Each SUVMax cutpoint prioritized either survival differences, total cohort comparison sample sizes, or sample size by stage. Patient outcomes and associations between SUVMax and cancer-associated weight loss were assessed by multivariate, categorical, and survival analyses.774 non-small cell lung cancer (NSCLC) patients with pre-treatment PET evaluated between 2006 and 2014 were identified. Using the international validated definition of cachexia, the presence of clinically significant pretreatment cancer-associated weight loss (WL) was retrospectively determined. Maximum Standardized Uptake Value . After stratifying the cohort by WL and dichotomized SUVMax, both factors were found to be relevant in predicting survival outcomes when the alternative variable was constant. Of note, the most striking survival differences contributed by WL status occurred in high SUVMax groups, where the presence of WL predicted a median survival time detriment of up to 10 months, significant regardless of cutpoint determination method applied to categorize high SUVMax patients. SUVMax classification was found to be most consistently relevant in both WL and no WL groups.Patients were found to have an increased likelihood of having WL at diagnosis associated with increasing primary tumor SUVMax underscores increased glucose uptake as a component of catabolic tumor phenotypes. This substantiates 18F‐FDG PET analysis as a prospective tool for assessment of cancer-associated weight loss and corresponding survival outcomes. Furthermore, the survival differences observed between WL groups across multiple SUVMax classifications supports the importance of weight loss monitoring in oncologic workups. Weight loss in the setting of NSCLCs with higher metabolic activity as determined by 18F‐FDG PET signal should encourage more aggressive and earlier palliative care interventions.The significant positive association between significant pretreatment cancer-associated weight loss and primary tumor SUV Cancer cachexia is a multifactorial syndrome characterized by skeletal muscle wasting and depletion of adipose stores , 2. Ther18F‐FDG), a positron emitting glucose molecule. 18F‐FDG accumulates in organs with high glucose utilization such as the brain, heart, and liver. Malignant cells demonstrate increased uptake of 18F-FDG on PET due to overexpression of GLUT receptors and elevated intracellular levels of hexokinase, which generate increased rates of glycolysis , a measurement of tracer uptake in a lesion normalized to injected activity and volume of distribution  is a functional imaging technique commonly used in the initial staging of patients with newly diagnosed solid tumors. PET is often performed with the intravenous administration of fluorine-18-fluoro-2-deoxy-D-glucose  and the presence of significant cancer-associated weight loss at the time of diagnosis, prior to any cancer-directed intervention, in a large cohort of NSCLC patients. We also aimed to investigate differential survival outcomes based on SUVMax and pretreatment weight loss. Further characterization of the effects of and potential associations between these factors may clarify metabolic disruptions exerted by tumors that lead to weight loss. Moreover, evaluating the prospective utility of weight assessment and tumor 18F‐FDG uptake might help providers identify NSCLC patients with poor prognoses.The clinical hallmark of cancer cachexia is unintentional weight loss. The presence of this finding at the time of detection of non-small cell lung cancer (NSCLC) portends poor survival, even after controlling for other factors such as age, sex, comorbidity, and tumor stage –23. Althth and 8th editions of the American Joint Committee on Cancer (AJCC) staging system were adopted in 2009 and 2018, respectively, but patient stage was not adjusted for the purpose of this study. This study was approved by our Institutional Review Board at UT Southwestern.Using a prospectively maintained tumor registry from a single tertiary care center and retrospective patient electronic medical record review, we identified 774 patients with NSCLC diagnosed between 1/1/2006 and 12/31/2013 with pre-treatment PET available for review. Exclusion criteria for this study included synchronous or metachronous malignancy and incomplete data. Patients with carcinoma in situ, carcinoid, neuroendocrine, lymphoma, melanoma, or sarcoma tumor histologies were excluded. Patient and tumor characteristics, including age, sex, race, date of death/last follow up, Charlson comorbidity index (CCI) score, primary tumor site, tumor grade, and cancer stage, were recorded. Descriptive statistics were used to summarize patient and tumor characteristics. The 72 or unintentional weight loss > 2% in patients with body mass index < 20 kg/m2. Patients with stable weight, weight gain, or purposeful weight loss were classified as not having WL. When multiple measures of weight were available in the pre-treatment period, a consistent weight decrease was required for a patient to be classified as having WL.We reviewed medical records—including vital signs, physician notes, and dietician notes at the time of cancer diagnosis but before any therapeutic measure—for documented weight loss and associated symptoms. At our health system, patients are routinely weighed as a part of each office visit, and this measurement is documented in the electronic medical record. Patients missing weight data before the initiation of therapy were excluded. Determination of clinically significant pretreatment cancer-associated weight loss (WL) was based on the validated international consensus definition of cancer cachexia . WL was 18F-FDG was obtained in all analyzed patients at the time of diagnosis and prior to any oncologic intervention. After a standard uptake phase of 60-90 minutes, each patient underwent computed tomography (CT) imaging from the level of the midbrain to the midthigh at 3 mm intervals with arms raised above the head. Following completion of the CT portion of the study, 3D emission images were obtained through the same distance. Oral contrast was used in all patients, unless otherwise contraindicated. The CT images were generated for the purpose of PET image optimization and for anatomical correlation of PET findings. Each PET report was generated by a trained nuclear medicine physician. SUVMax of the primary tumor, serum blood glucose level at the time of scan, and time interval between 18F‐FDG injection and scan initiation, when available, were recorded.PET with Max cutpoints. A cutpoint optimizing survival was calculated through the method of Contal and O’Quigley .Max. All variables were categorical with the exception of age at diagnosis and tumor SUVMax. Due to tumor grade limiting the sample size available for analysis, the parameter was not included in the primary multivariate analysis. The results from the limited sample (N=408) multivariate analysis including tumor grade as a covariate are included in Multivariate logistic regression (N=700) was conducted with WL at diagnosis as the dependent binary variable. Covariates included in this analysis were patient sex, age at diagnosis, race, alcohol history, tobacco history, and CCI score, as well as tumor characteristics including histology, stage and SUVMax groups. Tests were repeated for each cutpoint method.Chi square testing was carried out to evaluate differences between expected and observed incidence of WL in high and low SUVSurvival probability was plotted using Kaplan-Meier estimator. Event occurrence was defined by patient death. Time to censoring or event was provided by the time in months from the cancer diagnosis date to the date of patient death or last contact, respectively. Significance of survival differences were evaluated through log-rank testing.Max. Further analyses divided the cohort 4 groups on the basis of +/- WL at diagnosis and high/low SUVMax, in which log-rank testing was done across groups with one differing variable. All tests involving SUVMax were repeated for each cutpoint methodGroups evaluated for survival included the overall cohort, patients with or without WL at diagnosis, and patients with high or low SUVPatient characteristics summarized in Max demonstrated a statistically significant association with the incidence of cancer-associated weight loss at diagnosis with an odds ratio of 1.038 , SUVMax and 554 patients as high SUVMax. The incidence of WL at diagnosis was 15.00% in the low SUVMax group and 28.52% in the high SUVMax group . Median SUVMax values for ascending tumor stage values were 6.70, 9.50, 13.80, and 10.95. WL incidence was 19.64% and 29.84% in patients with stage-defined low and high SUVMax values respectively . Patients were grouped into one of four groups on the basis of WL status and SUVMax status utilizing the different cutpoint determination methods. Regardless of the cutpoint determining method implemented, both WL and SUVMax status demonstrated significant or near-significant associations with survival when the alternative variable was held constant. Notably, in the stage median cutpoint survival analysis, where sample size matching was prioritized by stage, WL significantly predicted for survival outcome in both low (P=0.0449) and high (P=0.0004) SUVMax groups.Max appeared to retain significance in lower stage tumors, with WL at diagnosis demonstrating associations with survival to a higher frequency in higher stage tumors  as well as conventional tumor classifications , further implicating a mechanistic association between these variables. By dichotomizing SUVMax across multiple cutpoint calculation methods, we provided experimental models for implementing SUVMax diagnostic cutoffs to assess for the presence of cancer-associated weight loss. Regardless of cutpoint method, patients with SUVMax values above a cutpoint had a 1.5 to 2 times increased incidence of pre-treatment cancer-associated weight loss qualifying for diagnosis of cachexia. Mechanistically, these findings underscore the relevance of heightened tumor glucose metabolism to the systemic metabolic derangements classically observed in cachexia. Clinically, our study supports both a heightened consideration of weight loss with increasing tumor SUVMax value and a crucial association between clinically significant pretreatment weight loss identified at diagnosis with survival detriment in patients with both high or low SUVMax tumors.Our study demonstrates a significant positive association between primary tumor SUVMax and WL at diagnosis in multivariate analysis suggest a potential prognostic value of PET imaging results in predicting presence or risk of cancer-associated weight loss. By applying multiple cutpoint strategies, we demonstrated that optimal PET measure cutoff values vary depending on