Baseline Circulating Tumor Cells Count Research Articles

Evaluation of clinical phenotype, survival, and circulating tumor cell (CTC) enumeration in men with metastatic castration-resistant prostate cancer (mCRPC).

5031 Background: The presence of ≥ 5 CTCs is prognostic for shorter survival in mCRPC men. However, many men have low CTCs despite widespread metastatic disease, suggesting heterogeneity in CTC phenotype or detection. We evaluated the association of baseline CTC enumeration with clinical characteristics and survival in mCRPC men at our institution. Methods: CTCs were enumerated with the standard CellSearch method in mCRPC men in a prospective correlative clinical study. The association between baseline CTC count and prostate-specific antigen (PSA), alkaline phosphatase (AP), lactate dehydrogenase (LDH) was explored using the Spearman correlation (r), and the relationship with Gleason sum, sites of metastasis (mets), and prior docetaxel exposure was explored using summary statistics. The overall survival probability (OS) was estimated by the Kaplan-Meier method. Results: In our cohort, 59/82 men had prior docetaxel exposure, and the median CTC count was 16.5. There were 25 men with visceral mets, 55 with bone-predominant mets, and 2 with lymph node only mets. We found a weak relationship between CTCs and PSA (r=0.20), but a more moderate and significant association with AP (r=0.48) and LDH (r= 0.50). We found no relationship between CTCs and Gleason sum, site of mets, or prior docetaxel. 66 men died, and the median OS was 11.2 months (95% CI: 9.2, 12.7). OS was improved in patients with <5 CTCs at baseline compared to ≥ 5 (8.9 vs. 16.6 months, HR=0.47 (95% CI: 0.27, 0.80)). CTC enumeration may further stratify outcomes in men with mCRPC and either visceral or bone metastasis as shown in the table below. Conclusions: The prognostic significance of CTCs is distinct from other established biomarkers and may reflect a unique biology of metastasis, dissociated in part from androgen receptor activity (as reflected by PSA values) but related in part to bone microenvironment, hypoxia, and tumor burden (as reflected by AP and LDH values). These associations need to be validated in larger trials. [Table: see text]

A combined EpCAM and MCAM circulating tumor cell (CTC) CellSearch enrichment to improve CTC capture rate in stage II/III breast cancer: A Dutch Breast Cancer Trialists' Group (BOOG) side study.

e22106 Background: Using the CellSearch System (Veridex, USA), ≥1 CTC/7.5 mL of peripheral blood is found in ~20% of primary breast cancer (BC) patients. However, epithelial cell adhesion molecule (EpCAM)-negative CTCs may be missed. We previously showed that a subset of BC cell lines lacks EpCAM, but does express melanoma cell adhesion molecule (MCAM) and that MCAM-positive CTCs (M-CTCs) do occur in metastatic breast cancer patients. In a side-study of the NEOZOTAC trial, a randomized study comparing neoadjuvant chemotherapy (NAC) with or without zoledronic acid in stage II/III BC, we investigated whether combined enrichment with EpCAM and MCAM increases the CTC detection rate. Besides, we hypothesized that CTC counts would correlate with pathological complete response (pCR) to NAC. Additionally, as exploratory study, circulated endothelial cells (CECs) were measured. Methods: At baseline and after the 1st chemotherapy cycle, blood was drawn from all patients in the NEOZOTAC trial who gave additional informed consent. The CellSearch System was used to count EpCAM-positive CTCs (E-CTCs) and M-CTCs. For the latter, the anti-EpCAM-ferrofluid was replaced by an anti-MCAM-ferrofluid (both Veridex). CECs were measured using flow cytometry. Results: At baseline, blood was received from 73 patients; ≥1 E-CTCs and M-CTCs were detected in 14/73 (19%, median 1) and 8/68 (12%, median 1), respectively. Combining both counts, ≥1 CTCs were detected in 17/68 (25%) of patients (p=0.01). After 1 chemotherapy cycle, E-CTCs, M-CTCs and overall CTCs were detected in 9/47 (19%), 7/45 (16%), and 13/44 (30%; p<0.01) patients. Median CEC count was 43 at baseline and 142 after cycle 1 (p<0.001). No differences in CTC and CEC count were found between the treatment arms. Baseline CTC counts, nor changes in counts, were associated with pCR. Conclusions: Combined enrichment of CTCs with EpCAM and MCAM significantly increased CTC detection rates in stage II/III BC with about 10%. CTC counts were not associated with pCR to NAC. Associations with disease free survival will follow when available.

MRNA expression profiles in circulating tumor cells (CTCs) of patients with metastatic breast cancer (MBC) treated with aromatase inhibitors (AI).

