The prognostic performance of pRiS was evaluated on two test sets  using concordance index. Patients from D2 and D3 who received either radiotherapy alone or chemoradiation were used to validate pRiS as predictive of added benefit of chemotherapy. Seven features were selected to construct pRiS, which was found to be prognostic of overall survival (OS) on univariate analysis in D2  and D3 . Chemotherapy was associated with improved OS for high-pRiS patients in D2  and D3 . In contrast, chemotherapy did not improve OS for low-pRiS patients, which indicates these patients did not derive additional benefit from chemotherapy and could be considered for treatment de-escalation. The proposed radiomic signature was prognostic of patient survival and informed benefit from chemotherapy for stage I and II HPV-associated OPSCC patients.Chemoradiation is a common therapeutic regimen for human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). However, not all patients benefit from chemotherapy, especially patients with low-risk characteristics. We aim to develop and validate a prognostic and predictive radiomic image signature (pRiS) to inform survival and chemotherapy benefit using computed tomography (CT) scans from 491 stage I and II HPV-associated OPSCC, which were divided into three cohorts D Based on data from 2015 to 2019, about 47,199 new HPV-associated cancers occurred in the United States each year, accounting for about 70% of cancers of the oropharynx1. It has been shown that patients with HPV-associated OPSCC demonstrate improved treatment response to chemoradiation and prognosis compared to patients with HPV independent OPSCC, whose tumors are much more often associated with alcohol and tobacco use2. A distinct tumor, node and metastasis (TNM) staging system for HPV-associated OPSCC patients was established in the 8th edition of the American Joint Commission on Cancer (AJCC) staging system to account for the unique clinical characteristics3. However, recent studies report that the differentiation of outcome between the AJCC 8th stage groups remains less than satisfactory5. New biomarkers to improve AJCC 8th staging in risk stratification within the HPV-associated OPSCC population is sorely needed.There has been a significant increase in the incidence of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) over the past several decades8. Skillington et al. compared the outcomes of 195 p16-positive, surgically managed OPSCC patients and found that receiving chemotherapy did not provide additional disease-free survival benefit and was associated with worse overall survival, potentially due to the lack of benefit from the additional chemotherapy in low-risk patients6. Furthermore, toxicity from the chemotherapy was also observed11. Significant deteriorations in swallowing outcome occurred in those who had chemotherapy in addition to radiotherapy9. To reduce treatment toxicity without compromising survival outcomes for HPV-associated OPSCC patients, recent clinical studies have focused on identifying patients suitable for treatment de-escalation with lower dose radiotherapy and also for potentially avoiding the use of chemotherapy without compromising survival outcome13. This highlights the need for developing a predictive biomarker to distinguish which low-risk OPSCC patients may not benefit from chemotherapy and thus for whom such toxic treatments could be avoided, without compromising patient outcomes.Previous studies have shown that using concurrent chemoradiation as definitive treatment confers lower local and regional failure rates compared with radiotherapy alone for high risk OPSCC. However, the addition of chemotherapy can cause increased treatment-related toxicity, a particular concern for patients with HPV-associated OPSCC who are at low risk for disease recurrence17. Radiomics, which seeks to identify subtle image-based attributes related to tumor phenotypes19 and prognosis21 is useful in not only identifying the presence of disease on radiographic scans but also in helping identify features that related to disease outcome and treatment response. Although a few studies have shown that machine learning based prognostic classifiers can predict molecular subtype19 and survival21 in head and neck cancer, there has been no methodology available to evaluate the predictive utilities of these approaches for chemotherapy benefit among HPV-associated OPSCC patients. Those patients with low risk of disease recurrence (AJCC 8th edition stage I and II patients) who would not derive additional benefit from chemotherapy could therefore potentially avoid the toxicity induced by chemotherapy.With the advent of computerized image analysis and an increased attention paid to machine learning, there is an opportunity for deep mining of computer-extracted image features to inform patients’ outcome and to guide treatment intensitiesIn this work, we interrogate a prognostic and predictive radiomic image signature (pRiS) that employs quantitative texture features derived from within and around the primary oropharyngeal tumor on treatment planning CT scans to predict survival and chemotherapy benefit. Using a total of 491 patients from 4 sites, treated either with radiotherapy + chemotherapy or radiotherapy alone, our goal is to investigate if pRiS is (a) prognostic of survival and (b) associated with chemotherapy benefit in AJCC stage I and stage II HPV-associated OPSCC patients.1 (n = 60), D2 (n = 162), and D3 (n = 269). Of the total 491 patients included in the study, 409 (83.3%) were men, and the median (interquartile range) age was 59 (53.8–64.2) years. 223 patients (45.4%) had AJCC 8th edition stage I OPSCC, of which 128 (57.4%) patients received chemotherapy. 268 patients (54.6%) had AJCC 8th edition stage II OPSCC, of which 201 (75%) patients received chemotherapy. More details regarding treatment are provided in Supplementary Table 1, D2 and D3 were 7.48, 7.76, and 5.16 years and the median values for DFS were 7.06, 7.63, and 5.13 years.Table 1 was −1.04 and was used for dividing patients into high- and low-pRiS groups. Clinical characteristics within the high- and low-pRiS groups are summarized in Supplementary Table 1, there were significant differences in OS , in D2  and in D3 . Point-biserial correlation analysis between each individual prognostic radiomic feature including pRiS and the clinicopathologic factors are provided in Supplementary Table Multivariate Cox analysis on OS in D1  and DFS  estimation  from X-tile software as the cutoff to define the pRiS groups and repeated the predictive experiments for OS , indicating a predictive effect of pRiS. On subset analysis by AJCC 8th overall stage (stage I and II), pRiS was predictive of chemotherapy benefit for stage II  95-01 randomized clinical trial6 demonstrated the survival benefit of concurrent chemoradiotherapy in high-risk head and neck cancer patients. This paved the way for chemoradiotherapy to become the standard treatment protocol23 for locally advanced OPSCC patients, regardless of HPV status. However, clinical characteristics and treatment response differ between various head and neck cancer subsites24 . As the HPV-associated OPSCCs have more favorable prognosis and are more responsive to radiation than the HPV negative OPSCC2, it is likely that some of these patients do not receive added benefit from chemotherapy and might have had a similar outcome if treated with radiotherapy alone. With the advent of HPV related cancers causing a significant epidemiological shift, there is a growing call for more specific and selective treatment planning strategies to be reconsidered for HPV-associated OPSCC22.The European Organization for Research and Treatment of Cancer (EORTC) 2293112 assigned HPV-associated OPSCC patients into one of two de-escalated treatment arms: (1) reduced dosage of radiotherapy with weekly cisplatin, or (2) accelerated radiotherapy (60 Gy) alone. Results showed that there were no significant differences (p = 0.23) in progression-free survival (PFS) rate between the two arms and the two-year OS rates were similar (96.7% and 97.3%). These results potentially indicate that not all selected candidates for treatment de-escalation benefited from the additional chemotherapy and they could have avoided the aggressive chemotherapy without compromising outcome. The interim analysis of NRG HN-005 trial failed to demonstrate the non-inferiority of 60 Gy of radiation plus cisplatin arm to the standard arm of 70 Gy with cisplatin. One possible explanation to this preliminary result is that the current selection criteria for lower risk HPV + OPSCC patients, which is based on clinical T/N stages and smoking status, remains insufficient for risk stratification. Development of novel and robust biomarkers to improve the accuracy of prognostication for HPV + OPSCC patients may aid in the design of future de-intensification trials. While definitive chemoradiation differs from the adjuvant treatment protocols, it still highlights the therapeutic importance of chemotherapy. Given these results, an individualized approach to accurately identify OPSCC patients who are most likely to benefit from chemotherapy would enable delivery of precision care to these patients.Currently, there are many treatment de-escalation strategies for HPV-associated OPSCC patients, including eliminating chemotherapy or reducing the dosage of radiotherapy, with no consensus on an optimal choice. Recently, a randomized phase II trial (NRG-HN002)25. The pRiS developed in this study was able to risk stratify these two populations, indicating that the true low-risk patients from these two clinically defined low-risk groups could be distinguished. On the other hand, high-pRiS patients were associated with worse outcome and thus should not be treated with de-escalation protocols. In addition, an integrated nomogram combining clinical factors and radiomic could improve the prognostic accuracy than using either alone27. These results indicate that pRiS could provide complementary information on OPSCC outcome beyond what is obtainable via currently known prognostic predictors.In this work, we present a prognostic CT-based radiomic signature (pRiS) which is also predictive of added benefit of chemotherapy using a cohort of 491 AJCC 8th edition stage I and II HPV-associated OPSCC. pRiS comprises 7 radiomic textural features, one of which captures spots and waves textural heterogeneity patterns from within the primary tumor and six from the peritumoral region characterizing the tissue microenvironment around the tumor. Within the 0–15 mm annular ring around the tumor, 2 Gabor feature quantifying filter response and four CoLlAGe features characterizing co-occurrence of gradient orientations and intensity disorders were found to be prognostic. In multivariable analysis, pRiS was found to be an independent prognostic predictor and was able to stratify patients into high- and low-risk groups with significant differences in OS. Clinically, AJCC 8th edition stage I and stage II HPV-associated OPSCC patients could be considered for treatment de-escalation. However, a subset of these patients still had poor survival outcome and need to be identified for better treatment managementth stage I and II HPV-associated OPSCC patients, which is the main innovative contribution of this work. To the best of our knowledge, there is no existing studies aimed to identify HPV-associated OPSCC patients who might not benefit from chemotherapy in addition to the definitive radiotherapy. Although previous work focused on risk-stratification and prognosis prediction, these studies could not provide an individualized treatment strategy based solely on the predicted risk profile21. In contrast, the pRiS developed in this study not only carry prognostic value but more importantly it could also convey which patients would derive additional benefit if treated with chemotherapy versus those who would not. This would enable a more granular and robust treatment de-escalation for OPSCC patient with low risk of recurrence. Currently, the criteria for selecting candidates suitable for chemotherapy are based on AJCC 7th edition staging31, which