Postsurgical Samples Research Articles

CABG Patients Develop Global DNA Hypermethylation, That Negatively Affect the Mitochondrial Function and Promote Post-Surgical Cognitive Decline: A Proof of Concept in Small Cohort.

Global DNA hypermethylation and mitochondrial dysfunction are reported to be associated with the development of mild cognitive decline (MCI). The present study aims to generate preliminary data that connect the above association with post-surgical coronary artery bypass grafting (CABG) cognitive decline in patients. Data were collected from 70 CABG patients and 25 age-matched controls. Cognitive function was assessed using the Montreal Cognitive Assessment (MOCA) test on day 1 (before surgery) and on the day of discharge. Similarly, blood was collected before and one day after the CABG procedure for mitochondrial functional analysis and expression of DNA methylation genes. Test analysis score suggested 31 (44%) patients had MCI before discharge. These patients showed a significant decrease in complex I activity and an increase in malondialdehyde levels (p < 0.001) from the control blood samples. Post-surgical samples showed a significant reduction in blood MT-ND1 mRNA expression from control and from pre-surgical samples (p < 0.005), along with elevated DNMT1 gene expression (p < 0.047), with an insignificant increase in TET1 and TET3 gene expression. Correlation analysis showed a significant positive relation between cognitive decline and elevated blood DNMT1 and declined blood complex I activity, signifying that cognitive decline experienced by post-surgical CABG patients is associated with increased DNMT1 expression and declined complex I activity. Based on the data, we conclude that both DNA hypermethylation and mitochondrial dysfunction are associated with post-CABG MCI, where the former is negatively correlated, and the latter is positively correlated with post-surgical MCI in CABG cases. Additionally, a multimarker approach that comprises MOCA, DNA methylation, DNMT, and NQR activities can be utilized to stratify the population that is sensitive to developing post-CABG MCI.

Effect of dynamic circulating tumor DNA minimal residual disease detection on recurrence surveillance and second resection rate after curative intent resection of colorectal liver metastasis: A prospective cohort study.

242 Background: Recurrence rates after curative intent resection of colorectal cancer liver metastases (CRLM) are nearly 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging and CEA measurements. However, recurrence detection often happens too late to resume a second resection. Previous studies have shown that circulating tumor DNA (ctDNA) detection, as a biomarker of minimal residual disease (MRD), is a robust prognostic factor for patients with colorectal cancers. Here, we performed a prospective study to explore the potential use for improving postoperative CRLM surveillance by dynamic ctDNA detection and its impact on the second resection of recurrent patients. Methods: Enrolled patients received curative intent resection of CRLM and were confirmed at the status of no evidence of disease (NED) by CT about one month after surgery. All patients received stand-of-care postoperative surveillance and serial plasma samples were collected, including presurgical and postsurgical samples (4-6 weeks after surgery) and every 3rd month until relapse or up to 24 months after resection. A personalized tumor-informed assay (Signatera/HuaJianwei bespoke multiplex-PCR NGS) was used to quantify ctDNA in plasma samples. Results: A total of 318 plasma samples from 60 patients recruited from July 2020 to July 2021 were included in our analysis. Twenty-five (41.6%) patients received neoadjuvant chemotherapy, and 46 patients (76.7%) received adjuvant chemotherapy after surgery. The median follow-up time was 18.0 months (range, 7.5 to 25.3 months). ctDNA was detectable in 58/60 (96.7%) patients before surgery, in 35/60 (58.3%) patients after surgery, and in 23/58 (39.7%) patients at the end of treatment (EOT). Patients with ctDNA/MRD positive status post-surgery or EOT experienced a significant shorter recurrence-free survival (RFS) than patients with negative ctDNA/MRD [hazard ratio (HR) 4.2; P &lt; 0.001 and HR 9.8; P &lt; 0.001]. The recurrence rate in MRD-positive patients (aka. positive predictive value, PPV) was 91.4% (32/35) at post-surgery and 91.3% (21/23) at EOT, respectively. In 93.0% of postoperative MRD-positive patients, ctDNA detected early relapse with a lead time up to 9.0 months (median 3.4 months) compared with standard radiological imaging. Among 40 patients with recurrence, the total secondary resection rate was 52.5% (21/40) with 40% (21/40) cases reached secondary NED. While among 24 recurrent cases with only liver metastases, the secondary resection rate was 66.7% (16/24) with 58.3% (14/24) cases reached secondary NED. Conclusions: Postoperative ctDNA status is strongly associated with shorter RFS. Serial ctDNA monitoring assists early detection of relapse, and therefore, improves patient outcomes by secondary resection. Clinical trial information: ChiCTR2000035677 .

