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25 Assessment of pathological response to neoadjuvant chemotherapy in locally advanced breast cancer using the Miller-Payne system and TUNEL Christina SHINTIA MD, Hardjolukito ENDANG MD and Kartini DIANI MD Anatomical Pathology Department, Faculty of Medical Universitas Indonesia Abstract Background: Responses to neoadjuvant (before surgery) chemotherapy in locally advanced breast cancer (LABC) consist of clinical and pathological responses. Evaluating chemotherapy response is essential to predict survival rate and guide future chemotherapy. Until now, the evaluation of pathological response mainly involves quantitative assessment and is often inconsistent with clinical response. We explored the evaluation of pathological responses by both quantitative and qualitative methods, i.e. by evaluating the cellularity of tumour cells and the percentage of apoptosis. Materials and method: A cross-sectional analytical retrospective study was conducted on tissue of LABC diagnosed between 2010 and 2014 at the Department of Anatomical Pathology, Faculty of Medicine, Universitas Indonesia Cipto Mangunkusumo Hospital and Division of Surgical Oncology, Cipto Mangunkusumo Hospital. Biopsy and resection specimens were compared to evaluate reduction in cellularity, which were subsequently categorized into stages of Miller-Payne (MP) classification. The resection specimens were stained with TUNEL and the percentage of apoptosis was calculated. Reduction in cellularity between biopsy and mastectomy specimens with TUNEL staining is evaluated as a modification of the MP method. Results: We found no association between clinical responses with percentage of apoptosis, MP pathological responses and modified MP. There was a correlation between the dead cell evaluated by MP and by modified MP (p=0.000). Conclusion: Modified MP increases the degree or grading of pathological responses, but it does not improve the correlation with clinical response. Keywords: locally advanced breast cancer, apoptosis, Miller-Payne, neoadjuvant therapy Address for correspondence: Shintia Christina, jl. Pisangan Baru No 6. Rt 002, Rw 011, Jakarta Timur, Indonesia. Tel: +62-856-8856-905, Fax: +62-215817490. E-mail: [email protected] ORIGINAL ARTICLE INTRODUCTION Breast cancer is the most common cancer in Indonesian women based on the 2010 Indonesian National Cancer Registry, contributing to 18.6% of cancers in Indonesian women. The American College of Surgeons reported in the National Cancer Data Base that 9% of female breast cancer in America are in stage III, which is in contrast many other countries (Latin America, Asia, Africa) where more than half of breast cancer patients are at stage III and IV. 1,2 Responses to neoadjuvant (before surgery) chemotherapy are categorised to clinical and pathological responses. Evaluating chemotherapy response is essential to predict survival rate and to guide future chemotherapy. Clinical responses can be evaluated by two criteria, i.e. the WHO and Response Evaluation Criteria In Solid Tumor (RECIST). Both criteria provide evaluation based on assessment of reduced tumour size. 3 There are some systems to evaluate the pathological responses, such as the systems of Miller-Payne (MP), Chevallier and NSABP B-18, Pinder, Sataloff and Smith. 4,5 In the MP system, the responses are evaluated based on reduction in tumour cellularity between biopsy and mastectomy specimens. 2,5-7 The system includes the following classification, i.e. Grade 1: no change, no significant reduction in malignant cells; Grade 2: a minor loss of tumour cells (30%); Grade 3: reduction in tumour cells between 30% and 90%; Grade 4: disappearance of tumour cells > 90%; Grade 5: no malignant cells identifiable, DCIS may be present. Grade 1-4 are categorized as partial pathological response (pPR) and grade 5 as pathological complete response (pCR). 2 Malaysian J Pathol 2016; 38(1) : 25 – 32

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Assessment of pathological response to neoadjuvant chemotherapy in locally advanced breast cancer using the Miller-Payne system and TUNEL

