Breast Cancer Relapse Research Articles

Targeting mitochondrial dynamics by AZD5363 in triple-negative breast cancer MDA-MB-231 cell–derived spheres

Breast cancer stem cells (BCSCs) have been suggested to contribute to chemotherapeutic resistance and disease relapse in breast cancer. Thus, BCSCs represent a promising target in developing novel breast cancer treatment strategies. Mitochondrial dynamics in BCSCs were recently highlighted as an available approach for targeting BCSCs. In this study, a three-dimensional (3D) cultured breast cancer stem cell spheres model was constructed. Mitochondrial dynamics and functions were analyzed by flow cytometry and confocal microscopy. We have demonstrated that the protein levels of FIS 1 and Mitofusin 1 were significantly increased in BCSCs. Moreover, Capivasertib (AZD5363) administration could suppress Mitofusin1 expression in BCSCs. Our use of MitoTracker Orange and annexin V double-staining assay suggested that AZD5363 could induce apoptosis in BCSCs. The sensitivity of stem cell spheres to doxorubicin was investigated by CCK8 assay, and our results indicated that AZD5363 could re-sensitize BCSCs to Doxo. Flow cytometry analysis identified doxo-induced CD44 and CD133 expression in BCSCs could be suppressed by AZD5363. In combination with AZD536, doxo-induced apoptosis in the BCSCs was significantly increased. In conclusion, our study explored, for the first time, that AZD5363 could target mitochondrial dynamics in 3D cultured stem cell spheres (BCSCs) by regulating Mitofusin.

Kruppel-like factor 8 regulates triple negative breast cancer stem cell-like activity.

Breast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties. Triple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice. Here, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells. These results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically.

Abstract 2442: KLF8 and OGT/O-GlcNAcylation regulate stem-like cell properties in breast cancer

Abstract Breast cancer is the most common cancer and the second leading cause of cancer death among women overall. One of the main challenges in fighting breast cancer is the intratumor heterogeneity as they are composed of different subpopulations of cancer cells. This heterogeneity of breast cancer is promoted and maintained by a subpopulation of cancer stem-like cells (CSC). CSC share properties of adult stem cells, capable of self-renewing, differentiating & repopulating a tumor. In addition to CSC role in promoting tumor growth, CSC are involved in metastasis, drug resistance and relapse of breast cancer. Therefore, understanding the mechanisms that drive and maintain CSC will help identify novel therapeutic targets in treating breast cancer. Functional activities of CSC are heavily influenced by modulations in the tumor microenvironment, such as alteration in nutrient status. One key component connecting signaling pathway and cancer cell metabolism is the enzyme O-GlcNAc Transferase (OGT), which utilize UDP-GlcNAc to modify nuclear and cytoplasmic proteins in a process termed O-GlcNAcylation. OGT and O-GlcNAc are upregulated in many cancers. Recently, we showed a direct connection between OGT/O-GlcNAc and CSC in breast cancer, in which OGT/O-GlcNAc is required and sufficient to promote the stemness of breast cancer cells in vitro, as well as tumorigenesis in vivo. Global transcriptomic analysis of CSCs-enriched mammospheres with OGT overexpression revealed Kruppel-like-factor 8 (KLF8) as a downstream target of OGT in CSC. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE), reduced CSC population, and tumor growth in vivo. In contrast, KLF8 overexpression increased MFE and CSC population. Importantly, KLF8 knockdown blocked the effect of elevated OGT and O-GlcNAcylation in promoting MFE, while KLF8 overexpression rescued OGT inhibition in regulating in breast cancer cells. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level, while KLF8 overexpression increased expression of these CSC factors. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O-GlcNAcylation, suggesting a possible feed-forward loop between OGT and KLF8 in promoting CSC. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple-negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem-like cells population. Citation Format: Giang Le Minh, Emily Esquea, Tejsi Dhamelia, Neha Akella, Mauricio J. Reginato. KLF8 and OGT/O-GlcNAcylation regulate stem-like cell properties in breast cancer [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 2442.

Identification of DYNLT1 associated with proliferation, relapse, and metastasis in breast cancer.

