Review Article
Breast Cancer Biomarker Changes after Neoadjuvant Chemotherapy: A Single Institution Experience and Literature Review
Opal L Reddy and Sophia K Apple*
Department of Pathology & Laboratory Medicine, David Geffen School of Medicine at UCLA, USA
*Corresponding author: Sophia K. Apple, Professor, Department of Pathology, City of Hope National Medical Center, 1500 E Duarte Rd, Duarte, CA 91010, USA, Emeritus Professor at David Geffen School of Medicine at UCLA, UCLA Center for the Health Sciences, USA
Published: 23 Mar, 2017
Cite this article as: Reddy OL, Apple SK. Breast Cancer
Biomarker Changes after Neoadjuvant
Chemotherapy: A Single Institution
Experience and Literature Review. Clin
Oncol. 2017; 2: 1245.
Abstract
Chemotherapy and hormonal treatment decisions for breast cancer are influenced by the expression
of tumor biomarkers, including estrogen receptor, progesterone receptor and human epidermal
growth hormone receptor 2 (HER2/neu). These biomarkers are most commonly assessed by
immunohistochemical staining and fluorescence in situ hybridization studies performed on breast
cancer biopsy specimens. Biomarker studies performed on excisional specimens may be discordant
with those performed on core needle biopsy, especially after neoadjuvant chemotherapy. We
evaluated 195 cases at our institution from 2002 to 2015 to determine the effects of neoadjuvant
chemotherapy on the expression of the aforementioned biomarkers and Ki-67. Forty-nine (25.1%)
cases showed complete pathologic response after neoadjuvant chemotherapy. Twelve cases (8.6%)
showed a change in estrogen receptor status after neoadjuvant chemotherapy. Twenty-four cases
(17.1%) showed a change in progesterone receptor status, predominantly from positive to negative
(p = 0.0002). HER2/neu expression was evaluated by both immunohistochemical staining and
fluorescence in situ hybridization, where two (1.5%) (p = 1.000) and eight (8.2%) (p = 0.4795)
cases changed expression status, excluding equivocal cases, respectively. Ki-67 was also evaluated,
with 49 cases (48%) (p <0.0001) showing altered proliferation indices after neoadjuvant therapy.
The differences were statistically significant for progesterone receptor and Ki-67. Overall survival
was also evaluated, and showed statistically significant differences between groups that changed
progesterone receptor status after neoadjuvant therapy. Given the therapeutic implications of
altered biomarker status after neoadjuvant chemotherapy, we recommend repeating biomarker
studies on resection specimens to most appropriately guide adjuvant chemotherapy decisions.
Keywords: Breast cancer; Biomarker changes; Neoadjuvant chemotherapy
Introduction
Breast cancer is the most common cancer affecting women in the United States. Neoadjuvant chemotherapy decisions for breast cancer are highly influenced by the expression of tumor biomarkers, including Estrogen Receptor (ER), Progesterone Receptor (PR), and human epidermal growth hormone receptor 2 (HER2/neu), that are performed on needle core biopsy specimens through immunohistochemical (IHC) staining or Fluorescence In Situ Hybridization (FISH) studies. While neoadjuvant chemotherapy for the treatment of breast cancer was historically reserved for patients with locally advanced, inoperable disease, neoadjuvant therapy is now being increasingly used for the initial treatment of smaller and operable tumors to downstage and perform conservative surgical excision [1,2]. After neoadjuvant chemotherapy and surgery, biomarker studies are frequently repeated on resection specimens, and may be different from biomarker studies performed on core needle biopsies. This finding may represent a variety of reasons, such as chemotherapy effect or intratumoral heterogeneity, a well-documented characteristic of breast carcinomas. Previous studies evaluating the effect of neoadjuvant chemotherapy on tumor biomarkers have not been entirely in agreement [3-5], and this information may affect treatment decisions following surgery. This study aims to add to the growing body of literature the effects of neoadjuvant chemotherapy on the expression of ER, PR, HER2/neu by IHC and FISH, and Ki-67, and includes clinical follow-up for our cohort, which most of the current literature did not report. We also present the most comprehensive literature review to date, and conclude with a summary of our statistically significant findings with recommendations.