Christina SHINTIA MD, Hardjolukito ENDANG MD and Kartini DIANI MD

Anatomical Pathology Department, Faculty of Medical Universitas Indonesia

Abstract

Background: Responses to neoadjuvant (before surgery) chemotherapy in locally advanced breast cancer (LABC) consist of clinical and pathological responses. Evaluating chemotherapy response is essential to predict survival rate and guide future chemotherapy. Until now, the evaluation of pathological response mainly involves quantitative assessment and is often inconsistent with clinical response. We explored the evaluation of pathological responses by both quantitative and qualitative methods, i.e. by evaluating the cellularity of tumour cells and the percentage of apoptosis. Materials and method: A cross-sectional analytical retrospective study was conducted on tissue of LABC diagnosed between 2010 and 2014 at the Department of Anatomical Pathology, Faculty of Medicine, Universitas Indonesia Cipto Mangunkusumo Hospital and Division of Surgical Oncology, Cipto Mangunkusumo Hospital. Biopsy and resection specimens were compared to evaluate reduction in cellularity, which were subsequently categorized into stages of Miller-Payne (MP) classification. The resection specimens were stained with TUNEL and the percentage of apoptosis was calculated. Reduction in cellularity between biopsy and mastectomy specimens with TUNEL staining is evaluated as a modification of the MP method. Results: We found no association between clinical responses with percentage of apoptosis, MP pathological responses and modified MP. There was a correlation between the dead cell evaluated by MP and by modified MP (p=0.000). Conclusion: Modified MP increases the degree or grading of pathological responses, but it does not improve the correlation with clinical response.

Keywords: locally advanced breast cancer, apoptosis, Miller-Payne, neoadjuvant therapy

Address for correspondence: Shintia Christina, jl. Pisangan Baru No 6. Rt 002, Rw 011, Jakarta Timur, Indonesia. Tel: +62-856-8856-905,Fax: +62-215817490. E-mail: [email protected]

ORIGINAL ARTICLE

INTRODUCTION

Breast cancer is the most common cancer in Indonesian women based on the 2010 Indonesian National Cancer Registry, contributing to 18.6% of cancers in Indonesian women. The American College of Surgeons reported in the National Cancer Data Base that 9% of female breast cancer in America are in stage III, which is in contrast many other countries (Latin America, Asia, Africa) where more than half of breast cancer patients are at stage III and IV.1,2

Responses to neoadjuvant (before surgery) chemotherapy are categorised to clinical and pathological responses. Evaluating chemotherapy response is essential to predict survival rate and to guide future chemotherapy. Clinical responses can be evaluated by two criteria, i.e. the WHO and Response Evaluation Criteria In

Solid Tumor (RECIST). Both criteria provide evaluation based on assessment of reduced tumour size.3 There are some systems to evaluate the pathological responses, such as the systems of Miller-Payne (MP), Chevallier and NSABP B-18, Pinder, Sataloff and Smith.4,5 In the MP system, the responses are evaluated based on reduction in tumour cellularity between biopsy and mastectomy specimens.2,5-7 The system includes the following classification, i.e. Grade 1: no change, no significant reduction in malignant cells; Grade 2: a minor loss of tumour cells (≤ 30%); Grade 3: reduction in tumour cells between 30% and 90%; Grade 4: disappearance of tumour cells > 90%; Grade 5: no malignant cells identifiable, DCIS may be present. Grade 1-4 are categorized as partial pathological response (pPR) and grade 5 as pathological complete response (pCR).2

