first_pagesettingsOrder Article Reprints Open AccessEditor’s ChoiceArticleSerum N-Glycosylation RPLC-FD-MS Assay to Assess Colorectal Cancer Surgical InterventionsbyAlan B. Moran 1,2, Georgia...Show morefirst_pagesettingsOrder Article Reprints Open AccessEditor’s ChoiceArticleSerum N-Glycosylation RPLC-FD-MS Assay to Assess Colorectal Cancer Surgical InterventionsbyAlan B. Moran 1,2, Georgia Elgood-Hunt 2, Yuri E. M. van der Burgt 1, Manfred Wuhrer 1, Wilma E. Mesker 3, Rob A. E. M. Tollenaar 3, Daniel I. R. Spencer 2 and Guinevere S. M. Lageveen-Kammeijer 1,4,*1Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands2Ludger Ltd., Culham Science Centre, Abingdon OX14 3EB, UK3Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, The Netherlands4Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands*Author to whom correspondence should be addressed.Biomolecules2023, 13(6), 896; https://doi.org/10.3390/biom13060896Received: 29 March 2023 / Revised: 16 May 2023 / Accepted: 24 May 2023 / Published: 27 May 2023(This article belongs to the Special Issue Protein Glycosylation and Human Diseases) Download keyboard_arrow_downBrowse FiguresReview ReportsVersions NotesAbstractA newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography–fluorescence detection–mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored. Show less
Moran, A.B.; Elgood-Hunt, G.; Burgt, Y.E.M. van der; Wuhrer, M.; Mesker, W.E.; Tollenaar, R.A.E.M.; ... ; Lageveen-Kammeijer, G.S.M. 2023
A newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was... Show moreA newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography–fluorescence detection–mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored. Show less
Liu, S.J.; Dinther, M. van; Hagenaars, S.C.; Gu, Y.Z.; Kuipers, T.B.; Mei, H.L.; ... ; Dijke, P. ten 2023
Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant... Show moreTriple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant metastasis is the main cause of death of TNBC patients. Better understanding of the mechanisms underlying TNBC metastasis may lead to new strategies of early diagnosis and more efficient treatment. In our study, we uncovered that the autophagy receptor optineurin (OPTN) plays an unexpected role in TNBC metastasis. Data mining of publicly available data bases revealed that the mRNA level of OPTN in TNBC patients positively correlates with relapse free and distance metastasis free survival. Importantly, in vitro and in vivo models demonstrated that OPTN suppresses TNBC metastasis. Mechanistically, OPTN inhibited the pro-oncogenic transforming growth factor-beta (TGF beta) signaling in TNBC cells by interacting with TGF beta type I receptor (T beta RI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGF beta gene response signature and expression of proto-typic TGF beta target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGF beta receptor/SMAD signaling and suppresses metastasis in TNBC. Show less
Diederiks, N.; Ravensbergen, C.J.; Treep, M.; Wezel, M. van; Kuruc, M.; Ruhaak, L.R.; ... ; Mesker, W.E. 2023
In the pursuit of personalized diagnostics and tailored treatments, quantitative protein tests contribute to a more precise definition of health and disease. The development of new quantitative... Show moreIn the pursuit of personalized diagnostics and tailored treatments, quantitative protein tests contribute to a more precise definition of health and disease. The development of new quantitative protein tests should be driven by an unmet clinical need and performed in a collaborative effort that involves all stakeholders. With regard to the analytical part, mass spectrometry (MS)-based platforms are an excellent tool for quantification of specific proteins in body fluids, for example focused on cancer. The obtained readouts have great potential in deter-mining tumor aggressiveness to facilitate treatment decisions, and can furthermore be used to monitor patient response. Internationally standardized TNM classifications of malignant tumors are beneficial for diagnosis, however treatment outcome and survival of cancer patients is poorly predicted. To this end, the importance of the tumor microenvironment has endorsed the introduction of the tumor-stroma ratio as a prognostic parameter in solid primary tumor types. Currently, the stromal content of tumor tissues is determined via routine diagnostic pathology slides. With the development of liquid chromatography (LC)-MS methods we aim at quantification of tumor-stroma specific proteins in body fluids. In this mini-review the analytical aspect of this developmental trajectory is further detailed. Show less
