HER2-positive Breast Cancer Subtypes Research Articles

Abstract PO2-25-04: Anti-TIGIT upregulate CD226 on CD8+T cell as a potential therapeutic manner in HER2 positive breast cancer

Abstract Purpose: HER2 positive breast cancer subtype shows high malignancy, aggressive invasion, frequent recurrence, and low efficacy to PD-1 inhibitor though enriches in immune microenvironment. TIGIT is an immune checkpoint which expresses on T/NK cells and participates in immune evasion of cancer cells. Here, we investigated the effect of anti-HER2 and anti-TIGIT combination regarding to HER2 breast cancer treatment and tumor microenvironment, further exploring mechanisms behind the combination utilization. Methods: We re-analyzed the untreated human and mouse HER2 breast cancer scRNA-seq dataset to explore proportions of unique cell clusters in tumor immune microenvironment and communicated signals between immune cells and tumor cells. Mouse HER2 non-sensitive model and 27 non-pCR patients were utilized to investigate the variation of TIGIT and its ligand CD112/CD155 in HER2 non-sensitive population. Relevance between CD112/CD155 and prognosis was verified by 200 patients tissue microarray IHC. We validated the efficacy of combination of anti-TIGIT and anti-Neu and delineated scRNA-seq landscape via mouse model. Multilayer analysis including CellChat and AuCell were used to investigate difference of immune network between ctrl group, monotherapy and combination. In vitro and in vivo studies were used to verify the efficacy of anti-TIGIT and pivotal clusters. Results: We found that CD8+T cells occupied major proportion in TILs in both human and mouse HER2 breast cancer tumor immune microenvironment. CD8+T cells showed TIGIT signal to malignant epithelial cells instead of PD-1 signal. Increased CD8+TIGIT+T cells were related to poor prognosis in 200-patients cohort. In HER2 non-sensitive mouse model, significantly increased CD8+TIGIT+T cells were found in HER2 non-sensitive group. Multiplex IHF of 27 non-pCR patients also confirmed that CD8+TIGIT+T cells increased after treatment. CD112, ligand of TIGIT on malignant cells, demonstrated higher expression after anti-HER2 treatment in HER2 non-sensitive population and negatively correlated with prognosis. In scRNA-seq about control group, monotherapy and combination group, we defined malignant cells as sensitive subclone, Neu resistant subclone and others according to cell ratio variation in ctrl, monotherapy and combination group. CD112 upregulated in all subclones in monotherapy group. GSVA analysis indicated Neu resistant subclone had highest IFN-γ response among subclones. In Neu resistant subclone, combination group showed highest expression of MHC-I genes and upregulated IFN-γresponse, suggested the restored antigen presenting ability of Neu resistant subclone. Higher CD226, MHC-I and IFN-γ signals between malignant epithelial cells and CD8+T cells was found in combination group via CellChat. AuCell manifested upregulated T cell activation and IFN-γ production pathways in combination group. Public dataset showed favorable results. Increased CD226 expression and promoted cytotoxicity on CD8+T cells can only be induced by anti-TIGIT therapy in vitro and in vivo. 200-patients cohort indicated the predictive values of CD8+CD226+T cells in HER2 breast cancer. Conclusion: In conclusion, engaged immune checkpoint TIGIT shades malignant cells from CD8+T mediated immune surveillance in HER2 breast cancer. Disruption of TIGIT inhibit tumor growth in vivo by immune elimination of malignant cells. Besides, CD8+CD226+T cells induced by combined therapy can predict patient prognosis. Citation Format: Liyi Zhang, Qi Zhang, Zehao Wang, Zhibo Shao, Jingyan Xue, Yayun Chi, Bingqiu Xiu, Jiong Wu. Anti-TIGIT upregulate CD226 on CD8+T cell as a potential therapeutic manner in HER2 positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-04.

Abstract 3940: Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8

Abstract Breast cancer is a highly complex multifactorial disease, which can be driven by the aberrant regulation of different signaling pathways including receptor tyrosine kinase (RTK) signaling. Notably, while RTKs have been classically described as transmembrane receptors, they can also translocate to the nucleus, even though the functional importance of this process remains largely unexplored for most RTKs. Here, we report a novel role for the nuclear form of the RTK human epidermal growth factor receptor 3 (HER3) in driving primary breast cancer growth and metastasis. Firstly, using different patient-derived organoids (PDOs) established from metastatic breast cancer patients, we demonstrated the robust translocation of the activated phosphorylated HER3 receptor (pHER3) from the plasma membrane to the nucleus in response to its ligand Neuregulin 1 (NRG1). Interestingly, the nuclear translocation was observed not only in the HER2-positive breast cancer subtype but also in luminal and triple-negative breast cancer-derived cells. In order to decipher the functional role of nuclear HER3 (nHER3), we identified and mutated its nuclear localization signal, resulting in decreased nuclear translocation of the receptor while its membrane form remained intact. This reduction in nHER3 remarkably impeded primary tumor growth and metastatic spread in different patient-derived xenograft (PDX) models. Conversely, selective overexpression of HER3 in the nucleus led to increased primary tumor growth and metastatic burden in vivo. In order to decipher the mechanism of action of nHER3, we performed co-immunoprecipitation followed by global mass spectrometric analysis to identify the protein binding partners of the receptor in the nucleus. Specifically, nHER3 was found to interact with different transcription factors such as the AP-2 family of transcription factors and with major chromatin remodeling complexes like the nucleosome remodeling and deacetylase (NuRD) complex, implicating its role in transcriptional regulation. Furthermore, gene expression analysis of PDOs expressing the wild-type or the mutated form of HER3 revealed the modulation of several key growth regulators such as early growth response 3 (EGR3) by nHER3. The nHER3-EGR3 axis further controlled downstream effectors like the immune chemoattractants CXCL1 and CXCL8. Accordingly, PDX from breast cancer cells overexpressing nHER3 showed significantly increased immune infiltration, suggesting a possible role of nHER3 in regulating the tumor microenvironment. Altogether, we report here a novel non-canonical role of the nuclear form of HER3 receptor in driving breast tumorigenesis, with key implications for our understanding and targeting of RTK signaling in breast cancer. Citation Format: Tasneem Cheytan, Roberto Würth, Nina Hahnen, Elisa Donato, Corinna Klein, Rebecca Weber, Daniele Colombo, Jeroen Krijgsveld, Martin Sprick, Andreas Trumpp. Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3940.