Malaysian J Pathol 2016; 38(1) : 25 – 32

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Complete clinical regression does not have direct correlation with pCR, therefore, the evaluation of specimens obtained from mastectomy is the gold standard for determining response to therapy.6 The evaluation of pathological response following neoadjuvant chemotherapy has now become an essential independent prognostic factor.4,5 Thus far, the evaluation of residual tumour after neoadjuvant chemotherapy is only based on decreased/ reduced tumour cellularity. Tumour regression following neoadjuvant therapy may also be caused by apoptosis.8,9 Morphological changes in cells undergo apoptosis are condensation of chromatin, nuclear fragmentation, cell shrinkage and blebbing of plasma membrane.10-12 Residual tumour cells can still be seen. However, the determination of apoptosis in haematoxylin-eosin (HE) stained cells during the first stage of DNA fragmentation can be uncertain.13,14 Chemotherapy initiates apoptosis through extrinsic and intrinsic pathways, in which both pathways activate caspase.11 Caspase activation is a marker of ongoing cell damage, an important marker of the cell’s entry point into the apoptotic signaling pathway and it can be detected by immunohistochemistry. Caspase-3 has an important role as the most distal effector of caspase in the apoptosis pathway. Moreover, it binds to the apoptosis inhibitors and therefore, can prevent the development of apoptosis.12,15-17 TUNEL (Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate in situ nick end labeling) is a technique used to detect the early stage of DNA fragmentation, which is a hallmark of apoptosis. The assay is very sensitive for detecting early DNA fragmentation before apoptosis can be identified morphologically.12,13,18 Clinical responses are often inconsistent with pathological responses, especially since the first stage of DNA fragmentation is difficult to evaluate with certainty. In this study we evaluated pathological responses by both quantitative and qualitative methods, i.e. by evaluating the

cellularity of tumour cells and the percentage of apoptosis.9

MATERIALS AND METHODS

A cross-sectional analytical study was performed. The study was conducted at the Department of Anatomical Pathology, Cipto Mangunkusumo Hospital Universitas Indonesia between December 2014 and March 2015. The selected sample was all cases of locally advanced breast cancer which had undergone biopsy and mastectomy at Cipto Mangunkusumo Hosptial and had been treated with neoadjuvant therapy between 2010 and 2014. After completing neoadjuvant chemotherapy, the clinical response was evaluated based on WHO criteria (complete, partial, stable and progressive).19 Pathological response was evaluated by two observers (SHI and ESR) independently. It was assessed based on the MP system by comparing the tumour cellularity between biopsy and mastectomy. The percentage difference was calculated and response categorized according to the MP grading system described earlier.2,5-7 A modified MP (MMP) evaluation was performed by calculating the percentage of apoptosis using TUNEL in the resected tissue divided by percentage of tumour cellularity in biopsy tissues and added with the number of death cells as scored by the MP system (Table 1). For cases with MP grade 5, AE1/AE3 staining was used when necessary to confirm whether the apoptotic cells were epithelial (tumour) cells instead of other cells.

Statistics The correlation between clinical response and apoptosis percentage was analyzed using Mann-Whitney test. Fisher test was undertaken to determine whether there was a difference in clinical response between MP and MMP. Spearman correlation test was performed to

TABLE 1: Criteria for Modified Miller-Payne Classification

Biopsy (b) Resection+ TUNEL (rT) Δ(b-rT)

Grade 1 100 100% 0Grade 2 100 70-99% 1%-≤30%Grade 3 100 10-69% 31%-90%Grade 4 100 1-9% 91%-99%Grade 5 100 0 100%

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determine the correlation between clinical reduction of tumour mass and the number of tumour cell death.

RESULTS

There were 203 cases of breast cancer that had received neoadjuvant chemotherapy in Cipto Mangunkusumo Hospital between 2010 and 2014. 42 of these cases with tissue obtained from both biopsy and mastectomy procedures, were retrieved from the archives of Department of Anatomical Pathology, Faculty of Medicine Universitas Indonesia which serves the Cipto Mangunkusumo Hospital.

Evaluation of pathological responses based on the Miller-Payne systemThe results of pathological responses based on the MP system are presented in Table 2. The evaluation of MP pathological responses in our study did not reveal any cases of grade 4. Grade 1 cases showed reduction in cellularity between 0-8.11%; while for grade 2 cases, there was reduced cellularity of 12.50-27.63%. Grade 3 cases demonstrated reduction in celularity between 31.51-86.88% and grade 5 cases showed reduction of as much as 100%. For two grade 5 cases, AE1/3 staining were performed on 2 slides each and demonstrated that there was no epithelial tumour left (Figures 1 & 2).

