Clinical, pathological, and PAM50 gene expression features of HER2-low breast cancer

Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression.


INTRODUCTION
HER2-positive breast cancer is currently defined according to the ASCO/CAP guidelines using immunohistochemistry (IHC) and/or in situ hybridization (ISH)-based techniques 1,2 . These guidelines identify a tumor as HER2-positive when there is a complete and intense circumferential HER2 IHC staining in ≥10% of cells (score 3+) and/or the gene is amplified with an HER2/CEP17 ratio ≥2.0 and an average HER2 gene (ERBB2) copy number ≥4.0 signals/cell using ISH-based techniques 1 . In breast cancer, 10-20% of tumors are HER2-positive and 80-90% are HER2-negative 3,4 .
Within HER2-negative disease, substantial heterogeneity exists regarding the expression of hormone receptors (HR) and HER2. For example, HER2-negative tumors can express some protein level of HER2 by IHC 5 (i.e., 1+ or 2+ and lack of ERBB2 amplification by in situ hybridization techniques) and are identified as HER2-low. Traditionally, patients with HER2-low-expressing tumors do not seem to benefit from HER2-targeted therapies, such as 1-year of adjuvant trastuzumab 6 . However, two HER2-directed antibodydrug conjugates (ADC) with chemotherapeutics, namely trastuzumab deruxtecan (T-DXd) and trastuzumab duocarmazine (SYD985) have shown very promising therapeutic activity in patients with HER2-low breast cancer [7][8][9] . A large pivotal randomized phase III trial of T-DXd in patients with pretreated HER2-low metastatic breast cancer is underway (i.e., NCT03734029/DESTINY-Breast04).
Owing to the recent and increased interest in the HER2-low group, there is an urgent need to better understand its clinicopathological and molecular features. Thus, we decided to collect clinicopathological and PAM50 gene expression data from multiple datasets [10][11][12][13][14][15][16][17] of HER2-negative disease and compare many features between HER2-low and HER2 0. Analyses were focused on the overall population and according to hormone receptor (HR) status and HER2 IHC expression.
Finally, we investigated if the distribution of PAM50 subtypes within HER2-low breast cancer differed according to ERBB2 mRNA levels. To approach it, we divided all patients with HER2-negative disease into tertiles (i.e., from low to high: T1, T2, and T3) based on ERBB2 expression (Table 3). As expected, subtype distribution differed in HER2-low breast cancer according to ERBB2 levels (p < 0.001) with the T2-3 group being more enriched with Luminal A, Luminal B and HER2-E subtypes (51.5%, 34.9%, and 6.3%) compared to the T1 group (31.7%, 15.8%, and 3.6%). On the contrary, the Basal-like subtype was more frequent in the T1 group compared to the T2-3 group (44.6% vs 2.9%). The results were similar when comparing either ERBB2 high/HER2-low and ERBB2 low/HER2-low tumors with the whole HER2-low population (p < 0.001 both) ( Table 3).
Gene expression profiles according to HER2 expression and HR status The previous results suggested that HR status is a key determinant of the underlying biology of HER2-low breast cancer. To further explore this, we evaluated the overall gene expression profile of HER2-negative breast cancer according to HER2 expression (i.e., HER2 0, 1+ and 2+) and HR status (i.e., positive and negative). The result clearly shows that HR status is the main driver of the underlying biology ( Fig. 4 and Supplementary Table 7). As expected, proliferation-related genes (e.g., CCNE1, MKI67 and EXO1) were found more expressed in TNBC compared to HRpositive, regardless of HER2 IHC status (i.e., HER2-low vs. HER2 0). On the contrary, luminal-related genes (e.g., ESR1, AR, and BCL2) and ERBB2 were found more expressed in HR-positive compared to TNBC, regardless of HER2 IHC status. Of note, the highest ERBB2 expression was found in the HR-positive/HER2-low group. Finally, concordant with the previous results, HER2-low tumors within HRpositive disease showed a relatively lower expression of proliferation-related genes and higher expression of luminalrelated genes compared to the HER2 0 group (Supplementary Fig. 4 and Supplementary Table 8).

