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Cytoglobin (Cygb) is a member of the hemoglobin family and is thought to protect against cellular hypoxia and oxidative stress. These functions may be particularly important in inflammation-induced cancer, e.g., in patients with ulcerative colitis (UC). In this study, we investigated the development of inflammation and tumors in a murine model of inflammation-induced colorectal cancer using a combined treatment of azoxymethane and dextran sulfate sodium. A bioinformatics analysis of genome-wide expression data revealed increased colonic inflammation at the molecular level accompanied by enhanced macroscopic tumor development in Cygb-deficient mice. Moreover, the expression of the UC-associated gene neurexophilin and PC-esterase domain family member 4 (Nxpe4) depended on the presence of Cygb in the inflamed colonic mucosa. Compared to wild type mice, RT-qPCR confirmed a 14-fold (p = 0.0003) decrease in Nxpe4 expression in the inflamed colonic mucosa from Cygb-deficient mice. An analysis of Cygb protein expression suggested that Cygb is expressed in fibroblast-like cells surrounding the colonic crypts. Histological examinations of early induced lesions suggested that the effect of Cygb is primarily at the level of tumor promotion. In conclusion, in this model, Cygb primarily seemed to inhibit the development of established microadenomas.

Incidence rates of prostate cancer in Denmark resemble those of countries that endorse prostate-specific antigen (PSA) screening. So far, no studies have described the consequences of PSA testing on diagnostic activity on a population level. The aim of this study was to describe the frequency of systematic transrectal ultrasound-guided biopsy (TRUS-gb) activity, including rebiopsy rates, in Denmark between 1995 and 2011.All men who underwent TRUS-gb during the period were identified in the Danish Prostate Cancer Registry. In total, 83,041 biopsy sets from 64,430 individuals were identified. The diagnostic rate and the frequency of rebiopsy were analyzed. Age, histology and PSA were compared at the time of biopsy.The number of TRUS-gb per 100,000 men per year increased 4.6-fold. The mean number of TRUS-gb procedures per individual increased from 1.08 in 1995 to 1.46 in 2011 (p = .0001), and the proportion of men with negative initial biopsy sets who underwent rebiopsy increased from 22% in 1995 to 41% in 2004, later decreasing to 31% in 2009.The diagnostic activity in Denmark and the rebiopsy rates in men with initial negative TRUS-gb have increased substantially, and guidelines for the management of men with a negative initial biopsy are highly warranted.

Purpose: To investigate the prognostic and predictive biomarker value of type IV collagen in colorectal cancer. Experimental Design: Retrospective evaluation of two independent cohorts of patients with colorectal cancer included prospectively in 2004–2005 (training set) and 2006–2008 (validation set). Plasma samples were available from 297 (training set) and 482 (validation set) patients. Type IV collagen determinations were performed using an ELISA. From the training set, 222 tumors were available for IHC. Clinical and follow-up data were retrieved from patient files and national registries. Results: High levels of type IV collagen showed independent prognostic significance in both cohorts with hazard ratios (HRs; for a one-unit change on the log base 2 scale) of 2.25 [95% confidence intervals (CIs), 1.78–2.84; P < 0.0001] and 2.24 (95% CI, 1.75–2.86; P < 0.0001) for the training and validation set, respectively. The prognostic impact was present both in patients with metastatic and nonmetastatic disease. The predictive value of the marker was investigated in stage II and III patients. In the training set, type IV collagen was prognostic both in the subsets of patients receiving and not receiving adjuvant antineoplastic therapy. However, in the validation set, the prognostic effect of the marker vanished when looking at patients who received adjuvant antineoplatic therapy (HR 0.90; 95% CI, 0.42–1.93) but was still present in the group not receiving adjuvant chemotherapy (HR 2.88; 95% CI, 1.98–4.21). Conclusions: The results indicate clinical validity of type IV collagen as a prognostic biomarker in colorectal cancer, although the suggested predictive role of the marker should be validated. Clin Cancer Res; 22(10); 2427–34. ©2015 AACR.

Objectives To investigate whether the International Society of Urological Pathology (ISUP) 2005 revision of the Gleason grading system has influenced the risk of biochemical recurrence (BCR) after radical prostatectomy (RP), as the new guideline implies that some prostate cancers previously graded as Gleason score 6 (3 + 3) are now considered as 7 (3 + 4). Patients and methods A matched-pair analysis was conducted. In all, 215 patients with Gleason score 6 or 7 (3 + 4) prostate cancer on biopsy who underwent RP before 31 December 2005 (pre-ISUP group), were matched 1:1 by biopsy Gleason score, clinical tumour category, PSA level, and margin status to patients undergoing RP between 1 January 2008 and 31 December 2011 (post-ISUP group). Patients were followed until BCR defined as a PSA level of ≥0.2 ng/mL. Risk of BCR was analysed in a competing-risk model. Results The median follow-up was 9.5 years in the pre-ISUP group and 4.8 years in the post-ISUP group. The 5-year cumulative incidences of BCR were 34.0% and 13.9% in the pre-ISUP and post-ISUP groups, respectively (P < 0.001). The difference in cumulative incidence applied to both patients with Gleason score 6 (P < 0.001) and 7 (3 + 4) (P = 0.004). There was no difference in the 5-year cumulative incidence of BCR between patients with pre-ISUP Gleason score 6 and post-ISUP Gleason score 7 (3 + 4) (P = 0.34). In a multiple Cox-proportional hazard regression model, ISUP 2005 grading was a strong prognostic factor for BCR within 5 years of RP (hazard ratio 0.34; 95% confidence interval 0.22–0.54; P < 0.001). Conclusion The revision of the Gleason grading system has reduced the risk of BCR after RP in patients with biopsy Gleason score 6 and 7 (3 + 4). This may have consequences when comparing outcomes across studies and historical periods and may affect future treatment recommendations.

Background Colorectal cancer (CRC) patients with metastatic disease can become cured if neoadjuvant treatment can enable a resection. The search for predictive biomarkers is often performed on primary tumours tissue. In order to assess the effectiveness of tailored treatment in regard to the primary tumour the differences in the genomic profile needs to be clarified. Methods Fresh-frozen tissue from primary tumours, synchronous liver metastases and adjacent normal liver was collected from 21 patients and analysed by whole-exome sequencing on the Illumina HiSeq 2500 platform. Gene variants designated as ‘damaging’ or ‘potentially damaging’ by Ingenuity software were used for the subsequent comparative analysis. BAM files were used as the input for the analysis of CNAs using NEXUS software. Results Shared mutations between the primary tumours and the synchronous liver metastases varied from 50 to 96%. Mutations in APC, KRAS, NRAS, TP53 or BRAF were concordant between the primary tumours and the metastases. Among the private mutations were well-known driver genes such as PIK3CA and SMAD4. The number of mutations was significantly higher in patients with right- compared to left-sided tumours (102 vs. 66, p = 0.004). Furthermore, right- compared to left-sided tumours had a significantly higher frequency of private mutations (p = 0.023). Similarly, CNAs differed between the primary tumours and the metastases. The difference was mostly comprised of numerical and segmental aberrations. However, novel CNAs were rarely observed in specific CRC-relevant genes. Conclusion The examined primary colorectal tumours and synchronous liver metastases had multiple private mutations, indicating a high degree of inter-tumour heterogeneity in the individual patient. Moreover, the acquirement of novel CNAs from primary tumours to metastases substantiates the need for genomic profiling of metastases in order to tailor metastatic CRC therapies. As for the mutational status of the KRAS, NRAS and BRAF genes, no discordance was observed between the primary tumours and the metastases. Electronic supplementary material The online version of this article (10.1186/s12885-018-4639-4) contains supplementary material, which is available to authorized users.

