Common Deletion Region Research Articles

Molecular Analyses of Deletion of the Long Arm of Chromosome 20 in Myelodysplastic Syndromes

Abstract 3834Del(20q), one of the common chromosome abnormalities in myeloid neoplasms, is observed in 5 to 10% of patients with myelodysplastic syndromes (MDS). However, the clinical and molecular biological significance of del(20q) has not been fully elucidated. We hypothesized that the genes involved in the molecular pathogenesis of MDS as tumor suppressor genes, are present within a common deleted region (CDR) of del(20q). Therefore, we attempted to determine CDR of del(20q), and, then, analyze the genes located in CDR.Microarray comparative genomic hybridization (CGH) analysis was performed using genomic DNA derived from bone marrow samples of five MDS patients (2 RA and 3 RCMD) with del(20q). In addition, five cases of acute myeloid leukemia with del(20q) were also included in the analysis. The results from microarray-CGH demonstrated that the size of CDR was 11.2 Mb. There are approximately 150 genes in CDR. Next, we performed mutation analysis of the genes located within the CDR using the next generation sequencing method, based on the “two-hit theory”, to identify TSGs which are involved in the molecular pathogenesis of MDS. We applied the SOLiD system to determine the sequences of genes located within CDR in eight patients of MDS with del(20q). At first, we selected and analyzed 32 genes located within or around CDR, which include candidate TSGs, or genes possibly involved in normal and/or malignant hematopoiesis. A total of 16 single nucleotide changes, which have not been reported as single nucleotide polymorphisms (SNPs), were found in the coding regions of the 32 genes. Of the 16 single nucleotide changes, two nonsynonymous nucleotide changes of the STK4 (R117Q) and NCOA3 (P467Q) genes were identified. After confirmation of the results by the Sanger sequencing method, we analyzed mutations for whole coding exons of the NCOA3 and STK4 genes in an additional 30 cases of MDS with del(20q) or monosomy 20. Two additional nonsynonymous single nucleotide changes of the NCOA3 gene, R353L and R1163W, which also have not been reported as SNPs, were found, while no additional mutations were found in the STK4 genes. Therefore, the nonsynonymous and non-SNPs single nucleotide changes in the NCOA3 gene were recurrently found in 3 (7.9%) of 38 cases of MDS with del(20q) or monosomy 20. The NCOA3 gene encodes a nuclear receptor coactivator that form coactivator complex with various molecules including nuclear receptors, and stimulates the transcriptional activities in multiple cellular pathways.Haploinsufficiency of TSGs may be another molecular mechanism in the pathogenesis of MDS. If target genes located within CDR of del(20q) exhibit haploinsufficiency, the loss of one allele as a result of del(20q) may be sufficient, and mutations of the remaining allele are not necessary. Therefore, we then examined the expression of 32 genes by quantitative RT-PCR, in 20 patients with MDS with del(20q) or monosomy 20, and compared it to those in 18 control subjects. In MDS patients with del(20q) or monosomy 20, expression of 8 out of the 32 genes was significantly reduced, compared to control subjects. We also examined expression of the 32 genes in 20 patients with MDS without chromosome 20 abnormalities. Interestingly, among the 32 genes, expression of 5 genes was significantly reduced in MDS patients without chromosome 20 abnormalities compared to control subjects. Expression of three genes (BCAS4, ADA, and ZNF335) was reduced in both MDS patients with del(20q) or monosomy 20 and those without chromosome 20 abnormalities, suggesting the significance of a decreased expression of those three genesin molecular pathogenesis of MDS. Molecular mechanisms other than chromosome deletion, including methylation of promoter regions may result in decreased expression of those genes.In the present study, we determined CDR of del(20q) and analyzed the genes located within or around CDR. Our present results showed recurrent mutations in the NCOA3 gene in MDS patients with del(20q), and decreased expression of genes within CDR in not only MDS patients with del(20q) or monosomy 20, but also in those without chromosome 20 abnormalities. The clinical and molecular biological significance of mutations of the NCOA3 gene and that of the decreased expression of the genes within CDR is also unclear. Further study is on going. Disclosures:No relevant conflicts of interest to declare.

Mutation Screening Associated with Chromosome 7 Abnormalities Using Next Generation Whole Exome Sequencing

