ER Expression Research Articles

Progesterone and Estrogen Receptors in Meningiomas – A Clinicopathological Analysis

Background – Meningiomas are common tumors of the Central Nervous System. Recurrence in meningiomas has been proven to be associated with factors like extent of resection and grade of tumor. Decrease in progesterone receptor expression has been linked to increased rate of recurrence. We undertook this study to know about progesterone and estrogen receptor expression in meningiomas and its association with different clinicopathological variables as data is lacking in the Indian population.
 Materials and Methods – Meningiomas operated in a tertiary referral centre of North Kerala, India in 2 years were taken into the study. The tumors were graded and immunohistochemistry was performed using antibodies to Estrogen and progesterone receptors (ER&PR) and Ki-67. The expression of PR and ER was correlated with clinicopathologic variables. The patients were followed up for 2 years.
 Results – Grade I and grade II tumors constituted 85.3% and 14.7% respectively of the total number of meningioma cases. There were no grade III tumors in the series. The average PR positivity in grade I tumors (60.77%) was higher than in grade II tumors (46.88%). There was no gender related difference in PR staining. ER positivity was found only in a few cases.
 Conclusion – PR deterioration was associated with increased cell turnover. Meningiomas occurring in this study population are similar in clinicopathological parameters to those tumors occurring in other parts of the country but different in some aspects like grade from tumors occurring in other parts of the world. Strict follow up of a larger cohort of patients for a longer time period will be required to draw conclusions about prognosis in this population.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Prevalence of TP53 gene Pro72Arg (rs1042522) single nucleotide polymorphism among Egyptian breast cancer patients

BackgroundThe P53 protein has an essential role in several cellular processes, including DNA repair, apoptosis, and cell cycle arrest. The pathophysiology of many cancer types has frequently been linked to polymorphisms in the TP53 locus. Over 200 single nucleotide polymorphisms (SNPs) have been identified in TP53. However, Pro72Arg (rs1042522) at codon 72, shows contradictory results in terms of cancer risk. In this study, we aimed to determine if the Pro72Arg (rs1042522) SNP in the TP53 gene would be linked to breast cancer (BC) risk among Egyptian patients.Materials and MethodsGenomic DNA was extracted from blood samples of 100 healthy volunteers and 100 breast cancer patients (50 familial and 50 non-familial). TP53 Genotyping was performed using tetra-primer amplification refractory mutation (Tetra-ARMS) PCR. Data were analyzed using SNPstat software.ResultsThe prevalence of TP53 (Pro72Arg) rs1042522 genotypes carrying the high-risk allele [Pro/Arg (CG) and Arg/Arg (GG)] were significantly higher in BC patients compared to healthy volunteers and were associated with BC susceptibility (OR 0.2; [95% CI 0.11–0.38]; P = 0.0001). However, there was no statistical significant difference in the prevalence of TP53 (Pro72Arg) rs1042522 genotypes carrying the high-risk allele between familial and non-familial BC patients. In addition, there was no association between the prevalence of TP53 (Pro72Arg) rs1042522 genotypes carrying the high-risk allele and BC patients’ clinical and pathological characteristics including tumor size, tumor grade, lymph node status, presence of lymphovascular invasion, expression of ER, PR and Her-2 in both of familial and non-familial BC patients.ConclusionsTP53 (Pro72Arg) rs1042522 is more prevalent among BC patients but not associated with disease progression.

Clinical characteristics and prognostic characterization of endometrial carcinoma: a comparative analysis of molecular typing protocols

BackgroundEndometrial carcinoma (EC) is one of the most common gynecological malignancies in China and globally, accounting for the fourth-prevalent cancer in women. Although numerous studies have confirmed prognostic value of The Cancer Genome Atlas (TCGA) molecular subgroups, it is unclear how they are combined with histological features. The main objective of this study was to compare ProMisE and TCGA classification for the rapid and accurate prediction of prognosis within EC patients, together with the provision of a revised strategy for individualized diagnosis and treatment of patients.MethodsWithin this study, 70 patients with EC from Beijing Tsinghua Changgeng Hospital (affiliated to Tsinghua University) were retrospectively examined between July 2015 and December 2021. Samples were processed for determination of clinical markers, together with ProMisE and TCGA classification.ResultsComparative analysis across four TCGA types (POLE, Low-CN, High-CN, and MSI-H) and age, was statistically significant (χ²= 7.000, p = 0.029). There was no significant difference observed among the four TCGA types and FIGO stage, vascular invasion and depth of invasion, or lymph node metastasis and tumor area. There was no significant association between the expression of Vimentin, Ki-67, PTEN, MSH2, PAX-8, β-catenin, CD10, ER, PR, P16, MLH1, and PMS2 with the four TCGA types. In addition, p63 expression (χ²= 11.09, p = 0.029) and p53 expression (χ²= 11.585, p = 0.005) were statistically significant. Numerous models demonstrated that patients with POLE mutations and low-CN had higher progression free survival (PFS) and overall survival (OS), whereas those with high-CN had lowest values. The log-rank test revealed that the survival rate of PR-positive and ER-positive patients was significantly higher (p < 0.001).ConclusionOverall, these results can be of additional benefit for clinical applications, in comparison to the ProMisE classification method. In addition, PR, ER, vascular infiltration, hyperlipidemia and atherosclerosis were found to be the key factors affecting EC prognosis.

Alterations in the balance of sex hormones may affect rat prostatic inflammation and fibrosis, and osteopontin might be involved in this process.

