Ligand-directed targeting of lymphatic vessels uncovers mechanistic insights in melanoma metastasis.

Metastasis is the most lethal step of cancer progression in patients with invasive melanoma. In most human cancers, including melanoma, tumor dissemination through the lymphatic vasculature provides a major route for tumor metastasis. Unfortunately, molecular mechanisms that facilitate interactions between melanoma cells and lymphatic vessels are unknown. Here, we developed an unbiased approach based on molecular mimicry to identify specific receptors that mediate lymphatic endothelial-melanoma cell interactions and metastasis. By screening combinatorial peptide libraries directly on afferent lymphatic vessels resected from melanoma patients during sentinel lymphatic mapping and lymph node biopsies, we identified a significant cohort of melanoma and lymphatic surface binding peptide sequences. The screening approach was designed so that lymphatic endothelium binding peptides mimic cell surface proteins on tumor cells. Therefore, relevant metastasis and lymphatic markers were biochemically identified, and a comprehensive molecular profile of the lymphatic endothelium during melanoma metastasis was generated. Our results identified expression of the phosphatase 2 regulatory subunit A, α-isoform (PPP2R1A) on the cell surfaces of both melanoma cells and lymphatic endothelial cells. Validation experiments showed that PPP2R1A is expressed on the cell surfaces of both melanoma and lymphatic endothelial cells in vitro as well as independent melanoma patient samples. More importantly, PPP2R1A-PPP2R1A homodimers occur at the cellular level to mediate cell-cell interactions at the lymphatic-tumor interface. Our results revealed that PPP2R1A is a new biomarker for melanoma metastasis and show, for the first time to our knowledge, an active interaction between the lymphatic vasculature and melanoma cells during tumor progression.

Metabolomics biomarkers of hepatocellular carcinoma in a prospective cohort of patients with cirrhosis.

Background & Aims: The effectiveness of surveillance for hepatocellular carcinoma (HCC) in patients with cirrhosis is limited, due to inadequate risk stratification and suboptimal performance of current screening modalities. Methods: We developed a multicenter prospective cohort of patients with cirrhosis undergoing surveillance with MRI and applied global untargeted metabolomics to 612 longitudinal serum samples from 203 patients. Among them, 37 developed HCC during follow-up. Results: We identified 150 metabolites with significant abundance changes in samples collected prior to HCC (Cases) compared to samples from patients who did not develop HCC (Controls). Tauro-conjugated bile acids and gamma-glutamyl amino acids were increased, while acyl-cholines and deoxycholate derivatives were decreased. Seven amino acids including serine and alanine had strong associations with HCC risk, while strong protective effects were observed for N-acetylglycine and glycerophosphorylcholine. Machine learning using the 150 metabolites, age, gender, and PNPLA3 and TMS6SF2 single nucleotide polymorphisms, identified 15 variables giving optimal performance. Among them, N-acetylglycine had the highest AUC in discriminating Cases and Controls. When restricting Cases to samples collected within 1 year prior to HCC (Cases-12M), additional metabolites including microbiota-derived metabolites were identified. The combination of the top six variables identified by machine learning (alpha-fetoprotein, 6-bromotryptophan, N-acetylglycine, salicyluric glucuronide, testosterone sulfate and age) had good performance in discriminating Cases-12M from Controls (AUC 0.88, 95% CI 0.83-0.93). Finally, 23 metabolites distinguished Cases with LI-RADS-3 lesions from Controls with LI-RADS-3 lesions, with reduced abundance of acyl-cholines and glycerophosphorylcholine-related lysophospholipids in Cases. Conclusions: This study identified N-acetylglycine, amino acids, bile acids and choline-derived metabolites as biomarkers of HCC risk, and microbiota-derived metabolites as contributors to HCC development. Impact and implications: The effectiveness of surveillance for hepatocellular carcinoma (HCC) in patients with cirrhosis is limited. There is an urgent need for improvement in risk stratification and new screening modalities, particularly blood biomarkers. Longitudinal collection of paired blood samples and MRI images from patients with cirrhosis is particularly valuable in assessing how early blood and imaging markers become positive during the period when lesions are observed to obtain a diagnosis of HCC. We generated a multicenter prospective cohort of patients with cirrhosis under surveillance with contrast MRI, applied untargeted metabolomics on 612 serum samples from 203 patients and identified metabolites associated with risk of HCC development. Such biomarkers may significantly improve early-stage HCC detection for patients with cirrhosis undergoing HCC surveillance, a critical step to increasing curative treatment opportunities and reducing mortality.