did not take HPV status into account for risk stratification. In our study, we found that patients with high-pRiS scores using median value from training set as cutoff (>−1.04) were estimated to benefit from chemotherapy with reduced hazard of dying while low-pRiS patients (<−1.04) showed non-significant hazard ratios between the two treatment arms when using OS as the endpoint. A slight change in the high- and low-pRiS membership using X-tile cutoff selection (−1.1) did not alter the statistical significance found in the radiotherapy alone cohorts, indicating the robustness of pRiS regarding predictive power . These results suggest that most of the high-pRiS patients in radiotherapy treatment arm are “truly high risk” patient populations with worse outcome and could have benefited from additional chemotherapy after radiation. This is strongly suggested by results in the chemoradiation arm, where we do not observe significant survival difference between high- and low-pRiS populations , potentially suggesting that high-pRiS patients have improved survival outcome due to receiving benefit from chemotherapy. When using DFS as the endpoint, high-pRiS patients derived statistically significant DFS benefit in D2 when treated with chemotherapy while did not yield the same significant DFS benefit in D3 (p = 0.074). However, we could observe a clear trend that patients who received chemoradiation had more favorable prognosis compared with those treated with radiation alone. Interestingly, low-pRiS patients in D3 showed detrimental effect of chemotherapy , regardless of the endpoint  models were all associated with survival benefits. It is known that DL-based approaches provide limited interpretability. On the other hand, the selected hand-crafted features in this study for pRiS includes descriptors characterizing CT pixel textural patterns . Kann et al.34 validated a DL algorithm in identifying Extranodal extension (ENE) on pretreatment CT scans in 144 patients with HNSCC. They demonstrated that the algorithm’s diagnostic performance surpassed that of specialized radiologists with head and neck cancer experience. Although pathologic ENE is an indication for adjuvant treatment escalation, to the best of our knowledge, there has been no studies on developing predictive imaging biomarker which could guide OPSCC clinical treatment decision in the definitive setting. A previous study by Leijenaar et al. externally validated the prognostic value of intratumoral radiomic signatures in a cohort of 542 OPSCC35 (C-index = 0.63). Our study is different from Leijenaar’s study in that we focused only on OPSCC patients with low-risk profile (HPV-associated stage I and II patients). Since HPV+ and HPV- patients are clinically considered different tumor entities with distinct outcome and treatment response, it is more meaningful to risk-stratify these two patient populations separately. Rather than combining these two populations together for analysis like Leijenaar et al study, we focused on prognosis and treatment response prediction specifically for HPV+ patients.Machine learning-based approaches have been applied to radiographic images for prognosticating outcome for head and neck cancer patients. Ou et al.35) either only constructing prognostic biomarker without investigating their association with treatment benefit21 or managed to quantify the treatment benefit for head and neck cancer with various disease subsites33 . Our study not only developed a prognostic radiomic biomarker which enabled individualized risk prediction of HPV-associated OPSCC patients, but also tackled a clinical-significant problem of identifying potential candidates not benefiting from chemotherapy and thus for whom such toxic treatments could be avoided.Within the HPV+ population, currently the main treatment strategy is to provide de-escalated treatment protocols . Existing studies  pRiS was shown to be not only prognostic for OPSCC patients’ outcome but also predictive to added benefits of chemotherapy; (b) rather than analyzing patients with different HNSCC subtypes, we specifically investigated the role of chemotherapy in the context of AJCC 8th stage I and II HPV-associated OPSCC patients.This work is significantly different from previous related publicationsWe acknowledge that our study did have its limitations. First, this is a retrospective study subject to confounders and biases. Second, radiation doses and type of chemotherapy are not strictly controlled for the two treatment arms. Third, we did not manage to assess the intra-observer and inter-observer agreement of the radiomic features, which might cause selected features susceptible to variation of the tumor annotations. Fourth, the training set D1 has a relatively small sample size with 20% of events, potentially limiting its generalizability to a broader population with a more diverse demographic distribution. This is suggested by the C-index discrepancy between the training and the validation datasets. More data is needed to construct a predictive biomarker with better generalizability. Nevertheless, the findings in this study could guide future studies in the design of more robust predictive biomarkers for HPV-associated OPSCC treatment de-escalation. Future investigations, such as a prospective clinical trial with a large patient cohort aimed at comparing survival benefit between high-pRiS OPSCC patients treated with and without chemotherapy, would be necessary to demonstrate its clinical utility.In summary, we have developed and validated a seven-feature prognostic and predictive signature for chemotherapy benefit in patients with HPV-associated OPSCC. Further work on larger populations will be needed to validate this preliminary biomarker. If prospectively validated, pRiS could serve as an inexpensive, tissue non-destructive prognostic and predictive companion diagnostic tool to identify patients most likely to be safely de-intensified.37 “OPC-Radiomic” cohort, 114 patients received chemoradiation and 108 patients received radiotherapy alone. 60 patients with radiotherapy alone from the “OPC-Radiomic” cohort formed the training set (D1) while the remaining 162 patients formed the internal validation cohort (D2). Among the 222 patients from the Cancer Imaging Archive (TCIA) “OPC-Radiomic” cohort, 114 patients received chemoradiation and 108 patients received radiotherapy alone. The fundamental rule is to unsure all patients in the training set received only radiation therapy (no chemotherapy), while maintaining descent number of patients treated with radiation alone in the internal validation set so as not to lose statistical power when comparing survival difference between the two treatment arms. We thought taking 60 out of the 108 patients treated with radiation alone to form the training set (D1) should be reasonable. Then all the remaining 48 radiation-treated patients plus 114 patients treated with chemoradiation  will be assigned to the internal validation set. We performed the following steps to determine the exact constitution of D1: we randomly selected 60 patients from the 108 patients treated with radiation alone and performed the statistical tests to see if there were statistically significant difference (p < 0.05) on clinicopathological variables  between the selected 60 patients and the remaining 162 patients (222-60). Differences between age were estimated using Wilcoxon rank-sum test and differences on gender, AJCC 8th staging, and disease recurrence were calculated using the chi-square test. We repeated this experiment for 500 iterations and documented the count for each patient which appears in iterations where there was no statistical difference between the selected 60 and the remaining 162 patients across all the four variables. Then the counts for all patients were re-sorted in descending order and we chose the top 60 patients to form the D1. The overall rationale is to keep balanced clinicopathological profile across the D1 and the D2. Three additional cohorts including cases from Cleveland Clinic Foundation , TCIA-HNSCC (N = 134), and TCIA-Head-Neck-PET-CT (N = 44) were combined to form the external validation set (D3), which consisted of 269 patients in total. A patient selection diagram is provided in Fig. 1 and D2 cohorts were determined using p16 immunohistochemistry (IHC). A combination of p16 IHC and/or HPV DNA in situ hybridization were used for the D3 cohort. The waiver of written informed consent was granted by the IRB at Cleveland Clinic since all images are de-identified. In addition, it is not practicable to obtain consent from patients for this study since most patients was not alive or available for consent.This retrospective study included 491 AJCC 8th edition stage I and II HPV-associated OPSCC patients from four different cohorts. Among the 222 patients from the Cancer Imaging Archive (TCIA)Inclusion criteria were: (i) non-metastatic (M0) AJCC 8th stage I and II HPV-associated  OPSCC, (ii) with available baseline pretreatment CT images covering the head and neck region, (iii) treatment with curative intent, (iv) follow-up continued for at least 20 months or until death and (v) matched clinical information . Patients with any of the following were excluded: (i) lack of identifiable tumors on CT scans or (ii) number of pixels within tumor being less than 200, this was the minimum tumor volume that was deemed to be necessary for feature extraction. Disease-free survival (DFS) was defined as time from radiotherapy end date to the date of following events: local, regional, distant failure or death whichever occurred earlier and censored at the date of last follow-up for those without event. Overall survival (OS) was defined as the time from radiotherapy end date to death from any cause and censored at the date of last follow-up for those alive. Clinical and outcome information from patients in the CCF cohort was obtained by chart review after approval from the Institutional Review Board (IRB number: CCF 14–551). The overall workflow is provided in Fig. All patients underwent an initial pretreatment CT scan with or without contrast agent injection for radiation therapy planning. CT images for the CCF cohort were acquired from GE  or Siemens  scanners. CT scans were acquired in helical mode with a slice thickness of 3 mm, at 120 kVp and 235 mAs tube current. Image resolution was between 0.4 mm - 0.5 mm for most of the patients, with image matrix of 512 × 512. The CT images for the three TCIA cohorts were acquired from one of the following CT scanners: General Electric Discovery ST; General Electric Lightspeed Plus; Toshiba Medical Systems Aquillion ONE. More details regarding the CT imaging parameters for the three TCIA cohorts included in this study could be found in Supplementary Table 38. The slices with dental artifacts were excluded for tumor annotations. The binary masks which comprise the outline of the gross primary tumor volume (GTV) for the three TCIA cohorts were obtained via the Radiation Therapy Structures (RTSTRUCT). RTSTRUCT is used to transfer anatomical structures related data between radiotherapy departments. It mainly comprises the information related to the regions of interest (ROIs) including GTV.For primary tumor annotations on CCF cohort, two board-certified head and neck radiologists J.L.  and S.S.  manually delineated the tumor boundaries across all two-dimensional CT axial slices with visible tumor present using the 3D Slicer software16. The binary tumor masks were then subtracted from the dilated masks to obtain the peritumoral regions, which were then sub-divided into three peritumoral rings of 5-mm-radius increments. For all patients, peritumoral masks were dilated 15 mm from the corresponding intratumoral masks in a two-dimensional fashion. For each CT axial slice with tumor, the number of pixels dilated in each peritumoral mask were calculated as follows:After obtaining the binary tumor mask for all patients, morphologic dilations were performed to define the annular ring region outside the tumor up to a radial distance of 15 mm based on a previous studyDuring this peritumoral feature extraction process, additional consideration