Katarina Cuk

Circulating microRNAs in plasma as early detection markers for breast cancer

In recent years, circulating miRNAs have attracted a great deal of attention as promising novel markers for various diseases. Here, we investigated their potential to serve as minimally invasive, early detection markers for breast cancer in blood plasma. We profiled miRNAs extracted from the plasma of early stage breast cancer patients (taken at the time‐point of diagnosis) and healthy control individuals using TaqMan low‐density arrays (TLDA). Selected candidates identified in the initial screen were further validated in an extended study cohort of 207 individuals including 127 sporadic breast cancer cases and 80 healthy controls via RT‐qPCR. Four miRNAs (miR‐148b, miR‐376c, miR‐409‐3p and miR‐801) were shown to be significantly upregulated in the plasma of breast cancer patients. ROC curve analysis showed that the combination of only three miRNAs (miR‐148b, miR‐409‐3p and miR‐801) had an equal discriminatory power between breast cancer cases and healthy controls as all four miRNAs together (AUC = 0.69). In conclusion, the identified miRNAs might be of potential use in the development of a multimarker blood‐based test to complement and improve early detection of breast cancer. Such a multimarker blood test might for instance provide a prescreening tool, especially for younger women, to facilitate decisions about which individuals to recommend for further diagnostic tests.

Circulating miRNAs as Surrogate Markers for Circulating Tumor Cells and Prognostic Markers in Metastatic Breast Cancer

Purpose: The use of circulating tumor cells (CTC) as a prognostic marker in metastatic breast cancer (MBC) has been well established. However, their efficacy and accuracy are still under scrutiny mainly because of methods of their enrichment and identification. We hypothesized that circulating miRNAs can predict the CTC status of patients with MBC, and tested for the same. Furthermore, we aimed at establishing a panel of circulating miRNAs capable of differentiating MBC cases from healthy controls. Experimental Design: Circulating miRNAs from plasma of CTC-positive and CTC-negative patients with MBC, and healthy controls, were profiled by TaqMan Human MicroRNA arrays. Candidates from the initial screen were validated in an extended cohort of 269 individuals (61 CTC-positive, 72 CTC-negative, 60 CTC-low MBC cases, and 76 controls). Results: CTC-positive had significantly higher levels of miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375, and miR-801 than CTC-negative MBC and controls (P < 0.00001), whereas miR-768-3p was present in lower amounts in MBC cases (P < 0.05). miR-200b was singled out as the best marker for distinguishing CTC-positive from CTC-negative patients (AUC 0.88). We identified combinations of miRNAs for differentiating MBC cases from controls (AUC 0.95 for CTC-positive; AUC 0.78 for CTC-negative). Combinations of miRNAs and miR-200b alone were found to be promising prognostic marker for progression-free and overall survival. Conclusion: This is the first study to document the capacity of circulating miRNAs to indicate CTC status and their potential as prognostic markers in patients with MBC. Clin Cancer Res; 18(21); 5972–82. ©2012 AACR.

Cancer diagnosis and prognosis decoded by blood-based circulating microRNA signatures.

In the recent years, circulating microRNAs (miRNAs) have garnered a lot of attention and interest in the field of disease biomarkers. With characteristics such as high stability, low cost, possibility of repeated sampling and minimal invasiveness, circulating miRNAs are ideal for development into diagnostic tests. There have been many studies reported on the potential of circulating miRNAs as early detection, prognostic, and predictive biomarkers in cancer. Here, we have reviewed the application of plasma and serum miRNAs as biomarkers for cancer focusing on epithelial carcinomas [prostate, breast, lung, colorectal, and gastric cancer (GC)] and hematological malignancies (leukemia and lymphoma). We have also addressed the common challenges that need to be overcome to achieve a successful bench to bedside transition.

Plasma microRNA panel for minimally invasive detection of breast cancer.

Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. Circulating miR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associated with breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levels are even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR-127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).

DNA methylation array analyses identified breast cancer‐associated HYAL2 methylation in peripheral blood

Breast cancer (BC) is the leading cause of cancer‐related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC‐associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome‐wide methylation screening and identified a BC‐associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10−9 adjusted for cell‐type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p < 0.0001; second validation round with 189 BC case and 189 controls: p < 0.0001). In addition to cg27091787, the decreased methylation of a 650‐bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p‐value of 0.006. To note, the BC‐associated decreased HYAL2 methylation was replicated in the T‐cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early‐stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age < 50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood‐based marker for the detection of early BC.

