HER2 intratumoral heterogeneity analyses by concurrent HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer patients

It is well established that 15–20 % of breast cancer patients are HER2 positive and it is associated with poor prognosis of breast cancer patients [24]. After the discovery of HER2 gene, the significance of HER2 gene role was demonstrated by the correlation of overall survival and time to relapse with the HER2 gene amplification in breast cancer [24]. Additionally, the association of the HER2 gene amplification with the HER2 protein overexpression was proved by immunohistochemical and Western blotting analyses [25]. In our current research using the HER2 GPA method for detecting both HER2 gene and protein targets, 21.4 % of breast cancer patients (n = 280) were classified as HER2 positive by HER2 gene status and HER2 DISH positive patients had significantly worse survival than HER2 gene negative patients. Also, 15.7 % of breast cancer patients were HER2 IHC positive and HER2 IHC positive patients had significantly worse survival than HER2 IHC negative patients. Consequently, our HER2 GPA study confirmed that HER2 positive breast cancer by either HER2 gene amplification or HER2 protein overexpression demonstrated a poor prognosis. Our cohort consisted with 11.8 % of HER2 IHC 2+ (equivocal) cases that require a reflex test if a traditional single HER2 IHC assay was conducted in a real world. Accurate and prompt HER2 status assessment of HER2 equivocal cases is the major challenge of HER2 testing for improving breast cancer patient care. The ASCO/CAP HER2 testing guideline states that HER2 equivocal breast cancer cases must be reexamined for the HER2 status with a different tissue block, if available, with the same HER2 testing method or by an alternative HER2 testing approach with the same tissue block [26]. Li et al. [27] reported that the HER2 GPA approach accurately divided breast cancer cases of a HER2 equivocal enriched cohort to HER2 positive, equivocal, and negative statuses without additional testing. Thus, HER2 GPA method can be utilized effectively for both HER2 positive and equivocal breast cancer cases.

Because HER2 IHC 0, 1+ (negative) cases would not be retested for the HER2 gene status with an ISH assay, if IHC assay was performed first, 1.8 % of patients who were IHC negative & DISH positive with the current cohort would not be eligible for a HER2-targeted therapy. Our statistical analyses showed the HER2 IHC negative & DISH positive group (Type E) had significantly worse survival than the HER2 IHC & DISH negative group (Type F). There are no solid data that if the patient group with HER2 protein negative and gene positive would respond to a HER2-targeted therapy. However, several studies suggested that a subgroup of HER2 negative patients benefit from HER2-targeted therapy [15, 16, 28]. In the near future, the results of the ongoing NSABP-B47 trial for adjuvant trastuzumab in low HER2 expression breast cancer will be reported and the efficacy of HER2-targeted therapy in low HER2 expression patients will be clarified [17].

The HER2 gene is localized on q21 of chromosome 17 [29]. Chromosome 17 polysomy sometimes exists in breast cancer cells [30] while the majority of CEN17 copy number gains are due to focal pericentromeric gains [18]. Copy number gain of CEN17 is thought to be a leading reason for discrepancy between HER2 IHC and HER2 ISH status [31, 32]. It has been suggested that the copy number of CEN17 is important for the accurate assessment of HER2 status [31, 33]. Even though the definition of CEN17 copy number gain has not been well-defined yet, in general, a mean of >3 CEN17 signals per nucleus is commonly adopted threshold [18]. The prevalence rates of CEN17 copy number gain in breast cancer were reported between 3 % and 46 % [18, 34‐49]. In our study, approximately 3 % of HER2 IHC & HER2 DISH negative tumors showed CEN17 copy number gain and the presence of CEN17 copy number gain was significantly associated with the presence of HER2 intratumoral heterogeneity. It is suggested that the HER2 intratumoral heterogeneity is caused by the chromosomal instability [20] and our study also demonstrated that CEN17 copy number gain was significantly correlated with the HER2 heterogeneity. The importance of HER2 gene copy numbers is stated for determining for HER2 status in addition to the ratio of HER2 gene/CEN17 in 2013 ASCO/CAP HER2 [26]. However, the CEN17 ISH signal copy numbers may still be significant for subtyping breast cancer cases with the HER2 heterogeneity.

