Cohort Of Metastatic Breast Cancer Patients Research Articles

Prognosis and tumor-infiltrating lymphocytes (TILs) in estrogen receptor (ER)-low metastatic breast cancer (MBC): Analysis from a large multi-institutional cohort and the TONIC phase II trial with nivolumab.

1100 Background: Although 1% as ER cutoff to discriminate ER+/HER2- from triple-negative (TN) BC serves the purpose of maximizing access to endocrine therapy, it may be suboptimally informative in terms of prognosis and immune checkpoint inhibitor (ICI) benefit. In the early setting, we reported that ER-low BC (ER 1-9%/HER2-) is immunologically and prognostically similar to TN (ER 0%) [Massa, 2023]. Here we focus on MBC and aim to assess prognosis of ER-low BC, including the conversion from ER+(≥10%)/HER2- to ER-low at relapse. We also compared ER-low and TN in terms of outcome after ICI, and TILs. Methods: We included two cohorts of MBC patients (pts): a multicentric retrospective cohort and pts with ER<10%/HER2- MBC enrolled in the TONIC trial testing nivolumab +/- induction therapy. All pts had ER and HER2 status assessed on metastatic tumor lesion; in the retrospective cohort ER and HER2 status on primary tumor was also available. Survival endpoints in the retrospective cohort: overall survival (OS, from BC diagnosis), post-relapse survival (PRS, from BC relapse). Exploratory endpoints from the TONIC trial: clinical benefit rate (CBR), OS and PFS (both from randomization). TILs were assessed on MBC samples (both cohorts) and on matched primary tumor (retrospective cohort). Results: We included 1112 pts in the retrospective cohort: the prevalence of ER-low BC was 3.6% in primary tumor and 5.9% in MBC lesions. In the TONIC trial, 15/110 pts had ER-low MBC. In the retrospective cohort, ER-low and TN MBC showed similar outcome, and significantly unfavorable vs ER+/HER2- MBC (Table). Among pts with ER+/HER2- primary BC, 6.7% converted to ER-low and 14.7% to TN at relapse. ER+/HER2- BC pts converting to ER-low had similar outcome than those converted to TN, and significantly poorer than pts with stable ER+/HER2- (Table). In the TONIC trial, ER-low MBC pts, compared to TN, showed similar response to ICI (CBR: 20.0% vs 22.1%, p=1), similar PFS and numerically worse OS (Table). TIL levels on MBC samples were similar between ER-low and TN both in the retrospective (median: 6% vs 5%, p=0.83) and the TONIC cohorts (median: 5.5% vs 5.0%, p=0.56) and were higher than ER+/HER2- (median: 3%). Conclusions: We confirmed that ER-low and TN MBC show similar poor outcome and the shift from ER+/HER2- to ER-low has a comparable unfavorable prognostic impact compared to conversion to TN. Our results support the hypothesis that ER-low and TN are immunologically akin and similarly prone to respond to ICI. [Table: see text]

Abstract 3705: Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies

Abstract Background. HER2 testing in tissue biopsies is extensively performed in breast cancer (BC) patients. However, tissue biopsy is invasive and repeated examination is impractical. Additionally, lack of concordance between primary tumor and metastatic sites are often observed. Liquid biopsy, on the other hand, enables less invasive and recurrent sample collection for ongoing monitoring. In this study, we developed an end-to-end Research Use Only assay for concomitant evaluation of HER2 gene amplification and protein overexpression in circulating tumor cells (CTCs). Methods. Blood was drawn from healthy volunteers into Streck Cell-Free DNA tubes, spiked with SKBR3 (HER2+) and Hs 578T (HER2-) breast cancer cells and processed on ANGLE's Parsortix® PC1 System, a microfluidic device that captures CTCs from metastatic breast cancer patients’ blood based on their size and deformability, without relying on specific markers. Captured cells were harvested and spun onto positively charged slides and stained with the Portrait HER2 immunofluorescence (IF) panel, consisting of a nuclear dye, epithelial and mesenchymal markers, leukocytes’ markers, and an antibody targeting HER2. Slides were then de-stained and subjected to HER2 FISH using a commercially available kit. All slides were imaged twice using BioView Allegro Plus, a platform equipped with artificial intelligence for automated imaging, CTC candidate identification and reporting. First, an automated scan after IF was performed to identify CTCs based on marker expression and morphology profiles. Second, an automated relocation of the CTCs based on IF results and reimaging using higher magnification to ensure precise analysis of the FISH signals in single cells. The workflow is being implemented in a cohort of BC patients. Results. Following IF, HER2 protein expression in SKBR3 cells was significantly higher (p<0.0001) than that of HER2-negative cells, with 100% analytical specificity and sensitivity. Following FISH, 100% of the cells were de-stained and presented sharp HER2/CEP17 foci, with 96% of the SKBR3 cells and only 4% of the Hs 578T cells displaying HER2 amplification. Additionally, in a small cohort of metastatic BC patients’, 100% of the identified CTCs are retained and evaluable from IF to FISH. Conclusions. An assay integrating HER2 identification by IF and FISH to characterize CTCs in metastatic BC patients was successfully developed. The assay enables the identification of both HER2 protein and gene copy information from each target cell with minimal cell loss, with the potential for minimally invasive patient monitoring during treatment and better classification of patients who may qualify and benefit from HER2-targeted therapies. An added value of the assay is the practicality of utilizing preserved blood samples, allowing streamline batch shipping of clinical samples. Citation Format: Mariacristina Ciccioli, Alex Young, Laila Vlietinck, Chassidy Johnson, Anne-Sophie Pailhes-Jimenez. Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies [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 3705.

