Persistent Leukemia Research Articles

Adoptive Immunotherapy with Cord Blood (CB) Grafts Targeting Shared Inherited Paternal Antigens (IPA) and/or Non-Inherited Maternal Antigens (NIMA) during Induction Treatment for High Risk Myeloid Malignancies: Safety and Preliminary Results

BackgroundSeveral groups have reported use of haploidentical peripheral blood donor cells as adjunct to chemotherapy in remission induction of myeloid leukemia (Colvin et al, BBMT 2009, Gue et al, Blood 2011). Moreover, CB grafts have been associated with greater disease control and enhanced graft-versus-leukemia responses, as a consequence of the fetal-maternal interactions during pregnancy (van Rood et al, PNAS 2009 and PNAS 2012).We evaluated prospectively the feasibility and tolerability of infusing partially HLA matched cord blood units (CBUs) with specific alloreactive properties after salvage chemotherapy to patients (pts) with refractory myeloid leukemia.Patients and MethodsPts with confirmed diagnosis of AML or MDS, according to WHO classification (excluding acute promyelocytic leukemia) who had received two or more previous induction/re-induction regimens were eligible. All pts were treated at NYPH and all CBUs were obtained from the NCBP. The study was performed under FDA IND and was approved by the IRBs of both institutions. Pts received induction chemotherapy followed by CBU infusion (1-3 days later) without any immunosuppression.CBUs eligible for the study contained a pre-cryopreservation Total Nucleated Cell (TNC) dose of 0.5-2.5x107/kg. They were HLA matched at 4-6/6, HLA-A, -B intermediate resolution, -DRB1 high resolution. CBUs had undergone HLA typing of the mother, thus allowing determination of the paternal and maternal antigens. CBUs with shared IPA targets and/or NIMA match were selected for cell therapy.ResultsA total of 34 patients underwent CBU search. Mean CBU number per search was 134 (range: 7-250) with 58% potentially eligible CBUs per pt (range: 5-159). A total of 143 CBUs underwent testing including HLA confirmatory typing. At least one eligible CBU was identified for each pt. Higher HLA allele level match was preferred in the final selection.Of the 34 pts, 23 did not receive treatment because of progressive disease and death (n=8); other clinical trials/treatments (n=6); hematopoietic stem cell transplants (HSCT) (n=9); while one is currently undergoing evaluation. Ten pts have been treated (Table); 8 had AML, 7 with primary induction failure (PIF); one had myeloid blast phase CML and one with high risk MDS. Nine pts had poor risk karyotype and 4 had prior allografts. Median age was 52 years (range: 34-67); median number of prior treatments was 11 (2-15) and median HSCT-CI was 3 (0-7).Of the CBUs infused, six were 5/6 and four were 4/6 HLA matched. The allele level compatibility and presence of shared IPA targets and/or NIMA matches are shown in the Table. CBU median TNC was 90x107 (range: 63-151) and they tended to be “older” with a median cryopreservation time of 10 years. CBUs provided a mean cell dose of 1.66 x 107 TNC/kg (range: 1.1-2.4). Mean post-thaw CD34+ and CD3+ cell doses were 52x103/kg (range: 20-90) and 0.34x107/kg (range: 0.24-0.52; SD: 0.08), respectively, with excellent post-thaw CD34+ viability (93.5%-98.3%).No CBU infusion reactions or cytokine release syndrome were observed. One pt, recipient of a prior allograft with no history of GVHD, experienced skin rash 1 week after CBU infusion. The skin biopsy was consistent with GVHD. No GVHD was observed in the other 9 pts. One pt died of disseminated adenovirus infection early after treatment; viremia was present at the initiation of chemotherapy.Transient CBU chimerism was detected in 5 of the 10 pts as early as one week and as late as 6 months after CBU infusion. Four pts with median pre-treatment BM blast counts of 56% (range 13-94) had objective hematologic responses (Cheson B JCO 2003) with median post-treatment blast counts of 0% (range 0-12), 3 with morphologic leukemia free state and one with a partial response. One patient had aplastic marrow but with flow cytometry evidence of persistent leukemia; while 4 pts recovered with morphologic persistent leukemia. Six pts went on to receive HSCT. Two subsequently relapsed and one died of disseminated adenovirus, while 3 pts remain in remission.ConclusionUse of CBUs as adoptive immunotherapy for pts with refractory myeloid leukemia represents a safe, well tolerated treatment, in which the potential for alloreactivity can be enhanced through the selection of CBUs by targeting shared IPA and NIMA present in the patients. Further, smaller CBUs, unlikely to be adequate grafts for adult transplantation, can be utilized for cell therapy. [Display omitted] DisclosuresRoboz:Cellectis: Research Funding; AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy. Ritchie:Celgene: Consultancy, Other: Travel, Speakers Bureau; Pfizer: Consultancy, Other: Research funding to my institution; Incyte: Consultancy, Speakers Bureau; Astellas Pharma: Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Bristol-Myers Squibb: Other: Research funding to my institution; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau. Lee:Amgen: Consultancy; Baxalta: Consultancy; Clinipace: Consultancy; Alexion Pharmaceuticals: Consultancy.

The Clinical Implication and Prognostic Impact of Dynamic Molecular Evolution in Acute Myeloid Leukemia

