Rafał Machowicz

mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5

Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV (‘Berlin patient’). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

Similar but not the same: Differential diagnosis of HLH and sepsis

Differential diagnosis of hemophagocytic lymphohistiocytosis (HLH; hemophagocytic syndrome) and sepsis is critically important because the life-saving aggressive immunosuppressive treatment, required in the effective HLH therapy, is absent in sepsis guidelines. Moreover, HLH may be complicated by sepsis. Hyperinflammation, present in both states, gives an overlapping clinical picture including fever and performance status deterioration. The aim of this review is to provide aid in this challenging diagnostic process. Analysis of clinical features and laboratory results in multiple groups of patients (both adult and pediatric) with either HLH or sepsis allows to propose criteria differentiating these two conditions. The diagnosis of HLH is supported by hyperferritinemia, splenomegaly, marked cytopenias, hypofibrinogenemia, low CRP, characteristic cytokine profile and, only in adults, hypertriglyceridemia. In the presence of these parameters (especially the most characteristic hyperferritinemia), the other HLH criteria should be assessed. Genetic analyses can reveal familial HLH. Hemophagocytosis is neither specific nor sensitive for HLH.

Demographic, Hematologic, and Clinical Features of Myelodysplastic Syndrome Patients: Results from the First Polish Myelodysplastic Syndrome Registry.

Epidemiological studies on myelodysplastic syndromes (MDS) in Middle-Eastern Europe are scarce. No data about the demographic, clinical, and laboratory features of Polish MDS patients have been published. The aim of this study was to assess the epidemiological data and toxic exposure of Polish MDS patients and their association with hematological parameters and clinical outcomes. For 15 months, 966 living MDS patients were enrolled at 24 centers (12 university and 12 community hospitals). Follow-up was conducted for the next 55 months. The percentage of patients older than 80 years (16%) was between the values for Eastern and Western countries. In patients younger than 55 years, a female predominance was observed (male/female ratio 0.70:1 vs. 1.29:1; p < 0.001). Female patients had higher platelet counts (160 × 109/l vs. 111 × 109/l; p < 0.001). Patients exposed to chemicals were younger than patients without such exposure; their median age at MDS diagnosis was 66 vs. 70 years (p = 0.037). Smokers had significantly lower hemoglobin concentrations (8.6 vs. 9.1 g/dl; p = 0.032) and lower platelet counts (99 × 109/l vs. 137 × 109/l; p < 0.001) than nonsmokers. We provide the first description of the characteristics of Polish MDS patients. Females predominated in the group aged <60 years and they had higher platelet counts. The course of the disease is affected by toxic exposure and smoking.

HLH in Adults

Hemophagocytic lymphohistiocytosis (HLH) in adolescent and adult patients (aHLH) is getting increasing attention due to an assumed rising incidence, a better pathophysiologic understanding, and a growing arsenal of therapeutic ammunition. For many years HLH used to be a domain of pediatrics and was almost totally neglected in adults. Nowadays, with raising awareness (although it is still severely underdiagnosed), more and more physicians face the challenges of diagnosis and treatment of aHLH. The aim of this chapter is to provide information on HLH, specifically in adults. The spectrum of underlying conditions, triggering factors, and genetic predisposition differs in age groups with enrichment of familial disease in children and enrichment of malignant disorders as underlying condition and/or trigger in adults. These differences plus differential tolerance of drugs due to age and comorbidity guide the recommended aHLH treatment approach. Simply copying the pediatric recommendations to adults may have deleterious consequences. Treatment of HLH requires individual tailoring of anti-inflammatory and immunosuppressive treatment components combined with disease-specific treatment such as chemotherapy in malignancy or antimicrobial drugs in infection-triggered HLH. The clinical overlap to sepsis with high risk of missed diagnosis on intensive care units will also be discussed.

