Standardization of the tumor-stroma ratio scoring method for breast cancer research.

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.

Serum-based measurements of stromal activation through ADAM12 associate with poor prognosis in colorectal cancer.

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.

Tumour-stroma ratio outperforms tumour budding as biomarker in colon cancer: a cohort study.

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.

The prognostic value of the tumor-stroma ratio is most discriminative in patients with grade III or triple-negative breast cancer.

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.

The intra-tumoural stroma in patients with breast cancer increases with age.

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.

The prognostic value of tumor-stroma ratio in tumor-positive axillary lymph nodes of breast cancer patients.

The tumor-stroma ratio (TSR) has previously been found to be a strong prognostic parameter in primary breast cancer tumors. Since the presence of tumor cells in lymph nodes is important for clinical decision making, the influence of TSR in the primary breast tumor combined with the TSR in tumor-positive lymph nodes on prognosis was evaluated. Women with invasive breast cancer without distant metastasis who underwent an axillary lymph node dissection between 1985 and 1994 at the Leiden University Medical Center were retrospectively analyzed. TSR assessment was performed on hematoxylin and eosin stained tissue slides. In total, 87 (45.5%) primary tumors were scored as stroma-low and 104 (54.5%) as stroma-high. Patients with a high stromal percentage in the primary tumors had a statistically significant worse relapse free period (RFP) compared to stroma-low tumors (HR 1.97, 95% CI 1.37-2.82, p < 0.001). A total number of 915 lymph nodes were assessed for TSR. In 101 (52.9%) patients, heterogeneity was observed between stroma percentage category in primary tumor and lymph nodes. The combination of TSR of the primary tumor combined with TSR of tumor-positive lymph nodes strengthened each other as independent prognostic parameter for RFP (p = 0.019). Patients with primary tumor stroma-low/lymph nodes stroma-low tumors showed strongly improved RFP rates compared to patients with primary tumor stroma-high/lymph node stroma-high tumors with 10-year percentages of 58 versus 8%, respectively. Assessing the TSR on tumor-positive lymph nodes can provide additional prognostic information. Stromal activation strongly differs between primary tumors and lymph node metastasis.

Predictive potential of tumour-stroma ratio on benefit from adjuvant bevacizumab in high-risk stage II and stage III colon cancer.

BACKGROUND: The tumour-stroma ratio (TSR) has proven to be an independent prognostic factor in colon cancer. METHODS: Haematoxylin eosin tissue slides of patients from the AVANT trial were microscopically scored for TSR and categorised as stroma -low or stroma -high. Scores were correlated to the primary and secondary endpoint disease-free survival (DFS) and overall survival (OS). RESULTS: Patients with stroma-high tumours (N = 339, 28%) had a significantly shorter DFS (p < 0.001) compared to stroma-low tumours (N = 824, 68%). In the bevacizumab-FOLFOX-4 arm, DFS was significantly shorter compared to FOLFOX-4 in stroma-low tumours, with a hazard ratio (HR) of 1.94 (95% CI 1.24-3.04; p = 0.004). In stroma-high tumours a trend for better DFS was seen in bevacizumab-FOLFOX-4 vs. FOLFOX-4 (HR 0.61 (95% CI 0.35-1.07; p = 0.08)). For bevacizumab-XELOX vs. FOLFOX-4, this was not seen (stroma-low HR 1.07 (95% CI 0.64-1.77; p = 0.80); stroma-high HR 0.78 (95% CI 0.47-1.30; p = 0.35)). OS showed the same pattern for bevacizumab-FOLFOX-4 vs. FOLFOX-4 with a HR of 2.53 (95% CI 1.36-4.71; p = 0.003) for stroma-low and HR 0.50 (95% CI 0.22-1.14; p = 0.10) for stroma-high tumours. For bevacizumab-XELOX vs. FOLFOX-4, HR 1.13 (95% CI 0.55-2.31; p = 0.74) for stroma-low tumours and HR 0.74 (95% CI 0.37-1.51; p = 0.41) for stroma-high tumours. CONCLUSIONS: This exploratory analysis suggests a significantly shorter DFS and OS in stroma-low tumours with addition of bevacizumab to intravenous oxaliplatin-based chemotherapy, contrary to stroma-high tumours, where a beneficial trend is observed.

The tumour-stroma ratio in colon cancer: the biological role and its prognostic impact.

The tumour microenvironment consists of a complex mixture of non-neoplastic cells, including fibroblasts, immune cells and endothelial cells embedded in the proteins of the extracellular matrix. The tumour microenvironment plays an active role in tumour behaviour. By interacting with cancer cells, it influences disease progression and the metastatic capacity of the tumour. Tumours with a high amount of stroma correspond to poor patient prognosis. The tumour-stroma ratio (TSR) is a strong independent prognostic tool in colon cancer and provides additional value to the current clinically used tumour-node-metastasis classification. The TSR is assessed on conventional haematoxylin and eosin-stained paraffin sections at the invasive front of the tumour. Here we review studies demonstrating the prognostic significance of the TSR in solid epithelial tumours with a focus on colon cancer. Moreover, the biological role of the tumour microenvironment during tumour progression and invasion will be discussed, as well as the attempts to target the tumour stroma for therapeutic purposes. We suggest that the TSR can be implemented with little effort and without additional costs in current routine pathology diagnostics owing to its simplicity and reliability.

