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Chromogenic in situ hybridization of topoisomerase 2 alpha in HER2
positive breast cancer – a more specific probe
Stian Wendelborg
Medical Student, University of Oslo
Abstract
Anthracycline-‐based treatment for HER2-‐positive breast cancer is associated with
serious side effects. The topoisomerase II alpha protein is the mechanical target of
anthracyclines, but its role as a biomarker has yet to be established. Amplification of
TOP2A is reported in 24-‐54% of HER2 positive tumours. Co-‐amplification has been
proposed as one explanation due to the close proximity of the two genes, but it is also
possible that the TOP2A DNA probes are too large, giving false positives. Chromosome
17 anomalies have also been suggested. In this study, we use new, smaller DNA probes
for in situ hybridization in 153 HER2-‐positive tumours. 33% were TOP2A-‐amplified,
and we found only three cases of CEP17 anomaly (deletions). A smaller DNA probe is
less likely to overlap with adjacent regions, and as such is more specific. The incidence
of TOP2A amplification in our material was consistent with current research, indicating
that DNA probe overlap cannot explain the amplification of TOP2A seen in HER2-‐
positive tumours.
Introduction
Breast cancer patients with HER2-‐amplified tumours are usually treated with
anthracycline-‐based chemotherapy. Such therapy is associated with serious negative
side effects such as cardiotoxicity (1), acute leukemia and myelodysplasia (2). Targeting
the right sub-‐group of patients has been the topic of much discussion. This is partly
because anthracyclines are inhibitors of DNA topoisomerase, a cleavage-‐ and re-‐ligation
enzyme, and not HER2 itself.
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HER2
HER2 (human endothelial growth factor receptor 2) is a receptor tyrosine kinase (RTK)
whose gene is amplified in 15-‐45% of invasive breast cancers (3-‐13). In our institution,
an average of 11 % of breast cancers are HER-‐2 positive (unpublished results). An
oncogene, located on the long arm of chromosome 17 (17q11.2-‐12), it encodes a
transmembrane glycoprotein of 185 kD. The RTKs are a family of receptors involved in
essential cellular processes like metabolism, migration, differentiation, proliferation,
survival and intercellular communication during development (14).
TOP2A
Topoisomerase II alpha (TOP2A), located on chromosome 17q12-‐21 (15), encodes an
ATP-‐dependent enzyme crucial in DNA replication. The 170 kD protein cleaves double-‐
stranded DNA, releasing the torsional stress of supercoiling during replication by letting
another DNA-‐duplex pass through the break, and subsequently religating the cleavage
site. Disturbance of the activity of topoisomerase II alpha leads to breaks in double
stranded DNA, and to cell death (16).
Recent studies indicate that there may be less co-‐amplification of HER2 and TOP2A than
previously assumed, and that the over-‐expression of TOP2A in HER2-‐positive breast
cancers may in part stem from probes containing the DNA sequences of both genes, and
in part from polysomy of chromosome 17 (17,18).
The aim of this study was to validate the presumed increased specificity of a new,
shorter DNA probe for TOP2A, using silver in situ hybridization (SISH).
Materials and methods
The pathology department files were searched for HER2-‐positive breast carcinomas
dating from 2005 until the first 6 months of 2011. HER2-‐positive cases were
immunohistochemically (IHC) 2+/3+ positive and/or amplified by in situ hybridisation.
In situ hybridisation was done either as FISH (fluorescent in situ hybridisation) or as
SISH. Amplification is defined as a minimum HER2-‐gene to CEP17 ratio larger than 2,0
as according to the Food and Drug Administration (19).
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A total of 265 cases were found. 73 were omitted due to sub-‐optimal tumour material,
missing paraffin embedded sections or failure to meet the HER2 positivity criteria. 39
were omitted due to time constraints. 153 cases were included in this study.
Data concerning subtype, grading, pTNM-‐staging, tumour size, estrogen-‐ and
progesterone receptor status, axillary lymph node status (ALN) and patient age were
likewise extracted from the data files. Estrogen and progesterone receptor positivity
was defined as nuclear positivity in > 10 % of the tumor cell nuclei (cut-‐off for ER
changed to > 1% later)
The formalin-‐fixed, paraffin-‐embedded tissue samples underwent in situ hybridization
for chromosome 17 and TOP2A according to the manufacturer’s procedures.
