Factor In Metastatic Breast Cancer Research Articles

Abstract 4854: Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy

Abstract Histone deacetylases (HDACs), originally described as histone modifiers, have recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immune-biology is incompletely understood. We have found that the pharmacological or genetic abrogation of a single HDAC, i.e. HDAC6, inactivates the STAT3 pathway and modulates the expression of immuno-regulatory proteins, including the down-regulation of PD-L1, PD-L2 and B7-H4, important negative regulators of immune function, often over-expressed in cancer cells; including breast cancer. HDAC6 has been also involved in a number of structural functions related to cellular motility, shape and intracellular transport through the regulation of the acetylation of numerous targets, including tubulin and cortactin. This function is strongly suggestive of HDAC6 being a key player in metastatic cancer progression. In our initial studies we observed that the selective HDAC6 inhibitor Nexturastat A is capable of reducing the tumor growth in a highly aggressive murine mammary carcinoma that mimics human triple negative breast cancer (TNBC), under both orthotopic and subcutaneous conditions of implantation. Additionally, we observed that the size and number of secondary tumor nodules in lungs were significantly diminished after the HDAC6i treatment. In order to boost the anti-tumor T-cell response, we also tested check-point inhibitors against the tumor (such as anti PD-1 and CTLA4 antibodies). While each of the standalone treatments showed a certain degree of success in reducing tumor growth and enhancing intra-tumoral IFNγ, we demonstrated that HDAC6i improves anti-tumor immune responses when combined with immune check-point blockade. Citation Format: Debarati Banik, Melissa Hadley, Jennifer Kim, Tessa Knox, Jayakumar Nair, Alan Kozikowski, Sida Shen, Charu Vyas, Ashleyn Donohue, Eduardo Sotomayor, Alejandro Villagra. Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4854. doi:10.1158/1538-7445.AM2017-4854

Combined peripheral natural killer (NK) cell and circulating tumor cell (CTC) enumeration to enhance prognostic efficiency in patients (pts) with triple-negative breast cancer (TNBC).

1105 Background: CTCs have emerged as an independent prognostic factor for metastatic breast cancer. However, the prognostic value of baseline CTCs regarding PFS in TNBC is still controversial, especially beyond first-line. We evaluated a novel combined NK cell/CTC detection system for enumeration to better understand the impact of cell count on prognosis in TNBC. Methods: 83 consecutive patients with metastatic TNBC were enrolled and received a new line of therapy (median=2, range: 1~5). Baseline circulating CTCs and NK cells were isolated and enumerated simultaneously prior to starting a new line of therapy using our previously published method targeting EpCAM (Bai et al. J Mater Chem B, 2014) and flow cytometry (antibodies against CD3-, CD45+, CD56+) respectively. The NK cell level was expressed as the proportion of total lymphocytes (%). Patients with normal to high NK cell proportion (≥ 9.15%) and > 2 CTC/2 ml were defined as NK resistant CTC, the rest were defined as non-NK resistant CTC. Results: 33 out of 83 TNBCs had first-line of therapy. Pts with ≤ 2 CTC/2 ml had a significantly longer median PFS than those with > 2 CTC/2 ml ( P = 0.028). The differences in median PFS were not significant ( P=0.110) for the entire group of pts with TNBC receiving different lines of therapy. Pts with non-NK resistant CTCs had a significantly longer median PFS than those with NK resistant CTCs ( P= 0.025), regardless of line of therapy. Conclusions: Including peripheral NK cell level into consideration can improve CTC prognostic efficiency in predicting PFS for TNBCs receiving different line of therapy. Further analysis of the unique biological features of NK-resistant CTCs and associated clinical consequence holds promise in guiding clinical practice. [Table: see text]

Feasibility study of multiple HLA-class IA restricted peptide vaccines (KRM-19) for metastatic triple negative breast cancer (TNBC).

