Cohort Of Colorectal Cancer Patients Research Articles

Cohort profile: The National Colorectal Cancer Cohort (NCRCC) study in China

PurposeThe National Colorectal Cancer Cohort (NCRCC) study aims to specifically assess risk factors and biomarkers related to endpoints across the colorectal cancer continuum from the aetiology through survivorship.ParticipantsThe NCRCC study...

Identification of liver-derived bone morphogenetic protein (BMP)-9 as a potential new candidate for treatment of colorectal cancer.

Colorectal cancer (CRC) is a high‐incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)‐9 and to find out whether the application of BMP‐9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP‐target gene ID1, especially in combination with low expression of the BMP‐inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T‐Orgs) and corresponding non‐malignant areas (N‐Orgs) of three patients. The N‐Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP‐9 stimulation of organoids promoted an enrichment of tumour‐suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP‐9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1.In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP‐9 enhances this ratio, even in the presence of noggin. Thus, BMP‐9 is identified as a novel target for the development of improved anti‐cancer therapies of patients with CRC.

TATI, TAT-2, and CRP as Prognostic Factors in Colorectal Cancer

Objectives: Colorectal cancer is the third most common cancer worldwide, with an obvious need for more accurate prognostics. Previous studies identified C-reactive protein (CRP) as a prognostic serum biomarker for colorectal cancer, whereas the biomarkers tumor-associated trypsin inhibitor (TATI) and tumor-associated trypsin-2 (TAT-2) are less well-known prognostic factors. Therefore, in this study, we aimed to compare the prognostic role of these biomarkers. Materials and Methods: Our cohort consisted of 219 women and 274 men who underwent colorectal cancer surgery at Helsinki University Central Hospital from 1998 through 2005. Serum and plasma samples were collected before surgery, aliquoted, stored at −80°C, and then analyzed using high-sensitivity methods with commercially available time-resolved immunofluorometric assay kits. Results: In univariate analysis, CRP (HR 1.67; 95% confidence interval [CI]: 1.25–2.23; p = 0.001), TATI (HR 1.87; 95% CI: 1.13–3.08; p = 0.014), and TAT-2 (HR 1.52; 95% CI: 1.13–2.06; p = 0.006) were significant prognostic biomarkers across the entire cohort. In subgroup analyses, TATI and TAT-2 represented significant negative prognostic factors among patients older than 66, while patients with left-sided disease, a high serum TAT-2, or a high plasma CRP experienced worse prognosis. None of the biomarkers emerged as important in the disease stage subgroup analysis nor did they serve as independent factors in the multivariate analysis. Conclusions: TATI and TAT-2 as well as CRP significantly, but not independently, served as prognostic factors in our cohort of colorectal cancer patients. Further research is needed to fully understand their clinical role in colorectal cancer.

Prognostic Value of C-Reactive Protein, Glasgow Prognostic Score, and C-Reactive Protein-to-Albumin Ratio in Colorectal Cancer.

Background: Emerging evidence suggests that inflammatory response biomarkers are predictive factors that can improve the accuracy of colorectal cancer (CRC) prognoses. We aimed to evaluate the prognostic significance of C-reactive protein (CRP), the Glasgow Prognostic Score (GPS), and the CRP-to-albumin ratio (CAR) in CRC.Methods: Overall, 307 stage I–III CRC patients and 72 colorectal liver metastases (CRLM) patients were enrolled between October 2013 and September 2019. We investigated the correlation between the pretreatment CRP, GPS, and CAR and the clinicopathological characteristics. The Cox proportional hazards model was used for univariate or multivariate analysis to assess potential prognostic factors. A receiver operating characteristic (ROC) curve was constructed to evaluate the predictive value of each prognostic score. We established CRC survival nomograms based on the prognostic scores of inflammation.Results: The optimal cutoff levels for the CAR for overall survival (OS) in all CRC patients, stage I–III CRC patients, and CRLM patients were 0.16, 0.14, and 0.25, respectively. Kaplan–Meier analysis and log-rank tests demonstrated that patients with high CRP, CAR, and GPS had poorer OS in CRC, both in the cohorts of stage I–III patients and CRLM patients. In the different cohorts of CRC patients, the area under the ROC curve (AUC) of these three markers were all high. Multivariate analysis indicated that the location of the primary tumor, pathological differentiation, and pretreatment carcinoembryonic antigen (CEA), CRP, GPS, and CAR were independent prognostic factors for OS in stage I–III patients and that CRP, GPS, and CAR were independent prognostic factors for OS in CRLM patients. The predictors in the prediction nomograms included the pretreatment CRP, GPS, and CAR.Conclusions: CRP, GPS, and CAR have independent prognostic values in patients with CRC. Furthermore, the survival nomograms based on CRP, GPS, and CAR can provide more valuable clinical significance.

