Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016

Tumor budding is a well-established independent prognostic factor in colorectal cancer but a standardized method for its assessment has been lacking. The primary aim of the International Tumor Budding Consensus Conference (ITBCC) was to reach agreement on an international, evidence-based standardized scoring system for tumor budding in colorectal cancer. The ITBCC included nine sessions with presentations, a pre-meeting survey and an e-book covering the key publications on tumor budding in colorectal cancer. The 'Grading of Recommendation Assessment, Development and Evaluation' method was used to determine the strength of recommendations and quality of evidence. The following 10 statements achieved consensus: tumor budding is defined as a single tumor cell or a cell cluster consisting of four tumor cells or less (22/22, 100%). Tumor budding is an independent predictor of lymph node metastases in pT1 colorectal cancer (23/23, 100%). Tumor budding is an independent predictor of survival in stage II colorectal cancer (23/23, 100%). Tumor budding should be taken into account along with other clinicopathological features in a multidisciplinary setting (23/23, 100%). Tumor budding is counted on H&E (19/22, 86%). Intratumoral budding exists in colorectal cancer and has been shown to be related to lymph node metastasis (22/22, 100%). Tumor budding is assessed in one hotspot (in a field measuring 0.785 mm2) at the invasive front (22/22, 100%). A three-tier system should be used along with the budding count in order to facilitate risk stratification in colorectal cancer (23/23, 100%). Tumor budding and tumor grade are not the same (23/23, 100%). Tumor budding should be included in guidelines/protocols for colorectal cancer reporting (23/23, 100%). Members of the ITBCC were able to reach strong consensus on a single international, evidence-based method for tumor budding assessment and reporting. It is proposed that this method be incorporated into colorectal cancer guidelines/protocols and staging systems.

Protein and modified vaccinia virus Ankara-based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice.

Because of the high variability of seasonal influenza viruses and the eminent threat of influenza viruses with pandemic potential, there is great interest in the development of vaccines that induce broadly protective immunity. Most probably, broadly protective influenza vaccines are based on conserved proteins, such as nucleoprotein (NP). NP is a vaccine target of interest as it has been shown to induce cross-reactive antibody and T cell responses. Here we tested and compared various NP-based vaccine preparations for their capacity to induce humoral and cellular immune responses to influenza virus NP. The immunogenicity of protein-based vaccine preparations with Matrix-M™ adjuvant as well as recombinant viral vaccine vector modified Vaccinia virus Ankara (MVA) expressing the influenza virus NP gene, with or without modifications that aim at optimization of CD8+ T cell responses, was addressed in BALB/c mice. Addition of Matrix-M™ adjuvant to NP wild-type protein-based vaccines significantly improved T cell responses. Furthermore, recombinant MVA expressing the influenza virus NP induced strong antibody and CD8+ T cell responses, which could not be improved further by modifications of NP to increase antigen processing and presentation.

Prognosis of stage II and III colon cancer treated with adjuvant 5-fluorouracil or FOLFIRI in relation to microsatellite status: results of the PETACC-3 trial.

BACKGROUND: Although colon cancer (CC) with microsatellite instability (MSI) has a more favorable prognosis than microsatellite stable (MSS) CC, the impact varies according to clinicopathological parameters. We studied how MSI status affects prognosis in a trial-based cohort of stage II and III CC patients treated with 5-fluorouracil (5-FU)/leucovorin or FOLFIRI. MATERIALS AND METHODS: Tissue specimens of 1254 patients were tested for 10 different loci and were classified as MSI-high (MSI-H) when three or more loci were unstable and MSS otherwise. Study end points were overall survival (OS) and relapse-free survival (RFS). RESULTS: In stage II, RFS and OS were better for patients with MSI-H than with MSS CC [hazard ratio (HR) 0.26, 95% CI 0.10-0.65, P = 0.004 and 0.16, 95% CI 0.04-0.64, P = 0.01). In stage III, RFS was slightly better for patients with MSI-H CC (HR 0.67, 95% CI 0.46-0.99, P = 0.04), but the difference was not statistically significant for OS (HR 0.70, 95% CI 0.44-1.09, P = 0.11). Outcomes for patients with MSI-H CC were not different between the two treatment arms. RFS was better for patients with MSI-H than with MSS CC in the right and left colon, whereas for OS this was significant only in the right colon. For patients with KRAS- and BRAF-mutated CC, but not for double wild-type patients, RFS and OS were significantly better when the tumors were also MSI-H. An interaction test was statistically significant for KRAS and MSI status (P = 0.005), but not for BRAF status (P = 0.14). CONCLUSIONS: Our results confirm that for patients with stage II CC but less so for those with stage III MSI-H is strongly prognostic for RFS and OS. In the presence of 5-FU treatment, stage II patients with MSI-H tumors maintain their survival advantage in comparison with MSS patients and adding irinotecan has no added benefit. CLINICALTRIALS.GOV IDENTIFIER: NCT00026273.

Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features.

BACKGROUND: Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications. MATERIALS AND METHODS: Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR). RESULTS: Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis. CONCLUSIONS: Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis.

Glutathione S-transferase M1-null genotype as risk factor for SOS in oxaliplatin-treated patients with metastatic colorectal cancer.

BACKGROUND: Oxaliplatin is used as a neo-adjuvant therapy in hepatic colorectal carcinoma metastasis. This treatment has significant side effects, as oxaliplatin is toxic to the sinusoidal endothelial cells and can induce sinusoidal obstruction syndrome (SOS), which is related to decreased overall survival. Glutathione has an important role in the defence system, catalysed by glutathione S-transferase (GST), including two non-enzyme producing polymorphisms (GSTM1-null and GSTT1-null). We hypothesise that patients with a non-enzyme producing polymorphism have a higher risk of developing toxic injury owing to oxaliplatin. METHODS: In the nontumour-bearing liver, the presence of SOS was studied histopathologically. The genotype was determined by a semi-nested PCR. RESULTS: Thirty-two of the 55 (58%) patients showed SOS lesions, consisting of 27% mild, 22% moderate and 9% severe lesions. The GSTM1-null genotype was present in 25 of the 55 (46%). Multivariate analysis showed that the GSTM1-null genotype significantly correlated with the presence of (moderate-severe) SOS (P=0.026). CONCLUSION: The GSTM1-null genotype is an independent risk factor for SOS. This finding allows us, in association with other risk factors, to conceive a potential risk profile predicting whether the patient is at risk of developing SOS, before starting oxaliplatin, and subsequently might result in adjustment of treatment.

Neoadjuvant chemoradiotherapy with or without panitumumab in patients with wild-type KRAS, locally advanced rectal cancer (LARC): a randomized, multicenter, phase II trial SAKK 41/07.

BACKGROUND: We conducted a randomized, phase II, multicenter study to evaluate the anti-epidermal growth factor receptor (EGFR) mAb panitumumab (P) in combination with chemoradiotherapy (CRT) with standard-dose capecitabine as neoadjuvant treatment for wild-type KRAS locally advanced rectal cancer (LARC). PATIENTS AND METHODS: Patients with wild-type KRAS, T3-4 and/or N+ LARC were randomly assigned to receive CRT with or without P (6 mg/kg). The primary end-point was pathological near-complete or complete tumor response (pNC/CR), defined as grade 3 (pNCR) or 4 (pCR) histological regression by Dworak classification (DC). RESULTS: Forty of 68 patients were randomly assigned to P + CRT and 28 to CRT. pNC/CR was achieved in 21 patients (53%) treated with P + CRT [95% confidence interval (CI) 36%-69%] versus 9 patients (32%) treated with CRT alone (95% CI: 16%-52%). pCR was achieved in 4 (10%) and 5 (18%) patients, and pNCR in 17 (43%) and 4 (14%) patients. In immunohistochemical analysis, most DC 3 cells were not apoptotic. The most common grade ≥3 toxic effects in the P + CRT/CRT arm were diarrhea (10%/6%) and anastomotic leakage (15%/4%). CONCLUSIONS: The addition of panitumumab to neoadjuvant CRT in patients with KRAS wild-type LARC resulted in a high pNC/CR rate, mostly grade 3 DC. The results of both treatment arms exceeded prespecified thresholds. The addition of panitumumab increased toxicity.

Altered expression of CD44 and DKK1 in the progression of Barrett's esophagus to esophageal adenocarcinoma.

Barrett's esophagus (BE) is an acquired condition in which the normal lining of the esophagus is replaced by intestinal metaplastic epithelium. BE can evolve to esophageal adenocarcinoma (EAC) through low-grade dysplasia (LGD) and high-grade dysplasia (HGD). The only generally accepted marker for increased risk of EAC is the presence of HGD, diagnosed on endoscopic biopsies. More specific markers for the prediction of EAC risk are needed. A tissue microarray was constructed comprising tissue samples from BE, LGD, HGD, and EAC. Marker expression was studied by immunohistochemistry using antibodies against CD44, DKK1, CDX2, COX2, SOX9, OCT1, E-cadherin, and beta-catenin. Immunostaining was evaluated semi-quantitatively. CD44 expression decreased in HGD and EAC relative to BE and LGD. DKK1 expression increased in HGD and EAC relative to BE and LDG. CDX2 expression increased in HGD but decreased in EAC. COX2 expression decreased in EAC, and SOX9 expression increased only in the upper crypt epithelial cells in HGD. E-cadherin expression decreased in EAC. Nuclear beta-catenin was not significantly different between BE, LGD, and HGD. Loss of CD44 and gain of DKK1 expression characterizes progression from BE and LGD to HGD and EAC, and their altered expression might indicate an increased risk for developing an EAC. This observation warrants inclusion of these immunohistochemically detectable markers in a study with a long patient follow-up.

