Phenotypic alterations in breast cancer associated with neoadjuvant chemotherapy: A comparison with baseline rates of change

Annals of Diagnostic Pathology 31 (2017) 14–19

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Phenotypic alterations in breast cancer associated with neoadjuvant chemotherapy: A comparison with baseline rates of change

MARK

Nosaibah Hariri, Andres A. Roma, Farnaz Hasteh, Vighnesh Walavalkar, Oluwole Fadare⁎ Department of Pathology, University of California San Diego, San Diego, CA, United States

A B S T R A C T Several studies have documented phenotypic alterations in breast cancer associated with neoadjuvant chemotherapy [NACT], but many of these studies are limited by the fact that they did not account for the baseline rate of expected phenotypic change between biopsies and resections in the absence of NACT. Herein, we assess whether the NACT-associated rate of phenotypic change is significantly different than would be expected in a control population of patients that did not receive NACT. From a pathologic database, we documented the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2/neu) phenotypes of consecutive invasive breast carcinomas (n = 826), as well as the subset in which at least one of these tests was assessed in both the biopsy and resection (n = 340). We then compared the rates of phenotypic change in the patients that did (n = 65) and did not (n = 275) receive NACT. Respectively, 49.2% and 36% of the NACT and non-NACT groups showed a biopsy-to-resection change in status for at least one biomarker (p = 0.0005). The NACT and non-NACT groups showed the following respective rates of a biopsy-to-resection change in phenotype: ER (9.2% vs 2.5%, p = 0.02); PR (30.7% vs 8%, p = 0.000006); Her2/neu-IHC (25% vs 22.3%, p = 0.7), Her2/neu-FISH (7% vs 3%, p = 0.6). The direction of change in the NACT group was positive in the biopsy to negative in the resection in > 70% of cases for all markers. For ER and PR, there was no statistically significant difference between cases that showed a biopsy-to-excision change in phenotype and those that were more phenotypically stable regarding a wide array of clinicopathologic variables. The average percentage of ER/ PR-immunoreactive tumor cells in the pre-NACT biopsies was significantly lower in the phenotypically altered cases as compared to the phenotypically stable cases. Our findings confirm that phenotypic alterations in breast cancer occur after NACT, and that these changes are more pronounced for hormone receptors (especially PR); Significant NACT-associated alterations were not apparent for HER2/neu. A distinct pathologic profile for cases displaying a phenotypic change within the NACT group was not demonstrable. The pre-NACT levels of ER and PR may affect the likelihood of a phenotypic change. These results highlight the need for repeat testing in residual tumors after NACT.

1. Introduction Neoadjuvant chemotherapy [NACT] has become an established approach in the management of a subset of patients with breast cancer. NACT serves to downstage locally-advanced tumors, thereby improving their operability and potentially reducing the extensiveness of surgery that is necessary to achieve an adequate excision [1-7]. For primarily operable cancers, NACT may also improve surgical options or eliminate the disease in its entirety [1-5]. For both groups, NACT facilitates an assessment of the tumoral responsiveness to the administered regimens, which may play a role in modulating the therapeutic approaches that are taken in the adjuvant setting for the same patient [8]. In some forms of breast cancer, such as inflammatory breast cancer, NACT is

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considered to be the standard of care, and there is evidence that NACTbased management approaches have improved overall patient outcomes for this notably aggressive tumor [9,10]. Similarly, for the larger group of patients with breast cancer, it is well established that patients with complete pathologic response to NACT show good long term outcomes [1,11-13]. More recently, however, immunohistochemicallydefined “molecular subgroups” of the broader array of breast cancers has allowed for more accurate prediction of tumor responsiveness for each subgroup and accordingly, a more “personalized” management approach. For example, HER2-positive (nonluminal) and triple-negative tumors generally display high chemosensitivity rates, and complete pathologic response in such tumors have been associated with excellent prognosis [14,15]. However, a failure to achieve complete pathologic

Corresponding author at: UC San Diego Medical Center, Department of Pathology, 200 West Arbor Drive, MC 8720, Room 2-120, San Diego, CA 92103, United States. E-mail address: [email protected] (O. Fadare).

http://dx.doi.org/10.1016/j.anndiagpath.2017.06.004

1092-9134/ © 2017 Published by Elsevier Inc.

