High BCR::ABL1 Expression Defines CD34+ Cells with Significant Alterations in Signal Transduction, Short-Proliferative Potential and Self-Renewal Ability.

Purpose: Chronic Myeloid Leukemia (CML) is a clonal disorder of the hematopoietic stem cell caused by expression of the BCR::ABL1 oncoprotein. High BCR::ABL1 levels have been associated to proliferative advantage of leukemic cells, blast crisis progression and tyrosine kinase inhibitors (TKIs) inefficacy. We have previously shown that high BCR::ABL1/GUSIS transcripts measured at diagnosis are associated with inferior responses to standard dose Imatinib (IM). However, the mechanisms underlying the higher rates of disease progression and development of TKIs resistance dependent on elevated BCR::ABL1 levels remain unclear. Methods: Leukemic cells were collected from CML patients showing, at diagnosis, high or low BCR::ABL1/GUSIS. BCR::ABL1 expression levels were measured using real-time PCR. Short-term culture and long-term culture-initiating cells assays were employed to investigate the role of BCR::ABL1 gene-expression levels on proliferation, clonogenicity, signal transduction, TKIs responsiveness and self-renewal ability. Cell division was performed by carboxyfluorescein-succinimidyl ester (CFSE) assay. Results: We found that BCR::ABL1 oncogene expression levels correlate in both PMNs and CD34+ cells. Furthermore, high oncogene levels increased both proliferation and anti-apoptotic signaling via ERK and AKT phosphorylation. Moreover, high BCR::ABL1 expression reduced the clonogenicity of leukemic CD34+ cells and increased their sensitivity to high doses IM but not to those of dasatinib. Furthermore, we observed that high BCR::ABL1 levels are associated with a reduced self-renewal of primitive leukemic cells and, also, that these cells showed comparable TKIs responsiveness with cells expressing lower BCR::ABL1 levels. Interestingly, we found a direct correlation between high BCR::ABL1 levels and reduced number of quiescent leukemic cells caused by increasing their cycling. Conclusion: Higher BCR::ABL1 levels improving the proliferation, anti-apoptotic signaling and reducing self-renewal properties cause an increased expansion of leukemic clone.

Single-Cell Analysis in the Omics Era: Technologies and Applications in Cancer.

Cancer molecular profiling obtained with conventional bulk sequencing describes average alterations obtained from the entire cellular population analyzed. In the era of precision medicine, this approach is unable to track tumor heterogeneity and cannot be exploited to unravel the biological processes behind clonal evolution. In the last few years, functional single-cell omics has improved our understanding of cancer heterogeneity. This approach requires isolation and identification of single cells starting from an entire population. A cell suspension obtained by tumor tissue dissociation or hematological material can be manipulated using different techniques to separate individual cells, employed for single-cell downstream analysis. Single-cell data can then be used to analyze cell-cell diversity, thus mapping evolving cancer biological processes. Despite its unquestionable advantages, single-cell analysis produces massive amounts of data with several potential biases, stemming from cell manipulation and pre-amplification steps. To overcome these limitations, several bioinformatic approaches have been developed and explored. In this work, we provide an overview of this entire process while discussing the most recent advances in the field of functional omics at single-cell resolution.

Impact of Different Cell Counting Methods in Molecular Monitoring of Chronic Myeloid Leukemia Patients.

BACKGROUND: Detection of BCR-ABL1 transcript level via real-time quantitative-polymerase-chain reaction (Q-PCR) is a clinical routine for disease monitoring, assessing Tyrosine Kinase Inhibitor therapy efficacy and predicting long-term response in chronic myeloid leukemia (CML) patients. For valid Q-PCR results, each stage of the laboratory procedures need be optimized, including the cell-counting method that represents a critical step in obtaining g an appropriate amount of RNA and reliable Q-PCR results. Traditionally, manual or automated methods are used for the detection and enumeration of white blood cells (WBCs). Here, we compared the performance of the manual counting measurement to the flow cytometry (FC)-based automatic counting assay employing CytoFLEX platform. METHODS: We tested five different types of measurements: one manual hemocytometer-based count and four FC-based automatic cell-counting methods, including absolute, based on beads, based on 7-amino actinomycin D, combining and associating beads and 7AAD. The recovery efficiency for each counting method was established considering the quality and quantity of total RNA isolated and the Q-PCR results in matched samples from 90 adults with CML. RESULTS: Our analyses showed no consistent bias between the different types of measurements, with comparable number of WBCs counted for each type of measurement. Similarly, we observed a 100% concordance in the amount of RNA extracted and in the Q-PCR cycle threshold values for both BCR-ABL1 and ABL1 gene transcripts in matched counted specimens from all the investigated groups. Overall, we show that FC-based automatic absolute cell counting has comparable performance to manual measurements and allows accurate cell counts without the use of expensive beads or the addition of the time-consuming intercalator 7AAD. CONCLUSIONS: This automatic method can replace the more laborious manual workflow, especially when high-throughput isolations from blood of CML patients are needed.

