Prognosis Of Metastatic Breast Cancer Research Articles

B2 Prognostic Index in Determining Prognosis in Metastatic Breast Cancer.

To determine the relationship between the B2 prognostic index (B2PI) scoring method and prognosis in metastatic breast cancer, and to create a formula based on parameters that can be easily accessed in daily practice. Descriptive study. Place and Duration of the Study: Department of Medical Oncology, Dokuz Eylul University, Izmir, Turkiye, between May 2010 and June 2021. The clinicopathological characteristics of the patients were compared between the groups. All female breast cancer patients over the age of 18 years with de novo metastatic and non-metastatic breast cancers who developed metastasis during follow-up, were included in the study. Those with a second solid cancer or haematological malignancy and with a life expectancy of less than 3 months were excluded from the study. Chi-square and Fisher's exact tests were used to compare categorical data between the groups. Overall survival evaluations were made using the Kaplan-Meier analysis method and Log-Rank test. Risk factors for mortality were evaluated in Cox regression analysis. In all statistical tests, p <0.05 was considered statistically significant. There were 176 patients in this study, out of which 111 (63.1%) were de novo metastatic. When the effect of B2PI risk groups on overall survival in intrinsic subtypes was analysed, significant differences were found in the overall survival of B2PI risk groups in all subtypes except HER2+ ER- (HER2 overexpression subtype). According to the B2PI scoring system, the median overall survival was higher for both low-risk and moderate-risk patients compared to those in the high-risk category. For metastatic breast cancer patients, the B2PI can be used to determine prognosis and develop treatment strategies, as it is a clinical decision-making tool based on parameters that are easily accessible in daily practice. Metastatic breast cancer, B2 prognostic index, Prognosis, Survival.

Perspectives for the clinical application of ctDNA analysis to breast cancer drug therapy.

Circulating tumor DNA (ctDNA) consists of DNA fragments released from cancer cells into the blood circulation with quick clearance. Analysis of ctDNA can enable real-time assessment of the presence of cancer cells and their genomic characteristics. Therefore, ctDNA is expected to be one of the most useful biomarkers for cancer. In recent years, several ultra-sensitive assays for ctDNA analysis have been developed, and many clinical trials are using these assays to investigate the efficacy of ctDNA-based therapeutic strategies. In the perioperative phase, real-time identification of minimal residual disease at the molecular level with ctDNA analysis can help evaluate the risk of recurrence to inform escalation or de-escalation of perioperative drug therapy. Many trials have examined whether therapeutic strategies using ctDNA analysis to predict treatment efficacy or resistance to molecular targeted agents can improve prognosis in metastatic breast cancer. In this review, we discuss the most recent ctDNA assays, the significance of introducing ctDNA assays to clinical practice, and the research on their application in perioperative and metastatic phases.

The clinical impact of plasma estrogen receptor-1 mutation in patients with metastatic breast cancer: A meta-analysis.

The relevance of the discovered plasma ESR1 mutations in positive metastatic breast cancer (BC) patients who had progressing disease after aromatase inhibitor (AI)-based therapy is still being debated. We conducted this meta-analysis to explore the prognostic and predictive role of plasma ESR1 mutations in patients with progressive BC who have previously received AI therapy. We searched for relevant studies in the PubMed, Embase and Cochrane Library databases to be included in the meta-analysis. This study was performed to compute combined hazard ratios (HRs) with 95% confidence intervals (95% CIs) for the progression-free survival (PFS) rate and overall survival (OS) rate. Subgroup and sensitivity analyses were also performed. The heterogeneity between studies was evaluated using the I2 statistic. In this meta-analysis, a total of 1,844 patients with metastatic BC and positive for estrogen receptors (ERs) were enrolled from 8 articles. The analysis revealed that patients with circulating ESR1 mutations had significantly worse PFS (HR: 1.34; 95% CI: 1.17-1.55; p < 0.001) and OS (HR: 1.59; 95% CI: 1.31-1.92; p < 0.001) compared to wild-type ESR1 patients. Subgroup analysis showed that the types of plasma ESR1 mutations were associated with differences in the prognosis of metastatic BC. The D538G mutation showed a statistically significant lower PFS (p = 0.03), while the Y537S mutation was not significantly correlated with PFS (p = 0.354). According to the findings of this meta-analysis, the assessment for plasma ESR1 mutations may provide prognostic and clinical guidance regarding subsequent endocrine therapy decisions for ER-positive, metastatic BC patients who had received prior therapy with AIs.

Clinical Profile and Prognosis of Metastatic Breast Cancer with Pseudocirrhosis: A Rare and Dismal Condition

Clinical Profile and Prognosis of Metastatic Breast Cancer with Pseudocirrhosis: A Rare and Dismal Condition

DEPDC1B-mediated USP5 deubiquitination of β-catenin promotes breast cancer metastasis by activating the wnt/β-catenin pathway.

