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Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer.

Author(s): Van der Auwera I, Peeters D, Benoy IH, Elst HJ, Van Laere SJ, Prové A, Maes H, Huget P, van Dam P, Vermeulen PB, Dirix LY

Publication: Br J Cancer, 2010, Vol. 102, Page 276-84

PubMed ID: 19953098 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared CTC detection rates of three different methods (CellSearch, AdnaTest, and real-time qRT–PCR) using peripheral blood of patients diagnosed with metastatic breast cancer and healthy volunteers. Blood was collected volunteers in CellSave tubes (10 mL), AdnaCollect blood collection tubes (2 x 5 mL), and EDTA tubes (9 mL) from 16 untreated cancer patients, 60 treated patients who received different cytostatic treatments mostly containing taxanes, vinorelbine, anthracyclines or capecitabine (N=40), endocrine therapy (N=17) or trastuzumab alone or in combination with other treatments (N=18) and 20 healthy. CellSave specimens were stored at room temperature and processed using the CellSearch Sytem within 72 h of collection. Blood collected into AdnaCollect tubes were immediately placed on ice, incubated with BreastSelect Beads, mRNA was recovered by magnetic separation using Olido(dT)-coated beads, and three breast cancer markers (Muc-1, HER2, and GA733-2) and a housekeeping gene (actin) were amplified by multiplex RT-PCR and visualized on a bioanalyzer. A specimen was classified as positive if a PCR fragment had a peak concentration ≥ of 0.30 ng/µl, while peaks with a concentration between 0.15–0.30 ng/ µl were classified as inconclusive, and a peak concentration <0.15 ng/ µl was classified as negative. EDTA blood was filtered for white blood cells with a LeukoLOCK filter, RNA was extracted using the LeukoLOCK system, analyzed by real-time quantitative multiplex RT-PCR for CK-19 and mammaglobin expression levels which were normalized to beta-actin (ACTB) and TATA-binding protein (TBP).

   Summary of Findings:

   Moderate concordance was observed for blood specimens analyzed by CellSearch CTC and the AdnaTest (81%, P<0.001) and between AdnaTest results and specimens determined to be positive by CK-19 levels as detected by real-time RT-PCR (78%, P<0.0001). Fair agreement was observed between CellSearch and positive results determined by CK-19 (72%, P=0.002), by mammaglobin (60%, P=0.04), and by CK-19 and/or mammaglobin (57%, P=0.04). Slight correlation was observed between the AdnaTest and mammaglobin results (53%, P=0.37) and CK-19 and/or mammaglobin levels (50%, P=0.189). Patients were more likely to be positive (for either CK-19 and/or mammaglobin) by real-time RT–PCR than by CellSearch (63 vs 36%, P=0.001) or the AdnaTest (61% vs 22%, P<0.001) and a significant difference in positive results was observed between CellSearch and the AdnaTest (36 vs 22%, P=0.013). Eight patients (10%) were positive for all three methods.

   With the CellSearch System, the median number of detectable CTCs in patients with progressive disease was significantly higher than in non-progressive patients (1 versus 0, P=0.004) and the number of detectable CTCs was positively correlated to both serum CA15.3 levels (P<0.001) and patient age (P=0.001); however, the presence of two or more CTCs was not correlated with ER, PR, HER2, or P53 expression within the primary tumor. With the AdnaTest, a CTC positive result (two positive blood samples) was positively correlated to high CA15.3 levels (P=0.001) and ER status of the primary tumor (P=0.02), but CTC results were not correlated to PR, HER2, or P53 status (P=0.53, P=0.17, and P=0.39, respectively), or tumor progression (P=0.22), nor was tumor progression associated with the number of CTC positive blood specimens (P=0.35). Real-time RT-PCR amplification of CK-19 and mammaglobin did not differ significantly between treated and untreated patient groups (P=0.87 and 0.91, respectively). Median levels of CK-19 and mammaglobin were significantly correlated (P=0.005) and both were correlated with CA15.3 levels (r=0.481, P<0.001 and r=0.386, P=0.001) but not with patient age. Of the 76 patient specimens, 20 (26%) patients were positive for CK-19, 41 (54%) for mammaglobin, 14 (18%) were positive for both, and 29 (38%) were negative for both. Forty-three percent of all patients (33/76) were positive for only one tumor marker (69% of untreated patients and 60% of treated patients, P=0.52). A positive real-time RT-PCR result for CK-19, mammaglobin, or either was not associated with tumor progression (P=0.19, 0.84, and 0.41, respectively), ER status (P=0.63, 0.47, and 0.55, respectively), PR status (P=0.57, 0.49, and 0.89, respectively), HER2 status (P=0.14, 0.74, and 0.40, respectively), or P53 status (P=0.11, 0.86, and 0.58, respectively).

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy
   RNA	Real-time qRT-PCR
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient age	Healthy volunteers: median age 39 years (range 25–54) Breast cancer patients: median age 62 years (range, 34–85) years
   Preaquisition	Diagnosis/ patient condition	Tumor progression ER status PR status HER2 status p53 status CA15.3 serum levels
   Real-time qRT-PCR Specific	Targeted nucleic acid	CK-19 Mammaglobin Beta-actin (ACTB) TATA-binding protein (TBP)
   RT-PCR Specific	Targeted nucleic acid	actin Muc-1 HER2 GA733-2
   Biospecimen Aliquots and Components	Cell capture method	CellSearch Adna Breast Cancer Select/Detect

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