Apoptosis and autophagy markers predict survival in neoadjuvant treated oesophageal adenocarcinoma patients.

BACKGROUND: Less than 20 % of patients with resectable oesophageal adenocarcinoma obtain a pathological response following neoadjuvant chemotherapy. Studies using oesophageal cancer cell lines have shown that drug sensitive tumour cells undergo apoptosis in response to drug treatment, whereas resistant cells induce autophagy and can recover following withdrawal of drug. In this study, we evaluated markers of apoptosis (active/cleaved caspase-3) and autophagy (LC3B) to establish whether these markers are useful prognostic indicators following neoadjuvant therapy. METHODS: Oesophageal adenocarcinoma tumour tissue from the Northern Ireland Biobank at Queens University Belfast was examined retrospectively. Tumours from 144 patients treated with platinum-based neoadjuvant chemotherapy followed by surgical resection were assembled into tissue microarrays prior to immunohistochemical analysis. Kaplan-Meier survival curves and log-rank tests were used to assess the impact of cleaved caspase-3 and LC3B expression on survival. Cox regression was used to examine association with clinical risk factors. RESULTS: High levels of cleaved caspase-3 were found in 14.6 % of patients and this correlated with a significantly better overall survival (p = 0.03). 38.9 % of patients had high cytoplasmic LC3B expression, which correlated with poor overall survival (p = 0.041). In addition, a distinct globular pattern of LC3B expression was identified in 40.3 % of patients and was also predictive of overall survival (p < 0.001). LC3B globular structures are also associated with tumour recurrence (p = 0.014). When these markers were assessed in combination, it was found that patients who showed low/negative cleaved caspase-3 staining and high/positive staining for both patterns of LC3B had the worst overall survival (p < 0.001). Multi-variate analysis also indicated that this marker combination was an independent predictor of poor prognosis (p = 0.008; HR = 0.046, 95% CI = (0.005-0.443). CONCLUSIONS: The expression of cleaved caspase-3 and specific LC3B staining patterns are associated with overall survival following neoadjuvant treatment. The combination of these markers is an independent indicator of outcome in neoadjuvant chemotherapy treated oesophageal adenocarcinoma.

Targeting the PI3K and MAPK pathways to improve response to HER2-targeted therapies in HER2-positive gastric cancer.

BACKGROUND: Aberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in overcoming trastuzumab resistance is unclear. METHODS: Using mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA and ERBB-family (EGFR, ERBB2, ERBB3 and ERBB4) genes in gastric tumour samples from 69 patients. A panel of gastric cell lines (N87, OE19, ESO26, SNU16, KATOIII) were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies. RESULTS: Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM-1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells (ED50: 0.83 ± 0.19 and 0.88 ± 0.13, respectively) and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05). CONCLUSIONS: PI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.

An Insight Into the Driver Mutations and Molecular Mechanisms Underlying Mucinous Adenocarcinoma of the Rectum.

