Biomarkers In Metastatic Breast Cancer Research Articles

Use of liquid biomarker thymidine kinase activity (TKa) to predict outcome and progression in patients with metastatic breast cancer (MBC) in the GEICAM/2014-12 FLIPPER trial.

1028 Background: There is a need for more precise blood-based tumor biomarkers in MBC. TKa is a liquid proliferation biomarker providing prognostic, predictive and monitoring information in MBC. TKa can be measured in blood using the DiviTum TKa assay. Here we report the results of TKa in a trial of endocrine therapy (ET) fulvestrant (F) + the CDK4/6 inhibitor (CDK4/6i) Palbociclib (Pa) vs. F + Placebo (Pl), the GEICAM FLIPPER trial (NCT02690480), a multicenter, double-blind, randomized (1:1) study. F+Pa showed 1-year PFS rates of 83.5% vs. 71.9% (p=0.064) and better median (m)PFS (31.8 vs. 22.0 months, p=0.001) than F+Pl in first line treatment of HR+/HER2- MBC patients. F+Pa also significantly improved ORR and CBR. ET+CDK4/6i are currently the cornerstone of treatment for HR+/HER2- MBC. However, 10-30% of these patients display primary resistance to treatment. Early identification of patient subsets with different risk of progression during treatment with CDK4/6i may help tailor clinical management and disease monitoring. Methods: Sequential plasma was collected from 187 patients (pts) at baseline (BL) and at 12, 24, 36, 48 weeks (w) on treatment, and at progression. 910 samples were analyzed for TKa with the FDA approved and CE labelled DiviTum TKa assay (Biovica, Sweden) using DuA (DiviTum unit of Activity) as measuring unit. The cutoff for categorizing in high vs low is 250 DuA. The Kaplan-Meier method was used to estimate mPFS and mOS. Unadjusted and adjusted hazard ratio (HR) and odds ratio (OR) with 95% confidence interval (CI) were calculated with the Cox proportional hazards regression model and with logistic regression respectively, considering relevant confounders. Results: Low BL TKa predicted better PFS (30.5 months mPFS in low vs 19.8 in high, HR 1.62; 95% CI 1.11-2.35, p=0.011) and OS (HR 1.81; 95% CI 1.15-2.85, p=0.0099). Higher TKa at BL and at 12w was detected in pts that progressed before 12 months. Conversely, low BL TKa, maintained throughout week 12-48, were associated with objective response (HR 2.58; 95% CI 1.38-4.81, p=0.0029) and OS (HR 5.74; 95%CI 2.06-16, p=0.0008). At progression, pts on F+Pa tended to have higher TKa levels than F+Pl (246 vs. 138 DuA), reflecting faster growing tumors, not achieving statistical significance (p=0.0585). TKa monitoring and dynamics from BL and on treatment timepoints (week 12-48) show that the group of pts with low TK at BL that remain low during treatment is enriched with non-progressors and pts that progress after 12 months (p=0.0108). High TKa at BL predicted shorter OS in the F+Pa arm (HR 4.39; 95% CI 2.15-8.99, p<0.0001). Conclusions: Measuring blood TKa levels can monitor and predict efficacy of F+/-Pa at BL and during therapy in MBC. TKa dynamics before and during therapy are associated with PFS and OS. Future studies will provide additional knowledge on the utility of TKa in MBC. Clinical trial information: NCT02690480 .

Abstract 7509: Evaluation ofHER-2 expression on circulating tumor cells as a real time biomarker in advanced breast cancer

Abstract Introduction: Breast cancer accounts for 12.5% of all new annual cancer cases worldwide. Cases classified as HER2-positive tend to proliferate, metastasize, and often lead to relapse. HER-2 is the most valid tumor marker, used as a diagnostics and prognostic biomarker in metastatic breast cancer (MBC). Thus, analysis of expression of HER-2 on CTCs can offer a real time dynamic biomarker for treatment options between endocrine therapy versus chemotherapy in advanced breast cancer. Functional assays on CTCs with HER-2 biomarker offer evolving biomarkers especially when the tissue is not accessible and inadequate. Methods: Retrospectively, 179 breast cancer patients' blood was analyzed for the presence of CTCs using the OncoDiscover® platform approved by Drug Controller General of India (CDSCO). Consisting of immunomagnetic multi-components mediated by anti EpCAM antibodies. The isolated cells were immunostained consisting of a nuclear dye DAPI, CK-18, CD45 and HER-2 antibodies. CTCs were identified by the presence of HER-2 + ve, CK 18 + ve, DAPI + ve and CD 45 -ve staining. Validation of HER-2 expression on the CTCs was analyzed based on the linear intensity gradients of the fluorescence signals. CTCs were termed as HER-2 -ve based on weak or no detectable fluorescence signal and termed +ve based on high fluorescence signal. Results: Among the cohort of 179 patient samples, 63.68 % samples showed the presence of CTC (CTC ranged from 1-7). 47.84% of these CTCs showed the expression of HER-2 signal. The mean value for fluorescence intensity for expression of HER-2 in CTCs was found to be 3.23. The observed mean fluorescence intensity for HER-2 expression further emphasizes the robustness of CTCs as a viable source for molecular characterization. Conclusion: Integration of HER-2 analysis on CTCs into the clinical assessment of metastatic breast cancer, may offer a non-invasive, real-time strategy for profiling and possibly paving the way for more precise and tailored therapeutic interventions. Citation Format: Jayant Khandare, Gourishankar Aland, Saloni Andhari, Ganesh Khutale, Prathamesh Patil, Tanvi Deshpande, Meghana Garbhe, Vikas Jadhav, Aravindan Vasudevan, Sreeja Jayant. Evaluation ofHER-2 expression on circulating tumor cells as a real time biomarker in advanced breast cancer [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 7509.