This study aimed to investigate the effects of sex hormone imbalance on rat prostatic inflammation and fibrosis and identify the key molecules involved. Castrated Sprague-Dawley (SD) rats were treated with a constant dose of oestradiol (E2) and different doses of dihydrotestosterone (DHT) to achieve different oestrogen/androgen ratios. After 8weeks, serum E2 and DHT concentrations, relative seminal vesicle weights, histopathological changes and inflammation were measured, collagen fiber content and oestrogen receptor (ER) and androgen receptor (AR) expression were detected, mRNA sequencing and bioinformatics analysis were performed to identify differentially expressed genes (DEGs). The severity of inflammation in the rat dorsolateral prostate (DLP) was higher, collagen fibre content and ER expression in the rat DLP and prostatic urethra were increased and AR expression in the rat DLP was decreased in the 1:1 E2/DHT-treated group than that in the 1:10 E2/DHT-treated group. RNA-seq analysis identified 487 DEGs, and striking increases in the expression of mRNAs encoding collagen, collagen synthesis and degradation enzymes, growth factors and binding proteins, cytokines and chemokines, and cell-surface molecules were confirmed in the 1:1 E2/DHT-treated group compared to the 1:10 E2/DHT-treated group. mRNA expression of secreted phosphoprotein 1 (Spp1) and protein expression of osteopontin (OPN, encoded by Spp1) were increased in the 1:1 E2/DHT-treated group compared to the 1:10 E2/DHT-treated group, and Spp1 expression correlated positively with Mmp7, Cxcl6 and Igfn1 expression. The imbalance in the oestrogen/androgen ratio may affect rat prostatic inflammation and fibrosis, and OPN might be involved in this process.

Evolution of biological features of invasive lobular breast cancer: Comparison between primary tumour and metastases

BackgroundInvasive lobular carcinoma (ILC) has unique clinical-biological features. Phenotypical differences between primary tumours (PTs) and metastases (M) have been described for invasive ductal carcinoma, but data on ILC are limited. MethodsWe retrospectively analysed patients with recurrent ILC from our institution from 2013 to 2020. We evaluated the discordance of the oestrogen receptor (ER), progesterone receptor (PgR) and HER2 between PT and M, to understand prognostic and therapeutic implications. ResultsThirteen percent (n = 91) of all patients had ILC. We observed 15%, 44% and 5% of ER, PgR and HER2 status discordance between PT and M. ER/PgR discordance was related to receptor loss and HER2 mainly due to gain. PT presented a luminal-like phenotype (93%); 6% and 1% were triple-negative (TNBC) and HER2-positive. In M, there was an increase in TNBC (16%) and HER2-positive (5%). Metastasis-free survival and overall survival (OS) were different according to clinical phenotype, with poorer prognosis for HER2+ and TNBC (p < 0.001); OS after metastatic progression did not differ across phenotypes (p = 0.079). In luminal-like ILC (n = 85) at diagnosis, we found that OS after relapse was poorer in patients experiencing a phenotype switch to TNBC but improved in patients with HER2 gain (p = 0.0028). Poorer survival was reported in patients with a PgR and/or ER expression loss of ≥25%. There was HER2-low enrichment in M1 (from 37% to 58%): this change was not associated with OS (p > 0.05). ConclusionOur results suggest that phenotype switch after metastatic progression may be associated with patients’ outcomes. Tumour biopsy in recurrent ILC could drive treatment decision-making, with prognostic implications.

Abstract P3-11-01: Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance

Abstract Background: New treatment paradigms are needed to overcome resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) in ER+ breast cancer (BC). ET resistance is due to survival of breast cancer stem cells (BCSCs) that contribute to relapse of ER+ BC. Notch signaling drives BCSCs. In order to identify Notch specific biomarkers for the purpose of patient selection for anti-Notch therapy, we conducted a pre-surgical biomarker window study combining ET plus MK-0752, a -secretase inhibitor (GSI). Death Associated Protein 6 (DAXX) was discovered to be a novel Notch1 target gene and necessary for GSI-mediated inhibition of BCSCs. Subsequently, we found that DAXX alone was sufficient to inhibit BCSCs. In this current study, we investigated the mechanism by which high DAXX expression inhibited growth of ET resistant ER+ BC cells in vitro and in vivo. Methods: Isogenic ER+ BC cell lines (parental MCF-7, ET resistant MCF-7/5C, parental T47D, and ET resistant T47D-ED) were used. Cells were cultured in estrogen-deprived medium for more than 1 year to mimic AI use. DAXX was depleted using siRNA or overexpressed using a pCMV-expression vector. Bulk cell proliferation was analyzed in response to estrogen depletion or increasing concentrations of 17-estradiol. BCSC survival was measured using the mammosphere-forming assay. Tumor onset and burden were measured by injecting DAXX-expressing or depleted mammospheres into mammary fat pads of female, athymic nude mice. Recurrence of an ER+ PDX tumor (BCM 5097) was measured after withdrawal of estrogen. RNA sequencing identified enriched genes and pathways that required DAXX. Based on these results, cell death was assessed using Annexin V/7-AAD flow cytometry, PARP-1 and Caspase 8 cleavage, phosphorylation of JNK, and expression of apoptotic protein regulators, BIM, BAX, Bcl-2, and Bcl-xL. JNK signaling was inhibited using SB600125. Results: Estradiol stimulated proliferation of parental, ER+ MCF-7 and T47D cells. In contrast, estradiol inhibited proliferation of isogenic ET resistant MCF-7/5C and T47D-ED cells in a concentration and ER-dependent manner. Estradiol induced the DAXX protein in both ET sensitive and resistant cells. DAXX was required for BCSC survival in ET sensitive cells. However, once ER+ cells acquired resistance to ET, DAXX was necessary and sufficient to inhibit both bulk cell proliferation and BCSC survival, suggesting that increasing DAXX might be a novel approach to overcome ET resistance. In mice, high DAXX expression significantly inhibited tumor onset and burden of ET resistant tumors compared to DAXX-depleted tumors. Low DAXX expression was significantly associated with recurrence of an ER+ PDX tumor (BCM 5097) after withdrawal of estrogen. RNA sequencing revealed that DAXX activated an anti-neoplastic gene signature, including transcription factors that regulate cell death genes including the Bcl2-family. DAXX was required for high BIM expression and low levels of Bcl-xL. DAXX was necessary and sufficient to induce apoptosis, PARP-1 cleavage, and phosphorylation of JNK in ET resistant cells. A selective JNK inhibitor, SB600125 rescued DAXX-mediated inhibition of ET resistant bulk cell proliferation and BCSC survival, suggesting that high DAXX expression activates JNK signaling to regulate apoptotic proteins to induce cell death of BCSCs-derived from ET resistant BC. Conclusions: Expressing high DAXX levels is a potent method to inhibit ET-resistant BC cell proliferation and BCSC survival. The mechanism by which DAXX inhibits ET-resistant BC is through activation of JNK signaling, regulation of pro-apoptotic genes, and induction of apoptosis. The translational impact of this research is to identify novel agents that can increase DAXX expression and test them pre-clinically and in clinical trials for patients with ET-resistant breast cancer. Citation Format: Clodia Osipo, Kathy S. Albain, Daniel Peiffer, Debra Wyatt. Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance [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 P3-11-01.