Materials and Methods
Ethics statement
This project was conducted with the approval of the University of
California, Los Angeles (UCLA) Institutional Review Board.
Study group
A retrospective chart review of our institution’s pathology
database was conducted to identify patients with invasive breast
carcinoma who underwent neoadjuvant chemotherapy prior to
surgical resection between 2002–2015. Patients who had both
pre- and post-treatment pathology reports with biomarker studies
available were included. Male patients and patients with multifocal,
bilateral, and recurrent disease were excluded. Clinical and
pathological data were collected via review of medical records and
extracted to the Research Electronic Data Capture (REDCap) [6]
database. Clinicopathologic data collected included patient age at the
time of surgery, follow-up data, and tumor characteristics, such as
histologic type, grade, and ER, PR, HER2/neu, and Ki-67 status. The
modified Bloom-Richardson scoring system was used to determine
histologic grade.
Immunohistochemical (IHC) methods and evaluation
Formalin-fixed, paraffin-embedded human breast cancer tissue
specimens from 195 patients with invasive breast carcinoma were
used in the study. IHC stains were performed with ER and PR
antibody clones with appropriate positive and negative controls.
Slides were baked for 1-4 hours in a 60oC oven and then deparaffinized
in xylene and brought into tap water through graded alcohols (100%
x4, 95%x2). Slides were then treated with 0.5% hydrogen peroxide
in absolute methyl alcohol for 10 minutes to quench endogenous
peroxidase activity, and then washed with tap water and deionized
water. Heat-induced antigen retrieval was then performed by placing
slides in 0.01 M citrate buffer, pH 6.0 preheated to 95oC in a vegetable
steamer (Black & Decker); heat-treatment lasted for 25 minutes,
followed by a 15-minute room temperature cool-down period.
Following rinse in PBS, slides were immunostained on a DAKO
Autostainer, using a goat anti-mouse Poly-HRP secondary antibody
(MACH2 Mouse HRP-Polymer, Biocare Medical, Concord, CA) as
the detection system. The staining procedure involved a 45minute
primary antibody incubation with mouse monoclonal antibodies
against Estrogen Receptor clone SP1 (1/50 dilution, Biocare Medical,
Concord, California) and Progesterone Receptor clone 636 (1/300
dilution, DAKO Corp, Carpenteria, CA), followed by a PBS wash and a
30 minute incubation in MACH2 mouse HRP-Polymer. The negative
control reagent was mouse IgG1 or diluent alone. Diaminobenzidine
was used as the chromogen to effect visualization of the peroxidase
enzyme. Slides were then counterstained with Harris hematoxylin.
Using appropriate positive and negative controls, the test for the
presence of these hormone receptor proteins was performed by the
immunoperoxidase method, and reported according to the 2009
CAP-ASCO Guidelines for Hormone Receptor testing. Tissues were
fixed between 6-72 hours in 10% neutral buffered formalin. A positive
ER or PR tumor showed greater than or equal to 1 percent of cells
staining, and results were semi-quantitated with percentage and
intensity of positive tumor cells.
For Ki-67, clone MIB-1 (Mouse Monoclonal, DAKO) was diluted
1:50 in buffered Calcium Chloride (0.074 gm Calcium Chloride
dihydrate in 50 ml of 0.05M Tris-buffered saline, pH 7.4, containing
25ul of Tween 20). Percentage of cells positive for Ki-67 was evaluated,
and the Ki-67 index was then categorized into a three-grade system:
<10% (low), 10-20% (intermediate), and ≥20% (high).
Both IHC analysis for HER2 protein and fluorescence in-situ
hybridization for ERBB2 gene were performed on all specimens from
our facility at the David Geffen University of California at Los Angeles
(UCLA) Medical Center from years 2002 through 2015. Optimal
tissue handling requirements (e.g. time to fixation) were followed
and recorded, particularly after publication of the American Society
of Clinical Oncology/College of American Pathologists guidelines,
after 2007.
The US Food and Drug Administration FDA-approved
HercepTest™ was performed using DAKO A0485 polyclonal antibody
kit (DAKO Corp, Carpenteria, CA). The results were scored by
US Food and Drug Administration (US FDA) guidelines prior to
publication of the American Society of Clinical Oncology/College
of American Pathologists guidelines, with immunohistochemistry
scoring of 3+ cases staining >10% of tumor cells.