The hybridized slides were compared to their hematoxylin-‐eosin counterparts in order
to assure that signals were counted from cells in the tumour tissue. An average number
of signals for CEP17 and TOP2A for each tumor were calculated counting 40 cells in a
bright field microscope at 40x magnification. Single signals of TOP2A counted as 1 (fig.
1), small clusters counted as 5 (fig. 2) whereas larger clusters counted as 10 signals (fig.
3). Each slide was primarily examined by a student and checked by an experienced
pathologist. Upon discrepancy, a pathologist would do another full signal count (3
cases). The TOP2A:CEP17 ratio for each cell was calculated to determine amplification,
deletion or polysomy. The cut-‐offs were set to 2.0 for amplification and 0.8 for deletion
(17,20). Ratios of >1,5 for amplification and 0,5-‐1,0 for deletion have also been used in
other studies (ibid). Defining polysomy is complicated by a number of factors, nuclear
truncation during sectioning being an important one (21). Also, chance decides wether
the centromere is included in the optical section or not. The colour points using SISH are
less distinct from each other than those seen in FISH, making it easier to confuse signals.
Therefore, we used a relatively low threshold in order to increase the sensitivity of
discovering polysomies on behalf of decreased specificity. Many studies use a CEP17
copy number threshold of 2-‐5, 3 being the most common, to define polysomy 17 (7,22-‐
24), but our data material had no such cases. Polysomy of chromosome 17 was therefore
defined using the lowest previously reported CEP17 copy number of ≥1,86 (25).
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Silver in situ hybridization of TOP2A
A dinitrophenol (DNP)-‐labelled probe (Ventana Medical Systems’ TOP2A DNA Probe)
spanning 67,400 base pairs of the TOP2A gene binds to the target DNA and subsequently
binds to rabbit anti-‐DNP antibody. Goat anti-‐rabbit antibody is conjugated to
horseradish peroxidase (HRP) which functions as a chromogenic enzyme, reducing
silver ions to metallic silver atoms by the addition of silver acetate, hydroquinone and
hydrogen peroxide (ultraView SISH Detection Kit, see fig. 4). The precipitate is deposited
in the nuclei, and after counterstaining the TOP2A gene can be visualized as a black
silver dot using bright field microscopy.
Red in situ hybridization of chromosome 17
The Ventana Medical Systems' INFORM Chromosome 17 Probe with DNP label is a 42
base-‐pair synthetic oligonucleotide that binds to the centromeric region of chromosome
17. Rabbit anti-‐DNP antibody is used as with SISH for TOP2A. The ultraViewTM Alkaline
Phosphatase Red ISH Detection Kit contains an alkaline phosphate-‐labelled secondary
antibody. The complex is visualized as a red dot in the microscope after utilization of the
naphtol and Fast red chromogens.
Results
Patients
All patients (n=153, 152 female, 1 male) included in the study had histopathologically
proven invasive breast carcinoma. Average patient age at diagnosis was 57,5 years
(range 28-‐92 years). Average tumour size was 23,6 mm (range 3-‐100 mm). The
majority (144 cases, 93%) were of ductal subtype, the remainder were either lobular (7
cases, 5%), mucinous (1 case, 0,6%) or of a mixed ductal and lobular subtype (1 case,
0,6%). 142 cases tested 2+ or 3+ on immunhistochemistry for HER2 (94%, 21 and 121
cases, respectively). 127 cases were amplified for the HER2 gene (83%). 21 cases (14%)
were not registered with ISH for HER2, but in all instances there was a 3+ IHC result for
HER2. 3 cases (2%) showed no HER2 amplification with ISH but 3+ on IHC. 83 tumours
(54%) were estrogen receptor positive. 66 tumours (43%) were progesterone receptor
positive. An overview of SISH-‐results, HER2 status, age, tumour size, grade, stage and
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receptor status for each sample is shown in table 2. A summary of data is shown in table
1.
SISH
Of the 153 HER2 positive tumours undergoing dual chromogenic in situ hybridization,
we found that 33% were amplified for TOP2A (51 cases) using a cut-‐off ratio of ≥2,0. 5%
had TOP2A deletion (8 cases, cut-‐off ratio ≤0,8). The remaining 62% had normal TOP2A
gene copy number (TOP2A:CEP17 ratio 0,8-‐2,0). Using a cut-‐off of CEP17>1,86 gives a
total of 3 tumours polysomic for chromosome 17.