164 Background: Our previous phase II clinical trial has indicated that the therapeutic selected personalized peptide vaccines (PPV) were effective for boosting anticancer immunity and the immune response after PPV was associated with the clinical outcome as a prognostic factor for metastatic breast cancer (mBC). Based on the data from the PPV studies, we have conducted an early phase II study to evaluate the safety and the efficacy of a new regimen using multiple peptide vaccines (KRM-19) for pts with metastatic TNBC. Methods: KRM-19 consisted of 19 mixed peptides which were chosen from the previously reported 31 PPVs according to their anti-tumor immunologic effect and safety profile for mBC pts. All patients had histologically confirmed measurable ER-PgR-Her2- mBC and their human leukocyte antigen (HLA)-A molecules should be each of -A2, A3, A11, A24, A26, A31, or A33. KRM-19 (19mg/ml) was administrated subcutaneously in order with schedule of every week for a total of 6 doses. The concurrent conventional chemo- and/or endocrine therapy was not permitted for the combination, but was available for post-study treatment. Measurement of peptide-specific cytotoxic T lymphocyte (CTL) and IgG responses were conducted before and after vaccination. Levels of IgG reactive to each of the 19 peptides in the pre- and post-vaccination plasma were measured using the LUMINEX system at every 6 vaccinations. Peptide-specific CTL responses were examined by INF-ɤ ELISPOT. Clinical response was evaluated by RECIST criteria. Results: 10 patients with refractory metastatic TNBC received all 6 vaccines and 10/10 patients experienced Grade 1-2 skin reaction at injection site, 5/10 pts had Grade 2-3 liver function disorder, 3/10 and 1/10 pts had Grade 2 bone marrow suppression and nausea, respectively. The clinical responses were assessed in 8 pts included 1 PR case, 4 SD cases, and 3 PD cases. The evaluation of other clinical and immunologic data is underway. Conclusions: Subcutaneous KRM-19 vaccine administration was safe and resulted in a 12.5% objective response and 62.5% clinical benefit rate in TNBC pts, warranting further larger scale study. Clinical trial information: UMIN000014616.

Abstract P2-02-08: Initial circulating tumor cell count and venous thrombosis-free interval in the course of metastatic breast cancer

Abstract Background: Circulating tumor cells (CTC) count is a major prognostic factor in metastatic breast cancer. It has been also reported to be associated with venous thrombosis in short-term studies on advanced metastatic breast cancer patients. Methods: We assessed whether an early CTC detection (CellSearch®), before the start of the first line chemotherapy, impacts Thrombosis-Free Interval during the whole course of metastatic breast cancer. Electronic medical files of all patients included in the large prospective IC 2006-04 CTC detection study (NCT00898014, Pierga et al, Ann Oncol 2012) and treated at Institut Curie (Paris, France) were manually and automatically searched for thrombosis events in the course of their metastatic breast cancer. Superficial venous thromboses were not taken into account.Thrombosis-Free Interval (TFI) was defined as the first event occuring between venous or arterial thrombosis. Results: In the 142 patients studied, with a median follow-up of 64 months [25-81 months], venous (deep venous thrombosis, pulmonary embolism, catheter thrombosis and portal vein thrombosis) and arterial thrombosis occurred in 21.8% and 4.2% of patients, respectively. Twenty-four (30%) of the 80 patients with ≥1 CTC/7.5ml of blood before the start of first line chemotherapy experienced at least one thrombotic events in the course of their disease, while only 10 (16%) of the 62 CTC-negative patients experienced a similar event (p=0.06). Baseline positive CTC was associated with a higher risk of experiencing a venous or arterial thrombosis (TFI: HR=2.6 [1.2-5.5], p=0.009). The median TFI was 64 months in CTC-positive patients and not reached in CTC-negative patients. Among the other patients characteristics tested at baseline, Performance Status (PS=1 vs PS=0: HR=0.85 [0.4-1.9], p=NS; PS=2 or 3 vs PS=0: HR=3.7 [1.5-8.7], p=0.003) and LDH levels at baseline (LDH>UNL vs LDH<UNL HR=2.4 [1.2-4.9], p=0.012) were also associated with TFI. Conclusions: Metastatic breast cancer patients with ≥1 CTC before the start of chemotherapy are at higher risk of venous thrombosis; the clinical relevance of thrombo-prophylaxis should be investigated in this population. Citation Format: Beinse G, Berger F, Cottu PH, Dujaric M-E, Kriegel I, Guilhaume M-N, Dieras V, Beuzeboc P, Pierga J-Y, Bidard F-C. Initial circulating tumor cell count and venous thrombosis-free interval in the course of metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-08.

Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients

A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

Circulating tumor cells in breast cancer.