The utility of serum osteopontin levels for predicting postoperative complications after colorectal cancer surgery.

Osteopontin (OPN) is a secretory glycoprotein, which is expressed not only in osteoblasts, but immune cells including macrophages and activated T cells. Its pleiotropic immune functions, such as bone remodeling, cancer progression, immune response, and inflammation have been reported previously. However, the association between OPN and postoperative complications (POC) after colorectal cancer (CRC) surgery has not been studied, so far. Peripheral blood samples were collected before (pre) and immediately after surgery (post), and on postoperative days (POD) 1, 3, 5, and 7. Serum OPN levels were measured by ELISA. In total, 78 patients who underwent elective CRC surgery were divided into the No-POC (n = 54) and POC (n = 24) groups. The POC group had significantly higher OPN levels than the No-POC group throughout the postoperative observation period. The maximum OPN levels from pre- to postsurgical samples showed the best predictive potential for POCs (cut off: 20.75ng/mL, area under the curve: 0.724) and were correlated with length of postoperative stays. OPN values were significantly correlated with C-reactive protein on POD3 and were identified as an independent predictive marker for POCs (odds ratio: 3.88, 95% CI: 1.175-12.798, P = 0.026). The severity of POCs was reflected in increased OPN levels. Increased postoperative OPN was associated with increased postoperative inflammatory host responses and POC after CRC surgery. Serum OPN level may be a useful biomarker for early prediction of POC and it may provide additional information for treatment decisions to prevent POC.

Abstract 2796: ctDNA prognosis in adjuvant triple-negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, even in early stages. Evidence of molecular residual disease (MRD), after treatment with curative intent (surgery, chemotherapy), predating macroscopic recurrence can provide rationale for early therapeutic intervention, potentially improving patient outcomes. Longitudinal evaluation of circulating tumor DNA (ctDNA) is emerging as a promising early marker of treatment efficacy and recurrence, validated to pre-date recurrence by radiological imaging. However, data in early TNBC (eTNBC) are limited. Here we investigate the prognostic value of longitudinal ctDNA monitoring in eTNBC patients post-surgery and after adjuvant chemotherapy (ACT) using a custom bespoke ctDNA assay. Methods: Tumor tissue and longitudinal post-surgical plasma samples were collected and analyzed from 186 patients enrolled in the phase 3 BEATRICE clinical study (NCT00528567). Samples from each patient were whole exome sequenced to identify up to 16 SNVs for ctDNA monitoring. ctDNA status was measured longitudinally and correlated with baseline prognostic factors as well as invasive disease free survival (iDFS) and overall survival (OS). Results: Baseline ctDNA (b-ctDNA), evaluated post-surgery and prior to chemotherapy was detected in 19.9% (36/181) of patients. b-ctDNA was positively associated with large tumors and lymph node (LN) involvement, and negatively correlated with presence of stromal tumor infiltrating lymphocytes (TILs). b-ctDNA presence was a stronger predictor of shorter IDFS and OS compared to LN involvement (HR IDFS: 4.36 [2.47-7.7] vs 1.86 [1.08-3.19]; HR OS: 4.01 [1.6-10.07] vs 2.89 [1.39-6]]), respectively). Remarkably, b-ctDNA prognostic value was restricted to LN+ pts (HR IDFS: 10.94 [3.2-37.41]) vs LN- pts (HR IDFS 1.61 [0.49-5.36]). ctDNA positivity after ACT was observed in 21.5% (40/186) of patients and was associated with reduced IDFS and OS (HR: 8.36 [4.62-15.1] and 18.45 [6.79-50.17]), independent of LN involvement. Upon chemotherapy treatment, the median time to first ctDNA positivity occurrence was 13 months (range 3-42.3 mo) and the median lead time from ctDNA detection to radiographic recurrence was 6.1 months (range 0-30.5 mo). Akaike information criterion (AIC, p&amp;lt;0.05) indicated that ctDNA detected post-surgery or post-chemotherapy identifies patients at the highest risk of disease progression even after adjustment for LN and TILs status as well as the tumor size. Conclusions: ctDNA, both at post-surgery and post-ACT, provides additional prognostic value beyond the known risk factors of LN involvement, tumor size and TILs. Post-op ctDNA+ provides an independent and stronger indicator of poor prognosis than any other evaluated baseline covariates. Our data show that TNBC pts that are ctDNA+ post-surgery are at the highest risk of recurrence and death and are underserved by current SOC treatment. Citation Format: Luciana Molinero, Derrick Renner, Hsin-Ta Wu, Nina Qi, Rajesh Patel, Ching-Wei Chang, Himanshu Sethi, Alexey Aleshin, Carlos Bais, David Cameron. ctDNA prognosis in adjuvant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2796.

