|MDS
The 12th International Symposium on Myelodysplastic Syndromes (MDS) was held in Berlin, Germany, from May 8-11, 2013. The symposium is held every two years and focuses on research findings related to myelodysplastic syndromes (MDS). The symposium brought together experts in the field of MDS from all over the world to discuss topical issues in the field in depth. At the same time, many latest research advances in the field of MDS were announced, covering the biology, diagnosis, prognosis and treatment of MDS. Liu Xinjian, Department of Hematology, Henan Cancer Hospital
Molecular mechanism of MDS pathogenesis, prognosis and epigenetic treatment progress
▪ MDS gene mutations
MDS is a group of clonal hematopoietic stem cell disorders characterized by hematocrit reduction, abnormal development of one or more lineages of myeloid cells (dysplasia), ineffective haematopoiesis and increased risk of evolution to acute myeloid leukaemia (AML).The main pathophysiological nature of MDS is.
(i) a clonal disease of hematopoietic stem cell origin.
(ii) abnormal development of one or more lines of granulocyte, red and megakaryocyte lines.
(iii) Ineffective hematopoiesis. MDS is a multi-step pathological process involving multiple genes.
Approximately 25-30 genetic mutations involved in MDS development have been identified by single nucleotide polymorphism (SNP) microarray technology and whole genome or exome sequencing. Following the previously identified mutations in oncogenes, oncogenes and transcription factor-encoding genes, 2 major classes of mutations have been newly identified, involving epigenetic regulator-encoding genes and shedder complex protein-encoding genes, respectively. These mutations can co-exist in different combinations for different patients. Epigenetic regulator-encoding genes overlap significantly between MDS and AML, whereas mutations in RNA shear complex protein-encoding genes are more common in MDS.
TET2 gene mutations are widely present in hematologic malignancies such as MDS (25%), AML (10%), myeloproliferative neoplasms (MPN, 10%-30%) and chronic granulomatous leukemia (CMML, 50%). The protein encoding the TET2 gene induces demethylation reactions, a process that also requires the participation of ferrous ions (Fe2+) and α-ketoglutarate (α-KG), the latter being the product of oxidative decarboxylation of isocitrate catalyzed by isocitrate dehydrogenase (IDH), and the presence of mutations in IDH1 or 2 genes can inhibit the function of the TET2 protein; therefore, mutations in the TET2 gene and mutations in IDH1 or 2 genes are often mutations in TET2 and IDH1 or 2 genes tend to be mutually exclusive. Studies have confirmed that TET2 mutations may be associated with effective demethylation drug therapy. In addition, a large cohort study has confirmed that EZH2 gene mutations are associated with poor prognosis in MDS patients. Among the genes found to encode the shear complex proteins SF3B1, SRSF2, U2AF1 and ZRSR2, only mutations in the SF3B1 gene are strongly associated with cyclic iron-granulocytosis anemia, which is the main causative gene for this subtype, and patients with mutations in this gene have a better prognosis.
▪ Revised International Prognostic Scoring System (IPSS-R)
The International Prognostic Scoring System (IPSS) for MDS was proposed in 1997, and the WHO Prognostic Scoring System (WPSS) was proposed in 2005 based on the World Health Organization (WHO) staging criteria (2001). In recent years, the cytogenetic prognostic groupings in these 2 systems have been revised to
(i) very good: deletion of the long arm of chromosome 11 [del(11q)], -Y.
(ii) Good: normal, der(1;7), del(5q), del[12 short arm (p)], del(20q), with 2 abnormalities of del(5q).
(iii) moderate: 7q-, +8, i(17q), +19, any other separate abnormality or 2 independent clones.
(iv) poor: -7, inv(3)/t(3q)/del(3q), 2 abnormalities containing -7/7q-, 3 abnormalities.
⑤ very poor: 3 or more anomalies.
The WPSS was revised in 2011 to change whether red blood cell transfusion-dependent to whether there was severe anemia (<90 g/L in men and <80 g/L in women). 2012 saw the introduction of the IPSS-R: primitive cells were classified by ≤2%, >2% to <5%, 5% to 10%, and >10% to 30%; the prognostic grouping of karyotypes used the aforementioned 5-group method; the refinement of the hematocrit The IPSS-R prognostic risk grouping was divided into 5 groups: very low risk, low risk, intermediate risk, high risk, and very high risk, with median survival of 6.8, 4.3, 2.3, 1.5, and 0.9 years, respectively, and median time to AML transformation of The median time to AML transformation was not reached, not reached, 15.7, 4.8 and 2.6 years, respectively.
