Clinical and Pathologic Features Associated With Invasive Breast Carcinoma With 2018 American Society of Clinical Oncology/College of American Pathologists In Situ Hybridization Group 2 Results (Human Epidermal Growth Factor Receptor 2 [HER2]/Chromosome 17 Centromere [CEP17] Ratio ≥2.0 and Average HER2 Copy Number <4.0) Open Access

The study was conducted under institutional review board approval. The institutional database was interrogated. All patients diagnosed between January 1, 2012, and December 31, 2014 with invasive, recurrent, or metastatic breast carcinoma with HER2 FISH testing performed at our institution were identified. All cases with HER2/CEP17 ratio of 2.0 or more and average HER2 copy number less than 4.0 were further analyzed. When available, age, primary tumor size, pathologic tumor stage, lymph node status, histologic subtype, Nottingham grade, status of hormone receptors (ie, estrogen and progesterone receptors), score of HER2 IHC, specimen type and site analyzed by FISH, results of additional FISH tests, if performed, treatment regimen, including targeted anti-HER2 therapy, cytotoxic, hormone, and radiation therapies, and clinical follow-up for patients were recorded. Estrogen and progesterone were assessed using the most recent ASCO/CAP guideline, wherein immunoreactivity in less than 1% or 0% tumor cell nuclei are considered “negative” and immunoreactivity in 1% to 100% of tumor cell nuclei are considered “positive”; estrogen staining of 1% to 10% tumor cell nuclei were classified as “estrogen low positive.”¹²  Cases were considered hormone receptor-positive if estrogen and/or progesterone were positive. Specimen types included biopsies, resections, and cytology samples. Biopsy specimens included core-needle biopsies and punch biopsies. Resection specimens encompassed wide local excisions, mastectomies, and excisions of metastatic tumor deposits.

As per our institutional standard during the study period, FISH was routinely performed as the primary testing methodology on all primary invasive, recurrent, and metastatic breast carcinoma cases to determine HER2 status. All cases with equivocal FISH results, according to the relevant guidelines at the time,^9,10  underwent additional FISH testing with an alternate chromosome 17 probe (D17S122) and/or reflex IHC testing to further define the HER2 status. In all cases where FISH was done, deparaffinized tissue sections were examined using a HER2 dual-probe FISH assay (PathVysion HER2 DNA Probe Kit; Vysis, Abbott Molecular, Des Plaines, IL, USA) as the primary methodology and when results were equivocal, a HER2:D17S122 (Empire Genomics, Buffalo, NY, USA) Probe Set was employed as previously described in Donaldson et al.¹³ HER2 FISH results were reported as negative, equivocal, or positive, according to the 2007 or 2013 ASCO/CAP guidelines, where applicable.^9,10 HER2 FISH results were reclassified into 5 ISH categories, as defined by the updated 2018 ASCO/CAP guideline that include the following: HER2/CEP17 ratio of 2.0 or more and an average HER2 copy number of 4.0 or more (Group 1); HER2/CEP17 ratio of 2.0 or more and an average HER2 copy number less than 4.0 (Group 2); HER2/CEP17 ratio less than 2.0 and an average HER2 copy number of 6.0 or more (Group 3); HER2/CEP17 ratio less than 2.0 and an average HER2 copy number of 4.0 or more and less than 6.0 (Group 4); and HER2/CEP17 ratio less than 2.0 and an average HER2 copy number less than 4.0 (Group 5).⁸  Monosomy of chromosome 17 was defined as less than 1.5 average CEP17 signals per cell, as established in prior studies.^14–16  Where available, HER2 IHC slides were reviewed and scored as negative (0, 1+), equivocal (2+), or positive (3+).⁸  In cases wherein HER2 IHC was not initially performed, HER2 IHC was conducted on the same formalin-fixed, paraffin-embedded tissue block analyzed by FISH testing. HER2 IHC was scored as negative (0, 1+), equivocal (2+), or positive (3+), accordingly, by a single board-certified surgical pathologist, blinded to the patient's treatment history and clinical outcome.

Differences between groups in categoric variables were assessed with the X² test and Fisher exact test, where applicable, and in noncategoric variables by using Student's t test. Statistical significance was established at P < .05.

