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514 Am J Clin Pathol 2009;132:514-520514 DOI: 10.1309/AJCPXY3MJ6GSRCYP

American Society for Clinical Pathology

Anatomic Pathology / HER2 SISH VS FISH

Chromogenic In Situ Hybridization

A Multicenter Study Comparing Silver In Situ HybridizationWith FISH

J.M.S. Bartlett, PhD, FRCPath,1,2 Fiona M. Campbell, MSc,1 Merdol Ibrahim, PhD,2 Peter Wencyk,3

Ian Ellis, MD,3 Elaine Kay,4 Yvonne Connolly, MSc,5 Anthony OGrady, PhD,4 Silvana Di Palma, MD,6

Jane Starczynski, PhD,7 John M. Morgan, PhD,8 Bharat Jasani, PhD, FRCPath,9 and Keith Miller, FIBMS2

Key Words: HER2; Amplification; Breast; Chromogenic in situ hybridization; Silver in situ hybridization; Fluorescence in situ hybridization;

Consistency; Intraobserver; Intersite; UK National External Quality Assessment Scheme

DOI: 10.1309/AJCPXY3MJ6GSRCYP

A b s t r a c t

Our purposes were to perform a robust assessment

of a new HER2 chromogenic in situ hybridization test

and report on concordance of silver in situ hybridization

(SISH) data with fluorescence in situ hybridization

(FISH) data and on intraobserver and interlaboratory

scoring consistency. HER2 results were scored from

45 breast cancers in 7 laboratories using the Ventana

(Tucson, AZ) INFORMHER-2 SISH assay and in

1 central laboratory using a standard FISH assay.Overall, 94.8% of cases were successfully analyzed

by SISH across the 6 participating laboratories

that reported data. Concordance for diagnosis of

HER2 amplification by SISH compared with FISH

was high (96.0% overall). Intraobserver variability

(8.0%) and intersite variability (12.66%) of absolute

HER2/chromosome 17 ratios appear to be tightly

controlled across all 6 participating laboratories. The

Ventana INFORMHER-2 SISH assay is robust and

reproducible, shows good concordance with a standard

FISH assay, and complies with requirements in nationalguidelines for performance of diagnostic tests.

According to guidelines for breast cancer management,

all patients with breast cancer must be tested for HER2 status

at initial diagnosis or at the time of recurrence.1,2 Establishing

tumor HER2 status supports treatment decisions by pre-

dicting responses to trastuzumab (Herceptin)1-5 and other

drugs, including tamoxifen, taxanes, and anthracyclines.2-4,6

Accurate and robust diagnostic testing of HER2 expression or

amplification is supported through additional guidelines and

external quality assurance schemes.1-5

Amplification of the HER2 gene drives overexpressionof the oncoprotein,7,8 and in situ hybridization (ISH) is an

essential component of HER2 testing in most countries.1,2,9-12

ISH tests measure HER2 with or without chromosome 17

copy number by using fluorescence in situ hybridization

(FISH) or chromogenic in situ hybridization (CISH) detection

methods, including silver staining (also known as silver ISH

[SISH]).1,13 Currently, FISH is regarded as the most accurate,

reproducible, and precise predictor of HER2 overexpres-

sion.5,7,8 The PathVysion system (Abbott UK, Kent, England)

comprises 2 fluorescently labeled probes for detection of the

HER2 gene and chromosome 17. ThisHER2 test is approvedby the US Food and Drug Administration and represents

the most widely used FISH test in the United Kingdom.1,2,5

However, a number of alternative probes and systems are

also available for the detection ofHER2 gene amplification.

HER2 assay systems must be reliable and reproducible across

multiple sites if they are to be applied within routine diagnos-

tic laboratories forHER2 testing. The UK National External

Quality Assessment Scheme (UK NEQAS) for ISH (UK

NEQAS ISH)5 monitors the quality of technical interpretation

relevant to routine diagnostic testing on a quarterly basis; data

returned by the participating laboratories are scored against

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Am J Clin Pathol 2009;132:514-520 515515 DOI: 10.1309/AJCPXY3MJ6GSRCYP 515


Anatomic Pathology / ORIGINALARTICLE

data on sequential sections produced by the UK NEQAS ISH

reference laboratories.

