Hydroxymethylcytosine (5hmC) methylation is a well-known epigenetic mark that is involved in gene regulation and may impact genome stability. To investigate a possible role of 5hmC in cancer ...development and progression, one must be able to detect and quantify its level first. In this paper, we address the issue of 5hmC detection at a single base resolution, starting with consideration of the well-established 5hmC measure Δβ and, in particular, with an analysis of its properties, both analytically and empirically. Then we propose several alternative hydroxymethylation measures and compare their properties with those of Δβ. In the absence of a gold standard, the (pairwise) resemblance of those 5hmC measures to Δβ is characterized by means of a similarity analysis and relative accuracy analysis. All results are illustrated on matched healthy and cancer tissue data sets as derived by means of bisulfite (BS) and oxidative bisulfite converting (oxBS) procedures.
Summary Background Lynch syndrome is an inherited tumour predisposition syndrome caused by germline mutations of DNA mismatch repair ( MMR ) genes. Mutation carriers have a high risk of developing ...colorectal cancer, but do not present with polyposis, a typical feature of other colorectal cancer syndromes such as familial adenomatous polyposis, in which polyposis reflects the high frequency of biallelic APC gene inactivation. We asked whether in Lynch syndrome biallelic inactivation of MMR genes occurred at a similar frequency to that of APC gene, and whether MMR inactivation resulted in detectable lesions within the intestinal mucosa. Methods Resections done for small and large bowel cancer between January, 2002, and January, 2011, were retrieved. We systematically analysed non-tumorous mucosa from carriers of a Lynch syndrome mutation (set 1: ten patients) and control patients without Lynch syndrome (set 1: nine patients) for MMR protein expression (MLH1, MSH2, and EPCAM) with immunohistochemistry. We validated the findings in an independent sample set (set 2: 30 Lynch syndrome patients, 79 controls). We did an analysis of microsatellite instability by PCR analysis to test lesions for mismatch repair deficiency. We applied a Poisson regression model to analyse the distribution of MMR-deficient crypt foci counts and a Fisher's exact test to compare the prevalence of these foci between mutation carriers and control patients. Findings 20 crypt foci with no MMR protein expression were detected in 20·1 cm2 of non-tumorous mucosa from Lynch syndrome patients (set 1), an additional five were detected upon resectioning of two samples. In an independent validation set (set 2), two MMR-deficient crypt foci were noted in 2·2 cm2 of mucosa. No MMR-deficient crypt foci were noted in non-tumorous mucosa from control patients without evidence for Lynch syndrome (set 1: 3·7 cm2 , set 2: 4·8 cm2 ). Microsatellite instability was detected in all seven MMR-deficient crypt foci analysed. A subset of these foci displayed unusual architectural and cytological abnormalities, although they had no polypous or adenomatous appearance. Interpretation We identified a novel type of lesion, the MMR-deficient crypt focus, as the manifestation of biallelic MMR gene inactivation in Lynch syndrome. The abundance of MMR-deficient crypt foci indicates a high frequency of biallelic MMR gene inactivation, which is in sharp contrast with the low number of clinically manifest cancers in Lynch syndrome. This discrepancy suggests that most MMR-deficient crypt foci do not progress to cancer. We propose Lynch syndrome as a unique model syndrome for studying initial steps of MMR deficiency, tumour initiation and, possibly, elimination. Funding German Cancer Aid and German Research Foundation.
