SHIP1, but not an AML-derived SHIP1 mutant, suppresses myeloid leukemia growth in a xenotransplantation mouse model.

Constitutive activation of the PI3K/AKT signaling pathway is found in ~50-70% of AML patients. The SH2-containing inositol 5-phosphatase 1 (SHIP1) is a negative regulator of PI3K/AKT signaling in hematopoietic cells. SHIP1 knockout mice develop a myeloproliferative syndrome and concomitant deletion of SHIP1 and the tumor suppressor PTEN leads to the development of lethal B-cell lymphomas. In the study presented here, we investigated the role of SHIP1 as a tumor suppressor in myeloid leukemia cells in an in vivo xenograft transplantation model. NSG Mice transplanted with UKE-1 cells derived from a secondary AML showed a significantly extended lifespan after lentiviral-mediated overexpression of SHIP1 in comparison to the vector control cohort. In contrast, the AML-derived SHIP1Y643H mutant, which has a strongly reduced enzymatic activity showed a significant reversion of the SHIP1-induced prolongation of the survival time. In addition, the analysis of 290 AML patients revealed a correlation between expression of SHIP1 and overall survival of the AML patients. These results indicate that SHIP1 can act as a tumor suppressor in acute myeloid leukemia cells and that higher SHIP1 expression is associated with prolonged overall survival in AML patients. SHIP1 may be an interesting candidate for gene therapy.

Epidemiological, genetic, and clinical characterization by age of newly diagnosed acute myeloid leukemia based on an academic population-based registry study (AMLSG BiO).

We describe genetic and clinical characteristics of acute myeloid leukemia (AML) patients according to age from an academic population-based registry. Adult patients with newly diagnosed AML at 63 centers in Germany and Austria were followed within the AMLSG BiO registry (NCT01252485). Between January 1, 2012, and December 31, 2014, data of 3525 patients with AML (45% women) were collected. The median age was 65 years (range 18-94). The comparison of age-specific AML incidence rates with epidemiological cancer registries revealed excellent coverage in patients < 70 years old and good coverage up to the age of 80. The distribution according to the European LeukemiaNet (ELN) risk categorization from 2010 was 20% favorable, 31% intermediate-1, 28% intermediate-2, and 21% adverse. With increasing age, the relative but not the absolute prevalence of patients with ELN favorable and intermediate-1 risk (p < 0.001), with activating FLT3 mutations (p < 0.001), with ECOG performance status < 2 (p < 0.001), and with HCT-CI comorbidity index < 3 (p < 0.001) decreased. Regarding treatment, obesity and favorable risk were associated with an intensive treatment, whereas adverse risk, higher age, and comorbidity index > 0 were associated with non-intensive treatment or best supportive care. The AMLSG BiO registry provides reliable population-based distributions of genetic, clinical, and treatment characteristics according to age.

Safety and preliminary activity results of the GATTO study, a phase Ib study combining the anti-TA-MUC1 antibody gatipotuzumab with the anti-EGFR tomuzotuximab in patients with refractory solid tumors.

BACKGROUND: The phase I GATTO study (NCT03360734) explored the feasibility, tolerability and preliminary activity of combining gatipotuzumab, a novel humanized monoclonal antibody binding to the tumor-associated epitope of mucin 1 (TA-MUC1) and an anti-epidermal growth factor receptor (anti-EGFR) antibody in refractory solid tumors. PATIENTS AND METHODS: Initially the study enrolled primary phase (PP) patients with EGFR-positive metastatic solid tumors, for whom no standard treatment was available. Patients received gatipotuzumab administered at 1400 mg every 2 weeks, 6 weeks after the start of the glyco-optimized anti-EGFR antibody tomuzotuximab at 1200 mg every 2 weeks. As this regimen was proven safe, enrollment continued in an expansion phase (EP) of patients with refractory metastatic colorectal cancer, non-small-cell lung cancer, head and neck cancer and breast cancer. Tomuzotuximab and gatipotuzumab were given at the same doses and gatipotuzumab treatment started 1 week after the first dose of the anti-EGFR antibody. Additionally, investigators could use a commercial anti-EGFR antibody in place of tomuzotuximab. RESULTS: A total of 52 patients were enrolled, 20 in the PP and 32 in the EP. The combined treatment was well tolerated and no dose-limiting toxicity was observed in the whole study, nor related serious adverse event or death. Preliminary activity of the combination was observed, with one and four RECIST partial responses in the PP and EP, all in colorectal cancer patients. The trial was accompanied by a comprehensive translational research program for identification of biomarkers, including soluble TA-MUC1 (sTA-MUC1) in serum. In the EP, patients with baseline sTA-MUC1 levels above the median appeared to have improved progression-free survival and overall survival. CONCLUSIONS: Combination of a TA-MUC1-targeting antibody and an EGFR-targeting antibody is safe and feasible. Interesting antitumor activity was observed in heavily pretreated patients. Future studies should test this combination together with chemotherapy and explore the potential of sTA-MUC1 as a companion biomarker for further development of the combination.