The aim of this study was to perform a robust assessment

of a new CISH assay, the Ventana INFORM HER-2 SISH

assay (Ventana, West Sussex, England), which detectsHER2

and chromosome 17 copy number. The technology has been

developed and recently released as an alternative bright-field

fully automated ISH assay, which is performed in approxi-

mately 6 hours. We provide data demonstrating that the SISH

assay results are equivalent to FISH and that the assay can be

run reliably on the BenchMark series of instruments (Ventana,

Tucson, AZ), in line with the quality requirements outlined by

the UK NEQAS-HER2 ISH scheme. We provide data on the

multicenter evaluation of 45 breast cancers using the SISH

assay compared with evaluation of the same cancers using a

FISH assay in a central reference laboratory. We document

concordance of SISH data from each laboratory with FISH

data from the central reference laboratory and intraobserver

and interlaboratory scoring consistency.

Materials and Methods

Study Design

The concordance ofHER2 determination by CISH using

the Ventana INFORM HER-2 SISH assay and by FISH, an

established method used routinely for clinical diagnosis,1,9

was determined on the basis of intrasite variation between

the 2 assays at a UK NEQAS ISH reference laboratory. Inaddition, the interlaboratory reproducibility of the INFORM

HER-2 SISH method was determined across 7 laboratories.

A commercially available tissue microarray (TMA; Stretton

Scientific UK, Stretton, England) containing 2 replicate

cores from 45 breast cancers was circulated to 7 laboratories

(randomly numbered 1-7) with experience in ISH methods.

All laboratories were reference laboratories from the UK

NEQAS ISH scheme (performing diagnostic and/or research-

based FISH). Each laboratory received the same material

and performed independent blinded analysis ofHER2 and

chromosome 17 using the INFORM HER-2 SISH assay. Inaddition, laboratory 1, a UK NEQAS ISH reference labora-

tory performed an independent blinded analysis ofHER2 and

chromosome 17 using FISH.

Determination ofHER2 and Chromosome 17 by SISH

and FISH

HER2 and chromosome 17 were determined by bright-

field automated CISH using the Ventana INFORM HER-2

SISH assay using the same batch of reagents in all centers.

Automated staining was performed on consecutive slides

using the Ventana BenchMark XT, which was installed and

validated in all sites, and all staff received appropriate training

in assay performance and analysis before the commencement

of the study. The assay protocol consisted of extended pretreat-

ment with CC2, pH 6.0, followed by protein digestion with

ISH protease 3 for 4 minutes for the xenograft control samples

and 12 minutes for the test TMA slides. Initial validation at

one center demonstrated more consistent staining for HER2

when the digestion time for the test TMA slide was reduced

to 8 minutes with ISH protease 3, whereas the remaining

laboratories used the recommended protocol of 12 minutes.

This procedure was followed by incubation with the specific

2,4-dinotrophenol-labeled DNA probes. Detection was per-

formed with the ultraView SISH Detection Kit and accessory

reagents (Ventana, West Sussex, England). This consisted of,

briefly, incubation with 2 consecutive antibodies followed by

the addition of 3 sequential silver reagents. The silver precipi-

tate is deposited in the nuclei, and a single copy of the chro-

mosome or theHER2 gene is visualized as 1 black dot. The

slides were then counterstained using Haematoxylin II (Fisher

Scientific, Loughborough, England) and a bluing reagent.

HER2 and chromosome 17 were determined in a single

central laboratory by dual-color FISH (PathVysion FISH

assay, Abbott UK) using UK NEQAS scoring guidelines.

FISH-stained TMA sections were analyzed at 630 to 1,000

magnification, and areas of carcinoma within each core were

identified. The number of chromosome 17 andHER2 signals

was counted in 20 nonoverlapping nuclei per core. The mean

HER2/chromosome 17 copy ratio was calculated per core,

and the meanHER2 and mean chromosome 17 copy numbers

observed were recorded on a core-by-core basis.

Analysis of Results

All data reported were collated centrally and analyzed in

the Edinburgh, Scotland, reference laboratory. Satisfactory data

were obtained from the CISH assay (INFORMHER-2 SISH)

in 6 of 7 participating laboratories; some laboratories repeated

the assay once or twice to obtain satisfactory data because

of failure to stain either the control or test slides for either

chromosome 17 or HER2. Because for some laboratories the

Ventana BenchMark XT systems were only installed for this

study, the cause of these failures was difficult to ascertain.The success rate for determination of HER2 using the

SISH assay was determined for each laboratory by case and

by core. The success rate for determination of HER2 using

the FISH assay was also determined for the central reference

laboratory (laboratory 1) by case and by core. Data are report-

ed as HER2/chromosome 17 ratios except where intrasite/

intersite variability is assessed for HER2 and chromosome 17

copy numbers.

