Pure red cell aplasia is an orphan disease, and as such lacks rationally established standard therapies. Most cases are idiopathic; a subset is antibody-mediated. There is overlap between idiopathic ...cases and those with T-cell large granular lymphocytic leukemia, hypogammaglobulinemia, and low-grade lymphomas. In each of the aforementioned, the pathogenetic mechanisms may involve autoreactive cytotoxic responses. We selected 62 uniformly diagnosed pure red cell aplasia patients and analyzed their pathophysiologic features and responsiveness to rationally applied first-line and salvage therapies in order to propose diagnostic and therapeutic algorithms that may be helpful in guiding the management of prospective patients, 52% of whom were idiopathic, while the others involved large granular lymphocytic leukemia, thymoma, and B-cell dyscrasia. T-cell-mediated responses ranged between a continuum from polyclonal to monoclonal (as seen in large granular lymphocytic leukemia). During a median observation period of 40 months, patients received a median of two different therapies to achieve remission. Frequently used therapy included calcineurin-inhibitors with a steroid taper yielding a first-line overall response rate of 76% (53/70). Oral cyclophosphamide showed activity, albeit lower than that produced by cyclosporine. Intravenous immunoglobulins were effective both in parvovirus patients and in hypogammaglobulinemia cases. In salvage settings, alemtuzumab is active, particularly in large granular lymphocytic leukemia-associated cases. Other potentially useful salvage options include rituximab, anti-thymocyte globulin and bortezomib. The workup of acquired pure red cell aplasia should include investigations of common pathological associations. Most effective therapies are directed against T-cell-mediated immunity, and therapeutic choices need to account for associated conditions that may help in choosing alternative salvage agents, such as intravenous immunoglobulin, alemtuzumab and bortezomib.
Somatic mutations in TET2 are common in myelodysplastic syndromes (MDS), myeloproliferative, and overlap syndromes. TET2 mutant (TET2
) clones are also found in asymptomatic elderly individuals, a ...condition referred to as clonal hematopoiesis of indeterminate potential (CHIP). In various entities of TET2
neoplasia, we examined the phenotype in relation to the strata of TET2 hits within the clonal hierarchy. Using deep sequencing, 1781 mutations were found in 1205 of 4930 patients; 40% of mutant cases were biallelic. Hierarchical analysis revealed that of TET2
cases >40% were ancestral, e.g., representing 8% of MDS. Higher (earlier) TET2 lesion rank within the clonal hierarchy (greater clonal burden) was associated with impaired survival. Moreover, MDS driven by ancestral TET2
is likely derived from TET2
CHIP with a penetrance of ~1%. Following ancestral TET2 mutations, individual disease course is determined by secondary hits. Using multidimensional analyses, we demonstrate how hits following the TET2 founder defect induces phenotypic shifts toward dysplasia, myeloproliferation, or progression to AML. In summary, TET2
CHIP-derived MDS is a subclass of MDS that is distinct from de novo disease.
Myelodysplastic syndromes are typically diseases of older adults. Patients in whom the onset is early may have distinct molecular and clinical features or reflect a demographic continuum. The ...identification of differences between "early onset" patients and those diagnosed at a traditional age has the potential to advance understanding of the pathogenesis of myelodysplasia and may lead to formation of distinct morphological subcategories. We studied a cohort of 634 patients with various subcategories of myelodysplastic syndrome and secondary acute myeloid leukemia, stratifying them based on age at presentation and clinical parameters. We then characterized molecular abnormalities detected by next-generation deep sequencing of 60 genes that are commonly mutated in myeloid malignancies. The number of mutations increased linearly with age and on average, patients >50 years of age had more mutations.
,
, and
were more commonly mutated in patients >50 years old compared to patients ≤50 years old. In general, patients >50 years of age also had more mutations in spliceosomal, epigenetic modifier, and RAS gene families. Although there are age-related differences in molecular features among patients with myelodysplasia, most notably in the incidence of
mutations, our results suggest that patients ≤50 years old belong to a disease continuum with a distinct pattern of early onset ancestral events.