Analysis of Septin 9 Gene Hypermethylation as Follow-Up Biomarker of Colorectal Cancer Patients after Curative Surgery.

Background: The Septin 9 test analyzes the methylation status of the SEPT9 gene, which appears to be hypermethylated in patients with colorectal cancer (CRC). This has been validated as a colorectal cancer screening test. Due to the high sensitivity and specificity found, the justification was to use it as a biomarker tool for monitoring minimal residual disease after radical surgery and recurrence. Methods: A prospective study was carried out at the Fundación Jiménez Díaz University Hospital extracting peripheral blood from 28 patients and 4 healthy donors. Free circulating DNA was obtained and subsequently a PCR reaction to quantify the number of methylated genes. Samples were obtained preoperatively and postoperatively at five to seven days, one and three months after surgery. Results: A total of 32 preoperative samples were analyzed. The sensitivity of the test to detect CRC was 55.6% and specificity was 100%. There were 22 postsurgical samples obtained at 5–7 days after surgery, the sensitivity to detect tumor recurrences was 100% and specificity was 75%. There were 21 samples analyzed 1 month after surgery exhibiting a sensitivity and specificity of 100% and 94.7%, respectively. At 3 months, 31 postsurgical samples were analyzed and the sensitivity and specificity were 66.7% and 80%. Conclusions: Detection of methylation of Septin 9 gene in circulating plasma DNA, obtained from a peripheral blood sample, may be a useful, non-invasive and effective method for detecting minimal residual disease and could therefore predict CRC tumor recurrences. The optimal time in our series to obtain the best prediction results based on Septin 9 methylation levels was one month after surgery. Despite these considerable findings, a study with more patients is necessary to obtain more robust conclusions.

Characterization of circulating molecules and activities in plasma of patients after allogeneic and autologous intraoral bone grafting procedures: a prospective randomized controlled clinical trial in humans

BackgroundThe objective was to assess whether intraoral bone augmentation procedures have an impact on the patient’s plasma levels of circulating nucleic acids, exosomes, miRNA levels and caspase activities. The null hypothesis was tested, that no significant differences between the two groups will be found.MethodsIn this prospective randomized controlled clinical trial 35 systemically healthy non-smoking participants were randomly allocated using sealed envelopes by a blinded clinician not involved in the clinical setting. Plasma samples were collected preoperatively and 3 times postoperatively (immediately, 5 weeks and 4 months postoperatively). The test group consisted of twenty-five patients who received allogeneic bone grafting material and the control group of ten patients who received autologous bone grafts. Levels of cell-free DNA (cfDNA) and microRNAs (miR-21, miR-27a, miR-218) were quantified by real-time PCR, caspase activities and exosome concentrations were determined by ELISA.ResultsStatistical evaluation reveled a significantly higher exosome level before surgery (p = 0.013) and the first postsurgical sample (p = 0.017) in the control group compared to the test group. The levels of miR-27a and miR-218 significantly differed between the plasma samples before surgery and after surgery in both groups. The levels of miR-21 only significantly differed between the pre- and postsurgical plasma samples in the test group, but not in the control group. All patients completed the study, no adverse events were recorded.ConclusionsOur data show the diagnostic potential of the plasma levels of miR-27a, miR-218 and miR-21 in detecting changes in bone metabolism after alveolar bone augmentation. Our very promising results indicate that there might be a high diagnostic potential in evaluating the plasma levels of the before mentioned miRNAs in order to detect bone resorption activities before they become clinically relevant.Trial registration Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) on 11/03/2016 as well as by the German Registry of Clinical Studies (DRKS 00,013,010) on 30/07/2018 (http://apps.who.int/trialsearch/).