Different views of the pathway's impact on Chemoresistance and Relapse Advanced Breast Cancer and type 2 Diabetes Mellitus

Advanced breast cancer (ABC) with type 2 diabetes mellitus could be an uncommon, however profoundly forceful frame of breast cancer, which accounts for less than 5% of all locally progressed introductions. The clinical introduction of advanced breast cancer regularly contrasts altogether from that of non-advanced breast cancer; immunohistochemistry uncovers a few recognizing highlights. The more forceful triple-negative and HER2-positive breast cancer subtypes are overrepresented in advanced breast cancer compared to non-advanced breast cancer, with a poorer guess in reaction to routine treatments and plasma glucose level control with hyperglucosemia modification. Current understanding of breast tumor chemoresistance - breast cancers localized at essential breast areas and treated early can still relapse because of the presence of cancer cells and the change of cancer cells into a moderately forceful phenotype. This audit summarizes the current proof recommending that inflammatory signaling pathways are up-regulated, which may give a road for novel therapeutics against the background of diabetes. The part of the tumor microenvironment, through tumor-associated macrophages and infiltrating lymphocytes, is additionally examined, recommending that these tumors' outward variables may offer assistance in accounting for the contrasts in behavior between advanced breast cancer with high plasma glucose levels and non-advanced breast cancer. There are different novel treatment techniques currently underway in clinical trials; they require encouraging the improvement of preclinical models of this uncommon but forceful disease is vital.

Dynamic light scattering microscopy sensing mitochondria dynamics for label-free detection of triple-negative breast cancer enhanced by deep learning.

We established a deep learning-based dynamic light scattering (DLS) microscopy sensing mitochondria dynamic for label-free identification of triple-negative breast cancer (TNBC) cells. The capacity of DLS microscopy to detect the intracellular motility of subcellular scatters was verified with the analysis of the autocorrelation function. We also conducted an in-depth examination of the impact of mitochondrial dynamics on DLS within TNBC cells, employing confocal fluorescent imaging to visualize the morphology of the mitochondria. Furthermore, we applied the DLS microscopy incorporating the two-stream deep learning method to differentiate the TNBC subtype and HER2 positive breast cancer subtype, with the classification accuracy achieving 0.89.

Biomarker dynamics affecting neoadjuvant therapy response and outcome of HER2-positive breast cancer subtype

HER2+ breast cancer (BC) is an aggressive subtype genetically and biologically heterogeneous. We evaluate the predictive and prognostic role of HER2 protein/gene expression levels combined with clinico-pathologic features in 154 HER2+ BCs patients who received trastuzumab-based neoadjuvant chemotherapy (NACT). The tumoral pathological complete response (pCR) rate was 40.9%. High tumoral pCR show a scarce mortality rate vs subjects with a lower response. 93.7% of ypT0 were HER2 IHC3+ BC, 6.3% were HER2 IHC 2+/SISH+ and 86.7% of ypN0 were HER2 IHC3+, the remaining were HER2 IHC2+/SISH+. Better pCR rate correlate with a high percentage of infiltrating immune cells and right-sided tumors, that reduce distant metastasis and improve survival, but no incidence difference. HER2 IHC score and laterality emerge as strong predictors of tumoral pCR after NACT from machine learning analysis. HER2 IHC3+ and G3 are poor prognostic factors for HER2+ BC patients, and could be considered in the application of neoadjuvant therapy. Increasing TILs concentrations, lower lymph node ratio and lower residual tumor cellularity are associated with a better outcome. The immune microenvironment and scarce lymph node involvement have crucial role in clinical outcomes. The combination of all predictors might offer new options for NACT effectiveness prediction and stratification of HER2+ BC during clinical decision-making.

