Sudhir Tauro

Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2

BACKGROUND Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA–peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 × 103/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy.

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

Allogeneic Stem-Cell Transplantation Using a Reduced-Intensity Conditioning Regimen Has the Capacity to Produce Durable Remissions and Long-Term Disease-Free Survival in Patients With High-Risk Acute Myeloid Leukemia and Myelodysplasia

PURPOSE The toxicity of allogeneic stem-cell transplantation can be substantially reduced using a reduced-intensity conditioning (RIC) regimen. This has increased the proportion of patients with myeloid malignancies eligible for allogeneic transplantation. However, the capacity of RIC allografts to produce durable remissions in patients with acute myeloid leukemia (AML) and myelodysplasia (MDS) has not yet been defined, and consequently, the role of RIC allografts in the management of these diseases remains conjectural. PATIENTS AND METHODS Seventy-six patients with high-risk AML or MDS received an allograft using a fludarabine/melphalan RIC regimen incorporating alemtuzumab. The median age of the cohort was 52 years (range, 18 to 71 years). RESULTS The 100-day transplantation-related mortality rate was 9%, and no patient developed greater than grade 2 graft-versus-host disease. With a median follow-up of 36 months (range, 13 to 70 months), 27 patients were alive and in remission, with 3-year actuarial overall survival (OS) and disease-free survival (DFS) rates of 41% and 37%, respectively. The 3-year OS and DFS rates of patients with AML in complete remission at the time of transplantation were 48% and 42%, respectively. Disease relapse was the most common cause of treatment failure and occurred at a median time of 6 months after transplantation. All but one patient destined to relapse did so within 24 months of transplantation. CONCLUSION The extended follow-up in this series identifies a high risk of early disease relapse but provides evidence that RIC allografts can produce sustained DFS in a significant number of patients with AML who would be ineligible for allogeneic transplantation with myeloablative conditioning.

Myelodysplastic Syndromes Are Propagated by Rare and Distinct Human Cancer Stem Cells In Vivo.

Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.

Δ160p53 is a novel N-terminal p53 isoform encoded by Δ133p53 transcript

p53 gene expresses several protein isoforms modulating p53‐mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, Δ160p53, lacking the first 159 residues. By knockdown experiments and site‐directed mutagenesis, we show that Δ160p53 is encoded by Δ133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of Δ160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and Δ133p53 protein expression but not the one of Δ160p53. Overall, these results show that the Δ133p53 transcript generates two different p53 isoforms, Δ133p53 and Δ160p53.

Functional disturbance of marrow stromal microenvironment in the myelodysplastic syndromes

The potential contribution of abnormal marrow stromal function to ineffective haemopoiesis in the myelodysplastic syndromes is unclear. We have compared the ability of stromal layers from normal (n = 7) and myelodysplastic (n = 9) marrow to alter proliferation and survival of the granulocyte–macrophage colony-stimulating factor/interleukin-3-dependent cell line F-36P. Co-cultures for 72 h in the absence of exogenous cytokines were either in direct contact with stroma or separated by transwell inserts. On normal stromal layers, the ratio of adherent F-36P cells relative to stromal cells increased from a mean of 0.2 ± 0.01 (s.d.) at 4 h of co-culture to 0.34 ± 0.08 after 72 h (n = 7). Corresponding values on myelodysplastic stroma (0.2 ± 0.02 at 4 h and 0.35 ± 0.05 at 72 h; n = 9) indicated that the ability of myelodysplastic stromal layers to regulate short-term proliferation of F-36P cells may be similar to normal. Apoptosis of F-36P cells was quantified after co-culture with normal or myelodysplastic stroma: results from myelodysplastic co-cultures were standardized as a fraction of values from co-cultures with paired normal stroma (apoptotic ratio). Augmented apoptosis of F-36P cells was detected in 8/9 co-cultures with myelodysplastic stroma (mean = 15.7 ± 9.7%, n = 9), compared with corresponding normal stroma (mean = 12.4 ± 4.6%, n = 7, P < 0.05) with a mean apoptotic ratio of 1.4 ± 0.5 (P < 0.05). There was no correlation between stroma-related apoptosis and FAB type, tumour necrosis factor-α concentrations in the culture supernatant or numbers of stromal macrophages, and no evidence of involvement of the Fas pathway. Increased apoptosis was detected in cells grown in transwell inserts over stroma (23.8 ± 3%, n = 5) compared to adherent cells in cultures with normal stromal layers, but this survival difference was not observed in co-cultures with myelodysplastic stroma. These results suggest that abnormal stromal function in patients with myelodysplastic syndromes may contribute to increased apoptosis of haemopoietic cells within the marrow microenvironment. The effect appears to be dependent on close cellular contact, rather than the release of soluble factors, but the exact mechanism remains unclear.

Rituximab is effective in the management of refractory autoimmune cytopenias occurring after allogeneic stem cell transplantation

Summary:Autoimmune haemolytic anaemia (AIHA), immune thrombocytopenia (ITP) and autoimmune neutropenia (AIN) are well-recognised complications of allogeneic stem cell transplantation (SCT), but have previously only been reported in the context of myeloablative conditioning regimens. Management of AIHA, ITP or AIN occurring after allogeneic SCT is unsatisfactory since they frequently prove refractory to conventional therapies including splenectomy. As a consequence, autoimmune cytopenias occurring after allogeneic SCT are associated with substantial morbidity and mortality. We report four patients who developed AIHA or ITP after allogeneic transplantation – three of which occurred after a reduced-intensity conditioning (RIC) regimen. All patients demonstrated a complete response to Rituximab, having failed to respond to conventional treatment including high-dose prednisolone and intravenous immunoglobulin (IVIg). We conclude that Rituximab can be a valuable agent in the management of autoimmune cytopenias occurring after allogeneic SCT and that autoimmune cytopenias may be a hitherto unrecognised complication of RIC regimens.

Factors predicting long-term survival after T-cell depleted reduced intensity allogeneic stem cell transplantation for acute myeloid leukemia

Background Reduced intensity conditioning regimens permit the delivery of a potentially curative graft-versus-leukemia effect in older patients with acute myeloid leukemia. Although T-cell depletion is increasingly used to reduce the risk of graft-versus-host disease its impact on the graft-versus-leukemia effect and long-term outcome post-transplant is unknown. Design and Methods We have characterized pre- and post-transplant factors determining overall survival in 168 patients with acute myeloid leukemia transplanted using an alemtuzumab based reduced intensity conditioning regimen with a median duration of follow-up of 37 months. Results The 3-year overall survival for patients transplanted in CR1 or CR2/CR3 was 50% (95% CI, 38% to 62%) and 44% (95% CI, 31% to 56%), respectively compared to 15% (95% CI, 2% to 36%) for patients with relapsed/refractory disease. Multivariate analysis demonstrated that both survival and disease relapse were influenced by status at transplant (P=0.008) and presentation cytogenetics (P=0.01). Increased exposure to cyclosporine A (CsA) in the first 21 days post-transplant was associated with an increased relapse risk (P<0.0001) and decreased overall survival (P<0.0001). Conclusions Disease stage, presentation karyotype and post-transplant CsA exposure are important predictors of outcome in patients undergoing a T-cell depleted reduced intensity conditioning allograft for acute myeloid leukemia. These data confirm the presence of a potent graft-versus-leukemia effect after a T-cell depleted reduced intensity conditioning allograft in acute myeloid leukemia and identify CsA exposure as a manipulable determinant of outcome in this setting.