Breast Cancer Research and Treatment

https://doi.org/10.1007/s10549-020-05772-6

PRECLINICAL STUDY

Differential prognostic value of positive HER2 status determined

by immunohistochemistry or fluorescence in situ hybridization
in breast cancer
Albina Stocker1,2 · Andreas Trojan1 · Constanze Elfgen1,4 · Marie‑Louis Hilbers1 · Linda Moskovszky3 ·
Zsuzsanna Varga3

Received: 18 April 2020 / Accepted: 23 June 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Purpose Human epidermal growth factor-receptor-2 (HER2) is a membrane-tyrosine-kinase that is amplified/overexpressed
up to 20% in breast cancer. HER2 positive status is associated with faster disease progression, higher metastatic potential,
and shorter disease-free/overall survival and also has emerged as an important therapeutic target in breast cancer. HER2
status can be determined by in-situ-hybridization (ISH) or immunohistochemistry (IHC). Although the concordance rate
between ISH and IHC is well-known, the prognostic power of both technologies if tested in parallel on the same tumor has
not been studied extensively.
Methods In this study we retrospectively analyzed a large HER2 positive breast cancer cohort tested both with fluorescence
labeled ISH (FISH) and IHC in parallel on each case. We stratified HER2 positive results by FISH and IHC with long-term
overall survival, 5-year survival and metastases/recurrence rates. Positive HER2 status both FISH and IHC was available
in 364 patients.
Results The number of HER2 FISH-positive and FISH-negative patients was 342 and 22, respectively. The number of HER2
IHC 0/1 + , IHC 2 + , and IHC 3 + patients was 12, 42, and 310, respectively. Among the patients with IHC 3 + status, 288
were FISH-positive and 22 FISH-negative. HER2 status determined by FISH correlated with clinical outcomes (overall
survival and with metastases/recurrence, p = 0.036, p = 0.039), whereas HER2 status determined by IHC did not.
Conclusion Our results indicate that prognostic information in HER2 positive breast cancer also depends on the methodology
of how positivity was determined. In our cohort, FISH was superior to IHC based positive HER2 status.

Keywords Positive HER2 status · Assessment method · Prognosis · Breast cancer

Introduction

Breast cancer is the most frequent and leading cause of

cancer-related deaths in female individuals worldwide [27].
Electronic supplementary material The online version of this
article (https​://doi.org/10.1007/s1054​9-020-05772​-6) contains The stratification of breast cancer cases based on their patho-
supplementary material, which is available to authorized users. logical, biological, or clinical characteristics may facilitate
tailoring therapeutic approaches to achieve a better treatment
* Zsuzsanna Varga
response [24, 27].
zsuzsanna.varga@usz.ch
http://www.pathologie.usz.ch Human epidermal growth factor receptor 2 (HER2) is a
membrane tyrosine kinase that regulates cell survival and
1
Breast Center Seefeld and Oncocenter, Zurich, Switzerland proliferation [13]. The HER2 gene, located on chromo-
2
County Hospital Waid, Zurich, Switzerland some 17, is found to be amplified in about 20% of breast
3
Institute of Pathology and Molecular Pathology, University cancer cases. HER2 amplification is the main mechanism
Hospital Zurich, Schmelzbergstrasse 12., CH‑8091 Zurich, driving HER2 overexpression and is associated with faster
Switzerland disease progression, higher metastatic potential, and
4
University of Witten-Herdecke, Witten, Germany shorter disease-free survival and overall survival [20].

