All is not lost: learning from 9p21 loss in cancer.

The cancer research community continues to search for additional biomarkers of response and resistance to immune checkpoint treatment (ICT). The ultimate goal is to direct the use of ICT in patients whose tumors are most likely to benefit to achieve a refinement that is equivalent to that of a genotype-matched targeted treatment. Dissecting the mechanisms of ICT resistance can help us characterize ICT nonresponders more efficiently. In this opinion, we argue that there may be additional knowledge gained about immune evasion in cancer by analyzing the loss of the human 9p21.3 locus; as an example, we highlight findings of 9p21.3 loss from the investigator-initiated, pan-cancer INSPIRE study, in which patients were treated with pembrolizumab (anti-PD-1 antibody) ICT.

Somatic 9p24.1 alterations in HPV- head and neck squamous cancer dictate immune microenvironment and anti-PD-1 checkpoint inhibitor activity.

Somatic copy number alterations (SCNAs), generally (1) losses containing interferons and interferon-pathway genes, many on chromosome 9p, predict immune-cold, immune checkpoint therapy (ICT)-resistant tumors (2); however, genomic regions mediating these effects are unclear and probably tissue specific. Previously, 9p21.3 loss was found to be an early genetic driver of human papillomavirus-negative (HPV-) head and neck squamous cancer (HNSC), associated with an immune-cold tumor microenvironment (TME) signal, and recent evidence suggested that this TME-cold phenotype was greatly enhanced with 9p21 deletion size, notably encompassing band 9p24.1 (3). Here, we report multi-omic, -threshold and continuous-variable dissection of 9p21 and 9p24 loci (including depth and degree of somatic alteration of each band at each locus, and each gene at each band) and TME of four HPV- HNSC cohorts. Preferential 9p24 deletion, CD8 T-cell immune-cold associations were observed, driven by 9p24.1 loss, and in turn by an essential telomeric regulatory gene element, JAK2-CD274. Surprisingly, same genetic region gains were immune hot. Related 9p21-TME analyses were less evident. Inherent 9p-band-level influences on anti-PD1 ICT survival rates, coincident with TME patterns, were also observed. At a 9p24.1 whole-transcriptome expression threshold of 60th percentile, ICT survival rate exceeded that of lower expression percentiles and of chemotherapy; below this transcript threshold, ICT survival was inferior to chemotherapy, the latter unaffected by 9p24.1 expression level (P-values < 0.01, including in a PD-L1 immunohistochemistry-positive patient subgroup). Whole-exome analyses of 10 solid-tumor types suggest that these 9p-related ICT findings could be relevant to squamous cancers, in which 9p24.1 gain/immune-hot associations exist.

Severity of coronary artery disease is associated with diminished circANRIL expression: A possible blood based transcriptional biomarker in East Africa.

Antisense Noncoding RNA in the INK4 Locus (ANRIL) is the prime candidate gene at Chr9p21, the well-defined genetic risk locus associated with coronary artery disease (CAD). ANRIL and its transcript variants were investigated for the susceptibility to CAD in adipose tissues (AT) and peripheral blood mononuclear cells (PBMCs) of the study group and the impact of 9p21.3 locus mutations was further analysed. Expressions of ANRIL, circANRIL (hsa_circ_0008574), NR003529, EU741058 and DQ485454 were detected in epicardial AT (EAT) mediastinal AT (MAT), subcutaneous AT (SAT) and PBMCs of CAD patients undergoing coronary artery bypass grafting and non-CAD patients undergoing heart valve surgery. ANRIL expression was significantly upregulated, while the expression of circANRIL was significantly downregulated in CAD patients. Decreased circANRIL levels were significantly associated with the severity of CAD and correlated with aggressive clinical characteristics. rs10757278 and rs10811656 were significantly associated with ANRIL and circANRIL expressions in AT and PBMCs. The ROC-curve analysis suggested that circANRIL has high diagnostic accuracy (AUC: 0.9808, cut-off: 0.33, sensitivity: 1.0, specificity: 0.88). circANRIL has high diagnostic accuracy (AUC: 0.9808, cut-off: 0.33, sensitivity: 1.0, specificity: 0.88). We report the first data demonstrating the presence of ANRIL and its transcript variants expressions in the AT and PBMCs of CAD patients. circANRIL having a synergetic effect with ANRIL plays a protective role in CAD pathogenesis. Therefore, altered circANRIL expression may become a potential diagnostic transcriptional biomarker for early CAD diagnosis.