was taken to get rid of the region with air (<−900 HU). To avoid any edge artifacts that might arise during feature extraction, the filtered “dead” pixels of the CT scan were substituted by using an averaging filter across its 9 × 9 neighborhood.39, Laws energy, Gabor wavelet-based features, and intensity gradient orientations features (CoLlAGe)40. These features were extracted on a per-pixel basis across all 2D slices with tumor for all patients from both intratumoral and peritumoral regions . The feature values were averaged across all slices. Statistics of mean, median, standard deviation (std), skewness, and kurtosis were calculated from the feature responses of all pixels within the region of interest, which resulted in a total of 2045 features. All feature values were transformed into new scores with a mean of 0 and a standard deviation of 1 (z-score transformation). Detailed description of extracted features is provided below:Radiomic feature extraction was performed for each patient on the primary tumor using an in-house program developed with MATLAB 2020b , meeting IBSI criteria in terms of the reporting criteria for reproducible and transparent implementations. We resampled all CT images to an isotropic voxel size of 1 mm prior to feature extraction. The feature families utilized in this study included gray level intensity features, gray level co-occurrence matrix (GLCM) Haralick features2-dimensional Laws filters are derived by computing the outer product of combinations of the following 1-dimensional filter vectors focused on different texture patterns: Level (L5)—detects smoothness of intensity values, L5 = [1 4 6 4 1]; Edge (E5)—detects edges between regions with abrupt changes in intensity, E5 = [−1 −2 0 2 1]; Spot (S5)—detects speckled enhancement patterns, S5 = [−1 0 2 0 −1]; Wave (W5)—detects oscillating local intensity patterns, W5 = [−1 2 0 −2 1]; Ripple (R5)—detects oscillating intensity patterns centered at region of extreme intensity, R5 = [1 −4 6 −4 1].y and x axes, e.g., L5E5 is the product of a level detection filter in the y axis and an edge detection filter in the x axis.To obtain a feature vector, convolution is performed on the filters and the images within a window size neighborhood followed by summing up all the values. Features are named by the combination of filters applied in the Two-dimensional Gabor filters are computed by modulating a Gaussian kernel function with one of 48 sinusoidal plane waves. Each sinusoidal plane wave corresponds to a unique combination of one of four spatial wavelengths  and one of seven orientations . Each Gabor filter is then convolved with the original image and values corresponding to filter response within the region of interest are concatenated.The following Haralick gray level co-occurrence matrix (GLCM) descriptors were computed: entropy, energy, inertia, inverse difference moment, correlation, information measure of correlation 1, information measure of correlation 2, sum average, sum variance, sum entropy, difference average, difference variance, and difference entropy. GLCM statistics were concatenated within a window size neighborhood, yielding 13 descriptor vectors per region.x and y direction are computed. Within a window size neighborhood, the dominant intensity gradient orientation (between 0° and 360°) is computed via principal component analysis, resulting in a 2D array of equal size with the dominant gradient orientation value centered at the corresponding pixel of the original image. Metrics of the co-occurrence matrix are then applied to this gradient orientation image in the same manner as described above for Haralick GLCM features. The resulting 13 CoLlAGe descriptors quantify the homogeneity of intensity gradient directionality within an image.An image’s intensity gradients in the 41 on OS using D1. The optimal value of the tuning parameter in the LASSO Cox  model was determined by 10-fold cross validation to search for 100 values up to 1% of the estimated maximum. Once the parameter alpha is determined, the number of features was locked down to 7. pRiS was then constructed by linearly combining these 7 features weighted by their corresponding coefficients. The cutoff value for dividing patients into low- and high-risk patients was chosen using the median pRiS value from D1 based on OS. The potential association between the dichotomous pRiS group (high- vs low-pRiS group) and survival was first evaluated in D1 and then validated in D2 and D3 based on Kaplan-Meier (KM) survival analysis and Harrell’s concordance index (C-index).The top prognostic radiomic features were selected by applying a least absolute shrinkage and selection operator (LASSO) Cox Proportional Hazard modelrad+c) by combining pRiS with the prognostic clinical factors and compare it against a clinical nomogram (Mc). Variables significant in univariate analysis in D1 were included in the Mrad+c. Both the Mrad+c and Mc were developed in D1 based on the multivariable Cox regression analysis and tested in D2 and D3 with respect to calibration, discrimination (C-index), and clinical usefulness. Calibration curves along with the Hosmer–Lemeshow test were used to compare the predicted survival probabilities with the actual probabilities. Decision curve analysis was performed to quantify the net benefit at various threshold probabilities and to compare the clinical usefulness of the Mrad+c and Mc.To investigate whether pRiS adds incremental prognostic value to the clinical factors for individualized prediction of OS and DFS, we constructed an integrated radiomic nomogram  was compared using the one-way analysis of variance test (ANOVA) and the association of the categorical variables with 3 datasets was estimated using the chi-square test. Differences of OS and DFS among groups was examined using the log-rank test. Univariate analysis of the continuous pRiS value with the clinical and pathologic factors  were conducted. Multivariable Cox regression analysis was performed to assess the relationships between the various clinical factors and OS. We also performed the point-biserial correlation analysis to investigate potential association between each individual prognostic radiomic feature and the clinicopathologic factors. Interaction between pRiS and chemotherapy was assessed by adding an interaction term in the Cox model. Statistical analyses were performed using R version 3.4.0. The R packages used in this study included glmnet, survminer, rms, survival, Hmisc, survMisc, survey, and SvyNom. A threshold of 0.05 was used to define statistical significance.The difference of the continuous variables  among 3 datasets (DFurther information on research design is available in the REPORTING SUMMARYSupplemental materials


NHIP  through an epigenome-wide association in placenta, identified a common genetic risk for ASD that was modified by prenatal vitamin use. While epigenomic signatures are distinct between different genetic syndromic causes of ASD, bivalent chromatin and some convergent gene pathways are consistently epigenetically altered in both syndromic and idiopathic ASD, as well as some environmental exposures. Together, these epigenomic signatures hold promising clues towards improved early prediction and prevention of ASD as well genes and gene pathways to target for pharmacological interventions. Future advancements in single cell and multi-omic technologies, machine learning, as well as non-invasive screening of epigenomic signatures during pregnancy or newborn periods are expected to continue to impact the translatability of the recent discoveries in epigenomics to precision public health.Autism spectrum disorder (ASD) comprises a heterogeneous group of neurodevelopmental outcomes in children with a commonality in deficits in social communication and language combined with repetitive behaviors and interests. The etiology of ASD is heterogeneous, as several hundred genes have been implicated as well as multiple in utero environmental exposures. Over the past two decades, epigenetic investigations, including DNA methylation, have emerged as a novel way to capture the complex interface of multivariate ASD etiologies. More recently, epigenome-wide association studies using human brain and surrogate accessible tissues have revealed some convergent genes that are epigenetically altered in ASD, many of which overlap with known genetic risk factors. Unlike transcriptomes, epigenomic signatures defined by DNA methylation from surrogate tissues such as placenta and cord blood can reflect past differences in fetal brain gene transcription, transcription factor binding, and chromatin. For example, the discovery of  Autism spectrum disorder (ASD) refers to a complex group of neurodevelopmental disorders characterized by deficits in social communication and language and gains in repetitive and restrictive behaviors and interests. The prevalence of ASD has been steadily increasing over the past 20 years, from US child estimates of 0.66% in 2002 , 1.13% iEpigenetics, literally meaning “on top of genetics” is a field that investigates additional layers of relevant biological information for interpreting phenotypes that do not alter the genetic code. Poised at the interface of genetic and environmental influences, the investigation of epigenetic modifications such as DNA methylation can reveal novel insights that are not apparent in the DNA sequence. See Box MECP2, a gene encoding a known epigenetic player, methyl CpG binding protein 2 [The field of autism epigenetics began initially through genetics, specifically the investigation of known genetic neurodevelopmental disorders with ASD comorbidity that were epigenetic in their inheritance pattern and/or gene function , 28. Therotein 2 , 34. Canrotein 2 . More reDNA methylation is the most frequently studied epigenetic modification in genome-wide ASD studies for both practical and biological reasons. On a practical level, DNA is much easier to obtain from a variety of limited clinical samples and DNA methylation is a relatively stable mark in archived frozen tissues , relativBox 1: What is epigenetic? Definitions and termsEpigenetic: Modifications to DNA or chromatin that can alter gene expression and phenotypes without changing the DNA sequence.Epigenetic layers: Epigenetic modifications that exist as specific layers of molecular information on top of DNA, including (in increasing order of distance from DNA): DNA methylation, histone post-translational modifications, chromatin loops, higher-order chromatin compartments, noncoding RNAs acting as chromatin modifiersEpigenetic players (or factors): Protein factors that modify, recognize, or change epigenetic layers. This group represents over 800 proteins encoded in the mammalian genome [DNMT3A), demethylases , histone deacetylases , histone demethylases , and chromatin remodeling factors .n genome  and manyn genome , 150. SpDNA methylation base pair targets: CpG, CpH DNA methylation modifications: 5-methylcytosine (5mC), 5-hydroxymethylC (5hmC)CpG islands: clusters of 5’-cytosine-guanine-3’ dinucleotides in mammalian genomes that are frequently unmethylated when occurring at the promoters of active or ubiquitously expressed genes.Gene bodies: regions of genes defined as transcription start site to transcription end site.Bivalent chromatin: developmentally poised chromatin state characterized by both active and repressive histone marks.Epigenomic: Genome-wide analyses of specific epigenetic modifications or chromatin features using microarray (Illumina Infinium 450 K or EPIC) or sequencing  approaches.Epigenome wide association study (EWAS): an analysis of cases and controls for differentially methylated regions or probes by array or sequencing-based approaches (See Box  See Box .Variably methylated regions (VMR): genomic regions defined as having the most interindividual variability of all regions assayed on the array or sequencing platform.Differentially methylated probes (DMP): individual CpG sites detected by probes on the Illumina Infinium arrays : genomic clusters of ~3–20 CpG sites with coordinate differential methylation patterns between two comparison groups of samples (ASD cases versus controls) or alleles .Epigenomic signature: A profile of multiple changes to an epigenetic layer  that together separate ASD from control samples using a principal components or clustering analysis. Like a handwritten signature, each individual part of the signature may not be precisely reproducible every time, but the multiple parts together have a distinctive combination of features that allow bioinformatic clustering by diagnosis. While an epigenomic signature is likely to be secondary to the primary cause(s) of ASD, it can inform both diagnosis as well as dysregulated gene pathways for pharmacological interventions.Box 2: Genome-wide methylome approaches for detecting epigenomic signaturesArray-based methodsCommercially available platforms that include hybridization to probes over predefined genomic regions. Sample DNA undergoes bisulfite conversion prior to hybridization to methylated and unmethylated probes for individual CpG sites.Illumina Infinium 27k was designed to include probes mostly over known gene promoters.Illumina Infinium 450k included some known enhancers as well as promoters.Illumina Infinium EPIC expanded to 850k design to include FANTOM5 enhancers, ENCODE open chromatin and enhancers, DNase hypersensitive sites, and miRNA promoter regions.Advantages● For human epidemiology studies, having a uniform platform makes it easier for cross-comparisons comparisons and meta-analyses.