Spatial transcriptomic analysis reveals local effects of intratumoral fusobacterial infection on DNA damage and immune signaling in rectal cancer.

Mucinous colorectal cancer (CRC) is a common histological subtype of colorectal adenocarcinoma, associated with a poor response to chemoradiotherapy. The commensal facultative anaerobes fusobacteria, have been associated with poor prognosis specifically in mesenchymal CRC. Interestingly, fusobacterial infection is especially prevalent in mucinous CRC. The objective of this study was therefore to increase our understanding of beneficial and detrimental effects of fusobacterial infection, by contrasting host cell signaling and immune responses in areas of high vs. low infection, using mucinous rectal cancer as a clinically relevant example. We employed spatial transcriptomic profiling of 106 regions of interest from 8 mucinous rectal cancer samples to study gene expression in the epithelial and immune segments across regions of high versus low fusobacterial infection. Fusobacteria high regions were associated with increased oxidative stress, DNA damage, and P53 signaling. Meanwhile regions of low fusobacterial prevalence were characterized by elevated JAK-STAT, Il-17, Il-1, chemokine and TNF signaling. Immune masks within fusobacterial high regions were characterized by elevated proportions of cytotoxic (CD8+) T cells (p = 0.037), natural killer (NK) cells (p < 0.001), B-cells (p < 0.001), and gamma delta T cells (p = 0.003). Meanwhile, fusobacteria low regions were associated with significantly greater M2 macrophage (p < 0.001), fibroblast (p < 0.001), pericyte (p = 0.002), and endothelial (p < 0.001) counts.

Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis.

OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.

Predictive modelling of response to neoadjuvant therapy in HER2+ breast cancer.

HER2-positive (HER2+) breast cancer accounts for 20-25% of all breast cancers. Predictive biomarkers of neoadjuvant therapy response are needed to better identify patients with early stage disease who may benefit from tailored treatments in the adjuvant setting. As part of the TCHL phase-II clinical trial (ICORG10-05/NCT01485926) whole exome DNA sequencing was carried out on normal-tumour pairs collected from 22 patients. Here we report predictive modelling of neoadjuvant therapy response using clinicopathological and genomic features of pre-treatment tumour biopsies identified age, estrogen receptor (ER) status and level of immune cell infiltration may together be important for predicting response. Clonal evolution analysis of longitudinally collected tumour samples show subclonal diversity and dynamics are evident with potential therapy resistant subclones detected. The sources of greater pre-treatment immunogenicity associated with a pathological complete response is largely unexplored in HER2+ tumours. However, here we point to the possibility of APOBEC associated mutagenesis, specifically in the ER-neg/HER2+ subtype as a potential mediator of this immunogenic phenotype.

Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

Increased Fusobacterium tumoural abundance affects immunogenicity in mucinous colorectal cancer and may be associated with improved clinical outcome.

There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the association of Fusobacterium abundance with immune cell composition and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumour microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with an increased proportion of CD8 + lymphocytes (p = 0.018), regulatory T-cells (p = 0.001) and M2 macrophages (p = 0.001). In the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. High Fusobacterium abundance was associated with an increased proportion of CD4 + lymphocytes (p = 0.031) and M1 macrophages (p = 0.006), whilst M2 macrophages (p = 0.043) were under-represented in this cohort. Patients with increased Fusobacterium relative abundance in our mucinous CRC TCGA cohort tended to have better clinical outcomes (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance may be associated with improved outcomes in mucinous CRC, possibly due to a modulatory effect on the host immune response. KEY MESSAGES: • Increased Fusobacterium relative abundance was not found to be associated with microsatellite instability in mucinous CRC. • Increased Fusobacterium relative abundance was associated with an M2/M1 macrophage switch, which is especially significant in mucinous CRC, where M2 macrophages are overexpressed. • Increased Fusobacterium relative abundance was associated with a significant improvement in disease specific survival in mucinous CRC. • Our findings were validated at a protein level within our own in house mucinous and non-mucinous rectal cancer cohorts.

Development, Validation, and Clinical Utility of a Six-gene Signature to Predict Aggressive Prostate Cancer.

