Marker Of Relapse Research Articles

Activation of the PI3K-AKT-mTOR cascade in uterine leiomyosarcoma: a retrospective study of 31 patients

Background. Taking into account the literature data, the activation of the PI3K-AKT-mTOR cascade plays a role in the development of leiomyosarcoma (LMS), and the expression of markers of this signaling pathway in the tumor is associated with a more aggressive course of the disease. In addition, there is some data on the effective use of mTOR inhibitors in LMS.
 Aim. To study the frequency of expression of mTOR and phospho-mTOR markers in relapse of high-grade uterine LMS and to evaluate the effect of phospho-mTOR expression on the immediate and long-term results of the use of doxorubicin-containing chemotherapy regimens and the combination of gemcitabine + docetaxel.
 Materials and methods. The study included 31 patients with high-grade uterine LMS operated on for relapse. IHC was performed using mTOR and phospho-mTOR antibodies on ready-made histological preparations of the surgical material. The expression level of these markers was estimated as a percentage from 0 to 100%. The immediate and long-term results of the most commonly used drug treatment regimens (doxorubicin-containing regimens and gemcitabine + docetaxel combinations) were analyzed depending on the level of phospho-mTOR expression in the tumor.
 Results. The expression of mTOR in the tumor was detected in 4 patients with an expression level from 1 to 2%, and the expression of phospho-mTOR was detected in 20 patients with an expression level from 1 to 70%. Median OS in the group without phospho-mTOR expression was 135 months, in the group with expression 104 months, p=0.732. When using doxorubicin-containing regimens in the group with phospho-mTOR expression, 12% of patients had a partial response (PR), median PFS was 26.7 months, in the group without expression there was no PR, median PFS was 8.7 months, p=0.67. When using a combination of gemcitabine + docetaxel, in the group with phospho-mTOR expression, 24% of patients had PR, median PFS was 11.1 months, in the group without expression, 14% of patients had PR, median PFS was 12.4 months, p=0.372.
 Conclusion. Phospho-mTOR expression was detected in 2/3 of patients with high-grade uterine LMS. There was no effect of the expression of this marker on the OS of all included patients and on PFS in patients who received chemotherapy according to the gemcitabine + docetaxel regime. There was a tendency to an increase in PFS in the group with phospho-mTOR expression in patients who received doxorubicin-containing chemotherapy regimens.

Abstract 10211: Longitudinal Changes in Clinical and Imaging Variables During Withdrawal of Heart Failure Therapy in Patients with Recovered Dilated Cardiomyopathy: An Analysis from TRED-HF

Introduction: In TRED-HF, 40% of patients with recovered dilated cardiomyopathy (DCM) relapsed during withdrawal of therapy. Non-invasive markers of relapse may be used to monitor patients who wish a trial of therapy withdrawal. Purpose: To profile the changes in clinical and imaging variables amongst patients with recovered DCM undergoing therapy withdrawal in TRED-HF. Methods: Patients with recovered DCM were randomised to phased withdrawal of therapy or to continue therapy for 6 months. After 6 months of continued therapy, those in the control arm underwent withdrawal of therapy in a crossover phase. Clinical variables were measured at each study visit and imaging variables were measured at baseline, 16 weeks and 6 months. Mean values and 95% CIs were calculated at each time point. Clinical variables were modelled using fractional polynomials. Results: Of 51 patients, 26 were assigned to continue therapy and 25 to withdraw therapy over the first 6 months. Of the former, 24 of 26 patients, withdrew therapy in the cross-over phase between 6-12 months. An increase in mean heart rate was noted within 4 weeks of beginning therapy withdrawal and was the earliest change observed. Heart rate and change in heart rate from baseline were closely associated with the occurrence of relapse in multivariable analysis. A rise in systolic and diastolic blood pressure was apparent by 8 weeks. A rise in mean log NT-pro-BNP was the latest change to occur and not observed until 6 months after starting therapy withdrawal. ( Figure 1 & Table 1) . A rise in mean LV mass and LVEDV and a reduction in mean LVEF was seen by 16 weeks ( Figure 2 & Table 1) . Conclusion(s): A rise in heart rate may be the earliest sign of relapse during withdrawal of medication. This is accompanied or closely followed by changes in LV structure and function, with changes in natriuretic peptide concentrations occurring later. This informs monitoring strategies in cases where it may be necessary to reduce heart failure therapy.

Stratification of Intermediate-Risk Acute Myeloid Leukemia According to the Expression Level of WT1 mRNA at Diagnosis