the intended prognostic measure. In this project, these measures included WL at diagnosis and survival prediction. Furthermore, our stage stratification of both survival analyses and cutpoint determination indicate differential utility of PET imaging results based on the depth of stratification applied within the cancer type.The associations observed between tumor SUVMax groups by stage. Still, the presence of WL at diagnosis significantly predicted a survival detriment independent of SUVMax status, as median survival time was decreased by approximately 17 months in patients with SUVMax below the stage-tailored cutpoint, and 10 months in patients with higher SUVMax values. This trend was consistently observed when analyses were conducted separately for each stage, albeit with diminished significance likely attributable to the limited statistical power permitted by multiple divisions of our cohort. Notably, within this stage stratified cohort, SUVMax surpassed WL as the primarily significant determinant of survival as indicated by the group comparisons in patients with stage 1 tumors. Meeting criteria for WL at diagnosis exceeded SUVMax as the primary determinant factor towards survival detriment. Of note, even when the cutpoint selected favored the relevance of SUVMax in survival , WL significantly predicted a detriment in survival within the high SUVMax group, which retained significance in stage 3 tumors after further stratification (with near-significant results in stage 1 tumors). The relevance of weight loss at presentation to survival of patients with high SUVMax tumors underscores an objective detriment in patient outcomes exerted by cancer-associated weight loss in patients with severe NSCLC tumor phenotypes.To control for the potential bias of later stage tumors  being disproportionally classified as having high PET imaging results, our stage-specific median cutpoint group balanced SUVMax and cancer-associated weight loss is concordant with results observed in murine models of cancer cachexia. In a prior study by Penet et al., in-vivo cachexia-inducing MAC16 tumors were characterized by higher 18F‐FDG uptake than histologically similar non-cachexia-inducing MAC13 tumors (18FDG uptake) in the induction or presentation of tumor phenotype capable of inducing weight loss.The observed positive association between primary tumor SUV3 tumors . With va18F‐FDG PET analysis to measure the activity of brown adipose tissue (The study by Penet et al. also found that mice with cachectic MAC16 tumors underwent significant depletion of lipid tissue in their adipose tissue and skeletal muscle, with sparing of lipid at tumor sites . These ee tissue  further This analysis has a number of limitations. For example, WL was retrospectively assessed from medical records rather than a formal prospective protocol. Although WL was based on the consensus definition of cancer cachexia, more specific diagnostic criteria indicative of a cachectic state - such as specific biomarkers, enhanced lipolysis, and sarcopenia- were omitted due to limitations on what is routinely obtained in patient management . Cachexi18F‐FDG uptake.Our study supports a positive association between increased tumor glucose utilization and the development of cancer-associated weight loss that is independent of various potential confounding patient and tumor characteristics. Moreover, we identified a vulnerability of patient survival time to significant weight loss prior to cancer diagnosis, particularly for patients with tumors demonstrating high 18F‐FDG uptake. Our study validates this function by consistently demonstrating the relevance of SUVMax obtained from this technique in survival prognosis. Given the maladaptive metabolic changes that underpin both cancer-associated weight loss and PET imaging enhancement by 18F‐FDG uptake (18F‐FDG PET analysis within the workup of cancer-associated weight loss and cachexia.PET imaging is routinely used for prognosing NSCLC patients using radiolabeled The original contributions presented in the study are included in the article/SO BG RI PI designed the study. DX, BG, and SO contributed to and organized the database. SO and BG performed statistical analyses. BG and SO wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by National Institutes of Health [P30 CA142543]; Burroughs Wellcome Fund Career Awards for Medical Scientists [1019692]; American Gastroenterological Association Scholar Award [2019AGARSA3]; American Cancer Society grant [133889-RSG-19-195-01-TBE]; Cancer Prevention and Research Institute of Texas [RP200170]; and V Foundation Scholar Award [V2019-014].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cytosine methylation is an epigenetic mark that participates in regulation of gene expression and chromatin stability in plants. Advancements in whole genome sequencing technologies have enabled investigation of methylome dynamics under different conditions. However, the computational methods for analyzing bisulfite sequence data have not been unified. Contention remains in the correlation of differentially methylated positions with the investigated treatment and exclusion of noise, inherent to these stochastic datasets. The prevalent approaches apply Fisher’s exact test, logistic, or beta regression, followed by an arbitrary cut-off for differences in methylation levels. A different strategy, the MethylIT pipeline, utilizes signal detection to determine cut-off based on a fitted generalized gamma probability distribution of methylation divergence. Re-analysis of publicly available BS-seq data from two epigenetic studies in Arabidopsis and applying MethylIT revealed additional, previously unreported results. Methylome repatterning in response to phosphate starvation was confirmed to be tissue-specific and included phosphate assimilation genes in addition to sulfate metabolism genes not implicated in the original study. During seed germination plants undergo major methylome reprogramming and use of MethylIT allowed us to identify stage-specific gene networks. We surmise from these comparative studies that robust methylome experiments must account for data stochasticity to achieve meaningful functional analyses. However, the language of the methylome 5 has remained much more elusive to interpretation than that of the underlying nucleotide sequence. If we follow the DNA code analog, we must achieve proficiency in methylome decoding in at least three ways: (i) reading at single site resolution, (ii) interpretation of downstream effects of single position methylation status, and (iii) understanding the meaning of different methylation patterns in their local and global DNA context. While it is currently feasible to read cytosine methylation at single base resolution, owing to advances in whole genome sequencing through bisulfite conversion6, and to interpret high-density methylome changes, such as in transposable elements (TEs) during major developmental events or in methylation machinery mutants7, our proficiency in decoding of the methylome remains lacking.Cytosine methylation has been described as the fifth letter of the genetic code10. This methodology was likely a consequence of experimental emphasis on datasets deriving from DNA methylation machinery mutants, which produced extremely robust methylation signal11. These units of change were termed differentially methylated regions (DMRs) and varied across studies for window size, requisite differentially methylated position (DMP) number, and uniform directionality for hyper/hypo-methylation14. DMR analysis can identify genomic sites likely to undergo, or be released from, gene silencing15 and, therefore, often produce datasets rich in TE and heterochromatic genomic intervals. With these analytical approaches, however, the function of low density, intragenic methylation repatterning during development and in response to environmental stimuli remains unapproachable. While there is an extensive understanding of the evolutionary and mechanistic origin of gene body methylation (GbM)16, debate on its functionality remains17.Much of the early analysis of methylome variation focused on high-density methylation changes within defined intervals across the genome18. Variation is thought to arise from thermodynamic fluctuations of cellular machinery and the DNA molecule itself22. There is inherent stochasticity to methylome remodeling with each cell division, giving rise to ‘spontaneous epimutations’ in all methylation contexts24. Noise in BS-seq datasets is also amplified by tissue pooling as the plant epigenome appears to be developmental stage-, tissue- and cell-type specific31. Therefore, a full picture of environmental or developmental epigenetic responses is only possible by separating treatment signal from noise in the system. This type of approach would make interpretation of methylome data feasible at the organ or organism level, even as single cell BS-seq becomes feasible in plants32, as it is in animals33.The plant methylome is often described as stochastic34. However, recent advances in computational biology demonstrate that interpreting existing methylome data at single read35 or single cytosine36 resolution yields new information. Single cytosine changes in methylome patterns have been shown to have important phenotypic effects38 and we are beginning to recognize the importance of gene body methylation in plants39. These observations point to a gap between the prevalent methodology and our current understanding of methylome biology.Stochastic methylome variation is often dismissed as information-free noise, inherent to the study of biological systems41. To test the relative efficacy of a signal detection-based method versus conventional methods for methylome data analysis, datasets produced in studies with robust experimental design and depth of sequencing can be re-analyzed with MethylIT, potentially increasing their utility. Here, we apply MethylIT, a signal detection pipeline, to two open access datasets of high quality; a study of methylome remodeling in response to phosphate starvation42 and an analysis of epigenetic changes during normal seed germination29. This report aims to demonstrate that applying novel approaches to existing high-quality methylome data can reveal meaningful additional information.Signal detection with machine learning is one approach to methylome data that bridges this gap. The MethylIT tool, based on physical statistics approaches, has been effective in identifying treatment-associated differential methylation in multiple study systemsBisulfite deamination of DNA, by conversion of cytosine into uracil to be read as thymine, combined with whole-genome sequencing, has enabled the study of methylome variation in many organisms where an assembled reference genome