| Objectives: HER2 positive breast cancer is a subtype of breast cancer molecular typing,accounting for about 15-20% of breast cancer.General chemotherapy drugs have poor effect and poor prognosis.In 1998,the clinical application of Herceptin,an anti HER2 targeted drug,greatly improved the prognosis of HER2 positive breast cancer patients,and improved the survival rate and life span of patients.However,in clinical treatment,it is found that primary or secondary Herceptin resistance will occur in targeted therapy of anti HER2.About 7%-10% of HER2(1+)patients were also FISH positive,some patients with negative HER2 are effective to lapatinib,a small TKI drug.Therefore,we speculate that HER2 status is not immutable,and some factors may cause HER2 expression to change from positive to negative.Therefore,timely and accurate understanding of HER2 expression status and dynamic changes is necessary and important.Human epidermal growth factor receptor 2(HER2)consists of extracellular domain(ECD),lipophilic transmembrane domain and intracellular domain(ICD).HER2 positive breast cancer is highly invasive.Compared with other subtypes,this subtype is more prone to internal organs metastasis and brain metastasis,and its response to some hormones and chemotherapy drugs is different from other subtypes.This subtype is related to the poor differentiation and high proliferation rate of tumor,at the same time,it will reduce the levels of estrogen receptor(ER)and progesterone receptor(PR).Importantly,HER-2 status is an independent prognostic indicator of breast cancer disease progression and death.Many chemotherapy schemes are designed based on HER2 status.The method of detecting the status of HER2 in clinical application is immunohistochemistry(IHC)to detect the ECD of HER2.When IHC indicates HER2(2+),it will further do immunofluorescence in situ hybridization(FISH)to determine whether the HER2 gene has been amplified.However,when IHC results are negative or HER2(1+),FISH test will not be carried out in general.When the results of IHC showed that HER2(3+),it was directly determined that HER2 was positive.Both IHC and FISH need invasive operation to obtain tissue samples for pathological examination,it is impossible to determine HER2 status for advanced breast cancer metastases that cannot be obtained from tissue samples,especially biopsy,and it is unable to dynamically monitor the HER2 status of patients.The biggest drawback of IHC is the inability to dynamically monitor the HER2 status of patients.For patients who cannot obtain tissue samples,their HER2 status cannot be judged.The free HER2-ECD has been detected in the peripheral blood of breast cancer patients.If the ECD of HER2 molecule is cut into the blood enough,the result of IHC test may be false negative.If the ECD of HER2 molecule is cut into the blood,there may be false negative results of IHC test,which will cause some patients to lose the opportunity of anti HER2 treatment.The mechanism of cutting HER2-ECD into blood is very complex,previous studies have shown that calpain-10 may play an important role in this process.Calpain-10 is a non lysosomal cysteine protease belonging to the calpain family,Some studies have shown that HER2-ECD is detected in 35% of HER2 negative breast cancer patients' peripheral blood,and calpain-10 is over expressed in these patients' tissues,whose HER2-ICD is positive,it can be concluded that HER2-ECD was cut into blood,resulting in HER2 negative expression,and Calpain-10 over expressed may be involved in this process and play an important regulatory role.The purpose of this study is to detect the HER2-ECD level in tumor tissue and peripheral blood of breast cancer patients,to determine the degree of coincidence and correlation,as well as the relationship with clinicopathology;to detect the expression of calpain-10 in breast cancer tissue,to determine the relationship with serum HER2-ECD,and to provide basis for clinical treatment.Methods: 1 Case selection From April 2016 to October 2016,289 breast cancer patients admitted to the breast center of the fourth hospital of Hebei Medical University were selected.Before treatment,these patients did not receive any radiotherapy,chemotherapy,drugs and surgery,and had no other history of cancer.2 Test method 2.1 At the first admission,5ml of peripheral venous blood was taken from the patients on an empty stomach state.At room temperature,1912 g centrifugal force was used to centrifuge the venous blood for 10 min.500ul of the upper serum was collected and divided into two EP tubes,frozen in the refrigerator at-80 ?.The serum HER2-ECD(extracellular)was detected by the two-point sandwich direct chemiluminescence method domain of s HER2-ECD(human epidermal growth factor receptor-2)in serum.2.2 0.5g of hollow needle biopsy specimens or tumor tissue specimens were collected and stored in a refrigerator at-80 for pathological diagnosis and ?IHC detection of HER2 and Calpain-10.2.3 Five breast cancer cell lines(MCF-7,MDA-MB-231,BT-549)with different HER2 status were selected,MDA-MB-453 and SKBR3),the expression of HER2 and Calpain-10 were measured by RT-qPCR and Western blot(WB),and the level of HER2-ECD in the culture medium was also measured.Two cell lines BT-549 and SKBR3,which were overexpressed by HER2 and Calpain-10,were selected.The expression of Calpain-10 was decreased or increased by transfection technology,and the expression of HER2 was measured again by RT-qPCR and Western blot(WB),The level of HER2-ECD in the medium was detected.3 Result judgment standard 3.1 If the s HER2-ECD is ? 15 ng / ml,it is positive;if it is < 15ng/ml,it is negative.3.2 judgment standard of HER2 results Using the Hercep Test score recommended by the American Society of clinical oncology and the American Society of pathologists,the results ranged from(-)to(3+).No staining or less than 10% of cell membrane staining is defined as negative(-);more than 10% of cancer cells are defined as weak and incomplete cell membrane staining as(+);more than 10% of cancer cells are defined as weak to medium complete cell membrane staining as medium positive(2+);more than 10% of cancer cells are defined as strong and complete cell membrane staining as strong positive(3+).(-)and(+)are considered to be low expression,i.e.HER2 negative,while(3+)is considered to be high expression,i.e.HER2 positive.The formalin fixed paraffin embedded tissues of HER2(2+)patients were used for fish test with HER2 DNA Probe Kit.When HER2 / neu gene was amplified,it was HER2 positive,when HER2 / neu gene was not amplified,it was HER2 negative.3.3 Calpain-10 result judgment standard When scoring the results of calpain-10 IHC,we need to consider both the staining intensity of tumor cells and the proportion of stained tumor cells.Each specimen was independently interpreted by two pathologists,who knew nothing about the patient's information,so they would not be interfered by the patient's disease status information.A positive reaction is defined as the presence of brown signals in the cytoplasm.For Calpain-10,the staining index(0-12)was determined by multiplying the staining intensity fraction by the proportion fraction of positive cells.The staining intensity score was set as: negative,0 point;weak positive,1 point;medium intensity positive,2 points;strong positive,3 points.The percentage of positive cells was < 5%,0 point;5-25%,1 point;26-50%,2 points;51-75%,3 points;> 75%,4 points.When the staining is uneven,the score is defined as follows: each component is scored independently,and the results are summarized.In statistical analysis,0-7 points were low expression of Calpain-10,8-12 points were high expression of Calpain-10.4 follow up All patients were followed up to January 2019,with disease progression(including recurrence,metastasis or death)as the end point,with a follow-up period of 23-30 months.The correlation of scher2-ecd and calpain-10 with overall survival rate(OS),disease free survival rate(DFS)and cumulative recurrence rate(CRR)was analyzed.DFS is defined as the time from radical surgery to the first event of disease recurrence or death for any reason.OS is defined as the time from the date of pathological diagnosis of breast cancer to the date of death for any reason.CRR is defined as the frequency of recurrence in patients with complete remission after a specific observation period(usually more than 1 year).5 Statistical treatment In the clinical study,chi square test was used for comparison between groups.Pearson correlation test was used for correlation analysis,and rank sum test was used for survival curve analysis.T-test was used in the in vitro experiment,including student T-test and paired T-test.Bonferroni test and one-way ANOVA were used for multiple comparisons.P<0.05 was considered statistically significant.Results: 1.Clinicopathological data and follow-up In this study,289 patients with primary breast cancer were all female,aged 26-80 years,median age 57 years,34 patients(11.76%),165 patients(57.09%)aged 35-55 years,and 90 patients(31.14%)aged over 55 years.Among them,159 were premenopausal women(55.02%),130 were postmenopausal women(44.98%).According to the TNM