Development of a CUB domain-containing protein 1 (CDCP1)-targeting antibody-drug conjugate for triple-negative and metastatic breast cancer.

e15012 Background: CUB-domain containing- protein 1 (CDCP1) is a transmembrane receptor involved in the progression of several cancers. Recent studies demonstrate that CDCP1 is a rational target for the development of innovative targeted therapies for cancer including theranostics agents and antibody-drug conjugates. Methods: To determine the therapeutic potential of CDCP1 in breast cancer, we investigated its expression in multiple cohorts of breast cancer tissues by immunohistochemistry, as well as in various preclinical models including cell lines, primary cells and patient-derived xenografts using flow cytometry, western blot and immunofluorescence staining. Then, we evaluated the capacity of the CDCP1-targeting chimeric antibody ch10D7 to specifically accumulate in breast cancer lesions in in vivo preclinical models including patient-derived xenografts and breast cancer metastasis models. Finally, we determined the efficacy of the ch10D7-MMAE antibody-drug conjugate to kill breast cancer cells in vitro and breast tumours ex-vivo and in vivo. Results: The CDCP1 receptor is expressed at targetable level in a significant proportion of breast cancer cases with high/intermediate expression detected in ~30% of localized ER-positive cases, ~50% of metastatic ER-positive cases and > 70% of Triple-negative or HER2-positive cases. Similar proportion of expression was detected in cellular models. We demonstrated that ch10D7 antibody labelled with the radionucleotide Zircodium-89 specifically accumulates in breast cancer lesions in vivo allowing the detection of mammary-fat pad implanted patient-derived xenografts and of breast cancer metastasis by PET/CT imaging. Finally, we confirmed that the ch10D7-MMAE antibody-drug conjugate is very efficient at inducing cell death in vitro as well as controlling primary tumour and metastatic tumour burden in pre-clinical models, conferring a significant survival advantage compared to classical therapy. Conclusions: Our work demonstrates that CDCP1 is a potential target to detect and limit the progression of breast tumours in both the primary and metastatic disease setting, particularly in triple-negative and HER2-positive breast cancer subtypes. Furthermore, we show that biomolecules specifically recognising this receptor are promising therapeutic agents which could improve survival of patients.

Abstract P5-02-53: P95HER2 Expression in HER2-Positive Breast Cancer

Abstract Background: -HER2-positive breast cancer subtype accounted for around 15-20% of all breast cancer. -The introduction of HER2-targeted therapy such as trastuzumab and pertuzumab has remarkably increased the patients’ prognosis of HER2-positive breast cancer. -However, resistance exists due to impaired drug binding to HER2 receptor and constitutive activation of HER2 downstream signaling pathways. -P95HER2 isoform is a truncated form of HER2 that retains the C terminal domain but lacks an N terminal trastuzumab binding site, leading to trastuzumab resistance in HER2-positive breast cancer. -A new P95HER2 antibody is developed to target the extracellular domain of p95HER2 in formalin-fixed paraffin-embedded (FFPE) HER2-positive breast cancer tissues by using hematoxylin and eosin (HE) staining method. Objectives: To evaluate the expression of P95HER2 and its clinicopathological characteristics in HER2-positive breast cancer. Methods: We assessed 68 HER2-positive patients (IHC 3+ or IHC 2+/in situ hybridization [ISH]+) from Fudan University Shanghai Cancer Center (FUSCC) who underwent breast cancer surgery and were treated with adjuvant chemotherapy (taxane or anthracycline or combination) plus trastuzumab from 2014 to 2016. P95HER2 HE antibody is provided by Simcere Pharma. In this study, we compared 27 patients with primary trastuzumab resistance with 41 non-relapse breast cancer patients. 14 patients have not received trastuzumab targeted therapy. P95HER2 staining of either 1+, 2+ or 3+ observed in any tumor area in HE slides was considered to be P95 HER2 positive. Chi-square test was used to determine the relationship between P95HER2 expression of patients’ characteristics. The main outcome measures were disease free-survival (DFS), distant disease-free survival (DDFS) and overall survival (OS) by using log-rank test. Univariable and multivariable Cox regression analyses were used to identify independent factors related to prognosis. Results: From 2014 to 2016, we assessed the expression of P95HER2 expression in 68 HER2 positive breast cancer patients from FUSCC. Median follow-up was 45 months. In our study, 19 (27.9%) were P95HER2 positive. P95HER2 positive expression rate is higher in premenopausal patients than in postmenopausal patients (68.4% vs 38.8%, P= 0.028). Univariable analysis showed that higher T-stage (P= 0.018), higher N-stage (P= 0.001) and P95HER2 positive expression (P= 0.033) were associated with worse DDFS. Multivariable analysis showed that higher T-stage (hazard ratio, 6.019; 95% CI, 1.205-30.078; P= 0.029) and P95HER2 positive (hazard ratio, 2.349; 95%CI, 1.03-5.358; P= 0.042) independently predicted worse DDFS. P95HER2 positive was significantly associated with shorter 5-year DDFS (42.1% vs 67.6%, P= 0.028), but has no significant difference in DFS (36.8% vs 59.5%, P= 0.072) and OS (74.8% vs 81.2%, P= 0.685). Conclusions: P95HER2 positive was found more in premenopausal patients and was associated with a higher metastasis rate, indicating that P95HER2 expression tends to be a more aggressive isoform type of HER2-positive breast cancer. P95HER2 may serve as a therapeutic target for anti-HER2 therapy. Citation Format: Chih Wan Goh, Liyi Zhang, Wei-Ru Chi, Bingqiu Xiu, Jiajian Chen, Wenqing Yang, Chunxia Ao, Jianxing Tang, Jingyan Xue, Yayun Chi, Jiong Wu. P95HER2 Expression in HER2-Positive Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-53.

