Autofluorescence-Raman Spectroscopy for Ex Vivo Mapping Colorectal Liver Metastases and Liver Tissue.

INTRODUCTION: Identifying colorectal liver metastases (CRLM) during liver resection could assist in achieving clear surgical margins, which is an important prognostic variable for both disease-free and overall survival. The aim of this study was to investigate the effect of auto-fluorescence (AF) and Raman spectroscopy for ex vivo label-free discrimination of CRLMs from normal liver tissue. Secondary aims include exploring options for multimodal AF-Raman integration with respect to diagnosis accuracy and imaging speed on human liver tissue and CRLM. METHODS: Liver samples were obtained from patients undergoing liver surgery for CRLM who provided informed consent (15 patients were recruited). AF and Raman spectroscopy was performed on CRLM and normal liver tissue samples and then compared to histology. RESULTS: AF emission spectra demonstrated that the 671 nm and 775/785 nm excitation wavelengths provided the highest contrast, as normal liver tissue elicited on average around eight-fold higher AF intensity compared to CRLM. The use of the 785 nm wavelength had the advantage of enabling Raman spectroscopy measurements from CRLM regions, allowing discrimination of CRLM from regions of normal liver tissue eliciting unusual low AF intensity, preventing misclassification. Proof-of-concept experiments using small pieces of CRLM samples covered by large normal liver tissue demonstrated the feasibility of a dual-modality AF-Raman for detection of positive margins within few minutes. CONCLUSIONS: AF imaging and Raman spectroscopy can discriminate CRLM from normal liver tissue in an ex vivo setting. These results suggest the potential for developing integrated multimodal AF-Raman imaging techniques for intraoperative assessment of surgical margins.

Comparing Expansion of Maxillary Antrum between Periapical Surgery and Extraction of Permanent Maxillary First Molar in Pediatric Patients Using CBCT.

AIM: To compare the expansion of maxillary antrum between periapical surgery and extraction of permanent maxillary first molar in pediatric patients using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: In this study, 136 participants in the age-group of 11-18 years were included. The participants were divided into two groups. Group A consisted of patients who underwent extraction of the permanent maxillary first molars. Group B consisted of patients who underwent endodontic microsurgery in the periapical area. Group A included 68 participants while group B also included 68 study subjects. The expansion of the maxillary antrum was obtained after evaluating the change in volume of maxillary antrum at 6 months and 24 months in relation to the volume of maxillary antrum at the time of the procedure (baseline). For calculating the volume of the maxillary antrum, three parameters were taken into consideration. These parameters were an anteroposterior (AP) dimension, mesiodistal dimension (MD), and superoinferior (SI) dimension. Cone-beam computed tomography was used for carrying out these measurements with the help of Dolphin software. RESULTS: An expansion of 675.27 ± 32 mm3 was observed in group A between baseline and 6 months of extraction, while the expansion of 765.47 ± 24 mm3 was observed between 6 months and 24 months of extraction. This intragroup difference was statistically significant (p = 0.001). On the other hand, an expansion of 652.28 ± 43 mm3 was observed in group B between baseline and 6 months after periapical surgery and expansion of 969.43 ± 12 mm3 was observed between 6 months and 24 months after periapical endodontic surgery. This intragroup difference was statistically significant. In the control group, an expansion of 152.11 ± 12.101 mm3 was observed between baseline and 6 months after procedures while an expansion of 347.01 ± 6.781 mm3 was observed between 6 months and 24 months of procedures. The intragroup difference was significant statistically. CONCLUSION: In this study, expansion of maxillary antrum was observed in both extraction of the maxillary permanent first molar in pediatric patients and the periapical endodontic surgery, and the expansion of maxillary antrum was more in cases of periapical endodontic surgery; however, the difference was non-significant statistically. CLINICAL SIGNIFICANCE: Maxillary antrum expansion is clinically important during maxillary permanent tooth extraction or endodontic periapical surgery in pediatric patients because the growth of maxillary bones is in the growing stage in these patients. There are certain limitations of conventional two-dimensional (2D) radiographic techniques such as shortening, elongation, and superimposition of images. Recently, three-dimensional technique (3D) such as CBCT has been introduced in which these disadvantages have been eliminated.