BackgroundAlthough the majority of acute myeloid leukemia (AML) patients may achieve complete remission (CR) after induction chemotherapy, about half of these patients relapse with a grave prognosis. Serial studies to assess the dynamic changes of molecular aberrations during follow-ups can help clarify the underlying mechanisms of leukemia progression. The advance and progress in the high throughput next generation sequencing provide us a good chance to comprehensively analyze gene mutations longitudinally during clinical follow-ups.MethodWe used TruSight myeloid panel (Illumina, USA), focusing on 54 genes related to myeloid neoplasms, to investigate the gene mutations of the bone marrow mononuclear cells from 154 adult de novo AML patients who obtained CR but relapsed and had adequate samples for paired samples analyses. The HiSeq platform (Illumina) was applied which had a median reading depth of 12000X. The variant calling was accomplished by CallSomaticVariants 3.6.2 (Illumina) or Genome Analysis Tool Kit (GATK) HaplotypeCaller. For detection of the large insertion of FLT3 and KMT2A, Pindel v0.2.5b8 was used specifically for chromosome 13 and 11, respectively, with default setting. Complete serial cytogenetic data were available in 92 (59.7%) patients.ResultsAt diagnosis, 98% patients had at least one genetic alteration including 44.7% patients had both cytogenetic abnormalities and molecular gene mutations, 48.0% patients had only gene mutations and 5.3% patients had only cytogenetic abnormalities. The most prevalent gene mutations at diagnosis were NRAS mutations (19.4%), followed by FLT3 -ITD (16.9%), NPM1 (16.9%), and DNMT3A (16.2%) mutations. The first relapse of these patients occurred at a median of 7.5 months (range, 1.2-64.4 months) after achieving CR and 75.3% of them had early relapses within one year from CR. At relapse, 77.3% patients had genetic evolution, including 12.0% with cytogenetic evolution, 37.0% with molecular gene evolution, and 28.3% with both cytogenetic and molecular gene evolution. Comparing the gene mutations between diagnosis and relapse, IDH1 (stability 100%), SRSF2 (100%), STAG2 (100%), SMC3 (100%), NPM1 (96%), ASXL1 (90%), and TP53 (85.7%) mutations remained stable during disease evolution; in contrast, some gene mutations were unstable, such as NRAS (28%), SF3B1 (50%), FLT3 (60%), and KRAS (60%) mutations. Sixty-seven (43.5%) of 154 patients had CR samples at 30±15 days after the first standard induction chemotherapy. Among them, 23 (34.3%) patients with persistent leukemia associated gene mutations (variant allele frequency at median 3.3%, range, 1.3-18.3%) at CR after first induction chemotherapy; these patients had a significant poorer overall survival (OS) than those without detectable gene mutations at CR (19.5±3.0 vs. 25.1±8.4 months, P=0.019). The 154 patients could be separated into two groups according to the patterns of clonal genetic evolution at disease evolution: Group 1 of patients had stable mutations (n=57, 37.0%) or gain of novel mutations (n=32, 20.8%) at relapse (type 1 of evolution); Group 2 had clonal sweeping, such as loss of previously harbored mutations (n=43, 27.9%) or mixed gain and loss of mutations (n=22, 14.3%), at disease progression (type 2). Type 2 evolution was positively associated with inv(16) (P=0.010), NRAS (P<0.001), and CEBPA mutations (P<0.001) but negatively associated with FLT3 -ITD (P=0.032) at diagnosis. In univariate analysis, type 1 evolution pattern predicted a shorter OS than type 2 (19.5 ±2.1 vs. 25.1±3.9 months, P= 0.034). In multivariate Cox proportional hazards regression analysis for OS, which included those prognostic factors with P < 0.1 in univariate analysis, type 1 was still an independent poor prognostic factor (relative risk 1.58, 95% confidence interval 1.03-2.44, P = 0.038).ConclusionThe majority of de novo AML patients had genetic evolutions at relapse, including cytogenetic changes, molecular genetic evolutions, or both. The persistence of leukemia associated gene mutations at CR was associated with a significant poorer OS. Genetic evolution patterns were heterogeneous in AML and type 2 clonal evolution was associated with distinct genetic alterations and better outcomes. DisclosuresChen:Celgene International Sàrl: Research Funding. Tsai:Celgene International Sàrl: Research Funding. Tang:Celgene International Sàrl: Research Funding. Kuo:Celgene International Sàrl: Research Funding. Lin:Celgene International Sàrl: Research Funding. Yao:Celgene International Sàrl: Research Funding. Li:Celgene International Sàrl: Research Funding. Huang:Celgene International Sàrl: Research Funding. Ko:Celgene International Sàrl: Research Funding. Hsu:Celgene International Sàrl: Research Funding. Lin:Celgene International Sàrl: Research Funding. Wu:Celgene International Sàrl: Research Funding. Chen:Celgene International Sàrl: Research Funding. Tsai:Celgene International Sàrl: Research Funding. Hou:Celgene International Sàrl: Research Funding. Tien:Celgene International Sàrl: Research Funding.

Mechanisms of GVL-Resistance: T Cell Exhaustion and a Failure of Antigen Cross-Presentation

In MHC-matched allogeneic hematopoietic stem cell transplantation (alloSCT), minor histocompatibility antigens (miHAs) are the major targets of alloreactive CD8+ T cells that mediate the GVL effect. Despite the development of alloreactive CD8 cells in most if not all patients, relapsed leukemia is the greatest single cause of treatment failure. Why relapse occurs has been elusive. Previously we tested adoptive immunotherapy with bona fide CD8+ memory T cells (TM) from donors vaccinated against a single miHA expressed by leukemia cells. Vaccination against the Kb-restricted miHA H60 (LTFNYRNL) greatly augmented TM-mediated GVL against mouse chronic phase and blast crisis chronic myeloid leukemia (BC-CML). In TMH60 recipients, despite a rapid expansion of H60-reactive T cells (measured by tetramer staining and IFN-g production) and an impressive GVL effect, we consistently observed persistent leukemia and later death in a subset of mice. We used this system to test hypotheses on GVL failure as we could track and enumerate H60-reactive CD8 cells, the only cells that mediate GVL in this system, by tetramer staining, which is not possible in traditional polyclonal GVL models. We hypothesized that GVL resistance could be due to immune selection of GVL-resistant leukemia cells and/or an insufficient T cell response. However, we found no evidence of immune selection. H60+ leukemia cells harvested from mice with relapsed BC-CML after an initially effective TMH60 GVL response and H60+ leukemia cells from transplanted mice that did not receive donor T cells were similarly sensitive to in vivo killing by both H60-reactive T cells and by OT-1 effector T cells when leukemia cells were pulsed with SIINFEKL. An insufficient T cell response could have been due to insufficient presentation of H60, as H60 is largely restricted to host hematopoietic cells, which are eliminated by the transplant. Indeed, H60 presentation was inefficient as fresh congenically marked TMH60, transferred 14 days post initial transplantation with TMH60, proliferated poorly in mice with or without leukemia. However, infusion of an agonist anti-CD40 antibody, also on day 14, promoted the proliferation and GVL activity of fresh TMH60 but not by the TeffH60+ (H60-reactive CD8 effectors) derived from TMH60 infused on day 0. The activity of anti-CD40 was mostly abrogated when donor BM was CD40-/-. Hence, cross-presentation by donor-derived cells is limiting, but T cells that initially mediated GVL were also poorly responsive. Relative to progeny from TMH60 infused on day 14, these cells had higher levels of PD-1, TIM-3, and TIGIT and a lower level of IFN-g production; this was even more marked in mice with residual leukemia. The increased exhaustion in mice with residual leukemia required the leukemia cells to present H60, as it did not occur when leukemias were H60+ but Kb-/-. Using the H60+Kb-/- BC-CML we found little evidence for cross-presentation of leukemia-derived H60 indicating that T cell responses against leukemia-restricted antigens is inefficient. Consistent with this, the anti-CD40 effect promoted cross-presentation of recipient and not leukemia-derived H60. In vivo treatment with anti-PD-1, but not anti-TIM-3 mAb, greatly augmented TMH60 mediated GVL without causing clinical GVHD as the TMH60 only target host hematopoietic cells. Day 0 treatment with anti-CD40 also augmented TMH60 mediated GVL without an increase in GVHD; but in this case the activity was on host cells. Taken together, our data support both exhaustion and a lack of antigen presentation later post BMT as factors that contribute to an insufficient T cell response, thus leading to GVL resistance. DisclosuresNo relevant conflicts of interest to declare.