ERBB2 expression analysis
The previous observation that ERBB2 levels differ according to HER2 IHC expression (HER2 0, 1+, and 2+) and HR status was somewhat unexpected. To further explore this finding, we formally compared the abundance of ERBB2 in HR-positive disease and TNBC based on HER2 IHC expression. ERBB2 levels were statistically significantly higher in HR-positive tumors compared to TNBC regardless of HER2 IHC expression (p < 0.001; Fig. 5A, B). Within HR-positive disease, ERBB2 levels were significantly higher in HER2-low tumors compared to HER2 0 (1.4-fold mean difference, p < 0.001, Fig. 5C), with the highest amount observed in HER2 IHC 2+ tumors, followed by 1+ and 0 (Fig. 5D), in decreasing order (1.7-fold mean difference between HER2 2+ vs. HER2 0). Within TNBC, there was no statistically significantly difference in ERBB2 levels across the three HER2 IHC groups (p = 0.080, Fig. 5E); however, TNBC/HER2-low tumors showed statistically significantly higher levels of ERBB2 compared to HER2 0 tumors (p = 0.027), although the absolute mean difference was very small (Fig. 5F).
Prognosis of HER2-low in advanced HER2-negative breast cancer We conducted an exploratory overall survival (OS) analysis in 1,304 patients with advanced breast cancer across two datasets (i.e., Memorial Sloan Kettering Cancer Center database 18 and Hospital Clinic internal database). OS was defined from the date of the first diagnosis of breast cancer. The median follow-up for the overall population was 90.3 months (95% confidence interval [CI]: 84.6-99.4). In all patients, no statistically significantly differences in OS were observed between the HER2-low and HER2 0 groups (p = 0.787). Similar results were obtained according to HR status and HER2 IHC levels (Fig. 6).