Zanubrutinib-induced liver injury: a case report and literature review.

BACKGROUND: Zanubrutinib is a Bruton's tyrosine kinase inhibitor that has been recently licensed in refractory mantle cell lymphoma and under assessment in phase 3 clinical trials for other B cell malignancies. To date, there are no reported cases of hepatotoxicity secondary to zanubrutinib. We report the first case of severe liver injury due to zanubrutinib. CASE PRESENTATION: A 56-year-old Caucasian male with a history of relapsed lymphoplasmacytic lymphoma was admitted to the hospital with new-onset jaundice, choluria, and pruritus for 10 days. He had been on zanubrutinib as part of a clinical trial for 30 months. His blood profile showed a severe hepatocellular injury with jaundice (alanine transaminase 2474 IU/L and total bilirubin 141 umol/L with mild coagulopathy). He had an extensive work-up including virology, autoimmune, and metabolic profiles in addition to abdominal ultrasound with no alternative explanation found for his liver injury. Zanubrutinib-induced liver injury was suspected, and causality assessment by the updated Roussel Uclaf Causality Assessment Method score showed a probable causal relationship with zanubrutinib. His liver histology was also consistent with drug-induced liver injury. His liver biochemistry improved following cessation of zanubrutinib and normalised after 8 weeks. CONCLUSION: We report the first case of severe liver injury secondary to zanubrutinib after 30 months of treatment. This case raises clinical awareness regarding zanubrutinib-induced liver toxicity and the importance of drug withdrawal in the event of liver injury.

Mediator complex (MED) 7: a biomarker associated with good prognosis in invasive breast cancer, especially ER+ luminal subtypes.

BACKGROUND: Mediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance. METHODS: MED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated. RESULTS: High MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001). CONCLUSIONS: MED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

Saccharomyces cerevisiae-like 1 (SEC14L1) is a prognostic factor in breast cancer associated with lymphovascular invasion.

Lymphovascular invasion is strongly related to breast cancer metastasis. However, the underlying mechanisms of lymphovascular invasion and its driver molecules in breast cancer remain to be defined. In this study, we explore differential expression of genes in large molecularly characterized and clinically annotated datasets of invasive breast cancer patients (n = 8056) coupled with histological review and strict definition for lymphovascular invasion status. The METABRIC series was used to identify genes associated with lymphovascular invasion, as defined using hematoxylin and eosin staining supplemented by immunohistochemistry, at the genomic/transcriptomic levels. Saccharomyces cerevisiae-like 1 (SEC14L1) was identified as one of the most significant genes associated with lymphovascular invasion. The prognostic significance of SEC14L1 gene copy number and mRNA expression was further investigated in the METABRIC series and externally validated using the Breast Cancer Gene-Expression Miner v4.0. Protein expression of SEC14L1 was also assessed using immunohistochemistry in series of early stage breast cancer using tissue microarrays. SEC14L1 gene copy number gain was significantly associated with high histological grade and poor outcome. SEC14L1 mRNA expression showed positive association with higher grade, lymph node metastasis, and poor outcome. SEC14L1 protein overexpression was significantly associated with lymphovascular invasion (p < 0.0001), higher grade (p = 0.011), HER2 positivity (p = 0.036), and shorter survival (p = 0.00075). Our findings specify SEC14L1 as an independent prognostic factor in breast cancer. Its association, at both transcriptome and protein expression levels, with lymphovascular invasion and outcome could imply an important role in tumor progression. A further mechanistic insight into its molecular roles including potential therapeutic utility is warranted.

HER2 challenge contest: a detailed assessment of automated HER2 scoring algorithms in whole slide images of breast cancer tissues.

