Relationship Between XRCC1 Arg399gln Polymorphism and Risk of Luminal Subtype Breast Cancer in Bali, Indonesia.

Introduction: Many studies have shown that the ALDH1 and Ki67 markers can accurately identify different types of cells in the breast. ALDH1 has proven to be a biomarker of breast cancer stem-like cells, while Ki67 has considered a proliferative marker. However, although Ki67 has been correlated with breast cancer grading, little attention has been given to the relationship between ALDH1 and histological grading. On the other hand, although ALDH1 has been correlated with intrinsic molecular subtype of breast cancer, there is an absence of literature between Ki67 and breast cancer subtypes. Thus, the goals of this study are to address the relationship between ALDH, Ki67 and histological grading in breast cancer. Methods and Material: Immunohistochemistry staining (IHC) for ALDH1 was performed on tissue micro array (TMA) of 200 patient samples with different histological grades of breast cancer. Similarly, IHC was used to identify and assess the expression of Ki67 on TMA of 66 patient samples of various breast cancer subtypes (40 Luminal and 26 TNBC). Both biomarkers were compared to respective normal breast tissue using t-test statistical analysis. Results: results showed that 51/129 (39.5%) of cancerous breast tissues were ALDH1 positive. ALDH1 positivity differed significantly between different grades cancerous breast tissues and normal tissue (p=0.0007). In addition, a direct relationship exists between increasing grade and increasing ALDH1 positivity, with grade 3 having the highest frequency and intensity of ALDH1 positivity (p=0.005). In normal tissue, 6/25 (24%) cores on the tissue microarray showed presence of Ki67. The average percentage of cells containing in Ki67 positive cores for normal tissue was 2.54%. In luminal subtypes, 30/38 (78.9%) patients on the tissue microarray showed presence of Ki67. The average percentage of cells containing Ki67 in luminal breast cancer, for patients with Ki67 positive cores was 3.93%, with a median of 2.5%. Ki67 was also present in 24/26 (92.3%) patients with triple negative breast cancer. Among the 24 patients with Ki67 present, an average of 6.91% of cells exhibited Ki67 in each core, with a median of 4.25%. A one-tailed student's T-test was used to determine statistical significance of the difference in Ki67 expression (including the patients with no Ki67 expression) between Luminal and Triple Negative subtypes of breast cancer with a p-value of 0.026 Conclusion: Our findings suggest that higher grades of breast cancer may contain higher quantities of breast cancer stem like cells. Given the role of Ki67 in cell proliferation, the increased presence of Ki67 in triple negative breast cancer, when compared to Luminal subtypes of breast cancer, may suggest a correlation with a worse prognosis. Identification of Ki67 positivity in breast cancer cells may aid in the development of prognosis and novel treatment techniques. Citation Format: Yahya I. Elshimali, Pierre Tamer, Sami Dwabe, Yanyuan Wu, Marianna Sarkissyan, Jaydutt V. Vadgama. The association between expression of ALDH1, KI67 and grading system in breast adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3887. doi:10.1158/1538-7445.AM2014-3887