● Bioinformatic pipelines are well-established and data storage is minimal.● Data integrate with array-based genotyping for determining methylation quantitative trait loci (meQTLs).● Cost is generally less expensive than sequencing-based approaches, but not always (see below).Disadvantages● Cover <3% of all CpGs in the human genome.● Only available for human studies.● Can require higher input DNA than current sequencing-based approaches.● Identifying differentially methylated regions (DMRs) is challenging.Sequencing-based methodsWhole Genome Bisulfite Sequencing (WGBS)Using the most current library preparation methods, genomic single stranded DNA is fragmented and bisulfite converted prior to ligation of truncated adapters, DNA synthesis, and ligation of indexed bar codes . After aLow pass WGBS varies from 1x-10x coverage genome-wide, which is sufficient for DMR-based analyses, network analyses of comethylated regions [ regions , as wellHigh coverage WGBS varies from 30x-50x coverage genome-wide, which is sufficient for single CpG resolution [solution .Reduced-representation bisulfite sequencing (RRBS) uses a either a methyl-sensitive restriction enzyme or hybridization-based capture approach to enrich DNA fragments for specific genomic regions prior to bisulfite conversion, library preparation, and sequencing.Advantages● WGBS covers >20 million CpGs and RRBS genomic coverage is greater than arrays.● Any species for which a reference genome is available can be analyzed.● WGBS libraries can be generated from 1 ng of DNA.● WGBS CpG coverage is ideal for DMR analyses, as well as comparison to all available epigenomic sequencing maps of transcription factor binding sites and chromatin states.● The cost of genomic sequencing continues to drop, making low-pass WGBS and RRBS approaches comparable to arrays.Disadvantages● Comparison of results to published array-based EWAS studies is challenging. For instance, in a recent WGBS cord blood analysis of cord blood, >80% of ASD DMRs did not overlap with even a single probe represented on the Infinium EPIC array .● Relatively large data storage and computational needs for bioinformatics● The cost is higher than array-based approaches, but only for high-coverage WGBS.New developments in bisulfite-free sequencing approachesEnzymatic methyl-seq(EM-seq) EM-seq utilizes two biological enzymes, first to convert 5mC to 5hmC with TET2, then to deaminate unmethylated cytosine to uracil with APOBEC3A [Advantages40● By eliminating the use of bisufite, DNA is less damaged, allowing DNA inputs down to 100 pg.Disadvantages40● Enzymatic conversion efficiency is lower than bisulfite, leading to genome complexity problems resulting in low mapping rates and uneven genome coverageAPOBEC3A , 154AdvaOxford Nanopore sequencing and Nanopolish directly detects 5 mC from long reads using a Hidden Markov model that generates a log-likelihood value for the ratio of probability of methylated to unmethylated CGs at a specific k-mer. This approach was used to map DNA methylation in the latest telomere to telomere (T2T) human genome [Advantages● Long reads processed without bisulfite allows repetitive regions of the genome to be investigated.Disadvantages● Nanopore sequencing has less accuracy of methylation quantitation at base pair resolution.n genome , 155.AdvGenetic and environmental factors may act independently on DNA methylation, or in additive or multiplicative interactions. At the simplest level, some genetic variants including single nucleotide polymorphisms (SNPs) can directly determine DNA methylation, for instance, if they cause a gain or loss of a CpG. These are expected to be rare (<1%) based on a study of neonatal genotype-methylation comparisons from cord blood . In thisVariably methylated regions (VMRs) have been investigated in more detail across different human tissues and environmental conditions, revealing some interesting insights. An array-based study examined polymorphic human methylation patterns across five cell types and ages, identifying both unique and common VMRs . InteresTwin studies have been critical to understanding the heritability of DNA methylation patterns in humans. Array-based comparisons of monozygotic (MZ) to dizygotic twins (DZ) using 27k and 450k platforms  or DNA methylation . Approximately 50 different syndromes caused by genes encoding epigenetic regulators, dubbed “chromatinopathies”, have been clinically defined and the clinical utility of using methylation data and EpiSign for improved diagnosis has been validated by independent teams [DNA methylation patterns have been used for decades to diagnose imprinted and X-linked disorders such as Prader-Willi/Angelman syndromes (PWS/AS) and fragile X syndrome because they offered specific tests that would encompass different genetic and epigenetic causes of gene loss or repression. However, a recent study demonstrated that four different imprinted syndromes  could be distinguished from each other and 364 reference controls with 100% specificity and sensitivity using the 450k array platform from blood DNA . What isnt teams , 50. WhiDnmt3a mutant that no longer binds to active chromatin results in progressive DNA hypermethylation across bivalent chromatin domains marked by H3K27me3 and de-repression of developmental regulatory genes in the adult hypothalamus [Dnmt3a knockout specifically targeted to excitatory neurons resulted in stunted maturation of synapses, elevated H3K27me3 levels, and fetal-like DNA methylation patterns in the postnatal period [In addition to the syndromic forms of ASD caused by rare mutations in single genes, epigenomic signatures have also provided some novel insights into the molecular pathogenesis into syndromes caused by copy number variants (CNVs), such as 15q11-q13 duplication , 52 or 1thalamus . An isofthalamus . Furtherl period . TogetheUBE3A is maternally expressed exclusively in neurons due to the paternal expression of the UBE3A antisense [15q11-q13 duplication syndrome (Dup15q) is one of the most common CNVs identified in ASD cases and ASD comorbidity is observed in 85% of Dup15q cases . Due to ntisense . Becausentisense . The oppntisense , reminisntisense . The enrntisense . This 45ntisense , a findintisense .Functional follow-up analyses using human neuronal cell line models revealed further insights into the potential mechanism responsible for the epigenomic signature of Dup15q syndrome and how multiple “hits” may impact the strength of the epigenomic signature. A human neuronal cell line model of Dup15q syndrome  was cultRUNX1 locus [RUNX1 encodes a developmental transcription factor important in both hematopoiesis and neurodevelopment [RUNX1.Down syndrome (DS) is another neurodevelopmental disorder caused by a chromosomal copy number change, trisomy 21. Estimates of ASD incidence is DS have ranged from 5–39% dependent on the study . MultiplX1 locus , which hX1 locus –76. RUNXelopment , 78. Intelopment . Mechanielopment . TherefoDNMT3L has the most accumulated evidence supporting its role in the hypermethylated loci observed across tissue types in DS [DNMT3L in in human neurons at three stages of differentiation demonstrated a hypermethylated signature shared with that of DS brain and other DS tissues [DNMT3L-induced hypermethylation occurred predominantly at regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation. In DNMT3L overexpressing cells, hypermethylation of RUNX1 was observed in the neuroblast and differentiated neurons, but not those in process of differentiation [DNMT3L overexpression recreates a facet of the genome-wide DS DNA methylation signature, specifically the gene loci that consistently display hyper-methylation in DS such as RUNX1. These results also suggest that DNMT3L may be a major contributor to the hypermethylated bivalent chromatin signature in DS, while RUNX1 binding and demethylation may be a contributor to the blood-specific hypomethylated signature of DS. However, there are also consequences to cellular viability, DNA repair, and metabolism associated with aneuploidy independent of chromosome 21 dosage effects that have been demonstrated in fibroblasts from DS in common with other trisomies [Of the potential known epigenetic regulators encoded on chromosome 21, the DNA methyltransferase gene, es in DS , 81. DNMes in DS , 83. Overisomies  that arerisomies , 73, 81.PRRT1, ZNF57, C11orf21) and one in cerebellum (SDHAP3). Furthermore, Wong et al. 2019 used a systems approach of grouping comethylated gene loci into modules, identifying a number of significant gene ontologies associated with idiopathic ASD, including homophylic adhesion, synapse part, and calcium ion binding [While the discovery of distinct epigenomic signatures distinguishing syndromic forms of ASD is possible with a relatively low sample size and different tissue types besides brain, this same approach has been more challenging in finding a robust DNA methylation signature for idiopathic ASD. Postmortem brain samples from individuals with idiopathic ASD have been the tissue type used for initial EWAS, because of the predicted involvement in ASD symptomology. Several studies have used the Illumina Infinium 450k platform to investigate different brain regions  from 6–12 ASD samples compared to matched controls, but no individual differentially methylated probe (DMP) reached genome-wide significance , 85–87.  