11045 Background: Enumeration of CTCs can be used to assess prognosis in MBC and to evaluate treatment response. Besides enumeration, molecular CTC characterization is a promising tool to develop a more personalized treatment approach. Here, we evaluated the association between mRNA expression of currently known CTC-specific genes and response to first-line AI in MBC patients with estrogen receptor (ER)+ primary tumors. Methods: CTCs were isolated and enumerated from blood of 25 MBC patients before first-line therapy with an AI. Fourteen patients received a non-steroidal AI (8 letrozole, 6 anastrozole) and 11 patients were treated with exemestane. mRNA expression levels of 96 genes were measured by quantitative RT-PCR as previously described (Sieuwerts et al. Clin Cancer Res. 17:3600-3618, 2011). Expression levels of these genes were studied for their association with time to progression (TTP) after start first-line AI. Results: Median TTP was 338 (range 14–1,239) days. Median baseline CTC count for the 25 patients was 14 (range 0–753). In this relatively small cohort, the clinically relevant cut-off level of ≥5 CTCs in association with TTP did not reach statistical significance (Hazard Ratio [HR] 4.76, 95% Confidence Interval [CI]: 0.59–38.22, P=0.14). For type of AI, when comparing steroidal with non-steroidal AI, the measures in Cox univariate regression analysis were HR 2.54 (95% CI: 0.67–9.64), P=0.17. A 10-gene CTC profile was constructed based on the Wald statistics of the contribution of the individual genes in univariate Cox regression analysis of TTP. To identify patients with good and poor outcome, the Wald corrected sum of the 10 genes was used to dichotomize the continuous 10-gene predictor (HR 12.87 [95% CI: 1.60–103.56], P=0.016). In multivariate analysis, corrected for the clinically relevant variables type of AI and CTC count, only the 10-gene CTC profile was an independent factor associated with TTP (HR 12.46 [95% CI: 1.29-120.08], P=0.029). Conclusions: A 10-gene CTC predictor was constructed which distinguishes good and poor outcome to first-line AI in MBC patients. This profile is currently being validated in an independent group of patients.

Three-arm randomized phase II study of cisplatin and etoposide (CE) versus CE with either vismodegib (V) or cixutumumab (Cx) for patients with extensive stage-small cell lung cancer (ES-SCLC) (ECOG 1508).

7508 Background: Targeted inhibition of the Hedgehog (HH) pathway by V & Insulin-like Growth Factor type-1 Receptor (IGF-1R) by Cx enhances efficacy of chemotherapy, and also demonstrates activity against the tumor cell fraction responsible for disease recurrence in SCLC. Methods: Patients (Pts) with newly diagnosed ES-SCLC with measurable disease, ECOG PS 0-1 were randomized to receive (four 21-day cycles) CE alone [(C 75 mg/m2 D1 & E 100 mg/m2 D1-3) Arm A] or in combination with either V [(150 mg/day PO) Arm B] or Cx [(6mg/kg weekly IV) Arm C]. Pts with responsive or stable disease on Arms B & C were continued on V or Cx respectively until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), stratified on gender. Circulating tumor cells (CTCs) were isolated/enumerated by Veridex Cell Search Platform at baseline, after 1or 2 cycles of chemotherapy, at completion of 4 cycles & 3 months (m). thereafter for correlation with efficacy parameters. The study was designed to detect a PFS HR of 0.58 with 90% power & an overall one-sided type I error rate of 0.10 for each of the comparisons of the V and Cx arms to CE alone. Results: 155 eligible pts were treated; 136 have died & 149 have experienced a PFS event. Pt. demographics & disease characteristics are well-balanced between the three arms except higher rate of PS 0 on Arm B (p=0.03). The median PFS on Arms A, B & C are 4.7, 4.4 & 4.6 m, the median OS 9.1, 9.8 & 10.1 m, & the RR are 43%, 52% & 49% respectively. None of the comparisons of these outcomes are statistically significant. PFS HR for V+CE vs. CE: 1.32, p=0.21, and for Cx+CE vs. CE: 1.12, p=0.58. The median OS among those with low CTC count (≤100 per 7.5ml) at baseline is 10.7 m vs. 7.2 m for those with high CTC count [HR1.70, p=0.01]. Toxicities in all three arms are as expected with the combination of CE alone. Conclusions: There is no significant improvement in PFS or OS with the addition of either V or Cx to CE vs.CE alone in pts with ED-SCLC. Low baseline CTC count is associated with improved OS, suggesting its role as a prognostic biomarker in SCLC. Clinical trial information: NCT00887159.