Ultrasensitive detection of BRAF mutations in circulating tumor DNA of non-metastatic melanoma

BackgroundImplementation of adjuvant therapies in non-metastatic melanoma improved treatment outcomes in some patients; however, adjuvant therapy can be associated with significant cost and risk of toxicity. Therefore, there is an unmet need to better identify patients at high risk of recurrence.Patients and methodsWe carried out an ultrasensitive droplet digital PCR (ddPCR)-based detection of BRAFV600E-mutated circulating tumor DNA (ctDNA) from blood samples prospectively collected before surgery, 1 hour after surgery, and then serially during follow-up.ResultsIn 80 patients (stages ≤III), BRAFV600E mutations were detected in 47.2% of tissue, in 37.7% of ctDNA samples collected before surgery, and in 25.9% of ctDNA samples collected 1 hour after surgery. Patients with detected ctDNA in blood collected 1 hour after surgery compared to patients without detected ctDNA had higher likelihood of melanoma recurrence (P < 0.001) and shorter median disease-free survival (P = 0.001) and overall survival (P = 0.003).ConclusionsUltrasensitive ddPCR can detect ctDNA in pre- and post-surgical blood samples from patients with resectable melanoma. Detection of ctDNA in post-surgical samples is associated with inferior treatment outcomes.

176P Type of TP53 alteration may correlate with the extent of lymphocytic infiltration in high-grade serous ovarian carcinomas

176P Type of TP53 alteration may correlate with the extent of lymphocytic infiltration in high-grade serous ovarian carcinomas

High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

BackgroundDNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery.ResultsThe MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR.ConclusionsThe MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

Perioperative circulating tumor DNA as a potential prognostic marker for operable stage I to IIIA non-small cell lung cancer.

Circulating tumor DNA (ctDNA) has emerged as a noninvasive biomarker for dynamically monitoring tumors. However, published data on perioperative ctDNA in patients with operable non-small cell lung cancer (NSCLC) are currently limited. This prospective study recruited 123 patients with resectable stage I to IIIA NSCLC. Preoperative and postoperative plasma samples and tumor tissue samples were subjected to next-generation sequencing with a panel of 425 cancer-related genes. Peripheral blood samples were collected before surgery, postoperatively within 1 month, and every 3 to 6 months for up to 3 years. After 4 exclusions, 119 eligible patients were enrolled from June 2016 to February 2019. Presurgical ctDNA was detectable in 29 of 117 patients (24.8%) and was associated with inferior recurrence-free survival (RFS; hazard ratio [HR], 2.42; 95% CI, 1.11-5.27; P = .022) and inferior overall survival (OS; HR, 5.54; 95% CI, 1.01-30.35; P = .026). Similarly, ctDNA was detected in 12 of 116 first postsurgical samples (10.3%) and was associated with shorter RFS (HR, 3.04; 95% CI, 1.22-7.58; P = .012). During surveillance after surgery, longitudinal ctDNA-positive patients (37 of 119; 31.1%) had significantly shorter RFS (HR, 3.46; 95% CI, 1.59-7.55; P < .001) and significantly shorter OS (HR, 9.99; 95% CI, 1.17-85.78; P = .010) in comparison with longitudinal ctDNA-negative patients. Serial ctDNA detection preceded radiologic disease recurrence by a median lead time of 8.71 months. These results suggest that perioperative ctDNA analyses can predict recurrence and survival, and serial ctDNA analyses can identify disease recurrence/metastasis earlier than routine radiologic imaging in patients with resectable NSCLC. The utility of serial circulating tumor DNA (ctDNA) monitoring for predicting disease recurrence and survival for early-stage non-small cell lung cancer (NSCLC) has not been well characterized. The detection of ctDNA before and after surgery is associated with the identification of a high risk of disease recurrence and long-term patient outcomes for resectable NSCLC. Perioperative ctDNA analyses identify disease recurrence earlier than routine radiologic imaging. ctDNA analyses can detect minimal residual disease for resectable NSCLC and thus can facilitate early intervention.

Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study.

BackgroundIn patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study.Methods and findingsWe prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient’s tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients’ tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/− adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing.ConclusionsWe confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM.Trial registrationACTRN12612000345886.

Physiology of Cultured Human Microglia Maintained in a Defined Culture Medium.