13
Vol.:(0123456789)
 Breast Cancer Research and Treatment

Therefore, HER2 has emerged as an important therapeu- Patient characteristics

tic target in breast cancer; several therapeutic agents for
HER2-positive breast cancer have been developed that are We retrospectively analyzed data from patients with stage
associated with improved clinical outcomes [25]. Tras- I–IV breast cancer in the databank of the Breast Center See-
tuzumab is a recombinant monoclonal antibody against feld in Zurich between 2001 and 2011. In this study, 364
HER2, which has been applied successfully in women patients with positive HER2 status (first tumor diagnosis
with HER2-positive breast cancer, increasing both disease- between 1995 and 2011) were included. HER2 status was
free survival and overall survival [25]. Several clinical considered as either IHC score 3 + or amplified by FISH.
studies showed improved long-term disease-free survival HER2 status in primary breast cancer specimens was ana-
after anti-HER2 therapy such as Trastuzumab, Lapatinib lyzed on formalin fixed paraffin embedded (FFPE) tissues
or Capecitabin treatment in patients with HER2-positive within the routine diagnostic service. The specimens were
early or metastatic breast cancer [3, 9, 24, 25, 32, 5]. obtained via core or vacuum-assisted biopsies as well as
The stratification of breast cancer based on their HER2 during surgery. HER2 status in metastatic lesions in case of
status is an important consideration for therapeutic deci- relapse or metastases was assessed on FFPE specimens and
sions [3, 9, 24, 25, 27]. The HER2 status of breast cancer in some cases on cytology samples. Breast specimens (pre-
cases can be determined using immunohistochemistry or operative core and vacuum biopsies and surgical specimens)
in situ hybridization (ISH) technologies labeled with sil- including HER2 testing were all processed in the Institute
ver (SISH), fluorescence (FISH) or chromogenic (CISH) of Pathology and Molecular Pathology, University Hospital
detection tags [28, 31, 35]. Guidelines for HER2 testing in Zurich, Switzerland. Reporting of retrospective data analysis
breast cancer are established, and the significance of high on biomarkers followed the REMARK recommendations.
concordance between IHC and ISH testing is strongly rec-
ommended in current guidelines [35]. For therapeutic deci-
sions, both IHC score 3 + and/or an amplified HER2 ISH Fluorescence in situ hybridization analysis
score are sufficient [34, 35]. There are several studies in the
literature showing concordance data between HER2 IHC FISH analysis was conducted as described previously and
and HER2 ISH methodologies [15, 22, 28–31]. However, was conducted according to the current time-valid scoring
only a few studies have analyzed the prognostic power of guidelines [30]. Briefly, a dual fluorescence kit (PathoVy-
HER2 positive breast cancer applying stratification based sion, Vysis, Abbott AG, Diagnostic Division Baar, Switzer-
on ISH amplification or IHC score 3 + score [8, 15, 22, 31]. land) was used to analyze the HER2 gene on 2-µm thick par-
In these few studies it was shown, that there are prognostic affin sections. A hematoxylin–eosin (HE) stain accompanied
power variations between IHC score3 + and ISH amplified each case for tumor identification. For samples processed
status if both methodologies are used in parallel on the same until 2010, probes were hybridized for 14–20 h at 37° C,
cohort [8, 15, 18, 19, 31]. washed twice in rapid wash solutions in accordance with
In the current study, we determined how HER2 status the manufacturer’s instructions, dried, and counterstained
stratification in patients with HER2-positive breast cancer using DAPI. For samples processed after 2011, an auto-
correlates with long-term overall survival (up to 20 years), mated Leica Bond autostainer was applied (Leica Biosys-
5-year survival and recurrence/metastases months after the tems, Nunningen, Switzerland). Slides were analyzed with
initial diagnosis based on the methodology used for stratifi- an Olympus computer-guided fluorescence microscope.
cation (FISH and IHC testing in all cases). FISH HER2 results were interpreted applying the current
time-valid guidelines such as the FDA guidelines and the
ASCO-CAP guidelines until 2007 and from 2008 onwards,
respectively [4, 10, 12, 22, 33].
Materials and methods

Ethical considerations HER2 immunohistochemistry

The current study was a part of a larger project focusing on HER2 immunohistochemistry and the determination of
investigating patients with breast cancer. Ethical permission HER2 protein expression was carried out as described ear-
for the project was granted by the Ethical Commission of lier [30]. The FDA-approved antibody PATHWAY (initially
the Canton Zurich (No KEK-ZH-2012-553). Patients pro- the anti-HER2 clone CB11 and later on the clone 4B5) was
vided written or oral-informed consent for participation in applied in all cases (Ventana, Switzerland) on 2-µm thick
the study at the time of data retrieval or during follow-up sections. Initially, a Ventana Benchmark semi-automated
consultations. system and Ventana reagents were used for the staining

13
Breast Cancer Research and Treatment

procedure, which was supplemented with a signal enhance- Table 1  Summary of clinic-pathological characteristics of the HER2
ment procedure. Since 2011, all specimens were processed positive cohort assessed with in-situ hybridization (FISH) and Immu-
nohistochemistry (IHC)
by a fully automated Leica Bond autostainer (Leica Biosys-
tems, Nunningen, Switzerland) without signal enhancement. Cohort n = 364 Number (in
IHC reactions were assessed on a light microscope. For the percentage)
interpretation of HER2 IHC, the time current FDA approved Age
DAKO guidelines and the time–current ASCO-CAP guide- < 50 years 129 (35%)
lines were applied [4, 10, 22, 33]. > 50 years 235 (65%)
Gender
Clinical data Male 4 (1%)
Female 360 (99%)
Clinical endpoints included: overall survival, 5-year sur- Histological type
vival, and the presence of metastases or recurrence/metas- Ductal carcinoma 316 (87%)
tases months after the initial diagnosis. Other histological type 48 (13%)
Histological grading
Statistical analyses G1 8 (2%)
G2 119 (33%)
SPSS Statistics, Version 22.0 was used to assess the G3 186 (51%)
data. The disease-free survival, five-year survival and the NA 51 (14%)
time to metastasis/recurrences were estimated using the Estrogen receptor status
Kaplan–Meier method, log-rank test and the Breslow and Positive 237 (65%)
Tarone-Ware tests. p < 0.05 based on two-sided statistical Negative 127 (35%)
tests was considered statistically significant. Progesterone receptor status
Positive 194 (53%)
Negative 170 (47%)
Results HER2 status by IHC
IHC score 0/1 + 12 (3%)
Patient characteristics IHC score 2 + 42 (11.5%)
IHC score 3 + 310 (85.5)
Patients’ median age was 54 years (range 23–91 years). The HER2 status by FISH
mean survival for the patients in this study was 56.14 months Amplified 342 (94%)
(median 41 months). 113 patients (36%) reached the five- Non-amplified 22 (6%)
year survival indicator and 251 did not. Metastases or tumor Lymphatic vessel invasion
recurrence during the disease course were present in 128 L1 190 (52%)
patients (35%). Patients were all treated with neo-/adjuvant L0 174 (48%)
settings containing time current combination therapy regi- Distant metastases/recurrence
ments including anti-HER2 therapy as well. Present 128 (35%)
The clinical characteristics of the patients are presented Absent 236 (65%)
in Table 1 and in Supplementary Table 1. Tumor stage (pT)
pT1 123 (34%)
Concordance of HER2 status assessed by FISH pT2 124 (34%)
or immunohistochemistry pT3 35 (10%)
pT4 23 (6%)
Patients with HER2-positive status determined with at least NA 59 (16%)
one of the used methods were included in the analysis (either Nodal stage (pN)
FISH positive independently from immunohistochemistry pN0 133 (37%)
scores or IHC score 3 + independently from FISH ampli- pN1 172 (47%)
fication status). The number of patients with IHC status NA 59 (16%)
0/1 + , 2 + , and 3 + was 12, 42, and 310, respectively. The
FISH HER2 status was positive and negative in 342 and 22 NA not available, pT pathological tumor stage, pN pathological nodal
stage, L lymphatic vessel invasion present (L1), absent (L0)
patients, respectively. The concordance between IHC score
3 + and FISH amplification status was 92%. Among patients
with IHC 3 + status, 288 were FISH HER2-positive and 22