Weakly supervised annotation-free cancer detection and prediction of genotype in routine histopathology.

Deep learning is a powerful tool in computational pathology: it can be used for tumor detection and for predicting genetic alterations based on histopathology images alone. Conventionally, tumor detection and prediction of genetic alterations are two separate workflows. Newer methods have combined them, but require complex, manually engineered computational pipelines, restricting reproducibility and robustness. To address these issues, we present a new method for simultaneous tumor detection and prediction of genetic alterations: The Slide-Level Assessment Model (SLAM) uses a single off-the-shelf neural network to predict molecular alterations directly from routine pathology slides without any manual annotations, improving upon previous methods by automatically excluding normal and non-informative tissue regions. SLAM requires only standard programming libraries and is conceptually simpler than previous approaches. We have extensively validated SLAM for clinically relevant tasks using two large multicentric cohorts of colorectal cancer patients, Darmkrebs: Chancen der Verhütung durch Screening (DACHS) from Germany and Yorkshire Cancer Research Bowel Cancer Improvement Programme (YCR-BCIP) from the UK. We show that SLAM yields reliable slide-level classification of tumor presence with an area under the receiver operating curve (AUROC) of 0.980 (confidence interval 0.975, 0.984; n= 2,297 tumor and n= 1,281 normal slides). In addition, SLAM can detect microsatellite instability (MSI)/mismatch repair deficiency (dMMR) or microsatellite stability/mismatch repair proficiency with an AUROC of 0.909 (0.888, 0.929; n= 2,039 patients) and BRAF mutational status with an AUROC of 0.821 (0.786, 0.852; n= 2,075 patients). The improvement with respect to previous methods was validated in a large external testing cohort in which MSI/dMMR status was detected with an AUROC of 0.900 (0.864, 0.931; n= 805 patients). In addition, SLAM provides human-interpretable visualization maps, enabling the analysis of multiplexed network predictions by human experts. In summary, SLAM is a new simple and powerful method for computational pathology that could be applied to multiple disease contexts. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The Prevalence of Pathogenic or Likely Pathogenic Germline Variants in a Nationwide Cohort of Young Colorectal Cancer Patients Using a Panel of 18 Genes Associated with Colorectal Cancer.

Simple SummaryThis study reveals that pathogenic or likely pathogenic germline variants are detected in one fourth of all young patients with colorectal cancer in a nationwide cohort. Immunohistochemistry staining for mismatch repair deficiency is an easy way to detect Lynch syndrome in the young colorectal cancer patient.Introduction: The prevalence of pathogenic or likely pathogenic germline variants (PGV) in colorectal cancer (CRC) in young patients is seen in approximately one in five patients, with the majority of cases having gene variants associated with Lynch syndrome (LS). The primary aim was to describe the prevalence of 18 genes, all associated with hereditary polyposis and CRC, in a nationwide population of young CRC (yCRC) patients, and outline disease characteristics in patients with or without germline variants. Methods: We screened 98 patients aged 18–40 with CRC diagnosed in 2010–2013 for variants in MSH2, MSH6, MLH1, PMS2, EPCAM, APC, MUTYH, SMAD4, BMPR1A, STK11, PTEN, POLE, POLD1, NTHL1, AXIN2, MSH3, GREM1 and RNF43 using Next Generation Sequencing. Comparisons between patients’ characteristics in patients with PGV, and patients without germline variants (NPGV) were analyzed. Results: PGV were detected in twenty-four patients (24.5%), and twenty-one patients (21.1%) had variants in the mismatch repair (MMR) genes associated with LS. Variants in the APC and MUTYH genes were detected in 1% and 4%, respectively. Patients with NPGV had more advanced disease with adverse histopathological features. Conclusion: PGV was detected in one in four yCRC patients, and one in five yCRC patients had disease causing variants in the mismatch repair genes associated with LS.

