of long-term responders of 4,249 trial patients from the UK MRC Myeloma IX (M-IX) and NCRI Myeloma XI (M-XI) trials. PATIENTS AND METHODS In M-IX patients were randomised between alkylating therapy (CVAD or MP) and thalidomide-based induction therapy (CTD). M-XI patients were randomised between thalidomide and lenalidomide based induction (CTD vs CRD) and a response-based bortezomib (CVD) intensification. Fitter patients received HD-Mel+ASCT consolidation. Patients were then randomised to thalidomide (M-IX) or lenalidomide (M-XI) maintenance or observation. Trials included symptomatic, newly diagnosed patients based on CRAB criteria. This analysis included 1,921 My-IX and 2,328 My-XI patients with median follow-up of 73 and 61 months (m), respectively. Genetic profiling was available for 1,866 patients. Patients with a long-term response post induction (PFS≥48m) were identified and their baseline characteristics, responses and treatment compared to those with PFS RESULTS In M-IX, 283 (25.8%) of transplant-eligible (TE) patients had PFS ≥48m whereas 58 (7%) of transplant non-eligible (TNE) patients reached PFS≥48m. In M-XI 410 (34.2%) patients had PFS≥48m for TE and 116 (10.2%) for TNE. Extended progression free survival translated to overall survival (OS) benefit with a median post progression OS of 36.9m for PFS≥48m vs 16.7m for PFS Clinical factors including ISS I (P The proportion of patients with a high risk lesion (Adverse translocation, Gain(1q) or Del(17p)) were lower in the PFS≥48m group than The majority of patients with PFS ≥48m showed ≥VGPR after induction +/- consolidation: 211 (76.4%) and 340 (84%) of PFS ≥48m patients in the TE arms and 26 (49.1%) and 87 (76.3%) in the TNE arms of M-IX and M-XI, respectively. 86.7% of patients who achieved a ≥VGPR had a PFS ≥48m in the absence of high risk lesions compared to 72.8% with any high risk lesion present (P=0.004). Some patients with PFS≥48m had only reached PR after induction; 56 (20.3%) and 57 (14.1%) of PFS ≥48m patients in the TE arm and 15 (28.3%) and 24 (21.1%) in the TNE arms of M-IX and M-XI, respectively. Baseline factors that were associated with still being able to achieve PFS≥48m from induction after only achieving a PR included the lack of high risk genetic lesions (P CONCLUSIONS Response assessment after induction+/-HD-Mel consolidation with baseline factors can define a patient group with superior outcomes in both TE and TNE patients and may influence future treatment strategies of MM patients undergoing first line therapy. Further analyses including modelling of predictors of response duration are ongoing and will be presented at the conference. Disclosures Shah: Celgene: Other: Travel, Accommodation expenses; Sanofi: Other: Travel and Accommodation expenses. Striha:Janssen: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; MSD: Research Funding; Amgen: Research Funding. Hockaday:Celgene: Research Funding; Amgen: Research Funding; Abbvie: Research Funding; Janssen: Research Funding; MSD: Research Funding; Millenium: Research Funding. Pawlyn:Celgene Corporation: Consultancy, Honoraria, Other: Travel support; Amgen: Consultancy, Honoraria, Other: Travel Support; Janssen: Honoraria, Other: Travel support; Takeda Oncology: Consultancy, Other: Travel support. Jenner:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Drayson:Abingdon Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Owen:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Other: Travel Support; Janssen: Consultancy, Other: Travel Support. Gregory:Celgene: Consultancy, Honoraria, Research Funding; Merck Sharp and Dohme: Research Funding; Janssen: Honoraria; Amgen: Research Funding. Morgan:Janssen: Research Funding; Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Cook:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Seattle Genetics: Honoraria; Glycomimetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Cairns:Celgene: Research Funding; Amgen: Research Funding; Merck Sharp and Dohme: Research Funding. Jackson:Roche: Consultancy, Honoraria, Speakers Bureau; Merck Sharp and Dohme: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel Support, Research Funding, Speakers Bureau. Kaiser:Amgen: Consultancy, Honoraria; Takeda: Consultancy, Other: travel support; Janssen: Consultancy, Honoraria; Chugai: Consultancy; Bristol-Myers Squibb: Consultancy, Other: travel support; Celgene: Consultancy, Honoraria, Research Funding.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material. Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis. Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor. Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Chronic lymphocytic leukemia (CLL) with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable region (IGHV) displays different states of anergy, indicated by reduced surface immunoglobulin M (sIgM) levels and signaling, consequent to chronic (super)antigen exposure. The subsets also differ in the incidence of high-risk genetic aberrations and in DNA methylation profile, preserved from the maturational status of the original cell. We focused on sIgM expression and function, measured as intracellular Ca(2+) mobilization following stimulation, and probed correlations with clinical outcome. The relationship with genetic features and maturation status defined by DNA methylation of an 18-gene panel signature was then investigated. sIgM levels/signaling were higher and less variable in U-CLL than in M-CLL and correlated with disease progression between and within U-CLL and M-CLL. In U-CLL, increased levels/signaling associated with +12, del(17p) or NOTCH1 mutations. In M-CLL, there were fewer genetic lesions, although the methylation maturation status, generally higher than in U-CLL, varied and was increased in cases with lower sIgM levels/signaling. These features revealed heterogeneity in M-CLL and U-CLL with clear clinical correlations. Multivariate analyses with phenotype, genetic lesions, or DNA methylation maturation status identified high sIgM levels as a new potential independent factor for disease progression. Multiple influences on sIgM include the cell of origin, the clonal history of antigen encounter in vivo, and genetic damage. This simple marker compiles these different factors into an indicator worthy of further investigations for prediction of clinical behavior, particularly within the heterogeneous M-CLL subset.