binding  that wer binding , 85, 86.OTX1, GRIK4, and ADCY5), as well as also differentially expressed in ASD  [The use of a sequencing-based WGBS approach allowed the discovery of 483 DMRs in idiopathic ASD prefrontal cortex, although this number of regions was 5–10x lower that those identified in two syndromic forms of ASD, Dup15q and Rett syndromes by the same analyses . In addi RBFOX1) . Further RBFOX1) . TogetheWhile these biological insights gained from brain EWAS studies have been important, there are many limitations to these studies, including the limited number of samples and the inability to control for confounding variables, such as medications and time and cause of death. Furthermore, the inaccessibility of sampling brain DNA from living individuals makes it not useful as a predictive biomarker. Therefore, multiple studies have attempted to use EWAS in accessible tissues such as blood, with limited success. Hannon et al performed a 450k EWAS on DNA samples isolated from whole blood matched to 4 brain regions  from 122 typically developing individuals, concluding that interindividual variation in whole blood was not a strong predictor of interindividual variation in the brain . This fi39 that have led to new mechanistic insights into ASD  and Early Autism Risk Longitudinal Investigation (EARLI). Both studies collect and store maternal biospecimens during pregnancy, cord blood and placenta at delivery, and follow up children through 36 months with biospecimen collections and quantitative evaluations of social and cognitive outcomes , 94. BotASD Fig. .Fig. 2MeLOC105373085 which additional functional studies showed to be expressed in brain and induced in response to neuronal differentiation and oxidative stress. This transcript was therefore renamed NHIP for neuronal hypoxia inducible, placenta associated. Using RNAseq on both postmortem ASD and control brain samples as well as NHIP-overexpressing cell lines, NHIP was shown to regulate expression of other known ASD-risk genes involved in chromatin and transcriptional regulation. A common structural variant within the NHIP locus was independently associated with increased ASD risk, reduced expression of NHIP, and reduced methylation. Interestingly, placentas from mothers who took a folic acid vitamin in the first month of pregnancy, a known protective anti-oxidant methyl-donor [NHIP methylation, essentially counteracting the genetic risk at NHIP [NHIP encodes a 20 amino acid peptide that localizes to neuronal nuclei in human brain. While this peptide is well conserved amongst primates, NHIP is not detected in other mammals, suggesting a recent evolved function in the response to hypoxia and oxidative stress in primates [Using MARBLES as the discovery cohort and EARLI as the replication cohort, Zhu et al 2022 analyzed a total of 204 placenta samples  using WGBS to identify ASD-associated methylation changes . 134 ASDyl-donor , 105, sh at NHIP . Functioprimates . Within primates . TogetheXACT [MECP2 and CDKL5 (Rett syndrome) [HDAC8 (Cornelia de Lange syndrome 5) [In contrast to placenta which is derived from the trophectoderm layer of the preimplantation embryo in all mammals, cord blood is derived from the hematopoietic cell lineage of the embryo proper . So perhXACT , which iXACT , 110. FuXACT , includiyndrome) , as welldrome 5) .The multiple lines of evidence presented above have established that epigenetic changes in the form of distinct DNA methylation signatures characterize both syndromic and idiopathic neurodevelopmental disorders on the autism spectrum. There is also strong evidence that in general, most inter-individual variation in DNA methylation is best explained by GxE models, meaning that common genetic risk is predicted to dynamically interact with common but variable environmental factors. Since most ASD cases are unlikely to have a single genetic or environmental cause, the use of epigenomic signatures to detect the multivariate intersection is likely to improve understanding of common genetic risk for ASD. Importantly, the DNA methylome signature of ASD polygenic risk is highly supportive of most common risk involving GxE more than other neuropsychiatric disorders . This isSince ASD is currently diagnosed by behavioral testing that does not detect all cases until 3 years of age , there iAdvances in machine learning are already beginning to be used to determine the most sensitive and specific predictors of ASD diagnosis from large-scale EWAS datasets –129. TheNHIP from cffDNA within maternal blood could provide an early marker of ASD risk in the child. Early identification of risk could prompt existing interventions during pregnancy that reduce medical complications and environmental exposures. Some preventative epigenomic screenings could even be performed pre-conception, as there has be some predictive success within small studies of sperm methyome differences in fathers of ASD offspring [Moving to an even earlier stage of potential preventative intervention that the newborn stage, prenatal screening from cell free fetal DNA within maternal blood holds potential for early identification and interventions to improve brain development during pregnancy for at risk fetuses. Currently, non-invasive prenatal screening is performed in the first trimester as an alternative to chorionic villus sampling for prenatal screening of Down syndrome. This procedure involves sampling and sequencing the cell-free fetal DNA (cffDNA) fraction from blood of pregnant mothers , 131. cfffspring , 142. ThFollowing up on specific mechanistic insights provided by epigenomic signatures from both syndromic and idiopathic ASD early life samples is expected to inform future therapies and early interventions. Future investigations should seek to understand the overlap between ASD-associated DNA methylation signatures with additional epigenetic layers, including histone acetylation and long noncoding RNAs identified as differential in ASD brain , 144. CoNHIP regulatory gene locus that was discovered in placenta, but highly expressed in brain and responsive to hypoxia in differentiated human neurons [NHIP expression in response to oxidative stress appears to be protective for the development of ASD, the NHIP gene or encoded micropeptide could be promising as a therapeutic approach during pregnancy or early infancy in high-risk populations. In summary, the discoveries made from epigenomic investigations using the latest genomic approaches are likely to yield clinically relevant advancements in the future that would not have been possible through traditional genetic approaches such as GWAS.But in addition to the cell type specific mechanisms, there may also be some tissue independent and universal defining mechanisms across different etiologies of ASD that are revealed from epigenomic signatures of syndromic and/or idiopathic ASD. An example discussed in this review is the  neurons . Hypoxia


Soliman et al. integrate recent molecular discoveries to bring insights and offer future directions to further our understanding of Topoisomerase IIA checkpoints. DNA Topoisomerase IIA (Topo IIA) is an enzyme that alters the topological state of DNA and is essential for the separation of replicated sister chromatids and the integrity of cell division. Topo IIA dysfunction activates cell cycle checkpoints, resulting in arrest in either the G2-phase or metaphase of mitosis, ultimately triggering the abscission checkpoint if non-disjunction persists. These events, which directly or indirectly monitor the activity of Topo IIA, have become of major interest as many cancers have deficiencies in Topoisomerase checkpoints, leading to genome instability. Recent studies into how cells sense Topo IIA dysfunction and respond by regulating cell cycle progression demonstrate that the Topo IIA G2 checkpoint is distinct from the G2-DNA damage checkpoint. Likewise, in mitosis, the metaphase Topo IIA checkpoint is separate from the spindle assembly checkpoint. Here, we integrate mechanistic knowledge of Topo IIA checkpoints with the current understanding of how cells regulate progression through the cell cycle to accomplish faithful genome transmission and discuss the opportunities this offers for therapy. Chromosome segregation errors result in aneuploidy that causes infertility, birth defects, and cancer . AneuploDNA replication produces physically entangled (catenated) sister DNA helices that must be resolved to allow chromosome segregation . TopoisoThe SPR is performed by a symmetrical homodimeric Topo IIA holoenzyme requiring hydrolysis of two ATP molecules . Topo IIThe DNA damage response (DDR) pathway is a G2 checkpoint responsible for arresting cells in G2 in response to DNA damage . The DDRBox 1Definitions and scope of the reviewTopo II is the only enzyme in eukaryotes that can disentangle intertwined DNA duplexes, i.e., catenated DNA. Many inhibitors have been identified directed at Topo II  bodies. The colocalization of these proteins is likely important in promoting NSMCE2-directed SUMOylation of Topo IIA, although no formal evidence of higher-order complex formation/physical contact has yet been seen within these nuclear structures. Nevertheless, given the pattern of S-phase chromatin loading and M-phase unloading of the SMC5/6 complex , a role Interestingly, in transformed cells engaging alternative lengthening of telomeres (ALT cells), ICRF-193 induces accumulation of Topo IIA and the SMC5/6 complex in a subset of PML bodies, termed APBs (ALT-associated promyelocytic leukemia nuclear bodies), and this is associated with a G2 arrest . HoweverWhile untransformed cells can engage a long-term G2 arrest in response to ICRF-193, cells have also been reported to trigger a short-term, caffeine-sensitive (requiring ATM or ATR) delay in G2 progression even in the absence of p53 . It has Irrespective of the details that have yet to be resolved, the evidence indicates that there is a checkpoint in G2 that monitors the Topo IIA closed clamp occupation of chromatin. The specific circumstances evidently impact the particular pathways engaged but the implication remains the same; if Topo IIA in this conformation is sensed on chromatin associated with the SMC5/6 complex, then there is a delay to G2 progression. The existence of these regulatory processes suggests that in the absence of any experimental manipulation, the steady-state association of Topo IIA with chromatin is exploited by cells to sense ongoing decatenation, the associated potential for non-disjunction, and hence the need for cell cycle delay and catenane resolution.The mitotic apparatus, the spindle and kinetochores, is mechanically complex, but without cell cycle controls, chromosome segregation is error prone. In early mitosis, the SAC senses chromosome biorientation, deciding when anaphase can proceed accurately . These ctop2 mutants that activate the metaphase Topo IIA checkpoint were isolated and found to be defective in ATPase activity  specifically on mitotic chromosomes and that in both interphase and mitotic cells, SUMOylation initiates repair pathways that have evolved to remove trapped Topo IIA complexes from DNA . This leThe evidence implicating Topo IIA SUMOylation in the metaphase checkpoint activation is important because it provides a direct link to Aurora B activation. In yeast and Xenopus egg extracts, Topo IIA SUMOylation recruits Haspin kinase to chromosomes in mitosis , and HasAdditional evidence has indicated that nucleosome binding of Topo IIA may facilitate activation of the metaphase Topo IIA checkpoint. Aurora B recruitment seems to rely on H3T3 phosphorylation by Haspin, leading to the prediction that Haspin must not only be recruited to chromosomes but must be in close proximity to nucleosomes. The discovery of a nucleosome binding region of Topo IIA, the chromatin tether (ChT) domain , could pAnother major gap in understanding is how Aurora B activates the metaphase Topo IIA checkpoint via Mad2 activation. Because there is no direct connection between Aurora B and Mad2, the relevant substrates of Aurora B during metaphase Topo IIA checkpoint activation are unknown. There may be novel Aurora B substrates involved since Aurora B is recruited to a distinctive location at chromosome cores where established kinetochore substrates are not known to be present. Further, metaphase Topo IIA checkpoint activation requires PKCε, which triggers a switch in Aurora B substrate selection . PKCε phGiven that the effectors of Aurora B remain to be identified, further studies are needed to determine how Mad2 is activated. The identity of the Topo IIA SUMO E3 ligase that activates the checkpoint is also unknown. Several E3 ligases have been shown to SUMOylate Topo IIA, including PIASy and RanBP2 , ZNF451/Repair mechanisms have evolved to disperse trapped Topo IIA complexes ; howeverThere is evidence that the resolution of excessive catenation appears to engage additional/distinct mitotic pathways that are determined by the specific G2 trigger. This is exemplified by the engagement of PKCε during mitosis, where loss of the p53-dependent checkpoint pathway in G2 is associated with a subsequent dependence on PKCε for effective cell division . By contBypassing the Topo IIA checkpoints engenders DNA bridging, which persists into telophase and cytokinesis, activating the Aurora B–dependent abscission checkpoint to delay cytokinesis see . InteresTP53 is mutated in >50% of all human tumors , there are druggable targets that are unusually engaged in these conditional pathways. These could be exploited in a synthetic lethal approach