IntroductionDespite advances in the genetic analysis of acute myeloid leukemia (AML), Wilms' tumor oncogene 1 (WT1) mRNA remains an important pan-marker of AML. A log reduction in WT1 mRNA expression after chemotherapy is a predictor of prognosis, and WT1 mRNA can be used as a marker of minimal residual disease or relapse. In the 1990s, a high expression level of WT1 mRNA at diagnosis was considered a poor prognostic factor. However, recent analyses have found that WT1 mRNA expression varies with AML type. Since the prognosis is affected by cytogenetic abnormalities and therapeutic intensity, we re-evaluated the clinical significance of WT1 mRNA expression at diagnosis in the context of the European Leukemia Net (ELN) risk classification and treatment.MethodWe retrospectively analyzed 216 patients at five institutions between 2011 and 2020. The expression level of WT1 mRNA was measured for all patients at diagnosis using bone marrow (BM) samples from 191 patients and peripheral blood (PB) samples from 25. WT-1 mRNA expression was measured using real-time quantitative polymerase chain reaction and the measured values were converted in normal logarithm. The median age at diagnosis was 62 (range: 23-93) years. The cytogenetic risk of ELN was classified as favorable (n = 41), intermediate (n = 123), and adverse risk (n = 41). Selected therapeutics were standard chemotherapy (n = 182, 84.3%, including CAG regimen; cytarabine, aclarubicin and G-CSF), hypomethylating agents or low-dose cytarabine (n = 19, 8.8%), and best supportive care (n = 15, 6.9%). Also, 143 patients (66.2%) received one or more courses of standard consolidation chemotherapy and 61 (28.3%) underwent allogeneic hematopoietic stem cell transplantation (allo-HCT). The median observation period was 518 [1-3418] days (Table 1). The overall survival (OS) and event-free survival (EFS) rates were assessed. Relapse or death was defined as an event, and OS was evaluated on the date of death.ResultThe median expression level of WT1 mRNA was 4.68 (range: 1.0-5.72) [log copies/µg RNA, units are omitted thereafter] in BM and 3.66 (range: 1.34-5.20) in PB (Table 1). Favorable-risk AML had the highest WT1 mRNA expression level in BM (4.95, p = 0.0054), whereas there was no difference between the expression levels in intermediate- and adverse-risk AML in BM (4.63 and 4.47, p = 0.711, Fig. 1A). WT1 mRNA expression in PB were 4.04, 3.84, and 3.05 for favorable-, intermediate-, and adverse-risk AML, respectively, and were higher for those with a favorable-risk AML (vs. adverse risk, p = 0.048, Fig. 1B). When WT1 mRNA expression level in BM was compared between the two groups, i.e., <4.0 (BM-WT1 low) vs. ≥4.0 (BM-WT1 high), 2-year EFS rates were 26.8% and 49.7% (p = 0.0035) and 2-year OS rates were 44.9% and 61.5% (p = 0.00053), respectively. When WT1 mRNA expression level in PB was compared between the two groups, i.e., <3.0 (PB-WT1 low) vs. ≥3.0 (PB-WT1 high), 2-year EFS rates were 0% and 51.5% (p = 0.023) and 2-year OS rates were 0% and 73.1% (p = 0.01), respectively. PB- or BM-WT1 low showed a worse prognosis at diagnosis.Since AML prognosis is affected by ELN risk and selected therapeutics, we evaluated 109 intermediate-risk patients who had received at least one course of standard chemotherapy. Fifty of them (45.9%) underwent subsequent allo-HCT. The 2-year EFS rates of PB or BM-WT1 low (n = 25, PB = 2 and BM = 23) vs. PB or BM-WT1 high (n = 84, PB = 23 and BM = 61) were 23.9% and 50.4%, respectively (p = 0.023, Fig. 2A) and the 2-year OS rates were 51.2% and 64.6%, respectively (p = 0.03, Fig. 2B). PB or BM-WT1 low of intermediate-risk AML had a poor prognosis even with standard chemotherapy. Multivariate analysis adjusted for ages, the reduction rate of WT1 mRNA after induction chemotherapy, duration of receiving consolidation chemotherapy, allo-HCT, and PB or BM-WT1 low at diagnosis were independent poor prognosis factors for EFS in intermediate-risk AML (HR: 3.32, 95%CI: 1.29-8.53, p = 0.013). The OS rate of PB or BM-WT1 low also worsened (HR: 2.33, 95%CI: 0.88-6.18, p = 0.089) (Table 2). Whereas, the outcome of adverse-risk AML was not affected by PB or BM-WT1 low/high.ConclusionThe expression level of WT1 mRNA at diagnosis was negatively correlated with prognosis. Favorable-risk AML had higher expressions of WT1 mRNA. PB or BM-WT1 low in intermediate-risk AML was associated with a poor prognosis. In standard-risk AML, WT1 mRNA may be a useful pan-marker to predict prognosis at diagnosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single Cell Sequencing of Pediatric Acute Myeloid Leukemia Reveals Clonal Evolution to Relapse on Combination Chemotherapy with Sorafenib

Introduction: Relapse of pediatric acute myeloid leukemia (AML) remains a leading cause of childhood cancer mortality, and leukemias with activation of the Fms-like tyrosine kinase 3 (FLT3) are particularly susceptible to relapsed disease. Risk-directed therapy to prevent relapse is based both on genetic changes known to drive drug resistance, and measurable residual disease (MRD) at the end of induction therapy (EOI). In adult AML, resistance to type II FLT3-inhibitors, like sorafenib, is primarily driven by on-target FLT3 kinase domain (KD) mutations. However, the resistance mechanisms for pediatric leukemias, which are treated on combination therapies, have not been fully elucidated. MRD is considered the among the most predictive markers of future relapsed disease. It has been assumed that the major clone at the time of MRD assessment will predict the majority clone at relapse. However, this assumption has not been proven. The definition of the most specific genetic and MRD markers of relapse are essential to prognosticate and personalize therapy to prevent relapsed disease.Methods: We performed single cell sequencing (SCS) with a high-throughput DNA sequencing platform, Mission Bio Tapestri, on bone marrow or peripheral blood samples from 24 samples from 8 pediatric patients treated on COG AAML1031 with serial samples from diagnosis, EOI, and relapse.Results: We analyzed a total of 94,833 cells from 8 pediatric patients (median cells per patient 12,428) all treated on AAML1031. SCS revealed a sensitive and specific description of clonal evolution on the combination of sorafenib with cytotoxic chemotherapy. The FLT3 internal tandem duplication (ITD) was controlled by the therapy in only half of the patients. In five of the patients, the FLT3-ITD was present in multiple clones. The FLT3-ITD co-mutated with additional mutations (NRAS, SH2B3, WT1, TET2, or NPM1) in half of the patients. However, the presence of a co-mutation did not necessarily correlate with whether or not the ITD-containing clone persisted at the time of relapse. Of the leukemias whose relapse was not driven by FLT3, the most likely mutational driver of resistance was NRAS. Notably, however, despite the fact that FLT3 KD mutations make up the bulk of mutational resistance to type II FLT3i such as sorafenib in adult patients, there were no on-target FLT3 mutations found in any of these pediatric patients. Further, SCS allows for an unprecedented depth of analysis of the genetic complexity of pediatric AML. Phylogenic analysis revealed that the same mutations may arise independently in different cells (NPM1 W288fs, NRAS G60E). Additionally, the same gene may be mutated twice within the same cell (WT1, TET2). These data, consistent with our prior work, suggest that some leukemias may have a predilection to mutations within specific loci. Finally, although there is a standing assumption that the dominant MRD population will proliferate into relapsed disease, in 3/8 patients, the dominant MRD clone did not predict the dominant relapse clone.Conclusions: SCS allows for direct measurement of clonal hierarchy and evolution, phylogeny, co-mutational status, and zygosity, which can only be inferred through traditional bulk NGS. The mutational mechanisms of resistance seen in adult leukemias treated with sorafenib monotherapy are not necessarily relevant to the pediatric population; rather than on-target FLT3 mutations, off target mutations including NRAS are found. This corroborates prior findings that off-target RAS pathway mutations may drive resistance to FLT3i. Non-RAS off-target mutations found in this cohort do not necessarily predict sorafenib resistance, so may be passenger mutations. The lack of consistent resistance mutations suggests that other mechanisms of resistance such as epigenetic modifications may also drive resistance to combination chemotherapy with FLT3i in pediatric leukemia. Further, SCS exposes more genetic complexity in pediatric AML than has previously been appreciated: the same mutation may independently arise in more than one cell or the same cell may have multiple mutations within the same gene. Finally, the sensitivity of SCS reveals that the major clone at the time of MRD assessment is not necessarily the major clone at relapse. This suggests a benefit of more frequent MRD monitoring to track clonal evolution in real time. DisclosuresSmith: Daiichi Sankyo: Consultancy; Revolutions Medicine: Research Funding; AbbVie: Research Funding; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Glucocorticoid-Resistant B-Cell Acute Lymphoblastic Leukemic Cells Can be Targeted By BCR-Signaling Inhibition

