PR and HER2 expression – Implications for the practising oncologist

European Journal of Cancer 60 (2016) 40e48

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.ejcancer.com

Original Research

Effect of neoadjuvant chemotherapy on breast cancer phenotype, ER/PR and HER2 expression e Implications for the practising oncologist* Renu Gahlaut a, Aneliese Bennett a, Hiba Fatayer b, Barbara J. Dall c, Nisha Sharma c, Galina Velikova d, Tim Perren d,e, David Dodwell d, Mark Lansdown b, Abeer M. Shaaban f,g,* a

Histopathology, St James’s Institute for Oncology, St James’s University Hospital, Leeds, UK Breast Surgery, St James’s Institute for Oncology, St James’s University Hospital, Leeds, UK c Imaging and Breast Screening, St James’s University Hospital, Leeds, UK d St James’s Institute for Oncology, St James’s University Hospital, Leeds, UK e Leeds Institute of Cancer Medicine and Pathology, St James’s Institute of Oncology, St James’s University Hospital, Leeds, LS9 7TF, UK f Pathology, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre and the University of Birmingham, Birmingham, UK g University of Tanta, Egypt b

Received 25 September 2015; received in revised form 2 March 2016; accepted 9 March 2016

KEYWORDS Neoadjuvant chemotherapy; Breast cancer; Oestrogen receptor; Progesterone receptor; HER2

Abstract Purpose: To assess the effect of neoadjuvant chemotherapy (NACT) on breast cancer characteristics, hormone receptors and human epidermal growth factor receptor 2 (HER2) expression and whether testing should be repeated on residual tumours. Material and methods: Patients with primary operable breast cancer who received NACT at a single United Kingdom tertiary referral centre were included. Tumour type, grade (including details of mitotic grade, tubule formation and pleomorphism), oestrogen receptor (ER), progesterone receptor (PR) and HER2 status were compared between pre-treatment and posttreatment residual samples using tissue microarrays. A control group of paired core and excision tumours from patients who did not receive NACT was also assessed. Results: Two hundred forty-six cases and 113 controls were included. Pathological complete response (path CR) was achieved in 21.5% of patients. In those patients failing to achieve a path CR, a change in the histological type was noted in 29 out of 178 cases (16.3%, p < 0.001) with increase in the lobular and metaplastic types. Downgrading occurred in

* Part of this work was presented at San Antonio Breast Cancer Symposium, San Antonio, Texas 2013 (poster) and at The British Breast Group Meeting, York, UK, 21st January 2014 (oral presentation). * Corresponding author: Department of Cellular Pathology, Queen Elizabeth Hospital Birmingham, Edgbaston, Birmingham B15 2WB, UK. Tel.: (þ44) 01213713356; fax: (þ44) 01213713333. E-mail address: [email protected] (A.M. Shaaban).

http://dx.doi.org/10.1016/j.ejca.2016.03.006 0959-8049/ª 2016 Elsevier Ltd. All rights reserved.

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28.8%, due to significant reduction in mitotic rate and prominent tubule formation. A change in ER/PR/HER2 status occurred in 12%, 14.5% and 7.1% of cases, respectively, predominantly as a switch from negative to positive status for ER and from positive to negative status for HER2. Further alterations in expression levels were also noted. Minimal changes in the low ER/PR expressors and the HER2 2þ tumours were found in the control group. Conclusion: Significant changes in tumour morphology, grade, hormone receptors and HER2 status occur following NACT. We recommend testing on residual invasive carcinoma. A switch from negative to positive status warrants offering endocrine/trastuzumab-based therapy to this group of patients. ª 2016 Elsevier Ltd. All rights reserved.

1. Introduction Neoadjuvant chemotherapy (NACT) has traditionally been the standard management for inflammatory and locally advanced cancer and is increasingly being used for other types of primary breast carcinoma with the aim of downstaging and facilitating conservative surgery [1]. Testing the tumour core biopsy samples for oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) expression is a prerequisite for selecting patients into the neoadjuvant route and deciding on drug combinations [2]. There is paucity of data in the literature on whether the primary tumour characteristics, hormone receptors and HER2 expression change as a result of chemotherapy. Previous studies of small patient cohorts reported conflicting results ranging from non-significant or no change [3], to up to 46% switch in ER status following treatment [4]. Similarly, reported data on HER2 status ranged from no alteration [5] to up to 43% change [6]. Due to the patchy and contradictory results, there has been inconsistency in practice worldwide with uncertainty as to whether testing on residual carcinoma is warranted and if treatment options should be modified based on the final molecular profile of the tumour. A change in tumour profile following NACT may have important implications into the management of those patients and the requirement for repeat testing on residual carcinoma. The aim of this study, therefore, was to test the effect of neoadjuvant chemotherapy on tumour type, grade and molecular profile through analysing a large, wellcharacterised cohort of tumour samples from patients who underwent neoadjuvant chemotherapy in a single large tertiary referral centre. This is to assess whether retesting should be done on residual carcinoma and inform future management strategies for these patients. 2. Patients and methods Ethical approval was sought and obtained (REC ref, 06/ Q1206/180). Patients who underwent NACT for primary and operable invasive carcinoma, including inflammatory and locally advanced breast cancer, at a

