Risk Factors And Molecular Characteristics Of Secondary Lung Cancer In Breast Cancer Patients

Background and objective:Breast cancer and lung cancer are common cancers in women with the first and second incidence rates.With the popularization of early breast cancer screening and technical improvements of clinical diagnosis and treatment,the survival rate of breast cancer patients has increased obviously.Due to the prolonged survival time,increasing incidence of lung cancer and gradual popularity of high-resolution computerized tomography(HRCT),the number of patients with breast cancer and secondary lung cancer had increased significantly in recent years.However,we are unclear about the risk factors and unique molecular characteristics of subsequent primary lung cancer in breast cancer patients up to now.Thus,we aimed to explore the risk factors of second lung cancer in breast cancer patients by Meta-analysis and case control study.Meanwhile,the pulmonary tumor and normal pulmonary tissue specimens of lung adenocarcinoma patients with previous breast cancer and single primary lung adenocarcinoma patients were collected.Then,the differences of gene mutations and expression between these two groups of patients were compared using the whole exome sequencing(WES)and transcriptome sequencing(RNA-seq),aiming to find ideal biomarkers.We hope that above works could provide some evidence for the clinical screening and management of high-risk breast cancer patients for second primary lung cancer,as well as the formulation of appropriate treatment and follow-up strategies,and try to conduct clinical interventions in these patients according to excavated biomarkers,thereby reducing the incidence of multiple primary breast and lung cancers.Methods:The study was divided into four chapters as follows:Chapter Ⅰ:Part one:the PubMed,EMBASE and Web of Science databases were searched for studies reporting the risk of second lung cancer in breast cancer patients from the establishment date to October 11,2020.The STATA 12.0 software was used for the Meta-analysis and the standardized incidence ratio(SIR)of second primary lung cancer and 95%confidence interval(CI)were combined.Part two:Above electronic databases were search for studies exploring the risk factors for second lung cancer of breast cancer patients from the establishment date to October 11,2020.The STATA 12.0 software was applied for statistical analysis and the relative risk(RR)or odds ratio(OR)with 95%CI were combined.Chapter Ⅱ:Breast cancer patients with second lung cancer who were admitted to our hospital from January,2010 to August,2020 and single primary breast cancer patients who received the surgery in the same medical group of Department of Breast Surgery,West China Hospital from March,2020 to November,2020 were retrospectively reviewed.The clinicopathological characteristics of patients in these two groups were compared,indirectly exploring the influencing factors for second lung cancer of breast cancer patients by multivariate Logistic regression analysis,and the OR was applied for evaluation.Meanwhile,the univariate and multivariate Logistic regression analysis were conducted to identify prognostic risk factors of breast cancer patients with second lung cancer and the hazard ratio(HR)was applied for evaluation.All statistical analysis were performed by SPPS 22.0 software.Chapter III:Genome analysis.We prospectively collected the lung tumor specimens from 10 patients with second primary lung adenocarcinoma and breast cancer and 10 single primary lung adenocarcinoma patients as samples,and the normal pulmonary tissues were used for comparison.The WES technology was applied to identify the differences in genomic characteristics between the two groups,including the single nucleotide variants(SNV),insertion and deletion mutations(InDel),base substitution,mutation feature and high-frequency mutation gene analysis.Chapter Ⅳ:Transcriptome analysis.The specimen selection was the same as that in Chapter Ⅲ.The RNA-seq technology was applied to identify the differences in transcriptome characteristics between the two groups.The Hallmarks,gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)gene sets were used as predefined gene sets to perform gene set enrichment analysis(GSEA).Significant gene sets were defined as |NES|≥1,FDR q-value<0.05 and NOM p-value<0.05.Besides,the leading edge analysis was also conducted to find high-frequency genes in significant gene sets.Results:Chapter Ⅰ1.A total of 15 studies were included in the first Meta-analysis,involving 1,161,979 participates.The pooled results indicated that compared to general population,female breast cancer patients showed significantly higher risk for second primary lung cancer(SIR=1.25,P<0.001).Although the subgroup analysis manifested that this phenomenon was only observed in patients who received radiotherapy(SIR=1.33,P=0.009)or were younger than 50 years old(SIR=2.15,P<0.001).2.A total of 24 studies were included in the second Meta-analysis,involving 3,090,620 participates.The pooled results indicated that the history of smoking(OR=9.73,P<0.001)and radiotherapy(RR=1.40,P<0.001)were risk factors for second primary lung cancer of breast cancer patients,but the chemotherapy(RR=0.69,P=0.002),estrogen receptor(ER)+(RR=0.93,P=0.014)and progesterone receptor(PR)+(RR=0.86,P<0.001)were protective factors.3.Subgroup analysis stratified by the relative position of lung tumor to breast tumor(ipsilateral vs contralateral)was conducted