Study Of The Clinical And Biology Features In Acute Lymphoblastic Leukemia With The 9p Abnormality

Part one: Study of the clinical and biology features in 174 acute lymphoblastic leukemia with the 9p abnormalityObjectiveTo evaluate the frequency and reveal the characteristics in acute lymphoblastic leukemia with the 9p abnormality in our hospital.MethodsCytogenetic analysis: chromosomes were prepared routinely by the direct or short-term culture of bone marrow cells.Karyptypes were analysed on R-banded metaphases,cases of ALL with 9p abnormality from 2005 to 2016 were selected.Results1.Of 2231 ALL cases that were examined cytogenetically in our hospital between 2005 and 2016.174 cases of abn(9p)ALL were detected and accounted for 7.8%(174/2231)of ALL.The male to female ratio was 106:68,the median age was 33 years(range 4-78 years),15-39 age group accounted for 52.9%,the median number of white blood cells was 32.4×10E9/L[(0.92-650)×10E9/L].The median lactate dehydrogenase was 461U/L[(136-8310)U/L].2.Karyotype anaylsis showed that the abnormality of 9p deletion accounted for 29.9%(52 cases),dicentric translocation 13.2%(23 cases),t(9p;V)abnormalities 8.6%(15 cases),inv(9p)4.6%(8 cases),+9/9p+ 1.1%(2 cases),among them complex chromosomal abnormalities(more than or equal to 5 abnormalities)accounted for 45.6%(89 cases).The most common additional karyotype abnormality was t(9;22),followed by del(20q).ConclusionsALL with abn(9p)had the following common features:(1)the frequency of abn(9p)among our cases was approximately 7.8%;(2)a elevated WBC and large liver and spleen lymph nodes were observed;(3)most of them were younger patients with a median age of 33 year old;(4)del(9p)was most common abnormality and the most common additional abnormality was t(9;22),followed by del(20q).Part two: Detection of common genes deletion,mutation and analysis prognostic factors in abn(9p)ALLObjectiveTo determine the frequency and the prognostic significance of gene deletions and mutations in abn(9p)ALL,and establish the prognostic scoring system to evaluate the prognosis of the disease.MethodsFISH analysis was performed on fixed cells from diagnostic bone marrow.Four commercial probes were used to detect CDKN2 A,PAX5,P53,RB1 genes deletions,amplification and mutation screening of exons of four genes(IKZF1,NOTCH1,P53 and HOX11)were performed.The EFS and OS were estimated by Kaplan-Meier,and the differences in proportion of variables were compared by the Log-rank test or COX regression.The level of significance was 0.05.All calculations were carried out using SPSS software version 21.0.Results1.In our cohort,161 cases were newly diagnosed ALL,13 CML-BC.CDKN2 A gene deletion was detected in 66 cases(70.2%),PAX5 gene in 34 cases(58.6%),RB1 gene in 7 cases(6.3%),P53 gene in 13 cases(11.8%).2.Four gene mutations were detected within target region in 31 cases of ALL.The IKZF1 gene mutation were found in 5 cases(5/31,16.1%),NOTCH1 gene in 3 cases(3/31,9.7%),P53 gene in 1 cases(1/31,3.2%),HOX11 gene in 1 cases(1/31,3.2%).3.Analysis of EFS factors: univariate analysis showed that the karyotype(P=0.010),ECOG score(P=0.035)and P53 gene(P=0.009),induced remission(P=0.007),and depth of molecular remission(MRD)(P=0.002),immunophenotyping(P=0.025)with statistical difference.Multivariate analysis showed that complex karyotype(abnormal karyotype more than 5 abnormalities),MRD is more than 10E4,and BCR-ABL fusion gene had significant adverse impact on EFS(P<0.05).Establish of EFS scoring system according to the above factors,can distinguish the different length of EFS [low risk factors(less than 2 points),risk factors(more than 3 points):P=0.002].4.OS factors analysis: the abn(9p)ALL with poor prognosis,5 year overall survival rate was only 17.55%.Univariate analysis showed that age(P=0.012),WBC(P=0.039),LDH(P=0.025)and BCR-ABL fusion gene(P=0.007),immunophenotype(P < 0.001),gene mutation(P=0.002),P53 gene(P=0.008),risk stratification(P < 0.001),treatment(P=0.006),PAX5 gene deletion negative with CDKN2 A homo-gene deletion group(P=0.013)showed a statistically significant difference.Multivariate analysis showed that age greater than 31 years,WBC>25×10E9/L,LDH>375U/L,BCR-ABL fusion gene positive,T-lymphoid expression,mutation,deletion of P53 gene,high-risk group,chemotherapy alone had adverse impact(P<0.05).Based on the above factors,the OS scoring system was established to distinguish the different prognosis of the disease [low risk group(1-4 points)VS high risk group of(5-6 points),P=0.011)].Conclusions1.The deletions of CDKN2 A,PAX5,P53 and RB1 genes were detected more often in abn(9p)ALL.2.Patients with CDKN2 A deletion and without PAX5 deletion belonged to unfavorable biological category.3.The EFS and OS scoring system established using the clinical and biological parameters can predict EFS and OS,and distinguish the different prognosis.Part three: Clinical and experimental study of 23 cases of ALL with 9p dicentric alterationObjectiveTo analyze the clinical and laboratory features of ALL with 9p dicentric alteration.MethodsFISH analysis was performed using probes for chromosomes 1,4,7,8,9,12,17,20 to confirm the dicentric chromosomes.Molecular technologies(a CGH/SNP/RNA-seq)were used to refine the breakpoint and identify the formation of new fusion genes.Results1.A total of 23 cases of dicentic abnormal ALL,male and female 16:7,with a median age of 38 years(10-78 years).The dic(9;20)was abnormal in 9 cases(39.1%);dic(1;9)was abnormal in 1 cases(4.3%);dic(4;9)was abnormal in 1 cases(4.3%);dic(7;9)was abnormal in 5 cases(21.7%);dic(8;9)was abnormal in 1 cases(4.3%);dic(9;12)was abnormal in 5 cases(21.7%);dic(9;17)was abnormal in 1 cases(4.3%).2.Of the 4 cases,a CGH and SNP were detected,9p and 20 q deletions were found in all 4 cases,and the fracture sites showed heterogeneity and instability.For 3 patients were submitted for transcription,and sequencing showed that case 3 existed a new fusion gene for EXOSC3-PIGU,and PCR and sequencing were confirmed the fusion of EXOSC3 and PIGU genes.ConclusionsThe dicentric abnormalities involved chromosome 9p in ALL had been described in rare cases.The frequent dicentric chromosomes in ALL with abn(9p)were dic(7;9),dic(9;12)and dic(9;20).The most common additional abnormality was t(9;22).Although breakpoint heterogeneity was characteristic of dic(9;20),a new fusion gene(EXOSC3-PIGU)by the formation of dicentric chromosomes was identified using molecular technologies and the function of fusion genes will be characterized.