Supplementary Figures 1-2, Tables 1-8 from Distinct Genomic Alterations in Prostate Cancers in Chinese and Western Populations Suggest Alternative Pathways of Prostate Carcinogenesis

The polycomb repressive complex 2 (PRC2) is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. Inactivating mutations of the catalytic component of PRC2, EZH2, are seen in myeloid disorders. We reasoned that the other 2 core PRC2 components, SUZ12 and EED, may also be mutational targets in these diseases, as well as associated factors such as JARID2. SUZ12 mutations were identified in 1 of 2 patients with myelodysplastic syndrome/myeloproliferative neoplasms with 17q acquired uniparental disomy and in 2 of 2 myelofibrosis cases with focal 17q11 deletions. All 3 were missense mutations affecting the highly conserved VEFS domain. Analysis of a further 146 myelodysplastic syndrome/myeloproliferative neoplasm patients revealed an additional VEFS domain mutant, yielding a total mutation frequency of 1.4% (2 of 148). We did not find mutations of JARID2 or EED in association with acquired uniparental disomy for chromosome 6p or 11q, respectively; however, screening unselected cases identified missense mutations in EED (1 of 148; 1%) and JARID2 (3 of 148; 2%). All 3 SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase activity in vitro, demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes.

AbstractProstate cancer is significantly more common in Western men than in Asian men, but the basis for this difference remains unknown. Because genomic studies of Asian prostate cancer are very limited, we used a genome-wide approach to reveal the genomic alterations in Chinese prostate cancers. We found a significant reduction in the frequency of certain somatic genomic changes that are commonly found in Western prostate cancers, including the 21q22.2-22.3 deletion, which involves the TMPRSS2:ERG fusion gene, and 10q deletion, which causes PTEN inactivation. Array results were confirmed by PCR-based molecular copy-number counting in selected samples. The different frequencies of these genomic changes were further evaluated by fluorescent in situ hybridization and immunohistochemistry analyses of tissue microarray samples. These alterations might be key genetic changes underlying the regional/ethnic difference in clinical incidence and might be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Our findings suggest that tumors arise in Western and Chinese populations by alternative pathogenetic mechanisms. Cancer Res; 70(13); 5207–12. ©2010 AACR.

Abstract Abstract 799 Introduction The accurate prediction and optimal management of transformed follicular lymphoma (t-FL) remains a significant clinical challenge. Investigation of the prognostic significance of genetic factors and the immune microenvironment in FL provides insight into the molecular pathogenesis of transformation and should aid in the development of robust molecular predictors of disease. Patient samples To address this issue, molecular profiling was performed on 91 samples including paired FL/t-FL samples from each of 31 patients (with additional germline (n=19) and relapse (n=10) samples where available). Median age at FL diagnosis was 46 years (range 22–71) with a median time to transformation of 4 years (1-16). Median follow-up of surviving patients was 10 years (2-19). Methods and Results The two most frequently mutated genes detected in FL/t-FL were TNFRSF14 (chr1p36.32) and EZH2 (chr7q36.1). Twelve TNFRSF14 mutations were detected in 10 patients (32%) with 3 arising in t-FL and one rendered homozygous on transformation. All were confined to exons 1–6 with 7/12 mutations causing premature termination of the protein. Heterozygous non-synonymous mutations in codon 641 of EZH2 occurred in 9 patients (29%) with 2 arising upon transformation. In mutated cases, the TNFRSF14 and EZH2 loci were subject to recurrent copy number (CN) loss/uniparental disomy (6/12) and gain (6/9) respectively. Genome-wide profiling was performed using the Affymetrix SNP 6.0 array to search for co-operating genetic events. A total of 1120 CN aberrations (CNAs) were identified across the cohort with a median of 5 gains (range 0–13) and 7 losses (0-42) in the earliest available FL (FL1) samples and 8 gains (0-33) and 10 losses (4-32) in the initial t-FL (t-FL1) samples. Recurrent CNAs included loss of chr1p, 6q, 9p, 17p and gain of 1q, 6p, 18 and × in both FL1 and t-FL1. In 18 cases, FL1 samples had CN changes absent from subsequent relapsed FL/t-FL samples. This is consistent with t-FL evolving from a common progenitor cell (CPC). Divergent changes seen in both FL and t-FL at relapse after t-FL1 further augment this hypothesis. CNAs common among different samples identify early events in FL/t-FL development. Recurrent regions of loss and gain affecting ≥20% of cases ranged in size from 4 kb to 60 Mb. Of these, gain 2p16.1-p15 (including REL and BCL11A) in FL1, predicted worse clinical outcome (survival from FL diagnosis; logrank p = 0.004, hazard ratio = 7). No cases with gain 2p16.1-p15, gain 8q24.13-q24.3, del 9p21.3 or gain 18q21.2-q21.33 were alive. Novel CNAs included gain 16p12.1 (containing IL4-R, IL21-R, NSMCE1) in 5 t-FL1 cases, recurrent deletion of ETS1 (n=4) on chr11q24.3 and further micro-deletions targeting CREBBP (chr16p13.3) and ALK (chr2p23.2). Conclusions The high mutational frequency of TNFRSF14 together with recurrent gain of IL4-R suggests a link between intra-cellular events and B-T cell interactions in the microenvironment. Enrichment of EZH2 mutations, the prognostic impact of gain REL/BCL11a and recurrent CN changes in CPC cases complete a core set of genetic changes that are potential molecular markers in t-FL. Disclosures: No relevant conflicts of interest to declare.