The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high... Show moreThe prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities. To this end, protein cluster patterns were evaluated to form a conceptual basis for further clinical analyses. Using a mass spectrometry-based bottom-up proteomics strategy, the protein abundance of 30 samples was analyzed: five sequential serum samples from six high-risk women; three who developed a breast malignancy (cases) and three who did not (controls). Serum samples were chromatographically fractionated and an in-depth serum proteome was acquired. Cluster analyses were applied to indicate differences between and within protein levels in serum samples of individuals. Statistical analyses were performed using ANOVA to select proteins with a high level of clustering. Cluster analyses on 30 serum samples revealed unique patterns of protein clustering for each patient, indicating a greater interpatient than intrapatient variability in protein levels of the longitudinally acquired samples. Moreover, the most distinctive proteins in the cluster analysis were identified. Strong clustering patterns within longitudinal intrapatient samples have demonstrated the importance of identifying small changes in protein levels for individuals over time. This underlines the significance of longitudinal serum measurements, that patients can serve as their own controls, and the relevance of the current study set-up for early detection. The TESTBREAST study will continue its pursuit toward establishing a protein panel for early breast cancer detection. Show less
Weerd, S. van de; Smit, M.A.; Roelands, J.; Mesker, W.E.; Bedognetti, D.; Kuppen, P.J.K.; ... ; Krieken, J.H.J.M. van 2022
The purpose of this study was to evaluate the association between four distinct histopathological features: (1) tumor infiltrating lymphocytes, (2) mucinous differentiation, (3) tumor-stroma ratio,... Show moreThe purpose of this study was to evaluate the association between four distinct histopathological features: (1) tumor infiltrating lymphocytes, (2) mucinous differentiation, (3) tumor-stroma ratio, plus (4) tumor budding and two gene expression-based classifiers-(1) consensus molecular subtypes (CMS) plus (2) colorectal cancer intrinsic subtypes (CRIS). All four histopathological features were retrospectively scored on hematoxylin and eosin sections of the most invasive part of the primary tumor in 218 stage II and III colon cancer patients from two independent cohorts (AMC-AJCC-90 and AC-ICAM). RNA-based CMS and CRIS assignments were independently obtained for all patients. Contingency tables were constructed and a chi 2 test was used to test for statistical significance. Odds ratios with 95% confidence intervals were calculated. The presence of tumor infiltrating lymphocytes and a mucinous phenotype (>50% mucinous surface area) were strongly correlated with CMS1 (p < 0.001 and p = 0.008) and CRIS-A (p = 0.006 and p < 0.001). The presence of mucus (>= 10%) was associated with CMS3: mucus was present in 64.1% of all CMS3 tumors (p < 0.001). Although a clear association between tumor-stroma ratio and CMS4 was established in this study (p = 0.006), still 32 out of 61 (52.5%) CMS4 tumors were scored as stroma-low, indicating that CMS4 tumors cannot be identified solely based on stromal content. Higher budding counts were seen in CMS4 and CRIS-B tumors (p = 0.045 and p = 0.046). No other associations of the measured parameters were seen for any of the other CRIS subtypes. Our analysis revealed clear associations between histopathologic features and CMS or CRIS subtypes. However, identification of distinct molecular subtypes solely based on histopathology proved to be infeasible. Combining both molecular and morphologic features could potentially improve patient stratification. Show less
The prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high... Show moreThe prospective, multicenter TESTBREAST study was initiated with the aim of identifying a novel panel of blood-based protein biomarkers to enable early breast cancer detection for moderate-to-high-risk women. Serum samples were collected every (half) year up until diagnosis. Protein levels were longitudinally measured to determine intrapatient and interpatient variabilities. To this end, protein cluster patterns were evaluated to form a conceptual basis for further clinical analyses. Using a mass spectrometry-based bottom-up proteomics strategy, the protein abundance of 30 samples was analyzed: five sequential serum samples from six high-risk women; three who developed a breast malignancy (cases) and three who did not (controls). Serum samples were chromatographically fractionated and an in-depth serum proteome was acquired. Cluster analyses were applied to indicate differences between and within protein levels in serum samples of individuals. Statistical analyses were performed using ANOVA to select proteins with a high level of clustering. Cluster analyses on 30 serum samples revealed unique patterns of protein clustering for each patient, indicating a greater interpatient than intrapatient variability in protein levels of the longitudinally acquired samples. Moreover, the most distinctive proteins in the cluster analysis were identified. Strong clustering patterns within longitudinal intrapatient samples have demonstrated the importance of identifying small changes in protein levels for individuals over time. This underlines the significance of longitudinal serum measurements, that patients can serve as their own controls, and the relevance of the current study set-up for early detection. The TESTBREAST study will continue its pursuit toward establishing a protein panel for early breast cancer detection. Show less