Atypical chronic myeloid leukaemia: A case of an orphan disease—A multicenter report by the Polish Adult Leukemia Group

Atypical chronic myeloid leukaemia (aCML) belongs to myelodysplastic/myeloproliferative neoplasms. Because of its rarity and changing diagnostic criteria throughout subsequent classifications, data on aCML are very scarce. Therefore, we at the Polish Adult Leukemia Group performed a nationwide survey on aCML. Eleven biggest Polish centres participated in the study. Altogether, 45 patients were reported, among whom only 18 patients (40%) fulfilled diagnostic criteria. Among misdiagnosed patients, myelodysplastic/myeloproliferative syndrome unclassifiable and chronic myelomonocytic leukaemia were the most frequent diagnoses. Thirteen patients were male, median age 64.6 years (range 40.4‐80.9). The median parameters at diagnosis were as follows: white blood cell count 97 × 109/L (23.8‐342) with immature progenitors amounting at 27.5% (12‐72), haemoglobin 8.6 g/dL (3.9‐14.9), and platelet count 66 × 109/L (34‐833). Cytoreductive treatment was used in all patients, and 2 patients underwent allogeneic hematopoietic stem cell transplantation. The median overall survival was 14.1 months (95% CI, 7.2), with median acute myeloid leukaemia‐free survival of 13.3 months (95% CI, 3.6‐22.6). Cumulative incidence of acute myeloid leukaemia transformation after 1 year in aCML group was 12.5% (95% CI, 0%‐29.6%). To conclude, aCML harbours a poor prognosis. Treatment options are limited, with allogeneic hematopoietic stem cell transplantation being the only curative method at present, although only a minority of patients are transplant eligible. Educational measures are needed to improve the quality of diagnoses.

Red Blood Cell Transfusion Dependency and Hyperferritinemia Are Associated with Impaired Survival in Patients Diagnosed with Myelodysplastic Syndromes: Results from the First Polish MDS-PALG Registry

BACKGROUND Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis, cytopenias and a risk of progression to acute myeloid leukemia (AML). Anemia is the most frequent cytopenia diagnosed in patients with MDS. Regular RBC transfusions are the only treatment option for about 40% of patients. Transfusion-dependent patients develop secondary iron overload. The influence of serum ferritin (SF) concentration on survival and acute myeloid leukemia transformation in MDS patients remains controversial. The data for the Central European population is scarce and so far there is no description for Poland. OBJECTIVES The aim of this study was to perform a retrospective analysis of the relationship of SF concentration with red blood cell transfusion dependency, survival and transformation to acute myeloid leukemia. MATERIAL AND METHODS We retrospectively evaluated the data of the 819 MDS patients (58% male; median age 70 years) included in the MDS Registry of the MDS Section of the Polish Adult Leukemia Group (PALG). RESULTS Analyses were performed on 190 patients diagnosed with MDS, maximal 6 months before inclusion to the registry in order to avoid selection bias (a shorter survival of higher risk MDS patients). Patients with hyperferritinemia higher than 1000 ng/L vs. patients with SF concentration lower than 1000 ng/L had a median survival of 320 days vs. 568 days, respectively (p log-rank = 0.014). The following factors were found to significantly worsen survival: RBC-transfusion dependence (p = 0.0033; HR 2.67L), platelet transfusion dependence (p = 0.0071; HR 3.321), hemoglobin concentration lower than 10 g/dL (p = 0.0036; HR 2.97), SF concentration higher than 1000 ng/L (p = 0.0023; HR = 2.94), platelet count lower than 10 G/L (p = 0.0081 HR = 5.04), acute leukemia transformation (p = 0.0081; HR 1.968). CONCLUSIONS Taking into account the relatively low number of patients in previous studies exploring hyperferritinemia in MDS, the results of the first Polish MDS Registry provide important insights. Hyperferritinemia higher than 1000 ng/L can be an important indicator of poor prognosis in MDS.