AIMS: Evaluating expression of the human epidermal growth factor receptor 2 (HER2) by visual examination of immunohistochemistry (IHC) on invasive breast cancer (BCa) is a key part of the diagnostic assessment of BCa due to its recognized importance as a predictive and prognostic marker in clinical practice. However, visual scoring of HER2 is subjective, and consequently prone to interobserver variability. Given the prognostic and therapeutic implications of HER2 scoring, a more objective method is required. In this paper, we report on a recent automated HER2 scoring contest, held in conjunction with the annual PathSoc meeting held in Nottingham in June 2016, aimed at systematically comparing and advancing the state-of-the-art artificial intelligence (AI)-based automated methods for HER2 scoring. METHODS AND RESULTS: The contest data set comprised digitized whole slide images (WSI) of sections from 86 cases of invasive breast carcinoma stained with both haematoxylin and eosin (H&E) and IHC for HER2. The contesting algorithms predicted scores of the IHC slides automatically for an unseen subset of the data set and the predicted scores were compared with the 'ground truth' (a consensus score from at least two experts). We also report on a simple 'Man versus Machine' contest for the scoring of HER2 and show that the automated methods could beat the pathology experts on this contest data set. CONCLUSIONS: This paper presents a benchmark for comparing the performance of automated algorithms for scoring of HER2. It also demonstrates the enormous potential of automated algorithms in assisting the pathologist with objective IHC scoring.

Rho-GTPase activating-protein 18: a biomarker associated with good prognosis in invasive breast cancer.

BACKGROUND: The prognostic value of lymphovascular invasion (LVI) in breast cancer (BC) has been demonstrated in several independent studies. However, identification of driver molecules for LVI remains a challenging task. Large-scale transcriptomic profiling of histologically validated LVI can potentially identify genes that regulate LVI. METHODS: Integrative bio-informatics analyses of the METABRIC study were performed utilising a subset of strictly defined LVI using histological and immunohistochemical (IHC) criteria. ARHGAP18 was among the top differentially expressed genes between LVI+ and LVI- BC with a 1.8-fold change. The prognostic impact of ARHGAP18 gene expression was assessed in the METABRIC data set (n=1980) and externally validated using the online BC gene expression data sets utilising bc-GenExMiner v4.0 (n=2016). Subsequently, ARHGAP18 protein expression was assessed on a large cohort of invasive BC (n=959) with long-term follow-up using IHC. RESULTS: Pooled analysis of ARHGAP18 mRNA expression showed that overexpression was associated with better outcome (P<0.001, hazard ratio (HR)=0.82, 95% CI 0.75-0.90). ARHGAP18 protein was expressed in the cytoplasm and nuclei of the tumour cells and its expression was positively associated with good prognostic variables. Lack of cytoplasmic expression showed associations with LVI (P=0.006), epithelial-mesenchymal transition and the HER+ subtype (P=0.01). Loss of nuclear expression was associated with higher grade, HER2+ and high Ki67LI (P=0.001). Cytoplasmic and nuclear expression showed a positive association with improved survival independent of other variables (P=0.01, HR=0.74, 95% CI 0.60-87). CONCLUSIONS: ARHGAP18 expression at transcriptomic and protein levels is associated with improved patients' outcomes whose deregulation may play a role in tumour progression and the development of LVI in BC. Further assessment of its potential therapeutic value in BC is warranted.

Phenotypic characterisation of breast cancer: the role of CDC42.