Abstract P3-06-14: Whole exome analysis of extreme long-term survivors with metastatic breast cancer

Abstract Background: Metastatic breast cancer (MBC) is incurable, yet some extreme long-term survivors (ELTS) live for a decade or more. Mechanisms underlying extreme survival are incompletely known, yet are important to improve prognosis and treatment. Extreme survival also provides an opportunity for cancer cells to evolve over decades. To evaluate the genomic characteristics of ELTS, we identified a cohort representative of the extreme tail of the metastatic survival curve and performed genomic analysis of blood and matched metastatic tumor. Methods: We identified a cohort of ELTS at our institution in an IRB-approved study. Eligible subjects had MBC and had lived a ≥10 years from the original breast cancer (BC) diagnosis with hormone receptor (HR)-positive disease or ≥5 years if HR-negative. Those with available archived tumor tissue were enrolled for genomic analyses. Archived samples were obtained from metastatic sites obtained late in the clinical course. Whole exome sequencing (WES) was performed on matched blood and tumor. Mutational profiles were qualitatively compared with publicly available reference somatic alterations in primary BC (Ctrl; cbioportal.org). Frequency of mutations is reported for known BC drivers. In an exploratory analysis to identify novel genes, the frequency of mutations in ELTS vs. Ctrl was compared for 15,659 well-covered genes. These were further evaluated for the ratio of non-synonymous to synonymous mutations, which is indicative of selective pressure. Results: 53 ELTS were identified; 13 had available archived tumor tissue and consented. The median survival from original BC diagnosis was 22 years, which corresponds to 99.8th percentile for survival, benchmarked to a cohort of MBC patients identified from the Surveillance, Epidemiology, and End Results-Medicare linked database. The 13 subjects had lived with metastatic disease for a median of 9 years (range 4-23). One subject had HR-/HER2+ BC and had lived 16 years with MBC. Three additional subjects had HR+/HER2+ BC with survival ranging from 19-25 years from original BC diagnosis. The remaining 9 subjects had HR+/HER2- BC with survival of 13-40 years from original diagnosis. Matched tumor/somatic WES achieved a mean tumor read depth of ˜600x and 96% coverage. Somatic mutations revealed near-expected rates of known driver alterations in commonly mutated genes such as PIK3CA, TP53, and CDH1 (Table). No mutations were found in PTEN or ESR1 in ELTS. Novel gene candidates were identified as having a high prevalence of mutations in this cohort including Androgen Receptor (10 of 13), MUC4 (12 of 13) MUC12 (10 of 13), and HRNR (12/13). Pathway analysis is ongoing. Conclusions: ETLS tumors obtained late in the clinical course have mutations in known cancer drivers near rates expected from primary BC. However, we have identified additional genes with an increased number of mutations either related to distinct genetic features of these cancers, or due to accumulated mutations that occur over the extended time for tumor evolution. Frequency of driver mutations in control and extreme long-long term survivorsGeneCtrl freqELTS freqGeneCtrl freqELTS freqPIK3CA0.350.38MAP2K40.020.38TP530.350.23NF10.040.15GATA30.120.08CBFB0.030.15CDH10.100.23ERBB20.030.08 Citation Format: Yan RE, Longhurst C, Lee K, Gilbert AD, Alexandridis RA, McGregor SM, Laffin JJ, Rocque G, Burkard ME. Whole exome analysis of extreme long-term survivors with metastatic breast cancer [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 P3-06-14.