Epithelial Cell Adhesion Molecule-positive Circulating Tumor Cells as Predictive Biomarker in Patients With Prostate Cancer

To assess the use of circulating tumor cells (CTCs) as a longitudinal endpoint factor for clinical monitoring of patients with prostate cancer and to evaluate the association among the baseline CTC number, various clinical characteristics, and survival. The CTCs were enumerated using the CellSearch Food and Drug Administration-cleared CTC kit in 202 patients with prostate cancer. Variables, including metastatic site, prostate-specific antigen level, Gleason score, testosterone level, and use of androgen treatment, were tested for association with the CTC number. The probability of patient survival over time was estimated using the Kaplan-Meier method. The baseline CTC numbers were strongly associated with survival (P<.0001), with overall survival significantly poorer in patients with≥5 CTCs. Significantly greater CTC numbers were observed in patients with bone metastasis (mean 41.12 CTCs) than in those with lymph node metastasis (mean 2.53 CTC, P= .026). Analysis of the association between the CTC count and prostate-specific antigen level revealed a weak positive correlation (correlation coefficient r=0.2695, P= .0007). The CTC number also correlated with the Gleason score (P= .0138) and lower testosterone level (P<.0001). Patients without androgen depletion had significantly lower CTC numbers (mean 2.70) than those with androgen depletion (mean 26.39, P<.0001). The baseline CTC counts were predictive of patient survival and correlated significantly with the clinical characteristics of patients with prostate cancer. Our study results have confirmed previous findings that support the use of CTC enumeration as a prognostic biomarker for patients with prostate cancer.

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.

Circulating tumor cell (CTC) counts and CTC telomerase activity (TA) as prognotic markers of overall survival (OS) in SWOG S0421: Docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer (mCRPC).

1 Background: CTCs are promising biomarkers in mCRPC, and telomerase activity (TA) is a recognized cancer marker. In this phase III trial we analyzed CTCs using 2 methods: CellSearch for fixed cell enumeration, and a novel Parylene-C slot filter for live CTC capture and TA measurement. Methods: Blood samples (7.5 ml) were drawn at baseline (d1) and pre-cycle 2 (d21) of Rx and shipped overnight for central processing. For CellSearch enumeration, Cox regression was used to evaluate the association between OS, baseline CTC counts, and CTC dynamics (d1 to d21). For TA, filter-trapped cells were lysed and assayed for TA using qPCR-based telomeric repeat amplification. Cox regression evaluated the association between OS and TA overall and within subgroups characterized by good vs. poor (&lt;5 vs. &gt;=5) prognosis baseline CTC counts. For all measurements, receiver operator characteristic (ROC) analysis and characteristics and regression trees (CART) were used to explore further prognostic cutpoints. Results: Samples were obtained from 263 men. Median d1 CTC count was 5, and there was a significant difference in OS for d1 CTC &lt; vs. &gt;=5, hazard ratio (HR) 2.92 (p&lt;0.001) after adjustment for other factors. D1 CTC and OS had ROC AUC of 0.781. In men with low d1 CTC (&lt; 5), an increase in CTC was associated with shorter OS, HR 4.04 (p=0.004); in men with high d1 CTC (&gt;=5), a &gt;=2-fold decrease in CTC was associated with longer OS, HR 0.45 (p=0.012); adjusting for risk factors. For TA, men with baseline CTC &gt;=5 (41% of cohort) who had high CTC TA had HR 1.14 (p&lt;0.005) for OS after adjustment for other factors including CTC counts. CART identified additional risk subgroups based on CTC counts and TA. Conclusions: In this phase III trial, d1 CTC and d1 to d21 CTC dynamics were prognostic of OS after risk factor adjustment, comprising the largest docetaxel-based prospective cohort to date which validates a 5 CTC prognostic threshold and identifies new potentially useful enumeration subgroups. In men with CTCs &gt;=5, TA from cells live-captured on a new slot filter constitutes the first CTC-derived biomarker prognostic of OS in a prospective clinical trial.

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.

Results of telomerase activity measurements from live circulating tumor cells captured on a slot microfilter in a phase III SWOG-coordinated prostate cancer trial (S0421).