Because 5 of 7 participating laboratories reported SISH

data from both cores for each case, the intraobserver variation

at each laboratory was analyzed. This analysis determined the

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Bartlett et al / HER2 SISH VS FISH

variation between duplicate cores for each case and was com-

pared with data obtained centrally for FISH testing.

The intersite variation between each of the individual sites

performing the SISH assay was determined: mean intersite

variation for each result reported was assessed, the percentage

variation was documented, and statistical differences in inter-

site variation were determined by using the Student ttest. An

additional analysis using the central laboratory (laboratory 1)

as the comparator investigated the correlation of absolute SISH

and FISH scores (all core data) between laboratory 1 and other

participating laboratories. Regression analysis was performed

on SISH results between each site and FISH results from

laboratory 1, and the slopes and intercepts between different

pairings were assessed using z statistics. The mean slope and

intercept, with SEs of these estimates, were compared with the

ideal slope (1.00) and intercept (0.0), with the average of SEs

from other slopes being assigned to the test situation.

To evaluate the concordance between FISH and SISH, a

similar analysis was performed on SISH results between each

site and FISH results from laboratory 1.

Results

Ventana INFORMHER-2 SISH Assay and Concordance

With the Standard FISH Assay

Table 1 shows the rate of successfulHER2determination

in the same TMA using the Ventana INFORMHER-2 SISH

assay across 7 UK NEQAS reference laboratories, comparedwith the FISH assay in reference laboratory 1. Satisfactory

data were obtained from the CISH assay (INFORM HER-2

SISH) in 6 of 7 participating laboratories. In 1 laboratory, the

SISH analysis for chromosome 17 failed on the first attempt,

and reagents expired before this could be repeated. The data

from this laboratory were therefore excluded because com-

plete data could not be provided.

Of the 45 cases determined in each laboratory using the

SISH assay (a maximum of 270 results in total), HER2 was

successfully determined by SISH in 89% to 100% of cases,

with an overall success rate of 94.8%. Similarly, of the 90duplicate cores scored by each laboratory (except the labora-

tory that determined only 45 cores, 1 per case), HER2 was

successfully determined by SISH in 83% to 96% of cores,

with an overall success rate of 89.4%. Of the 45 cases and 90

cores determined by FISH in the central laboratory (labora-

tory 1), HER2 was successfully determined by FISH in 39

(87%) of 45 cases and 76 (84%) of 90 cores.

Evaluation of the concordance of the Ventana INFORM

HER-2 SISH assay with the standard PathVysion FISH assay

is shown in Figure 1 and Table 2. Figure 1 shows examples

of the correlation of absolute SISH scores (all core data)

between individual laboratories compared with absolute FISH

scores (all core data) from the central laboratory (laboratory

1). Figure 1A shows that the absolute HER2/chromosome

17 data from the SISH assay for laboratory 1 are highly con-

sistent and very similar to the data obtained using the FISH

assay in the same laboratory (laboratory 1; slope of 1.02,

intercept of 0.06, andR value of 0.95). Figure 1B shows that

laboratory 5 had a number of cases with poor correlation of

absolute SISH scores with the FISH scores obtained using the

FISH assay in the central laboratory (laboratory 1), although

the overall correlation was adequate (slope of 1.01, interceptof 0.32, and R value of 0.92). The slope, intercept, and R

value for comparisons between all SISH scores from indi-

vidual laboratories and FISH scores from the central labora-

tory (laboratory 1) are shown in Table 3, which also includes

the concordance for diagnosis by SISH between each of these

laboratories and diagnosis by FISH in laboratory 1. Although

there were some minor differences between laboratories with

some individual cases showing poor correlation, concordance

for diagnosis ofHER2 amplification was high. Overall, there

was 96.0% (range, 88.9%-100%) concordance for diagnosis

ofHER2 amplification using SISH results from all 6 labo-ratories that reported data compared with FISH results from

laboratory 1. With the exception of laboratory 5, which had


for diagnosis between SISH and FISH was more than 95% in

all participating laboratories.

