Tumor Suppressor Gene FHIT Research Articles

Recent advances in small cell lung cancer: the future is now?

Small cell lung cancer is a relevant clinical issue as it is a highly malignant cancer, often diagnosed in advanced stage. Similarly to non-small cell lung cancer, tobacco smoking is currently the main risk factor. Its incidence, at least in males, has declined over the past decades, due to the worldwide decreased percentage of active smokers. The typical small cells of this tumor type are characterized by a high Proliferation Index, chromosomal deletions such as 3p(14-23) involving the tumor-suppressor gene FHIT, alterations of the MYC or Notch family proteins and the frequent expression of neuroendocrine markers. The combination of thoracic radiotherapy and chemotherapy is the standard treatment for limited stage disease, while platinum-based chemotherapy is the most effective choice for extensive stage disease. Unfortunately, whatever chemotherapy is used, the results are disappointing. No regimen has proved to be effective in the long run, indeed small cell lung cancer rapidly progresses after a frequent initial strong response, and the mortality rate remains still high. The advent of immunotherapy is actually changing the landscape in oncology. As well as in other cancers, recent trials have demonstrated the efficacy of the combination of immune checkpoint inhibitors and chemotherapy, opening new perspectives for the future of our patients.

Synthesis of Fluorescent Probes Targeting Tumor-Suppressor Protein FHIT and Identification of Apoptosis-Inducing FHIT Inhibitors.

For the early diagnosis of cancer, leading to a better chance of full recovery, marker genes whose expression is already altered in precancerous lesions are desirable, and the tumor-suppressor gene FHIT is one candidate. The gene product, FHIT protein, has a unique dinucleoside triphosphate hydrolase (AP3Aase) activity, and in this study, we designed and synthesized a series of FHIT fluorescent probes utilizing this activity. We optimized the probe structure for high and specific reactivity with FHIT and applied the optimized probe in a screening assay for FHIT inhibitors. Screening of a compound library with this assay identified several hits. Structural development of a hit compound afforded potent FHIT inhibitors. These inhibitors induce apoptosis in FHIT-expressing cancers via caspase activation. Our results support the idea that FHIT binders, no matter whether inhibitors or agonists of AP3Aase activity, might be promising anticancer agents.

Recent advances in small cell lung cancer: the future is now?

Small cell lung cancer is a relevant clinical issue as it is a highly malignant cancer, often diagnosed in advanced stage. Similarly to non-small cell lung cancer, tobacco smoking is currently the main risk factor. Its incidence, at least in males, has declined over the past decades, due to the worldwide decreased percentage of active smokers. The typical small cells of this tumor type are characterized by a high proliferation index, chromosomal deletions such as 3p(14-23) involving the tumor-suppressor gene FHIT, alterations of the MYC or Notch family proteins and the frequent expression of neuroendocrine markers. The combination of thoracic radiotherapy and chemotherapy is the standard treatment for limited stage disease, while platinum-based chemotherapy is the most effective choice for extensive stage disease. Unfortunately, whatever chemotherapy is used, the results are disappointing. No regimen has proved to be effective in the long run, indeed small cell lung cancer rapidly progresses after a frequent initial strong response, and the mortality rate remains still high. The advent of immunotherapy is actually changing the landscape in oncology. As well as in other cancers, recent trials have demonstrated the efficacy of the combination of immune checkpoint inhibitors and chemotherapy, opening new perspectives for the future of our patients.

Comprehensive, integrated, and phased whole-genome analysis of the primary ENCODE cell line K562.

K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and indels (both corrected for CN in aneuploid regions), loss of heterozygosity, megabase-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs), including small and large-scale complex SVs and nonreference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT. Taking aneuploidy into account, we reanalyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.

