Patient-Specific Measurable Residual Disease Markers Predict Outcome in Patients With Myelodysplastic Syndrome and Related Diseases After Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clinical relapse is the major threat for patients with myelodysplastic syndrome (MDS) undergoing hematopoietic stem-cell transplantation (HSCT). Early detection of measurable residual disease (MRD) would enable preemptive treatment and potentially reduced relapse risk. METHODS: Patients with MDS planned for HSCT were enrolled in a prospective, observational study evaluating the association between MRD and clinical outcome. We collected bone marrow (BM) and peripheral blood samples until relapse, death, or end of study 24 months after HSCT. Patient-specific mutations were identified with targeted next-generation sequencing (NGS) panel and traced using droplet digital polymerase chain reaction (ddPCR). RESULTS: Of 266 included patients, estimated relapse-free survival (RFS) and overall survival (OS) rates 3 years after HSCT were 59% and 64%, respectively. MRD results were available for 221 patients. Relapse was preceded by positive BM MRD in 42/44 relapses with complete MRD data, by a median of 71 (23-283) days. Of 137 patients in continuous complete remission, 93 were consistently MRD-negative, 39 reverted from MRD+ to MRD-, and five were MRD+ at last sampling. Estimated 1 year-RFS after first positive MRD was 49%, 39%, and 30%, using cutoff levels of 0.1%, 0.3%, and 0.5%, respectively. In a multivariate Cox model, MRD (hazard ratio [HR], 7.99), WHO subgroup AML (HR, 4.87), TP53 multi-hit (HR, 2.38), NRAS (HR, 3.55), and acute GVHD grade III-IV (HR, 4.13) were associated with shorter RFS. MRD+ was also independently associated with shorter OS (HR, 2.65). In a subgroup analysis of 100 MRD+ patients, presence of chronic GVHD was associated with longer RFS (HR, 0.32). CONCLUSION: Assessment of individualized MRD using NGS + ddPCR is feasible and can be used for early detection of relapse. Positive MRD is associated with shorter RFS and OS (ClinicalTrials.gov identifier: NCT02872662).

Machine Learning of Bone Marrow Histopathology Identifies Genetic and Clinical Determinants in Patients with MDS.

In myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN), bone marrow (BM) histopathology is assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, other morphologic findings may elude the human eye. We used convolutional neural networks to extract morphologic features from 236 MDS, 87 MDS/MPN, and 11 control BM biopsies. These features predicted genetic and cytogenetic aberrations, prognosis, age, and gender in multivariate regression models. Highest prediction accuracy was found for TET2 [area under the receiver operating curve (AUROC) = 0.94] and spliceosome mutations (0.89) and chromosome 7 monosomy (0.89). Mutation prediction probability correlated with variant allele frequency and number of affected genes per pathway, demonstrating the algorithms' ability to identify relevant morphologic patterns. By converting regression models to texture and cellular composition, we reproduced the classical del(5q) MDS morphology consisting of hypolobulated megakaryocytes. In summary, this study highlights the potential of linking deep BM histopathology with genetics and clinical variables. SIGNIFICANCE: Histopathology is elementary in the diagnostics of patients with MDS, but its high-dimensional data are underused. By elucidating the association of morphologic features with clinical variables and molecular genetics, this study highlights the vast potential of convolutional neural networks in understanding MDS pathology and how genetics is reflected in BM morphology. See related commentary by Elemento, p. 195.

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia.

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

Nitrous oxide analgesia for bone marrow aspiration and biopsy - A randomized, controlled and patient blinded study.