Evaluation of the results of neoadjuvant treatment in patients with HER2 positive breast cancer subtype

Background. In women, breast cancer is the most common malignant tumor. Based on current clinical guidelines for HER2 positive tumor growth, it is possible to carry out preoperative chemotherapy for further organ-preserving operations. However, a prognostic scale for assessing neoadjuvant treatment has not yet been developed.
 Aim. To create a mathematical model on the basis of which a computer program was developed to determine the likelihood of the neoadjuvant treatment effectiveness in patients diagnosed with HER2 positive breast cancer for further organ-preserving operations.
 Material and methods. A planned retrospective study was performed at the Samara Regional Clinical Oncology Dispensary to assess the results of combined treatment of 93 patients diagnosed with breast cancer with HER2-positive tumor growth subtype with organ-preserving operations. The age of the patients was from 31 to 62 years, the mean age was 47.119.78 years. Three (15.53%) patients were diagnosed with stage I of the disease according to the TNM system, and 90 (84.47%) with stage II. A search for statistically significant predictors of achieving morphological regression as a result of preoperative chemotherapy was carried out.
 Results. The mathematical model was created in the logistic regression module according to Wald's algorithm. Using SPSS 10.0, stepwise exclusion of predictors was performed. As a result of the multivariate analysis, a mathematical model and the corresponding program for the electronic computer Calculation of the achievement of complete morphological regression in patients diagnosed with primary operable breast cancer with epidermal growth factor receptors after neoadjuvant chemotherapy were created. In the future, the evaluation of the effectiveness of this program was compared with the results of 206 patients treatment.
 Conclusion. The creation of a mathematical model and a computer program made it possible to personalize the most effective treatment regimen for patients diagnosed with HER2 positive breast cancer, depending on the initial characteristics of the tumor.

A rapid rise in hormone receptor-positive and HER2-positive breast cancer subtypes in Southern Thai women: A population-based study in Songkhla.

The incidence of breast cancer is increasing in low- and middle-income countries, including Thailand. However, its molecular immunohistochemical (M-IHC) subtypes have not been summarized in a population-based cancer registry. Thus, we aimed to estimate the breast cancer incidence and trends based on the hormone receptor and human epidermal growth factor receptor 2 (HER2) status. This cross-sectional study included 2,883 women diagnosed with primary invasive breast cancer between 2009 and 2018 from the Songkhla Cancer Registry. After imputing the missing values of estrogen receptor (ER), progesterone receptor (PR), and HER2 status, the cases were classified into four subtypes: HR+/HER2-, HR+/HER2+, HR-/HER2-, and HR-/HER2+. The age-specific incidence rate of 5-year age groups and age-standardized incidence rate (ASR) were calculated. An age-period-cohort (APC) model was used to describe the effects of age, birth cohort, and period of diagnosis. Finally, the incidence trends were extrapolated to 2030 based on the APC and joinpoint models. The results showed, HR+/HER2- had the highest ASR in breast cancer. The incidence trends of HR+/HER2- and HR+/HER2+ increased with an annual percent change of 5.4% (95%CI: 2.5% to 8.3%) and 10.1% (95%CI: 4.9% to 15.5%), respectively. The rate ratio was high in the younger generation and recent period of diagnosis. The joinpoint and APC model projections showed that the ASR of HR+/HER2- would reach 30.0 and 29.2 cases per 100,000 women, while ASR of the HR+/HER2+ would reach 8.8 and 10.4 cases per 100,000 women in 2030. On the other hand, the incidence trends of the HR-/HER2- and HR-/HER2+ subtypes were stable. The rising trends of HR-positive and a part of HER2-positive breast cancer forecast a dynamicity of the future health care budgeting, resource allocation, and provision of facilities.

Is simultaneous multi-slice readout-segmented echo-planar imaging valuable for predicting molecular subtypes of breast cancer?

