Tumors Of Breast Cancer Patients Research Articles

Immuno-hormonal regulation of tumor proliferation in breast cancer patients

It is well known that tumor cells proliferation is regulated by sex steroid hormones, estradiol and progesterone (E2 and Pg) in breast cancer patients (BCP). Moreover, specific auto-antibodies to estrogen receptor (ERα) were detected in blood serum of BCP. Their levels positively correlated with the percentage of Ki-67 expressing breast cancer cells. We proposed that antiidiotypic auto-antibodies to E2 and Pg (IgG2-E2 and IgG2-Pg) could act as antibodies against membrane ER and progesterone receptor (PR). Our study aimed for research of IgG2-E2 and IgG2-Pg according to E2 and Pg serum levels in association with Ki-67 positive tumors in BCP. Antiidiotypic antibodies were studied in ER-positive patients with breast cancer (stage I, n = 374, and stage II-IV, n = 379,) using ELISA technique with monoclonal antibodies against E2 and Pg as adsorbed antigens. Blood serum concentrations of E2 and Pg were measured using “ImmunoEA-Estradiol” and “ImmunoEA-Progesterone” test-systems (“Immunotech”, Russia). Tumor Ki-67 was studied by standard immunohistochemical technique at the Kemerovo Oncological Hospital. Higher percentage of Ki-67 positive breast cancer cells (Ki-67 30) was increased in BCP II-IV stages compared with stage I patients (50.7% vs 29.8%, p 0,001). Such increased values were detected for the BCP with low levels of both IgG2-E2 and IgG2-Pg antibodies in the following subgroups: 1) at low serum E2 concentration of ≤ 200 pmol/L (50.9% vs 21.3%, р 0.001); 2) at the E2 exceeding 200 pmol/L (74.2% vs 34.1%, р = 0.003); 3) at the Pg levels under 600 pmol/L (60.6% vs 22.2%, р 0.001); 4) at Pg values exceeding 600 pmol/L (58.5% vs 30.8%, р = 0.02). Similar differences were not revealed between stage II-IV and stage I BCP with low levels of both IgG2-E2 and IgG2-Pg. Corresponding Ki-67 30 indices were as follows: 1) 36.9% vs 22.0% (р = 0.14); 2) 48.4% vs 34.5% (р = 0.08); 3) 34.0% vs 27.7% (р = 0.75). Significant differences were detected in BCP with Pg 600 pmol/L and high IgG2-E2 and IgG2-Pg levels only: 48.1% vs 26.6%, (р = 0.01). Hence, antiidiotypic auto-antibodies to steroid hormones may participate in regulation of tumor proliferation in BCP. Immunoassay of IgG2-E2 and IgG2-Pg may be used for prognosis of tumor proliferation upon breast cancer progression.

The Expression Profiles of lncRNAs Are Associated with Neoadjuvant Chemotherapy Resistance in Locally Advanced, Luminal B-Type Breast Cancer.

lncRNAs are noncoding transcripts with tissue and cancer specificity. Particularly, in breast cancer, lncRNAs exhibit subtype-specific expression; they are particularly upregulated in luminal tumors. However, no gene signature-based laboratory tests have been developed for luminal breast cancer identification or the differential diagnosis of luminal tumors, since no luminal A- or B-specific genes have been identified. Particularly, luminal B patients are of clinical interest, since they have the most variable response to neoadjuvant treatment; thus, it is necessary to develop diagnostic and predictive biomarkers for these patients to optimize treatment decision-making and improve treatment quality. In this study, we analyzed the lncRNA expression profiles of breast cancer cell lines and patient tumor samples from RNA-Seq data to identify an lncRNA signature specific for luminal phenotypes. We identified an lncRNA signature consisting of LINC01016, GATA3-AS1, MAPT-IT1, and DSCAM-AS1 that exhibits luminal subtype-specific expression; among these lncRNAs, GATA3-AS1 is associated with the presence of residual disease (Wilcoxon test, p < 0.05), which is related to neoadjuvant chemotherapy resistance in luminal B breast cancer patients. Furthermore, analysis of GATA3-AS1 expression using RNA in situ hybridization (RNA ISH) demonstrated that this lncRNA is detectable in histological slides. Similar to estrogen receptors and Ki67, both commonly detected biomarkers, GATA3-AS1 proves to be a suitable predictive biomarker for clinical application in breast cancer laboratory tests.