single large United Kingdom (UK) tertiary referral breast centre (The Leeds Teaching Hospitals NHS Trust) were identified from the oncology and imaging databases. At the authors’ institution, all patients undergoing NACT are routinely offered baseline and follow-up magnetic resonance imaging (MRI) scans to assess response. 2.1. Study group Included in this study were patients with primary operable breast carcinoma who had received NACT between 1st January 2005 and 30th April 2013 followed by breast surgery, including patients who underwent diagnostic surgery to the axilla (sentinel node biopsy) before or after NACT. This period corresponds to the introduction of anti-HER2 therapy (from 2005 onwards). Exclusion criteria were - NACT patient who did not undergo surgery, - Metastatic breast cancer, and - NACT patients without MRI follow-up.

2.2. Data collection Comprehensive clinical data including chemotherapy regimen, type of surgery and imaging characteristics (mammography and MRI) were collected. The following pathological data, where available, were collected from the pathology reports on both pretreatment core biopsy sample and residual tumours: tumour type, tumour grade including individual scores for tubule formation, nuclear pleomorphism and mitoses and pathological response: classified into complete (no residual invasive carcinoma), partial (residual invasive carcinoma with histological evidence of tumour response), and no response (no evidence of tumour response) as previously described [7]. Slides of pre-treatment core biopsies were reviewed and representative block was selected for immunohistochemistry. Where only one paraffin block containing multiple cores was available, this was melted and one core transferred into a new paraffin block for further analysis.

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For residual invasive disease (pathological partial response and no response), tumour slides were retrieved from the pathology files, reviewed and marked for tissue microarray (TMA) construction. The corresponding tumour block was identified. 2.3. Control group As a control group, consecutive core biopsies and a representative tumour block from the surgical excision, from patients who did not undergo neoadjuvant chemotherapy within the same study period, were identified and stained for ER/progesterone receptor (PR)/HER2. 2.4. Tissue microarrays TMAs were constructed from the marked cores of the residual invasive carcinoma using a manual tissue microarrayer (MTA1; Beecher Instruments, USA) as previously described [8,9]. Briefly, two 0.6 mm core punches from the marked representative residual tumour section (central and peripheral where possible) were selected from each block and assembled into a TMA donor block with a 1-mm interval between cores. Of each TMA block, 3 mm sections were cut on a microtome (RM2255; Leica) onto Superfrost Plus slides (Surgipath) for immunohistochemistry. 2.5. Immunohistochemical staining For ER/PR and HER2 staining, optimised immunohistochemical protocols routinely used within the Leeds Teaching Hospitals NHS Trust diagnostic histopathology department and approved by UK National External Quality Assessment Scheme were followed in accordance with the standard operating procedures. The following primary antibodies were used on the DAKO Autostainer Link 48: ER (Novocastra Leica Microsystems, clone 6F11, 1:200), PR (Dako clone PgR 636, 1:800), and HER2 (Novocastra c-erbB-2 oncoprotein, clone CB11, 1:800). The Dako Envision Flexþ detection kit secondary detection system was used for the three antibodies. Each batch of immunostaining included positive controls of varying staining intensity and a negative control where the primary antibody was omitted. 2.6. Immunohistochemical scoring

ER and PR immunohistochemistry was scored using the Quick (Allred) score as per the standard diagnostic guidelines [10]. HER2 immunohistochemistry was reported as negative (score 0 or 1þ), equivocal (2þ) or positive (score 3þ). Equivocal cases were tested by FISH PathVysion probe to assess the amplification status of the HER2 gene. HER2 gene:chromosome 17 ratio of >2 was regarded as positive [10,11]. Where the tissue has cut out, ER/PR/HER2 status was obtained from the original pathology reports. 2.7. Statistical analysis Statistical analysis was done using the PAWS statistical package v.20. Analysis for pre- and post-treatment categorical variables including tumour type, grade, and Allred scores/HER2 was done using the chi-square test. Receptor status was also dichotomised into negative and positive using a cut-off value of Allred score 2 for ER/ PR and 3þ or 2þ fluorescence in situ hybridisation (FISH) positive for HER2 to define positivity. Fisher’s exact test was used to compare receptor conversion rate between study cases and the control group. All comparisons were two-sided and p value of 0.05 was considered significant. 3. Results A total of 246 patients fulfilled the inclusion criteria. Patients predominantly received anthracycline-based therapy for six cycles. The majority (73.3%) of the HER2-positive tumours received trastuzumab-based therapy (Herceptin) in addition to taxanes. None of the HER2-positive patients received concurrent pertuzumab. The clinicopathological characteristics of all patients, pre-treatment, are summarised in Table 1. Over twothirds of the patients were aged 50 or under with a mean age of 47.8 (10.3) and a range of 23e68 years. On pretreatment core biopsy, the tumours were predominantly of ductal no special type (NST, 86.5%) and grade 3 differentiation (48.6%). Almost two-thirds of the tumours (64.9%) were ER positive and 19.9% were HER2 positive. Cases with pathological complete response (path CR, 21.5%) were excluded from the analysis. Details of those cases are shown in Table 2. 3.1. Histological type