Phase I trial of SU14813 in patients with advanced solid malignancies.

BACKGROUND: this phase I, open-label, dose-escalation study investigated SU14813, an oral multitargeted tyrosine kinase inhibitor, in adults with solid tumors. PATIENTS AND METHODS: seventy-seven patients received once-daily SU14813, either for 4 weeks followed by 1 week off treatment (schedule 4/1) or continuously [continuous daily dosing (CDD)]. The primary end point was to determine the maximum tolerated dose (MTD). Safety, pharmacokinetics, pharmacodynamics, and efficacy were assessed. RESULTS: MTDs were 200 mg/day on schedule 4/1 and 100 mg/day with CDD. Adverse events included fatigue (64%), diarrhea (61%), nausea (44%), anorexia (43%), and vomiting (42%). SU14813 steady state was attained by day 8. Exposure increased in a generally dose-proportional manner and SU14813 was eliminated with a mean terminal half-life of 9-34 h. Target plasma concentrations (>100 ng/ml SU14813) were achieved and sustained over 12 h at ≥ 100 mg/day. Progression-free survival among the 1 complete responder and 12 partial responders was 1.4-53.2 months. Fifteen patients remained on treatment at 1 year and 3 patients at 2 years. CONCLUSION: SU14813 has manageable safety and tolerability and allows once-daily continuous oral dosing. SU14813 shows dose-proportional pharmacokinetics, with target plasma concentrations achieved at doses ≥ 100 mg/day. Clinically meaningful activity with durable responses was observed, meriting further study.

Increased frequency of TIGIT+CD73-CD8+ T cells with a TOX+ TCF-1low profile in patients with newly diagnosed and relapsed AML.

The inhibitory receptor TIGIT, as well as theectonucleotidases CD39 and CD73 constitute potential exhaustion markers for T cells. Detailed analysis of these markers can shed light into dysregulation of the T-cell response in acute myeloid leukemia (AML) and will help to identify potential therapeutic targets.  The phenotype and expression of transcription factors was assessed on different T-cell populations derived from peripheral blood (PB, n = 38) and bone marrow (BM, n = 43). PB and BM from patients with AML diagnosis, in remission and at relapse were compared with PB from healthy volunteers (HD) (n = 12) using multiparameter flow cytometry. An increased frequency of terminally differentiated (CD45R-CCR7-)CD8+ T cells was detected in PB and BM regardless of the disease state. Moreover, we detected an increased frequency of two distinct T-cell populations characterized by the co-expression of PD-1 or CD39 on TIGIT+CD73-CD8+ T cells in newly diagnosed and relapsed AML in comparison to HDs. In contrast to the PD-1+TIGIT+CD73-CD8+ T-cell population, the frequency of CD39+TIGIT+CD73-CD8+ T cells was normalized in remission. PD-1+- and CD39+TIGIT+CD73-CD8+ T cells exhibited additional features of exhaustion by decreased expression of CD127 and TCF-1 and increased intracellular expression of the transcription factor TOX. CD8+ T cells in AML exhibit a key signature of two subpopulations, PD-1+TOX+TIGIT+CD73-CD8+- and CD39+TOX+TIGIT+CD73-CD8+ T cells that were increased at different stages of the disease. These results provide a rationale to analyze TIGIT blockade in combination with inhibition of the purinergic signaling and depletion of TOX to improve T-cell mediated cytotoxicity in AML. Abbreviations: AML: Acute myeloid leukemia; pAML: newly diagnosed AML; rAML: relapse AML; lrAML: AML in remission; HD: healthy donor; PB: peripheral blood; BM: bone marrow; TIGIT: T-cell immunoreceptor with Ig and ITIM domains; PD-1: Programmed cell death protein 1; CD73: ecto-5'-nucleotidase; CD39: ectonucleoside triphosphate diphosphohydrolase 1; ATP: adenosine triphosphate; ADO: adenosine; CD127: interleukin-7 receptor; CAR-T cell: chimeric antigen receptor T cell; TCF-1: transcription factor T-cell factor 1; TOX: Thymocyte selection-associated high mobility group box protein; NFAT: nuclear factor of activated T cells; NA: Naïve; CM: Central Memory; EM Effector Memory; EMRA: Terminal Effector Memory cells; FMO: Fluorescence minus one; PVR: poliovirus receptor; PVRL2: poliovirus receptor-related 2; IFN-γ: Interferon-γ; IL-2: interleukin-2; MCF: multiparametric flow cytometry; TNFα: Tumornekrosefaktor α; RT: room temperature.

