Identification and Modulation of Drug Resistance in Acute Myeloid Leukemia

A continuous, strictly organised process of blood cell production or hematopoiesis normally takes place in the bone marrow. The human hematopoietic system is capable of replacing the normal daily loss of peripheral blood cells, and will adapt the blood cell formation to increased demands such as bleeding or infection. This system is capable of maintaining a balance between cell loss and formation. The different types of blood cells that are normally present in the peripheral blood, are derived from committed progenitor cells. The compartment of these committed progenitor cells is maintained by a small population of pluripotent stem cells. Besides the ability of the stem cells to give rise to committed progenitor cells, they are capable of self-renewal. The process of proliferation and differentiation is regulated by cellular interaction, i.e. the microenvironment in the bone marrow, and several regulatory glycoproteins, the hematopoietic growth factors (HGPs). Malignant transformation of hematopoietic cells somewhere during their development and leading to the accumulation of immature hemopoietic cells is referred to as leukemia. According to the clinical presentation, the leukemias are divided in acute and chronic leukemias. Acute leukemias, if untreated, will lead to death within several weeks or months, while patients with untreated chronic leukemias often may survive for several years. Depending on the cell lineages involved, a further distinction in myeloid or lymphoid leukemias can be made. In this thesis, acute myeloid leukemia is subject of investigation

Reversal of typical multidrug resistance by cyclosporin and its non-immunosuppressive analogue SDZ PSC 833 in Chinese hamster ovary cells expressing the mdr1 phenotype

Summary The new non-immunosuppressive cyclosporin derivative SDZ PSC 833 (PSC) is a potent agent used to overcome typical multidrug resistance (MDR) associated with overexpression of themdr1 gene encoding for a P-170 glycoprotein. In the present study, the efficacy of PSC as compared with cyclosporin was determined in Chinese hamster ovary cell lines exhibiting different levels of resistance to colchicine (0, 0.1, 0.2 and 10 μg/ml, respectively). Low concentrations of PSC (8.2nm) increased the cytotoxicity of colchicine in cell lines expressing low levels of drug resistance. The concentration resulting in 50% cell survival (LC50 value) found for colchicine alone or in combination with PSC in the CHO-A3 cell line that was resistant to 100 ng colchicine/ml decreased from >500 to 200 ng/ml at 8.2nm PSC and to 500 ng/ml for colchicine alone to 500 ng/ml for colchicine used in combination with 8.2nm PSC and to <100 ng/ml for colchicine combined with 82 or 820nm PSC. At a concentration of 82nm PSC, the maximal effect in MDR reversal was observed in the cell lines exhibiting moderate resistance. In the highly resistant cell line, PSC (820nm) also reversed colchicine resistance. In drug-accumulation experiments, we obtained a 4-fold increase in intracellular doxorubicin accumulation using 820nm PSC. A comparison of PSC with cyclosporin revealed that a cyclosporin concentration 20-fold that of PSC was required to obtain the same sensitising effect. On the basis of these data, it may be concluded that PSC is a most promising chemosensitiser

COVID-19-associated immune thrombocytopenia

Thrombocytopenia is a risk factor for increased morbidity and mortality in patients with the new severe acute respiratory syndrome corona virus, SARS-CoV-2 infection (COVID-19 infection).1 Thrombocytopenia in COVID-19 patients may be caused by disseminated intravascular coagulation (DIC), sepsis or drug-induced. Recently a single case report suggested immune thrombocytopenia (ITP) may be associated with COVID-19 infection.2 ITP is a rare autoimmune disease characterized by a platelet count < 100x109/L, leading to an increased bleeding risk.3 Several risk factors have been described for ITP including environmental (e.g. infection, malignancy and drugs) and genetic predisposition.4 We report here the first case series of three patients with ITP associated with COVID-19 infection

EXPAND, a dose-finding study of ruxolitinib in patients with myelofibrosis and low platelet counts: 48-week follow-up analysis