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome.Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it.To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance.The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment.To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells.Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002).Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. DisclosuresBiondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

A Dual Marker for Monitoring MDR-TB Treatment: Host-Derived miRNAs and M. tuberculosis-Derived RNA Sequences in Serum.

BackgroundIn the absence of a late marker of treatment failure or relapse in MDR-TB patients, biomarkers based on host-miRNAs coupled with M. tuberculosis-RNAs evaluated in extracellular vesicles (EVs) are an alternative follow-up for MDR-TB disease. Characterization of EVs cargo to identify differentially expressed miRNAs before and after treatment, and to identify M. tuberculosis-derived RNA in serum EVs from resistant TB patients.MethodsEVs were isolated from serum of 26 drug-resistant TB (DR-TB) patients and 16 healthy subjects. Differential expression of miRNAs in pooled exosomes from both untreated and treated patients was assessed and individually validated at different time points during treatment. In addition, M. tuberculosis RNA was amplified in the same samples by qPCR.ResultsA multivariate analysis using miR-let-7e-5p, -197-3p and -223-3p were found to be a more sensitive discriminator between healthy individuals and those with TB for both DR-TB (AUC= 0.96, 95%, CI=0.907-1) and MDR-TB groups (AUC= 0.95, 95%, CI= 0.89-1). Upregulation of miR-let-7e-5p were observed at the time of M. tuberculosis negative culture T(3-5) for MDR-TB group or for long-term T(9-15) for MDR-TB group without diabetes (T2DM). A second pathogen-based marker based on 30kDa and 5KST sequences was detected in 33% of the MDR-TB patients after the intensive phase of treatment. The miR-let7e-5p is a candidate biomarker for long-term monitoring of treatment for the group of MDR-TB without T2DM. A dual marker of host-derived miR-let7e-5p and M. tuberculosis-derived RNA for monitoring-TB treatment based in serum EVs.ConclusionA dual marker consisting of host-derived miR-let7e-5p and M. tuberculosis-derived RNA, could be an indicator of treatment failure or relapse time after treatment was completed.

Molecular Minimal Residual Disease Detection in Acute Myeloid Leukemia.

Simple SummaryAlthough the majority of patients with acute myeloid leukemia (AML) reach a morphologic complete remission after high-dose chemotherapy, the majority of them face a relapse within a few years. Detection of residual cells, persisting in a considerably small amount, has shown to be predictive of impending relapse in multiple studies. Whereas the gold standard in minimal residual disease (MRD) detection in AML is currently based on immunophenotypic approaches, the use of molecular MRD testing to predict AML relapse has been explored extensively in recent years. This review aims to provide an overview of the different studies that improve molecular MRD detection in AML, and to describe the limitations and challenges it faces.Initial induction chemotherapy to eradicate the bulk of acute myeloid leukemia (AML) cells results in complete remission (CR) in the majority of patients. However, leukemic cells persisting in the bone marrow below the morphologic threshold remain unaffected and have the potential to proliferate and re-emerge as AML relapse. Detection of minimal/measurable residual disease (MRD) is a promising prognostic marker for AML relapse as it can assess an individual patients’ risk profile and evaluate their response to treatment. With the emergence of molecular techniques, such as next generation sequencing (NGS), a more sensitive assessment of molecular MRD markers is available. In recent years, the detection of MRD by molecular assays and its association with AML relapse and survival has been explored and verified in multiple studies. Although most studies show that the presence of MRD leads to a worse clinical outcome, molecular-based methods face several challenges including limited sensitivity/specificity, and a difficult distinction between mutations that are representative of AML rather than clonal hematopoiesis. This review describes the studies that have been performed using molecular-based assays for MRD detection in the context of other MRD detection approaches in AML, and discusses limitations, challenges and opportunities.