4663 Background: Analysis of circulating tumor cells (CTC) is a promising biomarker strategy in advanced prostate cancer, and telomerase activity (TA) is a recognized cancer marker. To test whether CTC TA is prognostic for survival (OS), we developed a novel Parylene-C slot microfilter capable of capturing live CTC and used it to measure CTC TA as part of a Phase III SWOG-coordinated therapeutic trial in metastatic castration resistant prostate cancer (S0421). Methods: Blood samples were drawn into EDTA tubes and shipped overnight to a central processing site. After Ficoll centrifugation, low constant pressure was used to pass the mononuclear cell layer through two slot microfilters in series as published previously (filter1 captures CTC + background white blood cells; filter2 captures only background white blood cells). Filter-trapped cells were lysed in CHAPS buffer and assayed for TA using qPCR-based telomeric repeat amplification. In parallel, CTC were enumerated using CellSearch (J&amp;J). Cox regression was used to evaluate the association between baseline (pre-treatment) TA and OS overall, and within subgroups characterized by good prognosis (&lt;5) vs. poor prognosis (&gt;=5) baseline CTC counts. CART regression was used to explore potential prognostic subgroups based on baseline PSA, CTC, and TA cutpoints. Results: Samples were obtained from 263 patients. While no association was observed between TA and OS overall, in patients with baseline CTC &gt;=5 (108 of 263 or 41% of patients), TAfilter2 – TAfilter1 representing high CTC TA relative to background blood cells was associated with a hazard ratio (HR) of 1.14 (95% CI 1.05-1.23, p&lt;0.001) for OS after adjusting for risk factors and remained significant when also adjusting for CTC: HR 1.14 (95% CI 1.04-1.23; p=0.005). Exploratory CART regression assessing baseline PSA, CTC, and TA identified risk groups based only on CTC and TA values. Conclusions: Baseline TA from CTC live-captured on a new slot microfilter is the first CTC biomarker shown to be prognostic of OS in men with CTC counts &gt;=5 in a prospective clinical trial. CTC TA may be useful for further identifying prognostic groups in this population.

Circulating tumor cells (CTCs) in patients (pts) with small cell lung cancer (SCLC) as a marker of disease burden and therapeutic response, and predictor of disease relapse.

7092 Background: Currently there are no validated biomarkers for assessment or prediction of disease burden or activity in SCLC. Enumeration of CTCs by CellSearch is FDA approved, highly reproducible and validated in other malignancies. Application as a prognostic and predictive marker in SCLC is limited due to a lack of studies documenting serial monitoring in pts on therapy. Methods: We are conducting a prospective study serially enumerating CTCs in pts with newly diagnosed SCLC. CTC number (per 7.5 ml peripheral blood) and percentage of CTCs demonstrating DNA damage and apoptosis based on γH2AX and M30 staining respectively, are being assessed prior to initiation of chemotherapy, during each cycle and at relapse. We are correlating CTC number with disease stage, number of metastatic sites, response to therapy, and time to progression (TTP). Results: 21 SCLC pts are evaluable. 9 pts with limited disease (LD) had median baseline CTC value of 1 (0-8); only 2 of 9 pts had &gt;5 CTCs. 12 pts with extensive disease (ED) had median baseline CTC value of 80.5 (0-37780); 8 of 12 pts had &gt;5 detectable CTCs (p value 0.02). Amongst the 12 pts with ED, median baseline CTC count was higher in pts with ≥3 (n=3) compared to 1-2 sites of metastatic disease (n=9) (2668 vs 71; p value 0.52). Median percentage of CTCs positive for γH2AX and M30 was also higher for pts with ≥3 compared to 1-2 metastatic sites (83, 164.5 vs. 2, 7.5) (p-value 0.40, 0.69). Serial CTC data are available on 3 pts with ED; all had responsive disease and reduction in CTC number to 0-1 after 2 cycles of chemotherapy. As this protocol is ongoing, correlation of CTCs with updated response status and TTP will be presented at the ASCO meeting. Conclusions: CTCs can be isolated and serially enumerated in pts with SCLC. Baseline CTCs correlate directly with disease stage. In pts with ≥ 3 metastatic sites, baseline CTCs tend to be greater and show higher levels of DNA damage and apoptosis compared to those with 1-2 metastatic sites. Reduction in CTCs is associated with radiographic response to therapy. Correlation between absolute number at baseline and TTP is not yet known.

Circulating tumor cell counts (CTC) as prognostic of overall survival (OS) in SWOG S0421-docetaxel with or without atrasentan for metastatic castration resistant prostate cancer (mCRPC).