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response in these 2 groups puts the patient at a substantial risk for relapses and have generally been associated with a poor prognosis [15,16]. These factors make the pathologic determination of NACTresponsiveness as well as biopsy/excision consistency in phenotype, of notable significance in these 2 subgroups. Testing for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu) are routinely performed on the core biopsy in which most initial diagnoses of breast cancer are made. These results are necessary to assess for the possibility of taking a neoadjuvant therapeutic-based approach to a given patient. Several previous studies have documented a significant rate of biopsyto-resection change in ER, PR and HER2/neu status in breast cancers from NACT-treated patients, although the results have not been entirely congruent [17,18]. In their meta-analysis of 8 previously reported studies, Jabbour et al. [18] found the average biopsy-to-resection discordance rate for ER, PR, and HER2/neu to be 12.9%, 32.0%, and 8.9% respectively. With a few exceptions [19-29], many of the aforementioned studies are limited by the fact that they did not account for the baseline rate of expected phenotypic change between biopsies and resections in the absence of NACT. Herein, we assess whether the NACTassociated rate of phenotypic change is significantly different than would be expected in a control population of patients who did not receive NACT. We hypothesized that the use of a control group may be of analytic significance, since there is a known biopsy/excision discordance rate for ER, PR and HER2/neu that is related to tissue fixation, sampling errors, tumoral heterogeneity or other pre-analytic variables, independent of NACT use [30,31]. A secondary study goal is to determine whether residual tumors in the excision specimen that have undergone an NACT-associated change in phenotype for at least one marker are pathologically distinct from tumors that are more phenotypically stable. On this question, a finding in the affirmative may theoretically facilitate the morphologic delineation of patients with residual tumor after NACT that are more likely to have demonstrated a phenotypic change.

phenotype for each biomarker, using the Fisher's exact test. A wide array of clinicopathologic features were recorded for all patients within the NACT group, including patient age, tumor size, tumor histotype (ductal versus non-ductal), histologic grade (modified Scarff Bloom-Richardson grading system, recorded as grade I versus grade II/III), stage (TNM staging system), ductal carcinoma in situ (DCIS) component (present or absent), lymphovascular invasion (present or absent), lymph node status (positive or negative), tumor necrosis (present or absent) and margins status (positive or negative), all as was determined from the resection specimen. Cases that showed no biopsy-to-resection change in phenotype for any of the biomarkers were classified as phenotypically stable (PS). Cases showing such change for at least one biomarker were classified as phenotypically-altered (PA). PA and PS cases were then statistically compared regarding the aforementioned variables, using the Fisher's exact and Student t-tests. An alpha of 0.05 was used for all statistical analyses. For all of the study period, it was an institutional policy to perform ER, PR and HER2/neu testing by immunohistochemistry on all newly diagnosed cases of breast cancer. For the latter part of the study period, a dual testing HER2/neu testing approach, with both IHC and FISH on all cases [34], was employed. Additionally, for the last 2.5 years of the study period, it was an institutional policy to perform all of the aforementioned tests on the residual tumor after NACT. Immunohistochemical studies at the UC San Diego laboratory are performed with the Ventana Benchmark automation and the Ultra View detection kit (Ventana Medical Systems, Tucson, AZ) using the following primary antibodies: ER (Clone SP1; prediluted, Ventana), PR (clone IE2; prediluted; Ventana) and HER2/neu (clone 4B5; prediluted; Ventana). FISH is performed using the dual-color HER2/CEP17 probe (PathVysion Her2/neu DNA Probe Kit, Abbott Molecular, Inc.). The alternative dual-color HER2/LIS1 (17p13) probe is used only if equivocal results are obtained with HER2/CEP17.

2. Materials and methods

826 consecutive cases of invasive breast cancer were accessioned during the study period. ER, PR and HER2/neu testing were performed on both the biopsy and the resection in 340 cases (41%). 65 (19%) of these 340 patients received NACT, and accordingly formed the study (NACT) group. The control (non-NACT) group was composed of the remaining 275 patients who did not receive NACT. 32 (49.2%) of the 65 cases in the NACT group had a biopsy-to-resection change in status for at least one biomarker, as compared with 72 (36%) of the 275 cases in the non-NACT group (p = 0.0005) (Tables 1). ER: There was a biopsy-to-resection change in ER status in 6 (9.2%) of 65 cases in the NACT group and in 7 (2.5%) of 275 cases in the nonNACT group (p = 0.02). The direction of biopsy-to-resection phenotype change was positive to negative in 83.3% of the NACT group and 71.4% in the non-NACT group (p = 1). Within the NACT group, there was no significant difference between PA and PS cases regarding patient age, tumor size, lymph node status, tumor histotype, histologic grade, stage,