Levink, I.J.M.; Klatte, D.C.F.; Hanna Sawires, R.G.; Vreeker, G.C.M.; Ibrahim, I.S.; Burgt, Y.E.M. van der; ... ; Mesker, W.E. 2022
Background: Surveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities... Show moreBackground: Surveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities is challenging. We aimed to identify changes in serum glycosylation levels over time to earlier detect PDAC in high-risk individuals. Methods: Individuals with a hereditary predisposition to develop PDAC were followed in two surveillance programs. Those, of which at least two consecutive serum samples were available, were included. Mass spectrometry analysis was performed to determine the total N-glycome for each consecutive sample. Potentially discriminating N-glycans were selected based on our previous cross-sectional analysis and relative abundances were calculated for each glycosylation feature. Results: 165 individuals ("FPC-cohort" N = 119; Leiden cohort N = 46) were included. In total, 97 (59%) individuals had a genetic predisposition (77 CDKN2A, 15 BRCA1/2, 5 STK11) and 68 (41%) a family history of PDAC without a known genetic predisposition (>10-fold increased risk of developing PDAC). From each individual, a median number of 3 serum samples (IQR 3) was collected. Ten individuals (6%) developed PDAC during 35 months of follow-up; nine (90%) of these patients carried a CDKN2A germline mutation. In PDAC cases, compared to all controls, glycosylation characteristics were increased (fucosylation, tri-and tetra-antennary structures, specific sialic linkage types), others decreased (complex-type diantennary and bisected glycans).The largest change over time was observed for tri-antennary fucosylated glycans, which were able to differentiate cases from controls with a specificity of 92%, sensitivity of 49% and accuracy of 90%. Conclusion: Serum N-glycan monitoring may support early detection in a pancreas surveillance program.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of IAP and EPC. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Purpose: The recently developed fibroblast activation protein inhibitor (FAPI) tracer for PET/CT, binding tumour-stromal cancer-associated fibroblasts, is a promising tool for detection of positive... Show morePurpose: The recently developed fibroblast activation protein inhibitor (FAPI) tracer for PET/CT, binding tumour-stromal cancer-associated fibroblasts, is a promising tool for detection of positive lymph nodes. This study provides an overview of features, including sizes and tumour-stromal content, of lymph nodes and their respective lymph node metastases (LNM) in colorectal cancer (CRC), since literature lacks on whether LNMs contain sufficient stroma to potentially allow FAPI-based tumour detection. Methods: Haematoxylin and eosin-stained tissue slides from 73 stage III colon cancer patients were included. Diameters and areas of all lymph nodes and their LNMs were assessed, the amount of stroma by measuring the stromal compartment area, the conventional and total tumour-stroma ratios (TSR-c and TSR-t, respectively), as well as correlations between these parameters. Also, subgroup analysis using a minimal diameter cut off of 5.0 mm was performed. Results: In total, 126 lymph nodes were analysed. Although positive correlations were observed between node and LNM for diameter and area (r = 0.852, p < 0.001 and r = 0.960, p < 0.001, respectively), and also between the LNM stromal compartment area and nodal diameter (r = 0.612, p < 0.001), nodal area (r = 0.747, p < 0.001) and LNM area (r = 0.746, p < 0.001), novel insight was that nearly all (98%) LNMs contained stroma, with median TSR-c scores of 35% (IQR 20-60%) and TSR-t of 20% (IQR 10-30%). Moreover, a total of 32 (25%) positive lymph nodes had a diameter of < 5.0 mm. Conclusion: In LNMs, stroma is abundantly present, independent of size, suggesting a role for FAPI PET/CT in improved lymph node detection in CRC. Show less