Fragmentation patterns of cell-free DNA and somatic mutations in the urine of metastatic breast cancer patients.

Urinary cell-free deoxyribonucleic acid (DNA) (ucfDNA) holds promise as a biomarker; however, its potential remains largely unexplored. We examined the fragmentation pattern of ucfDNA and identified somatic mutations within urine samples from metastatic breast cancer (MBC) patients. Urine and blood specimens were collected before treatment from 45 MBC patients and posttreatment urine samples from 16 of the 45 patients at the China National Cancer Center. Somatic mutations and tumor mutational burden (TMB) in the urine and plasma of 10 patients were analyzed by next-generation sequencing (NGS). Fragmentation patterns of cfDNA were displayed using electropherograms. Differences in the extracted amount of cfDNA, length of cfDNA fragments, and TMB between urine and plasma were compared using a Wilcoxon test. The fragmentation patterns of ucfDNA were categorized as follows: (1) profile A (n = 26) containing a short peak (100-200 bp) and a long peak (>1500 bp); (2) profile B (n = 8) containing only a long peak; and (3) profile C (n = 11) containing flat pattern. For profile A patients, the short-peaked ucfDNA circulating in the bloodstream was much shorter compared with plasma cfDNA (149 vs. 171 bp, Wilcoxon test, P = 0.023). The fragmentation patterns in lung metastasis patients exhibited a higher propensity toward profile C ( P = 0.002). After treatment, 87.5% of the patients exhibited consistent fragmentation patterns. The concordance rate for somatic mutations in the plasma and urine was 30%, and the median TMB of urine and plasma was not significantly different. This study established a fragmentation pattern for ucfDNA and detected somatic mutations in the urine of MBC patients. These results suggest the potential application of ucfDNA as a biomarker for MBC.

Analysis of the association of HER-2 low carcinomas and PAM50 assay in hormone receptor positive early-stage breast cancer.

HER2-low has emerged as a new predictive biomarker in metastatic breast cancer. However, its prognostic value in early-stage carcinomas needs to be revisited. We aimed to evaluate the association of HER2-low carcinomas with PAM50 risk groups combined with clinicopathological variables in early breast cancer. We conducted a retrospective analysis of 332 patients with early-stage breast cancer that underwent PAM50 signature analysis between 2015 and 2021at Hospital Universitario 12 de Octubre (Madrid, Spain). Clinical and pathological variables were collected from medical records. After adjusting for potential confounders, we estimated Odds Ratio (OR) and 95% confidence interval for high-risk PAM50 subgroup, comparing HER2-low versus HER2-zero carcinomas by multivariable logistic regression. P values below 0.05 were deemed statistically significant. 192 (57%) patients were classified as HER2-low carcinomas. Median follow-up was 34 months. Adjusted OR for high-risk PAM50 when comparing HER2-low versus HER2-zero carcinomas was 1.31 (95% CI: 0.75-2.30, p=0.33). The multivariable model detected significant associations for Ki-67% (≥20% vs. <20%: OR=4.03, 95% CI: 2.15-7.56, p<0.001), T staging category (T2/T3 vs. T1: OR=3.44, 95% CI: 1.96-6.04, p<0.001), progesterone receptor (PR≥20% vs. <20%: OR=0.44, 95% CI: 0.23-0.83, p=0.01), nodal staging category (N+ vs. N0: OR=3.8, 95% CI: 1.89-7.62, p<0.001) and histological grade (grade 2 vs. 1: OR=2.41, 95% CI: 1.01-5.73, p=0.04; grade 3 vs 1: OR=5.40, 95%CI: 1.98-14.60, p=0.001). In this early-stage breast cancer cohort, HER2-low was not associated with a high-risk PAM50 compared to HER2-zero carcinomas. Ki-67≥20%, T2/T3, histological grade 2/3, N+ and PR<20% were significantly associated to a high-risk PAM50.

Concurrent predictors of an immune responsive tumor microenvironment within tumor mutational burden-high breast cancer.