Recommendations for the Use of Etoposide-Based Therapy and Bone Marrow Transplantation for the Treatment of HLH: Consensus Statements by the HLH Steering Committee of the Histiocyte Society

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome requiring aggressive immunosuppressive therapy. Following 2 large international studies mainly targeting pediatric patients with familial disease and patients without underlying chronic or malignant disease, the HLH-94 protocol is recommended as the standard of care when using etoposide-based therapy by the Histiocyte Society. However, in clinical practice, etoposide-based therapy has been widely used beyond the study inclusion criteria, including older patients and patients with underlying diseases (secondary HLH). Many questions remain around these extended indications and published reports do not address several practical issues. To tackle these concerns, the HLH Steering Committee of the Histiocyte Society decided to issue guidance for use of the HLH-94 protocol. The group convened in a structured consensus finding process to define recommendations that are based largely on expert opinion backed up by available data from the literature. The recommendations address all main elements of HLH-94 including corticosteroids, cyclosporin, etoposide, intrathecal therapy, and hematopoietic stem cell transplantation (HSCT) and consider various forms of HLH and all age groups. Aspects covered include indications, applications, dosing, side effects, duration of therapy, salvage therapy, and HSCT. These recommendations aim to provide a framework to guide treatment decisions in this severe disease.

565. CCR5-Uco-TALEN – A Novel Transcription Activator-Like Effector Nuclease That Mediates High-Efficiency Knockout of HIV Co-Receptor CCR5 in Primary T Cells After mRNA Transfection

Besides its physiological role, the chemokine receptor CCR5 plays an essential part during HIV infection, acting as the co-receptor for so-called R5-tropic strains that usually mediate initial infection. Notably, homozygosity for CCR5D32, a natural deletion variant of CCR5, largely confers resistance towards HIV infection. Consequently, CCR5 has been proposed as a promising target for HIV therapy. The interest in using CCR5 as a therapeutic target has increased even further after the first report on the cure from HIV in an individual (“Berlin patient”) who received an allogeneic stem cell graft from a CCR5D32-homozygous donor to treat his acute leukemia. Since allogeneic transplantation is not a realistic treatment scenario for HIV, genetic disruption of CCR5 by the means of designer nucleases was proposed as an auspicious gene-therapy approach. We developed a novel transcription activator-like effector nuclease (TALEN), CCR5-Uco-TALEN for highly specific CCR5 knockout. We established efficient TALEN delivery into primary T lymphocytes using mRNA electroporation, a gentle and truly transient gene-transfer technique. After mRNA transfection, CCR5-Uco-TALEN routinely mediated high-rate CCR5 knockout (>90% in PM1 and >50 % in primary T cells) combined with low off-target activity, as demonstrated by flow cytometry, next-generation sequencing and a newly devised, very handy digital-PCR technique. Importantly, CCR5-edited cells were not only resistant towards HIV-derived lentiviral vectors, but also protected from infection with the wild-type CCR5-tropic HIV-1BaL strain. Indeed, 12-day exposure to HIV-1BaL resulted in almost complete suppression of viral replication as well as selection of CCR5-gene edited T cells. In summary, we have introduced a novel TALEN for high-efficiency targeted CCR5 knockout. The proposed mRNA-based gene-editing protocol does not rely on gene-modified organisms and is thus highly compatible with large-scale production and GMP requirements. Together, CCR5-Uco-TALEN and the proposed mRNA-based transfection strategy should therefore be excellently suited for clinical translation.

IgM multiple myeloma: diagnostic difficulties and results of bortezomib treatment – case report

The IgM paraprotein secretion is most frequently associated with Waldenstrom’s macroglobulinemia. In very rare cases it may also occur in multiple myeloma. The diagnostic and therapeutic process of a 84 year old male suffering from IgM multiple myeloma is described, including difficulties in establishing this diagnosis. Due to infectious complications, after the first cycle of melphalan– prednisone–bortezomib treatment, melphalan was stopped, subsequently, bortezomib and prednisone doses were also reduced. Two treatment interruptions have occurred. Despite reduced treatment intensity, the patient was in very good partial remission, while the toxicity profile was acceptable. IgM multiple myeloma should be included in the differential diagnosis of IgM paraproteinemia, also when osteolytic lesions and translocation 11:14 are not detected. Bortezomib-based therapy can be effective in elderly patients (also in those suffering from cardiac insufficiency), even when dose reduction is required.