| Objective The Tientsin Albino II(TA2)inbred mouse strain was established by the Animal Center of Tianjin Medical University,which was admitted by International Committee Standardized Genetic Nomenclature.TA2 has a high incidence of spontaneous breast cancer without external inducers or carcinogens.The morbidity of breast cancer in multiparous TA2 mice is 81%.Compared with other mouse models,TA2 can reflect the natural carcinogenesis of human breast cancers.Clonal evolution has been a foundational concept in cancer biology.Previous studies showed breast cancers with different phenotypes have different characteristics of clonal evolution.However,it is still unknown about the clonal evolution pattern of TA2 spontaneous breast cancer.Our study was designed to detect the clonal structure and clonal evolution of 4 cases of TA2 spontaneous breast cancer by whole genome sequencing.Furthermore,the defects of DNA double-strand breaks(DSBs)repairing and non-homologous end joining(NHEJ)in TA2 were also studied.Methods1.Seventeen cases of TA2 spontaneous breast cancer were collected in the Animal Center of Tianjin Medical University.Days of tumor onset,parity and sites of tumors were documented.Pathological subtypes were detected by HE staining.Molecular subtypes were detected by immunohistochemical staining of ER,PR,HER2 and EGFR.Cellular proliferation was accessed by immunohistochemical staining of Ki67.2.Four cases of TA2 spontaneous breast cancer(R01,R02,R03,R04)were selected for whole genome sequencing.R01 and R02 derived from the same TA2.R01 was the primary breast cancer,while R02 was the nape metastatic breast cancer.R03 and R04 derived from two different TA2,and both were primary breast cancers.All cases were infiltrating ductal carcinoma.Histological grade of R03 was grade 3,and the others were grade 2.The molecular subtype of all cases was basal-like breast cancer.The whole genomes of TA2 spontaneous breast cancers and mouse tails were sequenced by next generation sequencing.Reference sequence was the genome sequence of Mouse.C57BL/6J.(1)Genetic variations were detected,such as Single Nucleotide Variation(SNV),small In Del,Structure Variation(SV)and copy number variation(CNV).Genetic variations associated with TA2 spontaneous breast cancer were mined.(2)Clonal evolution was evaluated by Sci Clone and EXPANDS using SNV,CNV and LOH.3.Mouse embryonic fibroblasts(MEFs)of TA2 were cultured for DSBs study,and MEFs of TA1 and C57BL/6 were controls.MEFs were treated with bleomycin to induce DSBs.DSBs were detected by immunofluorescence of ?-H2 AX,which was the marker of DSBs.The foci of ?-H2 AX at different time were counted to evaluate DSB repair.The levels of serum Ig A of TA2,TA1 and B 6 were detected by ELISA to evaluate the non-homologous end joining(NHEJ).Results1.The average date of onset of TA2 spontaneous breast cancer was 315±71days.The average parity of TA2 was 3.06±1.12.The predilection sites of tumor were right prothorax(8 cases),right lower quadrant(4 cases),left lower quadrant(4 cases),left prothorax(1 cases),and two of them accompanied with nape tumor.There were no tumors in other sites.The pathological subtype of all TA2 spontaneous breast cancers was similar with human infiltrating ductal carcinoma.Histological grades of most cases were grade 2 except one case with grade 3.Molecular subtype of all cases was basal-like breast cancer with negative ER,negative PR,negative HER2,positive EGFR and high expression of Ki67.2.The bioinformatics analysis of the whole genome sequence consisted of detection of genetic variations and evaluation of clonal evolution.(1)There were many SNVs,In Dels,CNVs and SVs in TA2 spontaneous breast cancer specimens compared with reference sequence.Most genome-wide nucleotide substitutions in 4 cases were C>T or T>C transition mutations.The percentage of the same SNVs in 4 cases was99.02% by Venn analysis,and the percentage of the same In Dels in 4 cases was95.45%.Genes with variations in coding sequence(CDS)were classified into 25 classes according to gene function.The gene numbers of different classes in 4 cases had little changes.The same mutations of Brca2,Rad52,Rif1 and Trp53bp1 associated with homologous recombination(HR)and NHEJ were found in all 4 cases.Sixteen DNA repairing genes that associated with breast cancer were analyzed,and the same mutations of Trp53 and Kmt2 c genes were found in all 4 specimens.(2)There were 6 subclones in each case,and the subclonal architectures of each case were similar by Sci Clone analysis.There were 11 subclones in each case by EXPANDS analysis.All phylogenetic trees of 4 case depicted branched evolutionary trajectory.The evaluation of clonal structure and clonal evolution of 4 cases indicated the clonal evolution of TA2 spontaneous breast cancers were similar.3.(1)?-H2 AX foci were induced in MEFs nuclei by bleomycin.Numbers of?-H2 AX foci were counted at 4h and 24 h following bleomycin treatment.The average numbers of ?-H2 AX foci at 4h in TA2,TA1 and C57BL/6 MEFs were23.96±7.19,21.52±6.80 and 21.30±6.47,respectively.There were no statistical differences between TA2 and TA1,TA1 and C57BL/6,TA2 and C57BL/6 in ?-H2 AX foci at 4h(all P>0.05).The average numbers of ?-H2 AX foci at 24 h in TA2,TA1 and C57BL/6 MEFs were 5.98±2.27,1.22±0.98 and 1.32±0.79,respectively.There was no statistical difference between TA1 and C57BL/6 in ?-H2 AX foci at 24h(all P>0.05).The number of ?-H2 AX foci in TA2 MEFs was higher than TA1 or C57BL/6 MEFs at 24h(both P<0.05).The results showed that ?-H2 AX foci of TA2 MEFs resolved more slowly than TA1 or C57BL/6 MEFs,which indicated defects in DSB repairing in TA2 mice.(2)The average concentration of serum Ig A in TA2,TA1 and C57BL/6 was 418.60±47.18 ?g/m L,472.69±36.46 ?g/m L and 470.34±37.49?g/m L,respectively.There were no statistical differences between TA1 and C57BL/6in term of Ig A(P>0.05).The concentration of serum Ig A in TA2 was higher than TA1 or C57BL/6(both P<0.05),which indicated defects in NHEJ in TA2.Conclusion Our study showed that different TA2 spontaneous breast cancer specimens were similar with each other in pathological subtype,molecular subtype,genetic variation and clonal evolution.The similar aspects were as follows:(1)Pathological subtype of the TA2 spontaneous breast cancer was similar with human infiltrating ductal carcinoma.And the histological grade of most cases was grade 2.(2)Molecular subtypes of TA2 spontaneous breast cancers were basal-like breast cancer.(3)There were similar genetic variants,such as SNVs,In Dels,CNVs and SVs,in the whole genome,CDS or different gene classifications.Similar DSB associated gene mutations were found in different cases.(4)The similar clonal structure and clonal evolution was found in TA2 spontaneous breast cancers.We assumed that the clonal genome evolution of TA2 spontaneous breast cancers followed a constant trajectory,which requires further studies.Our study also showed that there were the defects of DSB repairing and NHEJ in TA2,which might contribute to carcinogenesis of TA2 spontaneous breast cancer. |
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