HER2 mutations are targetable alterations in patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC). In the SUMMIT basket study, patients with HER2-mutant MBC received neratinib monotherapy, neratinib + fulvestrant, or neratinib + fulvestrant + trastuzumab (N + F + T). We report results from 71 patients with HR+, HER2-mutant MBC, including 21 (seven in each arm) from a randomized substudy of fulvestrant versus fulvestrant + trastuzumab (F + T) versus N + F + T.
Patients with HR+ HER2-negative MBC with activating HER2 mutation(s) and prior cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) therapy received N + F + T (oral neratinib 240 mg/day with loperamide prophylaxis, intramuscular fulvestrant 500 mg on days 1, 15, and 29 of cycle 1 then q4w, intravenous trastuzumab 8 mg/kg then 6 mg/kg q3w) or F + T or fulvestrant alone. Those whose disease progressed on F + T or fulvestrant could cross-over to N + F + T. Efficacy endpoints included investigator-assessed objective response rate (ORR), clinical benefit rate (RECIST v1.1), duration of response, and progression-free survival (PFS). Plasma and/or formalin-fixed paraffin-embedded tissue samples were collected at baseline; plasma was collected during and at end of treatment. Extracted DNA was analyzed by next-generation sequencing.
ORR for 57 N + F + T-treated patients was 39% [95% confidence interval (CI) 26% to 52%); median PFS was 8.3 months (95% CI 6.0-15.1 months). No responses occurred in fulvestrant- or F + T-treated patients; responses in patients crossing over to N + F + T supported the requirement for neratinib in the triplet. Responses were observed in patients with ductal and lobular histology, 1 or ≥1 HER2 mutations, and co-occurring HER3 mutations. Longitudinal circulating tumor DNA sequencing revealed acquisition of additional HER2 alterations, and mutations in genes including PIK3CA, enabling further precision targeting and possible re-response.
The benefit of N + F + T for HR+ HER2-mutant MBC after progression on CDK4/6is is clinically meaningful and, based on this study, N + F + T has been included in the National Comprehensive Cancer Network treatment guidelines. SUMMIT has improved our understanding of the translational implications of targeting HER2 mutations with neratinib-based therapy.

BACKGROUND: The TROIKA trial established that HD201 and trastuzumab were equivalent in terms of primary endpoints (total pathological complete response) following neoadjuvant treatment. The objective of the present analysis was to compare survival outcomes and final safety.
METHODS: In the TROIKA trial, patients with ERBB2-positive early breast cancer were randomized and treated with either HD201 or the referent trastuzumab. Eligible patients received 8 cycles of either HD201 or referent trastuzumab (loading dose, 8 mg/kg; maintenance dose, 6 mg/kg) every 3 weeks in combination with 8 cycles of chemotherapy (4 cycles of docetaxel, 75 mg/m2, followed by 4 cycles of epirubicin, 75 mg/m2, and cyclophosphamide, 500 mg/m2) in the neoadjuvant setting. The patients then underwent surgery followed by 10 cycles of adjuvant HD201 or referent trastuzumab according to their initial randomization to complete one year of trastuzumab-directed therapy. Event-free and overall survival rates were calculated using Kaplan-Meier analysis. The hazard ratio for event-free survival was estimated by Cox proportional hazards regression.
RESULTS: The final analysis was performed after all patients completed the study at a median follow-up of 37.7 months (Q1-Q3, 37.3-38.1 months). A total of 502 randomized patients received either HD201 or the referent trastuzumab, and 474 (94.2%) were eligible for inclusion in the per-protocol set. In this population, the 3-year event-free survival rates were 85.6% (95% CI: 80.28-89.52) and 84.9% (95% CI: 79.54-88.88) in the HD201 and referent trastuzumab groups, respectively (log rank p = 0.938) (HR 1.02, 95% CI: 0.63-1.63; p = 0.945). The 3-year overall survival rates were comparable between the HD201 (95.6%; 95% CI: 91.90-97.59) and referent trastuzumab treatment groups (96.0%, 95% CI: 92.45-97.90) (log rank p = 0.606). During the posttreatment follow-up period, adverse events were reported for 64 (27.4%) and 72 (29.8%) patients in the HD201 and the reference trastuzumab groups, respectively. Serious adverse events were rare and none of which were related to the study treatment.
CONCLUSIONS: This final analysis of the TROIKA trial further confirms the comparable efficacy and safety of HD201 and trastuzumab.
BACKGROUND: ClinicalTrials.gov identifier: NCT03013504.