Interaction between polycyclic aromatic hydrocarbons and p16, FHIT gene CpG island methylation in patients with cervical intraepithelial neoplasias

Objective: To explore the effect of polycyclic aromatic hydrocarbons (PAHs) and p16, FHIT gene CpG island methylation, as well as their interaction in cervical intraepithelial neoplasias. Methods: Objects of this study were from a cohort of cervical lesions study in Yangqu county of Shanxi province. All the patients were diagnosed pathologically, that including 83 patients with high-grade cervical intraepithelial neoplasia (CINⅡ/Ⅲ), 86 patients with low-grade cervical intraepithelial neoplasia (CINⅠ) and another 91 women under normal cervical (NC) condition. 1-hydroxy pyrene in the urine was detected by high performance liquid chromatography (HPLC) while CpG island methylation status of tumor suppressor gene p16 and FHIT were measured by methylation-specifc polymerase chain reaction (MSP). Data were analyzed with Kruskal-Wallis H test, chi-square test and trend of chi-square test. Logistic regression models were used to estimate the odds ratio (OR) and corresponding 95% confidence intervals (95%CI) between influencing factors and the cervical disease by using the SPSS statistical software (version 20.0). The interaction under study was evaluated by using the generalized multifactor dimensionality reduction (GMDR) model. Results: Level of 1-hydroxy pyrene (H=50.743, P<0.001) and the high exposure rate of 1-hydroxy pyrene (trend χ(2)=20.146, P<0.001) were gradually increasing along with the severity of cervical intraepithelial neoplasia. The CpG island methylation rates of p16, FHIT in CINⅠand CINⅡ/Ⅲ group were higher than that in NC group, and gradually increasing along with the severity of cervical intraepithelial neoplasia (trend χ(2)=9.75, P=0.002; trend χ(2)=10.39, P=0.001). Results from the GMDR model showed that interaction existed among the high exposure of 1-hydroxy pyrene and the CpG island methylation of p16, FHIT in CINⅠ and CINⅡ/Ⅲ group. Conclusion: Under the high exposure of 1-hydroxy pyrene and the CpG island methylation of p16, FHIT appeared to have increased the risk of cervical intraepithelial neoplasia and causing synergistic effect in cervical intraepithelial neoplasia.

Abstract 4833: Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration

Abstract In human cervical carcinogenesis, the gain of the long arm of chromosome 3 is the decisive genetic event that determines progression of dysplasia to invasive disease. Other recurrent genomic imbalances, such as gains on 1q, 5p, 8q, and 20q, and losses on 2p, 3p, and 11q are also observed. To better understand the patterns of clonal evolution during disease progression we developed a multiplex FISH assay with three multicolor probe panels for sequential hybridizations of cervical intraepithelial neoplasia (CIN3) and invasive cervical carcinoma (ICC), including synchronous CIN3/ICC lesions. Our FISH assay targets oncogenes COX2 (1q), TERC (3q), TERT (5p), MYC (8q), ZNF217 (20q), and tumor suppressor genes ING5 (2p), FHIT (3p), CHEK1 (11q), and centromere 7 as a ploidy control. Our results confirmed the dominant role of the gain of 3q: nineteen out of twenty lesions showed a major clonal population with TERC amplification. Nine out of the 20 lesions showed a concomitant loss of FHIT (3p) suggesting an isochromosome 3q formation, which makes the loss of FHIT the second most frequent clonal event in this group of samples. Other imbalances were present, but, as expected, less common. The copy number increases of 3q can occur either in diploid cells or after tetraploidization of the genome. Interestingly, in our samples only three CIN3 lesions showed major clones with signal patterns consistent with near-tetraploidy, while all other lesions, including all ICC lesions and the two synchronous CIN3/ICC lesions showed major signal patterns consistent with near-diploid cell populations. Although tetraploidization can occur already in premalignant lesions, and there is independent evidence that such chromosome doublings increase the risk of chromosome segregation errors, and therefore promote the subsequent acquisition of genomic imbalances, our data indicate that a majority of cervical precancerous and cancerous lesions acquire aberrations in a diploid genome. Independent of whether the 3q gain and other aberrations are acquired in a diploid or tetraploid background, it selects cells with a proliferative advantage, which is supported by our observations that (i) cells with identical patterns of 3q gain are adjacent to each other, hence indicating a clonal proliferation, and (ii) that the only commonality between the major clones in the synchronous CIN3 and ICC lesions was a TERC gain. We further examined overall cell-to-cell heterogeneity in CIN3 versus ICC samples. While overall levels of clonal heterogeneity were indistinguishable between CIN3 and ICC, phylogenetic models of single tumors suggested greater average numbers of mutations per cell in ICC versus CIN3. These findings improve our understanding of clonal development during cervical carcinogenesis and with that our understanding of genome dynamics at early stages of carcinogenesis. Citation Format: Leanora S. Hernandez, Amanda Bradley, Timo Gaiser, Sonia Andersson, E. Michael Gertz, Salim A. Chowdhury, Russel Schwartz, Alejandro Schäffer, Kerstin Heselmeyer-Haddad, Thomas Ried. Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2015-4833

