Correction to: Assessment of individual tumor buds using keratin immunohistochemistry: moderate interobserver agreement suggests a role for machine learning.

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Assessment of individual tumor buds using keratin immunohistochemistry: moderate interobserver agreement suggests a role for machine learning.

Tumor budding is a promising and cost-effective biomarker with strong prognostic value in colorectal cancer. However, challenges related to interobserver variability persist. Such variability may be reduced by immunohistochemistry and computer-aided tumor bud selection. Development of computer algorithms for this purpose requires unequivocal examples of individual tumor buds. As such, we undertook a large-scale, international, and digital observer study on individual tumor bud assessment. From a pool of 46 colorectal cancer cases with tumor budding, 3000 tumor bud candidates were selected, largely based on digital image analysis algorithms. For each candidate bud, an image patch (size 256 × 256 µm) was extracted from a pan cytokeratin-stained whole-slide image. Members of an International Tumor Budding Consortium (n = 7) were asked to categorize each candidate as either (1) tumor bud, (2) poorly differentiated cluster, or (3) neither, based on current definitions. Agreement was assessed with Cohen's and Fleiss Kappa statistics. Fleiss Kappa showed moderate overall agreement between observers (0.42 and 0.51), while Cohen's Kappas ranged from 0.25 to 0.63. Complete agreement by all seven observers was present for only 34% of the 3000 tumor bud candidates, while 59% of the candidates were agreed on by at least five of the seven observers. Despite reports of moderate-to-substantial agreement with respect to tumor budding grade, agreement with respect to individual pan cytokeratin-stained tumor buds is moderate at most. A machine learning approach may prove especially useful for a more robust assessment of individual tumor buds.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer.

BACKGROUND: The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE: To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS: IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS: IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS: Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND: There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS: mRNA in situ hybridisation and immunostaining for E-cadherin, ß-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS: Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and ß-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous ß-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS: In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Prognostic role of FGFR1 amplification in early-stage non-small cell lung cancer.

BACKGROUND: Recently, fibroblast growth factor receptor 1 (FGFR1) was discovered in squamous cell carcinomas (SCC) of the lung with FGFR1 amplification described as a promising predictive marker for anti-FGFR inhibitor treatment. Only few data are available regarding prevalence, prognostic significance and clinico-pathological characteristics of FGFR1-amplified and early-stage non-small cell lung carcinomas (NSCLC). We therefore investigated the FGFR1 gene status in a large number of well-characterised early-stage NSCLC. METHODS: FGFR1 gene status was evaluated using a commercially available fluorescent in situ hybridisation (FISH) probe on a tissue microarray (TMA). This TMA harbours 329 resected, formalin-fixed and paraffin-embedded, nodal-negative NSCLC with a UICC stage I-II. The FISH results were correlated with clinico-pathological features and overall survival (OS). RESULTS: The prevalence of an FGFR1 amplification was 12.5% (41/329) and was significantly (P<0.0001) higher in squamous cell carcinoma (SCC) (20.7%) than in adenocarcinoma (2.2%) and large cell carcinoma (13%). Multivariate analysis revealed significantly (P=0.0367) worse 5-year OS in patients with an FGFR1-amplified NSCLC. CONCLUSIONS: FGFR1 amplification is common in early-stage SCC of the lung and is an independent and adverse prognostic marker. Its potential role as a predictive marker for targeted therapies or adjuvant treatment needs further investigation.

Intratumoural budding (ITB) in preoperative biopsies predicts the presence of lymph node and distant metastases in colon and rectal cancer patients.