A Novel System for Semiautomatic Sample Processing in Chronic Myeloid Leukaemia: Increasing Throughput without Impacting on Molecular Monitoring at Time of SARS-CoV-2 Pandemic.

Molecular testing of the BCR-ABL1 transcript via real-time quantitative-polymerase-chain-reaction is the most sensitive approach for monitoring the response to tyrosine-kinase-inhibitors therapy in chronic myeloid leukaemia (CML) patients. Each stage of the molecular procedure has been standardized and optimized, including the total white blood cells (WBCs) and RNA isolation methods. Here, we compare the performance of our current manual protocol to a newly semiautomatic method based on the Biomek i-5 Automated Workstations integrated with the CytoFLEX Flow Cytometer, followed by the automatic QIAsymphony system to facilitate high-throughput processing samples and reduce the hands-on time and the risk associated with SARS-CoV-2. The recovery efficiency was investigated in blood samples from 100 adults with CML. We observe a 100% of concordance between the two methods, with similar total WBCs isolated (median 1.137 × 106 for manual method vs. 1.076 × 106 for semiautomatic system) and a comparable quality and quantity of RNA extracted (median 103 ng/µL with manual isolation kit vs. 99.95 ng/µL with the QIAsymphony system). Moreover, by stratifying patients according to their BCR-ABL1 transcript levels, we obtained similar BCR-ABL1/ABL1IS values and ABL1 copies, and matched samples were assigned to the same group of molecular response. We conclude that this newly semiautomatic workflow has a performance comparable to our more laborious standard manual, which can be replaced, particularly when specimens from patients with suspected or confirmed SARS-CoV-2 infection need to be processed.

Impact of the Breakpoint Region on the Leukemogenic Potential and the TKI Responsiveness of Atypical BCR-ABL1 Transcripts.

Chronic Myeloid Leukemia (CML) is a hematological disorder characterized by the clonal expansion of a hematopoietic stem cell carrying the Philadelphia chromosome that juxtaposes the BCR and ABL1 genes. The ensuing BCR-ABL1 chimeric oncogene is characterized by a breakpoint region that generally involves exons 1, 13 or 14 in BCR and exon 2 in ABL1. Additional breakpoint regions, generating uncommon BCR-ABL1 fusion transcripts, have been detected in various CML patients. However, to date, the impact of these infrequent transcripts on BCR-ABL1-dependent leukemogenesis and sensitivity to tyrosine kinase inhibitors (TKIs) remain unclear. We analyzed the transforming potential and TKIs responsiveness of three atypical BCR-ABL1 fusions identified in CML patients, and of two additional BCR-ABL1 constructs with lab-engineered breakpoints. We observed that modifications in the DC2 domain of BCR and SH3 region of ABL1 affect BCR-ABL1 catalytic efficiency and leukemogenic ability. Moreover, employing immortalized cell lines and primary CD34-positive progenitors, we demonstrate that these modifications lead to reduced BCR-ABL1 sensitivity to imatinib, dasatinib and ponatinib but not nilotinib. We conclude that BCR-ABL1 oncoproteins displaying uncommon breakpoints involving the DC2 and SH3 domains are successfully inhibited by nilotinib treatment.

Opportunities and Challenges of Liquid Biopsy in Thyroid Cancer.

Thyroid cancer is the most common malignancy of the endocrine system, encompassing different entities with distinct histological features and clinical behavior. The diagnostic definition, therapeutic approach, and follow-up of thyroid cancers display some controversial aspects that represent unmet medical needs. Liquid biopsy is a non-invasive approach that detects and analyzes biological samples released from the tumor into the bloodstream. With the use of different technologies, tumor cells, free nucleic acids, and extracellular vesicles can be retrieved in the serum of cancer patients and valuable molecular information can be obtained. Recently, a growing body of evidence is accumulating concerning the use of liquid biopsy in thyroid cancer, as it can be exploited to define a patient's diagnosis, estimate their prognosis, and monitor tumor recurrence or treatment response. Indeed, liquid biopsy can be a valuable tool to overcome the limits of conventional management of thyroid malignancies. In this review, we summarize currently available data about liquid biopsy in differentiated, poorly differentiated/anaplastic, and medullary thyroid cancer, focusing on circulating tumor cells, circulating free nucleic acids, and extracellular vesicles.

