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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an average of 11 % of breast cancers are HER-‐2 positive (unpublished results).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.

10

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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.

46. O'Malley FP, Chia S, Tu D, Shepherd LE, Levine MN, Huntsman D, et al. Topoisomerase II alpha protein and responsiveness of breast cancer to adjuvant chemotherapy with CEF compared to CMF in the NCIC CTG randomized MA.5 adjuvant trial. Breast Cancer Res Treat. 2011 Jul. 1;128(2):401–9.

47. Mueller RE, Parkes RK, Andrulis I, O'Malley FP. Amplification of the TOP2A gene does not predict high levels of topoisomerase II alpha protein in human breast tumor samples. Genes Chromosomes Cancer. 2004 Apr.;39(4):288–97.

48. Mukherjee A, Shehata M, Moseley P, Rakha E, Ellis I, Chan S. Topo2α protein expression predicts response to anthracycline combination neo-‐adjuvant chemotherapy in locally advanced primary

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breast cancer. Br J Cancer. 2010 Dec. 7;103(12):1794–800.

49. Di Leo A, Larsimont D, Gancberg D, Jarvinen T, Beauduin M, Vindevoghel A, et al. HER-‐2 and topo-‐isomerase IIalpha as predictive markers in a population of node-‐positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide. Ann Oncol. 2001 Aug. 1;12(8):1081–9.

50. Tandon AK, Clark GM, Chamness GC, Ullrich A, McGuire WL. HER-‐2/neu oncogene protein and prognosis in breast cancer. J Clin Oncol. 1989 Aug.;7(8):1120–8.

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52. Yeh I-‐T, Martin MA, Robetorye RS, Bolla AR, McCaskill C, Shah RK, et al. Clinical validation of an array CGH test for HER2 status in breast cancer reveals that polysomy 17 is a rare event. Mod Pathol. 2009 Sep.;22(9):1169–75.

53. Bai Y-‐F, Ren G-‐P, Wang B, Teng L-‐S, Liu X. [Comparison between analysis of HER2 gene and chromosome 17 in breast cancer by dual-‐probe chromogenic in situ hybridization and fluorescence in situ hybridization]. Zhonghua Bing Li Xue Za Zhi. 2010 Mar.;39(3):161–5.

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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.

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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+


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+


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+


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+


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+


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+


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+


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+


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.

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