Methylthioadenosine Phosphorylase Genomic Loss in Advanced Gastrointestinal Cancers.

BACKGROUND: One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. MATERIALS AND METHODS: We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. RESULTS: The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). CONCLUSION: In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.

Loss of MTAP Expression by Immunohistochemistry Is a Surrogate Marker for Homozygous 9p21.3 Deletion in Urothelial Carcinoma.

Homozygous deletion of the chromosomal region 9p21.3 is common in urothelial carcinoma (UC) and leads to loss of several genes, including CDKN2A and MTAP, resulting in loss of MTAP protein expression. Here, we aimed to explore the diagnostic potential of MTAP immunohistochemistry (IHC) as a surrogate marker for homozygous 9p21.3 deletion (9p21 homozygous deletion [HD]) in UC. MTAP status was determined by IHC on 27 UC tissue specimens with known 9p21.3 status as defined by fluorescence in situ hybridization in matched cytological specimens, by IHC and fluorescence in situ hybridization on a tissue microarray (TMA) containing 359 UC at different stages, and by IHC on 729 consecutive UC from routine practice. Moreover, we analyzed a longitudinal series of matched specimens from 38 patients with MTAP-negative recurrent UC. MTAP loss by IHC was found in all 17 patients with 9p21 HD and in 2/8 cases without 9p21 HD. In the TMA, MTAP loss was more common in metastases (53%) than in muscle-invasive (33%) and non-muscle-invasive UC (29%) (P = .03). In the consecutive series, 164/729 (22%) cases showed loss of MTAP expression. In 41 of these 164 cases (25%), loss of MTAP expression was heterogenous. We also discovered loss of MTAP expression in flat urothelium adjacent to MTAP-negative low-grade UC, suggesting true flat low-grade neoplasia that could not be diagnosed by morphology alone. Longitudinal analysis of recurrences showed persistent negative MTAP status over time in 37/38 (97%) patients. MTAP IHC can serve as a surrogate marker for 9p21 HD in UC and as a diagnostic tool to differentiate reactive urothelium from urothelial neoplasia. It also provides a unique opportunity to study clinicopathological associations and the heterogeneity of 9p21 HD across the whole spectrum of UC manifestations.

Comparison of Immunohistochemistry, Next-generation Sequencing and Fluorescence In Situ Hybridization for Detection of MTAP Loss in Pleural Mesothelioma.

9p21 deletions involving MTAP/CDKN2A genes are detected in diffuse pleural mesotheliomas (DPM) but are absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study were to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam) and 1813 (NBP2-75730, Novus Biologicals) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted next-generation sequencing. 9p21 Copy number status was assessed by Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) analysis and also by CDKN2A fluorescence in situ hybridization in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining, and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or nonspecific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/fluorescence in situ hybridization calls (κ = 0.85; 95% CI, 0.71-0.99; P < .001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific, and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 because mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.

Technical comparison of Abbott's UroVysion® and Biocare's CytoFISH urine fluorescence in situ hybridization (FISH) assays.

BACKGROUND: This study aims to compare the technical performance of Abbott's UroVysion and Biocare's CytoFISH urine cytology probe panel and position the CytoFISH probe panel as an alternative to UroVysion. The CytoFISH probe panel was developed based on clinically sensitive chromosomes found to be amplified in bladder cancers, as well as a locus-specific probe also seen to be amplified in bladder tumors. After extensive testing comparing CytoFISH to UroVysion, we present here our findings for the two assays. MATERIALS AND METHODS: A total of 216 cases representing a mix of male (ages 36-99) and female (ages 46-91) patients were assayed with both probe sets. The CytoFISH and UroVysion probe panels were tested in accordance with the UroVysion procedure, as outlined in the manufacturer's supplied package insert with the following exception: the probe volume used was 3µL for UroVysion and 5µL for CytoFISH. RESULTS: The scoring used for the CytoFISH and UroVysion assays revealed a 95% concordance, suggesting that Biocare's CytoFISH Test has at least the same clinical sensitivity and specificity as claimed by the Abbott UroVysion Kit. We found that the CytoFISH 5p15.2 locus-specific probe was easier to score than UroVysion's 9p21 deletion. CONCLUSION: The high rate of concordance between the two assays suggests that Biocare's CytoFISH assay is a robust alternative to Abbott's UroVysion in the diagnosis and monitoring of bladder carcinoma.

