This study examines the prognostic significance of early molecular response using an expanded dataset in chronic myeloid leukemia patients enrolled in the International Randomized Study of Interferon ...and STI571 (IRIS). Serial molecular studies demonstrate decreases in BCR-ABL transcripts over time. Analyses of event-free survival (EFS) and time to progression to accelerated phase/blast crisis (AP/BC) at 7 years were based on molecular responses using the international scale (IS) at 6-, 12-, and 18-month landmarks. Patients with BCR-ABL transcripts > 10% at 6 months and > 1% at 12 months had inferior EFS and higher rate of progression to AP/BC compared with all other molecular response groups. Conversely, patients who achieved major molecular response MMR: BCR-ABL (IS) ≤ 0.1% by 18 months enjoyed remarkably durable responses, with no progression to AP/BC and 95% EFS at 7 years. The probability of loss of complete cytogenetic response by 7 years was only 3% for patients in MMR at 18 months versus 26% for patients with complete cytogenetic response but not MMR (P < .001). This study shows a strong association between the degree to which BCR-ABL transcript numbers are reduced by therapy and long-term clinical outcome, supporting the use of time-dependent molecular measures to determine optimal response to therapy. This study is registered at www.clinicaltrials.gov as NCT00006343.
The majority of patients with chronic myeloid leukemia in chronic phase gain substantial benefit from imatinib but some fail to respond or lose their initial response. In 2006, the European ...LeukemiaNet published recommendations designed to help identify patients responding poorly to imatinib. Patients were evaluated at 3, 6, 12, and 18 months and some were classified as “failure” or “suboptimal responders.” We analyzed outcomes for 224 patients with chronic myeloid leukemia in chronic phase treated in a single institution to validate these recommendations. Patients were followed for a median of 46.1 months. At each time point, patients classified as “failure” showed significantly worse survival, progression-free survival, and cytogenetic response than other patients; for example, based on the assessment at 12 months, the 5-year survival was 87.1% versus 95.1% (P = .02), progression-free survival 76.% versus 90% (P = .002), and complete cytogenetic response rate 26.7% versus 94.1% (P < .001). Similarly, the criteria for “suboptimal response” at 6 and 12 months identified patients destined to fare badly, although criteria at 18 months were less useful. The predictive value of some other individual criteria varied. In general, the LeukemiaNet criteria have useful predictive value, but a case could now be made for combining the categories “failure” and “suboptimal response.”
1 Immunologie, CHU & Nancy Université, France
2 Departamento de Hematologia, Centro Hospitalar de Coimbra, Portugal
Correspondence: Marie Christine Béné, Immunology, CHU & Faculté de Médecine de ...Nancy, Nancy Université, 9 allée du Morvan, 54500 Vandoeuvre les Nancy, France. E-mail: marie-christine.bene{at}medecine.uhp-nancy.fr
Resistance to therapeutic agents is a major factor in the failure of cancer treatments. In leukemia, the resistant cells remaining in the bone marrow and/or peripheral blood constitute minimal residual disease and are detectable by highly sensitive assays when the patient appears to be in complete remission. Early detection of the expansion of residual cells permits clinical intervention with the aim of reversing the proliferation of resistant leukemic cells. Therefore, accurate and precise measurement of minimal residual disease can greatly enhance optimization of oncology patients' clinical management. This notion is supported by a large body of data among chronic myeloid leukemia patients, but minimal residual disease detection and monitoring is increasingly applied to other types of leukemia, and is starting to be a factor in decision-making for some therapeutic trials in childhood acute lymphoblastic leukemia. Here, from the solid ground of minimal residual disease detection in chronic myeloid leukemia, the current state of the art and development of molecular techniques in other leukemias and the growing field of multiparameter flow cytometry are reviewed in two separate parts reporting on the respective advances, advantages and pitfalls of these emerging methods.
Key words: minimal residual disease, leukemia, flow cytometry, standardization, monitoring.
We sought kinase domain (KD) mutations at the start of treatment with dasatinib in 46 chronic myeloid leukemia (CML) patients resistant to or intolerant of imatinib. We identified BCR-ABL mutant ...subclones in 12 (26%) cases and used pyrosequencing to estimate subsequent changes in their relative size after starting dasatinib. Four patients lost their mutations, which remained undetectable, 3 patients retained the original mutation or lost it only transiently, 3 lost their original mutations but acquired a new mutation (F317L), and 2 developed another mutation (T315I) in addition to the original mutation within the same subclone. This study shows that expansion of a mutant Ph-positive clone that responds initially to a second generation tyrosine kinase inhibitor may be due either to late acquisition of a second mutation in the originally mutated clone, such as the T315I, or to acquisition of a completely new mutant clone, such as F317L.