10503 Background: CTC are promising biomarkers in mCRPC but have not been prospectively validated for docetaxel treatment (Rx). Using CellSearch technology (J&amp;J), we enumerated CTC in this Phase 3 trial &amp; assessed prognostic value for OS. The aim of this correlative study nested within 0421 was to compare CTC via CellSearch technology to newer microfilter technologies (Cote &amp; Goldkorn PIs RO1 CA141077). Comparative data analysis is ongoing. Methods: CTC were drawn at baseline (d1) &amp; pre-cycle 2 (d21) of Rx &amp; shipped overnight to a central site for enumeration (CTC/7.5 ml). Cox regression evaluated the association between OS and (i) baseline CTC counts &amp; (ii) CTC dynamics (d1 to d21) in pts with good (&lt;5) vs. poor (&gt;=5) baseline CTC counts. Receiver operator characteristic (ROC) analysis and Characteristics &amp; Regression Trees (CART) were used to explore further prognostic CTC cutpoints for 2-yr survival. Results: Of 263 patients (pts) consented, 238 were evaluable at d1 &amp; 232 at d21. At d1 median CTC was 5 (range 0-5916) &amp; d1 CTC &lt; vs. &gt;= 5 was associated with baseline PSA (mean 99 vs. 320 ng/ml, p=0.004) and worse bone pain (36% vs. 51%, p=0.03). There was a significant difference in OS for d1 CTC &lt; vs. &gt;=5, with a hazard ratio (HR) of 2.92 (95% CI 1.92-4.43, p&lt;0.001) after adjustment for PSA &amp; other factors. In pts with low d1 CTC (&lt; 5), an increase in CTC was associated with shorter OS, HR 4.04 (95%CI 1.56-10.44, p=0.004); in pts with high d1 CTC (&gt;=5), a &gt;=2-fold decrease in CTC was associated with longer OS, HR 0.45 (95%CI 0.24-0.84, p=0.012); adjusting for risk factors. D1 CTC and 2-year survival had ROC AUC of 0.781. CART analysis identified prognostic subgroups based on CTC of 0, 1-5, 6-53, and &gt;53: (HR 0.36, 0.77,1.3 and 2.8). Conclusions: In this phase 3 trial, d1 CTC was prognostic of OS after risk factor adjustment. CTC dynamics from d1 to d21 were also prognostic of OS. These data are an exploratory subset analysis of the overall study. Yet, they comprise the largest docetaxel-based prospective cohort to date, which validates a 5 CTC prognostic threshold for OS &amp; identifies new potential prognostic subgroups that may extend the clinical utility of CTC enumeration in mCRPC.

Relationship Between Lymphocytopenia and Circulating Tumor Cells as Prognostic Factors for Overall Survival in Metastatic Breast Cancer

IntroductionLymphocytopenia and circulating tumor cells (CTCs) have been reported as independent prognostic factors for overall survival (OS) in metastatic breast cancer (MBC), and both have been associated with bone metastases. Our objective was to compare the prognostic significance of lymphocytopenia, CTC count, and extensive bone metastases (> 2 lesions) assessed by fluorine-18 (18F) fluorodeoxyglucose positron emission tomography/computed tomography (FDG–PET/CT) in patients with MBC. Patients and MethodsThis is a retrospective study that included patients with MBC who were starting a new line of systemic therapy. The study population consisted of patients treated at the University of Texas MD Anderson Cancer Center between 2004 and 2008 for whom baseline CTC count, lymphocyte counts, and FDG–PET/CT scans were available. Patients were stratified according to estrogen receptor status (positive vs. negative), human epidermal growth factor receptor 2 (HER2) status (amplified vs. constitutive), baseline CTC counts per 7.5 mL of blood (< 5 CTCs/7.5 mL of blood vs. ≥ 5 CTCs/7.5 mL of blood), lymphocytopenia (< 1000 vs. ≥ 1000/μL), and extensive bone metastases (> 2 vs. ≤ 2 lesions). ResultsIn 195 assessable patients, the median OS was 27 months (range, 1 to > 45 months). In multivariate analysis, lymphocytopenia, ≥ 5 CTCs/7.5 mL of blood, estrogen receptor status, and line of therapy were the only predictive factors for progression-free survival (PFS) (2P = .001, 2P = .032, 2P = .029, and 2P = .002, respectively) and OS (2P = .001, 2P = .009, 2P = .004, and 2P = .024, respectively). ConclusionCTC measurement and lymphocytopenia are independent prognostic factors for PFS and OS in patients with MBC.

Evaluation of Circulating Tumor Cells and Circulating Tumor DNA in Non–Small Cell Lung Cancer: Association with Clinical Endpoints in a Phase II Clinical Trial of Pertuzumab and Erlotinib

Elevated levels or increases in circulating tumor cells (CTC) portend poor prognosis in patients with epithelial cancers. Less is known about CTCs as surrogate endpoints or their use for predictive biomarker evaluation. This study investigated the utility of CTC enumeration and characterization using the CellSearch platform, as well as mutation detection in circulating tumor DNA (ctDNA), in patients with advanced non-small cell lung cancer (NSCLC). Forty-one patients were enrolled in a single-arm phase II clinical trial of erlotinib and pertuzumab. Peripheral blood was analyzed for CTC enumeration, EGFR expression in CTCs, and detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC levels were correlated with 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging and survival endpoints. CTCs were detected (≥ 1 CTC) at baseline in 78% of patients. Greater sensitivity for mutation detection was observed in ctDNA than in CTCs and detected mutations were strongly concordant with mutation status in matched tumor. Higher baseline CTC counts were associated with response to treatment by Response Evaluation Criteria in Solid Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019) and longer progression-free survival (P = 0.050). These data provide evidence of a correlation between decreases in CTC counts and radiographic response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings require prospective validation but suggest a potential role for using CTC decreases as an early indication of response to therapy and ctDNA for real-time assessment of mutation status from blood.