ER-/PR+ breast cancer is controlled more effectively with an inflammatory inhibitor than hormonal inhibitor.

The anti-estrogen tamoxifen is a highly effective hormonal therapy for hormonal-positive (HR+) breast cancer patients; however, the estrogen receptor-negative, progesterone receptor-positive (ER-/PR+) subtype does not give the benefits of tamoxifen. Therefore ER-/PR+ breast cancer has a poor clinical outcome, and novel drug therapy for ER-/PR+ breast cancer could benefit these patients. 53,805 gene expressions were characterized into HR+ BC and triple-negative breast cancer (TNBC) and analyzed through Breast Cancer Gene Expression Miner in 4319 breast cancer patient samples. The clinical outcomes including overall survival, distant metastasis-free survival, and relapse-free survival were obtained from the PrognoScan database containing 1190 human breast cancer patient samples. To determine the function of ERα and inflammation-related genes such as USP1, CDC20, and CASP1, we used the CRISPR-Cas9 system or gene knockdown (KD) system. To check tumor cell proliferation and migration of ERα KO breast cancer cell line, we used tamoxifen and the inflammation inhibitor Ac-YVAD-CHO. For further confirmation, cancer growth was checked with the inflammation inhibitor in ERα KO breast cancer cell line using a three-dimensional (3D) organoid tissue culture system (ex vivo). We found that gene expression in ER-/PR+ hormonal-positive breast cancer is positively related to ER-/PR- very similar to TNBC, not other HR+ breast cancer using a 4319 breast cancer patient database. Especially, inflammation-related genes, USP1, CDC20, and CASP1, which are highly expressed in TNBC, are also upregulated in ER-/PR+ HR+ breast cancer. Suppression of USP1, CDC20, and CASP1 inhibited tumor cell growth and metastasis in ERα KO (ER-/PR +) cell lines. Interestingly, loss of ERα in HR+ cell lines is not responsive to tamoxifen, but highly sensitive to the inflammation inhibitor, Ac-YVAD-CHO. In in vitro and ex vivo (3D organoid) models, inflammation inhibitor-specific blocks ER-/PR+ tumor proliferation and migration. These findings suggest that an inflammation inhibitor might be a potential option for therapy for ER-/PR+ HR breast cancer patients.

Abstract P1-13-18: EGFR signaling contributes to acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells in vitro and in vivo

Abstract Breast cancer driven by different hormone receptors, including estrogen receptor (ER+), is responsible for approximately 70-80% of cases among women. While endocrine therapy (ET) of ER+ primary tumors with antiestrogens or aromatase inhibitors is an effective first-line therapy, its success is limited by intrinsic and acquired resistance. Response to ET can be limited due to overexpression of cyclin D1, which promotes the activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Selective inhibition of CDK4/6 and ER signaling is now standard-of-care therapy for ER+ metastatic breast cancer. CDK4/6 inhibitors (CDK4/6i), including palbociclib (PD), ribociclib, and abemaciclib, used in combination with ET, have shown improvement in progression-free survival compared to ET alone in the metastatic setting. However, the inevitable development of acquired resistance significantly limits the efficacy of this targeted therapy. Rewired signaling driven by different oncogenes, including the epidermal growth factor receptor (EGFR), overcomes the targeted inhibition of CDK4/6, which, in turn, allows cell cycle progression contributing to acquired resistance. We evaluated the expression and activity of EGFR in the panel of different matching pairs of ER- and ER+ CDK4/6i-sensitive (pS) and CDK4/6i-resistant (pR) breast cancer cell lines in vitro. Increased EGFR expression in ER+ MCF7/pR cells correlated with elevated ER phosphorylation, suggesting a direct cross-talk between EGFR and ER signaling in PD-resistant cells. Stimulation with EGF promoted ER activation, whereas estrogen stimulation promoted EGFR activation in ER+ pS and pR cell lines, indicating a direct cross-regulation between these molecular targets. Treatment of MCF7/pR cells with CDK4/6i resulted in elevated EGFR mRNA expression, confirming the dependency of resistant cells on EGFR signaling. Moreover, exposure to the EGFR inhibitors dramatically inhibited the proliferation of MCF7/pR cells compared to the parental cells, suggesting that EGFR could be an essential bypass signaling regulator contributing to resistance to CDK4/6i. We found that combined treatment with EGFRi and CDK4/6i significantly inhibited Rb phosphorylation in pR cells improving the effect of CDK4/6i on cell cycle arrest. We evaluated EGFR expression in MCF7/pS and pR mouse xenograft models in vivo. We observed a significant elevation in EGFR expression and phosphorylation in response to PD treatment, confirming the importance of EGFR signaling in maintaining the acquired resistance to CDK4/6i in ER+ breast cancer cells. Taken together, our findings suggest that EGFR signaling plays an important role in acquired resistance to CDK4/6i in ER+ breast cancer cells in vitro and in vivo and targeting EGFR with small-molecule inhibitors (like erlotinib or gefitinib) or chimeric monoclonal antibodies (i.e. cetuximab) can be a promising approach to improve CDK4/6i efficacy in breast cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan Daugherty, Jason Chesney, Yoannis Imbert-Fernandez. EGFR signaling contributes to acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells in vitro and in vivo. [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 P1-13-18.

