Biomarkers In Biopsies Research Articles

Circulating cell-free messenger RNA enables non-invasive pan-tumour monitoring of melanoma therapy independent of the mutational genotype.

Plasma-derived tumour-specific cell-free nucleic acids are increasingly utilized as a minimally invasive, real-time biomarker approach in many solid tumours. Circulating tumour DNA of melanoma-specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow-up of all genotypes, including wild-type. We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N=175 melanoma, N=20 normal skin, N=6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N=18 melanoma, N=8 benign nevi). Circulating cell-free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR. KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients' and healthy donors' plasma (AUC > 86%, p<.0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re-analysis of single-cell transcriptomes revealed a pan-tumour origin of cfRNA, including endothelial, cancer-associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression-free survival (PFS) (KPNA2 HR = .54, p=.0362; DTL HR = .60, p=.0349) and overall survival (KPNA2 HR = .52, p=.0237; BACE2 HR = .55, p=.0419; DTYMK HR = .43, p=.0393). Lastly, we found that cfRNA copies significantly increased during therapy in non-responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p=.0441) and DTYMK (HR = 1.82, p=.018) cfRNA during therapy was predictive of shorter PFS. In sum, we identified a new panel of cfRNAs for a pan-tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy-monitoring tool independent of the melanoma mutational genotype.

DNA-functionalized covalent organic framework capsules for analysis of exosomes

Exosomes have been deemed as hopeful liquid biopsy biomarkers for malignant tumor identification. However, proposing detection methods with high sensitivity still meets challenges. Herein, we have fabricated an electrochemical biosensor in this work via constructing nucleic acid functionalized covalent organic framework (COF) capsules for sensitive detection of exosomes. Firstly, nucleic acid functionalized COF capsules are prepared by modifying azide-labeled DNA onto alkynyl-containing COF capsules. Then, the capsules are used to fabricate an electrochemical biosensor for exosomes quantification. In this design of the sensor, the modification of DNA strands on COFs can greatly improve the binding efficiency, while the loading of plentiful horseradish peroxidase (HRP) facilitates signal amplification. So, the fabricated biosensor has effectively enhanced the sensitivity and lowered the detection limit to 87 particles/μL. Further studies show that the biosensor can be used for the identification of exosomes from non-small cell lung cancer. Moreover, by simply replacing the DNA sequences, the proposed strategy can generally be used to fabricate different DNA-COF capsules with diverse functions for broader biomedical applications.

Differential expression profiles of circRNAs in cancers: Future clinical and diagnostic perspectives

Since decades ago, circular RNAs (circRNAs) have been known for their critical role in RNA maturation, RNA transportation, epigenetic regulation, gene transcription, peptide/protein translation, and interaction with proteins to modulate their activities. At present, circRNAs are being extensively studied as oncotargets in the onset and development of several malignancies and treatment resistance by modulating various signaling pathways and cellular processes such as ubiquitination, degradation, invasion, proliferation, c-Myc oncoprotein stabilization, epithelial-mesenchymal transition, autophagy, and apoptosis. In addition, circRNAs are known to be highly conserved, have a longer life span than other RNAs, and their differential expressions are implicated in solid tumors and hematological malignancies. However, their potential diagnostic and therapeutic targets are not fully elucidated. Therefore, we sought to underline the opportunities and constraints associated with using these oncotargets in cancer therapy by outlining the functional mechanisms of circRNAs and their ectopic expression in distinct malignancies. The clinical applications of circRNAs in developing progressive markers like liquid biopsy biomarkers and for treating cancers are also prospected in this paper.

A LysLysLys-tag as trigger in polynorepinephrine epitope imprinting: The case study of soluble PD-L1 detection in serum by optical-based sensing