Data supporting high tumor mutational burden (TMB-H) as a lone biomarker for an immune-responsive tumor microenvironment (TME) in metastatic breast cancer (MBC) are weak, yet tumor agnostic approval in TMB-H advanced tumors provides immune checkpoint inhibition (ICI) as a clinical option. We evaluated concurrent predictors of immune-responsive and non-responsive TME within MBC. Tumor samples from patients with MBC (N=5621) were analyzed by next-generation sequencing of DNA (592-gene panel or whole exome) and RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). TMB-H threshold was set to ≥ 10 muts/Mb. PDL-1 was evaluated using SP142 antibody. Gene expression profiling and RNA deconvolution were used to estimate immune and stromal cell population abundance in the TME, and transcriptomic signature of immunotherapy response (T cell-inflamed score). 461 (8.2%) TMB-H MBC samples were identified. Consistent with prior studies, TMB-H tumors exhibited significant dMMR/MSI-H enrichment (7 vs. 0%, p<0.0001) and PD-L1+ expression (36 vs. 28%, p<0.05) compared to TMB-L. Across all samples, T cell-inflamed scores were weakly correlated with TMB. TMB-H was not associated with significantly increased immune responsive cell types (CD8+ T-cells, NK cells, or B cells) or immune response gene signatures (e.g. antigen presentation), yet positive trends were observed, while immunosuppressive fibroblasts were significantly decreased in TMB-H tumors (0.84-fold change compared to TMB-L, P<0.05). HR+/HER2- breast cancer was the only subtype in which TMB-H tumors exhibited increased T cell-inflamed scores vs. TMB-L. Concurrent PD-L1+ or dMMR/MSI-H with TMB-H was associated with high T cell-inflamed scores in both HR+/HER2- and TNBC. Among several associated biomarkers, B2M mutations and CD274 amplifications were positively associated with T-cell inflamed scores in TMB-H tumors; CDH1 and ERBB2 mutations were negatively associated. High TMB alone does not strongly correlate with immune infiltrate or immune-related gene signatures in MBC. TMB-H predicts T-cell inflamed signature compared to TMB-L in HR+/HER2- tumors only. Along with MSI-H and PD-L1+, several biomarkers, including B2M mutation and CD274 amplification, may help predict ICI benefit amongst TMB-H tumors. Co-occurring biomarkers within TMB-H breast cancer warrant evaluation in larger cohorts for response or resistance to ICI to develop composite predictive biomarkers in MBC.

Clinical and Translational Applications of Serological and Histopathological Biomarkers in Metastatic Breast Cancer: A Comprehensive Review.

Breast cancer is one of the most common malignancies worldwide and the most common form of cancer in women. A large proportion of patients begin with localized disease and undergo treatment with curative intent, while another large proportion of patients debuts with disseminated metastatic disease. In the last subgroup of patients, the prognosis in recent years has changed radically, given the existence of different targeted therapies thanks to the discovery of different biomarkers. Serological, histological, and genetic biomarkers have demonstrated their usefulness in the initial diagnosis, in the follow-up to detect relapses, to guide targeted treatment, and to stratify the prognosis of the most aggressive tumors in those with breast cancer. Molecular markers are currently the basis for the diagnosis of metastatic disease, given the wide variety of chemotherapy regions and existing therapies. These markers have been a real revolution in the therapeutic arsenal for breast cancer, and their diagnostic validity allows the classification of tumors with higher rates of relapse, aggressiveness, and mortality. In this sense, the existence of therapies targeting different molecular alterations causes a series of changes in tumor biology that can be assessed throughout the course of the disease to provide information on the underlying pathophysiology of metastatic disease, which allows us to broaden our knowledge of the different mechanisms of tissue invasion. Therefore, the aim of the present article is to review the clinical, diagnostic, predictive, prognostic utility and limitations of the main biomarkers available and under development in metastatic breast cancer.

Abstract P2-03-14: Tumor-agnostic measurement of ctDNA levels in first line ER+, HER2- metastatic breast cancer patients as a marker for survival

Abstract INTRODUCTION: Circulating tumor cell (CTC) count and levels of circulating tumor DNA (ctDNA) have proven their individual prognostic value in metastatic breast cancer (MBC). Whereas CTCs can be quantified by enumeration, levels of ctDNA are often expressed as the variant allele frequency of the dominant mutation. The modified fast aneuploidy screening test-sequencing system (mFAST-SeqS) estimates ctDNA-load by measuring chromosomal aneuploidy instead of mutations. The mFAST-SeqS method is cheap and provides a comprehensive overview of the tumor genome without prior knowledge on genomic tumor aberrations. Previously, we showed in a small MBC cohort (n=50), heterogeneous with regard to subtype and line of treatment, that the mFAST-SeqS-derived genome-wide aneuploidy score (GWA-score) was an independent prognostic biomarker (1). Our current aim was to provide evidence for the prognostic value of GWA-score in a homogeneous cohort of estrogen receptor positive (ER+), HER2 negative (HER2-) MBC patients, starting first line treatment with an aromatase inhibitor. MATERIALS &amp; METHODS: CTCs were enumerated using the CellSearch-system (Menarini) and screened for ctDNA fraction using mFAST-SeqS in ER+, HER2- MBC patients, starting with first line aromatase inhibitors. CTC-count was divided into high (CTChigh) and low (CTClow) using the validated cut-off of 5 CTCs/7.5 ml blood; similarly GWA-score was divided in high (GWAhigh)and low (GWAlow) based on the previously described cut-off of 5 (2). PFS and OS were estimated using Kaplan-Meier. Cox regression was used for uni- and multivariable analysis. RESULTS: One hundred and thirty-four patients were included in this analysis. Mean age was 68 (range 40-90). Median follow up was 26 months. Sixty-five samples had both scores low and 30 both high, 25 samples were CTChigh/GWAlow and 14 were CTClow/GWAhigh. PFS and OS were not significantly different between CTChigh versus CTClow groups (p=0.07 and p=0.26, median PFS 15 vs 24 months, median OS 45 vs 49 months). However, PFS and OS were significantly different between GWAhigh versus GWAlow groups (p &amp;lt; 0.01 for both, median PFS 11 vs 24 months, median OS 31 vs 51 months). In multivariate analysis, GWA-score was a significant prognostic factor for both PFS and OS while presence of visceral disease was also prognostic for OS (table 1). CONCLUSIONS: In patients treated with aromatase inhibitors in the first line of treatment, GWA-score was an independent prognostic marker for a worse PFS and OS and may have added value beyond CTC-count. 1. Verschoor, N., et al., Abstract P2-01-17: Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot study. Cancer Research, 2022. 82(4_Supplement): p. P2-01-17-P2-01-17. 2. Belic, J., et al., mFast-SeqS as a Monitoring and Pre-screening Tool for Tumor-Specific Aneuploidy in Plasma DNA. Adv Exp Med Biol, 2016. 924: p. 147-155. Table 1: Uni- and multivariate Cox regression for PFS and OS Citation Format: Noortje Verschoor, Jaco Kraan, Vanja de Weerd, Ngoc M Van, Agnes Jager, Stefan Sleijfer, John WM Martens, Saskia M Wilting. Tumor-agnostic measurement of ctDNA levels in first line ER+, HER2- metastatic breast cancer patients as a marker for survival [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 P2-03-14.

