Lymphodepletion chemotherapy followed by infusion of CD19-specific chimeric antigen receptor-modified (CAR) T cells has produced impressive antitumor responses in patients with refractory CD19+ B-cell malignancies but is often associated with cytokine release syndrome (CRS). Our understanding of CRS continues to evolve, and identification of the kinetics of CRS and predictive clinical and laboratory biomarkers of severity are needed to evaluate strategies to mitigate toxicity. We report the clinical presentation of and identify biomarkers of severe CRS in 133 adult patients who received CD19 CAR T cells. CRS developed in 70% of patients, including 62.5% with grade 1 to 3 CRS (grade 1, 26%; grade 2, 32%; grade 3, 4.5%), 3.8% with grade 4, and 3.8% with grade 5. A majority of cases of grade ≥4 CRS occurred during CAR T-cell dose finding. Multivariable analysis of baseline characteristics identified high marrow tumor burden, lymphodepletion using cyclophosphamide and fludarabine, higher CAR T-cell dose, thrombocytopenia before lymphodepletion, and manufacturing of CAR T cells without selection of CD8+ central memory T cells as independent predictors of CRS. Severe CRS was characterized by hemodynamic instability, capillary leak, and consumptive coagulopathy. Angiopoietin-2 and von Willebrand factor, which are biomarkers of endothelial activation, were increased during severe CRS and also before lymphodepletion in patients who subsequently developed CRS. We describe a classification-tree algorithm to guide studies of early intervention after CAR T-cell infusion for patients at high risk of severe CRS. These data provide a framework for early intervention studies to facilitate safer application of effective CD19 CAR T-cell therapy.

Chronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic stem cell transplantation with few effective options available after failure of corticosteroids. B and T cells play a role in the pathophysiology of cGVHD. Ibrutinib inhibits Bruton tyrosine kinase in B cells and interleukin-2-inducible T-cell kinase in T cells. In preclinical models, ibrutinib reduced severity of cGVHD. This multicenter, open-label study evaluated the safety and efficacy of ibrutinib in patients with active cGVHD with inadequate response to corticosteroid-containing therapies. Forty-two patients who had failed 1 to 3 prior treatments received ibrutinib (420 mg) daily until cGVHD progression. The primary efficacy end point was cGVHD response based on 2005 National Institutes of Health criteria. At a median follow-up of 13.9 months, best overall response was 67%; 71% of responders showed a sustained response for ≥20 weeks. Responses were observed across involved organs evaluated. Most patients with multiple cGVHD organ involvement had a multiorgan response. Median corticosteroid dose in responders decreased from 0.29 mg/kg per day at baseline to 0.12 mg/kg per day at week 49; 5 responders discontinued corticosteroids. The most common adverse events were fatigue, diarrhea, muscle spasms, nausea, and bruising. Plasma levels of soluble factors associated with inflammation, fibrosis, and cGVHD significantly decreased over time with ibrutinib. Ibrutinib resulted in clinically meaningful responses with acceptable safety in patients with ≥1 prior treatments for cGVHD. Based on these results, ibrutinib was approved in the United States for treatment of adult patients with cGVHD after failure of 1 or more lines of systemic therapy. This trial was registered at www.clinicaltrials.gov as #NCT02195869.

Critical clinical questions remain unanswered regarding diagnosis and management of patients with low von Willebrand factor (VWF) levels (30-50 IU/dL). To address these questions, the Low VWF Ireland Cohort (LoVIC) study investigated 126 patients registered with low VWF levels. Despite marginally reduced plasma VWF levels, International Society of Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT) confirmed significant bleeding phenotypes in the majority of LoVIC patients. Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30 to 50 IU/dL range. Furthermore, bleeding phenotypes could not be explained by concurrent hemostatic defects. Plasma factor VIII to VWF antigen (VWF:Ag) ratios were significantly increased in LoVIC patients compared with controls (P < .0001). In contrast, VWF propeptide to VWF:Ag ratios >3 were observed in only 6% of the LoVIC cohort. Furthermore, platelet-VWF collagen binding activity levels were both significantly reduced compared with controls (P < .05). In response to 1-desamino-8-D-arginine vasopressin (DDAVP), peak VWF:Ag levels exceeded 100 IU/dL in 88% of patients and was sustained >100 IU/dL after 4 hours in 72% of subjects. In conclusion, our novel data suggest that low VWF levels can be associated with significant bleeding and are predominantly due to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in some individuals, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. This trial was registered at www.clinicaltrials.gov as #NCT03167320.

