Danielle M. Townsley

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.).

Eltrombopag restores trilineage hematopoiesis in refractory severe aplastic anemia that can be sustained on discontinuation of drug

About a quarter of patients with severe aplastic anemia remain pancytopenic despite immunosuppressive therapy. We have previously demonstrated that eltrombopag has efficacy in this setting with 44% (11/25) of patients having clinically significant hematologic responses. We now report safety and efficacy data on a further 18 patients and long-term follow-up on the entire cohort of 43 patients. The overall response rate was 17 of 43 patients (40%) at 3 to 4 months, including tri- and bilineage responses. The majority of patients who remained on eltrombopag in an extension study (14/17) continued to show improvement, and 7 eventually had significant increases in neutrophil, red cell, and platelet lineages. Five patients with robust near-normalization of blood counts had drug discontinued at a median of 28.5 months after entry (range, 9-37 months), and all maintained stable counts a median of 13 months (range, 1-15 months) off eltrombopag. Eight patients, including 6 nonresponders and 2 responders, developed new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion. None evolved to acute myeloid leukemia to date. Eltrombopag is efficacious in a subset of patients with aplastic anemia refractory to immunosuppressive therapy, with frequent multilineage responses and maintenance of normalized hematopoiesis off treatment. This study is registered at www.clinicaltrials.gov as #NCT00922883.

Bone marrow failure and the telomeropathies

Our understanding of the pathophysiology of aplastic anemia is undergoing significant revision, with implications for diagnosis and treatment. Constitutional and acquired disease is poorly delineated, as lesions in some genetic pathways cause stereotypical childhood syndromes and also act as risk factors for clinical manifestations in adult life. Telomere diseases are a prominent example of this relationship. Accelerated telomere attrition is the result of mutations in telomere repair genes and genes encoding components of the shelterin complex and related proteins. Genotype-phenotype correlations show genes responsible for X-linked (DKC1) and severe recessive childhood dyskeratosis congenita, typically with associated mucocutaneous features, and others (TERC and TERT) for more subtle presentation as telomeropathy in adults, in which multiorgan failure may be prominent. Telomerase mutations also are etiologic in familial pulmonary fibrosis and cryptic liver disease. Detection of a telomere disease requires awareness in the clinic, appropriate laboratory testing of telomere content, and genetic sequencing. In treatment decisions, genetic screening of related donors for hematopoietic stem cell transplantation is critical, and androgen therapy may be helpful. Telomeres shorten normally with aging, as well as under environmental circumstances, with regenerative stress and oxidative damage. Telomere biology is complexly related to oncogenesis: telomere attrition is protective by enforcing senescence or apoptosis in cells with a long mitotic history, but telomere loss also can destabilize the genome by chromosome rearrangement and aneuploidy.

Danazol Treatment for Telomere Diseases

BACKGROUND Genetic defects in telomere maintenance and repair cause bone marrow failure, liver cirrhosis, and pulmonary fibrosis, and they increase susceptibility to cancer. Historically, androgens have been useful as treatment for marrow failure syndromes. In tissue culture and animal models, sex hormones regulate expression of the telomerase gene. METHODS In a phase 1-2 prospective study involving patients with telomere diseases, we administered the synthetic sex hormone danazol orally at a dose of 800 mg per day for a total of 24 months. The goal of treatment was the attenuation of accelerated telomere attrition, and the primary efficacy end point was a 20% reduction in the annual rate of telomere attrition measured at 24 months. The occurrence of toxic effects of treatment was the primary safety end point. Hematologic response to treatment at various time points was the secondary efficacy end point. RESULTS After 27 patients were enrolled, the study was halted early, because telomere attrition was reduced in all 12 patients who could be evaluated for the primary end point; in the intention-to-treat analysis, 12 of 27 patients (44%; 95% confidence interval [CI], 26 to 64) met the primary efficacy end point. Unexpectedly, almost all the patients (11 of 12, 92%) had a gain in telomere length at 24 months as compared with baseline (mean increase, 386 bp [95% CI, 178 to 593]); in exploratory analyses, similar increases were observed at 6 months (16 of 21 patients; mean increase, 175 bp [95% CI, 79 to 271]) and 12 months (16 of 18 patients; mean increase, 360 bp [95% CI, 209 to 512]). Hematologic responses occurred in 19 of 24 patients (79%) who could be evaluated at 3 months and in 10 of 12 patients (83%) who could be evaluated at 24 months. Known adverse effects of danazol--elevated liver-enzyme levels and muscle cramps--of grade 2 or less occurred in 41% and 33% of the patients, respectively. CONCLUSIONS In our study, treatment with danazol led to telomere elongation in patients with telomere diseases. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01441037.).

Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia

BACKGROUND Acquired aplastic anemia results from immune‐mediated destruction of bone marrow. Immunosuppressive therapies are effective, but reduced numbers of residual stem cells may limit their efficacy. In patients with aplastic anemia that was refractory to immunosuppression, eltrombopag, a synthetic thrombopoietin‐receptor agonist, led to clinically significant increases in blood counts in almost half the patients. We combined standard immunosuppressive therapy with eltrombopag in previously untreated patients with severe aplastic anemia. METHODS We enrolled 92 consecutive patients in a prospective phase 1–2 study of immunosuppressive therapy plus eltrombopag. The three consecutively enrolled cohorts differed with regard to the timing of initiation and the duration of the eltrombopag regimen (cohort 1 received eltrombopag from day 14 to 6 months, cohort 2 from day 14 to 3 months, and cohort 3 from day 1 to 6 months). The cohorts were analyzed separately. The primary outcome was complete hematologic response at 6 months. Secondary end points included overall response, survival, relapse, and clonal evolution to myeloid cancer. RESULTS The rate of complete response at 6 months was 33% in cohort 1, 26% in cohort 2, and 58% in cohort 3. The overall response rates at 6 months were 80%, 87%, and 94%, respectively. The complete and overall response rates in the combined cohorts were higher than in our historical cohort, in which the rate of complete response was 10% and the overall response rate was 66%. At a median follow‐up of 2 years, the survival rate was 97%; one patient died during the study from a nonhematologic cause. Marked increases in bone marrow cellularity, CD34+ cell number, and frequency of early hematopoietic progenitors were noted. Rates of relapse and clonal evolution were similar to our historical experience. Severe rashes occurred in two patients, resulting in the early discontinuation of eltrombopag. CONCLUSIONS The addition of eltrombopag to immunosuppressive therapy was associated with markedly higher rates of hematologic response among patients with severe aplastic anemia than in a historical cohort. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT01623167.)

GATA2 deficiency-associated bone marrow disorder differs from idiopathic aplastic anemia

Germ-line GATA2 gene mutations, leading to haploinsufficiency, have been identified in patients with familial myelodysplastic syndrome/acute myeloid leukemia, monocytopenia and mycobacterial infections, Emberger syndrome, and dendritic cell, monocyte, B-, and NK-cell deficiency. GATA2 mutations have also been reported in a minority of patients with congenital neutropenia and aplastic anemia (AA). The bone marrow (BM) from patients with GATA2 deficiency is typically hypocellular, with varying degrees of dysplasia. Distinguishing GATA2 patients from those with AA is critical for selecting appropriate therapy. We compared the BM flow cytometric, morphologic, and cytogenetic features of 28 GATA2 patients with those of 32 patients being evaluated for idiopathic AA. The marrow of GATA2 patients had severely reduced monocytes, B cells, and NK cells; absent hematogones; and inverted CD4:CD8 ratios. Atypical megakaryocytes and abnormal cytogenetics were more common in GATA2 marrows. CD34(+) cells were comparably reduced in GATA2 and AA. Using these criteria, we prospectively identified 4 of 32 patients with suspected AA who had features suspicious for GATA2 mutations, later confirmed by DNA sequencing. Our results show that routine BM flow cytometry, morphology, and cytogenetics in patients who present with cytopenia(s) can identify patients for whom GATA2 sequencing is indicated.

Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Hematologic Complications of Pregnancy

Pregnancy induces a number of physiologic changes that affect the hematologic indices, either directly or indirectly. Recognizing and treating hematologic disorders that occur during pregnancy is difficult owing to the paucity of evidence available to guide consultants. This review discusses specifically the diagnosis and management of benign hematologic disorders occurring during pregnancy. Anemia secondary to iron deficiency is the most frequent hematologic complication and is easily treated with oral iron formulations; however, care must be taken not to miss other causes of anemia, such as sickle cell disease. Thrombocytopenia is also a common reason for consulting the hematologist, and distinguishing gestational thrombocytopenia from immune thrombocytopenia (ITP), preeclampsia, HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), or thrombotic thrombocytopenic purpura (TTP) is essential since the treatment differs widely. Occasionally the management of mother and infant involves the expeditious recognition of neonatal alloimmune thrombocytopenia (NAIT), a condition that is responsible for severe life-threatening bleeding of the newborn. Additionally, inherited and acquired bleeding disorders affect pregnant women disproportionately and often require careful monitoring of coagulation parameters to prevent bleeding in the puerperium. Finally, venous thromboembolism (VTE) during pregnancy is still largely responsible for mortality during pregnancy, and the diagnosis, treatment options and guidelines for prevention of VTE during pregnancy are explored.

Moderate-dose cyclophosphamide for severe aplastic anemia has significant toxicity and does not prevent relapse and clonal evolution

First-line therapy of severe aplastic anemia (SAA) with high-dose cyclophosphamide causes toxicity and increased short-term mortality. We investigated cyclophosphamide at a lower, more moderate dose in combination with aggressive supportive care to determine whether severe infections might be avoided and hematologic outcomes defined for this regimen. From 2010 to 2012, 22 patients received cyclophosphamide at 120 mg/kg plus cyclosporine and antibacterial, antiviral, and antifungal prophylaxis. Toxicity was considerable, mainly due to prolonged absolute neutropenia, which occurred regardless of pretherapy blood counts, and persisted an average of 2 months. Granulocyte transfusions for uncontrolled infection were required in 5 patients, confirmed fungal infections were documented in 6, and 9 patients died. Nine patients (41%) responded at 6 months. After a median follow-up of 2.2 years, relapse occurred in 2 patients, and cytogenetic abnormalities (including monosomy 7) were observed in 4 patients. Although cyclophosphamide has activity in SAA, its toxicity is not justified when far less dangerous alternatives are available. This trial was registered at www.clinicaltrials.gov as #NCT01193283.

Memory Stem T Cells in Autoimmune Disease: High Frequency of Circulating CD8+ Memory Stem Cells in Acquired Aplastic Anemia

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4+ and CD8+ T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8+ TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8+ TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8+ TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8+ and CD4+ T cell subsets in AA patients, including CD8+ and CD4+ TSCMs. CD8+ TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8+ TSCM subset is a novel biomarker and a potential therapeutic target for AA.