is available. The cost of whole genome bisulfite sequencing (WGBS) continues to decline, enabling plant biologists to add epigenetic components to developmental and environmental response studies.43. The resulting short sequence reads are then aligned to the reference genome based on the three different bases that result from bisulfite conversion. A prevalent aligner for this step is Bismark44, and the output file contains methylated and unmethylated counts for each cytosine in the genome as well as its context.Upon completion of the bisulfite sequencing (BS-seq) runs, primary data must be converted to methylation counts for each cytosine in the genome. The first step, an overall quality check and the trimming of sequencing adaptors, is most commonly conducted with Trim Galore!, which performs both functions14 and they differ significantly in the statistical approaches implemented25. Without advanced statistical mathematics training, it is often easiest to adopt the most user-friendly, but not necessarily most powerful, options. Figure 29. This example demonstrates both the divergence of results when different methods are used and the bias of using a DMP-density-based approach  and regions (DMRs). There are more than 20 bioinformatic tools availableThe number of samples or biological replicates varies widely between methylome studies. The limiting factors include the cost per sample for WGBS and the amount of tissue needed (seedlings are often pooled into one sample). Whereas many common methylome analyses , or a sliding window. However, deciding on the number of DMPs sufficient for a DMR is a controversial step and another arbitrary filter.Some pipelines, including MethylSig, DMRcaller, and BSmooth5. The first step distinguishing this pipeline calculates the difference in methylation levels between all given samples, including the controls and the reference (centroid of controls) methylome. In MethylIT, differences are estimated in the form of Hellinger divergence and total variation distance. Next, the divergences are modeled for all samples based on a generalized gamma probability distribution model, the fitted probability distribution, yielding separate control and treatment group probability distributions5. Others have also proposed using information theory approaches for analyzing methylome32. In MethylIT, the potential treatment-associated signal is above the 95th percentile of the fitted probability distribution of the methylation, which becomes the initial cut-off for each individual sample. The following step uses machine learning to distinguish true DMPs by estimating the optimal cut-off sufficient for the classification of DMPs into control and treatment . Conversely, MethylIT assesses each potential DMP based on the fitted probability distribution of methylation in each biological replicate separately, accounting for methylation heterogeneity and inherent stochasticity41. This type of filtering avoids the application of arbitrary cut-offs that can eliminate meaningful DMPs or include DMPs that are the result of stochastic fluctuation, both impacting outcomes.MethylIT uses an information thermodynamics-derived approach to provide greater resolution of treatment-associated signals without consideration of DMP density Methylome analysis ends with feature annotation. The prevalent method yields a list of DMRs of arbitrary size that contain an arbitrary number of DMPs validated in the previous step. MethylIT also provides a list of DMRs; however, these are identified with the best generalized linear model selected from Poisson and Negative Binomial regression analyses, comparing control and treatment groups, and filtered with minimum methylation counts per cytosine and individual. The final step in both methodologies is to overlap or assess the proximity of DMR genomic addresses with genetic features of interest, including genes, TEs, and exons. Whereas prevalent methods look for overlaps of DMRs with annotated features, MethylIT statistically assesses all features of the selected category  as potential DMRs with no fixed set of DMRs to overlap with annotated features.14. Figure The resulting number of differentially methylated genes (DMGs) is generally higher using MethylIT than with the prevalent methodology Table , which mdescribe global changes in methylome levels. These changes generally include the percentage of methylated cytosines in each context, densely hypo- or hypermethylated or heterochromatic regions, TE silencing, and the global impact of epigenetic machinery mutants. MethylIT, as a signal detection method, aims to read global changes in methylome patterns at single cytosine resolution. The method, therefore, treats hypo- and hyper-methylation at each site equally, while evaluating each DMP based on the fitted probability distribution of each individual. The approach allows tracking of subtle changes in methylome repatterning without consideration of changes in methylome levels and discriminates variation within the treatment condition from the stochastic background effects present in both treatment and control conditions. Where most common approaches neglect to count DMPs that appear outside of “CpG islands”49, which have been shown to have important downstream and phenotypic effects38, the MethylIT procedure, by identifying only treatment-associated DMPs, incorporates all data regardless of methylation context or DMP density. Here, we focus our analysis on DMGs , particularly to demonstrate the importance of finding and interpreting intragenic methylation.By comparing pipelines and their data outputs Fig. , we can 42. We selected this study for re-analysis with the MethylIT signal detection pipeline based on several features of its experimental design. The depth of sequencing was sufficient, each condition had three individual biological replicates with no pooling, controls were rigorous and present in both short- and long-term starvation conditions (7 days and 16 days), and data were collected for roots and shoots separately.A 2015 study of epigenetic responses to phosphate starvation in Arabidopsis revealed that methylome repatterning occurs in response to low phosphate (Pi), with altered expression of a small number of differentially methylated genes responsive to low phosphate conditions. (2015) identified DMPs using an F-test and subsequently DMRs as regions where DMP density exceeded global DMP density. Yong-Villalobos et al. point out the arbitrary nature of defining a DMR and hence conduct further analysis using both DMRs and all DMPs. For valid comparison with MethylIT, we use their reported list of DMRs overlapped with TAIR10 genes  allowed the staggering number of DMGs to be converted into a Pi-starvation-associated subset of manageable size. Network analysis allowed potential key players in this tissue-specific stress response to be revealed.Genome Biology investigated methylome remodeling during embryogenesis and germination29. Narsai et al. (2017) analyzed five developmental stages and reported progressive demethylation of the Arabidopsis genome during the seed-to-seedling transition. This transition coincided with changes in mRNA and siRNA populations. We selected this dataset for re-analysis with MethylIT based on its robust biological replication, depth of sequencing, and limited discussion of the functional identity of DMRs determined. BS-seq read files were provided for dry seed, seed after 48 h stratification, 6 h after light exposure, 24 h after light exposure, and 48 h after light exposure. The investigators conducted methylome analysis using HOME (v0.1) with default parameters for time series analysis, and the added cut-off for the difference in methylation levels was 20%. In contrast to the original study’s hierarchical clustering, MethylIT analysis was conducted pairwise, with consecutive stages of development serving as the reference (control) methylome for the identification of DMGs in each following stage. As the original study did not include DMG analysis, we overlapped their reported 12,654 DMRs with TAIR10 genes to obtain 891 DMGs  that provide confidence in conclusions with a minimum of “cherry picking”The ability to derive data with unambiguous outcomes  and epigenetics, with C-5 methylation of cytosine always being one of the top scoring GO term enriched categories. MethylIT added numerous functionally important genes to the DMG list in both datasets, while removing stochastic variation-associated ones.Because MethylIT does not rely on DMP density for the detection of DMRs, the bias of assuming only methylation-dense regions as biologically impactful (present in most methods) is eliminated. Instead, signal detection allows the identification of only treatment-associated DMPs, regardless of the proximity or direction of neighboring DMPs. This allows meaningful analysis of intragenic methylation repatterning, which remains the frontier of methylome analysis as it eludes density-dependent approaches. Our re-analysis also demonstrates the power of data reuse with computational approaches that were not available when data were generated, highlighting the importance of FAIR principles in increasing the utility of expensive datasets, like methylomes.To avoid any changes due to discrepancies in the reference genome annotation versions, we overlapped the DMRs reported in the original studies with version 38 of the TAIR10 genome annotation to obtain DMGs.https://genomaths.github.io/methylit/articles/MethylIT.html), excluding modifications described in Supplemental Table p-value < 0.05. Gene annotation was done using version 38 of TAIR10.Raw sequencing reads were obtained from the GEO repository for both datasets. They were rimmed with TrimGalore and aligned the TAIR10 reference genome. DMPs were identified using MethylIT (version 0.3.2.4). Standard settings were used as described on the GitHub page  and > tenfold (seed germination) enrichment were plotted on heatmaps, using the ggplot2 package.DAVID Functional Annotation tool (6.8)51. The STRING database was used to construct the protein–protein interaction network from imported DMGs. The core hub was identified using the clusterMaker plug-in with the k-means cluster function. 3 clusters were calculated with 500 iterations, using Euclidean distance, betweenness centrality, closeness centrality, average shortest path length, clustering coefficient, degree, and eccentricity. The core hubs depicted in the figures were identified as the cluster with the highest centrality. The size of the node corresponds to the degree of connectivity score and the edge transparency corresponds to the STRING database score.DMGs were also functionally analyzed using Cytoscape (3.9.1)Supplementary Information 1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.