Aims: Breast cancer is one of the most common cancer diseases in women, with >1.67 million cases being diagnosed worldwide each year. In breast cancer, the sentinel lymph node (SLN) pinpoints the first lymph node(s) into which the tumour spreads, and it is usually located in the ipsilateral axilla. In patients with no clinical signs of metastatic disease in the axilla, an SLN biopsy (SLNB) is performed. Assessment of metastases in the SLNB, when using a conventional microscope, is performed by manually observing a metastasis and measuring its size and/or counting the number of tumour cells. This is done essentially to categorize the type of metastasis as macrometastasis, micrometastasis, or isolated tumour cells, which is used to determine which treatment the breast cancer patient will benefit most from. The aim of this study was to evaluate whether digital image analysis can be applied as a screening tool for SNLB assessment without compromising the diagnostic accuracy. Materials and results: Consecutive SLNBs from 135 patients with localized breast cancer receiving surgery in the period February to August 2015 were collected and included in this study. Of the 135 patients, 35 were received at the Department of Pathology, Rigshospitalet, Copenhagen University Hospital, 50 at the Department of Pathology, Zealand University Hospital, and 50 at the Department of Pathology, Odense University Hospital. Formalin-fixed paraffin-embedded tissue sections were analysed by immunohistochemistry with the BenchMark ULTRA Ventana platform. Rigshospitalet used a mixture of cytokeratin (CK) 7 and CK19, Zealand University Hospital used pancytokeratin AE1/AE3 and Odense used pancytokeratin CAM5.2 for detection of epithelial tumour cells. Slides were stained locally. SLNB sections were assessed in a conventional microscope according to national guidelines for SLNBs in breast cancer patients. The immunohistochemically stained sections were scanned with a Hamamatsu NanoZoomer-XR digital whole slide scanner, and the images were analysed with Visiopharm's software by use of a custom-made algorithm for SLNBs in breast cancer. The algorithm was optimized to the CK antibodies and the local laboratory conditions, on the basis of staining intensity and background staining. Conventional microscopy was used as the gold standard for assessment of positive tumour cells, and the results were compared with those from digital image analysis. The algorithm showed a sensitivity of 100% (that is, no false-negative slides were observed), including 67.2%, 19.2% and 56.1% of the slides from the three pathology departments being negative, respectively. This means that, on average, the workload could have been decreased by 58.2% by use of the digital SLNB algorithm as a screening tool. Conclusions: The SLNB algorithm showed a sensitivity of 100% regardless of the antibody used for immunohistochemistry and the staining protocol. No false-negative slides were observed, which proves that the SLNB algorithm is an ideal screening tool for selecting those slides that a pathologist does not need to see. The implementation of automated digital image analysis of SLNBs in breast cancer would decrease the workload in this context for examining pathologists by almost 60%.

Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level with biochemical recurrence (BR) following radical prostatectomy (RP). The study included 614 patients who had undergone RP. TRPM4 immunohistochemical staining was performed on samples of benign tissue, tissue containing PIN glands and PCa tissue using a commercially available polyclonal antibody. Staining intensity was recorded by two independent observers using a four-tired semi-quantitative grading system (0, 1+, 2+, 3+) converted into H-scores. Interobserver agreement was calculated by linear weighted kappa statistics. The association between staining intensity and BR was analysed using the Kaplan-Meier estimator and uni- and multiple Cox proportional hazard regression models. Significantly higher staining intensity was found in PCa glands compared to benign glands (p

Inverted papilloma (IP) of the urinary tract is classified by the World Health Organisation as a non-invasive urothelial tumour with normal to minimal cytological atypia of the neoplastic cells. During the 1980s, it came under suspicion of having a premalignant or malignant potential and of being concurrent with urothelial cell carcinoma (UCC). This quandary has been proven difficult to solve, due to the fact that IP is very rare and literature mostly consists of case reports with varying levels of information, making strong meta-analyses problematic. New immunohistochemical techniques and genetic approaches are more frequently being used in the attempt to achieve better classifications, prognosis and treatment of lesions hereunder IP. This review will, in our awareness, be the first to combine the knowledge from retrospective studies with these new approaches for determining a possible premalignant potential and concurrency with UCC and subsequently outline a recommendation for follow-up.

Early detection has increased prostate cancer (PCa) incidence. Randomized trials have demonstrated that early detection reduces the incidence of de novo metastatic PCa. Concurrently, life-prolonging treatments have been introduced for patients with advanced PCa. On a populations-based level, the authors analyzed whether early detection and improved treatments changed the incidence and 5-year mortality of men with de novo metastatic PCa.Men diagnosed with PCa during the periods 1980 to 2011 and 1995 to 2011 were identified in the US Surveillance, Epidemiology, and End Results (SEER) program and the Danish Prostate Cancer Registry (DaPCaR), respectively, and stratified according to period of diagnosis. Age-standardized incidence rates were calculated. Five-year mortality rates for de novo metastatic PCa were analyzed using competing risk analysis.Totals of 426,266 and 47,024 men were identified in SEER and DaPCaR, respectively. Of these, 29,555 and 6874 had de novo metastatic PCa. The incidence of de novo metastatic PCa decreased (from 12.0 to 4.4 per 100,000 men) in the SEER cohort (1980-2011), whereas it increased (from 6.7 to 9.9 per 100,000 men) in the DaPCaR cohort (1995-2011). Five-year PCa mortality in the SEER cohort was stable for men diagnosed with de novo metastatic PCa from 1980 to 1994 and increased slightly in the latest periods studied (P .0001), whereas it decreased by 16.6% (P .0001) in the DaPCaR cohort.Despite earlier detection, de novo metastatic PCa remains associated with a high risk of 5-year disease-specific mortality. The reduced 5-year PCa mortality in the Danish cohort is largely explained by lead-time. Early detection strategies do indeed decrease the incidence of de novo metastatic PCa, as observed in the SEER cohort. This achievement, however, must be weighed against the unsolved issue of overdetection and overtreatment of indolent PCa. Cancer 2018;124:2931-8. © 2018 American Cancer Society.

The diagnostic characteristics of men who eventually die from prostate cancer (PCa) and the extent to which early diagnostic strategies have affected these characteristics are unclear. We aimed to investigate trends in survival and clinical presentation at diagnosis in men who eventually died from PCa.Based on the national database, the Danish Prostate Cancer Registry, a nationwide population-based study of all 19,487 men who died from PCa in Denmark between 1995 and 2013 was conducted. Trends in median survival and trends in age, prostate-specific antigen (PSA), clinical stage, and Gleason score (GS) at diagnosis were analysed.A total of 46.9%, 16.8%, and 36.3% had metastatic (M+), locally advanced/lymph node positive (LaN+), and localised disease, respectively, at diagnosis. Only 0.15% had localised disease, GS ≤ 6 and PSA10. Over time, the proportion of men with M+ disease at diagnosis decreased from 54.0-38.3% (p 0.0001), whereas the proportion LaN + disease increased from 8.6-27.3% (p 0.0001). The proportion of localised disease remained stable at 33.2-41.9%. Median survival increased 2.11 years from 1.88 (95% CI: 1.68-2.08) in 1995 to 3.99 (95% CI: 3.71-4.28) years in 2013, p 0.0001.In a large population-based study, the results confirmed concurrent literature that the majority of men who eventually died from PCa had LaN+ or M+ disease at diagnosis. The proportion of men with M+ disease at diagnosis decreased significantly over time, parallelled by an increase in median survival. Taken together, this indicates a lead-time effect on survival, which presently, however, is not substantial enough to result in a reduced PCa-specific mortality.

Liver collagen proportionate area (CPA) assessed by computer-assisted digital image analysis has been proposed as an accurate and objective histological variable for subclassifying cirrhosis. The study aimed to examine the relationship between CPA and relevant clinical parameters in cirrhotic patients and to evaluate the sampling variability for CPA. The study included 48 consecutive liver transplantation patients with established cirrhosis. Hepatic venous pressure gradient (HVPG) and serum markers of liver failure were determined prior to transplantation. CPA was assessed in the explanted livers. In 20 of the livers, CPA was measured in more than one tissue sample. CPA showed significant correlations with HVPG and with various surrogate markers of hepatic dysfunction including albumin, bilirubin, INR, MELD score and Child-Pugh score. CPA reliably discriminated HVPG ≥10 mmHg, termed 'clinically significant portal hypertension' (area under receiver operator curve: 0.923, p0.001; odds ratio: 1.209, p = 0.003). CPA measured on tissue blocks showed no significant sampling variability (p0.5). In conclusion, the study correlated portal hypertension and hepatic dysfunction with the amount of collagen in cirrhotic livers. The findings support the presumption of CPA as a useful histological marker for subclassifying cirrhosis and as a helpful supplement to the qualitative description of hepatic architectural changes in routine pathology.

Transrectal ultrasound-guided biopsies (TRUS-gb) are associated with both mild and serious complications. Prophylactic antibiotics reduce the risk of septicaemia and mortality; however, no international consensus exists on the timing and duration of antibiotics, including the optimal drug strategy. We reviewed the current evidence supporting use of prophylactic antibiotics and the risk of complications following prostate biopsies.This review was drafted in accordance with the Prisma Guidelines. The PubMed, Embase and Cochrane databases were searched.A total of 19 eligible trials were identified. One trial demonstrated a significant reduction in the risk of infection after biopsy and reported that oral ciprofloxacin as either a single-dose or a three-day regimen was superior to oral chloramphenicol and norfloxacin. Of three studies investigating the timing of the first dose of antibiotic, one study found that administration 24 h before biopsy versus administration immediately before reduced the relative risk of post-biopsy infection by 55%. Seven studies compared different durations of antibiotic prophylaxis. None showed any benefit from continuing prophylaxis beyond a single dose or a one-day regimen.Evidence supporting a specific antibiotic regimen for TRUS-gb prophylaxis is scarce. Widespread use of fluoroquinolone prophylaxis may be associated with an increase in resistant Escherichia coli strains, posing a potentially major health issue in the future. .