The Molecular Predictive and Prognostic Biomarkers in Metastatic Breast Cancer: The Contribution of Molecular Profiling.

Simple SummaryWe propose in this article to review the state of knowledge about predictive and prognosis biomarkers in metastatic breast cancer through the prism of molecular profiling studies.The past decade was marked by several important studies deciphering the molecular landscape of metastatic breast cancer. Although the initial goal of these studies was to find driver oncogenic events to explain cancer progression and metastatic spreading, they have also permitted the identification of several molecular alterations associated with treatment response or resistance. Herein, we review validated (PI3KCA, ESR1, MSI, NTRK translocation) and emergent molecular biomarkers (ERBB2, AKT, PTEN, HRR gene, CD274 amplification RB1, NF1, mutational process) in metastatic breast cancer, on the bases of the largest molecular profiling studies. These biomarkers will be classed according the level of evidence and, if possible, the ESCAT (ESMO) classification. Finally, we will provide some perspective on development in clinical practice for the main biomarkers.

Circulating miR-200 Family and CTCs in Metastatic Breast Cancer before, during, and after a New Line of Systemic Treatment.

The extracellular circulating microRNA (miR)-200 regulates epithelial-mesenchymal transition and, thus, plays an essential role in the metastatic cascade and has shown itself to be a promising prognostic and predictive biomarker in metastatic breast cancer (MBC). Expression levels of the plasma miR-200 family were analyzed in relationship to systemic treatment, circulating tumor cells (CTC) count, progression-free survival (PFS), and overall survival (OS). Expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, and CTC status (CTC-positive ≥ 5 CTC/7.5 mL) was assessed in 47 patients at baseline (BL), after the first completed cycle of a new line of systemic therapy (1C), and upon the progression of disease (PD). MiR-200a, miR-200b, and miR-141 expression was reduced at 1C compared to BL. Upon PD, all miR-200s were upregulated compared to 1C. At all timepoints, the levels of miR-200s were elevated in CTC-positive versus CTC-negative patients. Further, heightened miR-200s expression and positive CTC status were associated with poorer OS at BL and 1C. In MBC patients, circulating miR-200 family members decreased after one cycle of a new line of systemic therapy, were elevated during PD, and were indicative of CTC status. Notably, increased levels of miR-200s and elevated CTC count correlated with poorer OS and PFS. As such, both are promising biomarkers for optimizing the clinical management of MBC.

Circulating extracellular vesicles provide valuable protein, but not DNA, biomarkers in metastatic breast cancer

AbstractDetection of cell‐free circulating tumour DNA (ctDNA) and cancer‐specific extracellular vesicles (EVs) in patient blood have been widely explored as non‐invasive biomarkers for cancer detection and disease follow up. However, most of the protocols used to isolate EVs co‐isolate other components and the actual value of EV‐associated markers remain unclear. To determine the optimal source of clinically‐relevant circulating biomarkers in breast cancer, we applied a size exclusion chromatography (SEC) procedure to analyse separately the content in nucleic acids of EV‐enriched and EV‐depleted fractions, in comparison to total plasma. Both cellular and mitochondrial DNA (cellDNA and mtDNA) were detected in EV‐rich and EV‐poor fractions. Analysing specific mutations identified from tumour tissues, we detected tumour‐specific cellular alleles in all SEC fractions. However, quantification of ctDNA from total plasma was more sensitive than from any SEC fractions. On the other hand, mtDNA was preferentially enriched in EV fractions from healthy donor, whereas cancer patients displayed more abundant mtDNA in total plasma, and equally distributed in all fractions. In contrast to nucleic acids, using a Multiplexed bead‐based EV‐analysis assay, we identified three surface proteins enriched in EVs from metastatic breast cancer plasma, suggesting that a small set of EV surface molecules could provide a disease signature. Our findings provide evidence that the detection of DNA within total circulating EVs does not add value as compared to the whole plasma, at least in the metastatic breast cancer patients used here. However, analysis of a subtype of EV‐associated proteins may reliably identify cancer patients. These non‐invasive biomarkers represent a promising tool for cancer diagnosis and real‐time monitoring of treatment efficacy and these results will impact the development of therapeutic approaches using EVs as targets or biomarkers of cancer.

