The Effect of Histopathological Growth Patterns of Colorectal Liver Metastases on the Survival Benefit of Adjuvant Hepatic Arterial Infusion Pump Chemotherapy.

BACKGROUND: Histopathological growth patterns (HGPs) are a prognostic biomarker in colorectal liver metastases (CRLM). Desmoplastic HGP (dHGP) is associated with liver-only recurrence and superior overall survival (OS), while non-dHGP is associated with multi-organ recurrence and inferior OS. This study investigated the predictive value of HGPs for adjuvant hepatic arterial infusion pump (HAIP) chemotherapy in CRLM. METHODS: Patients undergoing resection of CRLM and perioperative systemic chemotherapy in two centers were included. Survival outcomes and the predictive value of HAIP versus no HAIP per HGP group were evaluated through Kaplan-Meier and Cox regression methods, respectively. RESULTS: We included 1233 patients. In the dHGP group (n = 291, 24%), HAIP chemotherapy was administered in 75 patients (26%). In the non-dHGP group (n = 942, 76%), HAIP chemotherapy was administered in 247 patients (26%). dHGP was associated with improved overall survival (OS, HR 0.49, 95% CI 0.32-0.73, p < 0.001). HAIP chemotherapy was associated with improved OS (HR 0.61, 95% CI 0.45-0.82, p < 0.001). No interaction could be demonstrated between HGP and HAIP on OS (HR 1.29, 95% CI 0.72-2.32, p = 0.40). CONCLUSIONS: There is no evidence that HGPs of CRLM modify the survival benefit of adjuvant HAIP chemotherapy in patients with resected CRLM.

Infiltrating stromal immune cells in inflammatory breast cancer are associated with an improved outcome and increased PD-L1 expression.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare and rapidly progressive form of invasive breast cancer. The aim of this study was to explore the clinical evolution, stromal tumour-infiltrating lymphocytes (sTIL) infiltration and programmed death-ligand 1 (PD-L1) expression in a large IBC cohort. PATIENTS AND METHODS: Data were collected prospectively from patients with IBC as part of an international collaborative effort since 1996. In total, 143 patients with IBC starting treatment between June 1996 and December 2016 were included. Clinicopathological variables were collected, and sTIL were scored by two pathologists on standard H&E stained sections. PD-L1 expression was assessed using a validated PD-L1 (SP142) assay. A validation cohort of 64 patients with IBC was used to test our findings. RESULTS: Survival outcomes of IBC remained poor with a 5-year overall survival (OS) of 45.6%. OS was significantly better in patients with primary non-metastatic disease who received taxane-containing (neo)adjuvant therapy (P = 0.01), had a hormone receptor-positive tumour (P = 0.001) and had lower cN stage at diagnosis (P = 0.001). PD-L1 positivity on immune cells (42.9%) was higher in IBC than in non-IBC in both our patient samples and the validation cohort. Furthermore, PD-L1 expression predicted pCR (P = 0.002) and correlated with sTIL infiltration (P < 0.001). sTIL infiltration of more than 10% of the stroma was a significant predictor of improved OS (HR 0.47, 95% CI 0.27-0.81, P = 0.006) in a multivariate model. CONCLUSIONS: IBC is characterised by poor survival and high PD-L1 immunoreactivity on sTIL. This suggests a role for PD1/PD-L1 inhibitors in the treatment of IBC. Furthermore, we showed that PD-L1 expression predicts response to neo-adjuvant therapy and that sTIL have prognostic significance in IBC.

A geochemical weathering profile of a fine ash tailings dam and its impact on the underlying aquifers.

A weathering profile of an unlined fine ash tailings dam was established with the aid of static and kinetic tests to determine the leachate composition across a tailings dam profile and to evaluate its impact on the underlying aquifers. Kinetic leaching results indicated that SO42-, Ca2+, Na+, Cl-, Mg2+ and K+ ions were highly soluble in both, fresh and weathered fine ash. Leaching rates of elements including Ca, B, Ba and Al decreased with depth and age of the fine ash while V, Mo, Cu, Mn, Si, Li, Cr and SO4 had an increasing leaching rate. A constant leaching rate was measured for Se, Fe, Na, Mg, K, NO3 and Zn. At shallow depth, a weathering zone was identified by a decrease in leaching rates of most elements. Although the hydraulic conductivity of the fine ash dam was low (0.0067 m/d and 0.0367 m/d), long-term hydrochemical monitoring around the tailings dam showed alterations in groundwater types of the underlying aquifers due to seepage. Downstream of the tailings dam, the groundwater type of the shallow weathered aquifer changed from Ca/MgSO4/Cl to NaSO4/Cl and from Na-bicarbonate to Ca/MgSO4/Cl upstream. A reverse trend was observed in the deeper fractured aquifer indicating that the fine ash leachate plume migrated laterally and vertically from the shallower to the deeper aquifer. Hence, the natural clay layer below the tailings dam was insufficient in retaining soluble elements leached from the fine ash over two decades. Therefore, an impermeable liner is recommended prior to the tailings dam construction.

