Methylation of the hTERT Promoter: A Novel Cancer Biomarker for Leptomeningeal Metastasis Detection in Cerebrospinal Fluids.

PURPOSE: The diagnosis of leptomeningeal metastases is usually confirmed by the finding of malignant cells by cytologic examination in the cerebrospinal fluid (CSF). More sensitive and specific cancer biomarkers may improve the detection of tumor cells in the CSF. Promoter methylation of the human telomerase reverse transcriptase (hTERT) gene characterizes most cancer cells. The aim of this study was to develop a sensitive method to detect hTERT methylation and to explore its use as a cancer biomarker in CSF. EXPERIMENTAL DESIGN: In 77 CSF specimens from 67 patients, hTERT promoter methylation was evaluated using real-time methylation-sensitive high-resolution melting (MS-HRM) and real-time TaqMan PCR and MS-HRM in a single-tube assay. RESULTS: Real-time MS-HRM assay was able to detect down to 1% hTERT-methylated DNA in a background of unmethylated DNA. PCR products were obtained from 90% (69/77) of CSF samples. No false positive hTERT was detected in the 21 non-neoplastic control cases, given to the method a specificity of 100%. The sensitivity of the real-time MS-HRM compared with the cytologic gold standard analysis was of 92% (11/12). Twenty-six CSFs from 22 patients with an hTERT-methylated primary tumor showed cytologic results suspicious for malignancy; in 17 (65%) of them, a diagnosis of leptomeningeal metastases could be confirmed by the hTERT methylation test. CONCLUSION: The hTERT real-time MS-HRM approach is fast, specific, sensitive, and could therefore become a valuable tool for diagnosis of leptomeningeal metastases as an adjunct to the traditional examination of CSF. Clin Cancer Res; 19(8); 2216-23. ©2013 AACR.

Detection of an asymptomatic right-ventricle cardiac metastasis from a small-cell lung cancer by F-18-FDG PET/CT.

A right heart metastasis of a small-cell lung cancer was found on the whole-body F-fluoro-deoxy-glucose positron emission tomography/computed tomography (F-FDG-PET/CT) of a 69-year-old smoker investigated for a right pulmonary mass discovered on chest radiography after a fracture of the right humerus. The PET scan showed an increased FDG uptake by the mass in the right lung and an intense, atypical focal activity of the right ventricle strongly suggestive of a neoplastic process. CT-guided lung biopsy revealed a small-cell lung cancer and myocardial biopsy confirmed the presence of a cardiac metastasis. The patient was treated with six cycles of chemotherapy followed by radiation therapy, which included the heart lesion. At follow-up PET/CT 2 months after the end of treatment, the abnormal cardiac uptake had disappeared, whereas increased FDG uptake persisted in the pulmonary residual mass.

Detection of functional antigen-specific T cells from urine of non-muscle invasive bladder cancer patients.

To gain further insights into the role of T lymphocytes in immune responses against bladder tumors, we developed a method that monitors the presence of functional antigen-specific T cells in the urine of non-muscle invasive bladder cancer patients. As relatively few immune cells can usually be recovered from urine, we examined different isolation/amplification protocols and took advantage of patients treated with weekly intravesical instillations of Bacillus Calmette-Guérin, resulting in large amounts of immune cells into urine. Our findings demonstrate that, upon in vitro amplification, antigen-specific T cells can be detected by an interferon γ (IFNγ)-specific ELISPOT assay.

Early detection of lung cancer: a statement from an expert panel of the Swiss university hospitals on lung cancer screening.

The discussion about setting up a program for lung cancer screening was launched with the publication of the results of the National Lung Screening Trial, which suggested reduced mortality in high-risk subjects undergoing CT screening. However, important questions about the benefit-harm balance and the details of a screening program and its cost-effectiveness remain unanswered. A panel of specialists in chest radiology, respiratory medicine, epidemiology, and thoracic surgery representing all Swiss university hospitals prepared this joint statement following several meetings. The panel argues that premature and uncontrolled introduction of a lung cancer screening program may cause substantial harm that may remain undetected without rigorous quality control. This position paper focuses on the requirements of running such a program with the objective of harmonizing efforts across the involved specialties and institutions and defining quality standards. The underlying statement includes information on current evidence for a reduction in mortality with lung cancer screening and the potential epidemiologic implications of such a program in Switzerland. Furthermore, requirements for lung cancer screening centers are defined, and recommendations for both the CT technique and the algorithm for lung nodule assessment are provided. In addition, related issues such as patient management, registry, and funding are addressed. Based on the current state of the knowledge, the panel concludes that lung cancer screening in Switzerland should be undertaken exclusively within a national observational study in order to provide answers to several critical questions before considering broad population-based screening for lung cancer.

