A Nationwide Study of GATA2 Deficiency in Norway-the Majority of Patients Have Undergone Allo-HSCT.

PURPOSE: GATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT. METHODS: All medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records. RESULTS: Between 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively. CONCLUSION: Our main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described.

The use of eculizumab in Capnocytophaga canimorsus associated thrombotic microangiopathy: a case report.

BACKGROUND: The use of complement inhibition is well established for complement mediated thrombotic microangiopathy, but its role in secondary forms of thrombotic microangiopathy is debated. We here present a case of thrombotic microangiopathy triggered by Capnocytophaga canimorsus, illustrating the diagnostic difficulties in discriminating between different thrombotic microangiopathies, and the dilemmas regarding how to treat this disease entity. CASE PRESENTATION: A previously healthy 56-year-old woman presented with fever and confusion. She was diagnosed with sepsis from Capnocytophaga canimorsus and thrombotic microangiopathy. Marked activation of both T-cells, endothelium and complement were documented. She was successfully treated with antimicrobial therapy, the complement inhibitor eculizumab and splenectomy. After several weeks, a heterozygote variant in complement factor B was localized, potentially implying the diagnosis of a complement mediated TMA over an isolated infection related TMA. CONCLUSIONS: We discuss the possible interactions between complement activation and other findings in severe infection and argue that complement inhibition proved beneficial to this patient's rapid recovery.

Bendamustine plus rituximab for chronic cold agglutinin disease: results of a Nordic prospective multicenter trial.

Primary chronic cold agglutinin disease (CAD) is a well-defined clinicopathologic entity in which a bone marrow clonal B-cell lymphoproliferation results in autoimmune hemolytic anemia and cold-induced circulatory symptoms. Rituximab monotherapy and fludarabine-rituximab in combination are documented treatment options. In a prospective, nonrandomized multicenter trial, 45 eligible patients received rituximab 375 mg/m2 day 1 and bendamustine 90 mg/m2 days 1 and 2 for 4 cycles at a 28-day interval. Thirty-two patients (71%) responded; 18 (40%) achieved complete response (CR) and 14 (31%) partial response (PR). Among 14 patients previously treated with rituximab or fludarabine-rituximab, 7 (50%) responded to bendamustine-rituximab (3 CR and 4 PR). Hemoglobin levels increased by a median of 4.4 g/dL in the complete responders, 3.9 g/dL in those achieving PR, and 3.7 g/dL in the whole cohort. The 10th percentile of response duration was not reached after 32 months. Grade 3-4 neutropenia occurred in 15 patients (33%), but only 5 (11%) experienced infection with or without neutropenia. Thirteen patients (29%) had their dose of bendamustine reduced. In conclusion, bendamustine-rituximab combination therapy is highly efficient, sufficiently safe, and may be considered in first line for patients with CAD requiring therapy. The trial was registered at www.clinicaltrials.gov as #NCT02689986.

Mass cytometric single cell immune profiles of peripheral blood from acute myeloid leukemia patients in complete remission with measurable residual disease.

Measurable residual disease (MRD) is detected in approximately a quarter of AML chemotherapy responders, serving as a predictor for relapse and shorter survival. Immunological control of residual disease is suggested to prevent relapse, but the mechanisms involved are not fully understood. We present a peripheral blood single cell immune profiling by mass cytometry using a 42-antibody panel with particular emphasis on markers of cellular immune response. Six healthy donors were compared with four AML patients with MRD (MRD+) in first complete remission (CR1MRD+). Three of four patients demonstrated a favorable genetic risk profile, while the fourth patient had an unfavorable risk profile (complex karyotype, TP53-mutation) and a high level of MRD. Unsupervised clustering using self-organizing maps and dimensional reduction analysis was performed for visualization and analysis of immune cell subsets. CD57+ natural killer (NK)-cell subsets were found to be less abundant in patients than in healthy donors. Both T and NK cells demonstrated elevated expression of activity and maturation markers (CD44, granzyme B, and phosho-STAT5 Y694) in patients. Although mass cytometry remains an expensive method with limited scalability, our data suggest the utility for employing a 42-plex profiling for cellular immune surveillance in whole blood, and possibly as a biomarker platform in future clinical trials. The findings encourage further investigations of single cell immune profiling in CR1MRD+ AML-patients.

Molecular characterizations of derivatives of HCT116 colorectal cancer cells that are resistant to the chemotherapeutic agent 5-fluorouracil.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data.

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.