Circulating tumor cell (CTC) measurement in peripheral blood of patients with breast cancer offers prognostic information. In this review, we will try to identify evidence that could be used for prognosis, predictive power to draw this tool to clinical utility. We reviewed 81 manuscripts, and categorized those in discovery datasets, prognostic factors in metastatic breast cancer, identification of clinical utility in early breast cancer and in novel approaches. With each patient responding differently to chemotherapy, more efficient markers would improve clinical outcome. Current CTC diagnostic techniques use epithelial markers predominantly; however, the most appropriate method is the measurement of circulating DNA. It has been hypothesized that micrometastasis occurs early in the development of tumors. That implies the presence of CTCs in nonmetastatic setting. The origin of stimulus for malignant transformation is yet unknown. The role of microenvironment as a stimulus is also being investigated. It has been shown that CTCs vary in numbers with chemotherapy. The markers, which are followed-up in the primary tumors, are also being studied on the CTCs. There is discordance of the human epidermal growth factor receptor-2 status between the primary tumor and CTCs. This review summarizes our current knowledge about the CTCs. With genetic profiling and molecular characterization of CTCs, it is possible to overcome the diagnostic difficulties. Evidence for clinical utility of CTC as prognostic and predictive marker is increasing. Appropriate patient stratification according to CTC determination among other tests, would make personalized cancer therapy more feasible.

Circulating tumor cells and brain metastasis outcome in patients with HER2-positive breast cancer: the LANDSCAPE trial

BackgroundDecrease of circulating tumor cells (CTC) during treatment is an independent prognostic factor in metastatic breast cancer (MBC). We specifically evaluated the impact of CTC on brain metastasis outcome. MethodsHER2-positive MBC with brain metastasis not previously treated with whole-brain radiotherapy received first-line combination of lapatinib and capecitabine in a phase II study. CTC were detected at baseline and day 21 (CellSearch). ResultsMedian follow-up of the 44 analyzed patients was 21.2 months. The central nervous system objective response (CNS-OR) rate was 66%. At baseline, 20 of 41 assessable patients for CTC (49%) had ≥1 CTC (range 1–301, median 3) and 9 (22%) had ≥5 CTC. At day 21, 7 of 38 patients (18%) had ≥1 CTC (P = 0.006, versus baseline), and CTC had disappeared in 11 patients. CNS-OR rate was significantly higher in patients with no CTC at day 21 [25 of 31 (80%) versus 2 of 7 (29%), P = 0.01]. The 1-year overall survival rate was 83.9% in patients with no CTC at day 21 versus 42.9% in patients with ≥1 CTC (P = 0.02). ConclusionsThis is the first report showing a correlation between CNS metastasis response, outcome and early CTC clearance under targeted treatment of HER2+ MBC. Clinical Trials numberNCT00967031.

Establishment and Validation of Circulating Tumor Cell–Based Prognostic Nomograms in First-Line Metastatic Breast Cancer Patients

Circulating tumor cells (CTC) represent a new outcome-associated biomarker independent from known prognostic factors in metastatic breast cancer (MBC). The objective here was to develop and validate nomograms that combined baseline CTC counts and the other prognostic factors to assess the outcome of individual patients starting first-line treatment for MBC. We used a training set of 236 patients with MBCs starting a first-line treatment from the M.D. Anderson Cancer Center (Houston, TX) to establish nomograms that calculated the predicted probability of survival at different time points: 1, 2, and 5 years for overall survival (OS) and 6 months and 1 and 2 years for progression-free survival (PFS).The covariates computed in the model were age, disease subtype, visceral metastases, performance status, and CTC counts by CellSearch. Nomograms were independently validated with 210 patients with MBCs from the Institut Curie (Paris, France) who underwent first-line chemotherapy. The discriminatory ability and accuracy of the models were assessed using Harrell c-statistic and calibration plots at different time points in both training and validation datasets. Median follow-up was of 23 and 29 months in the MD Anderson and Institut Curie cohorts, respectively. Nomograms showed good c-statistics: 0.74 for OS and 0.65 for PFS and discriminated OS prediction at 1, 2, and 5 years, and PFS prediction at 6 months and 1 and 2 years. Nomograms, which relied on CTC counts as a continuous covariate, easily facilitated the use of a web-based tool for estimating survival, supporting treatment decisions and clinical trial stratification in first-line MBCs.

Pretreatment Thrombocytosis as a Prognostic Factor in Metastatic Breast Cancer

Background. An elevated platelet count is often associated with malignancies, and it has been confirmed as an adverse prognostic factor in various cancers including early stage breast cancer. We sought to determine if thrombocytosis is also a prognostic factor in metastatic breast cancer. Patients and Methods. The records of 165 metastatic breast cancer patients with complete follow-up that had thrombocytosis or normal platelet counts were reviewed. Kaplan-Meier curves were constructed, and the survivals of the two groups were compared using the LogRank test. A Cox regression analysis was used to determine if thrombocytosis is an independent factor for overall and progression free survival. Results. There was a statistically significant difference in overall and progression free survival favoring the normal platelets group (LogRank test P = 0.038 and 0.008, resp.). Thrombocytosis remained a significant adverse prognostic factor in multivariate analysis. Other independent prognostic factors for overall survival included age, ER/PR status, and grade. Conclusion. Thrombocytosis represents an independent adverse prognostic factor in patients with metastatic breast cancer. Thus metastatic breast cancer joins a range of cancers in which this easily measurable value can be used for clinical prognostication. Further use as a predictive value for specific treatments has a rationale and deserves to be investigated.