Discussion
Finding valid biomarkers for the benefit of anthracycline-‐based chemotherapy has so far
been unsatisfactory. Ongoing research concerning the role of TOP2A and chromosome
17 copy number aberrations shows no clear conclusion yet, but there are indications
that gene-‐adjacent regions may have an important role to play. Therefore, we wanted to
investigate the specificity of a new TOP2A DNA-‐probe, in the hope that better tools
might lead to more consistent results in future research. The DNP-‐labelled SISH probe
under investigation spans a considerably shorter segment of base pairs, respectively
30% and 42% the size of those targeted by two well-‐known probes used in FISH
analysis of TOP2A (20).
HER2 amplification has traditionally been regarded as a useful marker in identifying the
subset of patients who may benefit from anthracycline-‐based chemotherapy. This is,
however, not intuitively comprehensible, as anthracyclines do not target the receptor
coded by the HER2 gene. Rather, the topoisomerase II alpha protein is one of the
intracellular targets of such therapy, and the TOP2A gene is amplified in only 24-‐54% of
HER2-‐amplified tumors (15,26,27), creating some confusion regarding the mechanism
of action of the hitherto important anthracyclines. TOP2A and HER2 are believed to have
been co-‐amplified due to their very close location on the long arm of chromosome 17
(20,28,29). Studies by Arriola and Kauraniemi have observed common regions of
amplification around HER2 spanning 280-‐746 thousand base pairs, containing over 20
genes (28,30), with even more additional genes located next to these regions (29).
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TOP2A may be one such gene, potentially influencing amplification and expression of
HER2 and thus its clinical phenotype (14).
Chromosome 17, site of both genes in question, is the most gene dense chromosome in
our genome. Interestingly, chromosome 17 also contains the p53 and BRCA1 genes,
which are important in cancerogenesis. Some investigations have highlighted polysomy
17 as a possible mechanism of increased protein expression, including that of increased
topoisomerase II alpha. A trial published in 2011 comparing cyclophosphamide,
epirubicin (antracycline) and fluorouracil (CEF) against cyclophosphamide,
methotrexate and fluorouracil (CMF) suggested that tumours with duplication of CEP17
showed borderline responsiveness to anthracycline-‐based chemotherapy (17), whereas
eusomy 17 showed no apparent benefit. The authors proposed that CEP17-‐duplication
may indicate changes such as unbalanced translocations, sub-‐chromosomal
amplification or deletion, or duplication of the entire genome, and that CEP17 may be a
more reliable marker than HER2 and TOP2A. This remains to be confirmed by further
research.
Trying to determine the cause of increased benefit with anthracyclines depends on valid
techniques. In situ hybridization is widely accessible and has been the preferred method
for identifying changes in HER2 and TOP2A copy number (19,31,32). The close
proximity of the two genes, and the possible modifying regions neighbouring them,
postulates that a very specific DNA probe is necessary – one that overlaps as little as
possible with bases outside the gene itself. Moelans et al. used multiplex ligation-‐
dependent probe amplification-‐based copy number analysis (MLPA) as a control when
evaluating the DAKO pharmTM probe (228 kb of the TOP2A region) and the
PathvysionTM probe (160 kb of the TOP2A region). They found no difference in
detection of TOP2A deletions, but discovered that the larger of the probes resulted in
“overdetection” of HER2, most likely because of TOP2A overlap with the adjacent HER2
region (20).
Studies by Järvinen show that overexpression of HER2 alone does not influence the
sensitivity to anthracyclines (15). Indeed, patients with HER2-‐positivity who also have
TOP2A amplification have shown longer survival and increased response to
anthracyclines than those without TOP2A amplification (33-‐36). Moelans et al. (20)
discuss the wide variability of TOP2A amplifications (33-‐60% in some studies) and
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deletions (20-‐42%) (36-‐39), highlighting that although changes in TOP2A is very rare
when there is no HER2 amplification, some studies report a 10-‐20% TOP2A
amplification rate in HER2-‐negative breast cancer. According to Moelans, this variability
is likely related to the FISH technology used to determine gene status, a technique
whose reproducibility is questioned by other authors (18). A 2011 study of 1614 HER2-‐
negative cases discovered no TOP2A amplification, but 3% TOP2A deletion (19).
Elsewhere, results indicate that a significant number of HER2-‐negative tumours
exhibited high levels of TOP2A (40).