Abstract C9: Treatment response to abiraterone acetate (AA) in patients with castration-resistant prostate cancer (CRPC) based on circulating tumor cells (CTCs), prostate-specific antigen (PSA), and imaging

Abstract Background: AA, a potent oral irreversible inhibitor of CYP17A1, has been recently approved for patients with CRPC progressing after docetaxel. CTC count ≥ 5/7.5 ml correlates with worse survival in CRPC. In this analysis we evaluated PSA responses, radiological responses and CTC conversion rates in relation to patient outcome. Methods: 141 patients (ECOG Performance Status 0-2; Median Age: 69.7; 85 post-docetaxel, 56 pre-docetaxel) treated with AA at the Royal Marsden NHS Foundation Trusts between December 2005 and March 2011 were included in this retrospective analyses. CTCs were enumerated using CellSearch at screening, baseline, at multiple time points during the study and at treatment discontinuation. PSA and imaging (Computer Tomography and Bone Scans) were obtained at the same time points as CTC collection. The Kaplan-Meier method was utilized for these analyses. Results: Baseline CTCs of ≥ 5 cells/7.5 ml were detectable in 17/53 (32.1%) pre-docetaxel patients (3 inevaluable) and 58/77 (75.3%) post-docetaxel patients (8 inevaluable at baseline). CTC conversion (≥ 5 to &amp;lt; 5 cells/7.5 ml) was observed in 10/17 (58.8%) pre-docetaxel patients and in 12/52 (23.1%) (6 inevaluable) post-docetaxel patients. PSA responses by PCWG II were observed in 16/22 (72.7%) of patients who attained a CTC conversion. PSA responses were seen in 8/47 (17.0%) patients in the absence of a corresponding CTC conversion. The median duration of radiological SD was 12.13 months (n=55) and 5.4 months (n=75) in patients with a baseline CTCs count of ≤ 5 cells/7.5mls and ≥ 5 cells/7.5 mls respectively. Overall, radiological stable disease was observed for a median of 6 months (95% CI: 4.2, 8.4; range 0.3, 35.6) beyond PSA progression in the 105 patients who experienced PSA progression. Radiological and clinical disease stability during treatment of ≥ 1 year, ≥ 2 years and ≥ 3 years was observed in 43/141 (30.5%), 21/141 (14.9%) and 12/141 (8.5%) respectively. Conclusion: Radiological and clinical disease stabilization beyond PSA progression is seen in a significant proportion of patients treated with AA. CTCs conversion correlates with PSA response and radiological disease stability. Citation Format: Diletta Bianchini, Alison Reid, Gerhardt Attard, Johann De Bono, Shahneen Sandhu, Amy Cassidy Mulick, Deborah Mukherji, Carmel Pezaro, Andrea Zivi, Aurelius Omlin, Ajit Sarvadikar, Emilda Thompson. Treatment response to abiraterone acetate (AA) in patients with castration-resistant prostate cancer (CRPC) based on circulating tumor cells (CTCs), prostate-specific antigen (PSA), and imaging [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C9.

Abstract PR-3: Evaluation of circulating tumor cells and circulating tumor DNA in non-small cell lung cancer: Association with clinical endpoints in a phase II clinical trial of pertuzumab and erlotinib.

Abstract Purpose: Circulating tumor cells (CTCs) can be detected in the bloodstream of cancer patients in diverse epithelial cancers and elevated levels or increases in CTC counts portend poor prognosis. Less is known about the potential application of CTCs as surrogate endpoints, or as a source of cells for predictive biomarker evaluation in patients with advanced cancer enrolled in early phase clinical trials. This study investigated the feasibility of CTC enumeration, phenotypic characterization, as well as oncogenic mutation detection from CTCs and circulating tumor DNA (ctDNA) in patients with advanced lung cancer enrolled in a global multi-center, phase II clinical trial. Patients and Methods: Forty-one patients with advanced NSCLC who had received at least one prior chemotherapy regimen were enrolled in a phase 2, single-arm clinical study of the combination therapy with erlotinib and pertuzumab. Erlotinib is a small molecule inhibitor of epidermal growth factor receptor (EGFR), and pertuzumab is a monoclonal antibody that blocks HER2 dimerization with other HER family receptors. Peripheral blood was collected prior to the start of therapy and during the course of the study. Analyses in CTCs included enumeration, evaluation of EGFR expression and detection of oncogenic mutations in both CTCs and ctDNA and correlations were made to tumor response as measured by tumor FDG-PET changes or by computed tomography (CT) imaging. Results: CTC were detected (≥1 CTC) at baseline in 78% of patients (n=30). EGFR expression was evaluated in CTCs by immunofluorescence and revealed a range of expression levels and substantial heterogeneity for this biomarker in CTCs. Greater sensitivity for mutation detection was observed in ctDNA when compared to CTCs across a 6-gene panel (EGFR, KRAS, BRAF, NRAS, AKT1 and PIK3CA). Importantly, mutation detection in ctDNA was highly concordant with mutation status in patient tumor. Higher baseline CTC counts were observed in patients with EGFR mutations or with other oncogenic activating mutations, suggesting that CTC counts may be associated with underlying tumor genotype. A trend toward lower CTC counts was observed across on-treatment time-points, with statistically significant decreases in CTC counts (p=0.02) observed in either FDG-PET or RECIST responders. In comparison, no significant decreases were observed in non-responders. Conclusion: We demonstrate feasibility of enumeration and phenotypic characterization in CTCs and mutation detection in ctDNA from NSCLC patients in a global, multi-center Phase II study. In addition, our data provides the first evidence of a possible correlation between decreases in CTC counts and response by either FDG-PET or RECIST in patients with advanced NSCLC. These findings will require prospective validation but suggest a potential future role for using CTC decreases as an early indication of response to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-3.