Intrasite (Intraobserver) and Intersite Variation inHER2

Testing

Table 4 shows mean intraobserver variation for HER2

copy number, chromosome 17 copy number, andHER2/chro-

mosome 17 ratio determined by SISH in each center and the

Table 1SuccessfulHER2 Determination in the Same TissueMicroarray by Laboratory

No. (%) of Cases No. (%) of Cores

Laboratory Test (n = 45 per Laboratory) (n = 90 per Laboratory)*

1 FISH 39 (87) 76 (84)1 SISH 44 (98) 83 (92)2 SISH 41 (91) 78 (87)3 SISH 42 (93) 80 (89)4 SISH 44 (98) NR*5 SISH 40 (89) 75 (83)6 SISH NR NR7 SISH 45 (100) 86 (96)Overall SISH 256/270 (94.8) 483/540 (89.4)

FISH, fluorescence in situ hybridization; NR, not reported; SISH, silver in situhybridization.

* Only 1 core was scored for each case in laboratory 4. Data from laboratory 6 are not included because this laboratory measured only

HER2; it did not obtain scores for chromosome 17 or the ratio; therefore, data

from this laboratory were excluded from the analysis. The overall data are given as

number/total (percentage).

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data from the FISH assay in laboratory 1. Intraobserver varia-

tion was calculated based on duplicate analysis of both cores

for each of the 45 cases at 5 of 7 participating laboratories.

Mean intraobserver variation for all SISH results was 5.9%

for HER2, 5.4% for chromosome 17, and 8.0% for HER2/

chromosome 17 ratio.

Table 5 shows intersite variation for the SISH assay

across all participating laboratories. This variation represents

the compound of technical and observer variation between

pairs of sites and observers. Overall intersite variation between

laboratories (mean SE, 12.66% 4.03%) was similar to that

observed in previous studies. There were no significant differ-

ences in intersite variation.

Figure 2 shows an example of the correlation of absolute

SISH scores (all core data) between individual laboratories and

laboratory 1 as the comparator. The absolute HER2/chromo-

some 17 data for laboratory 2 are consistent and very similar

to the data obtained from laboratory 1 (slope of 1.22, intercept

of 0.05, andR value of 0.95). The slope, intercept, andR value

for comparisons between all 6 individual laboratories that

10.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

9.008.007.006.005.00

FISH

SISH

1

4.003.002.001.000.00

10.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

9.008.007.006.005.00

FISH

SISH5

4.003.002.001.000.00

A

B

Figure 1 Correlation of absolute silver in situ hybridization (SISH) scores from each laboratory with fluorescence in situ

hybridization (FISH) scores obtained from the central laboratory. A, Comparison of SISH for laboratory 1 with central laboratory

(laboratory 1) FISH. y = 1.019x 0.0643; R2 = 0.8998; R= 0.9486. B, Comparison of SISH for laboratory 5 with central

laboratory FISH. The absolute SISH scores from all core data obtained from each laboratory were compared with FISH scores

from all core data obtained from the central laboratory. The points represented by squares are discordant values between SISH

and FISH. The slope, intercept, and Rvalues were obtained from each plot and are summarized in Table 5. Laboratory 5 showed

a number of cases with poor correlation with FISH; all other laboratories showed high concordance. y = 1.0103x 0.3183; R2 =

0.8484; R= 0.9211.

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Table 3Comparison of Absolute SISH Scores Obtained in EachLaboratory With the SISH Scores Obtained in Laboratory 1*

Laboratory 1

Overall VariationLaboratory Slope Intercept R by Core (%)

2 1.2197 0.0447 0.9548 12.03 1.2398 0.571 0.8560 12.34 1.3173 0.4487 0.9069 11.15 0.9311 0.3969 0.9280 14.66 NR NR NR NR7 0.9311 0.3969 0.9280 7.1

NR, not reported; SISH, silver in situ hybridization.* The absolute SISH scores from all core data obtained from each laboratory were

compared with SISH scores obtained in the central laboratory (laboratory 1). The

slope, intercept, andR values were obtained from each plot (example shown inFigure 2).