During the study period, 4620 HER2 FISH tests were performed at our institution. A total of 39 of 4620 (0.84%) FISH results reclassified as 2018 ASCO/CAP ISH Group 2 from 39 patients, all of whom were women, were retrieved and reviewed. Median patient age was 62 years (mean, 61 years; range, 27–93 years). Median tumor size was 2.0 cm (mean, 2.4 cm; range, 0.5–10.9 cm). Of cases, 56% (22 of 39) were pathologic T1 (pT1) tumors, 33% (13 of 39) pT2, 8% (3 of 39) pT3, and 3% (1 of 39) were pT4. Of FISH study samples, 79% (31 of 39) were biopsy specimens, 18% (7 of 39) were resection specimens, and 3% (1 of 39) were cytology samples. FISH study samples included 90% (35 of 39) primary tumor samples, 5% (2 of 39) locally recurrent samples, and 5% (2 of 39) metastatic samples. Monosomy 17 was detected in 11 of 39 (28%) cases. The most common histologic type in this subset was invasive carcinoma of no special type (n = 29 of 39; 74%), followed by invasive carcinoma with mixed features (n = 5 of 39; 13%). The latter were composed of invasive carcinoma with mucinous features (n = 4) and invasive carcinoma with micropapillary features (n = 1). More than half (n = 20; 51%) of the cases were Nottingham grade III, and the majority had positive hormone receptor status (n = 27 of 39; 69%). Lymph node status was known for 32 patients; 12 (38%) had regional lymph node involvement at the time of initial surgery. At a median clinical follow-up time of 80 months, 25 of 39 (64%) patients showed no evidence of disease, 2 (5%) were alive with local recurrent disease, 2 (5%) were alive with metastatic disease, 7 (18%) succumbed to breast carcinoma, and 3 (8%) died of other causes.

HER2 immunohistochemistry was performed in 38 of 39 (97%) cases (Figure 1, A through H), of which 20 were performed during the initial case workup and 18 were conducted for the purposes of this study. Of 20 patients who received anti-HER2 therapy, 14 (70%) had negative (0 or 1+) HER2 IHC results, 4 (20%) had equivocal (2+) IHC results, and 1 (5%) had positive (3+) IHC results; 1 (5%) patient did not have HER2 IHC done. Of 19 patients who did not undergo HER2-targeted treatment, 13 (68%) had negative HER2 IHC results and 6 (32%) had equivocal IHC results. HER2 IHC was unable to be performed on the same tissue block sent for HER2 FISH in 1 of 39 (3%) cases.

Twenty of 39 (51%) patients with 2018 ASCO/CAP ISH Group 2 results received anti-HER2 therapy. Table 1 compares the clinical and pathologic characteristics between patients who received and did not receive HER2-targeted therapeutics. Potential cofounding baseline variables between patients treated with and without anti-HER2 therapy were assessed using X² test, Fisher exact test, or Student's t test, where applicable. Patient age, HER2/CEP17 ratio, HER2 copy number, monosomy of chromosome 17, pathologic tumor stage, involvement of regional lymph nodes, histologic grade, status of hormone receptor, tumor histology, specimen type or site analyzed by FISH, treatment with radiotherapy or endocrine therapy administration were similar among the 2 patient groups. The only significant difference between the 2 patient groups was receipt of cytotoxic chemotherapy therapy (P < .001). There was no significant difference in clinical status (P > .99) with similar length of clinical follow-up (P = .85).

Of 20 patients who received anti-HER2 therapy, 9 (45%) were treated in the neoadjuvant setting. Three (33%) of 9 patients achieved pathological complete response (pCR), all of whom were staged as ypT0ypN0 and showed no evidence of disease at follow-up (Figure 2, A through E). Of 3 patients with pCR, all 3 cases were invasive carcinoma of no special type, Nottingham grade II (n = 1) and III (n = 2). Two pCR cases were hormone receptor-negative, and 1 was hormone receptor positive. In neoadjuvant cases without pCR, 3 of 6 (50%) patients died of metastatic disease, and 3 (50%) showed no evidence of disease (median clinical follow-up time, 81 months; range, 75–82 months). Of 11 patients who received adjuvant HER2-targeted therapy, 7 (64%) patients showed no evidence of disease, 2 (18%) were alive with recurrent and/or metastatic disease, 1 (9%) succumbed to metastatic breast carcinoma, and 1 (9%) patient with a BRCA1 mutation died of metastatic ovarian carcinoma.