Abstract 969: Epithelial and mesenchymal CTC detection in triple negative breast cancer patients using ANGLE’s Parsortix®system

Abstract Introduction: Isolation of circulating tumor cells (CTCs) is widely recognised as a prognostic biomarker in metastatic breast cancer. Recently, it was shown that CTC clearance following specific therapies may predict chemosensitivity in triple negative breast cancer (TNBC) patients. However, a limited amount of CTC research is available in TNBC due to the inability of epitope-dependent CTC platforms to capture CTCs in a sufficient proportion of patients, especially when evaluating non-metastatic patients (Stage I-III). ANGLE has developed a Research Use Only (RUO) workflow including the Parsortix® system, an epitope-independent microfluidic device that isolates and harvests rare cells from blood based on their size and deformability, followed by an immunofluorescent (IF) assay for the identification and characterization of CTCs. This five-color multiplex IF assay enables the identification of both epithelial and mesenchymal CTCs as well as the investigation of other targets of interest on the CTCs. Methods: 7.5mL of peripheral blood drawn into a Streck CellFree DNA Blood Collection Tube from 11 TNBC patients (four Stage IV and seven Stage IIIa) was processed on Parsortix® system between 96 and 144 hours post collection. Harvested CTCs were cytospun onto a charged slide and immunofluorescently stained using an optimised antibody panel allowing for detection of epithelial and mesenchymal CTCs. Stained slides were imaged using a BioView Allegro Plus automated imaging system. Results: CTCs were identified in 9 (82%) of the 11 TNBC patients, with a median of 6 and mean of 30 CTCs identified per patient. CTC clusters were harvested by the Parsortix® system and were observed in the majority (8/9) of the CTC-positive patients. Cluster size ranged from 2-68 CTCs per cluster, and the number of clusters per patient ranged from 1-50. Phenotypically, a large proportion of the CTCs identified expressed only mesenchymal markers (80%), with the remainder expressing both epithelial and mesenchymal markers (20%). In 6 of the 7 non-metastatic (Stage IIIa) patients, high numbers of CTC clusters were detected. Lower numbers of CTC clusters were detected in 3 (75%) of the 4 metastatic (Stage IV) patients. Conclusions: This study demonstrated the ability of ANGLE’s multiplex IF assay to detect and phenotype CTCs. The proportion of non-metastatic TNBC patients found to have CTCs was much higher compared to previous reports using epitope-dependent systems (82% vs 25%). This highlights the advantages of epitope-independent CTC capture in TNBC. ANGLE’s RUO Parsortix® system and multiplex IF assay provide an efficient solution for the harvesting and characterization of multiple CTC phenotypes, with the added advantage of being able to process the blood between 96 and 144 hours post collection, allowing for the shipment of samples at room temperature for centralised analysis in support of global clinical trials. Citation Format: Amina Mezni, Matthew Hardy, Mariacristina Ciccioli, Anne-Sophie Pailhes-Jimenez. Epithelial and mesenchymal CTC detection in triple negative breast cancer patients using ANGLE’s Parsortix®system [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 969.

49P Analysis of HER2-low carcinomas and PAM50 signature in an early-stage breast cancer cohort

HER2-low (IHQ score 1+ or 2+ with negative in situ hybridization) has emerged as a new potential biomarker in metastatic breast cancer. However, its prognostic value in early-stage carcinomas needs to be revisited. We aimed to evaluate the association of HER2-low carcinomas with PAM50 risk groups combined with standard clinicopathological variables in early breast cancer.

