Custom gene expression panel for evaluation of potential molecular markers in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. It is a highly heterogeneous disease with poor prognosis and limited treatment options, which highlights the need for reliable biomarkers. This study aims to explore molecular markers that allow stratification of HCC and may lead to better prognosis and treatment prediction. MATERIALS AND METHODS: We studied 20 candidate genes (HCC hub genes, potential drug target genes, predominant somatic mutant genes) retrieved from literature and public databases with potential to be used as the molecular markers. We analysed expression of the genes by RT-qPCR in 30 HCC tumour and adjacent non-tumour paired samples from Vietnamese patients. Fold changes in expression were then determined using the 2-∆∆CT method, and unsupervised hierarchical clustering was generated using Cluster v3.0 software. RESULTS: Clustering of expression data revealed two subtypes of tumours (proliferative and normal-like) and four clusters for genes. The expression profiles of the genes TOP2A, CDK1, BIRC5, GPC3, IGF2, and AFP were strongly correlated. Proliferative tumours were characterized by high expression of the c-MET, ARID1A, CTNNB1, RAF1, LGR5, and GLUL1 genes. TOP2A, CDK1, and BIRC5 HCC hub genes were highly expressed (> twofold) in 90% (27/30), 83% (25/30), and 83% (24/30) in the tissue samples, respectively. Among the drug target genes, high expression was observed in the GPC3, IGF2 and c-MET genes in 77% (23/30), 63% (19/30), and 37% (11/30), respectively. The somatic mutant Wnt/ß-catenin genes (CTNNB1, GLUL and LGR5) and TERT were highly expressed in 40% and 33% of HCCs, respectively. Among the HCC marker genes, a higher percentage of tumours showed GPC3 expression compared to AFP expression [73% (23/30) vs. 43% (13/30)]. CONCLUSION: The custom panel and molecular markers from this study may be useful for diagnosis, prognosis, biomarker-guided clinical trial design, and prediction of treatment outcomes.

Occurrence, functionality and abundance of the TERT promoter mutations.

Telomere shortening at chromosomal ends due to the constraints of the DNA replication process acts as a tumor suppressor by restricting the replicative potential in primary cells. Cancers evade that limitation primarily through the reactivation of telomerase via different mechanisms. Mutations within the promoter of the telomerase reverse transcriptase (TERT) gene represent a definite mechanism for the ribonucleic enzyme regeneration predominantly in cancers that arise from tissues with low rates of self-renewal. The promoter mutations cause a moderate increase in TERT transcription and consequent telomerase upregulation to the levels sufficient to delay replicative senescence but not prevent bulk telomere shortening and genomic instability. Since the discovery, a staggering number of studies have resolved the discrete aspects, effects and clinical relevance of the TERT promoter mutations. The promoter mutations link transcription of TERT with oncogenic pathways, associate with markers of poor outcome and define patients with reduced survivals in several cancers. In this review, we discuss the occurrence and impact of the promoter mutations and highlight the mechanism of TERT activation. We further deliberate on the foundational question of the abundance of the TERT promoter mutations and a general dearth of functional mutations within noncoding sequences, as evident from pan-cancer analysis of the whole-genomes. We posit that the favorable genomic constellation within the TERT promoter may be less than a common occurrence in other noncoding functional elements. Besides, the evolutionary constraints limit the functional fraction within the human genome, hence the lack of abundant mutations outside the coding sequences.

Polymorphisms in CXCR3 ligands predict early CXCL9 recovery and severe chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is a major cause of mortality and morbidity after allogeneic stem cell transplantation (alloSCT). The individual risk of severe cGVHD remains difficult to predict and may involve CXCR3 ligands. This study investigated the role of single-nucleotide polymorphisms (SNPs) of CXCL4, CXCL9, CXCL10, and CXCL11, and their day +28 serum levels, in cGVHD pathogenesis. Eighteen CXCR3 and CXCL4, CXCL9-11 SNPs as well as peri-transplant CXCL9-11 serum levels were analyzed in 688 patients without (training cohort; n = 287) or with statin-based endothelial protection cohort (n = 401). Clinical outcomes were correlated to serum levels and SNP status. Significant polymorphisms were further analyzed by luciferase reporter assays. Findings were validated in an independent cohort (n = 202). A combined genetic risk comprising four CXCR3 ligand SNPs was significantly associated with increased risk of severe cGVHD in both training cohort (hazard ratio (HR) 2.48, 95% confidence interval (CI) 1.33-4.64, P = 0.004) and validation cohort (HR 2.95, 95% CI 1.56-5.58, P = 0.001). In reporter assays, significantly reduced suppressive effects of calcineurin inhibitors in constructs with variant alleles of rs884304 (P < 0.001) and rs884004 (P < 0.001) were observed. CXCL9 serum levels at day +28 after alloSCT correlated with both genetic risk and risk of severe cGVHD (HR 1.38, 95% CI 1.10-1.73, P = 0.006). This study identifies patients with high genetic risk to develop severe cGVHD.

