HER2-enriched Breast Cancer Research Articles

113 Poster - PD-L1 and HSP-70 molecules are part of immunosupressive environment in the deep layer of the lymphocyte predominant breast cancer (LPBC)

Background: Tumor infiltrating lymphocytes (TILs) are involved in host imunity against tumor cells. However, in later phases of the disease high TIL infiltration is related to disease progression. Tumor immunogenicity is strongly correlated with the higher tumor mutation burden. Triple negative (TN) and HER-2 enriched breast cancers have the highest immunogenic potential so the aim of our study was to investigate the TIL infiltration and expression of PD-L1, HSP-70 in such tumors.

108 Poster - Improvement of recurrent rates and survival in patients with primary breast cancer according to subtypes

Background: Previously we reported that the survival rate of patients with the HER2 enriched breast cancer subtype who experienced recurrence after 2001 had improved with the availability of trastuzumab in Japan. Moreover, there is an increase in the variability of therapeutic agents for luminal or triple negative (TN) subtypes. The aim of this study is to demonstrate that there was a change in the recurrence rate and survival time of patients who received these new therapeutic agents after 2001, and to show the efficacy of these agents according to the different breast cancer subtypes.

Clinicopathological characteristics and prognosis of breast cancer with special histological types: A surveillance, epidemiology, and end results database analysis

ObjectivesTo explore the clinicopathological features and prognosis of breast cancer with special histological types. Materials and methodsThe information of breast cancer patients was obtained from the Surveillance, Epidemiology, and End Results (SEER) database (2010–2016). Comparative analyses were performed to explore the difference in clinicopathological characteristics and propensity score matching (PSM) was used to weaken the effects from clinical profiles. Survival analysis was conducted to investigate the prognostic effects from histological types, and the prognostic factors of this group of patients were identified with the univariate COX proportional model. ResultsA total of 242863 breast cancer patients were eligible, of which 230213 individuals were ductal breast cancer (IDC) and 12650 individuals were special breast lesions, respectively. Comparatively, special breast cancer had a lower histological grade, a smaller tumor size, a lower proportion of nodal involvement and distant metastasis, in addition to a higher proportion of triple-negative subtype. The overall prognosis of special histological breast cancer was comparable to IDC, while the survival of HER2 enriched breast cancer was in favor of special breast cancer. With the PSM performance, the prognosis exhibited an inferior profile in the metaplastic breast cancer and was significantly favorable to apocrine, medullary, micropapillary, and papillary breast cancer. ConclusionThis study revealed that the special histological breast cancer presented distinct clinicopathological characteristics and great heterogeneity in the prognosis among diverse histological subtypes.

Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer

Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.

Impact of the molecular pathology of breast cancer on mortality rates and overall survival in a Haitian cancer clinic.

e13588 Background: Breast cancer in the most common malignancy among women in Haiti and is mostly diagnosed at an advanced stage. While it is well known that molecular subtype is a prognostic factor, it needs to be investigated among Haitian patients with breast cancer. This study aimed to evaluate the impact of molecular classification of breast cancer on the survival of patients managed in Haiti’s largest cancer clinic. Methods: A retrospective study was conducted on the breast cancer patients of Innovating Health International (IHI) in Port-au-Prince, Haiti from January 2014 to December 2018. Chart review included all patients with breast cancer and tested for molecular classification. Data on variables such as date of admission, age, TNM staging, molecular classification, outcome and date of death were collected for the analysis. Mortality rate and median overall survival (OS) were estimated as of December 31st, 2019 and stratified according to molecular subtypes. Results: Among the 948 breast cancer cases diagnosed for the study period, 234 (24.7%) of them had a complete molecular classification. The mean age was 51.5 years [range: 23-94]. 55.1% of the patients were ER-positive, among them 33.7% ER+/PR+/HER2-, 15.4% ER+/PR-/HER2-, 2.1% ER+/PR-/HER2+ and 3.8% ER+/PR+/HER+ (triple positive). There were overall 25.6% of luminal A and 29.5% of luminal B cases. 44.9% were ER-negative, among them 14.1% ER-/PR-/HER2+ (HER2-enriched) and 29.1% ER-/PR-/HER2- (triple-negative). 92.2% of the patients had advanced breast cancer (stages IIB to IV). 29.5% had metastatic breast cancer, 22.8% for luminal A cases, 27.0% for luminal B, 36.7% for HER2-enriched and 32.8% for TNBC. Overall mortality rate was 42.3%, respectively 33.3% for luminal A cases, 37.7% for luminal B, 42.4% for HER2-enriched cases and 55.9% for TNBC. Median OS was not yet reached for luminal A, luminal B and HER2-enriched breast cancer, with a respective mean survival of 52.4 months, 51.3 months and 51.6 months. However, OS was 30.6 months for triple-positive breast cancer and 23.7 months for TNBC. Conclusions: Patients with luminal A breast cancer were less likely to have metastatic disease and thus had lower mortality rate and better overall survival. This was likely due to its less aggressive biology and the availability of hormone therapy. Poor availability and inaccessibility of HER2-targeted drugs were the main cause of the higher mortality rate among HER2-enriched patients. TNBC remains the most aggressive subtype.