3. Results

This study was approved by the Human Research Protections Program at the University of California San Diego (Project number: 161362). A pathologic database was queried for consecutive patients with invasive breast carcinoma and who received a resection (mastectomy or breast conserving surgery) at our institution during a 6.5year period. For each patient, pathologic reports were reviewed, including those for the preceding core biopsies. We then determined the number of patients who that had ER, PR and/or HER2/neu testing was performed on both the biopsy and the resection, and recorded these results for both specimen types. The rate of biopsy-to-resection change in phenotype for each marker was determined. For ER and PR, a change in phenotype was determined to be present if a case changed from a positive result in the biopsy to negative result in the resection, or vice versa. All cases were scored using 2010 criteria of the American Society of Clinical Oncology and College of American Pathologists [32]; the percentage of immunoreactive cells that displayed any immunoreactivity was also documented for each case. For HER/2neu, change was assessed at the negative (scores 0 or 1 +) versus equivocal or positive (scores 2+ or 3+) threshold for immunohistochemistry (IHC) and at the amplified versus not amplified threshold for fluorescence in situ hybridization (FISH), using applicable scoring criteria [33]. The medical records for patients whose tumors received ER, PR and HER2/ neu testing on both the biopsy and resection were also searched, and the proportion of these patients who received NACT was determined. Patients who received NACT thereby formed the study group (NACT group) and patients who did not receive NACT (but whose tumors still had testing on both the biopsy and the resection) formed the control group (non-NACT group). The NACT and non-NACT groups were compared regarding their rates of a biopsy-to-resection change in

Table 1 Rates of biopsy-to-resection change in phenotype. Phenotype

ER PR HER2/neu by FISH HER2/neu by IHC

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Number of cases tested in both biopsy and resection

Change in phenotype (NACT group) n (%)

Change in phenotype (non-NACT group) n (%)

p Value

NACT

NonNACT

65 65 28

275 275 83

6 (9.2%) 20 (30.7%) 2 (7%)

7 (2.5%) 22 (8%) 3 (3.6%)

0.02 0.000006 0.6

60

229

15 (25%)

51 (22.3%)

0.71

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Table 2 A clinicopathologic comparison of carcinomas that showed a biopsy-to-resection change in phenotype (PA: phenotypically altered) and carcinomas cases that did not show a change in phenotype (PS: phenotypically stable). Pathologic features

ER

PR

HER2/neu by IHC

PA %

PS %

p Value

PA %

PS %

p Value

PA %

PS %

p Value

Age (mean) Tumor size (mean) Histotype – Ductal – Non-ductal

58 2.05

53.08 4.1

0.48 0.151

59 4.2

52.1 3.8

0.12 0.9

51.5 1.77

54.3 4.1

0.7 0.8

83.3% 16.7%

74.6% 25.4%

1

70% 30%

77.8% 22.2%

0.54

87.7 13.3

73.3 26.7

0.03

Histologic grade – I – II–III

33.3% 66.7%

22% 78%

0.6

35% 55%

17.8% 82.2%

0.2

20 80

20 80

1

Stage – I – II–IV

33.3% 66.7%

35.6% 64.4%

1

42.1% 57.9%

0.27

26.7 73.3

37.2 62.8

0.5

DCIS component – Present – Absent

16.7% 83.3%

61% 39%

0.08

35% 65%

66.7% 33.3%

0.03

46.7 53.3

55.6 44.4

0.8

Lymphovascular invasion – Present – Absent

33.3% 66.7%

39% 61%

1

40% 60%

37.8% 62.2%

1

43.75 56.25

55.5% 44.4

0.8

Lymph-node status – Positive – Negative

33.3% 66.7%

57.1% 42.9%

0.6

55% 45%

54.8% 45.2%

0.8

73.3 26.7

50 50

0.1

Tumor necrosis – Present – Absent

50% 50%

16.9% 83.1%

0.09

30% 70%

15.6% 84.4%

0.3

13.3 86.7

24.4 75.6

0.5

Margin status – Negative – Positive

100% 0%

91.5% 8.5%

1

95% 5%

95.6% 4.4%

1

93.3 6.7

97.8 2.2

0

profile. HER2/neu by FISH: HER2/neu status was determined by FISH in both the biopsy and resection in 28 cases in the NACT group and 83 cases in the non-NACT group. A biopsy-to-resection change was observed in 7% (2/28) of the NACT group and 3.6% (3/83) of the nonNACT group (p = 0.6). The direction of biopsy-to-resection phenotype change was amplified to non-amplified in both cases of the NACT group and in 2 of the 3 cases in the non-NACT group (p = 1).