Fluorescence in-situ hybridization analysis
Fluorescence in-situ hybridization (FISH) was performed using
the US Food and Drug Administration (FDA)-approved PathVysion™
HER2 DNA Probe Kit (PathVysion™ Kit), which is designed to detect
amplification of the ERBB2 gene via fluorescence in-situ hybridization
in formalin-fixed, paraffin-embedded human breast cancer tissue
specimens. Fluorescence in-situ hybridization analysis with VYSIS
dual-color probes specific for chromosome 17 centromere and
the ERBB2 gene (17q11.2) was performed and examined by two
independent technologists. Slides containing 4-micron sections were
submitted for fluorescence in-situ hybridization analysis. For each
slide, based on the corresponding hematoxylin & eosin slide, the
invasive tumor area(s) were circled with a secure line marker. Areas
containing ductal carcinoma in-situ or normal tissue were excluded
from fluorescence in-situ hybridization testing. Slides were baked
overnight at 600C and pretreated using the VP2000 tissue processor
as per manufacturer’s protocol (Abbott Molecular, Abbott Park,
IL). Amplification of the ERBB2 gene was detected by using the
PathVysion™ Kit; the instructions in the package insert were followed
for the hybridization, post-hybridization washing, and analysis
steps (Abbott Molecular, Abbott Park, IL). All cases were scored
according to US Food and Drug Administration guidelines for HER2
immunohistochemistry and used ERBB2 gene fluorescence in-situ
hybridization amplification cutoff value of 2.0 before 2007 and after
2013. Between 2008 – 2012, FISH and HER2 IHC cutoffs were used
from ASCO/CAP guidelines.
Statistical analysis
Results for the modified Bloom Richardson scoring parameters,
as well as ER, PR, HER2/neu statuses and Ki-67 levels were evaluated
with the Wilcoxon-signed rank test. Univariate survival analysis was
performed using log rank tests and visualized using Kaplan-Meier
survival curves. Overall survival was defined as the interval from the
date of resection to the date of death from any cause, or date of last
follow-up after known or suspected hospice. Patients were otherwise
censored at the date of last follow-up. Stata 12.0 software (Stata
Corporation, College Station, TX, USA) was used for all statistical
analyses. A p-value of <0.05 was considered statistically significant.
Literature review
A comprehensive search of the existing published literature
between 1991-2016 in PubMed was performed. MeSH terms included neoadjuvant, chemotherapy, breast, biomarker, ER, PR, and HER2.
The listed references in relevant articles retrieved were also reviewed.
Articles that met our criteria for review were included only if they
were published in the English language and were accessible online.
Table 1
Table 2
Table 2
Comparison of Mean Modified Bloom-Richardson Scores Pre- and
Post-Neoadjuvant Chemotherapy (n=89).
Results
Clinical and pathological data for 195 cases that fulfilled the
inclusion criteria were collected (Table 1). The median age at the time
of diagnosis was 49 (interquartile range, 41-58). All patients were
female. The majority of tumors were of invasive ductal carcinoma,
no specific type (90%), while the remaining were of invasive lobular
(4%) or other histologic types (6%). There were 14 (7%) Grade I
tumors, 66 (34%) Grade II tumors, and 105 (54%) Grade III tumors,
and the histologic grade for 10 (5%) tumors was not reported. Prior to neoadjuvant chemotherapy, most of tumors largely demonstrated
high proliferation indices based on Ki-67 staining of over 20% of
tumor cells (62%), while the remaining were either of low (<10%
staining of tumor cells) proliferation index (4%), intermediate (10-
20% staining of tumor cells) proliferation index (7%), or not reported.
