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Background/aim Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults. The aim of this study was to elucidate the molecular pathogenesis of sporadic RCC in Taiwan. Materials and methods Fifteen patients with RCC were screened for mutations in the von Hippel-Lindau (VHL) gene by PCR and Sanger sequencing. The methylation status of promoters of 24 tumor suppressor genes by methylation sensitive multiplex ligation-dependent probe amplification analysis was also determined. Results Inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, one promoter methylation, and one small deletion. In RCCs, methylation was most frequently observed in APC (100%), CDKN2B (92.9%), CASP8, MLH1_167, and KLLN (85.7.4%), but not in FHIT, MLH1_463, DAPK1, or HIC1 (0%). Conclusion In addition to VHL inactivation, promoter methylation of APC may be a universal pathognomonic event in the tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.

The incidence of anal squamous cell carcinoma (ASCC) has been increasing, particularly in populations with HIV. Human papillomavirus (HPV) is the causal factor in 85% to 90% of ASCCs, but few studies evaluated HPV genotypes and integrations in relation to genomic alterations in ASCC. Using whole-exome sequence data for primary (n = 56) and recurrent (n = 31) ASCC from 72 patients, we detected HPV DNA in 87.5% of ASCC, of which HPV-16, HPV-18, and HPV-6 were detected in 56%, 22%, and 33% of HIV-positive (n = 9) compared with 83%, 3.2%, and 1.6% of HIV-negative cases (n = 63), respectively. Recurrent copy-number variations (CNV) involving genes with documented roles in cancer included amplification of PI3KCA and deletion of APC in primary and recurrent tumors; amplifications of CCND1, MYC, and NOTCH1 and deletions of BRCA2 and RB1 in primary tumors; and deletions of ATR, FANCD2, and FHIT in recurrent tumors. DNA damage response genes were enriched among recurrently deleted genes in recurrent ASCCs (P = 0.001). HPV integrations were detected in 29 of 76 (38%) ASCCs and were more frequent in stage III–IV versus stage I–II tumors. HPV integrations were detected near MYC and CCND1 amplifications and recurrent targets included NFI and MUC genes. These results suggest HPV genotypes in ASCC differ by HIV status, HPV integration is associated with ASCC progression, and DNA damage response genes are commonly disrupted in recurrent ASCCs. Implications: These data provide the largest whole-exome sequencing study of the ASCC genomic landscape to date and identify HPV genotypes, integrations, and recurrent CNVs in primary or recurrent ASCCs.

BACKGROUND: The current cervical cancer screening strategies based on Papanicolaou (Pap) and Human papillomavirus (HPV) tests receive great achievement but still exhibit many limitations in clinical practice. Exploring new biomarkers as stratified management method in HPV primary screening is becoming the tendency of current research. METHODS: Immunocytochemistry (ICC) of FHIT and C-MYC were performed on exfoliated cervical cells from 197 eligible high-risk HPV positive women. Mann-Whitney U test, Pearson Chi-Square test, logistic regression analysis and receiver operating characteristic (ROC) curves were used to assess the diagnostic efficiency. RESULTS: ICC staining intensity of FHIT and C-MYC in high-grade cervical intraepithelial neoplasia (CIN) specimens was significantly different from low-grade CIN and normal specimens. Compared with Pap test, ROC analysis of ICC in detecting high-grade CIN resulted in a larger area under the curve (AUC) (0.805 and 0.814 vs 0.723, p< 0.001). FHIT achieved higher sensitivity than Pap test (79.41% vs 66.67%, p= 0.04). Logistic regression analysis of the combination of two biomarkers led to higher AUC value, specificity and PPV than any single biomarker. CONCLUSIONS: The utility of FHIT and C-MYC ICC analysis in cervical exfoliated cells of HPV-positive women displayed superior diagnostic potential and may improve clinical performance of cervical cancer screening.

Human papillomavirus (HPV) integration in the human genome is suggested to be an important cause of cervical cancer. With the development of sequencing technologies, an increasing number of integration “hotspots” have been identified. However, this HPV integration information was derived from analysis of whole cervical cancer tissue, and we know very little about the integration in different cancer cell subgroups or individual cancer cells. This study optimized the preparation of probes and provided a dual-color fluorescence in situ hybridization (FISH) method to detect HPV integration sites in paraffin-embedded cervical cancer samples. We used both HPV probes and site-specific probes: 3p14 (FHIT), 8q24 (MYC), 13q22 (KLF5/KLF12), 3q28 (TP63), and 5p15 (TERT). We detected HPV signals in 75 of the 96 cases of cervical cancer; 62 cases showed punctate signals, and 13 cases showed diffuse punctate signals. We identified 3p14 as a high-frequency HPV integration site in 4 cervical cancer cases. HPV integration at 8p14 occurred in 2 cases of cervical cancer. In the same cervical cancer tissue of sample No.1321, two distinct subgroups of cells were observed based on the HPV probe but showed no difference in cell and nucleus morphology. Our study provides a new method to investigate the frequent HPV integration sites in cervical cancer and reports the heterogeneity within cervical cancer from the perspective of HPV integration.

The loss of heterozygosity (LOH) is a genetic event that can change gene function. FHIT is a potential tumor suppressor gene. Although the precise FHIT molecular mechanism of action is not well understood, evidences suggest that Fhit protein reduced levels are involved in mammary carcinogenesis. The aim of this study was to investigate if FHIT LOH could influence on sporadic breast cancer (BC) biological behavior, through its association with prognostic factors for sporadic BC.Tumor tissue and peripheral blood samples were analyzed using the microsatellite marker D3S1300. The findings were associated with clinicopathological parameters including overall survival. LOH was detected in 31.1%(52/167) of the informative BC’ cases. Considering clinical and pathological characteristics we have found no significant association with FHIT LOH status. The mean follow-up time was 80 months. After the Cox regression analysis two parameters remained associated with BC’s risk of death: TNM stage III and IV - HR = 3.74(95% CI, 1.16-12.1) P=0.027 and disease relapse HR = 3.14(CI 95% 1.26-7.80) P =0.014. This study shows that FHIT LOH by itself is not a prognostic factor for sporadic BC. Further researches are required to elucidate the functional role of FHIT LOH concerning to BC.

Gynaecological carcinosarcomas are rare biphasic tumours which are highly aggressive. We performed molecular investigations on a series of such tumours arising in the uterus (n = 16) and ovaries (n = 10) to gain more information on their mutational landscapes and the expression status of the genes HMGA1/2, FHIT, LIN28A, and MTA1, the pseudogenes HMGA1P6 and HMGA1P7, and the miRNAs known to influence expression of the above-mentioned genes. In uterine carcinosarcomas (UCS), we identified mutations in KRAS, PIK3CA, and TP53 with a frequency of 6%, 31%, and 75%, respectively, whereas in ovarian carcinosarcomas (OCS), TP53 was the only mutated gene found (30%). An inverse correlation was observed between overexpression of HMGA1/2, LIN28A, and MTA1 and downregulation of miRNAs such as let-7a, let-7d, miR26a, miR16, miR214, and miR30c in both UCS and OCS. HMGA2 was expressed in its full length in 14 UCS and 9 OCS; in the remaining tumours, it was expressed in its truncated form. Because FHIT was normally expressed while miR30c was downregulated, not both downregulated as is the case in several other carcinomas, alterations of the epithelial-mesenchymal transition through an as yet unknown mechanism seems to be a feature of carcinosarcomas.