Purpose The tumor-stroma ratio (TSR) has repeatedly proven to be correlated with patient outcomes in breast cancer using large retrospective cohorts. However, studies validating the TSR often show... Show morePurpose The tumor-stroma ratio (TSR) has repeatedly proven to be correlated with patient outcomes in breast cancer using large retrospective cohorts. However, studies validating the TSR often show variability in methodology, thereby hampering comparisons and uniform outcomes. Method This paper provides a detailed description of a simple and uniform TSR scoring method using Hematoxylin and Eosin (H&E)-stained core biopsies and resection tissue, specifically focused on breast cancer. Possible histological challenges that can be encountered during scoring including suggestions to overcome them are reported. Moreover, the procedure for TSR estimation in lymph nodes, scoring on digital images and the automatic assessment of the TSR using artificial intelligence are described. Conclusion Digitized scoring of tumor biopsies and resection material offers interesting future perspectives to determine patient prognosis and response to therapy. The fact that the TSR method is relatively easy, quick, and cheap, offers great potential for its implementation in routine diagnostics, but this requires high quality validation studies. Show less
Hoorn, S. ten; Waasdorp, C.; Oijen, M.G.H. van; Damhofer, H.; Trinh, A.; Zhao, L.; ... ; Bijlsma, M.F. 2022
Background Recently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal... Show moreBackground Recently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal activation, A Disintegrin and Metalloprotease 12 (ADAM12) in colorectal cancer (CRC). Methods Using gene expression databases we investigated ADAM12 expression in CRC and delineated the source of ADAM12 expression. The clinical value of ADAM12 was retrospectively assessed in the CAIRO2 trial in metastatic CRC with 235 patients (31% of total cohort), and an independent rectal cancer cohort (n = 20). Results ADAM12 is expressed by activated CRC associated fibroblasts. In the CAIRO2 trial cohort, ADAM12 serum levels were prognostic (ADAM12 low versus ADAM12 high; median OS 25.3 vs. 17.1 months, HR 1.48 [95% CI 1.11-1.96], P = 0.007). The prognostic potential was specifically high for metastatic rectal cancer (HR 1.78 [95% CI 1.06-3.00], P = 0.030) and mesenchymal subtype tumors (HR 2.12 [95% CI 1.25-3.60], P = 0.004). ADAM12 also showed potential for predicting recurrence in an exploratory analysis of non-metastatic rectal cancers. Conclusions Here we describe a non-invasive marker for activated stroma in CRC which associates with poor outcome, especially for primary cancers located in the rectum. Show less
Strous, M.T.A.; Faes, T.K.E.; Gubbels, A.L.H.M.; Linden, R.L.A. van der; Mesker, W.E.; Bosscha, K.; ... ; Bruine, A.P. de 2022
Purpose: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in... Show morePurpose: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in tumour invasion and metastasis, the easy, low-cost and highly reproducible tumour-stroma ratio (TSR) could be a valuable prognostic marker, which is also believed to predict chemo resistance. Methods: Two independent series of patients with colon cancer were selected. TSR was estimated by microscopic analysis of 4 mu m haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low (<= 50%) or TSR-high (> 50%). Differences in overall survival and cancer-free survival were analysed by Kaplan-Meier curves and cox-regression analyses. Analyses were conducted for TNM-stage I-II, TNM-stage III and patients with an indication for chemotherapy separately. Results: We found that high TSR was associated with poor cancer-free survival in TNM-stage I-II colon cancer in two independent series, independent of other known high-risk features. This association was also found in TNM-stage III tumours, with an additional prognostic value of TSR in lymph node metastasis to TSR in the primary tumour alone. In addition, high TSR was found to predict chemo resistance in patients receiving adjuvant chemotherapy after surgical resection of a TNM-stage II-III colon tumour. Conclusion: In colon cancer, the TSR of both primary tumour and lymph node metastasis adds significant prognostic value to current pathologic and clinical features used for the identification of patients at high risk of cancer recurrence, and also predicts chemo resistance. Show less
Huang, Y.S.; Liu, S.J.; Shan, M.J.; Hagenaars, S.C.; Mesker, W.E.; Cohen, D.; ... ; Dijke, P. ten 2022