PURPOSE: The molecular landscape of breast cancer (BC), especially of the Luminal A subtype, remains to be fully delineated. Transcriptomic data show that Luminal A tumours are enriched for aberrant expression of genes in the cell division control 42 homolog (CDC42) pathway. This study aims to investigate the protein expression of CDC42 in BC and assess its clinicopathological significance. METHODS: Expression of CDC42 protein was examined by immunohistochemistry on tissue microarrays in a well-characterised cohort of 895 early-stage (I-IIIa) primary invasive BCs. RESULTS: CDC42 expression was observed in both the cytoplasm and the nucleus of BC cells. High nuclear CDC42 expression demonstrated a significant correlation with ER-positive, low-grade tumours and was more common in the lobular histological subtype (all p < 0.001). In contrast, cytoplasmic CDC42 showed increased expression in the ductal subtype (p < 0.001) and correlated with negative prognostic features such as larger size, higher grade (p < 0.05) and higher Ki67 labelling index (p = 0.001). Nuclear CDC42 expression was associated with a longer BC-specific survival in all cases (p = 0.025) and in luminal ER-positive tumours (p = 0.011). In multivariate analyses including size, grade, lymph node stage and intrinsic subtype, CDC42 was an independent prognostic factor (p = 0.032). CONCLUSION: The results indicate that CDC42 is an important molecule in luminal BC, with prognostic significance.

Prognostic significance of tumour infiltrating B lymphocytes in breast ductal carcinoma in situ.

AIMS: Tumour-infiltrating lymphocytes (TILs) are an important component of the immune response to cancer and have a prognostic value in breast cancer. Although several studies have investigated the role of T lymphocytes in breast cancer, the role of B lymphocytes (TIL-Bs) in ductal carcinoma in situ (DCIS) remains uncertain. This study aimed to assess the role of TIL-Bs in DCIS. METHODS AND RESULTS: Eighty DCIS cases (36 pure DCIS and 44 mixed with invasive cancer) were stained immunohistochemically for B lineage markers CD19, CD20 and the plasma cell marker CD138. TIL-Bs density and localization were assessed, including relation to the in-situ and invasive components. An association with clinicopathological data and patient outcome was performed. Pure DCIS showed a higher number of TIL-Bs and lymphoid aggregates than DCIS associated with invasion. In pure DCIS, a higher number of peri- and paratumoral TIL-Bs was associated significantly with large tumour size (P = 0.016), hormone receptor (ER/PR) negative (P = 0.008) and HER2+ status (P = 0.010). In tumours with mixed DCIS and invasive components, cases with high-density B lymphocytes, irrespective of their location or topographic distribution, were associated significantly with variables of poor prognosis, including larger size, high grade, lymphovascular invasion, lymph node metastases, ER/PR-negative and HER2+ status. Outcome analysis showed that pure DCIS associated with higher numbers of B lymphocytes had shorter recurrence-free interval (P = 0.04); however, the association was not significant with the CD138+ plasma cell count (P = 0.07). CONCLUSION: Assessment of TIL-B cells based on location and topographic distribution can provide prognostic information. Validation in a larger cohort is warranted.

The role of BUB and CDC proteins in low-grade breast cancers.

BACKGROUND: The drivers of neoplasia within low-grade luminal breast cancers remain undelineated. The BUB and CDC family are among kinase genes known recently to help to identify luminal breast cancers with poorer prognosis. Additionally, other CDC kinase genes (CDC42) are associated with luminal A breast cancers with good prognosis. We aimed to investigate the role of these kinases at the protein level within low-grade luminal breast cancers. METHODS: The Nottingham Tenovus Primary Breast Cancer Series (n=1858) microarrays were immunostained for BUB (BUB1, BUB1B, BUB3) and CDC proteins (CDC2, CDC42) and expression correlated with clinicopathological and molecular variables and patient outcome (SPSS, version 22). FINDINGS: On χ(2) analysis, cytoplasmic BUB1 and nuclear BUB3 were negatively associated with grade including pleomorphism, mitosis, and Nottingham Prognostic Index, whereas BUB1B was positively associated (p=0·05). BUB1 and BUB3 expression was positively correlated with oestrogen and progesterone receptor expression whereas BUB1B was negatively correlated (p=0·01). CDC42 had strong associations with tumour morphology within the low-grade luminal breast cancers, tubular and lobular (p=0·02). CDC42 nuclear expression revealed negative correlations with basal (CK5) and HER family biomarkers (p=0·02). By contrast, cytoplasmic CDC2 overexpression was associated with high-grade tumours (p=0·01). BUB1, BUB1B, and CDC42 showed significant associations (p=0·04) with breast-cancer-specific survival even at the 15-20-year range, indicating their long-term prognostic potential. INTERPRETATION: These results suggest that BUB1, BUB3, and CDC42 are key kinases for low-grade luminal tumours whereas BUB1B and CDC2 kinases are preferentially expressed in high-grade disease. High protein expression of BUB1, BUB3, and CDC42 in low-grade breast cancers was associated with longer overall survival whereas lower expression resulted in poorer outcome. FUNDING: Pathological Society of Great Britain and Northern Ireland, National Institute for Health Research.