CircRNF220, not its linear cognate gene RNF220, regulates cell growth and is associated with relapse in pediatric acute myeloid leukemia

BackgroundCircular RNAs (circRNAs) constitute a family of transcripts with unique structures and have been confirmed to be critical in tumorigenesis and to be potential biomarkers or therapeutic targets. However, only a few circRNAs have been functionally characterized in pediatric acute myeloid leukemia (AML).MethodsHere, we investigated the expression pattern of circRNAs in pediatric AML using a circRNA microarray. The characteristics, potential diagnostic value, and prognostic significance of circRNF220 were evaluated. A series of functional experiments were performed to investigate the role of circRNF220 in primary pediatric AML cells. Then we investigated the aberrant transcriptional networks regulated by circRNF220 in primary AML cells by RNA-seq. Furthermore, biotin RNA pulldown assays were implemented to verify the relationship between circRNF220 and miR-30a.ResultsWe identified a circRNA, circRNF220, which was specifically abundant in and accumulated in the peripheral blood and bone marrow of pediatric patients with AML. It could distinguish AML from ALL and other hematological malignancies with high sensitivity and specificity. Significantly, circRNF220 expression independently predicted prognosis, while high expression of circRNF220 was an unfavorable prognostic marker for relapse. Furthermore, we characterized the function of circRNF220 and found that circRNF220 knockdown specifically inhibited proliferation and promoted apoptosis in AML cell lines and primary cells. Mechanistically, circRNF220 may act as an endogenous sponge of miR-30a to sequester miR-30a and inhibit its activity, which increases the expression of its targets MYSM1 and IER2 and implicated in AML relapse.ConclusionsCollectively, these findings demonstrated that circRNF220 could be highly efficient and specific for the accurate diagnosis of pediatric AML, with implications for relapse prediction.

Оцінка ефективності хірургічного лікування АКТГ-залежного синдрому Кушинга в ранньому і пізньому післяопераційному періоді

Актуальність. АКТГ-залежний синдром Кушинга (СК) — тяжке ендокринне захворювання, що характеризується хронічним надлишком кортизолу внаслідок АКТГ-секретуючої аденоми гіпофіза. Метою роботи є оцінка ефективності проведеного хірурічного лікування АКТГ-залежного СК в ранньому і пізньому післяопераційному періоді. Матеріали та методи. Під спостереженням перебувало 234 пацієнти, середній вік чоловіків становив 26,38 ± 3,40 року, жінок — 27,58 ± 3,40 року. Усім хворим виконувався комплекс досліджень, що містив загальноклінічні, біохімічні, гормональні аналізи, а також магнітно-резонансна томографія головного мозку, рентгенографія органів грудної клітки, денситометрія. Результати. Трансназальна аденомектомія гіпофіза (ТАГ) була виконана первинно у 200 пацієнтів, з них повторно — у 34. При цьому ремісія у пацієнтів, які зазнали ТАГ з приводу АКТГ-залежного СК, настала у 42 %, рецидив розвинувся у 5 %, ремісія не досягнута у 46 %. Визначена вірогідність відмінностей між такими параметрами, як число пацієнтів в період ремісії і число рецидивів після ТАГ, рівень вільного кортизолу плазми і глікемії натще у хворих в період ремісії і рецидиву. Вивчений кореляційний зв’язок між рівнем вільного кортизолу плазми в ранньому постопераційному періоді після ТАГ і частотою рецидивів. Висновки. Найбільш вірогідним, інформативним прогностичним маркером рецидиву пухлини є рівень кортизолу плазми в ранньому післяопераційному періоді: чим нижче рівень кортизолу, тим стійкішою спостерігається ремісія. Безпосередні результати хірургічного лікування СК залежать від розмірів пухлини, ступеня тяжкості загального стану пацієнта, наявності нейроендокринних і кістково-метаболічних ускладнень.

Dissecting the Role of Individual Bcl-2 Members in Response and Resistance to Ibrutinib or Venetoclax in CLL

Introduction. BCL-2 family members are crucial determinants for survival of normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, is now successfully applied in Chronic Lymphocytic Leukemia (CLL). CLL cells cycle between the lymph node (LN) and peripheral blood (PB), and in those compartments display major changes in expression of BCL-XL, MCL-1 and BFL-1, which are reportedly not targeted by Venetoclax. Indeed, prolonged in vitro CD40 engagement also induces expression of these BCL-2 family members and renders CLL cells resistant to Venetoclax (Thijssen et al Haematologica 2015). The BTK inhibitor Ibrutinib drives CLL cells out of the LN and deprives them of survival and proliferative signals. After prolonged therapy, Ibrutinib resistance develops in certain patients, who then show fast disease progression. It is currently unknown how LN expulsion or development of resistance affects expression of BCL-2 members.In order to design effective, long-term therapies including rational combinations, it is crucial to understand the contribution of distinct BCL-2 family members to resistance against these single drugs. Recent LN emigrants (CD5hi/CXCR4lo) can be distinguished from ‘old’ returning cells (CD5lo/CXCR4hi; Calissano et al Mol med 2011), which allows for differential analysis of the two compartments using only PB samples. In addition, new BH3 mimetics targeting BCL-XL and MCL-1, and cysteine-reactive stapled BH3 peptides specific for BFL-1 (Huhn et al Cell Chem Biol 2016), constitute new tools for functional profiling of individual pro-survival BCL-2 members.Aims . We measured changes in prosurvival BCL-2 members in LN emigrants versus returning cells in PB samples of responding and relapsed patients on Ibrutinib treatment, and during Venetoclax ramp-up treatment. We also determined the contribution of individual BCL-2 members in determining CD40-induced resistance to Venetoclax by BH3 mimetic profiling (Peperzak, et al. CDD 2017), and by stapled peptides targeting BFL-1.Experimental procedures. Surface CD19/CD5/CXCR4 staining was combined with intracellular FACS staining for BCL-2, BCL-XL and MCL-1. Combinations of BH3 mimetics (ABT-199, WEHI-539, A1331852, A1210477, S63845), and a stapled BIM BH3 helix bearing an acrylamide warhead allowing selective covalent engagement of BFL-1, were applied in vitro to dissect Venetoclax resistance. Furthermore, RNA and siRNA nucleofection for individual BCL-2 members into CLL cells was applied.Results. In pretreatment blood samples, recent LN emigrants have higher BCL-XL and MCL-1 expression than returning cells. This clear distinction collapses in response to Ibrutinib (currently N=15), indicating that although peripheral blood cell counts rise, blocking access to the LN effectively lowers expression of protective BCLXL and MCL-1 in vivo . However, in patients that develop Ibrutinib resistance (N=9), the MCL-1 or BCL-XL pre-treatment profile reappears. For such patients the next therapy might be Venetoclax, and we are investigating retrospectively to what extent Ibrutinib resistance and the rebound in BCL-XL or MCL-1 levels affects sensitivity for Venetoclax in vitro .Venetoclax treatment associates with rapid reduction of CLL cells from PB and LN (N=8), but in contrast to Ibrutinib no changes in BCL-2 members in LN emigrants versus immigrants were observed. In fact, their levels can even increase in patients that show residual cells at T = 1-1.5 months (N=2).To dissect the role of BCL-XL, MCL-1 and BFL-1 in CD40-induced maximal resistance to Venetoclax, we combined BH3 mimetics with a stapled peptide targeting BFL-1. We found that Venetoclax resistance in CD40-treated CLL cells can be reverted to almost pre-stimulation susceptibility by combined antagonism of BCL-XL and BFL-1. This pharmacologic approach was complemented with siRNA suppression or RNA overexpression for individual BCL-2 members. Combined, our data indicate that BCL-XL and also BFL-1 contribute strongly to Venetoclax resistance, while MCL-1 unexpectedly seems less dominant.Conclusions . 1) BCL-2 members can be used not only as drug targets but also as markers for response and relapse. 2) Strategies that combine Venetoclax with Ibrutinib to prevent access to the protective LN environment, or tailored combinations with other BH3 mimetics, hold promise for successful long-term treatment of CLL. DisclosuresTam:Abbvie: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Walensky:Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Brown:Janssen Oncology: Honoraria; Redx: Consultancy; AstraZeneca: Consultancy; Sun BioPharma: Consultancy, Research Funding; Astellas Pharma: Consultancy; Janssen: Consultancy; Roche/Genentech: Consultancy; Pfizer: Consultancy; AbbVie: Consultancy, Honoraria; Celgene: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy. Kater:Celgene: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Eldering:Gilead: Research Funding; Celgene: Research Funding; Roche: Research Funding.