Abstract P2-10-12: How well predict the 2011 St Gallen early breast cancer surrogate phenotypes metastatic survival?

Abstract BACKGROUND: The five prognostic surrogate phenotypes, defined by the 2011 St Gallen consensus, predict metastatic relapse following early breast cancer treatment (Brouckaert et al Ann Oncol 2012). It is unknown to what extent these surrogate phenotypes defined on the primary tumor predict metastatic survival (MS) in a cohort of consecutive women with metachronous metastases. PATIENTS &amp; METHODS: All 4318 patients with primary operable breast cancer, diagnosed between 01–01-2000 and 31–12-2009 and treated in our center were prospectively entered in our institutional database. We included 345 patients with a (distant) metastatic relapse during their follow-up. We observed metastatic survival as the time between the diagnosis of the relapse and death. Tumor subtype was defined according to the 2011 St Gallen recommendations in 5 groups: luminal A (ER+/HER2−, grade 1–2), luminal B1 (ER+/HER2−, grade 3), luminal B2 or luminal-HER2 (ER+/HER2+), HER2-like (ER−/HER2−) and triple-negative. We focused on the value of the primary tumor subtype as a major prognostic determinant, but also assessed age at diagnosis, tumor size, lymph node involvement, tumor subtype, PR-status, primary detection (screening vs. palpation), histology, adjuvant therapy, distant-metastasis free interval (DMFI), first-line metastatic hormonal therapy, recurrence sites (isolate or multiple) and metastasis detection (clinical vs. biochemical). RESULTS: In our cohort, we recorded a median metastatic survival (MS) of 22.3 months (95% CI: 19.7–25.6) and a 5-year survival probability of 15%. Significant differences in median MS were noted when considering the primary tumor subtype (cf. table). We observed an independent prognostic effect for multiple recurrence sites (HR = 1.78, 95% CI 1.37–2.32), first-line metastatic hormonal therapy (HR = 0.30, 95% CI 0.18–0.49), DMFI (HR = 0.92 CI 0.85–0.98) and, finally, primary tumor subtype. CONCLUSION: Our results showed that primary tumor subtype, DMFI, solitary recurrence and first-line metastatic hormonal therapy act as independent prognostic factors in metastatic breast cancer. In order to overcome the biological complexity of metastatic breast cancer, we must appreciate the primary tumor subtype in our therapeutic approach. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-12.

Prognostic Value of Circulating Tumor Cells According to Immunohistochemically Defined Molecular Subtypes in Advanced Breast Cancer

BackgroundBreast cancer is a heterogeneous disease. Circulating tumor cell (CTC) enumeration might be useful to identify different risk categories within each molecular subtype. MethodsWe retrospectively analyzed 203 consecutive patients with metastatic breast cancer with baseline CTC enumeration performed with CellSearch (Veridex Corp, Warren, NJ) between March 2005 and July 2011. Patients were categorized into 3 prognostic groups based on the number of CTCs (0, 1-4, and ≥ 5) and into 5 categories based on tumor biological characteristics: luminal-A (estrogen receptor [ER] and progesterone receptor [PR] > 1%, grade 1/2, human epidermal growth factor 2 [HER2]-negative [HER2−], Ki67 value < 14%); luminal-B (ER and/or PR > 1%, grade 3, HER2−, Ki67 value > 14%); luminal-B HER2–positive [HER2+] (ER and/or PR > 1%, any grade, HER2+, Ki-67 value any); HER2+ (HER2 overexpressed/fluorescence in situ hybridization [FISH] amplified, ER and PR absent); triple negative (TN) (ER and PR 0%, HER2 not overexpressed/FISH not amplified). ResultsMedian age was 57 years (range 31-78 years). Twenty-seven patients (13.3%) had luminal-A category, 105 (51.7%) patients had luminal-B, 29 (14.3%) patients had luminal-B HER2+, 24 patients (11.8%) had HER2+, and 18 patients (8.9%) had TN. CTCs were mostly found in patients with luminal-A/luminal-B HER2− subtype. At multivariable analysis, CTC count was a significant predictive factor for overall survival (OS) in all molecular subtypes (log-rank P < .01). Patients with 0 CTCs/7.5 mL blood and all subtypes, except HER2+, seem to perform better compared with other categories. ConclusionThese findings confirm CTCs as an important prognostic factor for metastatic breast cancer in all molecular subtypes. Larger studies could help identify metastatic breast cancer subgroups in which CTC analysis would be particularly useful.

