Additional Genetic Alterations and Clonal Evolution of MPNs with Double Mutations on the MPL Gene: Two Case Reports.

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by abnormal megakaryocytic proliferation. Janus kinase 2 (JAK2) mutations are detected in 50-60% of ET and PMF, while myeloproliferative leukemia (MPL) virus oncogene mutations are present in 3-5% of cases. While Sanger sequencing is a valuable diagnostic tool to discriminate the most common MPN mutations, next-generation sequencing (NGS) is a more sensitive technology that also identifies concurrent genetic alterations. In this report, we describe two MPN patients with simultaneous double MPL mutations: a woman with ET presenting both MPLV501A-W515R and JAK2V617F mutations and a man with PMF displaying an uncommon double MPLV501A-W515L. Using colony-forming assays and NGS analyses, we define the origin and mutational landscape of these two unusual malignancies and uncover further gene alterations that may contribute to the pathogenesis of ET and PMF.

Multiple primary malignances managed with surgical excision: a case report with next generation sequencing analysis.

BACKGROUND: Multiple primary malignancies (MPM) are defined as tumors originating in the same individual without any correlation between them. In addition to morphological and immunohistochemical analyses, sensitive DNA sequencing methods such as next generation sequencing (NGS) may help to discriminate the common or different genetic alterations driving each malignancy, to better diagnose these uncommon cases. METHODS AND RESULTS: Here we report the case of a man who developed a poorly differentiated gastric adenocarcinoma invading the pancreas followed, two years later, by a colorectal cancer involving also the kidney and the diaphragm. Despite the advanced stage of both diseases, adjuvant chemotherapy was successful. While the second tumor was initially interpreted as a relapse of his stomach cancer, NGS-based mutation profiling of the two carcinomas revealed two distinct malignances, independently developing in different times and indicative of metachronous MPM. Indeed, sequencing of cancer-associated genes identified somatic mutations only in the first gastric cancer, besides germline variants on three different genes (PDGFRA, APC and TP53). However, analysis of both somatic and germline mutations with bio-informatics prediction tools failed to find a correlation between these variants and the unexpectedly good prognosis of both cancers. CONCLUSIONS: In summary, NGS analysis contributed to defined different molecular profiles for two tumors developed in the span of two years, thus allowing diagnosing the case as MPN. However, NGS was unable to establish a direct correlation between the identified alterations and cancer development.

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.

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.

Targeting BCL-2 as a Therapeutic Strategy for Primary p210BCR-ABL1-positive B-ALL Cells.

BACKGROUND/AIM: Philadelphia-positive acute lymphoblastic leukemia (Ph+ B-ALL) is caused by the malignant transformation of lymphoid cells induced by BCR-ABL1 constitutive catalytic activity. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML) cells, inducing durable hematological, cytogenetic and molecular responses. However, in Ph+ B-ALL - as in CML progressing to blast crisis - TKIs fail to maintain disease remission. We, therefore, wanted to investigate if dual targeting of BCL-2 and BCR-ABL1 would be more effective in killing Ph+ B-ALL cells. MATERIALS AND METHODS: p210-B-ALL CD34-positive cells were used to evaluate the BCR-ABL expression and pharmacological targeting of BCL-2, by venetoclax, alone or in combination with BCR-ABL1 inhibition. RESULTS: We demonstrated the cytotoxic effect of BCL-2 inhibition and that dual targeting of BCL-2 and BCR-ABL1 with venetoclax and nilotinib further increases this cytotoxicity. CONCLUSION: BCL-2 is a key survival factor for primary Ph+ B-ALL cells and its inhibition - alone or in combination with a BCR-ABL1 TKI - should be further investigated as a potential therapeutic strategy for these patients.

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.

Detection and Clinical Implications of a Novel BCR-ABL1 E12A2 Insertion/Deletion in a CML Patient Expressing the E13A2 Isoform.

BACKGROUND/AIM: The Philadelphia chromosome is the most frequent cytogenetic abnormality in chronic myelogenous (CML). More than 95% of CML patients are diagnosed with the e13a2 or e14a2 BCR-ABL1 fusion transcripts while, in about 1% of these individuals, the break generates the e1a2 rearrangement. Furthermore, about 5% of CML patients are diagnosed with rare BCR-ABL1 fusion transcripts, such as e19a2, e8a2, e13a3, e14a3, e1a3 and e6a2. However, there is limited evidence concerning the clinical and prognostic implications of these infrequent oncogenic variants for CML patients receiving tyrosine kinase inhibitors (TKIs). CASE REPORT: We describe a novel atypical e12a2 insertion/deletion (Ins/Del) BCR-ABL1 fusion identified in a CML 59-year-old man diagnosed with a common e13a2 BCR-ABL1 isoform. The use of primers recognizing more distant exons from the common BCR-ABL1 breakpoint region correctly identified and monitored in time the atypical e12a2 Ins/Del BCR-ABL1 fusion. CONCLUSION: Treatment with second- (nilotinib) and third-generation (ponatinib) TKIs was effective in suppressing leukemic clones exhibiting the atypical e12a2 Ins/Del BCR-ABL1.

