Mutant Allele Frequency Based Intratumoral Genetic Heterogeneity Related To The Tumor Shrinkage Mode After NAC In Breast Cancer Patients

Part Ⅰ Receptor status affects the accuracy of breast MRI in evaluating shrinkage mode after neo-adjuvant chemotherapyObjectsThis study aims to explore the accuracy and influencing factors of breast MRI in evaluating the shrinkage mode of breast cancer after N AC with pathological threedimensional reconstruction.MethodsOn the basis of previous studies,the sample size was increased.Between August 2014 to November 2018,105 patients with primary invasive breast cancer confirmed by histopathology with clinical stage Ⅱ and Ⅲ were enrolled in the first ward of breast surgery of Shandong cancer hospital.All the patients received full course of anthracycline and taxane-based chemotherapy regimens and underwent breast MRI examination before and after NAC.Trastuzumab treatment was delivered to human epidermal growth factor receptor 2（Her-2）positive breast patients.MR imaging was performed by 3D-DOCTOR software and serial pathological sections of breast cancer samples after radical surgery to construct the three-dimensional pathological and MRI shrinkage mode of breast cancer after NAC.The clinical data was collected.The accuracy and influencing factors of breast MRI in evaluating shrinkage mode of breast cancer after NAC were evaluated with pathological three-dimensional reconstruction.Results1.The sensitivity,specificity,positive predictive value,negative predictive value and overall accuracy of MRI in evaluating the shrinkage mode of breast cancer after NAC were 92.8%,97.2%,98.5%,87.5%and 94.3%,respectively.2.Receptor status such as ER（p=0.009）,PR（p=0.027）,molecular typing（p=0.022）and pathological stage after NAC（p=0.044）were correlated with the accuracy of MRI in evaluating the shrinkage mode after NAC,while there was no correlation between Her-2 status with the accuracy of MRI in evaluating the shrinkage mode after NAC（p=0.176）.3.The positive,negative predictive of MRI were 98.5%,87.5%.Conclusion1.There is a high consistency between MRI and the three-dimensional pathological model in evaluating the shrinkage mode of breast cancer after NAC.2.ER and PR receptor status were the factors that affected the accuracy of breast MRI in evaluating the shrinkage mode after NAC.While,there was no correlation between Her-2 status with the accuracy of MRI in evaluating the shrinkage mode after NAC.Molecular typing was associated with the accuracy of breast MRI in evaluating the shrinkage mode after NAC,especially the triple negative breast cancer patients.3.When MRI was used to evaluate the shrinkage mode of breast cancer after NAC,it is easier to cause overestimation in patients with non-concentric shrinkage mode or pCR.The diameter of residual tumor was related to the accuracy of breast MRI in evaluating the shrinkage mode of NAC.Part Ⅱ Mutant allele frequency based intratumoral genetic heterogeneity related to the tumor shrinkage mode after NAC in breast cancer patientsObjectiveAccurate detection of gene heterogeneity for predicting shrinkage mode after NAC based on breast cancer receptor heterogeneity.MethodsWe analyzed formalin-fixed,paraffin-embedded（FFPE）samples from 24 patients before and after NAC,to measure the mutations profile using the targeted nextgeneration sequencing approach with 456 cancer-related genes panel.Meanwhile,pathological shrinkage mode was reconstructed in three dimensions after surgery and the genetic heterogeneity level was estimated by mutant-allele tumor heterogeneity（MATH）.We measured the genetic intra-tumor heterogeneity（ITH）and explored its correlation with the shrinkage mode after NAC.Results1.The number of mutations in breast cancer samples with CSM decreased after NAC,and there were no new mutations appeared;The number of mutations in breast cancer samples with non-concentric shinkage mode increased significantly after NAC,and there were several new mutations occurred.In patients with CSM,the mutation frequency of TP53 decreased from 70%before NAC to 30%after NAC;While,in patients with NCSM,the mutation frequency of TP53 increased from 50%up to 100%.2.There was significant difference in the MATH value between breast cancer samples with CSM and NCSM before and after NAC（p=0.026,p=0.003）.There were significant differences in the overall MATH value between breast cancer samples with CSM and NCSM（39.66 vs 102.7,p<0.0001）.3.The discrimination of different shrinkage mode with MATH before（p=0.026）and after NAC（p=0.003）was evaluated,indicating that MATH before and after NAC were statistically significant in distinguishing shrinkage mode;Among the included samples,the MATH value of pre NAC samples or/and post NAC samples get a threshold within 48.5 and 59.7,and the tumor would show CSM after NAC.4.75%（3/4）of Luminal A breast cancer patients increased after NAC,2/3 of luminal B group（excluding Luminal Her-2 positive）patients decreased after NAC,and（7/8）of Her-2 positive patients were lower than the threshold before NAC.Conclusion1.Gene mutation characteristics varied in breast cancer with different shrinkage mode during NAC.TP53 mutation decreased after NAC in breast cancer patients with CSM after NAC,and increased in breast cancer patients with NCSM.2.The frequency of mutant allele based intratumoral genetic heterogeneity related to the tumor shrinkage mode after NAC in breast cancer patients.3.There was a nonlinear relationship between the baseline value of MATH before NAC and the shrinkage mode after NAC.4.We could accurately perform molecular detection based on receptor states to predict the shrinkage mode of breast cancer during NAC.For the patients with Luminal A and Her-2 positive breast cancer,it is suitable to detect the samples before NAC,and Luminal B group is suitable to select the samples after NAC.Part Ⅲ Analysis of gene heterogeneity of breast cancer before and after NAC based on high-throughput sequencingObjectiveDetection of gene heterogeneity of breast cancer and its changes before and after NAC based on high-throughput sequencing technology.MethodsThe FFPE samples were detected before and after NAC by NGS,and 456 related cancer genes were used.The variation of mutation and mutation fraction before and after NAC were measured respectively,and the level of gene heterogeneity in breast cancer with different receptor states was measured.Results1.In this group of breast cancer patients,TP53 and PIK3CA were the most common mutation genes in samples before and after NAC.A total of 113 mutation genes were detected in the pre-NAC samples,and 70 gene mutations only appeared before NAC;At the same time,29 gene mutations only appeared in the post-NAC samples;43 genes were detected both before and after NAC.2.There were significant differences in the baseline MATH value among breast cancer samples with different molecular types before NAC（p=0.027）;There were significant difference between samples with Luminal A and Her-2 positive and samples with Luminal B type（p=0.004,p=0.022）,and there were no significant differences between triple negative patients and other types（p=0.125,p=0.578,p=0.578）.The MATH value of breast cancer samples changed before and after NAC.3.We compared the MATH values before and after NAC,and found that the MATH values changed significantly after NAC.After paired t-test,there was no significant difference in MATH value after NAC（p=0.384）.4.There was no correlation between the overall MATH value and the number of tumor mutation sites（p=0.089）,and there was no correlation between the MATH and TMB（p=0.091）.Conclusion1.The gene mutation fraction and MATH value of breast cancer samples changed during NAC.2.There was difference in the baseline MATH value among breast cancer patients with different receptor status before NAC.3.The MATH value of breast cancer changed significantly after NAC,but did not decrease significantly after NAC.4.There was no correlation between the number of gene mutations and TMB in breast cancer with MATH value.