alongside catalytic Topo IIA inhibitors. There is already good evidence that targeting tumors with a defective Topo IIA–dependent G2 response may be a viable therapeutic strategy. Checkpoint deficient cell lines including hepatocellular and pancreatic ductal adenocarcinoma (PDAC)   and coloa (PDAC)  lines haa (PDAC) . Again, Box 2Context of checkpoint interventions in cancerThere are many cytotoxic agents, including the Topo IIA poisons, deployed in cancer treatments. These drugs induce DNA damage, which in cycling cells causes the malfunction of DNA replication, associated disjunction errors, failed cell division, and cell death. The narrow therapeutic window for these agents is a consequence of the rapid division of cancers relative to most tissues. Nevertheless, rapidly turning over tissues, e.g., gut and bone marrow, are associated with the “side effects” of such treatments. More selective approaches to the blockade of cell division through the specific targeting of cell cycle controls and checkpoints have promised greater selectivity and a better therapeutic index compared with these general DNA-damaging agents. This is the case for the cdk4/6 inhibitor Palbociclib, which is FDA-approved for deployment as a combination therapy in HR+ advanced breast cancer . While tTP53 mutant cancers. Topo IIA inhibition would arrest normal, healthy cells in the surrounding tissues in G2-phase, while the checkpoint-deficient tumor cells would be primed for targeting the checkpoint relay engaged in metaphase. Known targets of the metaphase pathway include PKCε (Topo IIA catalytic inhibitors are currently used in clinical practice alongside anthracycline chemotherapeutics . In thisude PKCε , Aurora ude PKCε , and PICude PKCε ; BLM helude PKCε  and Haspude PKCε  could alTo date, no combinations drawing upon these findings have been tested in patients, and although it is the case that previously available PKCε inhibitors lack the desired specificity for clinical use , new-genThere are many outstanding questions associated with the operation/inaction of these Topo IIA–dependent checkpoints and delays that have implications not only for our understanding of the mechanistic details but also for the deployment of interventions that can exploit the idiosyncratic dependencies of transformed cells. Some of these issues we have highlighted above, among others, is the question of timing—in G2, the engagement of a long-term arrest and commitment to a senescence program is not hard-wired, with some transformed cells delaying transit but then progressing through G2- to M-phase, albeit in a passage-dependent manner . What diIn bypassing the Topo IIA–dependent G2 checkpoint, cells engage control processes that are not usually required for cell cycle regulation. This is well characterized for the engagement of PKCε in the Topo IIA–dependent mitotic delay, where the reliance is not a function of somatic mutation as pharmacological bypass in otherwise G2-arrest competent cells still engages PKCε . It seem


Noninvasive cardiopulmonary exercise testing (CPET) provides the valuable capacity to analyze pulmonary gas exchange and cardiovascular responses that can be used to differentiate normal cardiopulmonary responses from abnormal. This case report highlights a proposed role for CPET in identifying potential cardiac pathologies in at-risk adolescents. An abnormal CPET response in an asymptomatic adolescent revealed a family history of early-age CAD. The significance of the abnormal CPET response was further supported by the presence of an elevated concentration of circulating high sensitivity C-reactive protein (hs-CRP). These findings emphasize the importance of a thorough clinical evaluation in at-risk adolescents, as CPET can aid in the early detection and management of cardiac pathologies, especially when combined with other relevant biomarkers such as plasma hs-CRP concentration, which can further suggest underlying pathology. Management considerations using serial CPET evaluations are recommended. Thus, CPET abnormalities combined with elevated hs-CRP should be taken seriously and provide justification for further evaluation and monitoring in adolescents at risk for cardiovascular disease. Cardiopulmonary exercise testing (CPET) is a well-recognized, but underutilized diagnostic and prognostic test for clinical evaluation in the symptomatic pediatric and adult populations. However, an effective preventive healthcare clinical workflow for apparently healthy asymptomatic individuals at-risk for future cardiovascular disease  is not well established, especially in adolescents. As a non-invasive and radiation-free test, CPET provides a simultaneous evaluation of cardiovascular, pulmonary, and skeletal muscle responses that may detect early-stage disease states. Therefore, a clinical workflow utilizing CPET has the potential to improve the current preventive healthcare strategy.2), which is the product of cardiac output and arterial-venous O2 difference. Cardiac output is measured indirectly by tracking HR and O2 pulse (surrogate of stroke volume) throughout incremental exercise. In healthy individuals, V̇O2 and HR increase linearly along with a linear or curvilinear O2 pulse when a linear ramping work rate (WR) protocol is used on a cycle ergometer. Key CPET parameters  reflect how V̇O2, HR, and O2 pulse respond to increasing workloads and provide important insight into cardiovascular function , and the usage of data for this Case Report was approved by the same IRB (#263026).The participant was enrolled in the original cohort study and was later recalled for a study visit when he was 14 years old, at which point the visit also included a CPET test. The case participant had no known previous or current medical history, was not taking any prescription medications, and was reportedly asymptomatic at the time of testing. via a maximum effort CPET using a metabolic cart  and cycle ergometer  with a linear ramp protocol of 20 watts per minute. The participant stopped the test due to leg fatigue and reached a respiratory exchange ratio of 1.16 (significant metabolic acidosis representing good effort), a peak HR of 93% of his age-predicted value (220-age), an anaerobic threshold (AT) of <40% of the predicted peak V̇O2, an O2 pulse at peak exercise (surrogate of peak stroke volume) of 58% of predicted, a ΔV̇O2/ΔWR of 6.4 ml·min−1·Watt−1 (64% of predicted) (2 of 21.3 ml·kg−1·min−1 (55% of predicted)  percent change were calculated by comparing the slope corresponding to the 2 min of exercise prior to test termination (slope 2) to the slope corresponding to the 2 min preceding AT . This quantitative analysis showed a 286% steepening in ΔHR/ΔWR in slope 2 compared to slope 1 and a 105% decrease in O2 pulse using the same slope comparison process, suggesting an underlying cardiac dysfunction. CPET slopes , with his father having a myocardial infarction before 50 years of age. The participant was seen by a pediatric primary care provider for further evaluation. A resting EKG was performed which showed normal sinus rhythm and no further testing was recommended by the provider. Thus, there was no intervention for the case. Unfortunately, we were not in a position to influence this decision, as it is our opinion that the case adolescent did not get sufficient thorough follow-up testing which should go beyond resting EKG measurements, as further discussed below.Because of the irregular findings reported here, early serum sample analyses were done for the case and a control participant. It was found that the case participant's serum hs-CRP was elevated at 5.3 mg/L vs. 0.1 mg/L in the control participant  .Previous CPET research has mostly focused on symptomatic individuals and few investigative studies have been published utilizing CPET to assess the cardiovascular and pulmonary function in asymptomatic at-risk individuals . However2/ΔWR, and peak V̇O2  — all w disease , 10. Int disease  detailedObesity is characterized with a low-grade systemic inflammation. However, as an adjunct screening tool to lipid screening, the predictive role of hs-CRP in the future occurrence of cardiovascular diseases in at-risk adults has been recognized . This is2 pulse in asymptomatic middle-aged adults found it was associated with cardiovascular risk factors  and specificity (98% vs. 55%) in detecting and excluding exercise-induced myocardial ischemia when nuclear SPECT imaging and coronary angiography were used to determine true CAD . Doppler2 and O2 pulse (stroke volume) as percent of predicted values may be used to assess to what extent the dysfunction is affecting health status. In general, peak V̇O2 and O2 pulse in the range of 70%–84% of predicted is classified as a mild cardiopulmonary impairment. However, if peak V̇O2 and O2 pulse are moderately to severely affected (<70% of predicted), a referral to a pediatric cardiologist could be considered for further assessment and treatment as needed. Serial CPET testing can be utilized to assess the effectiveness of the intervention with the goal of improving peak V̇O2 and O2 pulse compared to the initial CPET result. Even if a definitive diagnosis cannot be reached after a thorough clinical evaluation, an abnormal CPET result should not be ignored as it may be an early sign of an evolving disease process. Lifestyle interventions utilizing structured diet and individualized exercise prescriptions to target modifiable cardiovascular risk factors may prove to reduce the inflammatory environment and has the potential to normalize the cardiac dysfunction if the driver of the dysfunction is in a modifiable state. Serial CPET testing may help clinicians determine the optimum interval for further workup. Information gained from CPET may be a valuable tool for preventive medicine and should be made more accessible for asymptomatic adolescents who have an increased risk for cardiovascular disease. Collaborating with clinical exercise physiologists with experience in CPET testing and interpretation can optimize clinical workflow, and also underscores the important role of exercise physiology in healthcare. Currently these results, since from a unique test, cannot be effectively applied in a universal way due to barriers previously mentioned. However, we urge clinicians and policy makers not to underestimate the risk and consequences of early-age CAD. We call for studies aiming for a better understanding of early-age CAD with a particular focus on investigation of effective early detection testing and diagnoses in at-risk adolescents to inform treatment and reduce major adverse cardiac event risk.In adolescents with strong family histories of CAD, initial screening for cardiac dysfunction using CPET with simultaneous measures of hs-CRP has potential to provide valuable information on the cardiovascular status and prognosis for early-age CAD risk. At-risk adolescents with abnormal CPET responses and elevated hs-CRP should receive a structured management process, as it may indicate early asymptomatic cardiac dysfunction in the presence of a proinflammatory state. Inflammation has a well-established connection with atherosclerosis development in adults, further underscoring the importance of addressing these findings in a timely and appropriate manner. When abnormal CPET responses are revealed, peak V̇OCurrent resting tests and measures are often ineffective at detecting subtle cardiopulmonary abnormalities and cannot detect exercise-induced abnormalities. A detailed assessment of key CPET slopes may enhance sensitivity in detecting abnormalities in asymptomatic at-risk individuals such as the adolescent highlighted in this case report. Serial CPET measures might be important to perform in adolescents with strong family histories of CAD, especially early-age CAD. Research in this at-risk population should strive to better define the clinical role of CPET in the evaluation of cardiac dysfunction for the purpose of improving preventive healthcare. As the use of CPET grows, so will the need to improve healthcare practices and education to incorporate the pathophysiology that CPET may reveal in asymptomatic adolescents at-risk for cardiovascular events.