PD-1 Is Not a Reliable Marker of Impending Relapse after Allogeneic Stem Cell Transplantation

INTRODUCTIONPost-transplant relapse is a major obstacle to successful allogeneic stem cell transplantation (allo-SCT) in hematologic malignancies. Failure to maintain an effective graft versus leukemia effect (GVL) is one mechanism responsible for post-transplant relapse. There is recent interest in PD-1 and other T cell exhaustion markers as therapeutic targets in cancer immunotherapy, however PD-1 inhibitors might induce graft versus host disease (GVHD) after allo-SCT. Therefore, understanding PD-1 kinetics in allo-SCT is needed before using check point inhibitors to prevent or treat relapse. Here we evaluated the relationship between leukemia relapse and the kinetics of T-cell exhaustion markers during the early post-transplant period. We further analyzed the functions of leukemia associated antigen (LAA) specific T-cells from relapsed subjects and sought for the mechanism of post-transplant relapse at a single cell level. METHODSEighty-five patients with various hematologic malignancies received ex-vivo T-cell depleted HLA matched related sibling allo-SCT at a single center between 2006 and 2015. All subjects were treated with a myeloablative conditioning regimen of cyclophosphamide, fludarabine, and total body irradiation with GVHD prophylaxis of low-dose cyclosporine for 21 days. Peripheral blood mononuclear cells (PBMCs) were collected from donors and recipients at 30, 60, and 100 days following transplant and at relapse to characterize regulatory T cells and T cell exhaustion markers by flow cytometry. Samples from six relapsed subjects with acute leukemias were evaluated for 5LAAs (WT1, PRAME, NY-ESO1, MAGEA3, AURAK) by RT-PCR and LAA specific T-cells by Elispot and flow cytometry. LAA specific T cells in one subject were further analyzed for single cell RNAseq. RESULTSAt a median follow up of 3.4 years, 35 patients had relapsed with a 5-year cumulative relapse incidence of 44% at a median time of 226 days. Post-relapse survival was poor among subjects relapsing before 180 days post allo-SCT (median post-relapse survival 126 days vs. 329 days for patients relapsing after 180 days; P=0.004). In the early post-transplant period, PD-1 expression in CD4 and CD8 T-cells was significantly and comparably elevated in both relapsed and non-relapsed cohorts as compared to donors. No association was observed between relapse incidence and pattern of PD-1 expression. In contrast, Helios+Tregs frequency was significantly higher in the relapsed cohort at day 30 (Figure 1). Among six relapsed subjects, at least one LAA was detected in PBMCs before and after relapse. LAA specific T-cells were observed in four subjects, however all samples eventually expressed new LAA different from the ones targeted by their own LAA specific T-cells suggesting immunological escape of leukemic blasts. Of note, LAA specific T-cells were predominantly enriched in the PD-1 negative fraction (Figure 2). Single cell analysis in one patient revealed a distinct cluster with the other exhaustion marker, LAG3 in PRAME specific T-cells in comparison to CMV specific T-cells. CONCLUSIONOur findings contradict the prevailing view that PD-1+ T cell exhaustion is a prominent feature of leukemic relapse after SCT. We found that PD-1 was overexpressed in T-cells during the early post-transplant period in all patients and PD-1 expression patterns did not identify patients destined to relapse. Furthermore, the leukemia antigen specific T-cells did not overexpress PD-1, instead single cell analysis revealed LAG3 overexpression in one patient at relapse. These findings do not support PD-1 is the predominate marker for leukemia specific T cell exhaustion in relapsing patients but point to other exhaustion markers or suppressor Treg cells as causes of relapse. Further studies are required to identify predictive biomarkers for check point inhibitors in hematologic malignancies and the patterns of LAA expression in residual disease following allo-SCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of Novel Highly Specific FLT3 Targeted Inhibitors for Combination or Sequential Therapy of Acute Myeloid Leukemia

