Current Advances in Clinical Application of Liquid Biopsy

Liquid biopsy solutions are available for niche clinical applications. The patient benefits of such solutions are evident: ease of sampling, acceptable and repeatable. To date a number of solutions have received regulatory approval with more comprehensive, multi-cancer companion diagnostic approaches receiving approval in late 2020. Given these breakthrough advances and the ongoing clinical studies in early detection of cancer, the liquid biopsy field is making strides in technology. While circulating tumour DNA (ctDNA) solutions are quickly penetrating the market, strides in circulating tumour cells (CTC) and extracellular vesicles (EV) technologies is unlocking their potential for liquid biopsy. ctDNA solutions are paving the way towards clinical translation into the distinct applications across the cancer continuum. This chapter presents a detailed review of current approved liquid biopsy tests and provides a summary of advanced-stage prospective technologies within the context of distinctive clinical applications

The re-emergence of the B1 cell compartment : is this a pre-lymphoma stage?

Chronic Lymphocytic Leukemia (CLL) are in some cases stereotyped for immunoglobulin variants in different populations, suggesting emergence of B cell subsets following presentation of the same antigen. CLL cells may originate from CD5+ naïve cells and from CD5 memory cells. Gene expression studies characterized a common cell of origin of the two clinical categories of CLL; the unmutated aggressive type and the mutated indolent type. The aim of this study was to investigate the presence of CD5 positive B cells in the elderly and their potential stimulation with exosomes derived from tumor cells. The findings from this study is aimed to create a model to identify instigating carcinomatous factors that may stimulate B1 cells to transform into a CLL-like model. In this study we show that CD19\textsuperscript+ cells (B cells) in cord blood have a high expression of CD5. CD19/CD5 staining of blood samples from senior citizens showed the presence of B cells which also express the CD5 marker, though at a lower expression when compared to CLL cells (CD19+/CD5 dim B cells). Measurement of clonality using λ/Κ flow cytometry staining show a monoclonal origin of the human CD19+/CD5 dim B cells. Monoclonal B cell Lymphocytosis in the elderly is a potential cell compartment that represents the origin of B cell proliferative disorders. The origin of the B cell proliferative disease requires antigen stimulation. A preliminary experiment showed that sorted lymphocytes can be stimulated by exosomes isolated from 2 cancer cells lines, A549 (lung epithelial) and PC3 (prostate cell line). In comparison with phytohaemagglutinin (PHA) and phorbolmyristate acetate (PMA), known lymphocyte stimulators, the exosomes stimulated the proliferation of monocytic-like cells. Further characterization is required to know the origin of these cells. The result shows that one can speculate that exosomes present cancer-derived antigens and stimulate cell proliferation. Further studies are required to evaluate the potential transformation capacity of cancer-derived exosomes. In addition, various cytokines were measured in the sera of senior citizens to investigate a differential release of cytokines in the presence or absence of the CD19+/CD5 dim B cells. Cytokines examined were not significantly different between the 2 groups and further evaluation of cytokine levels is required.peer-reviewe

Phospho-Akt expression is high in a subset of triple negative breast cancer patients

The most commonly used biomarkers to predict the response of breast cancer patients to therapy are oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as anti-oestrogen therapy in the event of ER and PgR positivity, and trastuzumab, a monoclonal antibody, in the case of HER2 positive patients. Patients who are negative for all these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies. The PI3K/Akt pathway is activated in triple negative breast cancer cases, providing a possible target for therapy. The activation of Akt was investigated in Maltese triple negative breast cancer cases using an antibody detecting Akt phosphorylated at serine 473 (anti-Akt pS473). The study showed that 26\% of triple negative breast cancer patients had an elevated level of Akt (pS473). Furthermore, FTY720, a pharmacological activator of the phosphatase PP2A, was shown to block Akt activation at a concentration of 1\textmu M, in HCC1937 cells subjected to insulin-like growth factor 1 (IGF-1). Our data defined a subset of triple negative breast cancer patients based on high activity of AKT (pS473). This subset would be eligible for treatment using therapies which target the PI3K/Akt pathway, such as kinase inhibitors or phosphatase activators. In support of this, the BRCA1 mutant cells (HCC1937) were sensitive to the PP2a activator, FTY720. This suggests that FTY720 is a potential drug for use in adjuvant therapy in breast cancer cases having a high Akt (pS473).peer-reviewe

Molecular classification of breast cancer patients using formalin-fixed paraffin-embedded derived RNA samples

The use of archival formalin-fixed paraffin-embedded (FFPE) material to analyse gene expression is limited by the low quality of extracted RNA. In this paper, we utilised an RNA based assay to quantify expression of luminal and basal markers, together with ERBB2 probes, in FFPE archival tissue from 2009 to 2010, all of which had clinical and therapeutic information of more than 5 years. Receptor status of the patients was characterised using the QuantiGene® Plex assay with 100% concordance to immunohistochemical (IHC) and fluorescence in situ hybridisation (FISH) results. A panel of molecular markers known to classify luminal and basal tumours were used and correlated with receptor status of the tumours. As expected, the triple negative breast cancer (TNBC) samples were classified as basal and oestrogen receptor (ER) positive cases as luminal. In summary, the QuantiGene® Plex technology provides a platform to quantitate novel panels of biomarkers on archival material. Moreover, multiplex analysis allows the use of minimal amounts of material providing an opportunity to utilise laser micro-dissected material. FFPE tissue samples are an invaluable resource for retrospective studies to interrogate current novel biomarkers, particularly to generate disease free survival and overall survival graphs to measure predictive value using well annotated retrospective samples with full clinical and pharmacological outcomes.This study was funded by the Faculty of Medicine and Surgery, University of Malta and the Italia Malta Genome Breast Cancer Cross Border Risk Surveillance (ImaGenX) project financed under Operational Program Italia Malta 2007-2013 and co-financed by the University of Malta.peer-reviewe

