Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction.

1994 ◽  
Vol 12 (3) ◽  
pp. 475-482 ◽  
Author(s):  
Y H Datta ◽  
P T Adams ◽  
W R Drobyski ◽  
S P Ethier ◽  
V H Terry ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Mammary Carcinoma ◽  
Peripheral Blood ◽  
Carcinoma Cells ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mammary Carcinoma Cells ◽  
Polymerase Chain

PURPOSE Detection of occult carcinoma in patients with breast cancer may aid the establishment of prognosis and development of new therapeutic approaches. To improve on existing methods of detection, we have developed a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for keratin 19 (K19) transcripts to identify mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. PATIENTS AND METHODS Peripheral-blood or bone marrow samples obtained from 34 patients with stages I to IV breast cancer and 39 control subjects without breast cancer were screened for K19 mRNA by nested primer PCR. RESULTS In reconstitution experiments, K19 RT-PCR reliably detected 10 mammary carcinoma cells in 1 million normal peripheral-blood mononuclear (PBMN) cells. Four of 19 patients with stage IV breast cancer had detectable K19 transcript in peripheral blood. Five of six patients with histologically negative bone marrow biopsies following preablative chemotherapy and before autologous bone marrow transplant (BMT) were positive by this assay. Stem-cell apheresis harvests obtained from one of these patients and three additional patients immediately before BMT were all K19-negative. K19 RT-PCR analysis of CSF from a breast cancer patient with known carcinomatous meningitis was also positive. Thirty-eight of 39 non-breast cancer patients had negative K19 RT-PCR assays. The one exception was a patient with chronic myelogenous leukemia. CONCLUSION RT-PCR of K19 is a sensitive, specific, and rapid method for detection of occult mammary carcinoma cells in the peripheral blood and bone marrow of patients with breast cancer. The presence of residual breast cancer cells in histologically normal bone marrow aspirates but not in stem-cell apheresis harvests is a frequent finding. This assay may be useful in diagnosing metastatic disease, as well as in monitoring the effectiveness of systemic therapy.

Detection of Circulating Mammary Carcinoma Cells in the Peripheral Blood of Breast Cancer Patients Via a Nested Reverse Transcriptase Polymerase Chain Reaction Assay for Mammaglobin mRNA

1999 ◽  
Vol 17 (7) ◽  
pp. 2015-2015 ◽  
Author(s):  
Otto Zach ◽  
Hedwig Kasparu ◽  
Otto Krieger ◽  
Wolfgang Hehenwarter ◽  
Michael Girschikofsky ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Polymerase Chain Reaction ◽  
Estrogen Receptor ◽  
Tumor Cell ◽  
Peripheral Blood ◽  
Estrogen Receptor Status ◽  
Carcinoma Cells ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

PURPOSE: According to current medical research, mammaglobin (hMAM) is expressed exclusively in the mammary glands of adult women and in mammary tumor cell lines. Therefore, we examined hMAM expression as a marker for the detection of carcinoma cells in the peripheral blood of patients with breast cancer (BC). PATIENTS AND METHODS: Blood samples obtained from 114 BC patients at the various stages of their disease and from 68 individuals without BC were screened for hMAM mRNA by a nested reverse transcriptase polymerase chain reaction (RT-PCR) assay. RESULTS: The assay exhibited a calculated analytical limit of one tumor cell per 106 to 107 WBCs. None of the samples from peripheral blood of 27 healthy individuals were positive, whereas 29 (25%) of 114 samples from BC patients were positive for hMAM mRNA. hMAM mRNA expression was detected in five (28%) of 18 BC patients at diagnosis, in three (6%) of 53 with no evidence of disease, and in 21 (49%) of 43 with metastatic disease. These results correlate with patients' carcinoembryonic antigen (CEA) plasma level and, to some extent, with estrogen receptor status. Two of 41 samples from patients with malignancies other than BC were also positive. CONCLUSION: In contrast to healthy volunteers, hMAM transcripts were detected in the peripheral blood of BC patients. The percentage of positivity relates to the clinical stages of disease, CEA plasma level, and estrogen receptor status. Aberrant hMAM expression might occur occasionally in malignancies other than BC. The clinical relevance of hMAM RT-PCR–based tumor cell detection in the peripheral blood of BC patients should be further evaluated in prospective studies.

Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.