In myeloma, the prognostic impact of different strategies used to detect chromosome 13 deletion (Delta13) remains controversial. To address this, we compared conventional cytogenetics and interphase fluorescence in situ hybridization (iFISH) in a large multicenter study (n=794). The ability to obtain abnormal metaphases was associated with a poor prognosis, which was worse if Delta13, p53 deletion or t(4;14) was present, but only Delta13 remained significant on multivariate analysis. Patients with Delta13, by either cytogenetics or iFISH, had a poor prognosis. However, when cases with Delta13 detectable by both cytogenetics and iFISH were separated from those detected by iFISH only, the poor prognosis of iFISH-detectable Delta13 disappeared; their outcome matched that of patients with no detectable Delta13 (P=0.115). Addition of ploidy status to iFISH-Delta13 did not affect the prognostic value of the test. Indeed both cytogenetics and iFISH Delta13 divided both hyperdiploidy and nonhyperdiploidy into two groups with similar prognoses, indicating that the poor prognosis of ploidy is entirely due to its association with Delta13. We conclude that Delta13 detected by metaphase analysis is a critical prognostic factor in myeloma. Absence of Delta13, even in those patients yielding only normal or no metaphases, is associated with a relatively good prognosis.

A simple high throughput micro-fluorescence in situ hybridisation technique (FISH) was used to detect chromosome 13 deletions (delta13), immunoglobulin heavy chain (IgH) rearrangements, t(11;14)(q13;q32), t(4;14)(p16;q32), t(14;16)(q23;q32), p53 loss, and numerical changes of chromosomes 3, 6, 7, 9, 10, 11 and 17 in 228 cases of multiple myeloma (MM), including 33 asymptomatic/smouldering MM (SMM). The patients were not part of a clinical trial and were from 30 different hospitals. In all, 98.4% of cases were abnormal, with 43% having IgH rearrangements and 42% Delta13. The low incidence of IgH rearrangements was due to a decrease in this finding with age (P = 0.001) and the relatively high proportion of elderly patients in our study population (41% >70 years old). The incidence of specific IgH translocations was t(4;14) 11%, t(11;14) 16% and t(14;16) 3%. Univariate statistical testing showed delta13 (P = 0.002), and t(14;16) (P = 0.005) to be associated with shorter survival. This effect was exaggerated for patient's aged 70 years or under but no effect on survival was seen for those over 70 years. In younger patients t(4;14) (P = 0.044) and p53 deletion (P < 0.001) were also significant poor prognostic indicators. Multivariate analysis showed delta13 and t(14;16) to be independent prognostic variables when considered with age and clinical parameters.