Lysophosphatidic acid induces lymphangiogenesis and IL-8 production in vitro in human lymphatic endothelial cells.

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

Telomerase reverse transcriptase mutations in plasma DNA in patients with hepatocellular carcinoma or cirrhosis: Prevalence and risk factors.

Telomerase reverse transcriptase (TERT) mutation is the most frequent genetic alteration in hepatocellular carcinoma (HCC). Our aims were to investigate whether TERT mutations can be detected in circulating cell-free DNA (cfDNA) of patients with HCC and/or cirrhosis and characterize clinical parameters associated with these mutations. We retrieved data on TERT C228T and C250T promoter mutations in 196 HCCs from The Cancer Genome Atlas. We measured these TERT mutations in plasma cfDNA in 218 patients with HCC and 81 patients with cirrhosis without imaging evidence of HCC. The prevalence of TERT mutations in The Cancer Genome Atlas HCC specimens was 44.4%. TERT mutations were detected with similar prevalence (47.7%) in plasma cfDNAs from 218 patients with HCC. TERT mutations, either within the HCC or in cfDNA, were associated with male sex, hepatitis C virus (HCV), alcoholic cirrhosis, family history of cancer, and poor prognosis. The high prevalence of TERT mutations in HCCs in male patients with cirrhosis caused by HCV and/or alcohol was confirmed in an independent set of HCCs (86.6%). Finally, TERT mutations were detected in cfDNA of 7 out of 81 (8.6%) patients with cirrhosis without imaging evidence of HCC, including 5 male patients with cirrhosis due to HCV and/or alcohol. Genes involved in xenobiotic and alcohol metabolism were enriched in HCCs with TERT mutations, and vitamin K2 was identified as an upstream regulator. Conclusion: TERT mutations are detectable in plasma cfDNA. Long-term imaging surveillance of patients with cirrhosis with cfDNA TERT mutations without evidence of HCC is required to assess their potential as early biomarkers of HCC. (Hepatology Communications 2018;2:718-731).

Biological Validation of RNA Sequencing Data from Formalin-Fixed Paraffin-Embedded Primary Melanomas.

PURPOSE: Initiatives such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have generated high-quality, multi-platform molecular data from thousands of frozen tumor samples. While these initiatives have provided invaluable insight into cancer biology, a tremendous potential resource remains largely untapped in formalin-fixed, paraffin-embedded (FFPE) samples that are more readily available, but which can present technical challenges due to crosslinking of fragile molecules such as RNA. MATERIALS AND METHODS: We extracted RNA from FFPE primary melanomas and assessed two gene expression platforms -- genome-wide RNA sequencing (RNA-seq) and targeted NanoString -- for their ability to generate coherent biological signals. To do so, we generated an improved approach to quantifying gene expression pathways, in which we refine pathway scores through correlation-guided gene subsetting. We also make comparisons to the TCGA and other publicly available melanoma datasets. RESULTS: Comparison of the gene expression patterns to each other, to established biological modules, and to clinical and immunohistochemical data confirmed the fidelity of biological signals from both platforms using FFPE samples to known biology. Moreover, correlations with patient outcome data were consistent with previous frozen-tissue-based studies. CONCLUSION: FFPE samples from previously difficult-to-access cancer types - such as small primary melanomas - represents a valuable and previously unexploited source of analyte for RNA-seq and NanoString platforms. This work provides an important step towards the use of such platforms to unlock novel molecular underpinnings and inform future biologically-driven clinical decisions.

Tissue resources for clinical use and marker studies in melanoma.

The adequate procurement and preservation of high-quality tissue specimens from patients with melanoma is a critical clinical issue as patients' tumor samples are now used not only for pathological diagnosis but are also necessary to determine the molecular signature of the tumor to stratify patients who may benefit from targeted melanoma therapy. Tissue resources available for physicians and investigators include formalin-fixed paraffin-embedded (FFPE) tissue and frozen tissue, either preserved in optimal cutting temperature (OCT) media or snap frozen. Properly preserved tissue may be used to evaluate melanoma biomarkers by immunohistochemistry (IHC) with tissue microarray (TMA) technology, to perform genetic and genomic analyses, and for other types of translational research in melanoma.