AbstractAbstract 173One of the most common karyotypic abnormalities identified in myelodysplastic syndromes (MDS) is monosomy 7 (del7) or deletion of the long arm of chromosome 7 (del7q). The presence of del7/del7q carries a poor prognosis in MDS, MDS/myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML); the impact of these defects appears similar. Recently, a copy-neutral type of loss of heterozygozity (LOH also referred to as a somatic UPD) has been identified on 7q. Microdeletion on 7q corresponding to the EZH2 locus led to identification of inactivating mutations in this gene, though hemizygous EZH2 mutations are only rarely found and do not fully explain del7/7q pathogenesis. We performed a comprehensive analysis of myeloid neoplasms (N=189), using next generation whole exome sequencing technology, including MDS (N=34), MDS/MPN (N=26) or MPN (N=4) and 124 with AML (both primary and secondary). Among them, LOH7, involving del7/del7q were observed in 17% of cases (N=33). To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalyitic filtering approach, whereby paired DNA (tumor/CD3 lymphocyte) were sequenced and results aligned using Burrows-Wheeler Aligner and variants detected using GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on searching for del7/7q linked somatic mutational events involved comparisons of mutations in the area of del7q to cases diploid for this locus. We hypothesized that there may be heterozygous mutations of 7q, which could lead to functional haploinsufficiency that is also a result of del7q (haploinsuffcient theory, heterozygous mutations). Conversely, mutations may be either unique to del7q hemizygous inactivation, or shared between 7q diploid and haploid cases.In total, we found alterations in 12 genes located on chromosome 7 (6% of all alterations found). Using filtering strategies we narrowed the focus to “tier 1” mutations to avoid false positives; 11 mutated genes were found in cases with del7/7q and 2 in UPD7q. For example, novel hemzygous (but not heterozygous mutations) of an E3 ubiquitin ligase CUL1 gene were detected only in cases with del7/7q, suggesting that the wild type allele is protective. In cases with diploid 7q, 24 heterozygous alterations were observed (10 genes shared with del7/7q). The previously described EZH2 mutations were seen in heterozygous, homozygous and hemizygous configurations, but were most common in UPD7q (100%), while only 7% of del7/7q cases were positive. Notably, 5/12 mutant genes were located in commonly deleted regions (CDRs) either 7q22, 7q34 or 7q35–36. These CDRs also contain recurrently mutated lesions, including 7q22 (CUX1:n=4; STAG3:n=2), 7q34 (a splicing factor; LUC7L2: n=3) and 7q35–36 (EZH2: n=10). When we investigated the association between haploinsufficiency and heterozygous mutations, among those on del7/7q, cases with wild type forms of corresponding genes showed decreased expression. Similarly, such mutations were occasionally present in diploid configuration; here again the wild type cases showed a decreased expression. These findings suggest that mutated genes located in CDRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. EZH2 is a good example of such a gene. We also searched accessory genetic events observed on other chromosomes along with del7/7q and UPD7. By SNP-A, there were clear differences among 3 LOH7 groups, in which del7 was more associated with accessory chromosomal defects than cases with UPD7q or del7. Similarly, mutational patterns were specific to each LOH cohort. For example, while well known frequently mutated genes, such as U2AF1, TET2 and TP53, were commonly found in all 3 LOH7 groups, some specific genes, including the CSMD family, were uniquely observed in monosomy 7, not in del7q or UPD7. Similarly, LOH7q was associated with somatic mutations in SETBP1 and RUNX1. In conclusion, we detected several candidate genes that could be associated with del7/7q and UPD7. Some mutations were heterozygous in cases with diploid 7q and correlated with CDRs on del7/7q without mutation. Certain mutations are specifically observed with del7, while others are commonly observed in all categories of LOH7, including EZH2. Moreover, some genes outside of the chromosome 7 were coincidently mutated with LOH7. Disclosures:Makishima:Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

Clonal Chromosomal Anomalies Similar to CLL and Other Hematologic Malignancies Can Be Found in “Normal” Individuals

AbstractAbstract 873 Introduction:Clonal chromosomal anomalies have been well-characterized in the blood cells of patients with Chronic Lymphocytic Leukemia (CLL) and other hematological malignancies. However, the frequencies and types of chromosomal anomalies found in “normal” people (i.e. no history of hematological cancer) are much less well known. Here we compare chromosomal anomalies in untreated CLL patients with those found in a large sample of people (n>50,000) recruited for genome-wide association studies in the Gene Environment Association Studies (GENEVA) consortium. This comparison provides information about the extent to which people without a prior history of hematological cancer have chromosomal anomalies that are characteristic of CLL. Methods and Results:The anomaly detection method focused on acquired, rather than inherited, chromosomal anomalies identified in whole blood samples. The method and results for the GENEVA subjects were published recently (Laurie et al. 2012, Nat. Gen. 44:642–650). The same method was applied to pre-treatment samples from 220 CLL patients from US Intergroup Trial E2997 of the Eastern Cooperative Oncology Group (ECOG). Acquired anomalies were detected as mosaic mixtures of normal disomic cells along with cells containing autosomal anomalies consisting of large (generally > 1 Mb) deletions, duplications and/or acquired uniparental disomy (UPD). Detection requires a large fraction of cells with the same abnormal karyotype (>5–10%), which is presumably of clonal origin. The subjects (of all ages) came from 12 different GENEVA studies, which addressed a variety of health conditions not directly related to hematological cancer (e.g. addiction and glaucoma). DNA was extracted mainly from blood samples (or, in 8% of subjects, from buccal/saliva samples) and genotyped on Illumina SNP mircroarrays. Clonal mosaic anomalies were detected from change points in intensity and genotype data (B Allele Frequency and Log R Ratio). We found 514 anomalies in 404 GENEVA subjects out of 50,222 examined (0.8% of subjects), although the prevalence increased with age. Clinical data from about 75% of subjects with a clonal chromosomal abnormality revealed that only a small fraction (2.8%, 95% CI=1.0 – 4.7%) had a record of prior hematological cancer.In the ECOG study, blood samples from 220 CLL patients enrolled in protocol E2997 were genotyped on the Illumina HumanOmni1-Quad_v1 array and clonal mosaic chromosome anomalies were detected using the GENEVA method. Chromosomal anomalies typical of CLL also were identified using interphase FISH (i.e. 6q23.3-, 11q22-, 12q15+, 13q14-, 17p13-) as previously reported (Grever, JCO 25:799-804). We found 489 anomalies in 148 patients of the 220 examined (67% of patients). The anomalies detected by FISH and SNP microarray were highly concordant (>98%). Although, the most common recurrent anomalies occurred on chromosomes 6, 11, 12, 13 and 17 in regions that span the FISH probes (except for chromosome 6), a majority of the anomalies detected by SNP data (74% of 489) were not detected by the CLL FISH panel.The frequency of large chromosomal anomalies was much higher in CLL patients than in GENEVA (67% versus 0.8% of subjects) and the overall composition of anomaly types found was also different. The fraction of anomalies consisting of acquired UPD was substantially higher in GENEVA subjects than in ECOG (34.0% versus 10.6%), while deletions were less common in GENEVA (50.1% versus 72.4%) and duplications were similar (15.6% versus 17.0%) (Chi-squared test p-value=3 × 10−18). We compared the most commonly deleted regions (CDR) on each chromosome across studies. In the ECOG CLL patients, four chromosomes each had 10 or more overlapping deletions in their top CDR (6, 11, 13 and 17), which span the FISH probes (except for chromosome 6). The GENEVA subjects also had CDR on chromosomes 11, 13 and 17, which overlap those in ECOG. In addition, GENEVA had CDR spanning genes or regions implicated in myeloid malignancies, including DNMT3a on 2p, TET2 on 4q, PRAME on 22q, and a region on 20q13. Conclusions:These genotyping results of >50,000 subjects suggest clonal chromosomal anomalies may represent precursor states, or perhaps undiagnosed or unrecorded cases, of a variety of hematological malignancies. Further studies to validate these findings and explore the natural history of these genetic defects in individuals without hematologic malignancy are warranted. Disclosures:Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Haploinsufficiency of Mir-146a in High-Risk Del(5q) MDS/AML Requires an Intrachromosomal Gene Network Involving p62/TRAF6/NF-κB

