Accuracy of HER2 Status Determination on Breast Core-needle Biopsies (Immunohistochemistry, FISH, CISH and SISH vs FISH)

Laurent Arnould; Pascal Roger; Gaëtan MacGrogan; Marie-Pierre Chenard; André Balaton; Sophie Beauclair; Frederique Penault-Llorca

Disclosures

Mod Pathol. 2012;25(5):675-682. 

In This Article

Results

Analysis Population

Breast tumor specimens were tested between 14 April 2003 and 27 August 2007. The analysis population is described in Figure 1. In all, 261 patients were enrolled by 24 centers including 4 referent laboratories and 20 local centers; all the 20 local centers (83%) recruited the 11 expected patients. Among the 260 analyzed patients (1 exclusion for previous neoadjuvant treatment), 193 had also an SISH evaluation of their tumor, performed by 18 centers equipped with a Benchmark immunostainer (Ventana, Tucson, USA) according to the manufacturer guidelines.

Figure 1.

Study populations and data sets analyzed according to performed tests. SS, surgical specimen; CNB, core-needle biopsy; IHC, immunohistochemistry; FISH, fluorescence in-situ hybridization; CISH, chromogenic in-situ hybridization; SISH, silver in-situ hybridization. (1) As judged by referent center, surgical specimen was not sufficient for FISH in 12 cases. (2) As judged by referent center, preoperative biopsy specimen was not sufficient for FISH in 9 cases.

Patient and Tumor Characteristics

Women were aged 58±13 years at inclusion and the median time between the first diagnosis (based on the results of immunohistochemistry performed on preoperative biopsy specimen) and inclusion was 32 days.

On the basis of pretreatment and post-surgical histopathological analyses, main tumor characteristics were as follows: invasive ductal carcinoma (90% of cases), median tumor size (18mm (range: 3–80)), (p)TNM stage (T1 (60%) or T2 (35%), pN0 (61%)), SBR (Scarff Bloom and Richardson) grade (I 13%, II 43%, III 45%), hormonal status (ER+ (positive estrogen receptor) or PR+ (positive progesterone receptor) 73%, ER+ and PR+49%).

Technical Evaluation Method of HER2 Status

Fixation of Specimens Whatever the test performed (immunohistochemistry, CISH, FISH or SISH), analyzed core-needle biopsies specimens were mostly fixed with a formalin-based fixative (neutral buffered formalin in 47–54% or Hollande's fixative in 4 and 6%, depending on the test), and less frequently with alcohol-based fixative (alcohol-formalin-acetic acid in 34–44% or alcoholformalin in 4–6%, depending on the test).

In contrast, surgical specimens were mostly fixed with alcohol-based fixative (alcohol-formalin-acetic acid in 52–54% or alcohol-formalin in 4–6%, depending of the test) and less frequently with formalin-based fixative (neutral buffered formalin in 35–40% or Hollande's fixative in 4–6%, depending on the test).

Evaluation of HER2 Status With Immunohistochemistry and/or FISH Whatever the type of sample (core-needle biopsies or surgical specimens), the applied method was fully automated in 96% of cases and partially (not the deparaffinization, nor the antigen retrieval) in 69%. Slides were pretreated by using polyclonal A0485 anti-HER2 primary antibody (Dako, Glostrup, Denmark) in ~75% of samples; 4B5 (Ventana) and monoclonal CB11 (Novocastra, Newcastel, UK) antibodies were used less frequently (<20 and <10%, respectively).

Positive (3+) HER2 status was observed in 50–51% of cases and negative (0/1+) results were observed in 29–43% of cases, depending on the immunohistochemistry interpretation guidelines used and the type of specimen (Figure 2). The rate of equivocal cases (2+) ranged between 7 and 20% depending on the guideline used but it was lower for results observed on core-needle biopsies and surgical specimens when evaluated according to GEFPICS guidelines (7 and 8%, respectively). Most often, FISH could be used to determine any immunohistochemistry equivocal cases (2+), with <3% remaining doubtful (Figure 3).

Figure 2.

HER2 status according to immunohistochemistry results on preoperative biopsies and surgical specimens (n = 260). ASCO, American Society of Clinical Oncology; GEFPICS, Groupe d'Etude des Facteurs Pronostiques par Immunohistochimie dans le Cancer du Sein.

Figure 3.

HER2 status of equivocal immunohistochemistry results (2+) according to FISH on surgical specimens. ASCO, American Society of Clinical Oncology; GEFPICS, Groupe d'Etude des Facteurs Pronostiques par Immunohistochimie dans le Cancer du Sein.

Technical Failures Technical failure occurred in 6 and 5% of FISH tests, 5 and 3% of CISH tests, and 8 and 5% of SISH tests performed respectively on core-needle biopsies and surgical specimens.

Concordance of HER2 Status Determined by Immunohistochemistry, CISH and SISH (on Core-needle Biopsies) With FISH Results (on Surgical Specimens)

Whatever the immunohistochemistry interpretation guideline applied, excellent concordance (k: 0.92– 0.97) was shown between immunohistochemistry on core-needle biopsies and FISH on surgical specimens (discordance rates: 2–4%) ( Table 1 ). Specificity (97–98%) and sensitivity values (95–99%) of immunohistochemistry/FISH correlations did not differ significantly according to the immunohistochemistry interpretation guidelines used. Sensitivity was particularly high when results were determined by pathologists' scoring and using ASCO guidelines (sensitivity: 99 and 98%, respectively). Although lower, the sensitivity of immunohistochemistry results interpreted using GEFPICS guidelines (sensitivity: 95%) were mainly related to higher false negative results (7%) but did not differ significantly from immunohistochemistry results for sensitivity using the other guidelines.

Regarding ISH methods on core-needle biopsies, CISH and SISH showed a strong correlation with FISH on surgical specimens (k: 0.96 and 0.94, respectively), even if a lower false negative rate was observed for CISH than for SISH results (2 vs 5%). The characteristics of CISH/FISH and SISH/FISH correlations did not differ significantly, both methods being highly sensitive and specific in determining HER2 status on core-needle biopsies (sensitivity: 99 and 96%—specificity: both 98%).

Concordance of HER2 Status Determined by Each Test (Immunohistochemistry, FISH, CISH and SISH) on Core-needle Biopsies and Surgical Specimens

Whatever the test used, excellent concordance was shown between core-needle biopsies and surgical specimens (k 0.97 and discordance rates between 1 and 2%) ( Table 2 ).

For immunohistochemistry, although the discordance rate between core-needle biopsies and surgical specimens was higher for results interpreted using GEFPICS guidelines (2%), it did not differ significantly from discordance rates interpreted using pathologists' scoring and ASCO guidelines (both: 1%). Sensitivity results were similar between immunohistochemistry interpretation guidelines (98% for GEFPICS and 99% for pathologists' scoring and ASCO) and there was no significant difference in specificity results (specificity: 100% for pathologists' scoring and ASCO, 98% for GEFPICS).

For FISH, CISH and SISH, discordance rates were <1% and did not differ significantly. Correlations between core-needle biopsies and surgical specimens also showed excellent sensitivity values (99% for all three methods, always related to false negative rates: 1, 2 and 2%, respectively) and 100% specificity with no significant difference observed between these correlations.

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