Gene expression profiles of circulating tumor cells in metastatic breast cancer patients.

10504 Background: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer. Besides enumeration, CTC characterization promises to further improve outcome prediction and treatment guidance. We have previously shown the feasibility of measuring the expression of a panel of 96 clinically relevant genes in CTCs in a leukocyte background, and in the current study, we determined the prognostic value of CTC gene expression profiling in metastatic breast cancer. Methods: CTCs were isolated and enumerated from blood of 130 metastatic breast cancer patients prior to start of first-line systemic, endocrine or chemotherapeutic, therapy. Of these, 103 were evaluable for mRNA gene expression levels measured by quantitative RT-PCR in relation to time to treatment switch (TTS). Separate prognostic CTC gene profiles were generated by leave-one-out cross validation for all patients and for patients with ≥5 CTCs per 7.5 mL blood, and cut-offs were chosen to ensure optimal prediction of patients who might benefit from an early therapy switch. Results: In the total cohort, of whom 56% received chemotherapeutic and 44% endocrine therapy, baseline CTC count (≥5 versus &lt;5 CTCs/7.5 mL blood) predicted for TTS (Hazard Ratio (HR) 2.92 [95% Confidence Interval (CI) 1.71 – 4.95] P &lt;0.0001). A 16-gene CTC profile for all patients and a separate 9-gene CTC profile applicable for patients with ≥5 CTCs were identified, which distinguished those patients with TTS or death within 9 months from those with a more favorable outcome. Test performance for both profiles was favorable; the 16-gene profile had 90% sensitivity, 38% specificity, 50% positive predictive value (PPV) and 85% negative predictive value (NPV), and the 9-gene profile performed slightly better at 92% sensitivity, 52% specificity, 66% PPV and 87% NPV. In multivariate Cox regression analysis, the 16-gene profile was the only factor independently associated with TTS (HR 3.15 [95%CI 1.35 – 7.33] P 0.008). Conclusions: Two CTC gene expression profiles were discovered, which provide prognostic value in metastatic breast cancer patients. This study further underscores the potential of molecular characterization of CTCs.

Prognostic Value of EMT-Circulating Tumor Cells in Metastatic Breast Cancer Patients Undergoing High-Dose Chemotherapy with Autologous Hematopoietic Stem Cell Transplantation.

Background: Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer (MBC) patients treated by conventional dose chemotherapy. The aim of this study was to determine the role of CTCs and CTCs undergoing epithelial-mesenchymal transition (EMT) in metastatic breast cancer. We used the platform of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation (AHSCT) to study the CTCs and CTCs with EMT.Patients and methods: CTCs were enumerated in 21 MBC patients before apheresis and 1 month after AHSCT. CD34-depleted apheresis products were analyzed for CD326+ epithelial and Aldefluor+ cancer stem cells (CSC) by flow cytometry and were depleted of CD45+ cells and assessed for EMT-inducing transcription factors (EMT-TF) by quantitative RT-PCR.Results: Patients with ≥ 5 CTCs/7.5 mL of peripheral blood 1 month after AHSCT had shorter progression-free survival (PFS) (P=0.02) and overall survival (OS) (P=0.02). Patients with apheresis products containing high percentages of CD326+ epithelial cells or overexpressing EMT-TF had shorter PFS. In multivariate analysis, low percentage of CD326+ epithelial cells and response to HDCT with AHSCT were associated with longer PFS, whereas lower CTCs after AHSCT was associated with longer OS. High CTCs, 1 month after AHSCT correlated with shorter PFS and OS in MBC patients undergoing HDCT and AHSCT, while CTCs with EMT and CSCs phenotype in apheresis products are associated with relapse.Conclusion: Our data suggest that CTC and CTCs with EMT are prognostic in MBC patients undergoing HDCT followed by AHSCT.

