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Abstract: In this work, two novel background correction (BC) methods, along with several commonly used ones, are evaluated regarding noise reduction in eleven two-channel self-versus-self (SVS) hybridizations. The evaluation of each BC method is investigated under the use of four statistical criteria combined into a single measure, the polygon area measure. Overall, our proposed BC approaches perform very well in terms of the proposed measure for most of the cases and provide an improved effect regarding technical noise reduction. [Copyright &y& Elsevier]

Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts., (© 2024. The Author(s).)

Around 75% of breast cancer (BC) patients have tumors expressing the predictive biomarker estrogen receptor α (ER) and are offered endocrine therapy. One-third eventually develop endocrine resistance, a majority with retained ER expression. Mutations in the phosphatidylinositol bisphosphate 3-kinase (PI3K) catalytic subunit encoded by PIK3CA is a proposed resistance mechanism and a pharmacological target in the clinical setting. Here we explore the frequency of PIK3CA mutations in endocrine-resistant BC before and during treatment and correlate to clinical features. Patients with ER-positive (ER +), human epidermal growth factor receptor 2 (HER2)-negative primary BC with an ER + relapse within 5 years of ongoing endocrine therapy were retrospectively assessed. Tissue was collected from primary tumors (n = 58), relapse tumors (n = 54), and tumor-free lymph nodes (germline controls, n = 62). Extracted DNA was analyzed through panel sequencing. Somatic mutations were observed in 50% (31/62) of the patients, of which 29% occurred outside hotspot regions. The presence of PIK3CA mutations was significantly associated with nodal involvement and mutations were more frequent in relapse than primary tumors. Our study shows the different PIK3CA mutations in endocrine-resistant BC and their fluctuations during therapy. These results may aid investigations of response prediction, facilitating research deciphering the mechanisms of endocrine resistance., (© 2024. The Author(s).)

Although metabolic reprogramming within tumor cells and tumor microenvironment (TME) is well described in breast cancer, little is known about how the interplay of immune state and cancer metabolism evolves during treatment. Here, we characterize the immunometabolic profiles of tumor tissue samples longitudinally collected from individuals with breast cancer before, during and after neoadjuvant chemotherapy (NAC) using proteomics, genomics and histopathology. We show that the pre-, on-treatment and dynamic changes of the immune state, tumor metabolic proteins and tumor cell gene expression profiling-based metabolic phenotype are associated with treatment response. Single-cell/nucleus RNA sequencing revealed distinct tumor and immune cell states in metabolism between cold and hot tumors. Potential drivers of NAC based on above analyses were validated in vitro. In summary, the study shows that the interaction of tumor-intrinsic metabolic states and TME is associated with treatment outcome, supporting the concept of targeting tumor metabolism for immunoregulation., (© 2024. The Author(s).)

Background: Overexpression of human epidermal growth factor receptor 2 (HER2) caused by HER2 gene amplification is a driver in breast cancer tumorigenesis. We aimed to investigate the prognostic significance of manual scoring and digital image analysis (DIA) algorithm assessment of HER2 copy numbers and HER2/CEP17 ratios, along with ERBB2 mRNA levels among early-stage HER2-positive breast cancer patients treated with trastuzumab., Methods: This retrospective study comprised 371 early HER2-positive breast cancer patients treated with adjuvant trastuzumab, with HER2 re-testing performed on whole tumor sections. Digitized tumor tissue slides were manually scored and assessed with uPath HER2 Dual ISH image analysis, breast algorithm. Targeted ERBB2 mRNA levels were assessed by the Xpert® Breast Cancer STRAT4 Assay. HER2 copy number and HER2/CEP17 ratio from in situ hybridization assessment, along with ERBB2 mRNA levels, were explored in relation to recurrence-free survival (RFS)., Results: The analysis showed that patients with tumors with the highest and lowest manually counted HER2 copy number levels had worse RFS than those with intermediate levels (HR = 2.7, CI 1.4-5.3, p = 0.003 and HR = 2.1, CI 1.1-3.9, p = 0.03, respectively). A similar trend was observed for HER2/CEP17 ratio, and the DIA algorithm confirmed the results. Moreover, patients with tumors with the highest and the lowest values of ERBB2 mRNA had a significantly worse prognosis (HR = 2.7, CI 1.4-5.1, p = 0.003 and HR = 2.8, CI 1.4-5.5, p = 0.004, respectively) compared to those with intermediate levels., Conclusions: Our findings suggest that the association between any of the three HER2 biomarkers and RFS was nonlinear. Patients with tumors with the highest levels of HER2 gene amplification or ERBB2 mRNA were associated with a worse prognosis than those with intermediate levels, which is of importance to investigate in future clinical trials studying HER2-targeted therapy., (© 2024. The Author(s).)

