Presence Of Tumor Suppressor Gene Research Articles

IL-33 stimulates the anticancer activities of eosinophils through extracellular vesicle-driven reprogramming of tumor cells

Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.

Human chromosome 3p21.3 carries TERT transcriptional regulators in pancreatic cancer

Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.

A tumor profile in Patau syndrome (trisomy 13).

Individuals with trisomic conditions like Down syndrome and Edwards syndrome are prone to certain types of malignancy. However, for Patau syndrome (constitutional trisomy 13), which occurs in 1/10,000-1/20,000 live births, the tumor profile has not been well characterized. An awareness of susceptibility to malignancies can improve care of affected individuals, as well as further our understanding of the contribution of trisomy to carcinogenesis. Therefore, we conducted an extensive review of the literature; we found 17 malignancies reported in individuals with Patau syndrome. These comprised eight embryonic tumors, three leukemias, two malignant germ cell tumors, two carcinomas, a malignant brain tumor, and a sarcoma. Benign tumors were mainly extragonadal teratomas. The small number of reported malignant tumors suggests that there is not an increased risk of cancer in the context of trisomy 13. The tumor profile in Patau syndrome differs from that observed in Edwards syndrome (trisomy 18) and Down syndrome (trisomy 21), suggesting that the supernumerary chromosome 13 could promote particular tumor formations as it does particular malformations. No general and direct relationships of tumor occurrence with organ weight, congenital malformations, histological changes, or presence of tumor suppressor genes on chromosome 13 were observed. However, some tumors were found in tissues whose growth and development are controlled by genes mapping to chromosome 13. Recent reports of successful outcomes following surgical treatment and adapted chemotherapy indicate that treatment of cancer is possible in Patau syndrome.

The molecular pathogenesis of the myelodysplastic syndromes.

Recent studies have greatly illuminated the genomic landscape of the myelodysplastic syndromes (MDS), and the pace of discovery is accelerating. The most common mutations found in MDS occur in genes involved in RNA splicing (including SF3B1, SRSF2, U2AF1, and ZRSR2) and epigenetic modification (including TET2, ASXL1, and DNMT3A). The identification of spliceosome mutations in approximately half of all patients with MDS implicates abnormalities of RNA splicing, a pathway not previously known as a target for mutation, in the MDS pathogenesis. Several regulators of signal transduction (NRAS, JAK2) and transcription factors (RUNX1, TP53) are also frequently mutated in MDS. The complex patterns of associations between gene mutations identified have revealed epistatic interactions between spliceosome components and epigenetic modifiers in MDS. The cytogenetic abnormalities found in MDS are characterized by the loss of genetic material, whereas translocations are rare. The cytogenetic deletion maps of MDS (e.g., 5q-, 7q-, 20q-) provide us with circumstantial evidence for the presence of tumor suppressor genes. It is now recognized that haploinsufficiency (a gene dosage effect) resulting from gene deletions or inactivating mutations is an important disease mechanism in MDS. Haploinsufficiency of the ribosomal protein gene RPS14 plays a critical role in the development of anemia in the 5q- syndrome, and haploinsufficiency of CUX1 is important in some patients with MDS and AML with complete or partial loss of chromosome 7. Gene expression profiling has identified key deregulated genes and pathways and new prognostic gene signatures in MDS. Recent advances in the molecular pathogenesis of MDS are leading to new biological, clinical, and therapeutic insights.

Chromosome 7q31.1 deletion in myeloid neoplasms

We studied monosomy and deletions of chromosome 7 in 208 patients with myeloid disorders; we found 39 patients (19%) with monosomy or deletion of chromosome 7: 24 patients with chromosome 7 deletion and 15 with monosomy 7. In the 24 patients with chromosome 7 deletions, studied with copy-number variants, short-tandem repeats, microsatellites, single nucleotide polymorphisms, and deletion polymorphisms, the most common deleted region was 7q31.1 (20 patients). Deletion polymorphism studies performed in these 20 patients showed an interstitial deletion of at least 140 kilobase in 6 patients; the deletion spans between the genes forkhead box P2 and Myo D family inhibitor domain containing. Because both genes do not seem to be involved in leukogenesis, we suggest to look carefully into this deletion for the presence of tumor suppressor genes and microRNAs.

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions.

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.

Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.

MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma

Multiple myeloma is characterized by frequent chromosomal alterations. Deletion of chr 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. Here, we functionally validate the role of the microRNAs-15a/16-1 cluster, centered at the deleted region, as TSGs and delineate their downstream target genes in MM. Using "sponge" lentiviral vectors to competitive stably inhibit mature microRNAs in vitro and in vivo, we have documented enhanced proliferative and invasive capacity of cells with stably inhibition of miR-16. Importantly, miR-16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load and host angiogenesis. Expression profiling analysis of miR-16-deficient cells identified a large number of downstream target genes including FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. We validated designated genes showing binding sites within the conserved 3'-UTR and also within the mRNA coding region as direct miR-16 targets, thus indicating that the miRNAs may have many more targets than anticipated by conventional prediction methods. This loss-of-function system, which mimics the 13q chromosomal deletion, provides a valuable tool to investigate their function in MM pathogenesis and their potential use as therapeutic targets.

Epigenetic Inactivation of the Thyroid Hormone Receptor β1 Gene at 3p24.2 in Lung Cancer

Frequent allelic loss on chromosome 3p in various human cancers suggests the presence of tumor suppressor genes in this region. The thyroid hormone receptor beta1 (TRbeta1) gene is located at 3p24.2, where allelic loss frequently occurs in lung cancer, and aberrant TRbeta1 methylation was observed in several human cancers. We examined the expression, mutation, and promoter methylation of TRbeta1 in 18 small cell lung cancer (SCLC) and 29 non-small cell lung cancer (NSCLC) cell lines by reverse-transcription polymerase chain reaction (RT-PCR), direct sequencing, or methylation-specific PCR. Four lung cancer cell lines lacking TRbeta1 expression were treated with 5-aza-2-deoxy-cytidine and/or trichostatin-A, and the TRbeta1 expression was determined by RT-PCR. We also examined the TRbeta1 methylation in 116 NSCLC surgical specimens and analyzed the correlation between methylation status and clinicopathological parameters or mutations of KRAS and EGFR. TRbeta1 expression was absent in 61% of SCLCs and 48% of NSCLCs, and 67% of SCLCs and 45% of NSCLCs carried TRbeta1 promoter methylation, while no somatic mutation was found in all cell lines. TRbeta1 methylation status was significantly associated with loss of TRbeta1 expression. TRbeta1 expression was restored by treatment with 5-aza-2-deoxy-cytidine and/or trichostatin-A in four cell lines. TRbeta1 methylation was found in 47% of NSCLC surgical specimens; however, the methylation was not significantly associated with any clinicopathological parameters or mutations of KRAS and EGFR. This is the first study to demonstrate epigenetic inactivation of TRbeta1 through aberrant methylation in lung cancer, while TRbeta1 mutations are not common in lung cancer.

Recurrent Novel Regions of Uniparental Disomy and Homozygous Deletions in Mycosis Fungoides Identified Using High Resolution Single Nucleotide Polymorphism Analysis.