▪ Epigenetic treatments
mainly include DNA methylation, which can inhibit gene expression through methylation modification, and histone deacetylation, which affects DNA transcriptional activity by altering chromatin structure, due to epigenetic alterations in DNA and/or histones leading to “silencing” of genes associated with cell differentiation and inhibition of tumor growth “, these epigenetic alterations are reversible, i.e., they can be reversed with DNA methyltransferase inhibitors (e.g., decitabine) and deacetylase inhibitors, and epigenetic therapies are increasingly being studied with high priority.
Epigenetic abnormalities are one of the main molecular mechanisms in the development and evolution of MDS. 2010 a study showed that DNA methylation predicted survival and treatment response in MDS patients, which identified MDS patients with methylation phenotype by CpG island methylation phenotype (CIMP), and showed that the presence of CIMP was significantly associated with poor prognosis and risk of leukemic transformation in MDS patients. Baseline methylation levels were not significantly associated with response to decitabine treatment, and the duration of decreased methylation levels was associated with better clinical outcomes. In addition, it was shown that decitabine significantly prolonged progression-free survival (PFS) in patients with MDS in intermediate-to-high-risk patients. A recent phase II clinical study published in the Journal of Clinical Oncology evaluated the effectiveness of low-dose decitabine subcutaneously in patients with low- and intermediate-risk-1 MDS. The study included 65 patients divided into 2 groups treated with regimen A (decitabine 20 mg/m2 subcutaneously on days 1, 2, and 3, 1 course every 28 days) and regimen B (decitabine 20 mg/m2 subcutaneously on days 1, 8, 15, and 22, 1 course every 28 days). The median follow-up was 14.6 months. The results showed that 67% of patients in regimen A and 59% in regimen B were free from red blood cell and platelet transfusion dependence, and approximately 70% of patients survived up to 500 days.
Advances in epigenetic treatment of CMML
▪ Current status of CMML treatment
Chronic granulomatous monocytic leukemia (CMML) has features of both myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS); therefore, CMML is also defined as an MPN/MDS overlap syndrome. Patients have a diversity of clinical manifestations, survival and routine blood tests. In general, the median OS period of patients is about 20 months and may be related to the proportion of bone marrow primitive cells. 15%-30% of CMML patients may progress to AML. currently, treatment for CMML is mainly limited to anti-proliferative drugs, which target only the MPN subtype in CMML. In addition, although clonal cytogenetic abnormalities are present in 20%-30% of CMML, these abnormalities are not CMML-specific alterations, and most patients present with normal karyotype. Recent biological studies have also not had an effective impact on treatment options, and there are no better ways to achieve effective remission.
Advances in epigenetic therapy
A recent study presented at the 12th International Symposium on MDS showed that decitabine is effective in treating patients with CMML, with complete remission (CR) + myeloid complete remission (mCR) rates of up to 40.5%. Meanwhile, patients’ remission rates could be further improved by increasing the duration of decitabine treatment through sustained demethylation. The study included 44 patients with CMML given decitabine [20 mg/(m2・d) for 5 days every 28 days] for at least 6 cycles of treatment. After 4 and 6 cycles of treatment, patients were evaluated for remission according to the 2006 International Working Group (IWG) criteria. The results showed that of the 43 patients evaluated according to the World Health Organization (WHO) classification, 27 were CMML-type I and 16 were CMML-type II. The patients had a median age of 71 years and a median treatment duration of 8 cycles. Among them, 11 patients were treated for <4 cycles, 17 patients were treated for 4-6 cycles, and 15 patients were treated for >6 cycles. 81% of CMML-I patients were treated for >4 cycles. After 4 cycles of treatment, 14% of CMML cases had CR; 18.6% of CMML cases had mCR; 2.3% of CMML cases had partial remission (PR); and 34.9% of CMML cases had stable disease (SD). After continuing decitabine treatment for 2 cycles, the CR rate increased to 16.2%; the mCR rate increased to 24.3%; the SD rate decreased to 18.9%; and the CR+mCR rate reached 40.5%. The main reasons for patients to discontinue decitabine therapy were disease progression (35%), death (23%) and toxic reactions to the drug (7%). Currently, 7 (16.3%) patients are still on treatment. The duration of remission in patients who achieved remission after 6 cycles of treatment was 9.7 months. 7 of the 43 evaluable patients had one serious infection and one grade 3/4 cardiac and gastrointestinal toxic reaction each. From this small cohort study of CMML patients, the investigators did not obtain clinical features or cytogenetic alterations that might predict disease remission in patients.
