Flow cytometry potential applications in characterizing solid tumors main phenotype, heterogeneity and circulating cells

Flow cytometry (FCM) is a unique technique that allows rapid quantitative measurement of multiple parameters on a large number of cells at the individual level. FCM is based on immunolabelling with fluorochrome-conjugated antibodies, leading to high sensitivity and precision while time effective sample preparation. FCM can be performed on tissue following enzymatic or mechanical dissociation. The expression of epithelial antigens and cytokeratin isoforms help in distinguishing tumor cells from adjacent epithelial cells and from tumor infiltrating leukocytes. Tumor phenotypes can be characterized on expression intensity, aberrancies and presence of tumor-associated antigens as well as their cell proliferation rate and eventual heteroploidy. FCM can measure quantitative expression of hormone or growth factor receptors, immunoregulatory proteins to guide adjuvant therapy. Expression of adhesion molecules tells on tumor’s capacity for tissue invasion and metastasis seeding. Tumor heterogeneity can be explored quantitatively and rare, potentially emerging, clones with poor prognosis can be detected. FCM is easily applicable on fine needle aspiration and in any tumor related biological fluids. FCM can also be used to detect circulating tumor cells (CTC) to assess metastatic potential at diagnosis or during treatment. Detecting CTC could allow early detection of tumors before they are clinically expressed although some difficulties still need to be solved. It thus appears that FCM should be in the pathologist tool box to improve cancer diagnosis, classification and prognosis evaluation as well as in orientating personalized adjuvant therapy and immunotherapy. More developments are still required to better known tumor phenotypes and their potential invasiveness.

Clinical correlation of cadherin-17 (CA17 aka CDH17) marker with advanced tumor stages and poor prognosis of cholangiocarcinoma in a retrospective cohort of 180 patients.

344 Background: Aberrant expression of cadherin-17 (CA17, aka CDH17) cancer marker is often observed in major gastrointestinal (GI) cancers. In this retrospective study, we examined the CA17 tissue expression and analyzed its clinical significance in cholangiocarcinoma (CCA). Methods: A total of 180 patients with detailed demographic and clinical data were enrolled in this study and randomly divided into a training cohort (N = 120) and a validation cohort (N = 60). Biopsy samples (tumor and adjacent normal tissue) collected for tissue microarray were examined by immunohistochemistry using a mouse anti-CA17 monoclonal antibody (Clone 7C5). Image pro plus was applied to score the staining intensity and expression level of CA17 marker. Kaplan-Meier analysis, Cox's proportional hazards regression, and nomogram were applied to evaluate the prognostic significance of CA17. Results: CA17 cancer biomarker over-expression was significantly observed in CCA compared to their non-tumor counterparts (P < 0.001), and positively correlated with aggressive tumor phenotypes, like larger tumor size (P = 0.004), lymph node metastasis (P < 0.001), and advanced TNM stage (P < 0.001). Meanwhile, patients with high expression of CA17 correlated with worse postoperative overall survival (OS)(P = 0.002) and recurrence-free survival (P = 0.03). Besides, multivariate analysis identified that CA17 expression was an independent prognostic factor for cholangiocarcinoma patients (Training cohort: HR:1.716, 95% CI:1.116-2.638; P = 0.014; Validation cohort: HR: 1.811, 95%CI:1.028-3.892; P = 0.035), which indicated that the CA17 is much more efficient than serum CA19-9 (Training cohort: HR: 1.000, 95%CI: 1.000-1.001; P = 0.006; Validation cohort: HR: 1.000,95%CI:1.000-1.001; P = 0.013) in predicting the OS of CCA patients. Notably, the nomogram integrating CA17 expression had a better prognostic performance as compared with current TNM staging systems. Conclusions: CA17 cancer biomarker was aberrantly expressed in CCA tumor tissues, and its high expression level positively correlated with aggressive tumor phenotypes. Furthermore, CA17 was an independent adverse prognostic factor for CCA patients’ survival, which may serve as a potential drug target for CCA patients.