Breast cancer (BC) is the most common malignant disease worldwide. Although the survival rate is improved in recent years, the prognosis is still bleak once recurrence and metastasis occur. It is vital to investigate more efficient biomarkers for predicting the metastasis and relapse of BC. DYNLT1 has been reported that participating in the progression of multiple cancers. However, there is still a lack of study about the correlation between DYNLT1 and BC. In this study, we evaluated and validated the expression pattern and prognostic implication of DYNLT1 in BC with multiple public cohorts and BC tumor microarrays (TMAs) of paraffin-embedded tissues collected from the Affiliated Hospital of Jining Medical University. The response biomarkers for immune therapy, such as tumor mutational burden (TMB), between different DYNLT1 expression level BC samples were investigated using data from the TCGA-BRCA cohort utilizing public online tools. In addition, colony formation and transwell assay were conducted to verify the effects of DYNLT1 in BC cell line proliferation and invasion. The results demonstrated that DYNLT1 overexpressed in BC and predicted poor relapse-free survival in our own BC TMA cohort. In addition, DYNLT1 induced BC development by promoting MDA-MB-231 cell proliferation migration, and metastasis. Altogether, our findings proposed that DYNLT1 could be a diagnostic and prognostic indicator in BC.

Analysis of the breast cancer progression frequency depending on the amount of surgical treatment performed after neoadjuvant drug therapy

Purpose. To compare the incidence of breast cancer (BC) relapse after subcutaneous mastectomy and Madden radical mastectomy.Patients and methods. The study included 102 patients diagnosed with BC with stages IIB – III, who underwent neoadjuvant drug therapy with a subsequent surgical stage of treatment on the basis of the Tver Regional Clinical Oncology Dispensary from 2017 to 2020. The main group included 50 patients who underwent subcutaneous mastectomy with simultaneous installation of an endoprosthesis: stage IIB – 31 (62 %) patients; stage III – 19 (38 %) patients. The comparison group included 52 patients who underwent surgical treatment in volume – radical mastectomy according to Madden (RME): Stage IIB – 34 (65.4 %) patients; stage III – 18 (34.6 %) patients. Depending on the subtype of the tumor, patients received neoadjuvant drug therapy.Results. During the three-year follow-up period, the progression of the disease after subcutaneous RME was detected in 7 (14 %) patients. Locoregional relapses accounted for 4 cases (8 %), progression to distant organs 3 cases (6 %). After the Madden RME, disease progression was detected in 6 (11.5 %) patients. Locoregional relapses accounted for 3 cases (5.7 %), progression to distant organs 3 cases (5.7 %). The detected foci did not always coincide with the primary subtype of the tumor: in 5 cases the subtype was different (38.5 %), in 8 cases it coincided (61.5 %).Conclusion. The frequency of development of locoregional and distant metastases did not statistically depend on the volume of surgical intervention (p > 0.05). It is reasonable to conduct a histological examination in case of progression to determine the tactics of treatment.

Abstract P4-02-07: A large real-world study of circulating tumor DNA in early breast cancer patients