The predictive value of HER2 and TOP2A for anthracycline-‐based chemotherapy is still
the subject of much debate. Some retrospective analyses of randomized studies
indicated that adjuvant anthracycline therapy could be most effective when TOP2A was
amplified (19,31,32). In 2008, the results of two meta-‐analyses indicated that only
HER2-‐positive patients benefited from anthracycline-‐based chemotherapy (41,42).
However the same year, the BR9601/NEAT study (n=1870) (8,43) showed no consistent
predictive value of neither HER2 nor TOP2A. Furthermore, this study suggested that
polysomy 17 might have a part to play.
In a meta-‐analysis of four trials (n=1944), Di Leo et al. showed a modest and borderline
statistical predictive value using HER2 and TOP2A as biomarkers (44,45). In a new
meta-‐analysis in Lancet Oncology in 2011, Di Leo et al. once again compared CMF to
anthracycline-‐based therapy. Although HER2 amplification and combined amplification
or deletion of TOP2A may play a role in predicting response to anthracycline-‐based
therapy, their findings did not support the use of this therapy in only these two groups
of patients (18).
Two studies suggest that TOP2A deletion may provide increased sensitivity to
anthracyclines, but there is still no biological rationale for this (6,34).
Another problem in the search for valid biomarkers for breast cancer is the degree of
correlation between gene status and protein levels. Findings by O’Malley et al. suggest
that the expression of the topoisomerase protein is not regulated by the copy number of
the TOP2A gene, but rather at an RNA or post-‐translational level (46,47). Their study
found no significant correlation between TOP2A gene status and topoisomerase protein
levels (46). Some studies favor TOP2A expression over amplification as a positive
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predicitve marker of anthracycline benefit (24,48). It would be both convenient and
logical if TOP2A protein expression should show itself a valid biomarker, considering
the mechanical effect of protein inhibition believed to be offered by anthracyclines
(47,49-‐51).
There has been little progress regarding biomarkers that predict response to
chemotherapy in breast cancer. Results are inconsistent, and conclusions differ as to the
value of TOP2A regarding anthracyclines. If such treatment response cannot be
routinely predicted by HER2 and TOP2A, as according Di Leo and his associates (18),
who then should receive this widely used type of chemotherapy?
CEP17 copy number aberration was not often seen in our data. Using the most sensitive
criterion reported, we found only 3 tumours polysomic for chromosome 17. This may
indicate that increased number of TOP2A signals is not the result of increased number of
chromosomes, and is consistent with other studies that show that polysomy 17 is a rare
event in breast cancer (52). Abnormal signals of CEP17 is probably due to
pericentromeric gains or losses on chromosome 17 (53). In light of our results, CEP17
polysomy does not seem a good marker of anthracycline responsiveness.
A euploid human cell contains two chromosomes. However, in our material the average
number of CEP17 signals was 1,21 (0,53-‐2,55). This can be explained by the cutting of
nuclei in two, leaving only one chromosome in the slide. Alternatively, it can be
explained by an underestimation due to poor signal strength or partially overlapping
cells that obscure each other, combined with observer lack of experience in evaluation of
chromogenic in situ hybridization.
This study does not make a comparison of different TOP2A probes on the same tissue
samples. Such data would be necessary to determine with confidence any increased
probe specificity, as the number of TOP2A-‐amplified tumours in our data (33%) is
within the range reported (24-‐54%) in the literature using larger DNA probes. With
such differing rates of amplification of TOP2A, another investigation using larger probes
on our material could be helpful. However, it is reasonable to assume that much smaller
TOP2A DNA probes will overlap less with adjacent regions.
The majority of research being done on use of biomarkers and anthracyclines compares
CEF (with anthracycline) and CMF (without anthracycline). Such a study was beyond the
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scope of our investigation. A retrospective analysis of treatment results in our study is
theoretically possible, but with our small sample size, it uncertain wether there are
enough patients treated with anthracyclines versus non-‐anthracycline therapy to make
a statistically significant comparison.
In conclusion, a new TOP2A DNA probe of 67,400 base pairs detects amplification of
TOP2A in HER2-‐positive breast cancers at a rate comparable to that reported in
previous studies using larger DNA probes. A smaller DNA probe is less likely to overlap
with adjacent regions, and would be considered more specific. Our results suggest that
the amplification of TOP2A seen in HER2-‐positive tumours cannot be explained by DNA
probes spanning outside their designated target region. Our investigation cannot
conclude wether there is co-‐amplifiation or not, although it seems more likely now than
before. Certainly, a more specific DNA probe is no step backward.
