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Previous literature extensively explored biomarkers to personalize treatment for breast cancer patients. The clinical need is especially high in patients with triple-negative breast cancer (TNBC) due to its aggressive nature and limited treatment modalities. This review aims to evaluate the value of tumor-infiltrating lymphocytes (TILs) and tumor-stroma ratio (TSR) as prognostic biomarkers in TNBC patients and assess their clinical potential. A literature search was conducted in PubMed, Embase, Emcare, Web of Science, and Cochrane Library. Papers comparing survival outcomes of TNBC patients with low/high or negative/positive TSR and immune cells were included. The most frequently mentioned subgroups of TILs were selected and reported in this review. Data from 43 articles on TILs and eight articles on TSR were included. Among TNBC patients, high CD8 expression was generally associated with better survival. Notable, the poor survival outcomes were related to high intra-tumoral PD-L1 expression, whereas high stromal PD-L1 expression more often was correlated with favorable outcomes. For the TSR, a high amount of stroma in the primary tumor of TNBC patients was consistently associated with worse survival. This review highlights that a high number of CD8-positive T-cells is a promising prognostic factor for TNBC patients. PD-L1 expression analyzed for intra-tumoral and stromal expression separately reports strong but contrasting information. Finally, the TSR shows potential to be an important prognostic marker, especially for TNBC patients. Utilizing both biomarkers, either on itself or combined, could enhance clinical decision-making and personalization of treatment., Competing Interests: Declarations. Ethics approval: In this article, data were obtained from previously published studies and publicly available sources. Additional ethical approval was therefore not required for this review. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

Purpose: Tumour-stroma ratio (TSR) is an important prognostic and predictive factor in several tumour types. The aim of this study is to determine whether TSR evaluated in breast cancer core biopsies is representative of the whole tumour., Method: Different TSR scoring methods, their reproducibility, and the association of TSR with clinicopathological characteristics were investigated in 178 breast carcinoma core biopsies and corresponding resection specimens. TSR was assessed by two trained scientists on the most representative H&E-stained digitised slides. Patients were treated primarily with surgery between 2010 and 2021 at Semmelweis University, Budapest., Results: Ninety-one percent of the tumours were hormone receptor (HR)-positive (luminal-like). Interobserver agreement was highest using 100 × magnification (κ core  = 0.906, κ resection specimen  = 0.882). The agreement between TSR of core biopsies and resection specimens of the same patients was moderate (κ = 0.514). Differences between the two types of samples were most frequent in cases with TSR scores close to the 50% cut-off point. TSR was strongly correlated with age at diagnosis, pT category, histological type, histological grade, and surrogate molecular subtype. A tendency was identified for more recurrences among stroma-high (SH) tumours (p = 0.07). Significant correlation was detected between the TSR and tumour recurrence in grade 1 HR-positive breast cancer cases (p = 0.03)., Conclusions: TSR is easy to determine and reproducible on both core biopsies and in resection specimens and is associated with several clinicopathological characteristics of breast cancer. TSR scored on core biopsies is moderately representative for the whole tumour., (© 2023. The Author(s).)