Reliable and accurate epidemiological data is a prerequisite for a cost effective screening program for inherited disorders, which however, is lacking in a number of developing countries. Here we ...report the first detailed population study in the Republic of Guinea, a sub-Saharan West African country, designed to assess the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies, including screening for thalassemia.
Peripheral blood samples from 187 Guinean adults were screened for hemoglobin (Hb) variants by standard hematological methods. One hundred and ten samples from males were screened for G6PD deficiency by the fluorescent spot test. Molecular analysis was performed for the most common α-thalassemia (α-thal) deletions, β-globin gene mutations, G6PD variants B (376A), A (376G), A- (376G/202A) and Betica (376G/968C), using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) or sequencing.
Of the 187 subjects screened, 36 were heterozygous for Hb S β6(A3)Glu→Val, GAG>GTG (allele frequency 9.62%). Sixty-four subjects were heterozygous and seven were homozygous for the −α3.7 kb deletion (allele frequency 20.85%). β-Thalassemia alleles were detected in five subjects, four with the −29 (A>G) mutation (allele frequency 1.07%) and one with codon 15 (TGG>TAG) (allele frequency 0.96%). The G6PD A- and G6PD Betica deficient variants were highly prevalent with a frequency of 5.7 and 3.3%, respectively. While we did not test for ferritin levels or α0-thal, four females (5.2%) had red cell indices strongly suggestive of iron deficient anemia: Hb <9.7 g/dL; MCH <19.3 pg; MCV <68.2; MCHC <31.6 g/dl; RDW >19.8%.
Our results are consistent with high frequency of alleles such as Hb S, α-thal and G6PD deficient alleles associated with malaria resistance. Finding a 9.6% Hb S allele frequency supports the notion for a proficient neonatal screening to identify the sickle cell patients, who might benefit from early prophylactic treatment for infections. The incidence of significant iron deficient anemia in women is lower than expected in an under developed country.
Musashi 2 (also known as MSI2), a mRNA binding protein is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting ...translation. MSI2 is preferentially expressed in hematopoietic tissue, in particular early myeloid progenitors. Moreover, investigators suggest upregulated MSI2 disrupts regulatory pathway/s leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and worse clinical prognosis in CML and AML (Kharas et al. Nat Med. 2010; 16:903; Ito et al. Nature. 2010; 466:765). Indeed we have confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in chronic phase (CP), irrespective of lymphoid or myeloid transformation. Furthermore, we have shown MSI2 and BCR-ABL1 expressions correlate. Here we report data implying MSI2 functions viaTGFβ1 signalling pathway.
We retrospectively studied 54 CML cDNA samples from 34 patients (M:15 ; F:19) in CP with median age 55.5 years (range 12-74). Apart from 3 patients treated with interferon+AraC, the remainder were prescribed Imatinib mesylate. Of the 54 samples 29 were collected at diagnosis (Dx) and 25 were obtained at 3 months post therapy (3M). For 20 of the patients, samples collected at Dx and 3M were available. Eight of the patients failed to achieve major molecular response (MMR) within 12 months post therapy. In addition to patient samples we included 19 normal cDNA controls from healthy blood donors (M:10 ; F:9), with 41 years median age (range 20-61). We also included 20 cDNA samples derived from hematopoietic cell lines (lymphoma: 7; AML: 3; CML:6 ; essential thrombocythemia: 1 ; hyperesoinophilic syndrome: 1 ; Acute lymphoblastic leukemia: 1). The cDNA was synthesized using reverse transcriptase and random hexamers. All the samples were subjected to quantitative real time PCR using TaqMan assay to quantify MSI2, TGFRβ1 and GUSβmRNA levels. Furthermore, we subjected protein isolated from the cells lines to Western blot analysis to assess TGFβR1 expression.