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45. Di Leo A, C D, JMS B, al E. Final results of a meta analysis testing HER2 and topoisomerase II genes as pre-‐ dictors of incremental benefit from anthracyclines in breast can-‐ cer [abstract]. J Clin Oncol. 2010 Mar. 11.
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HER2 Ampl. No ampl. No ISH
IHC 3+ 95 3 23
IHC 2+ 21
IHC 1+ 2
IHC neg. 3
No IHC 6
Patient age Mean 57,5 (range 28-92)
Tumour size Mean 23,6mm (range 3-100)
TOP2A Amplification Deletion Polysomy
51 (32,9%) 8 (5,2%) 3 (1,9%)
PgR pos. PgR neg.
Estrogen receptor + 83 (54%) ER pos. 57 26
Progesterone receptor + 66 (43%) ER neg. 6 66
Tx 7 N0 71
T1a 4 Nx 70
T1b 16 N+ 12
T1c 56
T2 53
T3 15
T4 2
Table 1: Summary. N=153.
15
Figure 1: No TOP2A amplification. The signals can be counted individually as dark dots. The red dots represent CEP17.
Figure 2: TOP2A amplification where dark clusters count as f ive signals.
16
Figure 3: TOP2A amplification where dark clusters count as ten signals.
Figure 4: SISH reaction: TOP2A probe. Courtesy of Ventana.
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SISH Subtype
HER CEP17 TOP2A ratio Type Grade Size IHC FISH TNM stage ER PR Age
0,85 6,40 7,53 D 3 40 3+
pT2 pN1 pMx neg neg 71 0,90 6,15 6,83 D 3 18 3+ amplified pT1c pN0 pMx pos pos 60 0,95 6,40 6,74 D 2 35 2+ amplified pT2 pN1 pMx pos pos 46 1,00 6,70 6,70 D 2 12 3+ amplified pT1c pN1a pMx pos neg 54 0,98 6,50 6,67 D 3 17 3+ amplified pT1c pN0 pMx pos neg 56 1,03 6,60 6,44 D 3 25 3+ amplified pT2 pN0 pMx neg neg 65 1,05 6,50 6,19 D 2 23 3+
pT2 pN1 pMx pos pos 46
0,93 5,68 6,14 D 3 20 3+ amplified pT1c pN1m pMx pos pos 53 1,18 7,13 6,06 D 2 17 2+ amplified pTx pN0 pMx pos pos 57 0,95 5,58 5,87 D 3 34 3+ amplified pT2 pN0 pMx neg neg 81 0,90 5,00 5,56 D 2 10 3+ amplified pT1c pN0 pMx pos pos 42 1,05 5,55 5,29 D 2 15 3+
pT1c pN0 pMx pos neg 48
1,13 5,88 5,22 D 2 11 3+ amplified pT1c pN0 pMx pos neg 61 1,03 5,18 5,05 L 2 18 2+ amplified pT1c pN1 pMx pos pos 47 1,05 5,28 5,02 L 3 15 3+ amplified pT1c pN0 pMx pos pos 70 1,15 5,75 5,00 L 2 22 2+ amplified pT2 pNx pMx pos pos 65 1,75 8,68 4,96 D 2 15 3+ amplified pT1c pN0 pMx pos pos 43 1,45 6,90 4,76 D 2 15 3+ amplified pT1c pN1a pMx pos pos 83 0,98 4,45 4,56 M 3 30 3+ amplified pT3 pN3 pMx pos pos 64 1,38 6,25 4,55 D 1 17 3+ amplified pT1c pN0 pMx pos neg 62 1,13 5,05 4,49 D 3 13 3+
pT1c pN0 pMx neg neg 63
0,53 2,30 4,38 D 1 20 3+ amplified pT1a pNx pMx pos pos 92 0,95 4,13 4,34 D 3 12 3+ amplified pT2 pN0 pMx pos pos 56 1,08 4,35 4,05 D 2 15 3+ amplified pT1c pN0 pMx neg pos 52 0,83 3,30 4,00 D 3 27 3+