Microglia are the primary immune cell of the CNS, comprising 5-20% of the ∼60 billion neuroglia in the human brain. In the developing and adult CNS, they preferentially target active neurons to guide synapse maturation and remodeling. At the same time, they are the first line of defense against bacterial, fungal, and viral CNS infections. Although an extensive literature details their roles in rodents, less is known about how they function in humans because of the difficulty in obtaining tissue samples and the understandable inability to extensively study human microglia in situ. In this study, we use recent advances in the study of brain microenvironments to establish cultures of primary human microglia in a serum-free medium. Postsurgical samples of human brain were enzymatically and mechanically dissociated into single cells, and microglia were isolated at high purity by positive selection using CD11b Ab-coated microbeads. The CD11b+ cells were plated on poly-l-lysine-coated surfaces and bathed in serum-free DMEM/F12 supplemented with three essential components (TGF-β, IL-34, and cholesterol). Under these conditions, microglia assumed a ramified morphology, showed limited proliferation, actively surveyed their surroundings, and phagocytosed bacterial microparticles. In the presence of LPS, they assumed a more compact shape and began production of proinflammatory cytokines and reactive oxygen species. LPS on its own triggered release of TNF-α, whereas release of IL-1β required costimulation by ATP. Thus, human microglia maintained in a defined medium replicate many of the characteristics expected of native cells in the brain and provide an accessible preparation for investigations of human microglial physiology, pharmacology, and pathophysiology.

A microbial signature following bariatric surgery is robustly consistent across multiple cohorts

ABSTRACT Bariatric surgery induces significant shifts in the gut microbiota which could potentially contribute to weight loss and metabolic benefits. The aim of this study was to characterize a microbial signature following Roux-en-Y Gastric bypass (RYGB) surgery using novel and existing gut microbiota sequence data. We generated 16S rRNA gene and metagenomic sequences from fecal samples from patients undergoing RYGB surgery (n = 61 for 16S rRNA gene and n = 135 for metagenomics) at pre-surgical baseline and one, six, and twelve-month post-surgery. We compared these data with three smaller publicly available 16S rRNA gene and one metagenomic datasets from patients who also underwent RYGB surgery. Linear mixed models and machine learning approaches were used to examine the presence of a common microbial signature across studies. Comparison of our new sequences with previous longitudinal studies revealed strikingly similar profiles in both fecal microbiota composition (r = 0.41 ± 0.10; p < .05) and metabolic pathways (r = 0.70 ± 0.05; p < .001) early after surgery across multiple datasets. Notably, Veillonella, Streptococcus, Gemella, Fusobacterium, Escherichia/Shigella, and Akkermansia increased after surgery, while Blautia decreased. Machine learning approaches revealed that the replicable gut microbiota signature associated with RYGB surgery could be used to discriminate pre- and post-surgical samples. Opportunistic pathogen abundance also increased post-surgery in a consistent manner across cohorts. Our study reveals a robust microbial signature involving many commensal and pathogenic taxa and metabolic pathways early after RYGB surgery across different studies and sites. Characterization of the effects of this robust microbial signature on outcomes of bariatric surgery could provide insights into the development of microbiome-based interventions for predicting or improving outcomes following surgery.

Regulation of Cortisol in Patients Undergoing Total Joint Arthoplasty.

Osteoarthritis is a condition in which joint cartilage and bone degenerate progressively over time. Total joint arthroplasty is a definitive treatment. Cortisol is a hormone that is associated with pain and inflammation. This study aims to investigate the cortisol levels in patients undergoing total joint arthroplasty. Plasma samples were collected from 71 total joint arthroplasty (TJA) patients at baseline (pre-surgery), 24 hours post-operation, and 5 days post-operation. Cortisol levels were measured in each sample using a commercially available ELISA kit. All results were compiled as group means ± SD. The plasma cortisol level at baseline were 218.5 ± 12 ng/mL. The 24-hour post-surgical samples showed a marked increase in cortisol levels 240.7 ± 15 ng/mL. The blood samples drawn at the 5th day after surgery showed a downward trend (74 ± 12 ng/mL). At 5 days post-operation, cortisol levels were significantly lower than at baseline or 24 hours post-operation. These results point to the fact that prior to surgery, the patient’s emotional stress contributes to increased serum cortisol levels. The higher level of cortisol persists at 24 hours post-operation due to inflammation from the procedure. This data also suggests that at 5 days post-operation, the inflammatory response from the surgery and emotional stress subside, resulting in a near normalization of the cortisol levels. Cortisol is a hormone that plays a major role in the body’s response to surgery. The relevance between cortisol and different points in the surgical timeline has the potential to prognosticate and improve recovery measures.

Changes in circulating concentrations of testosterone and estrone sulfate after human chorionic gonadotropin administration and subsequent to castration of 2-year-old stallions

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48–96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred.

Circulating tumor DNA (ctDNA) analysis predicts recurrence following surgery in patients with stage I-IIIA non-small-cell lung cancer (NSCLC): Results of GASTO1035 and GASTO1018.