Polycatecholamines (pCAs)-based molecularly imprinted polymers (MIPs) represent the new performing generation of biocompatible ligand/receptor mimetics. In this context, dealing with MIPs synthesis for bio-macromolecules detection/extraction, one of the critical steps in ensuring effective binding affinity for the parent molecule is the selection of suitable epitopes for pCAs imprinting. To address this challenge, here we investigated the ability of lysine (K) residues to trigger the epitope imprinting process into a polynorepinephrine (PNE) matrix. To this aim, we first designed a set of model epitopes composed of three K and six alanine (A) residues to investigate the influence of each ‘KA’ combination on the imprinting process and the resulting binding performance by Surface Plasmon Resonance (SPR). Only the case of three flanking K residues in N-terminus arose as an excellent trigger for epitope imprinting. The efficacy of the 3K-tag strategy was then evaluated on two peptide templates belonging to soluble programmed cell death protein 1 ligand (PD-L1), which is of great interest as a cancer biomarker in liquid biopsies. These templates were selected due to their negligible natural ability to be imprinted into the PNE matrix and were modified with 3K-tags, in N-, C-, and N/C- positions, respectively. The SPR sensor developed by exploiting the N–3K tag strategy allowed us to achieve excellent sensitivity (0.31 ± 0.04 ng mL−1) and repeatability (avCV% = 4.5) in human serum samples. This strategy opens new insights both for epitopes' design for pCAs-based mimetics and as triggering tags when native epitopes display negligible imprinting capabilities.

The Immunohistochemical Expression of the Von Willebrand Factor: A Potential Tool to Predict Kidney Allograft Outcomes.

Few reports assessed endothelial activation biomarkers in kidney allograft biopsies using immunohistochemistry. This retrospective cohort study evaluated the association between posttransplant outcomes and the immunohistochemistry expression of Caveolin-1, Von Willebrand Factor (Vwf), and T-Cadherin in for-cause biopsies diagnosed as interstitial fibrosis and tubular atrophy of unknown etiology. Samples with antibody-mediated changes were excluded. The patients were followed for 3 years after the biopsy or until graft loss/death. Seventy-one (71) samples from 66 patients were included. Eighteen (25.4%) patients lost their grafts, mainly due to chronic rejection (33.3%). Caveolin-1 and T-Cadherin were not associated with graft loss. Vwf had good accuracy in predicting graft failure (AUC 0.637, 95% CI 0.486 to 0.788 P =0.101). The presence of more than 10% of Vwf positivity in the microvasculature (Vwf >10%) was associated with reduced death-censored graft survival (58.2% vs. 85.4% P =0.006), and this result was also observed in the subgroup presenting mild interstitial fibrosis (ci=1) (65.7% vs. 88.6% P =0.033). The multivariate analysis showed that Vwf >10% was an independent risk factor for graft loss (HR=2.88, 95% CI 1.03 to 8.02 P =0.043). In conclusion, Vwf might be an additional tool to predict allograft outcomes in kidney transplant recipients with interstitial fibrosis and tubular atrophy of unknown etiology, probably reflecting immune endothelial activation.

An integrated magneto-fluorescent nanosensor for rapid and sensitive detection of tumor-derived exosomes

Cancer released-exosomes containing specific surface proteins have high value in tumor diagnosis and are considered a novel liquid biopsy biomarker. However, the development of rapid and sensitive quantitative methods for their determination remains challenging because of the low abundance of exosome surface protein. Herein, we developed a new platform using an integrated magneto-fluorescent exosome (iMFEX) nanosensor for rapid and sensitive detection of exosomes derived from cancers. First, magnetic beads were coated with a DNA tetrahedral lipid probe to efficiently capture exosomes. The rationally designed bifunctional aptamer specifically recognized exosomal PD-L1 protein and initiated catalytic hairpin assembly, thereby converting protein signals into H1/H2 duplexes. Finally, the H1/H2 duplexes activated the Cas12a protein trans-cleavage reporter FAM-TTATT-BHQ1 for fluorescence signal amplification. By integrating magnetic separation and fluorescence signal amplification, the proposed iMFEX nanosensor exhibited excellent specificity and sensitivity toward PD-L1 positive exosomes, ranging from 2.86 × 103 to 2.86 × 107 particles/μL, with a detection limit of 1.71 × 103 particles/μL. Using the iMFEX nanosensor, we were able to effectively track the dynamic changes in exosomal PD-L1 expression induced by reagents and to distinguish patients with non-small cell lung cancer from healthy individuals, thus demonstrating the platform’s potential for clinical application.