The main risk factor for esophageal dysplasia and adenocarcinoma (DAC) is Barrett’s esophagus (BE), characterized by intestinal metaplasia. The critical genomic mechanisms that lead to progression of non-dysplastic BE to DAC remain poorly understood and require analyses of longitudinal patient cohorts and high-resolution assays. We tested BE tissues from 74 patients, including 42 non-progressors from two separate groups of 21 patients each, and 32 progressors (16 in a longitudinal cohort before DAC/pre-progression-BE, and 16 with temporally concurrent but spatially separate DAC/concurrent-BE). We interrogated genome-wide somatic copy number alterations (SCNAs) at the exon level with high-resolution SNP arrays in DNA from formalin-fixed samples histologically confirmed as non-dysplastic BE. The most frequent abnormalities were SCNAs involving FHIT exon 5, CDKN2A/B, or both in 88% longitudinal BE progressors to DAC vs. 24% in both non-progressor groups (P=0.0004). Deletions in other genomic regions were found in 56% of pre-progression-BE but only in 1 non-progressor-BE (P=0.0004). SCNAs involving FHIT exon 5 and CDKN2A/B were also frequently detected in BE temporally concurrent with DAC. TP53 losses were detected in concurrent-BE but not earlier in pre-progression-BE tissues of patients who developed DAC. CDKN2A/p16 immunohistochemistry showed significant loss of expression in BE of progressors vs. non-progressors, supporting the genomic data. Our data suggest a role for CDKN2A/B and FHIT in early progression of BE to dysplasia and adenocarcinoma that warrants future mechanistic research. Alterations in CDKN2A/B and FHIT by high-resolution assays may serve as biomarkers of increased risk of progression to DAC when detected in BE tissues.

Breast cancer is the most common cancer in women worldwide with remarkable proportion of the patients in advanced stage. Recently the importance of genetic mutations in cancers are well established and also the role of tumor suppressor genes such as

Background Human T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation. Methods Here, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples. Results We found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients’ samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation. Conclusions These results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current “wait and see approach” in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

Introduction Renal cell carcinoma (RCC) accounts for 2–3% of all tumors being the most frequent solid lesion in the kidney. Objective To determine what genetic alterations and immunohistochemical (IHC) of clear cell renal carcinoma (ccRCC) are associated with prognosis and tumor aggressiveness. Patients and Methods Experimental analytical study with 57 patients who underwent radical and partial nephrectomy between 2005 and 2011, all with diagnosis of ccRCC and minimum post-operative follow-up of 36 months. The pathological study included IHC determination of biomarkers associated (CAIX, CAM 5.2, CD10, c-erbB-2, EGFR, HIF-1a, Ki67, MDM2, PAX-2 y 8, p53, survivin and VEGFR 1 and 2). Genetic analysis was carried out using multiplex ligation-dependent probe amplification (MLPA). Clinical data were collected and summarized using an access-type database, adding genetic analysis and IHC data of each patient's tumor sample. IHC statistical analysis included Chi-square, Kruskal-Wallis and multivariate analysis. The genetic analysis was performed using multivariate logistic regression (normal/deletion-duplication). Significance level p Results Pathologic stage was: pT1 (61.8%), pT2 (32.7%); pT3-T4 (5.4%); 16.3% were pN+ and 19.3% M1. 23.6% recurred being predominantly to distance in 83.3%. 27.3% of patients died (73.3% ccCCR). CAIX (Carbonic anhydrase IX) and tumor size were associated with worse Fuhrman grade (p = 0.035; p = 0.001 respectively). Deletion-duplication of genes increased the likelihood: of death (APC, Bcl-2 and CDKN2A by 11, 7 and 4 respectively and SMAD4 reduced the probability by 88%); tumor recurrence (CDKN2A by fifteen fold and VHL reduced the probability by 87%); pT greater than 2 (CCND2, MDM2 and WT1 multiplied by 6, 7 and 9); risk of N+ (CDK4 and EBF1 by 13); distant metastases (BRCA2 and DLEU1 by 5); Fuhrman grade ≥3 (BRCA1, BRCA2 and p53 by 40, 75 and 34 respectively, while that FHIT reduced by 96%). Deletion-duplication of CDK4 and DCC increased survival by a factor of 13 and 16, while that DLEU1 and RUNX1 decreased survival time by 80%. Conclusion CAIX and tumor size are associated with increased aggressiveness. The mutations to level 5q, 9p, 11p, 12, 13q, 17, 18q and 21q are associated with more aggressive tumors and with worse survival rate.

Lung cancer has a high mortality rate. Promoting early diagnosis and screening of lung cancer is the most effective way to enhance the survival rate of lung cancer patients. Through computer technology, a comprehensive evaluation of genetic testing results and basic clinical information of lung cancer patients could effectively diagnose early lung cancer and indicate cancer risks. This study retrospectively collected 70 pairs of lung cancer tissue samples and normal human tissue samples. The methylation frequencies of 6 genes (FHIT, p16, MGMT, RASSF1A, APC, DAPK) in lung cancer patients, the basic clinical information, and tumor marker levels of these patients were analyzed. Then, the python package “sklearn” was employed to build a support vector machine (SVM) classifier which performed 10-fold cross-validation to construct diagnostic models that could identify lung cancer risk of suspected cases. Receiver operation characteristic (ROC) curves were drawn, and the performance of the combined diagnostic model based on several factors (clinical information, tumor marker level, and methylation frequency of 6 genes in blood) was shown to be better than that of models with only one pathological feature. The AUC value of the combined model was 0.963, and the sensitivity, specificity, and accuracy were 0.900, 0.971, and 0.936, respectively. The above results revealed that the diagnostic model based on these features was highly reliable, which could screen and diagnose suspected early lung cancer patients, contributing to increasing diagnosis rate and survival rate of lung cancer patients.

DNA methylation is the main epigenetic event for gene silencing and is associated with carcinogenesis. In this meta-analysis, we evaluated the association between the methylation of the promoter regions of APC, CADM1, CCNA1, CDH1, DAPK, FHIT, HIC1, MAL, MGMT, hMLH1, P16, PAX1, RAR-β, and RASSF1 genes and the risk of cervical cancer development and progression. Overall, 194 eligible studies were identified assessing the associations of promoter methylation status of aforementioned genes with low- and high-grade squamous intraepithelial lesions (LSIL and HSIL) and cervical cancer development. The majority of studies were conducted on Caucasian and Asian populations, whereas rare studies were available on the African population. Promoter methylation frequencies were shown to be significantly higher in LSIL and HSIL cervical cancer cases as compared to control specimens for CADM1, CCNA1, CDH1, DAPK1, FHIT, MAL, P16, PAX1, RAR-β, and RASSF1 genes. A moderate association was found between HIC promoter methylation, whereas APC, MGMT, and hMLH1 promoter methylation was not correlated with cervical cancer development. Promoter methylation could be considered as a noninvasive biomarker for early cervical lesions, making them highly promising targets for a personalized therapeutic approach.

Aim: We studied the possible clinical significance of loss of heterozygosity (LOH) at key tumor suppressor genes loci in advanced renal cancer patients treated with nivolumab. Methods: LOH study was performed on 3p14.2 (FHIT gene); 3p21.3–21.2; 9p21 (BDMF gene); 9p22 (SH3GL2 gene). Results: Of 12 patients, 8 (67%) had LOH. The most affected gene was FHIT. All five patients with LOH at FHIT locus had good outcome, mean progression free survival of 6.8 months. The patients LOH negative at FHIT locus had mean progression free survival of 4 months, 67% were treatment refractory. Overall, 75% of patients with LOH of at least one gene had benefit; 75% of LOH negative cases were refractory. Conclusion: LOH at key tumor suppressor genes should be further investigated as predictive for immunotherapy.