9023 Background: Circulating tumor DNA can be detected in the plasma and serum of patients with solid tumors and has emerged as a noninvasive biomarker for dynamically monitoring tumor. Postsurgical ctDNA analysis of early-stage NSCLC may identify patients at high risk of recurrence and facilitate early intervention and personalized cancer therapy. Methods: These studies recruited 123 patients with newly diagnosed resectable stage I-IIIA NSCLC. Preoperative and postoperative plasma and postoperative tissue samples were subjected to next-generation sequencing (Nanjing Shihe Jiyin Biotechnology Inc.) using a 425 cancer-related genes panel. Peripheral blood samples were collected before surgery, postoperatively within 1 month, and every 3-6 months for up to 3 years. Plasma samples with at least 1 variants detected in tissue samples were defined as ctDNA positive. Results: After 4 exclusions, 119 eligible patients were enrolled from June 2016 to February 2019. Presurgical ctDNA was detectable in 31 of 117 (26.5%) patients and was associated with inferior recurrence-free survival (HR, 3.90, 95% CI, 1.44-10.58, P = 0.004). Similarly, ctDNA was detected in 13 of 116 (11.2%) of the first postsurgical samples and was associated with shorter RFS (HR, 3.54, 95% CI, 1.22-10.23, P = 0.002). During surveillance after surgery, ctDNA-positive patients (38/119, 31.9%) were more than 9 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 9.17, 95% CI, 2.60-32.42, P &lt;0.001). Serial ctDNA detection preceded radiologic disease recurrence by a median lead time of 4.23 months (95% CI, 0.91-7.54 months). We also observed a positive correlation between the ctDNA detection rate and the disease stage. Conclusions: These results suggest that detection of ctDNA before and after surgery is associated with the identification of a high risk of disease recurrence of resectable NSCLC. Perioperative ctDNA analyses identify disease recurrence earlier than standard-of-care radiologic imaging, and thus could facilitate personalized cancer treatment at early time points. Clinical trial information: NCT03465241 and NCT03172156.

The application of dynamic monitoring of circulating tumor DNA for detecting minimal residual disease and predicting recurrence in colorectal cancer patients.

e15531 Background: Although the prognosis of colorectal cancer (CRC) has improved in the past decade, a subset of CRC patients may still suffer from relapse due to the progression from minimal residual disease (MRD) after surgical resection. A sensitive and non-invasive method to detect MRD and early diagnosis of recurrence disease is warranted to start therapeutic interventions at an earlier timing and improving the overall survival rate. In this study, we have evaluated the feasibility of circulating tumor DNA (ctDNA) analysis to detect MRD and early detection of recurrence in CRC patients. Methods: Plasma samples were collected prospectively from 38 CRC patients (stage I to IV), who underwent surgical resection. Preoperative blood samples were obtained just before surgery and post-operative samples were collected at multiple time-points to monitor the changes of tumor mutation profiles. Tumor-derived mutations were detected in preoperative blood samples as well as surgically resected-tumor tissues using ultradeep targeted next generation sequencing. Patient-paired peripheral blood cells (PBCs) were sequenced concurrently to exclude clonal hematopoiesis-related mutations. Results: Among the 38 patients, 74 non-synonymous mutations were identified in tumor tissues and 64 mutations in the preoperative plasma samples. Paired PBCs sequencing identified 11 mutations in plasma samples to be clonal hematopoiesis-related mutations. After the exclusion of clonal hematopoiesis-related mutations, 34 (89.5%) of the 38 patients harbors at least one somatic mutation either from tumor tissues or plasma samples to be monitored longitudinally. ctDNA was detectable in 5 of 14 (36%) post-surgical samples of patients who did not receive adjuvant chemotherapy and in 9 of 18 (50%) post-chemotherapy samples. Up to date, 6 patients have been detected with clinical recurrence and ctDNA analysis identified all 6 recurrences before imaging. Serial ctDNA analyses were able to detect disease recurrence up to 6 months before imaging tests. Furthermore, all patients that were ctDNA negative post-operative or post-chemotherapy showed no signs of clinical relapse. Conclusions: Our current results indicate that ctDNA analysis allows the detection of MRD in CRC patients. The integration of ctDNA analysis with current standard monitoring guidelines holds great promise in early detection of recurrence to allow clinical intervention to be applied promptly.

