Microscopical evaluation of prognostic factors in colorectal cancer

Aims and outline of the thesis. Since Fearon and Vogelstein in 1990 presented the genetic model for the adeno-carcinoma sequence of colorectal cancer, many prognostic studies varying from early stage markers to markers involved in late progression and liver metastases have followed. As has become evident from this introduction there is an ongoing need for prognostic markers that can be used for individualized prediction of clinical outcome. Chapter 2. Many systems are available for the detection of occult tumor cells in the bone marrow, blood and lymph nodes of cancer patients. In this chapter an overview is given of the various commercially available automated microscopy systems, and their capabilities. Furthermore the current status of the application of these instruments for bone marrow, blood and lymph nodes is presented. Chapter 3. Spread to locoregional lymph nodes is one of the most important prognostic indicators of the TNM classification. Detection of micrometastases in node-negative patients might upstage patients in need for additional chemotherapy. In this chapter an approach is described by which immunohistochemical staining and multiple sectioning is combined and is subjected to novel high-throughput automated imaging. Chapter 4. The presence of tumor cells in the bone marrow (BM) of cancer patients has shown to be related to a worse prognosis. This paper describes the use of array-CGH to detect genome alterations (gains and losses) in primary tumor tissue from BM-positive patients compared to matched (on stage and site) BM-negative patients. A higher number of differential aberrations and a distinct chromosome pattern, confirmed by interphase FISH, were found in the BM-positive group as compared to the BM-negative group. Chapter 5. While analyzing primary tumor tissue for a pilot study for array-CGH it was noticed that the set of patients with bad prognosis could not be analyzed, due to the fact that the amount of tumor material was less than 50%. This lower threshold is important for array-CGH to obtain reliable DNA profiles of the tumor cells and to avoid contamination with normal cells. Morphological evaluation of H&E stained sections showed that these tumors with bad prognosis had a high proportion of stroma and few tumor cells. The tumors with good prognosis showed the opposite, abundant tumor and less stroma. This phenomenon has led to the prognostic evaluation of this parameter in a larger patient study of which the results are shown in this chapter. Chapter 6. In this chapter the work presented in chapter 4 was continued but now focused on stage I-II colon patients. This subgroup of patients is in need for additional markers to select specific __high risk____ patients. Immunohistochemical staining of three molecular markers known to be involved in stroma production was performed. SMAD4 expression status was found to further improve the prognostic value of the presence of stroma in the primary tumor. Chapter 7. The conclusions of the studies presented in this thesis and the future perspectives of the presented parameters are discussed in this chapter.Financial support for publication of this thesis was kindly provided by: Leica Microsystems B.V, Covidien Nederland B.V., Merck Serono. With special thanks for their generous support to Applied Imaging a Genetix CompanyUBL - phd migration 201

In reply to Hynes et al.: "Back to the future: routine morphological assessment of the tumour microenvironment is prognostic in stages II/III colon cancer in a large population-based study'

Surgical oncolog

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

Surgical oncolog

A high tumour-stroma ratio (TSR) in colon tumours and its metastatic lymph nodes predicts poor cancer-free survival and chemo resistance

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 mu m haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low ( 50%). 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

Combined assessment of the tumor-stroma ratio and tumor immune cell infiltrate for immune checkpoint inhibitor therapy response prediction in colon cancer

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.Experimentele farmacotherapi

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.Surgical oncolog

Serum N-glycan profiles differ for various breast cancer subtypes

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.Surgical oncolog

Application of a Serum Protein Signature for Pancreatic Cancer to Separate Cases from Controls in a Pancreatic Surveillance Cohort

BACKGROUND: Pancreatic cancer (PC) surveillance is currently offered to individuals with a genetic predisposition to PC, but routinely used radiological screening modalities are not entirely reliable in detecting early-stage PC or its precursor lesions. We recently identified a discriminating PC biomarker signature in a sporadic patient cohort. In this study, we investigated if protein profiling can accurately distinguish PC from non-PC in a pancreatic surveillance cohort of genetically predisposed individuals. METHODS: Serum samples of 66 individuals with a CDKN2A germline mutation who participated in the pancreatic surveillance program (5 cases, 61 controls) were obtained following a standardized protocol. After sample clean-up, peptide and protein profiles were obtained on an ultrahigh-resolution matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry platform. A discriminant score for each sample was calculated with a previously designed prediction rule, and the median discriminant scores of cases and controls were compared. Individuals with precursor lesions of PC (n = 4) and individuals with a recent diagnosis of melanoma (n = 4) were also separately considered. RESULTS: Cases had a higher median discriminant score than controls (0.26 vs 0.016; P = .001). The only individual with pathologically confirmed precursor lesions of PC could also be clearly distinguished from controls, and having a (recent) medical history of melanoma did not influence the protein signatures. CONCLUSIONS: Peptide and protein signatures are able to accurately distinguish PC cases from controls in a pancreatic surveillance setting. Mass spectrometry-based protein profiling therefore seems to be a promising candidate for implementation in the pancreatic surveillance program as an additional screening modality.Surgical oncolog

A Matrix-Assisted Laser Desorption/Ionization—Mass Spectrometry Assay for the Relative Quantitation of Antennary Fucosylated N-Glycans in Human Plasma

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.Surgical oncolog

Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and Ultrahigh Resolution MS

Surgical oncolog