Abstract 2644Poster Board II-620Mycosis Fungoides (MF) is the commonest form of primary cutaneous T-cell lymphoma but our understanding of the underlying molecular pathogenesis remains limited. Cytogenetic studies remain limited due to difficulties in obtaining analysable mitotic chromosomes for metaphase G-band analysis from MF patients. We have utilised high resolution 250k Affymetrix single nucleotide polymorphism (SNP) arrays to analyse tumour samples from 34 MF patients including 12 paired skin samples using and identified 156 chromosomal aberrations in ten patients (29%). Compared with previous studies using comparative genomic hybridization (CGH) in both MF and Sezary syndrome (SS), SNP array analysis detected a greater frequency of deletions and amplifications on chromosomes 1, 2, 3, 4, 5, 6, 11, and 16. Heterozygous deletions of chromosome 8 were also detected in 3 patients which have been undetected by CGH studies. A particular feature of SNP array technology is the detection of uniparental disomy (UPD) which are copy neutral regions of homozygosity. UPD (median size 32.3Mb (range: 6.6Mb – 159Mb)) was detected in 21% of our MF patients affecting either chromosome 7 (whole chromosome), 8p, 9p/9q, 10q, 12p, 14q, 16p/16q, 17q and 19q. Of note, two patients shared a common UPD region on chromosome 9p that specifically encompassed the p14 and p16 tumour suppressor genes. Thirteen regions of homozygous deletions were also observed in six patients (18%) on chromosomes 1p21.1, 7q34, 9p22.1-21.3, 10p12.1/p12.2/p14/q23.31/q23.1 and 16q23.1-q23.2. Common minimal homozygously deleted regions at 9p21-p22.3 and 16q23.1, suggest the presence of tumour suppressor gene in these regions. Interestingly, all the homozygous regions appeared within a larger region of the UPD or heterozygous deletion. Grouping patients with UPD/deletions or UPD/amplification identified eleven common minimal regions of genomic aberrations occurring in at least 3 patients; 3p24.1-14.2, 7p22.3-12.2, 7q31.1-31.2, 9p21.3-22.1, 10q21.2-26.13, 12p13.33-13.31, 13q14.2-21.1, 16p13.13, 16q23.1, 17p13.3-13.1 and 17q21.32-25.1. The detection of these recurrent abnormalities associated with UPD suggests a series of candidate genes selected for dysregulation through mutational or epigenetic modulation such as MLH, CDKN2A/B, RB1 and TP53. Paired plaque samples were also analysed from 12 patients. Of note, SNP analysis did not always show identical aberrations between these samples. Six patients had multiple aberrant regions in at least one of their samples. Of these, three patients had unique aberrations which were present in only one. This discordance in the distribution of genomic aberrations between the samples may reflect clonal evolution of the disease. To our knowledge, this is the first high resolution SNP array study in MF that has provided additional insight into the pathogenesis of MF. We have identified distinct aberrations not detected by previous studies as well as frequent and widespread UPD harbouring important oncogenes and tumour suppressor genes likely to play key roles in the dysregulation of MF cells. Disclosures:No relevant conflicts of interest to declare.

The Functional Role of Microrna 15a/16-1 as Tumor Suppressor Genes in Multiple Myeloma.

Abstract 1963Poster Board I-986 Background:Multiple Myeloma (MM), a cancer of plasma cells is characterized by frequent chromosomal alterations. Deletion of chromosome 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. When studied in the context of CLL, the miR 15a and 16-1 cluster was associated with a distinct miR signature and clinical outcome. Over-expression of miR16 caused induction of apoptosis and downregulation of the anti apoptotic gene BCL2 in a megakaryocytic leukemia cell line and induced growth arrest in MM cells. Nonetheless, being lost in CLL, MM, MCL and LPL, their functional role has not been studied using a loss-of-function approach in any of these lymphoid malignancies. Here, we describe the generation of an in vivo system for the long term, stable knockdown of miR 15a/ 16-1 expression in myeloma cells to recapitulate the conditions seen in chromosome 13q14 deleted MM. Methods:Using lentiviral vectors to stably express a competitive sponge miR16 inhibitor we set up a system to functionally validate the role of microRNA 15a/16-1 cluster. Pure populations of lentivirally transduced MM cell lines were sorted by flow cytometry using GFP marker. Decreased miRs 15a and 16 expression levels were assessed by Northern blot and R-luciferase reporter system. Cell growth rate was measured using trypan blue counting, and thymidine incorporation. Cell cycle analysis was measured by flow cytometry after staining with PI. Intracellular signal modulation was demonstrated by Western blotting. RNA from MM cell lines expressing the control sponge or sponge16 were hybridized on an Affymetrix U133A 2.0 array chip, and validated using quantitative real time PCR. Xenograft murine models were performed using the stable MM cell lines injected into 6-week old NOD.CB17-PrkdcSCID/J irradiated mice. Results:Selected stable miR knockdown MM cell lines exhibited significantly reduced expression of miRs15a/16-1 as assessd by both by mRNA levels and miR luciferase reporter assays. The knockdown cells showed a significant increase in growth rates (1.5-2 fold) compared to control cells, as measured by viable cell counts and proliferation by thymidine incorporation in vitro. Importantly, miR16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load, invasiveness and host angiogenesis. To further delineate the role of miR15a/16 in MM and to gain additional insight into the possible target genes regulated by this cluster, we performed gene expression-profiling analysis in controls and miR16 deficient MMS1 and RPMI cell lines. Since our sponge system produces downregulation of the miRs, we focused on the upregulated probes. Expression profiling analysis of miR16 deficient cells identified a surprisingly large number of downstream target-genes such as FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. These results were verified both at the mRNA level and the protein level, as well as in other MM cell lines. Moreover, we were able to show that these knockdown cells were partially addicted to some of these pathways using specific drug inhibitors. We further validated designated genes as direct miR16 targets by showing binding sites within the conserved 3' UTR and also within the mRNA coding region, thus indicating that the miRs may have many more possible targets than anticipated by conventional prediction methods. Conclusions:Using this loss-of-function system, which mimics the pleiotropic chronic effects of microRNA loss at the 13q chromosomal deletion, provides a valuable tool to investigate their function as tumor suppressor genes in MM pathogenesis, affecting multiple targets, and may represent a novel potential for therapeutic targeting in MM and other lymphoid malignancies. Disclosures:Munshi:Seattle Genetics, Inc.: Research Funding.