Transforming growth factor-beta (TGF-beta) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-beta signaling by binding to the inhibitory SMAD7... Show moreTransforming growth factor-beta (TGF-beta) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-beta signaling by binding to the inhibitory SMAD7 and inducing its proteasomal degradation. How RNF12 activity is regulated and its exact role in cancer is incompletely understood. Here we report that RNF12 was overexpressed in invasive breast cancers and its high expression correlated with poor prognosis. RNF12 promoted breast cancer cell migration, invasion, and experimental metastasis in zebrafish and murine xenograft models. RNF12 levels were positively associated with the phosphorylated AKT/protein kinase B (PKB) levels, and both displayed significant higher levels in the basal-like subtype compared with the levels in luminal-like subtype of breast cancer cells. Mechanistically, AKT-mediated phosphorylation induced the nuclear localization of RNF12, maintained its stability, and accelerated the degradation of SMAD7 mediated by RNF12. Furthermore, we demonstrated that RNF12 and AKT cooperated functionally in breast cancer cell migration. Notably, RNF12 expression strongly correlated with both phosphorylated AKT and phosphorylated SMAD2 levels in breast cancer tissues. Thus, our results uncovered RNF12 as an important determinant in the crosstalk between the TGF-beta and AKT signaling pathways during breast cancer progression. Show less
Ravensbergen, C.J.; Kuruc, M.; Polack, M.; Crobach, S.; Putter, H.; Gelderblom, H.; ... ; Mesker, W.E. 2022
Simple Summary Liquid biopsy offers a novel minimally invasive approach to tumor sampling and is believed to capture a comprehensive overview of the molecular tumor landscape. However, current... Show moreSimple Summary Liquid biopsy offers a novel minimally invasive approach to tumor sampling and is believed to capture a comprehensive overview of the molecular tumor landscape. However, current liquid biopsy analytes in cancer are principally derived from the malignant cells without regard to the tumor microenvironment. The Stroma Liquid Biopsy(TM) (SLB) proteomics panel contains proteins from key stromal pathways in cancer and was designed to address the tumor microenvironment in liquid biopsy. We aimed to explore and characterize SLB panel constituents using an in-silico transcriptomics approach in colon cancer. Additionally, the association between the SLB panel constituents and histologic intratumoral stromal content, a poor prognostic tumor characteristic, was investigated. This explorative study presents an alternative workflow to gene signature development and provides a molecular characterization of the SLB panel. We believe that our findings contribute to the ever-increasing appreciation of the tumor microenvironment in cancer. Liquid biopsy has emerged as a novel approach to tumor characterization, offering advantages in sample accessibility and tissue heterogeneity. However, as mutational analysis predominates, the tumor microenvironment has largely remained unacknowledged in liquid biopsy research. The current work provides an explorative transcriptomic characterization of the Stroma Liquid Biopsy(TM) (SLB) proteomics panel in colon carcinoma by integrating single-cell and bulk transcriptomics data from publicly available repositories. Expression of SLB genes was significantly enriched in tumors with high histologic stromal content in comparison to tumors with low stromal content (median enrichment score 0.308 vs. 0.222, p = 0.036). In addition, we identified stromal-specific and epithelial-specific expression of the SLB genes, that was subsequently integrated into a gene signature ratio. The stromal-epithelial signature ratio was found to have prognostic significance in a discovery cohort of 359 colon adenocarcinoma patients (OS HR 2.581, 95%CI 1.567-4.251, p < 0.001) and a validation cohort of 229 patients (OS HR 2.590, 95%CI 1.659-4.043, p < 0.001). The framework described here provides transcriptomic evidence for the prognostic significance of the SLB panel constituents in colon carcinoma. Plasma protein levels of the SLB panel may reflect histologic intratumoral stromal content, a poor prognostic tumor characteristic, and hence provide valuable prognostic information in liquid biopsy. Show less
Introduction: Pancreatic cancer is associated with a high risk of venous thromboembolism (VTE). However, comprehensive data on incidence, timing and relevant determinants of VTE in this particular... Show moreIntroduction: Pancreatic cancer is associated with a high risk of venous thromboembolism (VTE). However, comprehensive data on incidence, timing and relevant determinants of VTE in this particular population are scarce. Current study assesses incidence, timing and predictors of VTE in pancreatic cancer through different phases of disease. Methods: All pancreatic cancer patients treated in our tertiary referral center between 2013 through 2017 were studied. Occurrence of VTE was evaluated from diagnosis through end of follow-up or death. Relevant de-terminants of VTE were identified in logistic regression models. Hazard ratios were calculated to evaluate impact of VTE on overall survival. Results: In total, 361 patients were followed for a median period