By low-density lipoprotein (LDL) reduction, statins play an important role in cardiovascular risk modification. Incompletely understood pleiotropic statin effects include vasoprotection that might originate from mobilisation and differentiation of vascular progenitor cells. Data on the potentially differential impact of statin treatment intensity on circulating progenitor cells in patients undergoing percutaneous coronary intervention (PCI) are scarce. This study examines the potential association of different permanent statin treatment regimens on circulating progenitor cells in patients with coronary syndrome.In a monocentric prospective all-comers study, 105 consecutive cases scheduled for coronary angiography due to either (A) non-invasive proof of ischemia and chronic coronary syndrome (CCS) or (B) troponin-positive acute coronary syndrome (ACS) were included. According to the 2018 American College of Cardiology Guidelines on Blood Cholesterol, patients were clustered depending on their respective permanent statin treatment regimen in either a high- to moderate-intensity statin treatment (HIST) or a low-intensity statin treatment (LIST) group. Baseline characteristics including LDL levels were comparable. From blood drawn at the time of PCI, peripheral blood mononuclear cells were isolated, cultivated and counted and, by density gradient centrifugation, levels of circulating progenitor cells were determined using fluorescence-activated cell sorting (FACS) analysis. In ACS patients both absolute and relative numbers of circulating early-outgrowth endothelial progenitor cells (EPCs) concurrently were significantly lower in the HIST group as compared to the LIST group. This effect was more pronounced in ACS patients than in CCS patients. Both in ACS and CCS patients, HIST caused a significant reduction of the number of circulating SMPCs.In patients undergoing PCI, a dose intensity-dependent and LDL level-independent pro-differentiating vasoprotective pleiotropic capacity of statins for EPC and SMPC is demonstrated. Statin treatment plays a pivotal role in the prevention of coronary artery disease (CAD) both in patients with acute coronary syndrome (ACS) as well as chronic coronary syndrome (CCS). Endothelial dysfunction plays a crucial role in the initiation and progression of CAD and is potentially modulated by changes in number and function of circulating progenitor cell populations released from the bone marrow. Derived from the bone-marrow, circulating early-outgrowth endothelial progenitor cells (EPCs) substantially contribute to vascular repair –3. Due tSince the discovery of circulating EPCs, research has been aimed at the characterisation of the various subtypes of cells that differentiate from circulating progenitors, the development of protocols for EPC characterization and quantification, and the clarification of the clinical relevance of EPCs. With the expansion of the research field, the related nomenclature has become increasingly complex and the optimal definition of EPCs still causes ambiguity. Generally, two methods are used to identify EPCs. One of the approaches is based upon the confirmation, after a defined period under angiogenic culture conditions, of the specific mature endothelial morphology of endothelial precursors . Based oDue to the prevailing hypothesis that smooth muscle progenitor cell (SMPC) recruitment may contribute to plaque stabilisation –16, SMPCThis study aims to detect a potential impact of high- to moderate-intensity statin therapy (HIST) or low-intensity statin therapy (LIST) treatment regimens on EPCs and SMPCs in patients after PCI for the treatment of ACS or CCS, respectively. Thus, we hypothesize that intensified statin treatment has a capacity to optimize endothelial function and plaque morphology by modifying circulating progenitor cell migration and maturation, thereby potentially improving clinical outcome of CAD patients.In a monocentric prospective all-comers study design, consecutive cases with an indication for coronary angiography due to either ACS with positive blood marker troponin or CCS with non-invasive proof of ischemia were included. Prior to inclusion, informed consent was obtained from all patients. The study was approved by the Ethics Committee of the Medical Faculty at University Hospital Aachen (EK 164/07). All patients that received at least one coronary stent were included. All patients were on permanent statin treatment due to known CCS, hyperlipidaemia, or documented peripheral artery disease beginning at least four weeks before inclusion. According to the statin efficacy as defined in the 2018 American College of Cardiology Guidelines on Blood Cholesterol , patient6 PBMCs were plated in 6-well plates coated with 200μg/mL fibronectin  and maintained in Endothelial Cell Growth Medium MV2 Kit . For SMPC selection and cultivation, 106 PBMCs were plated in 6-well plates coated with 80μg/mL Collagen G  and maintained in Dulbecco´s Modified Eagle Medium  supplemented with 10% fetal calf serum (FCS)  and 10ng/mL PDGF-BB . On day 4 the non-adherent cells were removed; EPC medium was replaced completely while half of the SMPC medium was changed. At day 7 in culture, PBMCs developed the EPC-specific elongated spindle shape morphology or the SMPC “hill and valley morphology”, respectively. Thus, the fraction of EPCs and SMPCs, respectively, derived from a defined PBMC amount was measured by counting the adherent cells. In addition, the absolute whole blood EPC concentration was calculated depending on the number of PBMCs per ml in the respective blood volume.Twenty mL whole blood samples were taken from each patient directly after PCI using natriumcitrate-dextrane as anticoagulant and immediately processed. EPCs and SMPCs were cultured as previously described , 21. In TM, California) and phycoerythrin anti human KDR  antibodies were used. For detection of SMPCs, antibodies against human CD34  and PDGF receptor  were used. FITC- or PE-conjugated isotype control antibodies  from the same manufacturer served as controls. Each analysis included at least 10.000 events within the lymphocyte gate. Data were processed using Cellquest software .EPCs were quantified by FACS analysis as previously described , 21. BriBiochrom AG) at 37°C for 2h to detect the uptake of Dil-Ac-LDL. Samples were viewed with an inverted fluorescent microscope (Leica). Dual-stainined cells positive for both lectin and Dil-Ac-LDL were judged as EPCs.For further phenotype confirmation, cultivated EPCs were fixed using 2% paraformaldehyde for 10 minutes and stained with FITC-labelled lectin Ulex europaeus agglutinin I  for 1h. Cells were incubated with 1,19–dioctadecyl–3,3,39,39–tetramethyl indocarbocyanine–labelled acetylated low-density lipoprotein (Dil-Ac-LDL)  in Endothelial Cell Growth Medium MV2 Kit (PromoCell) and incubated at 37°C. After 30 minutes incubation time, the supernatant was carefully discarded and the wells were washed 3 times with phosphate buffered saline (PBS) (Biochrom AG). EPC adhesion was quantified using the fluorescence based CyQuant Assay Kit (Invitrogen) according to the manufacturer’s instructions. In addition, data was normalised on whole blood EPC concentration.t-test (two-sided) and ANOVA for multiple comparisons with normally distributed variables followed by Fischer’s LSD test. A point biserial correlation was used to analyse the association between progenitor cells and cardiovascular risk factors. A multivariate analysis of variance was performed to evaluate the risk factors for CAD. P values less than 0.05 were considered as statistically significant. All statistical analyses were performed with Prism .Data are expressed as mean ± standard error of the mean (SEM). Comparisons between groups were analysed by 105 consecutive cases  were included. All patients have been under statin therapy for at least four weeks prior to PCI. Baseline characteristics including LDL levels were comparable .No correlation was found between EPC or SMPC count, respectively, and cardiovascular risk factors (r from -0.21 to 0.19) . 58 patiThere was no significant difference between males and females regarding EPC counts  and SMPC counts  .Under HIST versus LIST and ACS versus CCS , respectIn the clinical context of ACS, patients receiving HIST compared to LIST showed significantly decreased counts of absolute  and relative EPC numbers . Similarly, in the clinical context of CCS, patients receiving HIST revealed significantly lower EPC counts compared to patients receiving LIST both in relative  and absolute numbers  .+ KDR+ EPC under HIST compared to LIST . In cases treated with HIST the percentage of CD34+ KDR+ EPC was lower in ACS compared to CCS   .In ACS patients, EPC adhesion capacity was more pronounced under LIST compared than HIST . Moreover, under HIST, a weaker capacity to adhere was observed in ACS compared to CCS , while under LIST, adhesion capacity was markedly higher in ACS compared to CCS patients  .Both ACS and CCS patients exhibited decreased numbers of absolute  and relative  SMPC counts under HIST compared to LIST .Similarly, irrespective of the clinical context, detection of SMPC surface markers revealed lower percentages of CD34/PDGFR positive cells under HIST compared to LIST  .In patients undergoing PCI for ACS versus CCS treatment, the present study revealed an inverse correlation between statin treatment intensity and circulating EPC counts. In patients treated with HIST levels of circulating EPCs were significantly reduced; this effect was more pronounced in ACS than CCS patients. In the context of ACS, although the higher counts of EPCs in patients with LIST might be explained in part by an increase in the number of released PBMCs, EPC counts remained significantly higher compared to HIST after adjusting for mononuclear cells which might be explained by a HIST-facilitated maturation, migration and incorporation at sites of injury in the context of PCI. Moreover, both in ACS and CCS patients, HIST caused a significant reduction of the number of circulating SMPCs.Statins are effectively used drugs in the treatment of CAD and several trials described pleiotropic effects including an anti-thrombotic effect and an improvement of endothelial function independent of the reduction of LDL-cholesterol –24. In tThe functional capacity of bone marrow mononuclear cells was described to be significantly reduced in patients with CAD . In patiAvailable data on pleiotropic effects of statins on EPCs are not restricted to mobilisation and homing to sites of vascular injury but include reports on statin-facilitated maturation and differentiation of EPCs , 21.As the present study included exclusively patients with coronary syndrome undergoing PCI, iatrogenic vascular injury due stent induced endothelial denudation and endovascular laceration with subsequent vascular dysfunction is the major pathophysiological correlate in this population . Bone maThe studied population markedly differs in clinical characteristics and context from previous studies. Thus, during the pathway of EPC-facilitated improvement of coronary stent healing, the ability of EPCs to maturate, incorporate, and differentiate might be more crucial than their circulating numbers after mobilisation from the bone marrow in the studied population. Conclusively, we hypothesize that in the present PCI patient population, the high-potent statin dosages ultimately might improve the healing of stented coronary lesions by promoting maturation, incorporation, and differentiation of mobilized EPCs at coronary lesions that, in turn, might contribute to depletion of circulating EPC numbers.Multiple studies have reported an association between cardiovascular risk factors and number of EPCs showing decreased counts in patients with diabetes, hypertension, hyperlipidaemia or smokers , 21, 29.By increased integrin expression, statins lead to an improvement of the migratory and adhesive activity of EPCs by upregulating the telomere-binding factor TRF2 in EPCs . At the Although the mechanisms of statin therapy impact on SMPC are still unclear, our findings demonstrate a potentially beneficial pleiotropic effect of statin therapy in this specific patient subset. For the first time, SMPC levels were quantified on a larger cluster of patients with distinctive statin treatment and documented CAD. Both in ACS and CCS patients, HIST caused a significant reduction of the number of circulating SMPCs. Considering the fact that SMPC are involved in the progression of atherosclerosis after vascular injury , 36, cirDespite an increasing body of evidence on the efficacy of anti-inflammatory drugs for the treatment of CV disease, the current study did not include the analysis of a potential association between inflammatory markers and progenitor cells. Taking into consideration the substantial iatrogenic vascular injury during PCI, this aspect might be of crucial impact. As the present study aimed to determine the immediate changes after PCI associated with statin treatment, repeated sampling at later time points after PCI was not performed.In summary, our results reveal a dose intensity-dependent and LDL level-independent pro-differentiating vasoprotective pleiotropic capacity of statins for EPCs in patients undergoing PCI. The observed reduction of EPCs was more pronounced in ACS than in CCS patients and might be explained by a HIST-facilitated maturation, migration and incorporation at sites of injury in the context of PCI. The present findings concerning statin therapy interaction with SPMCs might point towards at potential pathway of HIST mediated restenosis suppression and endogenous regenerative statin effect after MI. These findings could potentially help to optimize drug treatment strategies in patients with CAD by distinct statin dosage and timing to improve endothelial function and plaque morphology and ultimately, clinical outcome.S1 FigPearson r correlation.