The decision to treat patients with CMML with decitabine can be made in the 2007 article by Aribi et al. in Cancer. The article recommends that decitabine should be considered for the following conditions.
(i) The presence of clinical manifestations of CMML, such as fever, weight loss, splenomegaly, leukocytosis, and other signs and symptoms of severe degree.
(ii) Progressive reduction of blood cells, such as anemia, thrombocytopenia, transfusion dependence.
(iii) CMML involving organs, such as skin damage, renal insufficiency, and pulmonary symptoms.
④Increased number of primitive cells, with bone marrow primitive cells increasing to more than 5% or 10%.
▪ Studies related to CMML gene mutations
Molecular genetic abnormalities are present in 80% of CMML patients, and the most common mutations are TET2 (58%), SRSF2 (46%) and ASXL1 (40%). Recent studies have shown that mutations in RNA shear-related genes play an important role in the development of myeloid malignancies. SF3B1 gene mutations were most common (75%-80%) in MDS patients with ringed iron granulocytes (RS). In contrast, mutations in the SRSF2 shear gene are mainly associated with CMML. Another Chinese study presented at the 12th International Symposium on MDS to assess the frequency of SRSF2 mutations also confirmed that SRSF2 mutations were the predominant shear mutations in CMML patients and were associated with older age and poor prognosis. In this study, 20 CMML patients were enrolled, and mutations were detected by polymerase chain reaction (PCR) combined with direct sequencing. The results showed that 11 male patients and 9 female patients, with a mean age of 62 years, had SRSF2 mutations instead of SF3B1 mutations in 4 patients (20%). In addition, the investigators suggest that decitabine may alleviate anemia and thrombocytopenia in patients with SRSF2 mutations, but a larger sample is needed.
Several other studies related to CMML were also presented at the 12th International Symposium on MDS. One of these studies evaluated the role of single nucleotide polymorphism (SNP) arrays in the diagnosis and prognosis prediction of patients with low-risk CMML. This study showed that SNP arrays can detect chromosomal abnormalities that are not detectable by traditional cytogenetic methods. Applying this method to a series of patients helps to better understand the disease characteristics of CMML. In addition, another study found that regulatory T cells (Tregs) can be significantly elevated in CMML patients with mutations in the TET2 gene, the significance of which needs to be further explored. As the research on CMML progresses, the current situation of CMML treatment will gradually be improved.
Establishment of a murine model of MDS and observation of the efficacy of demethylation drugs
Molecular genetic alterations of MDS and establishment of murine animal model
According to the morphological changes and the proportion of primitive cells, the 2008 revised World Health Organization (WHO) classification criteria classify MDS into: refractory hematocrit with unilineage developmental abnormalities (RCUD), refractory anemia with ringed iron granulocytes (RARS), refractory hematocrit with multilineage developmental abnormalities (RCMD), refractory hematocrit with primitive cytosis-1 ( RAEB-1, primitive cells 5%-9%), refractory hematocrit with primitive cells-2 (RAEB-2, primitive cells 10%-19%), MDS with simple 5q-, and MDS unclassifiable (MDS-U).
Recent studies have shown that the acquisition of multiple inherited somatic mutations and/or epigenetic abnormalities (DNA methylation, histone acetylation) in normal hematopoietic stem or progenitor cells leading to abnormal cell self-renewal as well as differentiation and maturation and the formation of malignant hematopoietic stem cell clones underlie the pathogenesis of MDS and AML. Currently known molecular events are focused on transcription factors, signal transduction pathways, epigenetic modifications and stem cell microenvironment, including TET2, NRAS, KRAS, CBL, ETV6, EZH2, ASXL1, TP53, RUNX1, FLT3, MLL-PTD, WT1, SF3B1 and SRSF2.
Mutations in the transcriptional regulator RUNX1 have a high incidence in AML and MDS patients, 13.1% and 17.5%, respectively. the RUNX1 gene encodes the RUNX1 protein, which combines with CBFβ to form a core binding factor transcriptional complex that regulates the expression of several hematopoietic-related genes. There are currently 2 main categories of RUNX1 mutations involved.
(i) those occurring in the N-terminus of RUNX1 (common mutation RUNX1D171N), mainly in the RHD structural domain, which affects RUNX1 binding to DNA.