of 43 months; 64 were diagnosed with VTE (18%). Most were tumor related thrombosis (59%), incidental (75%) and occurred after anti-cancer treatment had been stopped (80%), only 1.6% occurred during remission phase. Stage IV pancreatic cancer was a predictor for VTE (hazard ratio (HR) 2.46, 95% confidence interval (CI) 0.9-6.8). Biliary drainage (HR 0.52, 95%CI 0.28-0.98) and tumor resection (HR 0.45, 95%CI 0.45-1.83) were protective factors. VTE was not associated with worse survival (HR 1.3; 95% CI 0.97-1.74). Conclusions: VTE in pancreatic cancer is disease-stage dependent, with 80% occurring in advanced phases of disease when patients no longer receive active treatment. We speculate that this is the main reason for the absence of a survival effect of VTE in our cohort. These practice-based findings should be taken into account when considering wide-spread introduction of primary thromboprophylaxis in patients with pancreatic cancer. Show less
The best current biomarker strategies for predicting response to immune checkpoint inhibitor (ICI) therapy fail to account for interpatient variability in response rates. The histologic tumor... Show moreThe best current biomarker strategies for predicting response to immune checkpoint inhibitor (ICI) therapy fail to account for interpatient variability in response rates. The histologic tumor-stroma ratio (TSR) quantifies intratumoral stromal content and was recently found to be predictive of response to neoadjuvant therapy in multiple cancer types. In the current work, we predicted the likelihood of ICI therapy responsivity of 335 therapy-naive colon adenocarcinoma tumors from The Cancer Genome Atlas, using bioinformatics approaches. The TSR was scored on diagnostic tissue slides, and tumor-infiltrating immune cells (TIICs) were inferred from transcriptomic data. Tumors with high stromal content demonstrated increased T regulatory cell infiltration (p = 0.014) but failed to predict ICI therapy response. Consequently, we devised a hybrid tumor microenvironment classification of four stromal categories, based on histological stromal content and transcriptomic-deconvoluted immune cell infiltration, which was associated with previously established transcriptomic and genomic biomarkers for ICI therapy response. By integrating these biomarkers, stroma-low/immune-high tumors were predicted to be most responsive to ICI therapy. The framework described here provides evidence for expansion of current histological TIIC quantification to include the TSR as a novel, easy-to-use biomarker for the prediction of ICI therapy response. Show less
The tumour-stroma ratio (TSR) and tumour budding (TB) are two high-risk factors with potential to be implemented in the next TNM classification. The aim of the current study was to evaluate the... Show moreThe tumour-stroma ratio (TSR) and tumour budding (TB) are two high-risk factors with potential to be implemented in the next TNM classification. The aim of the current study was to evaluate the practical application of the two biomarkers based on reproducibility, independency and prognostic value. Patients diagnosed with stage II or III colon cancer who underwent surgery between 2005 and 2016 were included. Both TSR and TB were scored on haematoxylin and eosin-stained tissue sections. The TSR, based on the relative amount of stroma, was scored in increments of 10%. TB was scored following the consensus guidelines; a bud was defined as <= 4 tumour cells. For analysis, three categories were used. Cohen's kappa was used for reproducibility. The prognostic value was determined with survival analysis. In total, 246 patients were included. The TSR distribution was N = 137 (56%) stroma-low and N = 109 (44%) stroma-high. The TB distribution was TB-low N = 194 (79%), TB-intermediate N = 35 (14%) and TB-high N = 17 (7%). The reproducibility of the TSR was good (interobserver agreement kappa = 0.83 and intraobserver agreement kappa = 0.82), whereas the inter- and intraobserver agreement for scoring TB was moderate (kappa 0.47 and 0.45, respectively). The survival analysis showed an independent prognostic value for disease-free survival for TSR (HR 1.57; 95% CI 1.01-2.44; p = 0.048) and for TB-high (HR 2.01; 95% CI 1.02-3.96; p = 0.043). Based on current results, we suggest the TSR is a more reliable parameter in daily practice due to better reproducibility and independent prognostic value for disease-free survival. Show less
Hagenaars, S.C.; Groot, S. de; Cohen, D.; Dekker, T.J.A.; Charehbili, A.; Kranenbarg, E.M.K.; ... ; Dutch Breast Can 2021