Metabolic alterations mediated by STAT3 promotes drug persistence in CML

Leukemic stem cells (LSCs) can acquire non-mutational resistance following drug treatment leading to therapeutic failure and relapse. However, oncogene-independent mechanisms of drug persistence in LSCs are incompletely understood, which is the primary focus of this study. We integrated proteomics, transcriptomics, and metabolomics to determine the contribution of STAT3 in promoting metabolic changes in tyrosine kinase inhibitor (TKI) persistent chronic myeloid leukemia (CML) cells. Proteomic and transcriptional differences in TKI persistent CML cells revealed BCR-ABL-independent STAT3 activation in these cells. While knockout of STAT3 inhibited the CML cells from developing drug-persistence, inhibition of STAT3 using a small molecule inhibitor sensitized the persistent CML cells to TKI treatment. Interestingly, given the role of phosphorylated STAT3 as a transcription factor, it localized uniquely to genes regulating metabolic pathways in the TKI-persistent CML stem and progenitor cells. Subsequently, we observed that STAT3 dysregulated mitochondrial metabolism forcing the TKI-persistent CML cells to depend on glycolysis, unlike TKI-sensitive CML cells, which are more reliant on oxidative phosphorylation. Finally, targeting pyruvate kinase M2, a rate-limiting glycolytic enzyme, specifically eradicated the TKI-persistent CML cells. By exploring the role of STAT3 in altering metabolism, we provide critical insight into identifying potential therapeutic targets for eliminating TKI-persistent LSCs.

Flow cytometry reveals the nuances of clonal haematopoiesis

Flow cytometry reveals the nuances of clonal haematopoiesis

Higher Dose of CPX-351 Is Associated with Prolonged Hematologic Recovery: Results from an Interim Safety Analysis of the Randomized, Phase III AMLSG 30-18 Trial

Higher Dose of CPX-351 Is Associated with Prolonged Hematologic Recovery: Results from an Interim Safety Analysis of the Randomized, Phase III AMLSG 30-18 Trial

Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence.

Acute myeloid leukemia (AML) is a highly heterogeneous hematopoietic malignancy characterized by excessive proliferation and accumulation of immature myeloid blasts in the bone marrow. AML has a very poor 5-year survival rate of just 16% in the UK; hence, more efficacious, tolerable, and targeted therapy is required. Persistent leukemia stem cell (LSC) populations underlie patient relapse and development of resistance to therapy. Identification of critical oncogenic signaling pathways in AML LSC may provide new avenues for novel therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway, is often hyperactivated in AML, required to sustain the oncogenic potential of LSCs. Growing evidence suggests that targeting key components of this pathway may represent an effective treatment to kill AML LSCs. Despite this, accruing significant body of scientific knowledge, PI3K/Akt/mTOR inhibitors have not translated into clinical practice. In this article, we review the laboratory-based evidence of the critical role of PI3K/Akt/mTOR pathway in AML, and outcomes from current clinical studies using PI3K/Akt/mTOR inhibitors. Based on these results, we discuss the putative mechanisms of resistance to PI3K/Akt/mTOR inhibition, offering rationale for potential candidate combination therapies incorporating PI3K/Akt/mTOR inhibitors for precision medicine in AML.

Gpr44 Mediates the Selenium-Dependent Anti-Leukemic Effect in Acute Myeloid Leukemia

Abstract Objectives The relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia stem cells (LSCs) that are not targeted by existing therapies. LSCs show sensitivity to endogenous cyclopentenone prostaglandin J (CyPG) metabolites that are increased by dietary trace element selenium (Se), which is significantly decreased in AML patients. We investigated the anti-leukemic effect of Se supplementation in AML via mechanisms involving the activation of the membrane-bound G-protein coupled receptor 44 (Gpr44) and the intracellular receptor, peroxisome proliferator-activated receptor gamma (PPARγ), by endogenous CyPGs. Methods A murine model of AML generated by transplantation of hematopoietic stem cells (HSCs- WT or Gpr44−/−) expressing human MLL-AF9 fusion oncoprotein, in the following experiments: To investigate the effect of Se supplementation on the outcome of AML, donor mice were maintained on either Se-adequate (Se-A; 0.08–0.1 ppm Se) or Se-supplemented (Se-S; 0.4 ppm Se) diets. Complete cell counts in peripheral blood were analyzed by hemavet. LSCs in bone marrow and spleen were analyzed by flow cytometry. To determine the role of Gpr44 activation in AML, mice were treated with Gpr44 agonists, CyPGs. LSCs in bone marrow and spleen were analyzed. Mice transplanted with Gpr44−/- AML cells were compared with mice transplanted with wild type AML cells and the progression of the disease was followed as above. To determine the role of PPARγ activation in AML, PPARγ agonist (Rosiglitazone, 6 mg/kg, i.p, 14 d) and antagonist (GW9662, 1 mg/kg, i.p. once every other day, 7 injections) were applied to Se-S mice transplanted with Gpr44−/- AML cells and disease progression was followed. Results Se supplementation at supraphysiological levels alleviated the disease via the elimination of LSCs in a murine model of AML. CyPGs induced by Se supplementation mediate the apoptosis in LSCs via the activation of Gpr44 and PPARγ. Conclusions Endogenous CyPGs produced upon supplementation with Se at supraphysiological levels improved the outcome of AML by targeting LSCs to apoptosis via the activation of two receptors, Gpr44 and PPARg. Funding Sources NIH DK 07,7152; CA 175,576; CA 162,665. Office of Dietary Supplements, USDA Hatch funds PEN04605, Accession # 1,010,021 (KSP, RFP).

