Jaspal Kaeda

Increased frequencies of CD4 + CD25 high T regs correlate with disease relapse after allogeneic stem cell transplantation for chronic myeloid leukemia

The therapeutic efficacy of allogeneic hemopoietic stem cell transplantation (SCT) for chronic myeloid leukemia (CML) largely relies on the graft-versus-leukemia (GvL) effect exerted by donor T cells. CD4+CD25high regulatory T cells (Tregs) have been shown to downregulate antitumor responses but their role on GvL has not been evaluated. We performed a cross-sectional study in which we enumerated and characterized CD4+CD25high Tregs in the peripheral blood of CML patients undergoing allogeneic SCT. We documented higher frequencies of Tregs in patients after transplant as compared to normal controls and newly diagnosed patients. The increment was particularly evident in patients who had received their SCT 18 months before. In vitro functional studies demonstrated that the Tregs purified from SCT patients exhibited a more potent suppressive activity than Tregs isolated from healthy volunteers. Patients in whom Tregs numbers were higher than controls more than 18 months after SCT showed evidence of disease relapse. Although the increment in Tregs might have an advantageous effect on graft rejection in the early phase post-transplant, our data suggest that Tregs exert an inhibitory effect on GvL.

BCR-ABL transcript dynamics support the hypothesis that leukemic stem cells are reduced during imatinib treatment

Purpose: Imatinib induces a durable response in most patients with Philadelphia chromosome–positive chronic myeloid leukemia, but it is currently unclear whether imatinib reduces the leukemic stem cell (LSC) burden, which may be an important step toward enabling safe discontinuation of therapy. In this article, we use mathematical models of BCR–ABL levels to make inferences on the dynamics of LSCs. Experimental Design: Patients with at least 1 BCR–ABL transcript measurement on imatinib were included (N = 477). Maximum likelihood methods were used to test 3 potential hypotheses of the dynamics of BCR–ABL transcripts on imatinib therapy: (i) monoexponential, in which there is little, if any, decline in BCR–ABL transcripts; (ii) biexponential, in which patients have a rapid initial decrease in BCR–ABL transcripts followed by a more gradual response; and (iii) triexponential, in which patients first exhibit a biphasic decline but then have a third phase when BCR–ABL transcripts increase rapidly. Results: We found that most patients treated with imatinib exhibit a biphasic decrease in BCR–ABL transcript levels, with a rapid decrease during the first few months of treatment, followed by a more gradual decrease that often continues over many years. Conclusions: We show that the only hypothesis consistent with current data on progenitor cell turnover and with the long-term, gradual decrease in the BCR–ABL levels seen in most patients is that these patients exhibit a continual, gradual reduction of the LSCs. This observation may explain the ability to discontinue imatinib therapy without relapse in some cases. Clin Cancer Res; 17(21); 6812–21. ©2011 AACR.

Immune haemolytic anaemia following T cell-depleted allogeneic bone marrow transplantation for chronic myeloid leukaemia : association with leukaemic relapse and treatment with donor lymphocyte infusions

Immune haemolytic anaemia (IHA) is a recognised complication after allogeneic stem cell transplantation (SCT) and occurs more frequently if marrow cells have been subjected to T cell depletion (TCD). Among 58 consecutive patients who underwent TCD-allogeneic SCT from volunteer unrelated donors for the treatment of CML at the Hammersmith Hospital during a 3-year period (1 March 1996 to 28 February 1999) we identified nine cases of IHA. All patients had a strongly positive direct and indirect antiglobulin test and in eight patients the serological findings were typical of warm-type haemolysis often with antibody specificities within the Rh system. All nine cases had clinically significant haemolysis and were treated initially with prednisolone and immunoglobulin. The onset of IHA coincided with the occurrence of leukaemic relapse in six cases, and the presence of host haemopoiesis confirmed by lineage-specific chimerism in all four cases studied. Five patients received donor lymphocyte infusions (DLI); in three molecular remission and the restoration of full donor chimerism coincided with resolution of haemolysis. We conclude that in the context of leukaemic relapse, DLI is an effective therapy for IHA following allografts involving TCD. Bone Marrow Transplantation (2001) 28, 581–586.

