Marzena Anna Lewandowska

Prognostic and Predictive Biomarkers: Tools in Personalized Oncology

Oncology indispensably leads us to personalized medicine, which allows an individual approach to be taken with each patient. Personalized oncology is based on pharmacogenomics and the effect of genetic differences in individuals (germline and somatic) on the way cancer patients respond to chemotherapeutics. Biomarkers detected using molecular biology tools allow the molecular characterization of cancer signatures and provide information relevant for personalized treatment. Biomarkers can be divided into two main subgroups: prognostic and predictive. The aim of the application of prognostic biomarkers, which provide information on the overall cancer outcome in patients, is to facilitate cancer diagnosis, usually with no need for putting invasive methods into use. Predictive biomarkers help to optimize therapy decisions, as they provide information on the likelihood of response to a given chemotherapeutic. Among the prognostic factors that identify patients with different outcome risks (e.g., recurrence of the disease), the following factors can be distinguished: somatic and germline mutations, changes in DNA methylation that lead to the enhancement or suppression of gene expression, the occurrence of elevated levels of microRNA (miRNA) capable of binding specific messenger RNA (mRNA) molecules, which affects gene expression, as well as the presence of circulating tumor cells (CTCs) in blood, which leads to a poor prognosis for the patient. Biomarkers for personalized oncology are used mainly in molecular diagnostics of chronic myeloid leukemia, colon, breast and lung cancer, and recently in melanoma. They are successfully used in the evaluation of the benefits that can be achieved through targeted therapy or in the evaluation of toxic effects of the chemotherapeutic used in the therapy.

Application of PCR methods to evaluate EGFR, KRAS and BRAF mutations in a small number of tumor cells in cytological material from lung cancer patients.

The epidermal growth factor receptor (EGFR) mutation status in the tyrosine kinase domain is known to be a predictor of the response to gefitinib or erlotinib in lung cancer; thus, a non-surgical procedure of tumor specimen collection is critical for mutation analysis. The aim of the present study was to analyze the EGFR, KRAS and BRAF status in limited cytological material. To the best of our knowledge, this is the first time that the quantitative scale of tumor cells and the percentage of tumor cells in cytological material were evaluated at the early stages of pathomorphological material qualification for EGFR, KRAS and BRAF mutation analysis. Our results revealed that even 100–1,000 tumor cells from fine needle aspiration (FNA) samples provided reliable results of mutation analysis when sensitive real-time polymerase chain reaction (PCR) methods were used. EGFR mutations were detected in 10% (7/71) and KRAS mutations were detected in 35% (19/54) of the lung adenocarcinoma cases. In addition, we reported the most common inhibiting mutation (p.T790M) found in coexistence with p.L858R in an FNA sample from a patient, for whom short-term improvement after erlotinib treatment was observed before further progression of the disease. Subsequently, mutual exclusion of EGFR and KRAS mutations was observed. Cytological samples with a small number of tumor cells obtained via FNA, endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) or brushing are suggested to be used for diagnostic purposes after careful selection by cytopathologists and analysis using a validated, sensitive real-time PCR method.

The Use of a Two-Tiered Testing Strategy for the Simultaneous Detection of Small EGFR Mutations and EGFR Amplification in Lung Cancer

Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.

Detection of somatic BRCA1/2 mutations in ovarian cancer – next-generation sequencing analysis of 100 cases

The overall prevalence of germline BRCA1/2 mutations is estimated between 11% and 15% of all ovarian cancers. Individuals with germline BRCA1/2 alterations treated with the PARP1 inhibitors (iPARP1) tend to respond better than patients with wild‐type BRCA1/2. Additionally, also somatic BRCA1/2 alterations induce the sensitivity to iPARP1. Therefore, the detection of both germline and somatic BRCA1/2 mutations is required for effective iPARP1 treatment. The aim of this study was to identify the frequency and spectrum of germline and somatic BRCA1/2 alterations in a group of Polish patients with ovarian serous carcinoma. In total, 100 formalin‐fixed paraffin‐embedded (FFPE) ovarian serous carcinoma tissues were enrolled to the study. Mutational analysis of BRCA1/2 genes was performed by using next‐generation sequencing. The presence of pathogenic variants was confirmed by Sanger sequencing. In addition, to confirm the germline or somatic status of the mutation, the nonneoplastic tissue was analyzed by bidirectional Sanger sequencing. In total, 27 (28% of patient samples) mutations (20 in BRCA1 and 7 in BRCA2) were identified. For 22 of 27 patients, nonneoplastic cells were available and sequencing revealed the somatic character of two BRCA1 (2/16; 12.5%) and two BRCA2 (2/6; 33%) mutations. Notably, we identified six novel frameshift or nonsense BRCA1/2 mutations. The heterogeneity of the detected mutations confirms the necessity of simultaneous analysis of BRCA1/2 genes in all patients diagnosed with serous ovarian carcinoma. Moreover, the use of tumor tissue for mutational analysis allowed the detection of both somatic and germline BRCA1/2 mutations.