Telomere length in peripheral blood lymphocytes related to genetic variation in telomerase, prognosis and clinicopathological features in breast cancer patients.

Disruption of telomere length (TL) homeostasis in peripheral blood lymphocytes has been previously assessed as a potential biomarker of breast cancer (BC) risk. The present study addressed the relationship between lymphocyte TL (LTL), prognosis and clinicopathological features in the BC patients since these associations are insufficiently explored at present. LTL was measured in 611 BC patients and 154 healthy controls using the monochrome multiplex quantitative Polymerase Chain Reaction assay. In addition, we genotyped nine TL-associated single-nucleotide polymorphisms that had been identified through genome-wide association studies. Our results showed that the patients had significantly (P = 0.001, Mann-Whitney U-test) longer LTL [median (interquartile range); 1.48 (1.22-1.78)] than the healthy controls [1.27 (0.97-1.82)]. Patients homozygous (CC) for the common allele of hTERT rs2736108 or the variant allele (CC) of hTERC rs16847897 had longer LTL. The latter association remained statistically significant in the recessive genetic model after the Bonferroni correction (P = 0.004, Wilcoxon two-sample test). We observed no association between LTL and overall survival or relapse-free survival of the patients. LTL did not correlate with cancer staging based on Union for International Cancer Control (UICC), The tumor node metastasis (TNM) staging system classification, tumour grade or molecular BC subtypes. Overall, we observed an association between long LTL and BC disease and an association of the hTERC rs16847897 CC genotype with increased LTL. However, no association between LTL, clinicopathological features and survival of the patients was found.

Chromosomal damage and telomere length in peripheral blood lymphocytes of cancer patients.

Accumulation of non­specific structural chromosomal aberrations (CAs) and telomere shortening contribute to genome instability, which constitutes as one of the hallmarks of cancer. CAs arise due to direct DNA damage or telomere shortening. CAs in peripheral blood lymphocytes (PBL), which are considered to be markers of exposure, have been previously reported to serve a role in the pathophysiology and progression of cancer through mechanisms that are poorly understood. In addition, the prognostic relevance of telomere length (TL) in patients with cancer remains to be elucidated. In the present study, CAs and TL in PBL isolated from patients with newly diagnosed cancer (151 breast, 96 colorectal, 90 lung) and 335 cancer­free control individuals were investigated. These results were then correlated with clinicopathological factors and follow­up data. The accumulation of CAs in PBL was observed with increased susceptibility to breast and lung cancer (P<0.0001), while individuals with longer TL were found to be at a higher risk of breast cancer (P<0.0001). Increased chromatid­type aberrations were also revealed to be associated with lower overall survival of patients with breast and colorectal cancers using a multivariate model. Compared with control individuals, no association was observed between TL and CAs or age in patients with cancer. In conclusion, the present study demonstrates the association between CAs/TL in PBL and the susceptibility, prognosis and survival of patients with breast, colorectal and lung cancer.

Association of HERV-K and LINE-1 hypomethylation with reduced disease-free survival in melanoma patients.

Aim: To evaluate CpG methylation of long interspersed nuclear elements 1 (LINE-1) and human endogenous retrovirus K (HERV-K) retroelements as potential prognostic biomarkers in cutaneous melanoma. Materials & methods: Methylation of HERV-K and LINE-1 retroelements was assessed in resected melanoma tissues from 82 patients ranging in age from 14 to 88 years. In addition, nevi from eight patients were included for comparison with nonmalignant melanocytic lesions. Results: Methylation levels were lower in melanomas than in nevi. HERV-K and LINE-1 methylation were decreased in melanoma patients with clinical parameters associated with adverse prognosis, while they were independent of age and gender. Hypomethylation of HERV-K (but not LINE-1) was an independent predictor of reduced disease-free survival. Conclusion: HERV-K hypomethylation can be a potential independent biomarker of melanoma recurrence.