The value of additional bevacizumab in patients with high-risk stroma-high colon cancer. A study within the QUASAR2 trial, an open-label randomized phase 3 trial.

INTRODUCTION: Patients with a high stroma percentage within the primary tumor have a poor prognosis. In this study, we investigate whether anti-angiogenic therapy might improve survival of patients with a stroma-high profile with potentially increased angiogenesis. MATERIALS AND METHODS: Tissue samples of the primary tumor of 965 colon cancer patients participating in the QUASAR2 trial were analyzed for tumor-stroma ratio (TSR). Stroma-high (>50%) and stroma-low (≤50%) groups were evaluated with respect to survival. RESULTS: Disease free survival (DFS) was significantly lower in the stroma-high group (HR 1.53, 95%CI 1.19-1.95, P = 0.001). No difference in DFS was seen with respect to treatment with capecitabine alone (CAP) or capecitabine with bevacizumab (CAPBEV) (Stroma-high HR 1.00, 95%CI 0.69-1.46, P = 0.996; stroma-low HR 1.02, 95%CI 0.75-1.41, P = 0.883). A significant difference in survival was seen comparing groups with or without vascular invasion (DFS P < 0.001). A correlation between vascular invasion and stroma-high was seen (χ2 -test P = 0.043). DISCUSSION AND CONCLUSIONS: The TSR confirmed to be a strong prognosticator for disease-free survival in a selected high-risk patient population. No benefit was found in response to treatment with bevacizumab when stratified for TSR. TSR showed to have an additional prognostic value in patients with vascular invasion present in the primary tumor.

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression.

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section.

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.

Linkage-Specific in Situ Sialic Acid Derivatization for N-Glycan Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded Tissues.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.

The prognostic value of apoptotic and proliferative markers in breast cancer.

Increasing ability of early breast cancer (BC) diagnosis leading to more early stage detection, better survival, and low relapse marks one of the milestones achieved over the decades. Foregoing poses a challenge for clinicians regarding optimal treatment, in which over- and under-treatment should be avoided. Classical prognostic and predictive factors fall short for individualized adjuvant therapy selection in this patient group. The key to better characterization may be found in the biology underlying individual tumors. We hypothesized that markers related to cellular proliferation and apoptosis and the balance between these two processes in tumor development will be predictive for clinical outcome. Our study population (N = 822) consisted of all early stage BC patients primarily treated with surgery in our center between 1985 and 1996. Sections of available tumor tissue (87 %, 714/822) were immunohistochemically stained for expression of p53, active-caspase-3, and Ki67. In 43 % (304/714) and 18 % (126/714) of this cohort, respectively, a biochemical C2P(®) risk prediction and caspase-3 assay were performed. Expression data of the mentioned markers, single, or combined, were analyzed. Results showed that both the single and combined markers, whether of apoptotic or proliferative origin had associations with clinical outcome. An additive effect was seen for the hazard ratios when data on p53, active caspase-3, and Ki67 status were combined. The assembled prognostic apoptotic-proliferative subtype showed significant association for both the overall survival (p = 0.024) and relapse-free period (p = 0.001) in the multivariate analyses of grade I breast tumors. Combined markers of tumor cell apoptosis and proliferation represent tumor aggressiveness. The apoptotic-proliferative subtypes that we present in this study represent a clinical prognostic profile with solid underlying biological rationale and pose a promising method for accurate identification of grade I BC patients in need of an aggressive therapeutic approach, thus contributing to precision medicine in BC disease.

Deep learning based tumor-stroma ratio scoring in colon cancer correlates with microscopic assessment.

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.

e-Learning for Instruction and to Improve Reproducibility of Scoring Tumor-Stroma Ratio in Colon Carcinoma: Performance and Reproducibility Assessment in the UNITED 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.

Tumour-stroma ratio has poor prognostic value in nonpedunculated T1 colorectal cancer: A multicentre case-cohort study.

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.

Correlation of the tumour-stroma ratio with diffusion weighted MRI in rectal cancer.

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 (rs). 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 ADCmeanrs= -0.162 (p= 0.38) and ADCminrs= 0.041 (p= 0.82) for the single-slice and rs= -0.108 (p= 0.55) and rs= 0.019 (p= 0.92) for the whole-volume measurements in the training cohort, respectively. Results from the validation cohort were consistent; ADCmeanrs= -0.022 (p= 0.86) and ADCminrs = 0.049 (p= 0.69) for the single-slice and rs= -0.064 (p= 0.59) and rs= -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.

Bidirectional tumor/stroma crosstalk promotes metastasis in mesenchymal colorectal cancer.

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-ß/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.

Tumour-stroma ratio has poor prognostic value in non-pedunculated T1 colorectal cancer: A multi-centre case-cohort study.

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: Hematoxylin and eosin (H&E)-stained tumour tissue slides from a retrospective multi-centre case cohort of patients with non-pedunculated 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.