A prospective phase II study of neoadjuvant therapy guided by personalized drug-testing on patient-derived tumor-like cell clusters for early breast cancer.

e12625 Background: Individual tumors of breast cancer patients possess unique characteristics that may influence their neoadjuvant therapy (NAT) response to specific cytotoxic agents and treatment choices. Therefore, it is essential to have a platform that can assist in the personalized selection of chemotherapy for individual patients. Previously, we reported an in vitro model called patient-derived tumor-like cell clusters (PTC), which can structurally and functionally recapitulate original tumors. Retrospective research has shown that PTC hold potential as reliable pre-clinical drug testing model for guiding NAT therapeutic options in early breast cancer patients. Methods: This prospective, phase II, open-label study aimed to validate the efficacy of drug screening on patient-derived tumor-like cell clusters (PTCs). TNBC and HER2 positive patients received neoadjuvant therapy based on subtype and personalized drug testing on PTCs. TNBC patients were treated based on drug testing, receiving either TA (taxanes combined with anthracyclines) or TP (taxanes combined with carboplatin). HER2 positive patients received treatment based on drug testing, including THP (taxanes, trastuzumab and pertuzumab) or TCbHP (taxanes, carboplatin, trastuzumab, and pertuzumab). Sample size calculation was performed using Fleming's two-stage design. For TNBC patients, if 10 or more out of 20 patients achieved pCR in the first stage, an additional 26 patients would be enrolled in the second stage. For HER2-positive patients, the sample size was same with TNBC patients. The study was approved by the Ethics Committee of Peking University People's Hospital in Beijing and was registered on ClinicalTrials.gov (NCT04750122, NCT04836156). This time, we report the results of the first stage. Results: Among 20 enrolled TNBC patients in the first stage, 15 achieved pCR with the rate of 75%. 17 patients (85%) had RCB 0-I score while 3 patients (15%) had an RCB-II score. 14 patients received TP as it demonstrated greater sensitivity in vitro. Out of these 14 patients, 10 patients (71.4%) achieved pCR. Six patients received TA as it was more effective in drug testing. Among these patients, 5 (83.3%) patients achieved pCR. Out of 20 enrolled HER2-positive patients, 16 achieved pCR, resulting in a pCR rate of 80%. Six patients received THP treatment based on their sensitivity to this regimen in drug testing. Among these patients, 5 (83.3%) achieved a pCR. 14 patients who were sensitive to TCbHP but not to THP in drug testing received TCbHP treatment. Among these patients, 11 (78.6%) achieved pCR. The non-pCR patients had 2 RCB-II score and the ORR was 100% for all 14 patients. Conclusions: Our results demonstrate that personalized drug testing on PTCs can effectively guide the choice of neoadjuvant therapy for early TNBC and HER2-positive breast cancer patients. Clinical trial information: NCT04750122 , NCT04836156 .

Abstract PS03-01: Single cell analysis of paired lymph nodes and primary tumors in breast cancer patients

Abstract A sentinel lymph node (SN) is the primary node draining the tumor and is assumed to be affected early in the metastatic process. The sentinel node holds a key position in the immune response against tumor in breast, and represents a unique connection between the tumor and the host immune response. We hypothesize that the immune profile in the primary tumor and the paired lymph node (LN) is different during tumor progression. 3.7 million single cells from paired primary tumor and lymph nodes from 33 breast cancer patients (Oslo2 cohort) was analyzed by single cell mass cytometry (CyTOF) with a 47 antibody immune panel and characterized by a semi-automatic gating approach (FlowSOM). Tumor cells were found in 11 LN. When analyzing the leucocytes from LN we identified a significant difference in immune cell type skewing towards higher abundance of memory CD4 and CD8 T cells expressing an exhausted phenotype in LN with metastasis. In addition, a higher abundance of activated Tregs and significantly lower abundance of resting Tregs was found in the metastatic LNs compared to the sentinel lymph nodes. The change in immune composition and exhaustion was correlated to the metastatic tumor burden. The skewing towards an exhausted immune profile was also found in larger primary tumors compared to smaller primary tumors. We further analyzed tumor cells from 8 patients with paired primary tumor and LN. No differences were identified in the primary tumor when stratified by LN status, but LNs with smaller metastasis expressed lower levels of epithelial markers essential in the Epithelial-to-Mesenchymal Transition such as E-cadherin, Pan Cytokeratin and EpCAM – this in contrast to the LN with manifested metastasis in the axilla which expressed higher levels of epithelial markers and lower levels of mesenchymal markers such as Vimentin and CD44. We identified a skewing in the immune profile from a naive phenotype towards a memory and exhausted phenotype in CD8 and CD4 T cell population in LN with manifested metastasis, as well as in larger primary tumors. We also identified that tumor cells in smaller metastatic tumors resembled a “mesenchymal like” phenotype compared to in the larger manifested tumors. These results suggest that the immune suppression is correlated with the tumor burden. Citation Format: Marit Fjørtoft, Inga Rye, Kanutte Huse, June Myklebust, Hege Russnes, Margit Riis, Ole Christian Lingjærde, Øystein Garred, Karin Teien Lande, Inger Riise Bergheim, Ellen Schlichting. Single cell analysis of paired lymph nodes and primary tumors in breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-01.