Abstract Background Around 30-40% of early breast cancer (EBC) patients relapse after curative surgery and systemic treatment. However, it is very difficult to identify the patients with high risks of relapse. Recently, circulating tumor DNA (ctDNA) is shown to be a sensitive method to evaluate the minimal residual disease (MRD) of solid tumors after surgery. Here, we explored the prognostic value of ctDNA in a large real-world study of EBC patients. Method 346 invasive EBC patients from three hospitals in China were enrolled in this study. All patients received radical surgery and were followed up for a median of 13.1 months from surgery. 17 patients developed local and/or distant relapse during the follow-up. Primary breast cancer samples (n=346) and plasma samples (n=1059) were subjected to deep targeted sequencing using a large next-generation sequencing panel that covers 1,021 cancer-related genes. Results Among the 346 patients, 153 patients had detectable ctDNA in at least one of the plasma samples and 15 of them relapsed. Only two of the 193 patients with consistent negative ctDNA relapsed, demonstrating the value of positive ctDNA in predicting RFS (p< 0.0001; HR=13.09; 95%CI: 2.98-57.45). Of 334 patients who had postoperative plasma samples tested, 108 patients with positive ctDNA in at least one of the postoperative plasma samples had 11 relapse during follow-up, which had a significantly worse RFS than the 226 patients with negative postoperative ctDNA and 4 relapse (p< 0.0005; HR=5.98; 95%CI: 1.90-18.79). In this study, 176 patients received neoadjuvant therapy (NAT) and 59 of them had twice or more ctDNA tests before surgery. Among them, 12 patients with negative ctDNA before NAT remained negative before surgery and had no relapse, 36 patients with positive ctDNA before NAT turned negative before surgery and had 4 relapse, while 11 patients kept positive ctDNA before surgery and had 2 relapse. 165 patients received NAT and had postoperative ctDNA tests. Of the 31 patients achieved pCR, 21 patients with negative postoperative ctDNA did not relapse and 10 patients with positive postoperative ctDNA had 1 relapse. Of the 134 non-pCR patients, 90 patients with negative postoperative ctDNA had 4 (4.4%) relapse, which is significantly better than the 44 patients with positive postoperative ctDNA and 8 (18.2%) relapse (p< 0.05), indicating the feasibility to further distinguish the real high-risk patients among the patients with non-pCR after NAT. Among the 17 patients with relapse or metastasis, 11 patients had positive ctDNA after surgery before relapse and the median lead time was 74 days and a maximum of 526 days. Four patients had negative ctDNA before relapse and two patients did not test after surgery. Conclusions Circulating tumor DNA is a sensitive assay to predict the relapse in early breast cancer, especially in the patients who received NAT and did not achieve pCR. This may provide a window of opportunity to personalize the escalated adjuvant treatment in patients with high relapse risk. Table 1. The association between patients’ characteristics and ctDNA positivity Citation Format: Qiang Liu, Mengzi Wu, Shunying Li, Yudong Li, Liang Jin, Qianfeng Shi, Yu Zhang, Chang Gong. A large real-world study of circulating tumor DNA in early breast cancer patients [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 P4-02-07.

Abstract P5-05-01: Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer

Abstract Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF< 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-01.

Abstract GS4-09: Pregnancy Outcome and Safety of Interrupting Therapy for women with endocrine responsIVE breast cancer: Primary Results from the POSITIVE Trial (IBCSG 48-14/BIG 8-13)