Background: The TNM (tumor-node-metastasis) Evaluation Committee of Union for International Cancer Control (UICC) and College of American Pathologists (CAP) recommended to prospectively validate the cost-effective and robust tumor-stroma ratio (TSR) as an independent prognostic parameter, since high intratumor stromal percentages have previously predicted poor patient-related outcomes., Patients and Methods: The 'Uniform Noting for International application of Tumor-stroma ratio as Easy Diagnostic tool' (UNITED) study enrolled patients in 27 participating centers in 12 countries worldwide. The TSR, categorized as stroma-high (>50%) or stroma-low (≤50%), was scored through standardized microscopic assessment by certified pathologists, and effect on disease-free survival (DFS) was evaluated with 3-year median follow-up. Secondary endpoints were benefit assessment of adjuvant chemotherapy (ACT) and overall survival (OS)., Results: A total of 1537 patients were included, with 1388 eligible stage II/III patients curatively operated between 2015 and 2021. DFS was significantly shorter in stroma-high (n = 428) than in stroma-low patients (n = 960) (3-year rates 70% versus 83%; P < 0.001). In multivariate analysis, TSR remained an independent prognosticator for DFS (P < 0.001, hazard ratio 1.49, 95% confidence interval 1.17-1.90). As secondary outcome, DFS was also worse in stage II and III stroma-high patients despite adjuvant treatment (3-year rates stage II 73% versus 92% and stage III 66% versus 80%; P = 0.008 and P = 0.011, respectively). In stage II patients not receiving ACT (n = 322), the TSR outperformed the American Society of Clinical Oncology (ASCO) criteria in identifying patients at risk of events (event rate 21% versus 9%), with a higher discriminatory 3-year DFS rate (stroma-high 80% versus ASCO high risk 91%). A trend toward worse 5-year OS in stroma-high was noticeable (74% versus 83% stroma-low; P = 0.102)., Conclusion: The multicenter UNITED study unequivocally validates the TSR as an independent prognosticator, confirming worse outcomes in stroma-high patients. The TSR improved current selection criteria for patients at risk of events, and stroma-high patients potentially experienced chemotherapy resistance. TSR implementation in pathology diagnostics and international guidelines is highly recommended as aid in personalized treatment., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Self-supervised learning (SSL) automates the extraction and interpretation of histopathology features on unannotated hematoxylin-and-eosin-stained whole-slide images (WSIs). We trained an SSL Barlow Twins-encoder on 435 TCGA colon adenocarcinoma WSIs to extract features from small image patches. Leiden community detection then grouped tiles into histomorphological phenotype clusters (HPCs). HPC reproducibility and predictive ability for overall survival was confirmed in an independent clinical trial cohort (N=1213 WSIs). This unbiased atlas resulted in 47 HPCs displaying unique and sharing clinically significant histomorphological traits, highlighting tissue type, quantity, and architecture, especially in the context of tumor stroma. Through in-depth analysis of these HPCs, including immune landscape and gene set enrichment analysis, and association to clinical outcomes, we shed light on the factors influencing survival and responses to treatments like standard adjuvant chemotherapy and experimental therapies. Further exploration of HPCs may unveil new insights and aid decision-making and personalized treatments for colon cancer patients., Competing Interests: Declaration of interests AT is a co-founder of Imagenomix. The remaining authors declare no competing interests.

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 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-β (TGFβ) signaling in TNBC cells by interacting with TGFβ type I receptor (TβRI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGFβ gene response signature and expression of proto-typic TGFβ target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGFβ receptor/SMAD signaling and suppresses metastasis in TNBC., (© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

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 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.