Level of polychlorinated biphenyls in tumor and blood serum of breast cancer patients and control subjects from Punjab, Pakistan

The current study examined the level of Polychlorinated biphenyls (PCBs) in tumor and blood serum of female breast cancer patients and control individuals recruited from Punjab, Pakistan. Breast tumor and blood serum from 40 patients and only blood serum from ten control subjects were obtained and concentration of 32 PCB congeners was analyzed through Gas chromatography coupled with Mass spectrophotometry. Sociodemographic variables of the patients along with essential clinical and haematological parameters were taken as covariates. Tumor reflects the highest median (min-max) concentration (ng g−1 lw) of ƩPCBs at 115.94 (0.05–17.75) followed by 16.53 (0.09–2.94) and 5.24 (0.01–0.59) in blood serum of cancer patients and control group respectively. Median concentrations (ng g−1 lw) of non-dioxine like ƩPCBs were considerably higher at 83.04, 32.89 and 4.27 compared to 13.03 and 3.50 and 0.97 for dioxin like ƩPCBs in tumor, serum of breast cancer patients and control subjects respectively. PCB-87 was most dominant congeners in tumor followed by PCB-170 and -82 whereas PCB-28 and -52 reflected greatest contribution in serum of breast cancer patients. Blood haemoglobin, potassium and chloride ions showed significant positive whereas body mass index reflect inverse relationship when regressed with ƩPCBs in tumor. This pioneer study depicts elevated concentrations of PCBs in patients compared to control, reflecting potential positive association of PCBs with breast cancer which need further confirmation. We concluded that chronic exposure to PCBs might be associated with an increasing number of breast cancer incidences in developing countries like Pakistan, which should be further elucidated through detail in vitro and in vivo studies.

Abstract 1275: In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis

Abstract Metastasis is responsible for about 90% of cancer-related mortality. Throughout the metastatic cascade, cancer cells requires substantial metabolic rewiring to adapt the nutrient micro-environmental changes enabling dissemination and seeding in distant organs. Recent insights into importance of the tumor-intrinsic metabolic reprograming has been implicated in immune escape, yet altered immune responses imposed by tumor metabolism during metastasis are poorly understood. Here, we generated genetic perturbations in triple-negative breast cancer cells using metabolism-focused CRISPR knockout library and performed in vivo screens in both immunocompetent and immunodeficient mice. By comparing enriched sgRNAs in lung metastases or primary tumors, we identify loss of one gene related to long chain fatty acid (LCFA) β-oxidation (FAO) potentiates metastatic dissemination in immunocompetent mice but no in immunodeficient mice, suggesting that impaired FAO in tumor cells promotes breast cancer lung metastasis through an lymphocytes-dependent manner. We confirmed individual sgRNAs targeting this FAO gene from the primary screen dramatically accelerate metastasis in different breast cancer orthotopic models. Specifically, we find that low expression of this gene in primary tumors of advanced breast cancer patients with high grade tumors and positive lymph nodes (LN+) is associated with reduced overall survival. In addition, deletion of this gene generates an immunosuppressive microenvironment in the lung metastatic tumors with increased neutrophils, monocytes and decreased TNFα+-IFNγ+ CD8+ T cells. Mechanistically, impaired FAO in tumor cells causes mitochondria imbalance under lipid metabolism stress, consequently triggers inflammatory response on establishing a neutrophil-rich metastatic niche via mitochondrial nucleic acid-driven innate immunity. Altogether, our data provides a new insight on how tumor cell lipid metabolism impacting immune interplay during metastatic progression, nominating potential anti-tumor immunotherapies. Citation Format: Xiaoyong Wang, Shu-ting Chou, Verra Ngwa, Yoonha Hwang, Breelyn Karno, Deanna Edwards, Jin Chen. In vivo functional CRISPR screens identifies metabolic dependencies mediating triple negative breast cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1275.

Abstract 1750: Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration

Abstract Breast cancer's complex transcriptional landscape requires a deep understanding of sample and cell diversity to identify effective treatments. In this study, we amalgamate single-cell RNA sequencing data from breast cancer patient-derived xenografts (PDX), organoids, cell lines, patient tumors and reduction mammoplasties resulting in a comprehensive dataset of 117 samples with 506,719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 enriched (HER2E), and triple-negative breast cancer (TNBC) subtypes. We aimed to delineate similarities and distinctions across model systems and patient samples while also exploring stratification of therapeutic drug efficacy based on subtype proportions within tumors. Mammary tumor PDXs, organoids, and established cell lines exhibited higher proliferation and lower heterogeneity observed via UMAP dimensionality reduction compared to most patient tumors or normal breast epithelium. TNBCs had elevated proliferative and pro-metastatic signatures compared to HR+ and HER2E samples. Interestingly, compared to matched PDX tumors, organoids from these same models were found to exhibit stark differences in gene expression, including upregulation of metabolically active aldo-keto reductase family genes, highlighting differences in the model systems with implications for pre-clinical drug testing. Single-cell tumor subtyping analyses with scSubtype and TNBCtype methods found that therapeutically treated samples had shifts in the proportions of cell-wise subtype annotations when compared to matched untreated samples. Similarly, patient lymph node metastasis when compared with matched primary tumors were significantly linked to decreases in Basal-like and HER2-enriched cell-wise annotations in untreated ER+ samples. In vitro assessment of anti-cancer compounds on PDX cells showed significant correlation of subtype proportion with cell viability following treatment with targeted therapeutic agents. This subtyping methodology offers a powerful tool to monitor the evolving gene expression landscape within samples and predict responses to therapeutic agents. We present here a dynamic approach to cell-wise sample annotation and a substantial multi-model dataset for use facilitating informed decision-making in preclinical research and therapeutic development. Citation Format: Julia E. Altman, Carson J. Walker, Emily K. Zboril, Nicole S. Hairr, Rachel K. Myrick, David C. Boyd, Jennifer E. Koblenski, Madhavi Puchalapalli, Bin Hu, Mikhail G. Dozmorov, Xi Chen, Yunshun Chen, Charles M. Perou, Brian D. Lehmann, Jane E. Visvader, Amy L. Olex, J. Chuck Harrell. Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1750.