AbstractAbstract 557Deletion of chromosome 5q (del(5q)) is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Commonly deleted regions (CDR) have been mapped to 5q31.1 and 5q33.1 in del(5q) AML and MDS, respectively. Although there has been extensive efforts to identify candidate genes within the CDRs and decipher which genes contribute to the high-risk versus low-risk phenotypes, more recent findings indicate that deletions involving the telomeric bands of 5q are associated with more aggressive forms of del(5q) MDS/AML (Jerez et al., JCO, 2012). To determine which genes on the telomeric extremes of chr 5q contribute to the aggressive phenotype associated with these patients, we performed a gene expression and functional analysis of candidate tumor suppressor (TS) genes located between ∼5q34 to 5q35.3. Among the 7 candidate TS genes localized on the telomeric extreme, expression of miR-146a is significantly reduced in del(5q) MDS/AML marrow cells with extended deletions as compared to ones with shorter deletions. In addition, low expression of miR-146a correlates with increased marrow blasts and shorter overall survival of patients with del(5q). To investigate the effects of miR-146a on hematopoietic stem/progenitor cell (HSPC) function, we stably knocked down miR-146a in human CD34+ cord blood and mouse HSPC-enriched cells. HSPC cells with reduced miR-146a exhibited features of malignant transformation, including increased survival, proliferation, and colony replating in methylcellulose. The mechanism underlying reduced miR-146a expression was investigated by performing gene expression profiling on marrow cells from MDS/AML patients with extended and short del(5q). Gene Ontology and Gene Set Enrichment Analysis revealed that patients with low expression of miR-146a exhibited a significant increase in cell cycle, immune response, and NF-kB target genes. These findings are consistent with the model that reduced levels of miR-146a results in derepression of TRAF6, a signaling protein within the innate immune pathway and activator of NF-kB (Starczynowski et al., Nature Medicine 2010). TRAF6 forms a signaling complex with Sequestosome 1 (SQSTM1/p62), a gene located within the telomeric deleted region in del(5q) on band 5q34. Surprisingly, expression of p62 was not reduced, but rather overexpressed in del(5q) MDS/AML patients with the extended deletions and inversely correlated with miR-146a expression. The compensatory expression of p62 is due in part by loss of miR-146a. RNAi-mediated knockdown revealed that p62 and TRAF6 are essential for NF-kB activation and survival of human MDS/AML cell lines with low miR-146a expression. In summary, we identified that miR-146a is a TS-like gene in MDS/AML patients with extended deletions of chr 5q, and that reduced miR-146a expression increases HSPC survival/proliferation and NF-kB signaling via p62 and TRAF6. Therefore, we propose that inhibiting the p62/TRAF6/NFκB intrachromosomal gene network represents a novel targeting strategy in high-risk MDS/AML patients with extended chr 5q deletions. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

The Type of Genetic Abnormalities Causing Loss of 5q Varies Between MDS and AML and Is Associated with Worse Prognosis in MDS