Prognostic and biological significance of peroxisome proliferator-activated receptor-gamma in luminal breast cancer.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is an adopted orphan receptor that belongs to the nuclear receptor superfamily of transcription factors. PPARγ is regarded as a differentiation factor and it plays an important role in regulating adipogenesis, cell growth, proliferation and tumour progression. In breast cancer (BC), PPARγ agonists were reported to inhibit proliferation and growth invasion and promote phenotypic changes associated with a less malignant and more differentiated status. This study aims to assess the prognostic and biological roles of PPARγ protein expression in a large cohort of BC patients (n = 1100) with emphasis on the luminal oestrogen receptor (ER) positive class. Immunohistochemistry was used to assess the levels of PPARγ expression in BC series prepared as tissue microarrays (TMAs). PPARγ antibody specificity was confirmed using Western blotting. PPARγ nuclear expression was detected in 79 % of the cases and its expression was positively correlated with the hormonal receptors (ER, progesterone receptor and androgen receptor). PPARγ levels were significantly higher in tumours with lobular subtype, smaller size and lower grade, while HER2-positive, ductal or medullary tumours were associated with lower PPARγ levels. Survival analysis showed that PPARγ is associated with better outcome in the whole series as well as in luminal ER-positive class. Cox regression model showed that PPARγ is an independent predictor of outcome. Higher PPARγ was associated with longer survival in patients with ER-positive tumours who did not receive hormone therapy. PPARγ is a good prognostic marker associated with hormone receptors. In patients with luminal BCs, PPARγ is a marker of better prognosis and is associated with longer survival.

Clinical and biological significance of glucocorticoid receptor (GR) expression in breast cancer.

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.

Molecular Mechanisms Underlying Lymphovascular Invasion in Invasive Breast Cancer.

Lymphovascular invasion (LVI), the presence of malignant cells within lymphovascular channels, is a crucial step in the invasion-metastasis cascade. LVI, when identified morphologically in the peritumoural area, is regarded as an indicator of metastatic potential and is strongly associated with a poor prognosis in many solid tumours, including breast cancer (BC). Although molecular mechanisms associated with the development of LVI have been extensively studied, details of driver genes, and molecular pathways and mechanisms involved in its development in BC, remain poorly defined. Although invasive BC cells have the ability to invade surrounding stroma, only those that can interact with endothelial cells, penetrate the vascular wall and withstand the intravascular stress will develop LVI and complete metastatic dissemination. Identification of additional molecular events associated with LVI in the primary tumour and characterisation of the contribution of the tumour micro-environment to modulating biological processes leading to LVI in BC remain a challenging task. This stems not only from the complexity of the molecular alterations in the primary tumour and the interactions with different components of its micro-environment but also from the subjective nature of LVI assessment in human BC. In this review, we discuss the clinicopathological features and the current knowledge of the molecular mechanisms underlying LVI in BC.

Advantages of automated immunostain analyses for complex membranous immunostains: An exemplar investigating loss of E-cadherin expression in colorectal cancer.