Genetic abnormalities in leukaemia range from single gene defects to chromosomal translocations, inversions, losses and gains. While conventional technologies can detect macroscopic abnormalities, finding smaller regions remained a challenge until the recent introduction of high-resolution genomic platforms. Microarrays based on single nucleotide polymorphisms is one such technology. It has made possible genome-wide allelic association studies of predisposition to common clinical problems. This approach is also being used to identify somatic changes in cancer, such as loss, gain and copy-neutral loss of heterozygosity (CN-LOH), which are below the level of detection by conventional systems. Such arrays have been used to identify key genes involved in paediatric acute lymphoblastic leukaemia. We have used these arrays to identify regions of CN-LOH on a genome-wide scale in a large series of acute myeloid leukaemia samples, which so far has not been possible through any other technology.

Acquired homozygosity in the form of segmental acquired uniparental disomy (aUPD) has been described in follicular lymphoma (FL) and is usually due to mitotic recombination. SNP array analysis was performed with the use of the Affymetrix 10K 2.0 Gene-chip array on DNA from 185 diagnostic FL patients to assess the prognostic relevance of aUPD. Genetic abnormalities were detected in 118 (65%) of 182 patients. Number of abnormalities was predictive of outcome; more than 3 abnormalities was associated with inferior overall survival (OS; P < .03). Sites of recurrent aUPD were detected on 6p (n = 25), 16p (n = 22), 12q (n = 17), 1p36 (n = 14), 10q (n = 8), and 6q (n = 8). On multivariate analysis aUPD on 1p36 correlated with shorter OS (P = .05). aUPD on 16p was predictive of transformation (P = .03) and correlated with poorer progression-free survival (P = .02). aUPD is frequent at diagnosis of FL and affects probability of disease transformation and clinical outcome.

