Atypical Co-amplification with Co-localization of HER2 Gene in Breast Cancer: Combined IHC/FISH Approach as per ASCO/CAP 2018 Guidelines for Targeted Therapy Eligibility

Abstract

Breast cancer patients with HER2 gene amplification as assessed by FISH are eligible for HER2-targeted therapy. However, in a small subset of patients, unusual FISH pattern of co-localization and co-amplification can pose challenges in interpretation of the HER2 status and hence to assess the HER2 status accurately; our aim was to report their incidence and analyze them based on latest ASCO/CAP 2018 guidelines. We present seven cases with HER2/CEP17 co-amplification and co-localization from a total 4040 cases referred during the year 2017 to 2021 at Mumbai Reference Laboratory, SRL Diagnostics. Core needle biopsy/excision invasive breast carcinoma specimens from metastatic sites were tested for IHC for expressions of ER, PR, and HER2. The ones which came equivocal on HER2 IHC were then evaluated for HER2 amplification by FISH. Co-amplification and co-localization of HER2 and centromeric 17 was observed with a frequency of 0.1% that falls in the range of 0.5–0.1% as reported from other large-scale studies. Our study showed that implementation of a binary inhouse concurrent assessment with IHC as per the ASCO/CAP 2018 helps to reach the most definitive and accurate HER2 status. Our study is an attempt to report such challenging FISH patterns and their work-up for a better understanding on the interpretation. Cumulative data along with follow-up in these cases would bring an insight into exact therapeutic outcome.

Introduction

Approximately 15–20% of breast cancers are characterized by the amplification and overexpression of the human epidermal growth factor receptor type 2 gene (HER2/ERBB2) which is an established prognostic and therapeutic target in breast and gastric carcinomas [1].

HER2-amplified breast cancers are aggressive tumors that have shown benefits when treated with targeted therapy of monoclonal antibody—trastuzumab (Herceptin) and/or newer HER2-targeted drugs in both early and metastatic HER2-positive breast cancers. Due to its high cost and dose-limiting toxicities, precise assessment of HER2 status is an essential step for treatment of breast cancer [2].

HER2 testing is based on the HER2 protein expression and HER2 gene amplification using FDA-approved immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods [3].

Currently laboratories have adopted HER2 immunohistochemistry (IHC) as a screening test and in situ hybridization as a confirmation test for HER2 IHC equivocal cases.

In order to eliminate the equivocal category of HER2 the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines on HER2 testing were released in 2007 and later updated in 2013 and 2018 [4, 5].

ASCO/CAP 2018 focused HER2 guidelines emphasize on coordination and concomitant review with IHC and ISH results to eliminate the equivocal results for clearly defining the HER2 status.

Still 5–7% cases pose diagnostic dilemma as they do not reveal a clear status. Co-amplification and co-localization of CEP17 and HER2 is one such category with rare occurrence [6–13]. In these cases, FISH signals appear as yellow “merged” due to overlap of amplified centromeric 17 region and amplified HER2 gene signals. Although the HER2 copy numbers show clear amplification, but due to centromeric 17 co-amplification, the ratio is less than 2 and hence grouped in the Group 3 category. This pattern can be misleading to define HER2 status and thus lead to under-reporting of HER2 amplification. As per the 2018 guidelines, the Group 3 category cases with HER2/CEP17 ratio < 2.0 cells and an HER2 signals of > 6.0 should be tested for IHC concurrently and follow a binary assessment system by counting the tumour spot with most enriched area as assessed by both IHC and FISH.

We present seven cases of breast cancer with HER2/CEP17 co-amplification and co-localization by FISH from a total 4040 cases referred during the year 2017 to 2021. These cases were analyzed as per ASCO/CAP 2013 and 2018 guidelines.

Materials and Methods

Core needle biopsy/excision invasive breast carcinoma specimens were tested for immunohistochemistry for expressions of estrogen (ER), progesterone receptors (PR), and HER2. These cases showed HER2 IHC equivocal status and hence were referred to SRL laboratory for confirmation of HER2 gene amplification by FISH.

FISH was performed using PathVysion HER2 DNA Probe Kit from Vysis, Abbott molecular. The cases were reviewed by pathologist and cytogeneticist independently for FISH.

To distinguish the co-amplification signal pattern from a possible polysomy of chromosome 17, as per ASCO/CAP 2013 guidelines, a repeat HER2 testing using an alternative probe for another gene in chromosome 17 was recommended. This countercheck was possible on Case 4 due to tissue availability. FISH was done with application of Vysis TP53/CEP 17 FISH Probe Kit, Abbott molecular, that uses spectrum green fluorochrome to label the alpha satellite DNA material at the centromeric region of chromosome 17 (CEP 17) while the spectrum orange fluorochrome labels TP53 gene located at 17p13.1. Details of histopathology, tumor grade, and immunohistochemistry (IHC) were available for few of the cases as per Table 1.

Table 1.

