Significance of PI3K signalling pathway in clear cell renal cell carcinoma in relation to VHL and HIF status.

Renal cell carcinoma (RCC) includes diverse tumour types characterised by various genetic abnormalities. The genetic changes, like mutations, deletions and epigenetic alterations, play a crucial role in the modification of signalling networks, tumour pathogenesis and prognosis. The most prevalent RCC type, clear cell RCC (ccRCC), is asymptomatic in the early stages and has a poorer prognosis compared with the papillary and the chromophobe types RCCs. Generally, ccRCC is refractory to chemotherapy and radiation therapy. Loss of von Hippel-Lindau (VHL) gene and upregulation of hypoxia-inducible factors (HIF), the signature of most sporadic ccRCC, promote multiple growth factors. Hence, VHL/HIF and a variety of pathways, including phosphatase and TEnsin homolog on chromosome 10/phosphatidylinositol-3-kinase (PI3K)/AKT, are closely connected and contribute to the ontogeny of ccRCC. In the recent decade, multiple targeting agents have been developed based on blocking major signalling pathways directly or indirectly involved in ccRCC tumour progression, metastasis, angiogenesis and survival. However, most of these drugs have limitations; either metastatic ccRCC develops resistance to these agents, or despite blocking receptors, tumour cells use alternate signalling pathways. This review compiles the state of knowledge about the PI3K/AKT signalling pathway confined to ccRCC and its cross-talks with VHL/HIF pathway.

DNA methylation associates with survival in non-metastatic clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype among renal cancer and is associated with poor prognosis if metastasized. Up to one third of patients with local disease at diagnosis will develop metastasis after nephrectomy, and there is a need for new molecular markers to identify patients with high risk of tumor progression. In the present study, we performed genome-wide promoter DNA methylation analysis at diagnosis to identify DNA methylation profiles associated with risk for progress. METHOD: Diagnostic tissue samples from 115 ccRCC patients were analysed by Illumina HumanMethylation450K arrays and methylation status of 155,931 promoter associated CpGs were related to genetic aberrations, gene expression and clinicopathological parameters. RESULTS: The ccRCC samples separated into two clusters (cluster A/B) based on genome-wide promoter methylation status. The samples in these clusters differed in tumor diameter (p < 0.001), TNM stage (p < 0.001), morphological grade (p < 0.001), and patients outcome (5 year cancer specific survival (pCSS5yr) p < 0.001 and cumulative incidence of progress (pCIP5yr) p < 0.001. An integrated genomic and epigenomic analysis in the ccRCCs, revealed significant correlations between the total number of genetic aberrations and total number of hypermethylated CpGs (R = 0.435, p < 0.001), and predicted mitotic age (R = 0.407, p < 0.001). We identified a promoter methylation classifier (PMC) panel consisting of 172 differently methylated CpGs accompanying progress of disease. Classifying non-metastatic patients using the PMC panel showed that PMC high tumors had a worse prognosis compared with the PMC low tumors (pCIP5yr 38% vs. 8%, p = 0.001), which was confirmed in non-metastatic ccRCCs in the publically available TCGA-KIRC dataset (pCIP5yr 39% vs. 16%, p < 0.001). CONCLUSION: DNA methylation analysis at diagnosis in ccRCC has the potential to improve outcome-prediction in non-metastatic patients at diagnosis.

Novel variants in Nordic patients referred for genetic testing of telomere-related disorders.

Telomere-related disorders are a clinically and genetically heterogeneous group of disorders characterized by premature telomere shortening and proliferative failure of a variety of tissues. This study reports the spectrum of telomere-related gene variants and telomere length in Nordic patients referred for genetic testing due to suspected telomere-related disorder. We performed Sanger sequencing of the genes TERT, TERC, DKC1, and TINF2 on 135 unrelated index patients and measured telomere length by qPCR on DNA from peripheral blood leukocytes. We identified pathogenic or likely pathogenic variants in 10 index patients, all of which had short telomeres compared to age-matched healthy controls. Six of the 10 variants were novel; three in TERC (n.69_74dupAGGCGC, n.122_125delGCGG, and n.407_408delinsAA) and three in TERT (p.(D684G), p.(R774*), and p.(*1133Wext*39)). The high proportion of novel variants identified in our study highlights the need for solid interpretation of new variants that may be detected. Measurement of telomere length is a useful approach for evaluating pathogenicity of genetic variants associated with telomere-related disorders.