The prognostic value of androgen receptor (AR) in HER2-enriched metastatic breast cancer.

The significance of androgen receptor (AR) in metastatic breast cancer (MBC) remains unclear, and it is still largely unknown how AR expression level influences HER2-positive tumors. This study aimed to investigate the prognostic and predictive value of AR in HER2-enriched MBC. Primary and/or paired metastatic tumors of 304 patients with pathologically confirmed HER2-enriched MBC were collected and immunohistochemically assessed for AR expression. The associations of AR and other clinicopathological characteristics were compared using the Chi-square test. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method and log-rank test. Cox regression analysis was used to determine independent prognostic factors. AR-positivity with a cut-off value of 10% was observed in 237 (78.0%) cases and was associated with longer PFS, 13.2 months, as compared to that of 8.2 months (P = 0.004) in patients with AR-negativity. Moreover, a significant increase in the 5-year OS rate (65.3% vs 36.2%, P < 0.001) was also observed for patients with AR-positive tumors. Cox regression analysis identified AR-positivity as an independent prognostic factor of both PFS (hazard ratio = 0.71, P = 0.039) and OS (HR = 0.53, P = 0.013). Additionally, for those who received first-line Trastuzumab therapies, prolonged PFS (15.8 months vs 8.2 months, P = 0.005) and 5-year OS rate (66.2% vs 26.2%, P = 0.009) were observed in AR-positive tumors compared to AR-negative ones. In conclusion, AR was identified as an independent prognostic factor for favorable PFS and OS and could also predict the efficacy of first-line Trastuzumab treatment in patients with HER2-enriched MBC.

Specific chromatin landscapes and transcription factors couple breast cancer subtype with metastatic relapse to lung or brain

BackgroundFew somatic mutations have been linked to breast cancer metastasis, whereas transcriptomic differences among primary tumors correlate with incidence of metastasis, especially to the lungs and brain. However, the epigenomic alterations and transcription factors (TFs) which underlie these alterations remain unclear.MethodsTo identify these, we performed RNA-seq, Chromatin Immunoprecipitation and sequencing (ChIP-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) of the MDA-MB-231 cell line and its brain (BrM2) and lung (LM2) metastatic sub-populations. We incorporated ATAC-seq data from TCGA to assess metastatic open chromatin signatures, and gene expression data from human metastatic datasets to nominate transcription factor biomarkers.ResultsOur integrated epigenomic analyses found that lung and brain metastatic cells exhibit both shared and distinctive signatures of active chromatin. Notably, metastatic sub-populations exhibit increased activation of both promoters and enhancers. We also integrated these data with chromosome conformation capture coupled with ChIP-seq (HiChIP) derived enhancer-promoter interactions to predict enhancer-controlled pathway alterations. We found that enhancer changes are associated with endothelial cell migration in LM2, and negative regulation of epithelial cell proliferation in BrM2. Promoter changes are associated with vasculature development in LM2 and homophilic cell adhesion in BrM2. Using ATAC-seq, we identified a metastasis open-chromatin signature that is elevated in basal-like and HER2-enriched breast cancer subtypes and associates with worse prognosis in human samples. We further uncovered TFs associated with the open chromatin landscapes of metastatic cells and whose expression correlates with risk for metastasis. While some of these TFs are associated with primary breast tumor subtypes, others more specifically correlate with lung or brain metastasis.ConclusionsWe identify distinctive epigenomic properties of breast cancer cells that metastasize to the lung and brain. We also demonstrate that signatures of active chromatin sites are partially linked to human breast cancer subtypes with poor prognosis, and that specific TFs can independently distinguish lung and brain relapse.