Epithelial cells acquire mesenchymal phenotypes through epithelial-mesenchymal transition (EMT) during cancer progression. However, how epithelial cells retain their epithelial traits and prevent malignant transformation is not well understood. Here, we report that the long noncoding RNA LITATS1 (LINC01137, ZC3H12A-DT) is an epithelial gatekeeper in normal epithelial cells and inhibits EMT in breast and non-small cell lung cancer cells. Transcriptome analysis identified LITATS1 as a TGF-β target gene. LITATS1 expression is reduced in lung adenocarcinoma tissues compared with adjacent normal tissues and correlates with a favorable prognosis in breast and non-small cell lung cancer patients. LITATS1 depletion promotes TGF-β-induced EMT, migration, and extravasation in cancer cells. Unbiased pathway analysis demonstrated that LITATS1 knockdown potently and selectively potentiates TGF-β/SMAD signaling. Mechanistically, LITATS1 enhances the polyubiquitination and proteasomal degradation of TGF-β type I receptor (TβRI). LITATS1 interacts with TβRI and the E3 ligase SMURF2, promoting the cytoplasmic retention of SMURF2. Our findings highlight a protective function of LITATS1 in epithelial integrity maintenance through the attenuation of TGF-β/SMAD signaling and EMT., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background: The amount of stroma within the primary tumor is a prognostic parameter for colon cancer patients. This phenomenon can be assessed using the tumor-stroma ratio (TSR), which classifies tumors in stroma-low (≤50% stroma) and stroma-high (>50% stroma). Although the reproducibility for TSR determination is good, improvement might be expected from automation. The aim of this study was to investigate whether the scoring of the TSR in a semi- and fully automated method using deep learning algorithms is feasible., Methods: A series of 75 colon cancer slides were selected from a trial series of the UNITED study. For the standard determination of the TSR, 3 observers scored the histological slides. Next, the slides were digitized, color normalized, and the stroma percentages were scored using semi- and fully automated deep learning algorithms. Correlations were determined using intraclass correlation coefficients (ICCs) and Spearman rank correlations., Results: 37 (49%) cases were classified as stroma-low and 38 (51%) as stroma-high by visual estimation. A high level of concordance between the 3 observers was reached, with ICCs of 0.91, 0.89, and 0.94 (all P < .001). Between visual and semi-automated assessment the ICC was 0.78 (95% CI 0.23-0.91, P-value 0.005), with a Spearman correlation of 0.88 (P < .001). Spearman correlation coefficients above 0.70 (N=3) were observed for visual estimation versus the fully automated scoring procedures., Conclusion: Good correlations were observed between standard visual TSR determination and semi- and fully automated TSR scores. At this point, visual examination has the highest observer agreement, but semi-automated scoring could be helpful to support pathologists., Competing Interests: Jeroen van der Laak is member of the scientific advisory boards of Philips, the Netherlands and ContextVision, Sweden and receives research funding from Philips, the Netherlands and Sectra, Sweden. Francesco Ciompi is member of the scientific advisory board of TRIBVN, France. All other authors declare they have no conflicts of interest., (© 2023 The Authors.)

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 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 determining 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., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.K. is the vice president of the issuing organization of the patent-pending Stroma Liquid BiopsyTM product, the Biotech Support Group (BSG). All remaining authors have declared no conflict of interest., (© 2022 THE AUTHORS.)

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, 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 χ2 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.

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-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.

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 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., (© 2022. The Author(s).)

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 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., (© 2022. The Author(s).)

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 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 µm 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., (© 2021. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

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 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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 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., (© 2022. The Author(s).)

Transforming growth factor-β (TGF-β) acts as a pro-metastatic factor in advanced breast cancer. RNF12, an E3 ubiquitin ligase, stimulates TGF-β 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-β and AKT signaling pathways during breast cancer progression., (© 2022. The Author(s).)

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.

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 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., (© 2021. The Author(s).)

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 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 determinants 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., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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-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.

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 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., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

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 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.

Background: The amount of stroma in the primary tumor is an important prognostic parameter. The tumor-stroma ratio (TSR) was previously validated by international research groups as a robust parameter with good interobserver agreement., Objective: The Uniform Noting for International Application of the Tumor-Stroma Ratio as an Easy Diagnostic Tool (UNITED) study was developed to bring the TSR to clinical implementation. As part of the study, an e-Learning module was constructed to confirm the reproducibility of scoring the TSR after proper instruction., Methods: The e-Learning module consists of an autoinstruction for TSR determination (instruction video or written protocol) and three sets of 40 cases (training, test, and repetition sets). Scoring the TSR is performed on hematoxylin and eosin-stained sections and takes only 1-2 minutes. Cases are considered stroma-low if the amount of stroma is ≤50%, whereas a stroma-high case is defined as >50% stroma. Inter- and intraobserver agreements were determined based on the Cohen κ score after each set to evaluate the reproducibility., Results: Pathologists and pathology residents (N=63) with special interest in colorectal cancer participated in the e-Learning. Forty-nine participants started the e-Learning and 31 (63%) finished the whole cycle (3 sets). A significant improvement was observed from the training set to the test set; the median κ score improved from 0.72 to 0.77 (P=.002)., Conclusions: e-Learning is an effective method to instruct pathologists and pathology residents for scoring the TSR. The reliability of scoring improved from the training to the test set and did not fall back with the repetition set, confirming the reproducibility of the TSR scoring method., Trial Registration: The Netherlands Trial Registry NTR7270; https://www.trialregister.nl/trial/7072., International Registered Report Identifier (irrid): RR2-10.2196/13464., (©Marloes A Smit, Gabi W van Pelt, Elisabeth MC Dequeker, Raed Al Dieri, Rob AEM Tollenaar, J Han JM van Krieken, Wilma E Mesker, UNITED Group. Originally published in JMIR Formative Research (http://formative.jmir.org), 19.03.2021.)