PDL1 Positivity By FISH in Patients Not in Complete Remission at Transplantation

Introduction Failure to achieve morphological complete remission occurs in 30% AML patients resulting in dismal outcomes. Data addressing influence of pre-transplant PDL-1 status on post-transplant outcomes is limited. We hypothesize that PDL-1 expression by immunohistochemistry (IHC) or Fluorescent in situ hybridization (FISH) prior to transplantation may predict a poor outcome. Methods This was a retrospective, observational single center pilot study involving refractory AML patients who underwent allogeneic stem cell transplantation (ASCT) at Karmanos Cancer Institute from 01/01/2012 to 01/01/2018. A planned sample size of 24 patients were selected and assigned to two cohorts based on their respective responses to ASCT: a) poor responders: persistent leukemia by bone marrow biopsy within 30 days of ASCT, and b) good responders: no evidence of leukemia at 1-year post-transplant. Descriptive analyses evaluated differences in demographic, disease and treatment variables. FISH assay was used to evaluate PDL-1 alterations in the pre-transplant archival, formalin-fixed, paraffin-embedded bone marrow biopsy specimens including frequency and magnitude of 9p24.1 alterations— low-level disomy, polysomy, copy gain, and amplification. The expression of PD-L1 was evaluated by IHC in a semi-quantitative (0 through +3) manner. Results Of the total 24 patients, 12 patients were poor responders and 12 were good responders; among poor responders, median age was 58 years (range, 31- 72) and gender distribution was 1:1. Seven patients (58%) had prior MDS, Myelofibrosis or CMML, while five patients had de-novo AML. Treatment information is provided in table 1. At pre-transplant bone marrow biopsy assessment, median number of bone marrow and peripheral blood blast counts were 8.1% (1.7-37) and 2% (0-37), respectively. Bone marrow cytogenetics assessed prior to transplant included intermediate risk in 2 (17%) and high risk disease in 9 (75%) patients and one patient without cytogenetic evaluation. PD-L1 expression was negative by IHC in all samples, but FISH testing was positive in 3 of 12 (25%) poor responders, characterized by copy gain. In good responders, median age was 59 years (range, 43-70) with a 3:2 male predominance. Four patients (25%) had prior MDS or Myelofibrosis and 8 had de-novo AML. Five patients (42%) had intermediate risk and 7 (58%) had high-risk cytogenetics. At pre-transplant bone marrow biopsy assessment, median number of bone marrow and peripheral blast counts were 6% (1-40) and 1% (0-7), respectively. PD-L1 expression was negative by both IHC and FISH examination in this cohort. Conclusion Our study demonstrates that PDL-1 expression by FISH was specifically detected in 25% of patients with poor responders following ASCT, which could possibly provide an insight into the poor outcome. A larger sample will be required to validate this observation in this population.

Cytological and Histopathological Correlation of Feline Giant Cell-Rich Osteosarcoma

Background: Primary bone tumours are uncommon and poorly reported in cats but osteosarcoma (OSA) is the most frequent, mostly in elderly animals. Giant cell-rich OSA is considered rare in the literature representing 3% of all OSA in humans. The mitotic index seems to have a significant effect on the survival time of cats affected by this neoplasm as well as the tumour histopathological grade. The objective of this study was to report the cytological and histopathological findings of a giant cell rich OSA in a 4-year old cat with persistent feline leukemia virus (FeLV) antigenaemia.Case: A 4-year-old male neutered cat was referred with a history of persistent FeLV viraemia and pelvic limb lameness with a firm swelling. Previous radiographs of the affected limb revealed bone lysis in the third and fourth metatarsals and increased soft tissue radiopacity in the tarsal region. The referral veterinary assumed it to be osteomyelitis and initiated clinical treatment with antibiotic and anti-inflammatory. The cat was referred after there was no response to medical treatment. The cat was presented with a 5cm diameter ulcerated mass, with putrid odor in the pelvic limb. Complementary exams were performed, and abnormalities were found, including increased urea, creatinine, calcium and potassium, and decreased sodium and phosphorus. A new radiograph showed exuberant bone proliferation, with increased radiopacity involving tarsal, metatarsal, distal third of tarsal I and II, and distal diaphysis of metatarsal V, without compromising the metaphyseal region of distal diaphysis of metatarsal IV. Chest radiographs and abdominal ultrasound were unremarkable. Fine-needle aspiration was performed for cytological analysis, which reavealed a moderate amount of pleomorphic mesenchymal cells with moderate adhesion, cytoplasm with a format ranging from fusiform to stellate, pronounced anisocytosis and cellular pleomorphism, and elevated nucleus:cytoplasm ratio. Nucleus was oval and presented loose chromatin, single to double large and evident nucleolus, frequent karyomegaly, along with marked anisocariosis and nuclear pleomorphism. Multinucleated giant cells were and there was a single mitotic figure in 12 high power fields (0.196 mm2 FN20/400x). Therefore, it was suggestive of malignant mesenchymal neoplasia with possibility of OSA, fibrosarcoma oe undifferentiated sarcoma. Limb amputation with femoral disarticulation was performed uneventfully. The material was conserved in 10% formalin, submitted to macroscopic and microscopic evaluation, which showed a large number of fusiform and stellate cells, with indistinct edges, scarce eosinophilic cytoplasm, high nucleus:cytoplasm ratio and oval nucleus, presenting moderate to marked anisocariosis, loose chromatin, with unique and evident nucleoli, besides of bone trabeculae. Nuclear pleomorphism was moderate and there was four mitotic figures in three random high-power fields (400x). It was observed areas of vascular ectasia, and neoplastic embolization in lymphatic and blood vessels. Among the cells, collagenous stroma was predominant but in some areas there was an eosinophilic amorphous material with the possibility of osteoid matrix or collagen. There was also a large numbers of multinucleated giant cells. The histopathological result was compatible with a grade III giant cell rich OSA.Discussion: Although bone tumours are uncommon in cats, OSA is the most frequent, affecting maingly middle-aged to elderly cats, with a mean age of 10 years, which is different from the present report in a 4-year old cat, with FeLV persistent viraemia. Retroviral status may have influenced the development of the disease at na early onset. FeLV induces uncontrolled cell proliferation through insertional mutagenesis (usually near myc) inducing malignant neoplasias, mainly lymphoma, but also multiple cartilaginous exostosis, which, along with osteomyelitis and bone cyst were included in this patient´s list of differential diagnoses. Despite the macroscopic and radiographical andagressiveness there was no metastasis identified through chest radiographs or abdominal ultrasound, and feline OSA is associated with a lower metastatic rate, if compared to canine OSA. Cytological analysis was compatible with malignant mesenchymal neoplasia, being suggestive of giant cell rich OSA. There is no cytological classification for OSA, however cytological findings of malignancy may be correlated with the patient's clinical course. Cytological features were in agreement with the histopathological findings, compatible with a grade III giant cell rich OSA. Therefore, it is concluded that the characteristics of malignancy presented by cytology were sufficient for the recommendation of amputation of the affected limb, once there was no imaging sign of metastases in the chest or abdomen.