frequency of DCIS component, lymphovascular invasion, tumor necrosis and margins status (Tables 2). The average percentage of ERimmunoreactive tumor cells in the pre-NACT biopsies was not significantly different than the average percentage of the ER-immunoreactive tumor cells in the post-NACT resections (73% [ ± 33] versus 87% [ ± 39] respectively, p = 0.8). However, there were significant differences between the PA and PS groups in this regard: the average percentage of ER-immunoreactive tumor cells in the pre-NACT biopsies was significantly lower in the PA group as compared the PS groups (41% [ ± 19] vs 81% [ ± 28.6] respectively, p = 0.002). PR: A biopsy-to-resection change in PR status was observed in 20 (30.8%) of 65 cases in the NACT group and in 22 (8%) of 275 cases in the non-NACT group (p = 0.000006). The direction of biopsy-to-resection phenotype change was positive to negative in 75% of the NACT group and 73% in the non-NACT group (p = 1). Within the NACT group, PS cases had a higher frequency of a DCIS component in the excision (66.7%) than PA cases (35%, p = 0.03). PA and PS showed no significant differences regarding all of the other clinicopathologic variables that were assessed. However, the average percentage of PRimmunoreactive tumor cells in the pre-NACT biopsies was significantly lower in the PA group as compared the PS groups (34% [ ± 13] vs 69% [ ± 26] respectively, p = 0.03). HER2/neu by IHC: HER2/neu status was determined by immunohistochemistry in both the biopsy and the resection in 60 cases from the NACT group and 229 cases from the non-NACT group. 15 (25%) of the 60 cases in the NACT group showed a biopsy-to-resection change in results, as compared with 51 (22.3%) of the 229 cases in the non-NACT group (p = 0.7). The direction of biopsy-to-resection phenotype change was positive to negative in 87.5% of the NACT group and 90% in the non-NACT group (p = 1). Within the NACT group, PA tumors were of a ductal (vs non-ductal) histotype more frequently than PS tumors (87.7% versus 73.3%, p = 0.026), but otherwise, PS and PA cases showed no statistically significant differences in clinicopathologic

4. Discussion A number of studies have demonstrated that residual breast cancers post-NACT show a substantial rate of a change in ER, PR and HER2/neu phenotypes. However, the studies have not been entirely congruent regarding the frequency, direction and significance of these changes [19-29,35-62]. At present time, there are no fixed recommendations regarding whether residual, post-NACT tumors should uniformly be tested or under what circumstances such testing may be omitted. In the current study, we performed an analysis of tumors in paired pre-NACT biopsy/post-NACT resection specimens to determine the discordance rates between these specimens regarding their tumoral ER, PR and HER2 phenotypes. Our study also used a large control group to discern whether any changes in phenotype in the NACT group can truly be attributed to the NACT, rather than a variety of other potential factors. We demonstrated that there was a biopsy-to-resection change in ER and PR status in 9.2% and 30.7% of cases respectively. These changes were substantially above the observed rates in the control groups (2.5% for ER, 8% for PR; p < 0.05 for both). For HER2/neu status, whether determined by IHC or FISH, the observed rates of biopsy-to-resection change (25% for IHC; 7% for FISH) was not significantly above the control groups (22.3% for IHC; 3% for FISH; p > 0.05 for both). For all biomarkers, biopsy-to-resection change in phenotype went from positive to negative in > 70% of cases showing any changes. The average 16

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PR and HER2/neu. This was accomplished by comparing NACT-associated breast cancer cases that showed biopsy-to-resection phenotypic change to those that were more stable. The pre-study hypothesis was that there is a distinct pathologic profile for post-NACT tumors that may allow the morphologic recognition of cases that are most likely to have undergone a change in phenotype, which would in turn reduce the overall number of post-NACT testing that is necessary. However, we did not find a distinct pathologic profile that may be associated with cases that are phenotypically altered. There was no significant difference between phenotypically labile and phenotypically stable cases regarding patient age, tumor size, lymph node status, tumor histotype, histologic grade, stage, lymphovascular invasion, tumor necrosis and margins status. We did identify a higher frequency of a DCIS component in the phenotypically stable cases for PR (66.7% versus 35%, p = 0.03), although the significance of this finding is unclear. The current study uses one of the most robust control groups amongst the previously reported studies on this topic, and this represents a strength of the analysis. However, there are two major limitations to this study that may have potentially affected our results, and our findings should be evaluated within the context of these potential limitations. Our study did not address the specific types of neoadjuvant therapeutic regimens that were administered, and did not control for trastuzumab or endocrine therapy-containing regimens. In addition to the intuitive significance of the latter, even non-directed therapies may have a confounding effect because some regimens or medications, at least theoretically, may have greater modulatory effect on the expression of the studied biomarkers than others. Second, our NACT group is not of a sufficient size to allow us to control for other non-traditional variables within the group, such as menopausal status, biopsy to resection duration, chemotherapeutic schedules, number of cores obtained and patient ethnicity. The presence of a control group may have mitigated some of the analytic artifacts that may be introduced by incompleteness of these statistical adjustments. Nevertheless, this remains a study limitation. In summary, phenotypic alterations associated with NACT occur with a significant frequency in breast cancer. In this study, such changes occurred above baseline in a statistically significant fashion only for ER and PR and not for HER2/neu. Changes were most commonly from positive in the biopsy to negative in the resection. The preNACT levels of ER and PR may affect the likelihood of a phenotypic change to negative. The residual tumors in cases that had undergone a phenotypic change did not demonstrate a clinicopathologic profile that was clearly distinct from cases that showed more biopsy-to-resection phenotypic stability. Our findings support the necessity for repeat testing in residual tumors after NACT.