BACKGROUND: In colorectal cancer (CRC), tumour budding at the invasion front is associated with lymph node (LN) and distant metastasis. Interestingly, tumour budding can also be detected in biopsies (intratumoural budding; ITB) and may have similar clinical importance. Here we investigate whether ITB in preoperative CRC biopsies can be translated into daily diagnostic practice. METHODS: Preoperative biopsies from 133 CRC patients (no neoadjuvant therapy) underwent immunohistochemistry for pan-cytokeratin marker AE1/AE3. Across all biopsies for each patient, the densest region of buds at × 40 (high-power field; HPF) was identified and buds were counted. RESULTS: A greater number of tumour buds in the biopsy was associated with pT stage (P=0.0143), LN metastasis (P=0.0007), lymphatic (P=0.0065) and venous vessel invasion (P=0.0318) and distant metastasis (cM1) (P=0.0013). Using logistic regression, a 'scale' was developed to estimate the probability of LN and distant metastasis using the number of tumour buds (e.g. 10 buds per HPF: 64% chance of LN metastasis; 30 buds per HPF: 86% chance). Inter-observer agreement for ITB was excellent (intraclass correlation coefficient: 0.813). CONCLUSION: Tumour budding can be assessed in the preoperative biopsy of CRC patients. It is practical, reproducible and predictive of LN and distant metastasis. Intratumoural budding qualifies for further investigation in the prospective setting.

Geographic analysis of RKIP expression and its clinical relevance in colorectal cancer.

BACKGROUND: This study evaluates the geographic expression pattern of Raf-1 Kinase Inhibitor Protein (RKIP) in colorectal cancer (CRC) in correlation with clinicopathological and molecular features, markers of epithelial-mesenchymal transition (EMT) and survival outcome. METHODS: Whole-tissue sections of 220 well-characterised CRCs were immunostained for RKIP. NF-κB and E-Cadherin expression was assessed using a matched multi-punch tissue microarray. Analysis of mismatch repair (MMR) protein expression, B-Raf and KRAS mutations was performed. RKIP expression in normal mucosa, tumour centre, invasion front and tumour buds was each assessed for clinical relevance. RESULTS: RKIP was diffusely expressed in normal mucosa and progressively lost towards tumour centre and front (P<0.0001). Only 0.9% of tumour buds were RKIP-positive. In the tumour centre, RKIP deficiency predicted metastatic disease (P=0.0307), vascular invasion (P=0.0506), tumour budding (P=0.0112) and an invasive border configuration (P=0.0084). Loss of RKIP correlated with NF-κB activation (P=0.0002) and loss of E-Cadherin (P<0.0001). Absence of RKIP was more common in MMR-deficient cancers (P=0.0191), while no impact of KRAS and B-Raf mutation was observed. RKIP in the tumour centre was identified as a strong prognostic indicator (HR (95% CI): 2.13 (1.27-3.56); P=0.0042) independently of TNM classification and therapy (P=0.0474). CONCLUSION: The clinical relevance of RKIP expression as an independent prognostic factor is restricted to the tumour centre. Loss of RKIP predicts features of EMT and correlates with frequent distant metastasis.

Tumour budding: a promising parameter in colorectal cancer.

In 2011, the Tumour Node Metastasis (TNM) staging system still remains the gold standard for stratifying colorectal cancer (CRC) patients into prognostic subgroups, and is considered a solid basis for treatment management. Nevertheless, there is still a challenge with regard to therapeutic strategy; stage II patients are not typically selected for postoperative adjuvant chemotherapy, although some stage II patients have a comparable outcome to stage III patients who, themselves do receive such treatment. Consequently, there has been an inundation of 'prognostic biomarker' studies aiming to improve the prognostic stratification power of the TNM staging system. Most proposed biomarkers are not implemented because of lack of reproducibility, validation and standardisation. This problem can be partially resolved by following the REMARK guidelines. In search of novel prognostic factors for patients with CRC, one might glance at a table in the book entitled 'Prognostic Factors in Cancer' published by the International Union against Cancer (UICC) in 2006, in which TNM stage, L and V classifications are considered 'essential' prognostic factors, whereas tumour grade, perineural invasion, tumour budding and tumour-border configuration among others are proposed as 'additional' prognostic factors. Histopathology reports normally include the 'essential' features and are accompanied by tumour grade, histological subtype and information on perineural invasion, but interestingly, the tumour-border configuration (i.e., growth pattern) and especially tumour budding are rarely reported. Although scoring systems such as the 'BRE' in breast and 'Gleason' in prostate cancer are solidly based on histomorphological features and used in daily practice, no such additional scoring system to complement TNM staging is available for CRC. Regardless of differences in study design and methods for tumour-budding assessment, the prognostic power of tumour budding has been confirmed by dozens of study groups worldwide, suggesting that tumour budding may be a valuable candidate for inclusion into a future prognostic scoring system for CRC. This mini-review therefore attempts to present a short and concise overview on tumour budding, including morphological, molecular and prognostic aspects underlining its inter-disciplinary relevance.