Low expression of CD24 is associated with poor survival in colorectal cancer

In this study we analyzed expression of CD24 in a cohort of colorectal cancer patients using immunohistochemistry staining of CD24. We found a significant association between absence or low expression of CD24 (10% of membranous and 55% of cytoplasmic staining) and shortened patient survival. Protein localization played a crucial role in the prognosis: membranous form was the major and prognostic one in primary tumors, while cytoplasmic expression was elevated in liver metastases compared to the primary tumors and contained prognostic information. Then, using The Cancer Genome Atlas Colon Adenocarcinoma (TCGA-COAD) RNA-seq data, we showed that CD24 mRNA level was two-fold decreased in primary colorectal cancers compared to adjacent normal mucosa. Like the protein staining data, ten percent of patients with the lowest mRNA expression levels of CD24 in primary tumors had reduced survival compared to the ones with higher expression. To explain these findings mechanistically, shRNA-mediated CD24 knockdown was performed in HT-29 colorectal cancer cells. It resulted in the increase of cell migration in vitro, no changes in proliferation and apoptosis, and a slight decrease in cell invasion. As increased cell migration is a hallmark of metastasis formation, this finding corroborates the association of a decreased CD24 expression with poor prognosis. Differential gene expression analysis revealed upregulation of genes involved in cell migration in the group of patients with low CD24 expression, including integrin subunit α3 and α3, β3 subunits of laminin 332. Further co-expression analysis identified SPI1, STAT1 and IRF1 transcription factors as putative master-regulators in this group.

Physical Activity after Colorectal Cancer Diagnosis and Mortality in a Nationwide Retrospective Cohort Study.

Simple SummaryPhysical activity can help to prevent colorectal cancer, but its importance after cancer diagnosis has not been validated. In this nationwide insurance data-based study of 43,596 colorectal cancer patients, a high level of physical activity after colorectal cancer diagnosis was negatively associated with a risk of death in both colon and rectal cancer patients, particularly in the surgically treated group. Our findings support the importance of the physical activity among colorectal cancer patients.Physical activity reduces the risk of colon cancer, but its prognostic impact after cancer diagnosis remains unclear. To evaluate the association between post-diagnosis activity and cause-specific mortality, we reconstructed a colorectal cancer patient cohort from the 2009–16 Korean National Health Insurance Service (NHIS) database. Subgroup analyses were performed by treatment group. In total, 27,143 colon cancer patients and 16,453 rectal cancer patients were included in the analysis (mean follow-up, 4.3 years; median 4.0 years). In the surgically treated group, a high level of activity (the weighted sum of the frequencies for walking, moderate, and vigorous activity greater than or equal to 3 times/week) was inversely associated with all-cause mortality (colon cancer: HR, 0.79; 95% CI, 0.72 to 0.88; rectal cancer: HR, 0.75; 95% CI, 0.66 to 0.86) and colorectal cancer-specific mortality (colon cancer: HR, 0.85; 95% CI, 0.76 to 0.97; rectal cancer: HR, 0.77; 95% CI, 0.66 to 0.90). No significant results were shown for cardiovascular disease-specific mortality. No association was shown in patients who received chemoradiotherapy without surgery. The present study may provide evidence for post-diagnosis physical activity as a prognostic factor in colorectal cancer, particularly in surgically treated early-stage patients.

B7-H5 blockade enhances CD8+ T-cell-mediated antitumor immunity in colorectal cancer