13
 Breast Cancer Research and Treatment

were FISH HER2-negative. The patients in this cohort with p = 0.036), whereas overall survival did not differ based on
IHC score 0/1 + , 2 + were all FISH positive. the presence of IHC scores 0/1 + , 2 + , and 3 + (p = 0.272).
The data and concordance on HER2 status determined by For 5-year survival, although a fewer HER2 FISH-posi-
immunohistochemistry and FISH are presented in Table 2. tive patients reached 5 years survival than HER2 FISH-nega-
tive, this difference was not significant (p = 0.133). Five-year
Prognostic analysis between all IHC scores (0/1 + , survival also did not differ based on the presence of HER2
2 + , 3 +) and FISH amplification status IHC scores 0/1 + , 2 + , and 3 + (p = 0.171) Fig. 1.

HER2 FISH and IHC prognostic power for overall survival Metastases and recurrence rate stratification by positive
and five‑year survival HER2 status by FISH vs IHC

There was a significantly shorter overall survival in patients The recurrence and/or metastases months after the initial
with FISH HER2-positive status than in patients with FISH diagnosis rate was dependent on HER2 status determined by
HER2-negative status (54 months versus 80.5 months, FISH (p = 0.039) but not by IHC (p = 0.188) Fig. 1.

Prognostic analysis between IHC scores

Table 2  Concordance in positive HER2 status assessed by in-situ excluding 2 + category (only with scores 0/1 + and
hybridization (FISH) and Immunohistochemistry 3 +) and FISH amplification status
FISH HER2 score 3 + HER2 score 2 + HER2 score
0/1 + HER2 FISH and IHC prognostic power for overall survival
and five‑year survival
Amplified 288 (79.1%) 42 (11.5%) 12 (3.4%)
Non-amplified 22 (6%) – –
There was a significantly shorter overall survival in patients
N = 364 with FISH HER2-positive status than in patients with

Fig. 1  HER2 positive status (either IHC 3 + or FISH positive) stratified by FISH (including IHC score 2 +), n = 364. Prognostic relevance of
positive HER2 status assessed by FISH or IHC on overall survival, 5-years survival and the presence of metastatic disease/recurrence

13
Breast Cancer Research and Treatment

FISH HER2-negative status (54 months versus 79 months, and ASCO/CAP guidelines using a cut-off until 2007 and
p = 0.033), whereas overall survival did not differ based on from 2008.
the presence of IHC scores 0/1 + , 2 + , and 3 + (p = 0.120). HER2 determined by IHC (including 0/1 + , 2 + ,
For five-year survival, HER2 FISH-positive and FISH 3 + scores) did not differ in these two time periods, prognos-
negative patients did not show significant differences tic difference based on the IHC scores were not significant:
(p = 0.119). Five-year survival also did not differ based Until 2007: OS: p = 0.754, 5-years survival: p = 0.655
on the presence of HER2 IHC scores 0/1 + , 2 + , and 3 + metastases/recurrences p = 0.216. From 2008: OS: p = 0.538,
(p = 0.071) Fig. 2. 5-years-survival: p = 0.661, metastases/recurrences
p = 0.616.
Subgroup analysis based on FISH amplification and prog-
Metastases and recurrence rate stratification by positive
nostic relevance could be performed only until 2007 (there
HER2 status by FISH vs IHC
were no FISH negative cases in the IHC score 3 + subset
after 2008). Prognostic difference based on FISH amplifi-
The recurrence and /or metastases months after the initial
cation until 2007 were not significant in this subgroup: OS:
diagnosis rate showed association to HER2 status deter-
p = 0.294, 5-years survival: p = 0.844, metastases/recur-
mined by FISH (p = 0.041) but not by IHC (p = 0.104) Fig. 2.
rences p = 0.189.
Statistical p-values are summarized in Table 3.