[Figure][1] A 68-year-old woman presented with pneumonia and acute kidney injury. White blood cell count was 133.4 × 109/L (lymphocytes, 92.5 × 109/L). Blood count a month previously had shown mild lymphocytosis (9.6 × 109/L). Peripheral blood film showed 2 cell populations of small

Purpose: Regions on 1p with recurrent deletions in presenting myeloma patients were examined with the purpose of defining the deletions and assessing their survival impact. Experimental Design: Gene mapping, gene expression, FISH, and mutation analyses were conducted on patient samples from the MRC Myeloma IX trial and correlated with clinical outcome data. Results: 1p32.3 was deleted in 11% of cases, and deletion was strongly associated with impaired overall survival (OS) in patients treated with autologous stem cell transplant (ASCT). In patients treated less intensively, del(1)(p32.3) was not associated with adverse progression-free survival (PFS) or OS. The target of homozygous deletions was CDKN2C, however its role in the adverse outcome of cases with hemizygous deletion was less certain. 1p22.1-21.2 was the most frequently deleted region and contained the candidate genes MTF2 and TMED5. No mutations were identified in these genes. 1p12 was deleted in 19% of cases, and deletion was associated with impaired OS in univariate analysis. The target of homozygous deletion was FAM46C, which was mutated in 3.4% of cases. When cases with FAM46C deletion or mutation were considered together, they were strongly associated with impaired OS in the intensive treatment setting. Conclusion: Deletion of 1p32.3 and 1p12 was associated with impaired OS in myeloma patients receiving ASCT. FAM46C was identified as a gene with potential pathogenic and prognostic significance based on the occurrence of recurrent homozygous deletions and mutations. Clin Cancer Res; 17(24); 7776–84. ©2011 AACR.

The association of genetic lesions detected by fluorescence in situ hybridization (FISH) with survival was analyzed in 1069 patients with newly presenting myeloma treated in the Medical Research Council Myeloma IX trial, with the aim of identifying patients associated with the worst prognosis. A comprehensive FISH panel was performed, and the lesions associated with short progression-free survival and overall survival (OS) in multivariate analysis were +1q21, del(17p13) and an adverse immunoglobulin heavy chain gene (IGH) translocation group incorporating t(4;14), t(14;16) and t(14;20). These lesions frequently co-segregated, and there was an association between the accumulation of these adverse FISH lesions and a progressive impairment of survival. This observation was used to define a series of risk groups based on number of adverse lesions. Taking this approach, we defined a favorable risk group by the absence of adverse genetic lesions, an intermediate group with one adverse lesion and a high-risk group defined by the co-segregation of >1 adverse lesion. This genetic grouping was independent of the International Staging System (ISS) and so was integrated with the ISS to identify an ultra-high-risk group defined by ISS II or III and >1 adverse lesion. This group constituted 13.8% of patients and was associated with a median OS of 19.4 months.

We used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal gammopathy of undetermined significance [MGUS], presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell–cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.

We report serial genetic studies on a young female patient initially diagnosed with asymptomatic smouldering myeloma who progressed to symptomatic myeloma 4.5 years after presentation. An unbalanced translocation, der(14)t(4;14)(p16;q32), was initially found in all plasma cells plus deletions of other chromosomal regions as detected by array-based comparative genomic hybridization. Deletion of chromosome 13 was observed in a minor population of plasma cells (

We performed fluorescent in situ hybridization (FISH) for 16q23 abnormalities in 861 patients with newly diagnosed multiple myeloma and identified deletion of 16q [del(16q)] in 19.5%. In 467 cases in which demographic and survival data were available, del(16q) was associated with a worse overall survival (OS). It was an independent prognostic marker and conferred additional adverse survival impact in cases with the known poor-risk cytogenetic factors t(4;14) and del(17p). Gene expression profiling and gene mapping using 500K single-nucleotide polymorphism (SNP) mapping arrays revealed loss of heterozygosity (LOH) involving 3 regions: the whole of 16q, a region centered on 16q12 (the location of CYLD), and a region centered on 16q23 (the location of the WW domain-containing oxidoreductase gene WWOX). CYLD is a negative regulator of the NF-κB pathway, and cases with low expression of CYLD were used to define a “low-CYLD signature.” Cases with 16q LOH or t(14;16) had significantly reduced WWOX expression. WWOX, the site of the translocation breakpoint in t(14;16) cases, is a known tumor suppressor gene involved in apoptosis, and we were able to generate a “low-WWOX signature” defined by WWOX expression. These 2 genes and their corresponding pathways provide an important insight into the potential mechanisms by which 16q LOH confers poor prognosis.

