Prashanth Prithviraj

Efficacy and toxicity of treatment with the anti-CTLA-4 antibody ipilimumab in patients with metastatic melanoma after prior anti-PD-1 therapy

Background:Recent phase III clinical trials have established the superiority of the anti-PD-1 antibodies pembrolizumab and nivolumab over the anti-CTLA-4 antibody ipilimumab in the first-line treatment of patients with advanced melanoma. Ipilimumab will be considered for second-line treatment after the failure of anti-PD-1 therapy.Methods:We retrospectively identified a cohort of 40 patients with metastatic melanoma who received single-agent anti-PD-1 therapy with pembrolizumab or nivolumab and were treated on progression with ipilimumab at a dose of 3 mg kg−1 for a maximum of four doses.Results:Ten percent of patients achieved an objective response to ipilimumab, and an additional 8% experienced prolonged (>6 months) stable disease. Thirty-five percent of patients developed grade 3–5 immune-related toxicity associated with ipilimumab therapy. The most common high-grade immune-related toxicity was diarrhoea. Three patients (7%) developed grade 3–5 pneumonitis leading to death in one patient.Conclusions:Ipilimumab therapy can induce responses in patients who fail the anti-PD-1 therapy with response rates comparable to previous reports. There appears to be an increased frequency of high-grade immune-related adverse events including pneumonitis that warrants close surveillance.

Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells

BackgroundThe metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell’s phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype.MethodsThe expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2’-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells.ResultsExpression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells.ConclusionsOur data provide evidence for an important role of TKTL1 in aerobic glycolysis and tumor promotion in melanoma that may result from defective promoter methylation. This epigenetic change may enable the natural selection of tumor cells with a metabolic phenotype and thereby provide a potential therapeutic target for a subset of melanoma tumors with elevated TKTL1 expression.

Identifying and targeting determinants of melanoma cellular invasion

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients.

Embryonic Chicken Transplantation is a Promising Model for Studying the Invasive Behavior of Melanoma Cells

Epithelial-to-mesenchymal transition is a hallmark event in the metastatic cascade conferring invasive ability to tumor cells. There are ongoing efforts to replicate the physiological events occurring during mobilization of tumor cells in model systems. However, few systems are able to capture these complex in vivo events. The embryonic chicken transplantation model has emerged as a useful system to assess melanoma cells including functions that are relevant to the metastatic process, namely invasion and plasticity. The chicken embryo represents an accessible and economical 3-dimensional in vivo model for investigating melanoma cell invasion as it exploits the ancestral relationship between melanoma and its precursor neural crest cells. We describe a methodology that enables the interrogation of melanoma cell motility within the developing avian embryo. This model involves the injection of melanoma cells into the neural tube of chicken embryos. Melanoma cells are labeled using fluorescent tracker dye, Vybrant DiO, then cultured as hanging drops for 24 h to aggregate the cells. Groups of approximately 700 cells are placed into the neural tube of chicken embryos prior to the onset of neural crest migration at the hindbrain level (embryonic day 1.5) or trunk level (embryonic day 2.5). Chick embryos are reincubated and analyzed after 48 h for the location of melanoma cells using fluorescent microscopy on whole mounts and cross-sections of the embryos. Using this system, we compared the in vivo invasive behavior of epithelial-like and mesenchymal-like melanoma cells. We report that the developing embryonic microenvironment confers motile abilities to both types of melanoma cells. Hence, the embryonic chicken transplantation model has the potential to become a valuable tool for in vivo melanoma invasion studies. Importantly, it may provide novel insights into and reveal previously unknown mediators of the metastatic steps of invasion and dissemination in melanoma.

Updated efficacy and toxicity of treatment with the anti-CTLA-4 antibody ipilimumab in metastatic melanoma patients previously treated with anti-PD-1 therapy

Meeting abstracts Immunotherapy with anti-CTLA-4 and anti-PD-1 antibodies has demonstrated overall survival benefits in patients (pts) with metastatic melanoma (MM) compared to previous standard therapy. Two randomised clinical trials indicate that combined anti-CTLA-4 and anti-PD-1 antibody

Exosome-based liquid biopsy in the management of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) commonly presents at an advanced stage due to the lack of efficient early screening tools. Early, non-invasive biomarkers useful in the diagnosis and prognosis of HCC would be of significant benefit for HCC management. Development of exosome-based liquid biopsy as a non-invasive method for the management of HCC has gained much traction. Exosomes are small membranous vesicles secreted by most cell types including HCC cells. Exosomes serve as couriers for the intercellular transfer of important biomolecules, including, protein, nucleic acids and lipids to nearby and distant cells in the body. The molecular cargos carried by exosome have been described to play significant roles in cancer progression. Herein, we will dissect how HCC-derived exosomes confer aggressive traits such as tumour growth, invasion, immune remodelling and drug resistance to HCC cells. We review the current literature concerning exosomes as biomarkers in a diagnostic setting, evaluating their prognostic, predictive and monitoring capabilities. This review will highlight and discuss emerging research in the utility of exosome-based liquid biopsies therapeutic tools in HCC management. Here we will also focus on advances in exosome biology in preclinical studies.

Monitoring Immune Checkpoint Regulators as Predictive Biomarkers in Hepatocellular Carcinoma

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3, and VISTA were independently associated with worse prognosis, while B7-H4, CD73, and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73, and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB.

Reply to 'Comment on 'Efficacy and toxicity of treatment with the anti-CTLA-4 antibody ipilimumab in patients with metastatic melanoma after prior anti-PD-1 therapy''.

Reply to ‘Comment on ‘Efficacy and toxicity of treatment with the anti-CTLA-4 antibody ipilimumab in patients with metastatic melanoma after prior anti-PD-1 therapy’’