pT2 pN0 pMx neg neg 74
1,35 5,33 3,94 D 3 15 3+ not amp. pT1c pN1a pMx neg neg 60 1,30 5,08 3,90 L 2 24 3+
pT2 pN0 pMx pos pos 75
0,95 3,60 3,79 L 3 90 3+ amplified pT4 pN3 pMx pos neg 76 0,63 2,35 3,76 D 2 8 2+ amplified pT1b pN0 pMx pos pos 35 0,90 3,35 3,72 D 2 28 2+ amplified pT2 pN3 pMx pos neg 42 1,90 6,93 3,64 D 2 35 3+ amplified pT2 pN1 pMx neg neg 78 1,13 4,05 3,60 D+L 3 20 2+ amplified pT2 pN0 pMx pos pos 42 1,38 4,73 3,44 D 3 36 3+ amplified pT1c pN1 pMx neg neg 50 3,60 12,20 3,39 D 3 40 3+ amplified pT2 pN1 pMx neg neg 42 0,98 3,10 3,18 D 3 38 1+ amplified pT2 pN0 pMx neg neg 60 1,30 3,95 3,04 D 2 12 3+ amplified pT1c pNx pMx neg neg 32 1,20 3,38 2,81 D 2 13 3+ amplified pT1c pN0 pMx neg neg 45 1,25 3,43 2,74 D 2 11
amplified pT1c pN0 pMx pos pos 64
1,08 2,93 2,72 D 3 15 2+ amplified pT1c pNx pMx pos pos 37 1,35 3,35 2,48 D 3 9 3+ amplified pTx pN0 pMx pos pos 41 4,10 10,10 2,46 D 3 60 3+
pT3 pN1 pMx neg neg 83
1,10 2,68 2,43 D 3 30 3+ amplified pT2 pN2 pMx neg neg 48 1,00 2,38 2,38 D 2 60 2+ amplified pT3 pN3 pMx pos pos 43 1,10 2,50 2,27 D 2 18 3+ amplified pT1c pNx pMx pos neg 57 1,30 2,93 2,25 D 2 18 2+ amplified pT1c pNx pMx pos neg 53 1,13 2,40 2,13 D 2 23 3+
pT2 pN0 pMx pos neg 57
1,15 2,45 2,13 D 3 18 3+
pT1c pN0 pMx pos pos 38 1,33 2,75 2,08 D 2 14 3+ amplified pT1c pN0 pMx pos pos 58 0,88 1,80 2,06 D 3 30 3+ amplified pT2 pN1 pMx neg neg 65 1,10 2,25 2,05 D 2 17 3+ amplified pT1c pNx pMx pos neg 86 1,05 2,13 2,02 D 3 18 2+ amplified pT1c pN0 pMx neg neg 74 0,60 1,20 2,00 D 3 20 3+ amplified pT1c pN3 pMx neg neg 32 0,85 1,65 1,94 D 2 30
amplified pT2 pN1 pMx pos pos 50
1,18 2,28 1,94 D
25 2+ amplified pTx pN1 pMx pos pos 50 1,28 2,45 1,92 D 2 3 3+ amplified pT2 pN1 pMx pos neg 56 0,90 1,70 1,89 D 3 50 neg amplified pTx pN2 pMx pos pos 40
18
1,10 2,08 1,89 D 3 5 neg amplified pT1a pNx pMx neg neg 73 1,40 2,50 1,79 D 3 18 3+
pT1c pN1 pMx pos pos 43
0,90 1,60 1,78 D 2 25 3+ amplified pT2 pN1a pMx neg neg 75 0,88 1,55 1,77 D 3 15 3+ amplified pT1c pN0 pMx neg pos 53 1,08 1,90 1,77 D 2 12 3+ amplified pT1c pN0 pMx pos pos 60 1,00 1,75 1,75 D 3 21 3+ amplified pT2 pN0 pMx neg pos 58 1,15 2,00 1,74 D 2 50 3+ not amp. pT3 pN1 pMx pos pos 41 1,10 1,90 1,73 D 2 20 3+ amplified pTx pN1 pMx pos pos 58 0,95 1,63 1,71 D 3 6 2+ amplified pT1b pN0 pMx pos pos 65 0,83 1,38 1,67 D 3 14 3+ amplified pT1c pN0 pMx neg neg 49 1,15 1,90 1,65 D 3 11 3+ amplified pT1c pN0 pMx neg neg 52 0,98 1,60 1,64 D 2 80 3+ amplified pT3 pN2 pMx pos pos 54 0,88 1,43 1,63 D 3 21 3+ amplified pT2 pN1a pMx neg neg 50 1,00 1,63 1,63 D 3 25 3+