Can chromatin conformation technologies bring light into human molecular pathology

Regulation of gene expression in eukaryotes involves many complex processes, in which chromatin structure plays an important role. In addition to the epigenetic effects, such as DNA methylation and phosphorylation or histone modifications, gene expression is also controlled by the spatial organization of chromatin. For example, distant regulatory elements (enhancers, insulators) may come into direct physical interaction with target genes or other regulatory elements located in genomic regions of up to several hundred kilobases in size. Such long-range interactions result in the formation of chromatin loops. In the last several years, there has been a rapid increase in our knowledge of the spatial organization of chromatin in the nucleus through the chromosome conformation capture (3C) technology. Here we review and compare the original 3C and 3C-based methods including chromosome conformation capture-on-chip (4C), chromosome conformation capture carbon copy (5C), hi-resolution chromosome confomation capture (HiC). In this article, we discuss different aspects of how the nuclear organization of chromatin is associated with gene expression regulation and how this knowledge is useful in translational medicine and clinical applications. We demonstrate that the knowledge of the chromatin 3D organization may help understand the mechanisms of gene expression regulation of genes involved in the development of human diseases, such as CFTR (responsible for cystic fibrosis) or IGFBP3 (associated with breast cancer pathogenesis). Additionally, 3C-derivative methods have been also useful in the diagnosis of some leukemia subtypes.

Transdifferentiation of Bone Marrow Mesenchymal Stem Cells into the Islet-Like Cells: the Role of Extracellular Matrix Proteins

Pancreatic islet implantation has been recently shown to be an efficient method of treatment for type 1 diabetes. However, limited availability of donor islets reduces its use. Bone morrow would provide potentially unlimited source of stem cells for generation of insulin-producing cells. This study was performed to evaluate the influence of extracellular matrix proteins like collagen, laminin, and vitronectin on bone marrow mesenchymal stem cells (BM-MSCs) transdifferentiation into islet-like cells (ILCs) in vitro. To our knowledge, this is the first report evaluating the importance of vitronectin in transdifferentiation of BM-MSCs into ILCs. Rat BM-MSCs were induced to ILCs using four-step protocol on plates coated with collagen type IV, laminin type I and vitronectin type I. Quantitative real-time PCR was performed to detect gene expression related to pancreatic β cell development. The induced cells expressed islet-related genes including: neurogenin 3, neurogenic differentiation 1, paired box 4, NK homeobox factor 6.1, glucagon, insulin 1 and insulin 2. Laminin but not collagen type IV or vitronectin enhanced expression of insulin and promoted formation of islet-like structures in monolayer culture. Laminin triggered transdifferentiation of BM-MSCs into ILCs.

Potential Role of Methylation Marker in Glioma Supporting Clinical Decisions

The IDH1/2 gene mutations, ATRX loss/mutation, 1p/19q status, and MGMT promoter methylation are increasingly used as prognostic or predictive biomarkers of gliomas. However, the effect of their combination on radiation therapy outcome is discussable. Previously, we demonstrated that the IDH1 c.G395A; p.R132H mutation was associated with longer survival in grade II astrocytoma and GBM (Glioblastoma). Here we analyzed the MGMT promoter methylation status in patients with a known mutation status in codon 132 of IDH1, followed by clinical and genetic data analysis based on the two statuses. After a subtotal tumor resection, the patients were treated using IMRT (Intensity-Modulated Radiation Therapy) with 6 MeV photons. The total dose was: 54 Gy for astrocytoma II, 60 Gy for astrocytoma III, 60 Gy for glioblastoma, 2 Gy per day, with 24 h intervals, five days per week. The patients with MGMT promoter methylation and IDH1 somatic mutation (OS = 40 months) had a better prognosis than those with MGMT methylation alone (OS = 18 months). In patients with astrocytoma anaplasticum (n = 7) with the IDH1 p.R132H mutation and hypermethylated MGMT, the prognosis was particularly favorable (median OS = 47 months). In patients with astrocytoma II meeting the above criteria, the prognosis was also better than in those not meeting those criteria. The IDH1 mutation appears more relevant for the prognosis than MGMT methylation. The IDH1 p.R132H mutation combined with MGMT hypermethylation seems to be the most advantageous for treatment success. Patients not meeting those criteria may require more aggressive treatments.

IDH1 mutation analysis – an example of putative glioma marker

The astrocytoma cancer represents CNS neoplasms in which the predominant cell type is derived from an immortalized astrocyte. The genomewide analysis of glioma identified somatic mutation at codon 132 of the IDH1 gene which encodes NADP+ dependent isocitrate dehydrogenase. Further studies indicated that patients with somatic, heterozygous R132H mutation have distinct clinical characteristic: younger age at astrocytoma diagnosis (WHO II and WHO III) and improved clinical prognosis. Location of the majority of point mutations in the IDH1 gene are localized at 132 codon - what simplifies the use of this mutation for potential diagnostic purposes. The presence of R132H IDH1 mutation was analysed in group of 38 patients diagnosed with: fibrillar astrocytoma, astrocytoma gemistocyticum, astrocytoma pilocyticum and astrocytoma anaplasticum. The IDH1 mutation status was determinated by immunohistochemistry using monoclonal antibody specific for the R132H mutation. Additional data verification was performed by HRM Cold-PCR and Sanger sequencing. For statistical evaluation we distinguished two subgroups of patients: with and without IDH1 R132H mutation. Presence of IDH1 mutation in Polish astrocytomas’ patients correlates with better clinical outcome and longer median overall survival. Our findings confirm overall tendency for better survival benefits in patients with IDH1 mutated tumors and indicates that presence or absence of IDH1 mutant proteins may become a potential target in personalized medicine.