EEXPAND (phase Ib, dose-finding study) evaluated the starting dose of ruxolitinib in patients with myelofibrosis with baseline platelet counts of 50-99×109 /L. The study consisted of dose-escalation and safety-expansion phases. Based on the baseline platelet counts, patients were assigned to stratum 1 (75-99x109 /L) or stratum 2 (50-74x109 /L), with the primary objective of determining the maximum safe starting dose (MSSD); key secondary objectives included safety and efficacy. At week 48 data cutoff (stratum 1, n=44; stratum 2, n=25), 24.6% (17 out of 69) of patients were still receiving treatment. The MSSD was established as ruxolitinib 10 mg twice daily in both strata. Thrombocytopenia [grade 4 (stratum 1, n=1; stratum 2, n=2)] was the only reported dose-limiting toxicity (study drug related) at 10 mg twice daily. In the MSSD cohort (stratum 1, n=20; stratum 2, n=18), adverse events (regardless of study drug relationship) led to treatment discontinuation in 15.0% and 33.3% of patients in stratum 1 and stratum 2, respectively, and dose adjustment/interruption in 45.0% and 66.7% of patients in stratum 1 and stratum 2, respectively. Three cases of on-treatment deaths were reported at the MSSD. Spleen response was achieved at week 48 in 33.3% and 30.0% of patients in stratum 1 and stratum 2, respectively. Improvements in the Total Symptom Score were also observed. In this study, ruxolitinib demonstrated acceptable tolerability in both the strata at the MSSD of 10 mg twice daily. (Registered at: clinicaltrials.gov identifier: 01317875)

The association between haemorrhage and markers of endothelial insufficiency and inflammation in patients with hypoproliferative thrombocytopenia: a cohort study

In daily haematological practice, predicting bleeding in thrombocytopenic patients is difficult, and clinicians adhere to transfusion triggers to guide patients through the aplastic phase of chemotherapy. Platelet count is not the only determinant of bleeding and additional mechanisms for impending haemostasis are likely. Beside clot formation, platelets are essential for the maintenance of integrity of vascular beds. We therefore prospectively investigated associations between biomarkers for endothelial damage (urine albumin excretion) and inflammation (C-reactive protein) and bleeding (WHO grading) in 88 patients with 116 on-protocol episodes. We found an increase in grade 2 bleeding with a higher urine albumin/creatinine ratio one day after the measurement [odds ratio (OR) 1·24 for every doubling of the ratio, 95% CI 1·05–1·46, P-value 0·01] and a 29% increase in the odds of grade 2 bleeding for every doubling of serum C-reactive protein (CRP) (95% CI 1·04–1·60, P-value 0·02) after correction for morning platelet count. The 24 h post-transfusion corrected count increment (CCI24) showed a significant association with these biomarkers: increasing urine albumin/creatinine ratio and CRP were associated with lower CCI24. We report two inexpensive and easy-to-apply biomarkers that could be useful in designing a prediction model for bleeding risk in thrombocytopenic patients

Therapeutic plasma exchange for anticoagulant-refractory antiphospholipid syndrome with severe ischemic and necrotic skin lesions

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by clinical findings including thrombosis and/or obstetric complication and laboratory findings, e.g. ≥1 positive antiphospholipid antibodies (aPL) (lupus anticoagulant, anticardiolipin IgG/IgM and/or anti-β2-glycoprotein IgG/IgM). A rare APS clinical entity is severe necrosis which is difficult to treat and often does not respond to anticoagulant therapy. Three consecutive patients with primary or secondary APS who presented with necrotic skin lesions secondary to APS were treated with therapeutic plasma exchange (TPE), glucocorticoids and low-molecular-weight heparin. All patients had a rapid-onset, either full or significant recovery of their APS-related necrotic lesions. Upon treatment, one patients showed resolution of lupus anticoagulant. Two patients had a decrease of at least 88 % in aPL titers after the initial treatment, and were kept on TPE maintenance every 5–6 weeks. None of the p

