Research On The Influence Of New Oncogenic CSF3R Mutations In Chronic Neutrophilic Leukemia

In this research we will be mainly focusing on the a new genetic findings, such as granulocyte-colony stimulating factor receptor(CSF3R) mutations, especially a novel mutation site CSF3R-T618 I, as well as with an unreported novel mutation site of CSF3R- H54 A, and their influence on the molecular pathogenesis of the Chronic neutrophilic leukemia.Part I. Case presentation Chronic neutrophilic leukemia with overexpression EVI-1 gene and concurrent CSF3 R and SETBP1 mutationBackground: Chronic neutrophilic leukaemia(CNL) is a rare myeloproliferative neoplasm(MPN) characterised by sustained neutrophilia, splenomegaly, bone marrow granulocytic hyperplasia without evidence dysplasia and absence of the Philadelphia(Ph) chromosome. The case presented herein was characterized by marked and sustained neutrophilia, increased NAP score, absence of Ph chromosome, absence of bcr-abl fusion transcripts on RT-PCR, and exclusion of underlying disorders causing leukemoid reaction. In the present case, on the first admission patient was no hepatosplenomegaly, but other laboratory findings satisfied the WHO diagnostic criteria. One year later of the first admission, the patient suffered from bleeding gums, splenomegaly, and lymphadenopathy due to of disease progression. In our patient the mean value of peripheral blood cell count during the clinical course were as follows; leukocyte count 28.36 X 109/l(range 7.67-51.28 X109/l) with 88.5% mature neutrophils(range 78.8-98.5%), platelet count 33.3 X 109/l(range 23-94 X 109/l). Thrombocytopenia was observed in our patient at presentation and followed a progressive course without responding to treatment with hydroxyurea and IFN-α.Materials/Methods: Peripheral Blood were collected from the CNL patient and PMNCs were collected using Ficoll Gradient density separation and grown in complete medium at 37°C in a humidified atmosphere composed of 5% CO2 and 95% air. Neutrophil function analysis, such as cell apoptosis, cell cycle and cell migration test were checked with LSR II Flow Cytometer. Neutrophils are cultured in RPMI 1640 medium with 10% FBS, at different time points, cells were collected and labeled with Annexin V and PI(BD Pharmingen), then test the apoptosis compared with normal control group. Purified neutrophils were cultured in the transwell insert(1x106/well), and chemotactic peptide N-formyl-methionyl-leucyl-phenylalan(f MLP, 1u M, SIGMA) were added to the lower compartment. At each time point, we counted the number of the cells in the lower compartment. Oncogenic CSF3 R mutation in a whole exon and intron by PCR-based DNA Pyrosequencing method on the CD34+, CD15+ cell fraction were sorted using a BD FACSAria III(BD Biosciences) flow cytometer.Result: In patient has 93.9% of the cells were mature neutrophils. Flow cytometric analysis of cell apoptosis revealed that our patient’s neutrophil still alive after 96 hours, but we found very few alive neutrophils on the normal control group after 72 hours. Flow cytometric analysis of cell cycle revealed as a normal. Therein, all cells were terminally differentiated and there is no cell division. Extent of migration activity by f MLP was lower in the patient neutrophils compared to the normal control cells with f MLP, but higher than to normal negative control /without f MLP/. An interesting observation, EVI-1 gene positive was found by qualitative screening test. Then we checked the expression level of EVI-1 gene by cell sorting CD 34+, CD 15+ positive cell fraction and bone marrow sample by quantitative RT-PCR screening test 3 times during the clinical course. Interestingly, those tests results were similar, especially on the bone marrow sample and CD 34+ cell fraction detected overexpression EVI-1 gene, but on the CD15+ cell fraction EVI-1 gene was also positive, but expression was low. The patient’s result was positively detected the membrane proximal mutation p.Thr618 Ile on the exon 14 of CSF3 R on the both CD15+ and CD34+ cell fraction, in addition, a novel mutation site revealed on the exon 4 of CSF3 R heterozygous mutation p.His54 Asp. We also evaluated somatic SETBP1 mutational alteration analysis by PCR amplification followed by Sanger sequencing for codons between 706-917 in exon 3. The experiment result is indicated that the patient’s somatic heterozygous SETBP1 mutation was encoding at p.Asp868 Asn, c.2602C>A.Conclusion: This is a case of a 67-years-old man presenting with sustained neutrophilia, persistent thrombocytopenia, absent BCR-ABL1 transcripts and JAK2 V617F mutation, in addition an interestingly overexpressed EVI-1 gene and a new concurrent mutation of CSF3 R and SETBP1, as well as with a unreported novel mutation site of CSF3R- H54 A, ultimately diagnosed as CNL. Our next studies are needed to elucidate the mechanism of the mutation sites on the molecular pathogenesis of the CNL.Part