Breast cancer has become the malignant disease with the highest morbidity and mortality among female cancer patients. The prognosis of metastatic breast cancer is very poor, and the therapeutic effects still need to be improved. The molecular mechanism of breast cancer has not been fully clarified. Bioinformatics analysis was used to find the differentially expressed gene that affects the occurrence and development of breast cancer. Furthermore, scratch assays, transwell assays, immunofluorescence and western blotting were used to determine the biological behaviour of breast cancer cells affected by DEPDC1B. The molecular mechanism was investigated by mass spectrometry analysis, coimmunoprecipitation and ubiquitin assays. Here, we found that DEPDC1B was highly expressed in breast cancer cells and tissues and was associated with lower overall survival (OS) in patients. We found that DEPDC1B interference significantly inhibited tumour invasion and migration in vitro and tumour metastasis in vivo. Mechanistically, DEPDC1B was first shown to activate the wnt/β-catenin signalling pathway as an oncogene in breast cancer cells. In addition, we also confirmed the interaction between DEPDC1B, USP5 and β-catenin. Then, we found that DEPDC1B mediates the deubiquitination of β-catenin via USP5, which promotes cell invasion and migration. Our findings provide new insights into the carcinogenic mechanism of DEPDC1B, suggesting that DEPDC1B can be considered a potential therapeutic target for breast cancer.

Multi-factor dynamic analysis of ctDNA and CTC to aid the diagnostic prognosis of patients with metastatic breast cancer (MBC).

1038 Background: The monitoring of circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) in patients with MBC predicts treatment resistance and prognosis. We previously reported that ESR1 alterations in ctDNA were associated with tumor tissue characteristics and CTCs which may predict metastasis and worse prognosis in MBC (2019 ASCO#1036, 2022 ASCO#1057). Furthermore, ctDNA can be used to evaluate tumor heterogeneity in MBC (2020 ASCO #1028). Here, we report a multi-factor analysis in ctDNA and CTC which may help to elucidate disease prognosis in MBC. Methods: This study included 391 MBC patients who received systemic treatment before 2022 under an IRB-approved clinical trial at NU Lurie Cancer Center. Whole blood samples (7.5ml/each) were collected from all patients at three time points including before treatment, three months, and six months after treatment for CTC enumeration using the CELLTRACKS system (Menarini). The corresponding ctDNA was analyzed by Guardant360 NGS evaluating 74-gene mutations. The maximum follow-up period was 380 months from first diagnosis. Causal Inference with Ensembel Learning was used for statistical analyses. Results: Of the 391 patients ctDNA analysis, TP53 mutations were found in 160 patients (TP53Mut, 40.92%), CDH1 mutations were found in 21 patients (CDH1Mut, 5.37%), Myc mutations were found in 53 patients (MycMut, 13.55%), and BRAF mutations were found in 35 patients (BRAFMut, 8.95%) inclusive of all timepoints. The corresponding ctDNA mutations were not detected in 231 patients for TP53 (TP53WT, 59.08%), in 370 patients for CDH1 (CDH1WT, 94.63%), in 338 patients for Myc (MycWT, 86.45%), or in 356 patients for BRAF (BRAFWT, 91.05%). There was shorter median overall survival (mOS) in the ctDNA mutated groups compared to the non-mutated groups: 88 months TP53Mut vs 184 months TP53WT, HR 1.91, P = 0.0002; 133 months CDH1WT vs 81 months CDH1Mut, HR 2.03, P = 0.02; 76 months Myc Mut vs 179 months MycWT , HR 3.24, P &lt; 0.0001, and 77 months BRAFMut vs 146 months BRAFWT HR 2.45, P = 0.007. Furthermore, ≥5 CTCs were found in 32.42% patients. These patient had a shorter mOS compared to patients had no CTC detected at any time point (70.1 months vs 131 months; P &lt; 0.001; HR 2.97). CTC-clusters were 50 times more likely to predict metastasis than single CTCs. We also found that greater than 5 CTC-clusters, clusters with 1-5, and no CTC-clusters were found in 12.55%, 11.25%, and 76.19% patients, respectively. Larger CTC-clusters had shorter mOS at 63 months, 121 months, and 163 months, respectively (P &lt; 0.0001). Conclusions: In this study, we identified multiple liquid biopsy factors including ctDNA and CTC-clusters at various time points, and found that these are associated with prognosis. The synergy of multiple ctDNA mutations and CTC-clusters during treatment may expand the predictive role of liquid biopsy for the monitoring of disease progression in patients MBC.