Mutat Res. 2003 Apr 20;536(1-2):61-7. Role of haemoglobin in the protection of cultured lymphocytes against diepoxybutane (DEB), assessed by in vitro induced chromosome breakage. Porto B, Chiecchio L, Gaspar J, Faber A, Pinho L, Rueff J, Malheiro I. Laboratory of Cytogenetics, Instituto Ciências Biomédicas Abel Salazar (ICBAS), Largo do Prof. Abel Salazar, No. 2, 4099-003, Porto, Portugal. Abstract Diepoxybutane (DEB) is an alkylating agent that can be used to assess chromosome instability in repair-deficient subjects. Previous authors investigated the role of red blood cells (RBC) in determining individual susceptibility to DEB in normal healthy donors, and demonstrated that a polymorphic enzyme in RBC, Glutathione S-transferase T1 (GSTT1), is involved in DEB detoxification. In the present work we studied the influence of individual GSTM1 and GSTT1 genotypes and the presence of RBC on the frequency of DEB-induced chromosome breakage in lymphocyte cultures from normal individuals and, in particular, the influence of isolated components of RBC: RBC membranes, RBC lysate, and haemoglobin. Our results confirm that individual GSTT1 genotypes modulate the level of genetic lesions induced by DEB; however, this effect was not sufficient to explain the highly significant variation in chromosome breakage between whole blood and RBC-depleted cultures. We showed that RBC can protect cultured lymphocytes against chromosome breakage induced by DEB and we demonstrated the particular role of haemoglobin in the protective effect. PMID: 12694746 [PubMed - indexed for MEDLINE]

The tumor B-cell receptor (BCR) is the key to survival and proliferation in chronic lymphocytic leukemia (CLL) and is now a therapeutic target of very effective BCR-associated kinase inhibitors. CLL cells are typically characterized by a variable state of anergy, defined by low surface IgM (sIgM) levels and signaling ability with consequent relatively low proliferation rate. The subset with unmutated IGHV (U-CLL) is generally less anergic with a poorer prognosis than that with mutated IGHV (M-CLL), and appears more sensitive to BCR-inhibitors (Byrd et al., NEJM, 2013). However it remains unclear if the variable anergy reflects the nature of the cell of origin and/or the functional state of the BCR, thereby determining clinical behavior. In this study we investigated if variations of BCR sIgM levels and signaling correlate with clinical behavior of CLL and assessed the phenotypic and genetic associations with the variations. The study included samples at diagnosis or prior to treatment from 222 patients with sIgM/D CLL diagnosed according to the iWCLL2008 criteria (99 months median follow up). sIgM levels on the CD19+/CD5+ CLL cells and signaling [% intracellular Ca2+ mobilization (iCa2+)] were determined using soluble F(ab’)2 polyclonal antibodies (Mockridge at al, Blood, 2007). sIgM levels were analyzed for their association with i) survival, ii) phenotype (CD38, ZAP70 and BCR regulators CD5, CD19, CD20, CD22) and iii) genetic lesions (Del13q, Trisomy12, Del11q/ATM, Del17p/TP53, or NOTCH1 and SF3B1 mutants). Time from diagnosis to progression requiring first treatment (TTFT) was used as primary endpoint, while overall survival (OS) was used as a secondary endpoint to avoid the influences of chemotherapy. Best cut-offs for progression requiring treatment were determined with ROC and Youden’s T-tests (treatment as a state variable). Levels of sIgM (5th-95th percentile 12-378, median 52) varied markedly between patients. Levels were higher at more advanced stage of disease and sIgM expression (MFI>56) and signaling (iCa2+>6%) associated with significantly more rapid TTFT. Although sIgD levels and signaling also associated with shorter TTFT, multivariate Cox regression adjusted for IGHV status, sIgM levels, sIgM signaling, sIgD levels and sIgD signaling revealed that only U-IGHV (p By focusing on sIgM, a multivariate analysis adjusted for IGHV status, sIgM levels, CD38 and ZAP70 confirmed U-IGHV (p=0.002, 95% CI 1.35-3.56) and high sIgM (p=0.003, 95% CI 1.25-3.04) as the only independent predictors of shorter TTFT. Consistently, high sIgM levels associated with a more aggressive CLL even when U-CLL or M-CLL were investigated separately. Phenotypic analyses showed that high sIgM CLL had higher CD20, CD38, ZAP70, and CD22, than low sIgM CLL. Genetic analysis revealed that CLL subsets harboring Trisomy12 or Del17p/TP53 had significantly higher sIgM levels and signaling capacity than Del13q, even when U-CLL or M-CLL were analyzed separately. Trisomy12 U-CLL cases were enriched for NOTCH1 mutations. Consistently, U-CLL with NOTCH1 mutations (with or without Trisomy12) expressed higher sIgM than Del13q U-CLL. This study has several biological and clinical implications. First, it supports the critical role of sIgM in CLL-associated anergy. Our previous studies had shown dynamic variations of sIgM on circulating CLL cells following (auto)antigen engagement in tissue (Mockridge et al., Blood, 2007; Coelho et al. Blood, 2013). The subpopulation with higher sIgM is potentially the most dangerous and the most sensitive to kinase-inhibitors. Second, it documents the dominant role of sIgM levels on cell signaling and on tumor progression in CLL, with relevance even within U-CLL and M-CLL. Third, this study suggests influences of altered genetics on sIgM levels. However BCR-inhibitors overcome the influence of genetic lesions like Del17p/TP53. This points to a dominant role of reversed sIgM anergy in the behavior of CLL in vivo and claims the need of studies to verify sensitivity to kinase-inhibitors in CLL patients with different sIgM levels. Disclosures No relevant conflicts of interest to declare.