Objectives To identify genomic variations in cell-free DNA (cfDNA) and evaluate their clinical utility in small cell lung cancer (SCLC). Materials and methods We performed whole genome sequencing using plasma cfDNAs derived from 24 SCLC patients for copy number variation (CNV) analysis, and targeted sequencing using 17 pairs of plasma cfDNA and their matched gDNA for mutation analysis. We defined somatic mutations by comparing cfDNA to its matched gDNA with 5% variant alleles as the cutoff for mutation calls. We applied Kaplan-Meier to correlate the genomic alterations with overall survival (OS) and progression-free survival (PFS). Results We observed widespread somatic copy-number alterations and mutations, including amplification of MYC at 8q24, FGF10 at 5p13, SOX2 at 3q26 and FGFR1 at 8p12, as well as deletion of TP53 at 17p13, RASSF1 at 3p21.3, RB1 at 13q14.2, FHIT at 3p14, and PTEN at 10q23. The most frequent mutations were genes involved in chromatin regulation (KMT2D, ARID1A, SETBP1 and PBRM1), PI3K/MTOR pathway(MTOR,PIK13G), Notch1 signalling pathway (NOTCH1), and DNA repair related gene ATRX. Kaplan-Meier analysis revealed poor OS and PFS in patients with somatic mutations in gene SETBP1 (P = 0.0061/0.0264, HR = 4.785/3.841, 95% CI = 2.014–28.25/1.286–16.58) and PBRM1 (P = 0.0276/0.0286, HR = 3.532/3.506, 95% CI = 1.275 to 25.34/1.26–24.87). Poor OS was also associated with somatic mutations in ATRX (P = 0.0099, HR = 4.024, 95% CI = 1.926–42.95), EP300 (P = 0.025/0.0622, HR = 3.382/2.891, 95% CI = 1.448–27.76/1.013-17.29), while poor PFS was associated with ATM mutation (P = 0.0038, HR = 4.604, 95% CI = 2.211–40.93). The mutation index produced by summing up the number of mutations in the five genes was significantly associated with the poor OS/PFS (P = 0.0185/0.0294) after adjusting the effect of the stage. Conclusions Our result supports blood plasma as a promising sample source for the genomic analysis in SCLC patients whose tumor tissues are scarcely available and demonstrates potential clinical utilities of cfDNA-based liquid biopsy for clinical management of this deadly disease.

Objective: To investigate the relationship between the hypermethylation of folic acid metabolism-related genes and the incidence and prognosis of esophageal cancer among ethnic Kazakhs in Xinjiang (China). Methods: According to the standard of esophageal cancer diagnosis, exclusion and epidemiological investigation of the experimental and control groups. Ion capture immunoassays were used to measure serum folic acid levels, while methylation-specific polymerase chain reaction was used to detect gene promoter methylation levels. Log-rank tests and Cox regression models were used to identify prognostic factors in the patient population. Results: Serum folic acid levels in the experimental (cancer) group were significantly lower than in the control (non-cancer) group (Z = -9.13, P < 0.001). Furthermore, the methylation rates of MTHFR, CBS, MGMT, P16, FHIT, and RASSF1A in the experimental group were significantly higher than in the control group. Multivariate analysis identified depth of tumor invasion, regional lymph node metastasis, tumor-node-metastasis stage, and CBS and RASSF1A gene methylation status as independent prognostic factors; female gender and high serum folic acid levels were favorable prognostic factors. Conclusions: Low serum folic acid level is a risk factor for esophageal cancer among ethic Kazakhs. Moreover, methylation of MTHFR, CBS, MGMT, P16, FHIT, and RASSF1A is closely related to esophageal cancer tumorigenesis.

// Stefano Volinia 1 , Teresa Druck 2 , Carolyn A. Paisie 3 , Morgan S. Schrock 2 and Kay Huebner 2 1 Department of Morphology, Surgery & Experimental Medicine, University of Ferrara, 44121 Ferrara, Italy 2 Department of Cancer Biology & Genetics, The Ohio State University Comprehensive Cancer Center & Wexner Medical Center, Biomedical Research Tower, Columbus, OH 43210, USA 3 University of Washington, Department of Biomedical Informatics & Medical Education, Center for Infectious Disease Research, Seattle, WA 98109, USA Correspondence to: Kay Huebner, email: kay.huebner@osumc.edu Keywords: exome sequences; mutational signatures; data mining; clock-like signatures; cancer genes Received: September 07, 2017 Accepted: October 25, 2017 Published: November 06, 2017 ABSTRACT The FHIT gene is located at the fragile FRA3B locus where activation by carcinogen-induced and endogenous replication stress causes FHIT deletions even in normal cells over a lifetime. Our lab has shown that loss of FHIT expression causes genome instability and provides single-strand DNA substrates for APOBEC3B hypermutation, in line with evidence that FHIT locus deletions occur in many cancers. Based on these biological features, we hypothesized that FHIT loss drives development of COSMIC mutational signature 5 and here provide evidence, including data mining of >6,500 TCGA samples, that FHIT is the cancer-associated gene with copy number alterations correlating most significantly with signature 5 mutation rate. In addition, tissues of Fhit-deficient mice exhibit a mutational signature strongly resembling signature 5 (cosine similarity value = 0.89). We conclude that FHIT loss is a molecular determinant for signature 5 mutations, which occur in all cancer types early in cancer development, are clock-like, and accelerated by carcinogen exposure. Loss of FHIT caretaker function may be a predictive and preventive marker for cancer development.

BACKGROUND There have been suggestions that fragile histidine triad protein (FHIT) expression and FHIT gene hypermethylation were crucial to the pathogenesis of liver cancer. However, the conclusions remained unclear because of small sample size, disease subtype, and different detection techniques. Therefore, we performed a meta-analysis to estimate the associations of FHIT expression and FHIT gene hypermethylation with liver cancer pathogenesis. METHODS Studies that were published in electronic databases, such as PubMed, Web of Knowledge, China National Knowledge Infrastructure (CNKI), VIP, and WanFang, were retrieved and selected for the meta-analysis. Relative risk (RR) and 95% confidence interval (CI) were calculated to determine the correlations of FHIT expression and FHIT gene hyper-methylation with liver cancer pathogenesis with Stata 12.0 software. RESULTS A total of 17 papers that evaluated the associations of FHIT expression (14 articles) and FHIT gene methylation (3 articles) with liver cancer pathogenesis were included in this meta-analysis. In the overall analysis, the pooled relative risk was 1.93 (95% CI =1.72-2.17), which indicated a significant association between FHIT low expression and liver cancer risk. According to the results of clinical information, there were significant associations of FHIT expression with TNM-stage (RR =2.13, 95% CI =1.72-2.64), tumor size (RR =1.67, 95% CI =1.36-2.05), and merger of cirrhosis (RR =1.34, 95% CI =1.06-1.69) of liver cancer in the Chinese population. In addition, the FHIT gene hypermethylation was significantly associated with the risk of liver cancer (RR =1.45, 95% CI =1.08-1.93). CONCLUSION The FHIT expression and hypermethylation of FHIT gene were significantly associated with the risk of liver cancer, especially in the Chinese population. Furthermore, the results indicated significant associations between FHIT low expression and TNM-stage, tumor size, and merging of cirrhosis of liver cancer in the Chinese population.

In the last decade, the incidence rate of detection rate of superficial head, neck and esophageal squamous cell carcinomas has increased with the development of endoscopic imaging techniques. These cancers are thought to arise independently subsequent to tissue exposure to a common carcinogen e.g. alcohol or tobacco. This phenomenon has been termed field cancerization. To determine the molecular background of the development of hypopharyngeal squamous cell carcinomas (HPSCCs) and double esophageal squamous cell carcinomas (DESCCs), the present study immunohistochemically assessed tumor-related protein expression [p53, Fhit (fragile histidine triad), E-cadherin and activation-induced cytidine deaminase (AID)], and subsequently determined the correlation between protein expression and clinicopathological data. Tumor specimens of 9 HPSCCs and 9 DESCCs were endoscopically obtained from 8 patients with HPSCC. The 9 DESCCs, including 5 synchronous and 4 metachronous lesions, were all obtained from four patients with HPSCC. The overexpression of p53 and loss of Fhit expression was immunohistochemically detected in 8 (88.9%) and 8 (88.9%) of the 9 HPSCCs and in 8 (88.9%) and 8 (88.9%) of the 9 DESCCs, respectively, which demonstrated the high frequency of such expression. Additionally, 7 out of 9 HPSCCs, and 7 out of 9 DESCCs demonstrated aberrant expression of p53 and Fhit. The rate of aberrant AID and E-cadherin expression was 67 and 44% in HPSCCs and 44 and 44% in DESCC, respectively. These results suggested that aberrant p53 and Fhit expression was involved in the development of HPSCC and their DESCC, and that their expression may be used for the prediction of DESCC development in patients with HPSCC, thereby acting as a biomarker of field cancerization.