P4-07-10: Circulating Tumor Cells in Newly Diagnosed Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer and is associated with special clinical and biological features. Circulating tumor cells (CTC) are an independent prognostic factor in metastatic breast cancer for progression-free and overall survival. Previously we showed that metastatic IBC patients treated with first and subsequent lines of chemotherapy had a lower number of CTC when compared with CTC levels of non-IBC patients; hence the prognostic value of CTC in mIBC patients is limited. In the present study, we investigated the prognostic value of baseline CTC in newly diagnosed IBC patients. Patients and methods: This retrospective study included 84 newly diagnosed IBC (37 stage III B or C and 47 stage IV) patients treated with neoadjuvant therapy or first line chemotherapy between January 2004 and July 2009 at the MD Anderson Cancer Center. The median age was 55 years (range, 23–78). Twenty-four (28.8%) patients were HER2 positive, 36 (42.9%) hormone receptor positive and 33 (39.3%) were triple receptor negative. CTC were detected and enumerated using the CellSearch® system before patients started chemotherapy. Progression free survival (PFS) and overall survival (OS) were calculated from the date of CTC measurement and estimated by the Kaplan-Meier product limit method. Results: At baseline, 64 (76.2%) patients had at least ≥1 CTC and 29 (34.5%) ≥5 CTC. Proportions of patients with stage III IBC with ≥1 CTC and ≥5 CTC were lower than those of newly diagnosed stage IV IBC patients (67.6% vs. 86%; p = 0.12 and 24.3% vs. 42.6%; p = 0.11, respectively). At a median follow up of 22.4 months (range: 4.8 - 58.3 months), 48 (57.1%) patients experienced disease progression and 26 (31.0%) had died. Patients with &amp;lt; 5 CTC had similar PFS (p = 0.36) and OS (p = 0.80) to those of patients with ≥5 CTC, respectively. We investigated the prognostic value of CTC ≥ 1 in stage III IBC patients, as well. There was no difference in PFS (p = 0.71) and OS (p = 0.15) in patients with CTC = 0 vs. those of patients with CTC ≥ 1, respectively. Moreover, we correlated CTC levels with prognosis, using different CTC threshold, but we observed no correlation between baseline CTC and patients’ outcome (Table 1). Conclusions: CTC can be detected in a large proportion of patients with newly diagnosed IBC; however, baseline CTC was not prognostic for PFS and OS. Collective dissemination of cancer cells via lymphovascular tumor emboli and/or epithelial-mesenchymal transition accompanied by inadequate detection of CTC due to lack of sensitivity of current assays may account for limited prognostic value of CTC in newly diagnosed IBC. Nevertheless, sample size and study power could influence study results as well. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-10.

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis.

Circulating tumor cells as a prognostic factor independent of obesity in metastatic breast cancer patients.

610 Background: Breast cancer is now recognized to be a heterogeneous disease with differences in outcome among various molecular subtypes. Approximately 30-40% of patients diagnosed with primary breast cancer eventually die of recurrent disease indicating the need for identification of prognostic factors. Several retrospective studies suggested that obesity adversely affects outcome particularly in hormone-receptor (HR) positive early breast cancer. Detection of circulating tumor cells (CTCs) by CellSearch before treatment is an independent predictor of survival in metastatic breast cancer (MBC). We therefore investigated the value of CTCs and body mass index (BMI) as independent prognostic factors in MBC. Methods: We retrospectively analyzed 255 consecutive MBC patients treated between 2004 and 2010 who had baseline CTC enumeration by CellSearch and BMI measurements. OS was calculated from the date of CTC measurement and analyzed with Kaplan-Meier survival plots. Patients with a BMI ≥ 30 were considered as obese. Cox multivariate proportional hazards model was also performed. Results: At a median follow-up of 24.6 months, 132 (51.7%) patients had died. Among all patients, 92 (36.1%) had CTCs ≥ 5 and 81 (31.8%) were obese. Median BMI was 26.4 kg/m2 (range 15.9 - 56.8) and all collected within 1year from CTC assay. We found a BMI ≥ 30 only in patients with age >50 years (P = .022). Patients with CTCs ≥ 5 had a higher risk of death than patients with CTCs < 5 (hr =1.78, 95% CI 1.27 – 2.51); the same difference according CTC value was observed both in normal and obese patients. In the HR+/HER2-subtype (n = 129), obese patients had the same risk of death as normal patients (obese patients hr = .77, 95% CI .44 - 1.35). In the multivariate model, considering age, HR and HER2 status, metastatic disease sites and obesity, patients with CTCs ≥ 5 had a hazard ratio of death of 2.01 (95% CI 1.41-2.87) compared with patients with CTC < 5. Conclusions: This study confirms the use of CTCs in MBC as a useful tool for the prediction of OS independent of obesity. Moreover, the data suggest a lack of prognostic value of obesity in women with metastatic disease, including endocrine-sensitive disease.