Abstract 1907 Introduction: Basing treatment decisions on the clinical variability known to exist in myeloma represents a way to improve patient outcome, however, a robust way of describing this variability is needed. We have investigated the association of tumour-specific genetic variables detected by FISH with survival in the MRC Myeloma IX trial with the aim of identifying high risk subgroups suitable for alternate treatment strategies. Methods: Patients followed an intensive or non-intensive pathway determined by a combination of performance status, patient and physician preference. The intensive pathway randomised patients to CVAD (cyclophosphamide, vincristine, adriamycin and dexamethasone) vs CTD (cyclophosphamide, thalidomide and dexamethasone), followed by HDM and ASCT. The non-intensive pathway randomised patients to MP (melphalan and prednisolone) vs attenuated CTD. The trial recruited 1960 patients with a median age of 59 in the intensive pathway and 73 in the non-intensive, with a median follow-up of 3.7 years. Plasma cells were purified from presenting diagnostic bone marrow samples using CD138 magnetic beads. FISH was performed to detect an IgH split, the common IgH translocation partners (4, 6, 11, 16, 20), hyperdiploidy, del(1p), del(13q), del(16q), del(17p) and gain of 1q. FISH results were available from 1180 patients. The lesions often co-segregated, and consequently we investigated their impact as single and combined lesions. Results: t(4;14), t(14;16), t(14;20), del(17p) and 1q+ were all associated with adverse overall survival (OS) in multivariate analysis, so these ‘adverse FISH lesions’ were examined in more detail. t(4;14) t(4;14) was identified in 11% of patients and was positively associated with 1q+ (p Del(17p) del(17p) was identified in 8% of patients, and in 38 patients where del(17p) occurred without other adverse lesions there was a trend towards adverse OS which did not reach significance (38 months vs 49 months, p=0.42). However, when del(17p) occurred in combination with 1 other adverse FISH lesion the median OS was 20 months, which shortened to 9 months with 2 lesions (p 1q+ 1q+ was found in 39% of cases and as an isolated lesion it was associated with adverse OS (42 vs 53 months, p=0.015), but again, the association was stronger when it co-segregated with another adverse lesion (median OS 25 months) or 2 adverse lesions (median OS 9 months) (p Combined: These data suggest that the accumulation of adverse FISH lesions was associated with progressive impairment of OS, and used this observation to define a group with no adverse lesions associated with a median OS of 61 months, an intermediate group with one adverse lesion (median OS 42 months) and a high risk group defined by the co-segregation of ≥2 adverse lesions (median OS 22 months). These groups retained association with adverse prognosis in the context of thalidomide or conventional induction chemotherapy, intensive or non-intensive therapy. In comparison, the ISS stratified the same group to give a median OS of 68 months (ISS1) vs 48 months (ISS2) vs 36 months (ISS3). Combining the genetic grouping with the ISS identified patients associated with very poor survival that constitute a high risk subgroup. Summary: We show that when genetic lesions such as t(4;14) and del(17p) occur in isolation, they are not strongly associated with short survival. However, combining the results of t(4;14), t(14;16), t(14;20), del(17p) and 1q+ can be used to define high risk myeloma. The true value of these lesions lies in interpreting them together, with the worst performing patients defined by the co-segregation of these lesions. Combining these tumour genetic groups with patient-specific variables can robustly identify patients suitable for alternative therapeutic strategies. More detailed statistical modelling incorporating patient-specific variables will be presented at the meeting. Disclosures: Boyd: Celgene: Honoraria. Davies:Ortho Biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 2980 Introduction: Deletion of chromosome 13, detected by conventional chromosome