Smoking and alcohol consumption are major risk factors for the development of esophageal squamous cell carcinoma (ESCC). Recent studies have demonstrated that smoking and alcohol consumption may be associated with altered DNA methylation in human cancer development. The aim of the present study was to evaluate methylation-modulated protein expression of tumor-related genes (TRGs) in the early stages of esophageal squamous neoplasia (ESN). ESN tissue samples (n=141) comprising 19 cases of low-grade intraepithelial neoplasia (LGIN), 70 of high-grade intraepithelial neoplasia/carcinoma in situ (HGIN/CIS) and 52 of invasive cancer, were endoscopically resected. The methylation-modulated protein expression of 5 TRGs [fragile histidine triad (FHIT), E-cadherin, MutL homolog 1 (MLH1) /MutS homolog 2 (MSH2) and cyclooxygenase-2 (COX-2)] as well as p53 was examined with immunohistochemistry, and their expression was compared with patient clinicopathological characteristics. Reduced or loss of FHIT, E-cadherin, MLH1/MSH2 and COX-2 expression was detected in 26.3 (5/19), 5.3 (1/19), 0 (0/19) and 63.2% (12/19) of LGIN cases, 61.4 (43/70), 18.6 (13/70), 7.1 (5/70) and 65.7% (46/70) of HGIN/CIS cases, and 78.8 (41/52), 50.0 (26/52), 11.5 (6/52) and 59.6% (31/52) of invasive cancer cases, respectively. Reduced or absent expression of FHIT and E-cadherin was significantly associated with neoplastic progression (FHIT, P=0.0007; E-cadherin, P=0.00014). The mean number of TRGs (FHIT, E-cadherin, MLH1/MSH2, and COX-2) that exhibited reduced or absent expression in LGIN, HGIN/CIS and invasive cancer specimens was 1.12±0.61, 1.66±0.93 and 2.09±0.96, respectively, demonstrating a significant stepwise increment from LGIN to HGIN/CIS and then to invasive cancer (P

// Antonio Agostini 1, 2 , Marta Brunetti 1, 2 , Ben Davidson 3, 4 , Claes G. Trope 5 , Sverre Heim 1, 2, 4 , Ioannis Panagopoulos 1, 2 , Francesca Micci 1, 2 1 Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway 2 Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway 3 Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway 4 Faculty of Medicine, University of Oslo, Oslo, Norway 5 Department of Gynecology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Correspondence to: Francesca Micci, email: francesca.micci@medisin.uio.no Keywords: HMGA2, miR-30c, let-7a, FHIT, LIN28A Received: July 05, 2016 Accepted: January 31, 2017 Published: March 01, 2017 ABSTRACT The High-mobility group AT-hook 2 protein (HMGA2) is involved in different processes during tumorigenesis. High expression levels of HMGA2 are found in various types of cancer, with recent studies highlighting the important role of miRNAs in the regulation of HMGA2 expression. We report a study of 155 ovarian tumors (30 sex-cord stromal tumors, 22 borderline tumors, and 103 carcinomas) analyzed for HMGA2 expression as well as the expression of two miRNAs targeting this gene, let-7a and miR-30c. We also evaluated the expression of the fragile histidine triad ( FHIT ) and lin28 homologues ( LIN28A/B ) genes which are known to be an enhancer of miR-30c expression and a repressor of let-7a, respectively. HMGA2 was found expressed at high levels in most samples analyzed, with clear cell carcinomas as the only exception. let-7a and miR-30c were highly deregulated in all tumor types. LIN28A and FHIT were found overexpressed in all examined tumor types. The chromosomal imbalances that might lead to loss of the genes expressing let-7a and miR-30c could be evaluated on the basis of previously generated karyotypic and high resolution comparative genomic hybridization (CGH) data on 103 tumors. 76% of the samples with an imbalanced genome had at least one chromosomal aberration leading to a deletion of a miRNA cluster for let-7a and miR-30c. FISH using locus specific probes for these clusters validate the aberrations at the gene level. Our study shows that genomic imbalances are involved in miR-30c and let-7a deregulation. One can reasonably assume that dysregulation of these miRNAs is a cause leading to HMGA2 upregulation in ovarian tumors.

Inactivation of the fragile histidine triad (Fhit) gene has been reported in the majority of human cancers, particularly in lung cancer. The role of Fhit as a tumor suppressor gene has been well documented, and restoration of Fhit expression suppresses tumorigenicity in tumor cell lines and in mouse models by inducing apoptosis and inhibiting proliferation of tumor cells. Autophagy is a catabolic pathway, whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Although autophagy is necessary for cell survival under stress conditions, recent studies have shown that autophagy can also promote cell death. Due to the fact that both autophagy induction and Fhit expression are commonly associated with nutrient starvation, we hypothesized that Fhit expression may be related to autophagy induction. In the present study, we assessed whether Fhit overexpression by gene transfer induces autophagy in Fhit-deficient non-small cell lung cancer (NSCLC) cells. The results of our study indicate that Fhit protein induces autophagy in NSCLC cells, and that this autophagy prevents apoptotic cell death in vivo and in vitro in a 14-3-3τ protein-dependent manner. To the best of our knowledge, this is the first report to describe Fhit-induced autophagy. Suppressing autophagy might be a promising therapeutic option to enhance the efficacy of Fhit gene therapy in NSCLC.

Oral squamous cell carcinoma (OSCC) is a common cancer in Taiwan and worldwide. To provide some clues for clinical management of OSCC, 72 advanced-stage OSCCs were analyzed using two microarray platforms (26 cases with Affymetrix 500 K and 46 cases with Affymetrix SNP 6.0). Genomic identification of significant targets in cancer analyses were used to identify significant copy number alterations (CNAs) using a q-value cutoff of 0.25. Among the several significant regions, 12 CNAs were common between these two platforms. Two gain regions contained the well-known oncogenes EGFR (7p11.2) and CCND1 (11q13.3) and several known cancer suppressor genes, such as FHIT (3p14.2&ndash, p12.1), FAT1 (4q35.1), CDKN2A (9p21.3), and ATM (11q22.3&ndash, q24.3), reside within the 10 deletion regions. Copy number gains of EGFR and CCND1 were further confirmed by fluorescence in situ hybridization and TaqMan CN assay, respectively, in 257 OSCC cases. Our results indicate that EGFR and CCND1 CNAs are significantly associated with clinical stage, tumor differentiation, and lymph node metastasis. Furthermore, EGFR and CCND1 CNAs have an additive effect on OSCC tumor progression. Thus, current genome-wide CNA analysis provides clues for future characterization of important oncogenes and tumor suppressor genes associated with the behaviors of the disease.