Informing patients about their mutation tests: CDKN2A c.256G>A in melanoma as an example.

BACKGROUND: When germline mutations are suspected as causal in cancer, patient DNA may be sequenced to detect variants in relevant genes. If a particular mutation has not been reported in reliable family studies, genetic counselors are facing a dilemma of appropriately informing patients. Many sequencing facilities provide an interpretation of the findings based on the available sequence databases or on prediction tools that are curated from bioinformatics and mechanistic datasets. The counseling dilemma is exacerbated if the pedigree data are not informative but the in silico predictions suggest pathogenicity. METHODS: We present here a real world example of the c.256G > A CDKN2A variant, which was detected in one melanoma patient where two siblings were diagnosed with melanoma in situ. We investigated a detailed family history of the affected siblings in order to survey probability of the cancer risks within the context to this mutation. RESULTS: This c.256G > A CDKN2A variant was detected in one of the brothers and in the melanoma-free mother while the other brother in the family tested negative. The variant had been previously described in one patient from a melanoma family. In the family under investigation, the mother's 16 first-and second-degree relatives had survived past the median onset age for melanoma and none presented melanoma. We tested the variant using multiple bioinformatic tools that all predicted deleteriousness of the variant. The genetic counseling report to the melanoma patient stated that the CDKN2A variant was 'likely pathogenic' and the disease was defined as 'likely hereditary melanoma'. CONCLUSIONS: The pedigree data showed at the most a low penetrance variant, which, if taken into consideration, might have altered the provided diagnosis. When dealing with 'practically' unknown variants the counselors would be advised to incorporate a detailed family history rather than basing predictions on functionality provided by sequencing facilities.

Coding and noncoding somatic mutations in candidate genes in basal cell carcinoma.

Basal cell carcinoma (BCC) represents the most commonly diagnosed human cancer among persons of European ancestry with etiology mainly attributed to sun-exposure. In this study we investigated mutations in coding and flanking regions of PTCH1 and TP53 and noncoding alterations in the TERT and DPH3 promoters in 191 BCC tumors. In addition, we measured CpG methylation within the TERT hypermethylated oncological region (THOR) and transcription levels of the reverse transcriptase subunit. We observed mutations in PTCH1 in 58.6% and TP53 in 31.4% of the tumors. Noncoding mutations in TERT and DPH3 promoters were detected in 59.2% and 38.2% of the tumors, respectively. We observed a statistically significant co-occurrence of mutations at the four investigated loci. While PTCH1 mutations tended to associate with decreased patient age at diagnosis; TP53 mutations were associated with light skin color and increased number of nevi; TERT and DPH3 promoter with history of cutaneous neoplasms in BCC patients. Increased reverse transcriptase subunit expression was observed in tumors with TERT promoter mutations and not with THOR methylation. Our study signifies, in addition to the protein altering mutations in the PTCH1 and TP53 genes, the importance of noncoding mutations in BCC, particularly functional alterations in the TERT promoter.

Telomeres and Telomere Length: A General Overview.

Telomeres are highly conserved tandem nucleotide repeats that include proximal double-stranded and distal single-stranded regions that in complex with shelterin proteins afford protection at chromosomal ends to maintain genomic integrity. Due to the inherent limitations of DNA replication and telomerase suppression in most somatic cells, telomeres undergo age-dependent incremental attrition. Short or dysfunctional telomeres are recognized as DNA double-stranded breaks, triggering cells to undergo replicative senescence. Telomere shortening, therefore, acts as a counting mechanism that drives replicative senescence by limiting the mitotic potential of cells. Telomere length, a complex hereditary trait, is associated with aging and age-related diseases. Epidemiological data, in general, support an association with varying magnitudes between constitutive telomere length and several disorders, including cancers. Telomere attrition is also influenced by oxidative damage and replicative stress caused by genetic, epigenetic, and environmental factors. Several single nucleotide polymorphisms at different loci, identified through genome-wide association studies, influence inter-individual variation in telomere length. In addition to genetic factors, environmental factors also influence telomere length during growth and development. Telomeres hold potential as biomarkers that reflect the genetic predisposition together with the impact of environmental conditions and as targets for anti-cancer therapies.