DISCUSSION
Our results provide preliminary insights of the clinical and molecular characteristics of HER2-low breast cancer. According to our results, patients with HER2-low disease represent the vast majority (59.7%) of patients with HER2-negative tumors. Clinically, HER2-low breast cancer is apparently more frequent in older and male patients and shows more axillary lymph-node involvement compared to HER2 0 disease. Importantly, we observed that HR status has an important role in HER2-low disease. For example, the frequency of HER2-low disease is higher in HR-positive breast cancer than TNBC (65.4% vs. 36.6%) and most HER2-low tumors  Interestingly, when HR-positive disease and TNBC are divided according to the HER2 IHC score, no significant difference in subtype distribution is observed in TNBC, which was characterized by a high prevalence of the Basal-like subtype (84.7%), followed by the HER2-E (8.5%) subtype. On the contrary, HR-positive/HER2-low tumors appeared to be characterized by a higher proportion of luminal subtypes compared to HER2 0 tumors. Of note, the HER2-E subtype was infrequent and similarly distributed in HER2-low and HER2 0 breast cancer. As expected, the differences in subtype distribution according to HER2 IHC expression and HR status are consistent with the observed changes in expression of individual genes. For example, the vast majority of proliferation-related genes and tyrosine-kinase receptor genes are found more expressed in HER2 0 tumors compared to HER2-low tumors, while HER2-low tumors have more expression of luminal-related genes. This finding is especially relevant in HR-positive disease. On the contrary, no clear biological differences are observed in TNBC according to HER2 IHC expression. Overall, these findings suggest that HR-positive/ HER2-low tumors are a more distinct biological entity compared to TNBC/HER2-low tumors.
The lack of enrichment of the HER2-E subtype within HER2-low disease is intriguing and somewhat unexpected. However, previous studies have shown that the HER2-E phenotype is not defined by the expression of a single gene such as ERBB2. In fact, we and others have previously shown that the two variables (i.e., HER2-E subtype and ERBB2 levels) provide independent predictive and prognostic information 19 . Overall, this finding clearly highlights the need to separate expression of single genes or receptors from the underlying tumor phenotype.
Recent studies have opened up a new therapeutic scenario by showing potent activity of HER2-targeted novel ADCs in HER2-low breast cancer 8 . To date, T-DXd, a trastuzumab conjugated to eight molecules of deruxtecan, a topoisomerase I inhibitor, is at the most advanced in clinical development. A recently published phase Ib study enrolling highly pretreated patients with advanced HER2-expressing/mutated solid tumors, including HER2-low breast cancer, revealed a remarkable overall response rate (ORR) of 37.0% (95% CI: 24.3-51.3%) in HER2-low breast cancer and an impressive median duration of response of 10.4 months (95% CI: 8.8 monthnot evaluable), with no apparent differences in ORR between 1+ and 2+ IHC tumors (35.7% vs. 38.5%) 9 . Interestingly, the ORR did seem to differ according to HR status (40.4% in HR-positive disease and 14.3% in TNBC). This result is concordant with our findings that ERBB2 levels are more expresses in HR-positive/HER2-low tumors than in TNBC/HER2-low tumors. A phase III trial specifically enrolling patients with HER2-low metastatic breast cancer (i.e., NCT03734029/DESTINY-Breast04) is ongoing. Importantly, we previously demonstrated in HER2-positive disease that ERBB2 mRNA levels might provide a better selection of patients that benefit to the ADC T-DM1 20 . This might also be the case for HER2low tumors and might be worth focusing on this aspect in further studies.
SYD985 is another ADC comprises trastuzumab covalently bound to a linker drug containing duocarmycin. This drug also showed a promising ORR of 28 and 40% in HR-positive/HER2-low and TNBC/HER2-low, respectively 21 . In addition, other anti-HER2  Fig. 4 Gene expression profiles of HER2-negative breast cancer according to HER2 expression and HR status. Supervised clustering of 55 genes across four tumor classes defined according to HER2 IHC expression and HR status. All samples and gene expression data in each category have been combined into a single group. For each gene in a group, we calculated the standardized mean difference between the gene's expression in that class vs. its overall mean expression in the dataset using a 4-class Significance Analyses of Microarrays. The red color represents relative high gene score, green represents relative low gene score, and black represents median gene score. HR-positive hormone receptor positive, TNBC triple-negative breast cancer. ADCs (i.e., PF-06804103, MEDI4276, and XMT-1522) have shown promising activity in HER2-low tumors in the preclinical setting 8,22 , and phase 1 clinical trials are ongoing (clinicaltrials.gov identifier: NCT03284723, NCT02564900, and NCT02952729, respectively). Tumors with high ERBB2 mRNA levels, but overall HER2negative, might also benefit from novel tumor vaccines targeted against the HER2 protein, as shown by a recent randomized phase II trial of HER2-targeted vaccine nelipepimut-S combined with trastuzumab as adjuvant treatment in HER2-low high-risk breast cancer 23 . In this direction, we observed higher levels of TILs in the HER2 2+ group compared to the HER2 0 and 1+ groups, although this analysis was based on a very restricted number of cases. Further studies are needed to study the immune compartment of HER2-low breast cancer.
Our study presents limitations that need attention. First, we retrospectively combined patients from databases pertaining to different studies, with different original purposes and inclusion/ exclusion criteria; therefore, patients were not consecutively enrolled and a large proportion of them had metastatic disease. These might explain some of the imbalances that we observed between groups. Additionally, HER2 IHC status was not evaluated centrally; thus, inter-pathologist variability might have affected the results. Moreover, criteria for defining negative or equivocal ERBB2 amplification have changed over time 1,2 and most ERBB2 amplification results were only available in qualitative form (i.e., amplified, not amplified or equivocal). Another limitation is that we did not address intra-tumor HER2 heterogeneity, which represents 1%-34% of all breast tumors 24 and has clinical and prognostic implications, with poor response to anti-HER2-based regimens and worse prognosis, compared to HER2-positive tumors 24 . However, this feature is more common in HER2 equivocal disease 24 , a condition that was an exclusion criteria in our study, somewhat mitigating this issue. Finally, we limited our genomic analysis to the PAM50 genes and five additional genes. Thus, broader genomic analyses are likely to shed more light on this topic.
To our knowledge, this is the first comprehensive study focused specifically on HER2-low breast tumors. We provided extensive comparisons among the three different IHC-based classes of HER2-negative breast cancer and according to HR status. We found that HER2-low breast tumors are complex and heterogeneous, with no specific prognostic implications and HR-positive/ HER2-low emerge as a more distinct biological entity compared to the other groups. In addition, the evidence of ERBB2 levels being higher in HER2-low/HER2 2+ tumors (especially in the HR-positive) compared to HER2 1+ /0 is in line with some previous findings from single institutions-based studies, and contributes to reassure about the reliability of our results 25,26 . Similarly, the high prevalence of luminal disease in HER2-low disease has also been observed in other studies 24 . Finally, the concordance analysis of HER2 scoring by different pathologists showed an almost perfect agreement for HER2 0 and 3+ scores; however, the agreement for the HER2 1+ and 2+ categories was only substantial, according to Landis and Koch interpretation 27 . This result clearly suggests that more efforts are needed to standardize the scoring of HER2-low disease and potentially implement new and more sensitive assays that can help better discriminate HER2 levels within HER2negative breast cancer.

