Combination Therapies Targeting Apoptosis in Paediatric AML: Understanding the Molecular Mechanisms of AML Treatments Using Phosphoproteomics.

Paediatric acute myeloid leukaemia (AML) continues to present treatment challenges, as no "standard approach" exists to treat those young patients reliably and safely. Combination therapies could become a viable treatment option for treating young patients with AML, allowing multiple pathways to be targeted. Our in silico analysis of AML patients highlighted "cell death and survival" as an aberrant, potentially targetable pathway in paediatric AML patients. Therefore, we aimed to identify novel combination therapies to target apoptosis. Our apoptotic drug screening resulted in the identification of one potential "novel" drug pairing, comprising the Bcl-2 inhibitor ABT-737 combined with the CDK inhibitor Purvalanol-A, as well as one triple combination of ABT-737 + AKT inhibitor + SU9516, which showed significant synergism in a series of paediatric AML cell lines. Using a phosphoproteomic approach to understand the apoptotic mechanism involved, proteins related to apoptotic cell death and cell survival were represented, in agreement with further results showing differentially expressed apoptotic proteins and their phosphorylated forms among combination treatments compared to single-agent treated cells such upregulation of BAX and its phosphorylated form (Thr167), dephosphorylation of BAD (Ser 112), and downregulation of MCL-1 and its phosphorylated form (Ser159/Thr 163). Total levels of Bcl-2 were decreased but correlated with increased levels of phosphorylated Bcl-2, which was consistent with our phosphoproteomic analysis predictions. Bcl-2 phosphorylation was regulated by extracellular-signal-regulated kinase (ERK) but not PP2A phosphatase. Although the mechanism linking to Bcl-2 phosphorylation remains to be determined, our findings provide first-hand insights on potential novel combination treatments for AML.

"Immuno-flowFISH" for the Assessment of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.

Intravenous thiotepa for treatment of breast cancer-related leptomeningeal carcinomatosis: case series.

Leptomeningeal carcinomatosis (LMC) secondary to metastatic breast cancer (MBC) has increased in incidence with improved systemic disease control. Current treatment options include radiation therapy (to symptomatic sites) and systemic treatment [intrathecal (IT) or intravenous (IV) chemotherapy]. Methotrexate (MTX), thiotepa and cytarabine are the most commonly used IT agents, while high-dose MTX is the most common IV regimen. While IT treatments are generally well tolerated, complications like chemical meningitis, leukoencephalopathy, etc. occur. LMC may cause a breakdown in the blood-brain barrier and thus allow systemic agents to penetrate; however, efficacy is reported only for agents administered at high doses (MTX). We report our institution's experience in using IV thiotepa as treatment for LMC secondary to MBC. We conducted a retrospective chart review of 13 patients with MBC who developed LMC and treated with IV thiotepa at our institution. It was administered at 40 mg/m(2) every 21 days; median number of thiotepa cycles administered was 5 with the major dose-limiting toxicity being myelosuppression. Four had partial response, 3 had stable disease and 6 had progressive disease. The 6-month survival rate was 69 % and 1-year survival rate was 31 %. Despite retrospective nature of our case series, we found the use of IV thiotepa as sole treatment for LMC in patients with MBC to be well tolerated, easily administered in the ambulatory setting, and with efficacy comparable to the other chemotherapeutic agents commonly used in the treatment of LMC. This regimen warrants further investigation in prospective studies.

A pilot study of estradiol followed by exemestane for reversing endocrine resistance in postmenopausal women with hormone receptor-positive metastatic breast cancer.

BACKGROUND: Endocrine resistance is a frequent complication, and strategies to reverse it are a high research priority for metastatic breast cancer (MBC) that is hormone receptor positive. Preclinical data suggest re-exposure to estrogen induces tumor regression in tamoxifen-resistant tumors. We conducted a pilot study to determine whether short-term estradiol exposure would reverse endocrine resistance and resensitize tumors METHODS: Postmenopausal women with estrogen receptor-positive MBC whose disease had progressed after receiving at least one prior endocrine therapy were eligible for the study. Patients were initially treated with 6 mg/day estradiol, and those who had not progressed after 3 months were then switched to exemestane. RESULTS: Thirteen patients were evaluable for toxicity and response. No grade 3 or 4 toxicities were observed. Of the 13 patients who initiated estradiol therapy, 6 patients (46%) had not experienced disease progression at month 3 and were switched to exemestane. On exemestane, disease progression was documented in five patients, with one having stable disease as best response. Median progression-free survival for all patients was 4.8 months (range: 0.6-9.5 months). CONCLUSION: Treatment with an estrogen prior to resuming antiestrogen treatments was not effective at reversing hormone resistance; however, low-dose estradiol treatment had measurable clinical activity with minimal toxicity and should be considered as a therapeutic option for hormone-refractory MBC.