Age-related phenotypes in breast cancer: A population-based study.

Breast cancer in young (<40 years) is associated with a higher frequency of aggressive tumor types and poor prognosis. It remains unclear if there is an underlying age-related biology that contributes to the unfavorable outcome. We aim to investigate the relationship between age and breast cancer biology, with emphasis on proliferation. Clinico-pathologic information, immunohistochemical markers and follow-up data were obtained for all patients aged <50 (Bergen cohort-1; n = 355, not part of a breast screening program) and compared to previously obtained information on patients aged 50 to 69 years (Bergen cohort-2; n = 540), who participated in the Norwegian Breast Cancer Screening Program. Young breast cancer patients presented more aggressive tumor features such as hormone receptor negativity, HER2 positivity, lymph-node metastasis, the HER2-enriched and triple-negative subtypes and shorter survival. Age <40 was significantly associated with higher proliferation (by Ki67). Ki67 showed weaker prognostic value in young patients. We point to aggressive phenotypes and increased tumor cell proliferation in breast cancer of the young. Hence, tumors of young breast cancer patients may present unique biological features, also when accounting for screen/interval differences, that may open for new clinical opportunities, stratifying treatment by age.

Abstract A015: Mutations in mismatch repair genes that alter subcellular localization of the protein predict resistance to standard therapy but response to cell cycle inhibitors

Abstract Hormone positive breast cancer is one of the most common cancer types in U.S. Every year, over 150,000 patients are detected with this cancer type of which, &amp;gt;40,000 patients will go on to die of their disease. We recently identified defects in a principal mismatch repair (MMR) gene, MLH1, as a driver of resistance to standard-care treatment and poor patient outcomes. Moreover, we demonstrated increased sensitivity to palbociclib, a CDK4/6 inhibitor, in experimental model systems in vitro and in vivo and in clinical trial data. MLH1 is mutated in 5-10% of treatment-resistant tumors, i.e., 4,000 patient tumors every year. However, most of these mutations are missense and variants of unknown significance (VUS). The impact of these mutations on mismatch repair capability and on treatment resistance remains uncertain. Here, we use experimental model systems (ER+/HER2- breast cancer cells, organoid models, and patient tumors) to study how variants of MLH1 and its subcellular localization predicts resistance to standard endocrine therapy but sensitivity to cell cycle inhibitors. We also assessed the ability of nuclear MLH1 to induce activation of G1/S cell cycle checkpoints by activating Chk2. Currently, there are few tests available in the clinic to predict response to therapies early in the timeline of hormone positive breast cancer patients. Due to the nature of the disease, this can result in years of overtreatment. Overtreatment comes at great financial, physical, and emotional cost to patients and their families. We for the first time show that stalling nuclear translocation of MLH1 inhibits Chk2 activation which counterbalances CDK4/6 activity. Therefore, cytoplasmic Mlh1 may be first in-class biomarker for CDK4/6 response in cancer types. Citation Format: Aloran Mazumder, Svasti Haricharan. Mutations in mismatch repair genes that alter subcellular localization of the protein predict resistance to standard therapy but response to cell cycle inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A015.

Up Regulation of Ezrin and Radixin with respect to Grade of Tumors in Breast Cancer Patients

Background: Breast cancer (BC) is one of the main causes of death among women in Iran. Biomarkers involved in promotion and progression of disease is very important in management and control of BC outcomes. In this research, we aim to estimate the expression levels of Ezrin and Radixin, as two important factors in morphogenesis, endocytosis, exocytosis, adherence, and migration of cells, in BC patients and their relationship with pathological factors.&#x0D; Methods: One hundred and thirteen BC patients were involved in this research. Relative expression of Ezrin and Radixin genes were estimated with quantitative real-time PCR. Pathological data include the histology, tumor size, grade, lymphovascular invasion and clinical TNM (Tumor, Node, and Metastasis) staging of patients were recorded based on the pathology report and their relationship with relative expression of Ezrin and Radixin were estimated.&#x0D; Result: According to result Ezrin were over expressed in tumor samples in comparison to adjacent normal tissue. There is a significant relationship between over expression of Ezrin and Radixin and grade of tumor and necrosis. Also there is a direct relationship between relative expression of Ezrin and Radixin expression.&#x0D; Conclusions: These data support the role of Ezrin and Radixin in the biology of breast cancer and additional studies needed that determine the Ezrin and Radixin associated with phenotype and may validate them as markers of cancer progression and as a potential target for cancer therapy.