Parallel determination of NeuroD1, chromogranin-A, KI67 and androgen receptor expression in surgically treated prostate cancers.

PURPOSE: Neuroendocrine differentiation is a hallmark of prostate cancer. The aim of our study was the detection of the parallel expression of neuroendocrine related markers using a prostate tissue microarray (TMA). MATERIALS AND METHODS: Our study was aimed at detecting the parallel expression of NeuroD1, Chromogranin-A (ChrA), Androgen Receptor (AR) and Ki-67 by immunohistochemistry on prostate cancer tissue microarray. The data was analyzed using SAS version 8.2 (SAS Inc, Cary, NC). The relationships between NeuroD1, ChrA and AR expressions and patients' characteristics were investigated by multivariate logistic regression analysis. Progression and Overall Survival (OS) distributions were calculated using Kaplan-Meier method. RESULTS: Tissue reactivity for NeuroD1, ChrA and AR concerned 73%, 49% and 77% of the available cases, respectively. Regarding overall survival, there were 87 deaths and 295 patients alive/censored (6 years of median follow-up). Seventy-seven disease progressions occurred at the median follow-up 5.4y. A significant correlation between NeuroD1, ChrA and AR expression was observed (p < 0.001 and p < 0.03, respectively). Additionally, ChrA was strongly associated in multivariate analysis to Gleason score and Ki67 expression (p < 0.009 and p < 0.0052, respectively). Survival analysis showed no association between markers neither for overall nor for cancer-specific survival. CONCLUSIONS: The results highlight that NeuroD1, Chromogranin-A and Androgen Receptor are strongly associated, however their expression does not correlate with overall survival or disease progression.

Integrated functional and spatial profiling of tumour immune responses induced by immunotherapy: the iPROFILER platform.

BACKGROUND: Cancer immunotherapy elicits functional activation and changes in immune cell distribution in cancer. Tumour heterogeneity is a reason for treatment failure but is difficult to capture in experimental settings. This proof-of-principle study describes the integrated functional and digital spatial profiling platform iPROFILER to capture in-situ immune activation patterns with high precision. MATERIALS AND METHODS: iPROFILER combines an algorithm-based image analysis approach for spatial profiling with functional analyses of patient-derived tumour fragments (PDTFs). This study utilized a folate receptor 1 (FOLR1)xCD3 bispecific antibody in dual-affinity re-targeting (DART) format as a tool for inducing T-cell responses in patient tumour samples, and an in-depth investigation of the immune perturbations induced in the tumour microenvironment was performed. RESULTS: Ex-vivo DART stimulation induces upregulation of multiple activation markers in CD4+ and CD8+ T-cell populations and secretion of pro-inflammatory cytokines in FOLR1-positive tumour specimens. This response was reduced or absent in tissue samples that did not express FOLR1. Immunological responses were driven by a strong induction of interferon gamma (IFNγ) and IFNγ-induced chemokines suggestive of activation of cytotoxic or Th1-like T cells. Ex-vivo DART treatment led to a numerical increase in effector T cells and an upregulation of immune activation markers in the tumour microenvironment as captured by digital image analysis. Analysis of immune activation in tumour and stromal regions further supported the potential of the platform to measure local differences in cell-type-specific activation patterns. CONCLUSIONS: iPROFILER effectively combines functional and spatial readouts to investigate immune responses ex vivo in human tumour samples.

