The Application Of Single-Cell WGS And RNA-seq To Cancer Evolution

Cancer is a major health problem over the world.In 2015,there were estimated 4,292,000 new cancer cases in China.Meanwhile,an estimated 2,814,000 cancer deaths would occur.As research continues,many kinds of target therapies and immunotherapies emerge as assistant methods of radiotherapy and chemotherapy.However,cancer cells are capable of high variations and adaptions.Although therapies could mitigate disease,it’s the cancer relapse and metastasis which lead to the patients’death in most cases.Cancer initiation,progression and metastasis/relapse is a dynamic evolutional process.Chronic lymphocytic leukemia(CLL)is a good research object to study this process.CLL is a frequent hematologic malignancy arising from a group of abnormal B lymphocytes characterized by a set of molecules on the cell surface.In most cases,CLL is a slowly progressing disease,and may not need any treatment at the early stage.Besides,cancer cells can be obtained directly from the blood,which allows researchers to monitor the cancer progression in a very long period.As the cost of the sequencing decreasing,a large numbers of cancer samples are sequenced.With the large-scale WGS/WTS analysis,several key mutations and signalings contributing to CLL are identified,and the evolution models are built based on these variations.However,the evolution models based on cancer samples from one or a few time points are not accurate.Therefore,we focus on these questions and study a single CLL patient.Cancer samples are selected from 16 time points,and performed WGS and WTS at bulk and/or single-cell level.The evolution process is reconstructed based on the CNVs and expression changes.The main contents of this work include the following aspects:1.The object of this work is a CLL patients with 29-year follow-up.The PBMCs are obtained from the 8th to the 28th year after diagnosis.After fluorescence activated cell sorting(FACS),the samples are performed WGS and microarray sequencing at bulk level,and single-cell WGS and single-cell RNA-seq.2.The WGS and microarray sequencing identify the chromosome alterations 13q14 deletion,6q deletion and 12q amplification in the time points before the first therapy.The results of single-cell DNA sequencing indicate one fundamental cell clone at the early stage.The cell clone is eliminated following therapy,followed by the appearance of a clone containing trisomy 12 and a chromosome 10 copy neutral loss of heterogeneity that last to the year 28.Meanwhile,a nonsynonymous single nucleotide variation(SNV)is detected in MYD88,which is reported as an driver gene in CLL.3.Single-cell RNA-seq is performed on 300 cells from 5 time points.The results identify several transcription factors,including FOS,JUN and KLF4.After the second therapy,the cancer cells develop a much great diversity of expression profiles.Besides,the pseudo-temporal analysis further characterize the complex changes in expression during the cancer initiation,proliferation and extinction,and relapse.Several signalings,including B cell receptor signaling,EGFR signaling,ERKs signaling and NF-kappa B signaling,show expression changes along evolution.