Annals of Oncology Advance Access originally published online on December 15, 2005
Annals of Oncology 2006 17(3):424-428; doi:10.1093/annonc/mdj109
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© 2005 European Society for Medical Oncology
RT-PCR determination of maspin and mammaglobin B in peripheral blood of healthy donors and breast cancer patients
1 Istituto Oncologico Romagnolo, Forlì; 2 Department of Medical Oncology, S. Maria delle Croci Hospital, Ravenna; 3 Department of Thoracic Surgery and 4 Division of Oncology and Diagnostics, Morgagni Pierantoni Hospital, Forlì, Italy
* Correspondence to: Dr E. Flamini, Division of Oncology and Diagnostics, Morgagni-Pierantoni Hospital, Via Forlanini 34, 47100 Forlì, Italy. Tel: +39-0543-731840; Fax: +39-0543-731736; E-mail: e.flamini{at}ausl.fo.it
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Abstract |
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Background: The aim of the present study was to evaluate the accuracy of two markers, maspin and mammaglobin B, singly or in combination, to detect breast cancer. To define better the potential and limits of the two markers for diagnostic purposes, blood positivity was analyzed in relation to clinical, pathological and biological tumor characteristics.
Patients and methods: The markers were determined in peripheral blood (PB) samples from 27 healthy donors and 140 previously untreated patients using nested reverse transcriptase polymerase chain reaction (RT-PCR).
Results: Positivity for maspin in blood samples was observed in 24% of patients with an 89% specificity. For mammaglobin B, positivity was observed in 7% of patients and never in healthy donors. The presence of maspin was correlated with cell proliferation of the primary tumor (P = 0.015), whereas mammaglobin B positivity correlated with pathological stage (P = 0.013). The presence of either marker was significantly related to nodal status.
Conclusions: Our results indicate that the two markers in association could represent a potentially useful non-invasive tool to detect breast cancer. The validation of these markers as indicators of high risk of relapse is ongoing in a series of patients with an adequate follow-up.
Key words: breast cancer, mammaglobin B, maspin, peripheral blood, RT-PCR
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
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Breast cancer incidence and mortality have decreased in recent years [1
], with early diagnosis through mammographic screening programs and the efficacy of adjuvant systemic treatment in drug-sensitive tumors largely contributing to this improvement. However, breast cancer remains the most common malignancy and the second leading cause of death among women in the Western world [2, 3]. A non-negligible fraction of women with node-negative primary breast cancer submitted to curative surgery or with node-positive operable breast cancer treated with surgery and systemic adjuvant therapy are destined to relapse and die, with a risk that is proportional to the number of involved nodes at time of diagnosis [4]. The early and possibly non-invasive diagnosis of preclinical tumors, therefore, remains an important area of research [5, 6]. In women with familiar predisposition, the analysis of BRCA1 and BRCA2 mutations or polymorphisms represents a valid surveillance tool [7]. In sporadic cancers, some circulating biomarkers such as CEA, CA15-3, CK-19 [8–10] and, more recently, angiogenic growth factors, have been investigated to monitor breast cancer progression or for diagnostic purposes [11–13].
Over the last few years, conventional methodological approaches have been largely replaced by reverse transcriptase polymerase chain reaction (RT-PCR) because of its higher sensitivity [14–18]. In the present study, attention was focused on two tissue-specific breast markers, maspin [8, 19–22] and mammaglobin B [9, 21–28]. The former is a protein related to the serpin family of protease inhibitors that plays a key role in tumor-suppressing activity [8, 19–22]. The latter, a protein of the uteroglobin gene family, is overexpressed in primary breast cancers [9, 21–28] and, albeit not widely investigated, seems to be highly tissue-specific.
The aim of the present study was to evaluate the presence of the two markers in the peripheral blood (PB) of healthy individuals and operable breast cancer patients, also in relation to a number of pathological and biological tumor characteristics. The two transcripts were determined using a highly sensitive nested RT-PCR assay.
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patients and methods |
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TopAbstractintroductionpatients and methodsresultsdiscussionReferences |
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case series
The study was performed with approval of the local Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Twenty-seven healthy females (median age 47 years, range 40–60 years) and 140 patients with previously untreated operable breast cancer (median age 60 years, range 25–88 years) were enrolled in the study. Patients with a past history of cancer or a concurrent second malignancy were excluded. All patients gave written informed consent.
biological samples
PB samples (2.5 ml in Paxgene tubes: PreAnalytix-Qiagen, Hilden, Germany) were collected via a peripheral vein puncture and the first 5 ml were discarded to avoid possible contamination by epidermal cells during collection. PB samples from patients were taken before surgery.
The human mammary carcinoma cell lines MCF-7 and SKBR3 (American Type Culture Collection, Rockville, MD) were used as positive controls. Cells were cultured in DMEM/HAM F12 (50/50) supplemented with 10% fetal calf serum (FCS), 2 mM of L-glutamine, 1% non-essential amino acids and 10 µg/ml of insulin. Cells were harvested from subconfluent cultures into phosphate buffered saline (PBS) containing 0.05% trypsin-0.02% EDTA [29].