RNA Sequencing-Based Identification of Ganglioside GD2-Positive Cancer Phenotype

The tumor-associated ganglioside GD2 represents an attractive target for cancer immunotherapy. GD2-positive tumors are more responsive to such targeted therapy, and new methods are needed for the screening of GD2 molecular tumor phenotypes. In this work, we built a gene expression-based binary classifier predicting the GD2-positive tumor phenotypes. To this end, we compared RNA sequencing data from human tumor biopsy material from experimental samples and public databases as well as from GD2-positive and GD2-negative cancer cell lines, for expression levels of genes encoding enzymes involved in ganglioside biosynthesis. We identified a 2-gene expression signature combining ganglioside synthase genes ST8SIA1 and B4GALNT1 that serves as a more efficient predictor of GD2-positive phenotype (Matthews Correlation Coefficient (MCC) 0.32, 0.88, and 0.98 in three independent comparisons) compared to the individual ganglioside biosynthesis genes (MCC 0.02–0.32, 0.1–0.75, and 0.04–1 for the same independent comparisons). No individual gene showed a higher MCC score than the expression signature MCC score in two or more comparisons. Our diagnostic approach can hopefully be applied for pan-cancer prediction of GD2 phenotypes using gene expression data.

Protein Expression in Metastatic Melanoma and the Link to Disease Presentation in a Range of Tumor Phenotypes

Malignant melanoma is among the most aggressive skin cancers and it has among the highest metastatic potentials. Although surgery to remove the primary tumor is the gold standard treatment, once melanoma progresses and metastasizes to the lymph nodes and distal organs, i.e., metastatic melanoma (MM), the usual outcome is decreased survival. To improve survival rates and life span, advanced treatments have focused on the success of targeted therapies in the MAPK pathway that are based on BRAF (BRAF V600E) and MEK. The majority of patients with tumors that have higher expression of BRAF V600E show poorer prognosis than patients with a lower level of the mutated protein. Based on the molecular basis of melanoma, these findings are supported by distinct tumor phenotypes determined from differences in tumor heterogeneity and protein expression profiles. With these aspects in mind, continued challenges are to: (1) deconvolute the complexity and heterogeneity of MM; (2) identify the signaling pathways involved; and (3) determine protein expression to develop targeted therapies. Here, we provide an overview of the results from protein expression in MM and the link to disease presentation in a variety of tumor phenotypes and how these will overcome the challenges of clinical problems and suggest new promising approaches in metastatic melanoma and cancer therapy.

Molecular underpinnings of RB status as a biomarker of poor outcome in advanced prostate cancer.

189 Background: There is emergent and compelling evidence to support RB status as a biomarker in advanced prostate cancer. RB loss is strongly associated with poor progression-free, disease-specific, and overall survival in prostate cancer (PCa). Preclinical studies in PCa have revealed RB positive tumors are more responsive to CDK4/6 inhibitors. An ongoing randomized Phase IB/II study of enzalutamide with and without ribociclib in patients with metastatic castration-resistant, chemotherapy naïve PCa has become a pioneer trial to include a positive RB status as inclusion criteria in a PCa study (NCT02555189). Beyond CDK4/6 inhibitors, therapeutic agents that target tumor metabolism have been introduced in the clinic. Current data suggests that RB status may be crucial to understand and predict therapeutic response to these agents within tumors. Methods: The biological significance of RB loss was studied utilizing isogenic model systems and human tumor xenografts of castration resistant prostate cancer (CRPC) with and without RB deletion. The mechanism that drives aggressive tumor phenotypes was identified through comprehensive transcriptomic, cistromic, and metabolomic analysis. Novel functions of RB were identified and the response to clinically-relevant therapeutics was examined. Results: Exclusively in CRPC, RB loss results in significant rewiring of cancer cell metabolism. Functional investigation revealed a causative link between RB loss and antioxidant production sufficient to alter responsiveness to genomic insult and selected chemotherapeutics. Observed changes in response to therapeutic intervention were attributed to RB-dependent modulation of intracellular reactive oxygen species. Conclusions: RB loss is strongly associated with poor outcome in advanced PCa. Molecular investigation identified RB-dependent rewiring of cancer cell metabolism as a significant consequence of RB loss, sufficient to alter response in model systems to therapeutic strategies of clinical relevance. These studies significantly advance understanding of the means by which RB loss enhances lethal tumor phenotypes, and are of relevance for development of RB status as a clinically actionable biomarker.