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) often presents with cystic cervical metastasis and a small primary tumor localized in the palatine tonsils or base of the tongue, which is diagnostically challenging. Testing for HPV DNA in fine-needle aspiration (FNA) smears from metastases may facilitate a targeted diagnostic workup for identifying the primary tumor. This study was designed to assess the ability to detect HPV DNA in FNA smears with polymerase chain reaction (PCR).May-Grünvald-Giemsa (MGG)-stained FNA smears from metastases and corresponding surgical specimens were collected from 71 patients with known HPV-positive OPSCC, 12 patients with oral squamous cell carcinoma (OSCC), 20 patients with branchial cleft cysts, and 20 patients with Warthin tumors. Thirty-eight patients with OPSCC and 7 patients with OSCC had FNA smears available from metastases and also surgical specimens from the primary tumor and the metastases. The scraped cell material from FNA smears and corresponding surgical specimens were analyzed for HPV DNA by PCR. p16 immunohistochemistry was performed on surgical specimens from the carcinomas.HPV DNA was detected in 68 of the 71 FNA smears from OPSCC metastases. All corresponding surgical specimens from primary tumors (n = 71) and metastases (n = 38) were p16- and HPV DNA-positive. All the surgical specimens and corresponding FNA smears from OSCCs, Warthin tumors, and branchial cleft cysts were HPV DNA-negative. The sensitivity and specificity were 94.7% and 100%, respectively.The detection of HPV DNA in MGG-stained FNA smears by PCR is a valid method that could be implemented in routine clinical practice. Cancer Cytopathol 2016;124:820-7. © 2016 American Cancer Society.

The aim of this study was to evaluate the impact of preoperative and surgical parameters, including nerve-sparing technique, on the risk of positive surgical margins (PSM) following radical prostatectomy for clinically localized prostate cancer.A prospective consecutive single-institution Danish cohort of 1148 patients undergoing RP between 1995 and 2011 was investigated. To analyse the impact of covariates on risk of PSM, a multivariate logistic regression model was used, including cT category, biopsy Gleason score, prostate-specific antigen (PSA), percentage positive biopsies for cancer (PPB), surgeon and surgical technique.The overall rate of PSM was 31.4%. The risk of PSM depended (p value for Wald χ(2)) on PSA (p0.0001), PPB (p = 0.003), nerve-sparing surgery (p = 0.03) and the surgeon (p0.0001). For every doubling of PSA, the risk of PSM increased by 56%, beginning at 0.5 ng/ml [odds ratio (OR) = 1.56, 95% confidence interval (CI) 1.3-1.9, p0.01], and every 10% increase in PPB resulted in an 11% increased risk of PSM (OR = 1.11, 95% CI 1.0-1.2, p = 0.002). Two out of the six surgeons had a 50% reduction of risk of PSM compared to the referent surgeon. Nerve-sparing surgery increased the risk of PSM by 50% compared to wide resection (OR = 1.5, 95% CI 1.0-2.1, p = 0.03).Both preoperative and surgical parameters affect the risk of PSM after radical prostatectomy. Surgeon and high preoperative PSA, PPB and cT category were confirmed as predictors of PSM. Surprisingly, nerve-sparing surgery was independently associated with increased risk of PSM. The correct selection of candidates for nerve-sparing surgery remains unresolved.

Purpose A Topoisomerase 1 (Top1) poison is frequently included in the treatment regimens for metastatic colorectal cancer (mCRC). However, no predictive biomarkers for Top1 poisons are available. We here report a study on the TOP1 gene copy number in CRC patients and its association with patient prognosis and tumor cell proliferation. Experimental design The study included TOP1 and CEN-20 fluorescence in situ hybridization ( FISH) analyses on formalin fixed paraffin embedded (FFPE) tissue sections from 154 stage III CRC chemonaive patients. The frequencies of aberration in the TOP1 gene copy number, the CEN-20 copy number and the TOP1 /CEN-20 ratio were analyzed and associated with overall survival (OS), time to recurrence (TTR) and in a subgroup analysis of rectal cancer patients only with time to local recurrence (LR in RC). Moreover, the TOP1 and CEN-20 copy numbers were correlated with the tumor Ki67 proliferation index. Results 35.7% of the tumors had an increased TOP1 copy number above 4 n gene copies per cell and 28.6% and 9.7% had a TOP1 /CEN-20 ratio ≥1.5 or ≥2.0, respectively. The TOP1 copy number and the TOP1 /CEN-20 ratios were separately added into multivariate analyses as continuous variables, in which also age, gender, primary tumor location and Ki67 status were added as covariates. In contrast to the TOP1 /CEN-20 ratio, the TOP1 copy number was significantly associated with OS (HR: 0.62; 95% CI: 0.42–0.90; p = 0.01). Neither the TOP1 copy number nor the ratio was significantly associated with TTR and only the TOP1 /CEN-20 ratio was significantly associated with LR in RC (HR: 0.25; 95% CI: 0.08–0.83; p = 0.02). No significant correlation was found between the TOP1 copy number and proliferation, while a weak and inverse correlation between the CEN-20 copy number and proliferation was observed. Conclusions This study showed that increased TOP1 gene copy numbers are frequent findings in cancer cells in stage III CRC tumors but unrelated to the proliferative status of the tumors. The association with prognosis is important to consider when planning and analyzing future studies investigating TOP1 as a potential predictive biomarker for Top1 poisons.

Whole slide imaging (WSI) has been used in conjunction with virtual microscopy (VM) for training or proficiency testing purposes, multicentre research, remote frozen section diagnosis and to seek specialist second opinion in a number of organ systems. The feasibility of using WSI/VM for routine surgical pathology reporting has also been explored. In this review, we discuss the utility and limitations of WSI/VM technology in the histological assessment of specimens from the prostate. Features of WSI/VM that are particularly well suited to assessment of prostate pathology include the ability to examine images at different magnifications as well as to view histology and immunohistochemistry side-by-side on the screen. Use of WSI/VM would also solve the difficulty in obtaining multiple identical copies of small lesions in prostate biopsies for teaching and proficiency testing. It would also permit annotation of the virtual slides, and has been used in a study of inter-observer variation of Gleason grading to facilitate precise identification of the foci on which grading decisions had been based. However, the large number of sections examined from each set of prostate biopsies would greatly increase time required for scanning as well as the size of the digital file, and would also be an issue if digital archiving of prostate biopsies is contemplated. Z-scanning of glass slides, a process that increases scanning time and file size would be required to permit focusing a virtual slide up and down to assess subtle nuclear features such as nucleolar prominence. The common use of large blocks to process prostatectomy specimens would also be an issue, as few currently available scanners can scan such blocks. A major component of proficiency testing of prostate biopsy assessment involves screening of the cores to detect small atypical foci. However, screening virtual slides of wavy fragmented prostate cores using a computer mouse aided by an overview image is very different from screening glass slides using a microscope stage. Hence, it may be more appropriate in this setting to mark the lesional area and focus only on the interpretation component of competency testing. Other issues limiting the use of digital pathology in prostate pathology include the cost of high quality slide scanners for WSI and high resolution monitors for VM as well as the requirement for fast Internet connection as even a subtle delay in presentation of images on the screen may be very disturbing for a pathologist used to the rapid viewing of glass slides under a microscope. However, these problems are likely to be overcome by technological advances in the future.

Aim: The purpose of this study was to investigate whether specific combinations of polymorphisms in 5-fluorouracil (5-FU) metabolism-related genes were associated with outcome in 5-FU-based adjuvant treatment of colorectal cancer. Methods: We analyzed two cohorts of 302 and 290 patients, respectively, one cohort for exploratory analyses and another cohort for validating the exploratory analyses. A total of ten polymorphisms in genes involved in 5-FU pharmacodynamics and pharmacokinetics were studied. End points were disease-free survival (DFS) and overall survival. Multifactor dimensionality reduction was used to identify genetic interaction profiles associated with outcome. Results: Low-expression alleles in thymidylate synthase (TYMS) were associated with decreased DFS and overall survival (DFS:hazard ratio [HR] exploration 2.65 [1.40–4.65]; p = 0.004, HR validation 1.69 [1.03–2.66]; p = 0.03). A specific multifactor dimensionality reduction derived combination of dihydropyrimidine dehydrogenase and TYMS polymorphisms was associated with increased DFS (HR exploration 0.69 [0.49–0.98]; p = 0.04, HR validation 0.66 [0.45–0.95]; p = 0.03). Specific combinations of functional polymorphisms in DPYD and TYMS were demonstrated to be associated with DFS and overall survival in patients receiving adjuvant 5-FU-based treatment. Specifically high TYMS expression alleles seem to be associated with decreased DFS. Original submitted 13 April 2011; Revision submitted 10 June 2011