Data from laboratory 6 are not included because this laboratory measured only

HER2; it did not obtain scores for chromosome 17 or the ratio; therefore, data fromthis laboratory were excluded from the analysis.

Table 2Comparison of Absolute SISH Scores Obtained in Each Laboratory With FISH Scores Obtained in the Central Laboratory(Laboratory 1)

Comparison With FISH*

Overall Variation Concordance forLaboratory Slope Intercept R by Core (%) Diagnosis by Case (%)

1 1.019 0.0643 0.9486 8.2 95.62 1.2382 0.0297 0.8998 13.0 97.83 1.2649 0.4812 0.8093 14.2 100.04 1.501 0.7125 0.9343 11.3 95.65 1.0103 0.3183 0.9211 11.3 88.96 NR NR NR NR NR7 0.9069 0.1396 0.9447 8.2 97.8Across all laboratories 11.0

FISH, fluorescence in situ hybridization; NR, not reported; SISH, silver in situ hybridization.* The absolute SISH scores from all core data obtained from each laboratory were compared with FISH scores obtained in the central laboratory (laboratory 1). The slope,

intercept, andR values were obtained from each plot (examples shown in Figure 1). Data from laboratory 6 are not included because this laboratory measured onlyHER2; it did not obtain scores for chromosome 17 or the ratio; therefore, data from this

laboratory were excluded from the analysis.

Table 4Intraobserver Variation From Analysis of Duplicate Coresfor Each Case by Laboratory

Intraobserver Variation (%)

HER2 Chromosome 17Laboratory Test Copy No. Copy No. Ratio

1 FISH 4.3 3.7 4.21 SISH 5.1 3.8 5.62 SISH 3.8 3.8 4.53 SISH 9.0 8.7 13.54* SISH NR NR NR5 SISH 6.0 4.7 7.16 SISH NR NR NR7 SISH 5.7 6.2 9.2Overall SISH 5.9 5.4 8.0

FISH, fluorescence in situ hybridization; NR, not reported; SISH, silver in situ

hybridization.* Only 1 core was scored for each case in laboratory 4. Data from laboratory 6 are not included because this laboratory measured only

HER2; it did not obtain scores for chromosome 17 or the ratio; therefore, data from

this laboratory were excluded from the analysis.

Table 5Site-to-Site Variation for Analysis by Silver In Situ Hybridization*

Variation (%)

Laboratory 1 2 3 4 5 6 7

1 12.0 12.3 11.1 14.6 NR 7.12 12.0 14.0 10.0 9.7 NR 10.43 12.3 14.0 18.8 23.0 NR 14.94 11.1 10.0 18.8 12.0 NR 10.85 14.6 9.7 23.0 12.0 NR 9.26 NR NR NR NR NR NR7 7.1 10.4 14.9 10.8 9.2 NR Overall mean (SE) 11.42 (2.74) 11.22 (1.79) 16.60 (4.30) 12.54 (3.57) 13.70 (5.62) NR 10.48 (2.86)

NR, not reported.* Data from laboratory 6 are not included because this laboratory measured onlyHER2; it did not obtain scores for chromosome 17 or the ratio; therefore, data from this

laboratory were excluded from the analysis. The total overall variation across all laboratories was a mean SE of 12.66% 4.03%. No significant differences between

laboratories were observed.

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reported data and laboratory 1 as the comparator are shown

in Table 3, which also includes the concordance for diagnosis

between each of these laboratories.

Discussion

The results of this UK NEQAS ISH multicenter ring

study from 7 UK NEQAS reference laboratories show thatthe rate of successfulHER2 determination using the Ventana

INFORM HER-2 SISH assay in the same TMA construct

was consistent with an overall success rate of 94.8% of cases

(89%-100% across all 6 laboratories that reported data). This

level of performance is very high for single TMAs, in which

some fallout is expected owing to the need for a single diges-

tion method for all tissue samples.14 The overall success rate

of 94.8% of cases across all laboratories for HER2 determi-

nation using SISH compares with the success rate of 86.7%

of cases for HER2 determination using the standard FISH

method in laboratory 1. This laboratory had a success rate of98% of cases forHER2 determination using SISH, which rep-

resents a difference of only 5 cases determined successfully

by SISH compared with FISH. The success rates between

FISH and SISH in this study are comparable and very high for

TMA analysis. For both assays, we would expect improved

success rates using whole sections. TMAs were used in the

current study for ease of comparison of large numbers of

samples across sites.