72P Liquid biopsy biomarkers utility in radiation oncology

Genomics has not yet been integrated into the clinical practice of Radiation Oncology. Liquid biopsy is a non-invasive tool based on the study of circulating tumor DNA (ctDNA), mainly applied to advanced cancer patients treated with chemotherapy. This work assesses the utility of liquid biopsy in early-stage/oligometastatic cancer patients undergoing stereotactic body radiation therapy (SBRT).

Detection of rare CTCs by electrochemical biosensor built on quaternary PdPtCuRu nanospheres with mesoporous architectures

Circulating tumor cells (CTCs) are promising liquid biopsy biomarkers for early cancer detection and anti-cancer therapy evaluation. The ultra-low abundance of CTCs in blood samples requires highly sensitive and accurate detection ways. In this study, we propose the design of a dual-recognition electrochemical biosensor to improve both the specificity and signal response. PdPtCuRu mesoporous nanospheres (PdPtCuRu MNSs) with excellent three dimensions (3D) nanopore structures were synthesized by one-pot method and connected to mucin 1 (MUC1) aptamer to serve as signal amplification probe. Besides, superconductive carbon black, Ketjen Black (KB), and gold nanoparticles (AuNPs) modified organometallic frame (CeMOF-Au) were combined to work as signal transducer. The characteristic branching structure of KB provides abundant contact points to load CeMOF-Au to heighten the interface electron transfer rate. In addition, AuNPs were reduced on the surface of CeMOF, which could effectively bind the capture antibody and further enhance the conductivity. Under the optimized condition, the limit of detection (LOD) of the as-constructed biosensor was less than 10 cells mL−1 for model A549 cells, and showed good specificity and accuracy in spiked serum samples. We envision the as-proposed electrochemical biosensor would alternate as a useful tool for the clinical detection of CTCs for cancer diagnosis.

Circulating microRNAs: Challenges with their use as liquid biopsy biomarkers.

Circulating microRNA (miRNA) is a major focus in liquid biopsy studies. The circulating levels of certain miRNAs have been suggested to reflect specific physiological conditions, and several studies have reported their potential use as biomarkers for the detection and prognosis of cancer, as well as for predicting responses to chemotherapy or radiotherapy. Alongside these biomarker studies, research into the effects of specific background factors on circulating miRNA levels is progressing. Indeed, several studies have shown that a number of factors, including blood sample collection and processing methods, as well as subject-specific factors such as age, sex, and other physiological conditions, can affect the normal levels of circulating miRNAs. Unfortunately, the evidence supporting these effects is not yet strong enough to support a definite conclusion and further research is warranted. Here, we summarize the findings of several studies that have addressed these concerns and identify important topics that should be considered when analyzing circulating miRNA levels in liquid biopsy studies.

Down-regulated lncRNA ROR in tumor-educated platelets as a liquid-biopsy biomarker for nasopharyngeal carcinoma

PurposesTo evaluate the diagnostic value of tumor-educated platelets (TEP) lncRNA ROR for nasopharyngeal carcinoma (NPC).MethodsQuantitative real-time PCR was used to determine the expression level of TEP lncRNA ROR in NPC patients (n = 50) as compared to normal subjects (n = 33). The ROC curve analysis was performed to assess the diagnostic value of TEP lncRNA ROR for NPC. Correlations between TEP lncRNA ROR and clinical parameters were further analyzed.ResultsThe median of TEP lncRNA ROR was significantly lower in NPC patients than that in normal subjects (0.0209 vs 0.0610, p = 0.0019), while no significant difference was found in plasma lncRNA ROR. ROC analysis showed that TEP lncRNA ROR had a sensitivity of 60%, specificity of 70%, and accuracy of 63.9% in diagnosing NPC, and the area under ROC curve (AUC) was 0.70. The expression level of TEP lncRNA ROR in NPC showed no significant difference among different TNM stages. However, low level of TEP lncRNA ROR correlated well with positive Epstein–Barr virus (EBV) DNA (kappa value = 0.314, p = 0.06), TEP lncRNA ROR and EBV DNA had similar diagnostic positive rate (58.3%) for NPC, and the combination of TEP lncRNA ROR and EBV DNA increased the positive rate to 74%.ConclusionThe expression level of TEP lncRNA ROR was down-regulated in NPC and the diagnostic value of TEP lncRNA ROR was similar to EBV DNA. Our study indicated that TEP lncRNA ROR might serve as a novel type of liquid biopsy biomarker in diagnosis of NPC patients.