Safety, tolerability, and pharmacokinetics of recombinant ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; BAX 930; SHP655) were investigated in 15 patients diagnosed with severe congenital ADAMTS-13 deficiency (plasma ADAMTS-13 activity <6%) in a prospective phase 1, first-in-human, multicenter dose escalation study. BAX 930 was well tolerated, no serious adverse events occurred, and no anti-ADAMTS-13 antibodies were observed. After single-dose administration of BAX 930 at 5, 20, or 40 U/kg body weight to adolescents and adults, there was approximate dose proportionality with respect to maximum plasma concentration (Cmax [U/mL]) and area under the concentration-time curve (AUC [h∙U/mL]). Dose-related increases of individual ADAMTS-13:Ag and activity were observed and reached a maximum within 1 hour. With escalating BAX 930 doses administered, a dose-dependent persistence of ADAMTS-13-mediated von Willebrand factor (VWF) cleavage products and reduced VWF multimeric size were observed. This study demonstrated that pharmacokinetic parameters of BAX 930 were comparable to those estimated in previous plasma infusion studies and provided evidence of pharmacodynamic activity. This study was registered at www.clinicaltrials.gov as #NCT02216084.

Chronic graft-versus-host disease (cGVHD) is the main cause of late nonrelapse mortality and morbidity after allogeneic stem cell transplantation (allo-SCT). To improve such patients' outcomes, we conducted a phase 2, prospective, multicenter trial to test the efficacy of the addition of rituximab to corticosteroids (CSs) and cyclosporine A (CsA) as first-line therapy for newly diagnosed cGVHD after allo-SCT. Twenty-four patients (median age, 47 years) with mild (n = 2), moderate (n = 7), or severe (n = 15) cGVHD were included. All patients received rituximab 375 mg/m2 weekly for 4 weeks, followed by a second course 1 month later for patients with partial response. Twenty of 24 patients (83%) were in response at 1 year. Furthermore, among 19 evaluable patients, 14 (74%) were off CSs. The estimated 1-year overall survival was 83%, and the 1-year cumulative incidence of nonrelapse mortality was 14%. One patient died of progressive multifocal leukoencephalopathy. Although PD-L1hi naive B cells were significantly decreased at diagnosis of cGVHD, they increased after anti-CD20 B-cell depletion. In contrast, activated ICOShi PD-1hi circulating T follicular helper (Tfh) cells decreased after rituximab treatment. Overall, the addition of rituximab to corticosteroid and CsA appeared to be safe and effective for first-line treatment of cGVHD. Furthermore, our data suggest that this efficacy may be in part related to an effect on PD-L1hi B cells and Tfh cells. This study was registered at www.clinicaltrials.gov as identifier NCT01135641.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this "NOTCH2-BCR axis" in cGVHD revealed imbalanced expression of the transcription factors IRF4 and IRF8, each critical to B-cell differentiation and fate. All-trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4-to-IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5, but not BLIMP1 (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.

The antiapoptotic proteins BCL-2 and myeloid cell leukemia sequence 1 (MCL-1) promote multiple myeloma (MM) cell survival. Venetoclax is a selective, orally bioavailable small-molecule BCL-2 inhibitor; bortezomib can indirectly inhibit MCL-1. In preclinical studies, venetoclax enhanced bortezomib activity, suggesting that cotargeting of BCL-2 and MCL-1 could be an effective treatment strategy in myeloma. This phase 1b trial studied patients with relapsed/refractory MM receiving daily venetoclax (50-1200 mg per designated dose cohort; 800 mg in safety expansion) in combination with bortezomib and dexamethasone. A total of 66 patients were enrolled (54 in the dose-escalation cohorts and 12 in the safety expansion). Patients had received a median of 3 prior therapies (range, 1-13); 26 (39%) were refractory to prior bortezomib and 35 (53%) to lenalidomide; 39 (59%) had prior stem cell transplant. The combination was generally well tolerated, and common adverse events included mild gastrointestinal toxicities (diarrhea [46%], constipation [41%], and nausea [38%]) and grade 3/4 cytopenias (thrombocytopenia [29%] and anemia [15%]). The overall response rate (ORR) was 67% (44/66); 42% achieved very good partial response or better (≥VGPR). Median time to progression and duration of response were 9.5 and 9.7 months, respectively. ORR of 97% and ≥VGPR 73% were seen in patients not refractory to bortezomib who had 1 to 3 prior therapies. Patients with high BCL2 expression had a higher ORR (94% [17/18]) than patients with low BCL2 expression (59% [16/27]). This novel combination of venetoclax with bortezomib and dexamethasone has an acceptable safety profile and promising efficacy in patients with relapsed/refractory MM. This trial was registered at www.clinicaltrials.gov as #NCT01794507.

