Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates.
BACKGROUND
The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA).


METHODS
Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR.


RESULTS
FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells.


CONCLUSIONS
The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.
Maas
R A
RA
Department of Pathology, Nederlands Kanker Instituut/Antoni van Leeuwenhoek Huis, Amsterdam.
Bruning
P F
PF
Breedijk
A J
AJ
Top
B
B
Peterse
H L
HL
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001483:Base Sequence; D001707:Biopsy, Needle; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002469:Cell Separation / Q000379:methods; D003110:Colonic Neoplasms / Q000473:pathology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase