Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study

A prospective cohort study design was adopted where, upon recruitment, eligible participants with Stage I, IIa, or IIb breast cancer were requested to consent to tissue sampling from axillary lymph nodes (ALN), sentinel nodes (SLN), bone marrow (BM), and peripheral blood (PBL). Tissue sampling was accomplished at the time of surgical intervention. The study was carried out in compliance with the Helsinki Declaration ethical principles in medical research involving human subjects. All specimens were collected through the Medical University of South Carolina Institutional Review Board for Human Research approved protocols (HR 9551, HR 8374, HR 8903, HR 8432). Informed consent was obtained in accordance with each participating center's Institutional Review Board guidelines. The design, enrollment criteria, tissue acquisition protocols, and determination of gene expression values for patients enrolled in the MIMS trial are described in more detail in a separate publication [33]. The current study focuses on the subset of 215 patients with PBL samples and the subset of 177 patients with BM samples. Real-time RT-PCR analyses for cancer-associated genes were performed on all specimens at the Central Molecular Diagnostics Laboratory at the Medical University of South Carolina (MUSC). The Clinical Innovation Group (TCIG, Charleston, SC) (later known as the Data Coordination Unit (DCU) in the Department of Biostatistics, Bioinformatics and Epidemiology at MUSC) served as the coordinating center, and all study data were collected, processed and analyzed at this central facility.

Bone marrow aspirates were obtained from patient's left and or right anterior or posterior iliac crests under anesthesia at the time of operation. A 10 or 20 cc syringe with a 16–18 gauge bone marrow aspirate needle was used to aspirate 3–6 ml of bone marrow into a syringe and then immediately transferred to a sterile EDTA vacutainer. Peripheral blood samples were obtained before surgery or following the induction of anesthesia. A total of 5–10 ml of blood was drawn from a peripheral vein into a sterile EDTA vacutainer. Blood and bone marrow samples were then shipped at room temperature to the Central Molecular Diagnostics Laboratory at the MUSC for immediate processing by Ficoll density gradient centrifugation (Ficoll-Paque Plus; Amersham Biosciences). All the specimens inside US arrived in 24 hours and international shipments arrived in 48 hours. One mL of bone marrow was used for Cytospin preparation and stained for ICC analysis. These bone marrow samples were evaluated by a cytopathologist for the presence of micrometastases using cytokeratin AE1/AE3. Please note that the specimen acquisition protocol was amended after the initiation of the MIMS trial and for that reason only a subset of patients was included in this analysis.

In order to define baseline expression levels for the molecular markers used in this study, PBL and BM samples from control subjects were procured. Informed consent was obtained for BM aspiration from 49 patients undergoing orthopedic surgery at MUSC and for PBL drawn from 49 healthy volunteers. None of the control subjects had any history or clinical evidence of malignancy. Four to six ml of BM aspirate or 5–10 ml of PBL was transferred to an EDTA vacutainer and sent to the Central Molecular Diagnostics Laboratory to be processed by Ficoll density gradient centrifugation and analyzed by real-time RT-PCR.

Buffy coats were obtained by Ficoll density gradient centrifugation, and total cellular RNA was isolated using a guanidinium thiocyanate-phenol-chloroform solution (RNA STAT-60™; TEL-TEST, Friendswood, TX). Briefly, cells were re-suspended in 1 ml of RNA STAT-60™. Total RNA was isolated as per the manufacturer's instructions with the exception that 1 μL of a 50 mg/mL solution of glycogen (Sigma, St. Louis, MO) was added to the aqueous phase prior to addition of isopropanol. Glycogen was used as a nucleic acid carrier to enhance RNA precipitation. The RNA pellet was dissolved in 50 μl of 1x RNA secure buffer (Ambion, Austin, TX). RNA was quantified by spectrophotometry at 260 nm. cDNA was made from 5 μg of total RNA using 200 U of M-MLV reverse transcriptase (Promega, Madison, WI) and 0.5 μg Oligo (dT)_12–16 in a reaction volume of 20 μl (10 min at 70°C, 50 min at 42°C, 15 min at 70°C).