MSI2 mRNA median levels in patient samples were significantly decreased compared with the NC group at Dx and at 3M, p=0.002 and p=0.013, respectively. But we found no significant difference in MSI2 mRNA levels at Dx nor at 3M, between those who achieved major molecular response and those who did not within 12 months of starting therapy, p=0.215 and p=1.871, respectively. Also we observed no significant difference between the patient samples and the NC group TGFβR1 transcript numbers at Dx nor at 3M, p=0.057 and p=0.097, respectively. Equally we observed no significant difference in TGFβR1 levels, either at Dx or 3M, between those who did and failed to achieve MMR. But we found strong to moderate correlation between MSI2 and TGFβR1 expression when we compared the Dx and 3M data as determined by Spearman correlation coefficient, r=0.6975 and 0.5715, respectively. More importantly, we observed 9 of the cell lines, with increased (>6.7%) or detectable MSI2 mRNA expression, of which 6 express BCR-ABL1 (BV173, Lama87, K562, KCL22, KU812 and SupB15), the TGFβR1 protein was considerably decreased. The BCR-ABL1 positive cell line Meg01 was the one exception with increased MSI2 mRNA levels (7.1%) and clearly detectable TGFβR1 protein. Conversely, of the 10 BCR-ABL1 negative cell lines with decreased or undetectable MSI2 transcripts the TGFβR1 protein was clearly detectable in 8. Of these 7 were lymphoid cell lines (L-248, KM-H2, L-1236, HDLM-2, BJAB, U-266, U-2932) and one was essential thrombocythemia, SET2. The 2 exceptions were AML cell lines OCI-AML-2 and OCI-AML-3 with considerably decreased MSI2mRNA levels and markedly decreased TGFβR1 protein expression.
We suggest MSI2 expression is significantly decreased in CML patients in CP because the early progenitors expressing it are masked by large bulk of mature myeloid cells in these patients. Therefore to assess the prognostic value of MSI2 levels at diagnosis it should be quantified in CD34+ enriched samples at diagnosis. More importantly, finding cell lines expressing MSI2, with the exception of 3 cell lines (OCI-AML-2, OCI-AML-3 and Meg01) had decreased TGFβR1 protein is consistent with its role as a post transcription regulator. These findings combined with the previous data showing MSI2 mRNA levels are increased in CML BC support the notion that it functions via TGFβR1 signaling pathway to influence CML transformation.
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Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting ...translation. MSI2 is preferentially expressed in hematopoietic tissue and in particular in early myeloid progenitors. Furthermore, investigators have shown MSI2 expression is upregulated by HOXA9 resulting in dysfunction of the regulatory pathway, leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and is associated with worse prognosis in chronic myeloid leukemia (CML) and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). We verified MSI2 levels are significantly increased in CML patients in blast crisis compared with those in chronic phase, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). Given these observations we sought to assess MSI2 and HOXA9 expression in BCR-ABL1negative myeloproliferative neoplasm (MPN) patients.
We retrospectively quantified MSI2 and HOXA9 expression in 38 samples of which 22 were from highly heterogeneous MPN patients, all of whom are alive. These 22 MPN patients were classified as polycythemia vera (PV) n= 5; essential thrombocythemia (ET) n=3; primary myelofibrosis (PMF) n=11 and unclassified MPN (U-MPN) n=3, based on clinical hematologic and molecular parameters in accordance with the World Health Organization 2008 classification. The MPN patients demographic features were (M: 13; F:9) with median age of 69.5 years (range 53-88). Of the 22 patients 17 had the V617F allele, in one it was undetectable and 4 were not tested. The remaining cDNA samples were unselected normal controls (NC) from healthy blood donors (M: 6; F:10) with median age of 41 years (range 34-61). MSI2 and HOXA9 mRNA levels were quantified by quantitative real time PCR (Q-PCR) and normalized to GUSβendogenous control gene, expression levels. Patient and control data were subjected to Mann-Whitney unpaired test two-tailed anaylsis using Graphpad Prism version 6.04 software.
MSI2 and HOXA9 expression was detectable in all the samples by Q-PCR. However, HOXA9 transcript numbers, median 0.115% (range 0.020-2.360) were significantly increased (p=0.0123) in MPN patients when compared with the NC Group, median 0.060% (range 0.030-0.120). Conversely, median MSI2 expression level 0.715% (range 0.370-2.030) in MPN patients, was significantly decreased, p<0.0001 compared to that observed in the NC Group, median 2.817% (range 1.445-7.533). But both MSI2 and HOXA9 were significantly increased, p<0.0001, when comparing NC Group (n=16) with the PMF patients (n=11). Of these 11 PMF patients 6 had the V617F allele, in one it was undetectable and 4 were not screened. Interestingly, we noted a difference between PV and PMF data with respect to HOXA9 and MSI2 expression. Specifically, the PMF patients’ HOXA9 median n=11; median age 62 years (range 53-81), 0.790% (range 0.03-2.36); was significantly higher, (p=0.0021), than the PV patients’ n=5; median age 77 years (range 69-88), 0.030% (range 0.10-0.40). By contrast no significant difference, p=0.0504, was detected when comparing MSI2expression between patients with PV and PMF. The sample size was too small to reliably evaluate the other MPN groups.
