The Study Of Mechanism On ETV6/RUNX1 Positive Childhood B-precursor Acute Lymphobalstic Leukemia

Part I The study on clinical prognosis among ETV6/RUNX1 positive childhood B-precursor acute lymphocyte leukemiaObjective ETV6/RUNX1 fusion gene is the most common chromosomal translocation in childhood B-precursor acute lymphoblastic leukemia(B-ALL). In this study, the purpose was to investigate that the incidence of the ETV6/RUNX1 fusion gene among chinese pediatric patients with B-ALL and its effect on the prognosis. Methods A total of 723 patients with B-ALL were enrolled in this study from January 1, 2007 to December 31, 2014. All patients were detected ETV6/RUNX1 fusion gene by FISH, then, the clinical data were analyzed combined with ETV6/RUNX1 to analyze the clinical prognosis. Results Among 723 patients with B-ALL, there were 151 patients with ETV6/RUNX1 positive, occurring in approximately 20.89%(151/723) of B-precursor cases; there were 91 patients with recurrence among all patients, including 10 patients with ETV6/RUNX1 positive, and the recurrence rate of ETV6/RUNX1 postive was 10.99%(10/91), which is significantly lower than the 20.89%(151/723) overall occurrence rate(P=0.0126). It suggests that ETV6/RUNX1 is associated with an excellent outcome really. Among ten recurrent patients with ETV6/RUNX1, the other 9 patients all relapsed after more than 300 days from diagnosis, the longest was 1662 days, and the average time was 907.3 days(SD±524.1) except one patient who had relapsed in 65 days after diagnosis, while the recurrence times among the patients with ETV6/RUNX1 negative, the shortest time was 45 days, and the longest time was 634.01 days, and the average time was 1996 days(SD±470.17). Although the recurrence times bewteen two groups had no significant difference(P=0.09), the recurrence times of ETV6/RUNX1 positive patients were mainly concentrated at the end of clinical chemotherapy, and the recurrence time of ETV6/RUNX1 negative were mainly at maintaining chemotherapy period, there was an significant difference bewteen the distribution of recurrence time(P<0.0001). Conclusion ETV6/RUNX1 fusion gene is also a favorable predictor of outcome in Chinese pediatric B-ALL, and its occurrence rate in Chinese pediatric B-ALL is slightly lower than 25% of the European and American countries.Part II Study on the effect of ETV6 on the growth of ETV6/RUNX1 fusion gene positive leukemia cells Objective ETV6/RUNX1 fusion gene t(12;21)(p13;q22) is the most common chromosomal translocation in childhood B-precursor acute lymphoblastic leukemia, and it is associated with an excellent outcome, but it is prone to late recurrence. In this study, we intended to investigate the effect of ETV6 gene on the growth of ETV6/RUNX1 fusion gene positive leukemia cells. Methods ETV6 overexpression vector and ETV6 interference vector(Sh RNA) were constructed into human leukemia cell line REH with ETV6/RUNX1 fusion gene positve, to study the difference in the growth of REH cells, and the apoptosis rate of REH cells, and the expression of ETV6/RUNX1 fusion protein. Results After the overexpression of ETV6 gene and Sh RNA interference with ETV6 gene, the growth rate of REH cells were all decreased, but the growth rate of REH cells after ETV6 overexpression was lower than that of Sh RNA. However, the apoptosis rate of REH cells increased more significantly after ETV6 overexpression, which increased from 1.5% to 5.7%, than after Sh RNA. The expression level of ETV6/RUNX1 fusion protein was significantly decreased after Sh RNA(P<0.001), which might be due to the interference vector of ETV6 gene also influence the ETV6/RUNX1 fusion gene. Conclusion The wild-type ETV6 can affect the growth and proliferation of the leukemia cells by apoptosis, and the effect of ETV6/RUNX1 on leukemia cells may be by the growth and proliferation of the cells, not by inhibition of apoptosis.Part III The study on the effect of GNAO1-R209 C mutation on ETV6/ RUNX1 positiveacute lymphoblastic leukemia Section I The whole exome sequencingbased on monozygotic twins with ETV6/ RUNX1 positive acute lymphoblastic leukemia to screen out "second hit" Objective Through the whole exome sequencing based on the monozygotic twin patients with ETV6/RUNX1 positive B-ALLto screen out the genes related with ETV6/RUNX1 positive childhood B-ALL, which are also called as the "second hit" events. Methods First, DNA samples of bone marrow from monozygotic twin patient A and patient B all with ETV6/RUNX1 positive B-ALL wree fragmented, then followed to capture all exon sequences by the human genome gene expressionprobe sequence-Sure Select XT human whole exome 50 m kit. Second, using the next generation of high-throughput sequencing platform-Hi Seq2000(Illumina) to sequence all exon sequences from above-captured, then obtaining the equivalent to human genomic exon region 120 times coverage of sequences, and the obtained alignments are matched to the corresponding human genomic sequence. Third, to analyze the biological informations by SAMtools, gatk and software tools,according to human genome database published sequence information hg19, and by comparison to find out the different from the reference sequence of SNV, CNV and In Del single nucleotide variation changes. Finally, to find the location in the genome, then to determine the influence of protein coding or regulatory information. At the same time, through comparing with samples of patient A between onset and remission(including comparing with samples of patient Bbetween before and after the onset), to screen outpathogenic genes, which are associated with the occurrence of ETV6/RUNX1 positive B-ALL. Results To find the same mutated gene between patient A and patient B, and the mutation reads ratio is more than 15%. Finally, only GNAO1 meets this requirement.In patient A, the reads ratio was 50%(25/50, 50%), because of the heterozygous mutation, so it closes to 100%,while a proportion of leukemia cells was 95%; and in patient B, thereads ratio was 46.43%(26/56, 46.43%). Especially in patient B, 2 months before the onset, the reads ratio at GNAO1-R209 C locus was only 0.35%,and the percentage of ETV6/RUNX1 positive cells is 12.5 %, but after 2 months, when the onset, the mutation reads ratio at GNAO1-R209 C locus reached 46.43%, while the percentage of ETV6/RUNX1 positive cells also increased from 12.5 % to 96.2%. Conclusion Between monozygotic twin patients with ETV6/RUNX1 positive B-ALL, GNAO1-R209 C mutation is closely related with the occurrence of leukemia, and it is very likely to be the "second hit" events which leads to the onset of ETV6/RUNX1 positive B-ALL.Section II The Study on the regulation of ETV6 on expression of GNAO1 Objective To definite that whether there is a regulatory mechanism of ETV6 onthe expression of GNAO1, then to facilitate understanding of GNAO1 gene as the possibility of "second hit", and the possible pathogenic mechanism.Methods ETV6 overexpression vector and ETV6 interference vector(Sh RNA) were constructed into human leukemia cell line REH with ETV6/RUNX1 fusion gene positve, to study the difference in the expression of GNAO1 gene.Results While overexpression of ETV6 gene compared with the control group, the expression level of GNAO1 gene was down regulated; After Sh RNA interference targeting ETV6 gene, GNAO1 gene expression is up-regulated.Conclusion ETV6 gene may directly or indirectly regulate the expression of GNAO1 gene.