Determination of HER2 status has now become of major clinical importance with the
advent of anti-HER2 therapy, the recombinant humanised anti-p185Her-2/neu antibody
trastuzumab (Herceptin®) (Pegram et al, 1998; Cobleigh et al, 1999; Slamon et al,
2001).
Immunohistochemistry (IHC) is expected to be the best method for the determination
of HER2 status, as IHC assesses the level of HER2 overexpression, which is the target
of Herceptin® therapy. Moreover, the patient's selection for Herceptin® therapy is
mainly based on IHC because previous studies demonstrated a good correlation between
IHC results and gene status, as determined by fluorescence in situ hybridisation (FISH)
(Pegram et al, 1998; Jacobs et al, 1999,2000; Couturier et al, 2000; Jimenez et al,
2000; Lebeau et al, 2001; Lehr et al, 2001). However, the HER2-IHC detection was criticised
because of a lack of interlaboratory reproducibility and, furthermore, Herceptest®,
a standardised IHC method, was shown to be a method with excessive sensitivity when
compared to FISH (Persons et al, 1997; Bartlett et al, 2001; Tubbs et al, 2001). Even
though HER2 overexpression without gene amplification was reported in 2.9–8.3% of
cases (Kallioniemi et al, 1992; Persons et al, 1997; Couturier et al, 2000; Jimenez
et al, 2000; Pauletti et al, 2000), discordant results between IHC and FISH were mainly
observed for tumours that were scored 2+ by IHC (Persons et al, 1997; Bartlett et
al, 2001; Tubbs et al, 2001). For this reason, and particularly in Europe, a confirmation
of HER2 gene amplification by FISH became mandatory for a patient's inclusion in a
clinical trial using Herceptin®, when the corresponding tumour is scored 2+ by IHC
(Hoang et al, 2000; Ridolfi et al, 2000; Diaz, 2001; Tubbs et al, 2001; Vogel et al,
2002).
Some authors found that HER2 status determined by FISH was more reproducible (Press
et al, 1994; Persons et al, 1997; Bartlett et al, 2001; Tubbs et al, 2001). Thus,
these authors thought that FISH had to be proposed as the only method to select patients
for Herceptin®. However, FISH is a long and expensive procedure that requires trained
personnel and fluorescence microscopy.
Chromogenic in situ hybridisation (CISH) is a recently introduced technique in which
the DNA probe is detected using an immunoperoxidase reaction (Tanner et al, 2000).
This method is very close to FISH but does not require the use of fluorescence microscopy.
Moreover, FISH signals fade within a few weeks and the FISH results have to be recorded
with expensive digital systems. This is not the case for CISH staining. Owing to the
similarity with IHC staining, CISH is also easier to interpret for pathologists who
are not trained with fluorescence. In one study (Tanner et al, 2000), CISH was demonstrated
to be well correlated with FISH.
The aims of our study were to: (a) confirm the good correlation between FISH and CISH
in a nonhomogeneous series of breast tumours coming from eight different laboratories
using different fixation procedures, (b) analyse this correlation according to the
expression of HER2 protein analysed by IHC (c) focus on IHC 2+ cases and analyse in
this situation if CISH gives the same information as FISH for the treatment of patients.
MATERIALS AND METHODS
Tumours
A total of 79 tumours were collected from eight French laboratories. Each laboratory
selected cases in which IHC and FISH were previously and successfully performed. In
order to analyse the discriminating power of CISH in difficult cases, tumours scored
as 2+ by IHC were chosen in priority for this study. Owing to differences in the fixative
procedure between the laboratories, 47 tumours were fixed in neutral-buffered formalin,
10 in Holland's bouin, and 22 in alcohol–formalin–acetic acid (AFA).
IHC
The monoclonal antibody CB11 (Novocastra, Newcastle, England) was used in 25 cases
and the polyclonal antibody A485 (Dako, Glostrup, Denmark) in 44 cases. For all slides,
immunostaining was scored according to the Herceptest® scoring system, which is also
used in clinical trials (Cobleigh et al, 1999; Slamon et al, 2001). Negativity of
normal glands was the prerequisite for interpreting the cases, according to the recommendations
of the College of American Pathologists (Fitzgibbons et al, 2000).