The real-time RT-PCR primers have been previously reported [31, 36, 37]: mglo: F 5'-GCCGTGTGAACCATGTGACTTT, R 5'-CCAAATGCGGCATCTTCAAA; PDEF: F 5'-AGTGCTCAAGGACATCGAGACG, R 5'-AGCCACTTCTGCACATTGCTG; mam: F 5'-CGGATGAAACTCTGAGCAATGT, R 5'-CTGCAGTTCTGTGAGCCAAAG; CK19: F 5'-CATGAAAGCTGCCTTGGAAGA, R 5'-TGATTCTGCCGCTCACTATCAG; muc1: F 5'-ACCATCCTATGAGCGAGTACC, R 5'-ACCATCCTATGAGCGAGTACC; PIP: F 5'-GCCAACAAAGCTCAGGACAAC, R 5'-GCAGTGACTTCGTCATTTGGAC; EpCAM: F 5'-CGCAGCTCAGGAAGAATGTG, R 5'-TGAAGTACACTGGCATTGACGA; ErbB2: F 5'-CTGGTGACACAGCTTATGCCCT, R 5'-ATCCCCTTGGCAATCTGCA. Analyses were performed on a PE Biosystems Gene Amp^® 5700 Sequence Detection System (Foster City, CA). All reaction components were purchased from PE Biosystems. The standard reaction volume was 10 μl and contained 1X SYBR Green PCR Buffer; 3.5 mM MgCl₂; 0.2 mM each of dATP, dCTP, and dGTP; 0.4 mM of dUTP; 0.25 U AmpliTaq Gold^®; 0.1 U AmpErase^® UNG enzyme; 0.7 μl cDNA template; and 0.25 mM of both forward and reverse primer. The initial step of PCR was 2 min at 50°C for AmpErase^® UNG activation, followed by a 10-min hold at 95°C. Cycles (n = 40) consisted of a 15 sec denaturation step at 95°C, followed by a 1 min annealing/extension step at 60°C. The final step was a 60°C incubation for 1 min. All reactions were performed in triplicate. The cycle of threshold (C_t) analysis was set at 0.5 relative fluorescence units.

Real-time RT-PCR data were quantified as C_t values that are inversely related to the amount of starting template: high C_t values correlate with low levels of gene expression, whereas low C_t values correlate with high levels of gene expression. Each gene was analyzed in triplicate. Results were normalized to an internal control reference gene, β2-microglobin, by subtracting the mean C_t value of β2-microglobin from the mean C_t value of each respective gene (ΔC_t value). Samples for which C_t values for β ₂ -microglobin were equal or higher than 22 were considered to contain inadequate RNA and were excluded from the analysis. Approximately 10% of samples we rejected from the analysis based on this criterion. If the mean C_t value for a gene of interest was higher or equal to 38, the gene expression was considered to be undetectable. In order to define baseline levels of gene expression and to define thresholds for marker positivity, 49 specimens of PBL and 49 specimens of BM obtained from patients with no evidence of malignancy were analyzed. To be consistent with the previous molecular analyses of lymph nodes, threshold values for each individual marker were set at three standard deviations from the mean ΔC_t value in the control group. A subject was considered to be positive for the molecular analysis if at least one marker in the panel was above the defined threshold. Data from real-time RT-PCR analyses were compiled in a Microsoft Access database and submitted to the DCU at MUSC for statistical analyses. The molecular analysis was generated blinded to clinical outcome and patients' clinicopathologic data.

Specimens were collected, washed in CytoLyt^® (Cytyc, Boston, MA) and then resuspended in PreservCyt^® (Cytyc). Two ThinPrep (TP) slides were prepared and stained with Papanicolaou stain, and one slide was used for immunocytochemistry (ICC). A monoclonal antibody for cytokeratin (AE1/AE3) was used in conjunction with an automated immunostaining system (DAKO Autostainer, DAKO Cytomation, Carpeteria, CA) and a Nexus immunohistochemistry slide staining apparatus (Ventana Medical Systems Inc, Tuscon, AZ). Immunostaining was performed with the avidin-biotin immunoperoxidase (ABC-peroxidase) method of Hsu et al [38]. Briefly, the slides were incubated with primary antibody for 30 minutes and then incubated with secondary biotinylated antibody for 4 minutes. To visualize the antibody, the TP was treated with diaminobenzidine (0.05%) in 0.05 M Tris-HCL buffer (pH 7.8) with 0.03% H₂O₂ for 6 minutes and then washed in H₂O. TP was counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted in Permount. The specimens were analyzed by a skilled cytopathologist.