Abstract Background: Pregnancy after breast cancer (BC) is of substantial importance for many young women at diagnosis and during follow-up. BC treatment including standard endocrine therapy (ET) (5-10 years) may reduce ovarian reserve and the chances of subsequent successful pregnancy, given conception is contraindicated during ET. A temporary interruption of ET to attempt and carry a pregnancy in this population has never been prospectively studied. Methods: POSITIVE is a single-arm, prospective, investigator-initiated, international trial evaluating the safety and pregnancy outcomes of interrupting ET for young women with early-stage hormone-receptor-positive (HR+) BC who desire pregnancy. The primary objective is to assess the risk of BC relapse associated with ET interruption for ~2 years to achieve pregnancy. Women ≤42 years with stage I-III HR+ BC who received adjuvant ET (SERM alone, GnRH analogue plus SERM or AI) for 18 to 30 months and wished to interrupt ET to attempt pregnancy were eligible. The primary endpoint is breast cancer free interval (BCFI) defined as the time from enrollment to the first BC event (local, regional, distant recurrence or a new invasive contralateral BC). Planned sample size was 500 patients. Three interim analyses of BCFI were reviewed by the Data Safety Monitoring Committee (DSMC) to assure a 95% chance of stopping the trial early if the annual BCFI event rate exceeded 4%; with primary analysis triggered after 1600 patient years of follow-up (pyfu) and no more than 46 BCFI events defined as the safety threshold. The DSMC recommended continuing the study following each interim analysis. We now report the primary results. Results: From 12/2014 to 12/2019, 518 women were enrolled. At enrollment, the median age of participants was 37 years (27-43 years); 75.0% were nulliparous, 93.4% had stage I/II disease, 66.3% node-negative. Median time from BC diagnosis to enrollment was 29 months (IQR: 25-32). Tamoxifen alone was the most prescribed ET (41.7%), followed by tamoxifen+ovarian function suppression (35.7%). 62.0% of participants had received neo/adjuvant chemotherapy. At a median follow-up of 41 months (1638 pyfu), 44 participants had experienced a BCFI event, not exceeding the pre-specified safety threshold of 46 events. The 3-year BCFI failure percent was 8.9% (95% CI: 6.3 to 11.6%), similar to the 9.2% (95% CI: 7.6 to 10.8%) calculated in the comparative external control cohort from the SOFT/TEXT trials (Sun et al, Breast 2020). Of 497 women followed for pregnancy status, 368 (74.0%) had at least one pregnancy, 317 (63.8%) had at least one live birth, with a total of 365 babies born. Based on competing risk analysis, 76.3% of patients resumed ET (half within 26 months), 8.3% had BCFI event/death before ET resumption, and 15.4% had not resumed ET yet. Conclusions: The POSITIVE trial demonstrates that for young women with early HR+ BC desiring pregnancy, temporary interruption of ET to attempt pregnancy does not confer a greater short-term risk of recurrence than that observed in a modern historical control group that did not interrupt ET. Most participants have had a live birth. Further follow-up is planned to confirm long-term safety. These results should be considered in counselling BC patients desiring future pregnancy. Citation Format: Ann Partridge, Olivia Pagani, Samuel M. Niman, Monica Ruggeri, Fedro Alessandro A. Peccatori, Hatem A. Azim, Marco Colleoni, Cristina Saura, Chikako Shimizu, Anna Saetersdal, Judith Kroep, Audrey Mailliez, Ellen Warner, Virginia F. Borges, Frédéric Amant, Andrea Gombos, Akemi Kataoka, Christine Rousset-Jablonski, Simona Borstnar, Junko Takei, Jeong Eon Lee, Janice Walshe, Manuel Ruiz Borrego, Halle Moore, Christobel Saunders, Vesna Bjelic-Radisic, Snezana Susnjar, Fatima Cardoso, Karen L. Smith, Teresa Ferreiro Vilarino, Karin Ribi, Kathryn Ruddy, Sarra El-Abed, Martine Piccart, Larissa A. Korde, Aron Goldhirsch, Richard D. Gelber. Pregnancy Outcome and Safety of Interrupting Therapy for women with endocrine responsIVE breast cancer: Primary Results from the POSITIVE Trial (IBCSG 48-14/BIG 8-13) [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 GS4-09.

Abstract P2-22-01: Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse breast cancer

Abstract Late-stage relapse also known as late recurrence breast cancer is a metastatic disease that occurs more than five years after initial adjuvant endocrine therapy treatment. The 5-year relative survival rate is less than 30% and there are limited treatment options for patients with late-stage relapse because of poor performance status. We hypothesize that long non-coding RNA (lncRNA) protein interactions regulate genes to promote late-stage relapse and these interactions can be targeted using RNA therapeutics. To address this, we used RNA-sequencing to identify LINC00355 to be up-regulated in 24 late-stage relapse samples compared to a unique cohort of 72 primary breast cancer samples. Analysis of 480 primary breast cancer samples from The Cancer Genome Atlas (TCGA) also shows decrease expression of LINC00355 compared to late-stage relapse. To better assess LINC00355 cell type expression, we analyzed 26 publicly available primary breast cancer single cell sequencing data samples and detected 0.90% or less of cells expressing LINC00355, which was restricted to cancer epithelial cells. Due to cellular localization playing an important role in lncRNA function, we isolated nuclear and cytoplasmic lysates and orthogonally validated with RNAscope in situ hybridization (RNA-ISH) to show that LINC00355 is located in the nucleus of MCF7 long-term estrogen deprived (MCF7 LTED) cells. LTED cells are deprived of estrogen for longer than three years meant to recapitulate the acquired resistance to aromatase inhibitors. Next, we determined that LINC00355 binds to MENIN protein to regulate p27KIP expression to promote proliferation with UV crosslinking immunoprecipitation and chromatin immunoprecipitation qPCR of LINC00355 overexpression and mutant constructs. Furthermore, RNA-ISH assays validated high nuclear expression of LINC00355 and RNA-ISH protein co-detection assays showed co-localization of LINC00355-MENIN interaction in a panel of late-stage relapse breast cancer patient tissues compared to primary tissues. Lastly to determine clinical significance, we treated MCF7 LTED cells with two locked nucleic acid antisense oligos (ASOs) targeting LINC00355 to observe a significant decrease in proliferation, cell viability, and invasion. Overall, this is the first study to show the importance of lncRNA-protein interactions and therapeutically target lncRNAs with ASOs in late-stage relapse breast cancer. Moving forward, we intend to further understand how LINC00355 contributes to the progression of late-stage relapse with the intent of creating novel cancer diagnostics and therapies. Citation Format: DeAnna Wells, Kyla Gelev, Prasanth Thunuguntla, Reyka Jayasinghe, Li Ding, Jieya Shao, Cynthia Ma, Jessica Silva-Fisher. Nuclear long non-coding RNA LINC00355 expression and treatment using antisense oligonucleotides in Estrogen Receptor positive late-stage relapse 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 P2-22-01.