Following neoadjuvant chemotherapy, 49 patients (25.1%)
showed pathologic complete response with no residual invasive
carcinoma identified after extensive sampling of resection specimens,
and four patients (2.1%) had only minimal or microinvasive
carcinoma. The changes to the modified Bloom-Richardson scores
(m-BRS) were evaluated for 89 patients (45.6%) with available data
(Table 2). The pre-therapy mean tubule formation score was 2.84,
while the post-therapy mean was 2.85 (p = 0.76). The pre-therapy
mean nuclear pleomorphism score was 2.51, while the post-therapy
mean was 2.56 (p = 0.26). The pre-therapy mean mitotic activity
score was 2.00, while the post-therapy mean was 1.79 (p = 0.005). A
significant decrease in mitotic rate was observed after neoadjuvant
chemotherapy. The pre-therapy mean total m-BRS score was 7.33,
while the post-therapy mean was 7.20 (p = 0.19). Downgrading of
m-BRS score occurred with 29 patients. (Figure 1) shows invasive
ductal carcinoma pre- (A) and post- (B) neoadjuvant chemotherapy.
Changes in hormonal receptor expression
Pre and post neoadjuvant treatment ER and PR statuses were
available for 140 patients (Table 3). Of these, ninety-six (69%) tumors
were initially ER positive, six (6%) of which became negative following
neoadjuvant therapy. Likewise, six (4%) initially ER-negative tumors
became positive post-treatment. The change in ER status was not
statistically significant. Eighty-two tumors (59%) were initially PR
positive, and 21 (26%) of these became PR negative after neoadjuvant
therapy (Figure 1C and D). Three (2%) initially PR-negative tumors
became positive for PR post-treatment. Overall, the change in PR
status was statistically significant (p = 0.0002).
Changes in HER2/neu IHC and FISH
HER2/neu status was evaluated both by IHC and FISH (Table
3). HER2/neu IHC data pre- and post-treatment was available
for 130 tumors. Of these, 2 tumors (1.5%) had a change in status
after treatment, including one (0.8%) that changed from negative
to positive, and one (0.8%) that changed from positive to negative
(Figure 1E and F). Data on HER2/neu FISH status was available for 97
patients, and eight (8.2%) changed statuses after neoadjuvant therapy.
These included five (5.2%) that changed from positive to negative
and three (3.1%) that changed from negative to positive (Figure 2).
Neither the change in HER2/neu expression by IHC (p = 1.000) nor
by FISH (p=0.4795) was statistically significant.
Changes in Ki-67 expression
The majority of tumors demonstrated high Ki-67 proliferation
indices (Table 4). Pre- and post-treatment data on Ki-67 proliferation
indices was available for 102 tumors, 83 (81.3%) of which had high
proliferation indices. Twelve (14.4%) of these changed to intermediate
proliferation index, and 25 (30.1%) changed to low proliferation
index. Overall, 49 (48.0%) changed Ki-67 proliferation indices after
neoadjuvant therapy, showing a statistically significant change (p
<0.0001).
Biomarker changes and patient outcome
At the time of last follow-up, there were 18 (9%) deaths or patients
in hospice care. Those patients in hospice care were considered dead
of disease for our analysis since there were no subsequent medical
follow-up records. Median follow-up was 36.0 months (interquartile
range, 18.4 to 70.1). (Tables 5 and 6) detail patient outcomes by
hormone receptors and HER2 phenotype, as determined on biopsy
and excision specimens, respectively. Patients were also grouped
by biopsy and excision biomarker statuses, and overall survival was
assessed between groups. (Table 7) lists the number of deaths in
each group, with differences assessed by logrank analyses. Statistical
significance was seen only with PR groups (p = 0.0319), as visualized
by Kaplan-Meier curves (Figure 3).
Literature review
We performed a comprehensive literature review of all studies
evaluating biomarker change after neoadjuvant chemotherapy in
breast cancer (Table 8). Thirty-one articles were identified. Reports of
ER changes ranged from 0%-42.4% and reports of PR changes ranged
from 13.2%-67%. A number of studies evaluated hormone receptor
changes together, and reported results that ranged from 5-30%.
HER2 changes, either by IHC or FISH, ranged from 0% - 35%. Lastly,
changes in Ki-67 proliferation index ranged from 7% to 92.4%.
Summary of our results
Twelve cases (8.6%) showed change in ER status after neoadjuvant
chemotherapy. Twenty-four cases (17.1%) showed change in PR
status, predominantly from positive to negative (p = 0.0002). HER2/
neu expression was evaluated by both IHC and FISH, where 35 cases
changed expression status by IHC (p=0.0130), and eight by FISH (p
= 0.4795). Excluding equivocal cases by both IHC and FISH testing,
only two cases (1.5%) by IHC and eight cases (8.2%) by FISH showed
alterations in HER2/neu status. Ki-67 was also evaluated, with 49
cases (48%) (p <0.0001) showing decreases in proliferation indices
after neoadjuvant therapy. In addition, overall survival was assessed
between groups based on pre- and post-neoadjuvant chemotherapy
biomarker statuses, and showed statistically significant differences
between PR groups (p = 0.0319).