The Prosigna 50-gene profile and responsiveness to adjuvant anthracycline-based chemotherapy in high-risk breast cancer patients

The DBCG89D trial randomized high-risk early breast cancer patients to adjuvant CMF (cyclophosphamide, methotrexate and fluorouracil) or CEF (cyclophosphamide, epirubicin and fluorouracil). Prosigna assays were performed by researchers with no access to clinical data. Time to distant recurrence (DR) was the primary endpoint, time to recurrence (TR) and overall survival (OS) secondary. Among the 980 Danish patients enrolled, Prosigna results were obtained in 686. Continuous ROR score was associated with DR for CMF (adjusted hazard ratio (HR) 1.20, 95% CI 1.09–1.33), and for CEF (HR 1.04, 95% CI 0.92–1.18), Pinteraction = 0.06. DR was significantly longer in CEF compared to CMF treated patients with Her2-enriched tumors (HR 0.58, 95% CI 0.38–0.86), but not in patients with luminal tumors. Heterogeneity of treatment effect was significant for TR and OS. In this prospective-retrospective analysis, patients with Her2-enriched breast cancer derived substantial benefit from anthracycline chemotherapy whereas anthracyclines are not an essential component of chemotherapy for patients with luminal subtypes. The benefit of CEF vs. CMF correlated with increasing ROR Score.

Abstract P6-15-05: Demographics and survival in metastatic inflammatory breast cancer. SEER data analysis

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive malignancy, distinguished by characteristic skin changes with/without breast mass. While new therapies have improved the survival of IBC, the prognosis of metastatic disease (stage IV) remains poorly described. The current analysis is aimed at investigating the demographics of metastatic IBC and the impact of chemotherapy use and tumor subtype on survival. Methods: Using ICD-O-3 histology code 8530/3 in the Surveillance Epidemiology and End Results database (SEER), demographics and survival data on metastatic IBC diagnosed between 2010 and 2015 were abstracted. JMP 14 statistical software was used for data analysis. The SEER database does not record specific age above 85 years and these cases were excluded. Results: Over five years, 378 patients were diagnosed with metastatic IBC. All were women and most were Caucasian (282, 75%). Mean age was 60±14 years. During the observation period 284 (75%) patients died and of which 218 deaths were attributable to IBC. Cancer subtype was unknown in 61 patients; for the rest, Luminal A was the most common (121, 38%), followed by triple negative (74, 23%), HER2 enriched (62, 20%) and luminal B (60, 19%). Median overall survival (OS) was 18 months (95% CI 14-20), with a survival rate of 39% at 24 months from diagnosis. In unadjusted analyses, patients receiving chemotherapy (n=191) had increased survival at every time point compared to those who did not (n=93), (median OS 22 (95% CI 18-26) vs 5 months (95% CI 2-11), p&amp;lt;0.0001; 24 month survival rate 46% vs 22%). Stratification by cancer subtype showed worst median OS for triple negative breast cancer (11 months, 95% CI 6-13), similar survival for HER2 enriched and luminal A (20 [95% CI 17-29] and 22 [95% CI 17-34] months, respectively) and best OS for luminal B (42 months [95% CI 26-61]). These differences were statistically significant (log rank p&amp;lt;0.0001). A proportional hazards model including age, ethnicity, tumor subtype and treatment identified Caucasian race (HR 0.72 [95% CI 0.53-0.98], p=0.03) and use of chemotherapy (HR 0.49 [95% CI 0.34-0.71], p=0.0002) as predictors of improved survival. Patients with tumor subtype Luminal B had the best OS; by comparison, Luminal A (HR 1.43 [95% CI 0.94-2.22], p=0.09), HER2 enriched (HR 1.7 [95% CI 1.06-2.7], p=0.02) and triple negative breast cancer (HR 3.7 [95% CI 2.4-5.8], p&amp;lt;0.0001) had excess mortality. Interestingly, increasing age at diagnosis was not significantly associated with a poor prognosis (HR 1.007 [95% CI 99.7-1.01], p=0.18). Conclusion: From our data, IBC is common in Caucasian women in their 7th decade of life. Luminal B was the least common cancer subtype and had the best overall survival. Chemotherapy appears to play an important part in increasing survival; but ethnicity and tumor subtype are also important predictors of mortality. Keywords: Inflammatory breast cancer, Metastatic, Survival, Demographics Citation Format: Nwabundo Anusim, Filip Ionescu, John Khoury, Anish Konde, Ishmael Jaiyesimi. Demographics and survival in metastatic inflammatory breast cancer. SEER data analysis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-15-05.