Background and aims Bone marrow aspiration and/or biopsy (BMAB), performed under local anaesthesia in adults, is a common and often painful procedure. Anxiety is known to intensify pain during the procedure. Nitrous oxide (N2O), known for its sedative and analgesic benefit in various short medical procedures and labour pain, could be advantageous also for pain relief during bone marrow examination. N2O acts rapidly and is eliminated in a couple of minutes once the inhalation is stopped, and occasional side effects (e.g. dizziness and nausea) are mild. The aim of this study was to compare the analgesic effects of inhaled 50% mixture of nitrous oxide and oxygen to 50% oxygen during bone marrow examination. Methods In this randomized, controlled, patient and observer blinded study patients received either 50% mixture of nitrous oxide and oxygen or 50% mixture of oxygen in air during bone marrow examination, in addition to local analgesia. Both patient groups comprised 35 adult patients. Pre-procedural anxiety and procedural pain were rated on the Numeral Rating Scale (NRS 0‒10). Cognitive function was measured before and 30 min after the procedure. Possible side effects were recorded. A telephone interview was performed 24 h later. Results There were no statistically significant differences in pain scores of the procedural steps (median NRS ranging 3.0‒4.0) between the study groups. High pain scores of 8‒10 comprised 0% vs. 8.6% of the scores during infiltration, 2.9% vs. 5.7% during puncture, 11.4% vs. 14.3% during aspiration and 2.9% vs. 2.9% during biopsy in N2O and 50% O2 groups, respectively (NS). Pre-procedural anxiety (median NRS 3.5 in both groups), measured in the outpatient clinic just prior to procedure, correlated with pain intensity during bone marrow aspiration (P = 0.045). There were no significant differences between side effects. During the BMAB four patients (3 in N2O group, 1 in 50% O2 group) reported dizziness and one patient in the N2O group reported nausea. Gas inhalation did not affect the cognitive function of the participants. In both groups the majority (>80%) of the patients was satisfied with the inhalation technique. During the 24 h interview, most of the participants were pain free and they did not report any serious adverse effects. Conclusions In spite of similar moderate to strong procedural pain in both groups and no benefit of N2O, most patients were satisfied with the inhalational techniques. We assume that the bedside presence of an anaesthesiologist and the distraction caused by the inhalational arrangements introduced positive context-sensitive therapeutic effect independent of the gas used. Pre-procedural anxiety predicted pain associated with bone marrow aspiration. Implications Inhaled 50% nitrous oxide was not an effective analgesic during bone marrow examination in our unselected outpatient population. Further studies should concentrate on its use with patients predicted to be at increased risk of suffering intense pain during the procedure, such as very anxious patients or those who have a painful history of previous bone marrow examinations.

Warmed and buffered lidocaine for pain relief during bone marrow aspiration and biopsy. A randomized and controlled trial.

Background and purpose Local infiltration anaesthesia is frequently painful due to low pH of the used anaesthetics, such as lidocaine. Usually pH of the solution is near 4.0, which causes tissue irritation and excitation of the pain mediating nerve endings. Warming and buffering the local anaesthetic solution have been shown to reduce the patient's experience of pain and unpleasantness during infiltration. Buffering reduces the dissociation of the local anaesthetic molecule and may enhance the anaesthetic's entrance into nerve cells. In this randomized placebo-controlled trial warmed and buffered lidocaine with adrenaline was compared to room temperature unbuffered lidocaine with adrenaline infiltrated before bone marrow aspiration and/or biopsy (BMAB). The aim was to find out to what extent warming and buffering would diminish pain during infiltration and whether this would be reflected in less pain also during subsequent steps of the BMAB procedure. Methods One hundred patients scheduled to undergo BMAB were interviewed regarding subjective experiences from previous medical procedures, current chronic and temporary medications, and their present state of anxiety before the BMAB procedure. They received local anaesthetic infiltration of lidocaine prior to BMAB. The solution used was either warmed lidocaine 20 mg/ml with adrenaline buffered with sodium bicarbonate 75 mg/ml (warmed and buffered group, 50 patients, pH approximately 7.3, 32°C) or unbuffered lidocaine 20 mg/ml with adrenaline mixed with NaCl 0.9% solution (control group, 50 patients, pH approximately 3.7, room temperature). The lidocaine concentration was similar in both groups. The bone marrow sampling needle was inserted 2 min after local anaesthetic infiltration. The grade of preprocedural anxiety, and pain sensations during the BMAB, both rated on NRS (numeral rating scale, 0-10) were compared between the groups. Results In comparison with the use of an unbuffered solution at room temperature warmed and buffered lidocaine with adrenaline caused less pain during infiltration (median NRS 4.0 vs. 2.0, P < 0.002) but it did not make performing the other phases of BMAB any less painful. As expected, painful experiences from previous medical, other than BMAB, or dental procedures and anxiety were associated with local anaesthetic infiltration pain during BMAB. Patients' own pain or anxiolytic medication did not lessen pain during BMAB. Conclusions By warming and buffering the lidocaine solution containing adrenaline it is possible to make the pain during infiltration less intense. Unfortunately, such benefit was not detected during the following steps of BMAB, initiated 2 min later. Preprocedural anxiety made procedural pain more intense including that of the local anaesthetic infiltration. Implications Warming and buffering the local anaesthetic prior to its administration is an effective and simple way of diminishing pain during infiltration. This benefit seems to be underutilized in the BMAB procedure. However, warming and buffering are not sufficient enough to diminish pain during bone marrow sampling and thus additional pain alleviating methods should be used, particularly in patients showing preprocedural anxiety.

Non-transferrin-bound iron in haematological patients during chemotherapy and conditioning for autologous stem cell transplantation.