Somatic TET2 mutations (TET2MT) are frequent in myeloid neoplasia (MN), particularly chronic myelomonocytic leukemia (CMML). TET2MT includes mostly loss-of-function/hypomorphic hits. Impaired TET2 ...activity skews differentiation of hematopoietic stem cells toward proliferating myeloid precursors. This study was prompted by the observation of frequent biallelic TET2 gene inactivations (biTET2i) in CMML. We speculated that biTET2i might be associated with distinct clinicohematological features. We analyzed TET2MT in 1045 patients with MN. Of 82 biTET2i cases, 66 were biTET2MT, 13 were hemizygous TET2MT, and 3 were homozygous TET2MT (uniparental disomy); the remaining patients (denoted biTET2− hereafter) were either monoallelic TET2MT (n = 96) or wild-type TET2 (n = 823). Truncation mutations were found in 83% of biTET2i vs 65% of biTET2− cases (P = .02). TET2 hits were founder lesions in 72% of biTET2i vs 38% of biTET2− cases (P < .0001). In biTET2i, significantly concurrent hits included SRSF2MT (33%; P < .0001) and KRAS/NRASMT (16%; P = .03) as compared with biTET2−. When the first TET2 hit was ancestral in biTET2i, the most common subsequent hits affected a second TET2MT, followed by SRSF2MT, ASXL1MT, RASMT, and DNMT3AMT. BiTET2i patients without any monocytosis showed an absence of SRSF2MT. BiTET2i patients were older and had monocytosis, CMML, normal karyotypes, and lower-risk disease compared with biTET2− patients. Hence, while a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather determined monocytic differentiation, consistent with its prevalence in CMML. Additionally, biTET2i showed lower odds of cytopenias and marrow blasts (≥5%) and higher odds of myeloid dysplasia and marrow hypercellularity. Thus, biTET2i might represent an auxiliary assessment tool in MN.
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Paroxysmal nocturnal hemoglobinuria (PNH) is usually associated with reduced bone marrow (BM) capacity caused by acquired idiopathic aplastic anemia (AA). PIGA mutations lead to a partial or total ...deficiency of glycosylphosphatidyl-inositol (GPI) anchor proteins (AP). AA is characteristically accompanied by the presence of often tiny GPI-AP deficient clones, which in a significant proportion of patients (10-15%), irrespective of the initial success of immunosuppressive therapy, will evolve to produce manifest hemolytic PNH. Indeed in our cohort of BM failure patients (n=319), 41% of AA patients had a PNH clone present (0.02-20% of granulocytes) (AA/PNH), 14% of patients had primary PNH (primary PNH), and 8% had a history of PNH post AA (secondary PNH).
To date, drug development for PNH has focused on designing supportive therapies to prevent transfusions due to hemolysis or thrombotic complications. In addition to the current FDA approved C5 inhibitor eculizumab, new, more convenient and effective complement blockers are under development. Apart from hematopoietic stem cell (HSC) transplantation, no direct strategies targeting basic pathophysiologic mechanisms of PNH have been ventured to prevent evolution of PNH clones and cure the disease. In early AA/PNH syndrome, the PIGA mutant HSCs are rare and unlikely contribute to significant blood cell production. While in later stages of manifest hemolytic PNH, hematopoiesis relies most frequently on mutant HSCs and thus elimination of these cells would result in AA.
We hypothesized that if a selective inhibitor of GPI-AP-deficient GPI-AP (-) cells can be developed, it could be used primarily in AA/PNH patients with a small clone size. The hope would be to prevent both later expansion of GPI-AP d(-) cells and development of manifest PNH.
To discover compounds acting selectively against GPI-AP (-) cells, we subjected wild type (WT) and GPI-AP (-) cell lines (K562, TF-1) to a high-throughput screen using a platform of 3000 bio-active molecules to identify hits and chemical compounds capable of selectively eliminating GPI-AP (-) cells. Our robotic screen yielded several top hits including GR -89696 fumarate, D-cycloserine and CGS-15943. Dose-response experiments confirmed CGS-15943 as a candidate growth inhibitor of GPI-AP (-) cells. CGS-15943 is an adenosine receptor antagonist and non-phosphodiesterase inhibitor which has previously been shown to inhibit cancer cell growth via PI3K/Akt pathway.
