| Objective Phyllodes tumours(PTs) are rare fibroepithelial breast tumors.PTs are classified into benign,borderline and malignant categories as recommended by the World Health Organization(WHO) classification of tumors of the breast.Most PTs are benign;however,recurrences are not uncommon,and a relatively small number of patients will develop hematogenous metastases,particularly following a diagnosis of malignant PTs.Recent research has focused on the identification of potential molecular biomarkers that may have prognostic value.Our previous research demonstrated a striking difference between chromosomal aberrations in benign,borderline and malignant PTs,as detected using comparative genomic hybridization(CGH).The analysis of most frequent abnormal chromosomal regions in the three groups demonstrated a difference between benign and borderline or malignant PTs in gains of chromosomal material in the region of 4q12,indicating that this region might play an important role in the transformation from benign PTs to borderline and malignant PTs.c-kit(also known as CD117) is a type Ⅲ receptor tyrosine kinase(RTK) encoded by the KIT gene,which is a proto-oncogene located at 4q12 that encodes a transmembrane RTK.We determined c-kit expression in the stroma and epithelia of benign,borderline and malignant phyllodes tumors,respectively,as well as the relationship between c-kit expression in stromal elements and KIT gene copy number variations(CNVs).Previously reported Surveillance,Epidemiology,and End Results(SEER) data estimated the average annual incidence of malignant phyllodes tumor to be 2.1 cases per million women.Metastasis was likely to occur in the malignant PT of the breast.The current study includes a population database supported by the Cancer Hospital of Tianjin Medical University to analyze a large series of women who diagnosed with malignant PT of the breast.The primary objective of this analysis was to identify clinical and pathologic factors that predict for DFS,DMFS and OS,and evaluate the role of malignancy grading relative to the survival endpoint.Methods 1.We included 348 female patients,who were diagnosed with PT and underwent surgical excision at the Cancer Hospital of Tianjin Medical University from March 2005 to June 2014.There were 113 malignant phyllodes tumors in this period.Other 120 benign phyllodes tumors and 115 borderline phyllodes tumors were all selected through random sampling.Hematoxylin and eosin(H&E)-stained sections of samples from each case were reviewed by three senior pathologists to confirm the diagnosis and classify all PTs according to the WHO classification.Patient cases analyzed included both primary tumors and recurrences.All 348 cases were evaluated for c-kit expression via IHC.2.To study the clinical relevance of c-kit stromal expression and KIT gene CNVs,these cases were divided into positive and negative groups based on stromal cells staining for c-kit.H&E-stained sections of each block were used to identify areas of the tumor that were suitable for DNA extraction.3.The KIT gene copy number was assessed in genomic PCR assays via comparisons with coamplified β-actin(ACTB) because there is no evidence that chromosome 7 is overrepresented or lost in PTs.For genomic analyses of the KIT gene,the 2-??Ct method was adapted using SYBR green-based quantitative PCR(qPCR).According to the primer-design principle of genomic PCR and standard of q PCR,we chose both KIT exon 15 amplicon and exon 18 amplicon to evaluate the KIT CNVs.4.We included patients who were diagnosed with malignant PT and underwent surgical excision at Tianjin Medical University Cancer Institute and Hospital(Tianjin,China) from April 1987 to December 2013.The study included 188 cases.All tissues were fixed in 10 % buffered formalin and embedded in paraffin.For each case,three pathologists reviewed all archival slides stained with hematoxylin and eosin(H&E).Malignat PTs are dignosed when the tumor shows a combination of marker nuclear pleomorphism of stromal cells,stromal overgrowth defined as absence of epithelial elements in one low-power microscopic field containing only stroma,increased mitoses(≥10 per 10 HPF),increased stromal cellularity which is usually diffuse,and infiltrative borders.It is also diagnosed when malignant heterologous elements are present even in the absence of other features.The tripartite grouping of malignancy for the malignant PT was a combination of stromal atypia,stromal overgrowth,mitotic count,tumor differentiation and necrosis.The stromal elements of PTs were considered to be oncogenic;therefore,we consult the grading system of adult soft tissue sarcomas.The clinicopathologic parameters that were evaluated for each case included patient age at initial diagnosis,tumor size,recent growing history,pain,tumor laterality,surgical method,tumor recurrence,distant metastasis,and patient survival.Results 1.C-kit was assessed using IHC in 348 cases.The overall rate of positivity in the stroma was 46.8%.In addition,the rate was 24.2%,53.1% and 64.6%,respectively,in three grade PTs.There was significant variation in the expression of c-kit in the stroma according to grade(P<0.001).The c-kit-positive expression of the stromal elements increased as the malignancy of PTs increased(Pearson correlation coefficient was 0.319,P<0.001).To further explore this phenomenon,the positive staining of c-kit in stromal cells was analyzed via pairwise comparison.There were significant differences between benign PTs and borderline or malignant PTs in c-kit-positive stromal cells(P<0.001),while no distinction was found between borderline and malignant PTs(P=0.102).2.The epithelia in the