HER2-positive breast cancer is an aggressive tumor subtype and it is usually associated with worse clinical outcomes. Given the advances in HER2-targeted therapies, we hypothesized that HER2 amplification is no longer a marker of poor prognosis.
We conducted a population-based observational study employing two independent cohorts of patients with breast cancer. Samples from the METABRIC cohort were collected before clinical availability of HER2-targeted therapies, whereas samples from the SCAN-B cohort were collected afterward. The primary endpoint was overall survival (OS).
A total of 5121 patients were included in the analyses. In both cohorts, HER2-positive tumors were more likely to be node-positive (P < .05) and high grade (P < .001). Before HER2-targeted agents, HER2 patients had a significantly worse 5-year OS than hormone receptor-positive (HR+) patients (63.4% vs. 83.0%, HR = 2.49, P < .001). In contrast, after HER2-targeted agents entered clinical practice, 5-year OS no longer differed (88.3% vs. 90.4%, HR = 1.24, P = .17). Additionally, in an exploratory analysis using PAM50 subtypes, we identified that, after HER2-targeted therapies were implemented, patients clinically HER2-negative but PAM50-HER2-enriched have a lower OS (HR = 1.99, P = .009) than those who are both HER2-positive and PAM50-HER2-enriched, since they have not benefitted from HER2-targeted therapies.
HER2-targeted therapies dramatically altered the natural history of HER2-positive breast cancer, with overall survival approaching those of luminal subtype. HER2 positivity is no longer a marker of poor prognosis if access to HER2-targeted therapies is granted. Future trials should assess whether HER2-negative PAM50-HER2-enriched patients may also benefit from such therapies.

ErbB family of receptor tyrosine kinases play significant roles in cellular differentiation and proliferation. Mutation or overexpression of these receptors leads to several cancers in humans. The family has four homologous members including EGFR, ErbB2, ErbB3, and ErbB4. From which all except the ErbB2 bind to growth factors via the extracellular domain to send signals to the cell. However, dimerization of the ErbB receptor occurs in extracellular, transmembrane, and intracellular domains. The ErbB receptors are known to form homodimers and heterodimers in the active form. Heterodimerization increases the variety of identified ligands and signaling pathways that can be activated by these receptors. Furthermore, glycosylation of the ErbB receptors has shown to be critical for their stability, ligand binding, and dimerization. Here, atomistic molecular dynamics simulations on the glycosylated and unglycosylated heterodimer showed that the EGFR-ErbB2 heterodimer is more stable in its dynamical pattern compared to the EGFR-EGFR homodimer. This increased stability is regulated by maintaining the dimeric interface by the attached glycans. It was also shown that the presence of various glycosylation sites within the ErbB2 growth factor binding site leads to occlusion of this site by the glycans that inhibit ligand binding to ErbB2 and participate in further stabilization of the heterodimer construct. Putting together, glycosylation seems to promote the heterodimer formation within the ErbB family members as the dominant molecular mechanism of activation for these receptors.

HER2 overexpression has been reported in various human and canine tumours. The aim of this study is to investigate the expression of HER2 protein in different histotypes of canine carcinomas in order to identify potential tumours that could benefit from the HER2-targeted therapy. Eighty-two (82) canine carcinomas (squamous cell, gastro-intestinal, rectal, pulmonary, prostatic, urothelial, and ovarian) from paraffin-embedded samp les were immunohistochemically evaluated. The degree of HER2 expression was scored based on the ASCO/CAP 2018 guidelines. Intestinal carcinomas were those with greater HER2 overexpression (3+) with 81% of positive cases, followed by 42% of rectal carcinomas and 28% of squamous cell carcinomas. These observations suggest that HER2 overexpression could be a driver in the oncogenesis of several types of canine carcinomas and lay the foundations for the identification of different types of canine carcinomas that could benefit from HER2-targeted therapy.