Pancreatic ductal adenocarcinoma (PDAC) is known as a highly aggressive malignant disease. Prognosis for patients is notoriously poor, despite improvements in surgical techniques and new (neo)adjuvant chemotherapy regimens. Early detection of PDAC may increase the overall survival. It is furthermore foreseen that precision medicine will provide improved prognostic stratification and prediction of therapeutic response. In this review, omics-based discovery efforts are presented that aim for novel diagnostic and prognostic biomarkers of PDAC. For this purpose, we systematically evaluated the literature published between 1999 and 2020 with a focus on protein- and protein-glycosylation biomarkers in pancreatic cancer patients. Besides genomic and transcriptomic approaches, mass spectrometry (MS)-based proteomics and glycomics of blood- and tissue-derived samples from PDAC patients have yielded new candidates with biomarker potential. However, for reasons discussed in this review, the validation and clinical translation of these candidate markers has not been successful. Consequently, there has been a change of mindset from initial efforts to identify new unimarkers into the current hypothesis that a combination of biomarkers better suits a diagnostic or prognostic panel. With continuing development of current research methods and available techniques combined with careful study designs, new biomarkers could contribute to improved detection, prognosis, and prediction of pancreatic cancer.

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 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. See related commentary by Sauter, p. 429 ., (©2020 American Association for Cancer Research.)

Background: Current risk stratification models for early invasive (T1) colorectal cancer are not able to discriminate accurately between prognostic favourable and unfavourable tumours, resulting in over-treatment of a large (>80%) proportion of T1 colorectal cancer patients. The tumour-stroma ratio (TSR), which is a measure for the relative amount of desmoplastic tumour stroma, is reported to be a strong independent prognostic factor in advanced-stage colorectal cancer, with a high stromal content being associated with worse prognosis and survival. We aimed to investigate whether the TSR predicts clinical outcome in patients with non-pedunculated T1 colorectal cancer., Methods: Haematoxylin and eosin (H&E)-stained tumour tissue slides from a retrospective multicentre case cohort of patients with nonpedunculated surgically treated T1 colorectal cancer were assessed for TSR by two independent observers who were blinded for clinical outcomes. The primary end point was adverse outcome, which was defined as the presence of lymph node metastasis in the resection specimen or colorectal cancer recurrence during follow-up., Results: All 261 patients in the case cohort had H&E slides available for TSR scoring. Of these, 183 were scored as stroma-low, and 78 were scored as stroma-high. There was moderate inter-observer agreement κ = 0.42). In total, 41 patients had lymph node metastasis, 17 patients had recurrent cancer and five had both. Stroma-high tumours were not associated with an increased risk for an adverse outcome (adjusted hazard ratio = 0.66, 95% confidence interval 0.37-1.18; p = 0.163)., Conclusions: Our study emphasises that existing prognosticators may not be simply extrapolated to T1 colorectal cancers, even though their prognostic value has been widely validated in more advanced-stage tumours., (© 2021 The Authors. United European Gastroenterology Journal published by Wiley Periodicals LLC. on behalf of United European Gastroenterology.)