Out-of-focus background subtraction for fast structured illumination super-resolution microscopy of optically thick samples.

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes.

BACKGROUND: The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer. METHODS: CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ≥5 CTCs per 7.5 ml blood. RESULTS: No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ≥1 and ≥5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ≥80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ≥5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B-HER2-negative and TN disease. CONCLUSION: The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.

Gene expression profiles of inflammatory breast cancer: correlation with response to neoadjuvant chemotherapy and metastasis-free survival.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive disease. To date, no molecular feature reliably predicts either the response to chemotherapy (CT) or the survival. Using DNA microarrays, we searched for multigene predictors. PATIENTS AND METHODS: The World IBC Consortium generated whole-genome expression profiles of 137 IBC and 252 non-IBC (nIBC) samples. We searched for transcriptional profiles associated with pathological complete response (pCR) to neoadjuvant anthracycline-based CT and distant metastasis-free survival (DMFS) in respective subsets of 87 and 106 informative IBC samples. Correlations were investigated with predictive and prognostic gene expression signatures published in nIBC (nIBC-GES). Supervised analyses tested genes and activation signatures of 19 biological pathways and 234 transcription factors. RESULTS: Three of five tested prognostic nIBC-GES and the two tested predictive nIBC-GES discriminated between IBC with and without pCR, as well as two interferon activation signatures. We identified a 107-gene signature enriched for immunity-related genes that distinguished between responders and nonresponders in IBC. Its robustness was demonstrated by external validation in three independent sets including two IBC sets and one nIBC set, with independent significant predictive value in IBC and nIBC validation sets in multivariate analysis. We found no robust signature associated with DMFS in patients with IBC, and neither of the tested prognostic GES, nor the molecular subtypes were informative, whereas they were in our nIBC series (220 stage I-III informative samples). CONCLUSION: Despite the relatively small sample size, we show that response to neoadjuvant CT in IBC is, as in nIBC, associated with immunity-related processes, suggesting that similar mechanisms responsible for pCR exist. Analysis of a larger IBC series is warranted regarding the correlation of gene expression profiles and DMFS.

Mechanisms of tumour vascularization in cutaneous malignant melanoma: clinical implications.

Malignant melanoma represents < 10% of all skin cancers but is responsible for the majority of skin-cancer-related deaths. Metastatic melanoma has historically been considered as one of the most therapeutically challenging malignancies. Fortunately, for the first time after decades of basic research and clinical investigation, new drugs have produced major clinical responses. Angiogenesis has been considered an important target for cancer treatment. Initial efforts have focused primarily on targeting endothelial and tumour-related vascular endothelial growth factor signalling. Here, we review different mechanisms of tumour vascularization described in melanoma and discuss the potential clinical implications.

Blood microvessel density, lymphatic microvessel density and lymphatic invasion in predicting melanoma metastases: systematic review and meta-analysis.

In malignant melanoma (MM) there is an urgent need to identify new markers with predictive value superior to the traditional clinical and histological parameters. Angiogenesis and lymphangiogenesis have been recognized as critical processes in tumour growth and metastasis development, and numerous studies have evaluated the significance of these parameters in predicting the prognosis in solid tumours, including MM. We set out to determine whether angiogenesis, lymphangiogenesis and lymphatic invasion (LI) are valuable prognostic markers in MM. We systematically reviewed the available literature and subsequently performed a meta-analysis on the compiled data. To be eligible for the systematic review, a study had to provide the microvessel density (MVD), the lymphatic vessel density (LVD) or information about LI, assessed by immunohistochemistry on the primary site in patients with MM. To be evaluable for the meta-analysis, a study also had to provide information on clinical outcome. We approached selected studies with the Reporting recommendations for tumour marker (REMARK) criteria, verifying whether they had followed the recommendations. In total, nine angiogenesis, seven lymphangiogenesis and 10 LI studies were included in our meta-analysis, representing 419, 474 and 802 patients, respectively. Using meta-analysis, we showed that peritumoral LVD and the presence of LI have prognostic value for patients with MM. In contrast, MVD and intratumoral LVD did not have prognostic value in these patients. LVD and LI seem to have prognostic value for patients with MM.