Explorative Analysis of Low-Dose Metronomic Chemotherapy with Cyclophosphamide and Methotrexate in a Cohort of Metastatic Breast Cancer Patients

Background: Low-dose metronomic chemotherapy (LDMC) is increasingly used in metastatic breast cancer (MBC). In this retrospective analysis, we examined the therapeutic effects and side effects of LDMC in a cohort of MBC patients. Methods: Patients with MBC were included when LDMC with oral cyclophosphamide (CTX) and methotrexate (MTX) was administered between 2009 and 2015. The primary endpoint was disease control rate (DCR) ≥ 24 weeks after the start of LDMC. Secondary endpoints were duration of progression-free survival (PFS), rates of discontinuation due to side effects, and DCR with regard to subgroups. Results: Retrospective data of 35 patients were available for this analysis. 31% patients achieved DCR. The median PFS was 12 weeks. 9% of patients discontinued LDMC due to adverse events. DCR was 37% in the first 2 lines and 25% in further lines of therapy. 22% of patients with multiple metastases and 35% with ≤2 different metastatic sites achieved DCR. DCR was achieved in 33% of hormone receptor(HR)-positive patients and 27% of HR-negative patients. Conclusion: The DCR of 31% is in line with the results of previous phase II studies. LDMC was well tolerated. Subgroup analysis was not able to identify a group in which LDMC was more efficient.

Abstract P2-02-21: The utility and correlation of circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) based on HER2 positivity

Abstract Background: CTCs are well-established prognostic and predictive biomarkers for metastatic breast cancer (MBC) and other solid tumors. ctDNA is emerging as a quantitative blood-based biomarker for monitoring genomic alterations and disease progression. We evaluated the clinical utility and correlation of these liquid biopsy molecular tools in a cohort of MBC patients. Methods: CTC samples were obtained from an ongoing, prospective study of blood based prognostic biomarkers for breast cancer patients. At this time, 71 patients and 98 total samples have been collected. CTC enumeration was performed using the CellSearchTM platform (Menarini, IT). Within this cohort, MBC patients who had ctDNA testing were identified. ctDNA testing was performed using Guardant360TM (Guardant Health, CA), a digital next-generation sequencing technology. Two groups were analyzed: (1) HER2-negative patients with CTC ≥ 5 in 7.5 ml of blood (2) HER2-positive patients who had been treated with HER2 targeted therapy. Results: 22 samples (N=16 patients) were found with CTC ≥ 5 (range 8-904) and concurrent ctDNA testing (median timeframe between collection 0 days, range 0-42 days). There was a significant association between number of CTCs and the total number of genomic alterations detected in ctDNA (paired two sample t-test, p=0.012). In addition, CTC enumeration was significantly correlated with somatic alteration burden of the dominant clone (paired two sample t-test, p=0.023). The most common alterations detected in the blood were TP53 (55% of patients, 18 total mutations), PIK3CA (41% of patients, 15 total mutations), and ESR1 (32% of patients, 14 total mutations). For patients with HER2 positivity receiving HER2-targeted therapies (N=16 samples from 11 patients), only 18.8% of samples had detectable CTCs (all less than 5) as compared to 75.0% of samples with detectable ctDNA alterations. In N=12 samples with detectable ctDNA mutations, mean number of genomic alterations was 4.4 with mean somatic mutation burden of 2.95%. CTCs detectedctDNA detectedCTC ≥ 5Mean number of ctDNA alterations+Mean somatic alteration burden+HER2- (only cases with CTC ≥ 5)100% (22/22)100% (22/22)100%6.716.1%HER2+ (all cases)18.8% (3/16)75.0% (12/16)0%4.42.95%+excludes ctDNA samples without detected genomic alterations Conclusions: In HER2-negative MBC patients, CTC enumeration was significantly correlated with the number of ctDNA genomic alterations and somatic alteration burden, indicating the potential for ctDNA as a prognostic, quantitative biomarker of tumor burden. In patients with HER2 positivity, ctDNA may be a more sensitive liquid biopsy tool given the rarity of detecting CTCs detection in this population using the CellSearchTM system. In HER2-positive patients, consideration of size-dependent selection of CTCs using filtration of cells that have undergone epithelial-mesenchymal transition may improve detection in this subgroup. Citation Format: Davis AA, Zhang Y, Behdad A, Taxter T, Strickland K, Santa-Maria C, Flaum L, Cruz MR, Platanias LC, Gradishar WJ, Cristofanilli M. The utility and correlation of circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) based on HER2 positivity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-21.