Abstract Abstract 166 Recurrence of acute myeloid leukemia AML has a poor prognosis with only 20% of adults surviving to 5 years. Therefore it is of importance to identify molecular changes that explain the pathogenesis of relapsed AML. Previous studies had not identified consistently acquired cytogenetic changes at relapse. Recently, acquired uniparental disomy due to mitotic recombination was described in 40% of relapsed AML (Raghavan et al 2008). Most of the events lead to homozygosity for FLT3 mutations. This study aimed to discover if there are further genetic abnormalities acquired at disease recurrence that cannot be identified by conventional cytogenetics, i.e. microdeletions or gains. Twenty-one presentation and relapse paired AML patient blood and marrow samples were stored with consent at St Bartholomew's Hospital, London. Eleven patient samples had a normal karyotype at diagnosis, two had favourable prognosis cytogenetics (inv(16) and t(8;21)) and others had varying numerical cytogenetic abnormalities and rearrangements associated with an intermediate prognosis. DNA from the samples was analysed by array based high-resolution single nucleotide polymorphism (SNP) genotyping (Affymetrix Human SNP array 6.0). Data was analysed using Partek Genome Browser (Partek, MO). In all cases, the leukemia infiltrate of the marrow or blood was greater than 60% and most cases were greater than 90% allowing accurate identification of DNA copy number changes. Abnormalities of a size that would be identified by cytogenetics were disregarded. Using segmentation analysis using a p-value less than 0.001, over 400 microdeletions and gains were detected that were acquired at relapse in the 21 pairs. Each of the copy number changes was less than 2 megabases in size. One AML sample with a normal karyotype at diagnosis and trisomy 8 and add(9)(q34) at relapse had not acquired any microdeletions or gains. In contrast, in other samples as many as 69 microdeletions/gains were detected. There was no correlation between increased complexity of the karyotype of the leukemia and the number of microdeletions/gains. Several of the acquired microdeletions/gains were in regions containing genes known to be involved in AML, including a deletion of 234Kb at 13q12.2 involving FLT3 and CDX2, and an acquired deletion at 21p11.2 of 150Kb involving exons encoding the runt domain of RUNX1. Another copy number gain was detected at the MLL locus, suggestive of partial tandem duplication. Other detected locations are in Table 1.Table 1Location by cytobandCopy number changeSize / KbP valueGene13q12.2Deletion23410−33FLT3, CDX221q22.12Deletion15010−13RUNX111q23.3Gain5.10.0099MLL11p15.4Gain830.00001NUP9817q21.31Deletion8.00.0007BRCA1The results indicate that recurrent AML may be associated with the deletion or gain of several genes involved in leukaemogenesis. Many other locations are involved throughout the genome, suggesting at least some of these are also involved in the clonal evolution of the leukaemia at recurrence. Further studies should identify novel genes from these regions involved in the pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

The acquisition of uniparental disomy (aUPD) in acute myeloid leukemia (AML) results in homozygosity for known gene mutations. Uncovering novel regions of aUPD has the potential to identify previously unknown mutational targets. We therefore aimed to develop a map of the regions of aUPD in AML. Here, we have analyzed a large set of diagnostic AML samples ( n = 454) from young adults (age: 15–55 years) using genotype arrays. Acquired UPD was found in 17% of the samples with a nonrandom distribution particularly affecting chromosome arms 13q, 11p, and 11q. Novel recurrent regions of aUPD were uncovered at 2p, 17p, 2q, 17q, 1p, and Xq. Overall, aUPDs were observed across all cytogenetic risk groups, although samples with aUPD13q (5.4% of samples) belonged exclusively to the intermediate‐risk group as defined by cytogenetics. All cases with a high FLT3 ‐ITD level, measured previously, had aUPD13q covering the FLT3 gene. Significantly, none of the samples with FLT3 ‐ITD ‐ / FLT3 ‐TKD + mutation exhibited aUPD13q. Of the 119 aUPDs observed, the majority (87%) were due to mitotic recombination while only 13% were due to nondisjunction. This study demonstrates aUPD is a frequent and significant finding in AML and pinpoints regions that may contain novel mutational targets. © 2008 Wiley‐Liss, Inc.

To the editor: We read with interest the paper from Whitman and colleagues[1][1] reporting an adverse impact on disease-free survival of FLT3 tyrosine kinase domain ( FLT3 /TKD) mutations in 19 of 217 patients with normal karyotype acute myeloid leukemia (AML). This contrasts with our data where