Distribution, dynamic evolution, and clinical outcomes of patients with advanced breast cancer according to HER2 expression

BackgroundNovel antibody‒drug conjugates (ADC) have shown great efficacy in HER2-low advanced breast cancer. However, the clinical features of HER2-low disease still need to be clarified. The current study aims to evaluate the distribution and dynamic change in HER2 expression in patients with disease recurrence and the clinical outcome of those patients.MethodsPatients with pathologically diagnosed relapsed breast cancer between 2009 and 2018 were included. Samples were considered HER2-zero when the immunohistochemistry (IHC) score was 0, HER2-low when the IHC score was 1 + or 2 + with negative fluorescence in situ hybridization (FISH) results, and HER2-positive when the IHC score was 3 + or the FISH results were positive. Breast cancer-specific survival (BCSS) was compared among the three HER2 groups. Changes in HER2 status were also evaluated.ResultsA total of 247 patients were included. Among recurrent tumors, 53 (21.5%) were HER2-zero, 127 (51.4%) were HER2-low, and 67 (27.1%) were HER2-positive. The HER2-low subtype represented 68.1% of the HR-positive breast cancer group and 31.3% of the HR-negative group (P < 0.001). This three-group classification of HER2 status was prognostic in advanced breast cancer (P = 0.0011), with HER2-positive patients having the best clinical outcome after disease recurrence (P = 0.024), while only marginal survival advantages were observed in HER2-low patients versus HER2-zero patients (P = 0.051). In the subgroup analysis, the survival difference was observed only in patients with HR-negative recurrent tumors (P = 0.0006) or with distant metastasis (P = 0.0037). The overall discordance rate of HER2 status between primary and recurrent tumors was 38.1%, with 25 (49.0%) primary HER2-zero patients and 19 (26.8%) HER2-positive patients shifting to HER2-low at recurrence.ConclusionNearly half of the advanced breast cancer patients had HER2-low disease, which indicates a poorer prognosis than HER2-positive disease and marginally better outcomes than HER2-zero disease. During disease progression, one-fifth of tumors convert to HER2-low entities, and the corresponding patients may benefit from ADC treatment.

Characteristics of recurrence, predictors for relapse and prognosis of rapid relapse triple-negative breast cancer.