3q27 aberrations in a childhood ovary teratoma with associated malignant germ cell component

Cytogenetic studies of childhood ovary tumors have been poorly described. In the present article, the cytogenetic findings of an ovarian teratoma with malignant germ cell (yolk-sac) component occurring in an 8-year-old female are detailed. GTG-banding showed a karyotype of 46,XX, t(3;20)(q27;q13.3) [4]/46,XX, del3q27 [3]/46,XX [30]. Previous studies have demonstrated common sites of loss of heterozygosity at 3q27-q28 region in different types of cancer, suggesting the presence of tumor suppressor genes within this region.

The chromosome 3p21.3-encoded gene, LIMD1 , is a critical tumor suppressor involved in human lung cancer development

Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1(-/-) mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-Ras(G12D) markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell-cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.

Quantitative microsatellite analysis to delineate the commonly deleted region 1p22.3 in mantle cell lymphomas

The molecular pathogenesis of mantle cell lymphomas (MCL), a subset of B-cell non-Hodgkin's lymphomas with a poor prognosis, is still poorly understood. In addition to the characteristic primary genetic alteration t(11;14)(q13;q32), several further genetic changes are present in most cases. One of the most frequent genomic imbalances is the deletion of 1p22.1-p31.1 observed in nearly one-third of MCL cases. This might indicate the presence of tumor suppressor gene(s) in this critical region of deletion. Quantitative microsatellite analysis (QuMA) is a real-time PCR-based method to detect DNA copy number changes. Since QuMA has the resolving power to detect subtle genomic alterations, including homozygous deletions, this may help to identify candidate tumor suppressor genes from deleted regions. To gain more insight into the molecular pathogenesis of MCL, QuMA was performed on genomic DNA from 57 MCL cases. Eight microsatellite loci mapping to the chromosomal region 1p22.3 were analyzed. Losses were observed in 51 of the 57 ( approximately 89.5%) samples. Two cases showed a homozygous deletion at the locus containing the gene SH3GLB1, which plays a key role in Bax-mediated apoptosis. Two hotspots with copy number losses were detected at chromosomal localizations 85.4 and 86.6 Mb encompassing BCL10 and CLCA2. Both the genes seem to be attractive candidates to study tumor suppressor function in MCL.

Small regions of overlapping deletions on 6q26 in human astrocytic tumours identified using chromosome 6 tile path array-CGH.

Deletions of chromosome 6 are a common abnormality in diverse human malignancies including astrocytic tumours, suggesting the presence of tumour suppressor genes (TSG). In order to help identify candidate TSGs, we have constructed a chromosome 6 tile path microarray. The array contains 1,780 clones (778 P1-derived artificial chromosome and 1,002 bacterial artificial chromosome) that cover 98.3% of the published chromosome 6 sequences. A total of 104 adult astrocytic tumours (10 diffuse astrocytomas, 30 anaplastic astrocytomas (AA), 64 glioblastomas (GB)) were analysed using this array. Single copy number change was successfully detected and the result was in general concordant with a microsatellite analysis. The pattern of copy number change was complex with multiple interstitial deletions/gains. However, a predominance of telomeric 6q deletions was seen. Two small common and overlapping regions of deletion at 6q26 were identified. One was 1,002 kb in size and contained PACRG and QKI, while the second was 199 kb and harbours a single gene, ARID1B. The data show that the chromosome 6 tile path array is useful in mapping copy number changes with high resolution and accuracy. We confirmed the high frequency of chromosome 6 deletions in AA and GB, and identified two novel commonly deleted regions that may harbour TSGs.

Loss of heterozygosity at 17p13.3-ter, distal to TP53, correlates with negative hormonal phenotype in sporadic breast cancer

Genetic alterations on chromosome 17p are frequent in a variety of human malignancies such as sporadic breast carcinomas. The clinico-pathological significance of it remains to be elucidated. The purpose of this study was to explore whether the allelic loss (LOH) in 17p13.3, which is suggested by the presence of tumour suppressor genes, independent of TP53, are related to current clinico-pathological criteria for characterising breast cancer. A group of sporadic breast carcinomas, with no alteration in TP53 locus, were analysed for the presence of LOH in D17S34 and D17S30/5 loci, mapped to the 17p13.3 region, distinct from and telomeric to TP53. LOH by at least one marker was observed in 13 of 47 informative cases (27.6%). Clinicopathological parameters such as age, menopausal status, histological type, tumour size, nodal status, grading, ploidy, labelling index, S-phase fraction and hormonal phenotype, were evaluated. LOH at distal 17p13.3 significantly correlated with the absence of oestrogen receptors (ER) (P=0.018), progesterone receptors (PgR) (P=0.009) and concordant absence of either ER or PgR (P=0.006). This may provide a basis for speculation as to the function of the putative tumour suppressor genes in 17p13-ter in sporadic breast cancer.

Frequent allelic imbalances at 8p and 11q22 in oral and oropharyngeal epithelial dysplastic lesions

Allelic imbalance is characteristic of oral squamous cell carcinoma (SCC) and contributes to the tumorigenesis of this disease. Our previous studies suggest that chromosome regions 8p and 11q22.2∼q22.3 are frequent sites of loss of heterozygosity (LOH) in head and neck SCC. Here, we explored the allelic imbalance pattern of these regions in 27 cases of oral epithelial dysplastic lesions. A previously reported frequent LOH (9p21) in head and neck dysplasia was also examined. Laser capture microdissection (LCM) technology was utilized to harvest homogenous cell populations from archived clinical tissues and thus greatly enhancing the sensitivity, accuracy and reliability of genetic assessment. The allelic imbalance (LOH and microsatellite instability) on 8p, 11q22.2∼q22.3, and 9p21 were observed at one or more loci in 66.7%, 63.0%, and 63.0% of cases, respectively. Our results demonstrate that 8p, 11q22.2∼q22.3, and 9p21 are frequent allelic imbalance regions in oral premalignant dysplasia and suggest the presence of tumor suppressor genes in these regions.

Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas

Frequent allelic losses on the long arm of chromosome 22 (22q) in gliomas indicate the presence of tumor suppressor gene (TSG) at this location. However, the target gene(s) residing in this chromosome are still unknown and their putative roles in the development of astrocytic tumors, especially in secondary glioblastoma, have not yet been defined. To compile a precise physical map for the region of common deletions in astrocytic tumors, we performed a high-density loss of heterozygosity (LOH) analysis using 31 polymorphic microsatellite markers spanning 22q in a series of grade II diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas, and secondary glioblastomas that had evolved from lower grade astrocytomas. LOH was found at one or more loci in 33% (12/36) of grade II diffuse astrocytomas, in 40% (4/10) of anaplastic astrocytomas, in 41% (26/64) of primary glioblastomas, and in 82% (23/28) of secondary glioblastomas. Characterization of the 22q deletions in primary glioblastomas identified two sites of minimally deleted regions at 22q12.3–13.2 and 22q13.31. Interestingly, 22 of 23 secondary glioblastomas affected shared a deletion in the same small (957 kb) region of 22q12.3, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. Investigation of the promoter methylation and expression of this gene indicated that frequent hypermethylation correlated with loss of TIMP-3 expression in secondary glioblastoma. This epigenetic change was significantly correlated to poor survival in eight patients with grade II diffuse astrocytoma. Our results suggest that a 957 kb locus, located at 22q12.3, may contain the putative TSG, TIMP-3, that appears to be relevant to progression to secondary glioblastoma and subsequently to the prognosis of grade II diffuse astrocytoma. In addition, the possibility of other putative TSGs on 22q12.3–13.2 and 22q13.31 that may also be involved in the development of primary glioblastomas cannot be ruled out.

A Novel Fusion, KIAA0379 (Ankyrin Repeat Domain 28) to NUP98 in an Adult MDS/AML with a t(3;5;11)(p25;q35;p15).

In hematologic malignancies, deletion of 3p25-26 is a rare but recurrent cytogenetic aberration, indicating the presence of tumor suppressor gene (TSG) on this choromosome arm.We revealed a t(3;5)(p25;q35) that was identified by conventional cytogenetics results in a novel fusion of KIAA0379 on 3p25 to NUP98 on 11p15 in a patient with refractory MDS/AML by the molecular analysis. Fluorescence in situ hybridization (FISH) analysis using whole chromosomal painting and telomeric probes unclosed a criptic three-way translocation t(3;5;11)(p25;q35;p15). FISH restriction using BAC contig revealed that the 3p25 breakpoint is involved in KIAA0379 which locates between BAC 792N12 and 259O19. Split signals of BAC 265K23 for 5q35 and 348A20 for 11p15 showed that NSD1 on 5q35 and NUP98 on 11p15 are the responsible genes of this translocation. As the results of this translocation, expression of three chimeric transcripts were identified by RT-PCR. In KIAA0379-NUP98, exon 18 of KIAA0379 was fused in-flame with exon 13 of NUP98. In NUP98-NSD1, exon 12 of NUP98 was fused in-flame with exon 7 of NSD1. NSD1-KIAA0379 resulted in out-flame fusion. To investigate the expression of KIAA0379 in leukemic cells, RT-PCR for full-length KIAA0379 transcript was performed on bone marrow cells from the patient and a healthy volunteer, and leukemic cell line K562, HL-60 and KG-1a cells. Full-lengh PCR product with expected size was found in all samples, however, short variant transcript was amplified significantly from KG-1a and HL-60 cells. Subsequent sequence analysis of the short PCR product from KG-1a cells revealed a direct fusion of a part of exon 10 to a part of exon 28 without any hidden splicing sites. Southern blot analysis using a cloned KIAA0379 cDNA fragment suggested an internal deletion of KIAA0379 in KG-1a cells. KIAA0379 locates at 3p25.1 about 5.5Mb centromeric to VHL and is predicted to have 28 ankyrin repeat motifs by computational analysis. It is thought to be involved in protein to protein interaction whereas its precise funtion remains unclear. Immunostaining for KIAA0379 protein in NIH3T3 cells transfected with the wild KIAA0379 and the short variant KIAA0379 revealed that the wild KIAA0379 protein localized in cytoplasm and nuclear membrane, on the other hand, the truncated KIAA0379 protein localized exclusively on nuclear membrane. KIAA0379-NUP98 fusion protein is expected to localize on nuclear membrane by sub-localization signal of C-terminal of NUP98. Therefore, dysfunction of KIAA0379 protein might lead to leukemogenesis and KIAA0379 may be potential candidate for a novel TSG. In addition, it has been reported that t(5;11)(q35;p15.5) results in NUP98-NSD1 fusion in childhood AML. To our knowledge, this is the first report in adult case.

Systematic multiplex polymerase chain reaction and reverse transcription‐polymerase chain reaction analyses of changes in copy number and expression of proto‐oncogenes and tumor suppressor genes in cancer tissues and cell lines

Systematic multiplex reverse transcription-polymerase chain reaction (SM RT-PCR) is distinguishable from other multiplex RT-PCR methods by (i) utilization of primers that amplify sequences that fall within a single exon of the genes, (ii) utilization of genomic DNA as a calibration standard, and (iii) optimized PCR conditions that allow amplification of bands of similar intensity using genomic DNA template. We previously developed the human experimental systems of 68 glycosyltransferase genes, 39 Hox genes, and 26 integrin subunit genes, and analyzed the expression of those genes in human adult tissues. Here we report the establishment of an SM RT-PCR system of proto-oncogenes and tumor suppressor genes and the analysis of gene expression in human cancer tissues and cell lines. We also demonstrate that the SM RT-PCR system, which was developed for cDNA expression analysis, could also be used successfully for more exquisite analysis of copy number changes in genomic DNA. We observed a decrease in band intensity of HRAS, TP73, CDKN2A, and CDKN2B genes in most of the breast and prostate cancer cell lines examined. The decrease in copy number of HRAS proto-oncogene leads us to suspect the presence of tumor suppressor genes in the vicinity of this gene on chromosome 11p15.5.