Abstract P2-24-08: Altersolanol B, a fungal metabolite, induces proteasome-dependent degradation of estrogen receptor α (ERα) and inhibits downstream signaling targets in ER+ breast adenocarcinoma cells

Abstract We recently reported the cellular signal-modulating properties of altersolanol B (AB), a minor fungal tetrahydroanthraquinone (THAQ) metabolite, in the estrogen receptor positive (ER+) human breast adenocarcinoma cell lines, MCF-7 and T47D. MCF-7 (IC50 5.5 µM) and T47D (IC50 8.8 µM) were observed to be 4- and 2.4-fold more sensitive to the antiproliferative effects of AB, respectively, compared to MDA-MB-231 (triple-negative, IC50 21.3 µM). AB disrupted both AKT and ERK1/2 signaling leading to intrinsic apoptosis in MCF-7. The clinical limitations of multi-agent combination therapy that targets multiple pathways in cancer may potentially be circumvented by using a single molecule, such as AB, that inhibits both AKT and ERK1/2 signaling. The THAQ pharmacophore, with its disrupted conjugated ring system and relative redox inactivity, may possess greater mechanistic advantage against ER+ breast cancer when compared to the fully conjugated ring systems of the anthracyclines (doxorubicin) and anthraquinone (mitoxantrone) that are associated with nonselective mechanisms. The present phase of our study provides evidence that AB downregulated ERα expression at the post-translational level through the induction of proteasome-dependent degradation similar to a classical selective estrogen receptor degrader (SERD). AB dose-dependently downregulated both wild-type (WT)- and 17β-estradiol (E2)-stimulated ERα protein expression without altering mRNA expression in ER+ cells. This was corroborated by the marked reduction in the expression of downstream ERα target genes (cathepsin D and pS2). AB also dose-dependently downregulated cyclin D1, the estrogen-independent activator of ER. Antiproliferative effects were enhanced when ER+ cells were exposed to a combination of AB and the ERα-selective antagonist, MPP dihydrochloride. The AKT activator, SC79, showed that the AB-induced ERα protein downregulation was independent of the inhibition of AKT-FOXO1 signaling. The expression of Hsp90 was not affected. Co-treatment with the protein synthesis inhibitor, cycloheximide, revealed that AB downregulated ERα expression at the post-translational level. The proteosome inhibitor, MG-132, effectively suppressed AB-induced ERα degradation and restored cellular proliferation. In silico approaches were adopted to probe the direct binding of AB to ERα. AB demonstrated significant binding affinity with human ERα (PDB ID - 3ert) compared to the binding of the standard SERD, fulvestrant. Non-covalent bonding interaction analysis further revealed that, much like fulvestrant, AB successfully interacted with 5 major amino acid residues (i.e., Ala350, Asp351, Leu525, Leu536 and Trp383) at the ligand-binding domain of ERα which is stabilized by 7 non-bonding interactions. Principal component analysis indicated that binding with AB improved the compactness of 3ert folding. Molecular dynamics simulation indicated that the 3ert-AB interaction was stable even in the 100 ns simulated physiological environment. Citation Format: Md Afjalus Siraj, Md Sajjadur Rahman, Aaron T. Jacobs, Ghee T. Tan. Altersolanol B, a fungal metabolite, induces proteasome-dependent degradation of estrogen receptor α (ERα) and inhibits downstream signaling targets in ER+ breast adenocarcinoma cells [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 P2-24-08.

Abstract P3-05-14: Clinicopathological features of early breast cancer in Japanese premenopausal women