Free iron induced hydroxyl radical formation is one possible mechanism for tissue injury during cytotoxic therapy. We studied the appearance of free, non-transferrin-bound iron (NTBI) at baseline and during the 20-d period after the onset of cytotoxic chemotherapy in patients with haematological malignancy undergoing intensive chemotherapy or conditioning for autologous stem cell transplantation (aSCT). NTBI was detected on average for 15.6 d in patients treated with chemotherapy only, and for 6.1 d in patients undergoing aSCT. The recovery of the bone marrow function coincided with the disappearance of NTBI. The type of the conditioning regimen was also associated with the appearance of NTBI. The timing of the presence of NTBI accords with the presence of the most important non-infectious complication of intensive chemotherapy and autologous transplantation, mucosal injury, and free iron is likely to contribute to this and probably other complications of the intensive treatments.

Appearance of ring sideroblasts in bone marrow during linezolid therapy.

A female patient with renal dysfunction developed thrombocytopenia after 2 weeks of linezolid treatment. Her bone marrow showed abnormal erythroblast morphology and abundant ring sideroblasts. No chromosomal abnormalities or other signs of myelodysplasia were seen in an early follow-up bone marrow aspirate, where the abnormal morphological findings had almost totally subsided.

Effect of repeated apotransferrin administrations on serum iron parameters in patients undergoing myeloablative conditioning and allogeneic stem cell transplantation.

Myeloablative conditioning prior to allogeneic stem cell transplantation causes a rapid increase in transferrin saturation and potentially toxic non-transferrin-bound iron (NTBI) in plasma. We have studied the ability of repeatedly administered apotransferrin to maintain this iron in a transferrin-bound form. Twenty adult patients undergoing myeloablative conditioning and allogeneic stem cell transplantation were enrolled to receive apotransferrin with one of three dosage regimens. Ten consecutive patients with the same preconditioning were studied as controls. At the highest dose level, full transferrin saturation and appearance of NTBI were prevented in five of the eight patients. Serum iron increased significantly more in the patients receiving apotransferrin than in the controls and remained elevated until erythropoietic recovery. From the increment of iron saturation and the amount of endogenous and administered apotransferrin, an average 180 mumol of iron per day was bound to transferrin during the first 4 d after the start of the conditioning therapy. Thereafter, iron accumulation levelled off in most patients. The results suggested that about half of the amount of iron normally transported to erythropoiesis was initially released to plasma after induction of the erythroid arrest. Complete iron binding with apotransferrin would apparently require very high apotransferrin doses.

Erratum to "Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron". [FEMS Immunol. Med Microbiol. 37 (2003) 45-51].

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron.

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Effective binding of free iron by a single intravenous dose of human apotransferrin in haematological stem cell transplant patients.

Myeloablative treatment results in iron accumulation and the appearance of non-transferrin-bound iron (NTBI) in the circulation, which may contribute to treatment-related organ damage and susceptibility to infections. The aim of this study was to investigate the efficacy of human apotransferrin in the binding of NTBI in patients receiving an allogeneic stem cell transplant after myeloablative conditioning. A single intravenous 100 mg/kg dose of apotransferrin was given to six adult patients on d 3 after the transplantation. Initially, all patients had serum transferrin saturation above 80% and NTBI in their serum. After the apotransferrin injection, serum NTBI became undetectable in all patients and transferrin saturation decreased to 30-50%. Serum transferrin increased by an average of 1.95 g/l. The administered apotransferrin was subsequently converted into monoferric and diferric transferrin forms. NTBI reappeared and transferrin saturation again exceeded 80% 12-48 h after the injection in four patients and after 6 d in one patient. NTBI remained non-detectable for the whole 12 d follow-up period in one patient. The apotransferrin injection was well tolerated and no adverse events with probable association with the apotransferrin were observed. Repeated apotransferrin infusions might completely eliminate NTBI and iron-induced toxicity during myeloablative therapy.

Efficacy of a high-purity factor IX concentrate in hemophilia B patients undergoing surgery

O concentrado de fator IX ( Bemofil ) , um derivado plasmático de alta pureza tratado com solventes- detergente e nano-filtrado , foi avaliado em 19 pacientes portadores de Hemofilia B .Quatro pacientes apresentavam a forma grave da moléstia, 13 a forma leve e moderada e dois portadores em um total de 31 atos cirúrgicos.A recuperação média "in vivo" foi de 52% (36-76%). A atividade plasmática média pré-operatória do fator IX após a dose inicial foi de 0,86 UI ml û1 , média de 0,59 - 1,32 UI ml -1. Em oito procedimentos ortopédicos extensos , a média de utilização do fator IX foi de 44.600 UI ou 574 UI kg û1 durante a hospitalização que teve a média de 11,6 dias. A tromboprofilaxia não foi utilizada. A eficácia hemostática avaliada em todos os casos foi boa ,e não ocorreu nenhum tipo de complicação tromboembólica. Concluímos que o Bemofil em bolus foi considerado seguro e eficaz para a hemostasia em pacientes portadores de hemofilia B que necessitam de um procedimento cirúrgico.