More than 25% of acute myeloid leukemia (AML) patients have an activating mutation of the fetal-liver tyrosine kinase 3 (FLT3) receptor. FLT3 mutations vary, can be multiple, are often unstable length mutations located in the receptor's juxtamembranous domain (internal tandem duplication (ITD) mutations), and represent the strongest single marker for relapse of AML. When developing novel inhibitors of FLT3, it should be taken into careful consideration that there is a high inter- and intra-patient variability in the length and sequence repeated within ITD mutations. Recently, the first FLT3 targeting kinase inhibitor midostaurin was approved for combination therapy in FLT3 mutated AML, and several other kinase inhibitors are at present also in preclinical development. These inhibitors share broad kinase specificity, in several cases co-inhibiting FLT3 with structurally related targets like c-Kit and AXL. These targets may add therapeutic effect as well as adverse effects. Highly specific kinase inhibitors against varying FLT3-ITDs are to our knowledge currently not in clinical development.We are developing a novel class of highly specific FLT3-ITD targeting kinase inhibitors based on a well-established chemical backbone previously used in well-tolerated anti-viral therapy. These inhibitors, the BiKin2 compounds, are specifically developed to have superior selectivity towards FLT3-ITD over closely related type III tyrosine kinases, minimizing the chances of adverse off-target effects. We are screening the compounds to identify analogues that target a wide variety of FLT3-ITD mutations, and that can be combined with other BiKin2 analogues and/or with other targeted therapies in order to avoid development of acquired resistance during therapy.By employing cell lines expressing wild type (wt) FLT3 and ITD mutations of varying lengths we are currently testing more than 40 analogues of the BiKin2 compounds in in vivo and in vitro settings. Among the leukemic cell lines being used are the FLT3-mutated cells MOLM-13 and MV4-11 and the FLT3-wt cells OCI-AML3. Additionally, we are using the BA/F3 cells as a model of FLT3-ITD oncogene addiction. The BA/F3 cells are stably transduced to express FLT3-ITD of different lengths cloned from an AML patient at diagnosis and relapse, in addition to specific point mutations affecting the kinase domain. These screens give us valuable information on how the BiKin2 compounds inhibit and affect ITDs of varying length and sequences. Thus far these novel compounds have shown promising results in ITD mutated cell lines, as shown in Table 1 and Figure 1A.We have piloted an in vivo toxicity test (MTD) and proof-of-principle-experiments with the first compound showing promising selectivity for FLT3-ITD, BCI-332. We found no dose-limiting toxicity at doses up to 10 mg/kg (Figure 1B), and the drug also showed significant inhibition of tumor growth in a subcutaneous model of FLT3-ITD expressing MV4-11 (ITD/null) cells (Figure 1C). The FLT3 selectivity of BCI-332 at 1 µM over 250 kinases in vitro is superior with 90% versus below 20% inhibition.In the clinic, patients often develop resistance to FLT3 inhibitors due to secondary mutations within the mutated gene as well as selection of resistant leukemic clones. Therefore, we used a high throughput in vitro drug sensitivity and resistance test (DSRT) approach to evaluate the efficacy of the model compound BCI-332 in a combination screen together with 527 clinically approved and investigational drugs. Drug combinations were tested in both FLT3-ITD expressing (MOLM-13 wt/ITD) and FLT3-wt expressing (OCI-AML3 wt/wt) cells, and we identified several potential drug candidates that showed additive effect in combination with BCI-332 in both conditions (Figure 2). Potential combination candidates need to be further validated in more AML cell lines and in primary cells from AML patients with defined ITD mutational status.In summary, we employed a novel approach to develop FLT3-ITD targeting drugs by screening inhibitors for selectivity against multiple different length ITD mutations, in an attempt to identify FLT3-ITD specific inhibitors with minimal off-target inhibition. Ultimately, our goal is to identify one or several drug candidate(s) to be tested in a clinical trial setting, in combination or in sequences combined with other targeted therapies. [Display omitted] DisclosuresAmiable:BCI Pharma: Employment. Popa:Kinn Therapeutics AS: Employment. Guillon:BCI Pharma: Employment. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; IMI2 project HARMONY: Research Funding. McCormack:Kinn Therapeutics AS: Other: Co-ownership. Surleraux:BCI Pharma: Employment, Equity Ownership.

Heart Rate as a Marker of Relapse During Withdrawal of Therapy in Recovered Dilated Cardiomyopathy

ObjectivesThe objective of this study was to determine the relationship between heart rate and relapse among patients in the TRED-HF (Therapy withdrawal in REcovered Dilated cardiomyopathy trial). BackgroundUnderstanding markers and mechanisms of relapse among patients with recovered dilated cardiomyopathy (DCM) may enable personalized management. MethodsThe relationship between serial heart rate measurements and relapse was examined among patients in the TRED-HF trial, a randomized trial which examined the safety and feasibility of withdrawing heart failure therapy from 51 patients with recovered DCM over 6 months. In total, 25 patients were randomized to therapy withdrawal and 26 to continue therapy, of whom 25 subsequently began therapy withdrawal in a single arm crossover phase. ResultsThe mean ± SD heart rate for those who had therapy withdrawn and did not relapse was 64.6 ± 10.7 beats/min at baseline and 74.7 ± 10.4 beats/min at follow-up, compared to 68.3 ± 11.3 beats/min at baseline and 86.1 ± 11.8 beats/min at follow-up for those who relapsed. After adjusting for differences in heart rate at baseline, patients who had therapy withdrawn and relapsed had a 10.4 beats/min (95% CI: 4.0–16.8) greater rise in heart rate than patients who had therapy withdrawn and did not relapse (P = 0.002). After data were adjusted for age, log N-terminal pro–B-type natriuretic peptide, and left ventricular ejection fraction (LVEF), heart rate (per 10 beats/min; hazard ratio [HR]: 1.65; 95% CI: 1.10-2.57; P = 0.01) and change in heart rate from baseline (per 10 beats/min; HR: 1.70; 95% CI: 1.12–2.57; p = 0.01) were associated with relapse. The results remained qualitatively the same after adjusting for beta-blocker dose. ConclusionsFor patients with DCM and improved LVEF, the rise in heart rate after treatment is withdrawn treatment identifies patients who are more likely to relapse. Whether the increase in heart rate is a marker or a mediator of relapse requires investigation. (Therapy withdrawal in REcovered Dilated cardiomyopathy trial [TRED]; NCT02859311)

Circulating tumor DNA as a noninvasive marker of resectability in ovarian carcinomas.