AbstractAbstract 697 Background:Deletions of the long arm of chromosome 5 are frequent abnormalities in MDS and AML. Their size varies considerably. Two commonly deleted regions (CDR) have been described on 5q: a distal CDR deleted in the 5q- syndrome and a proximal region lost in higher-risk MDS and AML. However, the majority of MDS and AML patients with del(5q) show large deletions encompassing both CDRs. Recently, Jerez et al. (JCO 2012) reported on commonly retained regions (CRRs) using SNP arrays and observed that deletions involving the centromeric and telomeric extremes of 5q are associated with a more aggressive clinical course. From a cytogenetic view loss of 5q either occurs due to interstitial deletions, unbalanced translocations or monosomy 5. While in interstitial deletions the telomeric region of 5q is retained, this region is lost in cases with unbalanced 5q translocations and monosomy 5. Aim:Analyze whether the type of 5q loss (interstitial deletion vs unbalanced translocation/ monosomy) is associated with other biological markers and prognosis in AML and MDS. Patients and Methods:In total, 1,200 patients (pts) with loss of 5q were studied including 627 AML pts (de novo: 454, s-AML: 101, t-AML: 72) and 573 MDS pts (de novo: 511, t-MDS: 62) with a median age of 71.7 yrs (range: 30–90) and 73.2 yrs (range: 34–93). Interphase FISH had been performed in all pts with a probe for EGR1 (5q31) and all showed a heterozygous EGR1 deletion. Further, all cases had been studied by chromosome banding analysis and in addition by 24-color FISH whenever necessary to resolve complex karyotypes. Data on TP53 mutation status was available in 263 pts (AML: 152, MDS: 111).Results: Type of 5q loss:AML and MDS cases were separated into 2 groups according to type of 5q loss: 1) interstitial 5q deletion (idel5q): AML: 341/627 (54.4%), MDS: 385/573 (67.2%), and 2) 5q loss due to an unbalanced translocation (ut5q): AML: 286/627 (45.6%), MDS: 188/573 (32.8%). Cases with complete loss of chromosome 5 (AML: 12, MDS: 1) were assigned to the second group. 530/627 (84.5%) AML pts and 303/573 (52.9%) MDS pts with 5q loss showed a complex karyotype (defined as 4 or more abnormalities). Further, in 345 AML (55.0%) and 233 MDS (40.7%) cases clonal evolution was evident as subclones with additional aberrations were detected. Impact of 5q loss in MDS:In MDS clonal evolution and complex karyotypes were more frequent in pts with ut5q as compared to idel5q (109/188 (58.0%) vs 124/385 (32.2%), p<0.0001; 179/188 (95.2%) vs 124/385 (32.2%), p<0.0001). In MDS TP53mut was more frequent in ut5q than in idel5q (8/10 (80%) vs 25/101 (24.8%), p=0.001), in pts with clonal evolution (9/14 (64.3%) vs 24/97 (24.7%), p=0.005) and in pts with complex karyotype (13/15 (86.7%) vs 20/96 (20.8%) p<0.0001). Further, median OS was shorter in pts with ut5q as compared to pts with idel5q (15.3 months (mo) vs not reached, p<0.0001). Impact of 5q loss in AML:Similar to MDS, in AML complex karyotypes were more frequent in pts with ut5q as compared to pts with idel5q (274/286 (95.8%) vs 256/341 (75.1%) p<0.0001). However, in contrast to MDS no association between ut5q and clonal evolution was observed in AML. Moreover, also in contrast to MDS no association between TP53mut and type of 5q loss was observed in AML (TP53mut 59/67 (88.1%) in ut5q pts and 66/85 (77.6%) in idel5q pts). In AML, median OS was comparable in pts with ut5q and pts with idel5q (5.9 mo vs 5.8 mo). However, complex karyotype and the presence of TP53mut were associated with shorter OS in AML (5.5 mo vs 14.0 mo, p=0.32 and 4.8 mo vs 25.1 mo, p<0.0001). In multivariable Cox regression analysis of AML pts including the parameters significant in univariable analyses (complex karyotype, TP53mut, age and WBC count) only TP53mut (HR: 3.28, p=0.011) and WBC count (HR: 1.15 per 10 G/L, p=0.044) were independently associated with shorter OS. Conclusions:1. Loss of 5q due to unbalanced translocations encompassing the telomeric 5q region is more frequent in AML (45.6%) than in MDS (32.8%) and in both entities associated with a complex karyotype. MDS patients with unbalanced 5q translocations show a shorter survival as compared to patients with interstitial 5q deletions. This data suggests that in MDS unbalanced 5q translocations reflect chromosomal instability which most probably is associated with progression to AML and adverse prognosis. 2. In AML with loss of 5q the presence of TP53 mutations is the strongest adverse prognostic factor. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Tifab, a Novel Candidate Gene in Deletion Chromosome 5q, Contributes to Deregulation of NF-κB Signaling in MDS/AML.