Treatment of a STAT5b::RARα positive case of APL in a patient not eligible for intensive chemotherapy.

Acute promyelocytic leukaemia (APL) with a STAT5b::RARα gene fusion is an extremely rare subtype of APL characterised by resistance to conventional therapies and extremely poor prognosis. This case highlights that whilst APL with variant RARα translocations are rare, they do pose significant challenges both diagnostically and in their clinical management. This case, in the first instance, demonstrates the importance of using a combination of molecular techniques including next generation sequencing (NGS) for diagnosis particularly in morphological and immunophenotypic typical APL which appears negative by confirmatory testing. Secondly, our patient represents, to the best of our knowledge, the first documented example of this rare disease that has been managed with, and shown sensitivity to low-dose cytarabine (LDAC) in combination with venetoclax (Ven). This case demonstrates that although treatment options are extremely limited for patients not eligible for intensive chemotherapy non-intensive options do show increasing promise.

Association between JAK2V617F variable allele frequency and risk of thrombotic events in patients with myeloproliferative neoplasms.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are a group of chronic disorders of the bone marrow characterised by the overproduction of clonal myeloid stem cells. The most common driver mutation found in MPNs is a point mutation on exon 14 of the JAK2 gene, JAK2V617F. Various studies have suggested that measuring the variable allele frequency (VAF) of JAK2V617F may provide useful insight regarding diagnosis, treatment, risks and outcomes in MPN patients. In particular, JAK2V617F has been associated with increased risk of thrombotic events, a leading cause of mortality in MPNs. AIMS: The aim of this study was to determine if JAK2V617F VAF was associated with clinical outcomes in patients with MPN. METHODS: JAK2V617F VAF was determined by quantitative PCR (qPCR) in a cohort of 159 newly diagnosed MPN patients, and the association of JAK2V617F VAF and risk of thrombosis was examined in this cohort. RESULTS: We observed a significantly higher JAK2V617F VAF in PV and PMF versus ET. A significant association was observed between JAK2V617F VAF and risk of thrombotic events. When patients were stratified by thrombotic events prior to and post diagnosis, an association with JAK2V617F VAF was only observed with post diagnosis thrombotic events. Of note, these associations were not observed when looking at each MPN subtype in isolation. CONCLUSIONS: We have shown that a higher JAK2V617F VAF is associated with thrombotic events post MPN diagnosis. JAK2V617F VAF may therefore provide a valuable prognostic indicator for risk of thrombosis in MPNs.

Multi-probe FISH Analysis of Immunophenotyped Chronic Lymphocytic Leukemia by Imaging Flow Cytometry.

Imaging flow cytometry is an automated method that enables cells and fluorescent signals to be visualized and quantified. Here, we describe a new imaging flow cytometry method whereby fluorescence in situ hybridization (FISH) is integrated with cell phenotyping. The method, called "immuno-flowFISH," provides an exciting new dimension for the analysis of genomic changes in cytological samples (e.g., blood, bone marrow). Cells are analyzed in suspension without any requirement for prior cell isolation or separation. Multiple antibodies and FISH probes, each with a unique fluorophore, can be added and many thousands of cells analyzed. Specific cell populations are identified by their antigenic profile and then analyzed for the presence of chromosomal defects. Immuno-flowFISH was applied to the assessment of chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm where chromosomal abnormalities predict prognosis and treatment requirements. This integrated immunophenotyping and multi-probe FISH strategy could detect both structural and numerical chromosomal changes involving chromosomes 12 and 17 in CLL cells. Given that many thousands of cells were analyzed and the leukemic cells were positively identified by their immunophenotype, this multi-probe method adds precision to the cytogenomic analysis of CLL. © 2021 Wiley Periodicals LLC.

Uses of a research diary: learning reflectively, developing understanding and establishing transparency.

The author of this paper started a research diary to provide transparency to her study and to help her clarify thoughts and feelings--as well as to acknowledge factors that may have influenced her analysis. She found it helped in her development as a novice phenomenological nurse researcher through creative and critical thinking. She recommends all novice researchers keep a diary.

The histone deacetylase inhibitor Romidepsin induces as a cascade of differential gene expression and altered histone H3K9 marks in myeloid leukaemia cells.