Relapse has emerged as the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT). To test the hypothesis that natural killer (NK) cells can decrease the risk of leukemia relapse, we initiated a phase 1 dose-escalation study of membrane-bound interleukin 21 (mbIL21) expanded donor NK cells infused before and after haploidentical HSCT for high-risk myeloid malignancies. The goals were to determine the safety, feasibility, and maximum tolerated dose. Patients received a melphalan-based reduced-intensity conditioning regimen and posttransplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. NK cells were infused on days -2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 105/kg to 1 × 108/kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients engrafted with donor cells. Seven patients (54%) developed grade 1-2 acute GVHD (aGVHD), none developed grade 3-4 aGVHD or chronic GVHD, and a low incidence of viral complications was observed. One patient died of nonrelapse mortality; 1 patient relapsed. All others were alive and in remission at last follow-up (median, 14.7 months). NK-cell reconstitution was quantitatively, phenotypically, and functionally superior compared with a similar group of patients not receiving NK cells. In conclusion, this trial demonstrated production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, and was associated with significantly improved NK-cell number and function, lower viral infections, and low relapse rate posttransplant.

Clonal chromosomal abnormalities in Philadelphia chromosome-negative (CCA/Ph-) metaphases emerge as patients with chronic phase chronic myeloid leukemia (CP-CML) are treated with tyrosine kinase inhibitors (TKIs). We assessed the characteristics and prognostic impact of 598 patients with CP-CML treated on clinical trials with various TKIs. CCA/Ph- occurred in 58 patients (10%); the most common were -Y in 25 (43%) and trisomy 8 in 7 patients (12%). Response to TKI therapy was similar for patients with CCA/Ph- and those without additional chromosomal abnormalities (ACAs). We further categorized CCA/Ph- into those in which -Y was the only clonal abnormality, and all others. We found that patients with non -Y CCA/Ph- had worse failure-free survival (FFS), event-free survival (EFS), transformation-free survival (TFS), and overall survival (OS) compared with those without ACAs with the following 5-year rates: FFS (52% vs 70%, P = .02), EFS (68% vs 86%, P = .02), TFS (76% vs 94%, P < .01), and OS (79% vs 94%, P = .03). In a multivariate analysis, non -Y CCA/Ph- increased the risk of transformation or death when baseline characteristics were considered with a hazard ratio of 2.81 (95% confidence interval, 1.15-6.89; P = .02). However, this prognostic impact was not statistically significant when achieving BCR-ABL <10% at 3 months was included in the analysis. In conclusion, non -Y CCA/Ph- are associated with decreased survival when emerging in patients with chronic-phase CML across various TKIs. This trial was registered at www.clinicaltrials.gov as #NCT00048672, #NCT00038649, and #NCT00050531 (imatinib); #NCT00254423 (dasatinib); #NCT00129740 (nilotinib); and NCT01570868 (ponatinib).