// Xin Geng 1, * , Weilin Pu 2, * , Yulong Tan 1 , Zhouyi Lu 3 , An Wang 3 , Lixing Tan 2 , Sidi Chen 2 , Shicheng Guo 4 , Jiucun Wang 2 , Xiaofeng Chen 1 1 Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032, China 2 State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200433, China 3 Department of Chest Surgery, Shanghai Pulmonary Hospital, Shanghai 200433, China 4 Department of Bioengineering, University of California at San Diego, La Jolla, CA 92093, USA * These authors have contributed equally to this work Correspondence to: Shicheng Guo, email: scguo@ucsd.edu Xiaofeng Chen, email: cxf3166@126.com Jiucun Wang, email: jcwang@fudan.edu.cn Keywords: FHIT, DNA methylation, non-small cell lung cancer, NSCLC, diagnosis Received: April 28, 2016 Accepted: December 12, 2016 Published: December 27, 2016 ABSTRACT Aberrant methylation of CpG islands acquired in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates FHIT gene promoter hyper-methylation is involved in non-small cell lung cancer (NSCLC). To test the diagnostic ability of FHIT methylation status on NSCLC, thirteen studies, including 2,119 samples were included in our meta-analysis. Simultaneously, four independent DNA methylation datasets from TCGA and GEO database were analyzed for validation. The pooled odds ratio of FHIT promoter methylation in cancer samples was 3.43 (95% CI: 1.85 to 6.36) compared with that in controls. In subgroup analysis, significant difference of FHIT gene promoter methylation status in NSCLC and controls was found in Asians but not in Caucasian population. In validation stage, 950 Caucasian samples, including 126 paired samples from TCGA, 568 cancer tissues and 256 normal controls from GEO database were analyzed, and all 8 CpG sites near the promoter region of FHIT gene were not significantly differentially methylated. Thus the diagnostic role of FHIT gene in the lung cancer may be relatively limited in the Caucasian population but useful in the Asians.

The FHIT gene, encompassing an active common fragile site, FRA3B, is frequently silenced in preneoplasia and cancer, through gene rearrangement or methylation of regulatory sequences. Silencing of Fhit protein expression causes thymidine kinase 1 downregulation, resulting in dNTP imbalance, and spontaneous replication stress that leads to chromosomal aberrations, allele copy number variations, insertions/deletions, and single-base substitutions. Thus, Fhit, which is reduced in expression in the majority of human cancers, is a genome "caretaker" whose loss initiates genome instability in preneoplastic lesions. To follow the early genetic alterations and functional changes induced by Fhit loss that may recapitulate the neoplastic process in vitro, we established epithelial cell lines from kidney tissues of Fhit-/- and +/+ mouse pups early after weaning, and subjected cell cultures to nutritional and carcinogen stress, which +/+ cells did not survive. Through transcriptome profiling and protein expression analysis, we observed changes in the Trp53/p21 and survivin apoptotic pathways in -/- cells, and in expression of proteins involved in epithelial-mesenchymal transition. Some Fhit-deficient cell lines showed anchorage-independent colony formation and increased invasive capacity in vitro. Furthermore, cells of stressed Fhit-/- cell lines formed s.c. and metastatic tumors in nude mice. Collectively, we show that Fhit loss and subsequent thymidine kinase 1 inactivation, combined with selective pressures, leads to neoplasia-associated alterations in genes and gene expression patterns in vitro and in vivo.

We evaluated the association between methylation of 9 genes, SCGB3A1, GSTP1, RARB, SYK, FHIT, CDKN2A, CCND2, BRCA1, and SFN in tumor samples from 720 breast cancer cases with clinicopathological features of the tumors and survival. Logistic regression was used to estimate odds ratios (OR) of methylation and Cox proportional hazards models to estimate hazard ratios (HR) between methylation and breast cancer related mortality. Estrogen receptor (ER) and progesterone receptor (PR) positivity were associated with increased SCGB3A1 methylation among pre- and post-menopausal cases. Among premenopausal women, compared with Stage 0 cases, cases of invasive cancer were more likely to have increased methylation of RARB (Stage I OR = 4.7, 95% CI: 1.1-19.0; Stage IIA/IIB OR = 9.7, 95% CI: 2.4-39.9; Stage III/IV OR = 5.6, 95% CI: 1.1-29.4) and lower methylation of FHIT (Stage I OR = 0.2, 95% CI: 0.1-0.9; Stage IIA/IIB OR = 0.2, 95% CI: 0.1-0.8; Stage III/IV OR = 0.6, 95% CI: 0.1-3.4). Among postmenopausal women, methylation of SYK was associated with increased tumor size (OR = 1.7, 95% CI: 1.0-2.7) and higher nuclear grade (OR = 2.0, 95% CI 1.2-3.6). Associations between methylation and breast cancer related mortality were observed among pre- but not post-menopausal women. Methylation of SCGB3A1 was associated with reduced risk of death from breast cancer (HR = 0.41, 95% CI: 0.17-0.99) as was BRCA1 (HR = 0.41, 95% CI: 0.16-0.97). CCND2 methylation was associated with increased risk of breast cancer mortality (HR = 3.4, 95% CI: 1.1-10.5). We observed differences in methylation associated with tumor characteristics; methylation of these genes was also associated with breast cancer survival among premenopausal cases. Understanding of the associations of DNA methylation with other clinicopathological features may have implications for prevention and treatment.

FHIT is a tumor suppressor gene that is frequently silenced in non-small cell lung cancer (NSCLC) and also in preneoplastic lesions. Promoter hypermethylation was previously observed in NSCLC, and its epigenetic silencing, observed on mRNA or protein level, was proposed to predict NSCLC outcome. In the present study we evaluated the relationship between FHIT expression on mRNA level and promoter methylation, or immunoexpression level. The aim of this study was to analyze the usefulness of FHIT as early differentiating biomarker in NSCLC patients. Lung tissue specimens were obtained from 59 patients with diagnosed NSCLC (SCC=34, AC=20, LCC=5). FHIT promoter methylation was assessed in methylation-specific PCR. Relative expression analysis of FHIT was performed in real-time PCR (qPCR) and protein immunoexpression by ELISA assay. Significant differences in FHIT expression between NSCLC histopathological groups (SCC, AC, LCC) were observed (p=0.000009), with the lowest level in SCC. FHIT expression was significantly higher (p=0.034) in men vs. women. Methylated FHIT alleles were present both in NSCLC and control specimens. Mean MI value was higher in control tissue vs. neoplasm, and in men vs. women and it increased with patient age. Significant increase in MI level was observed in N0 group vs. N1 and N2, according to the TNM staging (p=0.0073). Differences in FHIT expression levels between AC, LCC and SCC indicated the usefulness of this gene as a diagnostic marker for NSCLC subtype differentiation. FHIT promoter hypermethylation both in cancer and control tissue indicated the presence of epigenetic alterations in early stage of NSCLC development. Differences in gene promoter methylation between cancer patients with and without node infiltration might be considered as a prognostic marker. Significantly lower FHIT protein immunoexpression was revealed in the group with long and intense history of smoking assessed as PYs (PY