Myelodysplastic syndromes (MDS) are a heterogeneous, clonal haematopoietic disorder, with ~1/3 of patients progressing to acute myeloid leukaemia (AML). Many elderly MDS patients do not tolerate intensive therapeutic regimens, and therefore have an unmet need for better tolerated therapies. Epigenetics is important in the pathogenesis of MDS/AML with DNA methylation, and histone acetylation the most widely studied modifications. Epigenetic therapeutic agents have targeted the reversible nature of these modifications with some clinical success. The aim of this study was to characterise the molecular consequences of treatment of MDS and AML cells with the histone deacetylase inhibitor (HDACi) Romidepsin. Romidepsin as a single agent induced cell death with an increasing dose and time profile associated with increased acetylation of histone H3 lysine 9 (H3K9) and decreased HDAC activity. Gene expression profiling, qPCR, network and pathway analysis recognised that oxidation-reduction was involved in response to Romidepsin. ROS was implicated as being involved post-treatment with the involvement of TSPO and MPO. Genomic analysis uncoupled the differences in protein-DNA interactions and gene regulation. The spatial and temporal transcriptional differences associated with acetylated, mono- and tri-methylated H3K9, representative of two activation and a repression mark respectively, were identified. Bioinformatic analysis uncovered positional enrichment and transcriptional differences between these marks; a degree of overlap with increased/decreased gene expression that correlates to increased/decreased histone modification. Overall, this study has unveiled a number of underlying mechanisms of the HDACi Romidepsin that could identify potential drug combinations for use in the clinic.

A phenomenological hermeneutic study into unseen chronic pain.

Acute pain is often accompanied by physiological changes, such as increased heart rate, unlike chronic pain, which in sharp contrast is often unaccompanied by any physiological changes other than the patients' self report of pain. Coupled with the lack of physiological changes, chronic pain will outlive the usual healing period if an injury has been sustained thereby leading to the unseen nature of the chronic pain phenomenon. This study (n?=?8) adopted a multi-method approach - a low-structured interview coupled with patients' diaries - and was analysed hermeneutically. 'Unseen pain' was identified as a category with subsequent themes of 'isolation', 'needing to prove the existence of chronic pain', 'in their head' and 'depression'. The unseen nature of the chronic pain phenomenon caused further distress to patients as they could feel disbelieved by society as well as by healthcare professionals. This has a profound effect on patients who then channel valuable psychological resources into appearing credible to society and healthcare professionals in particular. The lack of clear explanations and a definitive diagnosis or acceptable label also adds to the invisible nature of the chronic pain phenomenon and as healthcare professionals we need to confirm the validity of the expressed pain narrative through means that do not enforce illness behaviour, but empower the patient to accept their chronic pain and live with it rather than be ruled by it.

Decitabine-Vorinostat combination treatment in acute myeloid leukemia activates pathways with potential for novel triple therapy.

Despite advancements in cancer therapeutics, acute myeloid leukemia patients over 60 years old have a 5-year survival rate of less than 8%. In an attempt to improve this, epigenetic modifying agents have been combined as therapies in clinical studies. In particular combinations with Decitabine and Vorinostat have had varying degrees of efficacy. This study therefore aimed to understand the underlying molecular mechanisms of these agents to identify potential rational epi-sensitized combinations. Combined Decitabine-Vorinostat treatment synergistically decreased cell proliferation, induced apoptosis, enhanced acetylation of histones and further decreased DNMT1 protein with HL-60 cells showing a greater sensitivity to the combined treatment than OCI-AML3. Combination therapy led to reprogramming of unique target genes including AXL, a receptor tyrosine kinase associated with cell survival and a poor prognosis in AML, which was significantly upregulated following treatment. Therefore targeting AXL following epi-sensitization with Decitabine and Vorinostat may be a suitable triple combination. To test this, cells were treated with a novel triple combination therapy including BGB324, an AXL specific inhibitor. Triple combination increased the sensitivity of OCI-AML3 cells to Decitabine and Vorinostat as shown through viability assays and significantly extended the survival of mice transplanted with pretreated OCI-AML3 cells, while bioluminescence imaging showed the decrease in disease burden following triple combination treatment. Further investigation is required to optimize this triple combination, however, these results suggest that AXL is a potential marker of response to Decitabine-Vorinostat combination treatment and offers a new avenue of epigenetic combination therapies for acute myeloid leukemia.

Survival of patients with melanoma brain metastasis treated with stereotactic radiosurgery and active systemic drug therapies.