Abiraterone plus prednisone improves survival in metastatic castration-resistant prostate cancer.

Abiraterone plus prednisone improves survival in metastatic castration-resistant prostate cancer.

Baseline Circulating Tumor Cell Counts Significantly Enhance a Prognostic Score for Patients Participating in Phase I Oncology Trials

High circulating tumor cell (CTC) counts are associated with poor prognosis in several cancers. Enrollment of patients on phase I oncology trials requires a careful assessment of the potential risks and benefits. Many patients enrolled on such trials using established eligibility criteria have a short life expectancy and are less likely to benefit from trial participation. We hypothesized that the incorporation of CTC counts might improve patient selection for phase I trials. This retrospective analysis evaluated patients who had baseline CTCs enumerated prior to their starting on a phase I trial. CTCs were enumerated using the CellSearch System. Between January 2006 and December 2009 a total of 128 patients enrolled in phase I trials had CTC counts evaluated. Higher CTC counts as a continuous variable independently correlated with risk of death in this patient population (P = 0.006). A multivariate point-based risk model was generated using CTCs as a dichotomous variable (≥3 or <3), and incorporated other established prognostic factors, including albumin <35 g/L, lactate dehydrogenase greater than upper limit of normal, and >2 metastatic sites. Comparison of receiver operating characteristic curves demonstrated that the addition of baseline CTC counts improved the performance of the prospectively validated Royal Marsden Hospital phase I prognostic score, which now identifies three risk groups (P < 0.0001): good prognosis [score 0-1, median overall survival (OS) 63.7 weeks], intermediate prognosis (score 2-3, median OS 37.3 weeks), and poor prognosis (score 4, median OS 13.4 weeks). CTC enumeration improved the performance of a validated prognostic score to help select patients for phase I oncology trials.

TMPRSS2-ERG Status in Circulating Tumor Cells as a Predictive Biomarker of Sensitivity in Castration-Resistant Prostate Cancer Patients Treated With Abiraterone Acetate

BackgroundAbiraterone acetate (AA) is an androgen biosynthesis inhibitor shown to prolong life in patients with castration-resistant prostate cancer (CRPC) already treated with chemotherapy. AA treatment results in dramatic declines in prostate-specific antigen (PSA) in some patients and no declines in others, suggesting the presence of molecular determinants of sensitivity in tumors. ObjectiveTo study the role of transmembrane protease, serine 2 (TMPRSS2)–v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion, an androgen-dependent growth factor, in circulating tumor cells (CTCs) as a biomarker of sensitivity to AA. Design, setting, and participantsThe predictive value of TMPRSS2-ERG status was studied in 41 of 48 men with postchemotherapy-treated CRPC enrolled in sequential phase 2 AA trials. InterventionPatients received AA 1000mg daily and continuously. MeasurementsTMPRSS2-ERG status was characterized by a sensitive, analytically valid reverse transcription polymerase chain reaction assay in CTCs enriched from ethylene-diaminetetraacetic acid anticoagulated blood obtained prior to AA treatment. Outcomes were measured by PSA Working Group 1 criteria. Results and limitationsStandard procedures for specimen acquisition, processing, and testing using the validated TMPRSS2-ERG assay on a multiplex platform gave intra-assay and interassay coefficients of variation <7%. TMPRSS2-ERG fusion was present in 15 of 41 patients (37%), who had a median baseline CTC count of 17 (interquartile range: 7–103 cells per 7.5ml). A PSA decline ≥50% was observed in 7 of 15 patients (47%) with the fusion and in 10 of 26 patients (38%) without the fusion. Although limited by the low number of patients, a posttherapy CTC count of less than five per 7.5ml was prognostic for longer survival relative to a CTC count five or more. TMPRSS2-ERG status did not predict a decline in PSA or other clinical outcomes. ConclusionsMolecular profiles of CTCs with an analytically valid assay identified the presence of the prostate cancer–specific TMPRSS2-ERG fusion but did not predict for response to AA treatment. This finding demonstrates the role of CTCs as surrogate tissue that can be obtained in a routine practice setting. Trial registrationClinicalTrials.gov: NCT00474383 (COU-AA-003), NCT00485303 (COU-AA-004).