// Eugenio Gaudio 1,2,3,* , Francesco Paduano 3,* , Apollinaire Ngankeu 1 , Francesco Ortuso 4 , Francesca Lovat 1 , Sandra Pinton 2 , Sabrina D’Agostino 3 , Nicola Zanesi 1 , Rami I. Aqeilan 1,5 , Pietro Campiglia 6 , Ettore Novellino 7 , Stefano Alcaro 4 , Carlo M. Croce 1 and Francesco Trapasso 3 1 Department of Molecular Immunology, Virology and Medical Genetics, The Ohio State University, Columbus, Ohio, USA 2 Lymphoma & Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, Switzerland 3 Dipartimento di Medicina Sperimentale e Clinica, University Magna Graecia, Campus “S. Venuta”, Catanzaro, Italy 4 Dipartimento di Scienze della Salute, University Magna Graecia, Campus “S. Venuta”, Catanzaro, Italy 5 The Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research, The Hebrew University, Jerusalem, Israel 6 Dipartimento di Farmacia, Universita di Salerno, Fisciano, Italy 7 Dipartimento di Farmacia, Universita degli Studi di Napoli “Federico II”, Napoli, Italy * These authors have contributed equally to this work Correspondence to: Carlo M. Croce, email: // Francesco Trapasso, email: // Keywords : fragile histidine triad, FHIT, annexin A4, ANXA4, mimetic peptides Received : January 07, 2016 Accepted : April 11, 2016 Published : May 04, 2016 Abstract We recently reported that Fhit is in a molecular complex with annexin A4 (ANXA4); following to their binding, Fhit delocalizes ANXA4 from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. Here, we demonstrate that Fhit physically interacts with A4 through its N-terminus; molecular dynamics simulations were performed on a 3D Fhit model to rationalize its mechanism of action. This approach allowed for the identification of the QHLIKPS heptapeptide (position 7 to 13 of the wild-type Fhit protein) as the smallest Fhit sequence still able to preserve its ability to bind ANXA4. Interestingly, Fhit peptide also recapitulates the property of the native protein in inhibiting Annexin A4 translocation from cytosol to plasma membrane in A549 and Calu-2 lung cancer cells treated with paclitaxel. Finally, the combination of Tat-Fhit peptide and paclitaxel synergistically increases the apoptotic rate of cultured lung cancer cells and blocks in vivo tumor formation. Our findings address to the identification of chemically simplified Fhit derivatives that mimic Fhit tumor suppressor functions; intriguingly, this approach might lead to the generation of novel anticancer drugs to be used in combination with conventional therapies in Fhit-negative tumors to prevent or delay chemoresistance.

Up to 50% of multiple sclerosis (MS) patients do not respond to interferon-beta (IFN-β) treatment and determination of response requires lengthy clinical follow-up of up to 2 years. Response predictive genetic markers would significantly improve disease management. We aimed to identify IFN-β treatment response genetic marker(s) by performing a two-stage genome-wide association study (GWAS). The GWAS was carried out using data from 151 Australian MS patients from the ANZgene/WTCCC2 MS susceptibility GWAS (responder (R)=51, intermediate responders=24 and non-responders (NR)=76). Of the single-nucleotide polymorphisms (SNP) that were validated in an independent group of 479 IFN-β-treated MS patients from Australia, Spain and Italy (R=273 and NR=206), eight showed evidence of association with treatment response. Among the replicated associations, the strongest was observed for FHIT (Fragile Histidine Triad; combined P-value 6.74 × 10-6) and followed by variants in GAPVD1 (GTPase activating protein and VPS9 domains 1; combined P-value 5.83 × 10-5) and near ZNF697 (combined P-value 8.15 × 10-5). Refereed/Peer-reviewed

Treatment with interferon beta (IFNβ) is one of the first-line treatments for multiple sclerosis. In clinical practice, however, many patients present suboptimal response to IFNβ, with the proportion of non-responders ranging from 20 to 50%. This variable response can be affected by genetic factors, such as polymorphisms in the genes involved in the disease state, pharmacodynamics, metabolism or in the action mechanism of IFNβ, which can affect the efficacy of this drug. This review assesses the impact of pharmacogenetics studies on response to IFNβ treatment among patients diagnosed with relapsing-remitting multiple sclerosis (RRMS). The results suggest that the detection of polymorphisms in several genes (CD46, CD58, FHIT, IRF5, GAPVD1, GPC5, GRBRB3, MxA, PELI3 and ZNF697) could be used in the future as predictive markers of response to IFNβ treatment in patients diagnosed with RRMS. However, few studies have been carried out and they have been performed on small sample sizes, which makes it difficult to generalize the role of these genes in IFNβ treatment. Studies on large sample sizes with longer term follow-up are therefore required to confirm these results.

The authors thank I. Linares, V. Sanz, A.B. Rodriguez, A.I. Rodriguez and E. Arias for technical advice and R. Davies for editorial assistance., The capacity of cytotoxic-T lymphocytes to recognize and destroy tumor cells depends on the surface expression by tumor cells of MHC class I molecules loaded with tumor antigen peptides. Loss of MHC-I expression is the most frequent mechanism by which tumor cells evade the immune response. The restoration of MHC-I expression in cancer cells is crucial to enhance their immune destruction, especially in response to cancer immunotherapy. Using mouse models, we recovered MHC-I expression in the MHC-I negative tumor cell lines and analyzed their oncological and immunological profile. Fhit gene transfection induces the restoration of MHC-I expression in highly oncogenic MHC-I-negative murine tumor cell lines and genes of the IFN-γ transduction signal pathway are involved. Fhit-transfected tumor cells proved highly immunogenic, being rejected by a T lymphocyte-mediated immune response. Strikingly, this immune rejection was more frequent in females than in males. The immune response generated protected hosts against the tumor growth of non-transfected cells and against other tumor cells in our murine tumor model. Finally, we also observed a direct correlation between FHIT expression and HLA-I surface expression in human breast tumors. Recovery of Fhit expression on MHC class I negative tumor cells may be a useful immunotherapeutic strategy and may even act as an individualized immunotherapeutic vaccine., FEDER funds (EU) from the Instituto de Salud Carlos III PI12/02031 PI14/01978 PI15/00528 PI17/00197 PI19/01179 PT13/0010/0039 PT17/0015/0041, Worldwide Cancer Research 15-1166, Junta de Andalucia CTS-143 CTS-3952 CVI-4740, Instituto de Salud Carlos III, Rio-Hortega Contract from ISCIII CM12/00033, ibs.Granada Fellowship 496

Defining the cancer genome is essential for understanding the pathogenesis, but it remains challenging due to the paucity of robust models for characterizing recurrent mutations found in cancer. To understand the molecular mechanisms underlying the development of small-cell lung cancer (SCLC), an aggressive type of lung cancer initiated by inactivation of both RB and P53, it is important to determine the functional impact of recurrent mutations found in the patient tumors. To this end, we developed a somatic engineering approach, using a CRISPR/Cre hybrid tool that expresses guide RNA, Cas9 endonuclease, and Cre recombinase from a single adenoviral vector (Ad-target of gRNA-CRISPR/Cre). Intratracheal instillation of this recombinant virus causes mutation in a candidate tumor suppressor gene while recombining floxed alleles of Rb1, Trp53, and Rbl2 in the same epithelial cell. To determine functions of Ep300, Slit2, and Fhit, whose orthologous genes are frequently mutated in SCLC patient tumors, we infect the Rb1/Trp53/Rbl2 conditional mice with the viral vectors carrying gRNAs against these genes. The mice infected with Ad-Ep300-CRISPR/Cre or Ad-Fhit-CRISPR/Cre have higher tumor burden in the lungs than those infected with Ad-lacZ-CRISPR/Cre used as control. Although Slit2 is significantly mutated in SCLC, the mice infected with Ad-Slit2-CRISPR/Cre do not increase tumor burden relative to those with Ad-lacZ-CRISPR/Cre. These results for the first time validate the oncogenic impact of Ep300 and Fhit mutations. This study demonstrates the feasibility and robustness of a new somatic engineering-based model for characterizing candidate mutations, which will facilitate the discovery of key oncogenic drivers and pathways from the large number of novel somatic alterations in SCLC. Ongoing efforts with this new approach are focused on validation of functionally unknown mutations alone or in combinations. This abstract is also being presented as Poster A39. Citation Format: Kee-Beom Kim, Dong-Wook Kim, Kwon-Sik Park. A new somatic engineering-based model of small-cell lung cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHA04.

