Michael J. Clemente

Somatic STAT3 mutations in large granular lymphocytic leukemia.

BACKGROUND T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

STAT3 mutations unify the pathogenesis of chronic lymphoproliferative disorders of NK cells and T-cell large granular lymphocyte leukemia

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia.

BACKGROUND In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. METHODS We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. RESULTS Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. CONCLUSIONS Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

Pre-Transplant IFN-γ ELISPOTs Are Associated with Post-Transplant Renal Function in African American Renal Transplant Recipients

Final crossmatch testing is routinely used to assess the risk of antibody‐mediated graft injury/rejection post‐transplant. Analogously, we postulated that quantitative measurements of anti‐donor effector/memory T cells pre‐transplant would independently assess post‐transplant risk. To address this hypothesis, we determined the frequencies of pre‐transplant, donor‐specific interferon‐γ (IFN‐γ) enzyme‐linked immunosorbent spots (ELISPOTs) and correlated the results with post‐transplant outcomes in 37 African American recipients of deceased donor kidney transplants treated with tacrolimus‐ and sirolimus‐based immunosuppression.

Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia

Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

Alloreactivity in Renal Transplant Recipients with and without Chronic Allograft Nephropathy

The pathogenesis of chronic allograft nephropathy (CAN) involves both immunologic (antigen-dependent) and nonimmunologic (antigen-independent) mechanisms. In order to provide further insight into the immunologic basis of this disease, a cross-sectional analysis of cellular and humoral immunity in human renal allograft recipients with or without deteriorating renal function and biopsy proven CAN was performed. Interferon-gamma enzyme-linked immunosorbent spot assays were used to assess cellular immunity to donor, or fully mismatched third-party stimulator cells (direct pathway), and to synthetic peptides derived from donor HLA molecules (indirect pathway). Anti-HLA antibodies were evaluated by flow cytometry using HLA-coated beads. Both the mean frequencies of donor-reactive peripheral blood lymphocytes and the number of individuals who responded to donor antigens per group were statistically higher in CAN patients versus control subjects (P < 0.02). Calculated ratios of donor/third-party enzyme-linked immunosorbent spot responses showed mean values of 2.61 +/- 3.0 in the CAN group, with ratios of 0.50 to 0.72 +/- 0.42 in control subjects (P < 0.001), confirming that direct, donor-specific cellular immunity predominated in patients with CAN. Fifty percent of CAN patients studied exhibited donor peptide reactivity compared with only 28.6% in control subjects. Finally, 33% of patients in the CAN group developed new posttransplantation anti-HLA antibodies compared with only 4% in the control group (P < 0.05). Overall, the results suggest that persistent cell-mediated and humoral alloimmunity contribute to the development of CAN and further demonstrate that anti-donor immunity in patients with CAN is heterogeneous. Immune monitoring to predict long-term outcome should include multiple measures of cellular and humoral immunity.

Pretransplant cellular alloimmunity as assessed by a panel of reactive T cells assay correlates with acute renal graft rejection

Background. The panel reactive antibody test (PRA) is an established method for assessing posttransplant risk of immune-mediated graft injury. The panel of reactive T cell assay (PRT) in which transplant candidates’ peripheral blood mononuclear cells are tested for reactivity to a panel of allogenic stimulator cells by the IFN-&ggr; enzyme-linked immunosorbent spot assay analogously assesses the strength of the pretransplant effector–memory alloreactive T cell repertoire. Methods. PRT assays were performed in 30 kidney transplant candidates and results were correlated with acute rejection (AR). A positive PRT assay was defined as a response to at least 75% of the stimulators tested. Results. A positive pretransplant PRT test was observed in 11 of 30 (37%) patients, and AR within 1 year posttransplantation was seen in 7 of 30 (23%) subjects. Six of the seven (86%) patients with AR were PRT-positive (P=0.01) whereas only one of seven (14%) patients with a PRA greater than 15% had AR. The mean pretransplant PRT percentage was 40% for patients with no AR versus 81% for patients with AR (P=0.01). Estimated glomerular filtration rate (mL/min/1.73 m2) showed a trend towards a lower value in PRT-positive (48±15) versus PRT-negative (55±13) individuals. Conclusions. The data suggest that pretransplant PRT screening can identify patients at risk for posttransplant cellular immune mediated graft injury despite the absence of humoral allosensitization. Once confirmed by larger prospective trials, PRT screening could be used to guide clinical decision-making with regard to choosing donor organs and individualizing immunosuppression regimens.

Efficacy of rabbit anti-thymocyte globulin in severe aplastic anemia

Background A combination of horse anti-thymocyte globulin and cyclosporine produces responses in 60–70% of patients with severe aplastic anemia. We performed a phase II study of rabbit anti-thymocyte globulin and cyclosporine as first-line therapy for severe aplastic anemia. Design and Methods Twenty patients with severe aplastic anemia treated with rabbit anti-thymocyte globulin were compared to 67 historical control cases with matched clinical characteristics treated with horse anti-thymocyte globulin. Results Response rates at 3, 6 and 12 months were similar for patients treated with rabbit anti-thymocyte globulin or horse anti-thymocyte globulin: 40% versus 55% (P=0.43), 45% versus 58% (P=0.44) and 50% versus 58% (P=0.61), respectively. No differences in early mortality rates or overall survival were observed. We then performed multivariable analyses of response at 6 months and overall survival and identified the presence of a paroxysmal nocturnal hemoglobinuria clone (P=0.01) and a pretreatment absolute reticulocyte count greater than 30×109/L (P=0.007) as independent predictors of response and younger age (P=0.003), higher pretreatment absolute neutrophil (P=0.02) and absolute lymphocyte counts (P=0.03) as independent predictors of overall survival. None of the immunogenetic polymorphisms studied was predictive of response to immunosupressive therapy. Conclusions Despite reports suggesting differences in biological activity of different anti-thymocyte globulin preparations, rabbit and horse anti-thymocyte globulin appear to have a similar efficacy for up-front treatment of severe aplastic anemia. Clinicaltrial.gov: NCT01231841)

STAT3-mutations indicate the presence of subclinical T cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.