Long leukocyte telomere length in prostate cancer patients at diagnosis is associated with poor metastasis-free and cancer-specific survival.

Previous studies have suggested that leukocyte telomere length is associated with risk of developing prostate cancer. Investigations of leukocyte telomere length as a prognostic factor in prostate cancer are, however, lacking. In this study, leukocyte telomere length was investigated both as a risk marker, comparing control subjects and patient risk groups (based on serum levels of prostate-specific antigen, tumor differentiation, and tumor stage), and as a prognostic marker for metastasis-free and cancer-specific survival. Relative telomere length was measured by a well-established quantitative polymerase chain reaction method in 415 consecutively sampled individuals. Statistical evaluation included 162 control subjects without cancer development during follow-up and 110 untreated patients with newly diagnosed localized prostate cancer at the time of blood draw. Leukocyte telomere length did not differ significantly between control subjects and patients, or between patient risk groups. Interestingly, however, and in line with our previous results in breast and kidney cancer patients, relative telomere length at diagnosis was an independent prognostic factor. Patients with long leukocyte telomeres (⩾median) had a significantly worse prostate cancer-specific and metastasis-free survival compared to patients with short telomere length. In contrast, for patients who died of other causes than prostate cancer, long relative telomere length was not coupled to shorter survival time. To our knowledge, these results are novel and give further strength to our hypothesis that leukocyte telomere length might be used as a prognostic marker in malignancy.

Decreased telomere length in children with cartilage-hair hypoplasia.

BACKGROUND: Cartilage-hair hypoplasia (CHH) is an autosomal recessive chondrodysplasia caused by RMRP (RNA component of mitochondrial RNA processing endoribonuclease) gene mutations. Manifestations include short stature, variable immunodeficiency, anaemia and increased risk of malignancies, all of which have been described also in telomere biology disorders. RMRP interacts with the telomerase RT (TERT) subunit, but the influence of RMRP mutations on telomere length is unknown. We measured relative telomere length (RTL) in patients with CHH, their first-degree relatives and healthy controls and correlated RTL with clinical and laboratory features. METHODS: The study cohort included 48 patients with CHH with homozygous (n=36) or compound heterozygous RMRP mutations (median age 38.2 years, range 6.0-70.8 years), 86 relatives (74 with a heterozygous RMRP mutation) and 94 unrelated healthy controls. We extracted DNA from peripheral blood, sequenced the RMRP gene and measured RTL by qPCR. RESULTS: Compared with age-matched and sex-matched healthy controls, median RTL was significantly shorter in patients with CHH (n=40 pairs, 1.05 vs 1.21, p=0.017), but not in mutation carriers (n=48 pairs, 1.16 vs 1.10, p=0.224). RTL correlated significantly with age in RMRP mutation carriers (r=-0.482, p<0.001) and non-carriers (r=-0.498, p<0.001), but not in patients (r=-0.236, p=0.107). In particular children (<18 years) with CHH had shorter telomeres than controls (median RTL 1.12 vs 1.26, p=0.008). In patients with CHH, RTL showed no correlation with genotype, clinical or laboratory characteristics. CONCLUSIONS: Telomere length was decreased in children with CHH. We found no correlation between RTL and clinical or laboratory parameters.

DNA Methylation Adds Prognostic Value to Minimal Residual Disease Status in Pediatric T-Cell Acute Lymphoblastic Leukemia.

BACKGROUND: Despite increased knowledge about genetic aberrations in pediatric T-cell acute lymphoblastic leukemia (T-ALL), no clinically feasible treatment-stratifying marker exists at diagnosis. Instead patients are enrolled in intensive induction therapies with substantial side effects. In modern protocols, therapy response is monitored by minimal residual disease (MRD) analysis and used for postinduction risk group stratification. DNA methylation profiling is a candidate for subtype discrimination at diagnosis and we investigated its role as a prognostic marker in pediatric T-ALL. PROCEDURE: Sixty-five diagnostic T-ALL samples from Nordic pediatric patients treated according to the Nordic Society of Pediatric Hematology and Oncology ALL 2008 (NOPHO ALL 2008) protocol were analyzed by HumMeth450K genome wide DNA methylation arrays. Methylation status was analyzed in relation to clinical data and early T-cell precursor (ETP) phenotype. RESULTS: Two distinct CpG island methylator phenotype (CIMP) groups were identified. Patients with a CIMP-negative profile had an inferior response to treatment compared to CIMP-positive patients (3-year cumulative incidence of relapse (CIR3y ) rate: 29% vs. 6%, P = 0.01). Most importantly, CIMP classification at diagnosis allowed subgrouping of high-risk T-ALL patients (MRD ≥0.1% at day 29) into two groups with significant differences in outcome (CIR3y rates: CIMP negative 50% vs. CIMP positive 12%; P = 0.02). These groups did not differ regarding ETP phenotype, but the CIMP-negative group was younger (P = 0.02) and had higher white blood cell count at diagnosis (P = 0.004) compared with the CIMP-positive group. CONCLUSIONS: CIMP classification at diagnosis in combination with MRD during induction therapy is a strong candidate for further risk classification and could confer important information in treatment decision making.