Evaluation of apoptosis percentage There was no significant difference of cellularity in grade 1 cases between the specimens obtained from mastectomy and biopsies whereas in grade 2 cases, there was reduction in cellularity up to 20.57%. Grade 3 cases showed reduced cellularity between 31.98-85.46%, while the reduction for grade 4 and grade 5 cases were between 92.30-95.74% and as much as 100% respectively. The evaluation of pathological responses by the MMP system had caused altered scores in the grading system, i.e. 24 cases had

increased scores and 18 cases did not have any change (Tables 3 and 4).

Clinical responses Clinical responses were evaluated based on the WHO criteria. We found partial responses in 33 cases (78.57%), stable response/non-responsive cases in 6 subjects (14.28%) and progressive response in 3 cases (7.14%).

Correlation between clinical responses and total cell death and apoptosis The results of statistics showed that there was no correlation between clinical response and total cell death and apoptosis (p=0.108).

Correlation between clinical responses and pathological responses based on Miller-Payne grading system and the responses according to the modified Miller-Payne systemThe Fisher’s exact statistical test showed that there was no correlation between clinical responses and MP grading (p=1.000), as well as with MMP grading (p=0.655). Clinical reduction of tumour mass was correlated to the number of dead tumour cells, which was analyzed using Spearman test to demonstrate the correlation. It was not correlated to the MP system (p=0.177; r=0.212) and there was a weak correlation to the MMP system (p=0.609; r=0.081). To identify the correlation between clinical response and the number of dead tumour cells, Mann-Whitney test was undertaken. The evaluation on the number of dead tumour cells between MP and MMP systems did not reveal any correlation (p=0.198; p=0.108). To consider whether there is a difference between number of dead cell evaluated by MP and Modified MP system, the Wilcoxon test was performed and showed significant difference (p=0.000).

TABLE 2: Pathological response based on MP system

MP grading Σ Case Cellularity reduction

Grade 1 15 (35.71%) 0-8.11%Grade 2 8 (19.05%) 12.50-27.63%Grade 3 17 (40.48%) 31.51-86.88%Grade 4 0 0Grade 5 2 (4.76%) 100%

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FIG. 1: MP responses. (A) MP grade 1, biopsy. (B) MP grade 1, mastectomy (400x) (C) MP grade 2, biopsy. (D) MP grade 2, mastectomy (400x) (E) MP grade 3, biopsy. (F) MP grade 3, mastectomy (400x). (G) MP grade 5, biopsy. (H) MP grade 5, mastectomy (400x).

A

C

E

G

B

D

F

H

29

BREAST CANCER TUNEL

TABLE 3: Comparison of grade using the MP and Modified MP systems

∑ cases Mean percentage death cell ∑ cases total apoptotic cells MP with MP MMP Grade 1 15 -71,94 5 0Grade 2 8 20,38 1 20,57Grade 3 17 54,85 27 63,25Grade 4 0 - 7 94,56Grade 5 2 100 2 100

FIG.2: AE1/AE3 staining in two cases with grade 5 MP. (A) MP grade 5 response- a complete histological response with no residual tumour cells (HE, 100x). (B) AE1/3 staining (100x). (C) MP grade 5 response with foam cells and no residual tumour cells (HE,400x). (D) AE1/3 staining (400x).

DISCUSSION

Most cases included in our study were stage III B breast cancer (97.62%) and there was a case in stage IIIA (2.38%). The majority of cases, i.e. 41 (97.62%) were at T4. The average age of patients was 49 years. We found that the most common histological type was invasive carcinoma (no special type), comprising 80% of cases (32 out of 42 cases). The findings are consistent with published literature, which report

that the incidence of ductal type (70-75%) is more common than the lobular type (5-15%).7,20

Clinical responses in Cipto Mangunkusumo Hospital were evaluated based on WHO criteria. 33 cases had partial clinical responses, 6 stable responses and 3 progressive responses. Some reports suggest that partial clinical and pathological responses are the dominant groups (60-80%) while complete responses have a lower prevalence (3-30%).21

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TABLE 4: Change in pathological response grade using the MP and modified MP systems Grade MP Grade MP modification Number of cases

A. No change Grade 1 Grade 1 5 Grade 2 Grade 2 0 Grade 3 Grade 3 11 Grade 4 Grade 4 0 Grade 5 Grade 5 2 B. Change Grade 1 Grade 2 1 Grade 2 Grade 3 7 Grade 3 Grade 4 6 Grade 1 Grade 3 9 Grade 2 Grade 4 1