Nineteen patients forwent HER2-targeted therapy for the following reasons: reflex HER2 IHC showed a negative (0 or 1+) result (n = 6 of 19; 32%), declined further systemic adjuvant therapy after surgical management (n = 5 of 19; 26%), determined to derive no perceived benefit from anti-HER2 therapy, either due to poor functional status or risk of cardiac toxicity (n = 5 of 19; 26%), and subsequent nonamplified FISH result on primary resection specimen (n = 3 of 19; 16%). Overall, 12 of 19 (63%) showed no evidence of disease, 2 (11%) developed recurrent disease, 3 (15%) succumbed to metastatic breast carcinoma, and 2 (11%) died of other causes, which included metastatic small cell carcinoma and squamous cell carcinoma, both of the lung. Of 3 patients who died of disease, 2 (67%) subsequently developed metastatic disease, despite various adjuvant therapies, and 1 (33%) had presented with distant metastatic disease involving the central nervous system. Five patients received neither adjuvant anti-HER2 treatment, radiation, cytotoxic chemotherapy, nor hormone therapy. Of 5 patients, 3 (60%) showed no evidence of disease (median clinical follow-up time, 99 months; range, 76–101 months) and 2 (40%) developed recurrent disease.

Eleven of 39 (28%) cases showed monosomy of chromosome 17 (Figure 3, A through C). Of 11 cases, 7 (64%) were treated with anti-HER2 therapy. Five of 7 (71%) patients treated with HER2-targeted therapeutics showed no evidence of disease, and 2 of 7 (29%) patients succumbed to breast carcinoma (median clinical follow-up time, 82 months; range, 75–103 months). Two of 4 (50%) patients treated without anti-HER2 therapy showed no evidence of disease, and 2 of 4 (50%) patients died of disease (median clinical follow-up time, 46 months; range, 3–89 months).

Seventeen of 39 (44%) patients with 2018 ASCO/CAP ISH Group 2 results had additional FISH testing performed (Figure 4, A through D). Table 2 details the results of additional FISH tests. Twenty additional FISH tests were performed on different samples from 16 patients, including the primary biopsy or resection (n = 13 samples), recurrent sample (n = 4 samples), and metastatic sample (n = 4 samples). One additional FISH test was performed on a different tissue block from the same specimen in 1 patient. Of 20 additional FISH tests performed, ISH Group 5 results were found in 12 (60%), ISH Group 2 results in 7 (35%) samples, and ISH Group 1 results in 1 (5%) sample. Seven patients had repeat FISH testing on the primary resection following an ISH Group 2 result on primary biopsy procedures. In these cases, additional FISH tests yielded ISH Group 2 results in 3 (43%) cases, ISH Group 5 results in 3 (43%) cases, and ISH Group 1 results in 1 (14%) case.

Of 7 patients with additional FISH testing showing ISH Group 2 results, 5 (71%) received anti-HER2 therapy. Two patients showed no evidence of disease, 1 progressed with metastatic disease, 1 succumbed to breast carcinoma, and 1 died of other causes. Of 2 patients in whom additional FISH tested revealed ISH Group 2 results and were not treated with HER2-targeted therapy, 1 showed no evidence of disease and 1 died of breast carcinoma.

Of 9 patients with additional FISH testing revealing ISH Group 5 results, 5 (55%) received anti-HER2 treatment. One of these patients showed no evidence of disease, 1 developed metastatic disease, and 3 succumbed to breast carcinoma. Of 4 patients in whom additional FISH testing revealed ISH Group 5 results and were not treated with HER2-targeted therapy, 3 showed no evidence of disease, and 1 died of breast carcinoma.

The patient with ISH Group 1 results on the primary resection was a 93-year-old woman with a 3.0-cm invasive carcinoma of no special type, Nottingham grade III. She was treated with neither anti-HER2 therapy, radiotherapy, cytotoxic chemotherapy, nor hormone therapy, as she was not deemed to benefit from such therapeutics due to her functional status. Despite not receiving targeted therapy or adjuvant treatment, she showed no evidence of disease with 99 months of clinical follow-up.

In this retrospective, single-institution study of invasive breast carcinoma with HER2/CEP17 ratio of greater than or equal to 2.0 and average HER2 copy number of less than 4.0 signals per cell, classified as 2018 ASCO/CAP ISH Group 2 results, we have demonstrated no significant difference in clinical outcome between patients treated with and without anti-HER2 therapeutics. Of the clinical and pathologic features evaluated in our study cohort, the only significant difference between patients with or without HER2-targeted therapy was treatment with cytotoxic chemotherapy (P < .001). This difference is unsurprising, as the recommended neoadjuvant and adjuvant treatment regimens for HER2-positive breast carcinoma include combination chemotherapy and anti-HER2 therapy.¹⁷  In all other regards, the 2 treatment arms of our study cohort were balanced.