Low range dose CGS-15943 (1uM) induced cell growth inhibition in K562 and TF-1 GPI-AP (-) cells by 4.7 fold and 3.2 fold, respectively. No cell growth arrest was observed in K562 WT and TF-1 WT cells, as the percentage of alive cells was >95% upon drug treatment. Mixed competition assays were conducted in vitro using equal ratios of K562 and TF-1 WT and GPI-AP (-) cells exposed to CGS-15943 (1uM). Six days after culture, flow cytometric analysis of CD59 surface expression revealed that CGS-15943 allowed for preferential survival of WT cells (84.7 % K562, 96.3% TF-1) vs. GPI-AP (-) cells (15.3% K562, 3.7% TF-1). CGS-15943 induced an increase in the % of AnnexinV+/PI- and AnnexinV+/PI+ in TF-1 GPI-AP (-) cells (12.04% and 44.82, respectively). Similar results were obtained in K562 GPI-AP (-) cells (15.84% and 21.08%). Mononuclear cells of a PNH patient were stimulated with CD3/28 beads in presence of CGS-15943. Flow cytometric analysis indicates a dose dependent growth inhibition effect on GPI-AP (-) lymphocytes after 3 days of culture. Previous reported observations from our group identified that the survival differences between GPI-AP (-) and WT cells largely depend on active PI3K signaling pathway. Our pilot investigation of CGS-15943 - indicates that CGS-15943 induces an decrease in the protein expression of the PI3K isoform - p110γ - exclusively in GPI-AP (-) cells possibly suggesting that CGS-15943 inhibits the catalytic subunit of- p110γ.
In sum, we describe that the small molecule compound CGS-15943 selectively eliminates GPI-AP (-) cells in vitro, in both cell lines and in primary PNH cells most likely interfering with the PI3K/AKT survival pathway.
Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.
Introduction. Bone marrow (BM) aspiration is a routine test for hematologists, but it is a painful procedure for the patients. Since BM cells primarily stay in BM, not in the peripheral blood, BM ...analysis is an unavoidable procedure to diagnose hematological disorders. However, we previously reported that BM cells produce exosomes and such exosomes are released into the blood stream. Thus, by quantifying BM-cell specific mRNAs in plasma exosomes, we demonstrated that the condition of the BM could be assessed by using peripheral blood in patients with BM transplantation (Aoki et al. Clin Chem 60:675, 2014, ref. 1). Here we applied this analysis to much wider population of hematological disorders.
Materials and Methods. According to the Institutional Review Board (IRB)-approved procedure, a total 231 plasma samples from either peripheral blood or BM aspirate were collected. Two hundred mL of plasma were used to isolate exosomes followed by poly(A)+ mRNA isolation and cDNA synthesis as described previously (ref. 1). The resultant cDNA was used to amplify 10 different BM cell-specific mRNAs as well as 2 housekeeping genes, beta-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), by real time PCR (ref. 1). Using 10 fold dilution of 107 copies of ACTB and GAPDH DNA, cycle threshold was converted to copies/mL plasma.
Results. We selected 12 aplastic anemia (AA) as a model of hypoplastic BM, 8 paroxysmal nocturnal hemoglobinuria (PNH) and 9 myeloproliferative neoplasms (MPN) as a model of hyperplastic BM for erythroid and myeloid cells, respectively. Then we analyzed various diseases including 53 myelodysplastic syndromes (MDS), 46 acute myeloid leukemia (AML), 27 large granular lymphocytic leukemia (LGL), etc., without subclassification of disease subtypes and staging. As shown in Fig. 1A, the levels of hemoglobin B (HBB) mRNA (copies/mL plasma) in PNH (each dot represents a single patient) were significantly higher than those of AA (p=0.0003) (AA area was blanketed), indicating erythroid hyperplasia during the hemolytic phase of the diseasese. Interestingly, MDS and AML demonstrated highly heterogeneous populations over PNH levels and much less than AA. The levels of myelocyte marker defensin A3 (DEFA3) mRNA in MPN were significantly higher than those of AA (p=0.0028) (Fig. 1B), indicating myeloid hyperplasia in MPN. Patients with MDS, AML, and LGL also showed highly heterogeneous populations, suggesting myeloid hypo- and hyperplastic BM dependent on the subtypes and stages of diseases.
Conclusion. This study indicates that plasma exosome mRNAs will become an interesting tool for hematologists to asses BM condition by use of a blood test. Although we only showed HBB and DEFA3 data in Fig. 1, this assay is a powerful platform for the biomarker discovery of various hematological disorders.