pT2 pN2 pMx pos neg 84
1,10 1,78 1,61 D 3 20 3+ amplified pT2 pN0 pMx neg neg 48 0,83 1,30 1,58 D 2 40 2+ amplified pTx pNx pMx pos pos 77 1,38 2,15 1,56 D 3 25 3+ amplified pT2 pN0 pMx neg neg 80 1,20 1,88 1,56 D 2 26 2+ amplified pT2 pN0 pMx pos pos 46 1,08 1,68 1,56 D 3 7 3+ amplified pT1b pN1a pMx neg pos 66 1,13 1,75 1,56 D 3 35 1+ amplified pT2 pN1 pMx pos pos 28 1,25 1,93 1,54 D 3 22 2+ amplified pT2 pN2a pMx pos pos 34 1,15 1,75 1,52 D 2 50 3+ amplified pT3 pN1 pMx neg neg 67 0,90 1,33 1,47 D 3 40 3+ amplified pT2 pN0 pMx neg neg 51 1,18 1,73 1,47 D 3 7 3+ amplified pT1b pN0 pMx neg neg 30 1,08 1,53 1,42 D 3 30
amplified pT2 pN3 pMx neg pos 80
1,20 1,70 1,42 D 3 20 3+ amplified pT1c pN0 pMx neg neg 48 0,98 1,38 1,41 D 3 50 3+ amplified pT3 pN3 pMx pos pos 47 1,10 1,55 1,41 D 1 8 3+ amplified pT1b pN0 pMx pos neg 58 1,15 1,58 1,37 D 3 4 3+ amplified pTx N1 pMx neg neg 60 1,03 1,40 1,37 D 2 25 3+ amplified pT2 pN1 pMx neg neg 40 1,05 1,43 1,36 D 3 20 3+ amplified pT1c pN1a pMx neg neg 53 1,15 1,55 1,35 D 2 8 2+ amplified pT1b pN0 pMx neg neg 68 1,10 1,48 1,34 D 3 40 3+
pT2 pN0 pMx neg neg 76
1,18 1,58 1,34 D 3 15
amplified pT1c pN0 pMx pos pos 53 1,05 1,40 1,33 D 3 30
amplified pT2 pN2 pMx pos pos 70
1,00 1,33 1,33 D 2 18 3+ amplified pT1c pN0 pMx neg neg 48 1,10 1,45 1,32 D 2 20 3+ amplified pT1c pN0 pMx neg neg 51 1,05 1,33 1,26 D 3 10 2+ amplified pT1b pN0 pMx neg neg 42 1,25 1,58 1,26 D 2 7 3+ amplified pT1b pN0 pMx neg neg 52 1,48 1,85 1,25 D 2 80 3+
pT3 pN1 pMx neg neg 82
1,20 1,50 1,25 D 3 25 3+ amplified pT2 pN0 pMx neg neg 51 0,90 1,13 1,25 D 3 21 3+
pT2 pN0 pMx neg neg 50
1,28 1,58 1,24 D 3 35 3+ amplified pT2 pN1 pMx neg neg 82 0,98 1,20 1,23 D 2 20 3+ amplified pT1c pN0 pMx pos pos 60 0,98 1,18 1,21 D 2 22 3+ amplified pT2 pNmi pMx pos pos 57 1,15 1,38 1,20 D 2 7 2+ amplified pT1b pN0 pMx neg neg 68 1,23 1,45 1,18 D 3 33 3+
pT2 pN1 pMx pos neg 54
1,40 1,65 1,18 D 2 10 3+ not amp. pT1b pN0 pMx pos pos 51 1,43 1,68 1,18 D 3 70 3+ amplified pT3 pN1 pMx neg neg 33 1,15 1,35 1,17 D 2 9 3+ amplified pT1b pN0 pMx neg neg 73 1,33 1,55 1,17 D 2 35 3+ amplified pT2 pN1 pMx neg neg 71 1,50 1,75 1,17 D 3 19 3+ amplified pT1c pN0 pMx pos neg 71 1,03 1,18 1,15 D 2 12 3+ amplified pT1c pN1a pMx pos pos 45 1,05 1,20 1,14 D 2 10 3+ amplified pT1c pN0 pMx pos pos 53 1,33 1,50 1,13 D 3 50 3+
pT2 pNx pMx neg neg 74