Abstract B033: Multiplex analysis of the tumor immune microenvironment during treatment with atezolizumab/pelareorep/letrozole reveals novel immune-tumor interactions

Abstract Background: Pelareorep (pela) is a non-modified intravenously administered oncolytic reovirus exhibiting effective tumor suppression through both innate and adaptive immune responses, as well as direct tumor lysis. Findings from the AWARE-1 window of opportunity study previously demonstrated the synergistic potential of combining pela with atezolizumab (atezo), showcasing promising immunological reactions within tumors of early breast cancer (eBC) patients. To gain deeper insights into the complex tumor immune microenvironment (TiME) pre- and post-treatment, we employed imaging mass cytometry (IMC) to conduct high-dimensional, single-cell analysis of tissue samples. Methods: Patients (n=10) received pela (days 1, 2 and 8, 9), atezolizumab (day 3), and letrozole (day 1 to 21). Tumor biopsies (FFPE samples) were collected pre-treatment on day 3 (prior to atezolizumab administration) and on day ~21 (surgical excision). Samples from 8 out of 10 patients were subjected to IMC with a comprehensive panel of 37 antibodies for single cell investigation of TiME changes. Tumor, proliferation, T cell, macrophage, NK, and immunoregulatory markers were included in the panel. Results: Our first pass analysis of IMC images, showed an increase in proliferating (Ki67+) cytotoxic T cells adjacent to apoptotic (caspase 3+) tumor cells post-treatment (both D3 and D21) samples indicating immunogenic cell death. Conversely, we observed a decrease in Ki67+ and ER-positive tumor cells after treatment indicating decreased tumor cell proliferation and hormone expression. In 3 out of 8 patients, treatment was associated with a shift in monocyte/macrophages from an M1 (CD68+/CD163-) to M2 (CD68+/CD163+) phenotype associated with significant tumor infiltration. Surprisingly, in these patients we also noted a significant increase in apoptotic M2 macrophages on D21. Consistent with the known immune priming effects of pela, both PD-1 and PD-L1 increased on D3. Subsequently, on day 21, post atezo, tumor PD-L1 decreased on tumor cells while PD-1 expression persisted. Pela induced IDO expression on both tumor and monocyte/macrophage cells. Unlike its effect on PD-L1, atezo did not appear to attenuate IDO expression on day 21. We were also able to detect a treatment related increase in immune cell perivascular localization on D3 providing further evidence of TiME priming. Conclusions: The immune system plays a crucial role in regulating cancer progression; however, our understanding of immune interactions with tumors remains limited. Our study utilizing IMC revealed complex immune-tumor interactions changes during treatment with atezo/pela/letrozole. These findings provide valuable insights into the intricate dynamics of the TiME and may inform new therapeutic combination approaches in breast cancer treatment. Citation Format: Homa Dadrastoussi, Julian Olea, Eduardo Fernandez Hernandez, Hugo Lara Martinez, Kaijin Wu, Houra Loghmani, Thomas Heineman, Richard Trauger, Matt Coffey, Akil Merchant, Kevin Kelly. Multiplex analysis of the tumor immune microenvironment during treatment with atezolizumab/pelareorep/letrozole reveals novel immune-tumor interactions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B033.

A direct comparison of the diagnostic efficacy of alternative scaffoldbased radiopharmaceuticals [&lt;sup&gt;99m&lt;/sup&gt;Tc]Tc-ADAPT6 and [&lt;sup&gt;99m&lt;/sup&gt;Tc]Tc-(HE)&lt;sub&gt;3&lt;/sub&gt;-G3 in patients with HER2-positive breast cancer

Aim. To perform a direct comparison of the diagnostic efficacy of [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE) -G3 in HER2-positive breast cancer patients before the systemic treatment.Materials and methods. The study included 11 patients with HER2-positive breast cancer (T1–4N0–2M0–1) before the initiation of systemic treatment. All patients underwent a radionuclide examination with [99mTc]TcADAPT6 and [99mTc]Tc-(HE) -G3 with the interval of 3–4 days. Single-photon emission computed tomography (SPECT) /computed tomography (CT) was performed 2 and 4 hours after [99mTc]Tc-ADAPT6 and [99mTc]Tc(HE)3-G3 administration, respectively.Results. The analysis of [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE) -G3 distribution showed their high uptake in the kidneys and liver. Breast tumors were visualized in all cases. The average tumor uptake of [99mTc]Tc-ADAPT6 was 4.7 ± 2.1, which was significantly higher than in the [99mTc]Tc-(HE)3-G3 injection (3.5 ± 1.7) (p &lt; 0.005, paired t-test). The tumor-to-background ratio (15.2 ± 7.4 and 19.6 ± 12.4, respectively) had no statistical differences in both cases (p &gt; 0.05, paired t-test). Liver metastases were visualized in patients 1 and 5 and corresponded to the projection of metastases according to contrast-enhanced abdominal CT. The accumulation of [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 in the projection of metastases in both cases was significantly higher compared to the primary tumor (1.3 and 1.7 times higher in patient 1; 2.2 and 3.5 times higher in patient 5, respectively).Conclusion. Both [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 demonstrated the diagnostic efficacy in visualizing a primary HER2-positive tumor in breast cancer patients. However, [99mTc]Tc-ADEPT6 had higher accumulation values, which makes it a more promising diagnostic agent.

