Clinical adoptive regulatory T Cell therapy: State of the art, challenges, and prospective.

Rejection of solid organ transplant and graft versus host disease (GvHD) continue to be challenging in post transplantation management. The introduction of calcineurin inhibitors dramatically improved recipients' short-term prognosis. However, long-term clinical outlook remains poor, moreover, the lifelong dependency on these toxic drugs leads to chronic deterioration of graft function, in particular the renal function, infections and de-novo malignancies. These observations led investigators to identify alternative therapeutic options to promote long-term graft survival, which could be used concomitantly, but preferably, replace pharmacologic immunosuppression as standard of care. Adoptive T cell (ATC) therapy has evolved as one of the most promising approaches in regenerative medicine in the recent years. A range of cell types with disparate immunoregulatory and regenerative properties are actively being investigated as potential therapeutic agents for specific transplant rejection, autoimmunity or injury-related indications. A significant body of data from preclinical models pointed to efficacy of cellular therapies. Significantly, early clinical trial observations have confirmed safety and tolerability, and yielded promising data in support of efficacy of the cellular therapeutics. The first class of these therapeutic agents commonly referred to as advanced therapy medicinal products have been approved and are now available for clinical use. Specifically, clinical trials have supported the utility of CD4+CD25+FOXP3+ regulatory T cells (Tregs) to minimize unwanted or overshooting immune responses and reduce the level of pharmacological immunosuppression in transplant recipients. Tregs are recognized as the principal orchestrators of maintaining peripheral tolerance, thereby blocking excessive immune responses and prevent autoimmunity. Here, we summarize rationale for the adoptive Treg therapy, challenges in manufacturing and clinical experiences with this novel living drug and outline future perspectives of its use in transplantation.

Single-cell analysis based dissection of clonality in myelofibrosis.

Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.

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 (GFI1(36N)) and has a prevalence of 3%-5% among Caucasians. Because GFI1 regulates myeloid development, we examined the role of GFI1(36N) on the course of MDS disease. To this end, we determined allele frequencies of GFI1(36N) in four independent MDS cohorts from the Netherlands and Belgium, Germany, the ICGC consortium, and the United States. The GFI1(36N) allele frequency in the 723 MDS patients genotyped ranged between 9% and 12%. GFI1(36N) 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, GFI1(36N) remained an independent adverse prognostic marker. GFI1(36S) homozygous patients exhibited a sustained response to treatment with hypomethylating agents, whereas GFI1(36N) patients had a poor sustained response to this therapy. Because allele status of GFI1(36N) is readily determined using basic molecular techniques, we propose inclusion of GFI1(36N) status in future prospective studies for MDS patients to better predict prognosis and guide therapeutic decisions.

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

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.

Effects of Jak2 type 1 inhibitors NVP-BSK805 and NVP-BVB808 on Jak2 mutation-positive and Bcr-Abl-positive cell lines.

Janus kinases are critical components of signaling pathways that regulate hematopoiesis. Mutations of the non-receptor tyrosine kinase JAK2 are found in many BCR-ABL-negative myeloproliferative neoplasms. Preclinical results support that JAK2 inhibitors could show efficacy in treating chronic myeloproliferative neoplasms. JAK2 has also been postulated to play a role in BCR-ABL signal transduction. Therefore, inhibitors of JAK2 kinases are turning into therapeutic strategies for treatment of chronic myelogenous leukemia (CML). In this study, the effects of two novel JAK2 inhibitors, NVP-BSK805 and NVP-BVB808, have been investigated in cell lines expressing either BCR-ABL or mutant JAK2. Possible synergies between NVP-BSK805/NVP-BVB808 and the kinase inhibitors imatinib and nilotinib were assessed. Proliferation and apoptosis tests with both substances showed response in the following cell lines: CHRF-288-11, SET-2 and UKE-1. All BCR-ABL-positive cell lines showed some reduction in proliferation, but with half-maximal growth-inhibitory values >1 µM. Combination of the JAK2 inhibitors with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL-positive cell lines responded well to both CML therapeutic agents. Interestingly, it seemed that the combination of imatinib with NVP-BSK805 had a protective effect on the cells. Combination treatment with nilotinib did not show this effect.

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

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.

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.

Long-term prognostic significance of early molecular response to imatinib in newly diagnosed chronic myeloid leukemia: an analysis from the International Randomized Study of Interferon and STI571 (IRIS).

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.

How and why minimal residual disease studies are necessary in leukemia: a review from WP10 and WP12 of the European LeukaemiaNet.

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.

European LeukemiaNet criteria for failure or suboptimal response reliably identify patients with CML in early chronic phase treated with imatinib whose eventual outcome is poor.

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."

Finding of kinase domain mutations in patients with chronic phase chronic myeloid leukemia responding to imatinib may identify those at high risk of disease progression.

PURPOSE: Kinase domain (KD) mutations in the BCR-ABL gene are associated with resistance to imatinib in chronic myeloid leukemia (CML) but their incidence and prognostic significance in chronic phase (CP) patients without resistance are unclear. PATIENTS AND METHODS: We analyzed outcome for 319 patients with CML-CP who were treated with imatinib; 171 were in early CP (ECP) and 148 were in late CP (LCP). Patients were screened routinely for mutations using direct sequencing regardless of response status. The 5-year cumulative incidence of mutations was 6.6% for ECP and 17% for LCP patients. RESULTS: Of the 319 patients, 214 (67%) achieved complete cytogenetic responses (CCyR). The identification of a mutation without other evidence of imatinib resistance was highly predictive for loss of CCyR (RR, 3.8; P = .005) and for progression to advanced phase (RR, 2.3; P = .01), though the intervals from first identification to loss of CCyR and disease progression were relatively long (median, 21 and 16 months, respectively). Mutations in the P-loop (excluding residue 244) were associated with a higher risk of progression than mutations elsewhere. CONCLUSION: We conclude that routine mutation screening of patients who appear to be responding to imatinib may identify those at high risk of disease progression.

Imatinib for newly diagnosed patients with chronic myeloid leukemia: incidence of sustained responses in an intention-to-treat analysis.

PURPOSE: Imatinib is remarkably effective in treating newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). To date, most of the available data come from a single multicenter study in which some of the patients were censored for diverse reasons. Here, we report our experience in treating patients at a single institution in a setting where all events were recorded. PATIENTS AND METHODS: A total of 204 consecutive adult patients with newly diagnosed CML in CP received imatinib from June 2000 until August 2006. Response (hematologic, cytogenetic, and molecular), progression-free survival (PFS) and survival were evaluated. RESULTS: At 5 years, cumulative incidences of complete cytogenetic response (CCyR) and major molecular response (MMR) were 82.7% and 50.1%, respectively. Estimated overall survival and PFS were 83.2% and 82.7%, respectively. By 5 years, 25% of patients had discontinued imatinib treatment because of an unsatisfactory response and/or toxicity. The 5-year probability of remaining in major cytogenetic response while still receiving imatinib was 62.7%. Patients achieving a CCyR at 1 year had a better PFS and overall survival than those failing to reach CCyR, but achieving a MMR conferred no further advantage. The identification of a kinase domain mutation was the only factor predicting for loss of CCyR. CONCLUSION: Imatinib is highly effective in most patients with CML-CP; patients who respond are likely to live substantially longer than those treated with earlier therapies. Achieving CCyR correlated with PFS and overall survival, but achieving MMR had no further predictive value. However, approximately one third of patients still need better therapy.

In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib.

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.