The tumor-stroma ratio (TSR) has proven to be a strong prognostic factor in breast cancer, demonstrating better survival for patients with stroma-low tumors. Since the role of the TSR as a... Show moreThe tumor-stroma ratio (TSR) has proven to be a strong prognostic factor in breast cancer, demonstrating better survival for patients with stroma-low tumors. Since the role of the TSR as a predictive marker for neoadjuvant chemotherapy outcome is yet unknown, this association was evaluated for HER2-negative breast cancer in the prospective DIRECT and NEOZOTAC trials. The TSR was assessed on 375 hematoxylin and eosin-stained sections of pre-treatment biopsies. Associations between the TSR and chemotherapy response according to the Miller-Payne (MP) grading system, and between the TSR and pathological response were examined using Pearson's chi-square, Cochran-Armitage test for trend and regression analyses. A stroma-low tumor prior to neoadjuvant chemotherapy was significantly associated with a higher MP score (P = .005). This relationship remained significant in the estrogen receptor (ER)-negative subgroup (P = .047). The univariable odds ratio (OR) of a stroma-low tumor on pathological complete response (pCR) was 2.46 (95% CI 1.34-4.51, P = .004), which attenuated to 1.90 (95% CI 0.85-4.25, P = .119) after adjustment for relevant prognostic factors. Subgroup analyses revealed an OR of 5.91 in univariable analyses for ER-negativity (95% CI 1.19-29.48, P = .030) and 1.48 for ER-positivity (95% CI 0.73-3.01, P = .281). In conclusion, a low amount of stroma on pre-treatment biopsies is associated with a higher MP score and pCR rate. Therefore, the TSR is a promising biomarker in predicting neoadjuvant treatment outcome. Incorporating this parameter in routine pathological diagnostics could be worthwhile to prevent overtreatment and undertreatment. Show less
Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the... Show moreBreast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored. Most of the analyses that aim for early detection focus on proteins in the bloodstream. In this study, the biomarker potential of total serum N-glycosylation analysis was explored with regard to detection of breast cancer. In an age-matched case-control setup serum protein N-glycan profiles from 145 breast cancer patients were compared to those from 171 healthy individuals. N-glycans were enzymatically released, chemically derivatized to preserve linkage-specificity of sialic acids and characterized by high resolution mass spectrometry. Logistic regression analysis was used to evaluate associations of specific N-glycan structures as well as N-glycosylation traits with breast cancer. In a case-control comparison three associations were found, namely a lower level of a two triantennary glycans and a higher level of one tetraantennary glycan in cancer patients. Of note, various other N-glycomic signatures that had previously been reported were not replicated in the current cohort. It was further evaluated whether the lack of replication of breast cancer N-glycomic signatures could be partly explained by the heterogenous character of the disease since the studies performed so far were based on cohorts that included diverging subtypes in different numbers. It was found that serum N-glycan profiles differed for the various cancer subtypes that were analyzed in this study. Show less
Patuleia, S.I.S.; Hagenaars, S.C.; Moelans, C.B.; Ausems, M.G.E.M.; Gils, C.H. van; Tollenaar, R.A.E.M.; ... ; Wall, E. van der 2021
Women identified with an increased risk of breast cancer due to mutations in cancer susceptibility genes or a familial history of breast cancer undergo tailored screening with the goal of detecting... Show moreWomen identified with an increased risk of breast cancer due to mutations in cancer susceptibility genes or a familial history of breast cancer undergo tailored screening with the goal of detecting tumors earlier, when potential curative interventions are still possible. Ideally, screening would identify signs of carcinogenesis even before a tumor is detectable by imaging. This could be achieved by timely signaling of altered biomarker levels for precancerous processes in liquid biopsies. Currently, the Nipple Aspirate Fluid (NAF) and the Trial Early Serum Test BREAST cancer (TESTBREAST), both ongoing, prospective, multicenter studies, are investigating biomarkers in liquid biopsies to improve breast cancer screening in high-risk women. The NAF study focuses on changes over time in miRNA expression levels both in blood and NAF samples, whereas the TESTBREAST study analyzes changes in protein levels in blood samples at sequential interval timepoints. These within-subject changes are studied in relation to later occurrence of breast cancer using a nested case-control design. These longitudinal studies face their own challenges in execution, such as hindrances in logistics and in sample processing that were difficult to anticipate. This article offers insight into those challenges and concurrently aims to provide useful strategies for the set-up of similar studies. Show less