Increased Levels of Cortisol in Patients Undergoing Total Joint Arthroplasty may be Associated with Stress and Inflammatory Processes

IntroductionOsteoarthritis is a condition in which joint cartilage and its adjacent bone degenerate progressively over time due to inflammatory processes involving activation of matrix degrading enzymes. Total joint arthroplasty (TJA) is a common surgical treatment for osteoarthritis of the hip or knee. Cortisol is a steroid hormone in the class of glucocorticoids which is produced in the adrenal gland. Cortisol is generated in response to inflammation and physical or emotional stress. This study aims to investigate the cortisol levels in patients undergoing TJA.Materials and MethodsPlasma samples were prospectively collected from 71 TJA patients at baseline (pre‐surgery), 24 hours post‐operation, and 5 days post‐operation. Normal controls represented plasma samples from drug free health individuals (n=50). Cortisol and TNFa levels were measured in each sample using a commercially available ELISA kit. All results were compiled as group means ± SD.ResultsThe plasma cortisol level in normal individuals were 67 ± 11 ng/mL. In contrast, the pre‐surgical and 24‐hour post‐surgical samples showed a marked increase in cortisol levels (&gt; 200ng/mL). The blood samples drawn at the 5th day after surgery showed a downward trend reaching near normal levels (74 ± 12 ng/mL). There was no significant difference between cortisol levels pre‐operatively and within 24 hours post‐TJA, however there was a significant difference in comparison to the levels on the 5th day and normal. The TNFa levels were elevated in TJA patients in the pre‐surgical (18.6 ±3.2 pg/ml), at 24 hours post‐surgical (24.2 ± 7.6 pg/ml) and on the 5th day (11.3 ± 3.1 pg/ml) in comparison to normals (5.1 ± 2.1 pg/ml).Discussion and ConclusionAt 5 days post‐operation, cortisol levels were significantly lower than at baseline or 24 hours post‐operation. This finding differs from previous findings, which reported increased levels post‐operatively relative to at baseline. The TNFa levels also showed elevated levels in the pre‐ and post‐surgical samples which followed a downward trend on the 5th day. These results are consistent with the previous reports that such biomarkers as IL‐6 and TNFa are upregulated in TJA patients prior to surgery suggesting a role of inflammatory process. Furthermore the increased level of cortisol may be related to not only the inflammation but pre‐surgical emotional stress in TJA patients. The near normalization of the cortisol levels on the 5th post‐surgical day is suggestive of differential down‐regulation of the inflammatory process and stress levels in TJA patients.

Impact of successful pediatric ureteropelvic junction obstruction surgery on urinary HIP/PAP and BD-1 levels

In the pediatric patient whose ureteropelvic junction obstruction (UPJO) is not always symptomatic, imaging is the most common means of detecting surgical success. There is interest, however, in other means of post-operative monitoring. A panel of antimicrobial peptides (AMPs) has been previously found to be elevated in UPJO, but the impact of surgical correction on these AMPs is unknown. To determine if elevated levels of candidate urinary AMP biomarkers of urinary tract obstruction decrease following UPJO repair. Pediatric patients undergoing surgical correction of an UPJO were recruited for participation. Bladder urine from uninfected consenting/assenting patients was collected immediately prior to surgery and then at least 6 months afterward. Based on prior studies demonstrating significant elevation of beta defensin 1 (BD-1), hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), cathelicidin (LL-37), and neutrophil gelatinase-associated lipocalin (NGAL) in patients with UPJO versus control patients, we performed enzyme-linked immunosorbent assays on these four AMPs to compare their expression before and after surgical intervention. If found to significantly decrease, AMP levels were compared to healthy controls. AMP levels were normalized to urine creatinine. Results were analyzed with paired t test or Wilcoxon test using Graphpad software. Correlation was calculated using Pearson or Spearman correlation. A p-value of <0.05 was considered significant. 13 UPJO patients were included in this study; 9 were male (69%). Age at surgery was a median of 4.3 years (average 6.1, range 0.4-18.4 years). Follow-up urine samples were collected a median of 27.4 months after surgery (average 27.4; range 7.8-45.3 months). All 13 patients had clinical improvement and/or signs of improved hydronephrosis on post-operative imaging. HIP/PAP and BD-1 significantly decreased in post-surgical samples compared to pre-surgical samples (p=0.02 and 0.01, respectively); NGAL and LL-37 did not significantly change. Overall, HIP/PAP decreased in 12 patients (92%) and BD-1 decreased in 11 patients (85%). BD-1 levels after successful repair were not different from healthy controls (p=0.06). Urinary biomarkers of obstruction should detect significant obstructive pathology as well as reflect its resolution. This would enable their use in post-operative monitoring and augment current methods of determining successful surgical outcome through imaging. The AMPs HIP/PAP and BD-1 are significantly elevated in UPJO but then significantly decrease after pyeloplasty, with BD-1 returning to healthy control levels. As a result, these AMPs could serve as markers of successful surgical intervention.