BACKGROUND: Mucinous adenocarcinoma of the rectum accounts for 10% of all rectal cancers and has an impaired response to neoadjuvant chemoradiotherapy and worse overall survival. To date, insufficient genomic research has been performed on this histological subtype. OBJECTIVE: This study aims to define the mismatch repair deficiency rate and the driver mutations underpinning mucinous adenocarcinoma of the rectum and to compare it with rectal adenocarcinoma not otherwise specified. DESIGN: Immunohistochemistry and sequencing were performed on tumor samples from our tumor biobank. SETTINGS: This study was conducted across 2 tertiary referral centers. PATIENTS: Patients with mucinous adenocarcinoma and rectal adenocarcinoma not otherwise specified who underwent rectal resection between 2008 and 2018 were included. MAIN OUTCOME MEASURES: Mismatch repair status was performed by immunohistochemical staining. Mutations in the panel of oncogenes and tumor suppressor genes were determined by sequencing on the MiSeq V3 platform. RESULTS: The study included 33 patients with mucinous adenocarcinoma of the rectum and 100 patients with rectal adenocarcinoma not otherwise specified. Those with mucinous adenocarcinoma had a mismatch repair deficiency rate of 12.1% compared to 2.0% in the adenocarcinoma not otherwise specified cohort (p = 0.04). Mucinous adenocarcinoma and adenocarcinoma not otherwise specified rectal tumors had similar mutation frequencies in most oncogenes and tumor suppressor genes. No difference was found in the KRAS mutation rate (50.0% vs 37.1%, p = 0.29) or BRAF mutation rate (6.7% vs 3.1%, p = 0.34) between the cohorts. No difference was found between the cohorts regarding recurrence-free (p = 0.29) or overall survival (p = 0.14). LIMITATIONS: The major limitations of this study were the use of formalin-fixed, paraffin-embedded tissue over fresh-frozen tissue and the small number of patients included, in particular, in the mucinous rectal cohort. CONCLUSIONS: Most mucinous rectal tumors develop and progress along the chromosomal instability pathway. Further research in the form of transcriptomics, proteomics, and analysis of the effects of the mucin barrier may yield valuable insights into the mechanisms of resistance to chemoradiotherapy in this cohort. See Video Abstract at http://links.lww.com/DCR/B464. UNA PERCEPCIN SOBRE MUTACIONES IMPULSORAS Y MECANISMOS MOLECULARES SUBYACENTES AL ADENOCARCINOMA MUCINOSO DEL RECTO: ANTECEDENTES:El adenocarcinoma mucinoso del recto, representa el 10% de todos los cánceres rectales y tiene una respuesta deficiente a la quimioradioterapia neoadyuvante y una peor supervivencia en general. A la fecha, se han realizado muy pocas investigaciones genómicas sobre este subtipo histológico.OBJETIVO:Definir la tasa de deficiencia en la reparación de desajustes y mutaciones impulsoras, que sustentan el adenocarcinoma mucinoso del recto y compararlo con el adenocarcinoma rectal no especificado de otra manera.DISEÑO:Se realizaron inmunohistoquímica y secuenciación en muestras tumorales de nuestro biobanco de tumores.AJUSTE:El estudio se realizó en dos centros de referencia terciarios.PACIENTES:Se incluyeron pacientes con adenocarcinoma mucinoso y adenocarcinoma no especificado de otra manera, sometidos a resección rectal entre 2008 y 2018.PRINCIPALES MEDIDAS DE RESULTADO:El estado de reparación de desajustes se realizó mediante tinción inmunohistoquímica. Las mutaciones en el panel de oncogenes y genes supresores de tumores, se determinaron mediante secuenciación en la plataforma MiSeq V3.RESULTADOS:El estudio incluyó a 33 pacientes con adenocarcinoma mucinoso del recto y 100 pacientes con adenocarcinoma del recto no especificado de otra manera. Aquellos con adenocarcinoma mucinoso, tenían una tasa de deficiencia de reparación de desajustes del 12,1% en comparación con el 2,0% en la cohorte de adenocarcinoma no especificado de otra manera (p = 0,04). El adenocarcinoma mucinoso y el adenocarcinoma no especificado de otra manera, tuvieron frecuencias de mutación similares en la mayoría de los oncogenes y genes supresores de tumores. No se encontraron diferencias en la tasa de mutación de KRAS (50,0% frente a 37,1%, p = 0,29) o la tasa de mutación de BRAF (6,7% frente a 3,1%, p = 0,34) entre las cohortes. No se encontraron diferencias entre las cohortes con respecto a la supervivencia libre de recurrencia (p = 0,29) o la supervivencia global (p = 0,14).LIMITACIONES:Las mayores limitaciones de este estudio, fueron el uso de tejido embebido en parafina y fijado con formalina, sobre el tejido fresco congelado y el pequeño número de pacientes incluidos, particularmente en la cohorte mucinoso rectal.CONCLUSIONES:La mayoría de los tumores rectales mucinosos se desarrollan y progresan a lo largo de la vía de inestabilidad cromosómica. La investigación adicional en forma transcriptómica, proteómica y análisis de los efectos de la barrera de la mucina, puede proporcionar información valiosa sobre los mecanismos de resistencia a la quimioradioterapia, en esta cohorte. Consulte Video Resumen en http://links.lww.com/DCR/B464.

Feature fusion of Raman chemical imaging and digital histopathology using machine learning for prostate cancer detection.