DNA methylation status defines clinicopathological parameters including survival for patients with clear cell renal cell carcinoma (ccRCC).

Epigenetic alterations in the methylome have been associated with tumor development and progression in renal cell carcinoma (RCC). In this study, 45 tumor samples, 12 tumor-free kidney cortex tissues, and 24 peripheral blood samples from patients with clear cell RCC (ccRCC) were analyzed by genome-wide promoter-directed methylation arrays and related to clinicopathological parameters. Unsupervised hierarchical clustering separated the tumors into two distinct methylation groups (clusters A and B), where cluster B had higher average methylation and increased number of hypermethylated CpG sites (CpGs). Furthermore, tumors in cluster B had, compared with cluster A, a larger tumor diameter (p = 0.033), a higher morphologic grade (p < 0.001), a higher tumor-node-metastasis (TNM) stage (p < 0.001), and a worse prognosis (p = 0.005). Higher TNM stage was correlated to an increase in average methylation level (p = 0.003) and number of hypermethylated CpGs (p = 0.003), whereas a number of hypomethylated CpGs were mainly unchanged. However, the predicted age of the tumors based on methylation profile did not correlate with TNM stage, morphological grade, or methylation cluster. Differently methylated (DM) genes (n = 840) in ccRCC samples compared with tumor-free kidney cortex samples were predominantly hypermethylated and a high proportion were identified as polycomb target genes. The DM genes were overrepresented by transcription factors, ligands, and receptors, indicating functional alterations of significance for ccRCC progression. To conclude, increased number of hypermethylated genes was associated with increased TNM stage of the tumors. DNA methylation classification of ccRCC tumor samples at diagnosis can serve as a clinically applicable prognostic marker in ccRCC.

Telomere length in peripheral leukocytes is associated with immune cell tumor infiltration and prognosis in colorectal cancer patients.

Telomeres are protective structures at the end of chromosomes, essential for chromosomal integrity. A large number of studies have investigated leukocyte telomere length as a possible risk marker for various cancers, colorectal cancer (CRC) included. In contrast, studies investigating leukocyte telomere length in relation to CRC survival are lacking. We previously reported that relative telomere length (RTL) of leukocytes collected at diagnosis predicted survival in patients with breast and kidney cancer. We suggested that these findings might reflect various immunological mechanisms, affected by the presence of a tumor. In the present study, leukocyte RTL was examined in relation to immune cell tumor infiltration and prognosis in 130 patients with CRC diagnosis. RTL was measured with a well-established qPCR method. We found that patients with the highest degree of lymphocyte tumor infiltration had shorter leukocyte RTL. Consistent with our previous findings, short RTL was a favorable prognostic marker in univariate survival analysis. In the current study, RTL did not remain as an independent predictor in multivariate survival analysis, when including metastatic status in the model. However, a non-significant trend towards a similar telomere-associated survival pattern was observed in patients with limited disease. In contrast, for patients who died of other causes than CRC, short RTL was associated with significantly shorter survival time. To our knowledge, this is the first study to investigate an association between leukocyte RTL, immune cell tumor infiltration, and cancer-specific survival in CRC patients. Larger studies are warranted to verify these findings.

Reproducibility of telomere length assessment: an international collaborative study.

BACKGROUND: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories. METHODS: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques. RESULTS: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy. CONCLUSIONS: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories.

A case of disseminated histoplasmosis diagnosed after oral presentation in an old HIV-negative patient in Sweden.