Cases with HER2/CEP17 ratio of 2.0 or more and average HER2 copy number of less than 4.0 signals per cell have posed a clinical challenge. Clinical trials for trastuzumab have demonstrated no apparent benefit with HER2-targeted therapy for patients with 2018 ISH Group 2 results in disease-free survival and overall survival.^4,18,19  In their analysis of the HERA trial by HER2 amplification, Dowsett et al¹⁹  reported 48 patients with breast carcinoma with average HER2 copy number of less than 4.0. The study found these cases, including a subgroup analysis of cases with HER2/CEP17 ratio between 2.0 and 4.0, showed no reduced benefit on disease-free survival derived from 1 year of trastuzumab after chemotherapy, compared with observation after chemotherapy.^10,19  Similarly, the Breast Cancer International Research Group 006 trial included 42 patients with ISH Group 2 results who demonstrated no apparent benefit on either disease-free survival or overall survival from 1 year of trastuzumab, in combination with doxorubicin and cyclophosphamide followed by docetaxel compared with docetaxel alone.^4,18  More recently, Wang et al²⁰  showed no significant difference in disease-free survival and overall survival among 30 ISH Group 2 patients treated with and without HER2-targerted therapy in their retrospective study (P = .72 and .15, respectively).

A limited number of patients in our cohort received anti-HER2 therapy in the neoadjuvant setting. Of these patients, one-third achieved a pCR. Singer et al²¹  showed similar results in their prospective analysis of the relationship between pCR and HER2/CEP17 ratio in 114 patients. Tumors with high levels of HER2 amplification—greater than 6.0—had a significantly higher rate of achieving ypT0ypN0 compared with those with low amplification (69.0% versus 30.4%, correspondingly; P = .001).²¹ 

Under the 2013 ASCO/CAP clinical practice guideline update, cases with HER2/CEP17 ratio of 2.0 or more and average HER2 copy number of less than 4.0 signals per cell were considered ISH positive, with a caveat that some cases could represent monosomy 17.¹⁰  In such cases, the low average CEP17 signal could lead to an overestimation of HER2 amplification, particularly with a concomitantly low average HER2 copy number.¹⁰  Monosomy of chromosome 17 composes approximately 4% of all HER2-amplified breast carcinoma cases—an estimated annual incidence of 2300 cases.^16,22,23  The role of anti-HER2 therapy in patients with breast carcinoma showing monosomy of chromosome 17 has not been firmly established. Limited data available regarding efficacy of such targeted treatment are conflicting. The North Central Cancer Treatment Group N9831 (Alliance) adjuvant trastuzumab trial found no significant difference in hazard ratio for benefit with anti-HER2 therapy among a small subset (n = 79 patients) with monosomy of chromosome 17 (hazard ratio, 1.21; 95% confidence interval, 0.43–4.62, P = .78).²²  Contrastingly, Page et al¹⁶  demonstrated in their retrospective analysis of 99 patients with monosomy of chromosome 17 significant improvement in overall survival, recurrence-free survival, and distant recurrence-free survival in patients receiving anti-HER2 therapy compared with those treated with or without cytotoxic chemotherapy (P = .001, .04, and .005, respectively). In a retrospective study of 43 patients with metastatic breast carcinoma and treated with HER2-targeted therapy, Risio et al²⁴ reported significantly inferior response rates to trastuzumab in patients with monosomy of chromosome 17 compared with those with eusomy or polysomy of chromosome 17 (response rate, 53% versus 92%, respectively; P = .005). While the authors did not provide granular copy number data, median HER2/CEP17 ratio for cases with monosomy of chromosome 17 was 5.2 (range, 2.8–8.2), compared with 4.4 (range, 3.7–6.0) and 3.3 (range, 2.4–3.6) for chromosome 17 eusomy and polysomy, respectively. In our limited experience with patients of monosomy of chromosome 17 (n = 11), patients treated with anti-HER2 therapy showed improved overall survival compared with those who did not receive HER2-targeted therapeutics (71% versus 50%, respectively); however, the limited number of patients in our cohort precludes further analysis.

Our study was limited by its retrospective nature. Furthermore, this study was limited by the number of patients with 2018 ISH Group 2 results. Comparatively, our study provided a cohort with similar numbers to that of prior large scale clinical trials and retrospective studies. This is likely due to the FISH-first approach of our institution during the study period, which uncovered 2018 ISH Group 2 results among cases with nonequivocal IHC results. IHC could not be performed in 1 case, as the corresponding block on which FISH was performed was unable to be located.

Despite these limitations, our small, retrospective single-institution study provides a unique insight into the natural history of 2018 ISH Group 2 disease with and without anti-HER2 therapy, supported by lengthy clinical follow-up. Herein, we demonstrated similar clinical outcomes in 2018 ASCO/CAP ISH Group 2 patients, irrespective of HER2-targeted treatment. Further investigation, particularly with prospective studies and molecular analysis, is warranted to identify potential predictors of anti-HER2 treatment response among such patients.