Abstract P2-01-17: Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot study

Abstract Background and aim Minimally invasive liquid biopsies, usually referring to the analysis of tumor-derived material such as circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), have already proven their individual prognostic value in metastatic breast cancer. Whereas CTCs can be quantified by enumeration, levels of ctDNA are often expressed as the variant allele frequency of the dominant mutation, determined by rather costly targeted next generation sequencing. The modified fast aneuploidy screening test-sequencing system (mFAST-SeqS) represents a fast and cost-effective assay to estimate the fraction of ctDNA by measuring chromosomal aneuploidy instead of mutations. Next to the low costs (EUR 50, USD 60) additional advantages of the mFAST-SeqS method include the independency of mutational status of the tumor and the comprehensive overview of the tumor genome. Our aim was to investigate the potential concordance and complementarity between CTC-count and the mFAST-SeqS-based genome-wide aneuploidy score (GWA score). Material and methods Consecutive blood samples obtained from ER positive, Her2 negative metastatic breast cancer patients, starting a new line of therapy, (n=23) were enumerated for CTCs using the CellSearch-system (Menarini Silicon Biosystems, Bologna, Italy) and screened for the percentage of ctDNA using mFAST-SeqS. This method provides a GWA-score by amplification of long interspaced nuclear elements (LINE-1 elements) throughout the genome and calculating the overall deviation from the average profile in normal controls. Spearman correlation between CTC-count and GWA-score was calculated. For survival analysis, CTC-count was divided into high (CTChigh) and low (CTClow) using 5 CTCs/7.5 ml blood as the cut-off as before; similarly GWA-score was divided in high (GWAhigh)and low (GWAlow) based on the previously described cut-off of 5. Kaplan-Meier analysis was performed to assess overall survival (OS) in subgroups. Results Mean patient age was 58 (range 42-78). Median follow up was 15 months (range 0-37). Median number of previous treatment lines of patients was 2 (range 0-11). Median CTC-count was 22 (range 0-4438) and median GWA-score was 3.94 (range 0.87-114.31). The Spearman correlation coefficient between CTC count and GWA-score was 0.44 (p=0.02). 3 out of 20 patients were alive at end of follow-up. OS was significantly different in the CTChigh group versus the CTClow group (p=0.01, log-rank test, median OS 31 and 7 months, respectively), as well as in the GWAhigh versus the GWAlow group (p =0.01, log-rank test, median OS 27 and 7 months). There was concordance in 12 out of 23 samples (52.2%). 4 samples had both scores low and 8 both high, 9 samples were CTChigh/GWAlow and 2 were CTClow/GWAhigh. OS was significantly different in these groups (p &amp;lt; 0.01, log-rank test), with a median OS of 6 months in the group with both scores high, a median OS of 18 months for the cases with a discordant score and median OS not reached in the group with both scores low.Conclusions Our pilot study confirmed that both CTC-count and mFAST-SeqS GWA-score are prognostic and indicate that a combination of these measurements may yield complementary prognostic value. It should be noted however that the sample size was limited thus far. Our current preliminary results will be extended to 98 cases, results of which will be presented at the meeting. Citation Format: Noraly Verschoor, Elisabeth M Jongbloed, Jaco Kraan, Vanja de Weerd, Ngoc M Van, Agnes Jager, Stefan Sleijfer, Saskia M Wilting, John WM Martens. Circulating tumor cell count and levels of circulating tumor DNA are complementary prognostic biomarkers in metastatic breast cancer - A pilot study [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 P2-01-17.

Identification of CD105+ Extracellular Vesicles as a Candidate Biomarker for Metastatic Breast Cancer

Extracellular vehicles (EVs) released by malignant tumor cells can mediate the immune response and promote metastasis through intercellular communication. EV analysis is an emerging cancer surveillance tool with advantages over traditional liquid biopsy methods. The aim of this pilot study is to identify actionable EV signatures in metastatic breast cancer. Under an IRB-approved protocol for the analysis of patient plasma, samples were collected from women with newly diagnosed or progressive metastatic breast cancer and from women without cancer. Enriched EVs were analyzed via a bead-based multiplex assay designed to detect 37 distinct tumor-relevant epitopes. The mean fluorescent intensity of EV epitopes meeting a minimum threshold of detectability was compared between groups via independent samples t-test. Subgroup analysis was conducted for metastatic breast cancer patients who were positive for estrogen and/or progesterone receptors and negative for HER2. Other variables potentially affecting CD105 levels were also analyzed. CD105 was found to have a significantly higher mean fluorescent intensity in participants with metastatic breast cancer compared to control participants (P=0.04). ER/PR+ subgroup analysis revealed a similar pattern compared to control participants (P=0.01). Other analyzed variables were not found to have a significant correlation with CD105 levels. CD105 EV levels were significantly higher in samples from participants with breast cancer compared to controls. Given that CD105 is known to mediate angiogenesis and promote metastasis, EV-associated CD105 in plasma represents a potential biomarker for diagnosis, surveillance and therapeutic targeting in patients with metastatic breast cancer.

Expression of NF-kB Regulators (BCL-3, NFKB1, NFKB2) in Metastatic Breast Cancer Patients

Background/aim: BCL-3, upregulated in several cancers, functions as a crucial player not only in cell cycle and apoptosis, but also in metastasis. The study aimed to analyse the expression profile of BCL-3 and its interacting partners in metastatic breast cancer. Materials and methods: mRNA expression levels were evaluated in blood samples of metastatic breast cancer patients (n=55) and in healthy control donors (n=50) by RT qPCR. SPSS 26 program was used for statistical analysis. Results: Expression levels of BCL-3 mRNA was downregulated in metastatic breast cancers patients compared to healthy control group (p= 0.0004). Subsequently, expression levels of the NFKB1 (p= 0.0005), NFKB2 (p=0.0001), CYLD (p= 0.00042) and TP53 (p= 0.0287) were significantly lower in metastatic breast patients compared to healthy controls. CCND1 and CDH2 gene expressions were significantly upregulated in metastatic group (p=0.0009; and p= 0.0030, respectively). GSK3B, SMAD3 and TGFB1 expressions were not significantly different between groups. Conclusion: This study indicates significant expression difference between patients and controls. The NF-κB regulators might induce the metastatic potential through interaction with the other molecules in the microenvironment, nevertheless, it is needed further research whether the importance of BCL-3 as a biomarker for metastatic breast cancer.

