Biomarkers of systemic inflammation provide additional prognostic stratification in cancers of unknown primary.

BACKGROUND: Biomarkers of systemic inflammation have been shown to predict outcomes in patients with cancer of unknown primary (CUP). We sought to validate these findings in patients with confirmed CUP (cCUP) and explore their role alongside existing clinicopathological prognostic categories. PATIENTS AND METHODS: CUP oncologist from across the United Kingdom were invited to include patients with cCUP referred to their local CUP multidisciplinary team. Patient demographics, clinical, pathological and outcome data were recorded and analysed. RESULTS: Data were available for 548 patients from four CUP services. 23% (n = 124) of patients met clinicopathological criteria for favourable-risk cCUP. On multivariate analysis c-reactive protein (CRP) (p < 0.001) and the Scottish Inflammatory Prognostic Score (SIPS: combining albumin and neutrophil count) (p < 0.001) were independently predictive of survival. CRP and SIPS effectively stratified survival in patients with both favourable-risk and poor-risk cCUP based on clinicopathological features. CONCLUSIONS: Biomarkers of systemic inflammation are reliable prognostic factors in patients with cCUP, regardless of clinicopathological subgroup. We recommend that CRP or SIPS are incorporated into routine clinical assessments of patients with cCUP as a tool to aid investigation and/or treatment decision-making across all groups. Established clinicopathological factors can then be used to inform management pathways and specific systemic anticancer therapy selection.

Stratification of chemotherapy-treated stage III colorectal cancer patients using multiplexed imaging and single-cell analysis of T-cell populations.

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVETM) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as 'high', 'moderate' or 'low', for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1-) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1- Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1-. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.

Integration of a patient-centred MUO/CUP service within a new acute oncology service: challenges and rewards.

BACKGROUND: Holistic approach to the clinical management pathway for malignancy of undefined primary origin (MUO)/carcinoma of unknown primary (CUP) patients remains an unmet clinical need. To address this, an MUO/CUP service was implemented during conception of a new acute oncology service (AOS). METHODOLOGY: Over a comparable 17 months' duration, patient outcomes pre-MUO/CUP service implementation was retrospectively analysed and compared prospectively with post-service implementation database. Performance measures of MUO/CUP service were compared against national recommendations. RESULTS: In the retrospective cohort (n=32), median age was 71.5 years and median length of hospital stay (LOS) was 11.25 days. In the prospective cohort (n=42), median age was 75.5 years, median LOS was 7.75 days (p=0.037). Post-service implementation, 100% patients were discussed in MUO/CUP multidisciplinary team meeting; 96% of inpatient referrals were reviewed by oncology within 24-48 hours. In the prospective group, median overall survival (OS) was 73 days vs 35 days in the retrospective group (p=0.045; hazard ratio (HR) 1.61). Out of 20 patients suitable for anti-cancer treatment in the prospective group, 85% were treated within 31 days from the decision-to-treat; 90% were treated within 62 days of referral. Within the prospective group, median OS was 214 days in the treated sub-group, compared with 44 days in patients receiving best supportive care only (p<0.0001; HR 3.19). CONCLUSION: Timely specialised input from AOS with a dedicated MUO/CUP team can achieve enhanced patient-centred and healthcare-centred outcomes, both in terms of survival and hospital stay. However, heterogeneity in both retrospective and prospective study groups, as well as discrepancies in coding, makes direct comparison between both groups challenging.

Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition.

Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.

A Stepwise Integrated Approach to Personalized Risk Predictions in Stage III Colorectal Cancer.