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

BACKGROUND: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation. METHODS: Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis. RESULTS: On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines. CONCLUSION: We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.

Circulating tumour cells are associated with histopathological growth patterns of colorectal cancer liver metastases.

Histopathological Growth Patterns (HGPs) have prognostic and predictive value in patients with Colorectal Liver Metastases (CRLM). This study examined whether preoperative measurement of Circulating Tumour Cells (CTCs) is associated with HGP. CTCs were prospectively enumerated in 7.5 ml of blood using the FDA-approved CellSearch system in patients who underwent local treatment of CRLM with curative intent between 2008 and 2021. All CTC samples were collected on the day of local treatment. Patients treated with neoadjuvant chemotherapy for CRLM or with extrahepatic disease at the time of CTC sampling were excluded. HGP was scored retrospectively following the current consensus guidelines. The association between CTCs and HGP was investigated through multivariable logistic regression. Data were available for 177 patients, desmoplastic HGP (dHGP) was observed in 34 patients (19%). There were no statistically significant differences in patient and tumour characteristics between dHGP and non-dHGP at baseline. Patients with dHGP had longer overall - and disease-free survival (logrank p = 0.003 and 0.003, respectively) compared to patients with non-dHGP. CTCs were not detected in 25(74%) of dHGP patients and in 68(48%) of non-dHGP patients (chi-squared p = 0.006). Preoperative absence of CTCs was the only significant predictor for dHGP in multivariable logistic regression (Odds Ratio 2.7, 95%CI 1.1-6.8, p = 0.028), Table 3. Preoperative absence of CTCs is associated with dHGP in chemo naive CRLM patients without extrahepatic disease. Based on our results, CTC count alone is not sufficient to preoperatively identify HGPs, but integration of CTC count in multivariable prediction models may aid the preoperative identification of HGPs of CRLM.

Histopathological growth patterns of neuroendocrine tumor liver metastases.

Histopathological growth patterns (HGPs) of liver metastases represent a potential biomarker for prognosis after resection. They have never been studied in neuroendocrine tumor liver metastases (NETLM). This study evaluated if distinct HGPs can be observed in resected NETLM and if they have prognostic value. Sixty-three patients who underwent resection of NETLM between 01-01-2001 and 31-12-2021 were retrospectively included. HGPs were scored on Haematoxylin&Eosin slides using light microscopy, distinguishing desmoplastic- (dHGP), pushing- (pHGP) and replacement HGP (rHGP). Average HGP scores were calculated per patient. Each patient was classified according to predominant HGP. Overall and Disease-Free Survival (OS and DFS) were evaluated through Kaplan-Meier analysis and Cox regression. Eighteen patients had predominant dHGP (29%), 33 had predominant pHGP (52%) and 11 had predominant rHGP (17%). One patient had mixed HGP (2%). Five-year OS was 76% (95%CI: 66-87%) for the overall cohort. Five-year OS was 92% (95%CI: 77-100%) for dHGP, was 73% (95%CI: 59-91%) for pHGP, 50% (95%CI: 25-100%) for rHGP. Five-year DFS was 39% (95%CI: 19-83%) for dHGP, 44% (95%CI: 27-71%) for rHGP and 50% (95%CI: 23-100%) for pHGP. There was no significant association between HGP and OS or DFS in multivariable analysis. Distinct HGPs could be identified in NETLM. In patients who underwent resection of NETLM, no association was found between HGPs and postoperative survival. Half of the patients with NETLM have a predominant pushing growth pattern, which is a rare growth pattern in liver metastases from breast and colorectal cancer.

Belgian recommendations on perioperative maintenance fluid management of surgical pediatric population.

The European recommendations on perioperative maintenance fluids in children have recently been adapted from hypotonic to isotonic electrolyte solutions with lower glucose concentrations. In Belgium, however, the commercially approved solutions do not match with these recommendations and there is neither consensus nor mandate about the composition and volume of perioperative maintenance fluids in children undergoing surgery despite the continuing controversy in literature. This paper highlights the significant challenges and shortcomings while prescribing fluid therapy for pediatric surgical patients in Belgium. It is sensible to the authors to address these issues with national guidance through an organization such as The Belgian Association for Paediatric Anaesthesiology, and to propose Belgian recommendations on perioperative fluid management in surgical children, with the intention of improving the quality of care in this population.

Circulating tumour cells in the central and the peripheral venous compartment in patients with metastatic breast cancer.

BACKGROUND: The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream. METHODS: In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy. RESULTS: The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 0-4036) than in PVB (median: 33; range: 0-4013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement. CONCLUSION: A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.