Detection of Multiple Breast Cancer ESR1 Mutations on an ISFET Based Lab-on-Chip Platform.

ESR1 mutations are important biomarkers in metastatic breast cancer. Specifically, p.E380Q and p.Y537S mutations arise in response to hormonal therapies given to patients with hormone receptor positive (HR+) breast cancer (BC). This paper demonstrates the efficacy of an ISFET based CMOS integrated Lab-on-Chip (LoC) system, coupled with variant-specific isothermal amplification chemistries, for detection and discrimination of wild type (WT) from mutant (MT) copies of the ESR1 gene. Hormonal resistant cancers often lead to increased chances of metastatic disease which leads to high mortality rates, especially in low-income regions and areas with low healthcare coverage. Design and optimization of bespoke primers was carried out and tested on a qPCR instrument and then benchmarked versus the LoC platform. Assays for detection of p.Y537S and p.E380Q were developed and tested on the LoC platform, achieving amplification in under 25 minutes and sensitivity of down to 1000 copies of DNA per reaction for both target assays. The LoC system hereby presented, is cheaper and smaller than other standard industry equivalent technologies such as qPCR and sequencing. The LoC platform proposed, has the potential to be used at a breast cancer point-of-care testing setting, offering mutational tracking of circulating tumour DNA in liquid biopsies to assist patient stratification and metastatic monitoring.

Type IV collagen as a potential biomarker of metastatic breast cancer

No reliable, non-invasive biomarker of metastatic breast cancer (mBC) exists: circulating CA15-3 (cCA15-3) is the marker mostly used to monitor mBC. Circulating collagen IV (cCOLIV) has been evaluated in other metastatic cancers and has been found to be a promising biomarker. The overarching aim of this study was to evaluate cCOLIV as a potential biomarker in patients with mBC. The first aim was to determine the levels of cCOL IV and cCA15-3 in patients with healthy controls, primary breast cancer (pBC) and mBC. The second aim was to compare levels of cCOLIV and cCA15-3 in patients with different metastatic sites of BC. The third aim was to investigate the prognostic value of cCOLIV and cCA15-3 for mBC patients. The fourth aim was to analyse whether a combination of the two biomarkers was more accurate in detecting mBC than a single marker. Lastly, we investigated the tissue expression levels of COLIV in BC bone metastases (BM) and liver metastases (LM). Plasma levels of cCOLIV and cCA15-3 from healthy controls and patients with pBC and mBC were measured. COLIV expression in tissue from patients with LM and BM was analysed using immunohistochemistry. Clinical and survival data were collected from medical charts. The levels of cCOLIV and cCA15-3 were significantly elevated in mBC patients compared with healthy controls and pBC patients. No differences in cCOLIV and cCA15-3 levels were found based on the metastatic site. High levels of cCOLIV, but not cCA15-3, correlated with poorer survival. cCOLIV alone and the combination of cCA15-3 and cCOLIV were superior to cCA15-3 at detecting mBC. COL IV was highly expressed in the tissue of LM and BM. Our study suggests that cCOLIV is a potential marker to monitor patients with BC.

First-Line Doublet Chemotherapy for Metastatic Triple-Negative Breast Cancer: Circulating Tumor Cell Analysis of the tnAcity Trial.

PurposeCirculating tumor cells (CTCs) are prognostic biomarkers in metastatic breast cancer, but their role in predicting treatment outcomes in metastatic triple-negative breast cancer (mTNBC) is less clear. The tnAcity trial demonstrated a significant progression-free survival (PFS) benefit with nab-paclitaxel (nab-P)/carboplatin (C) over nab-P/gemcitabine (G) or G/C in patients with mTNBC. We assessed the correlation between CTC dynamics and clinical benefit in all patients and by treatment arm.MethodsCTC enumeration, performed using CELLSEARCH technology (Menarini Silicon Biosystems, Huntingdon Valley, PA, USA), was a prespecified exploratory endpoint in the tnAcity trial. Patients with TNBC were categorized based on pre- and post-treatment CTC levels: Group 1 (+ + +; elevated CTCs at baseline and postbaseline), Group 2 (+ ± ±; CTCs elevated at baseline and cleared postbaseline [cycle 3 and/or cycle 5]), or Group 3 (−; no CTCs detected at baseline). The baseline cutoff was ≥1 CTC/7.5 mL for the main analysis; cutoffs of ≥2 and ≥5 CTCs were used for supporting analyses.ResultsThe main analysis included 126 patients (Group 1, n = 24; Group 2, n = 54; and Group 3, n = 48). The median PFS was longer in Group 2 vs Group 1 (8.5 vs 4.7 months; HR, 0.30 [95% CI, 0.17–0.54]). These results were supported by the ≥2- and ≥5-CTC cutoff analyses. The median overall survival rates were 17.8, 16.0, and 9.8 months in Groups 2, 3, and 1, respectively. The overall response rates were 79.6%, 43.8%, and 29.2%, respectively. A numerically higher percentage of patients had CTC clearance during nab-P/C treatment vs nab-P/G or G/C.ConclusionEfficacy outcomes trended positively with chemotherapy-induced elimination of CTCs, suggesting that CTC clearance may predict the chemosensitivity of mTNBC tumors.Trial registrationEudraCT Number: 2013-000113-20; ClinicalTrials.gov number: NCT01881230.