BACKGROUND: Molecular signatures in prostate cancer (PCa) tissue can provide useful prognostic information to improve the understanding of a patient's risk of harbouring aggressive disease. OBJECTIVE: To develop and validate a gene signature that adds independent prognostic information to clinical parameters for better treatment decisions and patient management. DESIGN, SETTING, AND PARTICIPANTS: Expression of 14 genes was evaluated in radical prostatectomy (RP) tissue from an Irish cohort of PCa patients (n = 426). A six-gene molecular risk score (MRS) was identified with strong prognostic performance to predict adverse pathology (AP) at RP or biochemical recurrence (BCR). The MRS was combined with the Cancer of the Prostate Risk Assessment (CAPRA) score, to create a molecular and clinical risk score (MCRS), and validated in a Swedish cohort (n = 203). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary AP outcome was assessed by the likelihood ratio statistics and area under the receiver operating characteristics curves (AUC) from logistic regression models. The secondary time to BCR outcome was assessed by likelihood ratio statistics and C-indexes from Cox proportional hazard regression models. RESULTS AND LIMITATIONS: The six-gene signature was significantly (p < 0.0001) prognostic and added significant prognostic value to clinicopathological features for AP and BCR outcomes. For both outcomes, both the MRS and the MCRS increased the AUC/C-index when added to European Association of Urology (EAU) and CAPRA scores. Limitations include the retrospective nature of this study. CONCLUSIONS: The six-gene signature has strong performance for the prediction of AP and BCR in an independent clinical validation study. MCRS improves prognostic evaluation and can optimise patient management after RP. PATIENT SUMMARY: We found that the expression panel of six genes can help predict whether a patient is likely to have a disease recurrence after radical prostatectomy surgery.

Exhaled Breath Condensate (EBC) analysis of circulating tumour DNA (ctDNA) using a lung cancer specific UltraSEEK oncogene panel.

INTRODUCTION: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. METHODS: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). RESULTS: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10-3, p = 3.14 × 10-5, p = 1.95 × 10-3) and EBC (p = 2.18 × 10-3, p = 2.28 × 10-4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. CONCLUSIONS: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

Transcriptional CDK inhibitors, CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation.

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

Tumour Genome Characterization of a Rare Case of Pulmonary Enteric Adenocarcinoma and Prior Colon Adenocarcinoma.

Pulmonary enteric adenocarcinoma (PEAC) is a rare variant of lung adenocarcinoma first described in the early 1990s in a lung tumour with overlapping lung and small intestine features. It is a rare tumour with fewer than 300 cases described in the published literature and was only formally classified in 2011. Given these characteristics the diagnosis is challenging, but even more so in a patient with prior gastrointestinal malignancy. A 68-year-old Caucasian female presented with a cough and was found to have a right upper lobe mass. Her history was significant for a pT3N1 colon adenocarcinoma. The resected lung tumour showed invasive lung adenocarcinoma but also features of colorectal origin. Immuno-stains were strongly and diffusely positive for lung and enteric markers. Multi-region, whole-exome sequencing of the mass and archival tissue from the prior colorectal cancer showed distinct genomic signatures with higher mutational burden in the PEAC and very minimal overlap in mutations between the two tumours. This case highlights the challenge of diagnosing rare lung tumours, but more specifically PEAC in a patient with prior gastro-intestinal cancer. Our use of multi-region, next-generation sequencing revealed distinct genomic signatures between the two tumours further supporting our diagnosis, and evidence of PEAC intra-tumour heterogeneity.

Prognostic value of the 6-gene OncoMasTR test in hormone receptor-positive HER2-negative early-stage breast cancer: Comparative analysis with standard clinicopathological factors.

AIM: The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (OncoMasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. METHODS: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. RESULTS: OMm and OM (both with likelihood ratio statistic [LRS] P < 0.001; C indexes = 0.84 and 0.85, respectively) were more prognostic for DRFS and provided significant additional prognostic information to all other assessment tools/factors assessed (all LRS P ≤ 0.002). In addition, the OM correctly classified more patients with distant recurrences (DRs) into the high-risk category than other risk classification tools. Similar results were observed for IDFS. DISCUSSION: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions.

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.

Development of a Novel Weighted Ranking Method for Immunohistochemical Quantification of a Heterogeneously Expressed Protein in Gastro-Esophageal Cancers.

High expression of Junctional Adhesion Molecule-A (JAM-A) has been linked with poor prognosis in several cancers, including breast cancers overexpressing the human epidermal growth factor receptor-2 (HER2). Furthermore, JAM-A expression has been linked with regulating that of HER2, and associated with the development of resistance to HER2-targeted therapies in breast cancer patients. The purpose of this study was to establish a potential relationship between JAM-A and HER2 in HER2-overexpressing gastro-esophageal (GE) cancers. Interrogation of gene expression datasets revealed that high JAM-A mRNA expression was associated with poorer survival in HER2-positive gastric cancer patients. However, high intra-tumoral heterogeneity of JAM-A protein expression was noted upon immunohistochemical scoring of a GE cancer tissue microarray (TMA), precluding a simple confirmation of any relationship between JAM-A and HER2 at protein level. However, in a test-set of 25 full-face GE cancer tissue sections, a novel weighted ranking system proved effective in capturing JAM-A intra-tumoral heterogeneity and confirming statistically significant correlations between JAM-A/HER2 expression. Given the growing importance of immunohistochemistry in stratifying cancer patients for the receipt of new targeted therapies, this may sound a cautionary note against over-relying on cancer TMAs in biomarker discovery studies of heterogeneously expressed proteins. It also highlights a timely need to develop validated mechanisms of capturing intra-tumoral heterogeneity to aid in future biomarker/therapeutic target development for the benefit of cancer patients.

