Efficiency of Ber-EP4 antibody for isolating circulating epithelial tumor cells before RT-PCR detection.
Author(s): Sabile A, Louha M, Bonte E, Poussin K, Vona G, Mejean A, Chretien Y, Bougas L, Lacour B, Capron F, Roseto A, Bréchot C, Paterlini-Bréchot P
Publication: Am J Clin Pathol, 1999, Vol. 112, Page 171-8
PubMed ID: 10439796 PubMed Review Paper? No
Purpose of Paper
This paper compared the sensitivity of four different methods for isolating circulating tumor cells (CTCs) from peripheral blood: immunocapture with Ber-EP4 magnetic beads, density gradient separation, ammonium chloride-mediated erythrocyte lysis, and distilled water-mediated erythrocyte lysis. Isolated CTCs were analyzed for cancer-specific transcripts by RT-PCR. The consistency of Ber-EP4 expression was assessed in tumor and normal adjacent liver and prostate tissues that were frozen and formalin-fixed paraffin-embedded (FFPE).
Conclusion of Paper
Immunocapture with Ber-EP4-coated magnetic beads and density gradient separation were more sensitive in isolation of CTCs spiked in peripheral blood specimens from healthy volunteers than ammonium chloride-mediated or distilled water-mediated erythrocyte lysed specimens. Density gradient separation was significantly more sensitive in isolation of CTCs from carcinoma patients than Ber-EP4 immunocapture. When frozen and FFPE tissue sections were assessed for Ber-EP4 staining by immunohistochemistry (IHC), 59% of prostate adenocarcinoma specimens displayed positive staining while only 18% (5/27) of hepatocellular carcinoma specimen specimens displayed positive staining. Importantly, nontumorous prostatic tissues were negative for Ber-EP4 expression but positive immunostaining was observed in some cirrhotic nodules and all biliary cells and ducts in nontumorous liver tissues.
Studies
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Study Purpose
This study compared the sensitivity of four different methods for isolating CTCs from peripheral blood: immunocapture with Ber-EP4 magnetic beads, density gradient separation, ammonium chloride-mediated erythrocyte lysis, and distilled water-mediated erythrocyte lysis. Peripheral blood was collected in EDTA tubes from 20 healthy volunteers (50 mL per volunteer) and patients (15 mL per patient) diagnosed with a hepatocellular carcinoma (19 patients); a prostate adenocarcinoma (10 patients); chronic hepatitis, liver cirrhosis, or both (13 patients); and prostatic adenoma (7 patients). Blood specimens from healthy volunteers were spiked with serial dilutions (1, 5, 10, 50, and 100/108 white blood cells) of cells from prostate (LNCaP) or liver (HepG2) cancer cell lines. CTCs isolated using one of the four different methods were then subjected to RNA extraction with Trizol and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of α-fetoprotein (AFP) or prostate-specific antigen (PSA). To evaluate the suitability of Ber-EP4 (an epithelial cell surface antigen) as a target for immunocapture of CTCs, Ber-EP4 expression was assessed by immunohistochemistry (IHC) in tumor and normal adjacent tissue sections from 27 case-matched frozen and FFPE liver specimens and 34 FFPE prostate specimens.
Summary of Findings:
Immunocapture with Ber-EP4-coated magnetic beads and density gradient separation were more sensitive in isolation of HepG2 and LNCaP cells spiked in peripheral blood from healthy volunteers (1 cell/107 white blood cells) than both ammonium chloride-mediated erythrocyte lysis and distilled water-mediated erythrocyte lysis (1 cell/106 white cells) (P<.05). Density gradient separation was significantly more sensitive than Ber-EP4 immunocapture for CTC isolation from blood collected from both prostate adenocarcinoma patients (4/10 versus 2/10 cases) and hepatocellular carcinoma patients (4/19 versus 0/19 cases) (8/29 versus 2/29 cases overall, P=.037). No CTCs were isolated by either method from the seven prostatic adenoma or thirteen hepatitis and/or cirrhosis control patients that served as negative controls. IHC analysis of Ber-EP4 expression revealed positive immunostaining in 90% of LNCaP cells and 75% of HepG2 cells. In clinical tissue specimens, Ber-EP4 immunohistochemical staining patterns were comparable among frozen and paraffin-embedded liver specimens. While 59% (20/34) of prostate adenocarcinoma specimens (range of 5-70% labeled cells) stained positive for Ber-EP4 expression, only 18% (5/27) of hepatocellular carcinoma specimens (range of 1-50% labeled cells) were positive for Ber-EP4 expression. Ber-EP4 immunohistochemical staining was exclusive to differentiated tumor cells and localized to the apical surface of the cell membrane. All normal adjacent prostate specimens stained negative for Ber-EP4 expression but positive Ber-EP4 immunostaining was observed in several nontumorous liver tissues, specifically some cirrhotic nodules and all biliary cells and ducts.
Biospecimens
Preservative Types
- Formalin
- Frozen
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Cell count/volume Fluorescent microscopy RNA RT-PCR Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Cell capture method Ber-EP4- coated magnetic beads
Density gradient separation
Ammonium chloride-mediated erythrocyte lysis
Distilled water-mediated erythrocyte lysis
RT-PCR Specific Targeted nucleic acid PSA
α-fetoprotein
Immunohistochemistry Specific Targeted peptide/protein Ber-Ep4
Biospecimen Aliquots and Components Cell number 1 cancer cell
5 cancer cells
10 cancer cells
50 cancer cells
100 cancer cells