1997 ◽  
Vol 15 (7) ◽  
pp. 2701-2708 ◽  
Author(s):  
A Zippelius ◽  
P Kufer ◽  
G Honold ◽  
M W Köllermann ◽  
R Oberneder ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Tumor Cells ◽  
Carcinoma Cells ◽  
Cell Detection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

PURPOSE This study was designed to evaluate the potential of reverse-transcriptase polymerase chain reaction (RT-PCR) analyses for the detection of micrometastatic carcinoma cells in bone marrow (BM). PATIENTS AND METHODS The specificity of RT-PCR assays with primers specific for various tumor-associated and organ-specific mRNA species was examined by analysis of 53 BM aspirates from control patients with no epithelial malignancy. In addition, BM samples from 63 patients with prostate cancer (n = 53) or breast cancer (n = 10) were analyzed by RT-PCR with primers specific for prostate-specific antigen (PSA) mRNA. As a reference method, all samples were analyzed simultaneously by an established immunocytochemical assay, using monoclonal antibodies (mAbs) against cytokeratins (CK) for tumor-cell detection. RESULTS Seven of eight marker species could be detected in a considerable number of BM samples from control patients: epithelial glycoprotein-40 (EGP-40; 53 of 53 samples), desmoplakin I (DPI I; five of five), carcinoembryonic antigen (CEA; five of 19), erb-B2 (five of seven), erb-B3 (six of seven), prostate-specific membrane antigen (PSM; four of nine), and CK18 (five of seven). Only PSA mRNA was not detected in any of the 53 control BM samples. In serial dilution experiments, the PSA RT-PCR assay was able to detect five LNCaP prostate carcinoma cells in 4 x 10(6) BM cells. CK-positive cells were found in 20 patients (37.7%) with prostate cancer, while PSA mRNA was found in only 15 (28.3%; P = .04). Moreover, despite the recent observation that PSA is also expressed in mammary carcinomas, none of the 10 CK-positive BM samples were PSA mRNA-positive. CONCLUSION Limiting factors in the detection of micrometastatic tumor cells by RT-PCR are (1) the illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoietic cells, and (2) the deficient expression of the marker gene in micrometastatic tumor cells.

Quantitative Polymerase Chain Reaction for the Detection of Micrometastases in Patients With Breast Cancer

1999 ◽  
Vol 17 (3) ◽  
pp. 870-870 ◽  
Author(s):  
Martin J. Slade ◽  
Brendan M. Smith ◽  
H. Dudley Sinnett ◽  
Nicholas C.P. Cross ◽  
R. Charles Coombes
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Metastatic Breast Cancer ◽  
Peripheral Blood ◽  
Primary Breast Cancer ◽  
Metastatic Breast ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain

PURPOSE: Previous reports have indicated that reverse transcriptase polymerase chain reaction (RT-PCR) for cytokeratin 19 (CK-19) may be useful in the management of patients with breast cancer. However, the specificity of this technique is low, principally because of a high rate of false-positive results. To improve the specificity of this assay, we developed a quantitative RT-PCR methodology that enables an estimate to be made of the number of CK-19 transcripts in blood and bone marrow samples. PATIENTS AND METHODS: We examined 45 peripheral-blood samples and 30 bone marrow samples from patients with a variety of nonneoplastic conditions using nested RT-PCR for CK-19. We also examined bone marrow and peripheral-blood samples from 23 patients with primary breast cancer and peripheral-blood samples from 37 patients with metastatic breast cancer. The number of CK-19 transcripts was estimated in positive specimens by competitive PCR and normalized to the number of ABL transcripts as an internal control for the quality and quantity of cDNA. RT-PCR results were compared with the numbers of CK-19–positive cells detected by immunocytochemistry. RESULTS: Analysis of samples from patients without cancer enabled us to define an upper limit for the background ratio of CK-19 to ABL transcripts (1:1,000 for blood samples and 1:1,600 for bone marrow samples). Using these figures as cut-off points, elevated CK-19: ABL ratios were detected in peripheral-blood samples of 20 of 37 (54%) patients with metastatic breast cancer and in bone marrow samples of 14 of 23 (61%) patients with primary breast cancer. Only three of 23 (13%) primary breast cancer peripheral-blood samples and none of the control samples were positive by these criteria. Only two of 23 patients (9%) with primary breast cancer showed immunocytochemically detectable cells in the blood; 10 of 23 (43%) showed immunocytochemically detectable cells in the bone marrow. Of 36 patients with metastatic breast cancer, eight (22%) showed positive events. CONCLUSION: Quantitative RT-PCR for CK-19 detects a percentage of patients with breast cancer and may enable the progression or regression of the disease to be monitored.

Sign in / Sign up

Export Citation Format

Share Document