Concordance for diagnosis ofHER2 amplification by

SISH compared with FISH was high. Overall, there was

96.0% (range, 88.9%-100%) concordance for diagnosis of

HER2 amplification using SISH results from all 6 laboratories

that reported data compared with FISH results from the cen-

tral laboratory. With the exception of laboratory 5, which had


for diagnosis between SISH and FISH was more than 95%

in all participating laboratories. According to ASCO/CAP

guidelines, more than 90% concordance should be achieved

to validate novel FISH or immunohistochemical procedures.2This research study demonstrates a high level of concor-

dance between FISH and SISH, suggesting that the Ventana

INFORM HER-2 SISH assay is robust, provides consistent

results across all participating laboratories, and the majority of

laboratories satisfy UK and ASCO/CAP guidelines for valida-

tion of novel FISH procedures.5

The data from this study suggest that intraobserver and

intersite variability of absolute HER2/chromosome 17 ratios

appears to be tightly controlled across all 6 participating labo-

ratories that reported data using the Ventana INFORMHER-2

SISH assay. The level of intraobserver variability was consis-tent across all laboratories. Mean intraobserver variability was

5.9% forHER2, 5.4% for chromosome 17, and 8.0% forHER2/

chromosome 17 ratio for this SISH assay, which is lower than

previously reported interobserver variation for FISH (approxi-

mately 10%).8,15-17 In laboratory 1, the intraobserver variabil-

ity was 5.1% forHER2, 3.8% for chromosome 17, and 5.6%

forHER2/chromosome 17 ratio for the SISH assay compared

with 4.3% forHER2, 3.7% for chromosome 17, and 4.2% for

HER2/chromosome 17 ratio for the FISH assay.

Site-to-site variation represents a compound of interob-

server variation due to differences in scoring and technical

12.00

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

8.00 10.006.00

SISH 1

SISH2

4.002.000.00

Figure 2 Correlation of absolute silver in situ hybridization (SISH) scores between laboratories 1 and 2. The absolute SISH

scores from all core data obtained from each laboratory were compared. The slope, intercept, and Rvalues were obtained from

each plot and are summarized in Table 3. The plot shows an example for the comparison between laboratories 1 and 2. y =

1.2197x 0.0447; R2 = 0.9114; R= 0.9548.

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2. Wolff AC, Hammond EH, Schwartz JN, et al. AmericanSociety of Clinical Oncology/College of American Pathologistsguideline recommendations for human epidermal growthfactor receptor 2 testing in breast cancer.J Clin Oncol.2007;25:118-145.

3. Bartlett JMS. Pharmacodiagnostic testing in breastcancer: focus on HER2 and trastuzumab therapy.Am JPharmacogenomics. 2005;5:303-315.

4. Faratian D, Bartlett J. Predictive markers in breast cancer:the future. Histopathology. 2008;52:91-98.

5. Bartlett JMS, Ibrahim M, Miller K, et al. External qualityassurance ofHER2 fluorescence in situ hybridisationtesting: results of a UK NEQAS pilot scheme.J Clin Pathol.2007;60:816-819.

6. Bartlett JMS, Ellis IO, Dowsett M, et al. Human epidermalgrowth factor receptor 2 status correlates with lymph nodeinvolvement in patients with estrogen receptor (ER)-negative,but with grade in those with ER-positive early-stage breastcancer suitable for cytotoxic chemotherapy.J Clin Oncol.2007;28:4423-4430.

7. Bartlett J, Mallon E, Cooke T. The clinical evaluation ofHER-2 status: which test to use? J Pathol. 2003;199:411-417.

8. Bartlett JMS, Going JJ, Mallon EA, et al. Evaluating HER2amplification and overexpression in breast cancer. J Pathol.2001;195:422-428.

9. Ellis IO, Dowsett M, Bartlett J, et al. Recommendations forHER2 testing in the UK.J Clin Pathol. 2000;53:890-892.

10. Ellis IO, Bartlett J, Dowsett M, et al. Best practice No. 176:updated recommendations for HER2 testing in the UK.JClin Pathol. 2004;57:233-237.

11. Dowsett M, Hanby AM, Laing R, et al. HER2 testing in theUK: consensus from a national consultation.J Clin Pathol.2007;60:685-689.