FISH
FISH was performed in three different referent laboratories. It was performed on frozen
tumour sections in 65 cases and on fixed-paraffin-embedded samples in the 14 remaining
cases. FISH experiments were performed according to the protocol given by the supplier
(PathVysion kit, Vysis, Downers Grove, IL or Ventana HER2 inform, Tucson, AZ). The
centromeric probe of chromosome 17 was included in FISH analyses in 62 cases. In these
62 cases, HER2 amplification was determined as a ratio of HER2 and chromosome 17 centromere
signal counts. As in the study previously published (Tanner et al, 2000), ratios <2
were determined as no amplification with FISH (NAF) (Figure 1B
Figure 1
(A) CISH, only one or two signals are present in the nucleus of tumour cells (NAC).
(B) Same case analysed with FISH (NAF). Pink dots correspond to HER2 probe and green
dots correspond to centromere 17 probe. (C) CISH, six signals are present in the nucleus
of tumour cells (LAC). (D) Same case analysed with FISH (LAF) with a ratio of HER2
dots/centromere 17 dots=3. (E) CISH, large gene copy clusters are present in the nucleus
of tumour cells (HAC). (F) Same case analysed with FISH (HAF).
), those between 2 and 5 as low-level amplification with FISH (LAF) (Figure 1D) and
those >5 as high-level amplification with FISH (HAF) (Figure 1F). In the 14 other
cases, without centromeric 17 analysis, like for the CISH analysis in the study previously
published (Tanner et al, 2000), HER2 gene was judged as NAF when 1–5 signals were
present per nucleus. When 6–10 signals were present in more than 50% of tumour cell
nuclei, the tumours were judged as LAF. Finally, tumours having more than 10 signals
in more than 50% of the nuclei were judged as HAF.
CISH
CISH experiments were performed according to the protocol given by the supplier (Zymed
Inc., South San Francisco, CA, USA). The interpretation of the signal was that used
by other authors (Tanner et al, 2000) and was only performed on invasive tumour patterns.
HER2 gene was judged as nonamplified with CISH (NAC) when 1–5 signals were present
per nucleus (Figure 1A).When 6–10 signals were present in more than 50% of tumour
cell nuclei, the tumours were judged as having a low level of amplification with CISH
(LAC) (Figure 1C). Finally, the tumours with more than 10 signals or with large gene
copy clusters in more than 50% of the nuclei were judged as having a high level of
HER2 gene amplification (HAC) (Figure 1E).
RESULTS
Evaluation of IHC staining, FISH and CISH signals were performed in a blinded manner.
IHC
A total of 27 (34%) tumours were defined as 0 or 1+, 29 (37%) tumours were defined
as 2+, and the remaining 23 (29%) tumours were defined as 3+ (Table 1
Table 1
HER2 gene amplification determined by FISH and CISH, according to the overexpression
of HER2 protein determined by IHC
IHC
0 or 1+
2+
3+
(N=27)
(N=29)
(N=23)
FISH
27 NAF
14 NAF
11 LAF
4 HAF
1 NAF
22 HAF
CISH
25 NAC
align="center"12 NAC
8 LAC
4 HAC
1 NAC
20 HAC
2 WS
1 LAC
2 HAC
1 NAC
1 HAC
1 WS
1 WS
NAF=no amplification with FISH; LAF=low level of amplification with FISH; HAF=high
level of amplification with FISH; WS=without any signal; NAC=no amplification with
CISH; LAC=low level of amplification with CISH; HAC=high level of amplification with
CISH.
).
FISH
As a result of the material chosen for this study, FISH analysis was necessarily successful
in all cases. In all, 41 tumours were determined as NAF, 11 tumours as LAF and 26
tumours as HAF (Table 1).
CISH
CISH was successful in 75 of the 79 tumours (94.9%). The four cases without any signal
(WS) corresponded to four of the 22 tumours fixed in AFA. In these tumours, despite
the use of a great variation of pretreatment procedures, no signal was present in
any tumour cell. In all, 39 tumours were determined as NAC, nine as LAC and 27 as
HAC (Table 1).