Molecular Alterations in Thyroid Cancer: From Bench to Clinical Practice.

Thyroid cancer comprises different clinical and histological entities. Whereas differentiated (DTCs) malignancies are sensitive to radioiodine therapy, anaplastic (ATCs) and medullary (MTCs) tumors do not uptake radioactive iodine and display aggressive features associated with a poor prognosis. Moreover, in a majority of DTCs, disease evolution leads to the progressive loss of iodine sensitivity. Hence, iodine-refractory DTCs, along with ATCs and MTCs, require alternative treatments reflective of their different tumor biology. In the last decade, the molecular mechanisms promoting thyroid cancer development and progression have been extensively studied. This has led to a better understanding of the genomic landscape, displayed by thyroid malignancies, and to the identification of novel therapeutic targets. Indeed, several pharmacological compounds have been developed for iodine-refractory tumors, with four multi-target tyrosine kinase inhibitors already available for DTCs (sorafenib and lenvatinib) and MTCs (cabozantib and vandetanib), and a plethora of drugs currently being evaluated in clinical trials. In this review, we will describe the genomic alterations and biological processes intertwined with thyroid cancer development, also providing a thorough overview of targeted drugs already tested or under investigation for these tumors. Furthermore, given the existing preclinical evidence, we will briefly discuss the potential role of immunotherapy as an additional therapeutic strategy for the treatment of thyroid cancer.

Successful Management of a Pregnant Patient With Chronic Myeloid Leukemia Receiving Standard Dose Imatinib.

BACKGROUND/AIM: As approximately 10% of individuals developing chronic myeloid leukemia (CML) are females aged 20-44 years, a considerable number will consider a planned pregnancy if disease is well controlled by pharmacological treatment. The management of these young patients during pregnancy represents a therapeutic dilemma due to the potential teratogen effects of several tyrosine kinase inhibitors (TKIs) and is a matter of continuous debate. Indeed, despite the existence of several studies, there is currently no consensus on how to manage different pregnancy situations in subjects with CML. PATIENTS AND METHODS: We describe a female patient diagnosed with Ph-positive CML one month after her first delivery who achieved excellent hematological, cytogenetic and molecular responses while on imatinib mesylate (IM) treatment. RESULTS: The excellent responses allowed the patient to suspend TKI treatment in order to plan a second pregnancy. Despite IM discontinuation, stringent molecular monitoring of her BCR-ABL1/ABL1 levels allowed the safe delivery of the child and, while the patient eventually developed a molecular relapse after four years of treatment discontinuation, upon restarting IM she quickly regained a deep molecular response that is still ongoing. CONCLUSION: Our case report demonstrates that, if the pregnancy is properly planned in CML patients, it can result in excellent management of the clinical therapeutic option for the benefit of both mother and child.

BCR-ABL1 Doubling-Times and Halving-Times May Predict CML Response to Tyrosine Kinase Inhibitors.

In Chronic Myeloid Leukemia (CML), successful treatment requires accurate molecular monitoring to evaluate disease response and provide timely interventions for patients failing to achieve the desired outcomes. We wanted to determine whether measuring BCR-ABL1 mRNA doubling-times (DTs) could distinguish inconsequential rises in the oncogene's expression from resistance to tyrosine kinase inhibitors (TKIs). Thus, we retrospectively examined BCR-ABL1 evolution in 305 chronic-phase CML patients receiving imatinib mesylate (IM) as a first line treatment. Patients were subdivided in two groups: those with a confirmed rise in BCR-ABL1 transcripts without MR3.0 loss and those failing IM. We found that the DTs of the former patients were significantly longer than those of patients developing IM resistance (57.80 vs. 41.45 days, p = 0.0114). Interestingly, the DT values of individuals failing second-generation (2G) TKIs after developing IM resistance were considerably shorter than those observed at the time of IM failure (27.20 vs. 41.45 days; p = 0.0035). We next wanted to establish if decreases in BCR-ABL1 transcripts would identify subjects likely to obtain deep molecular responses. We therefore analyzed the BCR-ABL1 halving-times (HTs) of a different cohort comprising 174 individuals receiving IM in first line and observed that, regardless of the time point selected for our analyses (6, 12, or 18 months), HTs were significantly shorter in subjects achieving superior molecular responses (p = 0.002 at 6 months; p < 0.001 at 12 months; p = 0.0099 at 18 months). Moreover, 50 patients receiving 2G TKIs as first line therapy and obtaining an MR3.0 (after 6 months; p = 0.003) or an MR4.0 (after 12 months; p = 0.019) displayed significantly shorter HTs than individuals lacking these molecular responses. Our findings suggest that BCR-ABL1 DTs and HTs are reliable tools to, respectively, identify subjects in MR3.0 that are failing their assigned TKI or to recognize patients likely to achieve deep molecular responses that should be considered for treatment discontinuation.