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Mitsuhashi:NanoSomiX, Inc.: Employment; Hitachi Chemical America Co., Ltd.: Consultancy. Murakami:Hitachi Chemical America Co., Ltd.: Employment.
Since the pivotal revelation of the PIGA gene mutations responsible for glycosylphosphatidylinositol (GPI) anchor deficiency over 20 years ago, molecular and clinical research into the evolution of ...Paroxysmal Nocturnal Hemoglobinuria (PNH) has significantly advanced the current understanding of the disease, expanding upon the foundational work by numerous investigators over the previous two centuries. The discovery of multiple PIGA mutations in normal individuals using the bacterial toxin aerolysin as well as with florescent activated cell sorting (FACS) clearly demonstrated that PIGA mutations are common in normal hematopoiesis. A strong association of PNH with Aplastic Anemia (AA) and the failure of PIGA clones to expand in animal models argued for the necessity of permissive conditions, largely understood to be immune mediated bone marrow failure. While GPI anchor deficiency may lead to escape of the PNH clone from autoimmunity, recent research has added to the body of knowledge by demonstrating that the PNH clone may acquire additional mutations in other genes that promote clonal expansion in the absence of competition from normal hematopoiesis as found in AA.
Sequencing studies of PIGA in PNH patients suggested that one, two, or at most three hematopoietic stem cells were sufficient to supply the necessary blood cells for survival. Furthermore, specific monoclonal antibodies combined with the fluorescently labeled inactive proaerolysin variant (FLAER) currently used to perform PNH diagnostic assays visualized the relatively frequent occurrence of both Type II and Type III PNH cells, suggesting the presence of two PIGA mutations in a significant number of patients. Using a combination of multiparameter FACS and a custom designed multiamplicon next generation sequencing (NGS) assay targeting PIGA, our results suggest that this may be an underestimate.
17 sequential patients with PNH (N=7, 3 Male, 4 Female) or AA/PNH (N=10, 5 Male, 5 Female) were enrolled in this study. A flow cytometry panel consisting of CD235a/CD59 for RBCs and FLAER/CD24/CD15/CD45 for granulocytes was used to assess PNH clone size and to sort WBCs into PNH positive and negative fractions. Mean RBC clone size was 31.6% (range 1.1-63.5%); mean WBC clone size was 53.2% (range 0.17-99.7%). Sort purity was confirmed at >98% in both fractions, DNA was extracted and subjected to analysis using an NGS assay and a stringent bioanalytic pipeline with an average depth of 18,000 reads.
At least one PIGA mutation was detected in the PNH positive fraction of every patient. A total of 68 PIGA mutations were observed, consisting of 31 nonsynonymous SNVs, 16 frameshift insertion/deletions, 12 stopgains, 7 splice site, and 2 nonframeshift deletions. 13/17 (76%) had more than one mutation, and 12/17 (70%) had 3 or more mutations (range 3-14). Analysis of variant allelic frequency (VAF) indicated that multiple clones with distinct PIGA mutations greater than 5% VAF of the PNH positive fraction were found in 9/17 (53%) patients with a median VAF of 11% (range 5-86%) and 5/9 demonstrating 3 mutations >5%. Repeat identical experiments from three patients were performed on samples obtained roughly one month apart with concordant results. Overall, our results suggest that a complex clonal hierarchy with multiple dominant and/or subdominant yet expanded clones is relatively common in PNH.
The clonal hierarchy in PNH patients can include up to 14 PNH clones with distinct frequencies and mutations. In addition, it is a widely held notion that PIGA mutations occur only in a hematopoietic stem cell, thus affecting all lineages, yet anomalous cases of PNH have been reported where the RBC PNH clone size is markedly higher than that of the granulocyte clone, and comparison between monocyte and granulocyte clone size is significantly different. We have identified two such cases in this cohort, with flow cytometry revealing RBC PNH clones of 18.63% and 35.61%, while granulocyte clones were 0.17% and 8.11%, and monocyte clones were 53.12% and 50.35%, respectively. Current experiments isolating and sequencing both sorted PNH positive cell fractions as well as hematopoietic precursors for PIGA and other commonly mutated genes found in hematologic malignancies are underway to confirm and elucidate the complex clonal hierarchy these results suggest.
No relevant conflicts of interest to declare.
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