Human Epidermal Growth Factor Receptor-3 Expression Is Regulated at Transcriptional Level in Breast Cancer Settings by Junctional Adhesion Molecule-A via a Pathway Involving Beta-Catenin and FOXA1.

The success of breast cancer therapies targeting the human epidermal growth factor receptor-2 (HER2) is limited by the development of drug resistance by mechanisms including upregulation of HER3. Having reported that HER2 expression and resistance to HER2-targeted therapies can be regulated by Junctional Adhesion Molecule-A (JAM-A), this study investigated if JAM-A regulates HER3 expression. Expressional alteration of JAM-A in breast cancer cells was used to test expressional effects on HER3 and its effectors, alongside associated functional behaviors, in vitro and semi-in vivo. HER3 transcription factors were identified and tested for regulation by JAM-A. Finally a patient tissue microarray was used to interrogate connections between putative pathway components connecting JAM-A and HER3. This study reveals for the first time that HER3 and its effectors are regulated at gene/protein expression level by JAM-A in breast cancer cell lines; with functional consequences in in vitro and semi-in vivo models. In bioinformatic, cellular and patient tissue models, this was associated with regulation of the HER3 transcription factor FOXA1 by JAM-A via a pathway involving ß-catenin. Our data suggest a novel model whereby JAM-A expression regulates ß-catenin localization, in turn regulating FOXA1 expression, which could drive HER3 gene transcription. JAM-A merits investigation as a novel target to prevent upregulation of HER3 during the development of resistance to HER2-targeted therapies, or to reduce HER3-dependent tumorigenic signaling.

Genomic and Transcriptomic Characterisation of Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

Standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (NACRT), followed by surgical resection. However, >70% of patients do not achieve a complete pathological response and have higher rates of relapse and death. There are no validated pre- or on-treatment factors that predict response to NACRT besides tumour stage and size. We characterised the response of 33 LARC patients to NACRT, collected tumour samples from patients prior to, during and after NACRT, and performed whole exome, transcriptome and high-depth targeted sequencing. The pre-treatment LARC genome was not predictive of response to NACRT. However, in line with the increasing recognition of microbial influence in cancer, RNA analysis of pre-treatment tumours suggested a greater abundance of Fusobacteria in intermediate and poor responders. In addition, we investigated tumour heterogeneity and evolution in response to NACRT. While matched pre-treatment, on-treatment and post-treatment tumours revealed minimal genome evolution overall, we identified cases in which microsatellite instability developed or was selected for during NACRT. Recent research has suggested a role for adaptive mutability to targeted therapy in colorectal cancer cells. We provide preliminary evidence of selection for mismatch repair deficiency in response to NACRT. Furthermore, pre-NACRT genomic landscapes do not predict treatment response but pre-NACRT microbiome characteristics may be informative.

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.

Caspase-11 regulates the tumour suppressor function of STAT1 in a murine model of colitis-associated carcinogenesis.

Murine inflammatory caspase-11 has an important role in intestinal epithelial inflammation and barrier function. Activation of the non-canonical inflammasome, mediated by caspase-11, serves as a regulatory pathway for the production of the pro-inflammatory cytokines IL-1ß and IL-18, and has a key role in pyroptotic cell death. We have previously demonstrated a protective role for caspase-11 during dextran sulphate sodium (DSS)-induced colitis, however the importance of caspase-11 during colorectal tumour development remains unclear. Here, we show that Casp11-/- mice are highly susceptible to the azoxymethane (AOM)-DSS model of colitis-associated cancer (CAC), compared to their wild type (WT) littermates. We show that deficient IL-18 production occurs at initial inflammation stages of disease, and that IL-1ß production is more significantly impaired in Casp11-/- colons during established CAC. We identify defective STAT1 activation in Casp11-/- colons during disease progression, and show that IL-1ß signalling induces caspase-11 expression and STAT1 activation in primary murine macrophages and intestinal epithelial cells. These findings uncover an anti-tumour role for the caspase-11 and the non-canonical inflammasome during CAC, and suggest a critical role for caspase-11, linking IL-1ß and STAT1 signalling pathways.

Germline single nucleotide polymorphisms in ERBB3 and BARD1 genes result in a worse relapse free survival response for HER2-positive breast cancer patients treated with adjuvant based docetaxel, carboplatin and trastuzumab (TCH).