Our aims were to analyze reporting of Gleason pattern (GP) 3 and 4 prostate cancer with the ISUP 2005 Gleason grading and to collect consensus cases for standardization. We scanned 25 prostate biopsy cores diagnosed as Gleason score (GS) 6-7. Fifteen genitourinary pathologists graded the digital slides and circled GP 4 and 5 in a slide viewer. Grading difficulty was scored as 1-3. GP 4 components were classified as type 1 (cribriform), 2 (fused), or 3 (poorly formed glands). A GS of 5-6, 7 (3 + 4), 7 (4 + 3), and 8-9 was given in 29%, 41%, 19%, and 10% (mean GS 6.84, range 6.44-7.36). In 15 cases, at least 67% of observers agreed on GS groups (consensus cases). Mean interobserver weighted kappa for GS groups was 0.43. Mean difficulty scores in consensus and non-consensus cases were 1.44 and 1.66 (p = 0.003). Pattern 4 types 1, 2, and 3 were seen in 28%, 86%, and 67% of GP 4. All three coexisted in 16% (11% and 23% in consensus and non-consensus cases, p = 0.03). Average estimated and calculated %GP 4/5 were 29% and 16%. After individual review, the experts met to analyze diagnostic difficulties. Areas of GP 4 and 5 were displayed as heat maps, which were helpful for identifying contentious areas. A key problem was to agree on minimal criteria for small foci of GP 4. In summary, the detection threshold for GP 4 in NBX needs to be better defined. This set of consensus cases may be useful for standardization.

Alpha-methylacyl coenzyme A racemase (AMACR or P504S) is a mitochondrial and peroxisomal protein present in a variety of human cells. Demonstration of increased expression is used diagnostically in prostatic adenocarcinoma. AMACR is also produced by normal hepatocytes and it has been postulated that the demonstration of AMACR expression or its pattern of distribution is useful in the diagnosis of hepatocellular carcinoma (HCC) (Jiang et al., Hum Pathol 2003;34, Guzman et al., Appl Immunohistochem Mol Morphol 2006;14, Li et al., J Exp Clin Cancer Res 2008;27). The aim of the present study was to evaluate whether immunohistochemical staining for AMACR can be used in a routine histopathologic setting. Immunohistochemical staining for AMACR was performed on paraffin-embedded tissue from livers resected for HCC during 1980-2006 at Rigshospitalet, Copenhagen, Denmark (n = 44). Tumor sections as well as surrounding non-neoplastic tissues were studied. In both tumor and non-tumor tissues, intracellular localization and staining pattern were assessed and the staining intensity of AMACR was graded. The fraction of stained tumor cells was not significantly different from that of stained non-tumor cells in the same patients (p = 0.97). A significantly lower staining intensity was observed in clear cell areas (p = 0.005), but the AMACR expression did not correlate with the HCC type and could not distinguish neoplastic from non-neoplastic liver cells. AMACR is not applicable as a tool in the histopathologic diagnosis of HCC.

Background: Methylenetetrahydrofolate reductase is a pivotal enzyme in folate metabolism and 5-fluorouracil (5-FU) cytotoxicity. Two common single-nucleotide polymorphisms (SNPs), MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), reduce enzyme activity. Initially, these SNPs were claimed to predict clinical efficacy, but further studies have yielded contradictory results. We tested whether these two polymorphisms are determinants of clinical outcome in a large patient group with a long follow-up time. Patients and methods: We included 331 patients who had been treated with adjuvant 5-FU/leucovorin chemotherapy after intended curative resection between 1997 and 2003. Clinical data, including relapse rates, overall survival, and tumor stage, were collected. DNA was extracted from formalin-fixed tumor tissue and analyzed for the MTHFR 677C>T and 1298A>C SNPs with real-time PCR. Results: The MTHFR 677C>T and 1298A>C polymorphisms were not associated with survival or relapse-free survival (P > 0.2). The 677 CC genotype was associated to toxicity (odds ratio = 1.83, P = 0.01). Conclusions: The MTHFR 677C>T and 1298A>C polymorphisms probably do not predict efficacy of adjuvant 5-FU treatment in colorectal cancer after complete resection; however, the 677C>T polymorphism may be associated with lower toxicity in 5-FU treatment. Implementation of SNP analysis for these polymorphisms for individualized treatment

Most human cancer cells have structural aberrations of chromosomal regions leading to loss or gain of gene specific alleles. This study aimed to assess the range of gene copies per nucleus of thymidylate synthase (TYMS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR) in colorectal cancer, and to evaluate its prognostic significance following adjuvant chemotherapy, since these enzymes are closely related to efficacy of 5-fluorouracil (5FU).Consecutive patients (n = 314), who were completely resected for colorectal cancer stages II-IV and adjuvantly treated with 5-FU were retrospectively evaluated. Paraffin embedded tumor specimens were assessed for gene copies per nucleus of TYMS, TP and DHFR by fluorescence in situ hybridisation (FISH) using specific peptide nucleic acid probes. Outcome according to gene copies per nucleus above and below the median were compared. Also TYMS expression, assessed by immunohistochemistry, was associated with TYMS copies per nucleus.The number of gene copies per nucleus were 1.7 (0.7-2.8), 1.8 (0.9-3.1) and 1.8 (1.1-2.7) median (range) for TYMS, TP and DHFR, respectively. TYMS expression was directly associated with TYMS genes per nucleus (p = 0.05). Cox multivariate analysis, adjusted for the prognostic impact of disease stage, vascular tumor invasion, and bowel obstruction at resection, revealed that high TYMS gene copy number was associated with significantly higher risk of recurrence (HR = 1.6; 95%CI 1.1-2.2; p = 0.02) and death (HR = 1.6; 95%CI 1.1-2.3; p = 0.01). No significant differences in outcome appeared according to TP and DHFR gene ratios.Aberration of TYMS gene is of significance to expression of TYMS, which may influence the biology and 5-FU sensitivity of colorectal cancer. This may be utilized in the allocation of patients for treatment approaches and for decision on follow-up programs.

Colitis has a wide range of etiologies and pathogenetic mechanisms. While acute infectious colitis and inflammatory bowel disease (e.g., ulcerative colitis and Crohn’s disease) represent the most common causes of colitis, this chapter focuses on the rarer forms of colitis occurring in older adults. An overview of the pathophysiology, epidemiology, clinical characteristics and treatment of microscopic colitis, ischemic colitis, uremic colitis, radiation colitis, diverticular colitis, and drug-induced colitis is provided. Since most of these disorders mentioned are uncommon, valid evidence for treatment is difficult to obtain. Thus, the chapter offers a current review of research including randomized clinical trials, if any, within the field and attempt to bring forth practical guidelines for the diagnosis and therapy of these rarer forms of colitis.

Long-term inflammation of the colonic mucosa during chronic inflammatory bowel disease with alternating periods of ulceration and healing may lead to the formation of finger-like projections, so-called filiform polyps. In rare cases, several filiform polyps form large tumour masses, termed giant filiform polyposis. We present a case of giant obstructing filiform polyposis in a patient without previous evidence of chronic bowel inflammation. The resected ascending colon from a 37-year-old woman was evaluated macroscopically and microscopically, and the presence of gene polymorphisms was studied by means of multiplex capillary electrophoresis single-strand conformation polymorphism assay, DNA sequencing, TaqMan analysis, and restriction enzyme cleavage. The giant filiform polyposis was restricted to a 15 cm segment of the ascending colon, and the remaining colonic mucosa was entirely without inflammatory changes. During the post-operative follow-up period, the patient developed symptoms and signs of distal bowel inflammation. Gene polymorphism studies were inconclusive as to Crohn's disease. In conclusion, we present an unusual pathological entity of giant filiform polyposis, which developed relatively rapidly in a colon without any history or macroscopic changes suggestive of chronic inflammatory bowel disease. Although the patient subsequently developed symptoms in keeping with Crohn's disease, studies of genetic polymorphism were unable to confirm this notion, and colorectal tissue has not been sampled postoperatively for histological evaluation.

It has been reported that colonocytes in ulcerative colitis (UC) upregulate anti-apoptotic cytoprotective pathways. An expression-profiling study of apoptosis-related genes suggested that the cellular inhibitor of apoptosis protein-2 (cIAP2) could be upregulated in epithelial cells in UC. The role of cIAP2 in active UC was therefore investigated. Fourteen patients with active UC and 12 control subjects who underwent routine colonoscopy for control of their disease or as part of their examination program for irritable bowel syndrome were included. cIAP1 and cIAP2 expression was investigated by polymerase chain reaction, Western blotting, and immunohistochemistry. The regulation and role of cIAP2 for apoptosis was further investigated in cell cultures. cIAP2, but not cIAP1, was upregulated during active UC in regenerative epithelial cells. A similar upregulation was found in cell lines stimulated with proinflammatory cytokines and was dependent on nuclear factor kappaB activation. Inhibition of cIAP2 increases the susceptibility of epithelial cells to Fas ligation. Inflammation during active UC thus causes an upregulation of cIAP2 in regenerating epithelium, which renders the cells less susceptible to Fas ligation. This might play a role in regeneration of the epithelium but might additionally be implicated in carcinogenesis of UC.