Background. Immunotropic drugs are widely used in the modern strategy of cancer treatment. Importance is given to immunological markers of the tumor, which determine the prognosis of the disease, the effectiveness of treatment. Therefore, the study of their expression is one of the leading scientific directions. Of particular interest is the study of monomorphic HLA determinants, transferrin receptor 1 (TfR1), depending on its biological subtype of breast cancer.Aim. To evaluate the frequency of expression of HLA class I, II, TfR1 molecules by breast cancer cells and determine their relationship with the molecular biological subtype of the tumor.Materials and methods. This study included 120 patients with breast cancer who received treatment at the N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia. Tumor stages II and III prevailed: 56.7 % and 33.4 %, respectively. A moderate degree of differentiation (G2) was more often noted. The luminal subtype was 58.3 % (n = 70), non-luminal – in 41.7 % (n = 50). Immunophenotyping of the primary tumor was performed by immunofluorescence on cryostat sections. The reaction was evaluated using a ZEISS Axioscope 5 luminescent microscope (Zeiss AG, Germany). The frequency of expression of HLA class I and II molecules were studied depending on the clinical and morphological characteristics of breast cancer. The frequency of expression of HLA class I, HLA-DR, TfR1, molecules, toumor infiltration of СD45+, CD38+, depending on the molecular subtype of breast cancer was studied.Results. It was found that the frequency of expression of monomorphic determinants of the HLA class I in luminal and non-luminal subtypes of breast cancer was comparable; HLA-DR was expressed significantly more often in the luminal subtype of breast cancer: 37.3 % and 18.0 %, respectively, p = 0.022. The frequency of TfR1 expression was significantly higher in the luminal subtype of cancer compared to non-luminal, p = 0.014. Predominantly monomorphic type of reaction was observed: in 76.5 % (n = 39) of cases. The mosaic type of the TfR1 reaction was noted in 7.8 % of the samples. TfR1 monomorphic expression was detected in 50.0 % (n = 30) of cases in non-luminal cancer, the mosaic expression – in 20.0 % (n = 12) of cases. A pronounced degree of lymphoid infiltration, in particular plasmacytic, was established in non-luminal subtype of breast cancer: 70.7 % (n = 29) and 35.0 % (n = 14), respectively, p = 0.001. An association was noted between the expression of HLA I class molecules and the severity of general leukocyte infiltration, p = 0.007.Conclusion. The frequency of expression of HLA class I monomorphic determinants did not differ in molecular subtypes of breast cancer. The expression of the HLA class II molecule was significantly more frequently observed in the luminal subtype of breast cancer. The expression of HLA class I monomorphic determinants is associated with the degree of lymphoid infiltration of the tumor. In the non-luminal subtype, plasmacytic infiltration is more pronounced. The expression of transferrin receptors is significantly more pronounced in the luminal subtype.

Objective: According to St Gallen recommendations from 2013, estrogen receptor (ER), progesterone receptor (PR), HER-2, and Ki-67 defines two subtypes of ER-positive and HER-2 normal breast cancer (BC): Luminal A-like and Luminal B-like. Patients with Luminal B-like BC are often recommended chemotherapy in addition to endocrine therapy, whereas endocrine therapy may be sufficient for patients with Luminal A-like BC. Histological grade (G) 1, 2 and 3 are not included in the St Gallen recommendations. Our unpublished data from a series of 161 premenopausal N0 BC patients with long-term follow-up show that the classification of Luminal A-like vs Luminal B-like HER-2 normal BC is strongly associated to G. Luminal A-like BC is often G1 or G2 and Luminal B-like BC is usually G2 or G3. We also found that the few G3 (n=6) Luminal A-like cases had a prognosis more similar to Luminal B-like and that the few G1 (n=2) Luminal B-like HER-2 normal cases had a prognosis more similar to Luminal A-like. The aim of this study is to evaluate in other cohorts if these findings can be confirmed. Methods: G and St Gallen subtypes were evaluated in three BC cohorts from altogether 547 pre- and postmenopausal chemotherapy naïve T1-2N0-N1M0 patients. The endpoint was distant disease-free survival with 10 years of follow-up. We compared the Luminal A-like and the Luminal B-like HER-2 normal subtype definition according to the original St Gallen recommendation from 2013 based on ER, PR, HER-2 and Ki-67 (St Gallen 2013) with our proposal, where ER-positive, HER-2 normal, G1 BC is defined as Luminal A-like, independent of Ki-67 and PR, and ER-positive, G3 BC is defined as Luminal B-like, independent of Ki-67 and PR (St Gallen 2013+G). The importance of Ki-67 and PR for subtyping was thus restricted to G2 BC. Results: The hazard ratio (HR) between Luminal B-like HER-2 normal (n=185) and Luminal A-like (n=362) defined according to St Gallen 2013+G, was 2.3 (95% confidence interval (CI) 1.6-3.4; p<0.0001) compared to 1.6 (95% CI: 1.1-2.4; p=0.025) according to St Gallen 2013. Twenty-five patients, classified as Luminal B-like HER-2 normal with St Gallen 2013 were G1 and consequently reclassified as Luminal A-like with St Gallen 2013+G. None of these twenty-five patients developed metastases during the follow-up period. Thirty-eight patients showed the opposite pattern (G3 Luminal A-like with St Gallen but Luminal B-like according to St Gallen 2013+G). Seventeen of these patients developed distant metastases already during the first five years. Conclusion: Our findings strongly suggest that ER-positive, HER-2 normal, and G1 BC is a good prognosis group, independent of Ki-67 and PR, and should be treated as Luminal A-like BC, whereas ER-positive, HER-2 normal, and G3 BC should be considered as a worse prognosis group, independent of Ki-67 and PR, and should be treated as Luminal B-like BC. Based on our findings the importance of Ki-67 and PR is restricted to G2 BC for the discrimination between Luminal A-like and Luminal B-like HER-2 normal subtypes of BC. Citation Format: Anna Ehinger, Per Malmström, Pär-Ola Bendahl, Christopher W Elston, Anna-Karin Falck, Carina Forsare, Dorthe Grabau, Lisa Rydén, Olle Stål, Mårten Fernö. Histological grade provides significant prognostic information in the discrimination between luminal A-like and luminal B-like HER-2 normal subtypes of breast cancer according to St Gallen 2013 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-43.