ERBB2 exon 16 skipping is an alternatively spliced isoform of ERBB2, which was reported to lead to oncogenic activation of ERBB2 and could potentially cause tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC) in case studies. In this study, we aimed to evaluate the frequency of ERBB2 exon 16 skipping in a large patient cohort and its function in cancer development.
A total of 38 680 Chinese patients with cancer whose tumour specimens and/or circulating cell-free DNA underwent targeted nextgeneration sequencing of cancer-related genes were retrospectively reviewed. Clinicopathological features and treatment history of patients harbouring ERBB2 exon 16 skipping were evaluated. RNA-sequencing was performed to validate the presence of exon 16 skipping in ERBB2 at the transcriptional level.
ERBB2 exon 16 skipping is rare and was identified in a total of 18 patients (0.046% of total patients), including 12 lung cancers, which were caused by large fragment deletion spanning the whole or partial region of exon 16 (13/18, 72.2%) and/or splice site variants (6/18, 33.3%). The majority of these variants have not been previously reported and three of them were confirmed by RNA-sequencing. Among the 12 patients with lung cancer, 9 had coexisting activating EGFR mutations (exon 19 deletions or L858R) and received prior-treatment with epidermal growth factor receptor TKIs. Further analysis of matched pre-treatment and post-treatment samples in three EGFR-mutated NSCLC patients confirmed that ERBB2 exon 16 skipping was newly acquired on resistance to TKI therapies. In 6 out of 18 patients, including colorectal, gastric and ovarian cancers, there were no mutations in known cancer driver genes detected, indicating that ERBB2 exon 16 skipping might be the oncogenic driver in these patients.
Our data suggest that ERBB2 exon 16 skipping is another mechanism of TKI resistance in EGFR-mutated patients with lung cancer, in addition to its role of being an oncogenic driver in other solid malignancies.

HER2 is a therapeutic target for metastatic colorectal cancer (mCRC), as demonstrated in the pivotal HERACLES-A (HER2 Amplification for Colo-rectaL cancer Enhanced Stratification) trial with trastuzumab and lapatinib. The aim of HERACLES-B trial is to assess the efficacy of the combination of pertuzumab and trastuzumab-emtansine (T-DM1) in this setting.
HERACLES-B was a single-arm, phase II trial, in patients with histologically confirmed RAS/BRAF wild-type and HER2+ mCRC refractory to standard treatments. HER2 positivity was assessed by immunohistochemistry and in situ hybridisation according to HERACLES criteria. Patients were treated with pertuzumab (840 mg intravenous load followed by 420 mg intravenous every 3 weeks) and T-DM1 (3.6 mg/kg every 3 weeks) until disease progression or toxicity. Primary and secondary end points were objective response rate (ORR) and progression-free survival (PFS). With a Fleming/Hern design (H0=ORR 10%; α=0.05; power=0.85), 7/30 responses were required to demonstrate an ORR ≥30% (H1).
Thirty-one patients, 48% with ≥4 lines of previous therapies, were treated and evaluable. ORR was 9.7% (95% CI: 0 to 28) and stable disease (SD) 67.7% (95% CI: 50 to 85). OR/SD ≥4 months was associated with higher HER2 immunohistochemistry score (3+ vs 2+) (p = 0.03). Median PFS was 4.1 months (95% CI: 3.6 to 5.9). Drug-related grade (G) 3 adverse events were observed in two patients (thrombocytopaenia); G≤2 AE in 84% of cycles (n = 296), mainly nausea and fatigue.
HERACLES-B trial did not reach its primary end point of ORR; however, based on high disease control, PFS similar to other anti-HER2 regimens, and low toxicity, pertuzumab in combination with T-DM1 can be considered for HER2+mCRC as a potential therapeutic resource.
2012-002128-33 and NCT03225937.

Cervical cancer is a major health problem worldwide. Identification of effective clinicopathologic and molecular markers is vital to improve treatment stratification.
The purpose of this study was to validate a set of well-defined clinicopathologic features in a large population-based, prospectively collected cervical cancer cohort to support their use in the clinic. Further, we explored p53 and human epidermal growth factor receptor 2 as potential prognostic markers in cervical cancer.
Tissue was collected from 401 patients with cervical cancer. Clinical data that included follow-up evaluations were collected from patient journals. Histopathologic data were evaluated and revised by an expert pathologist. The prognostic impact of selected clinicopathologic variables was analyzed in the whole cohort. Tissue microarrays were prepared from 292 carcinomas, and p53 and human epidermal growth factor receptor 2 protein levels were evaluated by immunohistochemistry. Fresh frozen samples from overlapping cervical carcinomas previously were subjected to human papilloma virus typing (n=94), whole exome (n=100) and RNA (n=79) sequencing; the results were available for our analyses.
Among the clinicopathologic variables, vascular space invasion, histologic type, and tumor size were verified as strong independent prognostic markers. High p53 protein levels were associated significantly with markers for aggressive phenotype and survival, also in multivariate survival analysis, but did not reflect TP53 mutational status. High human epidermal growth factor receptor 2 protein levels were identified in 21% of all tumors. ERBB2 amplification was associated with poor outcome (P=.003); human epidermal growth factor receptor 2 protein level was not.
Our findings support that the Féderation Internationale de Gynécologie et d\'Obstétrique s guidelines should include vascular space invasion and tumor size 2-4 cm and that careful selection of histologic type is essential for stratification of patient risk groups. High p53 levels independently predict poor survival yet do not reflect mutational status in cervical cancer. Amplified ERBB2 significantly links to poor survival, while HercepTest does not. With optimal stratification, human epidermal growth factor receptor 2-based therapy may improve cervical cancer treatment.