Prognostic analysis using HER2 IHC and FISH data Discussion

prior and after the 2008 ASCO/CAP guidelines
In the current study, we analyzed the prognostic power of
We performed a subgroup analysis on overall survival, positive HER2 status determined as FISH positive or IHC
5-years survival and the occurrence of recurrent or meta- score 3 + for clinical outcomes in breast cancer in patients
static lesions under consideration of the time current FDA with HER2-positive breast cancer by at least one of the two

Fig. 2  HER2 positive status (either IHC 3 + or FISH positive) stratified by FISH (excluding IHC score 2 +), n = 322. Prognostic relevance of
positive HER2 status assessed by FISH or IHC on overall survival, 5-years survival and the presence of metastatic disease/recurrence

13
 Breast Cancer Research and Treatment

Table 3  Summary of prognostic values for positive HER2 status assessed with in-situ hybridization (FISH) and Immunohistochemistry (IHC)
Positive HER2 status including IHC Positive HER2 status without
score 2 + category IHC score 2 + category
FISH positive vs IHC scores 0/1 + vs FISH positive vs IHC scores
negative 2 + vs 3 + negative 0/1 + vs 3 +

Overall survival Log rank (mantel-cox) p = 0.036* p = 0.272 p = 0.033* p = 0.120

Breslow (generalized wilcoxon) p = 0.121 p = 0.581 p = 0.107 p = 0.394
Tarone-ware p = 0.056 p = 0.473 0 = 0.049* p = 0.264
5-years survival Log rank (mantel-cox) p = 0.135 p = 0.171 p = 0.119 p = 0.071
Breslow (generalized wilcoxon) p = 0.150 p = 0.475 p = 0.135 p = 0.271
Tarone-ware p = 0.133 p = 0.347 p = 0.119 p = 0.170
Recurrence and/or metastases Log rank (mantel-cox) p = 0.039* p = 0.327 p = 0.041* p = 0.145
after initial diagnosis Breslow (Generalized wilcoxon) p = 0.179 p = 0.188 p = 0.158 p = 0.104
Tarone-ware p = 0.080 p = 0.211 p = 0.075 p = 0.098

*Indicates statistical significance

methods. HER2 status assessed by FISH demonstrated supe- FISH amplification was found to be different based on the
rior prognostic value to IHC score 3 + for overall survival two assessments. In our study only the FISH-based posi-
and for metastases and recurrence/metastases months after tive HER2 status had significant prognostic value regard-
the initial diagnosis. ing overall survival and in recurrences/metastases months
Proper identification of HER2 status is important for after the initial diagnosis. Contrarily, positive HER2 status
treatment decisions in breast cancer patients, as HER2 has determined with IHC score 3 + alone did not significantly
been correlated with worse clinical outcomes in patients correlate with any of the clinical outcomes. This observa-
with breast cancer, including faster disease progression, tion yet needs a careful interpretation within the context of
higher metastatic potential, and shorter disease-free survival the given cohort. Our study is a retrospective cohort includ-
and overall survival [22, 23]. HER2-directed therapeutic ing a heterogeneous patient population, assessments both
agents, the prototype of which is Trastuzumab, can improve on biopsy and surgical specimens and also different scoring
clinical outcomes in this patient population [2, 6, 18]. guidelines and cut-offs for positivity scores resulting in a
ISH and IHC methodologies are established and rou- large deviation of pre-analytical, analytical parameters and
tinely used tests to determine HER2 status in breast cancer interpretation issues. Additionally, the patients received
[8, 15, 17, 22, 28, 30, 33, 35]. The concordance rate of the treatment modalities based on the given time current clinical
two methods has been shown to be relatively high in sev- guidelines potentially having an impact on the correlation of
eral studies even though variations still occur [8, 15, 17, HER2 positivity scores with clinical outcome.
19, 22, 30]. In our study, the concordance between HER2 Differences or similarities in clinical outcome in HER2
FISH and IHC score 3 + results was relatively high, reaching IHC score 3 + versus in FISH positive breast cancer have
92%. Thus, among the patients with IHC 3 + status, 288 were been documented contradictorily in the literature [6, 14, 18,
FISH HER2-positive and 22 were FISH HER2-negative. On 19, 22, 37]. The prospective HERA trial could not prove any
the contrary, IHC score 0/1 + were FISH amplified in 12 prognostic benefit in disease-free survival between HER2
cases (3.4%). Reasons for discrepancies between IHC and positivity assessed with IHC or FISH [6, 37]. Furthermore,
FISH include a large spectrum of problematic factors such neither the degree of FISH amplification nor the HER2/chro-
as pre-analytical differences in fixation time, fixatives used mosome 17 (CEP17) ratio had an impact on the prognosis or
and the area choice respectively the presence of intratumoral on the response to adjuvant Trastuzumab in the HERA trial
heterogeneity [16, 22, 28–30]. In addition to these factors, [6, 37]. Similar results were reported earlier by the CALGB
analytical variables during laboratory procedures and also 8541 clinical trial comparing IHC, FISH, and polymerase
assay interpretation variations both in IHC and in FISH in chain reaction (PCR)-based HER2 status assessment [7].
the observers’ assessment are crucial factors in final HER2 In this study IHC, FISH, and PCR all predicted a benefit
status decisions [16, 22, 28–30]. Especially the subset HER2 from adjuvant therapy in the HER2-positive subset and none
IHC score 2 + can pose a problematic diagnostic category of the methods was superior alone or in combination, even
as a result of pronounced intratumoral heterogeneity [21]. though the maximal therapy benefit was detected in tumors
Despite good concordance between IHC and FISH results, being IHC- and FISH-positive [7]. The N9831 trial showed
the prognostic power stratified by IHC score 3 + versus that both IHC score 3 + and FISH amplification alone could