Circulating tumor cells as prognostic and predictive markers in metastatic breast cancer patients receiving first-line systemic treatment

IntroductionCirculating tumor cells (CTCs) represent an independent predictor of outcome in patients with metastatic breast cancer (MBC). We assessed the prognostic impact of CTCs according to different first-line systemic treatments, and explored their potential predictive value in MBC patients.MethodsWe retrospectively evaluated 235 newly diagnosed MBC patients, treated at the University of Texas MD Anderson Cancer Center. All patients had a baseline CTC assessment performed with CellSearch®. Progression-free survival and overall survival were compared with the log-rank test between groups, according to CTC count (< 5 vs. ≥ 5) and type of systemic therapy. We further explored the predictive value of baseline CTCs in patients receiving different treatments.ResultsAt a median follow-up of 18 months, the CTC count was confirmed to be a robust prognostic marker in the overall population (median progression-free survival 12.0 and 7.0 months for patients with CTC < 5 and ≥ 5, respectively; P < 0.001). Conversely, in patients with human epidermal growth factor receptor-2-overexpressed/amplified tumors receiving trastuzumab or lapatinib, the baseline CTC count was not prognostic (median progression-free survival 14.5 months for patients with CTC < 5 and 16.1 months for those with CTC ≥ 5; P = 0.947). Furthermore, in patients with human epidermal growth factor receptor-2 normal tumors, a baseline CTC count ≥ 5 identified subjects who derived benefit from more aggressive treatments, including combination chemotherapy and chemotherapy plus bevacizumab.ConclusionsThis analysis suggests that the prognostic information provided by CTC count may be useful in patient stratifications and therapeutic selection, particularly in the group with positive CTCs, in which various therapeutic choices may procure differential palliative benefit.

Abstract P3-02-03: Prognostic Value of Circulating Tumor Cells (CTC) in HER-2 Positive Metastatic Breast Cancer (MBC) Patients (pts) Treated with Anti-HER2-Based Therapy

Abstract Background. Detection of ≥5 CTC per 7.5 mL of blood before starting systemic treatment is a strong and independent predictor of worse progression-free survival (PFS) and overall survival (OS) in MBC. Trastuzumab (Herceptin®) was shown to be highly effective in reducing CTC number in all HER-2 amplified MBC cases after the first dose (De Giorgi et al., SABCS 2009). Furthermore, trastuzumab can target chemotherapy-resistant CTC in pts with breast cancer (Bozionellou et al. 2004). To date no significant analysis exploring the effect of another anti-HER2 therapy, lapatinib (Tykerb®) on CTC has been published.We hypothesized that anti-HER2-based therapy may modify the detection rate and prognostic value of CTC in pts with HER-2 amplified MBC. Patients and Methods. We retrospectively evaluated 91 HER-2 positive MBC pts, treated at the M. D. Anderson Cancer Center with trastuzumab-based (n=59), or lapatinib-based therapy (n=32). In all pts, CTC were enumerated before starting systemic treatments, using CellSearch™. Follow-up CTC counts were available for 41 (69%) pts treated with trastuzumab and 25 (78%) pts treated with lapatinib. PFS and OS were estimated by Kaplan-Meier product limit, and compared between groups according to baseline CTC count (&amp;lt;5 vs ≥5) by log-rank test. Results. Median follow-up was 27 months (range, 8-65) and 16 months (range, 3-33) for pts receiving trastuzumab and lapatinib, respectively. Treatments administered and CTC counts are reported in table 1. Two (5%) pts receiving trastuzumab and 4 (16%) pts treated with lapatinib had CTC ≥5 at the follow-up assessment. Estimated median PFS and OS according to baseline CTC value, for both treatments are shown in table 2. An elevated CTC count was associated with poor prognosis in pts treated with lapatinib (median OS: 25.8 vs 13.6 months in pts with CTC &amp;lt;5 and ≥5, respectively, P=.072). Surprisingly, in the trastuzumab group, PFS showed a non-statistically significant trend in favor of pts with CTC ≥5 compared to those with &amp;lt;5. Conclusions. According to previously published data, our study confirms that trastuzumab-based therapies can drastically reduce the number of CTC and neutralize their negative prognostic value. Furthermore, we found that lapatinib had a limited effect on the detection rate and prognostic value of CTC, although the low number of pts and the high percentage of pre-treated women do not allow us to draw definitive conclusions. Table 1 Table 2 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-03.