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30-40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

Methylation of CpG island promoters in uveal melanoma.

BACKGROUND: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. AIM: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. METHODS: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. RESULTS: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. CONCLUSIONS: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma.

Effect of virtual articulator settings on occlusal morphology of CAD/CAM restorations.

PURPOSE: Determinants of mandibular movements, like condylar inclination and incisal guidance, should be considered in the fabrication of restorations in occlusion to prevent posterior excursive occlusal interferences. The aim of this study was to investigate differences in the occlusal morphology of the right mandibular molar resulting from high, average, and low values of settings for determinants of anteroposterior and transverse mandibular movement using a virtual articulation model. MATERIAL AND METHODS: The articulation functionalities of computer integrated restorative technology by imaging and new acquisition (CYRTINA, Oratio B.V., Zwaag, The Netherlands) were used as a tool to examine the potential effect of determinants of mandibular movement on occlusal molar design. High, average, and low values for condylar guide inclination, incisal guide angle, and intercuspal contact area (antero-posterior determinants) and laterotrusion, mandibular lateral translation and intercuspal contact area (transverse determinants) were introduced and differences in molar morphology studied. The latter was done by comparing mesiodistal and buccolingual sections of the occlusal designs. These interocclusal differences were quantified as differences in frequency of occlusal distance intervals in an interocclusal range of 1 mm, measured from the occlusal surface of the molar model. The vertical distance with which a standard crown in occlusion had to be corrected to avoid interferences was calculated. RESULTS: Among all parameters, the ipsilateral and contralateral mandibular lateral translation, sagittal condylar guide inclination, the ipsilateral laterotrusion and the incisal guide angle give substantial occlusal surface corrections. The high setting for the ipsilateral mandibular lateral translation required most correction. CONCLUSION: High and low setting values of mandibular movement determinants require considerable adaptation of the occlusal surface of a crown to prevent occlusal disturbances.

Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene.

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

Osteopontin and bone metabolism in healing cranial defects in rabbits.

Non-collagen proteins such as bone sialoprotein and osteopontin (OPN) form 10% of the extracellular bone matrix. In this study, the influence of OPN on bone repair was investigated. Human OPN (Innogenetics) was produced by a recombinant technique and bonded onto the surface of hydroxyapatite (Interpore 200). Thirty rabbits were divided into six equal groups. A circular defect (10mm) was prepared in each parietal bone. In four rabbits of each group the left and right defects were filled with either OPN-coated hydroxyapatite (OPN-HA) or non-coated hydroxyapatite (HA). One sham animal of each group received no implants. The animals were killed after 1, 2, 6, 12, 18 and 30 weeks. The histological sections were scanned and analysed digitally. There were no statistically significant differences in total bone formation between defects filled with OPN-HA and HA. Bone formation at the borders of the healing area was significantly higher in defects filled with OPN-HA than in those filled with HA. Less bone formation was noted in the OPN-HA and HA groups at the centre of the healing area than at the borders of the healing area and the dural area. Although some animals in the sham group showed a high level of bone formation in the dural area, this was not significantly different to that in the dural area of the other groups. There was no sign of infection or tissue rejection of the graft.

Alterations of the Wnt signaling pathway during the neoplastic progression of Barrett's esophagus.

Aberrant activation of the Wnt signaling pathway has been reported during neoplastic progression in Barrett's esophagus (BE). However, mutations in APC and CTNNB1 genes were rarely observed. In this study, expression pattern of Wnt ligands, Frizzled receptors and APC, as well as the methylation status of the APC, SFRP1 and SFRP2 promoter genes were investigated in normal esophageal mucosa and in preneoplastic and neoplastic lesions of BE patients. Promoter methylation of APC was found in all BE samples and in 95% of esophageal adenocarcinomas (EAC). Full methylation of APC correlated with lack of expression. In EAC, nuclear translocation of beta-catenin was observed regardless of the expression of APC. WNT2 expression was higher in dysplasia and EAC than in BE, with 20/26 (77%) of the EAC showing high expression of WNT2. SFRP1 methylation occurred in all BE samples and in 96% of EAC, while SFRP2 was methylated in 73% of the normal squamous esophageal mucosa samples. In conclusion, (1) alterations of key regulators of the Wnt signaling are frequent in the pathogenesis of BE; (2) the APC and SFRP1 genes are inactivated by promoter methylation in BE; (3) the WNT2 gene is upregulated along the progression from low-grade dysplasia to EAC.