[28-year old female patient with respiratory insufficiency, elevated liver enzymes, pancytopenia and fever]. / 28-jährige Patientin mit respiratorischer Insuffizienz, erhöhten Leberwerten, Fieber und Panzytopenie.

We report on a 28-year old female patient with fever and severe respiratory insufficiency requiring mechanical ventilation. Cytomegalovirus pneumonia was diagnosed by bronchoalveolar lavage, and antiviral therapy was initiated. However fever persisted and laboratory workup showed pancytopenia and elevated liver enzymes. Additional blood tests demonstrated a markedly elevated ferritin level and high concentrations of inflammatory cytokines. A diagnosis of hemophagocytic lymphohistiocytosis was made and immunosuppressive therapy was started. The patient's condition and laboratory values improved rapidly.

Correction: Role of growth arrest-specific gene 6-mer axis in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reduced proliferation of CD34(+) cells from patients with acute myeloid leukemia after gene transfer of INPP5D.

Acute myeloid leukemia (AML) is a malignant disease characterized by deregulated proliferation of immature myeloid cells. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently detected in approximately 50-70% of AML patients. The gene INPP5D encodes the SH2-containing inositol 5-phosphatase 1 (SHIP1), which is a negative regulator of PI3K/AKT signaling. After lentiviral-mediated gene transfer of INPP5D into CD34(+) cells derived from AML patients (n=12) the granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent proliferation was reduced in all samples analyzed (average 86%; range 72-93%). An enzymatically inactive form of SHIP1 (D672A) had no effect. In addition, SHIP1 reduced the autonomous proliferation of CD34(+) cells from a patient with a secondary AML who had a very high peripheral blast count (300 x 10(9) l(-1)). These data show that SHIP1 can effectively block GM-CSF-dependent and autonomous proliferation of AML cells.

DNMT3A mutant transcript levels persist in remission and do not predict outcome in patients with acute myeloid leukemia.

We investigated the prognostic impact of minimal residual disease (MRD) monitoring in acute myeloid leukemia patients harboring DNA methyltransferase 3A-R882H/-R882C mutations (DNMT3Amut). MRD was determined by real-time quantitative PCR (RQ-PCR) in 1494 samples of 181 DNMT3Amut patients. At the time of diagnosis, DNMT3Amut transcript levels did not correlate with presenting clinical characteristics and concurrent gene mutations as well as the survival end points. In Cox regression analyses, bone marrow (BM) DNMT3Amut transcript levels (log10-transformed continuous variable) were not associated with the rate of relapse or death. DNMT3Amut transcript levels were significantly higher in BM than in blood after induction I (P=0.01), induction II (P=0.05), consolidation I (P=0.004) and consolidation II (P=0.008). With regard to the clinically relevant MRD time points, after two cycles of induction and at the end of therapy, DNMT3Amut transcript levels had no impact on the end point remission duration and overall survival. Of note, only a minority of the patients achieved RQ-PCR negativity, whereas most had constantly high DNMT3Amut transcript levels, a finding which is consistent with the persistence of clonal hematopoiesis in hematological remission.

Acute myeloid leukemia derived from lympho-myeloid clonal hematopoiesis.

We studied acute myeloid leukemia (AML) patients with lympho-myeloid clonal hematopoiesis (LM-CH), defined by the presence of DNA methyltransferase 3A (DNMT3A) mutations in both the myeloid and lymphoid T-cell compartment. Diagnostic, complete remission (CR) and relapse samples were sequenced for 34 leukemia-related genes in 171 DNMT3A mutated adult AML patients. AML with LM-CH was found in 40 patients (23%) and was associated with clonal hematopoiesis of indeterminate potential years before AML, older age, secondary AML and more frequent MDS-type co-mutations (TET2, RUNX1 and EZH2). In 82% of AML patients with LM-CH, the preleukemic clone was refractory to chemotherapy and was the founding clone for relapse. Both LM-CH and non-LM-CH MRD-positive AML patients who achieved CR had a high risk of relapse after 10 years (75% and 75%, respectively) compared with patients without clonal hematopoiesis in CR with negative MRD (27% relapse rate). Long-term survival of patients with LM-CH was only seen after allogeneic hematopoietic stem cell transplantation (HSCT). We define AML patients with LM-CH as a distinct high-risk group of AML patients that can be identified at diagnosis through mutation analysis in T cells and should be considered for HSCT.