Deregulation of the phosphatase, PP2A is a common event in breast cancer, predicting sensitivity to FTY720

We would like to thank Professor Andrew Hanby, Professor Valerie Speirs and Dr Thomas A Hughes for the breast cancer cell lines used in this study. This research was supported by the Faculty of Medicine and Surgery, University of Malta.Background: The most commonly used biomarkers to predict the response of breast cancer patients to therapy are the oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as endocrine treatment in the event of ER and PgR positivity, and the monoclonal antibody, trastuzumab, in the case of HER2-positive patients. Patients who are negative for these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. Deregulation of the protein serine/threonine phosphatase type 2A (PP2A) and its regulatory subunits is a common event in breast cancer, providing a possible target for therapy. Methods: The data portal, cBioPortal for Cancer Genomics was used to investigate the incidence of conditions that are associated with low phosphatase activity. Four (4) adherent human breast cancer cell lines, MDA-MB-468, MDA-MB-436, Hs578T and BT-20 were cultured to assess their viability when exposed to various dosages of rapamycin or FTY720. In addition, RNA was extracted and cDNA was synthesised to amplify the coding sequence of PPP2CA. Amplification was followed by high-resolution melting to identify variations. Results and conclusion: The sequence of PPP2CA was found to be conserved across a diverse panel of solid tumour and haematological cell lines, suggesting that low expression of PPP2CA and differential binding of inhibitory PPP2CA regulators are the main mechanisms of PP2A deregulation. Interestingly, the cBioPortal for Cancer Genomics shows that PP2A is deregulated in 59.6% of basal breast tumours. Viability assays performed to determine the sensitivity of a panel of breast cancer cell lines to FTY720, a PP2A activator, indicated that cell lines associated with ER loss are sensitive to lower doses of FTY720. The subset of patients with suppressed PP2A activity is potentially eligible for treatment using therapies which target the PI3K/AKT/mTOR pathway, such as phosphatase activators.peer-reviewe

Predictive markers in cancer patient diagnosis, classification and prediction of therapy outcome using leukaemia as a model

The characterisation of the molecular mechanism of disease allows classification of patients into subtypes and potentially identifies specific targets for therapeutic intervention. Tyrosine kinase mutations are central to specific targeted therapy. Investigation of kinase deregulation within particular patient groups, has led to identification of mutant tyrosine kinases associated with disease progression and therapy modulation. Biomarker-specific therapies emerged, taking a leading role in guided-therapy. The extensive use of the specific kinase inhibitors and the longevity of the treatment protocols due to the uncertainty of residual disease, gave rise to new challenges, namely secondary resistance to therapy. Although there are various mechanisms of acquired resistance, mutations in the drug target itself play a dominant role.peer-reviewe

Biomarkers predicting sensitivity to PP2A activation therapy in cancer

The invention relates to the treatment and classification of cancer, in particular breast cancer. It relates to identifying patients who are likely to respond to cancer therapy with a PP2 A activator. Background: The provision of new treatments for cancer is of high importance, including for cancers resistant to known treatments. There is also a need for identification of markers that allow for detection, prognosis and classification of cancer, and which can predict responsiveness of a patient to a given therapy. The deregulation of the protein phosphatase 2A (PP2A) complex is known to be a common event in cancer [Grech et al; Tumor Biol; DOl 10.1007/s13277-016-5145-4 (2016)].peer-reviewe

Molecular profiling of breast cancer intra-tumor heterogeneity for the development of novel biomarkers

Tumor heterogeneity often challenges cancer diagnosis. In addition, a lack of fresh frozen tissue specimens and nucleic acid degradation in archival tissue can negatively impact cancer diagnostics. However, as tumor heterogeneity is a known mechanism for the development of drug resistance, it is essential to characterize heterogeneous tumor tissue. Moreover, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation. In this study, our research team optimized quantification of RNA in archival material. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes. Heterogeneous tumors were subjected to laser microdissection using the MMI CellCut system to separate morphologically different tissue areas for subsequent expression analysis.peer-reviewe

Novel missense mutation in the phosphatase PP2A catalytic subunit characterised in a solid tumour cell line

Introduction: The protein serine/threonine phosphatase type 2A (pp2a) is a heterotrimer complex composed of a catalytic subunit (pp2aC), the scaffolding A subunit (pp2aA), and the variable B regulatory subunit. The pp2a complex attenuates the phosphorylation of various signals involved in cell survival and proliferation. Missense mutations in the coding sequence of the PPP2CA catalytic subunit were previously described, and studies of the pathological effects require further investigation. Of interest low pp2a activity was described as a potential mechanism of Chronic Myeloid Leukaemia blast crisis, hence the use of the CML cell line, K562, as a model of pp2a studies. Increasing evidence show that decreased pp2a activity is implicated in the progression of multiple tumour types and provides a potential therapeutic target.peer-reviewe

Amiloride induces alternative splicing of the PP2Acα mRNA in haematopoietic cell lines

Introduction: Targeting BCRABL1 by imatinib has proven successful for the treatment of Chronic Myeloid Leukemia (CML) patients. Nonetheless, a cumulative 5year failure rate of 17% still exists due to therapeutic resistance. Increased expression of BCRABL1 inhibits PP2A activity, promoting survival and proliferation. Previous studies established that an isoform of the catalytic subunit a of PP2A (PP2Aca) is predominant in 15% of CML. This study investigates alternate splicing of PP2Aca as a novel potential mechanism of therapeutic resistance in CML.peer-reviewe