Comparison of molecular cytokeratin 19 reverse transcriptase polymerase chain reaction (CK19 RT-PCR) and immunocytochemical detection of micrometastatic breast cancer cells in hematopoietic harvests.
Detection of small numbers of breast cancer cells in patient blood, aphereses, and bone marrow has become increasingly important as data have accumulated showing immunocytochemically (ICC) positive tumor cells in up to 50% of women with stage I and II breast cancer, who were initially thought to be cured of their disease but later relapsed. The ability to rule out the presence of micrometastatic disease at any stage of the clinical management protocol, whether before, during, or after therapy, would provide a useful monitoring and diagnostic tool for both the clinician and the scientist. Monitoring for the presence of minimal residual disease (MRD) is traditionally performed using ICC. A more recently established RT-PCR technique uses a molecular marker (the presence of the cytokeratin 19, CK19, transcript) to identify MRD in patient samples, with a level of sensitivity reported to be one tumor cell in 10(6) nucleated cells. This level of sensitivity is generally higher than that claimed for ICC. Based on the discriminating results of this first study, a number of laboratories have evaluated this technique for its diagnostic potential. Results from several laboratories showed a higher than expected false positive rate due to a variety of identified and unidentified sources. Therefore, the current study was designed to achieve two aims: to establish the level of sensitivity and specificity of the RT-PCR technique and to dissect out the possible variables that may contribute to a false positive result using this molecular approach. To accomplish the first goal, two simulation strategies were used, limited dilution of tumor cells into apheresis harvests and semi-quantitative PCR using stepwise dilutions of extracted RNA from tumor cells in apheresis harvests. The second goal was accomplished by performing sequential blood drawings with variably timed sample processing to identify some of the more common variables (time, anticoagulant, sample sequence) that may contribute to false positive results. Of three variables investigated, including type of blood preservative, sequence of blood tube collection, and time point of sample processing, each may contribute to a false positive result. In addition to these problems, known events involving illegitimate transcription of specific genes nonspecifically in tissue is also a potential source of false positive results. These issues may be further compounded by lack of attention to the more common methodologic problems encountered in any laboratory using the PCR technique. However, recommendations can be developed for the effective application of this technique, whose greatest strength is the demonstration of tumor negativity of the sample.
Traystman
M D
MD
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-5660, USA.
Cochran
G T
GT
Hake
S J
SJ
Kuszynski
C A
CA
Mann
S L
SL
Murphy
B J
BJ
Pirruccello
S J
SJ
Zuvanich
E
E
Sharp
J G
JG
D000925:Anticoagulants / Q000097:blood; D001800:Blood Specimen Collection; D001854:Bone Marrow Cells / Q000737:chemistry; D001943:Breast Neoplasms / Q000473:pathology*; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis* / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D009360:Neoplastic Cells, Circulating / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000737:chemistry
D000925:Anticoagulants; D007633:Keratins; D012194:RNA-Directed DNA Polymerase