Abstract 2099: Predicting the metastatic potential in breast cancer patients in Bangladesh

Abstract Background and Aims: Breast cancer (BC) is the leading cause of mortality among all cancers in Bangladeshi women. The prognosis of metastatic breast cancer depends on certain factors, like surgery, radiotherapy, chemotherapy, hormonal receptor content of the tumor or associated illness such as diabetes mellitus and obesity. Among the associated illness, diabetes mellitus is one of the most common metabolic disease worldwide, which is linked to an increased risk of developing breast cancer and an even higher risk of recurrence and dying from this disease. In this context, we studied diabetic and metabolic disease related growth and metabolic factors in breast cancer patients for assessing the prognosis of BC. Methods: A total of 180 mastectomy patients were purposively taken in the present study. Data were collected from retrospective information. Anthropometric and biochemical parameters were taken by standard methods. Growth and metabolic factors were determined by enzyme-linked immunosorbent assay (ELISA) method. Association of diabetes treatment modalities with breast cancer metastasis was evaluated by correlation analysis among 80 diabetic patients. Results: Serum level of insulin-like growth factor 1 (IGF1) was found to be significantly higher (p=0.001) in ER and PR negative breast cancer than their positive counterparts. Patients who had lymph node positive and Her2 borderline showed a significant reduction in serum adiponectin level (p&amp;lt;0.05) and this association was retained after adjusting the confounding variables. Furthermore, delayed treatment, IGF-1, ALP, sleeping hours were significant predictors for metastasis of breast cancer patients in Bangladesh. The Diabetic BC patients treated with both insulin and oral drug was found to be negatively associated with breast cancer metastasis compared to Diabetic-BC patients treated with only oral drugs (p&amp;lt;0.05). However, BC metastasis did not any have an association with serum fasting insulin or insulin resistance. Obesity also did not show any relation with BC prognosis. Conclusions: Serum IGF-1 and adiponectin may have an association with invasiveness and metastasis of breast cancer. Diabetes treatment with combination therapy ameliorates breast cancer metastasis.Breast cancer prognosis would be independently predict by serum levels of IGF-1 as well as IGF1 could be used as a screening biomarker when validated in a larger sample size. Citation Format: Salima Akter, Muhammad Saiedullah, Mousumi Bhowmik, Hajara Akhter, Shahina Ansary, Waheed Akhtar, Sufi Hannan Zulfiqur Rahman, Tae Gyu Choi, Sung Soo Kim. Predicting the metastatic potential in breast cancer patients in Bangladesh [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 2099.

Abstract 1031: Specific gene alterations of HER2 positive single circulating tumor cell (CTC) compared to autologous leukocytes in metastatic breast cancer (MBC)

Abstract Introduction: Monitoring of CTC in MBC showed ability to predict treatment resistance and outcome. HER2 is one of the most important tumor markers which is associated with rapid cell division and the metastasis and prognosis of MBC in the clinic. More recently, we reported single CTC gene mutation is a key point to distinguish metastasis capability in MBC (2022 AACR and 2022 ASCO). Herein, we report specific gene alterations in HER2+ single CTC compared to autologous leukocytes in MBC. Methods: Whole blood sample (7.5ml/each) was collected from MBC patient before therapy. CTC enumeration was performed in FDA approved CellSearch™ System and then the single HER2+ CTC and leukocytes were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and then the initial library was prepared by using Ampli1™ Whole Genome Amplification, Ampli1™ QC Kit (Menarini) and AMPure XP kit (Beckman Coulter). The exome capture was performed by TruSeq DNA Exome kit (illumine). The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). Results: We identified 107 CTCs including 55 HER2+ CTCs and 14 CTC-clusters. Autologous single HER2+ CTC (HER2+CK+CD45−, Group 1), HER2+CK−CD45+ leukocyte (Group 2) and HER2−CK−CD45+ leukocyte (Group 3) were isolated and sequenced respectively. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 486,119 counts (56.69%) for intron variants, 175,819 counts (20.51%) for intergenic variants, 70,334 counts (8.20%) for exon variants, 50,370 counts (5.87%) for downstream genes, 45,915 counts (5.36%) for upstream genes and others (3.37%) in HER2+ CTC. Meanwhile, there were 71,848 (8.76%) and 0 counts for exon variants found in Group 2 and Group 3 respectively. There were 79 gene variants ((SNP and Ins-Del) identified to have the highest impact effect (≥20) on HER2+ CTC chromosome, when there were 85 and 0 highest impact gene variants were identified in Group 2 and Group 3 respectively. The specific gene alterations in Group 1 compared to Group 2 includes CCNA2, FOLH1, BRD4, SAMHD1, CYP17A1, IDE, HPGDS and CTNNB1 Among the top 50 high impact gene variants, there were 26 genes alteration sites are same in both Group 1 and Group 2, including FECH, HDAC8, CYP11B2, TTN(6 sites), RAN, CD207, HK1, CASP1(2 sites), BRCA1, PIK3CG (3 sites), APEX1(4 sites), KIF11, SIRT5 (2 sites), XYLB and CHKA. Conclusion: Genomic characterization of HER2 positive single CTCs elucidated specific gene alterations associated with disease metastasis compared to autologous leukocytes in MBC. The newfound HER2+CK−CD45+ cells with gene alterations maybe a new kind of cancer transformed leukocytes which need further validation. Specific gene alterations will help to develop novel drugs aimed at the eradication of CTCs using molecularly driven therapies for disease metastasis. Citation Format: Qiang Zhang, Lorenzo Gerratana, Zhe Ji, Xinkun Wang, Paolo D’Amico, Seema Singhal, Youbin Zhang, Andrew A. Davis, Ami N. Shah, William Gradishar. Specific gene alterations of HER2 positive single circulating tumor cell (CTC) compared to autologous leukocytes in metastatic breast cancer (MBC) [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 1031.