Negative immune checkpoint blockade immunotherapy has shown potential for multiple malignancies including colorectal cancer (CRC). B7-H5, a novel negative immune checkpoint regulator, is highly expressed in tumor tissues and promotes tumor immune escape. However, the clinical significance of B7-H5 expression in CRC and the role of B7-H5 in the tumor microenvironment (TME) has not been fully clarified. In this study, we observed that high B7-H5 expression in CRC tissues was significantly correlated with the lymph node involvement, AJCC stage, and survival of CRC patients. A significant inverse correlation was also observed between B7-H5 expression and CD8+ T-cell infiltration in CRC tissues. Kaplan−Meier analysis showed that patients with high B7-H5 expression and low CD8+ T-cell infiltration had the worst prognosis in our cohort of CRC patients. Remarkably, both high B7-H5 expression and low CD8+ T infiltration were risk factors for overall survival. Additionally, B7-H5 blockade using a B7-H5 monoclonal antibody (B7-H5 mAb) effectively suppressed the growth of MC38 colon cancer tumors by enhancing the infiltration and Granzyme B production of CD8+ T cells. Importantly, the depletion of CD8+ T cells obviously abolished the antitumor effect of B7-H5 blockade in the MC38 tumors. In sum, our findings suggest that B7-H5 may be a valuably prognostic marker for CRC and a potential target for CRC immunotherapy.

The Clinical and Prognostic Significance of Protein Arginine Deiminases 2 and 4 in Colorectal Cancer

Introduction: Protein arginine deiminases (PADIs) are a family of enzymes that catalyse the post-translational modification of proteins. Association between PADI expression and clinicopathology, protein expression, and outcome was determined. Methods: PADI2 and PADI4 expression was assessed immunohistochemically in a cohort of colorectal cancer (CRC) patients. Results: CRC tissues expressed variable levels of PADI2 which was mainly localised in the cytoplasm and correlated with patient survival (p = 0.005); high expression increased survival time from 43.5 to 67.6 months. Expression of cytoplasmic PADI2 correlated with the expression of nuclear β catenin, PADI4, and alpha-enolase. In contrast, expression of nuclear PADI2 correlated with a decrease in survival (p = 0.010), with high expression decreasing survival from 76.4 to 42.9 months. CRC tissues expressed variable levels of PADI4 in both the nucleus and cytoplasm. Expression of cytoplasmic PADI4 correlated with survival (p = 0.001) with high expression increasing survival time from 48.1 to 71.8 months. Expression of cytoplasmic PADI4 correlated with expression of nuclear β catenin, alpha-enolase (p ≤ 0.0001, p = 0.002), and the apoptotic related protein, Bcl-2. Expression of nuclear PADI4 also correlated with survival (p = 0.011), with high expression of nuclear PADI4 increasing survival time from 55.4 to 74 months. Expression of nuclear PADI4 correlated with p53, alpha-enolase, and Bcl-2. Multivariate analysis showed that TNM stage, cytoplasmic PADI2, and PADI4 remained independent prognostic factors in CRC. Both PADI2 and PADI4 are good prognostic factors in CRC. Conclusion: High expression of cytoplasmic PADI2, PADI4, and nuclear PADI4 were associated with an increase in overall survival.

Patients with mesenchymal tumours and high Fusobacteriales prevalence have worse prognosis in colorectal cancer (CRC)

ObjectivesTranscriptomic-based subtyping, consensus molecular subtyping (CMS) and colorectal cancer intrinsic subtyping (CRIS) identify a patient subpopulation with mesenchymal traits (CMS4/CRIS-B) and poorer outcome. Here, we investigated the relationship between prevalence...

Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients’ tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients’ tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

Core Circadian Clock Proteins as Biomarkers of Progression in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common tumours in developed countries. Although its incidence and mortality rates have decreased, its prognosis has not changed, and a high percentage of patients with CRC develop relapse (metachronous metastasis, MM, or local recurrence, LR) during their disease. The identification of these patients is very important for their correct management, but the lack of prognostic markers makes it difficult. Given the connection between circadian disruption and cancer development and progression, we aimed to analyse the prognostic significance of core circadian proteins in CRC. We measured the expression of PER1-3, CRY1-2, BMAL1 and NR1D2 in a cohort of CRC patients by immunohistochemistry (IHC) and analysed their prognostic potential in this disease. A low expression of PER2 and BMAL1 was significantly associated with metastasis at the moment of disease diagnosis, whereas a high expression of CRY1 appeared as an independent prognostic factor of MM development. A high expression of NR1D2 appeared as an independent prognostic factor of LR development after disease diagnosis. Moreover, patients with a low expression of BMAL1 and a high expression of CRY1 showed lower OS and DFS at five years. Although these markers need to be validated in larger and different ethnic cohorts, the simplicity of IHC makes these proteins candidates for personalizing CRC treatment.