The role and application of small extracellular vesicles in breast cancer.

Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related deaths in women worldwide. Currently, patients’ survival remains a challenge in BC due to the lack of effective targeted therapies and the difficult condition of patients with higher aggressiveness, metastasis and drug resistance. Small extracellular vesicles (sEVs), which are nanoscale vesicles with lipid bilayer envelopes released by various cell types in physiological and pathological conditions, play an important role in biological information transfer between cells. There is growing evidence that BC cell-derived sEVs may contribute to the establishment of a favorable microenvironment that supports cancer cells proliferation, invasion and metastasis. Moreover, sEVs provide a versatile platform not only for the diagnosis but also as a delivery vehicle for drugs. This review provides an overview of current new developments regarding the involvement of sEVs in BC pathogenesis, including tumor proliferation, invasion, metastasis, immune evasion, and drug resistance. In addition, sEVs act as messenger carriers carrying a variety of biomolecules such as proteins, nucleic acids, lipids and metabolites, making them as potential liquid biopsy biomarkers for BC diagnosis and prognosis. We also described the clinical applications of BC derived sEVs associated MiRs in the diagnosis and treatment of BC along with ongoing clinical trials which will assist future scientific endeavors in a more organized direction.

Investigating the consistency of extracellular vesicle production from breast cancer subtypes using CELLine adherent bioreactors

AbstractExtracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in‐depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV‐associated proteins are relatively consistent, with few minor variations over time, but that tracking the production of EVs is a convenient method to indirectly monitor the bioreactor and consistency of the yielded EVs. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.

Circular RNAs in peripheral blood mononuclear cells are more stable than linear RNAs upon sample processing delay.

Circular RNAs (circRNAs) are a novel class of RNAs with closed loop structure. Blood circRNAs are widely acknowledged to be more stable than linear mRNAs, which show promising prospect to be liquid biopsy biomarkers for clinical applications. However, accumulating studies have demonstrated that sample processing delays have profound effects on blood transcriptome expression profiles, wherein knowledge remains elusive about the impacts of prolonged sample processing on blood expression profiles of circRNAs. We collected whole blood samples from three donors and isolated peripheral blood mononuclear cells (PBMCs) at six different incubation time points. We measured total RNA expression profiles using RNA sequencing (RNA‐seq) and investigated the differentially expressed circRNAs, mRNAs and lncRNAs upon blood processing delay. Meanwhile, we explored the underlying inducement of aberrant expression of circRNAs against their corresponding mRNA transcripts. Finally, we utilized rMATS‐turbo and CIRI‐AS, respectively, to screen out differential alternative splicing (AS) events in linear mRNAs and circRNAs. Sample incubation at 4°C lasting to 48 hours (h) led to minimal effects to circRNAs' expression. However, it induced extensive alterations for mRNAs and lncRNAs when the incubation time was beyond 12 h. Additionally, only 2 h processing delays may result in profound impacts on AS events of linear mRNAs, while less impact on the equivalence of circRNAs. Our results suggested that PBMC circRNAs are stable upon sample processing delay, which are more suitable to be liquid biopsy biomarkers.

Differential proteomic analysis of plasma-derived exosomes as diagnostic biomarkers for chronic HBV-related liver disease

Hepatitis B virus (HBV) infection is still a major public health problem worldwide. We aimed to identify new, non-invasive biomarkers for the early diagnosis of chronic HBV-related diseases, reveal alterations in the progression of chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Here, exosomes were isolated and characterized through size exclusion chromatography and nanoparticle tracking analysis. Profiles of differentially expressed proteins (DEPs) were analyzed through liquid chromatography-tandem mass spectrometry (LC–MS/MS), Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses. Results showed that the DEPs, including CO9, LBP, SVEP1, and VWF levels in extracellular vesicles (EVs) were significantly higher in CHB than in healthy controls (HCs). VWF expression levels in EVs were significantly lower in CHB than in those with LC. KV311 expression levels in EVs were significantly higher, whereas LBP levels were significantly lower in patients with CHB than in those with HCC. All biomarkers seemed to exhibit a high diagnostic capacity for HBV-related liver disease. Patients with HBV-induced chronic liver disease exhibit characteristic protein profiles in their EVs. Thus, serum exosomes may be used as novel, liquid biopsy biomarkers to provide useful clinical information for the diagnosis of HBV-related liver diseases at different stages.