Details of cases showing Her2 co-amplified and co-localised positive status

Case no	Age/sex	Histomorphology	Immunohistochemistry	HER2 FISH analysis
				Average Her2 copies	Average CEP17 copies	HER2/CEP17 ratio
Case 1	57/F	Modified radical mastectomy (MRM) showing invasive ductal carcinoma, grade 3 with axillary nodal metastasis	Immunohistochemistry for estrogen (ER) was score 2(moderate intensity) in 90% of tumor and progesterone receptors (PR) was score 3(strong intensity)  in 20% of tumor, while the Her2 status was 2 + (equivocal)	18.59	17.08	1.0
Case 2	38/F	Grade 3 breast carcinoma, infiltrating ductal carcinoma-NOS type with axillary nodal metastasis	Moderate positivity was seen for expression of both ER and PR and IHC HER2 was equivocal score 2 +	17.77	17.52	1.01
Case 3	50/F	Left breast lump-infiltrating ductal carcinoma	Moderate positivity was seen for expression of both ER and PR and IHC HER2 was equivocal Score 2 +	14.39	12.43	1.16
Case 4	52/F	Invasive mammary carcinoma	KI 67—20%, negative for expressions of both ER and PR, and IHC HER2 score 3 +	18.55	16.94	1.09
Case 5	73/F	Invasive mammary carcinoma	IHC equivocal Score 2 +	9.53	9.2	1.04
Case 6	54/F			15.77	15.59	1.01
Case 7	40/F			17.90	17.9	1

Results

Seven cases showed multiple signals for centromeric 17 (spectrum green labeled) and HER2 gene (spectrum orange labeled) resulting in co-amplified and co-localized yellow fusion signals (Table 1 and Fig. 1). As a tertiary referral center study, assessment of HER2 gene status by FISH on 4040 cases showed 7 cases with co-amplification and co-localization of HER2 and centromeric 17 with average HER2 copy numbers of 16 and 15.2 respectively, with a ratio of less than 2. To distinguish the co-amplification signal pattern from a possible polysomy of chromosome 17, using TP53/CEP17 alternate probe revealed presence of two normal copies of TP53 gene and multiple green signals of CEP 17. Multiple green signals of centromeric 17 confirmed amplification of the centromeric region, and hence, polysomy 17 was ruled out and thus indicated a segmental amplification of the HER2 gene along with the centromeric region of the chromosome 17.

Fig. 1.

Case showing multiple signals for CEP 17 (spectrum green labeled) and HER2 (spectrum orange labeled) seen as yellow signals representing co-amplification and co-localization

Discussion

In a total HER2 amplification study of 4040 cases, the frequency of HER2 co-amplification/co-localization in our study is 0.1%. Large-scale studies by Starczynski et al., Pai et al., Hu et al., and Ballard et al., in 1787, 1788, 4016, and 8068 cases, respectively, have reported co-amplified/co-localized cases under the category of “non-classical amplified” with a frequency ranging from 0.5 to 0.1% [6–12]. Additional one more study from India reported study on 2 cases with co-amplification/co-localization [13].

With application of 2013 ASCO/CAP guidelines, all the 7 cases would be categorized as positive based on more than 6 HER2 copies and ratio less than 2, but as per the revised ASCO/CAP 2018 guidelines, they are categorized as Group 3, where HER2/CEP17 ratio is < 2.0 with a HER2 signal frequency > 6.

As per the focused ASCO/CAP 2018 recommendations, combined interpretation of the ISH and IHC assay with a binary inhouse concurrent assessment on the right tumor spot with most enriched area as assessed by both IHC and FISH was performed to reach the most definitive and accurate HER2 status. The concurrent FISH and IHC evaluation together categorize these cases as positive based on ASCO/CAP 2018 guidelines.

Out of 7 cases, IHC evaluation of case 4 showed a 3 + status while remaining cases showed an equivocal 2 + status. This equivocal staining probably would be a result of tumor heterogeneity in HER2 protein expression, technical experimental variations, and/or inter-observer analytical variations. However, none of the cases showed inclined negativity by IHC.

Studies have explained the mechanism for co-amplification signal pattern as an extension and incorporation of the HER2 amplicon into the pericentromeric region, resulting in the co-amplification with co-localization. Initially, in 2013 guidelines, it was recommended to use alternate probes on chromosome 17 to define the co-amplification/polysomy status. In the present study, case no. 4 by alternate FISH probe assessment ruled out the possibility of polysomy.

The updated 2018 ASCO/CAP guidelines however do not recommend the use of alternative probe as standard practice due to limited evidence on its analytical and clinical validity.

Majority of cases reported with these results have HER2 amplifications that include regions encompassing the centromere rather than true polysomy for the entire chromosome 17. Concurrent IHC 3 + protein over-expression in these cases would clearly classify these cases as positive. IHC with equivocal status needs a thorough evaluation of the right area with assessment of cells with IHC equivocal status and FISH patterns along with additional counting by two observers which is necessary. In cases with IHC 2 + staining, an algorithm favoring a concurrent study to classify these cases is needed or a repeat testing of other tissue samples may also be appropriate in this setting for confirmation of results. Additionally, comparative genomic hybridization microarray studies could be done as alternative method to rule out the possibility of high level chromosome 17 “polysomy” as a result of sub-chromosomal duplication in these cases [6].

Assessment based on histopathological findings, tumor grading, and ER/PR status is additionally important while considering HER2 targeted therapies.

Studies have shown that even in cases with polysomy 17-associated HER2 co-amplification, patients show a favorable response to targeted therapy and it is suggested that IHC 3 + tumors should be considered for targeted therapy irrespective of the FISH result [14].

HER2 expression profile at the mRNA level can be studied to know the exact mechanism of HER2 status in non-classical HER2 amplified cases.

As the occurrence of co-amplification along with co-localization signal pattern of HER2 FISH is extremely rare and with limited clinical evidence regarding benefit from HER2-targeted therapy, reporting them as a separate entity is important. Many more studies with follow-up treatment response are needed to have a more confident approach in inclusion of these cases into the positive category. Awareness of various ambiguous FISH patterns is important in understanding the interpretation of HER2 FISH results and cumulative data along with follow-up would bring an insight into the exact therapeutic impact.

Declarations

Conflict of Interest

The authors declare no competing interests.