Relapse is a major problem in acute myeloid leukemia (AML) and adversely affects survival. In this phase 2 study, we investigated the effect of vaccination with dendritic cells (DCs) electroporated with Wilms' tumor 1 (WT1) messenger RNA (mRNA) as postremission treatment in 30 patients with AML at very high risk of relapse. There was a demonstrable antileukemic response in 13 patients. Nine patients achieved molecular remission as demonstrated by normalization of WT1 transcript levels, 5 of which were sustained after a median follow-up of 109.4 months. Disease stabilization was achieved in 4 other patients. Five-year overall survival (OS) was higher in responders than in nonresponders (53.8% vs 25.0%; P = .01). In patients receiving DCs in first complete remission (CR1), there was a vaccine-induced relapse reduction rate of 25%, and 5-year relapse-free survival was higher in responders than in nonresponders (50% vs 7.7%; P < .0001). In patients age ≤65 and >65 years who received DCs in CR1, 5-year OS was 69.2% and 30.8% respectively, as compared with 51.7% and 18% in the Swedish Acute Leukemia Registry. Long-term clinical response was correlated with increased circulating frequencies of polyepitope WT1-specific CD8+ T cells. Long-term OS was correlated with interferon-γ+ and tumor necrosis factor-α+ WT1-specific responses in delayed-type hypersensitivity-infiltrating CD8+ T lymphocytes. In conclusion, vaccination of patients with AML with WT1 mRNA-electroporated DCs can be an effective strategy to prevent or delay relapse after standard chemotherapy, translating into improved OS rates, which are correlated with the induction of WT1-specific CD8+ T-cell response. This trial was registered at www.clinicaltrials.gov as #NCT00965224.

Recent studies suggest that the evolutionary history of a cancer is important in forecasting clinical outlook. To gain insight into the clonal dynamics of multiple myeloma (MM) and its possible influence on patient outcomes, we analyzed whole exome sequencing tumor data for 333 patients from Myeloma XI, a UK phase 3 trial and 434 patients from the CoMMpass study, all of which had received immunomodulatory drug (IMiD) therapy. By analyzing mutant allele frequency distributions in tumors, we found that 17% to 20% of MM is under neutral evolutionary dynamics. These tumors are associated with poorer patient survival in nonintensively treated patients, which is consistent with the reduced therapeutic efficacy of microenvironment-modulating IMiDs. Our findings provide evidence that knowledge of the evolutionary history of MM has relevance for predicting patient outcomes and personalizing therapy.

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190BCR-ABL1, only the CD19+ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19+ cells and CD19- precursors should be targeted to avoid CD19- relapses in patients with BCR-ABL1-positive ALL.

E-selectin extends from the plasma membrane of inflamed endothelium and serves to capture leukocytes from flowing blood via long-lived catch-bonds that support slow leukocyte rolling under shear stress. Its ligands are glycosylated with the tetrasaccharide sialyl Lewisx (sLex), which contributes to bond affinity and specificity. E-selectin-mediated rolling transmits signals into neutrophils that trigger activation of high-affinity β2-integrins necessary for transition to shear-resistant adhesion and transendothelial migration. Rivipansel is a glycomimetic drug that inhibits E-selectin-mediated vaso-occlusion induced by integrin-dependent sickle-red blood cell-leukocyte adhesion. How Rivipansel antagonizes ligand recognition by E-selectin and blocks outside-in signaling of integrin-mediated neutrophil arrest while maintaining rolling immune-surveillance is unknown. Here, we demonstrate that sLex expressed on human L-selectin is preferentially bound by E-selectin and, on ligation, initiates secretion of MRP8/14 that binds TLR4 to elicit the extension of β2-integrin to an intermediate affinity state. Neutrophil rolling over E-selectin at precise shear stress transmits tension and catch-bond formation with L-selectin via sLex, resulting in focal clusters that deliver a distinct signal to upshift β2-integrins to a high-affinity state. Rivipansel effectively blocked formation of selectin catch-bonds, revealing a novel mechanotransduction circuit that rapidly converts extended β2-integrins to high-affinity shear-resistant bond clusters with intracellular adhesion molecule 1 on inflamed endothelium.