Abstract P1-01-02: Circulating tumor cells (CTC) and endothelial cells (CEC) prognostic value in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab: First results of a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study

Abstract Background: increased levels of circulating tumor cells (CTC) are associated with worse progression-free survival (PFS) and overall survival (OS) in patients (pts) with metastatic breast cancer (MBC). It has been hypothesized that bevacizumab could modify CTC prognostic value. CEC variations to predict benefit of anti-angiogenic treatment is still controversial. Predictive markers for response to bevacizumab combined to chemotherapy in MBC remains a clinical unmet need. Patients & methods: The French cohort COMET is a prospective study including first line HER2 negative patients (pts) receiving weekly paclitaxel and bevacizumab according to EMA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome (CTC, CEC, serum markers, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). We present here the first planned analysis on 203 pts evaluated for CTC and CEC using the FDA cleared CellSearch method. Results: For CTC substudy, 211 patients were included from 09/2012 to 5/2014. Median follow-up is 24 months. Median PFS was 10 months (CI95 9-12) and response rate was 57%. Median OS was not reached. 203 patients were evaluable for both CTC and CEC at baseline and first day of second cycle of CT (D1C2). At baseline, 97/203 (48%) pts had ≥ 5 CTC (median 4 (range 0-30,000). Median number CEC was 21 (0-2231) at baseline and 22 (1-881) at D1C2. LDH, CEA, CA15.3 and CYFRA 21 were above normal at baseline in 44%, 46%, 73% and 71% of the cases respectively. CTC level was not correlated with any patients' characteristics except a number of metastatic site >3. After one cycle of chemotherapy (D1C2) 37 pts (22%) had still ≥ 5 CTC: 36 pts with initial high level and only one patient with low CTC at baseline had increased CTC above 5. Prognostic factors for PFS at univariate analysis were visceral disease, number of metastatic sites (> 3), triple negative status, LDH, CTC level at baseline and CTC level after one cycle of chemotherapy (D1C2). None of serum marker nor CEC level at baseline or any variations had prognostic value. In multivariate analysis for PFS, CTC level after one cyle of chemotherapy predicts poor outcome Table 1 Multivariate analysis for PFSPts' characteristicsnRRCI 95%p-valueNumber of metastatic sites <3 sites971 >=3 sites1001.65[1.13 ; 2.41]0.010CTC D1C2 < 51321 >= 5372.17[1.43 ; 3.29]<0.001Hormonal status Luminal (HR+)1531 Triple negative372.86[1.85 ; 4.54]<0.001 Conclusion: We confirm in a large prospective series the lack of clinical validity of CEC to predict response to an antiangiogenic based treatment in MBC. A persistent elevated level of CTC after just one cycle of chemotherapy is a very strong and independent marker of poor outcome in a homogeneously bevacizumab-treated cohort of MBC patients. This marker could be used to stopped earlier an inefficient and costly treatment. Citation Format: Pierga J-Y, Tredant O, Chevrier M, Dubot C, Lorgis V, Romieu G, Goncalves A, Debled M, Levy C, Ferrero J-M, Jouannaud C, Luporsi E, Mouret-Reynier M-A, Dalenc F, Berger F, Lemonnier J, Proudhon C, Bidard F-C. Circulating tumor cells (CTC) and endothelial cells (CEC) prognostic value in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab: First results of a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-02.

Abstract 4509: Clinical genomic profiling of 1000 metastatic breast cancer patients: actionable targets, novel alterations, and clinical correlations