The diagnosis of prostate cancer is challenging due to the heterogeneity of its presentations, leading to the over diagnosis and treatment of non-clinically important disease. Accurate diagnosis can directly benefit a patient's quality of life and prognosis. Towards addressing this issue, we present a learning model for the automatic identification of prostate cancer. While many prostate cancer studies have adopted Raman spectroscopy approaches, none have utilised the combination of Raman Chemical Imaging (RCI) and other imaging modalities. This study uses multimodal images formed from stained Digital Histopathology (DP) and unstained RCI. The approach was developed and tested on a set of 178 clinical samples from 32 patients, containing a range of non-cancerous, Gleason grade 3 (G3) and grade 4 (G4) tissue microarray samples. For each histological sample, there is a pathologist labelled DP-RCI image pair. The hypothesis tested was whether multimodal image models can outperform single modality baseline models in terms of diagnostic accuracy. Binary non-cancer/cancer models and the more challenging G3/G4 differentiation were investigated. Regarding G3/G4 classification, the multimodal approach achieved a sensitivity of 73.8% and specificity of 88.1% while the baseline DP model showed a sensitivity and specificity of 54.1% and 84.7% respectively. The multimodal approach demonstrated a statistically significant 12.7% AUC advantage over the baseline with a value of 85.8% compared to 73.1%, also outperforming models based solely on RCI and mean and median Raman spectra. Feature fusion of DP and RCI does not improve the more trivial task of tumour identification but does deliver an observed advantage in G3/G4 discrimination. Building on these promising findings, future work could include the acquisition of larger datasets for enhanced model generalization.

A case-control study examining the association of smad7 and TLR single nucleotide polymorphisms on the risk of colorectal cancer in ulcerative colitis.

AIM: Ulcerative colitis (UC) is characterized by chronic mucosal inflammation and an increased risk of colorectal cancer. smad7, TLR2 and TLR4 modulate intestinal inflammation and their polymorphisms affect the risk of development of sporadic colorectal cancer. The aim of the current study was to examine the association between single nucleotide polymorphisms (SNPs) in smad7, TLR2 and TLR4 and the development of colorectal cancer in patients with UC. METHOD: DNA was extracted from formalin-fixed, paraffin-embedded tissue from 90 patients with UC who had undergone panproctocolectomy between 1985 and 2013 (30 with UC-associated colorectal cancer and 60 control UC patients). Control cases were matched 2:1 for age at diagnosis of colitis, duration of disease and gender. Genotyping was performed for the smad7 rs4464148, rs11874392, rs12953717 and rs4939827 SNPs, the TLR2 rs5743704 and rs5743708 SNPs and the TLR4 rs4986790 and rs4986791 SNPs. RESULTS: Sixty three of the 90 patients (70%) were men and the mean age at diagnosis of UC was 38.6 ± 1.6 years. The mean time to the diagnosis of UC-associated colorectal cancer was 13.5 ± 1.9 years. The 5-year recurrence-free and cancer-specific survival rates were 76% and 88%, respectively. All eight SNPs were in Hardy-Weinberg equilibrium. None of the eight SNPs assessed in smad7, TLR2 or TLR4 were associated with the development of UC-associated colorectal cancer at an allelic or genotypic level. CONCLUSIONS: These data do not support an association between polymorphisms in smad7, TLR2 or TLR4 and the development of UC-associated colorectal cancer.

Mucin glycoproteins block apoptosis; promote invasion, proliferation, and migration; and cause chemoresistance through diverse pathways in epithelial cancers.

Overexpression of mucin glycoproteins has been demonstrated in many epithelial-derived cancers. The significance of this overexpression remains uncertain. The aim of this paper was to define the association of mucin glycoproteins with apoptosis, cell growth, invasion, migration, adhesion, and clonogenicity in vitro as well as tumor growth, tumorigenicity, and metastasis in vivo in epithelial-derived cancers by performing a systematic review of all published data. A systematic review of PubMed, Embase, and the Cochrane Central Register of Controlled Trials was performed to identify all papers that evaluated the association between mucin glycoproteins with apoptosis, cell growth, invasion, migration, adhesion, and clonogenicity in vitro as well as tumor growth, tumorigenicity, and metastasis in vivo in epithelial-derived cancers. PRISMA guidelines were adhered to. Results of individual studies were extracted and pooled together based on the organ in which the cancer was derived from. The initial search revealed 2031 papers, of which 90 were deemed eligible for inclusion in the study. The studies included details on MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC13, and MUC16. The majority of studies evaluated MUC1. MUC1 overexpression was consistently associated with resistance to apoptosis and resistance to chemotherapy. There was also evidence that overexpression of MUC2, MUC4, MUC5AC, MUC5B, MUC13, and MUC16 conferred resistance to apoptosis in epithelial-derived cancers. The overexpression of mucin glycoproteins is associated with resistance to apoptosis in numerous epithelial cancers. They cause resistance through diverse signaling pathways. Targeting the expression of mucin glycoproteins represents a potential therapeutic target in the treatment of epithelial-derived cancers.

Characterizing caspase-1 involvement during esophageal disease progression.