Prognostic impact of the expression of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and ALDH1 in colorectal cancer.

BACKGROUND: The aim of this study was to elucidate the prognostic impact of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and aldehyde dehydrogenase-1 (ALDH1) in colorectal cancer. METHODS: A tissue microarray of 1420 primary colorectal cancers and 57 normal mucosa samples was immunostained for CD133, CD166, CD44s, EpCAM, and ALDH1 in addition to 101 corresponding whole tissue sections. Invasive potential of three colorectal cancer cell lines was tested. RESULTS: Differences between normal tissue and cancer were observed for all markers (P<0.001). Loss of membranous CD166 and CD44s were linked to higher pT (P=0.002, P=0.014), pN (P=0.004, P=0.002), an infiltrating growth pattern (P<0.001, P=0.002), and worse survival (P=0.015, P=0.019) in univariate analysis only. Loss of membranous EpCAM expression was also linked to higher pN (P=0.023) and infiltrating growth pattern (P=0.005). The CD44s, CD166, and EpCAM expression were lost towards the invasive front. The CD44-/CD166- cells from three colorectal cancer cell lines exhibited significantly higher invasive potential in vitro than their positive counterparts. CONCLUSIONS: Loss, rather than overexpression, of membranous CD44s, CD166, and EpCAM is linked to tumour progression. This supports the notion that the membranous evaluation of these proteins assessed by immunohistochemistry may be representative of their cell adhesion rather than their intra-cellular functions.

Prognostic and predictive value of TOPK stratified by KRAS and BRAF gene alterations in sporadic, hereditary and metastatic colorectal cancer patients.

BACKGROUND: Our aim was to investigate the prognostic and predictive value of the oncogenic MAPKK-like protein T-cell-originated protein kinase (TOPK) stratified by KRAS and BRAF mutations in patients with sporadic, hereditary and metastatic colorectal cancer (CRC) treated with anti-EGFR therapy. METHODS: Immunohistochemistry (IHC) for TOPK was performed on four study groups. Group 1 included two subgroups of 543 and 501 sporadic CRC patients used to test the reliability of TOPK expression by IHC. In Group 2, representing an additional 222 sporadic CRCs, the prognostic effect of TOPK stratified by KRAS and BRAF was assessed. The prognostic effect of TOPK was further analysed in Group 3, representing 71 hereditary Lynch syndrome-associated CRC patients. In Group 4, the predictive and prognostic value of TOPK was analysed on 45 metastatic patients treated with cetuximab or panitumumab stratified by KRAS and BRAF gene status. RESULTS: In both sporadic CRC subgroups (Group 1), associations of diffuse TOPK expression with clinicopathological features were reproducible. Molecular analysis of sporadic CRCs in Group 2 showed that diffuse TOPK expression was associated with KRAS and BRAF mutations (p<0.001) and with poor outcome in patients with either mutation in univariate and multivariate analysis (P=0.017). In hereditary patients (Group 3), diffuse TOPK was linked to advanced pT stage. In metastatic patients treated with anti-EGFR therapy (Group 4), diffuse TOPK expression was linked to dismal outcome despite objective response to treatment (P=0.01). CONCLUSIONS: TOPK expression is an unfavourable prognostic indicator in sporadic patients with KRAS or BRAF mutations and also in patients with metastatic disease experiencing a response to anti-EGFR therapies. The inhibition of TOPK, which could benefit 30-40% of CRC patients, may represent a new avenue of investigation for targeted therapy.

p16 expression in oropharyngeal cancer: its impact on staging and prognosis compared with the conventional clinical staging parameters.