Immunotherapy predictive biomarkers in metastatic breast cancer (MBC).

1023 Background: We queried whether comprehensive genomic profiling (CGP) of mBC subtypes could identify biomarkers that have been linked to responsiveness to immunotherapy (IO) treatments. Methods: DNA was extracted from 3,871mBC: 1,388 ER+/HER2-, 1,969 HER2 amplified (amp) and 643 TNBC. CGP was performed using a hybrid-capture assay. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci in 564 cases. PD-L1 was determined by IHC (Dako 22C3). Results: Patient ages were similar. Genomic alterations (GA)/tumor were similar ranging from 5.9 to 7.3. MTOR pathway targets were common; lowest in TNBC. CDH1 and ESR1 GA most frequent in ER+/HER2- cases. ERBB2 short variant (SV) mut were most frequent in ER+/HER2- and HER2 amp and not seen in TNBC. Other kinase targets were uncommon except for FGFR1 GA in ER+/HER2-. BRCA1/ 2 GA least frequent in HER2 amp. AR was amplified in 1% of all mBC. Markers of potential IO benefit: CD274 ( PD-L1) amp (1-3%), BRAF GA (1-4%), TMB of ≥10 mut/Mb (8-12%), TMB of ≥20 mut/Mb (2-3%), MSI-High (0.1-0.4%), PBRM1 GA (1%) and low (1-10%) or high (0-3%) PD-L1 staining. Potential markers of resistance: inactivating GA in STK11 (1-2%) and MDM2 amplification (3-6%). Examples of mBC patients responding to IO therapies will be presented. Conclusions: In addition to guiding targeted therapy selection, CGP shows potential to identify GA linked to response and resistance to IO in mBC. The demonstrations of clinical benefit of immunotherapy in mBC supports the need for the development of biomarkers used to guide the use of ICPI drugs for these patients. [Table: see text]

Abstract P2-08-20: Prognostic impact of SRC, CDKN1B and JAK2 expression in metastatic breast cancer patients treated with trastuzumab

Abstract Background-aim: SRC, CDKN1B and JAK2 play a crucial role in the coordination and facilitation of cell-signaling pathways controlling a wide range of cellular functions. In the present study, we investigated the prognostic significance and clinical utlity of these biomarkers in metastatic breast cancer (MBC) patients treated with trastuzumab (T). Methods: We assessed SRC, CDKN1B and JAK2 mRNA expression with qRT-PCR (Taqman-MGB assays) on 197 paraffin tumors. PIK3CA mutation status was previously assessed. Relapsed (RMBC) and de novo MBC (dnMBC) patients had received T for metastatic disease only. Tumors were centrally re-assessed for HER2 status. Results: Only 133/197 patients (67.5%) were found to be truly HER2(+). CDKN1B mRNA expression strongly correlated with SRC (rho = 0.71) and JAK2 (rho = 0.54); high CDKN1B was more frequent in RMBC compared to dnMBC (p = 0.001) and in PIK3CA wild-type tumors (p = 0.005). In HER2(+) patients, low CDKN1B conferred higher risk for progression (HR 1.58, 95% CI 1.08-2.32, p = 0.018). In HER2(-) patients, low SRC was associated with longer survival (HR 0.56, 95% CI 0.32-0.99, p = 0.045) and, as a trend, with increased progression-free survival (PFS) (p = 0.067). For PFS, in RMBC, we observed trends for unfavorable low CDKN1B (p = 0.068) and JAK2 (p = 0.086); similarly, in dnMBC for unfavorable low CDKN1B (p = 0.072). Low SRC showed a trend for better survival in RMBC (p = 0.087). Upon multivariable analyses, only PIK3CA mutations strongly predicted for unfavorable PFS in HER2(+) patients (HR 3.37, 95% CI 1.98-5.73, p &amp;lt; 0.001). Low CDKN1B and JAK2 mRNA expression remained unfavorable factors for PFS in dnMBC and RMBC patients (HR 2.36, 95% CI 1.01-5.48, p = 0.046 and HR 1.76, 95% CI 1.01-3.06, p = 0.047, respectively). Conclusions: Low CDKN1B and JAK2 mRNA expression were unfavorable prognosticators in a cohort of T-treated MBC patients previously unexposed to this agent, with distinct impact in de novo and RMBC. Our results highlight biological and clinical differences between de novo and RMBC and suggest that CDKN1B and JAK2, if validated, may serve as prognostic factors potentially implicated in T-resistance, which seems to be associated with distinct pathways in the two MBC settings. Citation Format: Economopoulou P, Kotoula V, Koliou G-A, Papadopoulou K, Christodoulou C, Pentheroudakis G, Koutras A, Bafaloukos D, Papakostas P, Pectasides D, Kotsakis A, Razis E, Samantas E, Kalogeras KT, Economopoulos T, Fountzilas G. Prognostic impact of SRC, CDKN1B and JAK2 expression in metastatic breast cancer patients treated with trastuzumab [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-20.