Breast cancer (BC) is a complex disease comprising multiple distinct subtypes with different genetic features and pathological characteristics. Although a large number of antineoplastic compounds have been approved for clinical use, patient-to-patient variability in drug response is frequently observed, highlighting the need for efficient treatment prediction for individualized therapy. Several patient-derived models have been established lately for the prediction of drug response. However, each of these models has its limitations that impede their clinical application. Here, we report that the whole-tumor cell culture (WTC) ex vivo model could be stably established from all breast tumors with a high success rate (98 out of 116), and it could reassemble the parental tumors with the endogenous microenvironment. We observed strong clinical associations and predictive values from the investigation of a broad range of BC therapies with WTCs derived from a patient cohort. The accuracy was further supported by the correlation between WTC-based test results and patients' clinical responses in a separate validation study, where the neoadjuvant treatment regimens of 15 BC patients were mimicked. Collectively, the WTC model allows us to accomplish personalized drug testing within 10 d, even for small-sized tumors, highlighting its potential for individualized BC therapy. Furthermore, coupled with genomic and transcriptomic analyses, WTC-based testing can also help to stratify specific patient groups for assignment into appropriate clinical trials, as well as validate potential biomarkers during drug development.

The relationship between stochastic transcriptional bursts and dynamic 3D chromatin states is not well understood. Using an innovated, ultra-sensitive technique, we address here enigmatic features underlying the communications between MYC and its enhancers in relation to the transcriptional process. MYC thus interacts with its flanking enhancers in a mutually exclusive manner documenting that enhancer hubs impinging on MYC detected in large cell populations likely do not exist in single cells. Dynamic encounters with pathologically activated enhancers responsive to a range of environmental cues, involved <10% of active MYC alleles at any given time in colon cancer cells. Being the most central node of the chromatin network, MYC itself likely drives its communications with flanking enhancers, rather than vice versa. We submit that these features underlie an acquired ability of MYC to become dynamically activated in response to a diverse range of environmental cues encountered by the cell during the neoplastic process., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gene and protein expression of programmed death-ligand 1 (PD-L1) are prognostic in early breast cancer (BC), but their prognostic information is inconsistent at least in some biological subgroups. The validated prognostic gene signatures (GS) in BC are mainly based on proliferation and estrogen receptor (ER)-related genes. Here, we aimed to explore the prognostic capacity of PD-L1 expression at the protein vs mRNA levels and to investigate the prognostic information that PD-L1 can potentially add to routinely used GS. Gene expression data were derived from two early BC cohorts (cohort 1: 562 patients; cohort 2: 1081 patients). Tissue microarrays from cohort 1 were immunohistochemically (IHC) stained for PD-L1 using the SP263 clone. GS scores (21-gene, 70-gene) were calculated, and likelihood-ratio (LR) tests and concordance indices were used to evaluate the additional prognostic information for each signature. The immune cell composition was also evaluated using the CIBERSORT in silico tool. PD-L1 gene and protein expressions were independently associated with better prognosis. In ER+/HER2- patients, PD-L1 gene expression provided significant additional prognostic information beyond that of both 21-GS [LR-Δχ 2  = 15.289 and LR-Δχ 2  = 8.812, P < 0.01 for distant metastasis-free interval (DMFI) in cohorts 1 and 2, respectively] and 70-GS score alone (LR-Δχ 2  = 18.198 and LR-Δχ 2  = 8.467, P < 0.01 for DMFI in cohorts 1 and 2, respectively). PD-L1 expression was correlated with IHC-determined CD3+ cells (r = 0.41, P < 0.001) and with CD8+ (r = 0.62, P < 0.001) and CD4+ memory activated (r = 0.66, P < 0.001) but not with memory resting (r = -0.063, P = 0.14) or regulatory (r = -0.12, P < 0.01) T cells in silico. PD-L1 gene expression represents a promising favorable prognostic marker and can provide additional prognostic value to 21- and 70-gene scores in ER+/HER2- BC., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via β-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver.