Patients datasets
All non-overlapping publicly available breast datasets (i.e., 12 studies and 6477 patients) were interrogated from the cBio Cancer Genomics Portal (http://cbioportal.org). From these databases, HER2-negative tumors with known IHC and HER2 amplification status were extracted [10][11][12][13] . Other patients were extracted from internal databases from the Hospital Clinic (Barcelona, Spain), from two SOLTI clinical trials (SOLTI 1501-VENTANA and SOLTI 1402-CORALEEN) 14,15 , from the Spanish Cancer Research Group (GEICAM)/CIBOMA study 16 and from a previously published collaboration between Hospital Clinic (Barcelona, Spain), Hospital Vall d'Hebron (Barcelona, Spain), University Campus Bio-Medico (Roma, Italy) and GEICAM 17 (see Supplementary Table 9 for study details). All studies had received proper ethical approval by the local institutional research ethics committee of all participating institutions and patients had given their consent to participate.

Inclusion criteria
Patients were included if they were HER2-negative with known IHC and HER2 amplification status and if they had at least one of the following information available: (1) clinicopathological features, (2) PAM50 gene expression data, and (3) PAM50 intrinsic subtype identified. The following clinical-pathological features were evaluated, when available: Ki67 IHC, histological grade, estrogen receptor and progesterone receptor status, age at diagnosis, menopausal status, tumor sample origin (primary vs. metastatic), histological subtype and TILs.

IHC-based classification
Tumors were divided into HR-positive (i.e., ER and/or PgR ≥1%) or TNBC, defined as ER < 1% and PgR<1%. In addition, tumors were classified into HER2 0, in case of an IHC score of 0, and HER2-low, defined as HER2 IHC of 1+ or 2+ with an HER2 amplification negative result by in situ hybridization (ISH) techniques. HER2 IHC 0 and 1+ were considered HER2 0 and HER2-low, respectively, unless ISH-based data was available and reported as HER2-amplified. HER2 status in each cohort had been previously determined using standard FDA-approved antibodies and ISH-techniques and classified according to the ASCO/ CAP guidelines 1,2 . Whenever available, we interpreted ISH-derived HER2/CEP17 ratio value and ERBB2 copy number results jointly with HER2 IHC score, according to last ASCO/CAP guidelines 1 . More specifically, tumors with an average HER2 copy number <4.0 signals/ cell, were considered HER2-negative, and also HER2-low in case of an IHC score of 1+ or 2+, irrespective of the HER2/CEP17 ratio. However, if the HER2/CEP17 ratio was ≥2.0 and HER2 IHC 3+, tumors were considered HER2-positive and excluded 1 .
In case of available average HER2 copy number ≥4.0 and <6.0 signals/ cell without HER2/CEP ratio and an IHC 3+, the tumor was considered positive and excluded. In case of IHC 0 or 1+, the tumor was considered HER2-negative, and also HER2-low in the latter case 1 . In case of IHC 2+, considering the unfeasibility of a retesting, in our case, if the categorization HER2-positive/negative was available from the original dataset, it was adopted and the tumor was considered HER2-negative and HER2-low. If the categorization was not provided, the sample was excluded.
In case of IHC score 0, 1+ or 2+, and a concurrent average HER2 copy number ≥4.0 and <6.0 signals/cell, with HER2/CEP17 ratio <2.0, the tumor was considered HER2-negative, and HER2-low in the last two cases. On the contrary, if the HER2/CEP17 watio was ≥2.0, the tumor was considered HER2-positive and excluded 1 .
In case of HER2 copy number ≥6.0 signals/cell, the tumor was considered HER2-positive and excluded in case of IHC of 2+ or 3+, regardless of the HER2/CEP17 ratio result, but in case of HER2/CEP17 ratio <2.0 and IHC 0 or 1+, the tumor was considered negative, and also HER2low in the second case 1 .
Patients with a persistent HER2 equivocal result were excluded 1 .
To evaluate the concordance of the HER2 IHC categories among pathologists, we performed an inter-pathologist concordance analysis across 100 independent cases of HER2 staining (HER2 0, 1+, 2+, and 3 +). Five independent breast cancer-specialized pathologists (i.e., BG, ES, RF, VP, and GP) from four institutions (i.e., Hospital Clinic, VHIO, HVH, and Campus Bio-Medico) were involved. Blinded scores were provided to FS and AP, who performed the concordance analysis.