② occurring at the RUNX1 C-terminus (common mutation RUNX1s291fs), which enhances RUNX1 binding to DNA but affects its transcriptional activity. abnormal MLL genes are another common genetic alteration in MDS and MDS/AML. the MLL protein relies on methyltransferase activity to mediate chromatin modifications that regulate the expression of related genes. In addition to chromosomal ectopics involving MLL, another type of MLL abnormality is mainly manifested as MLL partial tandem duplication (MLL-PTD). the incidence of MLL-PTD is 7.5% in AML patients and 4.2% in MDS. It was shown that both MLL-PTD and RUNX1 mutations were often present at the time of disease progression in MDS patients, and the proportion of coexisting mutations was higher in primary and secondary AML. This led to the hypothesis that RUNX1 mutations have a synergistic effect with MLL-PTD, leading to MDS development and AML transformation.
To further confirm the above hypothesis, the investigators constructed the following three MDS/AML mouse models based on MLL-PTD knock-in (knock-in) mice with different mutations of RUNX1.
(i) MLL-PTD/RUNX1-/- model.
②MLL-PTD/RUNX1- D171N BMT model.
③MLL-PTD/RUNX1-291fs BMT model. First, Mx1-Cre was applied and the RUNX1 allele was knocked out in MLL-PTD knock-in mice. 1 month after pIpC induction, PTD/RUNX1-/- mice showed significant platelet hypoplasia, and macrocytic anemia and leukocytopenia were observed after 2-4 months. Morphological analysis of bone marrow and peripheral blood revealed characteristic changes of abnormal development of the granulosa, erythrocyte and megakaryocyte lineages. Most PTD/RUNX1-/- mice died within 8 months, with a median survival of 149 days. Next, transplanted mouse models were established by constructing RUNX1D171N and RUNX1s291fs retroviral plasmids infected with bone marrow cells from wild-type mice (WT) and MLL-PTD knock-in mice. In addition to macrocytic anemia, thrombocytopenia, and morphological abnormalities, the bone marrow of transplanted mice often showed an increase in primitive cells (especially in MLL-PTD/RUNX1-291fs transplanted mice), and serial bone marrow transplants showed that the MLL-PTD/RUNX1-291fs MDS disease phenotype was transplantable but did not undergo AML transformation.
▪ Efficacy of demethylating drugs
Previous studies have shown that several genes are hypermethylated in MDS patients, and therefore, the U.S. Food and Drug Administration (FDA) approved the methylation transferase inhibitors 5-azacytidine and 5-azido-2′-deoxycytidine (decitabine) for MDS treatment. The latter reverses the aberrant hypermethylation of cytosine in DNA CpG islands and reactivates tumor suppressor genes that have been genetically “silenced” due to hypermethylation, thus exerting tumor suppressive effects. Clinical trials have shown that decitabine is effective in intermediate and high-risk MDS and MDS/AML, with efficiency rates ranging from 30% to 73%. Using a Dot Blot assay, the investigators examined the expression of 5-mc and 5-hmc in MLL-PTD/RUNX1-291fs transplanted murine lin-Kit+ hematopoietic stem cells and showed that compared with the control group, the expression of 5-mc in MLL-PTD/RUNX1-291fs transplanted murine lin-Kit+ hematopoietic stem cells was significantly enhanced, suggesting that their DNA methylation levels were increased. The survival of MLL-PTD/RUNX1-291fs transplanted rats treated with low-dose decitabine (0.3 mg/kg, subcutaneously twice a week) was significantly longer than that of saline control group [(94.5±6.4) days versus (53.5±3.5) days, P<0.001]. MLL-PTD/RUNX1-291fs transplanted murine bone marrow cells were cultured in an in vitro series of colonies and treated with decitabine (0.5 μM), which showed a significant reduction in the number of colonies in the decitabine-treated group (34±7.7 versus 619±30.5, P<0.001) and promoted cell differentiation; however, for MDS initial cells (MICs), the effect of decitabine alone was limited. However, for MDS initial cells (MICs), decitabine alone had limited effect and failed to significantly reduce the number of MICs and colony formation ability, which may be one of the possible mechanisms of clinical decitabine resistance.
In conclusion, the establishment of an animal model of MDS not only contributes to the study of the pathogenesis of MDS, but also provides a useful tool for the implementation of new therapeutic strategies for MDS. In addition, although low-dose decitabine treatment can prolong the survival of MLL-PTD/RUNX1-291fs transplanted mice by promoting cell differentiation, decitabine monotherapy has limited efficacy in MICs, and further combination with other drugs to target MICs can improve the efficacy. Source: China Medical Tribune