Regulatory effects of fragile histidine triad (FHIT) gene on proliferation and apoptosis of osteosarcoma cells were studied. The hFOB1.19 and Saos2 cells were routinely cultured, pcDNA3.1-FHIT overexpression vectors carrying FHIT gene fragments and blank pcDNA3.1 vectors were transfected into Saos2 cells, respectively, and the cells were divided into hFOB, Saos2, transfection and no-load transfection groups. After transfection for 48 h, the cells were collected and analyzed. The expression of FHIT messenger ribonucleic acid (mRNA) was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression of FHIT protein was detected by western blot analysis. Cell Counting Kit 8 (CCK8) was used to detect cell proliferation, and flow cytometry was used to detect apoptosis. The expression of FHIT mRNA was significantly decreased in Saos2 group compared with that in hFOB group, and the difference was statistically significant (P

Effects of interventional embolotherapy on the expression of fragile histidine triad (FHIT) and p16 in hepatocellular carcinoma (HCC) patients were investigated. Patients with primary HCC who were definitely diagnosed and treated in the Department of Gastroenterology in Qingdao Central Hospital from March 2014 to March 2016 were selected, and they underwent interventional embolotherapy. HCC and cancer-adjacent tissues of the patients were harvested for immunohistochemical staining. The correlation between the expression levels of FHIT and p16 was analyzed at the gene and protein level. Clinical data were collected, and whether they were correlated with the expression of FHIT and p16 was investigated. The expression levels of FHIT and p16 in primary HCC tissues were remarkably lower than that in cancer-adjacent tissues (P

The aim of this study was to investigate the predictive value of the biomarkers FHIT, p27, and pERK1/ERK2 in salivary gland carcinomas. Immunohistochemical staining of FHIT, p27, and pERK1/ERK2 of 265 patients with salivary gland carcinomas was conducted, and associations with clinico-histopathological data, overall survival, and disease-specific survival were examined. Expression of FHIT (quick score 98.7 vs. 206.4) and p27 (QS 187.3 vs. 244.8) was significantly lower in carcinomas compared to non-tumor control tissue. Loss of FHIT frequently occurred in ACC (55.2%), SDC (68.2%), and SCC (100%). In the totality of tumors, loss of FHIT expression was found in 46.7% (106/227) and was significantly associated with advanced T stage and UICC stage, high-grade histology, loss of p27, PI3K, and survivin. FHIT positivity went along with significantly better overall and disease-specific survival. Negativity of p27 occurred in 28.7% (70/244) of tumors, particularly in SDC (54.4%) and SCC (50%). In the totality of tumors, p27 was associated with advanced patient age, high-grade histology, PI3K, survivin as well as better overall and disease-specific survival (p

Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit‐deficient mouse models are exquisitely susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit‐deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit‐deficient tissues and cultured cells, we found 300 to >1000 single‐base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the “age at diagnosis” signature identified in human cancers. The Fhit‐deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in −/− exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit‐deficient cells. Fhit‐deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen‐induced preneoplastic changes.

Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFβ signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression. Cancer Res; 76(3); 749–61. ©2015 AACR.

BACKGROUND To investigate the expression of the fragile histidine triad (FHIT) gene in acute lymphoblastic leukemia and its clinical significance. MATERIALS AND METHODS The level of expressed FHIT mRNA in peripheral blood from 50 patients with acute lymphoblastic leukemia (ALL) and in 50 peripheral blood samples from healthy volunteers was measured via RT-PCR. Correlation analyses between FHIT gene expression and clinical characteristics (gender, age, white blood count, immunophenotype of acute lymphoblastic leukemia and percentage of blast cells) of the patients were performed. RESULTS The FHIT gene was expressed at 2.49±7.37 of ALL patients against 14.4±17.9 in the healthy volunteers. The difference in the expression levels between ALL patients and healthy volunteers was statistically significant. The rate of gene expression did not significantly vary with immunophenotype subtypes. Gene expression was also found to be correlated with increase of total leukocyte and decrease in platelets, but not with age, gender, immunophenotyping or percentage of blast cells. CONCLUSIONS FHIT gene expression is low in acute lymphoblastic leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute lymphoblastic leukemia.

The development of carcinoma has been found to be associated with epigenetic modifications. The aim of this study was to estimate the methylation levels of FHIT and BRCA1 promoters using the bisulphite sequencing method (BSP) and high-resolution melting curve analysis (HRM) in the serum of patients with ductal breast carcinoma as a biomarker for the possible application of early diagnosis of breast cancer. The results showed that the methylation levels of both BRCA1 and FHIT promoters were higher in the serum of the breast ductal carcinoma group (BDC group) than those of the breast fibroadenoma group (BFA group), and the healthy individuals group (HI group). However, the methylation levels of the BRCA1 promoters were very low in all three groups compared to the levels of FHIT. The advanced quantitative detection of the samples with HRM showed that the FHIT promoter methylation level of the cfDNA in each serum was also very high in the BDC group compared to the HI group. The methylation level of FHIT was found to be significantly associated with breast cancer (p < 0.05). In conclusion, the methylation quantitative detection of FHIT promoter in serum may be useful for the early diagnosis of ductal breast carcinoma.

High-risk human papillomavirus (HPV)16/18 infection in the development of lung cancer has previously been identified, and fragile histidine triad (FHIT) loss and p53 mutation are frequently observed in the disease. However, the association between these factors has not been well studied. The present study aimed to further investigate the significance of HPV infection, FHIT loss and p53 mutations in the development of lung cancer and their possible associations. DNA was extracted from paraffin-embedded specimens from 88 cases of squamous cell carcinoma (SCC), 56 of adenocarcinoma (AC), 36 of small cell lung carcinoma (SCLC) and 110 non-cancer control cases of lung neoplasms. The prevalence of HPV infection was determined by polymerase chain reaction analysis, and FHIT loss and p53 mutations were detected by immunohistochemistry. The χ2, Fisher's exact and Pearson correlation tests were applied for statistical analysis. The results of the present study demonstrated that HPVL1 (the major capsid protein of HPV), HPV16 and HPV18 infection were more prevalent in the lung cancer samples compared with the non-cancer controls (all P

Background: Fragile histidine triad (FHIT) gene is a tumor suppressor gene which involved in breast cancer pathogenesis. Epigenetics alterations in FHIT contributes to tumorigenesis of breast cancer. Objective: Our objective was to study FHIT promoter region hypermethylation in Egyptian breast cancer patients and its association with clinicopathological features. Materials and Methods: Methylation-specific polymerase chain reaction was performed to study the hypermethylation of FHIT promoter region in 20 benign breast tissues and 30 breast cancer tissues. Results: The frequency of hypermethylation of FHIT promoter region was significantly increased in breast cancer patients compared to bengin breast disease patients. The Odds ratio (95%CI) of development of breast cancer in individuals with FHIT promoter hypermethylation (MM) was 11.0 (1.22-250.8). There were also significant associations between FHIT promoter hypermethylation and estrogen, progesterone receptors negativity, tumor stage and nodal involvment in breast cancer pateints. Conclusions: Our results support an association between FHIT promotor hypermethylation and development of breast cancer in Egyptian breast cancer patients. FHIT promoter hypermethylation is associated with some poor prognostic features of breast cancer.

The aim of this study was a detailed clinicopathological investigation of sinonasal NUT midline carcinoma (NMC), including analysis of DNA methylation and microRNA (miRNA) expression. Three (5%) cases of NMC were detected among 56 sinonasal carcinomas using immunohistochemical screening and confirmed by fluorescence in situ hybridization. The series comprised 2 males and 1 female, aged 46, 60, and 65 years. Two tumors arose in the nasal cavity and one in the maxillary sinus. The neoplasms were staged pT1, pT3, and pT4a (all cN0M0). All patients were treated by radical resection with adjuvant radiotherapy. Two patients died 3 and 8 months after operation, but one patient (pT1 stage; R0 resection) experienced no evidence of disease at 108 months. Microscopically, all tumors consisted of infiltrating nests of polygonal cells with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. Abrupt keratinization was present in only one case. Immunohistochemically, there was a diffuse expression of cytokeratin (CK) cocktail, CK7, p40, p63, and SMARCB1/INI1. All NMCs tested negative for EBV and HPV infection. Two NMCs showed methylation of RASSF1 gene. All other genes (APC, ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDH13, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, TIMP3, and VHL) were unmethylated. All NMCs showed upregulation of miR-9 and downregulation of miR-99a and miR-145 and two cases featured also upregulation of miR-21, miR-143, and miR-484. In summary, we described three cases of sinonasal NMCs with novel findings on DNA methylation and miRNA expression, which might be important for new therapeutic strategies in the future.