13
Breast Cancer Research and Treatment

significantly predict a benefit from adjuvant Trastuzumab; FISH-based HER2 status assessment. These observations
however, the maximal therapy effect was reached in this and the superiority or equality of a given methodology how-
trial in patients whose breast cancer were tested positive ever can be verified only in prospectively planned clinical
with both IHC and FISH technology [18]. Both the N9831 trials.
trial and an earlier paper from our institution reported that
increasing HER2/CEP17 ratio (> 8 resp. > 15) does not nec-
essarily imply better response to Trastuzumab-containing Funding No funding was necessary for this study.
therapy, most likely pointing to induced HER2 resistance
[18, 26]. Interestingly, another HER2/CEP17 ratio (> 4) Compliance with Ethical Standards
was associated with shortened metastases-free survival in a
Conflict of interest ZV received consultancy and speaking fees from
retrospective study on 117 patients receiving Trastuzumab- Roche, Astra Zeneca and Genomic Health. There is no conflict of in-
containing therapy in a HER2 positive cohort [1]. Gullo terest to disclose from the other authors.
et al. described discordant HER2/CEP17 ratios between pri-
mary tumor and corresponding metastases, being higher in Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the insti-
the metastatic lesion (10.5 vs 7.0), pointing to Trastuzumab tutional and/or national research committee and with the 1964 Helsinki
resistance in HER2 positive disease [11]. declaration and its later amendments or comparable ethical standards.
Similar to our results, in an earlier study, Mass et al. Ethical permission for the project was granted by the Ethical Commis-
reported that only in IHC score 2 + /3 + cases with concom- sion of the Canton Zurich (No KEK-ZH-2012-553). Patients provided
written or oral-informed consent for participation in the study at the
itant FISH-positive tumors was there a clinical benefit for time of data retrieval or during follow-up consultations.
Trastuzumab therapy, pointing to higher robustness of FISH
technology [14]. The superiority of FISH to IHC in terms
of prediction of response to Trastuzumab and of overall sur-
vival was described in two clinical trials such as the H0648 References
trial and BCIRG-005/006/007 trial by Sauter et al. and by
Press et al. ( [19, 22]. In the H06498 trial, there was no 1. Adamczyk A, Kruczak A, Harazin-Lechowska A, Ambicka A,
significant benefit with Trastuzumab or with chemotherapy Grela-Wojewoda A, Domagala-Haduch M, Janecka-Widla A,
Majchrzyk K, Cichocka A, Rys J, Niemiec J (2018) Relation-
in metastatic disease in patients with IHC score 3 + without
ship between HER2 gene status and selected potential biologi-
simultaneous FISH amplification [22]. cal features related to trastuzumab resistance and its influence
Literature data are sparse regarding HER2 IHC score on survival of breast cancer patients undergoing trastuzumab
0/1 + in breast cancer being amplified with an ISH assay adjuvant treatment. Onco Targets Ther 11:4525–4535. https​://
doi.org/10.2147/OTT.S1669​83
[30, 36]. This phenomenon has been reported as an existing
2. Ahmed S, Sami A, Xiang J (2015) HER2-directed therapy: current
rare category occurring between 1 and 15% in breast cancer, treatment options for HER2-positive breast cancer. Breast Cancer
although the exact number of HER2 gene amplification in 22:101–116. https​://doi.org/10.1007/s1228​2-015-0587-x
this subset is unknown as reflex ISH testing is not obligatory 3. Baselga J, Cortes J, Kim SB, Im SA, Hegg R, Im YH, Roman
L, Pedrini JL, Pienkowski T, Knott A, Clark E, Benyunes MC,
in this subset [30, 36]. In our study, we found 12 cases with
Ross G, Swain SM, Group CS (2012) Pertuzumab plus trastu-
HER2 IHC score 0/1 + being amplified simultaneously with zumab plus docetaxel for metastatic breast cancer. N Engl J Med
FISH. The IHC scores 0/1 + , as was the case with IHC score 366:109–119. https​://doi.org/10.1056/NEJMo​a1113​216
2 + and 3 + , had no prognostic impact on clinical outcome 4. Bilous M, Dowsett M, Hanna W, Isola J, Lebeau A, Moreno A,
Penault-Llorca F, Ruschoff J, Tomasic G, van de Vijver M (2003)
if HER2 positivity was stratified by IHC alone. Whether
Current perspectives on HER2 testing: a review of national testing
pre-analytical /analytical errors or interpretation discrepan- guidelines. Mod Pathol 16:173–182. https​://doi.org/10.1097/01.
cies explain the existence of this rare subgroup, cannot be MP.00000​52102​.90815​.82
answered definitely at the current time and needs further 5. Cameron D, Piccart-Gebhart MJ, Gelber RD, Procter M, Gol-
dhirsch A, de Azambuja E, Castro G Jr, Untch M, Smith I, Gianni
analysis of such cases to explore this rare group.
L, Baselga J, Al-Sakaff N, Lauer S, McFadden E, Leyland-Jones
In conclusion, the results of our study indicate that at least B, Bell R, Dowsett M, Jackisch C (2017) 11 years’ follow-up of
in the retrospective study population, HER2 positive assess- trastuzumab after adjuvant chemotherapy in HER2-positive early
ment by FISH was superior to HER2-positive assessment by breast cancer: final analysis of the HERceptin Adjuvant (HERA)
trial. Lancet 389:1195–1205. https​: //doi.org/10.1016/S0140​
immunohistochemistry to predict long-term overall survival
-6736(16)32616​-2
and advanced disease course in patients with HER2 positive 6. Dowsett M, Procter M, McCaskill-Stevens W, de Azambuja E,
breast cancer. A limitation of this study is the heterogeneous Dafni U, Rueschoff J, Jordan B, Dolci S, Abramovitz M, Stoss O,
therapy regiments in addition to anti-HER2 therapy. Our Viale G, Gelber RD, Piccart-Gebhart M, Leyland-Jones B (2009)
Disease-free survival according to degree of HER2 amplification
results however corroborate with results from very few ear-
for patients treated with adjuvant chemotherapy with or without 1
lier studies such as the H0648 trial and BCIRG-005/006/007 year of trastuzumab: the HERA Trial. J Clin Oncol 27:2962–2969.
clinical trial pointing to more robustness and superiority of https​://doi.org/10.1200/JCO.2008.19.7939