Abstract P1-10-13: Examination of CCL26, CCL17 and CCL19 chemokines as biomarkers in HER2+ breast cancer (BC) in the neo-adjuvant setting

Abstract Background: Increased tumour infiltrating lymphocytes (TILs) are associated with a better prognosis in HER2+ BC patients treated with neo-adjuvant chemotherapy. The signalling mechanisms associated with increased TIL levels are not fully understood. Chemotactic cytokines (chemokines) and their respective receptors have a major role to play in tumour immune cell infiltrate. Analysis of plasma chemokine levels and TIL levels in HER2+ BC patients treated in the neo-adjuvant setting has identified three chemokines of interest - CCL26, CCL17 and CCL19. Examination of tumor mRNA expression levels of their corresponding receptors CCR3, CCR4, and CCR7 in publicly available datasets reveals a significant association with overall survival in PAM50-defined HER2-enriched BC patients. Methods: Pre-treatment (n=43) and post-treatment (n=29) (2 weeks pre-surgery) blood samples were collected from patients enrolled in ICORG 10-05 (neo-adjuvant chemotherapy (docetaxel/carboplatin) +/- trastuzumab, lapatinib or trastuzumab/lapatinib). Patients were classified as having a pathological complete response (CR) or a non-CR (nCR). Plasma chemokine levels were determined by Luminex xMAP assay and validated by ELISA. TIL levels were determined from H and E-, AE1/AE3- and CD45- stained FFPE tissue. Chemokine receptor mRNA expression was interrogated in publicly available datasets using BreastMark (http://glados.ucd.ie/BreastMark/index.html). The PAM50 HER2-enriched molecular signature and a median cut-off was used for all analyses. Results: Circulating CCL17 levels were significantly lower in patients achieving CR, pre- (p=0.015) and post-treatment (p=0.012). Baseline CCL17 levels correlated with baseline TIL count (r=0.582, p=0.011) for CR but not nCR. There was no association between pre- or post-treatment CCL19 and CCL26 plasma levels and treatment response. However, baseline CCL26 levels were inversely correlated with baseline TILs for patients achieving CR (r= -0.49, p=0.028) but not nCR. Baseline CCL19 displayed a similar trend that did not reach significance (r=0.414, p=0.077). Analysis of publicly available datasets reveals tumor mRNA expression of CCR3 (Hazard ratio (HR)=1.9, p=0.001) and CCR7 (HR=0.53, p=0.002) are associated with overall survival in patients with a HER2-enriched molecular signature. Conclusions: Our results suggest circulating chemokine levels may have value as biomarkers of response and TIL status in HER2+ BC. The strong correlation of chemokine receptors with overall survival in tumors with a HER2-enriched molecular signature suggests further examination of chemokine/chemokine receptor axes is warranted in a larger cohort of HER2+ BC patients. Citation Format: Denis Martin Collins, Alacoque Browne, Stephen F Madden, Nicola Gaynor, Elaine W Kay, Joanna Fay, Katherine Sheehan, Sinead Toomey, Alex J Eustace, William M Gallagher, Bryan T Hennessy, John Crown. Examination of CCL26, CCL17 and CCL19 chemokines as biomarkers in HER2+ breast cancer (BC) in the neo-adjuvant setting [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-13.

Case of HER2-Enriched Multifocal Breast Cancer Associated with Pregnancy

Pregnancy-associated breast cancer is less common and often presents in advanced stage. Management is similar to non-pregnant cases with trimester-specific considerations. In this case report, we present a case of worrisome mother with locally advanced multifocal pregnancy-associated breast cancer who was so much concerned about effects of neoadjuvant chemotherapy on fetus leading to its delayed initiation, and show why targeted therapy is still not within reach in a developing economy like Nepal. Short term data of chemotherapy though reveal relative safety, long term data are lacking. This may be a hindrance in counselling, when patients are more anxious and worrisome.

Case of HER2-Enriched Multifocal Breast Cancer Associated with Pregnancy

Pregnancy-associated breast cancer is less common and often presents in advanced stage. Management is similar to non-pregnant cases with trimester-specific considerations. In this case report, we present a case of worrisome mother with locally advanced multifocal pregnancy-associated breast cancer who was so much concerned about effects of neoadjuvant chemotherapy on fetus leading to its delayed initiation, and show why targeted therapy is still not within reach in a developing economy like Nepal. Short term data of chemotherapy though reveal relative safety, long term data are lacking. This may be a hindrance in counselling, when patients are more anxious and worrisome.