1,43 1,60 1,12 D 2 8 neg amplified pT1b pN0 pMx pos neg 77 1,55 1,70 1,10 D 3 11 3+ amplified pT1c pN3a pMx pos pos 49 1,05 1,15 1,10 D 3 26 3+ amplified pT2 pN2 pMx neg neg 39
19
1,23 1,33 1,08 D 3 21 3+ amplified pT2 pN1 pMx neg neg 49 1,10 1,18 1,07 D 2 40 3+
pT2 pN0 pMx pos neg 64
1,45 1,53 1,05 L 1 11
amplified pT1c pN0 pMx pos pos 63 1,03 1,08 1,05 D 3 4 3+ amplified pT1a pN0 pMx pos neg 68 1,48 1,53 1,03 D 3 20 3+ amplified pT1c pN2 pMx neg neg 46 1,60 1,65 1,03 L 2 22 3+
pT2 N0 pMx pos pos 61
1,08 1,10 1,02 D 3 50 3+
pT3 pN0 pMx pos pos 75 1,38 1,40 1,02 D 3 17 3+
pT1c pN0 pMx neg neg 58
1,43 1,45 1,02 D 2 35 3+ amplified pT2 pN1 pMx neg neg 66 1,33 1,33 1,00 D 2 23 3+ amplified pT2 pN2 pMx pos neg 1,73 1,73 1,00 D 3 16 3+ amplified pT2 pN0 pMx pos pos 51 1,48 1,48 1,00 D 2 50 3+
pT3 pN1 pMx neg neg 59
1,45 1,43 0,98 D 3 40 3+ amplified pT3 pN2 pMx neg neg 47 1,50 1,45 0,97 D 3 50 3+ amplified pT3 pN1 pMx neg pos 61 1,23 1,18 0,96 D 2 7 3+ amplified pT1b pN0 pMx pos pos 57 1,05 1,00 0,95 D 2 9 3+ amplified pT1b pN1m pMx neg neg 62 1,10 1,03 0,93 D 3 25 3+ amplified pT2 N0 pMx pos pos 38 1,40 1,30 0,93 D 2 12 3+ amplified pT1c pN1 pMx pos neg 61 1,68 1,53 0,91 D 2 100 3+ amplified pT3 pN2 pMx neg neg 40 1,63 1,48 0,91 D 3 12 2+ amplified pT1c pN0 pMx neg neg 55 1,80 1,63 0,90 D 2 15 3+ amplified pT1c pNx pMx pos pos 86 1,63 1,43 0,88 D 3 34 3+ amplified pT2 pN0 pMx neg neg 57 1,63 1,43 0,88 D 3 13 3+ amplified pT1c pN1 pMx neg neg 62 1,63 1,43 0,88 D 3 3 3+ amplified pT1a pN0 pMx neg neg 66 1,73 1,50 0,87 D 3 11 3+ amplified pT1c pN0 pMx neg neg 63 1,53 1,33 0,87 D 3 12 3+ amplified pT1c pN0 pMx pos neg 51 0,95 0,83 0,87 D 3 35 3+ amplified pT4 pN1 pMx neg neg 87 1,65 1,43 0,86 D 3 30 3+ amplified pT2 pN1 pMx neg neg 69 1,20 1,03 0,85 D 2 25 3+ amplified pT2 pN0 pMx pos pos 64 1,60 1,35 0,84 D 3 11 3+ amplified pT1c pN1 pMx neg neg 60 1,65 1,35 0,82 D 3 12 3+ amplified pT1c pN2 pMx neg neg 53 1,70 1,33 0,78 D 3 17 2+ amplified pT1c pN1a pMx pos pos 75 1,85 1,38 0,74 D 2 40 3+ amplified pT2 pN1 pMx pos pos 38 1,33 0,98 0,74 D 2 50 3+ amplified pT3 pN2 pMx pos neg 61 1,50 1,10 0,73 D 3 20 3+ amplified pT1c pN0 pMx neg neg 61 1,85 1,30 0,70 D 1 40 3+ amplified pT2 pN2 pMx pos pos 37 2,25 1,50 0,67 D 3 9 3+
pT1b pN0 pMx pos neg 62
1,68 1,00 0,60 D 3 9 3+ amplified PT1b pNx pMx pos pos 66 2,55 0,83 0,32 D 3 11 3+
pT1c pN1a pMx neg neg 41
Table 2: Data. Subtypes Ductal , Lobular and Mucinous. Estrogen receptor status ER. Progesterone receptor status PR. Age in years. Size in mm. All tumours with TOP2A amplification are marked with blue.