AbstractAbstract 2812Myelodysplastic syndromes (MDS) are hematologic disorders defined by blood cytopenias due to ineffective hematopoiesis, altered cytogenetics, and predisposition to acute myeloid leukemia (AML). The most common cytogenetic alteration in de novo and treatment-related MDS is deletion of chromosome 5q (del(5q)). There are two commonly deleted regions (CDR) mapped to chr 5q, however the gene(s) in these regions responsible for the manifestation of del(5q) MDS are not clearly defined. A search of annotated genes revealed that TRAF-interacting protein with forkhead-associated domain B (TIFAB), a known inhibitor of TRAF6 and a novel gene identified by an in silico search for TIFA-related genes, resides within the proximal CDR on band 5q31.1. We first determined whether TIFAB is expressed in normal hematopoietic stem/progenitor cell (HSPC) by qRT-PCR. We find that expression of TIFAB is enriched in human CD34+/CD38+ and mouse lineage-/cKit+ progenitors as compared to more differentiated populations, suggesting that it plays a role in normal HSPC function. To determine whether TIFAB is implicated in del(5q) MDS, we measured TIFAB expression in del(5q) MDS patients. According to a microarray analysis, TIFAB mRNA was significantly lower in CD34+cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q (Pellagatti, et al., 2006). In an independent subset of patients, we confirmed that TIFAB expression was lower in marrow cells isolated from del(5q) MDS patients. Therefore, we hypothesize that TIFAB loss results in hematopoietic defects contributing to del(5q) MDS. To determine whether deletion of TIFAB affects hematopoiesis, we used lentiviral shRNAs to knockdown TIFAB mRNA in human cord blood CD34+ cells. To mimic haploinsufficiency of TIFAB in del(5q) MDS, we selected shRNAs that result in ∼50% knockdown of TIFAB mRNA and protein. Knockdown of TIFAB in human CD34+ cells results in increased survival, a competitive growth advantage, and altered hematopoietic progenitor function. Conversely, overexpression of TIFAB in human leukemia cell lines (THP1 and HL60) results in increased basal apoptosis, delayed G1/S-phase cell cycle progression, and impaired leukemic progenitor function in methylcellulose. Since TIFAB is predicted to regulate TRAF6, we examined the role of TIFAB on TRAF6 signaling. TIFAB suppressed TRAF6 lysine (K)-63 autoubiquitination (a measure of TRAF6 activity), and decreased total TRAF6 protein levels, suggesting that TIFAB may simultaneously inhibit TRAF6 function and protein expression. Consistent with this finding, TIFAB suppressed lipopolysaccharide-induced (TRAF6-dependent) NF-kB activation, but not TNF-induced (TRAF6-independent) NF-kB activation. TIFAB-mediated inhibition of TRAF6 also coincided with reduced phospho-IKK-beta (a measure of NF-kB activation) in leukemic cells. In summary, we have identified TIFAB as a novel del(5q) MDS/AML gene involved in regulating HSPC survival, progenitor function, and cell cycle. We propose that haploinsufficiency of TIFAB results in malignant clonal cell expansion and may contribute to the MDS/AML phenotype as a consequence of increased TRAF6-mediated activation of NF-kB. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

De novo microdeletions of chromosome 6q14.1-q14.3 and 6q12.1-q14.1 in two patients with intellectual disability - further delineation of the 6q14 microdeletion syndrome and review of the literature

Interstitial 6q deletions can cause a variable phenotype depending on the size and location of the deletion. 6q14 deletions have been associated with intellectual disability and a distinct pattern of minor anomalies, including upslanted palpebral fissures with epicanthal folds, a short nose with broad nasal tip, anteverted nares, long philtrum, and thin upper lip. In this study we describe two patients with overlapping 6q14 deletions presenting with developmental delay and characteristic dysmorphism. Molecular karyotyping using array CGH analysis revealed a de novo 8.9 Mb deletion at 6q14.1-q14.3 and a de novo 11.3 Mb deletion at 6q12.1-6q14.1, respectively. We provide a review of the clinical features of twelve other patients with 6q14 deletions detected by array CGH analysis. By assessing all reported data we could not identify a single common region of deletion. Possible candidate genes in 6q14 for intellectual disability might be FILIP1, MYO6, HTR1B, and SNX14.

Functional dissection of the chromosome 13q14 tumor-suppressor locus using transgenic mouse lines

AbstractDeletion of chromosomal region 13q14 represents the most common genetic aberration in B-cell chronic lymphocytic leukemia (CLL). 13q14 deletions are commonly large and heterogeneous in size and affect multiple genes. We recently found that targeted deletion in mice of the 0.11 megabase (mb)–long minimal deleted region (MDR) encompassing the DLEU2/miR-15a/16-1 cluster recapitulates the spectrum of CLL-associated lymphoproliferations in humans, including CLL, CD5+ monoclonal B-cell lymphocytosis, and CD5− non-Hodgkin lymphomas. In the present study, we demonstrate that additional deletion of the 0.69-mb large genomic region telomeric to the MDR called the common deleted region (CDR) changed the spectrum of lymphoproliferations developing in CDR- versus MDR-deleted mice in that the number of CLL among B-cell lymphoproliferations was significantly elevated in the former. In addition, CDR-deleted mice seemed to succumb to their disease faster than MDR-deleted mice. Comparing HCDR3 regions of CD5+ lymphoproliferations derived from this and published CLL mouse models, 44% (29 of 66) of junctions could be assigned to 8 sets of highly similar HCDR3 regions, demonstrating that CLL developing in mice frequently expresses almost identical, stereotypic Ag receptors. These results suggest that the size of 13q14 deletions influences the phenotype of the developing lymphoproliferations and potentially the severity of disease, suggesting a tumor-suppressor function for genetic elements in addition to DLEU2/miR-15a/16-1.

Important Genes in the Pathogenesis of 5q- Syndrome and Their Connection with Ribosomal Stress and the Innate Immune System Pathway

Myelodysplastic syndrome (MDS) with interstitial deletion of a segment of the long arm of chromosome 5q [del(5q)] is characterized by bone marrow erythroid hyperplasia, atypical megakaryocytes, thrombocythemia, refractory anemia, and low risk of progression to acute myeloid leukemia (AML) compared with other types of MDS. The long arm of chromosome 5 contains two distinct commonly deleted regions (CDRs). The more distal CDR lies in 5q33.1 and contains 40 protein-coding genes and genes coding microRNAs (miR-143, miR-145). In 5q-syndrome one allele is deleted that accounts for haploinsufficiency of these genes. The mechanism of erythroid failure appears to involve the decreased expression of the ribosomal protein S14 (RPS14) gene and the upregulation of the p53 pathway by ribosomal stress. Friend leukemia virus integration 1 (Fli1) is one of the target genes of miR145. Increased Fli1 expression enables effective megakaryopoiesis in 5q-syndrome.