Stained TMA immunohistochemistry slides were scanned using the Scanscope XT (AperioTM) at 40 magnification scored digitally. All immunohistochemical sections, including pre-treatment cores and residual post-treatment samples, were scored by one pathologist (RG) and then reviewed by a specialist breast pathologist (AMS). Discordant cases, low expressors and selected random cores were jointly reported. Both pathologists were appropriately blinded.

There was a change in the histological type following chemotherapy in 29 out of 178 cases (16.3%) of those patients who failed to achieve a path CR with NACT and this was statistically highly significant (chi squared, p < 0.001). Fifteen ductal NST carcinomas were classified into different histological types following treatment. Of note, six NST carcinomas showed mixed lobular features on excision and four showed

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metaplastic phenotype. Details of the histological type before and after NACT treatment are shown in Table 3.

Table 1 Clinicopathological features of the study cohort. Parameters

Number (%)

3.2. Tumour grade

Age 50 >50 Type Ductal NST Lobular Mixed ductal/lobular Apocrine Metaplastic Mixed other N/A Grade 1 2 3 N/A ER status (Allred score) Negative Positive PR (Allred score) Negative Positive HER2 0/1þ 2þ 3þ N/A

136 (68.3) 63 (31.7) 180 12 9 2 3 1 1

(86.5) (5.8) (4.3) (1) (1.4) (0.5) (0.5)

9 85 101 13

(4.3) (40.9) (48.6) (6.3)

73 (35.1) 135 (64.9) 99 (47.6) 109 (52.4) 162 5 40 1

(77.9) (2.4) (19.2) (0.5)

ER, oestrogen receptor; N/A, not applicable; NST, no special type; PR, progesterone receptor.

Clinicopathological features of the cohort that showed path CR (n Z 53). Age Mean: 50.08  10.6 Type Ductal NST Lobular Mixed ductal/lobular Apocrine Metaplastic Other Grade 1 2 3 N/A ER status (Allred score) Negative Positive PR status (Allred score) Negative Positive HER2 Negative Positive Deceased Yes No

Table 4 shows the distribution of tumour grade pre- and post-NACT. Downgrading (decrease in overall grade by at least one grade) occurred in 28.7% of cases and upgrading (increase in overall grade by at least one grade) in 13.8%. The downgrading was predominantly due to a decrease in the mitotic count (p Z 0.002) with prominence of tubule formation (p Z 0.04) within the residual invasive carcinoma, Fig. 1A and B. Grade 1 and 2 tumours comprised 46.1% of cases on pre-treatment core biopsy. This increased to 64.1% following treatment. The proportion of grade 3 carcinoma decreased from 48.5% pre-treatment to 32.3% post-NACT. 3.3. Changes in ER expression Using a cut-off 2/8 to define positivity, there was a change in the ER status in 16 out of 133 available tumour pairs (12%). A total of seven cases changed from ER positive to negative and nine from negative to positive. This difference was statistically highly significant (p < 0.001). In addition changes in the level of expression, without alteration of the ER status, was observed as shown in Table 5. 3.4. Changes in PR expression

Table 2

Parameter

43

Number

%

Range: 24e76 years 43 3 2 1 2 2

81.13 5.66 3.77 1.89 3.77 3.77

1 16 32 5

1.89 30.19 60.38 9.43

34 19

64.2 35.8

36 17

67.9 32.1

41 12

77.36 22.64

11 42

20.75 79.25

ER, oestrogen receptor; N/A, not applicable; NST, no special type; path CR, pathological complete response; PR, progesterone receptor.

The PR status showed a significant change between preand post-treatment tumour samples in 14.5% of cases (p < 0.001). Thirteen cases changed status from PR positive to negative and five negative to positive. Further variation in the Allred score without affecting the PR status was noted in nine cases that showed change in the PR expression level of two or more Allred scores. 3.5. Change in HER2 expression Eighty-eight HER2-negative and 17 HER2-positive tumours showed the same profile following NACT treatment. A discordant HER2 status following treatment was noted in 8 out of 133 cases (7.1%). Five cases changed profile from 3þ overexpression to HER2 negative, of which three had a final score of 0, one scored 1þ and one scored 2þ, FISH negative. Two cases changed from negative to 3þ following treatment. This was statistically highly significant (p < 0.001). 3.6. Control group A total of 113 paired core biopsy and surgical excision samples from patients who did not receive NACT was identified.