Hierarchical Au nanoarrays functionalized 2D Ti2CTx MXene membranes for the detection of exosomes isolated from human lung carcinoma cells

Exosome is considered an important biomarker of liquid biopsy in early cancer screening, which can reflect the physiological and pathological status of cancer cells. Herein, we construct a novel electrochemical biosensor based on hierarchical Au nanoarray-modified 2D Ti2CTx MXene membranes for sensitive detection of exosomes. Ti2CTx MXene nanosheets were fabricated as the building blocks for preparing 2D membranes as the sensing platform via vacuum filtration. To enhance the conductivity of the MXene membrane, for the first time, hierarchical Au nanoarrays were further deposited in situ on the MXene membrane surface. The combination of MXene membrane with a large specific area and hierarchical Au nanoarrays with excellent conductivity make higher electrocatalytic and more active sites in aptamer immobilization. In this strategy, the composite membrane modified by EpCAM recognized aptamer can specifically capture target exosomes, meanwhile, these target exosomes anchor aptamer for CD63 to further enhance the sensing sensitivity and accuracy of the biosensor. As a result, the biosensor achieved high sensitivity and reliable performance for exosome sensing, with a low detection limit (58 particles/μL) in the linear range of 1 × 102 to 1 × 107 particles/μL. In addition, this biosensor showed satisfactory electrochemical stability and anti-interference ability for the detection of exosomes in real serum samples.

Breast Cancer-Delivered Exosomal miRNA as Liquid Biopsy Biomarkers for Metastasis Prediction: A Focus on Translational Research with Clinical Applicability.

Metastasis represents the most important cause of breast cancer-associated mortality. Even for early diagnosed stages, the risk of metastasis is significantly high and predicts a grim outcome for the patient. Nowadays, efforts are made for identifying blood-based biomarkers that could reliably distinguish patients with highly metastatic cancers in order to ensure a closer follow-up and a more personalized therapeutic method. Exosomes are nano vesicles secreted by cancer cells that can transport miRNAs, proteins, and other molecules and deliver them to recipient cells all over the body. Through this transfer, cancer cells modulate their microenvironment and facilitate the formation of the pre-metastatic niche, leading to sustained progression. Exosomal miRNAs have been extensively studied due to their promising potential as prognosis biomarkers for metastatic breast cancer. In this review, we tried to depict an overview of the existing literature regarding exosomal miRNAs that are already validated as potential biomarkers, and which could be immediately available for the clinic. Moreover, in the last section, we highlighted several miRNAs that have proven their function in preclinical studies and could be considered for clinical validation. Considering the lack of standard methods for evaluating exosomal miRNA, we also discussed the challenges and the technical aspects underlying this issue.

Liquid Biopsy

The molecular landscape of the tumors has been typically established using the surgical or biopsy tissue samples resulting in a sampling bias offering only a single snapshot of tumor heterogeneity from the tissue-based tumor profiles. A rapid understanding of such a bias over the years has helped in procuring a precise portrait of the tumors. This practice has positioned the employability of currently employed molecular analysis of the circulating markers in blood and several other body fluids, such as urine, saliva, and pleural effusions, using liquid biopsies. The genomic profiling of the circulating markers such as circulating circulating tumor DNA (ctDNA), circulating tumor cells, or even RNA, proteins, and lipids as part of exosomes has not only guided the monitoring of response to treatment but also the drug resistance and minimal residual disease. The tumor educated platelets (TEPs) and their biological mechanisms driving the influencing of platelets by tumor cells are beginning to unearth TEPS as dynamically predominant components of liquid biopsy. Here, the biology, methodology, and clinical applications of liquid biopsy biomarkers are highlighted. The article puts forth how technological advances have catapulted cancer diagnosis via liquid biopsy in the last decade to obtain a tumor-derived genetic information for its exploitation toward personalized patient care so that liquid biopsy can come into routine clinical practice.