Even though the presence of HER2 genetic heterogeneity has been described [18, 50‐52], there are no published data on simultaneous phenotypic and genotypic heterogeneity analyses in breast cancer. Hanna et al. [18] illustrated that HER2 genotypic heterogeneity is categorized into two types: 1) clustered HER2 genotypic heterogeneity and 2) intermixed HER2 genotypic heterogeneity. The clustered heterogeneity type is defined as a cell population of HER2 amplified tumor cells bordered by a HER2 nonamplified tumor cell population. The intermixed genotypic heterogeneity type is defined as comingled HER2 amplified and nonamplified tumor cells within a tumor. In an earlier study, Nitta et al. also reported the findings of two HER2 genotypic heterogeneity types using HER2 DISH assay [6]. The ASCO/CAP 2013 guidelines state that breast cancer containing between 5 % and 50 % of total cells with HER2/CEN17 ratio >2.0 or more than 6 HER2 signals per cell should be reported as the HER2 gene amplification heterogeneity [50]. Allison et al. [52] reported that HER2 heterogeneity defined by the ASCO/CAP guidelines was present in 23 % of breast cancer. Seol et al. [20] suggested that HER2 heterogeneity cases are mainly observed among low grade or equivocal HER2 status breast cancer cases. Furthermore, they reported that HER2 intratumoral heterogeneity is associated with short disease-free survival of the patients [52]. However, our study showed HER2 negative breast cancer cases also showed the HER2 heterogeneity (17 %), although we used a different approach for evaluating the heterogeneity to the ASCO/CAP guidelines. In the present study, initially we evaluated the presence of HER2 heterogeneity only in patients group determined as IHC 0, 1+/DISH negative using FDA criteria as to the routine examination. And then we examined the presence of heterogeneity revealing at least 2 of 6 patterns of HER2-GPA expression such as, (A) IHC 3+/DISH positive; (B) IHC 3+/DISH negative; (C) IHC 2+/DISH positive; (D) IHC 2+/DISH negative; (E) IHC 0, 1+/DISH positive; and (F) IHC 0, 1+/DISH negative, in focal regions less than 10 % area within one tumor. However according to the experts’ opinion, HER2 genetic heterogeneity is defined as “more than 5 % but less than 50 % of infiltrating tumor cells with HER2/CEN17 ratio higher than 2.2″ based on HER2 ISH assay results with dual probes [51]. Because HER2 GPA method allows investigating 6 different HER2 gene and protein combinations at individual cell levels, we anticipate that we will evolve the HER2 tumor heterogeneity definition to another dimension with simultaneous observations of HER2 protein and gene heterogeneity, such as the coexistence of HER2 DISH positive & IHC negative cells with HER2 DISH & IHC positive tumor cells (data not shown). Significance of such the HER2 heterogeneity at the gene and protein levels in breast cancer needs to be investigated for evaluating the efficacy of HER2 targeted therapy with further studies as the therapy target is the protein, not the gene.

Our HER2 GPA data analyses showed that the HER2 heterogeneous group had significantly worse survival than the HER2 nonheterogeneous group among the HER2 IHC & DISH negative breast cancer population. Particularly, TNBC patients with the HER2 intratumoral heterogeneity had significantly worse survival than TNBC patients without the HER2 heterogeneity. TNBC is characterized by ER and PgR IHC staining (<1 % of positive tumor cells) and HER2 IHC (0 and 1+ scores) or ISH (no gene amplification). TNBC is an aggressive metastatic breast cancer factor like targetable HER2 positive breast cancer, but there are no targeted therapies available for TNBC patients. A systematic review study reported that the 5 year relative survivals of TNBC patients and non-TNBC patients were 77 and 93 %, respectively, among 6370 breast cancer patients [53]. Unexpectedly, our current HER2 GPA study analyses showed that the HER2 heterogeneity is a significant factor for short patient survival of TNBC and the majority of HER2 heterogeneous TNBC patients died within 5 years. Therefore, we speculate that the HER2 heterogeneity has a significant role for tumor progression in TNBC. In our study, the HER2 heterogeneity of TNBC was defined by having individual tumor cells of non-HER2 IHC & DISH negative in HER2 IHC & DISH negative cell populations. Sweeney et al. reported that 20.3 % TNBC patients were subtyped as HER2-enriched type by PAM50 test for 50 breast cancer classifier gene expression analyses using RT-qPCR [54]. Intriguingly, our HER2 GPA data analyses showed that 17.9 % of TNBC cases exhibited the HER2 intratumoral heterogeneity. Because breast cancer cases that showed incomplete membrane staining within >10 % of tumor cells are considered as HER2 negative according to the ASCO/CAP HER2 testing guideline and some HER2 negative breast cancer cases have HER2 IHC staining, it is not a surprising observation to detect the expression of HER2 mRNA with TNBC. Thus, it might be possible that amplified HER2 gene tumor cells without obvious HER2 protein expression start overexpressing the HER2 protein later in the cancer progression of TNBC. A combination of HER2 mRNA and DNA ISH assays on the same tissue sections allows further analyses of HER2 gene expression at individual cell levels in TNBC. To our knowledge we are the first team to report that the HER2 intratumoral heterogeneity might be a poor prognosis factor in TNBC patients. If this is a true breast cancer biological phenomenon, we might be able to develop a new therapy strategy for TNBC patients comparing between HER2 heterogeneous and nonheterogeneous patient groups. It might be a reasonable approach to treat HER2 heterogeneous TNBC patients with HER2-targeted therapy. Further studies are commanded to confirm our observations with TNBC patients and investigate opportunity to develop a new therapy approach for TNBC with the heterogeneity.