The oral histone deacetylase (HDAC) inhibitor (vorinostat) is safe and results in low incidence of acute graft-versus-host disease (GVHD) after reduced-intensity conditioning, related donor hematopoietic cell transplantation (HCT). However, its safety and efficacy in preventing acute GVHD in settings of heightened clinical risk that use myeloablative conditioning, unrelated donor (URD), and methotrexate are not known. We conducted a prospective, phase 2 study in this higher-risk setting. We enrolled 37 patients to provide 80% power to detect a significant difference in grade 2 to 4 acute GVHD of 50% compared with a reduction in target to 28%. Eligibility included adults with a hematological malignancy to receive myeloablative HCT from an available 8/8-HLA matched URD. Patients received GVHD prophylaxis with tacrolimus and methotrexate. Vorinostat (100 mg twice daily) was started on day -10 and continued through day +100 post-HCT. Median age was 56 years (range, 18-69 years), and 95% had acute myelogenous leukemia or high-risk myelodysplastic syndrome. Vorinostat was safe and tolerable. The cumulative incidence of grade 2 to 4 acute GVHD at day 100 was 22%, and for grade 3 to 4 it was 8%. The cumulative incidence of chronic GVHD was 29%; relapse, nonrelapse mortality, GVHD-free relapse-free survival, and overall survival at 1 year were 19%, 16%, 47%, and 76%, respectively. Correlative analyses showed enhanced histone (H3) acetylation in peripheral blood mononuclear cells and reduced interleukin 6 (P = .028) and GVHD biomarkers (Reg3, P = .041; ST2, P = .002) at day 30 post-HCT in vorinostat-treated subjects compared with similarly treated patients who did not receive vorinostat. Vorinostat for GVHD prevention is an effective strategy that should be confirmed in a randomized phase 3 study. This trial was registered at www.clinicaltrials.gov as #NCT01790568.

Hypomethylating agents (HMAs) improve survival in patients with higher-risk myelodysplastic syndromes (MDS) but are less well-studied in lower-risk disease. We compared the safety and efficacy of low-dose decitabine vs low-dose azacitidine in this group of patients. Adults with low- or intermediate 1-risk MDS or MDS/myeloproliferative neoplasm (MPN), including chronic myelomonocytic leukemia, according to the International Prognostic Scoring System, were randomly assigned using a Bayesian adaptive design to receive either azacitidine 75 mg/m2 intravenously/subcutaneously daily or decitabine 20 mg/m2 intravenously daily for 3 consecutive days on a 28-day cycle. The primary outcome was overall response rate (ORR). Between November 2012 and February 2016, 113 patients were treated: 40 (35%) with azacitidine and 73 (65%) with decitabine. The median age was 70 years; 81% of patients were intermediate 1-risk patients. The median number of cycles received was 9. The ORRs were 70% and 49% (P = .03) for patients treated with decitabine and azacitidine, respectively. Thirty-two percent of patients treated with decitabine became transfusion independent compared with 16% of patients treated with azacitidine (P = .2). Cytogenetic response rates were 61% and 25% (P = .02), respectively. With a median follow-up of 20 months, the overall median event-free survival was 18 months: 20 and 13 months for patients treated with decitabine and azacitidine, respectively (P = .1). Treatment was well tolerated, with a 6-week mortality rate of 0%. The use of low-dose HMAs is safe and effective in patients with lower-risk MDS and MDS/MPN. Their effect on the natural history of lower-risk disease needs to be further studied. This trial was registered at clinicaltrials.gov (identifier NCT01720225).

A recent randomized trial, the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET), showed a higher risk of inhibitor development with recombinant factor VIII (rFVIII) than plasma-derived concentrates (pdFVIII). We investigated whether risk stratification by F8 mutation identifies patients who do not suffer this deleterious effect of rFVIII. Among 235 randomized patients with severe hemophilia A previously untreated with FVIII concentrate, 197 with null mutations were classified as high risk and 38 with non-null mutations were classified as low risk. With pdFVIII, no inhibitors occurred in those with low genetic risk, whereas high-risk patients had a cumulative incidence of 31%. The risk among low- and high-risk patients did not differ much when they were treated with rFVIII (43% and 47%, respectively). This implies that patients with low genetic risk suffer disproportionate harm when treated with rFVIII (risk increment 43%), as also shown by the number needed to harm with rFVIII, which was 6.3 for genetically high-risk patients and only 2.3 for low-risk patients. Risk stratification by F8 mutation does not identify patients who can be safely treated with rFVIII, as relates to immunogenicity. This trial was registered at the European Clinical Trials Database (EudraCT) as #2009-011186-88 and at www.clinicaltrials.gov as #NCT01064284.