High Prevalence of Hemoglobin Disorders and Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in the Republic of Guinea (West Africa)

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.

Up-regulated MSI2 is associated with more aggressive chronic myeloid leukemia

Abstract A better understanding of events triggering chronic myeloid leukemia progression is critical for optimized clinical management of chronic myeloid leukemia (CML). We sought to validate that increased expression of Musashi 2 (MSI2), a post-transcription regulator, is associated with progression and prognosis. Screening of 152 patients with CML showed that MSI2 was significantly decreased among patients with CML in chronic phase (CP) at diagnosis (p < 0.0001), but found no significant difference between the normal control group and treated patients with CML in CP. Moreover MSI2 was significantly increased (p < 0.0001) in patients with advance disease (AD) CML. Furthermore, our human hematopoietic cell line data imply that MSI2 and BCR-ABL1 mRNA expression are correlated. However, these data cast a doubt on earlier reports that MSI2 effects HES1 expression via NUMB–NOTCH signaling.

GFI1(36N) as a therapeutic and prognostic marker for myelodysplastic syndrome

Inherited gene variants play an important role in malignant diseases. The transcriptional repressor growth factor independence 1 (GFI1) regulates hematopoietic stem cell (HSC) self-renewal and differentiation. A single-nucleotide polymorphism of GFI1 (rs34631763) generates a protein with an asparagine (N) instead of a serine (S) at position 36 (GFI136N) and has a prevalence of 3%–5% among Caucasians. Because GFI1 regulates myeloid development, we examined the role of GFI136N on the course of MDS disease. To this end, we determined allele frequencies of GFI136N in four independent MDS cohorts from the Netherlands and Belgium, Germany, the ICGC consortium, and the United States. The GFI136N allele frequency in the 723 MDS patients genotyped ranged between 9% and 12%. GFI136N was an independent adverse prognostic factor for overall survival, acute myeloid leukemia-free survival, and event-free survival in a univariate analysis. After adjustment for age, bone marrow blast percentage, IPSS score, mutational status, and cytogenetic findings, GFI136N remained an independent adverse prognostic marker. GFI136S homozygous patients exhibited a sustained response to treatment with hypomethylating agents, whereas GFI136N patients had a poor sustained response to this therapy. Because allele status of GFI136N is readily determined using basic molecular techniques, we propose inclusion of GFI136N status in future prospective studies for MDS patients to better predict prognosis and guide therapeutic decisions.

Is the BCR-ABL/GUSB transcript level at diagnosis an early predictive marker for chronic myeloid leukemia patients treated with imatinib?

The development of the first target-specific tyrosine kinase inhibitor (TKI) and its introduction in the clinical practice radically changed chronic myeloid leukemia (CML) treatment. Monitoring therapeutic response to TKIs is a critical step in the management of CML.1 Recently, several follow-up studies upon which the European Leukemia Net 2013 (ELN) recommendations were based, pointed to the importance of early clearance of leukemic cells as demonstrated by molecular methods. Attaining a BCRABLIS transcript level ≤10% three months after initial imatinib mesylate (IM) treatment was found to be associated with a favorable outcome, including longer progression-free (PFS) and overall survival (OS), and higher probability of achieving complete cytogenetic response (CCyR) and major molecular response (MMR).1 Quantification of the BCR-ABL transcript level reflects leukemic burden, and is carried out by quantitative real-time polymerase chain reaction (RQ-PCR). Molecular response is based on the ratio of BCR-ABL transcript levels and a control gene. Results are expressed according to an international scale (IS) assigned to every patient at diagnosis, which is equal to 100% of BCR-ABL/control gene transcripts, regardless of the absolute amount of BCR-ABL transcripts. Thus, the actual leukemic burden of patients at diagnosis is not taken into account.2 An ideal control gene would be expected to be uniformly expressed in different cell types regardless of its proliferative status as well as be unaffected by therapeutic regimens, constant between individuals and expressed at a level similar to BCR-ABL. In fact, this control gene does not exist, and BCR and ABL are the most widely used control genes for quantifying BCR-ABL transcripts, mainly due to historical reasons. However, both BCR and ABL control genes do not show linearity with BCR-ABL transcript levels above 10% contrary to the GUSB gene that is not affected by high-level distortions which allow for better estimations of the BCR-ABL transcript level at diagnosis.3 In this study, the BCR-ABL transcript levels of 31 CML patients under IM treatment were analyzed by RQ-PCR in matinib?