BACKGROUND: Currently, staging of head neck squamous cell carcinoma (HNSCC) is on the basis of primary tumor extension (cT), lymph node involvement (cN) and distant metastasis (cM). The aim of cancer staging was to improve diagnosis, prognosis and to compare outcome results. A new subgroup of oropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV) infection is reported to show an increasing incidence. These HPV-positive OPSCC show distinct molecular differences, specific p16 overexpression and a significantly better prognosis. Therefore, the aim of this study was to evaluate the prognostic influence of p16 expression in OPSCC and compare its relevance with the established prognostic markers cT and cN classification and the clinical stages I-IV. PATIENTS AND METHODS: Immunohistochemistry for p16 was carried out on the basis of a tissue microarray including 102 OPSCC patients with corresponding retrospective clinicopathological and follow-up data. RESULTS: p16 is the strongest independent prognostic marker in OPSCC, surpassing the significance of cT and cN classification as well as the clinical stages I-IV. Prognosis of p16-positive OPSCC of an advanced stage reached or even exceeded prognosis of the next clinically smaller conventionally staged group of tumors. CONCLUSION: p16 is the most relevant prognostic marker in OPSCC and should be considered for inclusion into the official staging system of HNSCC.

CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a 'pro-/anti-tumour' approach to tumour host interaction in colorectal cancer.

BACKGROUND: The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial-mesenchymal transition and its hallmark 'tumour budding', is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a 'pro-/anti-tumour' approach defined by an established 'pro-tumour' (tumour budding) and host-related 'anti-tumour' factor of the adaptive immunological microenvironment (CD8+ lymphocytes). METHODS: Double immunostaining for CK22/CD8 on whole tissue sections (n=279; Cohort 1) and immunohistochemistry for CD8+ using tissue microarrays (n=191; Cohort 2) was carried out. Tumour buds, CD8+ and CD8+ T-lymphocytes : tumour buds indices were evaluated per high-power field. RESULTS: In Cohort 1, a low-CD8+/ buds index was associated with lymph node metastasis (P<0.001), vascular invasion (P=0.009), worse survival in univariate (P<0.001) and multivariable (P<0.001) analysis, and furthermore in lymph node-negative patients (P=0.002). In Cohort 2, the CD8+/ buds index was associated with T stage (P<0.001), N stage (P=0.041), vascular invasion (P=0.005) and survival in patients with TNM stage II (P=0.019), stage III (P=0.004), and adjuvantly untreated (P=0.009) and treated patients (P<0.001). CONCLUSION: The CD8+ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease.

[EGFR mutation analysis in non-small-cell lung cancer : Experience from routine diagnostics]. / EGFR-Mutationsanalyse beim nichtkleinzelligen Lungenkarzinom : Erfahrungen aus der Routinediagnostik.

BACKGROUND: Some patients with non-small cell lung cancer (NSCLC) respond well to therapy with tyrosine kinase inhibitors (TKI). Somatic mutation of the epidermal growth factor receptor (EGFR) gene is an important predictive marker for TKI response. PATIENTS AND METHODS: We performed EGFR mutation analysis in 307 NSCLC (exon 18-21). The data were analyzed for associations with clinical-pathological parameters. RESULTS: Relevant EGFR mutations were found in 25/307 NSCLC (8.1%; 178 biopsies and 129 cytologies). Most mutations were found in exon 19 (50%) followed by the L858R point mutation in exon 21 (12.5%). EGFR mutations were significantly more common in women than in men (16.8% vs. 2.7%; p<0.001) and in adenocarcinoma than in other carcinoma subtypes (11.4% vs. 3.8%; p=0.017). EGFR mutation was associated with TTF-1 positivity (p<0.041). Almost all (96%) mutated NSCLC were TTF-1 positive. CONCLUSION: In Central Europe, the prevalence of relevant EGFR mutations in NSCLC is <10% of patients with NSCLC. EGFR mutations are more common in women and TTF-1 positive adenocarcinomas. Mutation analysis can be performed both from biopsies and cytologies.