Abstract Most large genomic profiling efforts in breast cancer have focused on primary breast tumors. Profiling of metastatic breast cancer (MBC) could more accurately define actionable genomic alterations and reveal novel alterations that arise under the selective pressure and clonal evolution of prior therapy. Utilizing a 410-gene targeted capture-based sequencing platform (MSK-IMPACT), we analyzed 920 tumors (62% metastatic, 38% primary) from 874 MBC patients for somatic mutations, DNA copy number alterations, and structural rearrangements (planned final analysis 1000 patients). Detailed clinical data including treatment outcomes was collected for all patients. The cohort was representative of the well characterized clinical subtypes of breast cancer with 71% ER+ or PR+ and HER2-, 17% HER2+, and 12% triple negative breast cancer (TNBC). Our analysis revealed recurrent alterations in multiple pathways including PI3K/AKT/mTOR (56%), cell cycle regulation (42%), RTK signaling (40%), epigenetic regulation (33%) and MAPK/ERK (19%) pathways. The most frequent actionable alterations include: PIK3CA mutation (36%), ERBB2 amplification (15%), FGFR1 amplification (12%), ESR1 mutation (11%), PTEN mutation/deletion (10%), AKT1 mutation (6%), and ERBB2 mutation (5%). The genomic landscape was significantly different across breast cancer subtypes. For example, the PI3K/AKT/mTOR pathway was altered in ER+ MBC mainly through activating PIK3CA mutations whereas PTEN deletion/mutations were most common in TNBC. We also identified significant differences in the genomic profiles of metastatic and primary tumor samples. Gene enrichment analyses revealed a subset of genes more frequently altered in metastatic tumors (STK11: 13 vs 2; ROS1: 14 vs 2; FGFR4: 15 vs 1) suggesting that mutations in these genes may play a role in breast tumor metastasis and/or therapy resistance. ESR1 mutations were predominantly present in metastatic tumors (88%) and were significantly associated with duration of prior hormonal therapy (P<0.0001). ESR1 mutations were also associated with a poor response to the ER degrader fulvestrant (median PFS: 4.8 vs 13.7 months, mutated vs wild type; P = 0.01). Analysis for potentially novel hotspot mutations revealed recurrent RHOA G17 mutations in 6 MBC patients, nominating RHOA, a GTPase transducer of membrane receptors, as a candidate driver in a subset of breast cancers. While identified previously in other tumors, RHOA G17 has never been implicated in breast cancer. In summary, our genomic analyses of this large cohort of MBC patients revealed actionable alterations in over 60% of the patients. 29% of patients with these alterations were enrolled in clinical trials of matched targeted therapies to date (PIK3CA 27%, AKT1 30%, ESR1 23%, ERBB2 39%) suggesting that prospective genomic characterization can accelerate enrollment of patients with MBC onto therapeutic clinical trials. Citation Format: Pedram Razavi, Matthew T. Chang, Sumit Middha, Dara S. Ross, Ahmet Zehir, Tracy A. Proverbs-Singh, Cyriac Kandoth, Sarat Chandarlapaty, Maura N. Dickler, Jorge S. Reis-Filho, Sujata Patil, Venkatraman Seshan, Lillian Smyth, Neil M. Iyengar, Komal Jhaveri, Shanu Modi, Chau T. Dang, Mark E. Robson, Larry Norton, Clifford A. Hudis, Marc Ladanyi, Maurizio Scaltriti, Nikolaus Schultz, David Hyman, Michael F. Berger, Barry S. Taylor, David B. Solit, José Baselga. Clinical genomic profiling of 1000 metastatic breast cancer patients: actionable targets, novel alterations, and clinical correlations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4509.

Abstract P4-01-12: Circulating tumor cells (CTC) and endothelial cells (CEC) changes in HER2 negative metastatic breast cancer (MBC) patients treated with first line weekly paclitaxel and bevacizumab: Preliminary results of a prospective cohort from the French Breast Cancer

Abstract Background: increased levels of circulating tumor cells (CTC) are associated with worse progression-free survival (PFS) and overall survival (OS) in patients (pts) with MBC (Bidard FC et al, Lancet Oncol 2014). The failure of chemotherapy to reduce CTCs to levels below five CTCs per 7.5 mL whole blood at first follow-up after initiating a new systemic therapy for MBC predicts shorter time to progression and OS. It has been hypothesized that bevacizumab could modify CTC prognostic value due to extravasation or epithelio-mesenchymal transition induction. CEC variations to predict benefit of anti-angiogenic treatment is still controversial. Patients & methods: the French cohort COMET is a prospective study including first line HER2 negative pts receiving weekly paclitaxel and bevacizumab according to EMEA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome (CTC, CEC, VEGFA levels, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat assessed by initial CTscan, serum estradiol level, and quality of life). We present here the first planned analysis on 206 patients evaluated for CTC and CEC using the FDA cleared CellSearch method. Results: inclusions started in 09/2012. At time of analysis, 219 patients were included, 211 were evaluable for CTC at baseline (failure rate 4%) and 207 for CEC at baseline (failure rate 5%). Due to short follow-up, 173 pts and 166 pts were evaluable for both CTC and CEC at baseline and first day of second cycle of CT (D1C2) respectively. At baseline, 100/211 (47%) pts had ≥ 5 CTC (median 4 (range 0-30,000) and 30% had no detectable CTC (0 CTC). After one cycle of chemotherapy (D28) 38 pts (22%) had still ≥ 5 CTC: 37 pts with initial high level and only one patient with low CTC at baseline had increased CTC above 5. Median number CEC was 21 (0)-2231) at baseline and 22 (1-881) at D1C2. CEC increased in 16% and decreased in 15% of the cases. CTC & CEC changes after one cycleCTC BaselineCTC at D1C2CEC BaselineCEC at D1C2≥ 5≥ 537 (21%)>20>2065 (39%)≥ 5<545 (26%)>20≤ 2025 (15%)<5≥ 51 (<1%)≤ 20>2027 (16%)<5<590 (52%)≤ 20≤ 2049 (30%)Total173166 CTC number at baseline line and CTC D1C2 were correlated (p<0.01) (Spearman test). There was no correlation between CEC at baseline or at D1C2 with CTC or CTC changes. Final analysis will be completed when 206 couples for both CTC and CEC at baseline and D1C2 will be available. Accrual is still ongoing. Conclusion: this 22% rate of failure to reduce CTC < 5 after one cycle of first line CT in a homogeneously bevacizumab-treated cohort of MBC patients did not differ from previous series. As second lines of chemotherapy do not improve the poor prognosis of this group of patients according to the SWOG 500 study results (Smerage et al, JCO 2014), trials of novel therapeutic agents should be considered at the time of progression. Citation Format: Jean-Yves Pierga, Isabelle Vaucher, Maya Gutierrez, Olivier Tredan, Séverine Guiu, Gilles Romieu, Anthony Goncalves, Marc Debled, Christelle Levy, Jean-Marc Ferrero, Christelle Jouannaud, Elisabeth Luporsi, Marie-Ange Mouret-Reynier, Florence Dalenc, Bernard Asselain, Jerome Lemonnier, Francois-Clement Bidard. Circulating tumor cells (CTC) and endothelial cells (CEC) changes in HER2 negative metastatic breast cancer (MBC) patients treated with first line weekly paclitaxel and bevacizumab: Preliminary results of a prospective cohort from the French Breast Cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-12.