Purpose: Apoptosis is essential for chemotherapy responses. In this discovery and validation study, we evaluated the suitability of a mathematical model of apoptosis execution (APOPTO-CELL) as a stand-alone signature and as a constituent of further refined prognostic stratification tools.Experimental Design: Apoptosis competency of primary tumor samples from patients with stage III colorectal cancer (n = 120) was calculated by APOPTO-CELL from measured protein concentrations of Procaspase-3, Procaspase-9, SMAC, and XIAP. An enriched APOPTO-CELL signature (APOPTO-CELL-PC3) was synthesized to capture apoptosome-independent effects of Caspase-3. Furthermore, a machine learning Random Forest approach was applied to APOPTO-CELL-PC3 and available molecular and clinicopathologic data to identify a further enhanced signature. Association of the signature with prognosis was evaluated in an independent colon adenocarcinoma cohort (TCGA COAD, n = 136).Results: We identified 3 prognostic biomarkers (P = 0.04, P = 0.006, and P = 0.0004 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively) with increasing stratification accuracy for patients with stage III colorectal cancer.The APOPTO-CELL-PC3 signature ranked highest among all features. The prognostic value of the signatures was independently validated in stage III TCGA COAD patients (P = 0.01, P = 0.04, and P = 0.02 for APOPTO-CELL, APOPTO-CELL-PC3, and Random Forest signatures, respectively). The signatures provided further stratification for patients with CMS1-3 molecular subtype.Conclusions: The integration of a systems-biology-based biomarker for apoptosis competency with machine learning approaches is an appealing and innovative strategy toward refined patient stratification. The prognostic value of apoptosis competency is independent of other available clinicopathologic and molecular factors, with tangible potential of being introduced in the clinical management of patients with stage III colorectal cancer. Clin Cancer Res; 23(5); 1200-12. ©2016 AACR.

Transcriptional upregulation of c-MET is associated with invasion and tumor budding in colorectal cancer.

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

EphA2 Expression Is a Key Driver of Migration and Invasion and a Poor Prognostic Marker in Colorectal Cancer.

PURPOSE: EphA2, a member of the Eph receptor tyrosine kinases family, is an important regulator of tumor initiation, neovascularization, and metastasis in a wide range of epithelial and mesenchymal cancers; however, its role in colorectal cancer recurrence and progression is unclear. EXPERIMENTAL DESIGN: EphA2 expression was determined by immunohistochemistry in stage II/III colorectal tumors (N = 338), and findings correlated with clinical outcome. The correlation between EphA2 expression and stem cell markers CD44 and Lgr5 was examined. The role of EphA2 in migration/invasion was assessed using a panel of KRAS wild-type (WT) and mutant (MT) parental and invasive colorectal cancer cell line models. RESULTS: Colorectal tumors displayed significantly higher expression levels of EphA2 compared with matched normal tissue, which positively correlated with high CD44 and Lgr5 expression levels. Moreover, high EphA2 mRNA and protein expression were found to be associated with poor overall survival in stage II/III colorectal cancer tissues, in both univariate and multivariate analyses. Preclinically, we found that EphA2 was highly expressed in KRASMT colorectal cancer cells and that EphA2 levels are regulated by the KRAS-driven MAPK and RalGDS-RalA pathways. Moreover, EphA2 levels were elevated in several invasive daughter cell lines, and downregulation of EphA2 using RNAi or recombinant EFNA1 suppressed migration and invasion of KRASMT colorectal cancer cells. CONCLUSIONS: These data show that EpHA2 is a poor prognostic marker in stage II/III colorectal cancer, which may be due to its ability to promote cell migration and invasion, providing support for the further investigation of EphA2 as a novel prognostic biomarker and therapeutic target.

Capecitabine and sixth cranial nerve palsy.

Capecitabine is an oral chemotherapeutic agent converted to 5 fluorouracil (5-FU). Neurotoxicity associated with the medication encompasses both central and peripheral nervous systems. We describe a 60 year old man with colonic carcinoma who developed diplopia due to a sixth nerve palsy following the use of capecitabine which is an orally administered prodrug of 5-FU. An MRI of brain did not reveal a space occupying lesion or vascular insult to account for his cranial nerve palsy. The sixth nerve palsy resolved spontaneously once capecitabine was withdrawn. Physicians in all walks of life are increasingly likely to come across such patients and should familiarize themselves with toxicities consequent to chemotherapy. Further research is needed to elucidate the cause of capecitabine associated neurotoxicity.