Clinical Characteristics and Outcomes of Colorectal Cancer in the ColoCare Study: Differences by Age of Onset.

Simple SummaryThe number of new colorectal cancer cases continues to increase in individuals under 50 years of age in the Western world. Underlying reasons for this observation remain unclear. Here, we compare demographic, clinical, and lifestyle characteristics by age at diagnosis in a large cohort of newly diagnosed colorectal cancer patients. We aim to identify potential risk factors and clinical characteristics of colorectal cancer patients diagnosed under the age of 50 years, compared to those over 50. The results of this study will help elucidate factors related to colorectal cancer in younger patients, and may help guide future research on colorectal cancer in younger patients.Early-onset colorectal cancer has been on the rise in Western populations. Here, we compare patient characteristics between those with early- (<50 years) vs. late-onset (≥50 years) disease in a large multinational cohort of colorectal cancer patients (n = 2193). We calculated descriptive statistics and assessed associations of clinicodemographic factors with age of onset using mutually-adjusted logistic regression models. Patients were on average 60 years old, with BMI of 29 kg/m2, 52% colon cancers, 21% early-onset, and presented with stage II or III (60%) disease. Early-onset patients presented with more advanced disease (stages III–IV: 63% vs. 51%, respectively), and received more neo and adjuvant treatment compared to late-onset patients, after controlling for stage (odds ratio (OR) (95% confidence interval (CI)) = 2.30 (1.82–3.83) and 2.00 (1.43–2.81), respectively). Early-onset rectal cancer patients across all stages more commonly received neoadjuvant treatment, even when not indicated as the standard of care, e.g., during stage I disease. The odds of early-onset disease were higher among never smokers and lower among overweight patients (1.55 (1.21–1.98) and 0.56 (0.41–0.76), respectively). Patients with early-onset colorectal cancer were more likely to be diagnosed with advanced stage disease, to have received systemic treatments regardless of stage at diagnosis, and were less likely to be ever smokers or overweight.

Comprehensive aptamer-based screen of 1317 proteins uncovers improved stool protein markers of colorectal cancer.

To screen and validate novel stool protein biomarkers of colorectal cancer (CRC). A novel aptamer-based screen of 1317 proteins was used to uncover elevated proteins in the stool of patients with CRC, as compared to healthy controls (HCs) in a discovery cohort. Selected biomarker candidates from the discovery cohort were ELISA validated in three independent cross-sectional cohorts comprises 76 CRC patients, 15 adenoma patients, and 63 healthy controls, from two different ethnicities. The expression of the potential stool biomarkers within CRC tissue was evaluated using single-cell RNA-seq datasets. A total of 92 proteins were significantly elevated in CRC samples as compared to HCs in the discovery cohort. Among Caucasians, the 5 most discriminatory proteins among the 16 selected proteins, ordered by their ability to distinguish CRC from adenoma and healthy controls, were MMP9, haptoglobin, myeloperoxidase, fibrinogen, and adiponectin. Except myeloperoxidase, the others were significantly associated with depth of tumor invasion. The 8 stool proteins with the highest AUC values were also discriminatory in a second cohort of Indian CRC patients. Several of the stool biomarkers elevated in CRC were also expressed within CRC tissue, based on the single-cell RNA-seq analysis. Stool MMP9, fibrinogen, myeloperoxidase, and haptoglobin emerged as promising CRC stool biomarkers, outperforming stool Hemoglobin. Longitudinal studies are warranted to assess the clinical utility of these novel biomarkers in early diagnosis of CRC.

Can physical prehabilitation prevent complications after colorectal cancer surgery in frail older patients?