The rs1333040 and rs10757278 9p21 locus polymorphisms in patients with intracranial aneurysm: a meta-analysis.

INTRODUCTION: The formation of intracranial aneurysms (IAs) has been associated with genetic polymorphisms. A few genome-wide (GWAS) and candidate gene association studies (CGAS) have reported that single nucleotide polymorphisms (SNPs) in locus 9p21 have been associated with the formation of IAs. MATERIALS & METHODS: We performed a meta-analysis of case-control studies to investigate the association of two SNPs (rs1333040, rs10757278), located at the 9p21 locus, with the formation of IAs. MEDLINE, EMBASE, Google Scholar and CENTRAL databases were comprehensively searched. RESULTS: For the rs1333040 (C > T) polymorphism, a significant association with IA was observed in the dominant [OR (95% CI): 1.39 (1.24, 1.56); Pz < 0.00001], recessive [OR (95% CI): 1.38 (1.28, 1.49); Pz < 0.00001] and over-dominant [OR (95% CI): 0.85 (0.79, 0.91); Pz < 0.00001] models. For the rs10757278(A > G) SNP, we observed a statistically significant association with IAs in the dominant [OR (95% CI): 1.41 (1.28, 1.56); Pz < 0.01] and recessive [OR (95% CI): 1.42 (1.29, 1.56); Pz < 0.01] models, while statistical significance was not revealed in the over-dominant model [OR (95% CI): 1.01 (0.93, 1.10); Pz=0.83]. DISCUSSION: A possible association between the two SNPs and IAs was indicated. The associations reported by our meta-analysis need to be further studied and validated by larger CGAS and GWAS.

Melanocytic tumors with spitzoid morphology: correlation of clinicopathologic features and FISH analysis.

BACKGROUND: A subset of melanocytic tumors with spitzoid morphology may lead to potential inaccurate diagnosis and lack of assessment of malignancy potential. In this study, we aimed to evaluate melanocytic tumors with spitzoid morphology using conventional melanoma FISH (RREB-1, CCND1, MYB and CEP6) and 9p21 FISH (CDKN2A) probes and compare the probe results with clinical and histopathological features. METHODS: This study is a multicentric retrospective study including three centers, Istanbul University-Cerrahpasa, Cerrahpasa School of Medicine, Department of Pathology, Acibadem University, School of Medicine, Department of Pathology and ETA Pathology Laboratory. The pathology reports in archives of these three centers between 2015 and 2017 have been reviewed for cases diagnosed as atypical Spitz tumor or melanoma with Spitzoid features. These cases were selected for the study. We analyzed 39 cases of atypical Spitz tumor (AST), 10 cases of melanomas with spitzoid features for clinicopathological data and chromosomal alterations, targeting RREB-1 (6p25), CCND1 (11q13), MYB (6q23), together with 9p21 (CDKN2A), using FISH methodology. RESULTS: Thirty out of total 49 cases showed chromosomal alterations by 4-probe melanoma FISH assay, 22 (56.4%) cases were ASTs, and 8 (80%) cases were melanomas. Eighteen out of 49 cases showed homozygote deletion by 9p21 FISH assay, 12 (30.8%) cases were ASTs, and 6 (60%) cases were melanomas. When histopathological data were compared with FISH results, a statistically significant correlation was found between 9p21 FISH positivity (homozygous deletion) and presence of deep mitosis (p < 0.05). In addition, epidermal consumption (p = 0.07) and increased mitotic activity (p = 0.05) were more frequent in cases with homozygous 9p21 deletion, but these differences did not reach statistical significance. When the clinical features were considered, there was a statistically significant correlation between 9p21 FISH positivity and the diameter (p < 0.05). There was no statistically significant correlation between melanoma FISH assay and any of the histopathological or clinical data. DISCUSSION: These data suggest that 9p21 FISH positivity correlated with more worrisome histopathologic and clinical features, such as deep mitosis, increased mitotic activity, epidermal consumption, and larger lesion size, so these features are precious, pointing out spitzoid lesions with higher risk. However, there is a need for further studies using FISH or similar techniques in order to provide more accurate prognostic information in lesions Blank morphology.

Analysis of 61 SNPs from the CAD specific genomic loci reveals unique set of SNPs as significant markers in the Southern Indian population of Hyderabad.