Expression profile of Toll‑like receptors in human breast cancer.

Toll-like receptors (TLRs) are the most widely studied pattern recognition receptors. Mounting evidence suggests an important association between TLRs and the occurrence and development of breast cancer. Thus, targeting these receptors may be a potential strategy for breast cancer treatment. The current study analyzed the data of 1,215 patients with breast cancer obtained from The Cancer Genome Atlas (TCGA) database. It was observed that, in addition to TLR6, TLR7 and TLR8, the expression of the remaining TLRs in breast cancer tissues was lower than that in normal tissues. In addition, TLR3 and TLR9 displayed significantly different expression levels in ER-/PR-negative breast cancer compared with the control tissues, while TLR5 expression was significantly reduced in HER2-enriched breast cancer. Furthermore, TLR10 exhibited lower expression levels in advanced stages of the disease as compared with that observed in earlier stages. Survival analysis revealed that the expression of TLR4 and TLR7 had a significant impact on survival, and higher expression levels suggested worse prognosis. Finally, the expression levels of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 were correlated with those of the inflammatory cytokines interleukin-1β and tumor necrosis factor-α, while the expression levels of TLR3, TLR7, TLR8 and TLR9 were correlated with those of interferon-β and C-X-C motif chemokine ligand 10. Taken together, the current study results suggest that TLR expression may serve as a biomarker of cancer pathogenesis and progression, and may provide new insights for the treatment of breast cancer through the regulation and targeting of TLRs.

Paget's disease of the nipple in a Her2-positive breast cancer xenograft model.

Paget's disease (PD) of the breast is an uncommon disease of the nipple usually accompanied by an underlying carcinoma, often HER2 + , and accounting for 0.5-5% of all breast cancer. To date, histogenesis of PD of the breast remains controversial, as two theories-transformation and epidermotropic-have been proposed to explain this disease. Currently, animal models recapitulating PD of the nipple have not been described. HER2-enriched DT13 breast cancer cells were injected into the mammary fat pad of NOD scid gamma null (NSG) female mice. Immunohistochemical staining and pathological studies were performed on tumor samples, and diagnosis of PD of the nipple was confirmed by expression of proteins characteristic of Paget cells (epidermal growth factor 2 (HER2), androgen receptor (AR), cytokeratin 7 (CK7), cytokeratin 8/18 (CK8/18), and mucin 1 (MUC1)). In addition, DT13 cells grown in 2D culture and in soft agar assays were sensitive to in vitro treatment with pharmacological inhibitors targeting Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. Mice developed tumors and nipple lesions that were detected exclusively on the tumor-bearing mammary fat pad. Tumor cells were positive for proteins characteristic of Paget cells. In vitro, DT13 cells were sensitive to inhibition of Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. Our results suggest that injection of HER2 + DT13 cells into the mammary fat pad of NSG mice recapitulates critical aspects of the pathophysiology of PD of the nipple, supporting the epidermotropic theory as the more likely to explain the histogenesis of this disease.

JS1.1 Specific genetic alterations of breast tumors lead to Yo paraneoplastic cerebellar syndromes

Abstract BACKGROUND Paraneoplastic cerebellar degenerations with anti-Yo antibodies (Yo-PCD) are rare syndromes associated with ovarian or breast cancers and caused by an auto-immune response against neuronal antigens expressed by tumor cells. We previously demonstrated in Yo-PCD ovarian cancers an association between massive infiltration of ovarian tumors by activated immune effector cells and recurrent gains and/or mutations of onconeural Yo genes (CDR2L and CDR2), suggesting that such genetic alterations in ovarian tumor cells may trigger immune tolerance breakdown and initiation of the auto-immune reaction against Purkinje cells. MATERIAL AND METHODS We pursued the characterization of Yo-PCD tumors and specifically studied breast cancer by IHC, FISH, CGH array and RNA sequencing analysis of 17 breast Yo-PCD tumors and by comparing their genetic characteristics with 10 sporadic breast tumors and public databases. RESULTS We confirmed that specific genetic alterations were also present in breast cancers associated with Yo-PCD. Moreover, this study provides additional evidence for a role of tumor cell specificities in PCD immunopathology by revealing peculiarities in Yo-PCD breast tumors compared to Yo-PCD ovarian cancer. Indeed, not only the CDR2L Yo gene was amplified in 8/9 breast Yo-PCD cancers but also the Erb2/Her2 gene in 15/16 (both genes are on chromosome 17q). In addition to this original Her2 and Yo antigen amplification confirmed by FISH, we observed an overexpression of these proteins by IHC analysis. These Yo-PCD breast cancers are also all negative for hormone receptors (HR). Thus, Yo-PCD breast tumors seem to belong to the molecularly and clinically distinct class of HR-negative and Her2-enriched breast cancers, which represents less than 10 % of breast cancers in the general population. Transcriptomic analysis confirmed that breast Yo-PCD tumors differ by their expression programs from classical breast cancers molecular subtypes. CONCLUSION Understanding the tumor genetic features leading to the immune breakdown and anti-tumor immune response as well as nervous tissue attack remains challenging and seems to be specific according to the tumor subtypes. Herein, our results suggest that, despite sharing common genetic alterations (copy number variations and mutations affecting Yo genes), the Yo-PCD immunopathogenesis of breast and ovarian cancers differ by involving different tumor-specific molecular pathways.

TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment.

Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and metastasis-initiating cells, we examined TGLI1 for its involvement in breast CSCs and found TGLI1 to transcriptionally activate stemness genes CD44, Nanog, Sox2, and OCT4 leading to CSC renewal, and TGLI1 outcompetes with GLI1 for binding to target promoters. We next examined whether astrocyte-priming underlies TGLI1-mediated brain tropism and found that TGLI1-positive CSCs strongly activated and interacted with astrocytes in vitro and in vivo. These findings demonstrate, for the first time, that TGLI1 mediates breast cancer metastasis to the brain, in part, through promoting metastasis-initiating CSCs and activating astrocytes in BCBM microenvironment.

Abstract 3689: Tumor-specific gain-of-function tGLI1 transcription factor is a novel mediator of breast cancer stem cells and a novel transcriptional activator of cancer stemness genes

Abstract Breast cancer is the second leading cause of cancer-related mortality in women; metastasis to distant organs results in 90% of deaths for these patients. Cancer stem cells (CSCs) are considered the drivers of metastasis. Despite our current knowledge of breast CSCs, there still remains a significant challenge in managing patients with the metastatic breast cancer, underscoring the need for identifying novel regulators of breast CSCs. The hedgehog pathway is an important mediator of stem cells; however, the effect of truncated glioma-associated oncogene homolog 1 (tGLI1), a nuclear effector of the hedgehog pathway and a gain-of-function GLI1 transcription factor, on breast CSCs has never been investigated. Herein, we investigated whether tGLI1 is implicated in breast CSCs by evaluating tGLI1 expression levels in cells grown as monolayer versus mammospheres, as a representation of the stem cell population, and found tGLI1 to be induced in mammosphere culture. Overexpression of tGLI1 promoted mammosphere-forming ability of breast cancer cells, as well as, increased the breast CSC population defined by CD44high/CD24low expression. Further, tGLI1 overexpression transformed normal mammary epithelial cells resulting in increased mammosphere formation and enhanced anchorage-independent growth of immortalized human mammary epithelial HMLE cells. Functional and biochemical assays further showed that tGLI1 promotes breast CSC self-renewal by transcriptional activation of stemness genes including a novel tGLI1 target gene, OCT4, a recently reported tGLI1 target gene (CD44), and known GLI1 target genes (Nanog and SOX2). Bioinformatic analysis of breast cancer patient datasets revealed that activated tGLI1 is associated with shortened time to develop metastasis to the lung, bone, and brain. Furthermore, tGLI1 activation is enriched in HER2-enriched and triple-negative breast cancers, the subtypes with the highest propensity to metastasize, compared to luminal subtypes. Gene Set Enrichment Analysis showed that high tGLI1 activation is enriched in breast cancer with high gene signatures of breast CSCs, radioresistance, and metastasis. We further validated these results by immunohistochemical staining of paired primary breast tumors with lymph node metastases and found that that expression of tGLI1, but not GLI1, was increased in lymph node metastases and that tGLI1 was expressed at higher levels (84-91%) of lymph node-positive metastatic HER2-enriched and triple-negative breast tumors. Lastly, tGLI1 knockdown resulted in decreased mammosphere formation of breast cancer cells and decreased expression of stemness genes, OCT4, CD44, and Nanog. Taken together, these findings establish a novel role that tGLI1 plays in mediating breast CSCs and implicate tGLI1 in facilitating breast cancer metastasis. Citation Format: Sherona R. Sirkisoon, Richard L. Carpenter, Tadas Rimkus, Daniel Doheny, Dongqin Zhu, Noah R. Aguayo, Marlyn Anguelov, Austin Arrigo, Fei Xing, Michael Chan, Jimmy Ruiz, Linda J. Metheny-Barlow, Roy Strowd, Jiayuh Lin, Boris C. Pasche, Waldemar Debinski, Kounosuke Watabe, Hui-Wen Lo. Tumor-specific gain-of-function tGLI1 transcription factor is a novel mediator of breast cancer stem cells and a novel transcriptional activator of cancer stemness genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3689.