Comparison of CISH and FISH results
Only 75 tumours were available for this comparison (Table 1). When we compared all
CISH amplifications (LAC+HAC) to all FISH amplifications (LAF+HAF), an agreement was
found in 72 out of 75 (96%) tumours (Table 3
Table 3
Advantages and disadvantages of FISH and CISH
FISH
CISH
Pro
Con
• Possibility of multicolour (HER2 and chromosome 17)
• No multicolour detection
• Is the simplest, without any chromogen detection
• Needs a chromogenic procedure
• Is currently the standard hybridisation procedure for HER2
• Not yet a standard hybridisation procedure for HER2
Con
Pro
• Requires a modern and expensive fluorescence microscope
• An ordinary microscope is effective
• FISH is not routinely used in pathology, and pathologists are not trained to analyse
FISH signals
• Pathologists are familiar with IHC signals
• Fluorescence signals can fade within several weeks
• Chromogenic reaction is permanent
• Results have to be recorded with an expensive CCD camera
• Regular slide storage
• Morphology is not always easy to analyse
• Morphology is easier to analyse
). The kappa coefficient (κ) measuring agreement between the methods (0: no agreement,
1: agreement) was 0.97 (P<10−9), and if FISH was chosen as the gold standard, the
sensibility of CISH would be close to 97% with a specificity of 95%. In nonproblematic
IHC tumours (0, 1+ and 3+), this agreement was found in 46 out of 47 (98%) cases (κ=0.95
(P<10−9), sensibility=95% and specificity=100%). On the other hand, in problematic
tumours (2+), this agreement was found in 26 out of 28 (92.8%) cases (κ=0.85 (P<5
× 10−6), sensibility=100% and specificity=85%). When we compared the level of amplification
determined with the two methods, an agreement was found in 70 out of 75 (93.3%) with
a κ=0.88 (P<10−9).
Analysis of the cases with polysomy of chromosome 17
Centromeric probes of chromosome 17 were included in FISH analyses in 61 of the 75
cases successfully analysed with CISH. The result of the analysis of this centromere
is summarised in Table 2
Table 2
Analysis of the cases with or without polysomy of chromosome 17
IHC
0 or 1+
2+
3+
N=24
N=16
N=21
Normal chromosome 17 status
N=18
N=6
N=20
FISH
18 HAF
4 NAF
2 LAF
1 NAF
19 HAF
CISH
18 HAC
4 NAC
2 LAC
1 NAC
18 HAC
1 NAC
Chromosome 17 polysomy
FISH
N=6
N=10
N=1
6 NAF
6 NAF
1 LAF
3 HAF
1 HAF
CISH
6 NAC
5 NAC
1 LAC
3 HAC
1 HAC
1 LAC
NAF=no amplification with FISH; LAF=low level of amplification with FISH; HAF=high
level of amplification with FISH; WS=without any signal; NAC=no amplification with
CISH; LAC=low level of amplification with CISH; HAC=high level of amplification with
CISH.
. Using a χ
2 test, we found that polysomy was statically (P<0.005) more frequently observed in
IHC 2+ tumours (10 out of 16: 62.5%) than in other situations (7 out of 45: 15.55%).
DISCUSSION
Since the FDA approved Herceptin® for the treatment of metastatic breast cancer (Pegram
et al, 1998; Cobleigh et al, 1999; Roche and Ingle, 1999; Slamon et al, 2001), and
in order to determine whose patients might benefit from this new therapy, there has
been a need to evaluate precisely the HER2 status of breast cancer specimens. The
determination of this status will also be important to choose the adjuvant strategy
if clinical trials including Herceptin® as adjuvant therapy give a positive result
(Hortobagyi and Perez, 2001). Moreover, in the future, HER2 status may also help select
patients for tyrosine kinase inhibitor therapy (Moasser et al, 2001). Two major methods
(IHC and FISH) for the determination of this HER2 status have been developed all around
the world but there is no consensus up to now regarding the best methods to determine
this status (Thor, 2001). In two recent different clinical trials, poor concordance
was found between local and central or reference IHC testing for HER2 (Paik et al,
2002; Roche et al, 2002). This poor concordance being given, the authors of these
studies recommended that the HER2 status of patients included in clinical trials should
be done in large-volume reference laboratories. These data also suggested an urgent
need to improve the quality control programme in laboratories that use IHC testing
(Fitzgibbons et al, 2000; Hoang et al, 2000; Ridolfi et al, 2000; Tubbs et al, 2001;
Paik et al, 2002; Roche et al, 2002; Vogel et al, 2002). In a previous study, we showed
that FISH may be used to obtain successfully a calibration of the in-house IHC technique
(Vincent-Salomon et al, 2003).