Circulating tumor cells as a predictive marker for chemotherapy and prognostic marker in patients with metastatic non-small cell lung cancer.

e21020 Background: Circulating tumor cells (CTCs) are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas. The number of CTCs before treatment has been reported to be prognostic factor for metastatic breast cancer. We investigated the relationship of the number of CTCs with overall survival, progression free survival, and response rate in patients with metastatic non-small cell lung cancer (NSCLC). Methods: We prospectively evaluated CTCs in 7.5-mL samples of peripheral blood sampled from patients with previously untreated metastatic NSCLC. A semiautomated system was used to capture CTCs with an antibody against epithelial cell adhesion molecule. From May 2008 through August 2010, 33 patients (23 men and 10 women; median age, 64 years; range, 46 to 74 years) were enrolled. All patients received chemotherapy of the combination of gemcitabine and carboplatin. Results: CTCs were detected in 36.4% of patients. Patients with positive CTCs were trend toward to longer survival (23 months) and progression free survival (5 months) than patients with negative CTCs (7 months, 2.5 months), although there were not statistically significant differences. The stable disease rate to chemotherapy was significantly lower in patients with positive CTCs (18%) than in patients with negative CTCs (75%; p = 0.002). Conclusions: CTCs could be a useful predictive marker for chemotherapy and might be prognostic marker in patients with metastatic NSCLC.

Abstract P3-02-05: Evaluation of Gene Transcripts in Primary Tumors at Time of Diagnosis and Circulating Tumor Cells (CTCs) at Time of Metastatic Disease

Abstract Introduction: The enumeration of CTCs has already shown to bear clinical relevance as a prognostic and predictive factor in metastatic breast cancer. In addition to enumeration, isolation of CTCs enables their molecular characterization and thus holds great promise to establish association of their genetic profile with patient outcome and to identify potential drugable targets. In this study we established epithelial-specific mRNA and microRNA profiles in CTCs of patients with metastatic breast cancer, compared these profiles to the profiles measured in corresponding primary tumors, and determined their association with clinical parameters. Study design: For this study we included 50 breast cancer patients, of which 32 presented themselves with over 5 CTCs at the time of metastatic disease. From 14 of these patients with more than 5 CTCs at the time of metastatic disease also the primary tumor at time of breast cancer diagnosis was evaluated. Total RNA was extracted 1) from blood of the 50 patients with metastatic disease after EpCAM-based enrichment of 7.5 mL whole blood with the CellSearch™ Profile Kit [Veridex LCC], 2) from 14 unprocessed whole blood preparations from healthy blood donors, and 3) from 14 primary tumors. Gene transcript levels of CTC-specific and potentially clinically relevant mRNAs and microRNAs were compared in CTCs isolated at time of metastatic disease and the corresponding primary tumors. In addition, the association of these transcript levels with clinical data was assessed. Results: We identified 24 mRNA and 14 microRNAs more abundantly expressed in CellSearch-enriched fractions from patients with at least 5 CTCs compared with those without CTCs and/or compared with unprocessed whole blood prior to CellSearch enrichment (Mann-Whitney U-test P&amp;lt;0.05). In addition, when comparing transcript levels present in CTCs during metastatic disease and those measured in the corresponding primary tumor, potentially clinically relevant discrepancies were observed. Findings of interest included changes in transcript levels of genes such as ESR1, ERBB2, TOP2A and MGB1, and in genes associated with proliferation and EMT. Finally, associations were observed between transcript levels measured in CTC preparations and clinical data like nodal status and size of the primary tumor. Conclusion: Our results show that molecular characterization of CTCs is feasible and has potential for a more tailored clinical approach above CTC enumeration in the treatment of metastatic breast cancer patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-05.

Abstract S6-6: High Independent Prognostic and Predictive Value of Circulating Tumor Cells in a Large Prospective Multicenter Trial Including Serum Tumor Markers in First Line Chemotherapy Metastatic Breast Patients