Abstract Background: Breast cancer incidence is constantly increasing in Japan. The age-specific incidence rates (ASIRs) of breast cancer shows two peaks, at ages 45-49 and 60-64, in Japan. Therefore, the incidence rate of breast cancer in Japanese premenopausal women is higher compared to the Western countries. In our study, we clarified the clinicopathological characteristics of Japanese premenopausal breast cancer. Patients and Methods: The total of 1,220 premenopausal breast cancer patients treated for early breast cancer, at stage 0-IIII, between 2010 and 2021. The patients were divided into three age groups; aged under 40 (group A, n=258, 21.1%), aged 40 to 45 (group B, n=428, 35.1 %), and aged 46 and over (group C, n=533, 43.7%). Information regarding the clinicopathological characteristics was collected from the medical records. Result: Significantly longer periods form from menarche to the breast cancer onset (p&amp;lt; 0.0001), larger number of history of pregnancy (p&amp;lt; 0.0001), and higher BMI (25 or higher) (p=0.0091), and more habitual drinking histories (p=0.0029) were recognized in group C. In premenopausal onset, ER-positive status is higher (p=0.0022), the histological grade is lower (p=0.0094), as age increases, and more negative PgR status(p=0.0019), and more triple negative breast cancer (p=0.0002) were shown in group A. Hormone receptor-positive HER2-negative breast cancers under 45 years of age (group A, B) were more likely to receive neoadjuvant chemotherapy than those aged 46 years and older (group C) (p=0.0077), but there was no age-related difference for other subtypes. The association between clinicopathological factors and distant relapse-free survival (DRFS) in each age group was analyzed. is associated with higher grades (group A, p=0.0638、group B, p=0.0208, group C, p=0.0006) , negative PgR status (group A, p=0.0379,group B, p=0.0683,group C, p=0.0042), and lower ER expression (group B, p=0.0395 group C, p=0.0021) were significantly associated with poor DRFS. Similarly, in overall survival, higher grades (group A, p=0.0257,group B, p=0.0027,group C, p=0.0111) ,negative PgR status (group A, p=0.0162,group B, p&amp;lt; 0.0001,group C, p=0.0003), and lower ER expression (group C, p=0.0003) were significantly poor prognosis. In univariate analyses, higher grade, negative PgR status, and ER negative/low-positives were risk factors, and in multivariate analyses, higher grade was a risk factor for poor prognosis Conclusion: We found that there are many clinicopathological characteristics related to poor prognosis in premenopausal breast cancer patients aged under 40, such as low or negative ER expression, higher histological grade, and negative PgR status. clinicopathological characteristics-1 clinicopathological characteristics-2 Citation Format: Wakako Tajiri, Sumire Koh, Yoshiaki Nakamura, Junji Kawasaki, Yumiko Koi, Chinami Koga, Hideki Ijichi, Eriko Tokunaga. Clinicopathological features of early breast cancer in Japanese premenopausal women [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 P3-05-14.

Abstract P3-11-03: Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ Breast Cancer

Abstract Endocrine receptor-positive (ER+) breast cancers (BC) comprise over 70% of all breast cancers and are the leading cause of BC-related deaths in women. Despite available targeted therapies against ER+ BC, recurrence arises primarily due to the development of endocrine therapy resistance and metastases. Thus, there is an unmet need to identify novel biomarkers for treating ER+ BC patients with metastases. We have identified a neuroimmune molecule, Semaphorin 7a (SEMA7A), as a potential biomarker for endocrine therapy resistance, increased relapse and decreased overall survival in ER+ BC patients. We have also found that SEMA7A promotes tumor growth, angio- and lymphangiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis and endocrine therapy resistance in pre-clinical models. Specifically, we have shown that SEMA7A+ ER+ MCF7 tumors result in lung metastases that are resistant to the endocrine therapy, fulvestrant, in part, via downregulation of ER in vivo – posing the need to identify novel, druggable targets for SEMA7A+ ER+ BC. SEMA7A is a membrane-bound protein that we have shown can inhibit tumor cell death via integrin-mediated activation of PI3K/Akt pro-survival signaling. Thus, we hypothesized that that SEMA7A+ ER+ BC cells may be sensitive to PI3K inhibition in combination with fulvestrant. Our results demonstrate that SEMA7A+ MCF7 cells are sensitive to the combination of fulvestrant and PI3K inhibition by alpelisib, which is FDA approved for ER+ BC patients with activating mutations in PI3K; this combination also inhibited tumor cell growth and decreased tumor sphere formation. In complementary studies, we identified that Singleminded 2s (SIM2s), which is expressed in breast epithelial cells and inhibits EMT, is downregulated SEMA7A+ MCF7 tumors. Additionally, our studies show that while ER-mediated signaling turns on SEMA7A expression prolonged expression of SEMA7A results in downregulation of ER, and the loss of SIM2s causes downregulation of ER expression and upregulation of SEMA7A in MCF7 cells. Further, while SEMA7A-overexpressing (OE) tumor cells exhibit increased tumor-promoting PI3K/Akt survival signaling, we found that SIM2s plays a role in suppression of PI3K/Akt survival signaling. Therefore, we are investigating the interactions between SIM2s, SEMA7A and the survival signaling pathways involved in their crosstalk. Our recent results show that MCF7 SIM2-knockout (KO) cells exhibit increased SEMA7A promoter activity, SEMA7A expression, activation of Akt signaling, EMT phenotypes and decreased ER expression. Also, addition of exogenous SEMA7A protein reduces SIM2s expression and promoter activity. Since SIM2s correlates with inhibition of PI3K/Akt signaling and decreased SEMA7A, and SEMA7A promotes survival signaling, we are investigating additional therapeutic strategies to prevent endocrine therapy resistance via direct inhibition of SEMA7A and/or restoration of SIM2s, including inhibition of COX-2 and Akt signaling. Citation Format: Rachel Steinmetz, Garhett Wyatt, Traci Lyons, Weston Porter, Lyndsey Crump. Novel Targeting of Semaphorin 7a-Mediated Survival Signaling in ER+ 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 P3-11-03.

Abstract P2-23-09: Ultra-high Tumor Mutation Burden in Metastatic/Clinically Advanced Breast Cancer (MBC)