Evaluation of malignant breast masses with abbreviated breast magnetic resonance imaging

Objectives: Our study aimed to investigate the sensitivity of the abbreviated magnetic resonance imaging (MRI) in the detection of tumors in breast cancer patients. Methods: Patients who underwent breast MRI between March 2018 and October 2021 were reviewed retrospectively. Patients with a histologic diagnosis of breast malignancy were included in the study. Patients who underwent a biopsy or an interventional procedure before the MRI examination and who received neoadjuvant chemotherapy were excluded from the study. Abbreviated MR protocol included a pre-contrast T1-weighted, 1st minute contrast-enhanced T1-weighted, and 1st minute subtracted series. Additionally, 2nd minute post-contrast series were evaluated. Results: A total of 83 lesions with a histologic diagnosis of breast cancer were evaluated in 81 patients. The mean age of the patients included in the study was 51.08 years (range: 27-79 years). Seventy-four of the 83 breast lesions showed contrast enhancement in the 1st minute contrast-enhanced images and subtraction images (sensitivity 89.1%). When missed cases were re-evaluated all of them were visible in the second-minute contrast-enhanced series. Conclusions: In this study, malignant lesions could be detected with high-sensitivity abbreviated MRI protocol and the addition of second-minute contrast-enhanced series to the protocol significantly improve lesion detection. We believe that MRI with the abbreviated MRI protocol can be used for screening purposes in high-risk women with dense breasts.

Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

Doxorubicin resistance in breast cancer is mediated via the activation of FABP5/PPARγ and CaMKII signaling pathway.

Breast cancer is the most prevalent malignancy among women. Doxorubicin (Dox) resistance was one of the major obstacles to improving the clinical outcome of breast cancer patients. The purpose of this study was to investigate the relationship between the FABP signaling pathway and Dox resistance in breast cancer. The resistance property of MCF-7/ADR cells was evaluated employing CCK-8, Western blot (WB), and confocal microscopy techniques. The glycolipid metabolic properties of MCF-7 and MCF-7/ADR cells were identified using transmission electron microscopy, PAS, and Oil Red O staining. FABP5 and CaMKII expression levels were assessed through GEO and WB approaches. The intracellular calcium level was determined by flow cytometry. Clinical breast cancer patient's tumor tissues were evaluated by immunohistochemistry to determine FABP5 and p-CaMKII protein expression. In the presence or absence of FABP5 siRNA or the FABP5-specific inhibitor SBFI-26, Dox resistance was investigated utilizing CCK-8, WB, and colony formation methods, and intracellular calcium level was examined. The binding ability of Dox was explored by molecular docking analysis. The results indicated that the MCF-7/ADR cells we employed were Dox-resistant MCF-7 cells. FABP5 expression was considerably elevated in MCF-7/ADR cells compared to parent MCF-7 cells. FABP5 and p-CaMKII expression were increased in resistant patients than in sensitive individuals. Inhibition of the protein expression of FABP5 by siRNA or inhibitor increased Dox sensitivity in MCF-7/ADR cells and lowered intracellular calcium, PPARγ, and autophagy. Molecular docking results showed that FABP5 binds more powerfully to Dox than the known drug resistance-associated protein P-GP. In summary, the PPARγ and CaMKII axis mediated by FABP5 plays a crucial role in breast cancer chemoresistance. FABP5 is a potentially targetable protein and therapeutic biomarker for the treatment of Dox resistance in breast cancer.

O-GlcNAcylation promotes topoisomerase IIα catalytic activity in breast cancer chemoresistance.

DNA topoisomerase IIα (TOP2A) plays a vital role in replication and cell division by catalytically altering DNA topology. It is a prominent target for anticancer drugs, but clinical efficacy is often compromised due to chemoresistance. In this study, we investigate the role of TOP2A O-GlcNAcylation in breast cancer cells and patient tumor tissues. Our results demonstrate that elevated TOP2A, especially its O-GlcNAcylation, promotes breast cancer malignant progression and resistance to adriamycin (Adm). O-GlcNAcylation at Ser1469 enhances TOP2A chromatin DNA binding and catalytic activity, leading to resistance to Adm in breast cancer cells and xenograft models. Mechanistically, O-GlcNAcylation-modulated interactions between TOP2A and cell cycle regulators influence downstream gene expression and contribute to breast cancer drug resistance. These results reveal a previously unrecognized mechanistic role for TOP2A O-GlcNAcylation in breast cancer chemotherapy resistance and provide support for targeting TOP2A O-GlcNAcylation in cancer therapy.