Immune Checkpoint Inhibitors in Breast Cancer: A Narrative Review.

Breast cancer is the most frequently diagnosed malignancy in patients worldwide and the main cause of cancer-related death. Though still incurable, metastatic breast cancer's prognosis has been considerably improved in the past 10years due to the introduction of new targeted agents, such as immune checkpoint inhibitors (ICI). However, these medications are associated with unique side effects known as immune-mediated adverse events (irAE). In this paper, we review the clinical evidence for the use of ICIs in breast cancer, in both the metastatic as well as neoadjuvant/adjuvant setting, followed by a review of irAE most commonly seen, and the medications used to treat them. Our opinion is that any cancer specialist treating patients with breast cancer should be aware of these side effects for early detection and management, and oncologists should be the leaders of the multidisciplinary team that will take care of them.

Single progesterone receptor-positive phenotype has the similar clinicopathological features and outcome as triple-negative subtype in metastatic breast cancer.

The same clinicopathological features and prognosis have been reported between single progesterone receptor-positive (sPR-positive) and triple-negative phenotype in early-stage breast cancer, but such similarity has not been studied in metastatic breast cancer (MBC). Therefore, the purpose of this study was to estimate the difference between sPR-positive phenotype and other phenotypes in MBC. Patients with HER-2-negative MBC were selected from the Surveillance, Epidemiology and End Results database. Pearson's χ2 test was used to compare the difference of clinicopathologic factors between sPR-positive phenotype and other phenotypes. Univariate and multivariate analyses were performed to evaluate the effects of hormone receptor (HoR) phenotypes and other clinicopathologic factors on the cancer-specific survival (CSS) and overall survival (OS). Overall, 10877 patients including 7060 patients (64.9%) with double HoR-positive (dHoR-positive), 1533 patients (14.1%) with single estrogen receptor-positive (sER-positive), 126 patients (1.2%) with sPR-positive and 2158 patients (19.8%) with double HoR-negative (dHoR-negative) were analyzed. The patients with sPR-positive or dHoR-negative were more likely to be younger, higher grade and tumor stage, visceral and brain metastasis than ER-positive phenotypes (P<0.001). MBC with sPR-positive had the similar CSS (HR: 1.135, 95%CI: 0.909-1.417, P=2.623) and OS (HR: 1.141, 95%CI: 0.921-1.413, P=0.229) as dHoR-negative, but worse outcome than ER-positive phenotypes. Chemotherapy significantly improved the survival for MBC, especially for sPR-positive MBC (CSS, HR: 0.39, 95%CI: 0.213-0.714, P=0.002; OS, HR: 0.366, 95%CI: 0.203-0.662, P=0.001). Patients with sPR-positive and triple-negative have similar biological behavior and prognosis in MBC. Chemotherapy may be a preferred recommendation for MBC with sPR-positive.

Relationship between metastasis and second primary cancers in women with breast cancer

BackgroundBreast cancer (BC) survivors have an increased risk of developing second primary cancers (SPCs); however, it is still unclear if metastasis is a risk factor for developing SPCs. Usually, long-term cancer survivors face an increased risk of developing SPCs; however, less attention has been paid to SPCs in patients with metastatic cancer as the survival outcomes of the patients are greatly reduced.MethodsA total of 17,077 American women diagnosed with breast cancer between 2010 and 2018 were identified from Surveillance, Epidemiology, and End Results (SEER) database and were included in the study. The clinical characteristics, standardized incidence ratio (SIR), standardized mortality ratio (SMR), and patterns of SPCs in BC patients with no metastasis, regional lymph node metastasis, and distant metastasis were investigated. Kaplan-Meier method was used to compare the prognosis of BC patients after developing SPCs with different metastatic status. XGBoost, a high-precision machine learning algorithm, was used to create a prediction model to estimate the prognosis of metastatic breast cancer (MBC) patients with SPCs.ResultsThe results reveal that the SIR (1.01; 95% CI, 0.99–1.03, p>0.05) of SPCs in non-metastasis breast cancer (NMBC) patients was similar to the general population. Further, patients with regional lymph node metastasis showed an 8% increased risk of SPCs (SIR=1.08, 95%CI, 1.05–1.11, p<0.05), and patients with distant metastasis had a 26% increased risk of SPCs (SIR=1.26, 95%CI, 1.16–1.37, p<0.05). The SIR of SPCs in all patients below the age of 40 was the highest, which decreased with age. Patients with poorly differentiated cancers, large tumor size, and late N stage had an increased risk of SPCs. However, an increase in SIR of SPCs was observed in distant MBC patients, even at the early T1 (SIR=1.60, 95% CI, 1.22–1.98, p<0.05) and N1 (SIR=1.27, 95% CI, 1.10–1.44, p<0.05) stage. An increase in the SIR of SPCs was observed in patients with triple-negative BC, and the SIR of SPC increased with metastasis development in BC patients with luminal A subtype. The peak of SPCs risk occurrence was earlier in MBC patients (4-6 months and 10 months) compared to NMBC patients (12 months). The effect of metastasis on the prognosis of SPCs patients was dependent on the type of SPCs. Meanwhile, the XGBoost model was created to predict the 3-year (AUC=0.873) and 5-year survival (AUC=0.918) of SPCs in MBC patients.ConclusionsOur study provides novel insight into the impact of metastasis on SPCs in BC patients. Metastasis could promote the second primary tumorigenesis which further increased cancer-related deaths. Therefore, more attention should be paid to the occurrence of SPCs in MBC patients.