Breast cancer is the leading cause of cancer related deaths in women worldwide and is classified into subtypes based on the cancer's receptor status. Of these subtypes, those expressing the human epidermal growth factor receptor 2 (HER2) receptor were traditionally associated with poor prognosis. Several advances have been made in the treatment of HER2-positive breast cancer, yet issues of resistance and poor response to therapy remains prevalent. In this study we explored the impact of HER-family and homologous recombination deficiency SNPs on response to patients who received TCH-based (docetaxel (T), carboplatin (C), and trastuzumab (H)) treatment versus those who received other treatment regimens. Using Cox regression analysis, we identified 6 SNPs that correlate with recurrence free survival in our patients and supported our findings using support vector machines. We also used reverse phase protein array analysis to examine the impact ERBB3 SNPs may have on both the PI3K/AKT and MAPK/ERK signaling pathways. Finally, using cell line models, we correlated SNP status with sensitivity to platinum based drugs and docetaxel. We found that patients on a TCH based regimen with the minor allele of the ERBB3 (rs2229046 and rs773123) and BARD1 (rs2070096) SNPs, were significantly more likely to relapse than those women who were not. Additionally, we observed that patients with these ERBB3 SNPs had shown elevated protein expression/phosphorylation of Src kinase, c-MET (Y1234/1235), GSK-3ß (S9) and p27, indicating that these SNPs are associated with non-PI3K/AKT signaling. Finally, using cell line models, we demonstrate that the BARD1 SNP (rs2229571) is associated with greater sensitivity to both carboplatin and cisplatin. The BARD1 and ERBB3 SNPs can potentially be used to determine those patients that will have a worse response to TCH based treatment, an effect that may arise from the SNPs impact on altered cellular signaling.

Frequency, impact and a preclinical study of novel ERBB gene family mutations in HER2-positive breast cancer.

BACKGROUND: Somatic mutations in the ERBB genes (epidermal growth factor receptor: EGFR, ERBB2, ERBB3, ERBB4) promote oncogenesis and lapatinib resistance in metastatic HER2+ (human epidermal growth factor-like receptor 2) breast cancer in vitro. Our study aimed to determine the frequency of mutations in four genes: EGFR, ERBB2, ERBB3 and ERBB4 and to investigate whether these mutations affect cellular behaviour and therapy response in vitro and outcomes after adjuvant trastuzumab-based therapy in clinical samples. METHODS: We performed Agena MassArray analysis of 227 HER2+ breast cancer samples to identify the type and frequency of ERBB family mutations. Of these, two mutations, the somatic mutations ERBB4-V721I and ERBB4-S303F, were stably transfected into HCC1954 (PIK3CA mutant), HCC1569 (PIK3CA wildtype) and BT474 (PIK3CA mutant, ER positive) HER2+ breast cancer cell lines for functional in vitro experiments. RESULTS: A total of 12 somatic, likely deleterious mutations in the kinase and furin-like domains of the ERBB genes (3 EGFR, 1 ERBB2, 3 ERBB3, 5 ERBB4) were identified in 7% of HER2+ breast cancers, with ERBB4 the most frequently mutated gene. The ERBB4-V721I kinase domain mutation significantly increased 3D-colony formation in 3/3 cell lines, whereas ERBB4-S303F did not increase growth rate or 3D colony formation in vitro. ERBB4-V721I sensitized HCC1569 cells (PIK3CA wildtype) to the pan class I PI3K inhibitor copanlisib but increased resistance to the pan-HER family inhibitor afatinib. The combinations of copanlisib with trastuzumab, lapatinib, or afatinib remained synergistic regardless of ERBB4-V721I or ERBB4-S303F mutation status. CONCLUSIONS: ERBB gene family mutations, which are present in 7% of our HER2+ breast cancer cohort, may have the potential to alter cellular behaviour and the efficacy of HER- and PI3K-inhibition.

The BAX/BAK-like protein BOK is a prognostic marker in colorectal cancer.

The intrinsic or mitochondrial apoptosis pathway is controlled by the interaction of antiapoptotic and pro-apoptotic members of the BCL-2 protein family. Activation of this death pathway plays a crucial role in cancer progression and chemotherapy responses. The BCL-2-related ovarian killer (BOK) possesses three BCL-2 homology domains and has been proposed to act in a similar pro-apoptotic pathway as the pro-apoptotic proteins BAX and BAK. In this study, we showed that stage II and III colorectal cancer patients possessed decreased levels of BOK protein in their tumours compared to matched normal tissue. BOK protein levels in tumours were also prognostic of clinical outcome but increased BOK protein levels surprisingly associated with earlier disease recurrence and reduced overall survival. We found no significant association of BOK protein tumour levels with ER stress markers GRP78 or GRP94 or with cleaved caspase-3. In contrast, BOK protein levels correlated with Calreticulin. These data indicate BOK as a prognostic marker in colorectal cancer and suggest that different activities of BOK may contribute to cancer progression and prognosis.