Triple-negative breast cancer (TNBC) patients who recur at different times are associated with distinct biological characteristics and prognoses. Research on rapid-relapse TNBC (RR-TNBC) is sparse. In this study, we aimed to describe the characteristics of recurrence, predictors for relapse, and prognosis in rrTNBC patients. Clinicopathological data of 1584 TNBC patients from 2014 to 2016 were retrospectively reviewed. The characteristics of recurrence were compared between patients with RR-TNBC and slow relapse TNBC(SR-TNBC). All TNBC patients were randomly divided into a training set and a validation set to find predictors for rapid relapse. The multivariate logistic regression model was used to analyze the data of the training set. C-index and brier score analysis for predicting rapid relapse in the validation set was used to evaluate the discrimination and accuracy of the multivariate logistic model. Prognostic measurements were analyzed in all TNBC patients. Compared with SR-TNBC patients, RR-TNBC patients tended to have a higher T staging, N staging, TNM staging, and low expression of stromal tumor-infiltrating lymphocytes (sTILs). The recurring characteristics were prone to appear as distant metastasis at the first relapse. The first metastatic site was apt to visceral metastasis and less likely to have chest wall or regional lymph node metastasis. Six predictors (postmenopausal status, metaplastic breast cancer,≥pT3 staging,≥pN1 staging, sTIL intermediate/high expression, and Her2 [1+]) were used to construct the predictive model of rapid relapse in TNBC patients. The C-index and brier score in the validation set was 0.861 and 0.095, respectively. This suggested that the predictive model had high discrimination and accuracy. The prognostic data for all TNBC patients showed that RR-TNBC patients had the worst prognosis, followed by SR-TNBC patients. RR-TNBC patients were associated with unique biological characteristics and worse outcomes compared to non-RR-TNBC patients.

Mechanistic modeling of metastatic relapse in early breast cancer to investigate the biological impact of prognostic biomarkers

Background and objectiveEstimating the risk of metastatic relapse is a major challenge to decide adjuvant treatment options in early-stage breast cancer (eBC). To date, distant metastasis-free survival (DMFS) analysis mainly relies on classical, agnostic, statistical models (e.g., Cox regression). Instead, we propose here to derive mechanistic models of DMFS. MethodsThe present series consisted of eBC patients who did not receive adjuvant systemic therapy from three datasets, composed respectively of 692 (Bergonié Institute), 591 (Paoli-Calmettes Institute, IPC), and 163 (Public Hospital Marseille, AP-HM) patients with routine clinical annotations. The last dataset also contained expression of three non-routine biomarkers. Our mechanistic model of DMFS relies on two mathematical parameters that represent growth (α) and dissemination (μ). We identified their population distributions using mixed-effects modeling. Critically, we propose a novel variable selection procedure allowing to: (i) identify the association of biological parameters with either α, μ or both, and (ii) generate an optimal candidate model for DMFS prediction. ResultsWe found that Ki67 and Thymidine Kinase-1 were associated with α, and nodal status and Plasminogen Activator Inhibitor-1 with μ. The predictive performances of the model were excellent in calibration but moderate in discrimination, with c-indices of 0.72 (95% CI [0.48, 0.95], AP-HM), 0.63 ([0.44, 0.83], Bergonié) and 0.60 (95% CI [0.54, 0.80], IPC). ConclusionsOverall, we demonstrate that our novel method combining mechanistic and advanced statistical modeling is able to unravel the biological roles of clinicopathological parameters from DMFS data.

Examination of patients who completed Abemaciclib administration for advanced or relapsed breast cancer

Examination of patients who completed Abemaciclib administration for advanced or relapsed breast cancer

ELOVL5 and IGFBP6 genes modulate sensitivity of breast cancer cells to ferroptosis.

Introduction: Relapse of breast cancer is one of the key obstacles to successful treatment. Previously we have shown that low expression of ELOVL5 and IGFBP6 genes in breast cancer tissue corresponded to poor prognosis. ELOVL5 participates directly in the elongation of polyunsaturated fatty acids (PUFAs) that are considered to play an important role in cancer cell metabolism. Thus, in this work we studied the changes in lipid metabolism in breast cancer cells with reduced expression of either ELOVL5 or IGFBP6 gene. Methods: MDA-MB-231 cells with a stable knockdown of either ELOVL5 or IGFBP6 gene were used in this study. Transcriptomic and proteomic analysis as well as RT-PCR were utilized to assess gene expression. Content of individual fatty acids in the cells was measured with HPLC-MS. HPLC was used for analysis of the kinetics of PUFAs uptake. Cell viability was measured with MTS assay. Flow cytometry was used to measure activation of apoptosis. Fluorescent microscopy was utilized to assess accumulation of ROS and formation of lipid droplets. Glutathione peroxidase activity was measured with a colorimetric assay. Results: We found that the knockdown of IGFBP6 gene led to significant changes in the profile of fatty acids in the cells and in the expression of many genes associated with lipid metabolism. As some PUFAs are known to inhibit proliferation and cause death of cancer cells, we also tested the response of the cells to single PUFAs and to combinations of docosahexaenoic acid (DHA, a n-3 PUFA) with standard chemotherapeutic drugs. Our data suggest that external PUFAs cause cell death by activation of ferroptosis, an iron-dependent mechanism of cell death with excessive lipid peroxidation. Moreover, both knockdowns increased cells' sensitivity to ferroptosis, probably due to a significant decrease in the activity of the antioxidant enzyme GPX4. Addition of DHA to commonly used chemotherapeutic drugs enhanced their effect significantly, especially for the cells with low expression of IGFBP6 gene. Discussion: The results of this study suggest that addition of PUFAs to the treatment regimen for the patients with low expression of IGFBP6 and ELOVL5 genes can be potentially beneficial and is worth testing in a clinically relevant setting.