5548 Background: Ovarian cancer is the leading cause of death by gynecological cancer. Complete surgery remains one of the main prognostic factors. Laparoscopic exploration is mandatory to assess surgical resectability at diagnosis or after neoadjuvant chemotherapy. However, there is no clinical or biological marker that can correctly predict resectability and may be able to avoid a second laparoscopic exploration for initially unresectable diseases. Our aim was to assess circulating tumor DNA (ctDNA) value as a predictive non-invasive marker of evolution towards resectability for patients with epithelial ovarian cancer receiving first-line chemotherapy. Methods: We explored in this work one of the secondary objectives of the CIDOC study (NCT03302884). CIDOC is a multicenter prospective study aiming to explore ctDNA value as early marker of disease relapse after first-line treatment for epithelial ovarian cancer. Patients with mucinous histology or early stages not requiring chemotherapy are excluded. Plasma samples are collected at diagnosis, during neoadjuvant chemotherapy, and during follow-up. After DNA extraction, panel-based next generation sequencing is performed on both tumor samples and germline DNA, and somatic mutations of interest are selected for ctDNA monitoring. ctDNA analyses are conducted using droplet digital PCR (BioRad QX200) by measuring the variant allele fraction (VAF) of previously identified mutations. Results: This intermediary analysis has included 47 patients diagnosed between March 2017 and December 2019. Median age was 69 years old (48 – 84). Most of the patients had advanced disease (89.4% stage FIGO III or IV), serous histology (94.8%), and high grade tumor (92.3%). Most of the patients underwent complete interval cytoreductive surgery (76.3% vs 17.4% complete upfront surgery). Most of the tumors had TP53 mutations (85.1%), following by alterations involving DNA repair genes (38.3%). Median cell-free DNA concentration at baseline was 0.38 ng/µL (0 – 12.8). ctDNA was identified in 92.1% of patients at baseline with a median VAF of 1.84% (0 – 42.52%). ctDNA VAF was correlated to the peritoneal dissemination ( p= 0.039) assessed with the peritoneal cancer index. ctDNA clearance after preoperative chemotherapy tended to be correlated to achievement of complete interval surgery for patients receiving neoadjuvant chemotherapy ( p= 0.108). Conclusions: ctDNA may be a promising non-invasive marker to assess peritoneal cancer spreading and to predict surgical resectability after neoadjuvant chemotherapy. If confirmed in larger populations, this may enable to avoid additional surgical explorations for patients who remain ctDNA positive after chemotherapy. Clinical trial information: NCT03302884.

MiRNAs and radical prostatectomy: Current data, bioinformatic analysis and utility as predictors of tumour relapse

Studies of microRNAs (miRNAs) and genes have particular interest for cancer biology and medicine due to the discovery of new therapeutic targets and markers. These studies are extensively influenced by anticancer therapy, as miRNAs interfere with the therapy's efficacy in prostate cancer (PCa). In this article, we summarise the available data on the influence of radical prostatectomy (RP) and biochemical recurrence on miRNA expression. Molecular targets of these miRNAs, as well as the reciprocal relations between different miRNAs and their targets, were studied using the DIANA, STRING and TransmiR databases. Special attention was dedicated to the mechanisms of PCa development, miRNA, and associated genes as tumour development mediators. Combined analysis of the databases and available literature indicates that expression of four miRNAs that are associated with prostate cancer relapse and alter their expression after RP, combined with genes that closely interact with selected miRNAs, has high potential for the prediction of PCa relapse after RP. PCa tissues and biofluids, both immediately after RP for diagnostics/prognostics and in long-term (relapse) monitoring, may be used as sources of these miRNAs. An overview of the usefulness of published data and bioinformatics resources looking for diagnostic markers and molecular targets is presented in this article. The selected miRNA and gene panels have good potential as prognostic and PCa relapse markers after RP and likely could also serve as markers for therapeutic efficiency on a broader scale.

USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

Tumor cell dissemination in cancer patients is associated with a significant reduction in their survival and quality of life. The ubiquitination pathway plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions and its dysregulation has been associated with malignant transformation and invasive potential of tumor cells, thus highlighting its value as a potential therapeutic target. In order to identify novel molecular targets of tumor cell migration and invasion we performed a genetic screen with an shRNA library against ubiquitination pathway-related genes. To this end, we set up a protocol to specifically enrich positive migration regulator candidates. We identified the deubiquitinase USP19 and demonstrated that its silencing reduces the migratory and invasive potential of highly invasive breast cancer cell lines. We extended our investigation in vivo and confirmed that mice injected with USP19 depleted cells display increased tumor-free survival, as well as a delay in the onset of the tumor formation and a significant reduction in the appearance of metastatic foci, indicating that tumor cell invasion and dissemination is impaired. In contrast, overexpression of USP19 increased cell invasiveness both in vitro and in vivo, further validating our findings. More importantly, we demonstrated that USP19 catalytic activity is important for the control of tumor cell migration and invasion, and that its molecular mechanism of action involves LRP6, a Wnt co-receptor. Finally, we showed that USP19 overexpression is a surrogate prognostic marker of distant relapse in patients with early breast cancer. Altogether, these findings demonstrate that USP19 might represent a novel therapeutic target in breast cancer.

Evaluation of Retinal Layer Thickness Parameters as Biomarkers in a Real-World Multiple Sclerosis Cohort.