Abstract 6297: New biomarker in liquid biopsies from breast and ovarian cancers

Abstract Breast and ovarian cancer (BOC) are among the top ten common cancers in females worldwide and remain important causes of death in women. There are shared risk factors between breast and ovarian cancer such as, BRCA1 and BRCA2. Detecting those risk factors are not enough to detect cancer early as approximately half of breast cancers develop in women who have no identifiable breast cancer risk factor other than gender (female) and age (over 40 years). Identification of specific biomarkers in liquid biopsies is required for early detection of BOC. BOC secretes exosomes in large quantities to blood circulation to communicate with different cell types such as immune cells to support cancer growth and metastasis. We detect a significant and readily measurable expression of a specific peptide from the a2 isoform vacuolar ATPase (a2NTD) in the exosomes isolated from the plasma collected from BOC patients. We found that a2NTD is highly expressed in stage III ovarian cancer. In this stage, the tumor spread outside of the abdomen and/or nearby lymph nodes. a2NTD positively correlates with the CA125 levels in plasma. These data show that plasma a2NTD correlates with tumor cell growth and metastasis. In addition, a2NTD associated breast cancer exosome can contribute to the pathophysiology of the disease as it induces PBMCs to secrete cancer promoting factors such as IL-8, CCL-2, and MMP-9. Importantly, those secreted factors promote angiogenesis and tumor cell migration. a2NTD promotes the immunosuppressive properties of neutrophils by stimulating arginase-1 release. Together, this data shows that a2NTD can be a potential diagnostic biomarker and therapeutic target in breast and ovarian cancer. Citation Format: Safaa A. Ibrahim, Valerie Riehl, Kenneth D. Beaman. New biomarker in liquid biopsies from breast and ovarian cancers [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 6297.

Abstract LB551: Critical role of necroptosis in colorectal carcinogenesis

Abstract Background: Unlike apoptosis, necroptosis, a programmed necrosis discovered in the past decade, elicits robust adaptive immune responses, including inflammatory cascade, against bacterial infection. However, no human study has yet examined the role of necroptosis in colorectal carcinogenesis. Methods: To compare the roles of necroptosis (TRPM7, pMLKL) with inflammation (COX-2), apoptosis (BAX, TUNEL) and cell proliferation (Ki67) biomarkers across the colorectal carcinogenesis spectrum, we evaluated the trends of biomarker levels from normal colorectal mucosa, to small adenoma, large adenomas and early stages of CRC in tissue microarray (TMA). Furthermore, we compared whether these biomarkers in rectal biopsies can predict the risk of developing metachronous polyp/adenoma. Results: In TMA, we revealed that levels of TRPM7, did not apparently change from normal mucosa (median=1.1) to small adenoma (median=0.8), but then substantially dropped in large adenoma (median=0.1) only to rise dramatically increased in early colorectal cancer (median=7.4) (P for trend=0.004). pMLKL, tended to decrease during carcinogenesis, although not significantly. Ki67 linearly and significantly increased during carcinogenesis (Ptrend=0.002). Similar to Ki67, levels of BAX and TUNEL increased significantly during carcinogenesis, but BAX started to drop in early carcinogenesis. Thus, compared to TRPM7, a necroptosis biomarker, BAX, an apoptosis biomarker, had an almost completely opposite trend during carcinogenesis. COX-2 intensity in the epithelium, but not the stroma, increased in carcinogenesis (Ptrend=0.01). Of 124/227 participants who had follow-up colonoscopy within 5 years, 69 (55.6%) were identified as having metachronous polyp/adenoma. For single biomarker analysis, overexpression of COX-2 was significantly associated with an increased risk of metachronous polyp OR=3.61 ( 95% CI: 1.05-2.47, Ptrend=0.02) and adenoma (OR=3.58, 95% CI 0.96-3.36, Ptrend=0.04). Higher expression of TRPM7 was associated with risk of recurrent polyp and adenoma in a dose-response manner although it did not reaching statistically significance. According to the results of single biomarker analysis and biological plausibility, we combined COX-2 with two necroptosis biomarkers, i.e., pMLKL, TRPM7 and two apoptosis biomarkers (BAX and TUNEL) to generate composite index scores using factor analysis. Several of these composite scores were strongly associated with a 3- to 5-fold increased of metachronous polyp and/or adenoma risk in the highest quartile including COX-2 with pMLKL, COX-2 and TRPM7, BAX and TRPM7 and COX-2 with BAX. The results were similar after the exclusion of recurrence within one year after the completion of the PPCCT. Conclusion: Necroptosis plays a key role in colorectal carcinogenesis. Combining necroptosis biomarkers with inflammation and apoptosis biomarkers can improve the prediction of risk of metachronous polyp/adenoma compared to using necroptosis, inflammation or apoptosis biomarkers alone. Citation Format: Xiangzhu Zhu, Timothy Su, Yong Li, Chang Yu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Reid M. Ness, Martha J. Shrubsole, Qi Dai. Critical role of necroptosis in colorectal carcinogenesis [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 LB551.

