Value In Metastatic Breast Cancer Research Articles

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 & 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 < 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.

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 < 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.

Single-Cells Isolation and Molecular Analysis: Focus on HER2-Low CTCs in Metastatic Breast Cancer.

Simple SummaryWhile the concept of HER2-low expression is gaining momentum in the scientific landscape of breast cancer research, HER2-low circulating tumor cells (CTCs) also have promising relevance as biomarkers. Unveiling the biological features behind this recently highlighted evidence on CTCs might achieve both goals of speeding up the interpretation of the general understanding of HER2-low expressing breast cancer and declaring their independent biological and predictive value. If, on the one hand, studying CTCs allows to drill down more easily to a single cell resolution, on the other hand, CTC collection still remains a challenging procedure. In order to improve and standardize this process, we developed a structured pipeline for HER2-low CTC detection and collection. We defined and validated the optimal thresholds to select this specific subtype of CTCs using breast cancer cell lines of known HER2 expression. Our study represents the technical and procedural milestone that will allow a standardized collection process for future molecular investigations.Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT™ for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann–Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT™ were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses.

Technical Challenges for CTC Implementation in Breast Cancer.

Simple SummaryLiquid biopsy appears as a feasible opportunity to complement tissue biopsy within breast cancer research and real-time monitoring of the disease. In this review, we not only summaries cur-rent trends in the study of CTCs as a potential biomarker of response to systemic therapies but also the technical challenges for their implementation in clinical practice, including the role of free circulating DNA and new approaches based on the isolation of CTCs from body fluids. We also highlight the importance of a consensus on the standardization of liquid biopsy methodologies as a fundamental requirement for their implementation in clinical practice.Breast cancer is the most common neoplasm in women worldwide. Tissue biopsy, currently the gold standard to obtain tumor molecular information, is invasive and might be affected by tumor heterogeneity rendering it incapable to portray the complete dynamic picture by the absence of specific genetic changes during the evolution of the disease. In contrast, liquid biopsy can provide unique opportunities for real-time monitoring of disease progression, treatment response and for studying tumor heterogeneity combining the information of DNA that tumors spread in the blood (circulating tumor DNA) with CTCs analysis. In this review, we analyze the technical and biological challenges for isolation and characterization of circulating tumor cells from breast cancer patients. Circulating tumor cell (CTC) enumeration value is included in numerous clinical studies due to the prognostic’s role of these cells. Despite this, there are so many questions pending to answer. How to manage lymphocytes background, how to distinguish the CTCs subtypes or how to work with frozen samples, are some of the issues that will discuss in this review. Based on our experience, we try to address these issues and other technical limitations that should be solved to optimize the standardization of protocols, sample extraction procedures, circulating-tumor material isolation (CTCs vs. ctDNA) and the very diverse methodologies employed, aiming to consolidate the use of CTCs in the clinic. Furthermore, we think that new approaches focusing on isolation CTCs in other body fluids such as cerebrospinal or ascitic fluid are necessary to increase the opportunities of circulating tumor cells in the practice clinic as well as to study the promising role of CTC clusters and their prognostic value in metastatic breast cancer.

Neutrophil-lymphocyte ratio in metastatic breast cancer is not an independent predictor of survival, but depends on other variables

The prognostic impact of neutrophil-lymphocyte ratio (NLR) in metastatic breast cancer (MBC) has been previously evaluated in early and metastatic mixed breast cancer cohorts or without considering other relevant prognostic factors. Our aim was to determine whether NLR prognostic and predictive value in MBC was dependent on other clinical variables. We studied a consecutive retrospective cohort of patients with MBC from a single centre, with any type of first line systemic treatment. The association of NLR at diagnosis of metastasis with progression free survival (PFS) and overall survival (OS) was evaluated using Cox univariate and multivariate proportional hazard models. In the full cohort, that included 263 MBC patients, a higher than the median (>2.32) NLR was significantly associated with OS in the univariate analysis (HR 1.36, 95% CI 1.00–1.83), but the association was non-significant (HR 1.12, 95% CI 0.80–1.56) when other clinical covariates (performance status, stage at diagnosis, CNS involvement, visceral disease and visceral crisis) were included in the multivariate analysis. No significant association was observed for PFS. In conclusion, MBC patients with higher baseline NLR had worse overall survival, but the prognostic impact of NLR is likely derived from its association with other relevant clinical prognostic factors.

Circulating miRNAs with prognostic value in metastatic breast cancer and for early detection of metastasis.

