Semi-quantitative Immunohistochemical Assay versus oncotype DX® qRT-PCR Assay for Estrogen and Progesterone Receptors

An Independent Quality Assurance Study

James A Kraus; David J Dabbs; Sushil Beriwal; Rohit Bhargava

Disclosures

Mod Pathol. 2012;25(6):869-876. 

In This Article

Results

All cases sent for oncotype DX® testing were positive for ER on core biopsy except one. The exact reason for the test order on ER-negative case is unknown. Of the total 464 cases, the ER results were concordant on 457 cases (98.5%) between immunohistochemistry and Genomic Health qRT-PCR assay (Table 1). All ER discordant cases were positive by immunohistochemistry and negative by qRT-PCR with a mean H-score of 82 (median 25; range 10–225). In order to exclude the possibility of tissue heterogeneity, ER immunohistochemistry was repeated on all discordant cases on the same tissue block that was sent for oncotype DX® testing (see results after repeat testing in Table 2). Of these six discordant cases, five remained unequivocally ER positive on repeat immunohistochemistry testing with a mean H-score of 105.4 (median 40; range: 2 to 240). The one negative case on repeat testing showed occasional positive cells but was judged to be less than 1% of the entire tumor and therefore interpreted as negative. After repeat immunohistochemistry testing, the sensitivity of qRT-PCR in comparison to ER immunohistochemistry was 98.9% (457/462), specificity (based on 2 cases only) was 100%, positive predictive value of 100% (457/457), and negative predictive value of 28.6% (2/7).

The overall concordance between immunohistochemistry and qRT-PCR for PR was 91.2% (423/464). Of the 41 discordant cases, 7 cases were immunohistochemistry negative- but qRT-PCR positive- and 34 cases were immunohistochemistry positive but negative by Genomic Health qRT-PCR assay (Table 3). Of the 34 immunohistochemistry-positive/qRT-PCR-negative cases, 20 had H-scores of 10 or higher. In order to exclude the possibility of tissue heterogeneity accounting for the discordance, PR immunohistochemistry was repeated on all 41 discordant cases on the same tissue block that was sent for oncotype DX® testing (see results after repeat testing in Table 4). On repeat immunohistochemistry testing, the seven immunohistochemistry-negative/qRT-PCR-positive cases, six showed immunoreactivity with a mean PR H-score of 51 (median: 40.5; range: 2 to 150). The one negative case on repeat testing showed occasional positive cells but was judged to be less than 1% of the entire tumor and therefore interpreted as negative. However, the intermixed in-situ carcinoma and normal ducts showed unequivocal reactivity for PR (Figure 2).

Figure 2.

This case of invasive carcinoma was interpreted as PR positive by oncotype qRT-PCR assay (expression units of 6.7) but remained negative by immunohistochemistry even on repeat testing. Note that the ductal carcinoma in situ (a) is positive for PR (b). The intermixed normal duct (c) also shows unequivocal reactivity for PR (d).

On repeat immunohistochemistry testing of 34 PR immunohistochemistry-positive/qRT-PCR-negative cases, 26 remained unequivocally positive by immunohistochemistry with a mean PR H-score of 24.2 (median: 9.5; range: 1–110). These 26 cases were slightly more intensely positive on initial core biopsy testing with a mean H-score of 56.2 (median: 30; range 1–270). No tumor tissue was available on one case for repeat testing. The remaining seven initially immunohistochemistry-positive cases that tested negative on repeat testing, five showed occasional positive cells but were judged to be less than 1% of the entire tumor and therefore interpreted as negative. These seven cases were weakly positive on initial core biopsy testing with a mean H-score of 17.9 (median: 6; range: 2–90). After repeat immunohistochemistry testing, the sensitivity of qRT-PCR in comparison to PR immunohistochemistry was 93.9% (401/427), specificity was 97.2% (35/36), positive predictive value of 99.8% (401/402), and negative predictive value of 57.4% (35/61).

The data was also examined for semi-quantitative immunohistochemistry and qRT-PCR results (expression units) using Pearson correlation. A linear correlation was obtained between immunohistochemistry and qRT-PCR results for both ER and PR (Figures 3 and 4). Regarding ER, H-scores ranged from 0 to 300, RT-PCR units ranged from <3.7 to 12, and the correlation coefficient was 0.579. For PR, H-scores ranged from 0 to 300, RT-PCR units ranged from <3 to >10, and the correlation coefficient was 0.685.

Figure 3.

Graphical representation of immunohistochemical and polymerase chain reaction results for estrogen receptor.

Figure 4.

Graphical representation of immunohistochemical and polymerase chain reaction results for progesterone receptor.

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