Glutaminase (GLS) expression in primary breast cancer (BC): Correlations with clinical and tumor characteristics.

558 Background: Tumor cells rely on glutamine for growth. GLS is a mitochondrial enzyme that is necessary for glutamine catabolism, and is present as isoforms GLS1 and GLS2. A GLS1 inhibitor is being studied in triple-negative (TN) BC. We studied GLS1 expression in primary BC to understand associations with clinical and tumor characteristics in publically available databases. Methods: GLS1 mRNA levels were evaluated using expression data from the TCGA (n = 817) dataset, with confirmation in METABRIC (n = 1992). Associations between GLS1 levels and tumor subtype were assessed using ANOVA, followed by the post-hoc Tukey test for pairwise comparisons. Pearson correlations were used to study associations between GLS1 and selected genes. Correlations with overall survival (OS) were studied with Cox proportional hazard model. For all analyses, p &lt; 0.05 was considered significant. Results: In TCGA, the expression of GLS1 and its isoform GLS2 were significantly inversely correlated (r = -0.32). GLS1 expression was highest in TN compared to hormone receptor (HR)+ and HER2+ BC (p &lt; 0.001). In addition, GLS1 expression was higher in basal vs luminal A, luminal B, and HER2 enriched BC (p &lt; 0.001). GLS1 expression was significantly inversely correlated with ER (r = -0.45), PR (r = -0.34), and AR (r = -0.34), and these inverse correlations remained significant when restricted to TNBC (ER: r = -0.25, PR: r = -0.25, AR: r = -0.30). Consistent with previous reports of MYC upregulation of GLS1, GLS1 expression was significantly positively correlated with MYC (r = 0.26). Similarly, in METABRIC, GLS1 was most highly expressed in basal and TNBC, significantly inversely correlated with the expression of GLS2, ER, PR, and AR, and positively correlated with MYC expression. In METABRIC, higher GLS1 expression was associated with better OS (HR 0.91, p = 0.005); this association remained significant in the TN subset (HR 0.83, p = 0.03). Correlations between GLS1 and genes involved in metabolism and immune activation will be presented at the meeting. Conclusions: GLS1 expression is highest in basal and TNBC, is associated with MYC expression, and may have prognostic implications. These findings support ongoing trials of GLS1 inhibition in TNBC.

Survival following radiotherapy in young women with localized early-stage breast cancer according to molecular subtypes.

BackgroundTo evaluate the significance and benefit of radiotherapy (RT) in young early‐stage breast cancer patients according to different molecular subtypes.MethodsWe conducted a retrospective cohort study utilizing the Surveillance, Epidemiology, and End Results database with known hormone receptor (HoR) and human epidermal growth factor receptor 2 (HER2) status. Female patients aged 18‐45, received RT treatment, and diagnosed with stage T1‐3, N0‐3, M0 primary breast cancer between 2010 and 2013 were identified.ResultsOf all the 23 148 included patients, 14 708 (63.54%), 3385 (14.62%), 1225 (5.29%), and 3830 (16.55%) were diagnosed with luminal‐A (HoR + HER2‐), luminal‐B (HoR + HER2+), HER2‐enriched (HoR‐HER2+), and triple‐negative (HoR‐HER2‐) breast cancer, respectively. RT was significantly correlated with improved overall survival (OS, HR: 0.295; 95% CI:0.138‐0.63, P = 0.002) and breast cancer‐specific survival (BCSS, HR: 0.328; 95% CI: 0.153‐0.702, P = 0.004) in HER2‐enriched patients. In addition, a significantly prolonged OS was also observed when RT was given to luminal‐A (HR: 0.696; 95% CI: 0.538‐0.902, P = 0.006) and luminal‐B (HR: 0.385; 95% CI:0.199‐0.744, P = 0.005) breast cancer patients compared to those without RT. Multivariable‐adjusted analyses showed that HER2 was a significant favorable factor for RT benefit in breast cancer patients.ConclusionsRT could offer significant survival benefit in luminal‐A, luminal‐B, and especially HER2‐enriched young early‐stage breast cancer female patients. The results enabled clinicians to predict the benefits of RT and improve evidence‐based treatment for breast cancer patients.