13
 Breast Cancer Research and Treatment

7. Dressler LG, Berry DA, Broadwater G, Cowan D, Cox K, Griffin 17 effect on patient outcome in the N9831 adjuvant trastuzumab
S, Miller A, Tse J, Novotny D, Persons DL, Barcos M, Hender- trial. J Clin Oncol 28:4307–4315. https​: //doi.org/10.1200/
son IC, Liu ET, Thor A, Budman D, Muss H, Norton L, Hayes JCO.2009.26.2154
DF (2005) Comparison of HER2 status by fluorescence in situ 19. Press MF, Sauter G, Buyse M, Fourmanoir H, Quinaux E, Tsao-
hybridization and immunohistochemistry to predict benefit from Wei DD, Eiermann W, Robert N, Pienkowski T, Crown J, Mar-
dose escalation of adjuvant doxorubicin-based therapy in node- tin M, Valero V, Mackey JR, Bee V, Ma Y, Villalobos I, Cam-
positive breast cancer patients. J Clin Oncol 23:4287–4297. https​ peau A, Mirlacher M, Lindsay MA, Slamon DJ (2016) HER2
://doi.org/10.1200/JCO.2005.11.012 gene amplification testing by fluorescent in situ hybridization
8. Gancberg D, Di Leo A, Cardoso F, Rouas G, Pedrocchi M, Paes- (FISH): comparison of the ASCO-college of american patholo-
mans M, Verhest A, Bernard-Marty C, Piccart MJ, Larsimont gists guidelines with FISH scores used for enrollment in breast
D (2002) Comparison of HER-2 status between primary breast cancer international research group clinical trials. J Clin Oncol
cancer and corresponding distant metastatic sites. Ann Oncol 34:3518–3528. https​://doi.org/10.1200/JCO.2016.66.6693
13:1036–1043. https​://doi.org/10.1093/annon​c/mdf25​2 20. Ross JS, Fletcher JA (1998) The HER-2/neu oncogene in breast
9. Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pien- cancer: prognostic factor, predictive factor, and target for ther-
kowski T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, apy. Oncologist 3:237–252
Skarlos D, Campone M, Davidson N, Berger M, Oliva C, Rubin 21. Sapino A, Maletta F, Verdun di Cantogno L, Macri L, Botta C,
SD, Stein S, Cameron D (2006) Lapatinib plus capecitabine for Gugliotta P, Scalzo MS, Annaratone L, Balmativola D, Pietribi-
HER2-positive advanced breast cancer. N Engl J Med 355:2733– asi F, Bernardi P, Arisio R, Viberti L, Guzzetti S, Orlassino
2743. https​://doi.org/10.1056/NEJMo​a0643​20 R, Ercolani C, Mottolese M, Viale G, Marchio C (2014) Gene
10. Gown AM, Goldstein LC, Barry TS, Kussick SJ, Kandalaft PL, status in HER2 equivocal breast carcinomas: impact of dis-
Kim PM, Tse CC (2008) High concordance between immuno- tinct recommendations and contribution of a polymerase chain
histochemistry and fluorescence in situ hybridization testing for reaction-based method. Oncologist 19:1118–1126. https​://doi.
HER2 status in breast cancer requires a normalized IHC scor- org/10.1634/theon​colog​ist.2014-0195
ing system. Mod Pathol 21:1271–1277. https​://doi.org/10.1038/ 22. Sauter G, Lee J, Bartlett JM, Slamon DJ, Press MF (2009)
modpa​thol.2008.83 Guidelines for human epidermal growth factor receptor 2 test-
11. Gullo G, Bettio D, Zuradelli M, Masci G, Giordano L, Bareggi C, ing: biologic and methodologic considerations. J Clin Oncol
Tomirotti M, Salvini P, Runza L, La Verde N, Santoro A (2013) 27:1323–1333. https​://doi.org/10.1200/JCO.2007.14.8197
Level of HER2/neu amplification in primary tumours and metas- 23. Shui R, Liang X, Li X, Liu Y, Li H, Xu E, Zhang Z, Lian Y, Guo
tases in HER2-positive breast cancer and survival after trastu- S, Yao M, Yang H, Xu F, Liu Y, Liu J, Guo D, Wang K, Li J, Ma
zumab therapy. Breast 22:190–193. https:​ //doi.org/10.1016/j.breas​ Y, Wang J, Shi J, Bu H, Yang W (2020) Hormone receptor and
t.2013.01.005 human epidermal growth factor receptor 2 detection in invasive
12. Hicks DG, Tubbs RR (2005) Assessment of the HER2 status in breast carcinoma: a retrospective study of 12,467 patients from