Ki67 measured in metastatic tissue and prognosis in patients with advanced breast cancer.

The purpose of this study is to determine the prognostic role of Ki67 evaluated in relapse biopsies from patients with metastatic breast cancer (MBC). Two hundred and ten patients diagnosed with MBC in Stockholm, Sweden between 1998 and 2009 and with Ki67 assessed at time of first systemic relapse (mKi67) were retrospectively identified and divided into two groups according to mKi67 fraction (low≤20%, high>20%). Post-relapse survival was compared between the groups using Kaplan-Meier and Cox regression methods. Death rate as function of continuous mKi67 was also evaluated. Furthermore, the prognostic role of intra-individual change in Ki67 between primary tumor and matched metastasis was explored by Kaplan-Meier plots. One hundred and twenty-five patients had low and 85 had high mKi67. Median survival was 25 and 17months in low- and high-mKi67 group, respectively [hazard ratio (HR) 0.69, 95% confidence intervals (CI) 0.51-0.92, P=0.01]. In a multivariate model adjusted for prognostic confounders, low-mKi67 showed a non-significant trend toward better survival (HR 0.85, 95%CI 0.62-1.16, P=0.30). Nevertheless, mKi67 independently correlated with survival when compared with primary tumor proliferation (HR 0.56, 95%CI 0.38-0.81, P=0.002). The 2-year death rate steeply increased as mKi67 increased. Moreover, the change from high in primary tumor to low in metastasis significantly correlated with longer survival when compared with stable Ki67 levels (HR 0.48, 95%CI 0.31-0.76, P=0.002). In this cohort of MBC patients, mKi67 inversely but not independently correlated with survival. However, a significant association between mKi67 and survival was shown regardless of primary tumor proliferation.

Abstract P3-13-10: Treatment patterns and benefit of later lines of chemotherapy in metastatic breast cancer: A retrospective cohort study

Abstract Introduction: The treatment of metastatic breast cancer (MBC) lacks evidence beyond second or third line, but later lines are frequently used in clinical practice. The need for guidelines and studies with longer follow-up makes this area of particular interest for deeper investigation and analysis. We aimed to describe the clinical practice and outcome from later lines of chemotherapy of MBC in Sweden and to find out if the effect of the two first lines could foresee effects in later lines. Patients and Methods: Patients with MBC treated after year 2009 were identified in our treatment database (RealQ®) in Uppsala University hospital. Of 321 patients with MBC, 165 had received at least one line of chemotherapy and were included for a deeper analysis of tumor factors, response, time-to-treatment failure (TTF), reasons for termination of therapy and overall survival (OS). There were 93 deceased patients, who allowed for a complete analysis of treatment pattern and time from last treatment to death. Subgroup analyses were performed based on tumor biology, age, adjuvant therapy and response or TTF of the two first lines of chemotherapy. Survival times were calculated from diagnosis of metastatic disease and start of first and third line of chemotherapy, respectively. Results: The median age was 60 years at diagnosis of MBC and 13% were triple negative, 18% HER2-positive and 69% HER2-negative/ER-positive (luminal). The 165 patients received together 553 lines of chemotherapy with a median follow-up of 34 months. The deceased patients were treated with mean 3.6 lines (median 3, range 1-12) and the median time from last treatment to death was only 4.6 weeks. The most used regimens were capecitabine, paclitaxel, FEC, vinorelbine and docetaxel in that order, with an overall response rate (OR) of 47%, 53%, 57%, 23% and 63%, respectively. In HER2-positive patients, 82% of the lines were accompanied by trastuzumab or lapatinib. The OR in first line was 63% and between 23-48% in line two to seven. The mean TTF increased until line three and was relatively stable through the first seven lines (mean 103-182 days). Response to the first two regimens did not predict for better OS, but long TTF (>30 weeks) after the first two lines was associated with a significant survival benefit (median 77 vs. 29 months). So did age below 50 years compared to 50-69 and +70 years (median 90 vs. 54 and 32 months) and HER2-positive disease compared to luminal and triple negative MBC (median 80 vs. 49 and 15 months). Response pattern in early lines could not predict for the response in later lines in the individual patient. Conclusions: We here present a cohort of MBC patients treated in clinical routine with long follow-up of given chemotherapy lines. Later lines of chemotherapy (from the third to the seventh) created surprisingly good response and long TTF in clinical practice. Long TTF after the two first lines of chemotherapy, but not response, correlated with increased survival from the third line and on. Younger patients and HER2-positive patients showed the best OS with remarkable long survival times. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-13-10.