RNA extraction and amplification
Total RNA was extracted by PAX-Gene blood RNA kit (PreAnalytix-Qiagen) from control and patient blood samples, whereas RNA isolation from cell lines was performed with RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA was treated with DNAse I (Qiagen). The manufacturer's instructions were followed apart from the incubation time, which was increased from 15 to 30 min to eliminate all genomic DNA residue.
Five hundred nanograms of RNA were reverse-transcribed using the GeneAmp gold RNA PCR Core Kit (Applied Biosystems, Foster City, CA). The final reaction volume was 20 µl. Reaction-mix contained buffer 5 x, 2.5 mM of MgCl2, 10 mM of DTT, 1 mM of dNTPs, 1.25 µM of oligo-dT, 10 units of RNAse inhibitor (20 units/µl) and 15 units of reverse transcriptase enzyme (50 units/µl) (Applied Biosystems). The final mixture was incubated at 25°C for 10 min and then at 42°C for 15 min. Each retrotranscription reaction was set up with a sample of water and reaction mix as negative control. The cDNA was first checked for integrity by a single round RT-PCR using the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH primers were upstream primer 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and downstream primer 5'-CATGTGGGCCATGAGGTCCACCAC-3'. Five microliters of the amplified product were separated electrophoretically on 1.5% agarose gel, stained with ethidium bromide and visualized under UV lights. Maspin and mammaglobin B mRNA was detected by nested PCR based on modified protocols reported by Luppi [19] and Aihara [23]. The external mammaglobin B primers were designed in our laboratory using the Primer program (upstream: 5'-GAA GCT GCT GAT GGT CCT CAT GCT GGC-3' and downstream: 5'-TCT GGC CAT AGT CTG TAG CCC TCT GAG C-3'). After retrotranscription reactions, cDNA amplification was performed in a final volume of 25 µl containing 10x PCR buffer, 0.2 mM of dNTPs, 0.4 µM of primers, 1 unit of TAQ polymerase (Qiagen), and 2 µl and 4 µl of cDNA in the first and second rounds of PCR, respectively. The final fragment was 175 bp for maspin and 245 bp for mammaglobin B. The reaction mixture was subjected to 38 PCR cycles at 94°C for 1 min and at 60°C for 1 min for maspin PCRs and mammaglobin B nested PCR, at 64°C for 1 min for the first round of mammaglobin B PCR, at 58°C for 1 min for GAPDH, and at 72°C for 1 min. The last 3 min of each PCR round were run at 72°C. In all PCR assays, cDNA probes from MCF-7 or SKBR3 cell lines and from blood samples from healthy volunteers were used as positive and negative controls, respectively. A serial dilution assay was performed for each mRNA marker to detect the sensitivity of the RT-PCR experiments. RNA from normal PBMCs was mixed with decreasing amounts of RNA isolated from MCF-7 cells at a tumor cell:PMBC ratio ranging from 1/5 x 102 to 1/5 x 107. To convert the amount of RNA into the number of cells, we calculated the proportion of cells used in serial dilutions with respect to the number of cells used for RNA extraction [9, 25, 28].
One of the internal primers of the nested PCR was end-labeled with fluorochrome 6-FAM provided by Applied Biosystems. Electrophoresis was carried out using a 3100 Avant Genetic Analyzer (Applied Biosystems) equipped with Genescan Analysis 3.7.
All the RT-PCR experiments were run in duplicate. If the results from the two tests were discordant, a third assay was carried out. This occurred in about 15% of healthy donors and in 30% of patients. We also determined CK-19 [22], which is often used as internal control of the RT-PCR assay.
statistical analysis
The 
2 test and Fisher's exact test were performed to evaluate the homogeneity of marker positivity between patient and tumor characteristics. A P value (two-sided) <0.05 was considered statistically significant. Statistical analysis was performed with SPSS.
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results |
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maspin and mammaglobin B expression
In MCF-7 human breast cancer cell lines, we obtained a sensitivity of 1 tumor cell in 5 x 106 PBMCs for maspin and CK-19, and 1 tumor cell in 5 x 105 PBMCs for mammaglobin B. CK-19, tested in a preliminary series, was detected in five of eight healthy individuals. However, because of its lack of specificity and the low reproducibility of results, this marker was abandoned. In healthy individuals, maspin was present in three of 27 (11%) cases, whereas mammaglobin B was never found, indicating a specificity of 100%. In patients, maspin expression was detected in 34 out of 140 cases (24%), and mammaglobin B was expressed in 10 cases (7%) (Table 1). Positivity or negativity to both markers was observed in 89% of healthy donors and in 80% of patients, and at least one of the two markers was present in 41 (29%) patients. Both markers were detectable in only three (2%) cases (tumor
4 cm and positive lymph nodes in two of three patients).