Abstract 2303: Decitabine impact on immunohistochemistry scores for tumor suppressor genes FHIT, WWOX, FUS1 and PTEN in human tumor samples

Abstract Purpose: Since tumor suppressor gene (TSG) expression may be lost through promoter hypermethylation, we assessed whether the demethylating agent decitabine could increase TSG expression in tumor tissues of patients receiving decitabine therapeutically. Experimental Design: Patients on a phase I trial received intravenous 1-hour infusions of decitabine 2.5, 5, or 10 mg/m2/day on days 1-5 and 8-12 each 4-week cycle or 15 or 20 mg/m2/day on days 1-5 each cycle, with filgrastim added at higher doses. Tumor biopsies were done pre day 1 and on day 12 of the first cycle. For the putative TSGs FHIT, WWOX, FUS1 and PTEN, immunohistochemistry (IHC) scores (0-300) were calculated by multiplying the % tumor cells staining by the intensity (0-3+) of staining. IHC scores were correlated with methylation of the LINE-1 repetitive element (as a marker of global DNA methylation, determined by pyrosequencing) and with tumor regression. Results: Twenty-five patients had at least one pre- or post-decitabine biopsy evaluable for expression of at least 1 TSG, including 4 patients with breast cancers, 3 kidney, 3 head &amp; neck, 4 melanomas, 3 thymic and 8 others. With negative staining pre-decitabine (score = 0), number of patients converting to positive staining post-decitabine was 1 of 1 for FHIT, 3 of 6 for WWOX, 2 of 3 for FUS1 and 1 of 10 for PTEN. In tumors with low pre-decitabine TSG scores (&amp;lt;150), expression was higher post-treatment in 8 of 8 cases for FHIT (p=0.014, by Wilcoxon signed rank tests for paired comparisons), 7 of 17 for WWOX (p=0.0547), 7 of 12 for FUS1 (p=0.0726), and 1 of 16 for PTEN (p=0.2034). If FHIT, WWOX and FUS1 were considered together, median pre- vs post-decitabine scores were 60 vs 100 (p=0.0002). Overall, TSG scores did not correlate with LINE-1 methylation, but if pre-decitabine scores for FHIT, FUS1 and WWOX were considered together, tumors with IHC scores = 0 for one of these genes (8 observations) had higher pre-decitabine % LINE-1 methylation than did tumors with IHC scores &amp;gt;0 (58 observations) (median 61.6% vs 45.4%, p=0.0481). TSG expression change did not correlate with LINE-1 methylation change, although tumors converting from negative to positive had a median decrease in LINE-1 methylation of 24%, compared to 6% in those not converting (p=0.069). Five of 15 fully evaluable patients had reductions in tumor diameter (by 0.2% to 33.4%). Of these, 3 had simultaneous increases in 3 TSGs (including the 2 patients with the greatest tumor regression) compared to 2 of 10 with tumor growth (p=0.25). Conclusions: In tumors with low TSG expression, decitabine may be associated with increased expression for the TSGs FHIT, WWOX &amp; FUS1, but not PTEN. Further study will be needed to determine if increased TSG expression is associated with reduced DNA methylation and to determine if increased TSG expression may contribute to any decitabine therapeutic effect. Citation Format: David J. Stewart, Maria I. Nunez, Jaroslav Jelinek, David Hong, Sanjay Gupta, C. Marcelo Aldaz, Jean-Pierre Issa, Razelle Kurzrock, Ignacio I. Wistuba. Decitabine impact on immunohistochemistry scores for tumor suppressor genes FHIT, WWOX, FUS1 and PTEN in human tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2303. doi:10.1158/1538-7445.AM2014-2303