BACKGROUND: The present study is a part of the major project on coronary artery disease (CAD) carried out at Indian Statistical Institute, Hyderabad to investigate the pattern of association of SNPs selected from the CAD specific genomic loci. The study is expected to portray the genetic susceptibility profile of CAD specifically in the Southern Indian population of Hyderabad. METHODS: The study was conducted in a cohort of 830 subjects comprising 350 CAD cases and 480 controls from Hyderabad. A prioritized set of 61 SNPs selected from the NHGRI GWAS catalogue were genotyped using FluidigmNanofluidic SNP Genotyping System and appropriate statistical analyses were used in interpreting the results. RESULTS: After data pruning, out of 45 SNPs qualified for the association analysis, four SNPs were found to be highly significantly associated with increased risk for CAD even after Bonferroni correction for multiple testing (p < 0.001). These results were also replicated in the random subsets of the pooled cohort (70, 50 and 30%) suggesting internal consistency. The ROC analysis of the risk scores of the significant SNPs suggested highly significant area under curve (AUC = 0.749; p < 0.0001) implying predictive utility of these risk variants. CONCLUSIONS: The rs10455872 of LP(A) gene in particular showed profound risk for CAD (OR 35.9; CI 16.7-77.2) in this regional Indian population. The other significant SNP associations observed with respect to the pooled CAD cohort and in different anatomical and phenotypic severity categories reflected on the role of genetic heterogeneity in the clinical heterogeneity of CAD. The SNP rs7582720 of WDR12 gene, albeit not individually associated with CAD, was found to be conferring significant risk through epistatic interaction with two SNPs (rs6589566, rs1263163 in ZPR1, APOA5-APOA4 genes) of the 11q23.3 region.

A Functional Variant on 9p21.3 Related to Glioma Risk Affects Enhancer Activity and Modulates Expression of CDKN2B-AS1.

Genome-wide association studies have identified SNPs associated with glioma risk on 9p21.3, but biological mechanisms underlying this association are unknown. We tested the hypothesis that a functional SNP on 9p21.3 affects activity of an enhancer, causing altered expression of nearby genes. We considered all SNPs in linkage disequilibrium with the 9p21.3 sentinel SNP rs634537 that mapped to putative enhancers. An enhancer containing rs1537372 exhibited allele-specific effects on luciferase activity. Deletion of this enhancer in GBM cell lines correlated with decreased expression of CDKN2B-AS1. Expression quantitative trait loci analysis using non-diseased brain samples showed rs1537372 to be a consistently significant eQTL for CDKN2B-AS1. Additionally, our analysis of Hi-C data generated in neural progenitor cells showed that the bait region containing rs1537372 interacted with the CDKN2B-AS1 promoter. These data suggest rs1537372, a SNP at the 9p21.3 risk locus, is a functional variant that modulates expression of CDKN2B-AS1.

Association between rs3088440 (G > A) polymorphism at 9p21.3 locus with the occurrence and severity of coronary artery disease in an Iranian population.

BACKGROUND: Several genome-wide association studies showed that a series of genetic variants located at the chromosome 9p21 locus are strongly associated with coronary artery disease (CAD). RATIONALE AND PURPOSE OF THE STUDY: In the present study, the relationship of rs3088440 (G > A) in cyclin-dependent kinase inhibitor 2A (CDKN2A) gene site with the presence of coronary artery disease (CAD) and its severity was evaluated in an Iranian population. METHODS AND RESULTS: The presence of rs3088440 (G > A) genotypes was assessed by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) technique in 324 CAD patients and 148 normal controls. rs3088440 (G > A) polymorphism was associated with increased risk of CAD in the total population (adjusted OR = 1.76, 95% CI = 1.10-2.82; p-value = 0.017) or in women (adjusted OR = 2.96, 95% CI = 1.34-6.55; p-value = 0.007), but not in the men (adjusted OR = 1.35, 95% CI = 0.70-2.6; p-value = 0.368). CONCLUSIONS: Our findings suggest that the presence of rs3088440 (G > A) is potentially linked with the risk of CAD and its severity in whole study subjects or in women only, independent of CAD risk factors.

Analysis on the polymorphisms of site RS4977574, and RS1333045 in region 9p21 and the susceptibility of coronary heart disease in Chinese population.