Table 3
Figure 1
Figure 1
A-B. After neoadjuvant chemotherapy, invasive ductal
carcinoma shows marked treatment related changes, including fibrosis
and pleomorphism (B), as compared to untreated tumor (A) (H&E, original
magnification x200).
C-D. Twenty-one tumors that were initially positive for PR (C), lost expression
of PR after neoadjuvant chemotherapy (D) original magnification x400).
E-F.
One tumor that was initially HER2 positive by IHC (E) lost expression of
HER2 after neoadjuvant chemotherapy (F) (original magnification x400).
Table 4
Figure 2
Figure 2
Three tumors that were initially HER2 negative by FISH (A) were
found to be ERBB2 gene amplified by FISH after neoadjuvant chemotherapy
(B).
Table 5
Table 6
Table 7
Figure 3
Figure 3
Kaplan-Meier survival analysis showed significant differences in
overall survival when grouped by biopsy and excision PR status (p = 0.0319).
Table 8
Conclusion
Recently, the American Society of Clinical Oncology (ASCO)
published clinical practice guidelines recommending re-biopsy
of breast cancer metastases to re-evaluate ER, PR and HER2/neu
expression. However, evidence is still lacking on whether changes
to chemotherapy regimens should be made on the basis of altered
biomarkers in the adjuvant therapy setting. The Panel consensus was
to use biomarker testing from metastatic tumors to direct therapy
accordingly [7]. There are no ASCO guidelines on whether excisional
biopsy specimens should be re-tested after neoadjuvant therapy,
and if there are changes, whether adjuvant chemotherapy should
be altered. Hence, the practice differs worldwide. Neoadjuvant
chemotherapy is being increasingly used prior to surgical resection
of breast cancer, with treatment regimens guided by hormone
receptor status and HER2/neu expression from core needle biopsy
samples. Previous studies have shown that biomarker expression
may change after neoadjuvant chemotherapy in breast cancer.
Several possibilities may explain the discordance between hormone
receptors and HER2/neu expression and ERBB2 gene amplification
before and after neoadjuvant chemotherapy. Possible explanations
include: 1. Discordances between core needle biopsy and excisional
specimens due to sampling variability, 2. Intratumoral heterogeneity,
3. Interlaboratory variability, 4. Differences due to ER and PR cutoff
changes from 10% previously to >1% currently, as well as ASCO/CAP
HER2/neu guideline changes, and 5. Instability of tumor biomarkers
throughout tumor progression and with multiple relapses [8].
Discordances between core needle biopsy and excision
due to sampling variability
Previous studies evaluating biomarker concordance have ranged
from 0% to 42.4% for ER, 13.2% to 67% for PR, and 0% to 35% for
HER2/neu [9,10]. In general, there is excellent agreement between
biomarkers assessed in core needle biopsy and excisional specimens.
Chen et al. [11] reported high concordance with ER, PR, and HER2
at 93.6%, 85.9%, and 96.3%, respectively [11]. Another study showed
an almost perfect concordance for ER (k = 0.89; 95% CI: 0.65-1.0)
and a substantial concordance for PR (k = 0.70; 95% CI: 0.46-0.93),
HER2 (k = 0.61; 95% CI: 0.38-0.84) and Ki-67 (k = 0.74; 95% CI: 0.58-
0.98) obtained between core needle biopsy and excision [12]. We also
previously studied the reliability of core needle biopsy samples on
breast cancer when compared to excisional specimens and reported
high concordance rates for ER and PR, at 95% and 89%, respectively,
and HER2/neu by IHC at 96% and by FISH at 100% [13].
Intratumoral heterogeneity
Somewhat related to issue #1 is intratumoral heterogeneity,
which has also been well-documented in breast cancer.