Role of RHAMM within the hierarchy of well-established prognostic factors in colorectal cancer.

OBJECTIVE: To compare the independent prognostic effect of a panel of immunohistochemical protein markers in colorectal cancer (CRC) and determine their ranking among the established prognostic factors T stage, N stage, vascular invasion, tumour budding and tumour grade. DESIGN: A tissue microarray of 1420 CRCs was immunostained for 23 markers and mismatch repair (MMR) proteins. Immunoreactivity was assessed semi-quantitatively. Receiver operating characteristic (ROC) curves were used to determine cut-off scores for tumour marker positivity. Survival time was investigated for each marker in multivariable analysis with T stage, N stage, vascular invasion, tumour budding and tumour grade. The hazard ratio (HR) was used to compare the prognostic effect of each marker on 5 year survival. RESULTS: To the standard prognostic features, only six markers added independent prognostic information including receptor for hyaluronic acid mediated motility (RHAMM) (HR = 2.39 (1.88 to 3.05)), epidermal growth factor receptor (HR = 1.65 (1.31 to 2.09)), tumour infiltrating lymphocytes (HR = 0.7 (0.54 to 0.92)), urokinase plasminogen activator (HR = 1.38 (1.09 to 1.75)), Raf-1 kinase inhibitor protein (HR = 0.75 (0.58 to 0.96)) and mammalian sterile 20-like kinase 1 (MST1) (HR = 0.75 (0.58 to 0.95). Diffuse (>90% staining) expression of RHAMM ranked above T stage, vascular invasion, tumour budding and tumour grade in terms of adverse prognostic significance and was associated with distant metastasis (p = 0.012) and with worse outcome in patients with metastatic disease (p = 0.031). CONCLUSIONS: The strong adverse effect of RHAMM on outcome in addition to its position within the hierarchy of well-established prognostic factors suggest that RHAMM should be considered a more important prognosticator than tumour grade, tumour budding and vascular invasion in patients with CRC.

Potential clinical scenarios of tumour budding in colorectal cancer.

Tumour budding, defined as single tumour cells or clusters of 4 tumour cells or less detached from the main tumour body, is a wellestablished indicator of aggressive tumour biology in colorectal cancer. As a marker of tumour dissemination, evidence points towards tumour budding as a morphological correlate of epithelialmesenchymal type changes in the tumour microenvironment. Despite many studies in the literature going back decades, tumour budding has not been systematically integrated in colorectal cancer reporting protocols. The recently published proceedings of the International Tumour Budding Consensus Conference (ITBCC) have sparked the systematic implementation of tumour budding in routine reporting of colorectal cancer. Tumour budding may be particularly relevant to patient management in endoscopically resected pT1 colorectal cancer, stage II tumour and pre-operative biopsies. The present review focuses mainly on these three potential clinical scenarios with the aim to provide a concise and updated overview on tumour budding in CRC.

Two-marker protein profile predicts poor prognosis in patients with early rectal cancer.