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and multifaceted transcription factor involved in multiple cellular functions. Its role in modifying anti-tumor immunity has been recently recognized. In this study, the biologic effects of STAT3 on immune checkpoint expression and anti-tumor responses were investigated in breast cancer (BC). A transcriptional signature of phosphorylated STAT3 was positively correlated with PD-L1 expression in two independent cohorts of early BC. Pharmacologic inhibition and gene silencing of STAT3 led to decreased Programmed Death Ligand 1 (PD-L1) expression levels in vitro, and resulted as well in reduction of tumor growth and decreased metastatic dissemination in a mammary carcinoma mouse model. The hampering of tumor progression was correlated to an anti-tumoral macrophage phenotype and accumulation of natural-killer cells, but also in reduced accrual of cytotoxic lymphocytes. In human BC, pro-tumoral macrophages correlated to PD-L1 expression, proliferation status and higher grade of malignancy, indicating a subset of patients with immunosuppressive properties. In conclusion, this study provides evidence for STAT3-mediated regulation of PD-L1 and modulation of immune microenvironment in BC., Competing Interests: Ioannis Zerdes, Majken Wallerius, Emmanouil G. Sifakis, Tatjana Wallmann, Stina Betts, Margarita Bartish, Nikolaos Tsesmetzis, Nicholas P. Tobin, Christos Coucoravas, George Z. Rassidakis and Charlotte Rolny have no conflicts of interest to disclose. Theodoros Foukakis: institutional grants from Roche and Pfizer and personal fees from Novartis, Pfizer, Roche and UpToDate; Jonas Bergh has received research funding from Merck paid to Karolinska Institutet and from Amgen, Bayer, Pfizer, Roche and Sanofi-Aventis paid to Karolinska University Hospital. No personal payments. Payment from UpToDate for a chapter in breast cancer prediction paid to Asklepios Medicine HB.

Background: Use of cyclin D1 (CCND1) gene amplification as a breast cancer biomarker has been hampered by conflicting assessments of the relationship between cyclin D1 protein levels and patient survival. Here, we aimed to clarify its prognostic and treatment predictive potential through comprehensive long-term survival analyses., Methods: CCND1 amplification was assessed using SNP arrays from two cohorts of 1965 and 340 patients with matching gene expression array and clinical follow-up data of over 15 years. Kaplan-Meier and multivariable Cox regression analyses were used to determine survival differences between CCND1 amplified vs. non-amplified tumours in clinically relevant patient sets, within PAM50 subtypes and within treatment-specific subgroups. Boxplots and differential gene expression analyses were performed to assess differences between amplified vs. non-amplified tumours within PAM50 subtypes., Results: When combining both cohorts, worse survival was found for patients with CCND1-amplified tumours in luminal A (HR = 1.68; 95% CI, 1.15-2.46), luminal B (1.37; 1.01-1.86) and ER+/LN-/HER2- (1.66; 1.14-2.41) subgroups. In gene expression analysis, CCND1-amplified luminal A tumours showed increased proliferation (P < 0.001) and decreased progesterone (P = 0.002) levels along with a large overlap in differentially expressed genes when comparing luminal A and B-amplified vs. non-amplified tumours., Conclusions: Our results indicate that CCND1 amplification is associated with worse 15-year survival in ER+/LN-/HER2-, luminal A and luminal B patients. Moreover, luminal A CCND1-amplified tumours display gene expression changes consistent with a more aggressive phenotype. These novel findings highlight the potential of CCND1 to identify patients that could benefit from long-term treatment strategies.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Automatic segmentation of the arterial lumen from ultrasound images is an important task in clinical diagnosis. Carotid artery recognition, the first task in lumen segmentation, should be performed in a fully automated, fast, and reliable way to further facilitate the low-level task of arterial delineation. In this paper, a user-independent, real-time algorithm is introduced for carotid artery localization in longitudinal B-mode ultrasound images. The proposed technique acts directly on the raw image, and exploits basic statistics along with anatomical knowledge. The method's evaluation and parameter value optimization were performed on a threefold cross validation basis. In addition, the introduced algorithm was systematically compared with another algorithm for common carotid artery recognition in B-mode scans, separately for multi-frame and single-frame data. The data sets used included 2,149 images from 100 subjects taken from three different institutions and covering a wide range of possible lumen and surrounding tissue representations. Using the optimized values, the carotid artery was recognized in all the processed images in both multi-frame and single-frame data. Thus, the introduced technique will further reinforce automatic segmentation in longitudinal B-mode ultrasound images.