Discordances between pre- and post-treatment biomarker testing
may represent sampling variability during initial biopsy, though
marked intratumoral heterogeneity is rare [14,15]. Intratumoral
heterogeneity has been proposed to arise from genetic alterations
during the clonal evolution of tumor cells, that may subsequently
result in genetic subclones within a single tumor [14]. Even without
neoadjuvant chemotherapy, alterations of biomarkers have been
reported between primary breast cancer compared with synchronous
axillary metastasis or recurrent metastatic breast cancer [16]. Most
of the literature have studied HER2 rather than hormonal receptors
in regards to intratumoral heterogeneity. A study by Kurozumi et al.
[17], evaluated HER2 negative breast cancer cases with IHC scores of
0 and 1+ and showed HER2 heterogeneity in 17 % of cases, using a
more sensitive HER2 gene-protein assay. These patients were found
to have a poor prognosis [17]. In particular, low histologic grade
and hormone receptor positive tumors have been shown to have
HER2 regional and genetic heterogeneity in 6.2% and 6.8% of cases,
respectively [18]. ERBB2 gene amplification genetic heterogeneity has
also been reported and ranges from 5% to 30% in the literature [19].
Others, however, have shown intratumoral heterogeneity is
not a significant factor. The largest translational study conducted
to investigate the correlation of central IHC/FISH assessments
with microarray mRNA readouts of ER, PR, and HER2 status is
the Microarray In Node-negative and 1 to 3 positive lymph node
Disease may Avoid ChemoTherapy (MINDACT) trial, which
showed no support for intratumoral heterogeneity for the cause of
biomarker discordance. MINDACT is an EORTC 10041 (BIG3-04),
international, prospective, randomized, phase III trial with 6694
patients investigating the clinical utility of MammaPrint in selecting
patients with early breast cancer for adjuvant chemotherapy [20].
Interlaboratory variability
Interlaboratory variability in hormone receptors and HER2/neu
testing have been reported. In early 2000, Rhodes et al. [21], published
data from 200 laboratories in the UK quality assessment for ER IHC
testing and showed a considerable interlaboratory variability in low
ER positive samples with a false negative rate of between 30% and
60%. High ER samples had over 80% concordance rate.
For HER2/neu, a prospective, randomized, three-arm, phase III
trial was conducted by the Breast Intergroup (N9831) and found a
poor concordance (74%) between local and central testing for HER2
status [22].The MINDACT trial study showed that ER had a positive
agreement of 98% and a negative agreement of 95%, PR had a positive
agreement of 85% and a negative agreement of 94%, and HER2 had
a positive agreement of 72% and a negative agreement of 99% with
central pathology [20]. In the current study, 41% of pretreatment
biomarker testing was performed on core biopsies from outside
institutions that were not repeated at our institution. Accordingly,
this may play a factor in the discordant rate between pre- and post
neoadjuvant chemotherapy specimens.
Differences due to ER and PR cutoff changes from 10%
previously to >1% currently, as well as ASCO/CAP HER2/neu guideline changes
A change in cutoff valuesfor ER and PR may have resulted in
discordancese between pre and post neoadjuvant chemotherapy
hormonal receptor status. In 2010, the American Society of Clinical
Oncology (ASCO) and College of American Pathologists (CAP)
issued guidelines that tumors with ≥1% cells staining positive for ER should be considered ER positive [23]. Prior to 2010, the cutoff
for hormone receptor expression was 10% in many institutions,
including our own. As a result, prior to 2010, biomarker studies in
the literature had variable hormone receptor reporting, with negative
hormone receptor tumor staining that ranged from <1% to <10%.
For HER2, the American Society of Clinical Oncology and the
College of American Pathologists published guidelines in 2007
(ASCO/CAP 2007 guidelines), which included modifications to
IHC analysis and fluorescence in-situ hybridization (FISH) tests that
reverted back to the Food & Drug Administration (FDA) guidelines
in 2013 [24,25]. Previously, our group compared concordance rates
before and after the 2007 guidelines. Prior to 2007, the concordance
rate between the assays was 97.6% with a corresponding kappa
coefficient of 0.90. After 2007, the concordance rate was 97.6% with
a corresponding kappa coefficient of 0.89. The implementation of the
new ASCO/CAP 2007 HER2 guidelines did not show a significant
difference in concordance rates and did not decrease the number of
inconclusive cases [26].