Abstract Background: High (HTMB) tumor mutation burden (TMB) defined as ≥10 mutations/megabase (Mb) identifies breast cancer patients who could benefit from pembrolizumab. The higher the TMB the greater the likelihood of benefit. The goal of this analysis was to determine the frequency and genomic landscape of MBC with ultra-high TMB (UHTMB) defined as a TMB &amp;gt; 20 mutations/Mb. Design 2,049 MBC patients (pts) underwent hybrid capture based comprehensive genomic profiling for genomic alterations (GA) in at least 324 genes including determination of TMB to guide therapy decisions using the FoundationOne®CDx assay. ER, PR and HER2 expression were abstracted from submitted pathology reports. Results: 165 of 2049 MBC (8.1%) had HTMB &amp;gt; 10 mutations/Mb, among these 45 (2.2% of all cases) had UHTMB. When compared with the 2,004 non-UHTMB MBC pts with TMB &amp;lt; 20 mutations/Mb, the 45 UHTMB pts were older (mean 64.6 yrs vs 58.2 yrs; p&amp;lt;.0001), more often had lobular histology (40.00% vs 14.5%; p&amp;lt;.0001) and ER+ disease (86.6% vs 70.0%), had higher average driver GA/tumor (9.84 vs 5.7; p&amp;lt;.0001), and less often had TNBC (13.3% vs 27.0%; p=.041) compared to non-UHTMB high cancers. There were no significant differences in ancestry. Mutation signature analysis revealed that APOBEC was predominant in UHTMB samples (82.5%) with a minor portion with an MMR signature (10%), however, MSI-H status was significantly higher in UHTMB high cases (11.6% vs 0.40%; p&amp;lt;.0001). GA more frequently identified in UHTMB cases included CDH1 (45.50% vs 14.32%; p&amp;lt;.0001), PIK3CA (81.80% vs 37.86%; p&amp;lt;.0001), CDKN2A (11.40% vs 3.19%; p=.017), ARID1A (25.00% vs 5.01%; p&amp;lt;.0001) and NF1 (20.50% vs 5.94%; p=.0014). PD-L1 (CD274) gene amplification (2.3% vs 1.3%) or protein expression by the Ventana SP142 assay (57.14% vs 51.10%) were not significantly different. Conclusions: UHTMB MBC is a rare but clinically important subset in breast cancer that could have high response rates to single agent pembrolizumab. This phenotype is driven by APOBEC mutagenesis, more often seen in ER+ lobular cancers, and have higher frequencies of MSI-high status and mutations in CDH1 and PIK3CA. Citation Format: Kristina Fanucci, maryam lustberg, Neal Fischbach, Maureen Pelletier, Abirami Sivapiragasam, Prashanth Ashok Kumar, Mansi Kallem, Natalie A. Danziger, Ethan Sokol, Smruthy Sivakumar, Dean Pavlick, Jeffrey S. Ross, Lajos Pusztai. Ultra-high Tumor Mutation Burden in Metastatic/Clinically Advanced Breast Cancer (MBC) [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 P2-23-09.

Abstract P1-09-08: Comparisons of aesthetic outcomes and prognosis of conventional breast conserving surgery, oncoplastic breast conserving surgery and breast conserving surgery plus immediate lipofilling in early breast cancer

Abstract Background: Conventional breast conserving surgery (CBCS) considered as a confined alternative to mastectomy could lead to potentially breast deformities. Oncoplastic breast conserving surgery (OBCS) as well as breast conserving surgery (BCS) plus immediate lipofilling have been shown to be reliable techniques for maintaining the natural breast contours, however, few studies compared those three surgical options for oncological safety, complications and aesthetic outcomes in early breast cancer (EBC). Methods: We retrospectively reviewed the data of BC patients who underwent BCS between January 2017 and December 2021 from The First Affiliated Hospital of Chongqing Medical University. We included female patients who received BCS for unilateral stage 0-III BC. Those who had bilateral BC, incomplete surgical records, unfinished adjuvant therapy, multiple primary malignant tumors and lost to follow-up and were excluded. Patient-reported outcome measures (PROMs) were assessed using the BREAST-Q Version 2.0. We utilized a multivariable linear regression model to identify clinical factors correlated with the BREAST-Q score, and the log-rank test and Cox regression models were used to compare the survival difference between groups. Results: Of 268 patients, 90 (33.6%) underwent CBCS, 53 (19.8%) underwent OBCS and 125 (46.6%) underwent BCS plus immediate lipofilling. Patients in OBCS and BCS plus immediate lipofilling groups were younger than those in CBCS group (mean age: 43.9yrs vs 49.1yrs, P=0.001 and 45.7yrs vs 49.1yrs, P=0.008). The largest tumor size and heaviest BCS specimen were observed in the OBCS group compared with CBCS (mean tumor size: 23.4mm vs 17.3mm, P&amp;lt; 0.001 and median excised weight: 122.0g vs 36.5g, P&amp;lt; 0.001) and BCS plus immediate lipofilling group (mean tumor size: 23.4mm vs 17.4mm, P&amp;lt; 0.001 and median excised weight: 122.0g vs 37.0g, P&amp;lt; 0.001). It was balanced for pathological type, Ki-67, ER and PR expression between three groups. In the multivariable linear regression models, with the CBCS group as referent, either OBCS or BCS plus immediate lipofilling groups had a significantly higher score in satisfaction with breast (Estimate: 9.27, P=0.001 and Estimate: 13.08, P&amp;lt; 0.001) and psychosocial well-being (Estimate: 6.06, P=0.021 and Estimate: 9.34, P&amp;lt; 0.001). Additionally, sexual well-being was improved among patients receiving BCS plus immediate lipofilling (Estimate: 6.19, P=0.029). Nevertheless, patients in OBCS group harbored worse physical well-being compared with CBCS (Estimate: -15.89, P&amp;lt; 0.001). There was no significantly statistical difference among three groups on re-excision rate (P=0.721) and postoperative complications (P=0.663). After 37-month median follow-up, identical event-free survival (EFS) was revealed among three groups (HROBCS vs CBCS: 3.93; 95%CI, 0.78-19.81, P=0.098; HRBCS plus Lipofilling vs CBCS: 1.15, 95%CI, 0.28-4.67, P=0.847). Conclusion: This study demonstrated that OBCS as well as BCS plus immediate lipofilling shared equivalent oncological safety but better cosmetic outcomes and patient satisfaction when compared with CBCS for EBC patients, suggesting that further prospective randomized clinical trials are warranted to confirm our findings. Citation Format: JH Ren, Yuanyuan Wang, Xiang Zhang, Kang Wang, Renxi Tang, Ling Yang, Junge Gong, Jiawei Xu, Qing Li, WM Zhu, Qiao Cheng, Guosheng Ren, Hongyuan Li. Comparisons of aesthetic outcomes and prognosis of conventional breast conserving surgery, oncoplastic breast conserving surgery and breast conserving surgery plus immediate lipofilling in early 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 P1-09-08.