analysis (CC), has been suggested to be an independent indicator of poor prognosis in myeloma (MM). With the exception of a single study using conventional therapy this has not been confirmed in multicenter randomized controlled trials which have mostly used FISH analysis. We set out to assess the prognostic value of del(13) and compare its detection by FISH and CC. Methods: We have examined the effect of del(13) in a large multicenter randomized controlled phase III trial, MRC Myeloma IX (ISRCTN68454111), which tested the effect of induction therapy with a thalidomide combination in both an intensive (CTD vs CVAD prior to single HDM plus ASCT) and a non-intensive (CTDa vs MP) setting. From June 2003 to November 2007 samples from newly presenting MM patients were sent from hospitals throughout the UK to a central laboratory where CD138 purification for FISH was performed, with conventional cytogenetic culture(s), using unpurified cells, being set up wherever possible. Results: 1960 evaluable patients were entered into the trial (1111 intensive, 849 non-intensive) of which 1036 were evaluable for del(13) by FISH (613 intensive, and 423 non-intensive); del(13) was seen in 45% (470/1036) with a similar incidence in both pathways. With a median follow up of 3.7 years del(13) detected by FISH was associated with impaired OS (median, 42 vs 52 mo, p=0.004). The effect was stronger in the non-intensive pathway (median OS 25 vs 37 mo, p=0.001) than in the intensive one (median 58 mo vs not reached, p=0.095). However, this adverse prognostic impact was negated when the strong association of del(13) with the bad prognosis IGH translocations t(4;14), t(14;16) and t(14;20) (bad IGH) was taken into account. In comparison, 639 patients were evaluable for CC results (378 intensive, 261 non-intensive) with an overall abnormality rate of 35% (224/639). We note that the impact of detecting any abnormal karyotype was not significantly different from a normal/failed karyotype irrespective of intensive or non-intensive treatment being used (whole trial p=0.213, intensive p=0.249, non-intensive p=0.252) suggesting that the capacity to generate abnormal metaphases alone is not an adverse prognostic factor. Del(13) was seen in 45% of abnormal karyotype cases (102/224) and 16% of all cases tested for CC. Overall the detection of del(13) by CC was associated with an adverse prognostic effect on OS (median 34 vs 47 mo, p=0.039). However, the entire effect was in the non-intensive pathway (median 20 vs 34 mo, p=0.018) (Fig1A), with no detectable effect in the intensive pathway (median 69 vs not reached, p=0.679) (Fig1B). We addressed the impact of the treatment used, and in a comparison of the thalidomide and non-thalidomide regimes for all del(13) CC patients there was no significant impact on OS (p=0.538). However, in the non-intensive pathway there was a significant improvement in favour of thalidomide (median 33 vs 18 mo p=0.03). Multivariate analysis of the prognostic impact of genetic factors within abnormal metaphases showed that bad IGH (p=0.001), gain of 1q (p=0.001) and deletion of 17p (p=0.001) were the only independent prognostic factors. In further analyses including all FISH-detected abnormalities, the markers bad IGH (p Conclusions: We demonstrate that in a large multicenter trial carried out across the UK del 13 detected by FISH does not have adverse prognostic impact if its association with the poor prognosis translocations is taken into account. Similarly del(13) detected by CC is not an independent prognostic factor when bad IGH and gain of 1q are taken into acount. The trial shows that thalidomide improves the outcome for del(13) by CC in patients treated with conventional dose therapy. However, this beneficial effect of thalidomide is not enough to account for the lack of independent significance of this marker across the study. On the basis of these results we cannot recommend the use of conventional cytogenetic testing methods as a routine for patients entered into multicenter clinical trials. Disclosures: No relevant conflicts of interest to declare.