Coma in thrombotic thrombocytopenic purpura

Thrombotic thrombocy topenic purpura (TTP) is characterised by a thrombotic, haemolytic microangiopathy leading to microvascular occlusion, haemolysis and ischaemic dysfunction of various organs including the brain. TTP may present with a variety of neurological symptoms, including headache, focal deficits, seizures and coma. The authors describe a 55-year-old man presenting with abdominal pain and rapidly progressive deterioration into coma without focal neurological deficits or seizures. A concomitant, transient, rapid increase in blood pressure raised the suspicion of a hypertensive crisis. Yet, our patient did not improve after vigorous treatment with antihy pertensives. Brain imaging excluded a hypertensive leucoencephalopathy . Despite the absence of a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13 (ADAMTS13) deficiency, the diagnosis idiopathic TTP was made after excluding secondary causes of TTP. Upon treatment with plasma exchange, corticosteroids and vincristin our patient gradually improved. On discharge to a rehabilitation centre he was awake and alert, had minor cognitive deficits and a mild proximal tetraparesis consistent with a critical illness poly (neuro)myopathy

Patients treated with high-dose intravenous immunoglobulin show selective activation of regulatory T cells

Intravenous immunoglobulin (IVIg) is used to treat autoimmune and systemic inflammatory diseases caused by derailment of humoral and cellular immunity. In this study we investigated whether IVIg treatment can modulate regulatory T cells (Tregs) in humans in vivo. Blood was collected from IVIg-treated patients with immunodeficiency or autoimmune disease who were treated with low-dose (n=12) or high-dose (n=15) IVIg before, immediately after and at 7 days after treatment. Percentages and activation status of circulating CD4+CD25+forkhead box protein 3 (FoxP3+) Tregs and of conventional CD4+FoxP3- T-helper cells (Tconv) were measured. The suppressive capacity of Tregs purified from blood collected at the time-points indicated was determined in an ex-vivo assay. High-dose, but not low-dose, IVIg treatment enhanced the activation status of circulating Tregs, as shown by increased FoxP3 and human leucocyte antigen D-related (HLA-DR) expression, while numbers of circulating Tregs remained unchanged. The enhanced activation was sustained for at least 7 days after infusion, and the suppressive capacity of purified Tregs was increased from 41 to 70% at day 7 after IVIg treatment. The activation status of Tconv was not affected by IVIg. We conclude that high-dose IVIg treatment activates Tregs selectively and enhances their suppressive function in humans in vivo. This effect may be one of the mechanisms by which IVIg restores imbalanced immune homeostasis in patients with autoimmune and systemic inflammatory disorders

Generating human prostate cancer organoids from leukapheresis enriched circulating tumour cells

Background: Circulating tumour cell (CTC)–derived organoids have the potential to provide a powerful tool for personalised cancer therapy but are restrained by low CTC numbers provided by blood samples. Here, we used diagnostic leukapheresis (DLA) to enrich CTCs from patients with metastatic prostate cancer (mPCa) and explored whether organoids provide a platform for ex vivo treatment modelling. Methods: We prospectively screened 102 patients with mPCa and performed DLA in 40 patients with ≥5 CTCs/7.5 mL blood. We enriched CTCs from DLA using white blood cell (WBC) depletion alone or combined with EpCAM selection. The enriched CTC samples were cultured in 3D to obtain organoids and used for downstream analyses. Results: The DLA procedure resulted in a median yield of 5312 CTCs as compared with 22 CTCs in 7.5 mL of blood. Using WBC depletion, we recovered 46% of the CTCs, which reduced to 12% with subsequent EpCAM selection. From the isolated and enriched CTC samples, organoid expansion succeeded in 35%. Successful organoid cultures contained significantly higher CTC numbers at initiation. Moreover, we performed treatment modelling in one organoid cell line and identified substantial tumour heterogeneity in CTCs using single cell DNA sequencing. Conclusions: DLA is an efficient method to enrich CTCs, although the modest success rate of culturing CTCs precludes large scale clinical application. Our data do suggest that DLA and subsequent processing provides a rich source of viable tumour cells. Therefore, DLA offers a promising alternative to biopsy procedures to obtain sufficient number of tumour cells to study sequential samples in patients with mPCa. Trial registration number: NL6019.</p