Metastatic breast tumors downregulate miR-145 regulating the hypoxia-induced vasculogenic mimicry

Tumor cells grow in three-dimensional (3D) channels-like structures denoted as vasculogenic mimicry (VM), which provides a route for nutrients and oxygen acquisition. VM is activated by hypoxia and associated with metastasis and poor prognosis. MetastamiRs are microRNAs regulating metastasis, however, if they control VM in breast cancer remains poorly understood. The aim of this study was to evaluate the expression of VM-associated microRNAs in tumors of metastatic breast cancer patients. Firstly, we constructed microRNAs/mRNAs coregulation networks using expression data from TCGA databases. Dozens of microRNAs regulating genes involved in VM and metastasis were found. Of these, we selected 10 microRNAs for further characterization. The presence of VM in histological samples from patients with or without metastasis was evaluated using CD31-/PAS+ immunophenotyping. Remarkably, data showed that VM was significantly increased in tumors from patients with metastasis in comparison with no-metastatic group. Gene expression analysis indicated that miR-145, miR-142-3p, miR-31, miR-148a, miR-200b-3p and miR-526b were downregulated in primary tumors from patients with metastatic disease and positive for VM. Moreover, modulated microRNAs showed a predictive clinical value in overall survival in a cohort (n=1262) of breast cancer patients. Of these, we evaluated the role of miR-145 in formation of hypoxia-induced 3D channels-like using an in vitro model that recapitulates the early stages of VM. Data showed that miR-145 mimics was able to abolish the VM development in both metastatic Hs578t and MDA-MB-231 breast cancer cells. In conclusion, manipulation of miR-145 levels may represent a therapeutic approach in metastatic breast cancer patients that developed VM.

Abstract CT043: Feasibility study to evaluate performance of the LUM Imaging System for intraoperative detection of residual tumor in breast cancer patients receiving and not receiving neoadjuvant therapy

Abstract Background: Microscopically tumor-free lumpectomy margins are critical for safe breast conserving surgery (BCS). With current tools, 15%-25% of BCS have positive margins that require second surgical procedures and increase cost and patient discomfort. Better detection tools are needed to identify residual cancer during the initial BCS and reduce second operations. LUM015 is a protease-activated fluorescent imaging agent that accumulates in tumor cells and tumor associated macrophages after preoperative intravenous injection. The LUM Imaging System visualizes activated LUM015 in the lumpectomy cavity via a hand-held detector and tumor detection software. This system has been tested in multiple single-site studies and two prospective multi-site studies enrolling &amp;gt;600 patients and demonstrated successful detection of residual BCS cavity tumor. Initial studies excluded the ~20% of patients receiving neoadjuvant therapy. Patchy tumor cell death with preoperative therapy can leave small, multifocal deposits of tumor invisible on pre-operative imaging and not palpable or visible during surgery. We now evaluate the LUM Imaging System in patients with and without neoadjuvant therapy. Design: This prospective, multi-center study tests the LUM Imaging System in BCS after neoadjuvant therapy to evaluate potential impact of treatment-related tissue changes and tumor cell death on tumor detection algorithms. An initial cohort of 10 patients addressed the objective of algorithm development. A second cohort of 104 patients will further evaluate the feasibility of the LUM Imaging System after neoadjuvant therapy. A third cohort will enroll 208 patients who have not received neoadjuvant therapy. All cohorts are evaluated for safety and for reduction in residual tumor after LUM Imaging System guidance compared to standard BCS. After excision of the main lumpectomy specimen, patients are randomized 3:1. In the device arm, the cavity is imaged and margins with LUM015 signal are excised. Final comprehensive shaved margins are removed in both arms to evaluate extent of residual disease after the use of the LUM Imaging System or after standard lumpectomy. No LUM Imaging is performed in the control arm, however, all patients are injected with LUM015 to evaluate drug safety. Patient reported outcomes assessing re-excision concerns, breast appearance and preferences for treatment type are collected. Eligibility: This study enrolls women with histologically confirmed primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a BCS for their breast malignancy who have received any form of neoadjuvant treatment prior to surgery or who have not received any therapy prior to surgery. Patients allergic to polyethylene glycol or intravenous contrast agents are excluded. Use of blue node mapping dyes before imaging with the LUM015 is not allowed. Progress: Cohort 1 has completed enrollment and interim analysis. No new risks specific to the neoadjuvant population were identified. LUM015 fluorescent signals measured in neoadjuvant patients were within the expected range, and no changes to the tumor detection algorithm were required. Cohorts 2 and 3 have enrolled a total of 84 patients. This trial is registered as NCT04440982. Citation Format: Jorge M. Ferrer, Irene Wapnir, Peter Blumencranz, Kelly Hunt, Shelley Hwang, David Carr, Kate Smith, Heidi Santa Cruz, Julia Shanno, Alexander Pogrebinsky, Manna Chang, Barbara L. Smith. Feasibility study to evaluate performance of the LUM Imaging System for intraoperative detection of residual tumor in breast cancer patients receiving and not receiving neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT043.