IntroductionFrail patients with colorectal cancer (CRC) are at increased risk of complications after surgery. Prehabilitation seems promising to improve this outcome and therefore we evaluated the effect of physical prehabilitation on postoperative complications in a retrospective cohort of frail CRC patients. MethodsThe study consisted of all consecutive non-metastatic CRC patients ≥70 years who had elective surgery from 2014 to 2019 in a teaching hospital in the Netherlands, where a physical prehabilitation program was implemented from 2014 on. We performed both an intention-to-treat and per protocol analysis to evaluate postoperative complications in the physical prehabilitation (PhP) and non-prehabilitation (NP) group. ResultsEventually, 334 elective patients were included. The 124 (37.1%) patients in the PhP-group presented with higher age, higher comorbidity scores and walking-aid use compared to the NP-group. Medical complications occurred in 26.6% of the PhP-group and in 20.5% of the NP-group (p = 0.20) and surgical complications in 19.4% and 14.3% (p = 0.22) respectively. In all frailty subgroups, the medical complications were lower in the PhP-group compared to the NP-group (35.9% vs. 45.5% for patients with ≥2 comorbidities, 36.2% vs. 39.1% for ASA score ≥ III, 29.2% vs. 45.8% for walking-aid use). Differences were not significant. ConclusionsIn this study, patients selected for physical prehabilitation had a worse frailty profile and therefore a higher a priori risk of postoperative complications. However, the postoperative complication rate was not increased compared to patients who were less frail at baseline and without prehabilitation. Hence, physical prehabilitation may prevent postoperative complications in frail CRC patients ≥70 years.

Clinical performance of methylation-based liquid biopsy test COLVERA after optimization of test interpretation rules.

3546 Background: Clinical guidelines recommend surveillance for patients who complete primary treatment for colorectal cancer (CRC) with the aim of detecting recurrence when amenable to curative intent treatment. Currently recommended surveillance protocols, including imaging and CEA have limitations both in sensitivity and specificity, thus novel methods that detect circulating tumor DNA (ctDNA) have been introduced into clinical practice. COLVERA is a laboratory-developed, real-time PCR test that detects DNA methylation of BCAT1 and IKZF1 genes. These two genes are hypermethylated in 95% of CRC tissue and COLVERA showed improved sensitivity for detection of recurrent disease in comparison to CEA in several clinical populations. The current study evaluated the impact of optimizing the assay’s qualitative reporting method on actionability and clinical performance for recurrence detection in CRC surveillance setting. Methods: Two previously described cohorts of CRC patients (N=322 and N=144) who completed primary treatment and were undergoing surveillance were evaluated. Imaging and blood collections were performed at, or adjacent to, a standard of care visit. cfDNA was extracted from whole blood, bisulphite-treated and assayed in triplicates for BCAT1/IKZF methylation. Previously, any positive replicate of either target gene was reported as COLVERA “detected”. In the current study, COLVERA is “detected” when at least one replicate of IKZF1 or multiple replicates of either IKZF1 and/or BCAT1 are present. Sensitivity, specificity, and diagnostic odds ratio (DOR) for CRC recurrence detection from a single time-point blood sample was determined using radiological imaging as clinical reference standard. Results: In the first cohort (N=322), overall COLVERA test positivity was 6.5% (21/322) with a sensitivity of 59.3% (95% CI: 38.8 - 77.6) and specificity of 98.3% (96.1 - 99.5) for detecting recurrence at a time-point adjacent to imaging, representing improved specificity, from 91.5% (87.7 - 94.4%), with minimal decrease in sensitivity, from 63.0% (42.4 – 80.6). Similarly, in the second cohort (N=144) sensitivity was 62% (47.2 -75.4), compared to 66.0% (57.1 – 69.3) under the prior interpretation method, while specificity was 92.6% (85.3-97), compared to 90.4% (84.7 - 94.7). A high DOR of 84 (26 - 272) (previously 18 (7.6 – 44.4)) indicates that the revised COLVERA interpretation method is clinically more informative and differentiates with greater accuracy patients with and without the disease. Conclusions: This change in the COLVERA interpretation rule resulted in optimized clinical specificity with minimal impact on sensitivity. For an assay intended to aid in surveillance and early recurrence detection, improved accuracy allows the physician to have increased confidence in making actionable decisions based on test result, including further imaging or treatment.

Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays.