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high resolution single nucleotide polymorphism (SNP) mapping array analysis to examine changes in DNA copy number and loss of heterozygosity in 115 cases with matched peripheral blood controls. Identification

Chromosomal translocations involving the IGH locus on 14q32 are a hallmark of B-cell malignancies. These translocations are particularly frequent in non-hyperdiploid (NHD) multiple myeloma (MM), representing approximately 50% of myeloma cases. MM-associated primary t(14q32) target at least 7 partner genes including cyclins D (CCND1, −D3), MAF transcription factors (CMAF, MAF B and A) and MMSET/FGFR3. Some of the translocations are predictive of clinical outcome. Recently, we identified a novel interstitial del(14q) involving IGH and recurrent in chronic lymphocytic leukaemia and MM (Pospisilova et al, Leukemia, 2007). In spite of extensive studies, the mechanism(s) and molecular consequences of del(14q) remain unknown. We report here 34 cases of plasma cell (PC) malignancies with del(14q) involving IGH, as proven by FISH. Cases were collected in UK and Belgium. The estimated incidence of these aberrations in PC malignancies was 1.4%. There were 13 female and 21 male patients ranging in age from 49 to 86 years (average 68). Twenty seven patients had MM, one had SMM and 6 had MGUS. In almost all cases, the del(14q) was detected at diagnosis. Clinical data of the reported cases have been collected. The del(14q) were roughly mapped by FISH and grouped into 3 categories according to the proximal breakpoint: del(14)(q24.1q32.33) involving the ZFP36L1 region (11 cases); deletions proximal to ZFP36L1 (14 cases) and deletions distal to ZFP36L1 (7 cases). The size of del(14q) was not determined in 2 cases. Biallelic deletion of TRAF3/14q32.33 recurrently occurring in MM, was detected by FISH in 1 out of 9 analyzed cases. Additional FISH analysis showed that the del(14q) was predominantly associated with NHD tumors (62% vs 38% with hyperdiploid karyotypes) and frequently (60%) accompanied by del(13q), regarded as a poor prognostic factor. All reported cases were negative for t(4;14) and t(11;14); they also showed a normal status of CCND3, CMAF, MAFB and CMYC, when examined. A gain of 1q/CKS1B was found in 57% (8/14) of analyzed cases. The expression pattern of cyclin D1−D3 has been examined.