Survival analysis of breast cancer patients after diagnosis of second primary malignancies, focusing on the second primary hematologic malignancy.

Although the overall survival (OS) of breast cancer patients is increasing with improved detection and therapies, so is the risk of breast cancer patients developing subsequent malignancies. We investigated the OS of breast cancer survivors according to sites of second primary malignancies (SPM). The OS of the second primary hematologic malignancy (SPHM) was then compared with that of metastatic breast cancer (MBC). We retrospectively analyzed patients diagnosed with primary breast cancer between 1998 and 2019. Only those with SPM were eligible for analysis. First, the OS of patients with SPM diagnosed as the first event after the diagnosis of breast cancer was analyzed. Next, the OS of patients with SPHM, with or without breast cancer relapse, was compared with that of patients with MBC, matched using the propensity score. Patients diagnosed with SPM without breast cancer relapse as the first event had a significantly better OS than did patients with MBC, but the OS of those with SPHM as the first event did not differ significantly from that of patients with MBC (hazard ratio [HR], 1.558; 95% confidence interval [CI], 0.856-2.839; P = 0.147). The OS of patients with SPHM with or without breast cancer relapse was worse than that of the MBC group after propensity score matching (HR, 1.954; 95% CI, 1.045-3.654; P = 0.036). Prognosis of SPM diagnosed as the first event was statistically better than that of MBC, except in case of SPHM. Patients with SPHM, with or without MBC, showed poor OS before and after propensity score matching.

Prediction of relapses in breast cancer based on the study of proteasomal activity

Prediction of relapses in breast cancer based on the study of proteasomal activity

Breast cancer relapses considering molecular biological characteristics

We aimed to analyze the frequency of breast cancer relapses and their relationship with molecular and biological tumor characteristics. We studied 6,136 breast cancer patients, including 146 with relapses (Group 1) and 455 without relapses (Group 2). We divided the patients based on age, menstrual function, disease stage, histology form and grade, and molecular-biological subtype. The 5-year relapse-free rate for Group 1 was longer for Lum A and TN subtypes (60% and 40%, respectively) but shorter for Lum B and HER-2/neu-amplified subtypes (38% and 31%, respectively). Disease stage, tumor histology, and grade did not significantly affect relapse frequency in these patients. Relapses were more common in premenopausal patients and the Lum B subtype.

Evaluation of Pattern of Relapse in Luminal Breast Cancer

Background: The most common molecular subtype of breast cancer worldwide is luminal breast cancer. It has shown a good prognostic profile compared to other types. Despite the immense development of adjuvant treatment in the past years, around 20% of patients with early-stage disease relapse. Aim of the study: To evaluate the relapse pattern in luminal breast cancer and the correlation between luminal types and (AJCC 8th edition). Subject and Methods: A total of 230 luminal breast cancer patients treated at Al-Hussein University Hospital between 2013 and 2018 were analyzed. Patients have been divided into the following groups: luminal A (31%) and B (32.6%), luminal B HER2+ve (18.6%), and luminal UC (17.3%). Results: The median follow-up was 66.7 months, and the relapse rate of luminal breast cancer was 27.8%. Although statistically not significant, luminal A had the lowest relapse rate (19.4%), while luminal B (36%), HER2 positive luminal B (23.2%), and luminal UC (32.5%) (p=0.117). The predominant organ relapse was bone (29.6%) mainly observed in luminal A and B, 35.7% and 32.3% of patients respectively (p=0.048). Luminal Correlation with the AJCC staging system was significant, luminal A was most often observed in early stages with 81.9% presented in Stages I to II (p=0.008). Conclusions: Luminal breast cancer has a wide discrepancy in relapse rate and pattern. Luminal A seems to have the best prognosis for DFS. Ki-67 % and HER2 testing would give a prognostic factor in luminal subcategorization and could be beneficial in the intensification of adjuvant therapy.