Barrett's esophagus (BE) is an inflammatory condition and a neoplastic precursor to esophageal adenocarcinoma (EAC). Inflammasome signaling, which contributes to acute and chronic inflammation, results in caspase-1 activation leading to the secretion of IL-1ß and IL-18, and inflammatory cell death (pyroptosis). This study aimed to characterize caspase-1 expression, and its functional importance, during disease progression to BE and EAC. Three models of disease progression (Normal-BE-EAC) were employed to profile caspase-1 expression: (1) a human esophageal cell line model; (2) a murine model of BE; and (3) resected tissue from BE-associated EAC patients. BE patient biopsies and murine BE organoids were cultured ex vivo in the presence of a caspase-1 inhibitor, to determine the importance of caspase-1 for inflammatory cytokine and chemokine secretion.Epithelial caspase-1 expression levels were significantly enhanced in BE (p < 0.01). In contrast, stromal caspase-1 levels correlated with histological inflammation scores during disease progression (p < 0.05). Elevated secretion of IL-1ß from BE explanted tissue, compared to adjacent normal tissue (p < 0.01), confirmed enhanced activity of caspase-1 in BE tissue. Caspase-1 inhibition in LPS-stimulated murine BE organoids caused a significant reduction in IL-1ß (p < 0.01) and CXCL1 (p < 0.05) secretion, confirming the importance of caspase-1 in the production of cytokines and chemokines associated with disease progression from BE to EAC. Targeting caspase-1 activity in BE patients should therefore be tested as a novel strategy to prevent inflammatory complications associated with disease progression.

Mucinous adenocarcinoma is a pharmacogenomically distinct subtype of colorectal cancer.

Mucinous colorectal cancer is a unique histological subtype that is known to respond poorly to cytotoxic chemotherapy and radiotherapy. There are a number of genes known to be associated with resistance to 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The aim of this study was to compare the somatic mutation frequency and copy number variation (CNV) in these genes between mucinous and non-mucinous colorectal cancer. A systematic search of PubMed was performed to identify papers investigating drug resistance in colorectal cancer. From this review, a list of 26 drug-resistance-associated genes was compiled. Using patient data from The Cancer Genome Atlas (TCGA), the somatic mutation rate and CNV was compared between patients with mucinous and non-mucinous colorectal cancer. Statistical analysis was carried out using GraphPad PRISM® version 5.00. Data were available on 531 patients (464 non-mucinous, 67 mucinous). A statistically significant difference in the somatic mutation rate between the two cohorts was identified in the TYMP (p = 0.0179), ATP7B (p = 0.0465), SRPK1 (p = 0.0135), ABCB1 (p = 0.0423), and ABCG2 (p = 0.0102) genes. A statistically significant difference in CNV was identified between the two cohorts in the GSTP1 (p = 0.0405), CCS (p = 0.0063), and TOP1 (p = 0.0048) genes. Differences in somatic mutation rate and CNV in genes associated with resistance to 5-FU, oxaliplatin, and irinotecan may partly account for the pattern of resistance observed in mucinous colorectal cancers. These genetic alterations may prove useful when deciding on a personalized approach to chemotherapy and may also represent potential therapeutic targets going forward.

Identification and clinical impact of potentially actionable somatic oncogenic mutations in solid tumor samples.

BACKGROUND: An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance. METHODS: The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. RESULTS: Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. CONCLUSIONS: Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

Barrett's Registry Collaboration of academic centers in Ireland reveals high progression rate of low-grade dysplasia and low risk from nondysplastic Barrett's esophagus: report of the RIBBON network.

Barrett's esophagus (BE) is the main pathological precursor of esophageal adenocarcinoma (EAC). Progression to high-grade dysplasia (HGD) or EAC from nondysplastic BE (NDBE), low-grade dysplasia (LGD) and indefinite for dysplasia (IND) varies widely between population-based studies and specialized centers for many reasons, principally the rigor of the biopsy protocol and the accuracy of pathologic definition. In the Republic of Ireland, a multicenter prospective registry and bioresource (RIBBON) was established in 2011 involving six academic medical centers, and this paper represents the first report from this network. A detailed clinical, endoscopic and pathologic database registered 3,557 patients. BE was defined strictly by both endoscopic evidence of Barrett's epithelium and the presence of specialized intestinal metaplasia (SIM). A prospective web-based database was used to gather information with initial and follow-up data abstracted by a data manager at each site. A total of 2,244 patients, 1,925 with no dysplasia, were included with complete follow-up. The median age at diagnosis was 60.5 with a 2.1:1 male to female ratio and a median follow-up time of 2.7 years (IQR 1.19-4.04), and 6609.25 person years. In this time period, 125 (5.57%) progressed to HGD/EAC, with 74 (3.3%) after 1 year of follow-up and 38 (1.69%) developed EAC, with 20 (0.89%) beyond 1 year. The overall incidence of HGD/EAC was 1.89% per year; 1.16% if the first year is excluded. The risk of progression to EAC alone overall was 0.57% per year, 0.31% excluding the first year, and 0.21% in the 1,925 patients who had SIM alone at diagnosis. Low-grade dysplasia (LGD) progressed to HGD/EAC in 31% of patients, a progression rate of 12.96% per year, 6.71% with the first year excluded. In a national collaboration of academic centers in Ireland, the progression rate for NDBE was similar to recent population studies. Almost one in two who progressed was evident within 1 year. Crucially, LGD diagnosed and confirmed by specialist gastrointestinal pathologists represents truly high-risk disease, highlighting the importance of expertise in diagnosis and management, and providing indirect support for ablative therapies in this context.