12. Hanna W, OMalley FP, Barnes P, et al. Updatedrecommendations from the Canadian National Consensus

Meeting on HER2/neu testing in breast cancer. Curr Oncol.2007;14:149-153.

13. Dietel M, Ellis IO, Hfler H, et al. Comparison of automatedsilver enhanced in situ hybridisation (SISH) and fluorescenceISH (FISH) for the validation ofHER2 gene status in breastcarcinoma according to the guidelines of the AmericanSociety of Clinical Oncology and the College of AmericanPathologists. Virchows Arch. 2007;451:19-25.

14. Bartlett JMS, Munro A, Cameron DA, et al. Type I receptortyrosine kinase profiles identify patients with enhanced benefitfrom anthracyclines in the BR9601 adjuvant breast cancerchemotherapy trial.J Clin Oncol. 2008;26:5027-5035.

15. Bartlett JMS, Watters AD, Ballantyne SA, et al. Estimation ofchromosome 9 copy number using quantitative fluorescence

in situ hybridisation FISH as a marker of disease recurrence intransitional cell carcinoma of the bladder [abstract].J Pathol.1997;182:A20.

16. Watters AD, Going JJ, Cooke TG, et al. Chromosome 17aneusomy is associated with poor prognostic factors in invasivebreast carcinoma. Breast Cancer Res Treat. 2003;77:109-114.

17. Watters AD, Ballantyne SA, Going JJ, et al. Aneusomy ofchromosomes 7 and 17 predicts the recurrence of transitionalcell carcinoma of the urinary bladder. BJU Int. 2004;85:42-47.

variation between sites. The overall intersite variation (mean

SE) between all laboratories of 12.66% 4.03% is consistent

with that reported in previous research studies (approximately

10%).8,15-17 There were no significant differences between

any laboratories in intersite variation. Analyses of the correla-

tion of absolute SISH scores (all core data) between individual

laboratories and laboratory 1 as the comparator showed that

core scoring data from all laboratories were consistent with

the data obtained in laboratory 1. Overall, all laboratories

showed excellent performance in diagnostic accuracy and

site-to-site variation.

The Ventana INFORM HER-2 SISH assay is robust,

provides highly consistent results across all participating UK

NEQAS reference laboratories, and complies with require-

ments in national guidelines for performance of diagnostic

tests. Furthermore, concordance for diagnosis of HER2

amplification by SISH compared with FISH was very high.

Evidence from this UK NEQAS ring study provides support

for the use of the Ventana INFORMHER-2 SISH assay as a

potential alternative for analysis and reporting ofHER2 gene

status of patients in routine practice. This study has also pro-

vided multisite data demonstrating intraobserver and intersite

consistency in absolute diagnostic ratios for HER2 using

SISH, with very tight practice between laboratories. The high

level of consistency underlines the high quality ofHER2 test-

ing achievable and demonstrates the potential for extremely

robust and quantitatively reproducible SISH in routine prac-

tice. Clearly, continued quality assessment is essential to

continued good performance.

From the 1Endocrine Cancer Group, Edinburgh, Scotland; 2UK

National External Quality Assessment Scheme, University College

London, London, England; 3Department of Histopathology,

Nottingham City Hospital, Nottingham, England; 4Department of

Histopathology, Beaumont Hospital; and5Adelaide and Meath

Hospital, Dublin, Ireland; 6Department of Histopathology, the

RSCH, University of Surrey, Guildford, England; 7Birmingham

Heartlands Hospital, Birmingham, England; 8Department of

Histopathology, Cardiff & Vale NHS Trust; and9Department of

Pathology, School of Medicine, Cardiff University, Cardiff, Wales.

Supported by Ventana (now Ventana Medical Systems,

Burgess Hill, England).

Address reprint requests to Prof Bartlett: Endocrine Cancer

Group, Edinburgh University Cancer Research Centre, Western

General Hospital, Crewe Rd South, Edinburgh EH4 2XR, England.

J. Merritt, PhD, Merritt Science, St Albans, England, a

professional medical writer, drafted the manuscript and was paid

by Ventana Medical Systems.

References

1. Walker RA, Bartlett JMS, Dowsett M, et al. HER2 testing inthe UK: further update to recommendations. J Clin Pathol.2008;61:818-824.

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