(PDF)Click here for additional data file.S2 FigNo significant difference was observed between genders on EPC and SMPC counts.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Allogeneic hematopoietic cell transplant  for eligible patients with acute myeloid leukemia (AML) in first complete remission is a central treatment paradigm to achieve durable remission. However, disease relapse after allo-HCT remains a significant concern and generally portends a poor prognosis. There is significant interest regarding the role for maintenance therapy after allo-HCT for patients with high risk of relapse, regardless of the presence of measurable residual disease. While there are currently no therapies approved for maintenance therapy for AML after allo-HCT, there are a number of ongoing investigations examining the role of maintenance therapies that include targeted agents against FLT3-ITD or IDH mutations, hypomethylating agents, immunomodulatory therapies and cellular therapies. In this review, we examine the current landscape and future strategies for maintenance therapy for AML after allo-HCT. Allogeneic hematopoietic cell transplant  for eligible patients with AML in first complete remission is a central treatment paradigm to achieve durable remission for many patients. However, disease relapse remains the leading cause of long-term failure after allo-HCT, and the prognosis after relapse is dismal. With the advent of less toxic or targeted therapies for AML, significant interest has emerged regarding the role of maintenance therapy after allo-HCT to improve overall outcomes by preventing disease relapse.Historically, therapy after allo-HCT was difficult to administer due to the reluctance to cause cytopenias, immunosuppression or toxicity in the period immediately after recovery from the peri-allo-HCT period. With the advent of novel agents aimed at targetable mutations and significantly more tolerable chemotherapeutics, maintenance therapy given after allo-HCT has become a more feasible approach. However, the potential benefits of additional chemotherapeutic agents must be balanced against the costs to patients, specifically myelosuppressive and other adverse side effects/toxicities, impaired engraftment, encouragement of graft versus host disease (GVHD) through immunomodulation and overall impact on quality of life. Currently, the role of maintenance therapy after allo-HCT is debated in the field , 2, and Generally, the standard of care for patients after allo-HCT in remission has been close monitoring for relapse with individualized treatment approaches towards management of immunosuppression for each patient based on the perceived risk of relapse. The benefit of this approach is that patients who do not relapse are not encumbered by any effects from additional chemotherapeutics after transplant. However, the obvious downside is that up to 40% of patients with AML experience relapse after allo-HCT, and these patients face an extremely poor prognosis with historic 2-year overall survival (OS) rates of < 20% .As a result, there has been great interest in preventative treatment strategies after allo-HCT in patients with high risk of relapse including pre-emptive and maintenance therapy strategies. Pre-emptive therapy is generally understood as a treatment strategy where patients are monitored closely for evidence of measurable residual disease (MRD). At the time of detectable MRD, patients are started on treatment with the hopes to prevent frank morphologic relapse and subsequent complications. The benefit of this approach is that treatment is targeted to patients where there is evidence of impending relapse; however, a downside of waiting to treat until MRD is detected is that kinetics of the disease may prevent any intervention from being effective. Moreover, MRD testing in AML is nowhere near standard for every patient and, oftentimes, depends on local assays for specific mutations. Alternatively, a maintenance therapy approach, the focus of this review, is when treatment is initiated for patients in remission after transplant regardless of MRD status. Conceptually, the ideal maintenance therapy is convenient for patients to receive, able to be initiated soon after transplant for maximal efficacy, does not impair donor hematopoietic engraftment, and does not induce transplant related adverse effects, specifically GVHD.Around 20% of patients with AML harbor FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations which have traditionally portended a high risk of relapse and death despite allo-HCT . Not surWe published the first such trial – a phase I trial using sorafenib as maintenance therapy  in 22 paSubsequently, two randomized controlled trials have validated the efficacy of maintenance sorafenib for FLT3-ITD AML. The SORMAIN trail was a multicenter double-blind placebo-controlled trial in Germany and Austria that randomized 83 patients to sorafenib maintenance vs. placebo for 2 years after allo-HCT . The triXuan et al. recently published a multicenter open-label phase 3 randomized trial of 202 patients in China comparing sorafenib maintenance therapy for only 6 months to no intervention . They foHowever, limitations of these studies need to be acknowledged. This includes that the SORMAIN trial was prematurely closed due to challenges in recruiting patients , and a significant number of patients in the study had to discontinue sorafenib due to toxicity. Xuan et al. only studied sorafenib maintenance for 6 months, and most patients required dose reduction. The biggest issue is that the efficacy of FLT3 maintenance therapy in patients who received FLT3 inhibitors (such as midostaurin) prior to allo-HCT is still unknown; only a minority of patients in both of these studies received FLT3 TKI before allo-HCT. Moreover, there was very limited analysis of the impact of MRD on outcomes and its interaction with the use of maintenance TKI. Lastly, it is unclear what the optimal duration of FLT3 TKI maintenance therapy should be given similar benefits observed in all studies to date.Midostaurin, which is now approved in combination with induction and consolidation chemotherapy for patients with FLT3 mutated AML, has been studied as a maintenance therapy in a small open-label phase 2 randomized trial. The RADIUS trial randomized 60 patients to midostaurin versus standard of care for up to 1 year after transplant for FLT3-ITD AML . This stWhile it is presumed the efficacy seen in the sorafenib maintenance trials is driven by on-target effect of FLT3 inhibition, there is evidence that suggests sorafenib may also enhance graft-vs-leukemia activity, perhaps through an autocrine mechanism that increases IL-15 and more active cytotoxic T cells . It doesOverall, there is mounting evidence of the safety and efficacy of FLT3 targeted maintenance therapy, particularly for the use of sorafenib. Based on the strength of the prospective randomized clinical trials and previous retrospective trials, maintenance FLT3 TKI after allo-HCT has become the standard of care at many centers with the specific choice of TKI dependent upon access and availability .Mutations in either IDH1 or IDH2 are found in approximately 20% of patients with AML, and a recent multicenter retrospective analysis suggested that 2 year relapse rates after allo-HCT are 31% and 25% for IDH1 and IDH2 mutated AML, respectively . EnasideLarger prospective studies investigating the efficacy of IDH1/2 inhibitors as maintenance after allo-HCT will be necessary to truly understand the efficacy of using these agents to prevent relapse. Complicating issues include off-trial use due to commercial access, the relatively low incidence of each mutation and common co-mutations including FLT3-ITD.There is considerable interest in using hypomethylating agents (HMA) such as azacitidine and decitabine as maintenance therapy, although there is limited prospective evidence regarding their efficacy in this setting. HMA, which induce global hypomethylation/epigenetic modifications, have likely several mechanisms of anti-leukemic effect including direct leukemic cell cycle control and potential immunomodulatory effects , 18. HMANot surprisingly, many of the early maintenance studies have been challenging to interpret given small sample sizes, heterogeneous risk of relapse and lack of a prospective control group . A largeThe recent development of effective oral azacitidine (CC-486) and decitabine  formulations has made this class of agents significantly more attractive and feasible as a post allo-HCT maintenance regimen . There aIn addition, as other agents have been combined with HMA in the non-transplant setting, there are ongoing maintenance therapy trials investigating azacitidine in combination with other agents such as the bcl-2 inhibitor venetoclax , novel agents such as eprenetapopt (APR-246), which shows promise for TP53 mutated myeloid malignancies (NCT03931291), and in concert with donor lymphocyte infusions.Glasdegib, a small molecule inhibitor of sonic hedgehog (shh), has recently been approved by the FDA as part of induction therapy for patients with AML older than 75 who are unfit for an intensive induction regimen. It was recently studied as maintenance therapy in a single-arm phase 2 pilot study of 31 patients with AML or MDS after allo-HCT . ResultsVenetoclax is a selective small molecule inhibitor of the B-cell lymphoma-2 (Bcl-2) protein, a key regulator of apoptosis. A recent phase 1 feasibility trial of 23 patients post allo-HCT (high risk AML and MDS), showed that venetoclax was tolerable for up to 1 year for most patients, although 11% of patients discontinued due to adverse effects or other transplant complications and half required dose interruption and adjustments due to side effects including cytopenias and GI related side-effects . As ment+ AML, and there has been some interest in its use as post-transplant maintenance therapy. The main concerns of using GO after allo-HCT are the incidence of cytopenias and its known association with hepatic veno-occlusive disease (VOD)/sinusoidal obstruction syndrome (SOS) (CD33 is a myeloid differentiation antigen that is highly expressed in AML and is the target of certain antibody drug conjugates such as gemtuzumab ozogamacin (GO). GO has been approved as part of upfront standard induction chemotherapy for CD33me (SOS) . There hme (SOS) ] in combme (SOS) . Larger Histone deacetylase (HDAC) inhibitors are thought to possess anti-leukemic activity through epigenetic modifications and its theorized immunomodulatory activity may help graft vs. leukemia (GVL) and mitigate GVHD after allo-HCT. A phase 1/2 trial examined the use of panobinostat after allo-HCT for 42 patients with AML or MDS . The majThe use of lenalidomide for its immunomodulatory properties has been investigated for AML after allo-HCT. The exact mechanism of action is unknown but lenalidomide is thought to promote natural killer and cytotoxic T cell activity as well as inhibit regulatory T cells after transplant. There have been previous safety concerns with lenalidomide inducing GVHD as observed in patients