The use of telomerase activity for the detection of prostatic cancer cells after prostatic massage.

PURPOSE: Prostate cancer is the most commonly diagnosed cancer in the United States. The diagnosis or followup of prostate cancer in men older than 50 years is based on digital rectal examination, measurement of the free-to-total prostatic specific antigen ratio and transrectal ultrasound assisted needle biopsy of the prostate. We developed and evaluated a noninvasive method for diagnosing prostate cancer based on the measurement of telomerase activity after prostatic massage in fresh voided urine or after urethral washing. MATERIALS AND METHODS: We obtained 36 specimens of cells after prostatic massage in the fresh voided urine of 16 patients who subsequently underwent radical prostatectomy and after urethral washing in 20 who underwent prostate needle biopsies. Ethylenediaminetetraacetic acid was immediately added to the collected urine or washing to a final concentration of 20 mM. After protein extraction by CHAPS buffer each specimen was tested for telomerase activity in a 2-step modified telomeric repeat amplification protocol assay. The 2 prostate cancer cell lines PC-3 and LNCaP with high telomerase activity were used as a positive control. RESULTS: Telomerase activity was detected in 14 of 24 samples with known prostate cancer (sensitivity 58%). In contrast, no telomerase activity was found in the 12 cases without histological evidence of prostate tumor (specificity 100%). Eight of 9 poorly differentiated cancers expressed telomerase activity (89%), while only 6 of 15 well and moderately differentiated cancers showed telomerase activity (40%). CONCLUSIONS: Our data illustrate that telomerase activity may be detected in voided urine or washing after prostatic massage in patients with prostate cancer. Sensitivity was higher for poorly differentiated tumors. This approach is not currently available for detecting prostate cancer in clinical practice. However, these results are promising and further studies are ongoing.

Effectiveness of early detection on breast cancer mortality reduction in Catalonia (Spain)

Background: At present, it is complicated to use screening trials to determine the optimal age intervals and periodicities of breast cancer early detection. Mathematical models are an alternative that has been widely used. The aim of this study was to estimate the effect of different breast cancer early detection strategies in Catalonia (Spain), in terms of breast cancer mortality reduction (MR) and years of life gained (YLG), using the stochastic models developed by Lee and Zelen (LZ). Methods: We used the LZ model to estimate the cumulative probability of death for a cohort exposed to different screening strategies after T years of follow-up. We also obtained the cumulative probability of death for a cohort with no screening. These probabilities were used to estimate the possible breast cancer MR and YLG by age, period and cohort of birth. The inputs of the model were: incidence of, mortality from and survival after breast cancer, mortality from other causes, distribution of breast cancer stages at diagnosis and sensitivity of mammography. The outputs were relative breast cancer MR and YLG. Results: Relative breast cancer MR varied from 20% for biennial exams in the 50 to 69 age interval to 30% for annual exams in the 40 to 74 age interval. When strategies differ in periodicity but not in the age interval of exams, biennial screening achieved almost 80% of the annual screening MR. In contrast to MR, the effect on YLG of extending screening from 69 to 74 years of age was smaller than the effect of extending the screening from 50 to 45 or 40 years. Conclusion: In this study we have obtained a measure of the effect of breast cancer screening in terms of mortality and years of life gained. The Lee and Zelen mathematical models have been very useful for assessing the impact of different modalities of early detection on MR and YLG in Catalonia (Spain).

Cost-effectiveness of early detection of breast cancer in Catalonia (Spain)

Background: Breast cancer (BC) causes more deaths than any other cancer among women in Catalonia. Early detection has contributed to the observed decline in BC mortality. However, there is debate on the optimal screening strategy. We performed an economic evaluation of 20 screening strategies taking into account the cost over time of screening and subsequent medical costs, including diagnostic confirmation, initial treatment, follow-up and advanced care. Methods: We used a probabilistic model to estimate the effect and costs over time of each scenario. The effect was measured as years of life (YL), quality-adjusted life years (QALY), and lives extended (LE). Costs of screening and treatment were obtained from the Early Detection Program and hospital databases of the IMAS-Hospital del Mar in Barcelona. The incremental cost-effectiveness ratio (ICER) was used to compare the relative costs and outcomes of different scenarios. Results: Strategies that start at ages 40 or 45 and end at 69 predominate when the effect is measured as YL or QALYs. Biennial strategies 50-69, 45-69 or annual 45-69, 40-69 and 40-74 were selected as cost-effective for both effect measures (YL or QALYs). The ICER increases considerably when moving from biennial to annual scenarios. Moving from no screening to biennial 50-69 years represented an ICER of 4,469€ per QALY. Conclusions: A reduced number of screening strategies have been selected for consideration by researchers, decision makers and policy planners. Mathematical models are useful to assess the impact and costs of BC screening in a specific geographical area.