Abstract 2203: Towards prediction of breast cancer risk in benign biopsies with high-plex GeoMx spatial protein profiling

Abstract Background: Biopsy diagnoses of benign breast disease (BBD) confer a 1.5- to 4-fold increased risk of developing breast cancer (BC) compared with women without BBD. Previously, we reported that decreased numbers of specific immune cell types in lobules of BBD biopsies predicted increased BC risk, suggesting the promise of future tissue biomarker studies to define BC risk markers among BBD patients. Thus, we applied protein-based GeoMx® Digital Spatial Profiling (DSP) to BBD biopsies preceding BC (cases) and to BBD biopsies from cancer-free patients (controls) to identify possible BC risk markers, which we then evaluated in subsequent BC tissues and in surrounding normal lobules of cases. Methods: Archived pathology slides of BBD biopsies were reviewed and used to guide preparation of TMAs containing 1.0-mm diameter FFPE cores of lobules from an age- and cohort-period-matched set of 91 cases and 88 controls from the Mayo Clinic BBD Cohort. For patients who later developed BC, we prepared TMAs of BC tissue and surrounding mapped normal lobules. We applied GeoMx® DSP (immune and canonical signaling proteins) to both sets of TMAs. Following QC and data normalization, associations of case status with log-transformed biomarker expression in lobules of BBD biopsies were carried out using linear mixed modeling approaches, accounting for multiple ROIs per individual. Biomarkers significantly associated with case status (p&amp;lt;0.05) were further examined in BCs and adjacent normal lobules, using similar approaches. Results: The mean age at BBD biopsy was 52 years, and at BC diagnosis of cases, 61.4 years (mean time from BBD to BC was 10.2 years). A family history of BC was more frequent among cases (70% versus 43%; chi-square p=0.002). Of 72 biomarkers tested, 46 (64.4%) were evaluable after QC and normalization and 5 were associated with BC risk after adjustment for family history of BC: BCL2 (p=0.005), STING (p=0.006), CD44 (p=0.02), S100 protein (p=0.03) and pan-AKT (p=0.05); each showed higher levels in lobules of BBD biopsies of controls than cases. Three unique patterns appeared when examining these biomarkers across tissue type within cases: for BCL2 (p=5 x 10-9) and STING (p=2 x 10-19), levels were high in both BC and lobules surrounding BC but low in preceding BBD; for GAPDH (p=4 x 10-53) and pan-AKT (p=2 x 10-33), levels were high in BC, low in preceding BBD, and moderate in lobules surrounding BC; and for CD44 (p=2 x 10-6) and S100B (p=2 x 10-49), levels were low in BC, high in lobules surrounding BC and moderate in preceding BBD. Conclusions: Using a novel TMA of lobules in combination with DSP, we preliminarily identified immune-based and PI3 kinase-related protein biomarkers in BBD biopsies associated with BC risk. In case-only analyses, these markers demonstrated complex differences between lobules in BBD biopsies, subsequent BCs and adjacent normal lobules. Citation Format: Robert Alan Vierkant, Jodi M. Carter, Stacey J. Winham, Chen Wang, Jennifer M. Kachergus, Ji Shi, Raymond M. Moore, Bryan M. McCauley, Laura M. Pacheco-Spann, E A. Thompson, Derek C. Radisky, Amy C. Degnim, Mark E. Sherman. Towards prediction of breast cancer risk in benign biopsies with high-plex GeoMx spatial protein profiling [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 2203.