Digital measurements: a different approach to evaluate bone formation. A technical report.

In this study a digital measurement technique has been proposed to quantify bone formation on histological images. Two standard parietal defects were created in 30 adult rabbits. The animals were divided into six groups. Four animals of each group were randomly chosen as experimental group in which osteopontin-coated hydroxyapatite (OPN-HA) and hydroxyapatite (HA) were inserted alternatively in created defects. To observe the spontaneous healing process of defects, one animal of each group was used as control animal and these created defects did not receive any implants. The animals were sacrificed after 1, 2, 6, 12, 18 and 30 weeks. The histological sections were magnified (x100) and scanned digitally. The newly formed bone surfaces within the healing area were indicated and quantified by means of Adobe Photoshop 7 software. This measuring technique was found to be reliable and reproducible. The results of this study show no significant differences in bone formation between the OPN-HA and non-coated HA defects, although a significant difference in bone formation was measured at the margins of the defects treated with OPN-HA.

Osteopontin and bone metabolism: a histology and scintigraphy study in rats.

Osteopontin (OPN) is one of the major non-collagen proteins in extracellular bone matrix. To elucidate the function of OPN in bone metabolism, a cellular defect was created in parietal bone and tibia of 12 rats. In Group 1, the left defects were filled with OPN-coated hydroxyapatite (OPN-H). In Group 2, the right defects were filled with non-coated hydroxyapatite (N-H). In both groups, the contra lateral defects were used as control defects. In Group 3, OPN-H was inserted in the left defects and N-H in the right defects. Bone metabolism was measured by (45)Ca and technetium-99m methylene diphosphonate scintigraphy for 4 weeks. Scintigraphy did not show any significant differences in bone metabolism between the defects filled with OPN-H and N-H. A higher bone metabolism was measured between the parietal defects filled with OPN-H or N-H in comparison with the parietal control defects. This difference, however, was not significant and was less for tibia defects. Histological observation (7th week) shows less inflammatory cells at the tibia defects filled with OPN-H compared to the tibia defects filled with N-H. This study did not show any acceleration or inhibition of bone metabolism in parietal or tibia bone in rats, but there is some evidence that OPN might influence inflammatory cells in bone matrix.

The influence of orthognathic surgery on masticatory performance in retrognathic patients.

Surgical correction of retrognathism may influence chewing performance and its underlying mechanisms selection and breakage of food particles. In this study we examined the hypothesis that treatment of this anomaly improves chewing performance. Furthermore, we determined to what extent this change can be attributed to selection and breakage of food particles. Eleven patients were tested before and 1-1.5 years after surgery. To determine chewing performance, eight cubes of 8 mm of a silicone rubber (Optosil) were used as a test food. Selection and breakage were determined in one-chew experiments using three particle sizes. On average, no differences were found for chewing performance, selection or breakage. However, on an individual basis, patients with a poor chewing performance before surgery tended to improve, whereas no improvement was observed for patients with a good chewing performance. The change in chewing performance was mainly due to a change in breakage of the food particles.

Swallowing threshold and masticatory performance in dentate adults.

A variety of both natural and artificial foods are commonly used for the evaluation of masticatory function. We compared swallowing thresholds of three natural foods (peanuts, cheese and carrots) to those of a standardized artificial test food (Optocal Plus) and examined the relationship between masticatory performance and the swallowing threshold. Eighty-seven healthy dentate subjects participated (25 men and 62 women, aged 42.0+/-12.1 years). We evaluated the dental state, registered the number of chewing strokes used before swallowing, analyzed the chewed particles and determined median particle sizes (X50) for Optocal Plus after 15 chewing strokes and at the moment of swallowing. The results show that the number of strokes used before swallowing each natural food linearly increased with volume (P<0.001), and that carrots required more strokes than peanuts and cheese (P<0.001). The number of chewing strokes used before swallowing Optocal Plus was comparable to the number used for carrots. Masticatory performance was significantly influenced by dental state, but not by age or gender. Significant correlations were observed for: (1) the number of chewing strokes used before swallowing natural foods and Optocal Plus; (2) the median particle sizes after 15 strokes and before swallowing; (3) the number of chewing strokes before swallowing and the corresponding median particle size. However, median particle sizes as obtained after 15 strokes did not correlate with the number of strokes used before swallowing (r=0.02). Thus, bad chewers did not necessarily chew longer before swallowing than good chewers. As a consequence bad chewers would, on average, swallow larger food particles.