Telomere length, arsenic exposure and risk of basal cell carcinoma of skin.

Telomere length per se a heritable trait has been reported to be associated with different diseases including cancers. In this study, based on arsenic-exposed 528 cases with basal cell carcinoma (BCC) of skin and 533 healthy controls, we investigated effect of telomere length, measured by real-time PCR, on the disease risk. We observed a statistically significant association between decreased telomere length and increased BCC risk [odds ratio (OR) = 5.92, 95% confidence interval (CI) = 3.92 to 9.01, P < 0.0001]. Due to confounder effect of arsenic exposure, in a two-sample Mendelian randomization (MR), telomere length associated single-nucleotide polymorphisms as instrument variables violated valid assumptions; however, one-sample MR adjusted for arsenic exposure indicated an increased risk of BCC with short telomeres. The interaction between arsenic exposure and telomere length on BCC risk was statistically significant (P = 0.02). Within each tertile based on arsenic exposure, the individuals with shorter telomeres were at an increased risk of BCC, with highest risk being in the highest exposed group (OR = 16.13, 95% CI = 6.71 to 40.00, P < 0.0001), followed by those in medium exposure group and low exposure group. The combined effect of highest arsenic exposure and shortest telomeres on BCC risk (OR = 10.56, 95% CI = 5.14 to 21.70) showed a statistically significant departure from additivity (interaction contrast ratio 6.56, P = 0.03). Our results show that in the presence of arsenic exposure, decreased telomere length predisposes individuals to increased risk of BCC, with the effect being synergistic in individuals with highest arsenic exposure and shortest telomeres.

Author Correction: Genome-wide association study identifies susceptibility loci for B-cell childhood acute lymphoblastic leukemia.

The original version of this Article contained an error in the spelling of a member of the PRACTICAL Consortium, Manuela Gago-Dominguez, which was incorrectly given as Manuela Gago Dominguez. This has now been corrected in both the PDF and HTML versions of the Article. Furthermore, in the original HTML version of this Article, the order of authors within the author list was incorrect. The PRACTICAL consortium was incorrectly listed after Richard S. Houlston and should have been listed after Nora Pashayan. This error has been corrected in the HTML version of the Article; the PDF version was correct at the time of publication.

TERT promoter mutation subtypes and survival in stage I and II melanoma patients.

Mutations within the promoter of gene encoding telomerase reverse transcriptase subunit are frequent in many cancers including melanoma. Previously, the TERT promoter mutations were shown to associate with markers of poor outcome and reduced survival in patients with primary melanoma. In this study, we investigated the impact of the subtypes of TERT mutations on disease-free and melanoma-specific survival in 287 patients with stage I/II nonacral melanoma. Our results showed that of the three TERT promoter mutation subtypes, in multivariate models, the -138/-139 CC > TT tandem mutation associated with worst disease-free and melanoma-specific survival. In particular, in combination with BRAF/NRAS mutations, the -138/-139 CC > TT TERT promoter mutation associated with statistically significant poor disease-free and melanoma-specific survival with hazard ratios of 6.04 (95% CI 2.03-17.94, p = 0.001) and 12.59 (95% CI 2.18-72.70, p = 0.005), respectively. In contrast to the survival data, luciferase assays showed that the highest activity was observed in experiments with a promoter construct with -124 C > T mutation followed by the -138/-139 CC > TT and -146 C > T mutations, which showed similar activity. Based on previous reports, we speculate that the tandem mutation probably leads to greater genomic instability than the common TERT promoter mutations, hence the association with worst survival. However, the results from the study are only preliminary with limited patient data, therefore, require a cautious interpretation. The observations in this study, if confirmed, could have implications for melanoma patients treated with MAP-kinase inhibitors.

Asymmetric dimethylarginine serum levels are associated with early mortality after allogeneic stem cell transplantation.