Abstract P6-17-07: Gene signatures and subtype changes during HER2 dual blockade in PAM50 HER2-enriched HER2-positive breast cancer

Abstract Background: HER2-positive (HER2+) breast cancer (BC) is composed of 4 molecular subtypes: Luminal A and B, HER2-enriched (HER2-E) and Basal-like. Among them, the HER2-E is highly sensitive to anti-HER2 treatment. However, ˜60% of HER2-E tumors do not achieve a pathological complete response (pCR) following neoadjuvant dual HER2 blockade without chemotherapy. Here, we aimed to better understand the molecular changes of the HER2-E subtype during anti-HER2 treatment. Methods: Gene expression was evaluated in 101 patients with HER2-E tumors from the PAMELA neoadjuvant phase II trial (Lancet Oncol 2017). Briefly, women with HER2+ BC were treated with lapatinib and trastuzumab (and hormonal therapy if hormone receptor [HR]-positive) for 18 weeks. The median time between the last dose of treatment and surgery was 35 days (range=213; interquartile range=16). Expression of the PAM50 genes and 6 PAM50 signatures (Luminal A, Luminal B, HER2-E, Basal-like, normal-like and the PAM50 proliferation score) were determined using the nCounter platform at baseline (n=101), after 2 weeks of treatment (n=96) and in residual tumors (non-pCR) at surgery (n=57). Same analyses were done in 2 HER2+/HER2-E cell line models (BT474 [HR+] and SKBR3 [HR-]) following in vitro treatment with trastuzumab in combination with lapatinib. Biological changes between 2 time-points were determined by paired t-tests with a false discovery rate (FDR) &amp;lt;5%. Results: After 2 weeks of treatment, 85.7% and 94.6% of the 56 genes/signatures were found differentially expressed (FDR&amp;lt;5%) in HER2-E/HR+ (n=35) and HER2-E/HR- (n=61) tumors, respectively. The two gene lists were highly correlated (correlation coefficient=0.93). Overall, a significant relative increase in Luminal A and normal-like signature scores, and a relative decrease in proliferation, HER2-E and Luminal B signature scores, were observed between baseline and week 2. Interestingly, a PAM50 subtype switch to Luminal A was observed in 31.6% and 4.8% of HER2-E/HR+ and HER2-E/HR- tumors. In BT474 and SKBR3, all genes/signatures were also found differentially expressed (FDR&amp;lt;5%) following 72h of dual HER2 blockade. The in vitro findings recapitulated the in vivo findings in 80-86% of the genes/signatures. Similar to tumors, a switch to a Luminal A subtype following dual HER2 blockade was observed in BT474 but not in SKBR3. Finally, 92.9% of the 56 genes/signatures were found differentially expressed (FDR&amp;lt;5%) in residual tumors at surgery compared to week 2. Contrary to the findings in the first 2 weeks of treatment, a general rebound effect in gene expression was observed between week 2 and surgery. Similarly, a rebound effect was observed in 60% of the genes/signatures in BT474 after removing anti-HER2 therapy for 72h, leading to a subtype switch from Luminal A back to HER2-E. Conclusions: Dual HER2 blockade in the HER2-E subtype induces large biological changes that lead to a more low-proliferative Luminal A phenotype both in tumors and in vitro models, especially in HER2-E/HR+ disease. These phenotypic changes are reversible upon stopping anti-HER2 treatment. This finding supports the use of maintenance anti-HER2 treatment +/- endocrine therapy (if HR+) in advanced HER2+ BC. Citation Format: Brasó-Maristany F, Griguolo G, Llombart-Cussac A, Pascual T, Paré L, Bermejo B, Oliveira M, Morales S, Martinez N, Vidal M, Pernas S, Lopez R, Muñoz M, Galvan P, Garau I, Manso L, Alarcón J, Martínez E, Villagrasa P, Cortés J, Prat A. Gene signatures and subtype changes during HER2 dual blockade in PAM50 HER2-enriched HER2-positive breast cancer [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 P6-17-07.