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Table 3 The distribution of tumour type in pre-treatment core biopsy and post-treatment residual invasive carcinoma. Pre-NACT tumour type

Post-NACT tumour type Ductal NST

Lobular

Ductal NST Lobular Mixed ductal lobular Mixed ductal micropapillary Apocrine Metaplastic Tubular Mixed micropapillary and mucinous N/A Total

140 2 3 0

0 6 0 0

1 0 0 0 0 146

Total

Mixed ductal lobular

Mixed ductal micropapillary

Apocrine

Metaplastic

Mixed micropapillary/ mucinous

Tubular

N/A

6 3 4 0

2 0 0 0

1 0 0 0

4 0 0 0

1 0 0 0

1 0 0 0

1 0 1 0

156 11 8 1

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 3 0 0

0 0 0 0

0 0 0 0

0 0 0 0

1 3 0 0

0 6

0 13

0 2

1 2

0 7

0 1

0 1

0 2

0 180

Note that comparison is made with partial responders where residual invasive tumour was identified and could be reliably typed. NACT, neoadjuvant chemotherapy; N/A, not applicable; NST, no special type.

Of these, five cases (4%) showed a discordant HER2 profile. All discordant cases scored 2þ by HER2 immunohistochemistry. In two cases, only a small amount of tumour tissue was available for assessment on core biopsy. None of the discordant cases were of HER2 score 0, 1þ or 3þ on core biopsy. ER was discordant between core and excision in 4.5% of cases (3 out of 67 available pairs); all belonged to the lowexpressor category (two cases scored 4 on core and 0 on surgical excision and one case scored 0 on core and 4 on excision). Similarly for PR, the rate of discordance was 3.3% (2 out of 60); both classified as negative Allred score 0 on core but showed minimal weak staining on excision (score 2/8). 3.7. Comparison on marker conversion in both groups We observed a 12% conversion rate in ER status (6.77% changed from ER negative to positive) in patients receiving NACT. In the control group, the conversion rate was 4.5%. Based on Fisher’s exact test, the difference in the conversion rate between the two groups of patients was not statistically significant (difference Z 7.5%, p Z 0.124). For PR, the corresponding conversation Table 4 The distribution of tumour grade in pre-treatment core biopsy and post-treatment residual invasive carcinoma. Pre-NACT grade

1 2 3 Total

Post-NACT grade

Total

1

2

3

N/A

4 16 2 22

4 51 30 85

0 19 45 54

1 1 4 6

9 77 81 167

Cases with residual DCIS only or a tiny amount of invasive disease were excluded. NACT, neoadjuvant chemotherapy; N/A, not applicable; DCIS, ductal carcinoma in situ.

rates were 14.5% and 3.3%, and the difference was statistically significant (difference Z 11.2%, p Z 0.023). The difference in the conversation rate between the two patient groups for HER2 was 7.1% versus 4% (difference Z 3.1%, p Z 0.57). 4. Discussion In this study, we report significant changes in tumour morphology, grade and receptor expression following neoadjuvant chemotherapy. The duration of the study cohort, 2005e2013, was selected to reflect current neoadjuvant practice including anti-HER2 therapy and allow for long follow-up. To our knowledge, this is the largest and most comprehensive study to date to analyse the effect of neoadjuvant chemotherapy on tumour characteristics and the expression of ER, PR and HER2. Several pre-analytical and analytical factors (such as variation in fixation and ischaemic time, quality of staining, observer variation) can result in discrepancies in ER/PR and HER2 status. Our study includes a control group of tumour samples from the same study period and undergoing the same fixation, processing and staining protocols. The small proportion of marker change within this control group supports that the changes identified in the test group are due to genuine treatment effect. There is paucity of data in the literature on the effect of NACT on tumour characteristics including type and grade. Earlier observations showed that following chemotherapy, tumours tend to acquire a lobular morphology due to a combination of reduced cellularity and abundant fibrosis [12]. Our findings confirm those observations with a large proportion of cases classified as of mixed ductal and lobular type following therapy (13 post-treatment versus 4 pre-treatment). Furthermore, four ductal carcinomas were classified as

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Fig. 1. Changes in histological grade and HER2 status following neoadjuvant chemotherapy. (A) Pre-treatment grade 2 invasive grade 2 carcinoma of solid pattern and conspicuous mitoses. (B) The same tumour post-treatment is grade 1 with tubule formation and no mitotic figures. Note the associated areas of tumour regression with stromal fibrosis and vascular proliferation as a result of chemotherapy. (C) Pre-treatment HER2 sample showing membranous staining (score 2þ). (D) Same tumour post-treatment with trastuzumab showing loss of HER2 protein expression. (E) Pre-treatment HER2-negative tumour (score 1þ). (F) Same tumour following neoadjuvant therapy (score 3þ).