PurposeTo investigate the feasibility of simultaneous multi-slice readout-segmented diffusion-weighted echo-planar imaging (SMS rs-EPI DWI) in predicting invasive breast cancer molecular subtypes using whole-tumor histogram and texture analyses. MethodsIn our retrospective study, 125 patients (mean age, 52.81 ± 12.01 years; range, 24–84 years) with single invasive breast cancer who underwent preoperative MRI with SMS rs-EPI DWI and surgery at our institution were included. Two radiologists independently performed whole-tumor histogram and texture analyses on the apparentdiffusioncoefficient (ADC) map of SMS rs-EPI DWI (TR, 3800.0 ms; TE 90.0 ms; field of view, 340 mm × 206 mm; in-plane matrix, 244 × 148; section thickness, 5 mm; readout segments, 5, b = 0, 800 s/mm2; scan time, 3 mins; slice acceleration factor, 2). The Mann–Whitney U test or Kruskal-Wallis test were used to compare histogram and texture parameters to assess their potential in differentiating molecular subtypes of breast cancer and predicting axillary lymph node status. Receiver operating characteristic (ROC) curves were generated for parameters with significant differences, and the area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. ResultsCompared to luminal A breast cancer, the HER2-positive breast cancer subtype showed higher ADC_95th percentile values (p = 0.016). In addition, HER2-positive breast cancer yielded significantly higher ADC_mean (p = 0.025), ADC_median (p = 0.025), and ADC_5th percentile (p = 0.041) values than luminal B breast cancer. The ADC_skewness value was higher for triple-negative breast cancer (TNBC) than Luminal A breast cancer (p = 0.021). The difference in ADC_mean, ADC_median, ADC_5th percentile and ADC_95th percentile values between the HER2-positive breast cancer and luminal types was statistically significant, with a combined AUC for these parameters of 0.762 (sensitivity 72.41%, specificity 75.00%, PPV 27.27 %, and NPV 95.45 %). The ADC_skewness value for TNBC was higher than for luminal types (p = 0.012) and yielded an AUC value of 0.688 (sensitivity 96.15%, specificity 36.78%, PPV 31.25%, and NPV 96.97 %). Additionally, significantly higher ADC_skewness and ADC_95th percentile values (p = 0.003, 0.002, respectively) were reported for non-luminal types than luminal types yielding a combined AUC value of 0.712 (sensitivity 92.11%, and specificity 50.23%, PPV 41.18%, and NPV 92.50%). The AUC value for ADC_5th percentile for axillary lymph node status prediction was 0.659 (sensitivity 65.38%, specificity of 64.86%). ConclusionsThe whole-tumor histogram and texture analyses parameters based on SMS rs-EPI DWI may provide biological information on aggressive molecular subtypes of breast cancer. In addition, ADC_5th percentile values were significantly different between lymph node-positive and lymph node-negative breast cancer.

Selection of a new peptide homing SK-BR-3 breast cancer cells.

Breast cancer diagnosis remains a challenge, mostly due to its heterogeneity. This reality translates in delayed treatments, increasing treatment aggressiveness and lower chances of overall survival. The conventional detection techniques, although becoming increasingly sophisticated each year, still lack the ability to provide reliable conclusions without being time consuming, expensive, and uncomfortable for the patients. The identification of novel biomarkers for breast cancer research is therefore of utmost relevance for an early diagnosis. Moreover, breast cancer-specific peptide moieties can be used to develop novel targeted drug delivery systems. In this work, we used phage display to identify a novel peptide with specificity to the SK-BR-3 breast cancer cell line. Cytometry assays confirmed its specificity, while bioinformatics and docking studies predicted the potential biomarkers at the SK-BR-3 cells' surface. These findings can be potentially useful in the clinical context, contributing to more specific and targeted therapeutic solutions against HER2-positive breast cancer subtypes.

The histone H2B ubiquitin ligase RNF40 is required for HER2-driven mammary tumorigenesis

The HER2-positive breast cancer subtype (HER2+-BC) displays a particularly aggressive behavior. Anti-HER2 therapies have significantly improved the survival of patients with HER2+-BC. However, a large number of patients become refractory to current targeted therapies, necessitating the development of new treatment strategies. Epigenetic regulators are commonly misregulated in cancer and represent attractive molecular therapeutic targets. Monoubiquitination of histone 2B (H2Bub1) by the heterodimeric ubiquitin ligase complex RNF20/RNF40 has been described to have tumor suppressor functions and loss of H2Bub1 has been associated with cancer progression. In this study, we utilized human tumor samples, cell culture models, and a mammary carcinoma mouse model with tissue-specific Rnf40 deletion and identified an unexpected tumor-supportive role of RNF40 in HER2+-BC. We demonstrate that RNF40-driven H2B monoubiquitination is essential for transcriptional activation of RHO/ROCK/LIMK pathway components and proper actin-cytoskeleton dynamics through a trans-histone crosstalk with histone 3 lysine 4 trimethylation (H3K4me3). Collectively, this work demonstrates a previously unknown essential role of RNF40 in HER2+-BC, revealing the H2B monoubiquitination axis as a possible tumor context-dependent therapeutic target in breast cancer.

The LSD1 inhibitor iadademstat (ORY-1001) targets SOX2-driven breast cancer stem cells: a potential epigenetic therapy in luminal-B and HER2-positive breast cancer subtypes.

SOX2 is a core pluripotency-associated transcription factor causally related to cancer initiation, aggressiveness, and drug resistance by driving the self-renewal and seeding capacity of cancer stem cells (CSC). Here, we tested the ability of the clinically proven inhibitor of the lysine-specific demethylase 1 (LSD1/KDM1A) iadademstat (ORY-100) to target SOX2-driven CSC in breast cancer. Iadademstat blocked CSC-driven mammosphere formation in breast cancer cell lines that are dependent on SOX2 expression to maintain their CSC phenotype. Iadademstat prevented the activation of an LSD1-targeted stemness-specific SOX2 enhancer in CSC-enriched 3-dimensional spheroids. Using high-throughput transcriptional data available from the METABRIC dataset, high expression of SOX2 was significantly more common in luminal-B and HER2-enriched subtypes according to PAM50 classifier and in IntClust1 (high proliferating luminal-B) and IntClust 5 (luminal-B and HER2-amplified) according to integrative clustering. Iadademstat significantly reduced mammospheres formation by CSC-like cells from a multidrug-resistant luminal-B breast cancer patient-derived xenograft but not of those from a treatment-naïve luminal-A patient. Iadademstat reduced the expression of SOX2 in luminal-B but not in luminal-A mammospheres, likely indicating a selective targeting of SOX2-driven CSC. The therapeutic relevance of targeting SOX2-driven breast CSC suggests the potential clinical use of iadademstat as an epigenetic therapy in luminal-B and HER2-positive subtypes.