As pathology moves towards digitisation, biomarker profiling through automated image analysis provides potentially objective and time-efficient means of assessment. This study set out to determine how a complex membranous immunostain, E-cadherin, assessed using an automated digital platform fares in comparison to manual evaluation in terms of clinical correlations and prognostication. Tissue microarrays containing 1000 colorectal cancer samples, stained with clinical E-cadherin antibodies were assessed through both manual scoring and automated image analysis. Both manual and automated scores were correlated to clinicopathological and survival data. E-cadherin data generated through digital image analysis was superior to manual evaluation when investigating for clinicopathological correlations in colorectal cancer. Loss of membranous E-cadherin, assessed on automated platforms, correlated with: right sided tumours (p = <0.001), higher T-stage (p = <0.001), higher grade (p = <0.001), N2 nodal stage (p = <0.001), intramural lymphovascular invasion (p = 0.006), perineural invasion (p = 0.028), infiltrative tumour edge (p = 0.001) high tumour budding score (p = 0.038), distant metastasis (p = 0.035), and poorer 5-year (p= 0.042) survival status. Manual assessment was only correlated with higher grade tumours, though other correlations become apparent only when assessed for morphological expression pattern (circumferential, basolateral, parallel) irrespective of intensity. Digital assessment of E-cadherin is effective for prognostication of colorectal cancer and may potentially offer benefits of improved objectivity, accuracy, and economy of time. Incorporating tools to assess patterns of staining may further improve such digital assessment in the future.

Mathematical modelling and deep learning algorithms to automate assessment of single and digitally multiplexed immunohistochemical stains in tumoural stroma.

Whilst automated analysis of immunostains in pathology research has focused predominantly on the epithelial compartment, automated analysis of stains in the stromal compartment is challenging and therefore requires time-consuming pathological input and guidance to adjust to tissue morphometry as perceived by pathologists. This study aimed to develop a robust method to automate stromal stain analyses using 2 of the commonest stromal stains (SMA and desmin) employed in clinical pathology practice as examples. An effective computational method capable of automatically assessing and quantifying tumour-associated stromal stains was developed and applied on cores of colorectal cancer tissue microarrays. The methodology combines both mathematical models and deep learning techniques with the former requiring no training data and the latter as many inputs as possible. The novel mathematical model was used to produce a digital double marker overlay allowing for fast automated digital multiplex analysis of stromal stains. The results show that deep learning methodologies in combination with mathematical modelling allow for an accurate means of quantifying stromal stains whilst also opening up new possibilities of digital multiplex analyses.

Preclinical evidence for anaplastic lymphoma kinase inhibitors as novel therapeutic treatments for cholangiocarcinoma.

Introduction: Bile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA. Methods: ALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells. Results: ALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis. Discussion: These results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

Associations between AI-Assisted Tumor Amphiregulin and Epiregulin IHC and Outcomes from Anti-EGFR Therapy in the Routine Management of Metastatic Colorectal Cancer.

PURPOSE: High tumor production of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), predicted benefit from anti-EGFR therapy for metastatic colorectal cancer (mCRC) in a retrospective analysis of clinical trial data. Here, AREG/EREG IHC was analyzed in a cohort of patients who received anti-EGFR therapy as part of routine care, including key clinical contexts not investigated in the previous analysis. EXPERIMENTAL DESIGN: Patients who received panitumumab or cetuximab ± chemotherapy for treatment of RAS wild-type mCRC at eight UK cancer centers were eligible. Archival formalin-fixed paraffin-embedded tumor tissue was analyzed for AREG and EREG IHC in six regional laboratories using previously developed artificial intelligence technologies. Primary endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 494 of 541 patients (91.3%) had adequate tissue for analysis. A total of 45 were excluded after central extended RAS testing, leaving 449 patients in the primary analysis population. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS [median: 8.5 vs. 4.4 months; HR, 0.73; 95% confidence interval (CI), 0.56-0.95; P = 0.02] and OS [median: 16.4 vs. 8.9 months; HR, 0.66 95% CI, 0.50-0.86; P = 0.002]. The significant OS benefit was maintained among patients with right primary tumor location (PTL), those receiving cetuximab or panitumumab, those with an oxaliplatin- or irinotecan-based chemotherapy backbone, and those with tumor tissue obtained by biopsy or surgical resection. CONCLUSIONS: High tumor AREG/EREG expression was associated with superior survival outcomes from anti-EGFR therapy in mCRC, including in right PTL disease. AREG/EREG IHC assessment could aid therapeutic decisions in routine practice. See related commentary by Randon and Pietrantonio, p. 4021.