Table 5 Details of ER Allred score pre- and post-treatment. ER pre-NACT

0 2 3 4 5 6 7 8 Total

ER post-NACT

Total

0

2

3

4

5

6

7

8

36 3 2 2a 0 0 1a 2a 46

3b 0 0 0 0 0 0 1a 4

3 0 1 0 1b 0 0 0 5

2a 1a 0 0 0 0 3b 1b 7

1a 0 0 0 2 4 1b 2b 10

0 0 0 0 0 0 3 1b 4

1a 0 1b 0 0 2 3 7 14

2a 0 0 1b 2b 2b 9 27 43

48 4 4 3 5 8 20 41 133

ER, oestrogen receptor; NACT, neoadjuvant chemotherapy. a Change in ER status from positive to negative or negative to positive following treatment. b Change in the level of expression of two or more Allred scores without change to the ER final status.

metaplastic carcinomas following NACT which may reflect an epithelial mesenchymal transition (EMT) as a mechanism of resistance. EMT has been shown in vitro to be associated with chemoresistance [13]. To our knowledge, this is the first report on this phenomenon within patient samples. In a small study of 40 pre- and post-treatment samples, Adams et al. [14] showed a significant decrease in the mitotic index following NACT but this was not associated with a significant change in the histological

grade in their series. Our findings, however, highlight significant changes in the proportion of tubule formation and mitotic count leading to downgrading of tumours in approximately a third (28.74%) of the 167 paired cancers available for grade assessment. Excellent concordance in receptor status between core and excision has been reported in patients who proceed to surgery without prior chemotherapy. Dekker et al. [15] reported 99.1% concordance of ER results in their own series and 93.7% in pooled data from 20 studies. Therefore, testing for hormone receptors and HER2 on diagnostic core biopsies has become the standard practice. In the current study, a significant proportion of tumours that received NACT changed the molecular profile (12% for ER, 14.5% PR, and 7.1% HER2). Only a small percentage of discordance (less than 5% for all three markers) was seen in the control group. These latter cases were of the HER2 2þ immunohistochemical profile and of the low-level hormone receptor expressors. In the NACT group, however, changes from strong to negative staining and vice versa was noted in all three markers, in addition to changes in the levels of expression, supporting a genuine effect of NACT on tumour profile. The differences, in conversion rate, however were statistically significant only for PR immunohistochemistry. Thus, there was no statistically significance difference between ER, HER2 status between cases and the control group.

46

Table 6 Summary of the previous studies of comparison of hormone and HER2 status pre- and post-NACT. Total number (paired samples)

Method

ER

PR

HER2

Hawkins, 1990 [17]

30 (of whom 13 received prior endocrine therapy) 99, 45 of whom received concomitant endocrine treatment (71) 31 (FNA samples)

Retrospective, no control group

No change in expressiona

NT

NT

Prospective, no control group

NS

NS

NS

Prospective þ control group

42

Retrospective, no control group

58% change, 35% increase, 23% decrease (NS) NT

NT

Vincent-Salomon et al., 2002 [18] Taucher et al., 2003 [34]

46% change (p Z 0.04) 42% decrease, 4% increase 4.8% mainly pos to neg

180

Prospective, no control group

Pos to neg (p Z 0.005)

NT

Burcombe et al., 2005 [3]

90

Prospective, no control group

5.9% change neg to pos

Kasami et al., 2008 [5] Hurley et al., 2006 [6] Adams et al., 2008 [14]

173 23 57 (26)

Prospective þ control group Prospective trial Retrospective, no control group

Pos to neg (p Z 0.02) scored as neg, 1þ, 2þ, 3þ 2.5% change (3 patients (both way) 11% neg to pos (NS) NT 7.7% pos to neg

Mittendorf et al., 2009 [24] Kinsella et al., 2012 [20] Dede et al., 2013 [35]

25 35 63

Prospective trial, no control group Prospective, no control group Retrospective, no control group

20% neg to pos No change 5.7% pos to neg (NS)

Li et al., 2013 [21]

124

Retrospective, no control group

NT

NT

Guaneri et al., 2013 [30]

107

NT

NT

Yang et al., 2013 [22]

113 (113)

Retrospective (two groups: NACT alone and NACT with Herceptin) Retrospective þ control group

15.9% pos to neg and neg to pos

Cockburn et al., 2014 [19] Zhou et al., 2015 [36]