Condensed versus standard schedule of high-dose cytarabine consolidation therapy with pegfilgrastim growth factor support in acute myeloid leukemia.

The aim of this cohort study was to compare a condensed schedule of consolidation therapy with high-dose cytarabine on days 1, 2 and 3 (HDAC-123) with the HDAC schedule given on days 1, 3 and 5 (HDAC-135) as well as to evaluate the prophylactic use of pegfilgrastim after chemotherapy in younger patients with acute myeloid leukemia in first complete remission. One hundred and seventy-six patients were treated with HDAC-135 and 392 patients with HDAC-123 with prophylactic pegfilgrastim at days 10 and 8, respectively, in the AMLSG 07-04 and the German AML Intergroup protocol. Time from start to chemotherapy until hematologic recovery with white blood cells >1.0 G/l and neutrophils >0.5 G/l was in median 4 days shorter in patients receiving HDAC-123 compared with HDAC-135 (P<0.0001, each), and further reduced by 2 days (P<0.0001) by pegfilgrastim. Rates of infections were reduced by HDAC-123 (P<0.0001) and pegfilgrastim (P=0.002). Days in hospital and platelet transfusions were significantly reduced by HDAC-123 compared with HDAC-135. Survival was neither affected by HDAC-123 versus HDAC-135 nor by pegfilgrastim. In conclusion, consolidation therapy with HDAC-123 leads to faster hematologic recovery and less infections, platelet transfusions as well as days in hospital without affecting survival.

A study from the EORTC new drug development group: open label phase II study of sabarubicin (MEN-10755) in patients with progressive hormone refractory prostate cancer.

Sabarubicin (MEN-10755), a new synthetic anthracycline analogue, was evaluated for safety and efficacy in a multicentre phase II study in patients with advanced hormone refractory prostate cancer (HRPC). Thirty seven patients were included, of which 34 were evaluable for PSA response according to Bubley's criteria. Sabarubicin was administered as a short (30 min) intravenous infusion at a dose of 80 mg/m(2) every 3 weeks. The main toxicity consisted of grade 3/4 neutropenia in 24 patients (64.9%), with grade 3/4 febrile neutropenia occurring in one patient only. Grade 3/4 cardiotoxicity was observed in 4 patients including one ineligible. Other toxicities were mild. Nine patients achieved a PSA response (26.5%), 10 patients had stable disease (29.4%) and 14 patients disease progression (41.2%). One patient (2.9%) had a PSA response that was not confirmed by repeat PSA testing. The objective response rate according to RECIST criteria was 6.7% in 15 patients with measurable disease. The median duration of PSA responses was relatively long 7.1 months (95% CI 4.9-20.7) as was the median time to treatment progression in patients with stable disease. The median overall survival was 18.7 months (95% CI 9.1-N), comparable to results recently observed in taxotere-containing regimens. To confirm and extend these results, further testing of sabarubicin in larger trials is warranted.

Antiangiogenic treatment with endostatin inhibits progression of AML in vivo.

Increased vessel density in the bone marrow of patients with acute myeloid leukemia as well as elevated expression of proangiogenic factors by leukemic cells implies a central role of angiogenesis in hematological malignancies. Endostatin (ES), a fragment of collagen XVIII, is an endogenous inhibitor of angiogenesis that has shown therapeutic activity in solid tumors in various preclinical models. Using microencapsulation technology, we studied the therapeutic effect of ES in AML. While ES had no effect on proliferation of M1 murine leukemic cells in vitro, ES producing microbeads significantly inhibited growth of subcutaneous chloromas in SCID mice as compared to controls. In a leukemia model using M1 cells the concomitant treatment of mice with ES microbeads prolonged median survival significantly. Histological analysis revealed a decreased microvessel density and a reduced number of CD31-positive single cells, putatively endothelial progenitor cells, in the bone marrow of treated animals. Taken together, ES has inhibitory effects on neo-angiogenesis in the bone marrow and on progression of leukemia in vivo. These experiments suggest a possible therapeutic role of antiangiogenic gene therapy with ES in AML.

A phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas.

BACKGROUND: A phase I open-label dose-escalation study was conducted to define the safety, tolerability, and pharmacokinetics (PK) of PankoMab-GEX, a glyco-optimised humanised IgG1, with high affinity to a novel tumour-specific glycopeptide epitope of MUC1 (TA-MUC1) with excellent preclinical anti-tumour activity. PATIENTS AND METHODS: Seventy-four patients with advanced TA-MUC1-positive carcinomas received PankoMab-GEX intravenously every 3 (Q3W), 2 (Q2W), or 1 (QW) week in doses of 1-2200 mg in a three-plus-three dose-escalation design until disease progression (NCT01222624). RESULTS: No maximum tolerated dose was reached. Adverse events were mainly mild-to-moderate infusion-related reactions (IRRs) by the first infusion in 45% of patients. Only one dose-limiting toxicity, a grade III IRR, was observed. PankoMab-GEX exhibited linear PK over all doses. Mean terminal half-life was 189 ± 66 h (Q3W), without dose dependency. A target trough level ≥50 µg/mL was reached after one infusion with doses ≥1700 mg Q3W in 80% of patients. Clinical benefit in 60 evaluable patients included one complete response in a patient with ovarian cancer treated 483 d and confirmed disease stabilisation in 19 patients lasting a median (range) of 23 (10-109) weeks. All but two of the patients with clinical benefit had received a compounded total dose ≥700 mg over a 3-week period, including 8 of 12 (67%) patients with ovarian cancer. CONCLUSION: PankoMab-GEX is safe, well tolerated, and showed promising anti-tumour activity in advanced disease. A phase IIb study is ongoing evaluating the efficacy of PankoMab-GEX as a maintenance therapy in advanced ovarian cancer.

Multicentre EORTC study 16997: feasibility and phase II trial of farnesyl transferase inhibitor & gemcitabine combination in salvage treatment of advanced urothelial tract cancers.

In this study, the feasibility and activity of combined chemotherapy of the farnesyl transferase inhibitor SCH66336 and gemcitabine was evaluated. This therapy was used as second-line treatment in patients with advanced urothelial tract cancer and the influence of SCH66336 exposure on the pharmacokinetics of gemcitabine was also determined. Patients who had received one previous chemotherapy regime for advanced urothelial cancer were treated with a combination of SCH66336 (150 mg in the morning and 100 mg in the evening) and Gemcitabine (1000 mg/m2 on day 1, 8 and 15 per 28-day cycle). Dosages of gemcitabine and its metabolite dFdU were performed on day one of cycle 1 before exposure to SCH66336 and day one of cycle 2. A total of 152 cycles were administered in 33 patients (median 3, range: 1-15). No patients had severe hematological toxicity, defined as Grade 4 thrombocytopenia or febrile neutropenia. Nine partial responses and one complete response were achieved in 31 assessable patients and corresponded to an overall response rate of 32.3% [95% CI:17%-51%]. There was no influence of exposure to SCH66336 on the level of gemcitabine or dFdU in 11 assessable patients. In conclusion, a combination of SCH66336 and gemcitabine is feasible in terms of toxicity and active as second-line treatment in patients with advanced urothelial tract cancer. SCH66336 had no effect on the pharmacokinetics of gemcitabine. Randomised trials should be undertaken to clarify the role of SCH66336 in combination with gemcitabine in cancer treatment.

Dedifferentiated chondrosarcoma--a fatal disease.

BACKGROUND: We report on 13 patients with dedifferentiated chondrosarcomas. The mean age of the patients at diagnosis was 59.8 years. Nine patients were classified as stage IIB and four as stage III. METHODS: In 11/13 cases surgery was performed. Mostly, limb salvage with tumour resection and implantation of a megaprosthesis was done; three patients needed amputation or disarticulation. In one out of three patients with a pelvic tumour resection was followed by implantation of a pelvic replacement; the other two patients received tumour resection with autologous stabilisation of the pelvis. Surgical margins were wide in six patients, marginal in two and intralesional in three. Adjuvant chemotherapy was given to five patients. RESULTS: Recurrence was detected in 5/11 of the patients operated on: in two with wide, in one with marginal, and in two with intralesional resection. No recurrence was seen in 5/11 patients: in four after wide and in one after marginal resection. In one patient the stage was unknown. At follow-up 11 patients were dead of disease (DOD), one dead of unknown reason (DOU) and one alive with disease (AWD). The mean survival time was 9.7 months. Metastasis to different anatomical sites was evident after a period of 10 months. CONCLUSIONS: Our results resemble those reported in the literature. DDCS is rare and is the primary malignant bone tumour with the worst prognosis. Surgery is the most important procedure, although it is unclear whether a radical resection improves the long-term results. Information regarding neoadjuvant and/or adjuvant therapy with chemotherapy is very limited.

