Leukemia (2006) 20, 2034–2040 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE Prognostic value of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma R Fonseca1, SA Van Wier1, WJ Chng1, R Ketterling2, MQ Lacy3, A Dispenzieri3, PL Bergsagel1, SV Rajkumar3, PR Greipp3, MR Litzow3, T Price-Troska3, KJ Henderson3, GJ Ahmann1 and MA Gertz3 1Division of Hematology and Oncology, Mayo Clinic Comprehensive Cancer Center, Scottsdale, AZ, USA; 2Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA and 3Division of Hematology, Mayo Clinic, Rochester, MN, USA A specific role for increased level of expression of CKS1B,asa including deletions and mutations of p53, and mutations of ras consequence of chromosome 1q21 copy number gain, has have been associated with disease progression, but only for a been postulated as both pathogenic, as well as a powerful small fraction of cases, and with yet to be proven pathogenic clinical prognostic factor in multiple myeloma (MM). The 1 purpose of this study is to determine the clinical associations consequences. and prognostic impact of copy number gain at chromosome Recently the group from the University of Arkansas (UAMS) 1q21 (with a bacteria artificial chromosome clone containing identified strong prognostic associations with an increased level CKS1B) and CKS1B gene level of expression in MM. We studied of gene expression of a cell cycle associated gene, CKS1B, the chromosome region 1q21 for copy number change in a located on chromosome 1q21.9,10 CKS1B favors cell cycle cohort of myeloma patients treated by high-dose therapy with progression by promoting degradation of p27 with release of the stem-cell rescue (HDT) (n ¼ 159). A separate cohort of patients, 11,12 treated by HDT was studied for CKS1B messenger RNA cyclin dependent kinases and entry into mitosis. This expression by gene expression profiling (n ¼ 67). 1q21 gain increased level of expression is associated with copy number was then correlated with clinical parameters and survival. Gain gain of chromosome 1 at band q21 in a subset of patients. of 1q21 copy number was detected in about a third of MM and Subsequently, the same group showed that 1q21 copy gain by was associated with more proliferative disease and poor-risk Fluorescent in situ hybridization (FISH) using a bacteria artificial cytogenetic categories such as t(4;14), and chromosome 13 chromosome (BAC) probe for CKS1B was also an important deletion. Both 1q21 gain and increase gene expression level 13 were significantly associated with reduced survival. However, prognostic factor in MM patients entered into total therapy II. neither is an independent prognostic marker in MM on multi- Previously comparative genomic hybridization studies have variate Cox proportional hazard analysis. shown that gains of chromosome 1q consistently involving the Leukemia (2006) 20, 2034–2040. doi:10.1038/sj.leu.2404403; 1q21 region is one of the most common genetic abnormalities in published online 5 October 2006 MM.14–16 Gain of 1q21 can occur as isochromosomes, Keywords: multiple myeloma; stem cell transplantation; gene 17,18 amplification; CKS1B; chromosome 1 duplications or jumping translocations in MM. Many of these chromosome changes are structural and do not involve copy number changes. However, the association of 1q21 copy gain and expression with proliferation and recurrent genetic abnormalities, and their relative impact on survival have not been clearly established. In Introduction this study, we assessed gain of chromosome 1q21 by FISH and increase CKS1B gene expression by gene expression profiling Molecular cytogenetic studies have revealed that, to a great (GEP), their relationship, prognostic impact and clinical and extent, the clinical heterogeneity of multiple myeloma (MM) is biologic correlations. dictated by the underlying genetic and cytogenetic aberrations present in the clonal plasma cells (PCs). This greater under- standing has defined several molecular subtypes of myeloma Patients and methods characterized primarily by the presence of specific chromosome translocations and associated chromosome ploidy changes.1–3 Patient cohorts This characterization is also associated, and ratified, by the Group 1: Mayo Clinic patients treated with HDT studied existence of specific transcription patterns that emerge from by FISH. We studied a cohort of 159 patients treated at the 4,5 these cytogenetically defined categories. It is also clear that Mayo Clinic that had been treated with melphalan-based high- these major cytogenetic categories are already present in the dose therapy (HDT). Patients were treated with melphalan at 6–8 early stages of the disease (i.e., MGUS), and that additional 200 mg/m2 (n ¼ 63) or melphalan at 140 mg/m2 plus total body genetic insults must participate in the progression to the more irradiation (n ¼ 83), and other conditioning regimens in 13. Of aggressive forms of the disease. Several candidate aberrations these patients 89 (56%) had been transplanted