High-throughput DNA methylation profiling exploits microarray technologies thus providing a wealth of data, which however solicits rigorous, generic, and analytical pipelines for an efficient systems level analysis and interpretation. In this study, we utilize the Illumina's Infinium Human Methylation 450K BeadChip platform in an epidemiological cohort, targeting to associate interesting methylation patterns with breast cancer predisposition. The computational framework proposed here extends the--established in transcriptomic microarrays--logarithmic ratio of the methylated versus the unmethylated signal intensities, quoted as M-value. Moreover, intensity-based correction of the M-signal distribution is introduced in order to correct for batch effects and probe-specific errors in intensity measurements. This is accomplished through the estimation of intensity-related error measures from quality control samples included in each chip. Moreover, robust statistical measures exploiting the coefficient variation of DNA methylation measurements between control and case samples alleviate the impact of technical variation. The results presented here are juxtaposed to those derived by applying classical preprocessing and statistical selection methodologies. Overall, in comparison to traditional approaches, the superior performance of the proposed framework in terms of technical bias correction, along with its generic character, support its suitability for various microarray technologies.

Background: It has been shown previously that glucocorticoids exert a dual mechanism of action, entailing cytotoxic, mitogenic as well as cell proliferative and anti-apoptotic responses, in a dose-dependent manner on CCRF-CEM cells at 72 h. Early gene expression response implies a dose-dependent dual mechanism of action of prednisolone too, something reflected on cell state upon 72 h of treatment., Methods: In this work, a generic, computational microarray data analysis framework is proposed, in order to examine the hypothesis, whether CCRF-CEM cells exhibit an intrinsic or acquired mechanism of resistance and investigate the molecular imprint of this, upon prednisolone treatment. The experimental design enables the examination of both the dose (0 nM, 10 nM, 22 uM, 700 uM) effect of glucocorticoid exposure and the dynamics (early and late, namely 4 h, 72 h) of the molecular response of the cells at the transcriptomic layer., Results: In this work, we demonstrated that CCRF-CEM cells may attain a mixed mechanism of response to glucocorticoids, however, with a clear preference towards an intrinsic mechanism of resistance. Specifically, at 4 h, prednisolone appeared to down-regulate apoptotic genes. Also, low and high prednisolone concentrations up-regulates genes related to metabolism and signal-transduction in both time points, thus favoring cell proliferative actions. In addition, regulation of NF-κB-related genes implies an inherent mechanism of resistance through the established link of NF-κB inflammatory role and GC-induced resistance. The analysis framework applied here highlights prednisolone-activated regulatory mechanisms through identification of early responding sets of genes. On the other hand, study of the prolonged exposure to glucocorticoids (72 h exposure) highlights the effect of homeostatic feedback mechanisms of the treated cells., Conclusions: Overall, it appears that CCRF-CEM cells in this study exhibit a diversified, combined pattern of intrinsic and acquired resistance to prednisolone, with a tendency towards inherent resistant characteristics, through activation of different molecular courses of action.

Automatic segmentation of the arterial lumen from ultrasound images is an important task in clinical diagnosis. In this paper, the Hough transform (HT) was used to automatically extract straight lines and circles from sequences of B-mode ultrasound images of longitudinal and transverse sections, respectively, of the carotid artery. In 10 normal subjects, the specificity and accuracy of HT-based segmentation were on average higher than 0.96 for both sections, whereas the sensitivity was higher than 0.96 in longitudinal and higher than 0.82 in transverse sections. The intima-media thickness (IMT) was also estimated from images of longitudinal sections; the corresponding validation parameters were generally higher than 0.90. To further validate the results, arterial distension waveforms (ADW) were estimated from sequences of images using the HT technique as well as motion analysis using block matching (BM). In longitudinal sections, diastolic and systolic diameters and relative diameter changes using HT and BM were not significantly different. In transverse sections, diastolic and systolic diameters were significantly lower using the HT technique; the differences were <7%. Relative diameter changes in transverse sections were not significantly different from BM-estimated ones. The HT technique was also applied to four subjects with atherosclerosis, in which sensitivity, specificity and accuracy were comparable to those of normal subjects; the low values of sensitivity in transverse sections may reflect departure from the circular model because of the presence of plaque. In conclusion, the HT technique provides a reliable way to segment ultrasound images of the carotid artery and can be used in clinical practice to estimate indices of arterial wall physiology, such as the IMT and the ADW.