Quantitative measurements of p95HER2 (p95) protein expression in tumors from patients with metastatic breast cancer (MBC) treated with trastuzumab: Independent confirmation of the p95 clinical cutoff.

604 Background: Expression of p95 in HER2-positive breast cancer is potentially a major determinant of trastuzumab resistance because p95 lacks the trastuzumab binding site while retaining kinase activity. Previously, an optimal clinical cutoff for a continuous measurement of p95 expression was defined in a training set of trastuzumab-treated MBC patients (Clin Cancer Res, 16:4226, 2010). Methods: Quantitative H2T (HERmark HER2-total) and p95 assays (VeraTag, Monogram Biosciences) were retrospectively performed on formalin-fixed, paraffin-embedded tumors from an independent series of 240 trastuzumab-treated MBC patients. The pre-specified cutoff for p95 was tested to determine whether p95 above the cutoff in the HER2-positive subset correlated with worse progression-free survival (PFS) and overall survival (OS), as was observed in the training set. P95 expression was also assessed by immunohistochemistry (IHC) using the same antibody as the p95 VeraTag assay. Results: In the subset of tumors assessed as H2T-positive (N=190), p95 VeraTag values above the pre-defined cutoff correlated with shorter PFS (HR=1.41; p=0.043) and shorter OS (HR=1.72; p=0.021) where both outcomes were stratified by hormone receptor status and tumor grade. The hormone receptor positive patients (N=78) primarily drove the shorter PFS (HR=2.08, p=0.0026) and OS (HR=2.28, p=0.0099) observed in the p95-high subset. In contrast to the quantitative p95 VeraTag measurements, p95 IHC was not significantly correlated with outcomes. Conclusions: A clinical p95 cutoff (VeraTag assay) predictive of clinical outcomes derived from a previous training dataset was confirmed in a second independent clinical series. In contrast, p95 IHC did not correlate with outcomes. The observed consistency in the p95 VeraTag cutoff across different cohorts of MBC patients treated with trastuzumab justifies additional studies employing blinded analyses in larger series of patients. Clinical relevance of p95 protein expression remains to be established in a controlled clinical trial.

P3.13 Immune Status Stratification of Metastatic Breast Cancer Patients: Lympho-Divpenia Predicts Overall Survival

ABSTRACT Background Lymphopenia ( 1 Giga/L) before initiation of chemotherapy is a predictive factor for toxicity and death in metastatic phase for cancer patients. Combinatorial diversity of T Cell Receptor -beta chain (TCR-β), as a measure of T cell repertoire diversity, was investigated and tested either alone or in combination with lymphopenia as a prognostic factor for overall survival (OS) in first line treatment of metastatic breast cancer (MBC) patients. Methods Using semi quantitative multiplex PCR, the V-D-J combinatorial diversity of the TCR was measured on cryo-preserved blood samples from 2 cohorts of MBC patients before the initiation of the first line chemotherapy: in an experimental cohort (cohort A, n = 66) and in a validation series (cohort B, n = 67). A prognostic score, defined NDL (Number & Diversity of Lymphocytes) combining lymphocyte count and TCR diversity was delineated. Univariate and multivariate analysis of prognostic factors for OS were performed in both cohorts. Results Lymphopenia ( 1 Giga/L) was associated with shorter OS for cohort B while TCR diversity ≤33% (called divpenia) was associated with a reduced OS in cohort A. The combination of lymphopenia with low TCR diversity (called lympho-divpenia) was associated with poor OS compared to patients with either lymphocyte count ≥1 Giga/L or diversity >33% or both, in cohort A (median OS: 7.6 vs 24.5 months, p.value =0.0006) and cohort B (median OS 10.6 vs 22.9 months, p.value =0.0035). In a multivariate analysis, including all significant clinical factors from the univariate analysis (PS, liver metastasis, hemoglobin) lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (A + B) (p = 0.005) along with triple negative tumors (p = 0.011) and hemoglobin level (11.5 g/dL) (p = 0.009). Conclusion NDL score combining lymphopenia and reduced TCR diversity seems to be a strong prognostic factor for OS and could be use to improve care quality of MBC patients.