with high-risk multiple myeloma after allo-HCT , and a pin vivo T-cell depleted allografts. These characteristics as well as the setting being therapy for relapsed disease may explain the relatively low rates of lenalidomide induced GVHD that may not translate if lenalidomide is used as an upfront maintenance agent even with concurrent HMA therapy.An approach of using azactidine with lenalidomide in 29 patients with relapsed AML/MDS after allo-HCT had relatively low rates of GVHD, where only 3 patients developed GVHD . HoweverWhile there has been interest in the use of immune checkpoint inhibitors (CPI) for relapsed disease for enhanced GVL effect, there is considerable risk due to potential immune related adverse effects, specifically increased rates of GVHD. A phase 1 trial of 28 patients examining the use of ipilimumab to treat relapsed disease in a variety of hematologic malignancies after allo-HCT found that 5 patients had a complete response and 2 had a partial response. Notably, 4 of the patients with complete response had extramedullary AML . HoweverFor decades, there have been investigations using donor lymphocyte infusion (DLI) to treat patients with relapsed disease. In theory, DLI provides non-tolerant donor T cells which may enhance the GVL effect but at the potential cost of causing GVHD. The best data in support of the use of DLI has been its use as a salvage treatment to induce complete remission for patients with indolent malignancies such as relapsed chronic phase CML. The otherwise limited utility of DLI as primary therapy for frankly relapsed disease, particularly its limitations in the treatment of relapsed AML, has been reviewed elsewhere .Prophylactic DLI as maintenance therapy and pre-emptive DLI in the setting of MRD or mixed chimerism has remained of interest. One prospective study of low-dose prophylactic DLI administered soon after allo-HCT examined 15 patients with AML and acute lymphocytic leukemia (ALL) treated with alemtuzumab-based conditioning . While tA more recent registry-based survey from the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation examined the use of DLI in the preemptive setting for MRD or mixed chimerism or prophylactically for high-risk patients . This stThere is clearly a need for collaborative prospective trials on prophylactic or preemptive DLI after allo-HCT to truly elucidate a treatment effect. Certainly, more homogeneity in the transplant platform and state of immune reconstitution / immunosuppression at time of DLI administration would be preferred. Not surprisingly, there has been interest in investigating the combination of DLI with immunomodulatory agents as maintenance therapy including a recent phase 2 trial investigating prophylactic low dose azacitidine and DLI in high-risk AML/MDS .in vivo anti-leukemic effects were noted in the relapsed disease cohort with 2 patients showing partial or complete response.While CAR T-cell therapy has emerged as a major modality of therapy for certain B-cell malignancies, there has been limited utility in the treatment of AML, largely due to a lack of AML-specific targets . While tNatural killer (NK) cells are thought to play a role in the immunologic control of AML and the GVL response after allo-HCT, and there is growing interest in adoptive NK cell-based therapy to prevent relapse after transplant. After transplantation, NK cells are the earliest lymphocytes to engraft, and there is evidence that high donor NK cell chimerism at 14 days is associated with lower relapse rate. In addition, it has long been observed that donor NK cell alloreactivity mediated through killer-cell immunoglobulin-like receptors (KIRs) ligand  has also been associated with reduced risk of relapse and increased survival , though Active trials investigating adoptive NK cell-based therapy have broadly been characterized as trials of NK cell enriched DLI and the administration of cytokine-induced activated NK cells. One recent phase 1/2 study of 25 patients with myeloid malignancies undergoing haploidentical transplant examined the safety and feasibility of ex-vivo expansion of donor derived NK cells which was then delivered on days -2, +7 and +28 . This stVaccines that prime the immune system against leukemic cells have been an area of active interest for patients with active disease and in remission. Both peptide-based and whole tumor cell-based vaccines have been developed for AML , althougAlternative vaccine approaches that use whole tumor cells as a source of antigens have also been explored. This includes gene-transduced tumor cell vaccine (GVAX), where leukemic cells are manipulated to express GM-CSF, and dendritic cell/AML fusion vaccines, where donor-derived dendritic cells are fused with patient-derived AML cells. A phase 1 study examining GVAX for 15 patients with AML/MDS demonstrated safety of the approach with comparable rates of GVHD compared to a historical control with a 2-year Kaplan-Meier estimate of OS of 57%. Results from a phase 2 trial were recently published by the same group, which randomized 57 patients with AML/MDS to GVAX versus placebo after allo-HCT. GVAX was well tolerated with only injection site reactions noted as adverse events; however, no difference in overall survival or progression free survival were observed in GVAX recipients compared to placebo . ResultsOver the last decade, there has been considerable interest in the use of maintenance therapy for patients with AML after allo-HCT to potentially reduce the significant risk of disease relapse. The challenges in conducting clinical trials in the allo-HCT maintenance setting are numerous. First, patients have significant competing risks including cytopenias, conditioning regimen induced organ toxicity, opportunistic infection and GVHD. Second, while drugs may be approved in other contexts with well understood side effects, the adverse effects may be more difficult to predict in the post-transplant immunological environment, including the potential impact on GVHD, which necessitates first conducting thoughtful safety studies. Third, recruitment of patients for treatments with the potential for significant adverse effects may be challenging when there is no active disease. Lastly, for therapies that have been approved for other indications and there is off-label commercial access, there may be significant off-study use with patients and treating physicians being reluctant to participate in a randomized controlled trial.Currently, the only randomized clinical trial data supporting the use of maintenance therapy is for FLT3 inhibitors in patients with FLT3-ITD AML. The recent publication of two randomized trials that demonstrated the significant benefit in RFS and OS for sorafenib maintenance therapy for FLT3-ITD AML has generated significant excitement in the field. Outside of FLT3-ITD AML, there appears to be only limited phase 3 trials in progress. Most notably, the results of the AMADEUS trial, examining oral azacitidine (CC-486), will be of significant interest to follow. Perhaps surprisingly, while CC-486 has been FDA approved for maintenance therapy for patients who are not candidates for allo-HCT, the larger clinical trials studying HMA maintenance post allo-HCT have not demonstrated significant benefits to date. The use of oral therapy should hopefully improve adherence and tolerability, which seems to have been a major limitation of prior trials where a low proportion of patients actually completed the treatment per protocol for various reasons. In addition, results from a German phase 3 trial investigating the HDAC inhibitor panobinostat are eagerly anticipated. Given the potential immunomodulatory effects with both HMA and HDAC inhibitors, attention to adverse events such as GVHD will be important to follow in addition to effects on disease relapse.As more specifically targeted agents for AML are developed, trials of maintenance therapy targeting genomically defined subsets will only increase. To truly define the efficacy of such approaches, large collaborative efforts will be essential given the relatively low incidence of such mutations. For example, investigation on the use of IDH1/2 inhibitors as maintenance therapy for IDH1/2 mutated AML remains in very early stages with only phase 1 trials to date. While both enasidenib and ivosidenib appear to be well tolerated in the post allo-HCT setting, larger randomized trials designed to assess efficacy are sorely needed.While not explored in detail in this review, it should be mentioned that disease-specific serial MRD testing will play an increasingly important role in stratifying patients for the highest risk of relapse –59. UtilAllo-HCT for eligible patients with acute myeloid leukemia in first complete remission remains a central treatment paradigm to achieve sustained remission. Nevertheless, relapse after transplant remains the leading cause of long-term failure. Over the last decade, intense interest has emerged as to whether maintenance therapy after allo-HCT can reduce relapse and improve post-transplant survival. Here, we have reviewed the principles of this treatment paradigm and the most recent advances including ongoing trials. The most promising data supporting maintenance therapy is for FLT3-TKI after allo-HCT for patients with FLT3-ITD AML and this has become the standard of care at many institutions. There are numerous other promising agents, such as other genomically targeted agents, HMA, immunomodulatory therapies, and cellular therapies, although well-designed clinical trials need to be conducted. Toxicity (chemotherapeutic cytotoxicity vs. immunologic cytotoxicity/GVHD) is the limiting factor for many of these approaches, as many treatments are repurposed therapies from the relapsed/refractory disease setting and may not be acceptable for patients without active disease. In the coming years, clinical trial results for gilteritinib, IDH inhibitors, oral azacitidine, azacitidine and venetoclax, panobinostat, and various cellular therapy approaches are expected to greatly improve our understanding of the role of maintenance therapy for AML after allo-HCT.RN and Y-BC conceived of the topic, designed and wrote the manuscript. All authors contributed to the article and approved the submitted version.YBC has served as a consultant for Novartis, Incyte, Jasper, Celularity, Equilium, Actinium, Daiichi and Abbvie.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ORF22 or ORF29b, both important for virus replication. Repetitive sequences were additionally inserted, allowing the removal of the F-derived sequences from the viral DNA genome upon reconstitution in permissive epithelial cells. Moreover, these self-repair-enabled BACs were used to generate deletion variants of the transforming strain C488 in order to minimalize the virus genome. Using the en passant mutagenesis with two subsequent homologous recombination steps, the BAC was seamlessly manipulated. To ensure the replication capacity in permissive monkey cells, replication kinetics for all generated virus variants were documented. HVS variants with increased insert capacity reached the self-repair within two to three passages in permissive epithelial cells. The seamless deletion of ORFs 3/21, 12–14, 16 or 71 did not abolish replication competence. Apoptosis induction did not seem to be altered in human T cells transformed with deletion variants lacking ORF16 or ORF71. These virus variants form an important step towards creating a potential minimal virus vector for gene therapy, for example, in human T cells.Herpesvirus saimiri (HVS) is discussed as a possible vector in gene therapy. In order to create a self-repairing HVS vector, the F plasmid vector moiety of the bacterial artificial chromosome (BAC) was transposed via Red recombination into the virus genes  While its natural host, the squirrel monkey Saimiri sciureus, does not show symptoms of infection, other species suffer from lymphoproliferative disorders upon experimental infection ..56]. Stiiderably . HoweverIn the future, the combined deletion of the above-mentioned genes in one single vector might provide an optimized minimal vector. From our experiments and the literature, up to 50 kb of insert capacity within a minimal variant HVS vector seem feasible. With regard to the notion that gene silencing in a viral vector is best avoided by using the original cellular promoter of the specific transgene, such an increase in insert capacity might be an important contribution to the vector development .