Metastasis is the principal cause of high morbidity and mortality among breast cancer (BC) patients. Identification of markers that can be routinely monitored to predict onset of metastasis in BC patients and prognosis of metastatic breast cancer (MBC) patients would increase their median survival. In this study, plasma miRNAs of 40 MBC patients were profiled by TaqMan low density arrays and miRNAs with prognostic capacity were identified. The candidates were validated initially in the samples of 237 MBC patients and subsequently in 335 samples from an independent study cohort of BC patients. Sixteen miRNAs were established to be significantly associated with overall survival, and were termed as prognostic miRNA panel template (PROMPT). These included miR-141, miR-144, miR-193b, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-215, miR-365, miR-375, miR-429, miR-486-5p, miR-801, miR-1260 and miR-1274a. Additionally, 11 of these miRNAs were also associated with progression-free survival. Their prognostic significance was further confirmed in samples from a second study cohort of BC patients. In addition, miR-200a, miR-200b, miR-200c, miR-210, miR-215 and miR-486-5p were found to be significantly associated with onset of metastasis up to 2 years prior to clinical diagnosis in BC patients. We have thus identified panels of miRNAs, which include metastasis promoting miR-200 family and miR-203, as well as oncogenic and tumor-suppressive miRNAs, that can serve as prognostic markers for MBC, and early detection markers of metastasis in BC.

Abstract P3-06-34: Plasma (p) VEGF-A and VEGFR-2 biomarker (BM) results from the BEATRICE phase III trial of bevacizumab (BEV) in triple-negative early breast cancer (BC)

Abstract Background: Several candidate BMs have been explored in randomized trials of BEV across tumor types with the aim of identifying patients (pts) deriving the most substantial benefit from BEV therapy. In phase III trials, baseline pVEGF-A and pVEGFR-2 showed potential predictive value in metastatic BC (AVADO, AVEREL), pancreatic cancer (AViTA), and gastric cancer (AVAGAST; VEGF-A only). The randomized phase III BEATRICE trial, evaluating the addition of BEV to adjuvant chemotherapy in pts with triple-negative early BC, includes a comprehensive program to identify potential BMs predicting efficacy and toxicity of BEV therapy. We report results for baseline pVEGF-A and pVEGFR-2. Methods: After selection of chemotherapy (anthracycline and/or taxane), pts with T1a-T3 operable BC were randomized 1:1 to receive ≥4 cycles of chemotherapy either alone or with 1 year of BEV 5 mg/kg/wk equivalent. The primary endpoint is invasive disease-free survival (IDFS). pEDTA samples were collected from consenting pts at baseline (before treatment, after surgery), during study treatment, and at relapse. Pts were dichotomized using the median baseline concentration of each marker as the cut-off between high and low cohorts; further exploratory analyses were also performed by quartile. Results: Between Dec 2007 and Mar 2010, 2591 pts were enrolled. Of these, 1273 (49%) consented to the BM study and 1178 (45%) were included in the BM-evaluable population (BEP). Overall, the BEP was representative of the ITT population except for lower proportions of Asian pts (12% vs 24%). IDFS was similar in the BEP and ITT populations. Baseline characteristics were balanced between arms in the BEP. Baseline pVEGF-A showed neither prognostic nor predictive value using the median as the cut-off, although with a third quartile (Q3) cut-off there was a more pronounced but non-significant differentiation between treatments (HR 0.92 [low] vs 0.64 [high]). High baseline pVEGFR-2 showed potential predictive value for BEV efficacy (HR 1.24 [low] vs 0.61 [high]; p=0.029). Conclusions: Unlike trials in metastatic BC (AVADO, AVEREL), in the adjuvant setting, baseline pVEGF-A concentration did not show predictive value for BEV efficacy with a median cut-off. However, analyses using the Q3 cut-off suggest a trend toward predictive value. High baseline pVEGFR-2 was associated with greater BEV treatment effect, consistent with previous results in AVADO and AVEREL. The impact of differing biology in the adjuvant setting, lower median VEGF-A concentration than in the metastatic setting (77.0 vs 125.0–129.1 pg/mL), and the possible influence of surgery immediately before treatment require further investigation. Additional exploratory analyses are ongoing to provide better understanding of the BEATRICE dataset and the complex biology of angiogenesis, including additional markers, changes over time, and combination signatures. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-34.

Circulating Tumor Stem Cells as Biomarkers for Cancer Progression

The identification of a minor cellular subpopulation responsible for the initiation, maintenance and expansion of tumors (the cancer stem cells -CSCs-) meant a radical paradigm shift, but at the same time the selection of specific biomarkers may have extraordinary prognostic value. Recently, circulating epithelial cells (CTCs) have been identified in peripheral blood of solid tumor patients, demonstrating their prognostic value in metastatic breast cancer and positive predictive value for treatment response. This identification is based on epithelial cell markers in peripheral blood through immunocytochemistry technology or PCR, with a clear prognostic goal. Since CSCs are responsible for the initiation of new tumor lesions, we propose that a subpopulation of CTCs is the CSCs that will initiate metastasis. It is reasonable to speculate that a subpopulation of CTCs with stem cell features, such as self-renewal, multipotency, tumorigenicity and likely showing markers of epithelial-mesenchymal transition (EMT), may be the relevant players during metastasis. Further research into molecular characterization of CSC specific CTCs will create opportunities for novel therapeutic intervention, but at the same time facilitate the detection of clinically relevant CTCs. In this review, recent patents on CTC biomarkers are summarized.