PurposeRetinal layer thickness parameters measured by optical coherence tomography (OCT) are emerging biomarkers of neuroaxonal degeneration and inflammation in multiple sclerosis (MS). We aimed to evaluate the value of retinal layer thickness for prediction of disability worsening and relapse in a real-world MS cohort.Patients and MethodsFor this longitudinal observational study, we included MS patients with spectral-domain OCT scans available and ≥1 year of clinical follow-up. The value of peripapillary retinal nerve fiber layer (pRNFL), macular ganglion-cell-and-inner-plexiform-layer (GCIPL) and inner nuclear layer (INL) thickness for prediction of disability worsening and relapse during the observation period was tested by multivariate models.ResultsWe analyzed 60 MS patients during a mean observation period of 2.9 years (SD 1.8). Lower baseline thickness of GCIPL (cut-off <77µm; HR 4.1, p=0.001) and pRNFL (cut-off ≤88µm; HR 3.1, p=0.019) were associated with an increased risk of disability worsening. Longitudinally, mean thinning rates were −0.8µm/year (SD 1.6) for GCIPL, −0.6µm/year (SD 3.5) for pRNFL. GCIPL thinning ≥1.0µm/year and pRNFL >1.5µm/year is associated with higher likelihood of disability worsening (HR 5.7, p=0.009 and HR 6.8, p=0.003, respectively). INL thickened in patients with relapse by a mean 0.9µm while thinning by 0.3µm in patients without relapse (p=0.04). In multivariate analyses, INL thickening was associated with an increased probability of relapse (OR 17.8, p=0.023).ConclusionCross-sectional and longitudinal measurement of GCIPL and pRNFL thinning is reliable as a biomarker of disability worsening in a real-world setting. Change of INL thickness is a promising marker of relapse, i.e. inflammatory activity.

Changes in cerebrospinal fluid interleukin-10 levels display better performance in predicting disease relapse than conventional magnetic resonance imaging in primary central nervous system lymphoma

BackgroudEstablishing diagnostic and prognostic biomarkers of primary central nervous system lymphoma (PCNSL) is a challenge. This study evaluated the value of dynamic interleukin (IL)-10 cerebrospinal fluid (CSF) concentrations for prognosis and relapse prediction in PCNSL.MethodsConsecutive 40 patients newly diagnosed with PCNSL between April 2015 and April 2019 were recruited, and serial CSF specimens were collected by lumbar punctures (LP) or by Ommaya reservoir at diagnosis, treatment, and follow-up phase.ResultsWe confirmed that an elevated IL-10 cutoff value of 8.2 pg/mL for the diagnosis value of PCNSL showed a sensitivity of 85%. A persistent detectable CSF IL-10 level at the end of treatment was associated with poor progression-free survival (PFS) (836 vs. 481 days, p = 0.049). Within a median follow-up of 13.6 (2–55) months, 24 patients relapsed. IL-10 relapse was defined as a positive conversion in patients with undetectable IL-10 or an increased concentration compared to the last test in patients with sustained IL-10. IL-10 relapse was detected a median of 67 days (28–402 days) earlier than disease relapse in 10/16 patients.ConclusionThis study highlights a new perspective that CSF IL-10 relapse could be a surrogate marker for disease relapse and detected earlier than conventional magnetic resonance imaging (MRI) scan. Further evaluation of IL-10 monitoring in PCNSL follow-up is warranted.

Haemoglobinuria for the early identification of aHUS relapse: data from the ItalKId-HUS Network.

Atypicalhaemolytic uremic syndrome(aHUS) is at high risk of relapse at any time, therefore patients require lifelong monitoring. The most appropriate way to monitor patientsis not yet clear. Patients could be monitored for relapse by urine dipstick testing for haemoglobinuria based on thehypothesisthat thrombotic microangiopathy involving the glomerulus and associated with renal damage (like aHUS) cannot occur without haematuria. The aim of this retrospective study is to analyse our experience with this approach in aHUS patients who have never previously been treated, who are currently on treatment or who have discontinued C5 inhibition.The records of all aHUS patients (children and adults) managed by or referred to our Centre from January 2009 to March 2020 were included and the analysisfor the presence of haemoglobinuriawas restricted to the period followingprimary remission. A positive test was defined as haemoglobin ≥ 1 + .Patients reporting positive urine dipstick tests underwent laboratory investigations to rule in or out the diagnosis of aHUS relapse. Eighty-four patients were included with 1517 determinations of haemoglobinuria during a cumulative observation period of 8904 patient-months. Haemoglobinuria for the early diagnosis of ongoing aHUS relapse shows a sensitivity of 100% and a specificity of 87.4% with a positive predictive value (PPV) of 10.5% and a negative predictive value (NPV) of100%. The accuracy of the test was 87.6%. Haemoglobinuria is a very sensitive and acceptably specific marker of aHUS relapse. This finding and its validation may have a positive impact on patients' quality of life and on the outcome of this life threatening disease via early diagnosis of relapse.

Parameters of the glutathione system and thioredoxin in blood plasma and ascites and GSTP1 Ile105Val gene polymorphism as factors of resistance to platinum-containing chemotherapy in ovarian cancer patients

Background . Chemotherapy is one of the main types of treatment in ovarian cancer. Standard first-line treatment includes platinum drugs. Every fifth patient develops chemoresistance after platinum-containing first line therapy. Glutathione detoxification systems play an important role in platinum drugs utilization. Purpose. To assess the redox status of blood plasma and ascitic fluid in ovarian cancer patients before and after neoadjuvant platinum-containing chemotherapy (NACT). Materials and methods . We determined the activity of the glutathione system and thioredoxin levels in blood plasma before and after NACT and in the ascitic fluid before NACT, and the presence of GSTP1 gene polymorphism (Ile105Val (rs1695), Ala114Val (rs1138272) in 30 III–IV FIGO stage ovarian cancer patients. Patients were divided into 3 groups: NR – no relapse in 2 years after last chemotherapy course; R1 – relapse in less than 6 months; R2 – relapse in more than 6 months. Results. We established an increase of the glutathione-transferase activity and a decrease of the GSH level in plasma after chemotherapy in R1 patients, and an opposite dynamic of glutathione-transferase and GSH in the R2 group. Thioredoxin level in plasma of all patients was lower than in the control group; differences in levels between groups were not statistically significant. GSTP1 105Val allele was more frequently present in patients than in the control group, and more frequently in R2 than in R1. Conclusion . The increase in plasma glutathione-transferase and glutathione-reductase levels can be a prognostic marker of early relapse. Thioredoxine dynamics do not correlate with the chemotherapy response. The presence of the GSTP1 105Val allele is a risk factor for ovarian cancer development, but a protective factor against early relapse.