Objective: This study evaluated the correlation between intratumoural stroma proportion, expressed as tumour-stroma ratio (TSR), and apparent diffusion coefficient (ADC) values in patients with rectal cancer., Methods: This multicentre retrospective study included all consecutive patients with rectal cancer, diagnostically confirmed by biopsy and MRI. The training cohort (LUMC, Netherlands) included 33 patients and the validation cohort (VHIO, Spain) 69 patients. Two observers measured the mean and minimum ADCs based on single-slice and whole-volume segmentations. The TSR was determined on diagnostic haematoxylin & eosin stained slides of rectal tumour biopsies. The correlation between TSR and ADC was assessed by Spearman correlation (r s )., Results: The ADC values between stroma-low and stroma-high tumours were not significantly different. Intra-class correlation (ICC) demonstrated a good level of agreement for the ADC measurements, ranging from 0.84-0.86 for single slice and 0.86-0.90 for the whole-volume protocol. No correlation was observed between the TSR and ADC values, with ADC mean r s = -0.162 (p= 0.38) and ADC min r s = 0.041 (p= 0.82) for the single-slice and r s = -0.108 (p= 0.55) and r s = 0.019 (p= 0.92) for the whole-volume measurements in the training cohort, respectively. Results from the validation cohort were consistent; ADC mean r s = -0.022 (p= 0.86) and ADC min r s = 0.049 (p= 0.69) for the single-slice and r s = -0.064 (p= 0.59) and r s = -0.063 (p= 0.61) for the whole-volume measurements., Conclusions: Reproducibility of ADC values is good. Despite positive reports on the correlation between TSR and ADC values in other tumours, this could not be confirmed for rectal cancer., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background &aims: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco-regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel., Methods: Total serum N-glycome analysis was applied to a discovery set (86 PDAC cases/84 controls) followed by independent validation (26 cases/26 controls) using in-house collected serum specimens. Protein N-glycan profiles were obtained using ultrahigh resolution mass spectrometry and included linkage-specific sialic acid information. N-glycans were relatively quantified and case-control classification performance was evaluated based on glycosylation traits such as branching, fucosylation, and sialylation., Results: In PDAC patients a higher level of branching (OR 6.19, P-value 9.21 × 10 -11 ) and (antenna)fucosylation (OR 13.27, P-value 2.31 × 10 -9 ) of N-glycans was found. Furthermore, the ratio of α2,6- vs α2,3-linked sialylation was higher in patients compared to healthy controls. A classification model built with three glycosylation traits was used for discovery (AUC 0.88) and independent validation (AUC 0.81), with sensitivity and specificity values of 0.85 and 0.71 for the discovery set and 0.75 and 0.72 for the validation set., Conclusion: Serum N-glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than single N-glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood-based diagnostic test., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Therapy resistance is a known problem in breast cancer and is associated with a variety of mechanisms. The role of the tumor microenvironment in cancer development and resistance mechanisms is becoming increasingly understood. Tumor-stroma is the main component of the tumor microenvironment. Stromal cells like cancer-associated fibroblasts (CAFs) are believed to contribute to chemotherapy resistance via the production of several secreted factors like cytokines and chemokines. CAFs are found to influence disease progression; patients with primary tumors with a high amount of tumor-stroma have a significantly worse outcome. Therefore the role of CAFs resistance mechanisms makes them a promising target in anti-cancer therapy. An overview of recent advances in strategies to target breast cancer stroma is given and the current literature regarding these stromal targets is discussed. CAF-specific proteins as well as secreted molecules involved in tumor-stroma interactions provide possibilities for stroma-specific therapy. The development of stroma-specific therapy is still in its infancy and the available literature is limited. Within the scope of personalized treatment, biomarkers based on the tumor-stroma have future potential for the improvement of treatment via image-guided surgery (IGS) and PET scanning.