staging standard of AJCC 7th Edition,75 patients(25.95%)were in stage I,160 patients(55.36%)in stage II,42 patients(14.53%)in stage ? and 12 patients(4.15%)in stage IV.158 patients had no lymph node metastasis(54.67%),131 patients had lymph node metastasis(45.33%);227 patients had no angioma thrombus(78.55%),62 patients had angioma thrombus(21.45%).The number of pathological grade ?,? and ? cases was 29,166 and 94 respectively,accounting for 10.03%,57.44% and 32.53% respectively.There were 209 ER positive patients(72.32%),80 ER negative patients(27.68%);179 PR positive patients(61.94%),110 PR negative patients(38.06%);167 HER2 positive patients(57.79%),122 HER2 negative patients(42.21%).Peripheral blood HER2-ECD was detected in 289 patients at first admission.The results showed that the expression of HER2-ECD was negative in 77.5%(224/289)patients and positive in 22.5%(65/289)patients.As a negative control group,20 healthy women's s HER2-ECD were detected at the same time.The detection value was 4.6-11.8ng/ml,with an average of 7.6ng/ml.All healthy adult women's s HER2-ECD were negative.2.The relationship between s HER2-ECD and clinicopathological features and prognosis Among 159 premenopausal patients,120 were in the negative group,accounting for 75.47%;39 were in the positive group,accounting for 24.53%;among 140 postmenopausal patients,104 were in the negative group,accounting for 80%;26 were in the positive group,accounting for 20%;there was no significant difference in menstrual status between the negative group and the positive group of s HER2-ECD(P > 0.05).There were 34 patients aged ? 35 years,10(29.41%)with s HER2-ECD positive,24(70.59%)negative;165 patients aged 35-55 years,48(29.09%)with s HER2-ECD positive,117(70.91%)negative;90 patients aged > 55 years,12(13.33%)with s HER2-ECD positive,78(86.67%)negative,and none among the three age groups Statistical difference(P = 0.199).75 patients in stage ?,72 in stage ?,121 in stage ?,and 39 in stage ?,accounted for 75.63% and 24.37% respectively;42 patients in stage ?,28 in stage ?,and 14 in stage ?,3 in stage ?,and 9 in stage ?,accounted for 75% and 4% respectively;the later the stage of clinical stage,the later the stage of clinical stage,the later the stage of clinical stage The higher the positive rate of HER2-ECD was(P <0.001).In 158 cases without lymph node metastasis,140 cases were negative in s HER2-ECD(88.61%),18 cases were positive in s HER2-ECD(11.39%),84 cases were negative in s HER2-ECD(64.12%),47 cases were positive in s HER2-ECD(35.88%).Among 227 patients without thrombus,188 were negative in s HER2-ECD(82.82%),39 were positive(17.18%),36 were negative in s HER2-ECD(58.06%),26 were positive(41.94%).The proportion of patients with thrombus was higher(P<0.001).Of the 29 patients with histological grade I,27(93.10%)were negative and 2(6.9%)were positive.Of the 166 patients with grade II,132(79.52%)were negative and 34(20.48%)were positive.Of the 94 patients with grade ?,65(69.15%)were negative and 29(30.85%)were positive.The higher the histopathological grade,the higher the s HER2-ECD The higher the proportion of positive patients was,the difference was statistically significant(P=0.017).Among 209 ER positive patients,43(20.57%)were positive for s HER2-ECD,166(79.43%)were negative.Among 80 ER negative patients,22(27.5%)were positive for s HER2-ECD and 58(72.5%)were negative.There was no significant difference between the ER positive group and the negative group(P=0.207).Among 179 PR positive patients,40(22.35%)were positive for s HER2-ECD,139(77.65%)were negative.Among 110 PR negative patients,25(22.73%)were positive for s HER2-ECD and 85(77.27%)were negative.There was no significant difference in the proportion of s HER2-ECD between PR positive group and negative group(P=0.359).Among 167 HER2 positive patients,50(29.94%)were positive and 117(70.06%)were negative.Among 122 HER2 negative patients,15(12.30%)were positive and 107(87.70%)were negative.The positive rate of HER2-ECD in HER2 positive group was significantly higher than that in HER2 negative group(P<0.001).DFS(P=0.0012)and OS(P=0.0136)in the negative group were significantly higher than those in the positive group.The cumulative recurrence rate(CRR)in the positive group was significantly higher than that in the negative group(P=0.0004).3.Expression of Calpain-10 and its relationship with prognosis Among 289 cases of breast cancer,54.33%(157/289)had low expression of Calpain-10,and 45.67%(132/289)had high expression of Calpain-10.Among 167 HER2 positive breast cancer patients,82(49.10%)had low expression