Our data are consistent with the notion that MSI2 plays a significant role in the biology of myeloid malignancies. While PV and ET patient numbers in this study were too small for evaluation, we did note finding increased HOXA9 expression among PMF subjects is consistent with reports that up to 30% of PMF patients harbor ASXL1 mutations, which are reported to lead to increased HOXA9 expression. Furthermore, the ASXL1 mutations are associated with worse prognosis, such that stem cell transplant (SCT) is recommended for patients harboring these mutations. In this study 2 of the 11 PMF patients had SCT and were among three patients with the highest HOXA9 levels determined. Given the findings it would be interesting to screen the PMF patients for ASXL1 mutations. Equally, finding MSI2 and HOXA9 increased expression in PMF patients is supportive of the later regulating the former. However, we observed no correlation between MSI2 and HOXA9 levels. An expanded study is required to assess these preliminary data implying HOXA9 expression is a prognostic marker in PMF patients.
le Coutre:Novartis: Honoraria.
To date no single molecular biomarker, with proven clinical utility, that is predictive and identifies patients with more aggressive chronic myeloid leukaemia (CML) at diagnosis, has been reported. ...Such a molecular biomarker would enable stratification of patients according to aggressiveness of the malignancy and therefore at greater risk of progressing to blast crisis (BC). If these patients could be identified a more potent TKI could be prescribed at presentation or be considered for stem cell transplant up front. Recently a common polymorphic variant of growth factor independent 1 (GFI1), a zinc finger DNA binding transcriptional repressor, whereby serine is substituted by asparagine at residue 36 (S36N), was reported (Khandanpour et al. Blood. 2010; 115:22462) to be associated with a 1.6 fold increased risk of developing acute myeloid leukemia (AML). In addition, investigators reported a frequency of 11% of the S36N GFI1 allele among AML patients, compared with 3-7% among the European populations. Reports suggest S36N GFI1 variant fails to repress the target genes, including HOXA9, and as a consequence their expression levels are elevated. These observations are consistent with a critical role for GFI1 in myeloid differentiation. In view of these findings we hypothesized that the GFI1 S36N allele might influence CML progression, via up-regulation of HOXA9, from a relatively indolent chronic phase (CP) to invariably fatal BC, characterized by presence of leukaemic stem cells, resistant to tyrosine kinase inhibitors in peripheral blood. Therefore, we assessed if S36N allele frequency was increased in CML and quantified GFI1expression.
We retrospectively studied 106 cDNA samples, synthesized using 1 µg RNA extracted from nucleated cellular pellets isolated from peripheral blood or bone marrow aspirate. Total RNA was reverse transcribed using reverse transcriptase primed with random hexamers oligonucleotides. Of the 196 sample 89 were from highly heterogeneous CML patients and the remaining 17 comprised of randomly selected normal control samples from healthy adult volunteer blood donors. The 89 CML patients included 24 (accelerated phase: 2 ; BC:22) with 51 years median age (range 20-75) who had progressed to advanced disease (AD) of these 13 were males. The remaining 65 CML patients (M:43 ; F: 22) were in chronic phase (CP) with median age 74 years (range 7-90). Polymerase chain reaction products, containing GFI1 exons 2, 3 and 4 were subjected to restriction fragment length polymorphism using BfaI restriction enzyme to test for the S36N GFI1 allele. Samples with the S36N allele were confirmed by Sanger sequencing. Total GFI1 (i.e. serine and asparagine alleles), HOXA9 and GUSß (endogenous control gene) mRNA transcripts were quantified by quantitative real time using Taqman assay. Each Taqman assay included a standard curve constructed using 2 plasmids containing HOXA9 or GUSß insert. Serially diluted cDNA derived from MEG01 hematopoietic cell line was used to prepare the standard curve for GFI1quantification.
S36N GFI1 allele frequency was significantly increased (p<0.001) among the CML patients (11/89) compared with the normal control group (1/17), but we detected no significant difference between AD and CP samples. However, the total GFI1 mRNA expression was significantly increased in CP samples compared with the control group (p<0.0001). But its expression was significantly decreased in AD compared with those in CP (p=0.0096). Interestingly HOXA9 expression was significantly increased (p=0.018) among individuals expressing the asparagine allele (i.e. heterozygous and homozygous subjects). But we observed no correlation between HOXA9 and GFI1 expression in CML patients.
These data suggest an association of S36N GFI1 allele with CML. Furthermore, the study implies the variant allele is associated with increased HOXA9 expression, which may influence disease progression viaMusashi 2. HOXA9 is reported to upregulate Musashi 2, which we and others have shown is increased in CML BC patients. The observed inverse expression of GFI1 levels between CP and AD is consistent with its reported role in myeloid cellular differentiation. These observations imply two mechanisms by which GFI influences CML disease kinetics: decreased GFI1 expression and/or elevated HOXA9 levels due to the presence of the S36N GFI1 allele.
le Coutre:Novartis: Honoraria.
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