After a very successful year in 2019 with 34 publications, our Topic collection “Drug Resistance and Novel Therapies in Cancers” guaranteed another productive year with the publication of 17 research articles and 4 review articles in 2020. These research articles covered many different types of cancers, including colorectal cancer (CRC) , epidermThe first research article of the year focused on the effects of sitravatinib, a novel multitargeted receptor tyrosine kinase inhibitor, in reversing multidrug resistance (MDR) . In thisIn a search for effective therapeutic agents treating gastric cancer (GC), Ke et al. examine the efficacy of a known cancer drug, tenovin-6, in treating GC . They foCarbon-ion radiotherapy is a highly advanced form radiotherapeutic treatment and is frequently used to treat osteosarcoma (OSA). Although this treatment has yielded promising results, the prognosis of OSA still remains unsatisfactory. In a study published in this collection, scientists from Korea and Japan joined hands to test the efficacy of carbon-ion beam irradiation, both alone and in combination with zoledronic acid (ZOL) on OSA cells . ZOL is Elegant research by Czech scientists uncovered novel mechanisms underlying the mechanism of nelfinavir in overcoming resistance to multiple myeloma . ProteasAnother article we promoted aimed to repurposing erdafitinib for the treatment of cancers with multidrug resistance. Erdafitinib is the first fibroblast growth factor receptor (FGFR) kinase inhibitor to be approved by the FDA. They observed that erdafitinib significantly re-sensitizes ABCB1-overexpressing multidrug-resistant cancer cells by inhibiting ABCB1 . Recent Cisplatin is a widely used and important anticancer drug. However, it is well-known that cisplatin causes renal salt and water-wasting syndrome (RSWS) as a side effect. The origin of RSWS is obscure. In a study published here, the authors indicate that cisplatin induces RSWS by inhibiting the epithelial Na+ channel . AnotherThis collection also published four review articles in 2020. One review article was focused on cannabinoids and its application in cancer therapy . Cannabi


A growing body of literature has increased our understanding of interpersonal autonomic synchrony (IAS), or exchanges across biological systems resulting in physiological covariation. While research suggests that IAS is more likely in relationships characterized by emotional intimacy, no research to date has examined the connection between IAS and the quality of sexual relationships.The current study seeks to elucidate the relationship between IAS and sexual satisfaction (SS) using tasks that have previously been used to assess synchrony , as well as several conversation-based tasks .Couples (n=28) completed procedures in a laboratory-based setting where they completed survey measures of SS before connecting to an electrocardiogram. Subsequently, heart rate (HR) data for each dyad were analyzed using a moderated multi-level modeling approach.IAS was detected, with men reliably predicting the HR of female partners, and women reliably predicting the HR of male partners . AIC values indicate a better fitting model for men predicting female HR. As such, moderation analyses were conducted for that model by study task, finding a significant interaction between SS and observed IAS during the mirroring task  and neutral-conversation task .These findings reflect evidence that IAS may be relevant to SS at the couple-level. The ability of couples to coregulate while attempting to synchronize (as in the mirroring task) or exchange information  may meaningfully change how they experience their sexual relationship.No significant relationships.


Papilledema is the main ocular finding in patients with idiopathic intracranial hypertension (IIH) although several chorioretinal abnormalities may also occur and contribute to visual loss. The purpose of this paper is to describe two cases of chorioretinal abnormalities associated with idiopathic intracranial hypertension: one with choroidal folds and another with polypoidal choroidal vasculopathy, an extremely unusual ocular complication in the disease.Case 1: A 47-year-old woman previous diagnosed with idiopathic intracranial hypertension treated with weight loss and acetazolamide that over the following 6 months had optic disc edema gradually resolved. The patient was follow-up for a period of 10 years and the papilledema disappeared, but choroidal folds remained unchanged. Case 2: A 61-year-old female patient was seen as a follow-up examination of a 5-year history of IIH that presented with papilledema. The patient was asymptomatic but fundoscopy evaluation revealed a yellowish white peripapillary subretinal nodular lesion temporally in OD. Multimodal imaging studies were made, and the patient was diagnosed with a rare and just recent described association of IIH and polypoidal choroidal vasculopathy.Papilledema, RNFL and retinal ganglion cell loss are the most common structural complications of IIH, but chorioretinal complications are important findings and should be carefully evaluated in such patients. Awareness of such occurrence and the use of appropriated clinical and multimodal imaging studies are of great importance for its early detection, leading to proper treatment and prevention of further visual loss. Idiopathic intracranial hypertension (IIH) is characterized by signs and symptoms of increased intracranial pressure of unknown etiology. Headache, transient visual obscuration, pulsatile tinnitus, and neck pain are the most typical symptoms although uncommon presentations can also occur , 2. The 2O in lateral decubitus) with normal cerebral spinal fluid (CSF) composition; (ii) normal brain parenchyma without hydrocephalus, mass or structural lesion and no meningeal enhancement or venous sinus thrombosis seen on neuroimaging; (iii) clinical evidence of papilledema with normal neurological examination except for sixth nerve palsy [The diagnosis of IIH is based on the modified Dandy criteria and, in addition to clinical symptoms, include: (i) an elevated lumbar puncture opening pressure  and retinal ganglion cells. Consequently, chronic IIH can produce an array of visual field (VF) abnormalities, including blind spot enlargement, inferior nasal depression, and generalized VF constriction . TherefoIn addition to papilledema and retinal neural loss, IIH may also be associated with several secondary retinal lesions, such as macular exudates, retinal hemorrhages, subretinal neovascular membrane, central retinal vein occlusion, and retinal folds . ChoroidThe purpose of the present paper is to describe two cases of choroidal abnormalities associated with IIH and to review chorioretinal findings associated with this disease. We also first report a multimodal imaging analysis of PCV in a patient with IIH, including the use of optical coherence tomography angiography (OCT-A).2O) with normal biochemical and cytological analysis.A 47-year-old woman complained over the last 3 months of occasional flashes and glare in the temporal side of both eyes (OU), lasting for a few seconds. Past medical history was significant only for obesity  and hyperopia OU. On examination, best-corrected visual acuity (VA) was 20/25 in the right eye  and 20/25 in the left eye . Pupillary examination, extraocular movements, and slit lamp examination were unremarkable. Fundoscopy revealed a marked papilledema and choroidal folds extending from the optic disc to the macula in OU. Standard automated perimetry revealed an enlarged blind spot and nasal sensitivity loss OU. Magnetic resonance imaging (MRI) and magnetic resonance venography (MRV) of the brain only demonstrated an empty sella turcica. CSF opening pressure revealed raised intracranial pressure  with normal CSF composition, normal MRI and MRV. At the diagnosis, she denied visual or systemic complains. Past medical history was significant for systemic arterial hypertension and grade II obesity at presentation. Treatment was accomplished with significant weight loss and oral acetazolamide leading to complete resolution of papilledema over the following 2 years.A 61-year-old female patient was seen as a follow-up examination of a 5-year history of IIH that presented with papilledema, elevated intracranial pressure  in OU and a yellowish white peripapillary subretinal nodular lesion temporally in OD (Fig. Neurologic examination and extensive laboratory investigation were normal. A diagnosis of a peripapillary PCV associated with IIH was made. A closer follow up was adopted without further treatment since no signs of active PCV or IIH were detected.Retinal lesions are important causes of vision loss in IIH. Subretinal neovascular membrane with subretinal fluid and hemorrhage are the most common chorioretinal complication in IIH associated with visual loss , 9. MacuThe cases here presented exemplify one common and another extremely unusual choroidal abnormality that may occur in patients with IIH. Choroidal folds, as shown in our first case, is the most common choroidal manifestation associated with papilledema in IIH and frequently persists after resolution of papilledema and normalization of the intracranial pressure , as obseIt is also important to consider that choroidal abnormalities in a scenario of raised intracranial pressure may not only involves structural changes, exemplified as wrinkles and folds. Angiogenic stimulus in a hypoxically choroidal environment are reported in chronic IIH and neovascularization can occur . PeripapPCV is a vascular disease of the choroid first described in 1990 . The polAs seen in our second case, detecting chorioretinal complications in IIH depends in part of multimodal analysis. OCT, OCT-A, fluoresceine angiography and ICGA are useful diagnostic modalities that help clinician to detect alterations not seen on retinography and ophthalmic examination. Besides it’s noninvasive nature, OCT and OCT-A has been widely used to access optic neuropathy. RNFL thickness measurement is helpful in quantifying changes in papilledema, even in the milder ones . GanglioIn conclusion, papilledema, RNFL and retinal ganglion cell loss are the most common structural complications of IIH, but chorioretinal complications are important findings and should be carefully evaluated in such patients. Awareness of such occurrence and the use of appropriated clinical and multimodal imaging studies are of great importance for its early detection, leading to proper treatment and prevention of further visual loss.