Contribution of early detection and adjuvant treatments to breast cancer mortality reduction in Catalonia, Spain

Background: Reductions in breast cancer (BC) mortality in Western countries have been attributed to the use of screening mammography and adjuvant treatments. The goal of this work was to analyze the contributions of both interventions to the decrease in BC mortality between 1975 and 2008 in Catalonia. Methodology/Principal Findings: A stochastic model was used to quantify the contribution of each intervention. Age standardized BC mortality rates for calendar years 1975-2008 were estimated in four hypothetical scenarios: 1) Only screening, 2) Only adjuvant treatment, 3) Both interventions, and 4) No intervention. For the 30-69 age group, observed Catalan BC mortality rates per 100,000 women-year rose from 29.4 in 1975 to 38.3 in 1993, and afterwards continuously decreased to 23.2 in 2008. If neither of the two interventions had been used, in 2008 the estimated BC mortality would have been 43.5, which, compared to the observed BC mortality rate, indicates a 46.7% reduction. In 2008 the reduction attributable to screening was 20.4%, to adjuvant treatments was 15.8% and to both interventions 34.1%. Conclusions/Significance: Screening and adjuvant treatments similarly contributed to reducing BC mortality in Catalonia. Mathematical models have been useful to assess the impact of interventions addressed to reduce BC mortality that occurred over nearly the same periods.

Discovery of a 29-gene panel in peripheral blood mononuclear cells for the detection of colorectal cancer and adenomas using high throughput real-time PCR.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.

Detection of Clinically Significant Prostate Cancer Using Magnetic Resonance Imaging-Ultrasound Fusion Targeted Biopsy: A Systematic Review.

CONTEXT: The current standard for diagnosing prostate cancer in men at risk relies on a transrectal ultrasound-guided biopsy test that is blind to the location of the cancer. To increase the accuracy of this diagnostic pathway, a software-based magnetic resonance imaging-ultrasound (MRI-US) fusion targeted biopsy approach has been proposed. OBJECTIVE: Our main objective was to compare the detection rate of clinically significant prostate cancer with software-based MRI-US fusion targeted biopsy against standard biopsy. The two strategies were also compared in terms of detection of all cancers, sampling utility and efficiency, and rate of serious adverse events. The outcomes of different targeted approaches were also compared. EVIDENCE ACQUISITION: We performed a systematic review of PubMed/Medline, Embase (via Ovid), and Cochrane Review databases in December 2013 following the Preferred Reported Items for Systematic reviews and Meta-analysis statement. The risk of bias was evaluated using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. EVIDENCE SYNTHESIS: Fourteen papers reporting the outcomes of 15 studies (n=2293; range: 13-582) were included. We found that MRI-US fusion targeted biopsies detect more clinically significant cancers (median: 33.3% vs 23.6%; range: 13.2-50% vs 4.8-52%) using fewer cores (median: 9.2 vs 37.1) compared with standard biopsy techniques, respectively. Some studies showed a lower detection rate of all cancer (median: 50.5% vs 43.4%; range: 23.7-82.1% vs 14.3-59%). MRI-US fusion targeted biopsy was able to detect some clinically significant cancers that would have been missed by using only standard biopsy (median: 9.1%; range: 5-16.2%). It was not possible to determine which of the two biopsy approaches led most to serious adverse events because standard and targeted biopsies were performed in the same session. Software-based MRI-US fusion targeted biopsy detected more clinically significant disease than visual targeted biopsy in the only study reporting on this outcome (20.3% vs 15.1%). CONCLUSIONS: Software-based MRI-US fusion targeted biopsy seems to detect more clinically significant cancers deploying fewer cores than standard biopsy. Because there was significant study heterogeneity in patient inclusion, definition of significant cancer, and the protocol used to conduct the standard biopsy, these findings need to be confirmed by further large multicentre validating studies. PATIENT SUMMARY: We compared the ability of standard biopsy to diagnose prostate cancer against a novel approach using software to overlay the images from magnetic resonance imaging and ultrasound to guide biopsies towards the suspicious areas of the prostate. We found consistent findings showing the superiority of this novel targeted approach, although further high-quality evidence is needed to change current practice.

Detection and analysis of apoptosis in peripheral blood cells from breast cancer patients

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.