172 (133) 107

Retrospective þ control group Retrospective, no control group

12.4% pos to neg and neg to pos 14 pos to negb 14% more pos to neg

8% change (5 cases 2þ to 3þ and 4 cases 3þ to 2þ) No change 43% loss by FISH 26.9% from neg (score 0 or 1) to pos (score 2 or more) 32% loss by FISH 18.8 pos to neg (NS) 17% change (neg to 2þ in 3 cases, from 2þ and 1þ to 0 in 2 patients) Change in 23.4% by IHC only, stable by FISH 40% loss in NACT only group and 14.7% in NACTþherceptin 15% pos to neg and neg to pos

Current study

246 (133)

Retrospective þ control group

12% more neg to posc

Bottini et al., 1996 [16]

Makris et al., 1999 [4]

15.6% pos to neg (p Z 0.045) NT 19.2% pos to neg 3.8% neg to pos NT 23.6% pos to neg (significant) 21.7% from pos to neg and 9% pos to neg (NS)

29 pos to negb 14.9% significant decrease, mainly in large and node pos cancer 14.5%c more pos to neg

NT

27.2% pos to neg 4.7% more pos to neg 7.1% mainly pos to neg

ER, oestrogen receptor; FISH, fluorescence in situ hybridisation; NACT, neoadjuvant chemotherapy; neg, negative; NS, not significant; NT, not tested; pos, positive; PR, progesterone receptor; FNA, fine needle aspiration; IHC, immunohistochemistry. a Measured by cytosol concentration. b Authors used a cut-off value of 5% to define positivity. c Using a cut-off of Allred score 2 to define positivity.

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Authors, year

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Previous studies on comparison between pre- and post-treatment molecular marker results have yielded conflicting results. Some, generally earlier, studies reported non-significant changes [16e18], whereas others [6,14,19] showed significant changes in hormone receptors and HER2 status. Some authors reported that PR was the only marker with significant change in expression levels following treatment [5,20] and that changes in HER2 status were noted on testing by immunohistochemistry only with a stable profile by FISH [5,21]. Others, however, reported up to 43% change in HER2 status by FISH testing following neoadjuvant treatment including trastuzumab [6]. There are several possible explanations for the differences in the conclusions of previous studies. First, patients received different chemotherapy regimens, for various duration and some studies, particularly earlier ones, did not include trastuzumab for HER2-positive tumours. Furthermore, the assessment of ER/PR/HER2 in tumour tissue has evolved remarkably over the last few years. Earlier studies analysed the concentration of ER in whole samples in cytosol of whole tissue extracts [17] which included non-tumorous components such as normal breast, stroma, inflammatory cells and also in situ disease. The cut-off values to define hormone positivity was variable at 1% (22), 5% (19) and 10% (14) [23,20] with some studies using the Allred score [23] as per the current study. Finally, the number of patients varied considerably in previous series from only few patients [17,18,24] to larger cohorts [19,22] and the majority did not include a control group to adjust for pre-analytical issues and interobserver variation, see Table 6. It has been postulated that NACT exerts a modulatory effect on hormone receptors and HER2 as well as on other markers’ expression. It is plausible that chemotherapy targets sensitive cells leaving insensitive clones behind. The change in receptor status may be a survival mechanism for cancer cells [25]. It has also been suggested that low circulating levels of oestrogen, as a result of chemotherapy, may lead to down-regulation of hormone receptors in tumour cells and oestrogen independent growth [26]. Furthermore, ER, PR, and HER2 are highly inter-dependent and modulating one receptor can change the others [27]. NACT has also been shown to induce a switch in somatic mutations for TP53 and PIK3CA [28] which is associated with better prognosis. Biologically, a change in hormone receptor status, for example, from ER/PR negative to positive, would provide a treatment option for patients who would otherwise be denied endocrine therapy if management decisions were to be made solely on the pre-treatment biopsy. In the metastatic setting, an unstable ER and HER2 status correlated with poor survival [29]. Previous, albeit small, studies have shown that change in the molecular profile following neoadjuvant treatment was associated with better prognosis. Tumours that switched from HER2 positive to negative following treatment

47

had significantly shorter recurrence-free survival [24] and a higher risk of relapse [30] compared with tumours of stable profile. Change to positive hormone receptor status after NACT was significantly correlated with better OS and DFS [31]. Hirata et al. [23] showed that patients with hormone receptor conversion that received endocrine therapy had significantly better overall survival than those with conversion without adjuvant endocrine therapy. Those authors therefore recommended that endocrine therapy should be administered to tumours that are hormone receptor positive on either pre- or post-treatment biopsy. Current American Society of Clinical Oncology/ College of American Pathologist [32] and UK [33] guidelines have not addressed this increasingly used mode of therapy and no guidance has been provided on testing of tumour samples in the neoadjuvant setting. The practice for neoadjuvant patients is currently variable worldwide. Some centres, including the authors’ institutions following this study, repeat testing routinely on residual tumour samples following NACT as is the practice for recurrent and metastatic tumours. Others rely on pre-treatment profiling only. Based on our findings, we recommend repeat testing of hormone receptors and HER2 following neoadjuvant treatment. This is particularly important for ER/PR and/or HER2negative pre-treatment tumours as these may switch to a positive status following therapy. We do not recommend withholding treatment for positive tumours if the status changes to negative following NACT. Offering patients adjuvant endocrine and/or trastuzumab-based therapy based on the initial pre-treatment or final post-treatment profile appears appropriate. It would be vital to collect data on those patients, their response to therapy and survival to provide evidence for future clinical practice. Conflict of interest statement The authors have no conflict of interest.