Identification of a Tumor-Suppressive Human-Specific MicroRNA within theFHITTumor-Suppressor Gene

Loss or attenuated expression of the tumor-suppressor gene FHIT is associated paradoxically with poor progression of human tumors. Fhit promotes apoptosis and regulates reactive oxygen species; however, the mechanism by which Fhit inhibits tumor growth in animals remains unclear. In this study, we used a multidisciplinary approach based on bioinformatics, small RNA library screening, human tissue analysis, and a xenograft mouse model to identify a novel member of the miR-548 family in the fourth intron of the human FHIT gene. Characterization of this human-specific microRNA illustrates the importance of this class of microRNAs in tumor suppression and may influence interpretation of Fhit action in human cancer.

Genetic instability of the tumor suppressor gene FHIT in normal human cells

Common fragile sites are hotspots for chromosome instability and co-localize to cancer genomic rearrangements. Whether these loci may be considered stable in human subjects under physiological conditions remains an open question. Here we show by molecular combing that a small but significant percentage of normal human cells carry an abnormal sequence pattern within the tumor suppressor gene FHIT (3p14.2) at FRA3B. Each sequence variation represents a unique pattern within a normal cell population, and therefore it would remain undetected or not interpreted by genome-wide analyses. Remarkably, the region is the same as in FHIT rearrangements described in tumors. By analyses on several normal cell lines (proliferating and resting primary lymphocytes, primary fibroblasts, lymphoblastoid cells including clonal cell cultures) we verified that: (a) each cell type displays altered sequence patterns at FHIT; (b) the presence of abnormal sequence patterns is specific for the FHIT locus; and (c) FHIT instability occurs de novo during cell proliferation, and heterogeneous sequence variants progressively accumulate in the cell populations. FHIT has been widely investigated in cancer cells, but to our knowledge this is the first direct evidence of spontaneous and recurrent occurrence of genomic instability at this gene in human subjects, at the same region involved in cancer rearrangements. Our results suggest that common fragile site activity is not restricted to in vitro cell culture and that genomic instability may pre-exist in normal cells in the absence of exogenous replication stress.

Abstract 5121: Acquired inactivating ARID2 mutations in lung non small cell carcinoma

Abstract The genetic alterations involved in the development lung adenocarcinoma (ADC) have been widely characterized allowing the identification of the most frequent oncogenes or tumor suppressor genes linked to this malignancy. Most studies have focus on proliferation pathway, anti-apoptotic factors or cell cycle alterations. Here we show that alterations in chromatin remodeling complexes are frequent in lung ADC. Chromatin structure plays a key role in the regulation of tissue-specific gene expression during development and differentiation therefore it is not surprising that proteins implicated in remodeling complexes play a role in cancer development. For example, mutations have been found in genes belonging to the SWI/SNF complex in different tumors. In lung cancer BRG1 knockout was shown to potentiate tumor development and mutations were recently identified in tumors by the use of a very sensitive method: massive parallel DNA pyrosequencing that suggests the presence of the alteration in cell subclones. In the course of identifying novel chromatin remodeling complex genes implicated in lung ADC development we analyzed a series of 82 lung adenocarcinomas by SNP array and looked for the presence of homozygous deletions on the entire genome. Three regions of homozygous deletions in known tumor suppressor genes CDKN2A, FHIT and PTEN and in JARID2, which is involved in chromatin remodeling, were found in more than 2 samples. Another ARID-domain containing gene; ARID2 was found deleted in one sample and we therefore decided to screen both ARID-domain genes for mutations by direct sequencing on cryopreserved tumor tissues. One missense somatic mutation was found in JARID2 and 6/82 tumors had ARID2 alterations predicted to inactivate the protein in at least 4 out of 6 samples. Indeed 3 mutations were frameshift or non-sense, one specimen had a homozygous deletion and 4 tumors demonstrated loss of heterozygosity associated to the mutation. Our results indicate that ARID2 inactivation is a common feature of lung cancer development and provide new insight on the impact of chromatin remodeling alteration in lung ADC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5121. doi:1538-7445.AM2012-5121