Abstract Background: Circulating tumor cells (CTCs) in peripheral blood and decrease during treatment has been reported as an independent prognostic factor in metastatic breast cancer (MBC) (Cristofanilli M., NEJM 2004). Data comparing CTC with circulating tumor markers are limited. We conducted a multicenter prospective study to validate these markers in a large series of patients receiving specifically first line chemotherapy (CT) for MBC including assessment of CA15.3, CEA and LDH. Methods: CTC were measured in 7.5ml of whole blood at baseline, before cycle 2, at clinical and/or radiological evaluation (C3-C4) and at tumor progression. Analysis was performed using the CellSearch™ System, combining EpCAM immunomagnetic selection (IMS) followed by anti-cytokeratin (A45B/B3). Double labeling for HER2 was performed in a subgroup of patients. Tumor markers were assessed locally with ELISA kit and high if above normal. Results: From 06/07 to 9/09, 267 patients with measurable or evaluable disease starting first line CT for MBC were included in five French Cancer Centers. Median follow-up was 16 months, 57 deaths and 160 progressions were observed. Median age was 57 years, 39 patients (15%) had HER2+ tumor and received trastuzumab, 55 (21%) had triple negative tumor, 125 received bevacizumab associated with CT, 260 were evaluable for CTC levels at baseline, 170 (65%) had ≥1CTC and 115 (44%) ≥5CTC. CTC HER2 status was assessed in 113 patients at baseline and was positive in 64% of the cases in at least one cell. CA15.3 was high in159/247 (64%), CEA in 109/212 (51%) and LDH in 99/220 (45%). CTC were highly correlated with tumor markers, tumor burden, performans status (PS), number of metastatic sites, liver metastasis) but were independent of tumor biology (HER2 status, Triple Negative BC (TNBC), grade). At first evaluation (C3-C4), 110/225 patients (49%) had objective response. At univariate analysis, high CTC level with a threshold of 1 or 5 at base line, before C2 (3-4w) and at radiological evaluation (C3-C4, 9-12w) was predictive of poor progression-free survival (PFS) and overall survival (OS) (p&amp;lt;10-5). Baseline CA15.3, CEA and LDH were prognostic for PFS and LDH only for OS. At multivariate analysis, TNBC, PS ≥2, CTC ≥5, and high CEA were prognostic for PFS and TNBC, high LDH, CTC ≥5 and PS ≥2 for OS. Multivariate analyses including CTC and tumor markers variations during treatment are ongoing. Conclusions: This is the largest prospective series validating the prognostic value of CTC in first line chemotherapy MBC independently from serum tumor marker for PFS and OS. Persistence of a high level of CTC before second cycle of chemotherapy is a strong and early predictive marker of poor PFS, before radiological evaluation. A prospective multicenter randomized trial (CIRCE01) to assess the use of CTC to modify treatment after second line chemotherapy is ongoing. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S6-6.

Abstract PD04-05: Prognostic Value of Circulating Tumor Cells According to Immunohistochemical Defined Molecular Subtype in Advanced Breast Cancer Patients

Abstract Background: Breast cancer (BC) is not a single disease with variable morphologic features and biomarkers but, rather, a group of molecularly distinct neoplastic disorders. Four main molecular classes of BC have been distinguished by gene expression profiling, called luminal-A, luminal-B, HER2-positive and triple-negative (TN) cancers. These subgroups correspond reasonably well to clinical characterization on the basis of estrogen receptor (ER) and HER2 status, as well as proliferation markers or histologic grade. Correlating circulating tumor cells (CTCs) with molecular subtypes might identify subgroup of patients (pts) at different risks and groups that might benefit more from prognostic evaluation by CTCs. Patients and methods: We retrospectively analyzed 187 consecutive metastatic BC pts who had baseline CTCs enumeration performed between March 2005 and December 2009 by CellSearch. Pts were categorized in 3 prognostic groups in accordance to the number of CTCs, 0 CTC, &amp;lt;5 CTCs and ≥5 CTCs, and in 4 categories based on the tumour biology: luminal-A with ER &amp;gt;1%, any progesterone receptor (PgR), G1/G2, Ki67&amp;lt;30%; luminal-B with ER &amp;gt;1%, any PgR, G3, Ki67&amp;gt;30%; HER-2 positive with HER-2 overexpressed or FISH amplified; TN with both ER and PgR 0% and HER-2 not overexpressed/FISH not amplified. Kaplan-Meier method for survival analysis was used. Results: Median age at baseline was 56 years (range 31-78). CTCs were a strong predictive factor for overall survival (OS) in all pts (Log-Rank P&amp;lt;0.001), in pts with bone metastases (MTS) only (n=43; p=0.045), visceral MTS only (n=42; borderline significant p=0.08) and both bone and visceral MTS (n=74; P&amp;lt;0.001). The same figure was not observed in the few pts with only soft tissue MTS (n=28; p=0.315). Sixty-one (33%) pts had Luminal-A tumour, 58 (31%) Luminal-B, 53 (28%) HER-2 positive and 15 (8%) TN. CTCs were a significant prognostic factor in all molecular subtypes as shown in Figure 1. Conclusion: This large series confirms CTCs as an important prognostic factor for metastatic BC in all different molecular subtypes, but pts with 0 CTCs at baseline and TN or luminal B tumors seem to perform better as compared to other categories. Furthermore, pts with metastatic disease including bone and/or visceral sites benefited most from CTCs prognostic evaluation. Further studies with larger series and longer follow-up time could help identify metastatic BC subgroups in which CTCs analysis would be particularly useful and should be recommended. Figure available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-05.