Abstract OT2-02-03: NSABP FB-13: An assessment of the biological and clinical effects of palbociclib with ovarian suppression and letrozole in the neoadjuvant treatment of pts (pts) with premenopausal (preM) estrogen-receptor positive/HER2-negative primary breast cancer

Abstract Background: There have been few studies of neoadjuvant endocrine therapy in premenopausal (preM) women despite low pathologic complete response (pCR) rates using neoadjuvant chemotherapy in ER+/HER2− pts. Suppression of Ki67, particularly complete cell cycle arrest (CCCA) defined as Ki67 &amp;lt;2.7% after 2 wks of neoadjuvant endocrine therapy has been demonstrated to correlate with recurrence-free survival. PALLET, a randomized, neoadjuvant endocrine trial evaluated letrozole (AI) v palbociclib (pal) in combination with AI for 14 wks in post-menopausal women. This study demonstrated a modest improvement in clinical response (54.3% with the combination v 49.5% with AI alone) but showed a significant difference in achievement of CCCA (Ki67&amp;lt;2.7%) with the combination of pal and AI v AI alone (90% v 59%). We seek to evaluate this combination, given over a longer duration in the neoadjuvant setting, in preM women with early-stage breast cancer. Trial design: FB-13 is a phase II, open-label study to examine the biological and clinical effect of neoadjuvant endocrine therapy with AI, pal, and ovarian suppression in preM pts with ER+, HER2-negative early invasive breast cancer. 76 pts will be stratified into one of two approximately equal sized cohorts based on Oncotype DX Breast Recurrence Score (RS) (cohort 1: pts with RS &amp;lt;11 or cohort 2: pts with RS 11 to &amp;lt;26). A baseline tumor biopsy prior to start of therapy (tx) for RS and Ki67 will both be centrally determined. Pts in both cohorts will receive letrozole 2.5 mg by mouth daily, pal 125 mg by mouth daily for 21 days of a 28-day cycle, and goserelin 3.6 mg SQ injection every 28 days. At wk 6, pts will have core-cut biopsies for real-time evaluation of Ki67. Pts from both cohorts who have Ki67 &amp;lt;10% will continue tx for a total of 6 cycles. Pts with a persistent Ki67 ≥10% will permanently discontinue tx and begin neoadjuvant chemotherapy or proceed to surgery at physician discretion. Eligibility: FB-13 is enrolling preM women newly diagnosed with ER+/HER2− early breast cancer who are candidates for neoadjuvant endocrine therapy. Pts must be preM based on standard definitions. Primary tumor size must be &amp;gt;2 cm. RS must be &amp;lt;26. Mandatory pre-treatment and post-surgical samples will be collected and analyzed for RS and Ki67, as well as additional biomarkers to correlate with response. Blood will be collected at baseline, 4 wks, and at 24 wks for estradiol levels to demonstrate ovarian suppression. A 4-wk estradiol level in the postmenopausal range will be required to receive further treatment. Specific aims: The primary aim is to determine complete cell cycle arrest as indicated by a Ki67 &amp;lt;2.7% at 6 wks. Secondary aims are to determine overall response rate (ORR) by clinical exam and ultrasound, the pCR, to generate a Preoperative Endocrine Prognostic Index (PEPI score), to compare the change in Ki67 at baseline, after 6 wks and 24 wks, and to determine the rate of breast-conservation therapy. Statistical methods: We anticipate that the addition of palbociclib to ovarian suppression and AI will result in a measurable drop in Ki67 and a high ORR. The null hypothesis is ORR &amp;lt;40% and alternative hypothesis is ORR &amp;gt;60%. We anticipate that &amp;gt;60% of pts in each cohort will have 6 wk Ki67 &amp;lt;2.7% and clinical response &amp;gt;35%. Present accrual and target accrual: Currently 12 of a planned 76 pts have been enrolled. NSABP Operations contact information: Diana Gosik, Diana.Gosik@nsabp.org NCT03628066 SUPPORT: Genomic Health Inc, Pfizer, NSABP Foundation Citation Format: Shannon Puhalla, Greg Yothers, Amy P Sing, Thomas B Julian, Norman Wolmark, Samuel A Jacobs. NSABP FB-13: An assessment of the biological and clinical effects of palbociclib with ovarian suppression and letrozole in the neoadjuvant treatment of pts (pts) with premenopausal (preM) estrogen-receptor positive/HER2-negative primary breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-02-03.