Abstract 885: Integrated analysis of colorectal cancer cell lines reveals an amplicon driver which may be involved in platinium resistance

Abstract Background: First line adjuvant treatment for colorectal cancer (CRC) is 5-fluorouracil and the platinum compound oxaliplatin. New targeted therapies such as cetuximab and PI3K/mTOR inhibitors are being developed in order to improve therapeutic outcomes. However, intrinsic and acquired resistance is common and selecting patients who are likely to respond remains a clinical and pathological challenge. The aim of this study is to identify new targets for therapy using an integrated genomic, transcriptomic, proteomic, and functional analysis of CRC cell lines. Methods: Cell proliferation assays (AlamarBlue) were carried out on a panel 15 colorectal cancer cell lines. IC50s were established for 5-fluoruracil, oxaliplatin, and BEZ-235, a PI3K/mTOR inhibitor. Cell lines were profiled using reverse phase protein arrays (RPPA) for protein expression, Illumina gene expression analysis, array comparative genomic hybridization (aCGH; NimbleGen 135k chips) and targeted sequencing of hotspot mutations. Associations of mutations, transcript, copy number variations and proteome with drug sensitivity were established using correlation coefficients and integrated network analysis. Validation of copy number gains and losses was performed in primary tumors using Oncomine to interrogate public databases. Results: IC50s varied from resistant to highly sensitive for all three compounds. BRAF V600E mutation was significantly associated with response to 5 fluorouracil (p = 0.006). There were 17 regions of recurrent amplification across 14 cell lines and 48 regions of loss. The frequently and highly amplified regions included 8q24.13 - 8q24.21 which was significantly correlated with oxaliplatin response (p = 0.031), and amplified in 11% of primary CRCs on interrogation of public databases. This amplified region contains the putative oncogene TRIB1, a cell signaling regulator, which was correlated with gene expression across the cell lines and was found to be significantly correlated to the AKT (p=0.026), phosphor-mTOR (Ser2481) (p = 0.017) and phosphoSRC (Thr24) (p = 0.020) protein expression. The recurrently deleted regions included 3p14.2 which was significantly associated with response to the PI3K/mTOR inhibitor BEZ235 (p=0.031). This region was found to be significantly correlated with MEK (p=0.042) and phosphoCDK2 (Thr160) (p=0.018) protein expression, and contained the putative tumor suppressor gene FHIT, a hydrolase involved with purine metabolism. Conclusions: TRIB1 is a potential oncogenic driver in CRC which may be associated with therapeutic resistance to platinum compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 885. doi:1538-7445.AM2012-885

Tumor suppressor genes FHIT and WWOX are deleted in primary effusion lymphoma (PEL) cell lines

AbstractPrimary effusion lymphoma (PEL) is a diffuse-large B-cell lymphoma with poor prognosis. One hundred percent of PELs carry the genome of Kaposi sarcoma–associated herpesvirus and a majority are coinfected with Epstein-Barr virus (EBV). We profiled genomic aberrations in PEL cells using the Affymetrix 6.0 SNP array. This identified for the first time individual genes that are altered in PEL cells. Eleven of 13 samples (85%) were deleted for the fragile site tumor suppressors WWOX and FHIT. Alterations were also observed in the DERL1, ETV1, RASA4, TPK1, TRIM56, and VPS41 genes, which are yet to be characterized for their roles in cancer. Coinfection with EBV was associated with significantly fewer gross genomic aberrations, and PEL could be segregated into EBV-positive and EBV-negative clusters on the basis of host chromosome alterations. This suggests a model in which both host genetic aberrations and the 2 viruses contribute to the PEL phenotype.