NUP98 - a novel predictor of response to anthracycline-based chemotherapy in triple negative breast cancer.

BACKGROUND: Triple Negative breast cancer (TNBC) is a poor outcome subgroup of breast cancer defined based on the absence of expression of ERα and PR and HER2 amplification. These hard to treat cancers lack targeted treatment options and are therefore treated with a standard of care (SoC) generic cocktail of DNA damaging chemotherapy, with a wide range of clinical responses. While a subset of TNBC patients respond very well to this treatment, others receive no clinical benefit and die from their disease within a short time period. We currently lack biomarkers to prospectively identify patients likely to relapse and we lack alternate treatment options. METHODS: NUP98 protein expression was investigated in patient samples using two independent tissue microarrays (TMAs), as well as a normal breast TMA. Correlation with pathological response to various chemotherapy regimens was investigated. RESULTS: We have shown that high NUP98 is significantly associated with poor outcome in TNBC patient samples both by gene expression and IHC-based protein analysis. While trends linking NUP98 expression with poorer outcomes were observed in breast cancer overall (and more specifically in the LuminalB Her2- subgroup), significant correlations were observed in TNBC. This appeared to be specific to anthracycline based regimens as the association between NUP98 and response was not observed in patients treated with taxane-based chemotherapy. CONCLUSIONS: We have identified a novel biomarker, NUP98, that can predict response to anthracycline based chemotherapy in TNBC. The ability to prospectively identify patients who are less likely to respond to SoC chemotherapy is a vital step in improving the overall survival of these patients.

Correction to: LC3B globular structures correlate with survival in esophageal adenocarcinoma.

Following publication of the original article [1], the authors reported an omission in the affiliations.

Mucinous adenocarcinoma of the colon and rectum: A genomic analysis.

BACKGROUND AND OBJECTIVES: Mucinous adenocarcinoma is a distinct subtype of colorectal cancer (CRC) with a worse prognosis when compared with non-mucinous adenocarcinoma. The aim of this study was to compare somatic mutations and copy number alteration (CNA) between mucinous and non-mucinous CRC. METHODS: Data from The Cancer Genome Atlas-colon adenocarcinoma and rectum adenocarcinoma projects were utilized. Mucinous and non-mucinous CRC were compared with regard to microsatellite status, overall mutation rate, the most frequently mutated genes, mutations in genes coding for mismatch repair (MMR) proteins and genes coding for mucin glycoproteins. CNA analysis and pathway analysis was undertaken. RESULTS: Mucinous CRC was more likely to be microsatellite instability-high (MSI-H) and hypermutated. When corrected for microsatellite status the single-nucleotide variation and insertion-deletion rate was similar between the two cohorts. Mucinous adenocarcinoma was more likely to have mutations in genes coding for MMR proteins and mucin glycoproteins. Pathway analysis revealed further differences between the two histological subtypes in the cell cycle, RTK-RAS, transforming growth factor-ß, and TP53 pathways. CONCLUSIONS: Mucinous CRC has some distinct genomic aberrations when compared with non-mucinous adenocarcinoma, many of which are driven by the increased frequency of MSI-H tumors. These genomic aberrations may play an important part in the difference seen in response to treatment and prognosis in mucinous adenocarcinoma.

Cleavage of the extracellular domain of junctional adhesion molecule-A is associated with resistance to anti-HER2 therapies in breast cancer settings.