A comprehensive immunohistochemical analysis of 26 markers in 250 cases of serous ovarian tumors

BackgroundWe examined a large cohort of serous tubo-ovarian tumors with 26 immunohistochemical markers, with the aim to assess their value for differential diagnosis and prognosis.MethodsImmunohistochemical analyses with 26 immunomarkers were performed on 250 primary tubo-ovarian tumors including 114 high grade serous carcinomas (HGSC), 97 low grade serous carcinomas (LGSC), and 39 serous borderline tumors (micropapillary variant, mSBT). The associations of overall positivity with clinicopathological characteristics were evaluated using the chi-squared test or Fisher’s Exact test.ResultsWe found significantly different expression of p53, p16, ER, PR, PTEN, PAX2, Mammaglobin, RB1, Cyclin E1, stathmin, LMP2, L1CAM, CD44, and Ki67 in HGSCs compared to LGSCs. No significant differences were found between LGSC and mSBT. None of the other included markers (PAX8, ARID1A, HNF1B, Napsin A, CDX2, SATB2, MUC4, BRG1, AMACR, TTF1, BCOR, NTRK) showed any differences between the investigated serous tumors. Regarding the prognosis, only PR and stathmin showed a statistically significant prognostic meaning in LGSCs, with better overall survival (OS) and recurrence-free survival (RFS) in cases positive for PR, and worse outcome (RFS) for stathmin. None of the study markers showed prognostic significance in HGSCs.ConclusionWe provided an extensive immunohistochemical analysis of serous ovarian/tubo-ovarian tumors. Although we found some differences in the expression of some markers in HGSCs compared to LGSCs, only p53, p16, and Ki67 seem to be useful in real diagnostic practice. We also suggested the best discriminative cut-off for Ki67 (10% of positive tumor cells) for distinguishing HGSC from LGSC. We found prognostic significance of PR and stathmin in LGSCs. Moreover, the high expression of stathmin could also be of predictive value in ovarian carcinomas as target-specific anti-stathmin effectors are potential therapeutic targets.

Purine Intake and All-Cause Mortality in Ovarian Cancer: Results from a Prospective Cohort Study

Current biological evidence suggests that purine involvement in purine metabolism may contribute to the development and progression of ovarian cancer (OC), but the epidemiological association is currently unknown. A total of 703 newly diagnosed patients with OC aged 18-79 years were included in this prospective cohort study. Utilizing a verified food-frequency questionnaire, the participants' dietary consumption was gathered. Using medical records and ongoing follow-up, the deaths up until 31 March 2021 were determined. To assess the hazard ratios (HRs) and 95% confidence intervals (CIs) of purine intake with OC mortality, Cox proportional-hazard models were utilized. During the median follow-up of 31 months (interquartile: 20-47 months), 130 deaths occurred. We observed an improved survival for the highest tercile of total purine intake compared with the lowest tercile (HR = 0.39, 95% CI = 0.19-0.80; p trend < 0.05), and this protective association was mainly attributed to xanthine intake (HR = 0.52, 95% CI = 0.29-0.94, p trend < 0.05). Additionally, we observed a curving relationship in which OC mortality decreased with total purine intake, and the magnitude of the decrease was negatively correlated with intake (p non-linear < 0.05). Significant inverse associations were also observed in subgroup analyses and sensitivity analyses according to demographic and clinical characteristics. Moreover, we observed that xanthine intake and hypoxanthine intake had a multiplicative interaction with ER and PR expression (p < 0.05), respectively. A high total purine and xanthine intake was linked to a lower risk of OC mortality. Further clarification of these findings is warranted.

Establishment and characterization of canine mammary tumoroids for translational research

BackgroundCancer heterogeneity is a main obstacle for the development of effective therapies, as its replication in in vitro preclinical models is challenging. Around 96% of developed drugs are estimated to fail from discovery to the clinical trial phase probably because of the unsuitability and unreliability of current preclinical models (Front Pharmacol 9:6, 2018; Nat Rev Cancer 8: 147–56, 2008) in replicating the overall biology of tumors, for instance the tumor microenvironment. Breast cancer is the most frequent cancer among women causing the greatest number of cancer-related deaths. Breast cancer can typically be modeled in vitro through the use of tumoroids; however, current approaches using mouse tumoroids fail to reproduce crucial aspect of human breast cancer, while access to human cells is limited and the focus of ethical concerns. New models of breast cancer, such as companion dogs, have emerged given the resemblance of developed spontaneous mammary tumors to human breast cancer in many clinical and molecular aspects; however, they have so far failed to replicate the tumor microenvironment. The present work aimed at developing a robust canine mammary tumor model in the form of tumoroids which recapitulate the tumor diversity and heterogeneity.ResultsWe conducted a complete characterization of canine mammary tumoroids through histologic, molecular, and proteomic analysis, demonstrating their strong similarity to the primary tumor. We demonstrated that these tumoroids can be used as a drug screening model. In fact, we showed that paclitaxel, a human chemotherapeutic, could kill canine tumoroids with the same efficacy as human tumoroids with 0.1 to 1 μM of drug needed to kill 50% of the cells. Due to easy tissue availability, canine tumoroids can be produced at larger scale and cryopreserved to constitute a biobank. We have demonstrated that cryopreserved tumoroids keep the same histologic and molecular features (ER, PR, and HER2 expression) as fresh tumoroids. Furthermore, two cryopreservation techniques were compared from a proteomic point of view which showed that tumoroids made from frozen material allowed to maintain the same molecular diversity as from freshly dissociated tumor.ConclusionsThese findings revealed that canine mammary tumoroids can be easily generated and may provide an adequate and more reliable preclinical model to investigate tumorigenesis mechanisms and develop new treatments for both veterinary and human medicine.

