Background: Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP’s transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) in vitro, but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer. Methods: We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up ( n = 371), and patient-matched breast and metastatic brain tumours ( n = 91; 30 pairs). Results: Membrane localisation of phospho-HER4 [pHER4(Y¹¹⁶²)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% versus 75% positive), where it was usually co-expressed with pHER3(Y¹²⁸⁹) ( p < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T²¹⁸/Y²¹⁰) ( p = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S¹²⁷) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53; p = 9.6E⁻⁰³), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78; p = 1.7E⁻⁰²), particularly when co-expressed with nuclear HER4-ICD ( p = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin; p < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S¹²⁷) in ER-negative cases ( p < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases. Discussion: Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S¹²⁷) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S¹²⁷) associations with breast cancer clinical outcomes were dependent on ER status. Conclusion: Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.

The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p < 0.01) as well as in their microenvironment (p < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (p < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (p < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2–3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

Aims: Invasive lobular carcinoma (ILC) may show targetable vulnerabilities secondary to the characteristic loss of the cell adhesion protein E‐cadherin. Specifically, a synthetic lethal interaction was identified between E‐cadherin loss and ROS1 inhibition. Several clinical trials are currently under way to assess the efficacy of ROS1 inhibitors in ILC; however, ROS1 expression has not been confirmed in ILC tumours and ROS1 has not been validated as a biomarker in the breast cancer setting. This study aimed to (i) examine ROS1 expression in a large cohort of breast cancer cases and (ii) investigate the biology and clinical significance of ROS1 positivity in breast cancer.Methods and results: ROS1 immunohistochemistry was performed on a large cohort of ILC (n = 274) and invasive carcinoma of no special type (NST; n = 431) cases with extensive clinicopathological data. The staining performance of four ROS1 antibody clones was compared. There was marked variation in ROS1 status according to antibody clone. D4D6 and SP384 were negative in almost all breast cancer cases, whereas EP282 and EPMGHR2 were positive in 37 and 47% of ILC cases, and 49 and 74% of NST cases, respectively. Only data from clones D4D6 and SP384 were highly concordant, while EP282 and EPMGHR2 were positive in distinct breast cancer subtypes.Conclusions: Assessment of ROS1 status in breast cancer appears to be highly antibody clone‐dependent. ROS1 antibody clone selection will be an important consideration in the design of clinical trials investigating the clinical validity of ROS1 as a predictive biomarker in breast cancer.

The heterogeneity of TILs are not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune cell subsets in brain metastasis tissues. We performed multiplex immunofluorescence (mIF) on a limited cohort of brain metastases arising from breast cancers (n=20). Using RNA-interference validated antibodies, we quantitated the subsets of immune cells in formalin-fixed paraffin embedded whole sections. The panel of proteins analyzed included PanCK, CD8, CD4, Vista and Iba1 and an average of 15000 cells per sample were analysed. We also analysed an independent publicly available cohort at the RNA level to corroborate our findings. We found that increased density of tils (high>30%; low <30%) correlated with survival and they were two distinct phenotypes. The tumours with low tils had significantly higher expression of the immune-checkpoint molecule Vista in tumour cells (p<0.01) as well as in their microenvironment (p<0.001). Contrastingly, the brain metastatic tumours with high tils displayed higher expression of microglia. Low tils-tumours display CD8+ T-cells that exclusively co-express Vista (p<0.01) compared to high tils group where CD8+ T-cells significantly co-express Iba1 (p<0.05). Interestingly no definite phenotypes were observed in CD4+ T-cells. These results were also found in an independent cohort where Vista was a highly ranked gene within the CD8+ T-cell population. Variety of immune escape routes may be involved in brain metastasis. This may be executed by increasing the expression of T-cell inhibitory molecule Vista or by increased activated microglia which may release immunosuppressive cytokines. Further studies are required to provide mechanistic insights into these phenomena.