Microarray CGH analyses of chromosomal 20q deletions in patients with hematopoietic malignancies

The chromosomal abnormality del(20q) is mostly found in various myeloid disorders, including myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemia. Here, microarray comparative genomic hybridization (aCGH) analyses of 14 patients cytogenetically confirmed to carry the del(20q) aberration in their bone marrow demonstrated that all deletions were interstitial and both the proximal and distal breakpoints varied among individuals. The centromeric breakpoints were located in the 20q11.21-12 region, and the telomeric breakpoints, in the 20q13.13-13.33 region. The extent of the deletion ranged from 11.2 to 27.3 Mb, and the commonly deleted region (CDR) was estimated to be 7.2 Mb in size. Two commonly retained regions were present, the proximal region adjacent to the centromere (20q11.1-11.21) and a subtelomeric one (20q13.33). The CDR of our study was more distal than reported previously. Furthermore, in three patients fluorescence in situ hybridization (FISH) demonstrated that del(20q) cells were detected at a higher frequency in the karyotype analyses than by interphase FISH and aCGH analyses. As the size and breakpoints of del(20q) have been reported to vary among patients, the presence of one or more tumor suppressor genes in the CDR has been suggested. Our study will contribute to the identification of candidate tumor suppressor genes on 20q.

Genetic aberrations on the short arm of chromosome 8 (8p) in tongue carcinomas

Aberrations on the short arm of chromosome 8 (8p) are frequently observed in several human cancers. In this study, 20 squamous cell carcinoma (SCC) specimens from the tongue were examined in order to evaluate the role of 8p in SCC of the tongue. Microsatellite analysis using 14 markers demonstrated two commonly deleted regions (CDRs) on 8p. Reverse transcription-polymerase chain reaction (RT-PCR) revealed frequent downregulation of the FEZ1 gene, mapped to 8p22, and frequent over-expression of the cathepsin B gene, mapped to 8p-21-22. These results suggested that genetic aberrations are involved in the development of SCC of the tongue. However, no significant relationship was observed to be established between the genetic alterations and clinicopathological features. Thus, further investigation is necessary in order to clarify the clinical role of 8p in carcinoma of the tongue.

Dgcr8 controls neural crest cells survival in cardiovascular development

DiGeorge syndrome (DGS), characterized genetically by a deletion within chromosome 22q11.2, is associated with a constellation of congenital heart defects. DiGeorge critical region 8 (Dgcr8), a gene that maps to the common deletion region of DGS, encodes a double stranded RNA-binding protein that is essential for miRNA biogenesis. To address the potential contribution of Dgcr8 insufficiency to cardiovascular development, we have inactivated Dgcr8 in cardiac neural crest cells (cNCCs). Dgcr8 mutants displayed a wide spectrum of malformations, including persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Interestingly, Dgcr8-null cNCCs that properly migrated into the cardiac outflow tract (OFT), proliferate normally and differentiate into vascular smooth muscle cells. However, loss of Dgcr8 causes a significant portion of the cNCCs to undergo apoptosis, causing a decrease in the pool of progenitors required for OFT remodeling. Our data uncover a new role of Dgcr8 in cardiovascular morphogenesis, plausibly as part of transmission mechanism for FGF-dependent survival cue for migrating cNCCs.

In Vivo Functional Dissection of the Chromosome 13q14 Tumor Suppressor Locus Reveals That Extent of Deletions Impacts Disease Course and Phenotype