It has become clear that carcinogenesis goes beyond tumor cell biology. Cancer research has acknowledged the importance of biological functions of the tumor-microenvironment, wherein not only cellular components seem to hold valuable information but also structural components like collagen fibers. Several studies have focused on the significance of stromal collagen fiber organization and reported on its role in cancer progression, invasiveness and treatment response. In this review, we discuss the different imaging methods for stromal collagen organization, followed by an in-depth discussion of current literature on in-vitro and animal experiments and human studies, highlighting its importance with respect to cancer progression, prognosis and prediction. We can conclude that collagen organization contains valuable information with regard to metastatic potential and clinical outcomes in cancer. However, the significance of an aligned versus disorganized collagen morphology differs between cancer types, implying more research is necessary before steps towards clinical implementation can be made., Competing Interests: Declaration of Competing Interest All authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

The tumor-stroma ratio (TSR) was evaluated as a promising parameter for breast cancer prognostication in clinically relevant subgroups of patients. The TSR was assessed on hematoxylin and eosin-stained tissue slides of 1,794 breast cancer patients from the Nottingham City Hospital. An independent second cohort of 737 patients from the Netherlands Cancer Institute to Antoni van Leeuwenhoek was used for evaluation. In the Nottingham Breast Cancer series, the TSR was an independent prognostic parameter for recurrence-free survival (RFS; HR 1.35, 95% CI 1.10-1.66, p = 0.004). The interaction term was statistically significant for grade and triple-negative status. Multivariate Cox regression analysis showed a more pronounced effect of the TSR for RFS in grade III tumors (HR 1.89, 95% CI 1.43-2.51, p < 0.001) and triple-negative tumors (HR 1.86, 95% CI 1.10-3.14, p = 0.020). Comparable hazard ratios and confidence intervals were observed for grade and triple-negative status in the ONCOPOOL study. The prognostic value of TSR was not modified by age, tumor size, histology, estrogen receptor status, progesterone receptor status, human epidermal growth factor receptor 2 status or lymph node status. In conclusion, patients with a stroma-high tumor had a worse prognosis compared to patients with a stroma-low tumor. The prognostic value of the TSR is most discriminative in grade III tumors and triple-negative tumors. The TSR was not modified by other clinically relevant parameters making it a potential factor to be included for improved risk stratification., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N - and O -glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N - and O -glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics.

Purpose: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies., Experimental Design: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo , we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations., Results: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFβ signaling-induced metastasis by protecting TGFβ type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression., Conclusions: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis and may provide a potential target for TNBC treatment., (©2019 American Association for Cancer Research.)

Patients with the mesenchymal subtype colorectal cancer (CRC) have a poor prognosis, in particular patients with stroma-rich tumors and aberrant SMAD4 expression. We hypothesized that interactions between SMAD4-deficient CRC cells and cancer-associated fibroblasts provide a biological explanation. In transwell invasion assays, fibroblasts increased the invasive capacity of SMAD4-deficient HT29 CRC cells, but not isogenic SMAD4-proficient HT29 cells. A TGF-β/BMP-specific array showed BMP2 upregulation by fibroblasts upon stimulation with conditioned medium from SMAD4-deficient CRC cells, while also stimulating their invasion. In a mouse model for experimental liver metastasis, the co-injection of fibroblasts increased metastasis formation of SMAD4-deficient CRC cells (p = 0.02) but not that of SMAD4-proficient CRC cells. Significantly less metastases were seen in mice co-injected with BMP2 knocked-down fibroblasts. Fibroblast BMP2 expression seemed to be regulated by TRAIL, a factor overexpressed in SMAD4-deficient CRC cells. In a cohort of 146 stage III CRC patients, we showed that patients with a combination of high stromal BMP2 expression and the loss of tumor SMAD4 expression had a significantly poorer overall survival (HR 2.88, p = 0.04). Our results suggest the existence of a reciprocal loop in which TRAIL from SMAD4-deficient CRC cells induces BMP2 in fibroblasts, which enhances CRC invasiveness and metastasis.