Acknowledgement This work was supported by a grant from Leeds CRUK (482442).

References [1] Thompson AM, Moulder-Thompson SL. Neoadjuvant treatment of breast cancer. Ann Oncol 2012;23(Suppl. 10):x231e6. [2] National Institute for Health and Care Excellence. Early and locally advanced breast cancer: diagnosis and treatment, NICE guidelines [CG80]. NICE; 2009. [3] Burcombe RJ, Makris A, Richman PI, Daley FM, Noble S, Pittam M, et al. Evaluation of ER, PgR, HER-2 and Ki-67 as predictors of response to neoadjuvant anthracycline chemotherapy for operable breast cancer. Br J Cancer 2005;92(1): 147e55.

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[4] Makris A, Powles TJ, Allred DC, Ashley SE, Trott PA, Ormerod MG, et al. Quantitative changes in cytological molecular markers during primary medical treatment of breast cancer: a pilot study. Breast Cancer Res Treat 1999;53(1):51e9. [5] Kasami M, Uematsu T, Honda M, Yabuzaki T, Sanuki J, Uchida Y, et al. Comparison of estrogen receptor, progesterone receptor and Her-2 status in breast cancer pre- and postneoadjuvant chemotherapy. Breast 2008;17(5):523e7. [6] Hurley J, Doliny P, Reis I, Silva O, Gomez-Fernandez C, Velez P, et al. Docetaxel, cisplatin, and trastuzumab as primary systemic therapy for human epidermal growth factor receptor 2positive locally advanced breast cancer. J Clin Oncol 2006; 24(12):1831e8. [7] Pinder SE, Rakha EA, Purdie CA, Bartlett JM, Francis A, Stein RC, et al. Macroscopic handling and reporting of breast cancer specimens pre- and post-neoadjuvant chemotherapy treatment: review of pathological issues and suggested approaches. Histopathology 2015;67(3):279e93. [8] Shaaban AM, Ball GR, Brannan RA, Cserni G, Di Benedetto A, Dent J, et al. A comparative biomarker study of 514 matched cases of male and female breast cancer reveals gender-specific biological differences. Breast Cancer Res Treat 2012;133(3):949e58. [9] Pinder SE, Brown JP, Gillett C, Purdie CA, Speirs V, Thompson AM, et al. The manufacture and assessment of tissue microarrays: suggestions and criteria for analysis, with breast cancer as an example. J Clin Pathol 2013;66(3):169e77. [10] National Coordinating Group for Breast Screening. Pathology Reporting in Breast Cancer Screening: NHSBSP Publication No 58. 2005. [11] Bartlett JM, Starczynski J, Atkey N, Kay E, O’Grady A, Gandy M, et al. HER2 testing in the UK: recommendations for breast and gastric in-situ hybridisation methods. J Clin Pathol 2011;64(8):649e53. [12] Carder P. Typing breast cancer following primary chemotherapy. Histopathology 1999;35(6):584e5. [13] Jiang L, He D, Yang D, Chen Z, Pan Q, Mao A, et al. MiR-489 regulates chemoresistance in breast cancer via epithelial mesenchymal transition pathway. FEBS Lett 2014;588(11):2009e15. [14] Adams AL, Eltoum I, Krontiras H, Wang W, Chhieng DC. The effect of neoadjuvant chemotherapy on histologic grade, hormone receptor status, and HER2/neu status in breast carcinoma. Breast J 2008;14(2):141e6. [15] Dekker TJ, Smit VT, Hooijer GK, Van de Vijver MJ, Mesker WE, Tollenaar RA, et al. Reliability of core needle biopsy for determining ER and HER2 status in breast cancer. Ann Oncol 2013;24(4):931e7. [16] Bottini A, Berruti A, Bersiga A, Brunelli A, Brizzi MP, Marco BD, et al. Effect of neoadjuvant chemotherapy on Ki67 labelling index, c-erbB-2 expression and steroid hormone receptor status in human breast tumours. Anticancer Res 1996;16(5B):3105e10. [17] Hawkins RA, Tesdale AL, Anderson ED, Levack PA, Chetty U, Forrest AP. Does the oestrogen receptor concentration of a breast cancer change during systemic therapy? Br J Cancer 1990;61(6): 877e80. [18] Vincent-Salomon A, Jouve M, Genin P, Freneaux P, SigalZafrani B, Caly M, et al. HER2 status in patients with breast carcinoma is not modified selectively by preoperative chemotherapy and is stable during the metastatic process. Cancer 2002; 94(8):2169e73. [19] Cockburn A, Yan J, Rahardja D, Euhus D, Peng Y, Fang Y, et al. Modulatory effect of neoadjuvant chemotherapy on biomarkers expression; assessment by digital image analysis and relationship to residual cancer burden in patients with invasive breast cancer. Hum Pathol 2014;45(2):249e58. [20] Kinsella MD, Nassar A, Siddiqui MT, Cohen C. Estrogen receptor (ER), progesterone receptor (PR), and HER2 expression