of Calpain-10 and 85(50.90%)had high expression;among 122 HER2 negative breast cancer patients,75(61.48%)had low expression of Calpain-10 and 47(38.52%)had high expression.The proportion of patients with high expression of Calpain-10 in HER2 positive group was significantly higher than that in HER2 negative group.The histological state of HER2 was significantly different between the two groups(P=0.037),and there was a positive correlation between Calpain-10 and t HER2.Among 224 cases of breast cancer with negative s HER2-ECD,75(61.48%)had low expression of Calpain-10 and 47(38.52%)had high expression of Calpain-10;among 65 cases of breast cancer with positive s HER2-ECD,8(12.31%)had low expression of Calpain-10 and 57(87.69%)had high expression of Calpain-10.The proportion of patients with high expression of Calpain-10 in the positive group of s HER2-ECD was significantly higher than that in the low expression group of Calpain-10.The difference of Calpain-10 protein expression between the positive group and the negative group of s HER2-ECD was statistically significant(P<0.001).The results showed that the expression of Calpain-10 protein in breast cancer was positively correlated with the expression of s HER2-ECD(r = 0.439).In 119 patients with low expression of Calpain-10,4 patients developed disease progression,accounting for 3.36%;in 158 patients with high expression of Calpain-10,19 patients developed disease progression,accounting for 12.03%;there was statistical difference between the high expression group and the low expression group of Calpain-10(P=0.0086),and the breast cancer patients with high expression of Calpain-10 were more likely to develop disease progression.It is suggested that Calpain-10 may be a prognostic indicator of breast cancer.In 122 patients with low expression of Calpain-10,2 died,accounting for 1.64%;in 167 patients with high expression of Calpain-10,7 died,accounting for 4.19%.There was no statistical difference in OS between the high expression group and the low expression group(P=0.2144),which may be due to the short follow-up time.In 122 patients with low expression of Calpain-10,5 patients had relapse,metastasis or death, accounting for 4.10%;in 167 patients with high expression of Calpain-10,25 patients had relapse,metastasis or death,accounting for 14.97%;there was a statistical difference in CRR between the high expression group and the low expression group(P=0.0024),and the survival time of breast cancer patients in the low expression group of Calpain-10 was longer.It is suggested that Calpain-10 may be a prognostic indicator of breast cancer.4.In vitro experiment of calpain-10 regulating HER2-ECD expression Firstly,the breast cancer cell lines overexpressing HER2 and Calpain-10 were screened.The HER2 and Calpain-10 of BT-549 and SKBR3 were overexpressed.The concentrations of HER2-ECD in the medium were 18.3 and 24.8ng/ml respectively.After transfection,the expression of Calpain-10 was down-regulated,and the expression of HER2 protein was up-regulated,while the concentration of HER2-ECD in the medium turned negative.The expression of Calpain-10 was up-regulated by transfection,and it was found that the expression of HER2 protein was down regulated by detection again,while the concentration of HER2-ECD in culture medium was increased.Conclusions: 1.The positive rate of s HER2-ECD was correlated with the later clinical stage,lymph node metastasis,poor tissue differentiation,overexpression of HER2 and short disease-free and total survival time,suggesting that the positive rate of HER2-ECD might be one of the predictors of poor prognosis in breast cancer patients.2.The high expression of Calpain-10 protein in breast tumor tissue can be accompanied by the high expression of t HER2 in tumor tissue and the positive expression of s HER2-ECD in peripheral blood,suggesting that Calpain-10 may be involved in the process of ECD in t HER2 being cut into blood.The low expression of Calpain-10 in tumor tissue is related to DFS in breast cancer patients.3.The expression of Calpain-10 and HER2 protein were down-regulated in HER2 high expression breast cancer cell lines,but the m RNA level did not change.HER2-ECD in the medium changed from positive to negative.The expression of Calpain-10 was up-regulated,the expression of HER2 protein was down regulated,but the m RNA level did not change,and the concentration of HER2-ECD in the medium increased.The results showed that Calpain-10 could cut off ECD from HER2 and regulate HER2 protein expression. |
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