Abnormalities of 16q are important recurrent events in multiple myeloma (MM). We performed FISH on CD138 selected plasma cells from 701 newly diagnosed MM patients from the LRF UKMF cytogenetics database. Gene mapping, including paired normal controls, and gene expression analysis was performed on 55 cases using the Affymetrix Human Mapping 500K Array Set and U133 Plus 2.0 Arrays respectively. 16q deletion (del16q) was identified by FISH using probes for cMAF (Abbott Diagnostics) in 131/701 cases (18.7%) and was significantly associated with deletion 17p (16.5% vs. 8.9%, p=0.006), deletion 13 (60.8% vs. 48.5%, p=0.009), deletion of IgH (22.1% vs. 11.1%, p=0.0003) and non-hyperdiploid status (58.3% vs. 42.7%, p=0.006). Del16q showed a trend to poor overall survival, mean survival 43 vs. 61 months (p=0.09), and was associated with significantly worse survival in combination with t(4;14) compared with either t(4;14) or del16q alone, mean survival 15 vs. 26 vs. 45 months respectively (p=0.006). t(14;16) was identified by FISH in 31/701 cases (4.4%) and was associated with poor prognosis, mean survival 29 vs. 54 months (p=0.005). Mapping arrays revealed loss of heterozygosity (LOH) involving all or part of 16q in 20 of 55 cases (36%) in 3 distinct patterns: uniparental disomy (UPD) of chromosome 16 or 16q in 4/55 cases (7%); deletion of chromosome 16 or the whole of 16q in 11/55 cases (20%); and interstitial deletion of small regions of 16q in 5/55 cases (10%), focused on 16q12, the location of CYLD, and 16q23, the location of WWOX. 16q LOH was distributed across translocation groups but was identified in all 4 mapping cases containing 17p deletion, supporting the association identified by FISH. As WWOX is the site of the common fragile site FRA16D and deletions at common fragile sites have been associated with DNA instability in human cancers, we assessed this using gene mapping in these 55 MM cases. Although deletions spanning other common fragile sites were identified, they were not restricted to those with 16q LOH. However, in 2 t(14;16) cases, hemizygous deletions of approximately 100kb could be identified within WWOX at the presumed translocation breakpoint. One of the t(14;16) cases had a similar hemizygous deletion within FHIT, another tumor suppressor gene located within common fragile site FRA3B, consistent with findings in other cancer types. Cases with 16q LOH or t(14;16) all had significantly reduced WWOX expression relative to cases without 16q abnormalities, confirming gene inactivation by either LOH or translocation. Cases with 16q LOH also had significantly reduced expression of two other potential tumor suppressor genes located on 16q, CYLD and RBL2. In summary, our data confirms the adverse prognosis associated with 16q translocation or deletion. Array data reveals 16q LOH occurs due to deletion or UPD with two regions involved, one defined by CYLD and the other by WWOX. WWOX is also inactivated by translocation and is associated with interstitial deletions at this and other common fragile sites. WWOX is a likely candidate gene in MM pathogenesis because of its interaction with TP53 and CYLD via its effects on NF-κB.

Among chromosomal abnormalities described in myeloma (MM), deletion of chromosome 13 (Δ13) has been linked to a poor outcome. However, prognosis appears to vary according to the detection method used: conventional cytogenetics (CC) or FISH. We have investigated this inconsistency in a large multi-center cytogenetic study. Bone marrow samples from 814 MM patients were provided from 95 UK hospitals. FISH on purified plasma cells (n=697) and/or CC (n=577) was performed centrally: 458 samples had both tests. Sample quality was often poor, with low cell counts (median 2x107 and low plasma cell (PC) content (median 9%), precluding both tests on all samples. FISH for Δ13, t(4;14), t(11;14), gain of chromosomes 5, 9, 15 and, where possible, 3, 7 &17 was carried out. Overall survival was used as the end point with median follow-up of 22mo (range 12–53mo). Abnormalities were detected by CC in 216 cases (37%). Non-hyperdiploidy (non-HRD) (≤47 chromosomes, n=82, 14% of total, 38% abnormal) was a stronger indicator of poor prognosis than hypodiploidy (≤45 chromosomes, n=55, 10% of total, 25% abnormal) (p96% and sensitivity >92%. There was complete agreement between CC and FISH for individual abnormalities. CC showed a highly significant difference in prognosis between abnormal karyotypes with (n=95, 44%) and without Δ13 (median 15 months vs not reached, p20% PC, n=322, 46%) to have the worst outcome among the individual abnormalities (median 30mo vs not reached, p50% or >80% PC, or by considering only Δ13 in non-HRD cases (median 29mo vs 49mo, p=0.001), consistent with our CC results. When patients with results from both CC and FISH were examined, the outcome for Δ13 by CC was significantly worse than by FISH only (median 15mo vs 33mo, p Fig 1. Survival curves for Δ13 by FISH (A) & by CC &/or FISH (B) Fig 1. Survival curves for Δ13 by FISH (A) & by CC &/or FISH (B)