Molecular Pathogenesis and Treatment Perspectives for Hypereosinophilia and Hypereosinophilic Syndromes.

Hypereosinophilia (HE) is a heterogeneous condition with a persistent elevated eosinophil count of >350/mm3, which is reported in various (inflammatory, allergic, infectious, or neoplastic) diseases with distinct pathophysiological pathways. HE may be associated with tissue or organ damage and, in this case, the disorder is classified as hypereosinophilic syndrome (HES). Different studies have allowed for the discovery of two major pathogenetic variants known as myeloid or lymphocytic HES. With the advent of molecular genetic analyses, such as T-cell receptor gene rearrangement assays and Next Generation Sequencing, it is possible to better characterize these syndromes and establish which patients will benefit from pharmacological targeted therapy. In this review, we highlight the molecular alterations that are involved in the pathogenesis of eosinophil disorders and revise possible therapeutic approaches, either implemented in clinical practice or currently under investigation in clinical trials.

Optimal Response in a Patient With CML Expressing BCR-ABL1 E6A2 Fusion Transcript With Nilotinib Therapy: A Case Report.

BACKGROUND/AIM: The Philadelphia chromosome is considered the hallmark of chronic myeloid leukemia (CML). However, although most patients with CML are diagnosed with the e13a2 or e14a2 breakpoint cluster region (BCR)-Abelson 1 (ABL1) fusion transcripts, about 5% of them carry rare BCR-ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3 and e6a2. In particular, the e6a2 fusion transcript has been associated with clinically aggressive disease frequently presenting in accelerated or blast crisis phases; there is limited evidence on the efficacy of front-line second-generation tyrosine kinase inhibitors for this genotype. CASE REPORT: We describe a case of atypical BCR-ABL1 e6a2 fusion transcript in a 46-year-old woman with CML. RESULTS: The use of primers recognizing more distant exons from the common BCR-ABL1 breakpoint region correctly identified the atypical BCR-ABL1 e16a2 fusion transcript. Treatment with second-generation tyrosine kinase inhibitor nilotinib was effective in this patient expressing the atypical e6a2 BCR-ABL1 fusion transcript.

ABL1-Directed Inhibitors for CML: Efficacy, Resistance and Future Perspectives.

The introduction of tyrosine kinase inhibitors (TKIs) directed against the catalytic activity of the ABL tyrosine kinase has considerably improved the outcome of chronic myeloid leukemia (CML) patients in the chronic phase of the disease. Indeed, these individuals currently show a life-expectancy comparable to those of healthy subjects. Currently, five TKIs (imatinib, dasatinib, nilotinib, bosutinib and ponatinib) are approved for the treatment of CML and can be used as first, second or further lines of treatment according to disease risk scores, patient comorbidities and the presence of known TKI resistance mechanisms. In fact, 15-20% of all CML patients fail to achieve optimal responses according to the current definitions of the European Leukemia Network and will require sequential TKI treatment to avoid disease progression. In this review, we present the state of art in several crucial areas of CML management by briefly: i) depicting the domain structure of the BCR-ABL1 oncoprotein; ii) describing pivotal data concerning TKI efficacy; iii) illustrating the diverse molecular mechanisms causing TKI resistance; and iv) summarizing new ABL1-directed therapeutic approaches that are presently under investigation.

Prognostic and Therapeutic Roles of the Insulin Growth Factor System in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain malignancy and is often resistant to conventional treatments due to its extensive cellular heterogeneity. Thus, the overall survival of GBM patients remains extremely poor. Insulin-like growth factor (IGF) signaling entails a complex system that is a key regulator of cell transformation, growth and cell-cycle progression. Hence, its deregulation is frequently involved in the development of several cancers, including brain malignancies. In GBM, differential expression of several IGF system components and alterations of this signaling axis are linked to significantly worse prognosis and reduced responsiveness to temozolomide, the most commonly used pharmacological agent for the treatment of the disease. In the present review we summarize the biological role of the IGF system in the pathogenesis of GBM and comprehensively discuss its clinical significance and contribution to the development of resistance to standard chemotherapy and experimental treatments.