Survival in surgically treated, nodal positive prostate cancer patients is predicted by histopathological characteristics of the primary tumor and its lymph node metastases

BACKGROUND: Histopathological risk factors for survival stratification of surgically treated nodal positive prostate cancer patients are poorly defined as reflected by only one category for nodal metastases. METHODS: We evaluated biochemical recurrence-free survival (RFS), disease-specific survival (DSS), and overall survival (OS) in 102 nodal positive, hormone treatment-naïve prostate cancer patients (median age: 65 years, range: 45-75 years; median follow-up 7.7 years, range: 1.0-15.9 years) who underwent radical prostatectomy and standardized extended lymphadenectomy. RESULTS: A significant stratification was possible, with the Gleason score of the primary and virtually all nodal parameters favoring patients with better differentiated primaries and metastases, lower nodal tumor burden, and without extranodal extension of metastases. In multivariate analyses, diameter of the largest metastasis (< or =10 mm vs. >10 mm) was the strongest independent predictor for RFS (P < 0.001), DSS (P < 0.001), and OS (P < 0.001) with a more than quadrupled relative risk of cancer related deaths for patients with larger metastases (Hazard ratio: 4.2, Confidence interval: 2.0-8.9; 5-year RFS/DSS/OS: 18%/57%/54%). The highest 5-year survival rates were seen in patients with micrometastases only (RFS/DSS/OS: 47%/94%/94%). CONCLUSION: The TNM classification's current allocation of only one category for nodal metastases in prostate cancers is unsatisfactory since subgroups with significantly different prognoses can be identified. The diameter of the patient's largest metastasis (< or =10 mm vs. >10 mm) should be used for substaging because of its independent prognostic value. The substage "micrometastasis only" is also useful in nodal positive prostate cancer since it designates the subgroup with the most favorable outcome.

A multifactorial histopathologic score for the prediction of prognosis of resected esophageal adenocarcinomas after neoadjuvant chemotherapy

BACKGROUND For esophageal adenocarcinoma treated with neoadjuvant chemotherapy, postoperative staging classifications initially developed for non-pretreated tumors may not accurately predict prognosis. We tested whether a multifactorial TNM-based histopathologic prognostic score (PRSC), which additionally applies to tumor regression, may improve estimation of prognosis compared with the current Union for International Cancer Control/American Joint Committee on Cancer (UICC) staging system. PATIENTS AND METHODS We evaluated esophageal adenocarcinoma specimens following cis/oxaliplatin-based therapy from two separate centers (center 1: n = 280; and center 2: n = 80). For the PRSC, each factor was assigned a value from 1 to 2 (ypT0-2 = 1 point; ypT3-4 = 2 points; ypN0 = 1 point; ypN1-3 = 2 points; ≤50 % residual tumor/tumor bed = 1 point; >50 % residual tumor/tumor bed = 2 points). The three-tiered PRSC was based on the sum value of these factors (group A: 3; group B: 4-5; group C: 6) and was correlated with patients' overall survival (OS). RESULTS The PRSC groups showed significant differences with respect to OS (p < 0.0001; hazard ratio [HR] 2.2 [95 % CI 1.7-2.8]), which could also be demonstrated in both cohorts separately (center 1 p < 0.0001; HR 2.48 [95 % CI 1.8-3.3] and center 2 p = 0.015; HR 1.7 [95 % CI 1.1-2.6]). Moreover, the PRSC showed a more accurate prognostic discrimination than the current UICC staging system (p < 0.0001; HR 1.15 [95 % CI 1.1-1.2]), and assessment of two goodness-of-fit criteria (Akaike Information Criterion and Schwarz Bayesian Information Criterion) clearly supported the superiority of PRSC over the UICC staging. CONCLUSION The proposed PRSC clearly identifies three subgroups with different outcomes and may be more helpful for guiding further therapeutic decisions than the UICC staging system.