International expert panel on inflammatory breast cancer: consensus statement for standardized diagnosis and treatment.

BACKGROUND: Inflammatory breast cancer (IBC) represents the most aggressive presentation of breast cancer. Women diagnosed with IBC typically have a poorer prognosis compared with those diagnosed with non-IBC tumors. Recommendations and guidelines published to date on the diagnosis, management, and follow-up of women with breast cancer have focused primarily on non-IBC tumors. Establishing a minimum standard for clinical diagnosis and treatment of IBC is needed. METHODS: Recognizing IBC to be a distinct entity, a group of international experts met in December 2008 at the First International Conference on Inflammatory Breast Cancer to develop guidelines for the management of IBC. RESULTS: The panel of leading IBC experts formed a consensus on the minimum requirements to accurately diagnose IBC, supported by pathological confirmation. In addition, the panel emphasized a multimodality approach of systemic chemotherapy, surgery, and radiation therapy. CONCLUSIONS: The goal of these guidelines, based on an expert consensus after careful review of published data, is to help the clinical diagnosis of this rare disease and to standardize management of IBC among treating physicians in both the academic and community settings.

Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype. METHODS: We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1. RESULTS: We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17,295 correlated miRNA-mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction. CONCLUSIONS: This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer.

BACKGROUND: The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice. METHODS: This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts. RESULTS: As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT-PCR for CK-19 and 54% using RT-PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT-PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001). CONCLUSION: We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients.

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients. We simultaneously obtained whole blood, plasma and serum samples from 80 patients and 20 controls. The CellSearch System was used to enumerate CTC in blood samples. Plasma total DNA levels were determined by a QPCR method. Sera were analysed by methylation-specific QPCR for three markers: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A) and oestrogen receptor 1 (ESR1). Total DNA levels in patients were significantly increased when compared with controls (P<0.001) and correlated with the number of CTC (r=0.418, P<0.001). Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in three (16%) serum samples from controls (P=0.003). APC was hypermethylated in 29%, RASSF1A in 35% and ESR1 in 20% of breast cancer cases. Detection of a methylated gene in serum was associated with the detection of CTC in blood (P=0.03). The detection of large amounts of circulating total/methylated DNA correlated with the presence of CTC in the blood from patients with breast cancer. This can be interpreted in two ways: (a) CTC are a potential source of circulating tumour-specific DNA; (b) high numbers of CTC and circulating methylated DNA are both a phenotypic feature of more aggressive tumour biology.

The VEGF pathway and the AKT/mTOR/p70S6K1 signalling pathway in human epithelial ovarian cancer.

Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples (N=86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) (R=0.29; P=0.002). Additional gene expression analysis in an independent cDNA microarray dataset (N=24) showed a negative correlation (R=-0.73, P<0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites (chi(2), P=0.002) and reduced overall survival of cisplatin-taxane-based patients with serous histology (N=32, log-rank test, P=0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

Histopathological growth patterns of colorectal liver metastasis exhibit little heterogeneity and can be determined with a high diagnostic accuracy.

Colorectal liver metastases (CRLM) exhibit distinct histopathological growth patterns (HGPs) that are indicative of prognosis following surgical treatment. This study aims to assess the reliability and replicability of this histological biomarker. Within and between metastasis HGP concordance was analysed in patients who underwent surgery for CRLM. An independent cohort was used for external validation. Within metastasis concordance was assessed in CRLM with ≥ 2 tissue blocks. Similarly, concordance amongst multiple metastases was determined in patients with ≥ 2 resected CRLM. Diagnostic accuracy [expressed in area under the curve (AUC)] was compared by number of blocks and number of metastases scored. Interobserver agreement (Cohen's k) compared to the gold standard was determined for a pathologist and a PhD candidate without experience in HGP assessment after one and two training sessions. Both the within (95%, n = 825) and the between metastasis (90%, n = 363) HGP concordance was high. These results could be replicated in the external validation cohort with a within and between metastasis concordance of 97% and 94%, respectively. Diagnostic accuracy improved when scoring 2 versus 1 blocks(s) or CRLM (AUC = 95.9 vs. 97.7 [p = 0.039] and AUC = 96.5 vs. 93.3 [p = 0.026], respectively), but not when scoring 3 versus 2 blocks or CRLM (both p > 0.2). After two training sessions the interobserver agreement for both the pathologist and the PhD candidate were excellent (k = 0.953 and k = 0.951, respectively). The histopathological growth patterns of colorectal liver metastasis exhibit little heterogeneity and can be determined with a high diagnostic accuracy, making them a reliable and replicable histological biomarker.

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype.

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.