Chromatin profile-based identification of a novel ER-positive breast cancer subgroup with reduced ER-responsive element accessibility

BackgroundOestrogen receptor (ER) signalling-dependent cancer cell growth is one of the major features of ER-positive breast cancer (BC). Inhibition of ER function is a standard and effective treatment for ER-positive tumours; however, ~20% of patients with ER-positive BC experience early or late recurrence. In this study, we examined intertumour heterogeneity from an epigenetic perspective based on the hypothesis that the intrinsic difference in epigenetic states around ER signalling pathway underlies endocrine therapy resistance.MethodsWe performed transposase-accessible chromatin sequencing (ATAC-seq) analysis of 42 BC samples, including 35 ER-positive(+) human epidermal growth factor receptor 2 (HER2)-negative(−) and 7 triple-negative tumours. We also reanalysed ATAC-seq data of 45 ER + /HER2 − tumours in the Cancer Genome Atlas (TCGA) BC cohort to validate our observations.ResultsWe conducted a comprehensive analysis of cis-regulatory elements (CREs) using ATAC-seq, identifying three subgroups based on chromatin accessibility profiles. We identified a subgroup of ER-positive BCs with a distinctive chromatin accessibility pattern including reduced accessibility to ER-responsive elements (EREs). The same subgroup was also observed in TCGA BC cohort. Despite the reduced accessibility to EREs, the expression of ER and potential ER target genes were not decreased in these tumours.ConclusionOur findings highlight the existence of a subset of ER-positive BCs with unchanged ER expression but reduced EREs accessibility that cannot be distinguished by conventional immunostaining for ER. Future studies should determine whether these tumours are associated with resistance to endocrine therapy.

Evaluation oncotype DX® 21-gene recurrence score andclinicopathological parameters: a single institutional experience.

Oncotype DX recurrence score (RS) is a clinically validated assay, which predicts the likelihood of disease recurrence in oestrogen receptor-positive/HER2-negative (ER+/HER2-) breast cancer (BC). In this study we aimed to compare the performance of Oncotype DX against the conventional clinicopathological parameters using a large BC cohort diagnosed in a single institution. A cohort (n= 430) of ER+/HER2- BC patients who were diagnosed at the Nottingham University Hospitals NHS Trust and had Oncotype DX testing was included. Correlation with the clinicopathological and other biomarkers, including the proliferation index, was analysed. The median Oncotype DX RS was 17.5 (range=0-69). There was a significant association between high RS and grade 3 tumours. No grade 1BC or grade 2 tumours with mitosis score 1 showed high RS. Low RS was significantly associated with special tumour types where none of the patients with classical lobular or tubular carcinomas had a high RS. There was an inverse association between RS and levels of ER and progesterone receptor (PR) expression and a positive linear correlation with Ki67 labelling index. Notably, six patients who developed recurrence had an intermediate RS; however, four of these six cases (67%) were identified as high-risk disease when the conventional clinical and molecular parameters were considered. Oncotype DX RS is correlated strongly with the conventional clinicopathological parameters in BC. Some tumour features such as tumour grade, type, PR status and Ki67 index can be used as surrogate markers in certain scenarios.

PTEN Deficiency in Tubo-Ovarian High-Grade Serous Carcinoma is Associated with Poor Progression-Free Survival and is Mutually Exclusive with CCNE1 Amplification

As a critical tumor suppressor, PTEN has gained much attention in cancer research. Emerging evidence suggests an association between PTEN status and clinical outcome in certain tumors, and may be predictive of response to several therapies. However, the significance of PTEN deficiency in tubo-ovarian high-grade serous carcinomas (HGSCs) is still poorly understood. We evaluated PTEN expression in HGSCs and determined its clinical relevance. A cohort of 76 HGSC specimens was profiled using tissue microarray. Immunohistochemistry (IHC) of PTEN, ER, PR, AR, CD8, FOXP3, and PD-L1 was performed. Targeted gene panel testing by massively parallel sequencing was performed in 51 cases. PTEN deficiency (complete or subclonal loss) detected by IHC was identified in 13 of the 62 HGSCs (21%) and was significantly correlated with reduced expression of ER and worse first progression-free survival (P < .05) but not with PD-L1 expression, the density of intratumoral T lymphocytes, or overall survival. In our cohort, tumor progression within 1 year of PARP inhibitor therapy was found more frequently in PTEN-deficient cases than in PTEN-intact cases (100% vs 52%). Molecular profiling showed that intragenic mutation or deletion was not the predominant mechanism for PTEN inactivation in HGSCs. In addition, CCNE1 amplification was found to be mutually exclusive with PTEN deficiency at both protein and DNA levels. An analysis of the genomic data from 1702 HGSC samples deposited with The Cancer Genome Atlas database obtained from cBioPortal confirmed the low rate of detection of PTEN gene alterations and the mutually exclusive nature of PTEN loss and CCNE1 amplification in HGSCs. These findings indicate that PTEN deficiency defines a distinct clinically significant subgroup of HGSCs with a tendency for ER negativity, wild-type CCNE1 status, inferior clinical outcomes, and potential drug resistance. These tumors may benefit from PI3K pathway inhibitors in combination with other ovarian cancer regimens, which deserves further investigation.