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relation between marker expression and clinicopathological factors
The relationship between marker expression and clinicopathological characteristics is shown in Table 2. The expression of the two markers was not related to patient age. Conversely, the frequency of maspin- and mammaglobin B-positive PB samples was about two-fold (P = 0.041) and three-fold (P = 0.04) higher in node-positive than in node-negative patients, respectively. Furthermore, a direct and significant correlation (P = 0.013) with stage was observed for mammaglobin B but not for maspin. No relation was observed between either of the markers and grading or hormone receptor expression, whereas a significant and direct association (P = 0.015) was observed between the frequency of maspin-positive PB samples and cell proliferation of the primary tumor.
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discussion |
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The relevance of circulating biomarkers for the diagnosis and prediction of early relapse in breast cancer has been widely investigated, but results are inconsistent and available markers have not yet convinced researchers of their usefulness in clinical practice. An important open problem, therefore, remains about their therapeutic implications. Several groups have developed assays based on RT-PCR for the detection of epithelial markers such as CK-19, which has traditionally been used as internal control for the detection of minimal residual disease in RT-PCR. Recently, however, CK-19 has been questioned as a marker of mammary carcinoma cells because of the documented expression of this gene in PBMCs and lymph nodes from healthy subjects [9
, 15, 21, 30, 31].
A large number of studies have reported 100% specificity of maspin RT-PCR [8, 19–21], but have also confirmed its low sensitivity. Conversely, other authors working on lymph nodes or leukapheresis products have obtained a higher sensitivity but have encountered problems with specificity, which, however, were not sufficiently serious to invalidate their results [31, 33].
One study [27] demonstrated the possibility of detecting tumor cells in PB of breast cancer patients through the determination of mammaglobin mRNA, a homolog of mammaglobin B. Patients with mammaglobin-negative PB samples at the time of relapse had a significantly longer survival than those with mammaglobin-positive PB samples. A sensitivity of about 30% was achieved in patients. Other studies using the maspin transcript have reported variable results ranging from a total absence to 38% of positive PB samples [8, 20, 21]. The highest positivity was observed after chemotherapy and this finding was interpreted as a mobilization of tumor cells by the antiblastic treatment [20]. In the last few years, some researchers have improved sensitivity by simultaneously analyzing a higher number of markers together [22, 24, 34–36]. In the present study, our aim was to evaluate CK-19, maspin and mammaglobin B. CK-19 was abandoned almost immediately because of its high rate of false-positives, already reported by other authors [9, 15, 21, 31, 37]. We thus confined our interest to two breast-specific mRNAs, maspin and mammaglobin B. For the assay, a small volume of blood was collected through a tube with a stabilization solution to ensure high quantity RNA. Thanks to the fluorescence evaluation and to the nested PCR conditions used, a lower quantity of RNA was needed in comparison with other methodological approaches [9, 20, 21].
We observed a maspin sensitivity up to 50-fold higher than that reported by authors who obtained a specificity of 100% [8, 19–21]. The higher sensitivity in our study, possibly due to the use of the fluorescence methodology, was one of the causes of false-positive samples. Another potential explanation can be found in the rationale of a recent work, which demonstrated that exposure of healthy donors to specific cytokines induces maspin expression in PB or bone marrow [38].
Conversely, mammaglobin B had a specificity of 100% but a low sensitivity, and its expression was directly related to unfavorable pathological factors such as nodal status and pathological stage. It must be highlighted that, in our case series, thanks to the screening program for the early detection of breast cancer, there were more stage I and II than stage III patients. It is likely that a higher number of stage III patients would have resulted in a higher sensitivity.
In conclusion, there are two important requisites for the success of RT-PCR assay to detect circulating tumor cells, the first being the choice of suitable markers. Although we believe that maspin and mammaglobin B are good markers, we intend to evaluate others, such as mammaglobin, to improve sensitivity and to enable us to carry out multivariate analysis. The second requisite is the use of an accurate quantitative method to have a continuous variable with which to identify the best cut-off to discriminate between healthy individuals and breast cancer patients. We are therefore developing a quantitative method, which will also hopefully enable us to resolve the specificity problems pertaining to CK-19.
After an adequate follow-up, we will perform multivariate analysis to investigate the independent role of these markers as prognostic factors. Finally, in addition to ongoing studies aimed at defining the diagnostic relevance of these biomarkers, other studies are currently investigating the potential of the markers to monitor response to treatment and to predict disease relapse before its detection by clinical evaluation.
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Acknowledgements |
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The authors wish to thank Professor Rosella Silvestrini for her invaluable scientific contribution and Gráinne Tierney for editing the manuscript. The work was supported by Istituto Oncologico Romagnolo, Forlì.
Received for publication July 29, 2005. Revision received November 4, 2005. Accepted for publication November 11, 2005.
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