Role of Circulating Tumor Cells in Determining Prognosis in Metastatic Breast Cancer.

Dinesh Chandra DovalBackground Circulating tumor cells (CTCs) in the peripheral blood may play a major role in the metastatic spread of breast cancer. This study was conducted to assess the role of CTCs to determine the prognosis in terms of survival in metastatic breast cancer patients. Methods This prospective study of 36 patients was conducted at the Hospital from April 2016 to May 2018. Details of each patient related to the demographic profile, tumor type, treatment, and follow-up information were recorded. The number of CTCs in the peripheral blood was measured by Celsee PREP 400 sample processing system and Celsee Analyzer imaging station. Results There was a positive correlation between the number of site of metastasis with number of CTCs ( p -value < 0.001). In the patients with clinical/partial response, a significant reduction in the number of CTCs after 1 month of therapy was observed ( p -value = 0.003). When the number of CTCs at baseline and 6 months were compared with the positron emission tomography response at 6 months, a statistically significant difference in CTCs in patients having partial response after 6 months was observed ( p -value = 0.001). On comparison with the responder groups, a statistically significant reduction in CTCs at baseline and 6 months was observed ( p -value = 0.001). Patients with CTCs less than 5 and more than or equal to 5 after 1 month of treatment had a mean progression-free survival of 11.1 months and 7.5 months ( p -value = 0.04) and a mean overall survival of 11.6 and 9.6 months ( p -value = 0.08), respectively. Conclusion Assessment of CTCs provides a more quantifiable response than radiographic evaluation and at a much earlier time point and is also a better predictor of survival.

Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis.

Early detection of metastatic breast cancer (MBC) is a serious issue for the healthcare system. It is essential to develop potential non-invasive, low-cost molecular biomarkers. The present study explored specific serum proteins of inflammatory, MAPK, and cytoskeletal signaling pathways involved in the progression of MBC to establish a panel of blood-based diagnostic and prognostic biomarkers. Healthy-control (HC), non-metastatic (NM), and metastatic (M) (pre- and post-therapy) breast cancer (BC) patients were recruited. LOX5, Rac1, Rac1b, p38α, phospho-p38α (Y182), LIMK1, phospho-LIMK1 (T508), cofilin1, and phospho-cofilin1 (S3) were quantified in the serum of the study group by real-time label-free surface plasmon resonance technology and verified by Western blot. Proteins were found to be significantly elevated in the serum of BC patients compared to HC and also higher in M compared to NM, which further downregulated in post-therapy M patients. Elevation of phospho-LIMK1 and phospho-cofilin1, which are critical for M, was also indicated in the serum level and can differentiate from NM. Receiver operating characteristics (ROC) derived area under the curve (AUC) (0.9) is very strong to differentiate between HC and BC. Moreover, the combined ROC of 3 molecules phospho-LIMK, p38α, and phospho-p38α were found to be a potent predictive panel of biomarkers between M and NM with AUC0.95. The panel of inflammatory cytoskeleton signaling regime proteins specified in this study can have significant clinical utility for diagnosis as well as prognosis of MBC at an early stage. The study may have a high translational value in a simple and cost-effective way by avoiding frequent CT/PET scans.

Abstract 1950: A novel ESR1 mutation assay for single circulating tumor cell by application of DEPArray࣪ System and Digital Droplet PCR