Breast Cancer is a heterogeneous disease, and tumors vary in pathological and clinical characteristics making it important that tumors be accurately characterized at diagnosis. Current methods reveal a number of breast cancer subtypes correlating with clinical factors; however, patients with the luminal subtype have a range of outcomes. Our hypothesis is that microRNA (miRNA) expression profiling may provide a method of discriminating tumors in this subtype into good and poor prognosis groups, as well as reveal potential biological factors affecting prognosis. Expression profiling was performed on miRNAs from 39 primary ER+, HER2- luminal breast tumors from a prospective cohort of women with node negative breast cancer with a median follow-up time of 116 months. Of the 39 tumors, 19 were from patients who eventually experienced a recurrence and 20 were from matched patients who remained disease-free. Total RNA was extracted from the specimens and the miRNAs quantified using TaqMan Array MicroRNA Cards. Results analyzed using significance analysis of microarrays (SAM) revealed nine miRNAs with a standard t-test p-value less than 0.05 and ranked within the top 20, designated as significantly differentially expressed between the two groups. Four of these, miR-135a, miR-140-5p, miR-200a, and miR-218, were selected for validation on the same samples using singleplex quantitative real-time PCR conducted in triplicate, and confirmed to be significantly less expressed in tumors in patients who had experienced recurrence compared to tumors from patients without recurrence. Functional analysis of miR-200a and miR-218 is being performed in MCF-7 breast cancer cells to determine the role they play in luminal breast cancer recurrence. Validated targets of these miRNAs suggest involvement in epithelial to mesenchymal transition and cellular migration, invasion, and proliferation. The activity of these miRNAs is being studied in vitro and altered expression of miRNA targets is being confirmed. Future studies to examine whether these modifications result in changes in tumorgenicity phenotypes will be addressed by assaying cell proliferation, migration, and invasion. This project has the potential to aid in improving breast cancer prognostication in the clinical setting, improve our knowledge of the role if miRNAs in luminal breast cancer, and identify miRNAs suitable as possible diagnostic biomarkers or novel therapeutic targets. Citation Format: Dylan A. Ehman, Dushanthi Pinnaduwage, Shelley B. Bull, Irene L. Andrulis. Investigating differential expression of microRNAs in the luminal breast cancer subtype. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A023.

Genomic analyses have revealed that many RNA species other than protein-coding mRNAs are transcribed from mammalian genomes and may have profound effects on cellular physiology and pathology. One such class of RNAs are the long noncoding RNAs (lncRNAs). In our previous work using Global Run-On and sequencing (GRO-seq), we identified and annotated ~1,900 lncRNAs in MCF-7 human breast cancer cells, which are differentially expressed across the distinct molecular subtypes of breast cancer. Further meta-analysis identified lncRNA152 as a predictor of increased metastasis-free survival. Thus, lncRNA152 levels may serve as a biomarker to track disease progression in breast cancer patients. However, the precise mechanisms by which lncRNA152 regulates these clinical outcomes remain largely unknown. Interestingly, lncRNA152 is highly expressed in the luminal subtypes of breast cancer, but downregulated in triple-negative breast cancers (TNBC), suggesting that lncRNA152 expression may be associated with more aggressive cancer features. Through multiple complementary experimental approaches, we found that (1) FoxA1 regulates lncRNA152 transcription, (2) knockdown of lncRNA152 inhibits the growth, but promotes cell migration and invasion, of luminal breast cancer cell subtypes, and (3) ectopic overexpression of lncRNA152 inhibits TNBC cell growth, migration, and invasion. Importantly, our xenograft studies provide evidence that lncRNA152 inhibits estrogen-dependent tumor growth of luminal and TNBC cells. In addition, transcriptome analysis of TNBC cell-derived xenografts indicates that lncRNA152 expression downregulates many genes that are involved in biological processes such as cell growth, migration, invasion, and angiogenesis. Collectively, these results suggest that lncRNA152 is a novel tumor suppressor in luminal and basal subtype of breast cancer. This work is supported by grants from the Cancer Prevention and Research Institute of Texas (CPRIT RP160318/RP190235) to W.L.K. Citation Format: Daeseok Kim. Functional characterization of lncRNA152 in ER+ and triple-negative breast cancers [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 P1-05-06.