BACKGROUND: Rs4977574 (A > G) and Rs1333045 (C > T) are both single nucleotide polymorphisms (SNPs) related with coronary artery disease, locating on chromosome 9p21.3. The study aimed to identify the correlation between rs4977574 and rs1333045 polymorphism genotypes and coronary heart disease (CHD) in a Chinese population. METHODS: Blood samples were collected from 855 subjects. A case-control study was used in this experiment, and 598 cases in the CHD group and 257 subjects in the control group were enrolled. Genotyping was identified by the Agena MassARRAY system. Statistical analysis was conducted by SPSS (Ver 16.0) and plink (Ver. 1.07, Shaun Purcell). Haplotype analysis was performed using Haploview software. RESULTS: Association analysis by plink indicated a significant difference in the allele distribution for single nucleotide polymorphisms between cases and controls (rs4977574 P = 0.003, rs1333045 P = 0.035). Fisher's exact test by plink proved that allele G may be associated with a higher risk of CHD (P = 0.003, odds ratio (OR) = 1.371) and the T allele was likely to reduce the risk of coronary events (P = 0.035, OR = 0.798). The serum levels of apolipoprotein A (ApoA) were higher in subjects with the AG + AA genotype of rs4977574 compared to those with the GG genotype (P = 0.028). In the dominant model of rs1333045, the levels of ApoA were higher and LDL levels were lower in the TC + TT genotype than in the CC genotype. CONCLUSIONS: The present study examined the association between the 9p21 chromosome rs4977574 and rs1333045 polymorphism genotypes and CHD in a population of Chinese patients. The G allele of rs4977574 and the C allele of rs1333045 are the susceptibility sites of CHD.

Evaluation of MTAP immunohistochemistry loss of expression in ovarian serous borderline tumors as a potential marker for prognosis and progression.

INTRODUCTION: Serous borderline tumors (SBT) are the most common subtype of ovarian borderline tumors with excellent clinical course. However, they can recur or progress to low-grade serous carcinoma (LGSC) in a small proportion of the cases. Beside BRAF and KRAS mutations, copy number alterations (CNA), particularly loss of chromosome 9p21 locus which results in deletion of genes CDKN2A and MTAP, have been suggested to be involved in disease progression. MTAP immunohistochemistry recently has been introduced for mesothelioma as a reliable surrogate marker for the homozygous deletion of chromosome 9p21 locus. Therefore, in the current study, we aimed to evaluate the MTAP loss of expression in serous borderline tumors and low-grade serous carcinomas to identify if it can be used as a marker for prognosis and progression. METHOD: Eighty-four total cases of ovarian serous lesions, including 21 cases of serous cystadenomas, 21 cases of serous borderline tumors, 12 cases of low-grade serous carcinomas and 30 cases of high-grade serous carcinomas were selected. MTAP immunohistochemistry was performed on the representative blocks and cytoplasmic staining was used for interpretation. The cases were labeled as positive (retained) if MTAP showed cytoplasmic granular staining and negative (loss of expression) if negative cytoplasmic staining was observed in the presence of positive internal control. RESULT: Ten of 21 cases of serous borderline tumors showed loss of MTAP expression (48%). Among those, 7 cases were bilateral, 2 cases had micropapillary features, one case had supraclavicular and cervical lymph node involvement by serous borderline tumor and 2 cases had progression to low-grade serous carcinoma, including one of micropapillary tumors. Also 8 out of 12 cases of LGSCs showed MTAP loss of expression (66%). Only 4 of 30 cases of high-grade serous carcinomas (13%) and none of the serous cystadenoma cases showed loss of expression of MTAP. CONCLUSION: To our knowledge, this is the first description of MTAP immunohistochemistry in serous borderline tumors and low-grade serous carcinomas. Our study was limited due to small sample size. However, it showed an association between MTAP loss of expression and adverse clinical behavior in ovarian serous borderline tumors. This supports the role for further investigations in larger series to evaluate the role of MTAP stain as a prognostic marker in these neoplasms.

A functional variant rs1537373 in 9p21.3 region is associated with pancreatic cancer risk.