Tumor Regression Grading of Gastrointestinal Carcinomas after Neoadjuvant Treatment

Multimodal therapy concepts have been successfully implemented in the treatment of locally advanced gastrointestinal malignancies. The effects of neoadjuvant chemo- or radiochemotherapy such as scarry fibrosis or resorptive changes and inflammation can be determined by histopathological investigation of the subsequent resection specimen. Tumor regression grading (TRG) systems which aim to categorize the amount of regressive changes after cytotoxic treatment mostly refer onto the amount of therapy induced fibrosis in relation to residual tumor or the estimated percentage of residual tumor in relation to the previous tumor site. Commonly used TRGs for upper gastrointestinal carcinomas are the Mandard grading and the Becker grading system, e.g., and for rectal cancer the Dworak or the Rödel grading system, or other systems which follow similar definitions. Namely for gastro-esophageal carcinomas these TRGs provide important prognostic information since complete or subtotal tumor regression has shown to be associated with better patient's outcome. The prognostic value of TRG may even exceed those of currently used staging systems (e.g., TNM staging) for tumors treated by neoadjuvant therapy. There have been some limitations described regarding interobserver variability especially in borderline cases, which may be improved by standardization of work up of resection specimen and better training of histopathologic determination of regressive changes. It is highly recommended that TRG should be implemented in every histopathological report of neoadjuvant treated gastrointestinal carcinomas. The aim of this review is to disclose the relevance of histomorphological TRG to accomplish an optimal therapy for patients with gastrointestinal carcinomas.

Prognostic implications of the seventh edition of the international union against cancer classification for patients with gastric cancer: the Western experience of patients treated in a single-center European institution

PURPOSE Validity of the seventh edition of the American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) staging systems for gastric cancer has been evaluated in several studies, mostly in Asian patient populations. Only few data are available on the prognostic implications of the new classification system on a Western population. Therefore, we investigated its prognostic ability based on a German patient cohort. PATIENTS AND METHODS Data from a single-center cohort of 1,767 consecutive patients surgically treated for gastric cancer were classified according to the seventh edition and were compared using the previous TNM/UICC classification. Kaplan-Meier analyses were performed for all TNM stages and UICC stages in a comparative manner. Additional survival receiver operating characteristic analyses and bootstrap-based goodness-of-fit comparisons via Bayesian information criterion (BIC) were performed to assess and compare prognostic performance of the competing classification systems. RESULTS We identified the UICC pT/pN stages according to the seventh edition of the AJCC/UICC guidelines as well as resection status, age, Lauren histotype, lymph-node ratio, and tumor grade as independent prognostic factors in gastric cancer, which is consistent with data from previous Asian studies. Overall survival rates according to the new edition were significantly different for each individual's pT, pN, and UICC stage. However, BIC analysis revealed that, owing to higher complexity, the new staging system might not significantly alter predictability for overall survival compared with the old system within the analyzed cohort from a statistical point of view. CONCLUSION The seventh edition of the AJCC/UICC classification was found to be valid with distinctive prognosis for each stage. However, the AJCC/UICC classification has become more complex without improving predictability for overall survival in a Western population. Therefore, simplification with better predictability of overall survival of patients with gastric cancer should be considered when revising the seventh edition.

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.

Investigation of IL23 (p19, p40) and IL23R highlights nuclear IL23p19 expression as a marker of indolent tumor features and favorable outcome in colorectal cancer patients

Objective: IL23 is involved in chronic inflammation but its role in cancer progression is not fully elucidated. Here we characterize IL23 subunits p40, p19 and IL23 receptor (IL23R) in the normal-adenoma-carcinomametastasis cascade of colorectal cancers and their relationship to clinicopathological and outcome data. Method: Immunohistochemistry for IL23R, IL12p40, IL23 and IL23p19 (monoclonal) was performed on a multi-punch tissue microarray (n=213 patients). Expression differences between normal-adenomas-cancerslymph nodes were evaluated. Correlation with clinicopathological and outcome data was undertaken. Results were validated on an independent cohort (n=341 patients). Results: An increased expression from normal-adenoma-cancer was observed (p<0.0001; all) followed by a marked reduction in lymph nodes (p<0.0001; all). Cytoplasmic and/or membranous staining of all markers was unrelated to outcome. Nuclear IL23p19 staining occurred in 23.1%and was associated with smaller tumor diameter (p=0.0333), early pT (p=0.0213), early TNM (p=0.0186), absence of vascular (p=0.0124) and lymphatic invasion (p=0.01493) and favorable survival (univariate (p=0.014) and multivariable (p=0.0321) analysis). All IL23p19 positive patients were free of distant metastasis (p=0.0146). Survival and metastasis results could be validated in Cohort 2. Conclusion: The presence of nuclear IL23p19 is related to indolent tumor features and favorable outcome supporting a more ‘protective’ role of this protein in colorectal cancer progression

Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^

CD8/CD45RO T-cell infiltration in endoscopic biopsies of colorectal cancer predicts nodal metastasis and survival

BACKGROUND AND AIMS: Reliable prognostic markers based on biopsy specimens of colorectal cancer (CRC) are currently missing. We hypothesize that assessment of T-cell infiltration in biopsies of CRC may predict patient survival and TNM-stage before surgery. METHODS: Pre-operative biopsies and matched resection specimens from 130 CRC patients treated from 2002-2011 were included in this study. Whole tissue sections of biopsy material and primary tumors were immunostained for pancytokeratin and CD8 or CD45RO. Stromal (s) and intraepithelial (i) T-cell infiltrates were analyzed for prediction of patient survival as well as clinical and pathological TNM-stage of the primary tumor. RESULTS: CD8 T-cell infiltration in the preoperative biopsy was significantly associated with favorable overall survival (CD8i p = 0.0026; CD8s p = 0.0053) in patients with primary CRC independently of TNM-stage and postoperative therapy (HR [CD8i] = 0.55 (95% CI: 0.36-0.82), p = 0.0038; HR [CD8s] = 0.72 (95% CI: 0.57-0.9), p = 0.0049). High numbers of CD8i in the biopsy predicted earlier pT-stage (p < 0.0001) as well as absence of nodal metastasis (p = 0.0015), tumor deposits (p = 0.0117), lymphatic (p = 0.008) and venous invasion (p = 0.0433) in the primary tumor. Infiltration by CD45ROs cells was independently associated with longer survival (HR = 0.76 (95% CI: 0.61-0.96), p = 0.0231) and predicted absence of venous invasion (p = 0.0025). CD8 counts were positively correlated between biopsies and the primary tumor (r = 0.42; p < 0.0001) and were reproducible between observers (ICC [CD8i] = 0.95, ICC [CD8s] = 0.75). For CD45RO, reproducibility was poor to moderate (ICC [CD45i] = 0.16, ICC [CD45s] = 0.49) and correlation with immune infiltration in the primary tumor was fair and non-significant (r[CD45s] = 0.16; p = 0.2864). For both markers, no significant relationship was observed with radiographic T-stage, N-stage or M-stage, indicating that assessment of T-cells in biopsy material can add additional information to clinical staging in the pre-operative setting. CONCLUSIONS: T-cell infiltration in pre-operative biopsy specimens of CRC is an independent favorable prognostic factor and strongly correlates with absence of nodal metastasis in the resection specimen. Quantification of CD8i is highly reproducible and allows superior prediction of clinicopathological features as compared to CD45RO. The assessment of CD8i infiltration in biopsies is recommended for prospective investigation.

Investigation of IL-23 (p19, p40) and IL-23R identifies nuclear expression of IL-23 p19 as a favorable prognostic factor in colorectal cancer: a retrospective multicenter study of 675 patients.

IL-23 is a heterodimeric cytokine involved in inflammatory diseases; its role in cancer progression is controversial. Here we analyse the expression of IL-23 subunits (p40 and p19) and IL-23R in colorectal cancer with regard to disease progression, clinical-pathological and molecular aspects. Immunohistochemistry for IL-23p19, IL-23p40, IL-23R and CD8 was performed on a multi-punch tissue microarray of 195 colorectal cancers (cohort 1), matched normal tissue, adenoma and lymph node metastases. Results were compared with clinical-pathological features and CD8+ T-cell counts, then validated on two patient cohorts (cohort 2: n=341, cohort 3: n=139). Cytoplasmic/membranous expression of IL-23 (p19 and p40 subunits) and IL-23R, respectively were over-expressed in carcinomas versus adenomas and normal tissues (p<0.0001) but were reduced in lymph node metastases (p<0.0001). Nuclear IL-23p19 expression was observed in 23.1% and was associated with early TNM stage (p=0.0186), absence of venous (p=0.0124) and lymphatic invasion (p=0.01493), favorable survival (p=0.014) and absence of distant metastasis (p=0.0146; specificity: 100%). This unexpected cellular localization was confirmed by cell fractionation. The beneficial effect of nuclear IL-23p19 was restricted to tumours with CD8+ high counts. Results were validated on Cohorts 2/3. This multicenter study underlines the possible CD8(+)--dependency and beneficial effect of nuclear IL-23p19 on overall patient survival.