Local Manufacture of CD19 CAR-T Cells Using an Automated Closed-System: Robust Manufacturing and High Clinical Efficacy with Low Toxicities

Introduction CD19 CAR-T cell products were recently approved as therapy for B-lineage malignancies. We initiated an IIT trial where manufacture of CAR-T cells was performed locally using a unique CD19 CAR with potent anti-leukemic effects. Patients and methods A total of 37 pts with relapsed/refractory B-acute lymphoblastic leukemia (12 female, 25 male, median age 10 y) were screened, 27 pts were enrolled for a trial, 10 were eligible for compassionate use of CD19 CAR-T cell therapy. Sixteen patients had relapsed B-ALL after haploidentical HSCT, 19 pts refractory relapse, 2 induction failure, 13 patients had previous blinatumomab infusion. Eighteen patients had &gt;20% blast cells, median bone marrow leukemia burden for patients with full blown disease was 89%, 19 pts had minimal residual disease (MRD) &gt;0.1% in BM, 3 had skeletal involvement with multiple mass lesions, one had CNS involvement. The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells. The automated production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct and transduced with lentiviral vector expressing the CD19CAR gene (second generation CD19.4-1BB zeta with alternate transmembrane domain derived from the TNF superfamily) (Lentigen, Miltenyi Biotec company). T cells were expansion over 10 days in the presence of serum-free TexMACS GMP Medium supplemented with MACS GMP IL-7 and IL-15. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab at 8mg/kg before CAR-T cell infusion. Patients did not receive HSCT as consolidation after CAR-T therapy. Results Thirty-five manufacturing cycles were successful. Median transduction efficacy was 60% (20-80). Median expansion of T cells was x 46 (18-51). CD4:CD8 ratio in the final product was 0.73. The cell products were administered at a dose of 3*106/kg of CAR-T cells in 4 pts, 1*106/kg in 9 pts, 0.5*106/kg in 14 pts, 0.1*106/kg in 8 pts. Two patients received 0.1*106/kg of CAR-T cells produced from haploidentical donors. The cytokine release syndrome (CRS) occurred in 22 (59%) pts and was mostly mild and moderate: grade I - 15 pts, grade II- 4 pts, grade III - 2 pt, grade IV - 1 pt. CAR-T cell related encephalopathy occurred in 15 (40%). Grade I-II neurotoxicity developed in 10 pts, grade III - in 2 pt, grade IV - 1 pt, grade V - 2 pt. In one patient with grade V neurotoxicity concomitant K. pneumonia encephalitis was documented. Severe (grade 3-5) CRS and neurotoxicity were associated exclusively with large leukemia burden (&gt;20% in the bone marrow) at enrollment, p=0,002. Thirty-one patient was evaluable for response at day 28. Four pts had persistent leukemia. In 27 (87%) cases Flow MRD-negative remission was achieved. Disease relapse after initial response was registered in 9 (33%) cases (7 patients had CD19 negative, 2 had CD19 positive relapse). At the moment of reporting, 10 patients have died (3 due to sepsis, 1 due to brain edema, 1 due to brain edema and K. pneumonia encephalitis, 5 due to progression of disease or relapse). Twenty-seven pts are alive, 19 in complete remission with a median follow up of 223 days (41-516 days). Conclusion CliniMACS Prodigy TCT process is a robust CAR-T cell manufacturing platform that enables rapid and flexible provision of CAR-T cells to patients in need. Significant toxicity of CD19 CAR-T cells was associated exclusively with high leukemia burden at enrollment. In the absence of HSCT consolidation relapse rate exceeds 30%. Disclosures Schneider: Lentigen Technology, A Miltenyi Biotec Company: Employment. Preussner:Miltenyi Biotec: Employment. Rauser:Miltenyi Biotec: Employment. Orentas:Lentigen Technology Inc., a Miltenyi Biotec Company: . Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Maschan:Miltenyi Biotec: Other: lecture fee.

Yttrium-90-labeled anti-CD45 antibody followed by a reduced-intensity hematopoietic cell transplantation for patients with relapsed/refractory leukemia or myelodysplasia.