Immunotherapy in combination with chemotherapy in triple-negative breast cancer - the first “target” therapy for the “target” patients’ population

The only option of systemic therapy for triple-negative breast cancer (TNBC) was cytotoxic chemotherapy until recently, moreover the unitary standard of care for metastatic forms was not strictly defined. That is why paradigm of the treatment remains at the discretion of the doctor and it is empirical, consisting of precise combination of drugs. TNBC is infamous as the worst prognostic subtype. The absence of estrogen and progesterone receptors expression and HER2 amplification in this subtype of tumors made the usage of target therapy impossible, which is essential for successful treatment of luminal and HER2 positive breast cancer subtypes. The data about potential immunogenicity of tumor in TNBC launched the investigation of immunotherapy efficacy in this subtype of breast cancer. International clinical trial Impassion130 has not only created the basis in this field of TNBC treatment but also identified and determined the “benefiters”-cohort of patients concerning one of the most important goals - improving overall survival, that is so significant for patients with this subtype of breast cancer.

Abstract P5-17-03: How is inflammatory breast cancer (IBC) different? Integration of clinico-pathological features and circulating tumor cells (CTCs)-based biomarkers for disease and prognostic assessment

Abstract Background: Since IBC is rare and burdened by a particularly unfavorable prognosis, biomarkers able to enhance diagnosis and risk assessment are of pivotal importance and a current unmet need. The aim of this study is to integrate standard clinico-pathological features with CTCs-based biomarkers for a more objective and detailed characterization of IBC. Methods: This study analyzed retrospectively 251 Advanced Breast Cancer (BC) patients (pts) longitudinally characterized for CTCs and CTCs-based biomarkers at Thomas Jefferson University (Philadephia, PA) and Northwestern University (Chicago. IL). CTCs were enumerated through the CellSearch system (Menarini Silicon Biosystems), and characterized for HER2 expression using the CellSearch CXC Kit. Pts were defined as stage IV aggressive based on the previously reported ≥5 CTCs cut-off (Davis et al. 2018). Associations between clinical features, CTC-derived biomarkers and IBC were tested through uni and multivariate logistic regression. Survival was tested though log-rank test. Results: Within the analyzed cases, 46% were diagnosed with IBC and among them, 38% was stage IV aggressive. CTC clusters (CTC_CL) were detectable in 12.5% of pts and HER2 positive CTCs (HER2_CTC) in 29.5%. Notably, IBC patients (pts) had a significantly lower CTC count with respect to non-IBC (median 2.5 vs 0 respectively for non-IBC and IBC; P=0.019). BC subtype (HER2 positive BC: OR 2.97; Triple negative BC: OR 2.13), liver and bone involvement (liver: OR 0.46; bone involvement: OR 0.31) were the only significant clinico-pathological features associated with IBC at univariate logistic regression. Interestingly, a marginal significance was observed for soft tissue involvement (OR 1.65, 95%CI 0.95 - 2.87, P=0.07). Stage IV aggressive and presence of HER2_CTC at baseline were moreover inversely associated with IBC. The multivariate model confirmed the significant association between IBC and HER2 positive BC subtype (OR 2.64, 95%CI 1.08 - 6.48, P=0.034), absence of bone involvement (OR 0.31, 95%CI 0.14 - 0.68, P=0.003) and absence of HER2_CTC (OR 0.38, 95%CI 0.15 - 0.98, P=0.045). The baseline detection of CTC_CL was a strong predictor of prognosis for OS in IBC pts (median OS (mOS) 7.6 months (mts) vs not reached (NR) respectively for detectable vs non-detectable CTC_CL; P<0.0001), while a trend was observed for HER2_CTC (mOS 9.9 mts vs NR respectively for detectable vs non-detectable HER2_CTC; P<0.082). Pts negative for CTC_CL at baseline had higher odds of developing CTC_CL in later time-points if stage IV aggressive (OR 12.27, 95%CI 2.10 - 71.57, P=0.005). Despite no baseline factors were significantly associated with the onset of HER2_CTC in later time-points, a trend (P=0.05) was observed for patients without lymph node involvement (OR: 5) and with bone involvement (OR: 4.3). Conclusion: HER2_CTC and in particular CTC_CL are promising prognostic predictors in IBC. Stage IV aggressive IBC pts could benefit from a longitudinal CTCs assessment, being more prone to develop CTC_CL and therefore at higher risk of rapid disease progression. Probably due to the tropism for soft tissue, IBC is characterized by a lower number of HER2_CTC. Citation Format: Gerratana L, Zhang Q, Wang C, Shah A, Davis AA, Ye Z, Zhang Y, Abu-Khalaf M, Flaum L, Strickland K, Rossi G, Behdad A, Gradishar W, Platanias L, Yang H, Cristofanilli M. How is inflammatory breast cancer (IBC) different? Integration of clinico-pathological features and circulating tumor cells (CTCs)-based biomarkers for disease and prognostic assessment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-03.