Context: The majority of BRCA mutation carriers diagnosed with breast cancer (BC) are treated with chemotherapy. The effectiveness of standard neoadjuvant chemotherapy (NAC) in BRCA associated BC compared to noncarriers has been poorly explored. Objectives: To assess whether the BRCA mutation status modifies the immune infiltration, chemosensitivity, and prognosis of breast cancer. Methods: We retrospectively identified in our institutional database all consecutive patients with BRCA germline mutation status available treated with NAC between 2002 and 2012. Microbiopsy specimens and paired surgical samples were evaluated for pre-NAC and post-NAC immune infiltration (stromal TILs, str TILs; intratumoral TILs, IT TILs). Response to chemotherapy was assessed by pathological complete response (pCR) rates. Association of clinical and pathological factors with pCR, overall survival (OS), and relapse free survival (RFS) was assessed by univariate and multivariate analyses. Results: Overall, 267 patients were included in this study (46 BRCA carriers and 221 BRCA noncarriers). The median age at BC diagnosis was 40 years old, and most of the patients (n=227, 85%) were premenopausal. Patients repartition by subtype was as follows: luminal (n=90, 33.7%), TNBC (n=110, 41.2%), HER2-positive (n=67, 25.1%). BRCA mutation carriers were likely to have familial history of BC (73.9% vs. 52.3%, p = 0.012), and be diagnosed with TNBC (58.7% vs 37.6%; p = 0.006), than noncarriers. No pattern was significantly different between BRCA mutation subgroups regarding age, body mass index, histology, tumor size, grade or Ki67. TIL levels were available in 192 patients. Neither pre-NAC stromal TIL levels nor IT TILs were significantly different by BRCA status in the whole population, nor in each BC subtype. PCR rates were significantly higher in BRCA mutation carriers (p= 0.035), and this association remained statistically significant only in the luminal BC subtype (p=0.006) after stratification by BC subtype (Pinteraction= 0.056). After multivariate analysis, only BC subtype and pre-NAC str TILs were independent predictors of pCR. Post-NAC stromal and intra-tumoral TIL levels were significantly higher luminal subtype (p=0.009 and p=0.019, respectively). After a median follow-up of 90 months, RFS and OS were not different between BRCA carriers and noncarriers, neither in the whole population nor after stratification by BC subtype. Discussion: In our study, BRCA status was associated with an enhanced response to standard NAC, particularly in luminal BC patients, in addition to higher post-NAC TIL levels. Whether patients with luminal BC and BRCA mutation derive benefit from second line immunotherapy after NAC completion remains to determine. Citation Format: Beatriz Grandal, Clémence Evrevin, Eric Daoud, Elise Dumas, Nadir Sella, Clara Sebbag, Sonia Rozette, Isabelle Jardin, Lucie Laot, Florence Coussy, Claire Saule, Dominique Stoppa-Lyonnet, Sophie Franck, Claire Sénéchal, Enora Laas, Marick Lae, Diane De Croze, Fabien Reyal, Anne-Sophie Hamy. Impact of BRCA mutation status on immune infiltration, chemosensitivity, and prognosis of breast cancer patients treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-10.