A global gene expression profile of non-inflamed colonic mucosal cells from patients with Crohn's disease (CD) and of colonic mucosal cells from controls was performed.Tissue specimens from macroscopically non-inflamed descending colon were obtained colonoscopically from 33 CD patients and from 17 control subjects. All controls and 10 CD patients were medication-free at the time of colonoscopy. The Human Genome U133 Plus 2.0 GeneChip Array was used for gene profiling. Hybridization data were analysed with dChip software. Results were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Protein product expression of selected genes was assessed by immunohistochemistry using the Envision+ visualization technique.The expression profile was not homogeneous with the statistical cut-point settings applied. In comparison with controls, it was found that 19 CD patients had three differentially expressed genes, two of them related to the innate immune system: dual oxidase 2 on chromosome 15 (DUOX2, fold change 4.1) and lipocalin 2 on chromosome 9 (LCN2, fold change 3.1). The third gene, regenerating islet-derived 1 alpha (REG1A, fold change 3.9), codes for a mitogenic protein; this could not be confirmed by RT-PCR. Medication-free patients had no differentially expressed genes as compared with controls. Immunohistochemistry indicated that these proteins were produced by epithelial cells (REG1A, LCN2) and leucocytes (DUOX2 and LCN2).As compared with controls, non-inflamed colonic mucosal cells contain two up-regulated genes related to the innate immune system. Up-regulation of these genes, known to be induced by microorganisms, suggests either increased microflora antigenicity or an altered function in mucosal barrier defence.

In breast cancer, analysis of HER2 expression is pivotal for treatment decision. This study aimed at comparing digital, automated image analysis with manual reading using the HER2-CONNECT algorithm (Visiopharm) in order to minimize the number of equivocal 2+ scores and the need for reflex fluorescence in situ hybridization (FISH) analysis. Consecutive samples from 462 patients were included. Tissue micro arrays (TMAs) were routinely manufactured including two 2 mm cores from each patient, and each core was assessed in order to ensure the presence of invasive carcinoma. Immunohistochemical staining (IHC) was performed with Roche/Ventana’s HER2 ready-to-use kit. TMAs were scanned in a Zeiss Axio Z1 scanner, and one batch analysis of the HER2-CONNECT algorithm including all core samples was run using Visiopharm’s cloud-based software. The automated reading was compared to conventional manual assessment of HER2 protein expression, together with FISH analysis of HER2 gene amplification for borderline (2+) protein expression samples. Compared to FISH analysis, manual assessment of the HER2 protein expression demonstrated a sensitivity of 85.8 % and a specificity of 86.0 % with 14.0 % equivocal samples. With HER2-CONNECT, sensitivity increased to 100 % and specificity to 95.5 % with less than 4.5 % equivocal. Total agreement when comparing HER2-CONNECT with manual IHC assessment supplemented by FISH for borderline (2+) cases was 93.6 %. Application of automated image analysis for HER2 protein expression instead of manual assessment decreases the need for supplementary FISH testing by 68 %. In the routine diagnostic setting, this would have significant impact on cost reduction and turn-around time.

The treatment of patients with colorectal liver metastasis has improved significantly and first line therapy is often combined chemotherapy and bevacizumab, although it is unknown who responds to this regimen. Colorectal liver metastases grow in different histological growth patterns showing differences in angiogenesis. To identify possible response markers, histological markers of angiogenesis were assessed. Patients who underwent resection of colorectal liver metastasis at Rigshospitalet, Copenhagen, Denmark from 2007 to 2011 were included (n = 254) including untreated and patients treated with chemotherapy or chemotherapy plus bevacizumab. The resected liver metastases were characterised with respect to growth pattern, endothelial and tumour cell proliferation as well as microvessel density and tumour regression. Tumour regression grade of liver metastases differed significantly between untreated/chemotherapy treated patients in comparison to chemotherapy plus bevacizumab treated patients (both p 0.0001). Microvessel density was decreased in liver metastases from patients treated with bevacizumab in comparison to those from untreated/chemotherapy-treated patients (p = 0.006/p = 0.002). Tumour cell proliferation assessed by Ki67 expression correlated to a shorter recurrence free survival in the total patient cohort. In conclusion, liver metastases from patients treated with neo-adjuvant chemotherapy and bevacizumab had significantly lower microvessel densities and tumour regression grades when compared to liver metastases from untreated or chemotherapy treated patients. This may indicate that bevacizumab treatment results in altered vascular biology and tumour viability, with possible tumour reducing effect.

Biomarkers predicting response to primary androgen deprivation therapy (ADT) and risk of castration-resistant prostate cancer (CRPC) is lacking. We aimed to analyse the predictive value of ERG expression for development of CRPC.In total, 194 patients with advanced and/or metastatic prostate cancer (PCa) treated with first-line castration-based ADT were included. ERG protein expression was analysed in diagnostic specimens using immunohistochemistry (anti-ERG, EPR3864). Time to CRPC was compared between ERG subgroups using multiple cause-specific Cox regression stratified on ERG-status. Risk reclassification and time-dependent area under the ROC curves were used to assess the discriminative ability of ERG-status. Time to PSA-nadir, proportion achieving PSA-nadir ≤0.2 ng/ml, and risk of PCa-specific death were secondary endpoints.Median follow-up was 6.8 years (IQR: 4.9-7.3). In total, 105 patients (54.1%) were ERG-positive and 89 (45.9%) were ERG-negative. No difference in risk of CRPC was observed between ERG subgroups (P = 0.51). Median time to CRPC was 3.9 years (95%CI: 3.2-5.1) and 4.5 years (95%CI: 2.3-not reached) in the ERG-positive and ERG-negative group, respectively. Compared to a model omitting ERG-status, the ERG-stratified model showed comparable AUC values 1 year (77.6% vs. 78.0%, P = 0.82), 2 years (71.7% vs. 71.8%, P = 0.85), 5 years (68.5% vs. 69.9%, P = 0.32), and 8 years (67.9% vs. 71.4%, P = 0.21) from ADT initiation. No differences in secondary endpoints were observed.ERG expression was not associated with risk of CRPC suggesting that ERG is not a candidate biomarker for predicting response to primary ADT in patients diagnosed with advanced and/or metastatic PCa.

Cytotoxic effect of 5-fluorouracil 5-FU is mediated through inhibition of thymidylate synthase (TS), and 5-FU is catabolised by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity of colorectal cancer. Archival tumour specimens from 303 consecutive patients were analysed for the expression of TS and DPD using immunohistochemistry. All patients were completely resected for colorectal cancer stages II-III and have subsequently received adjuvant treatment with 5-FU. In a multivariate analysis adjusting for the impact of bowel obstruction and vascular tumour invasion, diffuse TS pattern was significantly associated with increased risk of recurrence (hazard ratio (HR) = 1.9; 95% confidence interval (CI): 1.1-3.2; p = 0.02), but without significant association to death (HR = 1.6; 95% CI: 0.9-2.8; p = 0.08). High TS intensity was not significantly associated with lower risk of recurrence (HR = 0.6; 95% CI: 0.3-1.1; p = 0.07) or death (HR = 0.6; 95% CI: 0.3-1.2; p = 0.2). High DPD intensity was significantly associated with increased risk of recurrence (HR = 1.5; 95% CI: 1.1-2.3; p = 0.03) and death (HR = 1.6; 95% CI: 1.1-2.5; p = 0.02). Patients with a combination of low TS and high DPD intensity were at significantly increased risk of both recurrence (HR = 2.1; 95% CI: 1.0-4.2; p = 0.04) and death (HR = 2.0; 95% CI: 1.0-4.0; p = 0.05). No relationship between tolerability and toxicity of 5-FU and TS and DPD expression was found. It is concluded that characterizing colorectal carcinomas by TS and DPD expression may disclose subsets of patients with significantly greater risk of disease recurrence and early death. This may be utilized in the selection of patients for treatment approaches and for decision on follow-up programs.