Outcomes of patients with persistent high-risk leukemia or myelodysplasia prior to allogeneic hematopoietic cell transplantation are dismal. We therefore conducted a phase I trial evaluating the use of CD45-targeted radiotherapy preceding hematopoietic cell transplantation with the goal of improving outcomes for this high-risk scenario. Fifteen patients, median age 62 (range 37-76) years, were treated: ten with advanced acute myeloid leukemia, five with high-risk myelodysplastic syndrome. All patients had evidence of disease prior to treatment including nine with marrow blast counts ranging from 7-84% and six with minimal residual disease. Patients received escalating doses of yttrium-90-labeled anti-CD45 antibody followed by fludarabine and 2 Gy total body irradiation prior to human leukocyte antigen-matched, related or unrelated hematopoietic cell transplantation. Although a maximum dose of 30 Gy was delivered to the liver, no dose-limiting toxicity was observed. Therefore, the maximum-tolerated dose could not be estimated. Treatment led to complete remission in 13 patients (87%). All patients engrafted by day 28. Six patients relapsed, median of 59 (range 6-351) days, after transplantation. The 1-year estimate of relapse was 41%. Eight patients (53%) are surviving with median follow up of 1.8 (range 0.9-5.9) years. Estimated overall survival at one and two years was 66% and 46%, respectively, with progression-free survival estimated to be 46% at each time point. In conclusion, the combination of 90Y-DOTA-BC8 with an allogeneic hematopoietic cell transplantation regimen was feasible and tolerable. This approach appears promising in this high-risk leukemia/myelodysplasia patient population with active disease. (Trial registered at clinicaltrials.gov identifier: NCT01300572.)

How close are we to incorporating measurable residual disease into clinical practice for acute myeloid leukemia?

Assessment of measurable residual disease, also called “minimal residual disease,” in patients with acute myeloid leukemia in morphological remission provides powerful prognostic information and complements pretreatment factors such as cytogenetics and genomic alterations. Based on data that low levels of persistent or recurrent residual leukemia are consistently associated with an increased risk of relapse and worse long-term outcomes, its routine assessment has been recommended by some experts and consensus guidelines. In addition to providing important prognostic information, the detection of measurable residual disease may also theoretically help to determine the optimal post-remission strategy for an individual patient. However, the full therapeutic implications of measurable residual disease are uncertain and thus controversy exists as to whether it should be routinely incorporated into clinical practice. While some evidence supports the use of allogeneic stem cell transplantation or hypomethylating agents for some subgroups of patients in morphological remission but with detectable residual leukemia, the appropriate use of this information in making clinical decisions remains largely speculative at present. To resolve this pressing clinical issue, several ongoing studies are evaluating measurable residual disease-directed treatments in acute myeloid leukemia and may lead to new, effective strategies for patients in these circumstances. This review examines the common technologies used in clinical practice and in the research setting to detect residual leukemia, the major clinical studies establishing the prognostic impact of measurable residual disease in acute myeloid leukemia, and the potential ways, both now and in the future, that such testing may rationally guide therapeutic decision-making.

Characterization of Genomic Landscape and Risk Stratification of De Novo Cytogenetically Normal Acute Myeloid Leukaemia

Background. Acute myeloid leukaemia (AML) is a group of heterogeneous diseases with distinct clinicopathologic, cytogenetic and genetic features, sharing in common an abnormal increase in myeloblasts in blood or bone marrow (BM). Induction and consolidation chemotherapy as well as allogeneic haematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However, treatment outcome is unsatisfactory and only 30-40% patients achieve durable remission. Disease heterogeneity in AML may account for different treatment responses. In this study, we examined the mutation spectrum of cytogenetically normal AML (CN-AML) patients in Hong Kong, where AML treatment and indications for allogeneic HSCT are uniform, and evaluated their clinical outcome with respect to the specific gene mutations.Patients. Young adult patients (18-60 years old) with AML diagnosed between August 2003 to September 2017 in 8 regional hospitals in Hong Kong were included. Their clinicopathologic and cytogenetic features as well as treatment outcome were examined.Treatment and endpoints. Treatment comprised induction (daunorubicin 60-90 mg/m2 for 3 days; cytarabine 100 mg/m2 for 7 days) and consolidation (cytarabine 3 gram/m2 for 4-6 doses) for 3-4 courses. BM examination was performed on day 28 or until white cell and platelet counts recovered. Non-remission or relapsed cases were treated with salvage chemotherapy comprising combinations of cytarabine with idarubicin/etoposide, fludarabine, mitoxantrone or clofarabine. Complete remission (CR) was defined as circulating and BM blasts of ≤5% with neutrophil (≥1x109/L) and platelet count (≥100x109/L) recovery. CR with incomplete haematological recovery (CRi) was defined as circulating and BM blasts of ≤5% without complete neutrophil or platelet recovery.Mutation profiling. Next generation sequencing (NGS) for 36 recurrently mutated genes in AML was performed in diagnostic BM samples. Sequencing data were analyzed by in-house bioinformatics pipeline.Statistical evaluation. Patient survivals were calculated by Kaplan-Meier analysis and compared by log-rank test. P-values of <0.05 were considered statistically significant.Results. One hundred and seventy five patients were studied, with CR achieved in 150 cases after 1 (N=105, 70%), 2 (N=27, 18%) or ≥ 3 (N=18, 12%) courses of induction chemotherapy; and not achieved in 25 cases (primary refractory). Allogeneic HSCT was performed in 64 patients (at CR1,N=35; CR2, N=21; ≥ CR3, N=2; relapsed / persistent leukaemia, N=6). Relapse-free-survivals (RFS) at 1, 2 and 5 years were 41.3%, 27.3% and 9.3%; with median at 0.84 year. Overall survival (OS) at 1, 2 and 5 years were 69.1%, 38.3% and 15.4%, with median at 1.51 years. Mutations of ≥ 1 genes occurred in 169 cases (96%); involving with highest frequencies NPM1 (44%), FLT3-ITD (35.3%), DNMT3A (31%), IDH2 R140Q + R172K (14.3%) and CEBPA (bi-allelic, 13.7%). Individually these gene mutations had no impact on CR. However, FLT3-ITD + wildtype NPM1 and FLT3-ITD + mutated DNMT3A portended extremely poor RFS and OS. Bi-allelic CEBPA mutations portended significantly better OS but not RFS, suggesting that these cases were susceptible to relapse but were salvageable with re-induction and allogeneic HSCT. Co-existing mutations of NPM1, DNMT3A and FLT3 were common. Other mutations were mostly solitary, including bi-allelic CEBPA and IDH2 R172K mutations. NRAS and FLT3 mutations were mutually exclusive.Conclusion. CN-AMLs were intrinsically heterogeneous. Mutational profile might be useful for personalized treatment. The cooperativity and mutual exclusivity of gene mutations may be of pathogenetic significance and can inform patient-specific therapeutic strategies. DisclosuresKwong:Bayer: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Leung:Daiichi: Research Funding; Novartis: Speakers Bureau.