Circulating microRNAs (miRNAs) have been proposed as minimally invasive prognostic markers for various types of cancers, including colorectal cancer (CRC), the third most diagnosed cancer worldwide. ...We aimed to evaluate the levels of circulating miRNAs that might serve as markers for CRC prognosis and survival. We included plasma samples of 543 CRC patients with stage I‐IV disease from a population‐based study carried out in Germany. After comprehensive evaluation of current literature, 95 miRNAs were selected and measured with Custom TaqMan® Array MicroRNA Cards. Plasma samples of non‐metastatic and metastatic colon cancer patients, each group consisting of ten patients with ‘good’ and ten patients with ‘bad’ prognosis were screened. Identified candidate miRNAs were further validated by RT‐qPCR in the whole study cohort. The association of the miRNA levels with patients' survival and the prognostic subtypes was analyzed with uni‐ and multivariate logistic regression and Cox proportional hazards regression models. Increased miR‐122 levels were associated with a ‘bad’ prognostic subtype in metastatic CRC (Odds ratio: 1.563, 95% confidence interval (CI): 1.038‐2.347) and a shorter relapse‐free survival and overall survival for non‐metastatic (Hazard ratio (HR): 1.370, 95% CI: 1.028‐1.825; HR: 1.353, 95% CI: 1.002‐1.828) and metastatic (HR: 1.264, 95% CI: 1.050‐1.520; HR: 1.292, 95% CI: 1.078‐1.548) CRC patients. Additionally, several members of the miR‐200 family showed associations with patients' prognosis and correlations to clinicopathological characteristics. The here identified miRNA markers, miR‐122 and the miR‐200 family members, could be of use in the development of a multi‐marker blood test for CRC prognosis.
What's new?
Metastasis is the major cause of death for colorectal cancer (CRC) patients. Prognostic markers that are constantly measurable using liquid biopsies could facilitate the earlier prediction of relapse events and metastasis formation. In a prospective study cohort of 543 colorectal cancer patients, the authors evaluated the role of circulating miRNAs as potential prognostic markers for CRC with a specific focus on miRNAs known to be involved in tumor progression and metastasis formation. MiR‐122 and several members of the miR‐200 family were identified as prognostic markers, suggesting their potential use in the development of a multi‐marker blood test for CRC prognosis.
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.
What's new?
Circulating microRNAs (miRNAs) have distinct expression profiles in cancer and are unusually stable in plasma, making them potentially useful for early cancer detection. Here, using the largest plasma sample size for miRNA profiling of breast cancer to date, four circulating miRNAs were found to be more abundant in breast cancer patients compared with healthy individuals. The identified miRNAs could be used to develop a multi‐marker blood‐based test to complement existing early breast cancer detection strategies.
The tumor suppressor P53 is a critical regulator of normal cellular homeostasis whose function is either distorted or lost in several cancer types including colorectal cancer (CRC). A small group of ...microRNAs have come to be recognized as essential mediators of P53 function. In a genome‐wide systematic approach, we explored miRNAs that are substantially altered by P53 loss and found miR‐30e to be the most significantly deregulated miRNA in P53‐knockout human CRC cells. We identified miR‐30e‐5p to be a novel direct transcriptional target of P53 with gain and loss of function experiments revealing miR‐30e‐5p to be a significant regulator of tumor cell migration, invasion and in vivo metastasis mediated in part by integrins alpha‐6 and beta‐1 as novel targets. MiR‐30e‐5p also significantly reduced tumor cell proliferation by causing G1/S cell cycle arrest, which was achieved by inducing P21 and P27 expression. Finally, we found miR‐30e‐5p to be lost in resected CRC tumors as compared to normal colon tissues. Taken together, miR‐30e‐5p is a novel effector of P53‐induced suppression of migration, invasion and metastasis.
What's new?
The P53 tumor suppressor plays a critical role in preventing tumor development. To carry out its functions, it is suspected of interacting with select microRNAs (miRNAs). Here, members of the miRNA‐30 family, notably miR‐30e‐5p, were found to be downregulated in P53 knockout human colorectal cancer cells. MiR‐30e‐5p bound directly to integrins alpha‐6 and beta‐1, suppressing cancer cell adhesion, migration, and invasion. Moreover, enhanced miR‐30e‐5p expression inhibited cell proliferation by upregulating cell cycle checkpoint inhibitor proteins P21 and P27, even in the absence of P53. The findings identify miR‐30e‐5p as a potential target for curbing metastatic spread in P53‐deficient tumors.