9p21.3 has been identified as an unexpected hot point in multiple diseases GWAS including cancers, and we performed a two-stage case-control studies integrating functional assay strategy to find the potential functional variants modified susceptibility to pancreatic cancer (PC). An expanded Illumina HumanExome Beadchip of PC including 943 cases and 3908 controls was used to examine 39 tagSNPs in 9p21.3 and the promising single nucleotide polymorphism (SNP) was validated in stage 2 comprising 624 cases and 1048 controls. The strongest signal was rs6475609 (Odds ratio, OR = 0.81, 95% confidence interval, CI = 0.72-0.91) maps to the long non-coding RNA ANRIL. Bioinformatics analysis revealed rs1537373 lies in the linkage disequilibrium (LD) block which the rs6475609 tagged might have potential function and was also associated with a decreased risk of PC in both stages (OR = 0.82, 95% CI = 0.75-0.90 in combined analysis). Dual luciferase reporter assay and the electrophoretic mobility shift assay (EMSA) verified rs1537373 as the best candidate causative variant for influencing the activity of enhancer through differential binding to certain transcription factor. The expression quantitative trait loci (e-QTL) analysis indicated the genotypes of rs1537373 were associated with expression of CDKN2B gene (P dominant = 6.00 × 10-4 ). In conclusion, our study provided evidence that rs1537373 in ANRIL may influence transcription factor binding and regulate CDKN2B expression, thus confer the susceptibility to PC.

Common SNP-based haplotype analysis of the 9p21.3 gene locus as predictor coronary artery disease in Tanzanian population.

Genome-wide association studies (GWAS) have recently confirmed a strong association of the 9p21.3 locus with Coronary Artery Disease (CAD) in different populations but no data has been reported for the Tanzanian population. This study aimed to investigate the 9p21.3 locus harboring the disease-causing hotspot variations in Tanzanian CAD patients and their associations with the risk factors. 135 patients with CAD and 140 non-CAD patients were enrolled into the study. Further the biochemical analysis, the genotyping assays were performed by the use of qRT-PCR. The genotype and allele frequencies of rs1333049, rs2383207, rs2383206, rs10757274, rs10757278, and rs10811656 were significantly different between the groups (p<0.005). The genotype distribution of rs1333049, rs10757278 and rs10811656 polymorphisms were significantly different among patients with one, two, three stenotic vessels (p<0.05). For rs10757274 and rs10757278, the GG genotype indicated a significant 3-fold and 4-fold increased risk of CAD (p<0.0001,respectively). Additionally, haplotype analysis revealed that AAGCAG, AAACAG, GGGTGC haplotypes of 9p21.3 locus polymorphisms are associated with CAD risk. The GGGTGC haplotype was over-represented while the other two underrepresented in patients as compared to controls (p<0.00001,respectively) suggesting the first one a high-risk and the other two low-risk haplotypes for Tanzanian population. The AUC of a risk model based on non-genetic risk factors was 0.954 (95% CI: 0.930-0.977) and the combination with genetic risk factors improved the AUC to 0.982 (95% CI: 0.954-0.985) (p<0.012), indicating good diagnostic accuracy. Our results are the first data reporting statistically significant associations between 9p21.3 polymorphisms and CAD, and the very first haplotype block harboring the disease-causing variations in Tanzanian population.

Morphological difference between pleural mesothelioma cells in effusion smears with either BAP1 loss or 9p21 homozygous deletion and reactive mesothelial cells without the gene alterations.

We previously characterized the morphological characteristics of malignant pleural mesothelioma (MPM) cells with 9p21 homozygous deletion (HD) using a combination of the virtual microscopy and fluorescence in situ hybridization (FISH). In this study, we investigated whether MPM cells with BRCA1-associated protein 1 (BAP1) loss show the same morphological characteristics identified in MPM cells with 9p21 HD. MPM cells with either BAP1 loss detected by immunocytochemistry (ICC) or 9p21 HD detected by FISH were identified via virtual microscopy prior to ICC or FISH, followed by analysis and quantification of their morphological characteristics. MPM cells with BAP1 loss or 9p21 HD exhibited significantly more frequent cell-in-cell engulfment, multinucleation, and larger multicellular clusters composed of more than 10 cells than reactive mesothelial cells. In conclusion, MPM cells with BAP1 loss or 9p21 HD share similar cytological features, indicating that the same morphological criteria can be used to detect MPM cells harboring such genetic aberrations.

Genetic Variation in 9p21 and the Plasma Proteome.