INTRODUCTION: With new systemic therapies demonstrating activity in melanoma brain metastasis, most of the previously reported stereotactic radiosurgery (SRS) data are superseded. In this study, we report the outcomes (overall survival [OS] and brain control [BC]) and identify factors that associate with such outcomes in the era of modern systemic therapy. METHOD: A total of 108 patients treated with SRS from 2010 to 2015 were included. Systemic treatment use within 6 weeks of SRS was noted. OS was defined as time from SRS to death or last follow-up, and BC was defined as absence of any active intracranial disease during follow-up. Univariate and multivariate Cox proportional hazard analyses were performed on clinico-pathological prognostic features associated with OS and BC. RESULTS: The median age was 64.3 years, and the median follow-up was 8.6 months. Seventy-nine (73.1%) patients received systemic treatment. The median OS were as follows: anti-CTLA4 - 7.5 months (95% CI: 4.4-15.6), anti-PD1 - 20.4 months (95% CI: 8.8 - N/A) and BRAF inhibitor (BRAFi) ± MEK inhibitor (MEKi) - 17.8 months (95% CI: 11.8 - N/A). Median BC was as follows: anti-CTLA4 - 7.5 months (95% CI: 4.0-15.6), anti-PD1 - 12.7 months (95% CI: 5.5 - N/A) and BRAFi ± MEKi - 12.7 months (95% CI: 8.3-18.5). In multivariate analysis, age and type of systemic therapy were strongly associated with OS. Age, Eastern Cooperative Oncology Group performance status, Graded Prognostic Assessment (GPA) score, and presence of symptoms were associated with BC. CONCLUSIONS: Favourable outcomes are seen in patients treated with SRS and with the best survival seen in patients treated with anti-PD1. Known independent prognostic factors for survival such as age and performance status and GPA score remain relevant in this setting.

Believing the patient with chronic pain: a review of the literature.

The aim of this article is to review the literature on studies on the effect of having health professionals treat a patient's pain experience as credible and to propose considerations for nursing practice. A systematic literature search was performed via Cinahl, Medline, British Nursing Index, Cochrane and PsychINFO from 1970-2003 using the key words 'chronic pain', 'attitude', 'confirmation', 'listening to' and 'belief'. Study findings were reviewed in relation to the effect of 'being believed'. There are no studies that look exclusively at the effects of having a health professional believe a patient's reported pain experience; however, within the literature there are several references to the need to examine this phenomenon. Future research in this area is needed to provide direction for clinical practice for nurses in this aspect of chronic pain management.

Combination capecitabine and bevacizumab in the treatment of metastatic hepatic epithelioid hemangioendothelioma.

Hepatic epithelioid hemangioendothelioma (HEHE) is a rare, often misdiagnosed vascular neoplasm with clinical behaviors that range from indolent to highly aggressive. Even when the appropriate diagnosis is achieved, the best treatment for HEHE has not been defined or standardized, further complicating the care of these patients. We present a diagnostically challenging case of HEHE where we utilized capecitabine and bevacizumab as another novel treatment option.

A phase I clinical trial of bavituximab and paclitaxel in patients with HER2 negative metastatic breast cancer.

Bavituximab is a chimeric monoclonal antibody that targets phosphatidylserine (PS). PS is externalized on cells in the tumor microenvironment when exposed to hypoxia and/or other physiological stressors. On attaching to PS, bavituximab is thought to promote antitumor immunity through its effects on PS receptors in monocytes, and myeloid-derived suppressor cells, as well as trigger antitumor effects by inducing an antibody-dependent cellular cytotoxicity on tumor-associated endothelial cells. We conducted a phase I clinical trial of bavituximab in combination with paclitaxel in patients with HER2-negative metastatic breast cancer. Patients were treated with weekly paclitaxel (80 mg/m(2) for 3/4 weeks) and weekly bavituximab (3 mg/kg for 4/4 weeks). Correlative studies included the measurement of circulating microparticles, endothelial cells, and apoptotic tumor cells by flow cytometry. Fourteen patients with metastatic breast cancer were enrolled; all were evaluable for toxicity and 13 were evaluable for response. Treatment resulted in an overall response rate (RR) of 85% with a median progression-free survival (PFS) of 7.3 months. Bone pain, fatigue, headache, and neutropenia were the most common adverse effects. Infusion-related reactions were the most common adverse event related to bavituximab therapy. Correlative studies showed an increase in the PS-expressing apoptotic circulating tumor cells in response to bavituximab, but not with paclitaxel. No changes in the number of circulating endothelial cells or apoptotic endothelial cells were observed with therapy. Platelet and monocyte-derived microparticles decreased after initiation of bavituximab. Bavituximab in combination with paclitaxel is well tolerated for treatment of patients with metastatic breast cancer with promising results observed in terms of clinical RRs and PFS. The toxicity profile of bavituximab is notable for manageable infusion-related reactions with no evidence for increased thrombogenicity. Recent preclinical data suggest that bavituximab can also promote antitumor immune activity that should be explored in future clinical trials.