Inhibition of CD38 Shows Anti-Leukemic Activity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an early failure of healthy hematopoiesis and a high relapse rate, caused by persistent leukemia initiating cells in the bone marrow niche. The hematopoietic stem cell niche in myeloid malignancies shows severe structural and functional alterations with an inflammatory response upon leukemia infiltration and impaired immunosurveillance. CD38 is a glycoprotein that is expressed on various immunoregulatory cells and commonly on AML blasts. Targeting CD38 by using the monoclonal antibody daratumumab showed high efficacy in multiple myeloma which is mediated by both cell-autonomous as well as immunomodulatory mechanisms. In this study we have investigated the anti-leukemic efficacy of daratumumab as well as its underlying mechanisms in AML.Using an in vitro co-culture model with different human AML cell lines together with a fibroblast cell line (MS-5) or human umbilical vein endothelial cells (HUVEC) revealed a significant reduction in AML cell growth upon daratumumab treatment in 5 of 8 AML cell lines. Interestingly, anti-leukemic activity of daratumumab was more pronounced when co-cultured with MS-5 stroma cells and to lower extent with HUVECs as compared to mono-cultured AML cells (mean cell reduction in MS-5 co-culture, range from 12.9 to 31%; in HUVEC co-culture, range from 1.9 to 24.1% and in mono-culture, range from 9.9 to 18%). To validate the anti-leukemic activity of daratumumab in human AML and to elaborate the microenvironment-mediated effects we created a 3D invitro model with HUVECs and human mesenchymal spheres cultured with primary AML or normal donor cells. We could demonstrate that this triple-culture model led to an increased growth support for both AML as well as normal donor samples as compared to mono-cultured cells. Adding daratumumab to mono-cultured AML cells reduced cell growth by only 7.2% (p<0.01), whereas the anti-leukemic efficacy increased to 35.6% in triple-cultured AML samples (p<0.05; n=10 replicates). Of note, daratumumab did not show any significant cytotoxicity in normal donor samples at any culture condition. The anti-leukemic efficacy of daratumumab in AML appeared to depend on the level of CD38 expression. Addition of tretinoin (ATRA) induced a ~4-fold increase of CD38 expression in AML blasts and to a lower extent in normal donor cells. In fact, combination of tretinoin and daratumumab led to an enhanced anti-leukemic activity in AML. As daratumumab has been shown to exert diverse effects in myeloma we next assessed the underlying mechanisms in AML. Upon daratumumab treatment there were no significant differences in blast proliferation and apoptosis as assessed by BrdU and Annexin V assays, nor did we observe any induction of differentiation. Given the strong immunological reactions induced by daratumumab we next performed effector function assays. While there was no increase in antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), we observed a significant induction of antibody dependent phagocytosis (ADCP) with daratumumab compared to IgG1 control. Human macrophages showed a ~1.7-fold increase in phagocytosis of AML cell lines and primary human AML cells after daratumumab treatment.To prove the anti-leukemic activity of daratumumab in AML in vivo we intravenously transplanted primary human AML cells into immunodeficient NOD scid gamma (NSG) mice. Leukemic mice were treated with ATRA over 4 weeks and once weekly with either daratumumab or IgG1 control. Preliminary results show a reduction in circulating blasts with a significant decrease of leukemia burden in peripheral blood by ~42% (p=0.0354), while there were no significant changes in bone marrow infiltration (n=5-6 mice per group). Ongoing experiments will further elucidate the anti-leukemic efficacy of daratumumab in combination with low-dose cytarabine.In summary, we found that targeting CD38 shows promising anti-leukemic activity with both cell-autonomous and microenvironment-mediated effects in AML. Together with tretinoin und putatively low-dose chemotherapy the efficacy of daratumumab in AML could be increased. This combination of well tolerated drugs could present a new option of niche-targeted therapies in AML. DisclosuresDuehrsen:Janssen: Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Research Funding.

Up-regulation of regulatory T cells, CD200 and TIM3 expression in cytogenetically normal acute myeloid leukemia.

The bone marrow immunosuppressive microenvironment of AML patients sustains and modulates proliferation, survival and drug resistance of AML through deregulation of both innate and adaptive immune response. We aimed to investigate the level of Tregs, expression of Tim-3 on peripheral blood T cells, expression of CD200 in myeloid blasts in newly diagnosed AML patients with normal cytogenetics (AML-NC) and their prognostic impact. This study included 40patients with de novo AML-NC and 20healthy controls. Flow-cytometry was used for detection of CD4+CD25+high FoxP3+ regulatory T cells, Tim-3 expression on peripheral blood T cells and CD200 expression on myeloid blasts. The percentages of CD4+CD25+high and CD4+CD25+high Foxp3+ Tregs were significantly increased in AML patients than controls. The levels of Tregs, Tim-3/CD4+, Tim-3/CD8+, CD200 and MFI of CD200 were significantly lower in responding patients than in those with persistent leukemia. Only high CD200 expression (> 50%) showed statistically significant worse OS with P< 0.04. The increased levels of Tregs, Tim-3 expression on peripheral blood T cells and CD200 expression in myeloid blast in AML patients could play a role in the development of AML. Analysis of these markers could serve as prognostic markers and might guide the therapy in AML patients in the future.