Mechanisms of growth factor expression in acute myeloid leukemia (AML).

To investigate possible mechanisms of growth factor expression in acute myeloid leukemia, genes for granulocyte macrophage colony-stimulating factor (GM-CSF) were analyzed by Southern blots in 20 patients, for M-CSF in 13, for interleukin-6 (IL-6) in 14, for IL-6 receptor in 14 and for G-CSF in five patients. Only in one patient a complex rearrangement of the G-CSF gene with possible amplification was noted indicating rarity of direct alterations of growth factor genes in acute myelogenous leukemia (AML). Spontaneous m-RNA expression for GM-CSF was found in only one of 20 patients, and for IL-6 in eight of 11 patients. In vitro incubation of AML cells of eight patients with recombinant tumor necrosis factor for 24 hr revealed induction of GM-CSF m-RNA expression in three cases and GM-CSF protein expression in two of them. These data suggest that spontaneous GM-CSF production occurs rarely in AML and that monokines, such as tumor necrosis factor, may induce GM-CSF in AML cells. Therefore, interactions of AML cells with normal or malignant accessory cells may be important for autocrine stimulation in AML. Our data suggest that ectopic growth factor secretion is not the primary cause of generating AML but may contribute to progression of the disease. Alternatively, AML may represent a heterogenous group of leukemias with different etiology but similar phenotype.

Absence of point mutations in a functionally important part of the extracellular domain of the c-kit proto-oncogene in a series of patients with acute myeloid leukemia (AML).

The proto-oncogenes c-fms and c-kit belong to a family of growth factor receptors possessing protein kinase activity. It has been shown that transfection of a c-fms gene carrying a point mutation at codon 301, leads to a ligand-independent transformation of mouse NIH3T3 cells. In human acute myeloid leukemia (AML), point mutations at codon 301 of the c-fms gene have been observed implying an important role in the transformation process. The possibility of a point mutation of the c-kit proto-oncogene was investigated. We sequenced a segment of the c-kit proto-oncogene coding for a part of the extracellular domain. This segment was 40.7% homologous to the c-fms region encompassing codon 301. c-DNA was prepared from peripheral blood or bone marrow cells from 25 patients with AML, from four patients with myelodysplastic syndrome (MDS) and from three human myeloid cell lines. The region of interest was amplified with two rounds of polymerase chain reactions (PCR) with nested primers and directly sequenced. No point mutations were found in the investigated samples. Thus, point mutations in this segment of the c-kit gene do not seem to play an important role in the transformation process of human acute leukemia.

Absence of point mutations in the extracellular domain of the alpha subunit of the GM-CSF receptor in a series of patients with acute myeloid leukemia (AML).

The GM-CSF receptor belongs to the cytokine receptor superfamily. The high-affinity receptors of this class are lacking intrinsic tyrosine kinase domains. The GM-CSF receptor consists of alpha and beta subunits. The beta subunit is shared with the receptors of IL-3 and IL-5. In addition to the membrane bound forms the receptors have been found to possess soluble isoforms. Since retroviral infection of the human GM-CSF dependent cell line, TF-1, leads to factor independent growth by increased expression of the GM-CSF receptor alpha chain in a subgroup of infected clones, we were interested in studying the role of this chain in human AML. Further considering that a point mutation in the extracellular domain of the erythropoietin receptor, also a member of the cytokine receptor superfamily, resulted in constitutive activation of a murine cell line, we investigated the possibility that a point mutation of the GM-CSF receptor was responsible for autonomous growth of AML cells. We sequenced a segment of the receptor coding for the extracellular domain of the alpha subunit. cDNA was prepared from peripheral blood or bone marrow cells from 24 patients with AML, from four patients with MDS and from three human myeloid cell lines. The region of interest was amplified with two rounds of PCR reactions with nested primers, covering five overlapping fragments, and directly sequenced using a non-radioactive technique. No point mutations were found in the investigated samples. Thus, point mutations in this segment of the GM-CSF receptor gene do not seem to play an important role in the transformation process of human acute leukemia.