D-Dimers Variability in the Perioperative Period of Breast Cancer Surgery Helps to Predict Cancer Relapse: A Single-Centre Prospective Study.

The importance of D-dimers (DD) assessment in the diagnostic algorithm of venous thromboembolic (VTE) disease is well known. Increase of DD concentration may be also associated with neoplastic disease. Many studies documented that high concentration of DD before solid tumour surgery indicates more advanced disease and poor life expectancy. The prognostic value of the DD concentration variability in the perioperative period, in women undergoing breast cancer surgery, has not been analysed so far. Thus, the aim of the present prospective study was to assess whether the trend of DD concentration changes in the perioperative period may predict cancer recurrence in women undergoing breast cancer surgery. 189 consecutive women with histopathological diagnosis of breast cancer (BC) referred for surgical treatment were included. DD concentration was measured twice in each patient: at the time of admission to hospital and at the time of discharge home. Enoxaparin in standard dose of 40mg daily s. c. was used as primary VTE prophylaxis in all of the patients. The recurrence of BC, within 1year observation time, occurred in 13 patients (6.8%), in 11 (5.8%) patients with DD increase after surgery and only in 2 (1.1%) without an increase in DD, P = .0179. Increase in DD concentration after BC surgery was an independent positive predictor of disease relapse (OR 8.600, LCI 1.451, UCI 96.80, P = .0371) together with the lack of postoperative radiotherapy (OR 6.009, LCI 1.305, UCI 31.95, P = .0245), whereas the lack of postoperative chemotherapy predicted no BC relapse (OR .07355, LCI .0056, UCI .58, P = .0245). Increase of DD in the early postoperative period may be considered as additional independent predictor of recurrence of BC within 1 year.

BAG2 drives chemoresistance of breast cancer by exacerbating mutant p53 aggregate.

Rationale: Chemoresistance is a major challenge in the clinical management of patients with breast cancer. Mutant p53 proteins tend to form aggregates that promote tumorigenesis in cancers. We here aimed to explore the mechanism for the generation of mutant p53 aggregates in breast cancer and assess its role in inducing chemoresistance. Methods: Expression of BCL2-associated athanogene 2 (BAG2) was evaluated by qRT-PCR, western blotting, and immunohistochemistry in breast cancer patient specimens. The significance of BAG2 expression in prognosis was assessed by Kaplan-Meier survival analysis and the Cox regression model. The roles of BAG2 in facilitating the formation of mutant p53 aggregates were analyzed by co-immunoprecipitation, immunofluorescence, and semi-denaturing detergent-agarose gel electrophoresis assays. The effects of BAG2 on the chemoresistance of breast cancer were demonstrated by cell function assays and mice tumor models. Results: In the present study, we found that BAG2 was significantly upregulated in relapse breast cancer patient tissues and high BAG2 was associated with a worse prognosis. BAG2 localized in mutant p53 aggregates and interacted with misfolded p53 mutants. BAG2 exacerbated the formation of the aggregates and recruited HSP90 to promote the propagation and maintenance of the aggregates. Consequently, BAG2-mediated mutant p53 aggregation inhibited the mitochondrial apoptosis pathway, leading to chemoresistance in breast cancer. Importantly, silencing of BAG2 or pharmacological targeting of HSP90 substantially reduced the aggregates and increased the sensitivity of chemotherapy in breast cancer. Conclusion: These findings reveal a significant role of BAG2 in the chemoresistance of breast cancer via exacerbating mutant p53 aggregates and suggest that BAG2 may serve as a potential therapeutic target for breast cancer patients with drug resistance.