PAM50 subtypes and gene expression data
We obtained PAM50 subtype information and individual gene expression data from 9 of the 13 retrospective cohorts (Hospital Clinic internal series, SOLTI and GEICAM trials reported in Supplementary Table 9). An nCounterbased research version of PAM50 had been previously used 28,29 . Intrinsic subtypes and raw gene expression data had been obtained from formalinfixed paraffin-embedded (FFPE) tumor samples. For RNA purification (Roche High Pure FFPET RNA isolation kit), at least 1 to 3 10-μm FFPE slides had been used for each tumor specimen, and macrodissection performed, when needed, to avoid normal breast tissue contamination. A minimum of 150 ng of total RNA had been used to measure the expression of 50 breast cancer-related genes, 4 immune-related genes, androgen receptor gene (full gene list included in Supplementary Table 5), and 5 housekeeping genes (ACTB, MRPL19, PSMC4, RPLP0, and SF3A1) using the nCounter platform (NanoString Technologies, Seattle WA) 28,30 . Data had been log base 2 transformed and normalized using the five housekeeping genes. Intrinsic subtyping (Luminal A, Luminal B, HER2-E, Basal-like and Normal-like) had been previously performed using the research-based PAM50 intrinsic subtype predictor 29 . We also retrieved intrinsic subtypes from the publicly available TCGA database (see "Data availability" section for further information).

Statistical analysis
Patient and tumor characteristics were analyzed using chi-square (χ 2 ) test, Fisher's exact test, Kruskalis-Wallis and Wilcoxon rank sum test with continuity correction, where appropriate. The concordance analysis among pathologists was performed using the Fleiss' Kappa. The agreement among pathologists was considered poor for k < 0, low for k = 0.01-0.20, fair for k = 0.21-0.40, moderate for k = 0.41-0.60, substantial for k = 0.61-0.80, and almost perfect for k = 0.81-1.00 27 .
All differences were considered significant at p < 0.05. Bonferroni-Holm method was used to control the family-wise error rate in case of multiple comparisons.
OS was evaluated for patients with homogeneous follow-up with available or computable survival data. Such patients pertained to the Memorial Sloan Kettering Cancer Center (MSKCC)'s subset of the cBio Cancer Genomics Portal group and to the Hospital Clinic of Barcelona subset. All patients were affected by metastatic disease and presented available information regarding primary tumor diagnosis.
The OS distributions were estimated using the Kaplan-Meier method and the log-rank test was used to assess the difference in survival distribution between the groups 31 . Censoring was done at the date of last available follow-up. Significance Analysis of Microarray (SAM) for unpaired samples (multiclass and two class) was used to compare gene expression profiles between groups 32 . Differences were considered significant at an FDR < 5%. All analyses were performed with R version 3.6.1 33 , Cluster 3.0, Javatreeview 1.1.6r4 34 and Microsoft Excel.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.