Increasing evidence suggests that endothelial cell distress is associated with mortality after allogeneic stem cell transplantation and acute graft-versus-host disease. Asymmetric dimethylarginine is an endogenous nitric oxide synthase inhibitor that induces endothelial cell dysfunction. We analyzed the impact of pre-transplant serum levels of asymmetric dimethylarginine on outcome after allogeneic stem cell transplantation. Since acute graft-versus-host disease and its treatment are major contributors to post-transplant mortality, the effect of asymmetric dimethylarginine on outcome measures was also assessed after onset of acute graft-versus-host disease. A total of 938 patients allografted at two centers between 2002 and 2013 were included in the retrospective study. In multivariable models, higher pre-transplant asymmetric dimethylarginine levels were significantly associated with an increased risk of non-relapse mortality (hazard ratio 1.43 per 1-log2 increase, P=0.005) but not with relapse (hazard ratio 1.21, P=0.109) within the first year after transplantation. This translated into worse overall survival (hazard ratio 1.45, P<0.0001) and shorter progression-free survival (hazard ratio 1.30, P=0.002) in the first year after transplantation. Higher pre-transplant asymmetric dimethylarginine levels were also associated with shorter overall survival (hazard ratio 1.46, P=0.001) and progression-free survival (hazard ratio 1.32, P=0.010) and higher non-relapse mortality (hazard ratio 1.36, P=0.042) within 1 year after the onset of acute graft-versus-host disease. Taken together, our data indicate an association between pre-transplant asymmetric dimethylarginine status and early non-relapse mortality in allografted patients, both overall and after the onset of acute graft-versus-host disease. These findings underline the relevance of endothelial dysfunction for transplant complications.

Author Correction: Telomere length and survival in primary cutaneous melanoma patients.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Telomere length, telomerase reverse transcriptase promoter mutations, and melanoma risk.

Telomere repeats at chromosomal ends, critical for genomic integrity, undergo age-dependent attrition and telomere length has been associated with different disorders including cancers. In this study, based on 1469 patients and 1158 healthy controls, we show a statistically significant (P = 6 × 10-10 ) association between increased telomere length and melanoma risk. Mendelian randomization, using 5 telomere length-associated polymorphisms, ruled out confounding factors or reverse causality and showed association between increased telomere length and melanoma risk with odds ratio of 2.66 (95% confidence interval: 2.07-3.25). Age-dependent telomere attrition was faster in melanoma cases than controls (P = .01). The carriers of a highly penetrant germline -57A>C TERT promoter mutation, in a previously reported melanoma family, had longer telomeres than the noncarriers. The mutation causes increased TERT and telomerase levels through creation of a binding motif for E-twenty six (ETS) transcription factors and the carriers develop melanoma with an early age of onset and rapid progression to metastasis. In analogy, we hypothesize that increased telomere length in melanoma patients reflects stochastic increased telomerase levels due to common genetic variation. Paradoxically, we observed shorter telomeres (P = 1 × 10-5 ) in primary tumors from unrelated melanoma patients with (121) than without (170) somatic TERT promoter mutations that similar to the germline mutation, also create binding motifs for ETS transcription factors. However, the age-dependent telomere attrition was faster in tumors with the TERT promoter mutations than in those without such mutations. Besides a robust association between increased telomere length and risk, our data show a perturbed telomere homeostasis in melanoma.

Telomere length and survival in primary cutaneous melanoma patients.

Telomere repeats at chromosomal ends, critical to genomic integrity, undergo age-dependent attrition. Telomere length, a polygenic trait, has been associated with risk of several disorders including cancers. In contrast to association of long telomeres with increased risk of several cancers, including melanoma, emerging reports suggest that short telomeres predict poor survival in patients with different cancers. In this study based on 1019 stage I and II cutaneous melanoma patients, we show an association between the patients with short telomeres and poor melanoma-specific survival (HR 2.05, 95% CI 1.33-3.16) compared to patients with long telomeres. Due to inverse correlation between age and telomere length (r -0.19, P < 0.0001), we stratified the patients into quantiles based on age at diagnosis and also carried out age-matched analysis. The effect of short telomeres on survival was determined by using multivariate Cox regression that included composite genetic risk score computed from genotyping of the patients for telomere-length associated polymorphisms. The effect of decreased telomere length on poor melanoma-specific survival was particularly strong in patients within the age quantile below 30 years (HR 3.82, 95% CI 1.10-13.30) and between 30-40 years (HR 2.69, 95% CI 1.03-7.03). Our study shows that in contrast to increased melanoma risk associated with increased telomere length, decreased telomere length predicts poor survival in melanoma subgroups.