Activation of the IGF Axis in Thyroid Cancer: Implications for Tumorigenesis and Treatment.

The Insulin-like growth factor (IGF) axis is one of the best-established drivers of thyroid transformation, as thyroid cancer cells overexpress both IGF ligands and their receptors. Thyroid neoplasms encompass distinct clinical and biological entities as differentiated thyroid carcinomas (DTC)-comprising papillary (PTC) and follicular (FTC) tumors-respond to radioiodine therapy, while undifferentiated tumors-including poorly-differentiated (PDTC) or anaplastic thyroid carcinomas (ATCs)-are refractory to radioactive iodine and exhibit limited responses to chemotherapy. Thus, safe and effective treatments for the latter aggressive thyroid tumors are urgently needed. Despite a strong preclinical rationale for targeting the IGF axis in thyroid cancer, the results of the available clinical studies have been disappointing, possibly because of the crosstalk between IGF signaling and other pathways that may result in resistance to targeted agents aimed against individual components of these complex signaling networks. Based on these observations, the combinations between IGF-signaling inhibitors and other anti-tumor drugs, such as DNA damaging agents or kinase inhibitors, may represent a promising therapeutic strategy for undifferentiated thyroid carcinomas. In this review, we discuss the role of the IGF axis in thyroid tumorigenesis and also provide an update on the current knowledge of IGF-targeted combination therapies for thyroid cancer.

Efficacy of Nilotinib in a CML Patient Expressing the Three-way Complex Variant Translocation t(2;9;22).

BACKGROUND/AIM: Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia chromosome, resulting from the reciprocal translocation involving chromosomes 9 and 22. About 5-10% of newly diagnosed patients in chronic-phase (CP) CML show complex additional chromosomal aberrations (ACA), that may involve one or more chromosomes in addition to 9 and 22. Data concerning the prognostic significance of ACA in CP-CML subjects at diagnosis are controversial. Furthermore, there is no evidence showing that selection of imatinib (IM) or second-generation tyrosine kinase inhibitors (2G-TKI) would be of benefit for these patients. CASE REPORT: We report the three-way complex variant translocation t(2;9;22) in a CP-CML patient. Conventional cytogenetic analysis was employed to identify the ACA. Multiplex reverse transcription-PCR was used to identify the BCR-ABL1 transcript and its levels were measured using quantitative real-time-PCR. This rare ACA t(2;9;22) in our young patient displayed primary resistance to IM, but was responsive to second-line treatment with nilotinib. CONCLUSION: CP-CML patients exhibiting this rare aberration at diagnosis may benefit from a 2G-TKI therapy compared to IM.

Colony-Forming Cell Assay Detecting the Co-Expression of JAK2V617F and BCR-ABL1 in the Same Clone: A Case Report.

BCR-ABL1-negative myeloproliferative disorders and chronic myeloid leukaemia are haematologic malignancies characterised by single and mutually exclusive genetic alterations. Nevertheless, several patients co-expressing the JAK2V617F mutation and the BCR-ABL1 transcript have been described in the literature. We report the case of a 61-year-old male who presented with an essential thrombocythaemia phenotype and had a subsequent diagnosis of chronic phase chronic myeloid leukaemia. Colony-forming assays demonstrated the coexistence of 2 different haematopoietic clones: one was positive for the JAK2V617F mutation and the other co-expressed both JAK2V617F and the BCR-ABL1 fusion gene. No colonies displayed the BCR-ABL1 transcript alone. These findings indicate that the JAK2V617F mutation was the founding genetic alteration of the disease, followed by the acquisition of the BCR-ABL1 chimeric oncogene. Our data support the hypothesis that a heterozygous JAK2V617F clone may have favoured the bi-clonal nature of this myeloproliferative disorder, generating clones harbouring a second transforming genetic event.

BCR-ABL nuclear entrapment kills human CML cells: ex vivo study on 35 patients with the combination of imatinib mesylate and leptomycin B.

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) localizes to the cell cytoplasm, where it activates proliferative and antiapoptotic signaling pathways. We previously reported that the combination of the ABL kinase inhibitor imatinib mesylate (IM) and the nuclear export inhibitor leptomycin B (LMB) traps BCR-ABL inside the nucleus, triggering the death of the leukemic cells. To evaluate the efficacy of the combination of IM and LMB on human cells we collected CD34-positive cells from 6 healthy donors and myeloid progenitors from 35 patients with CML. The sequential addition of IM and LMB generated the strongest reduction in the proliferative potential of the leukemic cells, with limited toxicity to normal myeloid precursors. Furthermore, nested reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on colonies representative of each experimental condition demonstrated that the combination of IM and LMB was the most effective regimen in reducing the number of BCR-ABL-positive colonies. The efficacy of the 2-drug association was independent of the clinical characteristics of the patients. Our results indicate that strategies aimed at the nuclear entrapment of BCR-ABL efficiently kill human leukemic cells, suggesting that the clinical development of this approach could be of significant therapeutic value for newly diagnosed and IM-resistant CML patients.