The aim of this study was to establish an immunohistochemical protein profile to complement preoperative staging and identify rectal cancer patients at high-risk of adverse outcome. Immunohistochemistry was performed on a tissue microarray including 482 rectal cancers for APAF-1, EphB2, MST1, Ki67, p53, RHAMM, RKIP and CD8(+) tumour infiltrating lymphocytes (TILs). After resampling of the data and multivariable analysis, the most reproducible markers were combined and prognosis evaluated as stratified by pT and pN status. In multivariable analysis, only positive RHAMM (P<0.001; HR=1.94 (1.44-2.61)) and loss of CD8(+) TILs (P=0.006; HR=0.63 (0.45-0.88)) were independent prognostic factors. The 5-year cancer-specific survival rate for RHAMM+/TIL- patients was 30% (95% CI 21-40%) compared to 76% (95% CI: 66-84%) for RHAMM-/TIL+ patients (P<0.001). The 5-year cancer-specific survival of T1/T2/RHAMM+/TIL- patients was 48% (20-72%) and significantly worse compared to T3/T4/RHAMM-/TIL+ patients (71% 95% CI 56-82%); P=0.039). Stratifying by nodal status, only N+/RHAMM+/TIL- patients demonstrated a significantly worse prognosis than N0/RHAMM+/TIL- patients (P=0.005). Loss of CD8(+) TILs was predictive of local recurrence in RHAMM+ tumours (P=0.009) only. RHAMM and CD8(+) TILs may assist in identifying early stage rectal cancer patients facing a particularly poor prognosis and who may derive a benefit from preoperative therapy.

Prognostic and predictive factors in colorectal cancer.

Prognostication of newly diagnosed colorectal cancer (CRC) predominantly relies on stage as defined by the UICC-TNM and American Joint Committee on Cancer classifications. Tumour extent, lymph node status, tumour grade and the assessment of lymphatic and venous invasion are still the most important morphological prognostic factors. Evidence suggests that tumour budding and tumour border configuration are important, additional histological parameters but are not regarded as essential in prognosis. Although several molecular features, such as LOH18q and TP53 mutation analysis, have shown promising results in terms of their prognostic value, the American Society of Clinical Oncology Tumor Markers Expert Panel does not currently recommend their use in routine practice. cDNA-microarray, PCR and fluorescence in situ hybridisation are now frequently used to identity potential prognostic indicators in CRC, but the applicability of these methods in routine use is likely to have limited impact. Reliable prognostic markers identified by immunohistochemical protein profiling have yet to be established. Randomisation of data sets, assessment of interobserver variability for protein markers and scoring systems, as well as the use of receiver operating characteristic curve analysis in combination with multimarker-phenotype analysis of several different markers may be an effective tactical approach to increase the value of immunohistochemical findings. This article reviews the well established and additional prognostic factors in CRC and explores the contribution of molecular studies to the prognostication of patients with this disease. Additionally, an approach to improve the prognostic value of immunohistochemical protein markers is proposed.

Immunophenotypic profile of tumor buds in breast cancer.

BACKGROUND: Tumor buds are associated with lympho-vascular invasion and lymph node metastases leading to the assumption that they are involved in the early metastatic process. Hence, it would be important to know if tumor buds can be targeted with the most widely used targeted therapies in breast cancer (BC) and if changes in hormone and Her2 status occur. The aim of this study was to answer these questions by determining whether hormone receptor (HR) and Her2 status are expressed in the tumor buds of a large cohort of BCs. DESIGN: We constructed a tumor bud next-generation tissue microarray (ngTMA) consisting of n = 199 BCs of non-special type. Generally, two 1 mm punches were taken from the tumor bud areas in the periphery (PTB) and within the tumor center (ITB). HR and Her2 status was assessed using immunohistochemistry and fluorescence in situ hybridization, respectively. HR status was positive if ≥1% of tumor bud cells were positive. Her2 status was considered positive if bud cells showed strong complete membranous Her2 over-expression or Her2 amplification. RESULTS: Most tumor buds were positive for estrogen (ER) (PTB: 86%; ITB: 88.3) and progesterone receptor (PgR) (PTB: 72%; ITB: 72.8%) and Her2 was positive in: PTB 11.5% and ITB 11%. A difference between the main tumor mass and tumor buds (PTB and ITB) was seen for PgR in 3.5% of cases (n = 7). No differences were seen for ER and Her2 between tumor buds and main tumor mass. CONCLUSION: Most tumor buds (96.5%) share the same HR and Her2 expression profile of the main tumor mass, implying that tumor buds relay on the same pathways as the main tumor mass and might be equally responsive to targeted therapies.