Pathological responses evaluated using the MP system were compared to the MMP system and showed increased grading of pathological response indicating better responses with MMP system. About 24 cases showed increase scores and 18 cases did not have any changes. There was a significant difference in the number of dead cells detected before TUNEL staining (MP) and after staining (Modified MP) (Figure 3).The results indicate that the detection of cells that have undergone apoptosis may reveal the true pathological responses. These findings are consistent with published literature suggesting that the mechanism of cell death in tumour following the administration of chemotherapy is actually through apoptosis.8,9

There was no statistical correlation found between clinical responses and apoptosis, which may be because most clinical responses were partial responses. Figure 4 shows that there is a tendency of positive correlation, i.e. the better the clinical responses, the more cells undergoing apoptosis. Chemotherapy induces cell death not only through the mechanism of apoptosis, but also through other non-apoptotic mechanisms such as necrosis, autophagy and mitotic catastrophe; however, apoptosis is the major mechanism that causes cell death.8,9,16,22

Therewasnosignificantcorrelationbetweenclinical responses and pathological responsesinourstudybecause(1)mostcaseshadpartialclinicalandpathologicalresponses,(2)thereis

A B

FIG. 3: (A) Tumour cells in a mastectomy with no obvious apoptosis on routine stain (H&E, 400x). (B) The same specimen. Apoptotic cells (brown nuclear staining) revealed with TUNEL (400x)

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awide rangeof eachgrade in theMP systemaswellasinclinicalresponsesand(3)differentmethodsforevaluatingclinicalandpathologicalresponses may cause undetected correlationbetween MP/Modified MP grading and theclinicalresponses.23Changesintissuesobtainedfrommastectomy (tumour bed) following theadministrationofneoadjuvantchemotherapyarestromal hyalinization, oedema, fibroelastosis,large sheets of stromal foamy histiocytes,lymphocytesaggregatesandhaemosiderophages.Areaoftumournecrosismaydevelopnodules,whichconsistofcollectionsofhistiocytesandcholesterolclefts.24Changesinnon-neoplastictissues obtained from mastectomy followingthe neoadjuvant treatment may result in nocorrelation between clinical and pathologicalresponses. Ourstudydidnotshowanycorrelation,eitherbetweenclinicalresponseandMP,orbetweenclinical response and Modified MP, as wellas between clinical reduction of tumourmassand the number of cell death (measured bothbyMP andModifiedMP techniques). Thesefindingsareconsistentwiththeresultsofsomestudies suggesting that clinical responses donot demonstrate the pathological responses;thereforepathologicalexaminationonspecimensobtained from mastectomy is essential toevaluatetherapeuticresponsesincetheclinicalresponsedoesnotcorrelatewiththepathologicalresponse.5,6,25 Therefore, the evaluation ofcellularity is more relevant to gauge tumour

response compared to measuring the tumoursize. We found two cases with partial clinical responses, but had complete pathological response and 1 case with increased tumour volume who had progressive clinical response and grade 3 MP system (the number of dead cell of 75.43%) and grade 4 Modified MP with the number of total dead cell and apoptosis as many as 92.30%. Changes in the stroma of mastectomy specimens due to chemotherapy, such as stromal fibrosis, fibromyxoid, fibroelastotic stroma and microcalcification, may also cause bias in clinical response evaluation.2,4,21,26 This phenomenon was found in two cases in our study.

ConclusionApoptosis does not correlate with clinical response. Modified MP increases the degree or grading of pathological responses, but it does not improve the correlation with clinical response. Pathological responses to neoadjuvant therapy as one of prognostic factors should be confirmed by histopathology examination using modified MP since the reduction of tumour size is not representative of the number of cell death. There are reactions in the non-neoplastic elements that may contribute to the discrepancy.

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FIG. 4: Correlation between clinical response and apoptotic percentage

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to�c

per

cent

age

Stable &progressive

Clinical response

Par�al

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