Identification of HER2 Immunohistochemistry-Negative, FISH-Amplified Breast Cancers and Their Response to Anti-HER2 Neoadjuvant Chemotherapy

I-sanna Gibbons-Fideler, MD; Hiroaki Nitta, PhD; Adrian Murillo, PhD; Gary Tozbikian, MD; Peter Banks, MD; Anil V. Parwani, MD, PhD; Zaibo Li, MD, PhD

Disclosures

Am J Clin Pathol. 2019;151(2):176-184. 

In This Article

Results

Clinical and Pathologic Characteristics of Study Cohort

During a 3-year period (November 2013 to December 2016), a total of 1,107 consecutive invasive BCs had been assessed by both HER2 IHC and FISH on core needle biopsy specimens in our institution. The overall HER2-positive rate (HER2 IHC positive and/or HER2 FISH amplified) was 14.5% (161/1,107). Among all cases, 17 were identified with discordant HER2 IHC and FISH results, and all were HER2 IHC negative and FISH amplified (1.5%). The mean age of these 17 patients was 57.1 years (range, 30–95 years), and all tumors were invasive ductal-type carcinomas. More than half were Nottingham grade 3 (64.7%) and nuclear grade 3 (76.5%). Eight of 17 cases showed lymph node metastasis. Six of these 17 cases showed positive estrogen receptor (ER) and progesterone receptor (PR) (35.3%). The mean HER2 gene signals per cell was 6.86 (range, 3.60–19.30), and the average ratio was 2.45 (range, 1.29–5.57) Table 1 .

HER2 IHC and FISH Results of 17 Cases With Discordant IHC and FISH Results

The HER2 IHC and FISH results of the 17 discordant cases are detailed in Table 2 . All showed HER2 IHC scoring as 1+. Four cases showed CEN17 signals between 1.5 and 2, suggesting some tumor cells had a monosomy 17–like pattern. Seven cases showed CEN17 signals greater than 3, suggesting coamplification/polysomy 17 (Table 2). Eight cases showed a HER2/CEN17 ratio of 2.0 or more and a HER2 copy number of 4.0 or more (group 1). Two patients (cases 1 and 3) showed a HER2/CEN17 ratio of 2.0 or more and a HER2 copy number of less than 4.0 (group 2), which are defined as HER2 negative based on the current ASCO/CAP 2018 HER2 guideline update.[14] Seven patients (cases 11–17) showed a HER2/CEN17 ratio less than 2.0 but a HER2 copy number of 6.0 or more (group 4), which are defined as HER2 positive based on the current ASCO/CAP 2018 HER2 guideline update.[14] Four cases with subsequent resection after NAC had HER2 IHC performed with three cases as 1+ and one case as 2+. HER2 FISH had been performed on two cases; one was amplified and the other was nonamplified after anti-HER2 neoadjuvant chemotherapy.

HER2 GPA, which merges HER2 IHC and dual ISH, was performed on freshly cut sections from original blocks and was found to confirm the HER2 status as originally determined with separate tests in all cases. Image 1 shows representative images of GPA from three different scenarios: a case with both IHC and ISH as negative (no protein membranous staining, no increased HER2 gene signals seen as black dots), a case with both IHC-positive staining and ISH amplification (complete strong protein membranous staining and clustering of HER2 gene signals), and a case with negative IHC (weak, incomplete protein membranous staining) but positive ISH (increased HER2 gene signals).

Image 1.

Representative images of gene protein assay from cases with different human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and in situ hybridization (ISH) results. A, B, A case with both IHC and ISH as negative (no protein membranous staining, no increased HER2 gene signals—black dots). C, D, A case with both IHC and ISH positive (complete strong protein membranous staining and clustering of HER2 gene signals). E, F, A case with negative IHC (weak, incomplete protein membranous staining but positive ISH [increased HER2 gene signals]). (×200)

The GPA results also confirmed the cases with coamplification/polysomy 17 Image 2A and the cases with monosomy 17–like pattern Image 2B . In addition, the GPA assay revealed that five cases showed HER2 intratumoral heterogeneity Image 2C , two of which showed both intratumoral heterogeneity (ITH) and coamplification/polysomy 17 Image 2D (Table 2).

Image 2.

Gene protein assay results in four cases with HER2 immunohistochemistry-negative and fluorescent in situ hybridization–amplified results. A, A case of coamplification/polysomy 17 with average chromosome 17 centromere (CEN17) signal more than three per nucleus. B, A case of monosomy 17–like pattern with average CEN17 signal less than two per nucleus. C, A case with intratumoral heterogeneity. D, A case with both intratumoral heterogeneity and coamplification/polysomy 17. (×200)

HER2 RNA ISH Results of Cases With Discordant IHC and FISH Results

HER2 RNA ISH was successfully performed on 10 of 17 HER2 IHC–/ISH+ cases, and increased HER2 RNA level was detected in eight HER2 IHC–/ISH+ cases compared with HER2 IHC–/ISH– cases Image 3.

Image 3.

Representative images of human epidermal growth factor receptor 2 (HER2) gene protein assay and RNA in situ hybridization from cases with different HER2 results ranging from immunohistochemistry (IHC) 3+/fluorescent in situ hybridization (FISH) high amplified to IHC 1+/FISH not amplified. An increased HER2 RNA level in a case with a HER2 IHC 1+/FISH-amplified result (fourth row) compared with a case with a HER2 IHC 1+/FISH–not amplified result (fifth row). (×200)

Clinical Follow-up Results

Eight patients had anti-HER2 NAC (four cycles of doxorubicin/cyclophosphamide together with paclitaxel/docetaxel and trastuzumab) with subsequent surgical resection. Of these, three patients demonstrated pathologic complete response (PCR) while the other five patients had residual tumor. All three patients achieving PCR were ER/PR negative; one of five patients with residual tumor was ER/PR positive. Residual cancer burden (RCB) was analyzed for all five cases, with two as RCB-I and three as RCB-II. Among these five cases with residual tumor, three cases showed coamplification/polysomy 17, and one case showed ITH Table 3 . Six patients tolerated the therapy well with only mild nausea, diarrhea, neuropathy with hand tingling, and back pain; however, two patients developed severe intractable nausea and vomiting, which required emergency room visit and treatment.

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