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

pre- and post- neoadjuvant chemotherapy in primary breast carcinoma: a single institutional experience. Int J Clin Exp Pathol 2012;5(6):530e6. Li P, Liu T, Wang Y, Shao S, Zhang W, Lv Y, et al. Influence of neoadjuvant chemotherapy on HER2/neu status in invasive breast cancer. Clin Breast Cancer 2013;13(1):53e60. Yang YF, Liao YY, Li LQ, Xie SR, Xie YF, Peng NF. Changes in ER, PR and HER2 receptors status after neoadjuvant chemotherapy in breast cancer. Pathol Res Pract 2013;209(12):797e802. Hirata T, Shimizu C, Yonemori K, Hirakawa A, Kouno T, Tamura K, et al. Change in the hormone receptor status following administration of neoadjuvant chemotherapy and its impact on the long-term outcome in patients with primary breast cancer. Br J Cancer 2009;101(9):1529e36. Mittendorf EA, Wu Y, Scaltriti M, Meric-Bernstam F, Hunt KK, Dawood S, et al. Loss of HER2 amplification following trastuzumab-based neoadjuvant systemic therapy and survival outcomes. Clin Cancer Res 2009;15(23):7381e8. van de Ven S, Smit VT, Dekker TJ, Nortier JW, Kroep JR. Discordances in ER, PR and HER2 receptors after neoadjuvant chemotherapy in breast cancer. Cancer Treat Rev 2011;37(6):422e30. Bines J, Oleske DM, Cobleigh MA. Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer. J Clin Oncol 1996;14(5):1718e29. Dati C, Antoniotti S, Taverna D, Perroteau I, De Bortoli M. Inhibition of c-erbB-2 oncogene expression by estrogens in human breast cancer cells. Oncogene 1990;5(7):1001e6. Jiang YZ, Yu KD, Bao J, Peng WT, Shao ZM. Favorable prognostic impact in loss of TP53 and PIK3CA mutations after neoadjuvant chemotherapy in breast cancer. Cancer Res 2014; 74(13):3399e407. Yang YF, Liao YY, Yang M, Peng NF, Xie SR, Xie YF. Discordances in ER, PR and HER2 receptors between primary and recurrent/metastatic lesions and their impact on survival in breast cancer patients. Med Oncol 2014;31(10):214. Guarneri V, Dieci MV, Barbieri E, Piacentini F, Omarini C, Ficarra G, et al. Loss of HER2 positivity and prognosis after neoadjuvant therapy in HER2-positive breast cancer patients. Ann Oncol 2013;24(12):2990e4. Tacca O, Penault-Llorca F, Abrial C, Mouret-Reynier MA, Raoelfils I, Durando X, et al. Changes in and prognostic value of hormone receptor status in a series of operable breast cancer patients treated with neoadjuvant chemotherapy. Oncologist 2007;12(6):636e43. Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 2013; 31(31):3997e4013. Rakha EA, Pinder SE, Bartlett JM, Ibrahim M, Starczynski J, Carder PJ, et al. Updated UK Recommendations for HER2 assessment in breast cancer. J Clin Pathol 2014. Taucher S, Rudas M, Gnant M, Thomanek K, Dubsky P, Roka S, et al. Sequential steroid hormone receptor measurements in primary breast cancer with and without intervening primary chemotherapy. Endocr Relat Cancer 2003;10(1):91e8. Dede DS, Gumuskaya B, Guler G, Onat D, Altundag K, Ozisik Y. Evaluation of changes in biologic markers ER, PR, HER 2 and Ki-67 index in breast cancer with administration of neoadjuvant dose dense doxorubicin, cyclophosphamide followed by paclitaxel chemotherapy. J Buon 2013;18(2):366e71. Zhou X, Zhang J, Yun H, Shi R, Wang Y, Wang W, et al. Alterations of biomarker profiles after neoadjuvant chemotherapy in breast cancer: tumor heterogeneity should be taken into consideration. Oncotarget 2015.