Comparison of anthracyclines used for induction chemotherapy in patients with FLT3-ITD-mutated acute myeloid leukemia

This retrospective analysis compared anthracyclines (as part of an induction regimen) in 128 newly diagnosed FLT3-ITD-mutated AML patients. Induction regimens comprised high-dose daunorubicin (HD-DN; 90 mg/m2/d × 3d; n = 44), standard-dose daunorubicin (SD-DN; 45 mg/m2/d × 3d; n = 51), or idarubicin (IDA; 12 mg/m2/d × 3d; n = 33) in combination with cytarabine (100–200 mg/m2/d × 7d). Fifty-three patients showing persistent leukemia on interim bone marrow examination received a second course of induction chemotherapy comprising 2 days of daunorubicin (45 mg/m2/d) or IDA (8 or 12 mg/m2/d) in addition to 5 days of cytarabine. Complete remission (CR) rates were 77.3%, 56.9%, and 69.7% for HD-DN, SD-DN, and IDA, respectively (P = 0.101; HD-DN vs. SD-DN, P = 0.036; HD-DN vs. IDA, P = 0.453; IDA vs. SD-DN, P = 0.237). The HD-DN showed higher overall survival (OS) and event-free survival (EFS) than SD-DN and IDA: the differences between HD-DN and SD-DN (P = 0.009 for OS and P = 0.010 for EFS) were statistically significant.Results of in vitro studies using FLT3-ITD-mutated cell lines supported these findings. In conclusion, HD-DN improved the CR rate, OS, and EFS of FLT3-ITD-mutated AML patients. HD-DN also tended to yield better outcomes than IDA, though the difference was not significant. The superiority of HD-DN over IDA should be confirmed in future studies.

Mechanisms of NT5C2 mutations Driving Thiopurine Resistance in Acute Lymphoblastic Leukemia

▪Relapse acute lymphoblastic leukemia (ALL) is associated with chemotherapy resistance and poor prognosis. Persistent leukemia initiating cells with increased self-renewal capacity, clonal heterogeneity and selection of resistance-driving genetic alterations have been proposed as drivers of leukemia relapse. Gain of function mutations in the 5'-Nucleotidase, Cytosolic II (NT5C2) gene are selectively present in relapsed ALL and confer resistance to chemotherapy with 6-mercaptopurine (6-MP). Yet, the specific mechanisms mediating constitutive activation of NT5C2 remain unknown. Here we performed detailed genetic, enzymatic, structural and functional analyses of relapsed leukemia-associated NT5C2 mutant alleles uncovering previously unrecognized functional heterogeneity in their mechanisms of action. Specifically, detailed nucleotidase assays in the presence of increased concentration of allosteric activators across 10 relapse-associated NT5C2 mutant proteins and analyses of the crystal structures of 7 distinct mutant proteins and wild type NT5C2 in the presence and absence of allosteric factors defined three classes of NT5C2 mutants with distinct mechanisms of action. Class I NT5C2 mutations force an active configuration of the NT5C2 catalytic center in the absence of allosteric activator. In contrast, class II NT5C2 mutations disrupt a novel switch-off mechanism mediated by the dynamic mobilization of the tip region of the arm segment responsible for returning the enzyme to its basal inactive configuration following allosteric activation. Finally, increased allosteric response in the class III p.Gln523* truncating mutation results from loss of the C-terminal acidic tail of NT5C2, which functions as a lock stabilizing the enzyme in its inactive basal configuration. The proposed role of the arm domain region and the C-terminal segment were verified in enzymatic assays incubating wild type NT5C2 with antibodies recognizing these regulatory elements, which phenocopied the increased enzymatic activity and response to allosteric effector of NT5C2 mutations involving these domains. In all, these results identify distinct molecular mechanisms mediating NT5C2 regulation targeted by activating NT5C2 mutant alleles and serve as a framework for the functional annotation of clinically-relevant NT5C2 mutations in relapsed ALL. Finally, the prominent role of the allosteric binding site as critical mediator of the mechanisms mediating thhe increased nucleotidase activity of NT5C2 mutant proteins point to this region as a preferred target for the design of NT5C2 inhibitors for the reversal of chemotherapy resistance in relapsed ALL.Figure 1 Relapse associated NT5C2 mutations and crystal structure of basal and activated wild type human NT5C2. (a) Graphical representation of relapse associated mutations in B-ALL (black) and T-ALL (red).(b-d) In vitro nucleotidase assays assessing the enzymatic activity of (b)class I, (c) class II, and (d)class IIINT5C2 mutations in the presence of increased concentrations of ATP. (e) A Ribbon diagram of the basal structure of wild type NT5C2-X537, displaying the HAD domain (cyan) and its three extensions: N-terminal segment (orange), arm (pink), and C-terminal segment (marine). The mutation sites are depicted as red solid spheres for Cα of each mutated amino acid. The phosphate (yellow for phosphorus) and Mg ions (dark green) are depicted as stick models and solid spheres respectively. (f) A Ribbon diagram of the activated structure of the full-length wild type NT5C2, in which the allosteric helix A (αA) is shown in dark purple. (g,h) Ribbon and surface (for subunit B) depictions of basal and activated dimers of (g) NT5C2-X537 and the (h) full-length, respectively. (i,j) Ribbon diagrams of the basal (i) NT5C2-X537 and activated (j) full-length wild type tetramers, respectively. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Achieving Minimal Residual Disease Negative Status in CLL - a Good Point to Start Thinking about Life without Treatment: How to Check MRD Status?

Patients diagnosed with chronic lymphocytic leukemia (CLL) do not necessarily have to undergo intensive treatment. The implementation of the therapy depends on patient condition and the form of CLL which is determined using predictive and prognostic factors. Both indolent and aggressive forms of CLL should be monitored. To improve survival and the quality of life it is very important to know how the organism responds to the treatment. For this purpose, the estimation of the minimal residual disease (MRD) - the pool of persistent leukemia cells after or at the time of treatment was introduced. These cells resistant to treatment can lead to recurrence of the disease. There are several methods for MRD assessment, however in every case it is necessary to know the initial status of the tested alterations (chromosome aberrations, surface protein expression, etc.). It allows for assessment whether the treatment eliminated the leukemic cells. The most common method for MRD evaluation in CLL is flow cytometry. Intensive development of molecular biology techniques can contribute to implementation of other extremely sensitive and specific methods. This review presents the most recent state of the art concerning minimal residual disease in CLL, including methods that are used for MRD assessment.

Comparison of Treatment Strategies in Patients over 60 Years with AML: Final Analysis of a Prospective Randomized German AML Intergroup Study

Comparison of Treatment Strategies in Patients over 60 Years with AML: Final Analysis of a Prospective Randomized German AML Intergroup Study