SAS Version 9.1 Software (SAS Institute Inc., SAS Campus Drive, Cary, North Carolina) was used for the analysis of pathological and molecular outcome. Chi-square analyses were conducted to explore the association between pre-defined baseline covariates that have been associated with pathological outcome in prior studies and PBL and BM RT-PCR positivity/negativity status. Pre-defined baseline covariates were tumor size, histological grade, estrogen receptor status, progesterone receptor status, her2neu status, and St. Gallen risk category (minimal/low risk: tumor size ≤ 1 cm, positive ER and/or PR status, grade I and age ≥ 35; intermediate risk: tumor size >1 or 2 cm, positive ER and/or PR status, and grade I; and high risk: lymph node positive, tumor size > 2 cm, negative ER and/or PR status, grade II or III, or age <35) [39]. Statistical significance was defined as p-values < 0.05.

The distribution of the demographic and clinicopathologic characteristics in Table 1 indicate that the subset of patients with PBL analysis (n = 215) and the subset of patients with BM analysis (n = 177) are representative of the entire study group of 489 [33].

We have previously shown that the majority of known breast cancer-associated genes have some background expression in normal lymph nodes [31, 36, 37]. For this study we selected seven breast cancer-associated genes [mam, CEA, CK19, PIP, muc1, PSE, Erb (BM only) and EpCAM (PBL only)] known to be over-expressed in metastatic breast cancer compared to control lymph nodes [31, 36, 37]. For this study, baseline gene expression was precisely quantitated in 49 normal PBL samples and 49 normal BM samples by real-time RT-PCR (Figure 1A and 1B; horizontal lines indicate the ΔCt thresholds). To obtain maximum specificity, a threshold value for marker positivity, i.e. abnormal expression was set at three standard deviations from the mean ΔC_t value for each gene. Out of seven cancer-associated gene-markers used to detect tumor cells in PBL and BM, CK19, muc1 and ErbB2 were not informative due to the high expression in normal control samples.

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Using the five-marker gene-panel (mam, PIP, CEA, PSE and EpCAM) at the threshold of three standard deviations above the mean expression level in normal control samples for each gene, 136 (63%) patients out of 215 were positive for at least one marker. On an individual marker basis (Table 2), the most frequently over-expressed markers were PSE (58/215; 27.0%) and CEA (51/215; 23.7%) followed by PIP (36/215; 16.7%), mam (29/215; 13.5%) and EpCAM (7/215; 3.3%). Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083; Table 3). Out of 136 RT-PCR positive patients 97 patients (71%) were positive for one, 33 patients (24%) for two and six patients (4%) for three markers. Interestingly, over-expression of PSE gene had statistically significant association with ER-positive and PR-positive tumors (p = 0.0123 and p = 0.0134, respectively) and showed a trend towards pathology-negative nodal status (31% vs. 19%; Table 3). However, overexpression of mam gene had statistically significant association with high grade (p = 0.0315) and showed a trend towards ER-negative tumors (22% vs. 11%) and a high risk category (15% vs. 6%; Table 3). Interestingly, there was no association between marker positivity in PBL and either pathologic (H&E) status or molecular (multi-marker qRT-PCR) status of axillary lymph nodes.

Using a four-marker gene-panel (mam, PIP, CEA, and PSE) at the threshold of three standard deviations above the mean expression level in normal control samples for each gene, 19 patients (11%) out of 177 were positive. All 19 were positive to one marker only. Marker positivity in bone marrow had no statistically significant association with any of the traditional prognostic indicators. Looking at individual markers separately (Table 2), the most frequently overexpressed marker was mam (7/177; 4.0%) followed by PIP (5/177; 2.8%), PSE (5/177; 2.8%) and CEA (2/177; 1.1%)

To determine whether there was an association between molecular analysis in PBL and molecular analysis in BM, we performed Chi-Square and Fisher's Exact test on 138 patients that had results from both PBL and from BM (Table 4). Comparison of the results using gene-panel data did not show statistically significant association, however, the results of mam and PIP gene expression in PBL had statistically significant association with the mam and PIP gene expression in BM (p = 2.5E-04 and p = 0.0188, respectively).

BM cytopathology assessment resulted in detection of no abnormal or suspicious cells. Eighty three BM samples were randomly selected for additional cytokeratin ICC staining. Five out of 83 (6%) samples were positive by ICC and two of these samples were also positive by RT-PCR (one positive for mam and other for PIP). Ten patients out of 83 (12%) that had inconclusive ICC results were all RT-PCR negative (Table 5). Although there was 84% agreement (excluding inconclusive ICC results) between 2 methodologies, this was mostly because of the concordance of dual negative findings. Overall there was no statistically significant association between ICC and PCR data (Chi-Square 0.1064; Fisher's exact test 0.1607; ICC inconclusive results excluded).