Histoplasmosis is an endemic disease in various regions such as North America and South-East Asia but remains rare in Europe. Disseminated histoplasmosis is unusual in HIV-negative patients. Here, we describe a case of disseminated histoplasmosis in an HIV-negative patient diagnosed after oral presentation.

TERT promoter mutations in clear cell renal cell carcinoma.

We screened promoter region of the telomerase reverse transcriptase (TERT) for activating somatic mutations in 188 tumors from patients with clear cell renal cell carcinoma (ccRCC). Twelve tumors (6.4%) carried a mutation within the core promoter region of the gene. The mutations were less frequent in high grade tumors compared to low grade tumors [odds ratio (OR) = 0.15, 95% confidence interval (CI) = 0.03-0.72, p = 0.02]. Multivariate analysis for cause specific survival showed statistically significant poor outcome in patients with TERT promoter mutations [hazard ratio (HR) = 2.90, 95% CI = 1.13-7.39, p = 0.03]. A common polymorphism (rs2853669) within the locus seemed to act as a modifier of the effect of the mutations on patient survival as the noncarriers of the variant allele with the TERT promoter mutations showed worst survival (HR = 3.34, 95% CI = 1.24-8.98, p = 0.02). We also measured relative telomere length (RTL) in tumors and difference between tumors with and without the TERT promoter mutations was not statistically significant. Similarly, no difference in patient survival based on RTL in tumors was observed. Our study showed a relatively low frequency of TERT promoter mutations in ccRCC. Nevertheless, patients with the mutations, particularly in the absence of the rs2853669 variant showed the worst disease-specific survival. Thus, it is possible that the TERT promoter mutations define a small subset of tumors with an aggressive behavior.

Specific genomic aberrations predict survival, but low mutation rate in cancer hot spots, in clear cell renal cell carcinoma.

Detailed genetic profiling of clear cell renal cell carcinoma (ccRCC) has revealed genomic regions commonly affected by structural changes and a general genetic heterogeneity. VHL and PBRM1, both located at chromosome 3p, are 2 major genes mutated at high frequency but apart from these aberrations, the mutational landscape in ccRCC is largely undefined. Potential prognostic information given by the genomic changes appears to depend on the particular cohort studied. We analyzed a Swedish ccRCC cohort of 74 patients and found common changes (loss or gain occurring in >20% of the tumors) in 12 chromosomal regions (1p, 3p, 3q, 5q, 6q, 7p, 7q 8p, 9p, 9q, 10q, and 14q). A poor outcome was associated with gain of 7q and losses on 9p, 9q, and 14q. These aberrations were more frequent in metastasized tumors, suggesting alterations of genes important for tumor progression. Sequencing of 48 genes implicated in cancer revealed that only VHL, TP53, and PTEN were mutated at a noticeable frequency (51%, 9%, and 9%, respectively). Shorter relative telomere length (RTL) has been associated with loss of specific chromosomal regions in ccRCC tumors, but we could not verify this finding. However, a significantly lower tumor/nontumor (T/N) RTL ratio was detected for tumors with losses in 4q or 9p. In conclusion, poor outcome in ccRCC was associated with gain of 7q and loss on 9p, 9q, and 14q, whereas the mutation rate overall was low in a screen of cancer-associated genes.

Long leukocyte telomere length at diagnosis is a risk factor for dementia progression in idiopathic parkinsonism.

Telomere length (TL) is regarded as a marker of cellular aging due to the gradual shortening by each cell division, but is influenced by a number of factors including oxidative stress and inflammation. Parkinson's disease and atypical forms of parkinsonism occur mainly in the elderly, with oxidative stress and inflammation in afflicted cells. In this study the relationship between blood TL and prognosis of 168 patients with idiopathic parkinsonism (136 Parkinson's disease [PD], 17 Progressive Supranuclear Palsy [PSP], and 15 Multiple System Atrophy [MSA]) and 30 controls was investigated. TL and motor and cognitive performance were assessed at baseline (diagnosis) and repeatedly up to three to five years follow up. No difference in TL between controls and patients was shown at baseline, nor any significant difference in TL stability or attrition during follow up. Interestingly, a significant relationship between TL at diagnosis and cognitive phenotype at follow up in PD and PSP patients was found, with longer mean TL at diagnosis in patients that developed dementia within three years.

Stand up for health--avoiding sedentary behaviour might lengthen your telomeres: secondary outcomes from a physical activity RCT in older people.