Abstract Introduction: Although circulating tumor cells (CTC) display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. ESR1 mutations are a major mechanism of acquired endocrine resistance in metastatic breast cancer (MBC). We previously reported that ESR1 mutations in circulating tumor DNA (ctDNA) was associated with worse prognosis in MBC (2020 ASCO). Herein, we report a new ultra-high sensitivity approach of ESR1 mutations assay for single CTC by using DEPArray system and Droplet Digital PCR (ddPCR). Methods: Whole blood sample (7.5ml/each) was collected from stage IV MBC patients and then CTC enumeration was performed in CellSearch™ System by targeting the EpCAM antigen (2020 AACR #3120). The single CTC and single white blood cell (WBC) were then isolated from CellSearch cartridges by using DEPArray࣪ System (Menarini). The single CTC DNA was isolated by QIAamp DNA Micro Kit (Qiagen). ESR1 mutations hotspots including p.E380Q, p.Y537S and p.D538G were analyzed by using QX200™ ddPCR System (Annealing/Extension=58 °C, 40 cycles) and the corresponding probes (Mutation-FAM and Wild type-HEX mixed) for p.E380Q-dHsaMDS500014536, p.Y537-dHsaMDS975379796, p.D538G- dHsaMDS460485301 respectively (Bio-Rad). Meanwhile, corresponding plasma ctDNA was analyzed by Guardant 360 Health NGS-based assay for a 73 genes panel for single nucleotide variants. Results: Positive controls (using 0.00000325ng pure positive DNA) and negative control (using water) were performed in each ddPCR assay. In cohort 1: total of 8 samples including 4 single CTC samples and 4 single WBC samples were collected using the DEPArray system. Wild type ESR1 (p.E380Q, p.Y537S and p.D538G) was detected in all 4 single CTC samples and 4 single WBC samples. There were 3 copies, 3.4 copies, 1.6 copies and 1.8 copies of p.E380Q mutation droplets found in 4 single CTC samples when there were no p.Y537S or p.D538G mutations found in these samples. There was no ESR1 mutation found in the corresponding ctDNA. In cohort 2: Two samples including 4 CTCs and 100 CTCs were collected from CellSearch cartridges. There were 0.09 vs 1 copies (8.25%) and 0.1 vs 0.31 copies (24.39%) of p.E380Q mutation droplets found in these two samples respectively, compared to ctDNA results of 1.80% and 0 respectively. Furthermore, there was 0.08 vs 0.67 copies (10.66%) of p.D538G mutation droplets were found in the sample including 100 CTCs compared to 12.70% mutations detected in the corresponding ctDNA. Conclusion: Our new approach verified the feasibility of ESR1 mutations assay in very low amount DNA from single CTCs with high sensitivity even when ESR1 mutation could be found in CTCs but not ctDNA. This approach may offer a strong evidence for early disease metastasis, efficacy of individualized treatment monitoring and personalizing cancer therapy for precision medicine. Citation Format: Qiang Zhang, Paolo D'Amico, Marwa Manai, Carolina Reduzzi, Andrew A. Davis, Lorenzo Gerratana, Weijun Qin, Jianhua Jiao, Saya L. Jacob, Jeannine Donahue, Youbin Zhang, Lisa Flaum, Massimo Cristofanilli, Leonidas C. Platanias, Ami N. Shah, William Gradishar. A novel ESR1 mutation assay for single circulating tumor cell by application of DEPArray࣪ System and Digital Droplet PCR [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 1950.

HBV Infection Status Does Not Influence the Initial Metastatic Pattern and the Prognosis of Breast Cancer Patients with de novo and Relapsed Metastatic Disease.

PurposeThis study aimed to evaluate the influence of hepatitis B virus (HBV) infection status on the initial metastatic pattern and prognosis in metastatic breast cancer (MBC).MethodsMBC patients admitted to Chongqing University Cancer Hospital between January 2011 and December 2019 were enrolled. The association of HBV infection status with clinicopathological features was analyzed. The impact of HBV infection status on initial metastatic pattern and survival was evaluated.ResultsA total of 1124 patients with MBC, including 310 with de novo (cohort A) and 814 with relapsed metastatic disease (cohort B), were eligible for this study. Seropositive HBsAg was identified in 28 (9.0%) and 68 (8.4%) patients in cohort A and B, respectively. The clinicopathological features are similar between HBsAg-positive and HBsAg-negative patients. There was no significant association of HBV infection status with the rate of metastasis at each site in de novo and relapsed MBC. HBsAg-positive patients tended to have longer metastasis-free survival (MFS) and/or overall survival (OS) time, but it was not the independent prognostic factor.ConclusionIn conclusion, HBV infection status does not influence the initial metastatic pattern and the prognosis of MBC patients.

Abstract P2-01-04: Esr1 hotspot mutations in circulating tumor DNA mutation are associated with endocrine therapy resistance in metastatic breast cancer