Common Fragile Site Tumor Suppressor Genes and Corresponding MouseModels of Cancer

Chromosomal common fragile sites (CFSs) are specific mammalian genomic regions that show an increased frequency of gaps and breaks when cells are exposed to replication stress in vitro. CFSs are also consistently involved in chromosomal abnormalities in vivo related to cancer. Interestingly, several CFSs contain one or more tumor suppressor genes whose structure and function are often affected by chromosomal fragility. The two most active fragile sites in the human genome are FRA3B and FRA16D where the tumor suppressor genes FHIT and WWOX are located, respectively. The best approach to study tumorigenic effects of altered tumor suppressors located at CFSs in vivo is to generate mouse models in which these genes are inactivated. This paper summarizes our present knowledge on mouse models of cancer generated by knocking out tumor suppressors of CFS.

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.

Clonal heterogeneity in childhood myelodysplastic syndromes—Challenge for the detection of chromosomal imbalances by array‐CGH

To evaluate whether copy number alterations (CNAs) are present that may contribute to disease development and/or progression of childhood myelodysplastic syndromes (MDS), 36 pediatric MDS patients were analyzed using array-based comparative genome hybridization (aCGH). In addition to monosomy 7, the most frequent chromosome aberration in childhood MDS, novel recurrent CNAs were detected. They included a loss of 3p14.3-p12.3, which contains the putative tumor suppressor gene FHIT, a loss of 7p21.3-p15.3, a loss of 9q33.3-q34.3 (D184) and microdeletions in 17p11.2, 6q23 containing MYB, and 17p13 containing TP53. In this small patient cohort, patients without CNA, patients with monosomy 7 only and patients with one CNA in addition to monosomy 7 did not differ in their survival. As expected, all patients with complex karyotypes, including two patients with deletions of TP53, died. A challenge inherent to aCGH analysis of MDS is the low percentage of tumor cells. We evaluated several approaches to overcome this limitation. Genomic profiles from isolated granulocytes were of higher quality than those from bone marrow mononuclear cells. Decreased breakpoint calling stringency increased recognition of CNAs present in small clonal populations. However, further analysis using a custom-designed array showed that these CNAs often did not confirm the findings from 244k arrays. In contrast, constitutional CNVs were reliably detected on both arrays. Moreover, aCGH on amplified DNA from distinct myeloid clusters is a new approach to determine CNAs in small subpopulations. Our results clearly emphasize the need to verify array-CGH results by independent methods like FISH or quantitative PCR.

Concordance of DNA methylation pattern in plasma and tumor DNA of Egyptian hepatocellular carcinoma patients

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the fourth leading cause of cancer mortality globally. HCC incidence has doubled in Egypt in the past 10 years, which could be attributed to the high prevalence of hepatitis C virus (HCV) and hepatitis B virus (HBV), although HBV rates have declined after the introduction of the vaccine in 1992. Aberrant DNA methylation may play an important role in hepatocarcinogenesis. Liver biopsy is the current gold standard for methylation studies; however, imaging techniques often suffice for diagnosis making tissue samples increasingly scarce. The efficacy of conducting DNA methylation studies in molecular epidemiology using plasma DNA is still unclear. We compared tumor methylation profile for the tumor suppressor genes APC, FHIT, p15, p16, and E-cadherin in tumor tissues and plasma to test the concordance between the two types of specimen from the same HCC patients. Twenty-eight HCC patients with matching tissue and plasma DNA were recruited from a case—control study in Gharbiah, Egypt. Concordance between the tissue and plasma was statistically significant in all five genes as follows: APC (23/28, 82.1%, p=0.001), FHIT (24/28, 85.7%, p=.0001), p15 (25/28, 89.2%, p=0.045), p16 (19/28, 67.9%, p=0.037), and E-cadherin (22/28, 78.5%, p=0.0008). The average specificity was 90%, 86%, 96%, 86%, and 100%, respectively. There was no significant association between methylation and hepatitis viral infection for any of the genes tested in this study. Plasma DNA can be reliable for testing methylation profile in liver cancer patients in this population. Future studies on a larger sample size should investigate methylation profile in populations with higher rates of HBV, HCV, and other risk factors.