Abstract 468Deletion of chromosomal region 13q14 represents the most common genetic aberration in chronic lymphocytic leukemia (CLL) and occurs with less frequency in other lymphoid malignancies. 13q14 deletions are commonly large and heterogeneous in size, affecting multiple genes. We recently demonstrated that the targeted deletion in mice of miR-15a/16-1 alone causes lymphoproliferations and recapitulates the spectrum of CLL-associated phenotypes in humans. However, both the penetrance and severity of the disease increased markedly upon deletion of the 110 kilobase (kb)-long minimal deleted region (MDR) that encompasses the DLEU2/miR-15a/16-1 cluster (Klein et al., Cancer Cell, 2010). Thus, we investigate here the extent to which the additional deletion of genetic material encoded in the 690 kb large genomic region telomeric of the MDR, including the DLEU7 and RNASEH2B genes, affects the phenotype and spectrum of lymphoproliferations and disease course. To this end, we generated a conditional allele that allows in vivo deletion of this 800 kb large region termed common deleted region (CDR) specifically in B cells. B-cell conditional deletion was necessary since homozygous CDR germ-line deletion led to embryonic lethality.We demonstrate the technical feasibility of mimicking a large chromosomal deletion (800 kb) occurring in human cancer in vivo and in a cell type-specific fashion, thus establishing an animal model for this genomic aberration. We found that despite a similar time-point of disease onset (∼12 months) and disease penetrance (∼40%), the spectrum of lymphoproliferations developing in CDR-deleted mice differed from those found in MDR-deleted mice in that the fraction of CLL among the various types of B-lymphoproliferations (including CLL, CD5+ monoclonal B-cell lymphocytosis (MBL) and CD5– non-Hodgkin lymphomas (NHL)) was significantly elevated. Specifically, 19/20 mice with homozygous deletion of the CDR developed lymphoproliferations that represented CLL (95% CLL; 5% MBL) vs. 12/22 of the corresponding MDR-deleted mice (55% CLL; 27% MBL; 18% NHL). Since we previously observed that MDR+/– mice compared to wild-type mice showed a trend towards developing clonal lymphoproliferations, we established and monitored cohorts of CDR+/– mice and wild-type littermates. 25% of CDR+/– mice developed CD5+ lymphoproliferations vs. 8% of control mice. Determination of event-free survival demonstrated that CDR+/– mice succumb to their disease earlier than wild-type littermates (P=0.0002). Compared to MDR+/– mice, the CDR+/– mice showed a trend towards a more aggressive disease course (70% of CDR+/– vs. 45% of MDR+/– mice have died at 20 months). In summary, these results suggest that the additional loss of genetic elements located telomeric of the MDR impacts the phenotype of the lymphoproliferations and potentially the severity of disease.Notably, lymphoproliferations derived from 4 independent CLL mouse models, i.e. TCL1-tg (Yan et al., PNAS, 2006) as well as miR-15a/16-1, MDR (Klein et al., Cancer Cell, 2010) and CDR-deleted mice, expressed antibody genes with nearly identical, stereotypic antigen binding regions. Specifically, comparing all HCDR3 regions derived from MBL or CLL cases of our own (n=46) and the TCL1-tg (n=20) studies, we observed that 44% (29/66) of the junctions can be assigned to 8 sets of highly similar HCDR3 regions among the sequence collection, 5 of which have previously been defined by Yan et al. These findings provide a rationale for defining clusters of stereotypic antigen receptors in CLL-prone mice. They also suggest that genetic aberrations disrupting the control of cell growth and survival and chronic antigen-stimulation co-operate in the clonal expansion of CLL tumor cells.In summary, published and the present results suggest that whereas deletion of the miR-15a/16-1 cluster is the critical mechanism in the pathogenesis of CLL with 13q14 aberrations, the additional loss of genetic elements encoded in the 13q14 tumor suppressor locus can significantly influence the penetrance of the phenotype, the spectrum of the lymphoproliferations, and the severity of the disease course. Since 13q14 deletions in humans are usually heterogeneous, these findings provide a clear rationale for determining the pathogenic role of the various genetic elements affected by deletions of the 13q14 locus. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Aberration Profile of Chronic Myeloid Leukemia and Its Dynamic Changes During Imatinib Therapy,

Abstract 3755Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by excessive production of myeloid cells and the presence of the BCR-ABL fusion oncogene resulting from the t(9;22) reciprocal translocation. The leukemic clone in CML often accumulates other somatic lesions that may collaborate with BCR-ABL oncogenicity or confer resistance to tyrosine kinase inhibitors. The chromosomal instability in CML is believed to be caused by the BCR-ABL oncogene. We hypothesized that the accumulation of cytogenetic lesions in CML are acquired after the t(9;22). Thus, imatinib therapy should not only cause the molecular remission of BCR-ABL positivity but also the remission of most cytogenetic lesions. If cytogenetic lesions preceded t(9;22) acquisition, a residual clone should be detectable after remission of BCR-ABL positivity. In order to test these predictions, we determined high-resolution karyotypes of CML patients at diagnosis and at time points with variable durations of imatinib therapy. At the same time, we aimed to characterize the overall cytogenetic aberration profile of CML at different disease stages.The copy number abnormalities and loss of heterozygosity coupled with acquired uniparental disomy (UPD) were determined using Affymetrix SNP 6.0 arrays. Overall 62 patients were included in the study.For 13 patients paired DNA samples were available, the first one taken at diagnosis and the second after treatment with imatinib. These samples were used to evaluate the cytogenetic remission after treatment. In 7 out of 13 patients we could detect an additional chromosomal aberration at diagnosis. Interestingly, the clone size assessment based on copy number signal intensity data was concordant with BCR-ABL burden. Analysis of the follow-up samples revealed that 6 patients with additional cytogenetic aberrations at diagnosis showed complete cytogenetic remission. In one patient imatinib therapy led to significant reduction of the BCR-ABL burden but a residual BCR-ABL negative clone persisted. This residual clone exhibiting del6p, del10q and del13q was detectable as a minor clone at diagnosis and it fully replaced the BCR-ABL positive clone after 8 months of imatinib therapy. This data indicates that the BCR-ABL fusion was either acquired through the clonal progression of the initial clone carrying described cytogenetic aberrations, or two distinct clones were present at diagnosis.In order to define the cytogenetic profile of CML samples we analyzed an additional number of 12 patient samples taken at diagnosis, as well as 37 samples taken after treatment with tyrosine kinase inhibitors. In 35.5% of the patients we could detect cytogenetic lesions, consisting of 28 deletions, 9 gains and 1 UPD. The most recurrent deletions were in the breakpoint region of the BCR (n=3) and ABL (n=5). Deletions of 13q (4 events) defined two common deleted regions (CDR) overlapping with previously defined CDR in other myeloproliferative neoplasms and chronic lymphoid leukemia. Other set of deletions clustered on chromosome 7p targeting 5 genes, among which was ABCB5, a member of ATP-binding cassette transporter family. One of the patients was also found to be a carrier of 11pUPD. In order to characterize the target gene within the UPD region we performed whole exome next generation sequencing of this sample and found 9 candidate mutations which are currently evaluated.Compared to other myeloproliferative neoplasms, CML exhibits low cytogenetic complexity. This is demonstrated by the fact that only 35.5% of patients have chromosomal aberrations in addition to t(9;22). Our data indicates that the majority of aberrations in CML occur after t(9;22) during the clonal evolution of the leukemic cells. Most of these aberrations disappear after treatment with tyrosine kinase inhibitors. However in a proportion of patients clonal hematopoiesis persists, even after successful targeting of BCR-ABL positive cells. In these patients imatinib therapy does not restore polyclonal hematopoiesis despite the BCR-ABL remission. The persistence of a clone prone to accumulation of other mutations can potentially lead to induction of other hematological phenotypes. Disclosures:No relevant conflicts of interest to declare.

Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q− syndrome

AbstractLarge chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q− syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q− syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q− syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q− syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q− syndrome.

Microdeletion at 4q21.3 is associated with intellectual disability, dysmorphic facies, hypotonia, and short stature

Chromosomal imbalances are a major cause of intellectual disability (ID) and multiple congenital anomalies. We have clinically and molecularly characterized two patients with chromosome translocations and ID. Using whole genome array CGH analysis, we identified a microdeletion involving 4q21.3, unrelated to the translocations in both patients. We confirmed the 4q21.3 microdeletions using fluorescence in situ hybridization and quantitative genomic PCR. The corresponding deletion boundaries in the patients were further mapped and compared to previously reported 4q21 deletions and the associated clinical features. We determined a common region of deletion overlap that appears unique to ID, short stature, hypotonia, and dysmorphic facial features.

2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

1 A mouse model highlights a role for p53 in 5q − syndrome

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

Mapping of Genetic Deletions on Chromosome 3 in Colorectal Cancer: Loss of 3p25-pter is Associated with Distant Metastasis and Poor Survival

There is no detailed analysis of loss of heterozygosity (LOH) on chromosome 3 in colorectal cancer (CRC). Our aim was to define frequently deleted loci on chromosome 3 and to explore novel prognostic markers and the locations of candidate tumor suppressor genes associated with CRC. LOH at 23 microsatellite markers spanning on chromosome 3 was determined in 112 sporadic CRC by automated fluorescence-based polymerase chain reaction. Genetic loss was assessed for the clinicopathological significance by univariate and multivariate analyses. Fifty-eight (51.8%) of 112 carcinomas exhibited LOH at one or more loci tested. Among seven loci with high LOH rates, allelic losses at D3S1297 and D3S1266 occurred more frequently in younger patients. A marked gender distortion for genetic deletion was observed at six loci, where LOH was identified more frequently in male cases. For clinical outcome, LOH solely at D3S1297 (3p26.3) was significantly associated with distant metastasis (P=0.001) and was indicative of a shorter overall survival (P=0.014). In addition, loss of one common deletion region at 3p25-pter was significantly correlated to distant metastasis (P=0.009) and had an adverse effect on patients' overall survival in univariate and multivariate tests (P=0.009 and 0.001, respectively). Loss of chromosome 3p25-pter could act as an independent predicator of poor prognosis in CRC, suggesting that microsatellite analysis is a useful means to stratify patients into different risk groups. In addition, inactivation of candidate tumor suppressor genes in this region might involve in CRC progression.

Both Gene Amplification and Allelic Loss Occur at 14q13.3 in Lung Cancer

Because loss of Nkx2-8 increases lung cancer in the mouse, we studied suppressive mechanisms in human lung cancer. NKX2-8 is located within 14q13.3, adjacent to its close relative TTF1/NKX2-1. We first analyzed LOH of 14q13.3 in forty-five matched human lung cancer and control specimens. DNA from tumors with LOH was then analyzed with high-density single-nucleotide polymorphism (SNP) arrays. For correlation with this genetic analysis, we quantified expression of Nkx2-8 and TTF1 mRNA in tumors. Finally, suppressive function of Nkx2-8 was assessed via colony formation assays in five lung cancer cell lines. Thirteen of forty-five (29%) tumors had LOH. In six tumors, most adenocarcinomas, LOH was caused by gene amplification. The 0.8-Mb common region of amplification included MBIP, SFTA, TTF1, NKX2-8, and PAX9. In 4 squamous or adenosquamous cancers, LOH was caused by deletion. In three other tumors, LOH resulted from whole chromosome mechanisms (14(-), 14(+), or aneuploidy). The 1.2-Mb common region of deletion included MBIP, SFTA, TTF1, NKX2-8, PAX9, SLC25A21, and MIPOL1. Most tumors had low expression of Nkx2-8. Nevertheless, sequencing did not show NKX2-8 mutations that could explain the low expression. TTF1 overexpression, in contrast, was common and usually independent of Nkx2-8 expression. Finally, stable transfection of Nkx2-8 selectively inhibited growth of H522 lung cancer cells. 14q13.3, which contains NKX2-8, is subject to both amplification and deletion in lung cancer. Most tumors have low expression of Nkx2-8, and its expression can inhibit growth of some lung cancer cells.