Intercellular adhesion molecule-1 (ICAM-1) is important in ulcerative colitis (UC) by mediating the arrest and further migration of neutrophils. In vitro studies have shown that colonocytes from chronically inflamed colon and cultured colon cancer cells are capable of expressing ICAM-1. The aim of this study was to assess the ICAM-1 expression in human colonic tissue representing UC, Crohn's disease (CD), adenomas, and adenocarcinomas, with special attention to the epithelium.Formalin-fixed and paraffin-embedded tissue from the archives of the Department of Pathology of Rigshospitalet University of Copenhagen was examined. Colonic tissue from 10 patients with UC, 10 with CD, 32 adenomas, 27 adenocarcinomas, and 10 lymph node metastases were included. The expression of ICAM-1 was assessed by using the EnVision(+)technique (DakoCytomation).Endothelial ICAM-1 was up-regulated in areas with dense lymphocyte infiltration and near crypt abscesses and ulcerations. Ulcerations were covered by a continuous layer of macrophages and epithelial cells expressing ICAM-1. Similar observations were made in the case of adenomas and adenocarcinomas, but in adenocarcinomas the epithelial ICAM-1 was more diffuse and not related solely to sites of surface destruction.In the colon, endothelial cells, macrophages, and epithelial cells are in certain conditions capable of expressing ICAM-1. Although the ICAM-1 expression was related to both the degree and the nature of inflammation, the data indicate increased susceptibility of cancer cells to express ICAM-1. Epithelial and macrophage ICAM-1 might be involved in the immune surveillance and the first-line defense of the diseased colon.

To study the distribution of the thiopurine methyltransferase (TPMT) genotype among azathioprine (Aza)-tolerant and -intolerant patients with various disorders, and to investigate a possible relationship with the Aza metabolite levels.Forty-six Aza-tolerant and six Aza-intolerant patients had the TPMT genotype distribution determined using a polymerase chain reaction (PCR) assay and the forty-six Aza-tolerant patients had the Aza metabolite levels determined using a high-pressure liquid chromatography (HPLC) analysis.One non-functional TPMT mutant allele was demonstrated in 2 of the 46 Aza-tolerant patients (4.4%) and one or two non-functional mutant alleles in 2 of the 6 Aza-intolerant patients (33.3%). Of the 4 patients, with one or two non-functional mutant alleles 2 (50%) were intolerant to Aza compared with 4 of the 48 patients (8.3%) with no mutations detected. The time to hepatotoxicity did not differ significantly between the 2 patients with one or two non-functional mutant alleles and the remaining 3 patients ( P=0.5). The TPMT genotype distribution differed slightly in the three different categories of disorders ( P=0.05). The median E-6-TGN level among the 2 TPMT heterozygous patients was 275 pmol/8x10(8) RBC (range 240-310), whereas the remaining 44 patients had a median E-6-TGN level of 110 pmol/8x10(8) RBC (range 0-440) ( P=0.07).Although TPMT genotyping cannot be recommended on behalf of the present study, it is to be expected that half of the patients with one or two non-functional TPMT mutant alleles will develop Aza intolerance leading to withdrawal of therapy. Thus, clinicians may anticipate about 5% of the patients to develop intolerance to Aza therapy solely for that reason.

A soluble form of intercellular adhesion molecule-1 (sICAM-1) shed from endothelial cells is present in the circulation. Whether the circulating molecules represent passive turnover of surface ICAM-1 or may have some active functions in the inflammatory process is unknown. Glucocorticoids (e.g. prednisolone) are cornerstones in the treatment of acute exacerbations of ulcerative colitis (UC), and influence of the leucocyte/endothelial interaction appears to be part of their mode of action. The aim of the present study was therefore to evaluate the ICAM-1-shedding through measurements of sICAM-1 concentrations during prednisolone treatment of UC patients.Prednisolone (40 mg) was prescribed to 15 patients with severe disease activity. At inclusion, and after 2 weeks of treatment, plasma sICAM-1 levels were measured using the ELISA technique.The concentrations of sICAM-1 were significantly decreased during treatment from median 256.2 (ng/ml) (interquartile range 239.7-321.0 ng/ml) to 220.4 ng/ml (196.0-276.3 ng/ml) (P0.01). This reduction correlated with a decrease in disease activity (r(s) = 0.8; P0.003).sICAM-1 seems to be a poor diagnostic tool, but since plasma sICAM-1 concentrations decreased during the treatment period, it might prove to be applicable as an activity marker in the individual patient.

Despite improved therapy of advanced colorectal cancer, the median overall survival (OS) is still low. A surgical removal has significantly improved survival, if lesions are entirely removed. The purpose of this retrospective explorative study was to evaluate the prognostic value of histological growth patterns (GP) in chemonaive and patients receiving neo-adjuvant therapy. Two-hundred-fifty-four patients who underwent liver resection of colorectal liver metastases between 2007 and 2011 were included in the study. Clinicopathological data and information on neo-adjuvant treatment were retrieved from patient and pathology records. Histological GP were evaluated and related to recurrence free and OS. Kaplan-Meier curves, log-rank test and Cox regression analysis were used. The 5-year OS was 41.8% (95% CI 33.8-49.8%). Growth pattern evaluation of the largest liver metastasis was possible in 224 cases, with the following distribution: desmoplastic 63 patients (28.1%); pushing 77 patients (34.4%); replacement 28 patients (12.5%); mixed 56 patients (25.0%). The Kaplan-Meier analyses demonstrated that patients resected for liver metastases with desmoplastic growth pattern had a longer recurrence free survival (RFS) than patients resected for non-desmoplastic liver metastases (p=0.05). When patients were stratified according to neo-adjuvant treatment in the multivariate Cox regression model, hazard ratios for RFS compared to desmoplastic were: pushing (HR=1.37, 95% CI 0.93-2.02, p=0.116), replacement (HR=2.16, 95% CI 1.29-3.62, p=0.003) and mixed (HR=1.70, 95% CI 1.12-2.59, p=0.013). This was true for chemonaive patients as well as for patients who received neo-adjuvant treatment.

Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal epithelial cells (IECs) was increased at the wound edge after 24 h ( P < 0.05), returned to normal after reepithelialization, and correlated with the inflammatory reaction in the experimental wounds ( P < 0.001). cIAP2 was induced in vitro in regenerating Caco2 IECs after wound infliction ( P < 0.01). Knockdown of cIAP2 caused a substantial impairment of the IEC regeneration through inhibition of migration ( P < 0.005). cIAP2 overexpression lead to formation of migrating IECs and upregulation of expression of RhoA and Rac1 as well as GTP-activation of Rac1. Transforming growth factor-β1 enhanced the expression of cIAP2 but was not upregulated in wounds in vivo and in vitro. NF-κB and MAPK pathways did not affect cIAP2 expression. cIAP2 is in conclusion a regulator of human intestinal wound healing through enhanced migration along with activation of Rac1, and the findings suggest that cIAP2 could be a future therapeutic target to improve intestinal wound healing.

Successful colonization by a cancer cell of a distant metastatic site requires immune escape in the new microenvironment. TNF signaling has been implicated broadly in the suppression of immune surveillance that prevents colonization at the metastatic site and therefore must be blocked. In this study, we explored how TNF signaling influences the efficiency of liver metastasis by colon and lung carcinoma in mice that are genetically deficient for the TNF receptor TNFR2. We found a marked reduction in liver metastases that correlated with a greatly reduced accumulation at metastatic sites of CD11b+GR-1+ myeloid cells with enhanced arginase activity, identified as myeloid-derived suppressor cells (MDSC). Reduced infiltration of MDSC coincided with a reduction in the number of CD4+FoxP3+ T regulatory cells in the tumors. Reconstitution of TNFR2-deficient mice with normal bone marrow, or adoptive transfer of TNFR2-expressing MDSC into these mice, was sufficient to restore liver metastasis to levels in wild-type mice. Conversely, treatment with TNFR2 antisense oligodeoxynucleotides reduced liver metastasis in wild-type mice. Clinically, immunohistochemical analysis of liver metastases from chemotherapy-naïve colon cancer patients confirmed the presence of CD33+HLA-DR−TNFR2+ myeloid cells in the periphery of hepatic metastases. Overall, our findings implicate TNFR2 in supporting MDSC-mediated immune suppression and metastasis in the liver, suggesting the use of TNFR2 inhibitors as a strategy to prevent metastatic progression to liver in colon, lung, and various other types of cancer. Cancer Res; 75(24); 5235–47. ©2015 AACR.

Medullary carcinoma of the colon is a rare variant of colorectal cancer claimed to have a more favorable prognosis than conventional adenocarcinomas. The histopathologic appearance may be difficult to distinguish from poorly differentiated adenocarcinoma. The study aimed to evaluate the diagnostic interobserver agreement and to characterize the immunohistochemical and molecular differences between these two subgroups. Fifteen cases initially classified as medullary carcinoma and 30 cases of poorly differentiated adenocarcinomas were included. Two pathologists reviewed the slides independently without knowledge of the original diagnosis and subgrouped the tumors into the two entities. Agreement was reached in 31 of 45 cases (69 %) with kappa = 0.32. An extensive immunohistochemical panel was performed, and KRAS, NRAS, and BRAF mutational status was assessed. Of the 31 cases with diagnostic agreement, the expression of only MLH-1 along with corresponding expression of PMS-2 differed significantly (p = 0.04). A high rate of BRAF mutations was detected in both subgroups without significant differences. Expression of MLH-1 was superior in dividing the tumors into two separate entities with significant differences in CK20 (p = 0.005) expression and in the rate of BRAF mutations (p = 0.0035). In conclusion, medullary carcinomas of the colon are difficult to discriminate from poorly differentiated adenocarcinoma even with the help of immunohistochemical and molecular analyses. This raises the question whether these morphological subtypes should be maintained or whether an alternative classification of poorly differentiated colorectal adenocarcinomas based on MLH-1 status rather than morphology should be suggested.