Abstract Introduction: Metastatic breast cancer (MBC) is a challenging clinical condition because most tumors will develop resistance to endocrine treatment as the cancer progresses. We previously reported the correlation of ESR1 mutations in circulating tumor DNA (ctDNA) and CTCs resulted in worse prognosis in MBC (ASCO-2020). In this study, we report a new finding that specific hotspots of ESR1 ctDNA mutations are associated with endocrine therapy resistance status in MBC and their allele frequency (AF) can be used to monitor response. . Methods: Plasma cfDNA were collected from a total of 299 MBC patients (2016-2019) after informed consent under an IRB-approved trial at the Lurie Cancer Center at Northwestern University. ER+ patients are 180 out of 297 (61%). Patients with HR+ MBC were evaluated before endocrine therapy (Anastrozole, Exemestane and Letrozole ) +/- CDK4/6 inhibitors (Palbociclib, Ribociclib, Abemaciclib), and every six months after initiation of therapy. Response to therapy was recorded according to the different time points. Endocrine resistance is defined as relapse during the first 2 years of adjuvant endocrine therapy or progressive disease within the first 6 months of first-line endocrine therapy for metastatic breast cancer. Plasma ctDNA was isolated using a Qiagen circulating nucleic acid kit, and then was analyzed using the Guardant360 Health next-generation sequencing (NGS)-based assay (Guardant Health). In this study, only patients with ESR1 mutations were included for statistical analyses of correlation between the hotspots mutation with the endocrine therapy resistance by using Causal Inference approach. All statistical analyses were conducted Mann-Whitney U test by IBM SPSS version 23.0. Results: There were total of 239 ESR1 ctDNA mutations detected in 18 hotspots from 42 patients at different time points, among which there were 131 endocrine therapy resistance status in all time points. The numbers of ESR1 hotspots mutations and the corresponding endocrine therapy resistance cases were found in the following hotspots respectively as Q314 (3 cases with mutation, 0 cases with resistance), T347T (4, 0), N359I (4, 0), K362N (4, 0), E380Q(32, 16), V392I (1, 1), G442R(3, 0), F461I(3, 0), S463P (3, 2), S464 (1, 0), I487M(14, 0), K520K (2, 0), M528V (5, 0), L536H (31, 19), Y537N/C/S (46, 34), D538G (77, 59), D545D(2, 0) and Q565 (4, 0). There were 50%, 61.2%, 73.9% and 76.6% patients developed endocrine therapy resistance when they had E380Q, L536H, Y537N/C/S and D538G mutations respectively at any time points. Furthermore, among these 4 hotspot mutations, the MBC patients with lack of response to endocrine therapy have significantly higher percentage of mutated DNA than the patients with sensitive response. For the patients with E380Q mutation, the mean percentage of mutation DNA was 8.44% for those with lack of response and lower than 0.01% for those with sensitive response (P&amp;lt;0.001). For the patients with L536H mutation, the mean percentage of mutation DNA was 3.48% for those with lack of response and lower than 0.01% for those with sensitive response (P&amp;lt;0.001). For the patients with Y537N/C/S mutation, the mean percentage of mutation DNA was 5.23% for those with lack of response and 0.02% for those with sensitive response (P&amp;lt;0.001). For the patients with D538G mutation, the median percentage of mutation DNA was 5.13% for those with lack of response and 0.01% for those with sensitive response (P&amp;lt;0.001). Conclusions: The outcome of this study demonstrated that patients with mutations on known ESR1 hotspots (E380Q, L536H, Y537N/C/S and D538G) develop clinical resistance to endocrine therapy. Evaluation of these ESR1 hotspots mutations by ctDNA could be reliably used to monitor the response to endocrine therapy and predict the prognosis for MBC patients. Citation Format: Qiang Zhang, Paolo D'Amico, Weijun Qin, Jianhua Jiao, Andrew A. Davis, Lorenzo Gerratana, Saya L. Jacob, Youbin Zhang, Jeannine Donahue, Wenan Qiang, Ami N. Shah, Amir Behdad, Lisa Flaum, William Gradishar, Leonidas C Platanias, Massimo Cristofanilli. Esr1 hotspot mutations in circulating tumor DNA mutation are associated with endocrine therapy resistance in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-04.

Landscape of HER2-low metastatic breast cancer (MBC): results from the Austrian AGMT_MBC-Registry

BackgroundAbout 50% of all primary breast cancers show a low-level expression of HER2 (HER2-low), defined as immunohistochemically 1+ or 2+ and lack of HER2 gene amplification measured by in situ hybridization. This low HER2 expression is a promising new target for antibody–drug conjugates (ADCs) currently under investigation. Until now, little is known about the frequency and the prognostic value of low HER2-expression in metastatic breast cancer (MBC).Patients and methodsThe MBC-Registry of the Austrian Study Group of Medical Tumor Therapy (AGMT) is a multicenter nationwide ongoing registry for MBC patients in Austria. Unadjusted, univariate survival probabilities of progression-free survival (PFS) and overall survival (OS) were calculated by the Kaplan–Meier method and compared by the log-rank test. Multivariable adjusted hazard ratios were estimated by Cox regression models. In this analysis, only patients with known HER2 status and available survival data were included.ResultsAs of 11/15/2020, 1,973 patients were included in the AGMT-MBC-Registry. Out of 1,729 evaluable patients, 351 (20.3%) were HER2-positive, 608 (35.2%) were HER2-low and 770 (44.5%) were completely HER2-negative (HER2-0). Low HER2-expression was markedly more frequent in the hormone-receptor(HR)+ subgroup compared to the triple-negative subgroup (40% vs. 23%). In multivariable analysis, low HER2 expression did not significantly influence OS neither in the HR+ (HR 0.89; 95% CI 0.74–1.05; P = 0.171) nor in the triple-negative subgroup (HR 0.92; 95% CI 0.68–1.25; P = 0.585), when compared to completely HER2-negative disease. Similar results were observed when HER2 IHC 2+ patients were compared to IHC 1+ or 0 patients.ConclusionLow-HER2 expression did not have any impact on prognosis of metastatic breast cancer in this real-world population.

Evaluating Serum Thymidine Kinase 1 in Patients with Hormone Receptor-Positive Metastatic Breast Cancer Receiving First-line Endocrine Therapy in the SWOG S0226 Trial.