BACKGROUND: Junctional adhesion molecule-A (JAM-A) is an adhesion molecule whose overexpression on breast tumor tissue has been associated with aggressive cancer phenotypes, including human epidermal growth factor receptor-2 (HER2)-positive disease. Since JAM-A has been described to regulate HER2 expression in breast cancer cells, we hypothesized that JAM-dependent stabilization of HER2 could participate in resistance to HER2-targeted therapies. METHODS: Using breast cancer cell line models resistant to anti-HER2 drugs, we investigated JAM-A expression and the effect of JAM-A silencing on biochemical/functional parameters. We also tested whether altered JAM-A expression/processing underpinned differences between drug-sensitive and -resistant cells and acted as a biomarker of patients who developed resistance to HER2-targeted therapies. RESULTS: Silencing JAM-A enhanced the anti-proliferative effects of anti-HER2 treatments in trastuzumab- and lapatinib-resistant breast cancer cells and further reduced HER2 protein expression and Akt phosphorylation in drug-treated cells. Increased epidermal growth factor receptor expression observed in drug-resistant models was normalized upon JAM-A silencing. JAM-A was highly expressed in all of a small cohort of HER2-positive patients whose disease recurred following anti-HER2 therapy. High JAM-A expression also correlated with metastatic disease at the time of diagnosis in another patient cohort resistant to trastuzumab therapy. Importantly, cleavage of JAM-A was increased in drug-resistant cell lines in conjunction with increased expression of ADAM-10 and -17 metalloproteases. Pharmacological inhibition or genetic silencing studies suggested a particular role for ADAM-10 in reducing JAM-A cleavage and partially re-sensitizing drug-resistant cells to the anti-proliferative effects of HER2-targeted drugs. Functionally, recombinant cleaved JAM-A enhanced breast cancer cell invasion in vitro and both invasion and proliferation in a semi-in vivo model. Finally, cleaved JAM-A was detectable in the serum of a small cohort of HER2-positive patients and correlated significantly with resistance to HER2-targeted therapy. CONCLUSIONS: Collectively, our data suggest a novel model whereby increased expression and cleavage of JAM-A drive tumorigenic behavior and act as a biomarker and potential therapeutic target for resistance to HER2-targeted therapies.

Stromal TRIM28-associated signaling pathway modulation within the colorectal cancer microenvironment.

BACKGROUND: Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment. METHODS: Reverse phase protein arrays were generated from laser capture micro-dissected carcinoma and stromal cells from fresh frozen colorectal cancer tissues. Phosphorylation and total protein levels were measured for 30 cancer-related signaling pathway endpoints. Strength and direction of associations between signaling endpoints were identified using Spearman's rank-order correlation analysis and compared to TRIM28 levels. Expression status of TRIM28 in tumor epithelium and stromal fibroblasts was assessed using IHC in formalin fixed tissue and the epithelium to stroma protein expression ratio method. RESULTS: We found distinct proteomic networks in the epithelial and stromal compartments which were linked to expression levels of TRIM28. Low levels of TRIM28 in tumor stroma (high epithelium: stroma ratio) were found in 10 out of 19 cases. Upon proteomic network analyses, these stromal high ratio cases revealed moderate signaling pathway similarity exemplified by 76 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). Furthermore, low levels of stromal TRIM28 correlated with elevated MDM2 levels in tumor epithelium (p = 0.01) and COX-2 levels in tumor stroma (p = 0.002). Low TRIM28 epithelium to stroma ratios were associated with elevated levels of caspases 3 and 7 in stroma (p = 0.041 and p = 0.036) and an increased signaling pathway similarity in stromal cells with 81 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). CONCLUSIONS: By dissecting TRIM28-associated pathways in stromal fibroblasts and epithelial tumor cells, we performed comprehensive proteomic analyses of molecular networks within the tumor microenvironment. We found modulation of several signaling pathways associated with TRIM28, which may be attributed to the pleiotropic properties of TRIM28 through its translational suppression of the family of KRAB domain transcription factors in tumor stromal compartments.

The significance of mucin pools following neoadjuvant chemoradiotherapy for locally advanced rectal cancer.