// Andreas Knopf 1 , Justine Lempart 1 , Murat Bas 1 , Julia Slotta-Huspenina 2 , Naglaa Mansour 1 , Marie Kristin Fritsche 1 1 Technische Universitat Munchen, Hals-Nasen-Ohrenklinik und Poliklinik, 81675 Munchen, Germany 2 Technische Universitat Munchen, Institut fur Allgemeine Pathologie und Pathologische Anatomie, 81675 Munchen, Germany Correspondence to: Andreas Knopf, e-mail: a.knopf@lrz.tum.de Keywords: Tongue, oncogenes, tumour suppressor genes, oral cancer, young patients Received: October 23, 2014 Accepted: December 03, 2014 Published: January 30, 2015 ABSTRACT Objectives : The occurrence of squamous cell carcinoma of the tongue (SCCT) of young patients increased. There are still controversies about patient prognosis. The underlying molecular mechanisms remain unclear. Methods : 276 patients (66 ≤45, 210 >45 years) with SCCT were included. Clinical parameters and survival data were assessed. Oncogenes and tumor suppressors were analyzed via immunohistochemistry (p53, CXCR4, p16, EGFR) and qPCR ( CDK4, CDKN2A, TP53, MDM2, AKT1, PIK3CA, NRAS, HRAS, KRAS, HGF, MET, EGF, ATM, BRCA1, E2F1, FHIT, RUNX3, STK11, BCL2, CTNNB1 ). Results : The median overall survival was 142 (≤45 years) and 34 months (>45 years) ( p 45 years ( p < 0.0001; HR [95%CI]: 0.33 [0.26–0.57]). Immunhistochemistry visualized a comparable expression of analyzed proteins. QPCR demonstrated in patients ≤45 years a higher expression of genes that are associated with carcinogenesis ( CTNNB1, STK11, CDKN2A, HGF, MET ) as well as tumor suppressors that constitute an enhanced radio-sensitivity ( ATM, BRCA1E2F1, FHIT ). Conclusion : Derogation of the WNT-CTNNB1-STK11 and CDKN2A-HGF-MET pathway can constitute the carcinogenesis, while the higher expression of radio-sensitizers ATM, BRCA1E2F1 and FHIT can explain the better OS/DSS in young patients.

MLL-rearranged acute lymphoblastic leukaemia (ALL) in infants is the most difficult-to-treat type of childhood ALL, displaying a chemotherapy-resistant phenotype, and unique histone modifications, gene expression signatures and DNA methylation patterns. MLL-rearranged infant ALL responds remarkably well to nucleoside analogue drugs in vitro, such as cytarabine and cladribine, and to the demethylating agents decitabine and zebularine as measured by cytotoxicity assays. These observations led to the inclusion of cytarabine into the treatment regimens currently used for infants with ALL. However, survival chances for infants with MLL-rearranged ALL do still not exceed 30–40%.Here we explored the in vitro potential of the novel nucleoside analogue clofarabine for MLL-rearranged infant ALL. Therefore we used both cell line models as well as primary patient cells. Compared with other nucleoside analogues, clofarabine effectively targeted primary MLL-rearranged infant ALL cells at the lowest concentrations, with median LC50 values of ∼25nM. Interestingly, clofarabine displayed synergistic cytotoxic effects in combination with cytarabine. Furthermore, at concentrations of 5–10nM clofarabine induced demethylation of the promoter region of the tumour suppressor gene FHIT (Fragile Histidine Triad), a gene typically hypermethylated in MLL-rearranged ALL. Demethylation of the FHIT promoter region was accompanied by subtle re-expression of this gene both at the mRNA and protein level. We conclude that clofarabine is an interesting candidate for further studies in MLL-rearranged ALL in infants.

Background FHIT is a genome caretaker/tumor suppressor that is silenced in >50% of cancers. Although it was identified more than 20 years ago, questions remain as to how FHIT loss contributes to cancer, and conversely, how FHIT acts to maintain genome integrity and suppress malignancy. Fhit belongs to the histidine triad family of enzymes that catalyze the degradation of nucleoside 5′,5′-triphosphates, including the m7GpppN ‘caps’ that are generated when mRNAs undergo 3′-5′ decay. This raised the possibility that Fhit loss might affect changes in the translation of cancer-associated mRNAs, possibly as a consequence of increased intracellular concentrations of these molecules. Results Ribosome profiling identified several hundred mRNAs for which coding region ribosome occupancy changed as a function of Fhit expression. While many of these changes could be explained by changes in mRNA steady-state, a subset of these showed changes in translation efficiency as a function of Fhit expression. The onset of malignancy has been linked to changes in 5’-UTR ribosome occupancy and this analysis also identified ribosome binding to 5′-untranslated regions (UTRs) of a number of cancer-associated mRNAs. 5’-UTR ribosome occupancy of these mRNAs differed between Fhit-negative and Fhit-positive cells, and in some cases these differences correlated with differences in coding region ribosome occupancy. Conclusions In summary, these findings show Fhit expression impacts the translation of a number of cancer associated genes, and they support the hypothesis that Fhit’s genome protective/tumor suppressor function is associated with post-transcriptional changes in expression of genes whose dysregulation contributes to malignancy. Electronic supplementary material The online version of this article (10.1186/s12943-017-0749-x) contains supplementary material, which is available to authorized users.

Aberrant DNA methylation is a recognized feature in various types of human cancer, and folate has a vital role in the epigenetics of mammalian cells by supplying methyl groups for DNA methylation reactions. Fragile histidine triad (FHIT) is a tumor suppressor gene that is frequently silenced in cervical cancer (CC) and preneoplastic lesions. Promoter hypermethylation was previously observed in CC, and its epigenetic silencing has been observed at mRNA or protein levels. Changes in folate intake to modulate DNA methylation may be a mechanistic link to cancer, but this remains to be elucidated. The aim of the present study was to evaluate the influences of folate on FHIT gene methylation and expression in the progression of cervical cancerization. In the present study, red blood cell (RBC) folate levels, FHIT gene methylation status, and mRNA and protein expression levels were detected in 254 women, including normal cervix (NC, n=80), cervical intraepithelial neoplasm grade 1 (CIN1, n=55; CIN2/3, n=55) and cervical squamous cell carcinoma (SCC, n=64) samples. The methylation status of FHIT gene and its mRNA and protein expression levels were measured in CaSki (HPV16 positive) and C33A (HPV16 negative) CC cells treated with different concentrations of folate. The results indicated that FHIT gene methylation rate increased with the severity of cervix lesions, however, RBC folate levels, FHIT mRNA and protein expression levels were reduced. The proliferation inhibition rate, apoptosis rate, and FHIT protein and mRNA expression levels increased along with rising concentrations of folate, whereas the degree of FHIT gene methylation gradually weakened in CaSki or C33A cell lines. The present findings indicated that folate deficiency, FHIT gene promoter hypermethylation and reduced expression were significantly associated with cervical carcinogenesis. The results indicated that folate was able to enhance apoptosis and inhibit the cervical cell proliferation while regulating FHIT gene methylation and expression. Adequate intake of folate to maintain normal DNA methylation status is an effective way for cervical lesions prevention, and demethylation treatment may offer a new strategy for therapy of CC.