BackgroundLuminal subtype breast cancer accounts for a predominant number of breast cancers. Considering the heterogeneity of the disease, it is urgent to develop novel biomarkers to improve risk stratification and optimize therapy choices. Long non-coding RNA (lncRNA) represents an emerging and understudied class of transcripts that play a significant role in cancer biology. Growing knowledge of cancer-associated lncRNAs contributes to the development of molecular markers for prognosis evaluation and gene therapy.Materials and methodsThree pairs of primary luminal subtype breast cancer tissues and adjacent non-cancerous tissues were collected and sequenced. EBseq algorithm was used to identify differentially expressed lncRNAs. RNA sequencing data from The Cancer Genome Atlas (TCGA) database were used to validate the robustness of our RNA-seq results. Kaplan–Meier and Cox regression analyses were utilized to assess the association between the lncRNAs and overall survival of patients in TCGA cohort.ResultsA total of 796 lncRNAs were significantly dysregulated in luminal subtype breast cancer, including 436 upregulated and 360 downregulated lncRNAs. Among them, FAM83H antisense RNA 1 (FAM83H-AS1) was the most upregulated lncRNA, whereas GSN antisense RNA 1 (GSN-AS1) was the most downregulated lncRNA. Moreover, we proved that the high expression level of FAM83H-AS1 indicated unfavorable prognosis not only in luminal subtype breast cancer but also in all subtype breast cancers. To the best of our knowledge, this is the first report indicating that FAM83H-AS1 was involved in luminal subtype breast cancer and was an independent prognostic indicator.ConclusionOur study provides a rich resource to the research community for further identifying lncRNAs with diagnostic and therapeutic potentials and exploring biological function of lncRNAs in luminal subtype breast cancer.

Background: Tumor growth and development is determined by the mutual interaction between the cancer cells themselves and the microenvironment. It contains various elements, including immune cells. Of all, mast cells have one of the most controversial roles. The aim of the present study was to evaluate the expression of mast cell tryptase in the luminal and non-luminal subtypes of breast cancer and establish a possible link between infiltration with MCs and expression of hormone receptors. Material and methods: The experimental study included 80 cases of breast carcinomas that were analyzed immunohistochemically in order to establish the molecular profile and the expression of tryptase, a specific marker of mast cells. The data were processed using the SPSS program. Pearson’s coefficient (r) and the other values were considered statistically significant in case of p≤0.05. Results: Both intratumoral mast cells (MCit) and peritumoral mast cells (MCpt) correlated with the expression of hormone receptors for estrogen (ER) and progesterone (PR). Thus, the following relations were established: MCit and ER (r=0.343, p=0.002), MCpt and ER (r=0.394, p=0.000295) and MCpt and PR (r=0.386, p=0.000409). Statistically significant correlations between HER2 expression and mast cells content have not been established. Conclusions: Mast cells invasion, peri- and intratumoral, is strongly influenced by the expression of hormone receptors. The luminal subtypes of breast cancer are characterized by a higher density of mast cells.

Identification of molecular signatures that allow detection of the transition from normal breast epithelial cells to malignant invasive cells is a critical component in the development of diagnostic, therapeutic, and preventative strategies for human breast cancer. Substantial efforts have been devoted to deciphering breast cancer etiology at the genome level, but only a limited number of studies have appeared at the proteome level. In this work, we compared individual in situ proteome profiles of nonpatient matched nine noncancerous, normal breast epithelial (NBE) samples with nine estrogen receptor (ER)-positive (luminal subtype), invasive malignant breast epithelial (MBE) samples by combining laser capture microdissection (LCM) and quantitative shotgun proteomics. A total of 12,970 unique peptides were identified from the 18 samples, and 1623 proteins were selected for quantitative analysis using spectral index (SpI) as a measure of protein abundance. A total of 298 proteins were differentially expressed between NBE and MBE at 95% confidence level, and this differential expression correlated well with immunohistochemistry (IHC) results reported in the Human Protein Atlas (HPA) database. To assess pathway level patterns in the observed expression changes, we developed protein set enrichment analysis (PSEA), a modification of a well-known approach in gene expression analysis, Gene Set Enrichment Analysis (GSEA). Unlike single gene-based functional term enrichment analyses that only examines pathway overrepresentation of proteins above a given significance threshold, PSEA applies a weighted running sum statistic to the entire expression data to discover significantly enriched protein groups. Application of PSEA to the expression data in this study revealed not only well-known ER-dependent and cellular morphology-dependent protein abundance changes, but also significant alterations of downstream targets for multiple transcription factors (TFs), suggesting a role for specific gene regulatory pathways in breast tumorigenesis. A parallel GOMiner analysis revealed both confirmatory and complementary data to PSEA. The combination of the two annotation approaches yielded extensive biological feature mapping for in depth analysis of the quantitative proteomic data.

TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.

Aims:Forkhead box A1 (FOXA1) is a forkhead family transcription factor expressed in breast cancer cells. It is essential for optimal expression of ∼50% of oestrogen receptor (ER)-related genes. This study...

BACKGROUND: Metastasis is the leading cause of mortality in luminal subtype breast cancer. While cluster of differentiation (CD)44 has been widely studied, the prognostic relevance of its isoforms particularly the CD44v6/CD44s ratio remains unclear. This study evaluates CD44v6, CD44s, vascular endothelial growth factor (VEGF), and the CD44v6/CD44s ratio as potential prognostic biomarkers for metastasis in luminal breast cancer.METHODS: This case-control study included 38 luminal subtype breast cancer patients (18 with metastasis, 20 without metastasis). Serum levels of CD44v6, CD44s, VEGF, and the CD44v6/CD44s ratio were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Statistical analyses included ROC analysis to determine optimal cut-off points, logistic regression to assess risk factors, and correlation analysis for biomarker relationships.RESULTS: A low CD44v6/CD44s ratio (<0.03) was identified as a significant independent factor for metastasis (adjusted OR 7.0, 95% CI: 1.2–40.6, p=0.03). While serum levels of CD44v6, CD44s, and VEGF were higher in the metastasis group, these individual markers showed a non-significant trend toward association with metastasis. A strong positive correlation was observed between CD44s and VEGF levels (r=0.7, p<0.01).CONCLUSION: The CD44v6/CD44s ratio showed a significant association with metastasis and may have potential as a prognostic marker in luminal breast cancer. Further studies with larger sample sizes are needed to confirm these findings.KEYWORDS: CD44v6, CD44s, VEGF, CD44v6/CD44s ratio, luminal breast cancer, metastasis

The definitive characterization of common cancer stem cell (CSCs) subpopulations in breast cancer subtypes with distinct genotypic and phenotypic features remains an ongoing challenge. In this study, we have used a non-biased genome wide screening approach to identify transcriptional networks that may be specific to the CSC subpopulations in both luminal and basal breast cancer subtypes. In depth studies of three CSC-enriched breast cancer cell lines representing various subtypes of breast cancer revealed a striking hyperactivation of the mevalonate metabolic pathway in comparison to control cells. The upregulation of metabolic networks is a key feature of tumour cells securing growth and proliferative capabilities and dysregulated mevalonate metabolism has been associated with tumour malignancy and cellular transformation in breast cancer. Furthermore, accumulating evidence suggests that Simvastatin therapy, a mevalonate pathway inhibitor, could affect breast cancer progression and reduce breast cancer recurrence. When detailing the mevalonate pathway in breast cancer using a single-cell qPCR, we identified the mevalonate precursor enzyme, HMGCS1, as a specific marker of CSC-enriched subpopulations within both luminal and basal tumour subtypes. Down-regulation of HMGCS1 also decreased the CSC fraction and function in various model systems, suggesting that HMGCS1 is essential for CSC-activities in breast cancer in general. These data was supported by strong associations between HMGCS1 expression and aggressive features, such as high tumour grade, p53 mutations as well as ER-negativity in lymph node positive breast cancer. Importantly, loss of HMGCS1 also had a much more pronounced effect on CSC-activities compared to treatment with standard doses of Simvastatin. Taken together, this study highlights HMGCS1 as a potential gatekeeper for dysregulated mevalonate metabolism important for CSC-features in both luminal and basal breast cancer subtypes. Pharmacological inhibition of HMGCS1 could therefore be a superior novel treatment approach for breast cancer patients via additional CSC blocking functions.