II. Study on the mutation sites of the CSF3 R, which were responsible for the clonal advantage of CNLObjective: To determine CSF3R-FL, CSF3R-T618 I, CSF3R- H54 A mutations have some significance on the molecular pathogenesis of CNLMaterials/Methods: 1. Plasmid construction. Plasmids were constructed by PCR amplification of the insert, restriction digestion and ligation using standard molecular biology methods, briefly: the host vectors p LV-EF1α-EYFP-N, p LP-2, p VSV-G, p LP-1 gag pol were purified with Omegabio-tek maxi prep kit and digested by restriction enzymes(ECO RI, NOTI). The linearized vectors were purified from agarose gel using a Axy Prep TM DNA Gel Extraction Kit and the concentration of the samples was estimated on an agarose gel stained with ethidium bromide. The inserts(CSF3R-FL, CSF3R-T618 I, CSF3R- H54A) were generated by PCR, the sequences of the primer-pairs used and the conditions of the PCR reactions. The amplified DNA fragments were purified from agarose gels using Axy Prep TM DNA Gel Extraction kits and digested by ECO RI, NOTI was used to linearize the acceptor vector. Enzymatic reactions in the case of the i) insert: 100–3000 ng purified PCR product was digested by appropriate amount of enzyme and 4 μl of 10 x reaction buffer in 40 μl of final volume for overnight at the appropriate temperature; ii) vector: 2000–4000 ng plasmid DNA was digested by appropriate amount of enzyme and 2 μl of 10 x reaction buffer in 20 μl of final volume for 4 hours at the appropriate temperature. When necessary the digested fragments were purified again and the concentrations of the inserts were estimated on agarose gels, as described above. A 1:3 vector:insert molar ratio was used for the ligation reactions. Chemically competent DH5α Escherichia coli bacteria were transformed with the products of the ligation reactions and were grown on Luria Bertrani(LB) agar plates containing the required antibiotic, such as ampicillin (Sigma). A day later single colonies were picked from the plate, inoculated into and grown overnight in LB medium containing with ampicillin. Plasmids were purified from the overnight cultures as above and tested by restriction mapping for the presence of the insert. Selected clones were sequenced by Sanger sequencing. 2. Lentiviral packaging system we used 3 main components, such as the lentiviral expression vector(Plasmid DNA of CSF3R-FL, CSF3R-T618I), the lentiviral packaging plasmids(p LP-1, p LP-2 plasmids that encode for gag, pol, and rev from the HIV or FIV genome and p VSV-G), 293 TNN producer cells. We seed 1X105 293 TNN cells per 10 cm2 culture plate in 2-3 ml of culture medium containing DMEM medium supplemented with 4 m M L-glutamine, 4.5 g/l glucose, and fetal bovine serum(10%) without antibiotics. Grow for 18-24 hours at 37 °C with 5% CO2 so that the cell density reaches ~60- 80% confluency at the time of transfection. We used a GFP as a positive control, to confirm transfection experiment was successful. Then we collected the cell culture supernatant, which is containing infectious pseudoviral particles. 3. Transduction of Pseudotyped Viral Particles into the primary cell of mouse. In the final step we have used the Mouse Colony Forming Unit Assay using Metho CultTM to assess the effects of CNL-associated oncogenes on the morphology and number of primary murine cells derived bone marrow. For this purpose cells are transduced with either control, which is without viral construct or a construct expressing the oncogene of interest(CSF3R-FL, CSF3R-T618I).Results: 1. On the Plasmid construction step we successfully extracted and purificated of recombinant plasmids of CSF3R-FL and CSF3R-T618 I cloning, but we still did experiment to obtain the recombinant plasmid CSF3R- H54 A cloning. 2. After 24 hours of transfection 293 TNN Cells with Packaging Plasmids and the Expression Construct, cells we visualized with green fluorescence protein under the fluorescence microscope. 3. The both two of CSF3 R cloning were capable of transforming murine colony forming cells. After transforming, CFU-GM colonies were counted manually by light microscopy seven days after plating. We found that the membrane proximal mutation(T618I) transformed CFU-GM colonies number was more than the full length non-mutants(CSF3R-FL), which indicates that T618 mutation of CSF3 R conferred the clonal advantage of CNL leukemia cells.Conclusion: 1. The establishment of the plasmid reconstruction, lentiviral packaging system and Mouse Colony Forming Unit Assay were done successfully. 2. We confirmed the transformation capacity of the CSF3 R mutations, especially CSF3R-T618 I was higher than CSF3R-FL. This result demonstrates that T618 mutation of CSF3 R conferred the clonal advantage of CNL leukemia cells. 3. Further studies will be continued and are needed to prove the effects of the novel mutation site CSF3R-H54 A on the transduced murine bone marrow progenitor cells by using CFC assay.