BACKGROUND: Telomere length has been associated with a healthy lifestyle and longevity. However, the effect of increased physical activity on telomere length is still unknown. Therefore, the aim was to study the relationship between changes in physical activity level and sedentary behaviour and changes in telomere length. METHODS: Telomere length was measured in blood cells 6 months apart in 49, 68-year-old, sedentary, overweight individuals taking part in a randomised controlled physical activity intervention trial. The intervention group received individualised physical activity on prescription. Physical activity was measured with a 7-day diary, questionnaires and a pedometer. Sitting time was measured with the short version of The International Physical Activity Questionnaire. RESULTS: Time spent exercising as well as steps per day increased significantly in the intervention group. Reported sitting time decreased in both groups. No significant associations between changes in steps per day and changes in telomere length were noted. In the intervention group, there was a negative correlation between changes in time spent exercising and changes in telomere length (rho=-0.39, p=0.07). On the other hand, in the intervention group, telomere lengthening was significantly associated with reduced sitting time (rho=-0.68, p=0.02). CONCLUSIONS: Reduced sitting time was associated with telomere lengthening in blood cells in sedentary, overweight 68-year-old individuals participating in a 6-month physical activity intervention trial.

Immortalization of T-cells is accompanied by gradual changes in CpG methylation resulting in a profile resembling a subset of T-cell leukemias.

We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and unlimited proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs), islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis.

A mutation in the H/ACA box of telomerase RNA component gene (TERC) in a young patient with myelodysplastic syndrome.

BACKGROUND: Telomeres are repeated sequences (the hexanucleotide TTAGGG in vertebrates) located at chromosome ends of eukaryotes, protecting DNA from end joining or degradation. Telomeres become shorter with each cell cycle, but telomerase, a ribonucleoprotein complex, alleviates this attrition. The telomerase RNA component (TERC) is an essential element of telomerase, serving as a template for telomere elongation. The H/ACA domain of TERC is indispensable for telomere biogenesis. Mutations in the telomerase components allow accelerated telomere loss, resulting in various disease manifestations, including bone marrow failure. To date, this is the first detailed report of an H-box mutation in TERC that is related to human disease. CASE PRESENTATION: A 26-year-old man with myelodysplastic syndrome (MDS) had very short telomeres. Sequencing identified a single heterozygous mutation in the H box of the patient's TERC gene. The same mutation was also present in his father and his son, demonstrating that it was germline in origin. The telomere length in the father's blood was shorter compared to age-matched healthy controls, while it was normal in the son and also in the sperm cells of the patient. In vitro experiments suggested that the mutation was responsible for the telomere shortening in the patient's leukocytes and contributed to the pathogenesis of bone marrow failure in our patient. CONCLUSION: We analyzed a mutation (A377G) in the H box of TERC in a young MDS patient who had significantly short-for-age telomeres. As telomeres protect chromosomes from instability, it is highly plausible that this genetic lesion was responsible for the patient's hematological manifestations, including marrow failure and aneuploidy in the hematopoietic stem cell compartment.

Gender and telomere length: systematic review and meta-analysis.

BACKGROUND: It is widely believed that females have longer telomeres than males, although results from studies have been contradictory. METHODS: We carried out a systematic review and meta-analyses to test the hypothesis that in humans, females have longer telomeres than males and that this association becomes stronger with increasing age. Searches were conducted in EMBASE and MEDLINE (by November 2009) and additional datasets were obtained from study investigators. Eligible observational studies measured telomeres for both females and males of any age, had a minimum sample size of 100 and included participants not part of a diseased group. We calculated summary estimates using random-effects meta-analyses. Heterogeneity between studies was investigated using sub-group analysis and meta-regression. RESULTS: Meta-analyses from 36 cohorts (36,230 participants) showed that on average females had longer telomeres than males (standardised difference in telomere length between females and males 0.090, 95% CI 0.015, 0.166; age-adjusted). There was little evidence that these associations varied by age group (p=1.00) or cell type (p=0.29). However, the size of this difference did vary by measurement methods, with only Southern blot but neither real-time PCR nor Flow-FISH showing a significant difference. This difference was not associated with random measurement error. CONCLUSIONS: Telomere length is longer in females than males, although this difference was not universally found in studies that did not use Southern blot methods. Further research on explanations for the methodological differences is required.