breast cancer by fluorescence in situ hybridization: a technical 19 chinese representative clinical centers. Clin Breast Cancer
review with interpretive guidelines. Hum Pathol 36:250–261. 20:e65–e74. https​://doi.org/10.1016/j.clbc.2019.07.013
https​://doi.org/10.1016/j.humpa​th.2004.11.010 25. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V,
13. Krishnamurti U, Silverman JF (2014) HER2 in breast cancer: Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M,
a review and update. Adv Anat Pathol 21:100–107. https​://doi. Baselga J, Norton L (2001) Use of chemotherapy plus a mono-
org/10.1097/PAP.00000​00000​00001​5 clonal antibody against HER2 for metastatic breast cancer that
14. Mass RD, Press MF, Anderson S, Cobleigh MA, Vogel CL, Dyb- overexpresses HER2. N Engl J Med 344:783–792. https​://doi.
dal N, Leiberman G, Slamon DJ (2005) Evaluation of clinical org/10.1056/NEJM2​00103​15344​1101
outcomes according to HER2 detection by fluorescence in situ 24. Slamon D, Eiermann W, Robert N, Pienkowski T, Martin M,
hybridization in women with metastatic breast cancer treated Press M, Mackey J, Glaspy J, Chan A, Pawlicki M, Pinter T,
with trastuzumab. Clin Breast Cancer 6:240–246. https​://doi. Valero V, Liu MC, Sauter G, von Minckwitz G, Visco F, Bee
org/10.3816/CBC.2005.n.026 V, Buyse M, Bendahmane B, Tabah-Fisch I, Lindsay MA, Riva
15. Middleton LP, Price KM, Puig P, Heydon LJ, Tarco E, Sneige A, Crown J, Breast Cancer International Research G (2011)
N, Barr K, Deavers MT (2009) Implementation of American Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J
Society of Clinical Oncology/College of American Pathologists Med 365:1273–1283. https​://doi.org/10.1056/NEJMo​a0910​383
HER2 Guideline Recommendations in a tertiary care facility 26. Stocker A, Hilbers ML, Gauthier C, Grogg J, Kullak-Ublick GA,
increases HER2 immunohistochemistry and fluorescence in situ Seifert B, Varga Z, Trojan A (2016) HER2/CEP17 ratios and
hybridization concordance and decreases the number of incon- clinical outcome in HER2-positive early breast cancer undergo-
clusive cases. Arch Pathol Lab Med 133:775–780. https​://doi. ing trastuzumab-containing therapy. PLoS ONE 11:e0159176.
org/10.1043/1543-2165-133.5.775 https​://doi.org/10.1371/journ​al.pone.01591​76
16. Moatamed NA, Nanjangud G, Pucci R, Lowe A, Shintaku IP, 27. Torre LA, Siegel RL, Ward EM, Jemal A (2016) Global cancer
Shapourifar-Tehrani S, Rao N, Lu DY, Apple SK (2011) Effect incidence and mortality rates and trends–an update. Cancer Epi-
of ischemic time, fixation time, and fixative type on HER2/neu demiol Biomark Prev 25:16–27. https​://doi.org/10.1158/1055-
immunohistochemical and fluorescence in situ hybridization 9965.EPI-15-0578
results in breast cancer. Am J Clin Pathol 136:754–761. https​:// 28. Tubbs RR, Hicks DG, Cook J, Downs-Kelly E, Pettay J,
doi.org/10.1309/AJCP9​9WZGB​PKCXO​Q Hartke MB, Hood L, Neelon R, Myles J, Budd GT, Moore HC,
17. Moelans CB, de Weger RA, Van der Wall E, van Diest PJ (2011) Andresen S, Crowe JP (2007) Fluorescence in situ hybridiza-
Current technologies for HER2 testing in breast cancer. Crit Rev tion (FISH) as primary methodology for the assessment of
Oncol Hematol 80:380–392. https​://doi.org/10.1016/j.critr​evonc​ HER2 Status in adenocarcinoma of the breast: a single insti-
.2010.12.005 tution experience. Diagn Mol Pathol 16:207–210. https​://doi.
18. Perez EA, Reinholz MM, Hillman DW, Tenner KS, Schroeder org/10.1097/PDM.0b013​e3180​64c72​a
MJ, Davidson NE, Martino S, Sledge GW, Harris LN, Gralow 29. Varga Z, Noske A (2015) Impact of modified 2013 ASCO/
JR, Dueck AC, Ketterling RP, Ingle JN, Lingle WL, Kaufman CAP guidelines on HER2 testing in breast cancer One Year
PA, Visscher DW, Jenkins RB (2010) HER2 and chromosome