Background: Cellular-cell free-DNA (ccfDNA) is being explored as a diagnostic and prognostic tool for various diseases including cancer. Beyond the evaluation of the ccfDNA mutational status, its fragmentation has been investigated as a potential cancer biomarker in several studies. However, probably due to a lack of standardized procedures dedicated to preanalytical and analytical processing of plasma samples, contradictory results have been published.Methods: ddPCR assays allowing the detection of KRAS wild-type and mutated sequences (KRAS p.G12V, pG12D, and pG13D) were designed to target different fragments sizes. Once validated on fragmented and non-fragmented DNA extracted from cancer cell lines, these assays were used to investigate the influence of the extraction methods on the non-mutated and mutated ccfDNA integrity reflected by the DNA integrity index (DII). The DII was then analyzed in two prospective cohorts of metastatic colorectal cancer patients (RASANC study n = 34; PLACOL study n = 12) and healthy subjects (n = 49).Results and Discussion: Our results demonstrate that ccfDNA is highly fragmented in mCRC patients compared with healthy individuals. These results strongly suggest that the characterization of ccfDNA integrity hold great promise toward the development of a universal biomarker for the follow-up of mCRC patients. Furthermore, they support the importance of standardization of sample handling and processing in such analysis.

CA125: A superior prognostic biomarker for colorectal cancer compared to CEA, CA19-9 or CA242.

The tumor stage represents the single most important prognostic factor for colorectal cancer (CRC), although more accurate prognostics remain much needed. Previously, we identified CA125 as an independent significant prognostic factor, which we have further validated along with CEA, CA19-9, and CA242 in a large cohort of CRC patients. Using enzyme-linked immunosorbent assays, we analyzed preoperative serum samples in 322 CRC patients operated on between 1998 and 2003. Using the Spearman's rho model, we calculated the correlation between our previous findings on MUC16 and CA125, for which the correlation coefficient was 0.808 (p < 0.001). The Cox regression analysis of the linear and logarithmic values of CEA, CA125, CA242, and CA19-9 identified only CA125 (hazard ratio [HR] 1.03; 95% confidence interval [95% CI] 1.02-1.04; p < 0.001) as significant when using the linear values. Survival among CRC patients with a high CA125 level was poor compared with CRC patients with a low CA125 level (HR 2.48; 95% CI 1.68-3.65; p < 0.001). In subgroup analyses, patients with high CA125 levels and aged ≤67 or >67, with stage I-II or III-IV, and both colon and rectal cancer exhibited poor prognoses. In the multivariate analysis, we used clinical pathological variables in the model, where age, gender, and stage served as the background characteristics. We dichotomized CA125 using the Youden maximal cutoff point, and the median values for CEA, CA19-9, and CA242. CA125 emerged as the only marker remaining significant and independent together with stage, location, and age (HR 1.91; 95% CI 1.24-2.95; p 0.003). CA125 represents a significant and independent prognostic factor in CRC patients, superior to CEA. Furthermore, CA242 served as a better prognostic marker than both CEA and CA19-9. We recommend including both CA125 and CA242 in prognostic clinical trials among CRC patients.

Serum carcinoembryonic antigen pre-operative level in colorectal cancer: revisiting risk stratification.

Biomarkers may play a role as predictive and prognostic factors in colorectal cancer patients. The aims of the study were to verify the prognostic role of pre-operative serum carcinoembryonic antigen (CEA) level in predicting overall survival and risk of recurrence in a cohort of colorectal cancer patients and to evaluate optimal cut-off values. A retrospective cohort analysis was performed on colorectal cancer patients undergoing elective curative surgery between 2004 and 2019 at an Italian Academic Hospital. Main outcomes were overall survival, disease-free survival at 3-years and risk of local, loco-regional and distant recurrence during follow-up. A receiver operating characteristic (ROC) curve analysis was plotted using CEA pre-operative values and follow-up data in order to estimate the optimal cut-off values. A total of 559 patients were considered. The mean CEA value was 12.1 ± 54.1ng/mL, and the median 29.3 (0-4995) ng/mL. The ROC curve analysis identified 12.5ng/mL as the best CEA cut-off value to predict the risk of metastatic development after surgery in stage I-III colorectal cancer patients, and 10ng/mL as the best CEA cut-off value to predict overall survival and disease-free survival in stage III-IV patients. These data suggest a stratification of colorectal cancer patients in three classes of risk: a low risk class (CEA <10 ng/mL), a moderate risk class (CEA 10-12.5 ng/mL) and a high risk class (CEA >12.5 ng/mL). In conclusion, pre-operative serum CEA measurements could integrate information to enhance patient risk stratification and tailored therapy.