Differential expression of SHP-1 in chronic myeloid leukemia

DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication. Just Accepted by Leukemia & Lymphoma

JAK2 V617F allele burden quantified by real time quantitative polymerase chain reaction and competitive polymerase chain reaction in patients with chronic myeloproliferative neoplasia.

Abstract Assessing the clinical significance of JAK2 V617F mutant allele burden is complicated by a myriad of techniques reported to detect and quantify the mutation. As a consequence, the level of sensitivity and how the data is reported vary. Harmonization of well-defined molecular studies would permit evaluation of the clinical significance of measuring allele burden and rapid determination of the efficacy of novel agents for the treatment of chronic myeloproliferative neoplasia via multicenter clinical trials, at the subclinical level. Here we report a comparison between the widely available TaqMan quantitative real time polymerase chain reaction (Q-PCR) and competitive PCR (C-PCR) assays. We found that the tumor load was invariably greater when measured by C-PCR compared to that recorded by Q-PCR. Furthermore, none of the samples converted from undetectable to detectable when the enriched granulocyte (GR) fraction was tested. While a difference in the V617F allele levels was detected between GR fraction and whole blood, this was not statistically significant.

Long-term follow-up of patients with Philadelphia chromosome-positive chronic myeloid leukemia after stem cell mobilization under imatinib

Based on the impressive results from the IRIS trial (International Randomized Interferon vs. STI-571), the tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) has become the recommended first-line therapy for patients with chronic myeloid leukemia (CML) [1]. IM targets the constitutively expressed oncoprotein, p210, encoded by the chimeric BCR–ABL1 gene resulting from the hallmark of CML, the Philadelphia (Ph) chromosome. A remarkable 88% of patients with newly diagnosed CML in chronic phase who were randomized to IM in the IRIS study are alive after 6 years [2]. However, a substantial number of patients develop resistance, and430% of newly diagnosed patients with chronic phase (CP) CML discontinue IM within 6 years. In the preimatinib era, autologous stem cell transplant (ASCT) or autografting was evaluated under the assumption of reducing tumor burden and the number of leukemic cells at risk for acquiring a second lesion which may trigger a blastic transformation, thus ‘setting back the clock’ [3]. In addition, the existence of Ph-negative hematopoietic progenitors in early CP offered the opportunity of cytogenetic conversions and restoring Ph-negative hematopoiesis, with longterm remissions in a proportion of patients. However, with the introduction of IM, all prospective studies comparing ASCT with non-transplant strategies were closed prematurely without reaching their recruitment targets [4]. Therefore, the data are limited and equivocal. With the advent and greatly enhanced efficacy of IM compared with other agents, autologous collection of stem cells was reevaluated. The stem cells harvested following in vivo purging with IM from patients in complete cytogenetic remission (CCyR) were expected to yield apheresis products with a markedly reduced number of Phpositive cells. Several groups have therefore assessed the safety and feasibility of stem cell mobilization during IM therapy [5–9]. Using granulocyte-colony stimulating factor (G-CSF) alone, mobilization for patients in CCyR yielded success rates comparable to those with previous chemotherapy-based approaches, in the range of 40–100%. Evaluation of stem cell harvests indicated that even polymerase chain reaction (PCR) negativity for BCR–ABL1 transcripts was possible in a number of patients. We have previously reported our experience with stem cell mobilization in 18 patients who achieved CCyR on IM therapy [8,10,11]. Here, we present their long-term outcome, with the inclusion of four additional patients. Between October 2000 and February 2006, all patients with Ph-positive CML achieving CCyR with IM therapy were offered stem cell mobilization. CCyR was confirmed at least twice by conventional cytogenetics on bone marrow samples. Patients gave their written informed consent and all procedures were followed in accordance with the Declaration of Helsinki. Stem cell mobilization was achieved by administering 10 mg/kg body weight/day filgrastim (G-CSF; Amgen, Munich, Germany) subcuta-