CISH, a hybridisation procedure using a staining of the probe similar to IHC staining,
has been previously proposed as an alternative for FISH (Tanner et al, 2000). When
compared with FISH, CISH has been described as having several advantages (Tanner et
al, 2000; Zhao et al, 2002). It does not require an expensive fluorescence microscope
with multi-band-pass filters, CISH staining is permanent and it does not need to be
recorded with an expensive CDD camera. Moreover, morphology is easier analysed on
CISH slides, particularly for distinguishing invasive cancer cells and in situ components.
Finally, pathologists are more familiar with the IHC signal than with the FISH signal.
The advantages and disadvantages of these two hybridisation techniques are summarised
in Table 3.
In order to confirm the results of this study and to precise the place of this technique
in problematic IHC cases (2+), we performed a study on a group of 79 breast tumours
that contained an abnormal percentage (37%) of 2+. Our study was also different from
the study published earlier because the breast tissue came from different purveyor
laboratories using different fixatives. CISH procedures were successful in 95% of
our cases, which is identical to the results observed with FISH on paraffin sections
(Lebeau et al, 2001) and very close to the results (98%) published with CISH in an
unselected group of tumours (Tanner et al, 2000). It may be noticed that AFA seems
to be a less effective fixative procedure for CISH, as CISH was successful in only
18 (82%) of the 22 tumours fixed in AFA and successful in all (100%) of the tumours
fixed in neutral-buffered formalin or Holland's bouin. We found a very good concordance
between the CISH and FISH. In terms of amplification, there was indeed an agreement
between CISH and FISH in 96% of the tumours, which is almost the same as the agreement
previously published (93.6%) (Tanner et al, 2000). The agreement was a little higher
(98%) for nonproblematic (0, 1+, 3+) IHC cases than for problematic (2+) IHC tumours
(93%). Owing to the small number (3%) of 2+ cases in their study, Tanner et al did
not notice this small difference. In terms of sensitivity, when we compared the level
of amplification estimated by the two methods (no amplification, low level and high
level of amplification), we found that the results of CISH analyses were very close
to those given by FISH, with an agreement in 93% of the tumours. According to Tanner
et al (2000), the discrepancy between CISH and FISH may be because of a lower sensitivity
of CISH. However, this explains only one discordant case in our study, which was found
to be amplified with FISH but not with CISH. Other discrepancies may also be because
of differences in the sample materials or the thickness of the slides. The most difficult
situations with CISH are when 6–10 spots are present in tumour cells. Double-colour
FISH analyses may give more information, particularly the ratio between HER2 signal
and the number of chromosome 17, and may separate the high polysomy of chromosome
17 and the very low level of HER2 amplification. We found that polysomy of chromosome
17 is statically more frequent in IHC 2+ tumours, but only one of our discordant cases
could probably be linked to this phenomenon. In routine, these situations are very
infrequent and it is not proved that this distinction is relevant in terms of response
to Herceptin® therapy. Clinical trials, including a large number of IHC 2+ tumours
with a low level of amplification, are needed to confirm that the exact level of HER2
gene amplification is important for the patient's selection for specific therapy.
Anyway, double-colour staining CISH procedures, including HER2 and chromosome 17 probes,
will soon be available. The results of these new procedures would also have to be
compared with the double-colour FISH analysis.
Our study confirms that CISH may be an alternative to FISH for the determination of
HER2 gene status, particularly in laboratories that are not equipped or trained from
fluorescence analyses. In our opinion, CISH is too expensive and too sophisticated
to be an alternative to IHC screening of all the breast tumours. However, because
of the good correlation between CISH and FISH, even in ambiguous IHC results, we think
that it may be used for the determination of gene amplification status in IHC 2+ tumours.
Owing to the poor concordance between HER2 status established in local laboratories
in comparison to reference laboratories, we also think that, in many laboratories,
CISH may be an excellent method to calibrate IHC procedures or, as a quality control
test, to check regularly that the IHC signal is in agreement with gene status.