13
Breast Cancer Research and Treatment

Experience. PLoS ONE 10:e0140652. https​://doi.org/10.1371/ LM, Dowsett M (2018) Human Epidermal growth factor receptor
journ​al.pone.01406​52 2 testing in breast cancer: American Society of clinical oncol-
30. Varga Z, Noske A, Ramach C, Padberg B, Moch H (2013) ogy/college of American pathologists clinical practice guideline
Assessment of HER2 status in breast cancer: overall positiv- focused update. Arch Pathol Lab Med 142:1364–1382. https:​ //doi.
ity rate and accuracy by fluorescence in situ hybridization and org/10.5858/arpa.2018-0902-SA
immunohistochemistry in a single institution over 12 years: 35. Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB,
a quality control study. BMC Cancer 13:615. https ​ : //doi. Bartlett JMS, Bilous M, Ellis IO, Fitzgibbons P, Hanna W, Jenkins
org/10.1186/1471-2407-13-615 RB, Press MF, Spears PA, Vance GH, Viale G, McShane LM,
31. Varga Z, Tubbs RR, Moch H (2014) Concomitant detection of Dowsett M (2018) Human epidermal growth factor receptor 2 test-
HER2 protein and gene alterations by immunohistochemistry ing in breast cancer: American Society of clinical oncology/col-
(IHC) and silver enhanced in situ hybridization (SISH) identi- lege of american pathologists clinical practice guideline focused
fies HER2 positive breast cancer with and without gene ampli- update. J Clin Oncol 36:2105–2122. https​://doi.org/10.1200/
fication. PLoS ONE 9:e105961. https​://doi.org/10.1371/journ​ JCO.2018.77.8738
al.pone.01059​61 36. Yamashita H, Ishida N, Hatanaka Y, Hagio K, Oshino T, Take-
32. Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, shita T, Kanno-Okada H, Shimizu AI, Hatanaka KC, Matsuno
Pegram M, Oh DY, Dieras V, Guardino E, Fang L, Lu MW, Olsen Y (2020) HER2 gene amplification in ER-positive HER2 immu-
S, Blackwell K, Group ES (2012) Trastuzumab emtansine for nohistochemistry 0 or 1+ Breast cancer with early recurrence.
HER2-positive advanced breast cancer. N Engl J Med 367:1783– Anticancer Res 40:645–652. https​://doi.org/10.21873​/antic​anres​
1791. https​://doi.org/10.1056/NEJMo​a1209​124 .13994​
33. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred 37. Zabaglo L, Stoss O, Ruschoff J, Zielinski D, Salter J, Arfi M,
DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer Bradbury I, Dafni U, Piccart-Gebhart M, Procter M, Dowsett M,
A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Team HTS (2013) HER2 staining intensity in HER2-positive dis-
Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de ease: relationship with FISH amplification and clinical outcome
Vijver M, Wheeler TM, Hayes DF, American Society of Clinical in the HERA trial of adjuvant trastuzumab. Ann Oncol 24:2761–
O, College of American P (2007) American Society of Clinical 2766. https​://doi.org/10.1093/annon​c/mdt27​5
Oncology/College of American Pathologists guideline recommen-
dations for human epidermal growth factor receptor 2 testing in Publisher’s Note Springer Nature remains neutral with regard to
breast cancer. J Clin Oncol 25:118–145. https​://doi.org/10.1200/ jurisdictional claims in published maps and institutional affiliations.
JCO.2006.09.2775
34. Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB,
Bartlett JMS, Bilous M, Ellis IO, Fitzgibbons P, Hanna W, Jen-
kins RB, Press MF, Spears PA, Vance GH, Viale G, McShane

13