Abstract 4493: Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate for the treatment of sortilin-positive (SORT1+) TNBC and Her2-positive breast cancers

Abstract Over the last decade, the targeting of specific cell surface receptors such as human epidermal growth factor receptor 2 (Her2) in breast cancer cells has allowed the emergence of innovative strategies for the delivery of anticancer agents. High expression levels of the sortilin receptor (SORT1) have been reported in various tumors of breast cancer patients, including triple-negative breast cancer (TNBC), HR+ and Her2+ breast cancers. Given SORT1 function in ligand internalization, we addressed the sorting and trafficking of the newly designed TH19P01 peptide recognizing SORT1, and of TH1902, an anticancer TH19P01-docetaxel conjugate (PDC), and whether such PDC could target SORT1+ breast cancers. In vitro, high expression of SORT1 was found in several TNBC and Her2+ breast cancer cell lines as well as in in more than 88% of cases (H-score &amp;gt;100) from commercial breast cancer tissue microarrays. SORT1-mediated cell surface binding and internalization of TH19P01 was first investigated in TNBC-derived MDA-MB-231 cells. The binding and uptake of Alexa488-labeled TH19P01, at 4 and 37°C respectively, were found significantly reduced upon siRNA-mediated SORT1 silencing. This demonstrates that functional SORT1 processes are required for both cell surface recognition and internalization of TH19P01. Moreover, the internalization of TH19P01 and TH1902 in MDA-MB-231 cells were monitored by fluorescence microscopy using a polyclonal antibody generated against TH19P01. Both TH19P01 and TH1902 showed a rapid uptake and co-localized in the perinuclear region with the late endosomal marker Rab-7 and with the lysosomal marker Lamp-1 within 30 minutes indicating that both compounds are internalized through a receptor-mediated endocytosis pathway. In addition, TH19P01 internalization after 1 hour appeared significantly associated within intracellular compartments of MDA-MB-231 cells whereas Herceptin remained mostly localized at the cell surface of Her2+ BT-474 cells. In vivo, weekly administration of intravenous bolus of TH1902 (35 mg/kg) at an equivalent docetaxel MTD dose (15 mg/kg) led to complete tumor regression, while docetaxel only inhibited tumor growth by half in a murine MDA-MB-231 xenograft tumor model. Furthermore, in mice bearing Her2+ HCC-1954 breast tumor xenografts, TH1902 induced complete tumor regression in contrast to docetaxel and Herceptin. Taken together, these preclinical data demonstrate the high anticancer properties of TH1902 against SORT1+ TNBC and Herceptin resistant Her2+ breast cancers. These results demonstrate that TH1902 can be a promising avenue for personalized therapy in the treatment of all SORT1+ breast cancers. Citation Format: Cyndia Charfi, Michel Demeule, Jean-Christophe Currie, Alain Zgheib, Bogdan Alexandru Danalache, Richard Béliveau, Christian Marsolais, Borhane Annabi. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate for the treatment of sortilin-positive (SORT1+) TNBC and Her2-positive breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4493.

Abstract 2569: Investigation of the effect of chronic IL-1 exposure on breast cancer cell line response to external signaling factors

Abstract As part of our immune response, the body produces anti-tumorigenic inflammatory proteins to kill cancer cells. However, cancer cells can usurp chronic inflammation for their own survival. Interleukin-1 (IL-1) is an inflammatory protein found in breast cancer (BCa) tumors that is elevated in the BCa patient tumors and serum and promotes metastasis, indicating IL-1 is clinically relevant. But the molecular mechanisms underlying the effect of chronic IL-1 on BCa progression remain unknown. We previously discovered that acute exposure to IL-1 represses essential hormone receptors in cancers cells, but concomitantly upregulates other compensatory pro-survival proteins that contribute to tumor progression. To determine if chronic IL-1 inflammation has the same effect, we generated cell subline models using the human breast cancer cell line, T47D, by exposing T47D cells chronically to IL-1 for several months. Notably, we found that the T47D chronic IL-1 sublines stably evolve insensitivity to exogenous IL-1. This result suggests that chronic IL-1 causes stable molecular changes that may affect cancer cell response to exogenous factors.Therapeutic strategies include blocking exogenous hormone or growth factor signals in the tumor microenvironment to attenuate cancer cell growth, proliferation, and survival. Therefore, we grew T47D parental and chronic IL-1 sublines in the absence of serum to determine the effect of chronic IL-1 exposure on BCa cell response to hormone and growth factor inhibition. Preliminarily, we do not observe differences in parental versus subline response to serum starvation, suggesting that differential response to exogenous factors is specific to IL-1 signaling. To better understand how chronic IL-1 -induced molecular changes effect BCa cell response to the tumor microenvironment, we will perform sequencing and bioinformatics analysis to identify IL-1 -dependent and/or independent signaling pathways stably altered in the chronic IL-1 sublines. Citation Format: Roopal Dhar, Ola M. Olaleye, Rana Tarig Abdelaziz, Jyotsna Tera Reddy, Nikki Delk. Investigation of the effect of chronic IL-1 exposure on breast cancer cell line response to external signaling factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2569.