Revascularization via coronary artery bypass grafting (CABG) to treat cardiovascular disease is established as one of the most important lifesaving surgical techniques worldwide. But the shortage in functionally self-adaptive autologous arteries leads to circumstances where the clinical reality must deal with fighting pathologies coming from the mismatching biophysical functionality of more available venous grafts. Synthetic biomaterial-based CABG grafts did not make it to the market yet, what is mostly due to technical hurdles in matching biophysical properties to the complex demands of the CABG niche. But bacterial Nanocellulose (BNC) Hydrogels derived by growing biofilms hold a naturally integrative character in function-giving properties by its freedom in designing form and intrinsic fiber architecture. In this study we use this integral to combine impacts on the luminal fiber matrix, biomechanical properties and the reciprocal stimulation of microtopography and induced flow patterns, to investigate biomimetic and artificial designs on their bio-functional effects. Therefore, we produced tubular BNC-hydrogels at distinctive designs, characterized the structural and biomechanical properties and subjected them to in vitro endothelial colonization in bioreactor assisted perfusion cultivation. Results showed clearly improved functional properties and gave an indication of successfully realized stimulation by artery-typical helical flow patterns. But CABG also installs an artificial biophysical niche by cross bridging the environments of the ascending aorta and the occluded coronary artery. After revascularization, this artificial niche needs to fulfill a cluster of native biophysical requirements which control the complex myocardial mass transport of nutrients and metabolites down to capillary flow and maintain its vascular biological functions. However, clinical reality proved that the only design to fulfill these demands sufficiently is derived by nature, more precisely the active biological adaption of autologous arteries like the internal thoracic artery (ITA)4. Hence, no other graft, neither autologous veinous grafts like the great saphenous vein (GSV) nor any synthetic material overcomes functional impairments like competitive, low or turbulent flow5, low shear stress5 or overstressing vascular cells, which finally fosters pathological narrowing of the lumen by thrombosis, intimal hyperplasia or atherosclerosis6. In consequence, progress in the development of biomaterials allowing for biomimetic physiological performance of synthetic CABG grafts is highly called for and already created a wide variety of tools to defend causalities for the clinically observed pathologies15.While cardiovascular diseases constantly lead the causes of death worldwide16, tailoring biomimetic stimulation stresses can be realized by controlling mainly (i) elasticity and (ii) form &topography. On this matter, currently the generation of functional synthetic CAGB grafts still is blocked mainly by the unsolved challenge inmatching the biomechanical properties between CABG and the host in (i) and suffers from corresponding disturbances in flow and mechanotransduction18.As the vascular biophysicalniche can be defined by reciprocally involved contact and flow derived stresses, like topography and shear stress, curvature and pressure or stiffness and wall strain15. Consequently, integrated technologies in (i) and (ii) could greatly enhance proceedings towards clinical relevance20. At this level, biological hydrogels derived by growing bacterial nanocellulose (BNC) biofilms hold a naturally integrative character of both (i) and (ii) in its intrinsic architecture22.This makes it a modular technology that elegantly combines all relevant scaffold properties. Furthermore, hydrogel scaffolds can be directly harvested from BNC biofilms, which already closely resemble the typical structure of Extracellular Matrix (ECM).Such BNC hydrogels already passed the classification as a safe biomaterial, which is accepted for clinical use24.Interestingly, technical approaches in (ii) are seemingly much further progressed and provide a variety of structures to hamper the adhesion of thrombotic particles, prevent adverse flow events and guide endothelial colonizationxylinus at the air-liquid culture medium interface25. Kralisch et al. established a bioreactor-based production of medical-grade BNC directly from the bacterial culture broth at the headspace of the bioreactor25. Today this process is employed to produce BNC patches holding medical approval e.g. for wound dressing .It is produced as a nonwoven matrix of a biofilm by cellulose producers as Komagateibacter 19.Basically, the same procedure, but utilizing form giving templates which are mechanically dipped into the culture broth which are then subjected to air incubation at the headspace, to allow the bacteria to transform the wetting into a BNC biofilm, is used to produce tubular BNC nonwovens as vascular scaffolds. This coating technology is called Mobile Matrix Reservor Technology (MMR-Tech) and was developed by Prof. Dieter Klemm 28.Our earlier efforts in adapting MMR-Tech derived vascular BNC scaffolds for CABG grafting again proved biocompatibility, revealed good surgical handling and initially showed a multilayered ingrowth of host cells in a small animal model. But over a series of following large animal studies to assess modifications towards a smoother luminal surface, longer sized grafts continuously showed luminal narrowing29. For BNC Hydrogels the recipe can only be adjusted indirectly by balancing variables and parameters of the bacterial productivity31. Consequently, in this study we want to use the freedom over (i) and (ii) as given by the integral nature of the growing biofilms, by combining the MMR-Tech tools with bioprocess engineering methodology to combine promising biophysical stimulation patterns into integrated multifunctional approaches for CABG prostheses.For further development, the MMR Tech bioreactor delivers mutable tools to set up the properties of the formed intrinsic BNC matrix of the biofilm. This holds a very essential difference to manually produced hydrogels, which properties can be defined directly by ingredients at their recipe35, to prevent cross flow and to provide a cleaning effect36, and further consider that for fiber-scaffolds, larger fibers and denser scaffolds better stimulated endothelial growth and physiology37.Therefore, it appeared obvious to consider the known beneficial effects of mimicking artery typical axial oriented macro and microstructures which have been widely investigated and shown to direct endothelial cell growthA) was to find an integrated biomimetic design that can transfer a combination of these effects to the MMR-Tech technology, and which additionally provides sufficient artery-typical wall shear stress (WSS) stimulation under the artificial CAGB environment. Additionally, we tried to use findings from microfluidics, where angled micro topographies have been shown to induce helical flow patterns for micro-mixing applications38, to introduce helical flow patterns to BNC-CABG grafts via micro-topographic designs into our integrated approach. Inducing helical flow patterns is a relatively new motivation based on the growing evidence about the essential role of helical flow along arterial walls for maintaining its healthy physiology. Significance substantiates increasingly on its function to stabilize the mass transport, suppress adverse flow events, enhance oxygen supply to the wall, prevent the accumulation of low-density lipoprotein (LDL) particles and reduce the adhesion of blood cells39 in arteries of the systemic circulation as well as in coronary arteries41. At the same time spiral flow is under debate to improve the immediate clinical performance of CABG grafts by preventing flow disturbances after anastomosis, but technical realization remained rather elaborate and mostly relies on macroscopic shovels or swirling geometries33.But next to this, the listed beneficial effects on vascular biology and the respectively expected impact on long-term patency, especially findings on reduced energy dissipation by aortic helical flow in vivo, potentially leading to optimal myocardial loading43, drives our interest to find a suitable transfer to BNC-CABG technology. Here, a direct functional link to the biomechanical properties is given, so that both qualities may interact to either correct disturbances or eventually enhance their effects on damping remaining energy dissipation. Therefore, the introduction of helical flow while vectoring endothelial alignment for BNC hydrogel tubes that biomechanically matches to artery typical properties, represents our motivation (B) on finding an integrated artificial design that transforms the artificial CABG niche to the mentioned biofriendly functions. Therefore, we consider our motivation (B) rather as bioinspired than to be biomimetic.Accordingly, our first motivation  strategies. Consequently, we evaluated the standard pO2 gas-mix control cultivation strategy at a setpoint of 4 vs. 10% pO2. First cultivations revealed high formation of BNC at all available surfaces in the bioreactor for the 10% setpoint, while decreasing it to 4 % resulted in largely suppressed BNC formation.Here, the MMR Tech procedure requires conditions that inhibit BNC production in the culture broth but induce it only by air incubation. Consequently, enzymatic cellulose digestion and genetically engineered manipulations e.g. by inducible expression vectors, are not applicable. But the K. 6 cells ml–1 by a 20 ± 3.8-fold expansion in about 300 hours of processing. The process courses showed that after controlling the pO2 robustly at the setpoint for about 24 hours, it dropped down and remained at zero. Here, the range of oxygen feed into the manipulated gas-mix was maxed out and the process turned to stable oxygen limitation processing what then continued BNC suppression and growth-control via the gassing rate. The redox potential kept stable while pO2control but showed a parabolic pattern in the oxygen limitation phase, having its vertex at glucose concentrations below 15 gl–1. Subsequently it decreased again when cS fell below 6 gl–1 and the pH reached values below 4  and – 0.35% strain (R2 = 0.5) per million K. xylinus cells in cX_w. This clearly shows that for physiological wall stresses, the risk of clinically problematic overdistensions leading to aneurism and thrombosis can be counteracted by increasing cX_w.Uniaxial tensile testing of BNC hydrogel tube walls, produced by increasing cues Fig. . Apparenark Fig. a,b and uA) of longitudinally arranged lamellar structures, we chose a regular groove and ridge pattern of 50µm in width and height. Here, the motivation was to try to combine the effects of cell-alignment in oriented clusters at this size41 and impacts on WSS stimulation. The respective form giving templates were prepared by 3D printing and subjected to MMR-Tech coating.To realize the integrated biomimetic approach (B) to combine a guided and vectored cell alignment with inducing helical flow along the wall, we prepared templates using a