The tumor border configuration of colorectal cancer as a histomorphological prognostic indicator.

Histomorphological features of colorectal cancers (CRC) represent valuable prognostic indicators for clinical decision making. The invasive margin is a central feature for prognostication shaped by the complex processes governing tumor-host interaction. Assessment of the tumor border can be performed on standard paraffin sections and shows promise for integration into the diagnostic routine of gastrointestinal pathology. In aggressive CRC, an extensive dissection of host tissue is seen with loss of a clear tumor-host interface. This pattern, termed "infiltrative tumor border configuration" has been consistently associated with poor survival outcome and early disease recurrence of CRC-patients. In addition, infiltrative tumor growth is frequently associated with presence of adverse clinicopathological features and molecular alterations related to aggressive tumor behavior including BRAFV600 mutation. In contrast, a well-demarcated "pushing" tumor border is seen frequently in CRC-cases with low risk for nodal and distant metastasis. A pushing border is a feature frequently associated with mismatch-repair deficiency and can be used to identify patients for molecular testing. Consequently, assessment of the tumor border configuration as an additional prognostic factor is recommended by the AJCC/UICC to aid the TNM-classification. To promote the assessment of the tumor border configuration in standard practice, consensus criteria on the defining features and method of assessment need to be developed further and tested for inter-observer reproducibility. The development of a standardized quantitative scoring system may lay the basis for verification of the prognostic associations of the tumor growth pattern in multivariate analyses and clinical trials. This article provides a comprehensive review of the diagnostic features, clinicopathological associations, and molecular alterations associated with the tumor border configuration in early stage and advanced CRC.

Towards the introduction of the 'Immunoscore' in the classification of malignant tumours.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

The impact of neural invasion severity in gastrointestinal malignancies: a clinicopathological study.

OBJECTIVES Because neural invasion (NI) is still inconsistently reported and not well characterized within gastrointestinal malignancies (GIMs), our aim was to determine the exact prevalence and severity of NI and to elucidate the true impact of NI on patient's prognosis. BACKGROUND The union internationale contre le cancer (UICC) recently added NI as a novel parameter in the current TNM classification. However, there are only a few existing studies with specific focus on NI, so that the distinct role of NI in GIMs is still uncertain. MATERIALS AND METHODS NI was characterized in approximately 16,000 hematoxylin and eosin tissue sections from 2050 patients with adenocarcinoma of the esophagogastric junction (AEG)-I-III, squamous cell carcinoma (SCC) of the esophagus, gastric cancer (GC), colon cancer (CC), rectal cancer (RC), cholangiocellular cancer (CCC), hepatocellular cancer (HCC), and pancreatic cancer (PC). NI prevalence and severity was determined and related to patient's prognosis and survival. RESULTS NI prevalence largely varied between HCC/6%, CC/28%, RC/34%, AEG-I/36% and AEG-II/36%, SCC/37%, GC/38%, CCC/58%, and AEG-III/65% to PC/100%. NI severity score was uppermost in PC (24.9±1.9) and lowest in AEG-I (0.8±0.3). Multivariable analyses including age, sex, TNM stage, and grading revealed that the prevalence of NI was significantly associated with diminished survival in AEG-II/III, GC, and RC. However, increasing NI severity impaired survival in AEG-II/III and PC only. CONCLUSIONS NI prevalence and NI severity strongly vary within GIMs. Determination of NI severity in GIMs is a more precise tool than solely recording the presence of NI and revealed dismal prognostic impact on patients with AEG-II/III and PC. Evidently, NI is not a concomitant side feature in GIMs and, therefore, deserves special attention for improved patient stratification and individualized therapy after surgery.