majority of cases were c-kit-positive(98.2%),and the intensity was strong.The epithelial positivity rates were 100%,99.1% and 95%,respectively.There was significant variation in the expression of c-kit in the epithelia according to grade(P=0.014).The c-kit-positive expression of the epithelial elements decreased as the malignancy of PTs increased(Pearson correlation coefficient was 0.319,P<0.001).To further explore this phenomenon,the positive staining of c-kit in epithelia was analyzed via pairwise comparison,a slight difference was observed between benign and malignant PTs(P=0.013).3.We extracted DNA from all PTs;however,because of the poor stability and integrity of DNA in FFPE tissue and the requirements of q PCR,only 42 of these cases were successfully assessed for KIT gene CNVs via genomic PCR.The stromal elements of PTs were considered to be oncogenic;therefore,all the 42 cases were divided into either a positive or negative group in accordance with c-kit stromal staining status.In the experiment that used the primer of the KIT exon 15 amplicon,34(80.9%)cases had an increase in KIT gene copy number(in contrast with 5 normal breast tissue).Twenty-seven(93.1%)c-kit-positive cases and 7(53.8%) negative cases were verified by KIT gene copy number increases,and there were significant differences between the two series(P=0.003).4.The outcome was similar in the experiment that used the exon 18 sequence: 36(85.7%)of 42 cases was KIT gene copy number increased.There were 27(93.1%)c-kit-positive cases and 9(69.2%) negative cases among those with an increase in KIT gene copy number(P=0.041).5.The FNCLCC grading system is based on 3 parameters: tumor differentiation,mitotic index,and tumor necrosis.This system is similar to the condition of malignant PTs;Therefore,we consider the rule of FNCLCC grading system and the characteristics of malignant PTs as an integrated system in malignancy grading.These parameters are scored 1 to 2 for tumor stromal atypia and overgrowth,1 to 3 for tumor differentiadtion and mitotic index,and 0 to 2 for necrosis.A 3-grade system is obtained by summing the scores obtained for each of these parameters.Grade 1 is defined as a total of 4 to 6;grade 2 as a total of 7 to 9;and grade 3 as a total of 10 to 12.According to the malignancy grading system,of the 188 malignant PT cases,88(46.8%) were classified as low grade,77(41%) as intermediate grade,and 23(12.2%) as high grade.6.In our study population,the features of distant metastasis and death associated with the malignancy grade of malignant PT(P<0.001).In addition,the malignancy grading of malignant PT could influence the choice of surgical procedure(P<0.001).Although there were no significant differences between patient age,tumor size and malignancy grade,bigger tumor size(≥ 5 cm) and younger age(< 35 years) were more frequently present in the high-grade malignant PT.7.A total of 37 women(19.7%)were documented histologically to have recurrences during the follow-up period.Tumor size,recent growth,pain,history of benign or borderline PTs and laterality were not associated with recurrent disease.Younger age(P=0.040)and tumor with malignant heterologous elements(P=0.013)were statistical associated with recurrence.The patients who undergo mastectomy are more frequently occur the local recurrence than that with the wide excision(P=0.031).The surgery procedure was rejected after multivariate analysis.8.A total of 10 women(5.3%)were documented histologically to have distant metastasis during the follow-up period,1(1.1%) were classified as low grade,3(3.9%)as intermediate grade,and 6(26.1%) as high grade.The distant metastases were associated with younger age(P=0.033),tumor size(P=0.032),recent growth(P=0.047) and malignancy grade(P<0.001).For multivariate analysis,the distant metastases were associated with malignancy grade(P<0.001),recent growth(P=0.029) and tumor laterality(P=0.046).9.In this study,the follow-up period was from 15 to 251 months,and the median follow-up period was 43.5 months.In the DFS curves,there was a significant association of the present of heterologous elements(P=0.001) and younger age(P=0.008)with a better prognosis.Patients with wide excision had a more favorable disease-free survival than those with mastectomy(P=0.025).Tumor with heterologous elements(P=0.003)and younger age(P=0.012)retained independent prognostic significance after COX multivariate survival analysis.In the DMFS curves,the survival time was related to the younger age(P=0.029)and malignancy grade(P<0.001).The malignancy grade retained independent prognostic significance merely(P<0.001).To evaluate the factors for the OS,the malignancy grade was a significant influence factor for both univariate and multivariate survival curves(P<0.001,P=0.009).Conclusions In this study,we demonstrated a positive association between malignancy and c-kit expression in PT stromal elements.C-kit epithelial positivity was negatively associated with PT malignancy.C-kit overexpression in the stromal elements was related to KIT gene copy numbers increases.KIT regulates cell differentiation and proliferation,resists cell apoptosis,and plays a key role in tumor occurrence,development,migration and recurrence.KIT gene copy number increases may play a large role in PT generation and deterioration.The poor prognosis was associated with the malignant grading of malignant phyllodes tumors.The malignancy grading system should be generalized in the clinical practises.The present of heterologous elements,younger age,large tumor size,resent rapidly growth and higher malignancy grade were indicated the poor prognosis. |
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