Methylation of the PTEN Promoter Leads to PTEN Inactivation in Multiple Myeloma

Aberrant methylation of CpG island of the promoter regions of genes is an important epigenetic mechanism, alternative to deletions or mutations, that cause the silencing of genes involved in carcinogenesis. We therefore investigated epigenetic events involved in multiple myeloma by screening 6 genes of interest for evidence of promoter hypermethylation.PATIENTS AND METHODS: The methylation patterns of the tumor suppressor genes p16INK4a(p16), E-cadherin (ECAD), FHIT and PTEN (negative regulator of AKT/PKB signalling pathway), Wnt signalling pathway antagonist genes WIF-1 and DKK-3 were determined in the bone marrow aspirates of 50 patients with Multiple Myeloma (MM) (37 at diagnosis; male 22; female 28; 68.8 median age. ISS stage: 47% stage I, 8% stage II and 45% stage III.), using the methylation-specific polymerase chain reaction after DNA bisulphite modification. Ten patients with monoclonal gammopathy of undetermined significance (MGUS) and 4 peripheral blood from controls were also evaluated. For statistical analysis Student t and Chi squared or Fisher exact tests were used.RESULTS: We found at least one hypermethylated gene in all MM patients. Gene methylation frequencies varied from 6% to 74%. ECAD, FHIT and p16 genes demonstrated a relatively high frequency of aberrant methylation with frequencies of 74%, 69% and 42%, respectively. WIF-1, PTEN, DKK-3 genes showed a low frequency of methylation with values of 28%, 14%, 6%, respectively. The methylation frequencies detected in MGUS were 91.6% for ECAD and FHIT, 41.6% for WIF-1, 16,6% for p16 and DKK3. The difference in methylation profile between MM and MGUS was statistically significant (X2= 37, df=5; p=0.0000). PTEN was found hypermethylated in 7/50 (14%) MM but none of MGUS or control samples were methylated. Of these 6 patients had IgG K isotype, 4 had abnormal cariotype with 13q deletion, one hyperdiploidia, one IgH rearrangements. Two patients were simultaneously hypermethylated in p16 gene promoter. No statistically significant correlation between methylation data and clinical parameters: gender, age, isotype of M component, type of light chain, haemoglobin, serum albumin level, calcium, β2 microglobulin, serum creatinine level, LDH, lytic bone lesions were found for any of examined genes. When correlation with Durie-Salmon Stage disease was tested hypermethylation of p16 and WIF-1 resulted more frequently associated with stage IIIA/B (p values 0.006 and 0.000, respectively). When gene methylation status and cytogenetics abnormalities were compared, we found that aberrant methylation of p16 and ECAD were significantly more frequent in MM patients with chromosome 13 abnormalities as sole entity or with 13q deletion associated to IgH translocations. By contrast, promoter hypermethylation of Wnt signalling antagonist genes was associated with 13q deletion co-occurring with a hyperdiploide cariotype. (X2=17; df=8 p value: 0.000). According to the number of methylated genes observed in each sample 48% MM had 3–4 hypermethylated genes. When differences between methylator phenotype and cytogenetics abnormalities were analyzed we found that 3–4 hypermethylated genes have the tendency to be associated with chromosome 13 abnormalities more often than with hyperdiploide cariotype (p value 0.05).CONCLUSION: Our results indicate that the accumulation of epigenetic events affecting genes regulating cell cycle control, cell adhesion and apoptosis is a common phenomenon in plasma cell disorders and may further contribute to the phenotype of MM cells. This is the first demonstration of PTEN inactivation as a result of promoter hypermethylation in MM patients. Since talidomide seems to have a role in the PTEN/PI3K/AKT pathway, the PTEN function is worthy to be further investigated in Multiple Myeloma.