machine-grinding setup. Here, we aimed to arrange colonized cells to a locally predominantly longitudinally oriented arrangement in a rather acute angle that still can induce spiral flow patterns.To realize the bioinspired approach  biomimetic or (B) bioinspired functional topographies were evaluated on their effectivity by in vitro colonization of endothelial cells and short-term bioreactor assisted perfusion cultivation. Here, after discussion with local clinical perfusionists, the flow was set to 20 ml min–1 representing a low flow that is considered as still acceptable for clinical performance. The resulting flow patterns and the distribution of WSS stimulation were complemented by computational fluid dynamics (CFD). For the topography in (A) this proved 2-fold increased WSS at lamellar structures when compared to smooth surfaces, but decreased stimulation at the groove bottoms  as previously reported41 and integrate regions of increased WSS exposure, targeting to induce artery-typical levels of shear induced anti-thrombotic signaling like e.g. NO production, what also is considered as an one important factor for the clinical success of ITA in contrast to GSV3. But at the same time, the groove cavities showed to reduce flow and WSS stimulation, so our initial design should be further optimized.Thereby, for the integrated biomimetic approach in Balancing of biological effects in future studies will require to use viscous media and comprehensive bioanalytical evaluations. Given the artificial nature of the CABG environment it may be useful to deviate from pure biomimicry and prioritize on significant impacts to clinical functions. Artificial topography designs with distributed but high NO production may be feasible when adverse flow advents can be prevented at the same time.B, the angled topography showed indication to be effective on inducing artery typical helical flow patterns while also guiding a native-like cell shape and arrangement in endothelial colonization. Though the chosen geometry reveals even higher local WSS stimulation at lamellar structures than the longitudinal structures in A, it also shows a large drop for the grooves and therefore should be subjected to comprehensive further optimization as well. Nevertheless, after perfusion cultivation, colonized groove structures still showed a pronounced longitudinal shape of endothelial cells and a vectored alignment to large scale interconnected cell clusters. Interestingly, when contrasted to cardiac cells which populated the spatially highly organized myocardial extracellular matrix, the OD´s in both topographies, A and B show an increase of about 50 % in the resulting orientation density after colonization52.Following this idea generally in our bioinspired approach in 54, these approaches can be combined in following studies. Further, the low wall shear stress stimulation by the low media viscosity and low flow rates blocks in depth investigations of cellular adaption towards an atherogenic niche. This is warranted to be investigated in comprehensive future studies as well.Of course, as this study mainly investigates the integrated impact of the biophysical properties of a scaffold, the presented functional evaluations still hold several limitations, especially regarding the translation towards its clinical relevance. In this context, we used a high cell density seeding method to observe the effects of the topography to endothelial alignment and induced flow in short model cultivations. Therefore, the simulation of in vivo like dynamics of endothelial growth is excluded from the study. But as the aspect of attracting cells in the clinical environment is investigated in parallel research of our group by post production modificationsxylinus bacteria were pre-cultivated using standard microbiological techniques. Therefore, colonies were grown on Hestrin Schramm (HS) agar plates and subsequently subjected to a series of pre-cultivations in test tubes and Erlenmeyer flasks before banking them in 50% glycerol on – 80 °C for further inoculations . For standard MMR-Tech BNC inoculum preparation, aliquots then were given to 500 ml Hestrin Schramm media in an Erlenmeyer flask.BNC producing Komagateibacter 2 –, pH -, and redox-potential, BlueSens off gas sensors for CO2 and O2  and a clamp on heating jacket.Dynamic bioreactor assisted mass cultivation was realized using an Infors HT Multifors  bioreactor system providing two parallel glass vessels of 1 liter working volume. The system was equipped by blade stirrers (removed after cultivation 1) rings sparger for gassing, in-line sensors for temperature, pO2 value was decreased to zero by nitrogen gassing. After inoculation, pO2-gasmix control was started at a setpoint of 4% and a fix gassing rate. Cultivations were sampled daily and stopped after about 300 hours.Before inoculation by adding 2 to 4 of the banked std-aliquots to 1 liter HS media, the pOFor harvesting, the culture broth was centrifuged at 2800 rpm and 4 °C using 50 ml falcon tubes. Cell pellets were pooled and re-suspended in 50 ml of 50 % glycerol HS media. 5 ml each were frozen at – 80 °C for usage as high cell density aliquots (hc-aq).Thereby, we ran a series of 6 cultivations under full analytical monitoring.X_w. Thereby, for each production process a batch 5 liters of HS media was inoculated and given to the bioreactor. The bioreactor system runs a series of mechanically dipping the installed arrangement of form giving templates to the culture broth and incubating the wetted templates against air. Induced by the high oxygen availability, K. xylinus bacteria then transform the fluid film of culture broth into a BNC stabilized biofilm. For standard productions, bamboo rods of 5 mm diameter were used as templates. Productions for tubes of type A  were equipped by self-prepared templates of medical grade 3D printing filament and by stainless-steel rods for type B (angled micro topography).Tubular BNC hydrogels were produced using one unit of the MMR-Tech bioreactor system . Inoculations were prepared by 500 ml of HS media and either a varying number of std-aliquots for standard productions, or hc-aliquots for productions at high working cell densities cX_wFor all test, critical points at ultimate tensile stress UTS and ultimate strain US werepaired to the respective cX_W and used for correlations using excel.For uniaxial tensile testing of BNC tubes, a Tira Test machine was equipped with a 50 Newton force sensor. Tubes of approximately 5 cm length were longitudinally cut open and mounted to the grips by using grained paper strips to avoid slipping. Tensile testing was performed at a speed of 1 mm per second. The geometry of the samples was measured using a scale and used for normalization of distension to strain and measured force to cross-sectional wall stress. Thereby, we analyzed n=5 samples for standard productions under an n=3 for different working cell densities cTemplates for productions of type A tubes were prepared by 3D printing using a Formlabs medical grade printer and belonging surgical guide resin . Therefore, an axially oriented regular groove and ridge pattern design of 50 vs. 50 µm in width and depth was given to a rounded square rod design of about 16 cm length.Templates for production of type B tubes: Stainless steel cylinders of 5 mm diameter and 19 cm length were machine grinded using a self-established setup for micro-shaping. Therefore, the cylinders were axially mounted into a rotatable stand and grinded by fixed movable Dremel in a 60° angle using grain size 60.6 cell ml–1 ingrowth media for the seeding suspension.Commercially available primary human umbilical vein endothelial cells (HUVEC)  were expanded by standard cell culture techniques using Endothelial Cell Growth Media 2 (Promocell)supplemented by endothelial supplement mix, 10% fetal bovine serum and antibiotics. Before reaching a confluency level of 80%, cells were harvested using 1x trypsin-EDTA mix for 3 min at 37 °C and adjusted to density of 1 × 102. This procedure was repeated the next day, before colonized cells were allowed to adhere under static incubation over weekend. Subsequently, the colonized tubes were mounted to a specialized perfusion bioreactor setup  installed inside of a Heracell 240i Cell culture incubator (Thermofischer) and run at 20 ml min–1perfusion flow at 37 °C and 5% CO2. Perfusion was realized using a Reglo ICC peristaltic pump  installed outside of the incubator. Perfusion cultivation then was performed for 4 days.BNC tubes at a length of about 6 cm were used for in vitro colonization. For mounting to the given connectors inside of the perfusion chamber of the bioreactor, male luer-lock connectors were tied to both ends using surgical suture. Tubes were then filled by the seeding suspension and subjected to 6 hours of incubation under stepwise manual axial rotation of 45° in intervals of 45 minutes, at 37 °C and 5% COAfter stopping cultivation, tubes were washed in cold phosphate buffered saline and fixed in 4% buffered paraformaldehyde and 10% sucrose for 10 minutes. Fluorescent staining of nuclei and cell bodies was realized using combined DNA and RNA staining by acridine orange (AO) according to the manufacture’s recommendations . Fluorescent staining of the cytoskeleton was performed using phalloidin rhodamine (abcam), specifically interacting with F-Actin according to the manufacturer’s recommendations. Microscopic evaluation was done using an Evos FL Cell Imaging System (Thermofischer).–1.3D models of BNC tubes in Type A and B were designed using the commercial software AutoCAD and imported to STARCCM+ (CFD software from Siemens PLM) to conduct the simulations. Then STARCCM+ was used to generate the mesh, solve, undertake post processing and obtain the results. Based on the low Reynolds number, a laminar flow model was selected with steady state flow conditions to perform all the simulations. Special attention was given to estimate the respective distributions of WSS and flow velocities under perfusion at 20 vs 100 ml minThe shaping of single cells and cell clusters was evaluated using ImageJ particle analysis of stained cell bodies at microscopic images. Therefore, first scale bars were used for calibration and images were prepared by background subtraction and thresholding. Thereby, resulting particles represented single cells or aligned clusters. Circularity, surface area and overall orientation angle of particles were analyzed and used for the evaluation of the distributions.51. Briefly, the orientation angle of shaped cells was used to sort the digitalized cells and estimate the fraction of clearly oriented cells (FO). This fraction was divided by its angle range to estimate the O.D. to indicate the share of cells per degree of orientation that is trusted to represent the bulk orientation.The orientation density (O.D.) was calculated as previously described–1.We analyzed a n = 3 respectively for colonized and perfused BNC tubes of Type A and an n = 4 for tubes of Type B. Thereof, two tubes were colonized by only one repetition of cell seeding and perfused by 10 ml minNo primary human or animal material was used. The used cell line was commercially acquired.