Lymphopenia combined with low TCR diversity to predict overall survival in metastatic breast cancer patients.

10562 Background: Lymphopenia (<1 Giga/L) before initiation of chemotherapy is a predictive factor for toxicity and death in metastatic phase for cancer patients. Combinatorial diversity of T Cell Receptor beta chain (TCR-ß), as a measure of T cell repertoire diversity, was investigated and tested either alone or in combination with lymphopenia as a prognostic factor for overall survival (OS) in first line treatment of metastatic breast cancer (MBC) patients. Methods: Using semi quantitative multiplex PCR, the V-D-J combinatorial diversity of the TCR was measured on cryo-preserved blood samples from 2 cohorts of MBC patients before the initiation of the first line chemotherapy: in an experimental cohort (cohort A, n=66) and in a validation series (cohort B, n=67). A prognostic score, defined NDL (Number & Diversity of Lymphocytes) combining lymphocyte count and TCR diversity was delineated. Univariate and multivariate analysis of prognostic factors for OS were performed in both cohorts. Results: Lymphopenia (<1 Giga/L) was associated with shorter OS for cohort B while TCR diversity ≤33% (called divpenia) was associated with a reduced OS in cohort A. The combination of lymphopenia with low TCR diversity (called lympho-divpenia) was associated with poor OS compared to patients with either lymphocyte count ≥1 Giga/L or diversity >33% or both, in cohort A (median OS: 7.6 vs 24.5 months, p.value =0.0006) and cohort B (median OS 10.6 vs 22.9 months, p.value =0.0035). In a multivariate analysis, including all significant clinical factors from the univariate analysis (PS, liver metastasis, hemoglobin) lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (A+B) (p=0.005) along with triple negative tumors (p=0.011) and hemoglobin level (11.5 g/dL) (p=0.009). Conclusions: NDL score combining lymphopenia and reduced TCR diversity seems to be a strong prognostic factor for OS and could be use to improve care quality of MBC patients.

Circulating tumor cells (CTC), [18F]fluorodeoxyglucose positron emission tomography, and computed tomography (PET/CT) for treatment monitoring in patients with metastatic breast cancer (MBC)

e12007 Background: Use of [18F]-fluorodeoxyglucose PET /CT and/or CTC is being investigated to follow up response to treatment in patients with MBC. It is not clear if these tests can be a surrogate for one another. Methods: We retrospectively analyzed a database of female patients with MBC undergoing chemotherapy or hormonal therapy. Most of these patients received at least 2 lines of therapy. Standard CT scan tumor measurements were used to assess response to therapy. CTC were defined either low (0–5) or high (>5). Maximum standard uptake values (max SUV) on PET scan were defined either low (<3) or high (>3). Correlation between the max SUV and CTC counts was statistically analyzed. Sensitivity, specificity, positive and negative predictive values were calculated from 2 x 2 table. Results: A total of 9 female patients with MBC were identified (mean age of 52 years). The receptor status of these patients includes 67 % positive for ER and 33 % positive for HER-2/neu. Median follow up was 9.8 months. There were 59 time points (> or = 4 weeks apart) when either PET/CT or CTC were performed. The results of PET/CT scans were compared with CTC at 38 events. The sensitivity of CTC to detect metastatic disease shown on PET/CT was 32% and specificity of 100%. The positive and negative predictive values were 100% and 32% respectively. There was a positive correlation between the max SUV and CTC count (p = 0.001). However in three patients, despite of progression of disease per PET/CT, CTC were undetectable at three different time points. Interestingly, two out of these three patients were triple negative. Disease progression was confirmed by biopsy in two of these patients. Conclusions: Our data suggest positive correlation between PET/CT scan and CTC. However, CTC had poor sensitivity and negative predictive value to detect progressive metastatic disease. Normal CTC values have to be interpreted cautiously in patients with MBC. We are now planning to investigate the utility of these tests, prospectively, in a large cohort of MBC patients. No significant financial relationships to disclose.