Accurate determination of the predictive markers human epidermal growth factor receptor 2 (HER2/ERBB2), estrogen receptor (ER/ESR1), progesterone receptor (PgR/PGR), and marker of proliferation Ki67 (MKI67) is indispensable for therapeutic decision making in early breast cancer. In this multicenter prospective study, we addressed the issue of inter- and intrasite reproducibility using the recently developed reverse transcription-quantitative real-time polymerase chain reaction-based MammaTyper® test.
Ten international pathology institutions participated in this study and determined messenger RNA expression levels of ERBB2, ESR1, PGR, and MKI67 in both centrally and locally extracted RNA from formalin-fixed, paraffin-embedded breast cancer specimens with the MammaTyper® test. Samples were measured repeatedly on different days within the local laboratories, and reproducibility was assessed by means of variance component analysis, Fleiss\' kappa statistics, and interclass correlation coefficients (ICCs).
Total variations in measurements of centrally and locally prepared RNA extracts were comparable; therefore, statistical analyses were performed on the complete dataset. Intersite reproducibility showed total SDs between 0.21 and 0.44 for the quantitative single-marker assessments, resulting in ICC values of 0.980-0.998, demonstrating excellent agreement of quantitative measurements. Also, the reproducibility of binary single-marker results (positive/negative), as well as the molecular subtype agreement, was almost perfect with kappa values ranging from 0.90 to 1.00.
On the basis of these data, the MammaTyper® has the potential to substantially improve the current standards of breast cancer diagnostics by providing a highly precise and reproducible quantitative assessment of the established breast cancer biomarkers and molecular subtypes in a decentralized workup.

HER2 (ERBB2) gene amplification and its corresponding overexpression are present in 15-30% of invasive breast cancers. While HER2-targeted agents are effective treatments, resistance remains a major cause of death. The American College of Surgeons Oncology Group Z1041 trial (NCT00513292) was designed to compare the pathologic complete response (pCR) rate of distinct regimens of neoadjuvant chemotherapy and trastuzumab, but ultimately identified no difference.
In supplement to tissues from 37 Z1041 cases, 11 similarly treated cases were obtained from a single institution study (NCT00353483). We have extracted genomic DNA from both pre-treatment tumor biopsies and blood of these 48 cases, and performed whole genome (WGS) and exome sequencing. Coincident with these efforts, we have generated RNA-seq profiles from 42 of the tumor biopsies. Among patients in this cohort, 24 (50%) achieved a pCR.
We have characterized the genomic landscape of HER2-positive breast cancer and investigated associations between genomic features and pCR. Cases assigned to the HER2-enriched subtype by RNA-seq analysis were more likely to achieve a pCR compared to the luminal, basal-like, or normal-like subtypes (19/27 versus 3/15; P = 0.0032). Mutational events led to the generation of putatively active neoantigens, but were overall not associated with pCR. ERBB2 and GRB7 were the genes most commonly observed in fusion events, and genomic copy number analysis of the ERBB2 locus indicated that cases with either no observable or low-level ERBB2 amplification were less likely to achieve a pCR (7/8 versus 17/40; P = 0.048). Moreover, among cases that achieved a pCR, tumors consistently expressed immune signatures that may contribute to therapeutic response.
The identification of these features suggests that it may be possible to predict, at the time of diagnosis, those HER2-positive breast cancer patients who will not respond to treatment with chemotherapy and trastuzumab.
NCT00513292, NCT00353483.