Interval-Valued Intuitionistic Fuzzy Soft Rough Approximation Operators and Their Applications in Decision Making Problem.

It has been found that fuzzy sets, rough sets and soft sets are closely related concepts. Many complicated problems in economics, engineering, social sciences, medical science and many other fields involve uncertain data. These problems, which one comes in real life, cannot be solved using classical mathematical methods. There are several well-known theories to describe uncertainty, for instance, fuzzy set theory, rough set theory, and other mathematical tools. But all of these theories have their inherit difficulties as pointed out by D. Molodtsov. In 1999, D. Molodtsov introduced the concept of soft sets, which can be seen as a new mathematical tool for dealing with uncertainties. The concept of rough sets, proposed by Z. Pawlak as a framework for the construction of approximations of concepts. It is a formal tool for modeling and processing insufficient and incomplete information. Zhou and Wu first proposed the concept of intuitionistic fuzzy rough sets (IFrough sets). The aim of this paper is to introduce the concept of interval-valued intuitionistic fuzzy soft rough sets (IVIFS rough sets). We also investigate some properties of IVIFS rough approximation operators. Some basic operations and properties are studied. Lastly applications have been shown in decision making problems.

Anti-miR-135/SPOCK1 axis antagonizes the influence of metabolism on drug response in intestinal/colon tumour organoids.

Little is known about the role of microRNAs (miRNAs) in rewiring the metabolism within tumours and adjacent non-tumour bearing normal tissue and their potential in cancer therapy. This study aimed to investigate the relationship between deregulated miRNAs and metabolic components in murine duodenal polyps and non-polyp-derived organoids (mPOs and mNPOs) from a double-mutant ApcMinFbxw7∆G mouse model of intestinal/colorectal cancer (CRC). We analysed the expression of 373 miRNAs and 12 deregulated metabolic genes in mPOs and mNPOs. Our findings revealed miR-135b might target Spock1. Upregulation of SPOCK1 correlated with advanced stages of CRCs. Knockdown of miR-135b decreased the expression level of SPOCK1, glucose consumption and lactic secretion in CRC patient-derived tumours organoids (CRC tPDOs). Increased SPOCK1 induced by miR-135b overexpression promoted the Warburg effect and consequently antitumour effect of 5-fluorouracil. Thus, combination with miR-135b antisense nucleotides may represent a novel strategy to sensitise CRC to the chemo-reagent based treatment.

A passage through systems biology to systems medicine: adoption of middle-out rational approaches towards the understanding of therapeutic outcomes in cancer.

During the last few decades, conventional practices in medicine including oncology focus their interests towards the reductionism of molecular detailing and at the analytical level population-based assessment with stochastic principles, technically called 'evidence-based medicine', is generally practiced. Due to fluctuations in physiological parameters, the analysis and prediction of a therapeutic outcome in cancer on an individual level is uncertain. In recent times the well accepted opinion is that cancer should be looked upon as a systems disorder. This makes a paradigm shift - from a fragmented to a systems approach, linear to nonlinear methodology and from genome to physiome based analysis to understand the cancer. In the arena of systems biology, different groups have different views, namely, bottom-up (mechanistic), top-down (operational) and middle-out (rational). With respect to cancer each has a special relevance to serve the specific objectivity. In this article we have reviewed the views of the different schools, recent developments and controversies associated with the uncertainties in prediction of the therapeutic outcome of cancer. Recent advances in dynamical science and control theory may provide suitable analytical tools for capturing the uncertainties associated with cancer therapy through the development of middle-out rationalist (MORA) views.