at the time of relapse. Of the 159 patients 114 have died. Median and Correspondence: Dr R Fonseca, Division of Hematology and minimum follow-up of the 45 survivors is 55 and 43 months, Oncology, Mayo Clinic Comprehensive Cancer Center, Johnson respectively. Research Building 356, Mayo Clinic, 13400 E Shea Boulevard, In all cases studied a research bone marrow (BM) sample had Scottsdale, AZ 85259, USA. E-mail: [email protected] been obtained before HDT. All patients gave written informed Received 11 July 2006; accepted 17 August 2006; published online consent for the collection and long-term storage of additional 5 October 2006 cells at the time of the BM examination including separate Chromosome 1q21 copy change and prognosis in myeloma R Fonseca et al 2035 authorization for genetic studies and separate consent for review a matrix to define the actual proportion of cells exhibiting a of their charts in accord with both federal regulations and possible amplification or copy gain pattern, and determine a Health Insurance Portability and Accountability Act (HIPAA) final ratio (Supplementary Figure 1). A ratio greater than 1 guidelines. The consent was approved by the Institutional represents copy gain of 1q21. This strategy will allow the Review Board of the Mayo Clinic College of Medicine. Cytospin assessment of the effect of 1q21 gain independent of ploidy slides were made in all cases and the remaining sample was changes. The second strategy is similar to that used by purified using CD138 þ magnetic beads when possible and Hanamura et al.,13 where the absolute copy of 1q21 is counted. stored in liquid nitrogen for future use. All patients in whom We considered patients who have three or more signals in more available samples were obtained were used in this analysis. than 20% of cells counted as having gain of chromosome 1q21. One hundred cIg-positive cells were counted. Group 2: Mayo Clinic patients studied by GEP. Gene expression analysis was performed on CD138 þ selected PCs from pretreatment BM samples of 67 newly diagnosed MM GEP patients using the Affymetrix U133A chip (Affymetrix, Santa Gene expression analysis was performed on CD138 þ cells Clara, CA, USA). This group of patients was subsequently treated using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA, with various forms of therapy including HDT and stem cell USA). RNA isolation, purification and microarray hybridization rescue in 63% (none of these patients are in group 1), has been reported previously.23 Gene expression intensity melphalan-based oral chemotherapy in 21%, dexamethasone- values were log transformed, normalized to the median and based chemotherapy in 13% and novel agents in 3%. analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA, USA). Group 3: UAMS patients treated with HDT (Total Therapy II). These are newly diagnosed patients entered into Total Therapy II studies. These patients have FISH analysis for Statistics 1q21 copy number using the same probe as that used in our To describe the prevalence of abnormalities we used simple þ study, as well as GEP performed on CD138 selected PCs from descriptive statistics. The Wilcoxon rank sum test was used to pretreatment BMs using the Affymetrix U133plus chip. The FISH compare the difference in PC labeling index (PCLI) between 13,19 and GEP of these patients have been published. For our patients with and without 1q21 copy change. Correlation analysis we utilized a subset of patients (n ¼ 248) with both FISH between the level of CKS1B gene expression and the PCLI was and GEP results available (These data are available from the NIH assessed using the Pearson’s correlation coefficient. The Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/, relationship between 1q21 copy change and CKS1B gene under accession number GSE2658). These patients were expression and cytogenetic categories was assessed by the w2 assigned Translocations and Cyclin (TC) class and a gene test. Two-tailed p-values were reported with the significance expression-based proliferation index (PI) based on published level set at 0.05. Survival (overall and progression free) 4 methods. probabilities were determined using the methods of Kaplan– Meier. Cox proportional hazards models were used to assess the association of several prognostic factors with survival. Methods FISH. The cytoplasmic immunoglobulin light chain (cIg-FISH) technique20 that combined the interphase FISH strategy with fluorescent detection of cIg was used. For the detection of the Results common chromosome abnormalities (t(11;14)(q13;q32), t(4;14)(p16;q32), þ 14q32, chromosome 13 abnormalities Clinical features of patients studied for DNA copy gain (D13) and À17p13), we used the same probes and conditions A total of 159 patients, in whom we had stored research samples published previously by us.21 and had been treated with HDT and stem cell transplant To score for 1q21 copy gain by FISH we used a BAC RPCI between January 1990 and January 2001, were studied.