Triple Negative Breast Cancer (TNBC) is a molecularly complex and heterogeneous subtype of breast cancer, characterized by the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. TNBCs are often associated with an increased risk of metastasis and recurrence, however, the molecular mechanisms underlying TNBC metastasis and recurrence remail unclear. In this study, we present our findings of massively parallel RNA sequencing used to compare global gene expression profiles of primary tumors and their matched metastatic lesions. Our results shed light on the diverse genetic mechanisms underlying TNBC metastases and may provide potentially actionable therapeutic targets. n = 31) and the Stavanger cohort (n = 5). Among the 31 patients in the Zurich cohort, 18 had primary TNBC tumors that did not metastasize, and 13 had primary tumors that metastasized . The Stavanger cohort included five matched primary and metastatic TNBC tumors. Significantly differentially expressed genes  were identified and subjected to functional analyses. We investigated if there was any overlap between DEGs from both the cohorts with epithelial-to-mesenchymal-to-amoeboid transition (EMAT) gene signature. xCell was used to estimate relative fractions of 64 immune and stromal cell types in each RNA-seq sample. Results: In the Zurich cohort, we identified 1624 DEGs between primary TNBC tumors and matched metastatic lesions. xCell analysis revealed a significantly higher immune scores for metastatic lesions compared to paired primary tumors in the Zurich cohort. We also found significant upregulation of three MammaPrint signature genes  in primary tumors that metastasized compared to primary tumors that remained metastasis-free. In the Stavanger cohort, we identified 818 DEGs between primary tumors and matched metastatic lesions. No significant differences in xCell immune scores were observed. We found that 21 and 14 DEGs from Zurich and Stavanger cohort, respectively, overlapped with the EMAT gene signature. In both cohorts, genes belonging to the MMP, FGF, and PDGFR families were upregulated in primary tumors compared to matched metastatic lesions. Conclusions: Our results suggest that distinct gene expression patterns exist between primary TNBCs and matched metastatic tumors. Further studies are warranted to explore whether these discrete expression profiles underlie or result from disease status. Background: Although triple-negative breast cancer (TNBC) is associated with an increased risk of recurrence and metastasis, the molecular mechanisms underlying metastasis in TNBC remain unknown. To identify transcriptional changes and genes regulating metastatic progression in TNBC, we compared the transcriptomic profiles of primary and matched metastatic tumors using massively parallel RNA sequencing. Methods: We performed gene expression profiling using formalin-fixed paraffin-embedded (FFPE) TNBC tissues of patients from two cohorts: the Zurich cohort ( Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancers and exhibits a unique molecular profile ,4. The hMetastasis involves the dissemination and spread of cancer cells from the primary tumor site to distant organs, resulting in the formation of treatment-resistant clones capable of proliferation in multiple locations and hindering the success of the treatment ,7,8. SucThe aim of this study was to identify genes and pathways regulating the metastatic progression of TNBC and to compare the global gene expression between primary tumors and their matched metastatic lesions. Understanding the molecular alterations associated with metastatic progression may help identify actionable therapeutic targets in TNBC, which are urgently needed.Formalin-fixed paraffin-embedded (FFPE) primary and matched metastatic tumor tissues from 36 patients with TNBC were obtained from the University Hospital Zurich, Switzerland, and Stavanger University Hospital, Stavanger, Norway . The ZurHematoxylin and eosin (H&E) slides were prepared for all samples, and the tumor content was assessed by a pathologist. Total RNA was extracted from FFPE tissue sections using a NucleoSpin total RNA FFPE kit . RNA samples were subjected to optical density measurements using NanoDrop and Qubit . RNA purity and concentration were determined using 2200 Tapestation . RNA integrity number (RIN) according to Agilent 2100 assays depend on sample type and quality. In general, quality of RNA extracted from FFPE samples is poor. Typical RIN of RNA isolated with NucleoSpin RNA FFPE kits  are in the range of 2–6. The RIN of total RNA isolated from FFPEs for most of the samples (87%) was 2 or above and for a few samples was ~2 . Illuminp-value to <0.05. Significant DEGs were used to generate heatmaps and volcano plots. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to predict the biological roles of the DEGs. This analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8, which is a web-based functional annotation tool. We used web-based Venny 2.1 tool to generate the Venn diagrams [Raw FASTQ files were subjected to quality control analysis using FASTQC . Raw seqdiagrams . The lisdiagrams .Estimation of cell type abundance was performed using the bioinformatics tool xCell and normalized bulk RNA-seq expression data as input. xCell is a high-resolution gene-signature-based method for estimating the tumor’s immune and stromal cell composition . The relp < 0.05) between primary tumors and matched metastatic lesions in the Zurich and Stavanger cohorts, respectively. In the Zurich cohort, 253 genes were upregulated, and 1371 genes were downregulated in primary tumors compared to matched metastatic lesions  between primary tumors and metastatic or recurrent tumors in Zurich and Stavanger cohorts are shown in HRASLS, RASSF7, and TGFB3 (part of the MammaPrint assay) were significantly upregulated in metastatic primary TNBC tumors compared to non-metastatic primary tumors  (EMT plays a key role in development as well as in cancer cell invasion and metastasis . Elucida COL3A1)  that werThe tumor microenvironment (TME) is composed of malignant cells, stromal infiltrates, and immune cells. The complex interplay between these TME components can influence different tumor properties, including cancer cell invasion, metastasis, and therapy resistance ,26. To gIn this study, we compared the transcriptomic profiles of matched primary and metastatic tumors using RNA-seq to identify molecular changes and DEGs regulating metastasis in TNBC. Primary tumors and matched metastatic tumors showed distinct gene expression patterns as multiple DEGs and differentially regulated pathways and immune components were identified between the two groups. Discordance in the transcriptomic profiles of primary and matched metastatic TNBC tumors can be attributed to temporal and spatial differences between primary and metastatic lesions and the effects of neoadjuvant chemotherapy received by 4 of the 11 (~40%) patients with matched primary and locoregional recurrences (Zurich cohort); neoadjuvant chemotherapy may have led to changes in the gene expression profile of tumors in these patients. These distinct gene expression profiles may indicate a molecularly dynamic tumor adapting to a new microenvironment that supports metastatic selectivity. Comprehensive analyses integrating various omics techniques can be undertaken as further cohorts with paired primary and metastatic tumors become available. We also found genes belonging to the MMP, FGF, and PDGFR families to be commonly upregulated in both the cohorts. MMPs are known to contribute to each step of breast cancer metastatic cascade owing to their ability to cleave various non-matrix and matrix substrates ,28. It hPDGFRL, UCHL1, COL5A2, and COL3A1 that were common among all three-DEGs from the EMAT gene signature, the Zurich and the Stavanger cohort. Interestingly, all these genes have been implicated in promoting invasion and metastasis and associated with poor survival outcomes in breast cancer patients [COL3A1 were higher in primary TNBC tumors compared to lymph node metastasis [UCHL1 in promoting breast cancer progression has been well documented. It has been shown to promote breast cancer metastasis by maintaining TGF-β signaling pathway and promoting breast cancer cells invasion by activating Akt signaling [We investigated if there is any overlap between the DEGs from both the cohorts with EMAT gene signature which is implicated in considering both, the EMT and MAT continuum instead of either of the gene signatures to capture the heterogeneity of metastatic propensity. Since study by Emad et al. implicated the true clinical and prognostic significance of EMT as a driving process in cancer progression towards distant metastasis can be fully appreciated if it is complemented by the additional occurrence of MAT, a process that plays an important role in embryonic development and is similarly reawakened (as EMT) by cancers during the metastatic cascade ,31. Our patients ,33,34. Ptastasis . Role ofHRASLS, TGFB3, and RASSF7 in metastatic primary TNBC tumors compared to non-metastatic primary tumors. Interestingly, these genes are part of MammaPrint, a 70-gene signature that is used to predict the risk of recurrence and metastasis in breast cancer [TGFB3 contributes to aggressive phenotypes by promoting invasiveness, angiogenesis and creating an immunosuppressive environment [HRASLS and RASSF7, oncogenic transformation-related genes, have been implicated in contributing to at least three of the hallmarks of cancers  described by Hanahan and Weinberg [We found significant upregulation of t cancer ,22,36. Tt cancer . Specifiironment ,39. HRASWeinberg ,40. DEGsPreviously, studies have shown that the immune profile of tumor-draining nodes is predictive of survival outcomes in breast cancer patients . xCell aThere are some limitations to this study. First, even though these cohorts represent a unique resource of matched primary and metastatic tumors, their sample sizes were small. As most institutions do not routinely biopsy metastatic or recurrent tumors, obtaining matched sets of primary and metastatic or recurrent tumors is challenging. Second, the cases chosen for this study were based on sample availability, inadvertently introducing selection bias. Third, because this was a retrospective study and some FFPE samples were old, it was difficult to isolate high-quality RNA. Finally, the low number of significant DEGs in the Stavanger cohort could be attributed to the small sample size and may represent false positives.Our results demonstrate that metastatic cancer cells undergo a biologically significant transcriptomic shift upon colonization. Further studies are warranted to explore whether gene expression profiles associated with metastasis could serve as actionable therapeutic targets in TNBC.