BACKGROUND: Neoadjuvant chemo-radiotherapy is utilized for locally advanced rectal cancer to optimize local control. A subset of patients form mucin pools following radiotherapy but the association between mucin pools and pathological and oncological outcomes following curative proctectomy for rectal cancer remains unknown. OBJECTIVE: The aim of this study was to determine the significance of mucin pool formation after neoadjuvant chemoradiotherapy for rectal cancer. METHODS: This is a retrospective analysis of a prospectively maintained rectal cancer database. Patients who underwent curative proctectomy for rectal cancer following long course chemoradiotherapy between January 2007 and December 2016 were eligible for inclusion. RESULTS: A total of 297 patients were eligible for inclusion; of these 36 (12.1%) had mucin pools on final histopathology. Tumors with mucin pools were less likely to be ypT3/T4 (25.0 vs 51.0%, P = 0.003), were more likely to have a good response (83.3 vs 53.6%, P < 0.001) and more likely to have a pathologic complete response (41.7 vs 19.2%, P = 0.006) to radiotherapy. The presence of mucin pools was associated with less distant recurrence ( P < 0.05) and improved overall survival ( P = 0.02). CONCLUSIONS: The presence of mucin pools following neoadjuvant chemoradiotherapy for rectal cancer represents a surrogate marker of response to treatment and downstaging and is associated with improved survival.

BCL-2 system analysis identifies high-risk colorectal cancer patients.

OBJECTIVE: The mitochondrial apoptosis pathway is controlled by an interaction of multiple BCL-2 family proteins, and plays a key role in tumour progression and therapy responses. We assessed the prognostic potential of an experimentally validated, mathematical model of BCL-2 protein interactions (DR_MOMP) in patients with stage III colorectal cancer (CRC). DESIGN: Absolute protein levels of BCL-2 family proteins were determined in primary CRC tumours collected from n=128 resected and chemotherapy-treated patients with stage III CRC. We applied DR_MOMP to categorise patients as high or low risk based on model outputs, and compared model outputs with known prognostic factors (T-stage, N-stage, lymphovascular invasion). DR_MOMP signatures were validated on protein of n=156 patients with CRC from the Cancer Genome Atlas (TCGA) project. RESULTS: High-risk stage III patients identified by DR_MOMP had an approximately fivefold increased risk of death compared with patients identified as low risk (HR 5.2, 95% CI 1.4 to 17.9, p=0.02). The DR_MOMP signature ranked highest among all molecular and pathological features analysed. The prognostic signature was validated in the TCGA colon adenocarcinoma (COAD) cohort (HR 4.2, 95% CI 1.1 to 15.6, p=0.04). DR_MOMP also further stratified patients identified by supervised gene expression risk scores into low-risk and high-risk categories. BCL-2-dependent signalling critically contributed to treatment responses in consensus molecular subtypes 1 and 3, linking for the first time specific molecular subtypes to apoptosis signalling. CONCLUSIONS: DR_MOMP delivers a system-based biomarker with significant potential as a prognostic tool for stage III CRC that significantly improves established histopathological risk factors.

Impact of somatic PI3K pathway and ERBB family mutations on pathological complete response (pCR) in HER2-positive breast cancer patients who received neoadjuvant HER2-targeted therapies.

BACKGROUND: The Cancer Genome Atlas analysis revealed that somatic EGFR, receptor tyrosine-protein kinase erbB-2 (ERBB2), Erb-B2 receptor tyrosine kinase 3 (ERBB3) and Erb-B2 receptor tyrosine kinase 4 (ERBB4) gene mutations (ERBB family mutations) occur alone or co-occur with somatic mutations in the gene encoding the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (PIK3CA) in 19% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers. Because ERBB family mutations can activate the PI3K/AKT pathway and likely have similar canonical signalling effects to PI3K pathway mutations, we investigated their combined impact on response to neoadjuvant HER2-targeted therapies. METHODS: Baseline tumour biopsies were available from 74 patients with HER2-positive breast cancer who were enrolled in the phase II TCHL neoadjuvant study (ICORG 10-05) assessing TCH (docetaxel, carboplatin, trastuzumab) (n = 38) versus TCL (docetaxel, carboplatin, lapatinib) (n = 10) versus TCHL (docetaxel, carboplatin, trastuzumab, lapatinib) (n = 40), each for six cycles. Activating mutations in PIK3CA and ERBB family genes were identified using mass spectrometry-based genotyping. Phosphatase and tensin homolog (PTEN) expression was assessed by immunohistochemistry. RESULTS: PIK3CA and/or ERBB family mutations were detected in 23 (31.1%) tumour samples tested, whereas PTEN expression was low in 31.1% of cases tested. Mutation frequency was similar in each treatment arm (31.3% in TCH arm, 30% in TCL arm and 31.3% in TCHL arm) and was not influenced by oestrogen receptor (ER) status (27.6% in ER-negative patients, 33.3% in ER-positive patients) or progesterone receptor (PR) status (32.6% in PR-negative patients, 29% in PR-positive patients). There was no significant difference in pathological complete response (pCR) rates between 47 patients with wild-type (WT) tumours and 22 patients whose tumours carried mutations (in either PIK3CA or ERBB family genes) (42.5% vs. 54.5%; p = 0.439). Similarly, there was no significant difference in pCR rates between patients with PIK3CA/ERBB family mutated/PTEN-low (i.e., PI3K-activated) tumours and patients without PI3K activation (50% vs. 44%; p = 0.769). However, in the TCHL (but not the TCH) group, the pCR rate was higher for 9 patients with PIK3CA/ERBB family mutated tumours than for 20 patients with PIK3CA/ERBB family WT tumours (77.8% vs. 35%; p = 0.05). CONCLUSIONS: Our results indicate that patients who receive neoadjuvant TCHL and have PIK3CA/ERBB family mutated tumours may be more likely to have a pCR than patients with WT tumours. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01485926 . Registered on 2 December 2011.