Is There a Benefit of HER2-Positive Breast Cancer Subtype Determination in Clinical Practice?

Background Breast cancer (BC) with increased expression of human epidermal growth factor receptor 2 with tyrosine kinase activity (HER2+) is a clinically and bio-logically heterogeneous dis-ease. In terms of gene expression, there are four major molecular subtypes - Luminal A, Luminal B, HER2-enriched (HER2-E), and Basal-like. The most common subtype is HER2-E (50- 60%). In hormone-dependent (HR+) HER2-positive tumors, the subgroup HER2-E represents 40- 50% of cases; others are luminal A and B subtypes. Purpose The aim of this review is to provide information on the significance of the distribution of HER2-positive tumors accord-ing to subtype, which is considered a predictive parameter for guid-ing treatment decisions. For example, HER2-E subtype is characterized by a higher probability of achiev-ing complete pathological remission when treated with chemother-apy and antiHER2 ther-apy, and it is thought that it could be treated us-ing a dual HER2 blockade without chemother-apy. Currently, triple-positive tumors, a specific subtype of breast cancer characterized by HER2+ and HR+, are more often subjects of interest. Their unique bio-logical properties are due to complex interactions between HER2 and estrogen receptor (ER) signalling, which result in lower effectiveness of endocrine ther-apy in these patients than in HR+ and HER2-negative patients and, at the same time, the ER positivity in HER2+ tumors can result in resistance to antiHER2 ther-apy. This type of BC is a non-homogeneous group where the impacts of HER2 positivity on tumor malignant behavior and activity of the estrogen-driven signal-ing pathway are inconsistent. Current studies focus on test-ing new treatments such as dual HER2 block-ing or immunother-apy, in combination with antiHER2 targeted ther-apy with fulvestrant, aromatase inhibitors, cyclin dependent kinase 4/ 6 inhibitors, or inhibitors of the PI3K (phosphatidylinositol-3-kinase) pathway. Conclusion The distribution of HER2+ BC accord-ing to individual subtype provides information that can contribute to achiev-ing more accurate decisions about the most appropriate ther-apy. Key words breast cancer - subtype - HER2 - trastuzumab - HER2 positive - triple positive - HER2 enriched The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 27. 9. 2018 Accepted: 26. 11. 2018.

The Burden of HER-2 Positive Patients in Sub-Saharan Africa: A Caregiver Perspective

Background and context: Several studies showed that the use of the recombinant monoclonal antibody trastuzumab, in addition to adjuvant chemotherapy, for breast cancer with overexpression of the HER-2 protein or amplification of the HER-2/neu gene reduced the risk of recurrence by 50% in women irrespective of nodal status. In Nigeria, breast cancer is the leading and the most common cancer in women. A trastuzumab brand known as Herceptin is the popular brand usually prescribed by the oncologist in Nigeria. Once a histopathology/immune-chemistry result shows HER-2 positive and oncologist informs that patient of the diagnosis being HER-2 positive; most patients emotionally breaks down due to cost of the drugs. In Nigeria, as at 2015 the cost of herceptin 455mg was N650,000 ($1857). As at June 2016 that same drug increased in cost to N1,100,000 ($3142) due to the unstable exchange rate in Nigeria. In Nigeria 90% of patients pay-out of pocket for treatment and drugs as the health insurance system in my country does not cover chemotherapy drugs. Several cancer patients with HER-2 patients face serious financial challenges in Nigeria, as so many experience spread of the cancer to vital organ when they are unable to get finance to purchase the life-saving drug for their HER-2 positive breast cancer subtype. Aim: To understand the burden of HER-2 diagnosis in Nigeria and stimulate advocacy for the reduction of the cost of Herceptin in Nigeria and other sub-Saharan Africa where finance is a serious impediment to accessing medical care. Strategy/Tactics: The study will present case studies of three Nigerian women who were diagnosed of HER-2. Interview was used as a method of data collection, informed consent were also completed by the patients before the interview. Program/Policy process: Our study reveals that there is a need to engage the federal and state governments, corporate organizations, insurance schemes and most importantly pharmaceutical companies to reduce cost of cancer drugs and particularly Herceptin. HER-2 breast cancer patients need to lead the advocacy for reduction in cost of drug at all levels. Outcomes: Our qualitative findings reveal that caregivers are mostly affected by the cost of drugs such as Herceptin. In some cases caregivers, sell valuables, take loan and go public to raise funds for the cancer drugs. What was learned: Most HER-2 positive breast cancer patient could not afford Herceptin and some waited for several months to raise funds, which is probably a factor in metastasis and low-survival.