Circulating tumor cells (CTCs) in metastatic breast cancer (MBC): prognosis, drug resistance and phenotypic characterization

Background: The expression of ATP-binding cassette transporters on circulating tumor cells (CTCs) is predictive of response to chemotherapy in cancer patients. We tested the hypothesis that drug-resistant CTCs might have predictive value in metastatic breast cancer (MBC) and possibly retain stem-like properties.Patients and methods: CTCs obtained from 42 MBC patients were evaluated for multidrug-resistance-related proteins (MRPs), aldehyde dehydrogenase 1 (ALDH1), estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2/neu). Primary objective was to evaluate the prognostic and predictive value of CTCs profile. Secondary end points were the level of concordance in ERα and HER2/neu status between primary tumors and CTCs and the correlation in CTCs between ALDH1, drug resistance profile and number of MRPs.Results: A difference in progression-free survival (PFS) was found between CTCs-positive and CTCs-negative patients. PFS was shorter in patients with a ‘drug resistance’ CTCs profile and in patients whose CTCs expressed two or more MRPs. No correlation was found between tumor characteristics and ALDH1. ALDH1 correlated to negative ERα and positive HER2/neu status in CTCs. The correlation between the number of MRPs expressed in CTCs and ALDH1 was statistically significant.Conclusion: In MBC, the presence of CTCs expressing MRPs and ALDH1 is predictive of response to chemotherapy.

Changes over time of extracellular domain of HER2 (ECD/HER2) serum levels have prognostic value in metastatic breast cancer

Blood levels of the extracellular domain of HER-2/neu (ECD/HER2) have been suggested to have potential as a tumor marker in breast cancer. Our aim was to assess the prognostic value of baseline levels of ECD/HER2, but more importantly changes in levels over time, in women with metastatic breast cancer. Baseline and serial levels of ECD/HER2 were measured in 158 women with newly-diagnosed metastatic breast cancer, in whom we previously performed serial measurement of plasma osteopontin. ECD/HER2 was measured in 1,282 serum samples using a validated ELISA at baseline and every 3-12 weeks during and after therapy until death (median, n=8 samples per patient). Multivariate time-dependent survival analyses were conducted using models that right-censored patient outcomes 3, 6 and 12 months after last known ECD/HER2 measurement. Thirty-four patients (22%) had elevated baseline ECD/HER2 (median 10.2 ng/ml: range 4.1-40.4 ng/ml). In univariate analysis, elevated baseline ECD/HER2 was associated with short survival (P=0.001). In a multivariate model incorporating standard clinical prognostic factors, baseline ECD/HER2 was significantly associated with survival duration (RR 1.029; P=0.020). Presence of visceral metastases and ECOG status 2-4 also retained significance. In a multivariate model incorporating standard prognostic factors and changes in sequential ECD/HER2 levels, an ECD/HER2 increase of >12 ng/ml at any time was the variable with most prognostic value for poor survival (RR 6.10; P=0.0003); poor ECOG status also retained significance. Increases over time of ECD/HER2 levels were strongly associated with poor survival in this cohort of women with metastatic breast cancer.

Lapatinib: the evidence for its therapeutic value in metastatic breast cancer

Breast cancer is the most common cancer affecting women. Many patients ultimately progress to metastatic disease and optimal management of this disease remains a significant therapeutic challenge. Lapatinib, a dual tyrosine kinase inhibitor, is in clinical development for treatment of this disease. The objective of this article is to review the published evidence for the treatment of metastatic breast cancer with lapatinib, and assess its therapeutic potential. Most evidence has appeared in meeting abstract reports of phase I and II studies in healthy volunteers and cancer patients. Four studies have included patients with exclusively breast cancer. Complete and partial responses and stable disease has been reported in some patients. Emerging evidence indicates that complete and partial responses can be achieved in some patients with metastatic breast cancer. Lapatinib appears to be well tolerated in cancer patients and the maximum tolerated dose is in the region of 1800 mg/day. In addition, it has been used in combination with other cancer treatments. Five ongoing or planned phase II monotherapy and three phase III combination-therapy studies with lapatinib have been identified. The phase I and II studies reported to date have provided safety data and preliminary indications regarding efficacy. There is preliminary evidence that lapatinib can achieve objective response rates of 10-38% in patients with metastatic breast cancer. Patients with tumors overexpressing ErbB1 and/or ErbB2 are likely to benefit from lapatinib treatment.

A direct assay for carcinoembryonic antigen in serum and its diagnostic value in metastatic breast cancer

The development of an optimized direct assay for carcinoembryonic antigen (CEA) in serum is described. CEA was purified from liver metastases from colorectal carcinomas, and avid anti-CEA antibodies (Ka 1.0-1.6x10(11) L/mol) were produced in rabbits. Radiolabelling of CEA was performed by the "Iodo-Gen" method. Experiments performed in order to characterize antigen-antibody specificity and to work out assay conditions are described. In the separation step of the assay, problems associated with non-specific binding of radioligand were virtually eliminated by using a new solid-phase second antibody, based on monodisperse polymer particles. The clinical performance of the assay is demonstrated by its high ability to detect relapses in patients with mammary carcinoma.