Changes in the abundance of antennary fucosylated glycans in human total plasma N -glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N -linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of α2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer., (Copyright © 2020 Rebello, Nicolardi, Lageveen-Kammeijer, Nouta, Gardner, Mesker, Tollenaar, Spencer, Wuhrer and Falck.)

Objectives: The tumor-stroma ratio (TSR) is based on the relative amount of stroma in the primary tumor and has proven to be an independent prognostic factor in various solid tumors. The prognosis of patients and adjuvant treatment decision making in lung squamous cell carcinomas (SqCC) is based on the TNM classification. Currently, no other prognostic biomarkers are available. In this study we evaluated the prognostic value of the TSR in lung SqCC., Material and Methods: Patients undergoing lung surgery because of lung SqCC between 2000 and 2018 at the Leiden University Medical Center were included. The TSR was scored on hematoxylin & eosin stained tissue sections. Based on the amount of tumor-stroma, two groups were defined: ≤50% was classified as a stroma-low tumor and >50% as stroma-high. The prognostic value of the TSR was determined with survival analysis., Results: A total of 174 stage I-III patients were included. Of them, 79 (45%) were stroma-low and 95 (55%) stroma-high. Separately analyzed for tumor stages, the TSR showed to be an independent prognostic biomarker in stage II (n = 68) for 5-year overall survival (HR=3.0; 95% CI, 1.1-8.6; p = 0.035) and 5-year disease free survival (DFS) (HR=3.6; 95% CI, 1.3-9.9; p = 0.014). Patients with a stroma-high tumor had a worse 5-year DFS in the whole cohort (HR 1.6; 95% CI, 1.0-2.4; p = 0.048), but no independent prognostic value was found., Conclusion: In stage II lung SqCC patients, stroma-low tumors have a better prognosis compared to stroma-high tumors. Moreover, adjuvant chemotherapy could be spared for these stroma-low patients., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The tumour microenvironment in older patients is subject to changes. The tumour-stroma ratio (TSR) was evaluated in order to estimate the amount of intra-tumoural stroma and to evaluate the prognostic value of the TSR in older patients with breast cancer (≥ 70 years)., Methods: Two retrospective cohorts, the FOCUS study (N = 619) and the Nottingham Breast Cancer series (N = 1793), were used for assessment of the TSR on haematoxylin and eosin stained tissue slides., Results: The intra-tumoural stroma increases with age in the FOCUS study and the Nottingham Breast Cancer series (B 0.031, 95% CI 0.006-0.057, p = 0.016 and B 0.034, 95% CI 0.015-0.054, p < 0.001, respectively). Fifty-one per cent of the patients from the Nottingham Breast Cancer series < 40 years had a stroma-high tumour compared to 73% of the patients of ≥ 90 years from the FOCUS study. The TSR did not validate as an independent prognostic parameter in patients ≥ 70 years., Conclusions: The intra-tumoural stroma increases with age. This might be the result of an activated tumour microenvironment. The TSR did not validate as an independent prognostic parameter in patients ≥ 70 years in contrast to young women with breast cancer as published previously.

Body fluid N-glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N-glycosylation analysis aiming for high-throughput clinical applications. A protocol for facile N-glycosylation profiling from DBS is developed that includes sialic acid linkage differentiation. This protocol is based on a previously established total plasma N-glycome mass spectrometry (MS) method, with adjustments for the analysis of DBS specimens. After DBS-punching and protein solubilization N-glycans are released, followed by chemical derivatization of sialic acids and MS-measurement of N-glycan profiles. With this method, more than 80 different glycan structures are identified from a DBS, with RSDs below 10% for the ten most abundant glycans. N-glycan profiles of finger-tip blood and venous blood are compared and short-term stability of DBS is demonstrated. This method for fast N-glycosylation profiling of DBS provides a minimally invasive alternative to conventional serum and plasma protein N-glycosylation workflows. With simplified blood sampling this DBS approach has vast potential for clinical glycomics applications., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)