Abstract P3-10-09: The association age-related parity and HER2 over-expression in breast cancer

Abstract Backgrounds Reproductive factors, such as age at menarche, parity, age at first birth, and breast feeding, are known to be associated with risk of breast cancer. Recent studies show the association between the reproductive factors and the molecular subtypes of breast cancer, especially in luminal-like subtypes. Nulliparity is known a risk factor of breast cancer, multiparity is considered a protective factor. However, pregnancy associated breast cancers show the high expression of HER2, Therefore, we hypothesized the number of birth (multiparity) is correlated with the over-expression of HER2. Methods We reviewed the database of 2,594 breast cancer patients were over 20 years of age, diagnosed between January 2000 and April 2017 at Korea University of Anam Hospital, Seoul, Korea. According the number of birth, nulliparous (the number of birth = 0; 271 patients), primiparous (the number of birth = 1; 420 patients) and multiparous (the number of birth≥2; 1,903 patients) groups were classified. Results The median age (range) of nulliparous, primiparous and multiparous groups was 43 (22-77), 48 (27-87) and 51 (26-86) years (P<0.001). In univariate analysis, the number of birth was associated with the expression of ER and HER2, nulliparous patients showed higher ER positivity (P=0.013) and multiparous patients showed higher HER2 overexpression (P=0.048). There was no association with PR and Ki-67 level. In logistic regression with age, the parity was negative correlation with ER positivity (OR 0.83, 95% CI 0.72-0.95, P=0.010) and positive correlation with HER2 overexpression (OR1.18, 95% CI 1.02-1.36, P=0.021) P=0.009) and multiparity was higher HER2 overexpression (OR 1.41, 95% CI 1.02-1.94, P=0.033). By subgroup analysis with menopausal status, there was statistically significance in only premenopausal women, not in postmenopausal women. In premenopausal women, the parity was negatively associated with ER positivity (OR 0.78, 95%CI 0.64-0.94, P=0.010) and multiparity was associated with HER2 overexpression (OR 1.72, 95%CI 1.12-2.63, P=0.013), there was no difference in HER2 overexpression between nulliparous and primiparous patients. Conclusion Parity was associated with ER and HER2 expression, multiparous patients were associated with ER negativity and HER2 overexpression, especially in premenopausal women. This suggests that pregnancy could affect risk of breast cancer, especially in HER2 positive breast cancer subtype (ER- HER2+ tumors), before menopause. We need further investigation and evaluation. Citation Format: Bae SY, Kim J, You JY, Jung SP, Bae JW. The association age-related parity and HER2 over-expression in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-10-09.

Challenging a Misnomer? The Role of Inflammatory Pathways in Inflammatory Breast Cancer.

Inflammatory breast cancer is a rare, yet highly aggressive form of breast cancer, which accounts for less than 5% of all locally advanced presentations. The clinical presentation of inflammatory breast cancer often differs significantly from that of noninflammatory breast cancer; however, immunohistochemistry reveals few, if any, distinguishing features. The more aggressive triple-negative and HER2-positive breast cancer subtypes are overrepresented in inflammatory breast cancer compared with noninflammatory breast cancer, with a poorer prognosis in response to conventional therapies. Despite its name, there remains some controversy regarding the role of inflammation in inflammatory breast cancer. This review summarises the current molecular evidence suggesting that inflammatory signaling pathways are upregulated in this disease, including NF-κB activation and excessive IL-6 production among others, which may provide an avenue for novel therapeutics. The role of the tumor microenvironment, through tumor-associated macrophages, infiltrating lymphocytes, and cancer stem cells is also discussed, suggesting that these tumor extrinsic factors may help account for the differences in behavior between inflammatory breast cancer and noninflammatory breast cancer. While there are various novel treatment strategies already underway in clinical trials, the need for further development of preclinical models of this rare but aggressive disease is paramount.

Tumour-infiltrating lymphocytes and the emerging role of immunotherapy in breast cancer

Breast cancer has not previously been considered a highly immunogenic cancer. Observations of tumour-infiltrating lymphocytes (TILs) in and around neoplastic cells in tumour samples, and associations with improved pathological complete response and clinical survival end points have changed our perspective on this. Lymphocytic infiltrates have long been observed in breast cancer; however, more recently, retrospective analysis of prospectively collected tissue samples from clinical trials has demonstrated the potential role of host immunosurveillance in influencing the biology of breast cancer. This association appears to be strongest in triple negative and HER2 positive breast cancer subtypes. Contrastingly, the association in luminal tumours is less clear, and is potentially limited by substantial tumoural heterogeneity. Several methodologies have been employed to quantify, and describe the composition of TILs, each with its own advantages and disadvantages. The results of these analyses have been generally consistent, and valuable efforts are currently underway to standardise the evaluation of TILs toward a universal approach. More technical methods of TIL characterisation remain important in the research setting. The evaluation of TILs becomes increasingly relevant with the emerging role of immunotherapy in breast cancer. Early phase trials of checkpoint blockade show promising results; however, it is likely that some patients will require combination treatments to maximise therapeutic benefits. Equally, some patients may not derive any benefit from immunotherapies. This underscores the importance of the development of relevant predictive biomarkers. As a key representative of the immune interaction between host and tumour, lymphocytic infiltrates are ideally placed for continued research into the determinants of immunogenicity, and response to immunotherapeutic approaches. In this review, we will discuss the current methodologies of evaluation, and the clinical relevance of TILs. Additionally, we discuss the emerging role of immunotherapy in breast cancer, and the future of TIL characterisation in this context.