Impact of Updated HER2 Testing Guidelines in Breast Cancer—Re-evaluation of HERA Trial Fluorescence in Situ Hybridization Data

Oliver C Stoss; Andreas Scheel; Iris Nagelmeier; Hans-Ulrich Schildhaus; Thomas Henkel; Giuseppe Viale; Bharat Jasani; Michael Untch; Josef Rüschoff

Disclosures

Mod Pathol. 2015;28(12):1528-1534. 

In This Article

Results

A total of 6018 HERA screening cases have been initially evaluated by dual-color fluorescence in situ hybridization using the FDA-approved package insert of the PathVysion test kit. We applied the modified thresholds for HER2 dual-color in situ hybridization recommended in the ASCO/CAP guidelines published in 2007 (ref.[6]) and 2013 (ref.[7]) on the same raw data. The different thresholds are summarized in Table 1.

HER2/CEP17 Ratio: ASCO/CAP 2013 Criteria Applied to 6018 HERA Screening Cases

The HER2 gene amplified cases according to the updated ASCO/CAP 2013 guidelines can be divided into four distinct subgroups (Table 2). The first subgroup defined with a ratio of ≥2.0 and a gene count of ≥6 (48.7%, n=2931) includes 353 cases (12%) showing ≥3 CEP17 signal counts per cell and an average CEP17 count of 2.2. From 1546 cases of this subgroup, HER2 immunohistochemistry was assessed and two third of the cases were scored positive. From 720 of the 1546 cases, the percentage of stained cells was determined. A total of 445 (83.4%) of all 533 immunohistochemistry 3+ cases showed a homogenous HER2 expression over the tumor area. Twenty (3.8%) of the immunohistochemistry positive cases showed focal staining and 25 (13.4%) of all 187 immunohistochemistry 2+ cases were focally expressed.

The intermediate level amplification subgroup (ratio of ≥2.0 and a gene count of ≥4 and <6) consisted of 381 cases (6.3%). In comparison to the high-level amplification subgroup, no polysomic cases were identified and the average CEP17 count declined to 1.8. From 195 cases assessed by immunohistochemistry, the positive cases dropped to 13.3% (n=26) within this subgroup whereas the amount of equivocal cases increased to 85.7% (n=167). Assessment of the percentage of stained cells from 79 cases showed a decrease of homogenously stained tumor cells to 63.8% (7 out of 11 cases) that were scored 3+ whereas focal expression with a 3+ score increased to 18.1% (2 out of 11 cases). With 13 out of 68 cases (19.1%), focal expression with a 2+ score remained on a relatively high level.

The low-level amplification subgroup (ratio of ≥2.0 and a gene count of <4) included 47 cases (0.8%). Thirty-nine (83%) of these cases had a HER2/CEP17 ratio of <3. No polysomy was observed, the average CEP17 count declined to 1.5. Thirty cases were characterized by immunohistochemistry. An inversion of the relationship of positive and equivocal immunohistochemistry cases was observed in comparison to the high-level amplification subgroup, with 10 (33.3%) positive and 20 (66.7%) equivocal cases. An interesting characteristic of the low-level amplification subgroup is that the immunohistochemistry 3+ cases in this subgroup showed only homogenous protein expression of the tumor.

The last subgroup of the fluorescence in situ hybridization positive cases has been scored negative by the FDA guidelines since the ratio is <2.0. However, there were 21 cases with a gene count of ≥6 which are now defined as positive according to the ASCO/CAP guidelines of 2013. A common feature of this subgroup is that all cases also show an increased CEP17 count (average count of 5.0). Twenty cases were assessed by immunohistochemistry and 15 (75%) of these cases were scored as HER2 immunohistochemistry positive, supporting the ASCO/CAP recommendation. From nine of these positive cases, the percentage of stained tumor cells was assessed. Six cases (66.7%) were homogenously expressed and three patient samples (11.1%) showed focal HER2 expression.

From the 2525 patient samples (41.9%) that were tested to be HER2 negative by fluorescence in situ hybridization according to the ASCO/CAP guidelines of 2013, we observed 135 (5.3%) cases with an average CEP17 count of ≥3.0. Most cases were disomic (CEP17: 2.2±0.5). Immunohistochemistry was positive in 2% (24 of 1211 available immunohistochemistry stains) but none of these cases had a HER2 copy number >4. Fourteen of these cases had CEP17 counts ≥3.0. Most of the equivocal immunohistochemistry cases assessed in this subgroup showed a heterogenous protein expression (n=6; 50%) followed by a focal staining of tumor cells (n=4; 33.3%).

Comparison of HERA Screening Fluorescence in Situ Hybridization Results Obtained With the FDA, ASCO/CAP 2007 and ASCO/CAP 2013 Guidelines

Next, we compared the final fluorescence in situ hybridization results between the original HERA classification (FDA-approved kit guidelines), the ASCO/CAP guidelines of 2007 (ref.[6]) and the most recent ASCO/CAP guidelines of 2013.[7] In this comparison, the ASCO/CAP guidelines of 2013 produce the highest amount of fluorescence in situ hybridization positive cases (n=3380 cases; 56.2%) compared with 3359 cases (55.8%) using the FDA guideline (Table 3). The above mentioned 21 cases called positive according to the ASCO/CAP 2013 recommendations (average HER2 copy number ≥6) would have been scored negative according to the original HERA testing guidelines. However, since the HERA trial screening algorithm also allowed the site for requesting central immunohistochemistry testing as the first choice, the number of patients included into the HERA trial would have differed by only five cases if the new ASCO/CAP criteria would have been applied and if the sequential algorithm (immunohistochemistry first followed by in situ hybridization) would have been followed for equivocal cases which is widely implemented in routine pathology.

Investigation of Equivocal Cases

The most obvious difference between the FDA and the ASCO/CAP guidelines is the definition of the equivocal range. The FDA fluorescence in situ hybridization scoring criteria do not foresee a final equivocal result although the recommendation is given to re-assess cases with a ratio between 1.8 and 2.2 on 40 nuclei. The former ASCO/CAP guidelines published in 2007 defined cases with a ratio between 1.8 and 2.2 as equivocal. In total 44 cases (0.7%) of all 6018 HERA cases assessed by fluorescence in situ hybridization would have been determined equivocal (Table 3).

The definition of equivocal cases according to the recent ASCO/CAP guidelines of 2013 has changed by focusing on the copy number instead of the ratio. The amount of equivocal fluorescence in situ hybridization cases increased to 113 cases (1.9%). Sixty-nine (61.1%) of all cases had ≥3 CEP17 signal counts per cell with an average CEP17 count of 3.2. The equivocal group was analyzed by immunohistochemistry on 84 cases. Sixty-nine patient samples (82.1%) were again scored as equivocal (2+) and remained nondecisive. Twelve cases (14.3%) were HER2 positive based on immunohistochemistry.

For single-probe in situ hybridization assays where the decision is based on the gene count only, the amount of equivocal cases (gene count ≥4 and <6) would have increased dramatically to 494 cases (8.2%, Table 4). Interestingly, most of these cases (>80%) were nondecisive by immunohistochemistry (score 2+) irrespective of whether the ratio was <2.0>. HER2 status could be clarified in about 15% of these cases, only.

Another observation is that only 0.3% of all cases (n=17) fall into the equivocal subgroup both by HER2 gene counts and HER2 ratio (Table 4). A total of 5.9% of these cases had CEP17 counts ≥3.0, comparable to the negative group. One of eight available immunohistochemistry stains for this group was immunohistochemistry positive; this case had a HER2 gene count of 5.97.

processing....