Background: Molecular subtypes of breast cancer have been characterized by gene expression analysis. Luminal subtypes are hormone receptor (HR) positive and Her2/neu negative. Immunohistochemistry (IHC) has been used as a surrogate test for gene expression. Ki67 is a proliferation marker that identifies high-risk subtype of luminal breast cancer. Recently, the proposed Ki67 index (KI) of 14% was suggested as a cut-off to distinguish between luminal A and luminal B tumors (JNCI. 2009;101:736-750). The oncotype dx (ODx) is a 21- gene test that provides prognostic and predictive information in early stage HR-positive breast cancer patients. The test is reported as low (<18), intermediate (18-30) and high (>30) risk recurrence scores (RS) Design: We investigated the relationship between ODx RS and luminal subtypes of breast cancers using the KI of 14% as the cut-off for distinguishing luminal A and B tumors. Biomarker analysis (ER, PR, Her2/neu, Ki67 and p53) was performed as part of the diagnostic work-up using standard IHC procedures. Scoring was done by automated image analysis. Her2/neu FISH was performed on all IHC 2+ and 3 + results. Pathologic parameters such as, tumor size, grade, and presence of LVI were evaluated. Ploidy was performed by the Autocyte system (Tripath) on Feulgen stained paraffin sections. Results: We identified 106 patients with HR positive breast cancer who were tested for ODx from February 2006 to May 2010. 85/106 had Ki67 data available for analysis. 46/85 (54%) were luminal A and 39/85 (46%) luminal B tumors. The mean KI in luminal A was 6.93% versus 31.1% in luminal B (P<0.0001). The mean tumor size was 1.94 and 1.92 cm in luminal A and B respectively. Grade 1, 2 and 3 comprised 28/85 (32.9%), 49 (57.6%) and 8 (9.41%) of all tumors respectively. Of the grade1 tumors, 75% were luminal A, and grade 3 tumors were predominantly luminal B (p=0.013). LVI was present in 16 cases, 11 (68.8%) in luminal B and 5/16 (31.2%) in luminal A tumors (p=0.0416). Luminal A tumors were predominantly diploid 30/45 (66.6%) and luminal B were mostly aneuploid 21/32 (65.6%) (p=0.019). The overall mean RS in luminal A and B tumors was 14.67 and 20.15 respectively (P<0.0004). Luminal A tumors had low RS in 32/46 (66.6%). and luminal B tumors had predominantly intermediate/high RS in 23/39 (62.1%) (p=0.0082). ER Allred Scores were 7.1 and 7.3 and percent positivity was 88.1% and 91% respectively for luminal A and B subtypes. PR Allred scores were 5.6 and 5.8, and percent positivity was 62.1 and 52.9% for A and B tumors respectively. Information regarding treatment was available in 72 cases. 19 (26.3%) were treated by a combination of chemo and anti-hormonal therapy and 53 (73.6%) were treated by anti hormonal therapy alone, 8 of the 19 (42.1%) luminal A patients received combination therapy versus 11 (57.8%) in the luminal B category (p=0.277). Conclusion: Ki67 is a useful marker that showed significant correlation with RS by ODx. Luminal A tumors are more likely to be low grade, diploid with low RS compared to luminal B tumors. Ki67 in conjunction with other pathologic parameters may serve as a surrogate marker for the ODx RS. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-07.

Breast cancer is a heterogeneous collection of diseases grouped by hormone receptor status or by expression of key subtype-determining genes. Breast cancer subtypes, in particular basal-like breast cancer and the luminal breast cancer subtypes, differ by hormonal receptor status, proliferation, genomic instability and mutational signatures, treatment response, and prognosis. The highly distinct signatures of basal-like and luminal breast cancer suggest that they may have different cells of origin within the breast duct. As part of the Washington University Human Tumor Atlas Network (WU-HTAN) program, we generated multi-omic data for 53 samples from 37 breast cancer tumors and 4 normal adjacent tissues. Genomic subtyping was applied to both bulk and single-nucleus RNA sequencing. Analysis of single-nucleus gene expression and chromatin accessibility in epithelial cells underscores similarities between basal-like breast cancer and luminal progenitor cells within the breast duct, and between luminal breast cancer and mature luminal ductal cells. This study links distinct breast cancer subtypes to normal breast cell populations and suggests distinct cells of origin for these cancer types. Citation Format: Michael D. Iglesia, Reyka G. Jayasinghe, Daniel Cui Zhou, Nadezhda V. Terekhanova, John Herndon, Alla Karpova, Siqi Chen, Nataly Naser Al Deen, Kazuhito Sato, Feng Chen, Deborah J. Veis, Ryan C. Fields, William E. Gillanders, Li Ding. Multi-omic characterization of transitional cell populations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2936.