More recently a similar study by Overcast et al. [27], demonstrated
that by using the 2013 ASCO/CAP HER2 guidelines, there was
a significant shift in HER2 FISH recategorization on core needle
biopsy, reclassifying 9.9% of cases. Most (9.3%) changed from the
HER2 FISH negative category to the HER2 FISH equivocal category,
resulting in an increase of HER2 equivocal cases from 0.5% to 6.5%
[27].
The ASCO/CAP 2007 guidelines for HER2 also introduced
an average HER2 copy number criterion for HER2 amplification
of ≥6.0/cell as positive for amplification. New guidelines increased
HER2 positive cases by 2.6%, increased equivocal cases by 2.1% and
decreased HER2 negative cases by 4.1%, according to Singh et al.
[28]. Lim et al. [29] found similar results, showinga decrease in HER2
negative and an increase in HER2 positive as well as equivocal cases,
with an overall decrease in concordance rates between FISH and IHC
from 94.9% to 93.8%.
Instability of tumor biomarkers throughout tumor
progression and with multiple relapses
The mechanism behind alterations in biomarkers after
neoadjuvant chemotherapy is not well understood. It may be due
to chemotherapy effects on selectively killing sensitive cancer cells
and leaving insensitive cells behind in residual tumors, or it may
be secondary to intratumoral heterogeneity. It is documented that
alterations of biomarkers are more frequently seen after neoadjuvant
chemotherapy than in control groups. Gahlaut et al. [30], compared
neoadjuvant treated groups with control, non-neoadjuvant treated
groups and found more biomarker alterations after neoadjuvant
chemotherapy. Statistical significance in this study, however, was only
seen with PR, and not with ER or HER2 [30]. Overall, the literature
has suggested that Ki-67, followed by PR are altered more frequently
than HER2 and ER. Triple negative breast cancers are known to stay
as triple negative tumors after neoadjuvant chemotherapy [31]. Our
data also showed statistically significant differences in Ki-67 and
mitotic rates after neoadjuvant chemotherapy, as has been seen in
other studies [32,33].
Pathologic Complete Response (pCR) after neoadjuvant
chemotherapy occurs more frequently for patients with HER2
positive and triple negative breast cancers when compared to
hormonal receptor positive tumors. pCR is also a predictive factor of
better overall survival and disease free survival, especially when seen
with changes in Ki-67 expression and axillary lymph node response
[31].
As far as prognosis is concerned, loss of hormonal receptor
positivity and switching to triple negative phenotype were related to
worse overall survival and disease-free survival [8,34]. 18The current
study evaluated overall survival between groups based on pre- and
post-neoadjuvant chemotherapy biomarker statuses, and showed
statistically significant differences between PR groups. Alterations
in biomarkers are not unique to the neoadjuvant setting. Curtit et
al. [35], reported ER and PR changes in 17% and 29%, respectively,
between the primary breast tumor and the corresponding metastatic
lesion after adjuvant chemotherapy. Interestingly, in this paper, a
positive cut off for ER and PR was 10%. Currently HER2 remained
stable, and only 4% discordance was reported with HER2/neu in their
study.
Regardless of the mechanisms underlying discordances
between pre- and post-treatment biomarker statuses, most studies
have recommended re-testing hormone receptors and HER2/
neu expression on surgical specimens received after neoadjuvant
chemotherapy. A loss of ER and HER2 expression and amplification
could imply resistance to endocrine therapy or trastuzumab,
respectively. Likewise, gain in expression of ER or HER2 may introduce
additional therapeutic options, providing improved prognosis
[36,37]. Accordingly, given the treatment and prognostic implications
of changed biomarker status after neoadjuvant chemotherapy, we
recommend repeating biomarker studies on resection specimens to
most appropriately guide adjuvant chemotherapy decisions.
In summary, we present our cohort of 195 patients who
had received neoadjuvant chemotherapy and were re-tested for
biomarkers on residual tumor found in excision specimens, and
included clinical follow-up. We also performed an extensive
literature review of discordant biomarker rates after neoadjuvant
chemotherapy. Our findings are consistent with other studies, and
we agree in recommending repeating biomarkers after neoadjuvant
chemotherapy as in recurrent/metastatic tumors.
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