percentage of ER and PR-immunoreactive tumor cells in the pre-NACT biopsies was significantly lower in the cases showing a change in phenotype than in cases showing no such change, which suggests that the pre-NACT levels of ER and PR may affect the likelihood of a phenotypic change. For ER and PR, our findings are consistent with the previously published literature. Xian et al. [36] reviewed the literature and estimated that the median frequencies of biopsy-to-resection change in phenotype were 13% for ER, 21% for PR, and 12% for HER2/neu. In another meta-analysis of the literature [18], the average biopsy-to-resection discordance rate for ER, PR, and HER2/neu were found to be 12.9%, 32.0%, and 8.9% respectively. The mechanisms that underlie these phenotypic changes are unclear. van de Ven et al. [17] hypothesized 4 possible mechanisms: a) NACT targets cells with a particular phenotype, leaving viable cells with a different phenotype in the resection; b) the tumor cells actively change phenotype as a survival mechanism in response to NACT; c) NACT actively cause a regression in tumoral hormone receptors; and d) NACT-associated ovarian insufficiency causes lower levels of circulating estrogen in pre-menopausal women, which may in turn cause down regulation of the estrogen and/or progesterone receptors, and lead to a manner of tumoral growth that is entirely independent of a hormone receptor pathway [17]. Perhaps more significantly, the prognostic implications of a change in hormone receptor phenotype is similarly unclear. One study have found a change in hormone receptor positive status from negative to positive to be significantly correlated with better overall survival when compared with patients whose hormone receptor status was unchanged [23]. Somewhat similarly, a switch from hormone receptor positive to negative has been associated with decreased overall and/or disease free survival [46,48]. Other studies have found no significant survival differences based on the presence or direction of hormone receptor status switch following NACT [43,47]. Our finding that HER2/neu status appears to be relatively resistant to the modulatory effects of NACT is supported by a substantial body of prior literature [19,20,25,38,41,51,52,55,58,59,61,62] and is contradicted by a comparably sized body of literature [22,26,28,36,40,43,50,54,56,57,60]. Studies on both sides of the question have included retrospective and prospective analyses, and included analyses with and without a control arm. Since the possibility that NACT has a modulatory effect on HER2/neu status has at minimum, not been conclusively excluded, we recommend continued testing for HER2/neu in post-NACT tumors. Prior studies that have analyzed the significance of clinicopathologic features in NACT-treated breast cancer patients have focused on different areas of the topic. Some have been centered on predicting response to therapy by evaluating pathologic features in the biopsy and correlating those findings with pathologic response rates in the resections. In one such study, Alvarado-Cabrero et al. [63] found that neither histologic grade nor histologic type to correlate with a pathologic response. In contrast, Colleoni et al. [64,65] found that lobular carcinomas and larger tumors showed less responsiveness to NACT regimens than non-lobular and smaller tumors. Other studies have investigated whether clinicopathologic features pre-NACT can be used to predict which tumors will undergo a phenotypic change after NACT. In one recent study [36], the authors were unable to demonstrate any effect of patient age, histologic grade, or node status on the biopsy-to-resection stability of biomarkers after NACT. A third group of studies have emphasized the morphologic changes on the tumor itself that can be directly attributed to NACT, by comparing pathologic features of the residual tumor in the resection specimens and those in the pre-NACT core biopsies. Adams et al. [40], for example, found that NACT results in a significant decrease in mitotic count but had no significant effect on nuclear pleomorphism, tubule formation or the overall Bloom-Richardson score. In the current study, we attempt to discern any potential correlations between clinicopathologic features of NACT-treated tumors and phenotypic stability or lability regarding ER,

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