BACKGROUND & AIMS: Fibrogenesis results in release of certain extracellular matrix protein fragments into the circulation. We evaluated the diagnostic and prognostic performance of two novel serological markers, the precisely cleaved N-terminal propeptide of type III collagen (Pro-C3) and a peptide of helical collagen type III degradation (C3M), in chronic hepatitis C (CHC) patients. METHOD: Pro-C3 and C3M were measured by ELISA in plasma from CHC patients (n = 194) from a prior phase II antifibrotic trial (NCT00244751). Plasma samples and paired liver biopsies were obtained at baseline and after 1-year. Patients were stratified according to Ishak stages 2-4. Internal cross-validation was performed by bootstrap analysis. RESULTS: Pro-C3 levels were significantly higher in CHC patients in Ishak stage 4 compared to stage 2 (P

To investigate the clinical implications of interobserver variation in the assessment of re-biopsies obtained during active surveillance (AS) of prostate cancer.In all, 107 patients with low-risk prostate cancer with 93 diagnostic biopsy sets and 109 re-biopsy sets were included. The International Society of Urological Pathology 2005 Gleason scoring system was used for the histopathological assessment of all biopsies. Three different definitions of histopathological progression were applied. Unweighted and linear weighted Kappa (κ) statistics were used to compare the interobserver agreement.The overall Gleason score agreement was 68.8% with a weighted κ of 0.670. The interobserver agreement was 79.6% for meeting the AS selection criteria. According to the three progression definitions applied, overall agreement was between 80.7% and 89.0% with weighted κ values of 0.746-0.791. Treatment recommendations would have changed in up to 10.1% (95% confidence interval 5.4-17.7%) of the 109 re-biopsy sets.Kappa statistics showed strong agreement between the histological evaluations. However, up to 10% of patients on AS would receive a different treatment recommendation depending upon which histopathological evaluation of re-biopsies was used for treatment planning.

Udgivelsesdato: 2010-Nov

Compelling biomarkers identifying prostate cancer patients with a high risk of progression during active surveillance (AS) are needed.To examine the association between ERG expression at diagnosis and the risk of progression during AS.This study included 265 patients followed on AS with prostate-specific antigen (PSA) measurements, clinical examinations, and 10-12 core rebiopsies from 2002 to 2012 in a prospectively maintained database. ERG immunohistochemical staining was performed on diagnostic paraffin-embedded formalin-fixed sections with a ready-to-use kit (anti-ERG, EPR3864). Men were characterised as ERG positive if a minimum of one tumour focus demonstrated ERG expression.Overall AS progression was defined as clinical progression: increased clinical tumour category ≥cT2b by digital rectal examination and ultrasound, and/or histopathologic progression: upgrade of Gleason score, more than three positive cores or bilateral positive cores, and/or PSA progression: PSA doubling time3 yr. Risk of progression was analysed using multiple cause-specific Cox regression and stratified cumulative incidences (Aalen-Johansen method). Curatively intended treatment, watchful waiting, and death without progression were treated as competing events.A total of 121 of 142 ERG-negative and 96 of 123 ERG-positive patients had complete diagnostic information. In competing risk models, the ERG-positive group showed significantly higher incidences of overall AS progression (p0.0001) and of the subgroups PSA progression (p0.0001) and histopathologic progression (p0.0001). The 2-yr cumulative incidence of overall AS progression was 21.7% (95% confidence interval [CI], 14.3-29.1) in the ERG-negative group compared with 58.6% (95% CI, 48.7-68.5) in the ERG-positive group. ERG positivity was a significant predictor of overall AS progression in multiple Cox regression (hazard ratio: 2.45; 95% CI, 1.62-3.72; p0.0001). The main limitation of this study is its observational nature.In our study, ERG positivity at diagnosis can be used to estimate the risk of progression during AS. If confirmed, ERG status can be used to individualise AS programmes.The tissue biomarker ERG identifies active surveillance patients with an increased risk of disease progression.

To investigate how location of positive surgical margins (PSM) in pT2 tumors affect the risk of biochemical recurrence (BR).The study includes 1,133 consecutive patients from 1995 until end of 2011, who had organ-confined disease (pT2) following RP. The location of PSM was stratified into apical and non-apical. BR was defined as the first PSA ≥ 0.2 ng/ml after RP. Risk of BR was analyzed with Kaplan-Meier and Cox regression analysis.Median follow-up was 3.6 years (range: 0.5-15.5 years). The overall pT2 PSM rate was 26.3%. Overall, a pT2 with PSM had a 3.1-fold increased risk of BR compared to margin negative patients. Patients with pT2 apical and non-apical PSM had a 5-year biochemical recurrence-free survival of 84.9% (95% CI: 77.6-92.2%) and 78.6% (95% CI: 71.3-85.9%), respectively. In multivariate analysis, pT2 apical and non-apical PSM was individually associated with a 2.2- and 3.8-fold increased risk of BR compared to margin negative patients.In our cohort the location of pT2 PSM was associated with time to BR, that is, patients with non-apical pT2 PSM endured the highest risk of BR compared to apical PSM. This may indicate that not all patients with pT2 PSM should be offered adjuvant therapy.

A dysregulated local immune reaction with unbalanced cytokine expression seems essential in inflammatory bowel disease (IBD), i.e. ulcerative colitis (UC) and Crohn's disease (CD). Since the roles of interleukin (IL-)13 and IL-15 remain unclear, this study aimed at studying intestinal expression of IL-13 and IL-15 in IBD. Methods: In colonic biopsies from 24 UC, 18 CD, and 12 controls IL-13 and IL-15 were measured using ELISA, and their gene expressions were assessed by RT-PCR. Leukocytes were visualised histochemically. Results: Concentrations of IL-13 were decreased in UC (median 56 pg/mg tissue; interquartile range 30–99 pg/mg) compared to CD (82 pg/mg tissue; 41–122; P =0.004) and controls (83 pg/mg tissue; 18–134; P >0.05), and lower in active UC (53 pg/mg tissue; 33–96) than in inactive UC (80 pg/mg tissue; 65–99; P =0.02). IL-15 concentrations were higher in CD patients (34 pg/mg tissue; 24–53) as compared to controls (20 pg/mg tissue; 15–21; P =0.001) whilst being 22 pg/mg tissue (15–32) in UC. IL-13 mRNA and IL-15 mRNA were detected in 20% and 15%, respectively. Infiltration of leukocytes correlated inversely with IL-13 levels ( P =0.02). Conclusion: Active UC is associated with decreased colonic IL-13 suggesting that IL-13 levels are diminished as a part of UC exacerbations, or that exacerbations follow active downregulation of IL-13.

Neutrophil gelatinase-associated lipocalin (NGAL) is a newly described neutrophil lipocalin that may bind the proinflammatory bacterial tripeptide N-formylmethionyl-leucyl-phenylalanine. In situ hybridization and immunohistochemical studies have shown a strong NGAL expression in colonocytes and neutrophils in ulcerative colitis (UC). Because NGAL is highly protease resistant, it should be ideal for in vivo fecal and dialysate studies. Our aim was to investigate the potential of NGAL as a disease activity marker in UC and to compare it with IL-8 and TNF-alpha.Twenty-three patients with UC, 14 with Crohn's disease (CD), 19 patients with acute infectious enterocolitis, and 20 healthy controls were included. The disease activity of UC and CD was scored semiquantitatively. Concentrations of NGAL, IL-8, and TNF-alpha were determined in rectal dialysis fluid, feces, and serum using sandwich enzyme-linked immunosorbent assays. The total protein concentration in feces and dialysate fluid was measured, and the amount of markers was expressed as ng/mg protein.In healthy controls and non-IBD (irritable bowel disease) colitis, the median values for NGAL in feces were 183 ng/mg protein and 546 ng/mg protein (p0.01), respectively. When separating UC into clinical activity groups (remission, mild/moderate, and severe disease activity) the corresponding values of NGAL were 442 ng/mg (p0.05), 605 ng/mg (p0.02), and 3646 ng/mg (p0.001, compared with controls), respectively, and in quiescent colonic CD 368 ng/mg (p0.05) and in active stages 751 ng/mg (p0.01). NGAL levels in dialysis fluid listed in the same order were: 11 ng/mg for controls, 71 ng/mg (p0.05) for non-IBD colitis, 100 ng/mg (p0.02), 179 ng/mg (p0.01), and 2053 ng/mg (p0.001) for UC, and 14 ng/mg (p0.05) and 121 ng/mg (p0.02) for CD, respectively. Serum NGAL concentrations did not differ between UC and CD in quiescent versus active stages. A significant increase of NGAL in both feces and dialysate with increasing disease activity of UC was found (p = 0.02 and p = 0.003, respectively).The NGAL content in rectal dialysate and particularly in feces seems to be a reliable marker for severe disease activity in UC, whereas serum NGAL concentrations do not reflect disease activity.