in the second Meta-analysis.The pooled results demonstrated that radiotherapy only increased the risk of ipsilateral lung cancer(RR=1.27,P=0.001)but not contralateral lung cancer(RR=1.16,P=0.487).Chapter Ⅱ1.One hundred and thirty-seven breast cancer patients with second primary lung cancer and 189 single primary breast cancer patients were enrolled in the case group and control group,respectively.Most cases in the case group were asynchronous multiple primary carcinomas,with the median interval of 60(ranged from 2 to 456)months between the diagnosis of breast cancer and lung cancer.2.The results of univariate analysis in the case group indicated that radiotherapy for breast cancer(HR=2.340,P=0.035),T stage of lung cancer(HR=9.276,P<0.001),lymph node metastasis of lung cancer(HR=27.488,P<0.001),distant metastasis(HR=6.883,P<0.001)and TNM stage of lung cancer(HR=7.211,P<0.001)were related with overall survival(OS).Multivariate Logistic regression analysis demonstrated that only poor differentiation of lung cancer(HR=6.372,P<0.001)and TNM Ⅲ/Ⅳ stage(HR=5.830,P<0.001)were independent prognostic risk factors for patients in the case group.3.Significant differences of family history for malignant tumors,T stage of breast cancer,N stage,history of chemotherapy and history of radiotherapy between the case group and control group were observed.Multivariate Logistic regression analysis manifested that family history of malignant tumors(OR=2.694,P=0.010)and radiotherapy(OR=1.848,P=0.026)were risk factors for second primary lung cancer of breast cancer patients and chemotherapy was a protective factor(OR=0.491,P=0.020).Chapter Ⅲ1.C>T/G>A mutation was the most common type of base substitution in 20 patients,and it might be related to spontaneous cytosine deamination which was an important mutation process in lung adenocarcinoma.Signature 1 and Signature 15 mutation characteristics existed in all patients.Signature 1 is the result of an endogenous mutation process caused by spontaneous deamination of 5-methylcytosine,and Signature 6 is related to DNA mismatch repair defects.2.EGFR,KMT2D,KRTAP4-9,MUC5B,FLG,AHNAK2,KRTAP4-8,OR1S1,AHNAK and LOC101059915 were the TOP 10 mutation frequency genes in this study and the mutation frequency of EGFR was the highest,but the difference between the two groups did not reach statistical significance.3.After the high-frequency mutation gene analysis,we found that TRIM73,DLX6 and CNGB1 mutations only existed in the multiple primary cancer group with the mutation frequency of 40%(4/10),which suggested that they might be relatively distinctive genes of breast cancer patients with second primary lung adenocarcinoma.However,further studies should be conducted to verify this.Chapter Ⅳ1.No significant up-or down-regulated gene sets was found by GSEA after we selected the Hallmarks(h.all.v7.2.symbols.gmt)as the predefined gene set.2.When the all GO(c5.all.v7.2.symbols.gmt)was used as predefined gene set,365 gene sets significantly enriched and up-regulated in the multiple primary breast and lung cancer group,and 294,18 and 53 gene sets were related with biological process(BP),molecular function(MF)and cellular component(CC),respectively.In addition,there were 28 significantly down-regulated gene sets,and 17,2 and 9 gene sets were related with BP,MF and CC,respectively.After leading edge analysis we found 13 high-frequency genes with significantly higher expression levels in the multiple primary cancer group,including the EDN1,CAV1,VEGFA,AGTR2,FGF10,WNT3A,DAB2IP,TEK,WNT7A,AGER,PPARG,CD36,ADCY8.3.When the KEGG(c2.cp.kegg.v7.2.symbols.gmt)was used as predefined gene set,nine enriched and up-regulated pathways and two down-regulated pathways were found,of which eight pathways were related with tumor growth,invasion or metastasis,including the Pathways in cancer,Calcium signaling pathway,Phagosome,Regulation of actin cytoskeleton,PI3K-Akt signaling pathway,Focal adhesion,cAMP signaling pathway and Cytokine-cytokine receptor interaction.After leading edge analysis we found seven high-frequency genes with significantly higher expression levels in the multiple primary cancer group,including the CHRM1,PDGFA,ADCY8,ADRB1,EDNRB,FGF10,and VEGFA.Conclusion:1.The risk of second primary lung cancer in patients with breast cancer is significantly higher than that of the general population,and it is related with the family history of malignant tumors,smoking,radiotherapy,chemotherapy,ER and PR.2.Among the 20 lung adenocarcinoma patients,C>T/G>A was the most common type of base substitution and Signature 1 and 15 were important mutation features.3.EGFR,KMT2D,KRTAP4-9,MUC5B,FLG,AHNAK2,KRTAP4-8,OR1S1,AHNAK and LOC101059915 were the TOP 10 mutation frequency genes in this study and the mutation frequency of EGFR was the highest.4.TRIM73,DLX6 and CNGB1 may be relatively distinctive genes of breast cancer patients with second primary lung adenocarcinoma,but further studies should be conducted to verify this.5.The occurrence of multiple primary breast and lung cancers might be closely associated with cytokine-cytokine receptor interaction,autophagy,PI3K-Akt,cAMP and calcium signaling pathways.6.FGF10 and VEGFA genes may play an important role in the occurrence of multiple primary breast and lung cancers,and are expected to become significant biomarkers for the screening of high-risk populations of multiple primary breast and lung cancers and core targets for clinical interventions.Further studies about FGF10 and VEGFA should be conducted.