PD6-1-5: Dose-response relationships in extensive small cell lung cancer and implications for resistance mechanisms.

Copyright © 2007 by the International Association for the Study of Lung Cancer S426 Restoration of the tumor suppressor genes P53, Retinoblastoma, FHIT and FUS1 had only minimal effect on cell death, although the tumor suppressors are functional defective in >80 % of SCLC. Modulating the oncogene activity of Bcl-2 was attempted by introducing a truncated version of Bax, with increased apoptotic activity. Inhibition of Hsp70-mediated cancer survival was attempted by the introduction of Hsp40 J domain, a dominant negative gene inhibiting Hsp70 activity. Although the gene products activated cellular death pathways in transfected cells, no significant cell death was observed. In contrast suicide gene strategies showed great potential for SCLC gene therapy. The suicide gene system herpes simplex virus thymidine kinase (HSVtk) with the prodrug ganciclovir (GCV) had already been tested and implemented at the laboratory showing >50 % cell death in vitro and tumor regression in vivo. HSVtk efficacy was improved by the replacement of GCV with penciclovir (PCV), which demonstrated significant less toxicity to untransfected cells. Another suicide gene system was implemented for testing: the yeast cytosine deaminase (YCD)gene either alone or fused with the yeast uracil phosphoribosyltransferase (YUPRT) gene and combined with the prodrug 5-FC. The YCD/YUPRT fusion gene showed superior efficacy, mediating 90 % SCLC cell death. This dual suicide gene strategy targets in contrast to HSVtk/GCV or PCV, both DNA and RNA synthesis, also causing cell death to non-proliferating, dormant cancer cells. The strategy demonstrated significant bystander cytotoxicity due to the diffusion of toxic metabolites to nearby cancer cells, showing potential to overcome the low transfection efficiency currently characterizing in vivo gene therapy. An update will be presented.

0037: Alterations of the Tumor Suppressor Gene Fhit in Vulvar Cancer Related to Cigarette Smoking and Reduced Disease-Specific Survival

0037: Alterations of the Tumor Suppressor Gene Fhit in Vulvar Cancer Related to Cigarette Smoking and Reduced Disease-Specific Survival

Silencing of the tumor suppressor gene FHIT is highly characteristic for MLL gene rearranged infant acute lymphoblastic leukemia

MLL rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL), diagnosed predominantly in infants (<1 years of age). Since current chemotherapy fails in >50% of patients with MLL, new therapeutic strategies are desperately needed. For this, understanding the biological features characterizing MLL is necessary. Analysis of gene expression profiles revealed that the expression of the tumor suppressor gene FHIT is reduced in children with MLL rearranged ALL as compared to ALL patients carrying germ line MLL. This finding was confirmed by quantitative real-time PCR. In 100% of the infant MLL cases tested, methylation of the FHIT 5'CpG region was observed, resulting in strongly reduced mRNA and protein expression. In contrast, FHIT methylation in infant and non-infant ALL patients carrying germ line MLL was found in only approximately 60% (P< or =0.004). FHIT expression was restored upon exposing leukemic cells to the demethylating agent decitabine, which induced apoptosis. Likewise and more specifically, leukemic cell death was induced by transfecting MLL rearranged leukemic cells with expression vectors encoding wild-type FHIT, confirming tumor suppressor activity of this gene. These observations imply that suppression of FHIT may be required for the development of MLL, and provide new insights into leukemogenesis and therapeutic possibilities for MLL.