Infiltration of leukocytes into the bowel wall is a landmark of the inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD). The leukocyte movement is dependent on physical contact (adhesion) between the leukocytes and activated endothelial cells and can be divided into capturing, rolling, leukocyte flattening, and extra-vasation. The molecules shown to form the basis of leukocyte-endothelial binding are referred to as cell adhesion molecules (CAMs). Several of these molecules have additional properties, including interaction between leukocytes and proteins in the extracellular matrix, collaen in basement membranes, and stromal cells in lymphoid tissue and bone marrow. Furthermore, studies have indicated that CAMs interfere with the tumor cell's ability to metastasize. This paper will focus on a description of those CAMs that are either known or believed to be involved in the pathogenesis of IBD. Investigations of the presence and functions of these CAMs in IBD is reviewed, and potential new treatments are discussed.

OBJECTIVE. We propose a repurposing strategy where anthracyclines are reintroduced to a subgroup of patients with metastatic colorectal cancer with the highest likelihood of response. In breast cancer, DNA topoisomerase II alpha gene (TOP2A) alterations predict incremental benefit of anthracyclines, but this association has not been investigated in colorectal cancer. Frequency analysis of TOP2A gene alterations in colorectal cancer and the association with prognosis are evaluated and the challenges of using a TOP2A/CEN-17 FISH probe combination are addressed. MATERIAL AND METHODS. Formalin-fixed, paraffin-embedded material from 154 stage III colorectal cancer patients included in the RANX05 clinical trial was retrospectively assessed for TOP2A gene alterations using FISH. The TOP2A/CEN-17 ratio as well as the TOP2A gene copy number alone was used to define gene alterations and associations between gene status and outcomes were analyzed. RESULTS. TOP2A gene gain was a frequent finding with 9.8 % having a total of ≥4 TOP2A copies per cell. According to the TOP2A/CEN-17 ratio, 10.5 % had TOP2A gene gain. Polysomy or gain of the centromere region of chromosome-17 was not as frequent as reported in breast cancer. No prognostic characteristic of TOP2A was identified. CONCLUSION. TOP2A gene gain is present in numbers relevant to identify a subgroup of patients who may benefit from anthracycline therapy. Based on the present findings, we will initiate a prospective clinical trial designed to evaluate this hypothesis in patients with metastatic colorectal cancer who have failed 5-fluorouracil and oxaliplatin chemotherapy.

Colorectal metastases in the liver grow according to three histological patterns: a pushing pattern, a replacement pattern, and a desmoplastic pattern. The objective of the current study was to explore the prognostic significance of these three growth patterns for survival. The study included 217 consecutive patients, liver resected between 2007 and 2011 due to hepatic metastases from colorectal adenocarcinoma. The growth patterns were assessed on archival hematoxylin and eosin-stained tissue sections. In 150 metastases, the density of the immune cell infiltrate at the tumor periphery was judged by a semi-quantitative method. The prevalence of the pushing-type, the desmoplastic-type, and the replacement-type was 33%, 32%, and 11%, respectively; 24% of the metastases displayed a mixed pattern. Kaplan-Meier analysis and Cox regression demonstrated a prognostic significance of the growth patterns (P=0.0006, log-rank test), as the replacement pattern appeared as an independent predictor of poor overall survival. For patients with replacement growth, the hazard of death was 2-2.5 times higher than for patients with pushing growth (P=0.004, cox regression) or mixed growth (P=0.01), and nearly four times higher than for patients with desmoplastic growth (P

To investigate risk factors associated with positive surgical margins (PSM) and biochemical recurrence (BR) in organ confined tumors (pT2) after radical prostatectomy (RP) for localized prostate cancer (PCa).Between 1995 and 2011, 1,649 patients underwent RP at our institution. The study includes the 1,133 consecutive patients with pT2 tumors at final histopathology. Logistic regression analysis was used for risk of PSM. Risk of BR, defined as the first PSA ≥ 0.2 ng/ml, was analyzed with Kaplan-Meier and Cox regression analysis.Median follow-up was 3.6 years (range: 0.5-15.5 years). In logistic regression, NS surgery was independently associated with an increased risk of pT2 PSM (OR = 1.68, 95% CI: 1.3-2.0, P = 0.01) relative to non-NS surgery. NS surgery was not independently associated with BR but the interaction of PSM and NS surgery trended (P = 0.08) to increase the risk of BR compared to PSM and non-NS surgery.Several factors influence the risk of pT2 PSMs in radical prostatectomy. In our cohort pT2 PSM is associated with NS surgery and trend to increase risk of BR compared to non-NS surgery. The optimal selection of candidates for NS surgery is still not clear.

The aim of this study was to analyse whether primary Gleason pattern in biopsy Gleason score (GS) 7 predicted adverse histopathological features and had an impact on the risk of biochemical failure in a consecutive series of patients undergoing radical prostatectomy (RP).Between 2006 and 2010, 441 patients with biopsy GS 7 underwent RP at Rigshospitalet, Copenhagen, Denmark. Favourable histopathological features were defined as pT2 margin-negative cancer, RP specimen GS ≤ 3+4 and no lymph-node metastasis. Adverse histopathological features were defined as advanced pT3/4 cancer or pT2 margin-positive cancer and/or RP specimen GS ≥ 4+3 and/or positive lymph nodes. Biochemical failure was defined as the first prostate-specific antigen (PSA) ≥ 0.2 ng/ml.A total of 344 patients (78.0%) had GS 3+4 in biopsies, while 97 patients (22.0%) had GS 4+3. No difference in age, PSA, percentage of biopsies with cancer, clinical tumour stage or volume on transrectal ultrasonography was found. Primary Gleason pattern 4 was associated with worse pathological stage (p = 0.049). On multivariate analysis, primary Gleason pattern 4 (p0.0001), cT stage (p = 0.024), PSA (p0.0001) and age (p = 0.009) predicted adverse histopathological features. In univariate analysis, Gleason score 3+4 had a significantly lower biochemical failure rate compared with Gleason score 4+3 (p = 0.0035). PSA (p0.0001), primary Gleason pattern 4 (p = 0.001) and percentage of biopsies with cancer (p = 0.02) were independently associated with risk of biochemical failure.In biopsies with GS 7, a primary Gleason pattern 4 was associated with significantly elevated risk of adverse histopathological features and biochemical failure compared to pattern 3 in patients undergoing RP. This study underlines the heterogeneity of biopsy GS 7.

The gastrin and the gastrin/CCK-B receptor genes are co-expressed in several carcinomas. The primary translational product, progastrin, however, is processed to several peptides of which only those that are α-amidated at their C-terminus are receptor ligands. So far, characterization of the progastrin-derived peptides in gastric cancer has not been reported. The authors therefore examined the molecular nature of gastrin and its receptor in human gastric carcinomas.Twenty patients with adenocarcinoma underwent partial or total gastrectomy. In samples from each carcinoma, gastrin peptides were characterized, using a library of sequence-specific immunoassays. Expression was also demonstrated by immunohistochemistry. In addition, the gastrin and gastrin/CCK-B receptor gene expression was quantitated using real-time PCR, and the receptor protein demonstrated by western blotting.α-Amidated gastrins were detectable in 16 of 20 carcinomas (median concentration 2.1 pmol/g tissue; range 0-386 pmol/g tissue). The tissue concentrations correlated closely to the gastrin mRNA contents (r = 0.75, p0.0001). Moreover, progastrin and non-amidated processing intermediates, including glycine-extended gastrins, were detected in 19 carcinomas. Immunohistochemistry corroborated gastrin expression in carcinoma cells. Chromatography revealed extensive progastrin processing with α-amidated gastrin-34 and -17 (tyrosyl-sulfated as well as non-sulfated) as major products. Finally, gastrin/CCK-B receptor mRNA and protein were detected in all tumors.The results show that the elements for a local loop of α-amidated gastrins and their receptor are detectable in 80% of human gastric adenocarcinomas. Therefore, the results support the contention that locally expressed gastrin may be involved in the tumorigenesis of gastric adenocarcinomas.