Single nucleotide polymorphisms (SNPs) in the noncoding region of 9p21 have been associated with cardiovascular disease (CVD), but the mechanisms by which these genetic variants contribute to the pathogenesis of CVD remain unknown since no annotated proteins are present in this region of DNA. The objective of the current study was to determine if 9p21 genotypes are associated with distinct plasma proteins in young adults. Subjects were 1611 young adults aged 20-29 years from the Toronto Nutrigenomics and Health Study (1098 females and 513 males). DNA was isolated from fasting blood to analyze four SNPs in 9p21 (rs2383206, rs10757274, rs10757278, and rs1333049). Abundant plasma proteins ( n = 54) were measured using liquid chromatography multiple reaction monitoring mass spectrometry. Analysis of covariance was used to determine differences in plasma proteins between genotypes. In Caucasians ( n = 524), four SNPs were associated with apolipoprotein E and two with haptoglobin-ß-chain concentration. In East Asians ( n = 388), three SNPs were associated with α-1b-Glycoprotein, two with apolipoprotein B-100, one with apolipoprotein E and one with haptoglobin-ß-chain concentration. In South Asians ( n = 117), one SNP was associated with apolipoprotein B-100 concentration. Our findings suggest that 9p21 genotypes may play a role in various pathophysiological pathways that contribute to risk of CVD in early adulthood that might be distinct among different ethnocultural groups.

The 9p21 locus: A potential therapeutic target and prognostic marker in breast cancer.

Breast cancer is an important cause of cancer related mortality in women. Despite extensive efforts to identify valid biomarkers for risk stratification, there are relatively few with proven clinical utility. It is recognized that genetic factors play a major role in determining susceptibility to breast cancer. Recent genome-wide-association-studies and gene expression analysis have demonstrated that a locus on chromosome 9p21, which contains three genes; CDKN2B (encoding p15ink4b), CDKN2A (encoding p16ink4a and p14ARF) and the 3' end of CDKN2BAS (an antisense noncoding RNA in the INK4 locus [ANRIL]) are associated with an increased risk of this malignancy. ANRIL has a post transcriptional modulatory activity, which has been shown to perturb the expression of nearby genes and may play an important role in coordinating tissue remodeling through regulation of cell proliferation, apoptosis, aging, extra-cellular matrix remodeling, and inflammatory response. However, the role of ANRIL is not well understood in breast cancer. Hypermethylation of the p14ARF and p16INK4a genes is found in some tumor types. Nevertheless, further studies are necessary to confirm the clinical utility of these putative markers in risk stratification, or assessing prognosis. In this review, we have summarized the prognostic and therapeutic potential of the p14ARF and p16INK4a genes in patients with breast cancer.

BRAF V600E mutation and 9p21: CDKN2A/B and MTAP co-deletions - Markers in the clinical stratification of pediatric gliomas.

BACKGROUND: Genetic alterations in pediatric primary brain tumors can be used as diagnostic and prognostic markers and are the basis for the development of new target therapies that, ideally, would be associated with lower mortality and morbidity. This study evaluates the incidence and interplay of the presence of BRAF V600E mutation and chromosomal 9p21 deletions in a series of 100 pediatric gliomas, aiming to determine the role of these alterations in recurrence and malignant transformation, and to verify if they could be used in the clinical set for stratifying patients for tailored therapies and surveillance. METHODS: Sanger sequencing was used for the assessment of BRAF mutations at exon 15 and Fluorescent In Situ Hybridization (FISH) with BAC: RP11-14192 for the detection of 9p21 alterations. Expression levels of the CDKN2A and MTAP by real-time PCR were evaluated in cases with 9p21 deletions. Statistical analysis of genetic and clinical data was performed using Graph Pad Prism 5 and SPSS Statistics 24 software. RESULTS: In our cohort it was observed that 7 /78 (8,9%) of the low-grade tumors recurred and 2 (2,6%) showed malignant transformation. BRAF V600E mutations were detected in 15 cases. No statistically significant correlations were found between the presence of BRAF V600E mutation and patient's morphologic or clinical features. Deletions at 9p21 abrogating the CDKN2A/B and MTAP loci were rare in grade I gliomas (12.2%, p = 0.0178) but frequent in grade IV gliomas (62.5%, p = 0.0087). Moreover it was found that deletions at these loci were correlated with a shorter overall survival (p = 0.011) and a shorter progression-free survival (p = 0.016). CONCLUSIONS: It was demonstrated that in these tumors BRAF V600E mutated and that CDKN2A/B MTAP co-deletions may be used for stratifying patients for a stricter surveillance. The Investigating and defining if glial tumors with CDKN2A/B and MTAP homozygous loss may be vulnerable to new forms of therapy, namely those affecting the methionine salvage pathway, was proven to be of importance.