Investigating the role of the IGF axis as a predictor of biochemical recurrence in prostate cancer patients post-surgery.

BACKGROUND: Between 20% and 35% of prostate cancer (PCa) patients who undergo treatment with curative intent (ie, surgery or radiation therapy) for localized disease will experience biochemical recurrence (BCR). Alterations in the insulin-like growth factor (IGF) axis and PTEN expression have been implicated in the development and progression of several human tumors including PCa. We examined the expression of the insulin receptor (INSR), IGF-1 receptor (IGF-1R), PTEN, and AKT in radical prostatectomy tissue of patients who developed BCR post-surgery. METHODS: Tissue microarrays (TMA) of 130 patients post-radical prostatectomy (65 = BCR, 65 = non-BCR) were stained by immunohistochemistry for INSR, IGF-1R, PTEN, and AKT using optimized antibody protocols. INSR, IGF1-R, PTEN, and AKT expression between benign and cancerous tissue, and different Gleason grades was assessed. Kaplan-Meier survival curves were used to examine the relationship between proteins expression and BCR. RESULTS: INSR (P < 0.001), IGF-1R (P < 0.001), and AKT (P < 0.05) expression was significantly increased and PTEN (P < 0.001) was significantly decreased in cancerous versus benign tissue. There was no significant difference in INSR, IGF-1R, or AKT expression in the cancerous tissue of non-BCR versus BCR patients (P = 0.149, P = 0.990, P = 0.399, respectively). There was a significant decrease in PTEN expression in the malignant tissue of BCR versus non-BCR patients (P = 0.011). Combinational analysis of the tissue proteins identified a combination of decreased PTEN and increased AKT or increased INSR was associated with worst outcome. We found that in each case, our hypothesized worst group was most likely to experience BCR and this was significant for combinations of PTEN+INSR and PTEN+AKT but not PTEN+IGF-1R (P = 0.023, P = 0.028, P = 0.078, respectively). CONCLUSIONS: Low PTEN is associated with BCR and this association is strongly modified by high INSR and high AKT expression. Measurement of these proteins could help inform appropriate patient selection for postoperative adjuvant therapy and prevent BCR.

HLA-DR expression in tumor epithelium is an independent prognostic indicator in esophageal adenocarcinoma patients.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis, and incidence is increasing rapidly in the Western world. Measurement of immune markers has been shown to have prognostic significance in a growing number of cancers, but whether this is true for EAC has yet to be evaluated. This study aimed to characterize HLA-DR expression in the esophagus across the inflammation to cancer progression sequence and to assess the prognostic significance of HLA-DR expression in EAC. Tissue microarrays (TMA) were constructed from esophageal tissue taken from patients at different stages in the cancer progression sequence; normal, esophagitis, Barrett's esophagus (BE), low- and high-grade dysplasia (LGD, HGD) and EAC. HLA-DR expression in tissue epithelium and stroma was assessed by immunohistochemistry. HLA-DR expression increased early in the inflammation to cancer progression sequence; with higher expression detected in esophagitis and BE compared to normal tissue. Patients with low (<50%) HLA-DR expression in the EAC tumor epithelium had significantly worse survival outcomes, compared to those with high expression, in both the tumor core (hazard ratio, HR = 2.178, p = 0.024, n = 70) and leading edge (HR = 2.86, p = 0.013, n = 41). Multivariate analysis demonstrated that low HLA-DR expression in leading edge tumor epithelium was an independent predictor of poor survival, associated with a 2.8-fold increase in disease-associated death (p = 0.023). This study shows that HLA-DR is an independent prognostic marker in EAC tumor epithelium. This may have implications for patient stratification strategies as well as EAC tumor immunology.