Simulation is a crucial tool for the evaluation and comparison of statistical methods. How to design fair and neutral simulation studies is therefore of great interest for both researchers developing ...new methods and practitioners confronted with the choice of the most suitable method. The term simulation usually refers to parametric simulation, that is, computer experiments using artificial data made up of pseudo-random numbers. Plasmode simulation, that is, computer experiments using the combination of resampling feature data from a real-life dataset and generating the target variable with a known user-selected outcome-generating model, is an alternative that is often claimed to produce more realistic data. We compare parametric and Plasmode simulation for the example of estimating the mean squared error (MSE) of the least squares estimator (LSE) in linear regression. If the true underlying data-generating process (DGP) and the outcome-generating model (OGM) were known, parametric simulation would obviously be the best choice in terms of estimating the MSE well. However, in reality, both are usually unknown, so researchers have to make assumptions: in Plasmode simulation studies for the OGM, in parametric simulation for both DGP and OGM. Most likely, these assumptions do not exactly reflect the truth. Here, we aim to find out how assumptions deviating from the true DGP and the true OGM affect the performance of parametric and Plasmode simulations in the context of MSE estimation for the LSE and in which situations which simulation type is preferable. Our results suggest that the preferable simulation method depends on many factors, including the number of features, and on how and to what extent the assumptions of a parametric simulation differ from the true DGP. Also, the resampling strategy used for Plasmode influences the results. In particular, subsampling with a small sampling proportion can be recommended.
The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, ...microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.
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.
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).
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.
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.
High CD33 expression in acute myeloid leukemia (AML) with mutated
provides a rationale for the evaluation of gemtuzumab ozogamicin (GO) in this AML entity. We conducted a randomized trial to evaluate ...GO in combination with intensive induction and consolidation therapy in
-mutated AML.
Between May 2010 and September 2017, patients ≥ 18 years old and considered eligible for intensive therapy were randomly assigned up front for induction therapy with idarubicin, cytarabine, etoposide, and all-
-retinoic acid with or without GO. The early (
= .02) primary end point of event-free survival (EFS) was evaluated 6 months after completion of patient recruitment.
Five hundred eighty-eight patients were randomly assigned (standard arm, n = 296; GO arm, n = 292). EFS in the GO arm was not significantly different compared with that in the standard arm (hazard ratio, 0.83; 95% CI, 0.65 to 1.04;
= .10). The early death rate during induction therapy was 10.3% in the GO arm and 5.7% in the standard arm (
= .05). Causes of death in both arms were mainly infections. The cumulative incidence of relapse (CIR) in patients achieving a complete remission (CR) or CR with incomplete hematologic recovery (CRi) was significantly reduced in the GO arm compared with the standard arm (
= .005), with no difference in the cumulative incidence of death (
= .80). Subgroup analysis revealed a significant beneficial effect of GO in female, younger (≤ 70 years), and
internal tandem duplication-negative patients with respect to EFS and CIR.
The trial did not meet its early primary end point of EFS, mainly as a result of a higher early death rate in the GO arm. However, in patients achieving CR/CRi after induction therapy, significantly fewer relapses occurred in the GO compared with the standard arm.
Incomplete understanding of the metastatic process hinders personalized therapy. Here we report the most comprehensive whole-genome study of colorectal metastases vs. matched primary tumors. 65% of ...somatic mutations originate from a common progenitor, with 15% being tumor- and 19% metastasis-specific, implicating a higher mutation rate in metastases. Tumor- and metastasis-specific mutations harbor elevated levels of BRCAness. We confirm multistage progression with new components ARHGEF7/ARHGEF33. Recurrently mutated non-coding elements include ncRNAs RP11-594N15.3, AC010091, SNHG14, 3' UTRs of FOXP2, DACH2, TRPM3, XKR4, ANO5, CBL, CBLB, the latter four potentially dual protagonists in metastasis and efferocytosis-/PD-L1 mediated immunosuppression. Actionable metastasis-specific lesions include FAT1, FGF1, BRCA2, KDR, and AKT2-, AKT3-, and PDGFRA-3' UTRs. Metastasis specific mutations are enriched in PI3K-Akt signaling, cell adhesion, ECM and hepatic stellate activation genes, suggesting genetic programs for site-specific colonization. Our results put forward hypotheses on tumor and metastasis evolution, and evidence for metastasis-specific events relevant for personalized therapy.
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