Serum thymidine kinase 1 (sTK1) activity is associated with poor prognosis in metastatic breast cancer (MBC). We assessed the prognostic effect of sTK1 in patients with hormone receptor-positive MBC treated on a prospective randomized trial of anastrozole (A) versus A plus fulvestrant (A + F). sTK1 was assessed in 1,726 serums [baseline (BL), cycles 2, 3, 4, and 7] using the DiviTum assay. A prespecified cutoff of ≥200 Du/L was considered high. Progression-free survival (PFS) and overall survival (OS) were analyzed by Kaplan-Meier, log-rank tests, and Cox regression. BL sTK1 was elevated in 171 (40%) of 432 patients. Patients with high versus low BL sTK1 had significantly worse PFS [median 11.2 vs. 17.3 months, HR = 1.76; 95% confidence interval (CI; 1.43-2.16); P < 0.0001] and OS [median 30 vs. 58 months, HR = 2.38; 95% CI (1.91-2.98); P < 0.0001]. OS was significantly better for patients with high sTK1 who did not have prior adjuvant tamoxifen and who received A + F versus A alone [median 46 vs. 21 months, HR = 0.58; 95% CI (0.38-0.87); P = 0.0087]. Patients with low sTK1 had no difference in outcomes by therapy (P = 0.44). At serial timepoints, high versus low sTK1 had significantly worse subsequent PFS and OS [at cycle 2: PFS HR = 1.70, 95% CI (1.34-2.17); P < 0.0001, OS HR = 2.51, 95% CI (1.93-3.26); P < 0.0001]. High sTK1 at BL and subsequent timepoints is associated with worse prognosis in patients with MBC starting first-line endocrine therapy (ET). Patients with low sTK1 at BL have comparable outcomes on single-agent or combination ET.

323P Clinical profile and prognosis of metastatic breast cancer with pseudocirrhosis: A rare and dismal condition

Pseudocirrhosis (PC) is defined by radiological abnormalities suggesting cirrhosis in patients (pts) with liver metastasis (mets), especially metastatic breast cancer (mBC). The cause of PC can be due to liver disease progression (DP), treatment toxicity or response to systemic treatment. PC is associated with high mortality.

Abstract 589: CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer

Abstract Background: CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and endocytosis may identify highly aggressive breast cancer (BC) subtypes likely to spread via the circulatory system. We first studied the activation and endocytosis of CCR5 in response to its ligand CCL5 (RANTES) in the model BC cell line MDA-MB231. We then screened two types of circulating tumor-associated cells (TACs) with known negative clinical outcomes, 1) circulating tumor cells (CTCs) and 2) cancer-associated macrophage-like (CAMLs) cells, to evaluate CCR5 upregulation in relation to disease progression in metastatic breast cancer (mBC). Methods: MB231 cells were used to visualize CCR5 activation within the cell's surface and intracellular space using an anti-CCR5 antibody after stimulation with RANTES. MB231 cells were stained with LAMP-1A to verify co-localization of CCR5 and RANTES to endosomes. Anonymized peripheral blood samples from (n=54) mBC patients were obtained in accordance with local IRB regulations and with informed consent. TACs were isolated using a low-flow CellSieve Microfiltration system and stained for Cytokeratin, CD45, and CCR5. CCR5 expression signal and localization were evaluated on all TACs via fluorescence microscopy. Results: CCR5 in MB231 cells appeared as ~1μm clusters known as “CCR5 pools”, which can be found on the cell's surface, within the cytoplasm, and in the nuclear space. CCR5 pools were upregulated in the presence of RANTES, which appears to relate to endocytosis and nuclear translocation. Within the TACs of mBCs, we found similar patterns of CCR5 pooling with 70% of patients (n=38) having CCR5+ CAMLs and 41% (n=22) having CCR5+ CTCs. It was found that higher numbers of CCR5 pools (≥10pools/cell) were correlated to a 2-fold increase in both CTCs and CAMLs in circulation. In the analysis of outcome data, it was determined that patients having ≥10 CCR5 pools on their CAMLs was a highly significant predictor of worse progression free survival (PFS) and overall survival (OS) (PFS HR=4.8, CI 95% 1.9-11.6, p=0.0015) and (OS HR=3.6, CI 95% 1.3-10.0, p=0.0331). In CTCs ≥10 pools/cell was not a significant predictor for worse PFS and OS. However, ≥5 CCR5 pools/cell significantly predicted for worse PFS but not OS (PFS HR=2.9, CI 95% 1.2-7.4, p=0.0418 and OS HR=3.3, CI 95% 1.1-9.3, p= 0.0539). Conclusion: We observed the activation and endocytosis of CCR5 pools using RANTES in MD-MB231 cell lines, and then demonstrated that this upregulated motility pathway appears to be conserved in migratory cells in mBC patients. Further, this upregulation of CCR5 appears to correlate with worse clinical outcomes, which may provide an explanation for how tumor-associated cells move into circulation and acquire metastatic potential. Citation Format: Ashvathi Raghavakaimal, Massimo Cristofanilli, Cha-Mei Tang, R. K. Alpaugh, Kirby P. Gardener, Saranya Chumsri, Daniel L. Adams. CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 589.