STUDYING ABERRANT METHYLATION AND miRNA PROFILING FOR THE DETECTION OF LUNG CANCER BIOMARKERS

Lung cancer is a major chronic disease responsible for the highest mortality rate, among other types of cancer, and represents 29% of all deaths in Canada. The clinical diagnosis of lung carcinoma still requires a standard diagnostic approach, as there are no symptoms in its early stage. Therefore, it is usually diagnosed at a later stage, when the survival rate is low. With the recent advancement in molecular biology and biotechnology, a molecular biomarker approach for the diagnosis of early lung cancer seems to be a potential option. In this study, we aimed to investigate and standardize a promising Lung ,Cancer Biomarker by studying the aberrant methylation of two tumour suppressor genes, namely RASSFIA and RAR-B, and the miRNA profiling of four . commonly deregulated miRNA (miR-199a-3p, miR-182, miR-lOO and miR-221). Four lung cancer cell lines were used (two SCLC and two NSCLC), with comparisons being made with normal lung cell lines. Our results, we found that none of these genes were methylated. We then evaluated TP53, and found the promoter of this gene to be methylated in the cancer cell lines, as compared to the normal cell lines, indicating gene inactivation. We carried out miRNA profiling of the cancer cell lines and reported that 80 miRNAs are deregulated in lung cancer cell lines as compared to the normal cell lines. Our study was the first of its kind to indicate that hsa-mir-4301, hsa-mir-4707-5p and hsa-mir-4497 (newly discovered miRNAs) are deregulated in lung cancer cell lines. We also investigated miR-199a-3p, mir-lOO and miR-182, and found that miR-199a -3p and mir-l00 were down-regulated in cancer lines, whereas miR-182 was up-regulated in the cancer cell lines. In the final part of the study we observed that mir-221 could be a putative biomarker to distinguish between the two types of lung cancer because it was down-regulated in SCLC, and up-regulated in the NSCLC cell lines. In conclusion, we found four miRNA molecular biomarkers that possibly could be used in the early diagnosis of the lung cancer. More studies are still required with larger numbers of samples to effectively establish these as molecular biomarkers for the diagnosis of lung cancer

Potential Urinary Protein Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

Globally, Prostate cancer (PCa) is the most frequently occurring non-cutaneous cancer, and is the second highest cause of cancer mortality in men. Serum prostate specific antigen (PSA) has been the standard in PCa screening since its approval by the American Food & Drug Administration (FDA) in 1994. Currently, PSA is used as an indicator for PCa - patients with a serum PSA level above 4ng/mL will often undergo prostate biopsy to confirm cancer. Unfortunately fewer than similar to 30% of these men will biopsy positive for cancer, meaning that the majority of men undergo invasive biopsy with little benefit. Despite PSA's notoriously poor specificity (33%), there is still a significant lack of credible alternatives. Therefore an ideal biomarker that can specifically detect PCa at an early stage is urgently required. The aim of this study was to investigate the potential of using deregulation of urinary proteins in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the protein signatures specific for PCa, protein expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using LC-MS/MS. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This approach revealed that significant the down-regulation of Fibronectin and TP53INP2 was a characteristic event among PCa patients. Fibronectin mRNA down-regulation, was identified as offering improved specificity (50%) over PSA, albeit with a slightly lower although still acceptable sensitivity (75%) for detecting PCa. As for TP53INP2 on the other hand, its down-regulation was moderately sensitive (75%), identifying many patients with PCa, but was entirely non-specific (7%), designating many of the benign samples as malignant and being unable to accurately identify more than one negative.

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (similar to 22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression "signatures" are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.

Serum proteomic abnormality predating screen detection of ovarian cancer

Ovarian cancer is characterized by vague, non-specific symptoms, advanced stage at diagnosis and poor overall survival. A nested case control study was undertaken on stored serial serum samples from women who developed ovarian cancer and healthy controls (matched for serum processing and storage conditions as well as attributes such as age) in a pilot randomized controlled trial of ovarian cancer screening. The unique feature of this study is that the women were screened for up to 7 years. The serum samples underwent prefractionation using a reversed-phase batch extraction protocol prior to MALDI-TOF MS data acquisition. Our exploratory analysis shows that combining a single MS peak with CA125 allows statistically significant discrimination at the 5% level between cases and controls up to 12 months in advance of the original diagnosis of ovarian cancer. Such combinations work much better than a single peak or CA125 alone. This paper demonstrates that mass spectra from the low molecular weight serum proteome carry information useful for early detection of ovarian cancer. The next step is to identify the specific biomarkers that make early detection possible.