An arranged marriage for precision medicine: hypoxia and genomic assays in localized prostate cancer radiotherapy

Prostate cancer (CaP) is the most commonly diagnosed malignancy in males in the Western world with one in six males diagnosed in their lifetime. Current clinical prognostication groupings use pathologic Gleason score, pre-treatment prostatic-specific antigen and Union for International Cancer Control-TNM staging to place patients with localized CaP into low-, intermediate- and high-risk categories. These categories represent an increasing risk of biochemical failure and CaP-specific mortality rates, they also reflect the need for increasing treatment intensity and justification for increased side effects. In this article, we point out that 30-50% of patients will still fail image-guided radiotherapy or surgery despite the judicious use of clinical risk categories owing to interpatient heterogeneity in treatment response. To improve treatment individualization, better predictors of prognosis and radiotherapy treatment response are needed to triage patients to bespoke and intensified CaP treatment protocols. These should include the use of pre-treatment genomic tests based on DNA or RNA indices and/or assays that reflect cancer metabolism, such as hypoxia assays, to define patient-specific CaP progression and aggression. More importantly, it is argued that these novel prognostic assays could be even more useful if combined together to drive forward precision cancer medicine for localized CaP.

Prognostic value of matrix metalloproteinases in oral squamous cell carcinoma

The aim of this study was to investigate whether there is a correlation between the expressions of four matrix metalloproteinases (MMPs): MMP-2, MMP-7, MMP-9 and MMP-13, and the TNM (tumour-node-metastasis) stages of oral squamous cell carcinoma (OSCC); and to explore the implication of these MMPs in OSCC dissemination. Samples from 61 patients diagnosed with oropharyngeal tumour were studied by immunohistochemistry against MMP-2, MMP-7, MMP-9 and MMP-13. The assessment of immunoreactivity was semi-quantitative. The results showed that MMP-2 and MMP-9 had similar expression patterns in the tumour cells with no changes in the immunoreactivity during tumour progression. MMP-9 always had the highest expression, whereas that of MMP-2 was moderate. MMP-7 showed a significant decrease in expression levels during tumour evolution. MMP-13 had constant expression levels within stage T2 and T3, but showed a remarkable decline in immunoreactivity in stage T4. No significant differences in the MMPs immunoreactivity between tumour cells and stroma were observed. Although strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired, we believe that combining patients' MMPs expression intensity and clinical features may improve the diagnosis and prognosis. Strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired. Application of MMPs as prognostic indicators for the malignancy potential of OSCC might be considered in every case of tumour examination. We believe that combining patients' MMPs expression intensity and clinical features may improve the process of making diagnosis and prognosis.

Tumor budding in colorectal cancer revisited: results of a multicenter interobserver study.

Tumor budding in colorectal cancer (CRC) is recognized as a valuable prognostic factor but its translation into daily histopathology practice has been delayed by lack of agreement on the optimal method of assessment. Within the context of the Swiss Association of Gastrointestinal Pathology (SAGIP), we performed a multicenter interobserver study on tumor budding, comparing hematoxylin and eosin (H&E) with pan-cytokeratin staining using a 10 high power field (10HPF) and hotspot (1HPF) method. Two serial sections of 50 TNM stage II-IV surgically treated CRC were stained for H&E and pan-cytokeratin. Tumor buds were scored by independent observers at six participating centers in Switzerland and Austria using the 10HPF and 1HPF method on a digital pathology platform. Pearson correlation (r) and intra-class correlation coefficients (ICC) comparing scores between centers were calculated. Three to four times more tumor buds were detected in pan-cytokeratin compared to H&E slides. Correlation coefficients for tumor budding counts between centers ranged from r = 0.46 to r = 0.91 for H&E and from r = 0.73 to r = 0.95 for pan-cytokeratin slides. Interobserver agreement across all centers was excellent for pan-cytokeratin [10HPF: ICC = 0.83 and 1HPF: ICC = 0.8]. In contrast, assessment of tumor budding on H&E slides reached only moderate agreement [10HPF: ICC = 0.58 and 1HPF: ICC = 0.49]. Based on previous literature and our findings, we recommend (1) pan-cytokeratin staining whenever possible, (2) 10HPF method for resection specimens, and (3) 1HPF method for limited material (preoperative biopsy or pT1). Since tumor budding counts can be used to determine probabilities of relevant outcomes and as such more optimally complement clinical decision making, we advocate the avoidance of cutoff scores.