Fusobacterium and colorectal cancer: Causal factor or passenger? Results from a large colorectal cancer screening study.

Colorectal cancer is a leading cause of morbidity and mortality worldwide in both men and women. The gut microbiome is increasingly recognized as having an important role in human health and disease. Fusobacterium has been identified in former studies as a leading gut bacterium associated with colorectal cancer, but it is still not clear if it plays an oncogenic role. In the current study, fecal samples were collected prior to bowel preparation from participants of screening colonoscopy in the German BliTz study. Using 16S rRNA gene analysis, we examined the presence and relative abundance of Fusobacterium in fecal samples from 500 participants, including 46, 113, 110 and 231 individuals with colorectal cancer, advanced adenomas, non-advanced adenomas and without any neoplasms, respectively. We found that the abundance of Fusobacterium in feces was strongly associated with the presence of colorectal cancer (P-value < 0.0001). This was confirmed by PCR at the species level for Fusobacterium nucleatum. However, no association was seen with the presence of advanced adenomas (P-value = 0.80) or non-advanced adenomas (P-value = 0.80), nor were there any associations observed with dietary or lifestyle habits. Although a causal role cannot be ruled out, our observations, based on fecal microbiome, support the hypothesis that Fusobacterium is a passenger that multiplies in the more favorable conditions caused by the malignant tumor rather than a causal factor in colorectal cancer development.

Plasma hyaluronic acid level as a prognostic and monitoring marker of metastatic breast cancer

Conventional tumor markers have limited value for prognostication and treatment monitoring in metastatic breast cancer (MBC) patients and novel circulating tumor markers therefore need to be explored. Hyaluronic acid (HA) is a major macropolysaccharide in the extracellular matrix and is reported to be associated with tumor progression. In our study, we investigated plasma HA level with respect to progression free survival (PFS) and overall survival (OS), as well as the treatment monitoring value in MBC patients. The prognostic value of plasma HA level was investigated in a discovery cohort of 212 MBC patients with 2.5‐year follow‐up and validated in an independent validation cohort of 334 patients with 5‐year follow‐up. The treatment monitoring value of plasma HA level was investigated in 61 MBC patients from discovery cohort who had been radiographically examined after first complete cycle of chemo therapy. We found a robust association between high plasma HA level and poor prognosis of MBC patients in both discovery (pPFS = 7.92 × 10−6 and pOS = 5.27 × 10−5) and validation studies (pPFS = 3.66 × 10−4 and pOS = 1.43 × 10−4). In the discovery cohort, the plasma HA level displayed independent prognostic value after adjusted for age and clinicopathological factors, with respect to PFS and OS. Further, the decrease of plasma HA level displayed good concordance with treatment response evaluated by radiographic examination (AUC = 0.79). Plasma HA level displays prognostic value, as well as treatment monitoring value for MBC patients.

Cell-free circulating DNA integrity is an independent predictor of impending breast cancer recurrence

Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.

DNA methylation array analysis identifies breast cancer associated RPTOR , MGRN1 and RAPSN hypomethylation in peripheral blood DNA

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 × 10−6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

The association between breast cancer and S100P methylation in peripheral blood by multicenter case-control studies

Breast cancer (BC) is the leading cancer in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignant diseases. Making use of screening results by llumina 27K Methylation Assay, we validated demethylation of five CpG sites of S100P gene in blood cell DNA of BC patients by three independent retrospective studies with subjects from different centers (Validation I: 235 familial BC case and 206 controls, odds ratio per -1% methylation > 1.03, and P < 6.00 × 10-8 for all five CpG sites; Validation II: 189 sporadic BC case and 189 controls, odds ratio per -1% methylation > 1.03, P < 8.0 × 10-5 for four CpG sites; Validation III: 156 sporadic BC case and 151 controls, odds ratio per -1% methylation > 1.03, P < 6.0 × 10-4 for four CpG sites). In addition, the blood-based S100P methylation pattern was similar among BC patients with differential clinical characteristics regardless of stage, receptor status and menopause status. The observed BC-associated decreased S100P methylation in blood mainly originates from the leucocytes subpopulations but not B cells. The methylation levels of most S100P CpG sites were inversely correlated with the expression of S100P in leucocytes (P < 1.2 × 10-4) and in tissue (P < 1.1 × 10-4). This study reveals significant association between blood-based decreased S100P methylation and BC, and provides another proof for the application of altered DNA methylation signatures from blood cells as potential markers for the detection of BC, especially for the early stage.